Bacterial wilt on potato The South African Experience 2014.pdf

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Review

BACTERIAL WILT ON POTATO: THE SOUTH AFRICAN EXPERIENCE

A review written by P.F. Nortjé 2015

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PREFACE One of the most important crops under cultivation in South Africa today is the potato, and as in every other potato-producing country around the world, the successful production of potatoes in South Africa is threatened by a number of bacterial, fungal and viral diseases. The optimal production of potatoes has become a science, with scientific farming techniques and practices being essential in the successful delivery of an acceptable, topquality product to the highly discerning consumer market. Effective disease management is necessary to control and contain the many diseases plaguing the successful production of potatoes. The bacterial disease Ralstonia solanacearum, commonly known as bacterial wilt, was first discovered in South African potatoes in 1914, but only a few incidences of the disease were observed and reported during the five decades that followed. However, there was a sudden and inexplicable increase in the incidence of the disease in the late 1970s and early 1980s in registered seed potato plantings and also in commercial fields. The latent infection of seed potatoes in relatively cooler production areas must undoubtedly have played a significant role in the spread of the disease. The same applies to farm saved seed, which is a common practice in some production areas. The confirmation of many diagnostic samples served as a wake-up call for the South African potato industry. This report spans a period of 22 years, during which the potato industry as well as research institutions dug deep to address the serious threat posed by bacterial diseases affecting potatoes. Stringent management measures were introduced by the seed potato industry to detect and contain the presence of disease, with intensive research projects being launched to study the disease under local conditions in view of advising the industry on the characterisation and detection of the pathogen, the survival potential of the disease, and the efficacy of various control measures. The first chapter of this review deals with the role of the South African potato industry, including the management strategies employed, as well as the structural changes brought about in the seed potato industry during the 1990s, which played a crucial role in the containment of bacterial wilt. The chapters that follow contain summaries of three successfully completed MSc studies on bacterial wilt in potatoes, as well as several other scientific research studies. For practical reasons, these studies are presented on a subject basis in order to facilitate the reading of the report and to demonstrate what has been achieved thus far by research colleagues in the respective fields of interest. As a former Research Manager and CEO of the Potato Certification Service, I consider myself privileged to be given the opportunity to compile this report and to have been personally involved in the application of the management strategies developed by the seed potato industry. I gratefully acknowledge the tremendous support of all the role players involved, particularly the seed potato growers, the Department of Agriculture, Forestry and Fisheries and my respected colleagues who helped to ensure the success of our efforts to contain the spread of this dreaded disease. A word of caution, however, vigilant implementation of administrative and testing protocols and procedures should be upheld at all times. A special word of thanks goes to Dr Fienie Niederwieser (Research Manager at Potatoes South Africa) for her invaluable advice, recommendations and support throughout the process of compiling this report.

P.F. Nortje (PhD) Agrinor Consultants (December, 2014)

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CONTENT Page Preface

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Acknowledgements

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Abbreviations

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Executive summary

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Chapter 1. The role of the South African ptato industry in combating bacterial wilt

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Chapter 2. Literature review

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Chapter 3. Characterisation and detection studies on Ralstonia solanacearum

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Chapter 4. Host plant studies

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Chapter 5. Survival in soil and the influence of temperature on the development of bacterial wilt

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Chapter 6. Aspects of the management of bacterial wilt

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References

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Outputs

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Annexure 1

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Annexure 2

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Annexure 3

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Annexure 4

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Annexure 5

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Annexure 6

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ACKNOWLEDGEMENTS This report comprises the work of six researchers, with three of these studies having culminated in M.Sc. degrees, all awarded by the University of Pretoria:

1. A.E. Swanepoel: Vegetable and Ornamental Plants Institute of the Agricultural Research Council Scientific articles:  Swanepoel, A.E. 1988. Characteristics of South African strains of Pseudomonas solanacearum. Plant Disease, 72: 403-405.  Swanepoel, A.E. 1990. The effect of temperature on the development of wilting and on progeny tuber infection of potatoes inoculated with South African strains of biovar 2 and 3 of Pseudomonas solanacearum. Potato Research, 33: 287-290.  Swanepoel, A.E. 1992. Survival of South African strains of biovar 2 and biovar 3 of Pseudomonas solanacearum in the roots and stems of weeds. Potato Research, 35(3): 329-332.  Swanepoel, A.E. and Theron, D.J. 1999. Control measures for bacterial wilt, caused by Ralstonia solanacearum, as applied by the South African Potato Certification Scheme. In: A. Leone, S. Foti, P. Ranalli, A. Sonnino, V. Vecchio, A. Zoina, L. Monti and L. Frusciante (Eds.). Abstracts of Conference Papers, Posters and Demonstrations. 14th Triennial Conference of the European Association for Potato Research. Sorrento, Italy. 2-7 May 1999, pp. 241-242.

2. D.U. Bellstedt and K.J. van der Merwe: Stellenbosch University Paper read: Bellstedt, D.U. and Van der Merwe, K.J. 1989. The development of ELISA kits for the detection of Pseudomonas solanacearum bacterial wilt in potatoes. In: Potato Research Symposium. Warmbaths, South Africa. 1-2 August 1989, pp. 64-69. Chapter in book: BELLSTEDT, D.U. 2009. Enzyme-linked immunosorbent assay detection of Ralstonia solanacearum in potatoes: the South African experience. In Methods in Molecular Biology, Volume 508: Plant pathology: techniques and protocols, pp. 51-62, The Humana Press, New Jersey, USA.

3. N.J.J. Mienie: Vegetable and Ornamental Plants Institute of the Agricultural Research Council Mienie, N.J.J. 1998. An integrated management approach to the control of bacterial wilt in potatoes. M.Sc. Dissertation, University of Pretoria. Research team: Ms. L. Urquhart (ARC Roodeplaat), Ms K Phetla and Ms Anna Raphulo Prof. P.L. Steyn – Promoter (University of Pretoria) Prof. A.C. Hayward – Co-promoter Dr E. van Zyl – Co-promoter Completion date: 1998

4. E.I.M. Stander: University of Pretoria Stander, E.I.M. 2001. Cultural practices for the control of bacterial wilt of potato. M.Sc. Dissertation, University of Pretoria. Research team: Ms. W. von Broekhuizen (University of Pretoria)

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Prof. P.S. Hammes – Promoter (University of Pretoria) Prof. L. Korsten – Promoter (University of Pretoria) Starting date: 1994 Completion date: 2001

5. W. van Broekhuizen: University of Pretoria Van Broekhuizen, W. 2002. Detection, characterisation and suppression of Ralstonia solanacearum. M.Sc. Dissertation, University of Pretoria. Research team: Prof. L. Korsten – Promoter (University of Pretoria) Prof. P.S. Hammes – Co-promoter (University of Pretoria) Dr G. Swart (University of Pretoria) Dr M. Oosthuizen (University of Pretoria) Starting date: Mid-1990s Completion date: 2002

6. A. Espach: Coen Bezuidenhout Testing Laboratory (now known as Plantovita) Espach, A. 2008. Internal Report: Investigation into hosts for Ralstonia solanacearum, biovar 2 and biovar 3, as listed in Table 1 of the South African Seed Potato Certification Scheme. Pretoria: Potato Certification Service.

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ABBREVIATIONS ARC: Agricultural Research Council ICCSP: Independent Certification Council for Seed Potatoes PCS: Potato Certification Service PLS: Potato Laboratory Service PPO: Potato Producers’ Organisation PSA: Potatoes South Africa SU: Stellenbosch University TZC: Tetrazoliumchloride UP: University of Pretoria

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EXECUTIVE SUMMARY 1

The most destructive bacterial disease of potatoes, bacterial wilt, is caused by the pathogen Ralstonia solanacearum. This disease was relatively unknown in the South African potato industry until the mid-1970s, when there were confirmed visual detections in both seed potato and commercial potato plantings. The disease is prohibited in the seed potato industry, with zero tolerance status in the Seed Potato Certification Scheme. Interestingly, after the first incidence of the disease was reported in South Africa in 1914, only four incidences were reported in all the years leading up to 1970, in the Limpopo and Western Cape provinces, at which point the number of confirmed cases across the country inexplicably began to rise. This sudden increase in outbreaks was a cause of great concern in the potato industry at that time, with desperate appeals to the country’s research institutions to urgently find a means to control the disease.

2

Historically, bacterial wilt was known to occur primarily in tomatoes in the subtropical lowlands of the Eastern Transvaal (now known as Mpumalanga), the coastal regions of KwaZulu-Natal, and the Cape Flats region of the Western Cape, with the disease being confirmed in tobacco only in the 1980s. A survey conducted by the Vegetable and Ornamental Plant Research Institute from 1984 to 1986 was one of the early measures taken in an effort to determine the identity and distribution of the different biovars of the pathogen.

3

Several research projects were subsequently launched in the early 1990s in order to investigate the different aspects of the disease, including its survival capacity in the soil, the range of crop and weed hosts, the development of detection methods, characterisation studies and the evaluation of herbal species as bio-fumigation agents, the effects of low temperature on multiplication and survival of the pathogen, the investigation of bacterial antagonism, and the evaluation of the efficacy of disinfectants against R. solanacearum.

4

Several comprehensive surveys and characterisation studies have shown that biovar 2 (race 3) is the most prevalent biovar on potatoes in South Africa, causing infection in cooler regions and in high-altitude locations. Moreover, latent infection of tubers in cooler climates by biovar 2 can result in the increased spread of the pathogen, with potatoes, tomatoes and certain weed species being most susceptible. Biovar 3 (race 1) occurs mostly in the tropical and subtropical regions of the country, with a wide range of hosts in terms of crops and weed species.

5

The seed potato industry first started testing all registered seed potato plantings for the presence of bacterial wilt in 1995. This involves the sampling of fields by certification officials according to a sampling method approved for use in the United States of America to combat the dreaded ring rot (Clavibacter mitchiganensis) disease. The seed potato samples are subsequently tested for the presence of bacterial wilt by registered and approved laboratories, by means of an ELISA test developed and manufactured by Stellenbosch University. Any sample that tests positive for bacterial wilt with the ELISA test is then confirmed by the controlling laboratory by means of conventional plating-out techniques.

6

The Independent Certification Council for Seed Potatoes (ICCSP), the Potato Certification Service (PCS) and the Department of Agriculture, Forestry and Fisheries (DAFF) jointly compiled a protocol for the administration and management of seed potato farms where incidents of bacterial wilt have been confirmed. As such, a Bacterial Wilt Committee was formed with the purpose of investigating confirmed cases of wilt on farms, and to then recommend whether seed potato production should be allowed to continue on the infested farm, and if so, under what conditions. Should the Committee’s recommendations be approved by the ICCSP and DAFF, the PCS is then responsible for monitoring that the seed grower in question does in fact implement those recommendations.

7

The introduction of compulsory testing for all registered seed potato plantings in 1995 coincided with some major changes in the seed potato industry. A revised certification scheme and seed potato programme, consisting of a revolutionary (for South Africa) limited-generation concept with a dual phasing-out system, was introduced, to be administered by an advanced management database with traceability capabilities that would serve to promote sound and orderly management. At the same time, the necessary testing laboratories were established and registered, while

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testing protocols and procedures were drawn up and standardised. This new scheme, along with effective laboratory procedures and good support by seed growers, contributed greatly to the establishment of a successful management and containment programme for bacterial wilt disease. 8

Research projects on bacterial wilt in potatoes commenced in the early 1980s at the Vegetable and Ornamental Research Institute and the University of Pretoria, culminating in the awarding of three M.Sc.aaaaaaa degrees by the University of Pretoria between 1998 and 2002. The final research project, conducted by the controlling test laboratory, Plantovita, to investigate the host status of crops and weeds as listed in Table 1 of the Certification Scheme, was completed in 2008. The results of these research efforts were published in several international scientific journals, as well as in the local agricultural press. Details of the ELISA test kit developed and manufactured by Stellenbosch University, which is the test currently used for certification purposes in South Africa, were also published in an international scientific journal.

9

In South Africa, R. solanacearum was reported to survive in the soil for much longer periods than previously reported in other countries. In a soil survival experiment, milky exudates from potato stems of the potato monoculture treatments were observed eight years after the initial infestation of the soil. Maize monoculture, weed fallow and bare fallow treatments also realised positive results for the pathogen from potato indicator plants. After 10 years, the entire trial area was planted to potatoes, and the pathogen could not be isolated from any of the treatments. These results should be treated with caution, however, as the pathogen may have migrated into deeper soil layers in the dry and unfavourable conditions. The soil of the experimental site used in this instance was a clay-loam soil, and it is a known fact that survival of the pathogen is influenced by a variety of factors, including soil type.

10

The role of weeds in the perpetuation of bacterial wilt was also amongst the factors investigated. As such, biovar 2 was isolated from Datura ferox, D. stramonium, Portulaca oleraceae and Hibiscus trionum, although wilting was observed in only some of the P. oleraceae plants. The host range for biovar 3 was much wider and included Amaranthus spp., Bidens bipinnata, Chamaesyce prostata, Chenopodium album, Chenopodium carinatum, Cyprus rotundus, Datura ferox, Datura stramonium, Eragrostis curvula, Hibiscus trionum, Portulaca oleraceae, Sonchus oleraceus and Tragopogon dubius. Although biovar 3 was isolated from 14 species, only five species showed wilting symptoms. A study by Stander (2001) identified several weed species to be hosts of bacterial wilt for the first time in South Africa. In a similar study by Swanepoel (1992) biovar 2 was isolated from wild gooseberry (Physalis angulata). These studies confirmed the ability of the pathogen to survive in weeds, thus confirming the importance of weed control in infested soil. Both the aforementioned studies confirmed weeping love grass (E. curvula) to be a host of biovar 3, although no wilting symptoms were observed.

11

Each year sees the addition of more crop species to the list of R. solanacearum hosts. As in the case of weeds, the range of hosts is much wider for biovar 3 than for biovar 2. Some crops are listed as hosts of biovar 2 or biovar 3 in certain countries, but not in others. There are many different factors that can affect the outcome of such studies, with some strains of a specific biovar not necessarily infecting the same hosts in different countries. The crops listed in Table 1 of the Potato Certification Scheme were all confirmed by Espach (2008) as being hosts of either biovar 2 or biovar 3. Mienie (1998) evaluated the host status of 26 crops, finding cucumber, muskmelon, lettuce, beans, peas, lucerne, barley, maize, oats, sorghum, wheat, onions, beets, carrots, parsley, sweet potato and strawberry to be nonhosts of R. solanacearum and thus suitable for use as rotational crops. On the other hand, other countries have confirmed cucumbers, beans, peas, onions, beets and carrots as being hosts for bacterial wilt. It is evident that the overall issue of crop hosts in South Africa must be clarified as a matter of urgency, keeping in mind that the list of recommended rotational crops will become more limited, thus placing increasing economic pressure on seed potato growers. It may therefore become necessary to evaluate the risk of bacterial wilt infection caused by certain crops currently included in Table 1 of the Scheme, and also when considering the addition of crops to Table 1, seeing that seed potatoes are subjected to extensive testing before certification is concluded.

12

Preliminary in vitro studies aimed at determining the suppressiveness of certain weeds/grasses have found that microbial activity associated with certain weeds could be involved in suppression of the wilt organism, but further studies in this regard are needed. In addition, the effect of maize on R. solanacearum populations was evaluated in both a pot

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trial and in hydroponic culture, with results indicating that microbial populations present in the maize plant could play a role in the susceptibility of maize to bacterial wilt infection. As such, antagonistic bacteria associated with some maize plants or with the maize rhizosphere could be partly responsible for suppression of wilt. An antagonist, subsequently tentatively identified as Chromobacterium violaceum, was consistently found in the solution, as well as in the maize plants. 13

Thirteen herbal species were evaluated for their potential use as bio-fumigation agents to suppress R. solanacearum in soil. Unfortunately, certain problems were experienced with soil inoculation, meaning that suppression of the pathogen could not be evaluated successfully. There could be several possible reasons why no wilt symptoms developed and why all tissue isolations tested negative for the presence of R. solanacearum, such as low inoculum concentration, low soil temperatures, the dilution factor created by the soil, the soil in pots being too dry, or the loss of virulence.

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According to Mienie (1998), biological control has the potential to be included as an integral part of an integrated control strategy for bacterial wilt. The antagonist Pseudomonas resinovorans isolated from maize exhibited promising results in terms of reducing the percentage of wilt in potato plants.

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The effect of incubation temperature, storage period and inoculum concentration on the multiplication and survival of R. solanacearum in tubers was investigated. It must be noted that cold storage (4°C) of tubers (with latent infection) for 10 weeks is no guarantee that the potatoes will be free from bacterial wilt. Viable counts dropped and remained relatively low whilst in cold storage, but increased exponentially after removal from storage.

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In vitro evaluation of disinfectants against bacterial wilt found Chlorox, Jeyes Fluid, HTH and Sporekill to be bactericidal products suitable for recommendation for sanitation purposes.

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A trapping technique based on the use of indicator plants was successfully developed to detect R. solanacearum from artificially infected soil suspensions. However, it was found to not be rapid enough for commercial use and thus not a suitable replacement for the existing ELISA and selective media diagnostic methods.

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Various molecular techniques were evaluated for the identification and characterisation of different R. solanacearum isolates. The ERIC-PCR, which was used on eight biovar 2 and biovar 3 isolates, successfully distinguished between the two groups. The RISA-PCR and RFLP with Sau3A were used to characterise 44 R. solanacearum isolates. Although the techniques were able to distinguish between the two groups of biovars, it proved impossible to draw a correlation between the isolates and the different regions from which they were isolated.

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Chapter 1

THE ROLE OF THE SOUTH AFRICAN POTATO INDUSTRY IN COMBATING BACTERIAL WILT 1.1

BACKGROUND

Bacterial wilt, also known as brown rot or “vrotpootjie”, is a destructive and widespread disease caused by the Ralstonia solanacearum pathogen that affects potatoes in temperate, subtropical and tropical regions throughout the world. It also results in substantial losses in other crops like tomatoes, eggplants, peppers, tobacco and bananas (Moko disease). Bacterial wilt was first detected in South Africa in 1914, firstly in potatoes and thereafter in several other crops such as tomatoes, peppers, eggplants, peanuts and tobacco (Granville disease). In the eastern and central African regions, bacterial wilt is considered one of the most limiting factors for potato production. As the causal organism is capable of surviving in the soil for long periods of time, and with a wide host range that includes several different weed species, it poses a severe threat to the South African potato industry if not contained. It is of the utmost importance that effective control measures are implemented in an effort to curb and prevent the spread of the disease. South Africa is a country of diverse soil and climatic conditions, varying from sandy to heavy soils, and from temperate to subtropical growing conditions. Potatoes are produced in 14 different regions, with seed potatoes being grown in at least 10 of these regions. Growing conditions for potatoes in South Africa are generally warmer than in most other countries, but it is in the cooler areas that latent tuber infection presents a problem, because wilting symptoms are not pronounced or are totally absent, and harvested tubers appear healthy. It should also be kept in mind that it is not compulsory in South Africa to plant only certified seed potatoes. It is also a commom (not recommended) practice for ware-potato growers to retain seed from commercial plantings and even to multiply seed more than once. After being detected for the first time in 1914, very few incidences of bacterial wilt on potatoes were documented in South Africa up until 1980, but thereafter the number of confirmed incidences of the disease on potatoes increased. At that time, bacterial wilt had already been declared a prohibited organism by the Department of Agriculture, and the then-voluntary Seed Potato Certification Scheme had specific regulations in place to limit the spread of bacterial wilt. However, control and management were limited to visual field inspections in registered seed-potato plantings. If wilt symptoms on plants happened to be encountered during such field inspections, the plants were then subjected to laboratory testing, and if the causal organism for bacterial wilt was subsequently confirmed, the registered planting was then rejected and certain isolation requirements were imposed on the infested field. A serological test (ELISA), developed locally in the 1980s, is still in use today as the method of testing for the presence of bacterial wilt in all registered seed-potato plantings. Compulsory statistical sampling and testing for the presence of bacterial wilt in registered seed-potato plantings was introduced in the Nineties, forming part of the strict regulatory process in effect. The increase in confirmed bacterial wilt incidences in potato plantings during the late Seventies and Eighties resulted in a concentrated research focus on this disease. Several scientific articles were subsequently published, with three M.Sc. degrees awarded for research on the disease. This chapter is aimed at presenting a review of the bacterial wilt experience in terms of South African potatoes, with a discussion of the management strategy introduced by the potato industry – and specifically the Independent Certification Council for Seed Potatoes – to curb the spread of bacterial wilt. The research conducted on the disease will be discussed in later chapters. The report will serve to provide potato farmers, seed growers, researchers and technical advisors with information on the status of the disease in South Africa.

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1.2

OCCURRENCE OF BACTERIAL WILT IN SOUTH AFRICA

Bacterial wilt in potatoes was considered an extremely rare disease in South Africa up until the late Seventies, with the disease having been confirmed only in the vicinity of the Bon Accord Dam near Pretoria and perhaps one or two other areas in South Africa, but with the exact locations unknown. The first incidence of the disease in potatoes was reported in 1914, with the disease subsequently being detected and confirmed in two locations in the town of Stellenbosch, but with no recording of the actual year in each case. Thereafter, one incidence of the disease was reported in the area now known as Gauteng, and five incidences were reported in the Western Cape region. It is not known whether these incidences occurred in seed-potato plantings or in ware-potato plantings. Since 1979, incidences of the disease have been confirmed in different regions, especially in the Western Cape, the Limpopo Province, the Gamtoos Valley in the Eastern Cape, and in KwaZulu-Natal. The diagnostic samples in these cases originated mainly from ware-potato plantings (Figure 1.2.1). The practice of retaining seed from ware-potato plantings was extremely popular amongst ware-potato farmers at that time, and it deserves mention that ignorance regarding the risk of seed-borne bacterial diseases was commonplace. Confirmed incidences of bacterial wilt originating from registered seed-potato plantings were reported as from 1989 (Fig 1.2.1). It cannot be stated as fact that all such positive cases were as a result of infected soils, since in some cases the seed potatoes that were planted had originated from registered seed-potato plantings, pointing to the possibility of latent infections in certified seed potatoes. The Roodeplaat Vegetable and Ornamental Plant Research Institute was responsible for the testing of most of those early diagnostic samples that were collected by farmers, extension officers and certification officers during the process of conducting field inspections. These incidences of the disease sparked a renewed interest in bacterial wilt amongst researchers, and caused great concern in the seed potato industry in the 1990s. During this period the seed potato industry developed a new certification system, which later resulted in a marked improvement in orderly seed testing procedures and seed production regulations. The ‘basic’ seed certification scheme implied that seed could be multiplied for an unlimited number of generations, as long as the seed was visually in compliance with the certification standards. The limited-generation scheme and compulsory testing for R. solanacearum in all seed-potato plantings were proposed to seed growers at open meetings in all seed potato production regions. Seed growers subsequently approved the proposed seed programme and committed themselves to the effort to combat this “new threat” of bacterial wilt disease, by means of every control measure possible. The revised Certification Scheme, which became effective in 1995, was based on limited-generation multiplication and included a management database to improve traceability. The new limited-generation Certification Scheme was promulgated under the Plant Improvement Act of 1976 (Act No. 53 of 1976) during May 1998, and it undoubtedly played a role in reducing the incidence of bacterial wilt in the late Nineties, in both registered seed-potato plantings and the ware-potato industry. It should be acknowledged that it was impossible to trace the source of any bacterial wilt incidence in seed potatoes during those early days, as the “basic” Certification Scheme did not provide traceability options unless there were more than one recipient of the same seed source experiencing bacterial wilt disease. The traceability function of the database that forms the foundation of the Certification Scheme enables the Certification Service to trace the origin of any seed potato lot back to the original greenhouse where the test tube plantlets had been grown. The detection of bacterial wilt during field inspections is effective where there are visible symptoms of the disease. However, during cooler periods especially, the disease is able to be present in latent form in plants and developing tubers. It is impossible to visually detect the disease under such conditions and where it is in a latent state. In response to this problem, the potato industry funded the development of a serological test procedure (ELISA) by Stellenbosch University in the 1980s. After intensive evaluation and validation of the technique by the Potato Laboratory Services, the ELISA test was implemented in 1996 for the testing of all registered seed-potato plantings.

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Figure 1.2 1. Occurrence of confirmed incidences of bacterial wilt in South African seed potatoes and commercial plantings from 1979 – 2014, as well as notable milestones and developments in the seed industry 2014. Revision of role of Bacterial Wilt Committee 1

2014 2013 2012 2011 2010 2009

2008. Investigation into hosts in Table 1

2008 2007 2006 2005 2004 2003

2002. MSc Van Broekhuizen 2002 2001. MSc Stander

2001 2000 1999

1998. Promulgation of limited-generation scheme: MSc Mienie

1998

1997. Formation of ICCSP, PCS, PLC

1997

1996. Introduction of ELISA testing for bacterial wilt 1996 1995. Introduction of limited-generation scheme 1995 introduced 1994. Privatisation of Potato Board to become PPO, with compulsory 1994 testing for R. solanacearum

1993 1992

1991. Potato Board responsible for managing the Scheme 1991 1990

1989. Potato Board responsible for inspection and certification

1989

1988. Identification of R solanacearum by Swanepoel 1988 1987 1986 1985 1984

1983. Introduction of ‘Basic Seed’ Scheme, along with in vitro propagation 1983 1982

1981. Potato Board responsible for multiplication of basic seed Pre 1981. Department of Agriculture responsible for the Scheme and the multiplication of basic seed

1981 1980 1979

Pre 1979

1914. Identification of bacterial wilt for the first time in South Africa

0

10

20

30

40

Number of positive bacterial wilt incidences

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The prodigious increase in confirmed cases in 1994 coincides with the compulsory testing of all registered seed-potato plantings introduced in 1994. This increase points to the detection of latent infections that would not have been possible to detect through visual inspection. The positive incidences of bacterial wilt in seed-potato plantings prior to 1994 were found in diagnostic samples collected on the basis of visual detection. All confirmed cases of bacterial wilt in ware-potato plantings were found in diagnostic samples. Figure 1.2.2 shows the geographical distribution of confirmed cases of bacterial wilt in South Africa for the period 1980 to 2014. The high incidence of cases in Limpopo and the Eastern Cape, with tomatoes, tobacco and ware potatoes being planted in rotation, can probably be ascribed to infected seed sources, as well as the planting of uncertified seed (seed retained by ware growers) and the presence of infected soils. The decrease in the number of incidences of bacterial wilt in both registered seed-potato plantings, and ware-potato plantings as from 2008 (Fig. 1.2.1), points to the successful management strategies implemented by the authorities. It should be kept in mind that all confirmed incidences prior to 1991 were found in diagnostic samples (suspicious plants or tubers), and there may in fact have been many more incidences than reported.

Figure 1.2.2 Geographical distribution of incidences of bacterial wilt in South Africa from 1980 to 2014 (Potato Certification Service)

1.3 DEVELOPMENT AND IMPLEMENTATION OF MANAGEMENT STRATEGIES 1.3.1

Organisational structures in the potato industry

A brief review of the establishment of relevant organisational structures in the potato industry is considered necessary to give the reader of this report a clearer understanding of the functioning and responsibilities of the various role players (see Annexure 1). 1951: The Potato Board was established as being responsible for instituting stabilisation measures to efficiently manage surplus situations (including export), to promote market development in the potato industry, and to develop and distribute production information. 1981:

The Potato Board accepted responsibility for the multiplication of Elite, Foundation I and II seed production, which until that point had been the responsibility of the Department of Agriculture in the Sabie area.

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1983:

In terms of a seed potato certification system as implemented by the Directorate of Plant and Seed Control, the production of seed potatoes would in future take place as follows:  Elite and Foundation Seed by the Potato Board;  Basic Seed by registered seed-potato growers; and  Ordinary Certified Seed by registered seed-potato growers

1983:

In vitro propagation was initiated at the Foundation Seed Unit near Lydenburg in Mpumalanga.

1989:

The Potato Board agreed to a request by the State to take over the inspection and certification of seed potatoes. The Department of Agriculture had been responsible for administrating the Certification Scheme for the preceding 45 years.

1991:

The Scheme came into operation, under the auspices of the Potato Board.

1994:

The Potato Board was privatised and became known as the Potato Producers’ Organisation, with the name subsequently being changed to Potatoes South Africa in 1996.

1997:

The Independent Certification Council for Seed Potatoes (ICCSP) was established by South African seed-potato growers, for the purpose of making decisions regarding all aspects of seed-potato certification, as well as appointing a company to administer the Certification Scheme. The Department of Agriculture designated the ICCSP as the authority to exercise the Scheme, with the council consisting of democratically elected seed-potato growers from each seed production region. The Department of Agriculture, the Agricultural Research Council, the Potato Laboratory Services and the Potato Certification Service are all represented on the council. The independent chairperson of the ICCSP is nominated and appointed by the council members.

1997:

An Article 21 company, namely the Potato Certification Service, was formed and tasked with the certification of seed potatoes and the general administration of the Scheme. Similarly, an Article 21 company known as the Potato Laboratory Services was established to be responsible for conducting all laboratory tests necessary for the certification of seed potatoes. Both these companies report to the Independent Certification Council for Seed Potatoes with regard to all certification and laboratory issues. The directors of each company are selected by the seed growers from each seed production region conforming to the requirements regarding the number of bags certified and hectares registered.

1998:

The limited-generation Potato Certification Scheme is promulgated.

1.3.2

Sampling of tubers

Compulsory testing for bacterial wilt in registered seed potato plantings commenced in 1994 (see Annexure 2). Since a suitable statistical method had to be found for the sampling of tubers, the decision was made to adopt the method described by Clayton and Slack (1988), which was the sampling method implemented by the Seed Potato Industry of the United States of America to test seed-potato fields for the presence of ring rot (Clavibacter michiganensis). The relationship between field size and sample size using the hypergeometric formula suggested by Clayton and Slack is illustrated in Table 1.3.2.1.

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Table 1.3.2.1 Relationship between field size and sample size using the hypergeometric formula (Clayton and Slack, 1988) N1

Approximate field size (ha)2

n3

5 000 10 000 25 000 100 000 500 000 1 000 000

0.1 0.2 0.5 2.0 10.0 20.0

3 009 3 689 4 204 4 499 4 582 4 593

1) N = Total number of plants in a field, 2) based on density of 50 000 plants/ha, 3 = sample size required to ensure PEA = .01, using the hypergeometric formula.

The decision was subsequently made to draw a sample of 4605 tubers from every registered field, cultivated and irrigated as a unit (see reasoning in American Potato Journal 1988, volume 65, page 719). The sample is only drawn once all plant foliage has died. Tuber samples must be drawn randomly across the field, with no more than one tuber per plant. Although tuber samples should be drawn across the entire field, sampling should be more concentrated in areas that might be suspect. Should a field be level with no confirmed cases of bacterial wilt in the vicinity, tuber samples should be drawn randomly across the entire field. If the topography of the land is sloping, or the land is adjacent to an area where bacterial wilt has previously been encountered, sampling should be more concentrated in the lower part of the field and in the area closest to the infected field. Tuber samples are bagged and sealed according to the prescribed protocol of the Certification Scheme, with comprehensive instructions for such statistical sampling discussed in the specified protocol. Only officials of the Certification Service are authorised to draw these statistical tuber samples. Samples are transported to testing laboratories approved by the Department of Agriculture, Forestry and Fisheries, as well as the Independent Certification Council for Seed Potatoes.

1.3.3

Preparation and testing of samples

A general serological test method, known as the enzyme-linked immunosorbent assay (ELISA), is used to test for the presence of Ralstonia solanacearum. The specific ELISA test kit was developed by the Department of Biochemistry of Stellenbosch University, and is currently manufactured locally by that same Department. Since variations are possible during the testing procedure and the interpretation of the results, it is important that a standard procedure is followed nationally by all registered laboratories so as to ensure consistency amongst technicians and tests. Standardisation is based on both “Good Laboratory Practice (GLP)” and the test protocol outlined by the manufacturer, and is further refined by the controlling laboratory of the Potato Laboratory Services (i.e. Plantovita). The Plant Improvement Act of 1976 requires that all relevant laboratories must be registered with the Department of Agriculture, Forestry and Fisheries in order to carry out tests for certification purposes. A certificate of approval is also granted by the Independent Certification Council for Seed Potatoes (ICCSP), based on the outcome of an audit conducted by the technical manager of the controlling laboratory, i.e. Plantovita. A detailed protocol regarding the handling of samples is prescribed, with a register to be completed upon the receipt, processing and completion of the samples. Should any sample not conform to the protocol, a new sample must be drawn by the Certification Service. Samples are stored according to the protocol requirements with regard to hygiene, isolation and sealing. All samples are washed to limit background readings by the ELISA test. Rotten tubers are removed and kept separate, while suspicious tubers are tested in a single well, as per the ELISA procedure. A positive result must be confirmed with conventional testing at the controlling laboratory. In the case of a negative ELISA test, the remainder of the sample is tested as per normal. A portion of the skin, approximately 10 mm in diameter, is cut from the stolon end of the tuber to expose the vascular bundle. A conical piece, containing the entire vascular bundle, is cut from the exposed area, and all tuber pieces from the subsamples are used for further testing. After the conical piece has been removed as described, a diagonal cut is made underneath the sample area

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to check for ring symptoms. Should ring symptoms be observed (Figure 1.3,3.1), only that particular tuber is subjected to the ELISA test. In the case of a positive ELISA result, the entire tuber is sent to the controlling laboratory for confirmation. Test results must be submitted to the regional office of PCS, and results may not be communicated telephonically or be revealed to any other party besides the certification offial concerned. All relevant documentation must be kept for five years. A retest is done in cases where the controls do not meet the requirements or in the case of an apparent test error, with the decision resting with the technologist concerned. In such a case, the remaining tubers are processed using the Chip method as per the specified protocol.

Fig. 1.3.3.2

1.3.4

Tuber with vascular ring symptoms.

Summary of testing procedure

During the first year of compulsory testing of seed-potato fields for the presence of bacterial wilt, seed growers were concerned about the time taken for results to become available, especially where colonies appeared suspect. The diagram that was subsequently drawn up to explain the testing procedure to the growers is contained in Annexure 3.

1.3.5

Control and administrative measures

In the event of bacterial wilt being confirmed in a registered seed-potato planting, the Certification Service was required to immediately inform the relevant seed-potato grower in writing that the planting was being withdrawn from certification and that all potatoes harvested from the planting would have to be sold as ware potatoes. The Certification Service would then immediately make use of the management database to identify the source of the seed planted and to determine whether seed from that same source had also been planted by other seed growers. Annexure 4 contains a diagrammatic presentation of the procedure to be followed in the event of a suspicious plant or tuber being found in a seed-potato or ware-potato planting. The seed-potato industry subsequently decided to establish a committee to be responsible for investigating incidences of bacterial wilt. As such, the Department of Agriculture and the Independent Certification Council for Seed Potatoes approved the establishment of the proposed committee, after which the Potato Certification Service compiled the guidelines for the functioning of the committee. The comprehensive guidelines for the Bacterial Wilt Committee, as contained in Annexure 5, are considered pertinent to this report, as the Committee played an important role in the success of the management strategy for the control of bacterial wilt.

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The Bacterial Wilt Committee (currently functioning as the Potato Quarantine Pest Committee) conducted numerous investigations during the Nineties and up until 2005. As a result, seed-potato production was discontinued on several seedpotato farms due to confirmed bacterial wilt infestations. In all cases, however, the Committee evaluated the risk of further seed-potato production and made every effort to assist growers to contain the spread of bacterial wilt without having to close down the valuable seed-potato farms. This was done by means of establishing isolation areas and permanent pastures, while seed growers also committed themselves to abide by the decisions and recommendations of the Bacterial Wilt Committee.

1.3.6

Regulations regarding bacterial wilt in the Certification Scheme

Bacterial wilt in potatoes is still considered a prohibited organism in South Africa and is treated accordingly. The stringent measures in effect since the early Nineties have proved so successful that the disease is now treated as being manageable rather than as an extreme threat to the potato industry. Seed growers have, however, accepted that although the cost of statistical sampling and laboratory testing of all registered seed-potato plantings contributes towards their production costs, these precautionary measures do form an integral part of the management strategy for bacterial wilt. The industry is also aware of the importance of having these measures in place to ensure the effective management of bacterial wilt on a continuous basis. It can be expected that the ELISA test may be supported or replaced by molecular techniques in future, but due to the sensitivity of these techniques, the seed-potato industry has requested that the seed-potato laboratories first conduct the necessary research to investigate the possible implementation of molecular techniques for the testing of both bacterial and viral diseases. The regulations regarding bacterial wilt in the Seed Potato Certification Scheme are presented in Annexure 6. The following additional management procedures regarding bacterial wilt were introduced to control and curtail the spread of bacterial wilt: 1 All potato fields of seed-potato growers where incidences of bacterial wilt have been detected are recorded on GPS. 2 Infected fields (race 3, biovar 2; and race 1, biovar 3) may not be planted for a period of eight years. Should an infected field be planted for seed potatoes after eight years of no potato cultivation and the planting of non-host crops, the crop will only be certified after a negative test. The seed tuber sample is warmed up for 14 days at 30°C before the test is conducted. 3 The infected field in question is noted on the tuber inspection report. 4 Should more than one incidence of bacterial wilt be confirmed on a specific farm, PCS may request that the Bacterial Wilt Committee investigates the risk of continued seed-potato production on the property. The Committee may recommend that no further registrations be accepted on that particular farm. 5 In the case of a confirmed incidence of bacterial wilt (race 1, biovar 3), said unit may never again be considered for purposes of seed-potato production.

1.3.7

General recommendations for the control of bacterial wilt

Recommendations for the management of the disease were compiled and subsequently made available to the potato industry, with the following being included (Mienie, 1997; Hammes, 2006 and Carlson, 2012): Preventative control measures  Only certified seed potatoes should be planted. Ascertain that only certified seed potatoes are planted on farms/lands that are rented out, as well as in the gardens of farmworkers.  Avoid the cutting or dipping of seed tubers.  Do not use irrigation water from an infected or potentially infected source, e.g. where runoff water from an infected field flows into an irrigation dam.  Effective control measures against root-knot nematodes, including the planting of resistant varieties, the chemical treatment of soil, and the fumigation of soil, should form part of an integrated management approach.

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 

 

Implements used to work an infected field must first be disinfected before being used on an uninfected field. This can be done by removing and rinsing off all soil and plant residue from the implements, onto the infected land itself. Once all soil has been removed from an implement that has come into contact with the soil or infected plant matter, all parts of the implement must be thoroughly rinsed with a disinfectant. Effective disinfectants include 0.5% Jeyes Fluid, formalin or bleach such as Jik (containing 3.5% sodium hypochlorite). During sunny, dry weather conditions, an effective method is to leave the implements in the sun for two to three days after removing all soil and plant residue. If infected plant material has been handled (stored, washed or sorted), everything – including all sorting machines, crates and floor surfaces – must be disinfected, as they can serve as sources of inoculum.

Restrictive control measures  If the disease has been confirmed in a potato field and the crop is marketable, it is strongly advised that the bags be marked “Not to be planted”.  An infected field should be cordoned off to prevent unnecessary movement through that field and thus prevent the disease being spread through soil clinging to the hooves of animals and the shoes and feet of humans.  If the disease has been confirmed in a field with a slope, and if the runoff from this field could potentially contaminate lower-lying fields, dams or rivers, it is essential that contours be built.  The control of weeds (especially solanaceous weeds), as well as volunteer potato plants, is essential.  The fallowing of an infected field, as well as the ploughing of the field during the dry seasons, will reduce the population of the pathogen in the soil. It has been documented that the pathogen can be irradiated if the soil reaches 43°C for a minimum of six hours for four consecutive days. Keep in mind that the pathogen can still survive in deeper soil layers. The survival period of biovar 2 was found to be between two and three years in Australia under bare fallow or pasture. In South Africa, biovar 2 was found to survive for up to eight years in bare fallow soil (clay loam).  Crop rotation with non-host crops will also greatly reduce the population of the pathogen. Crops recommended in the literature include maize, sorghum and wheat, as well as grasses or pasture.  If a disease is confirmed in a crop that is still growing, it is important to prevent uncontrolled movement through that particular field. All implements and workers’ shoes must be cleaned and disinfected after being in contact with the field.  In cases of low-level infection, the removal and burning of infected plants will prevent further spread of the disease.  There are no effective chemicals available for the control of bacterial wilt. General biocides (such as methyl bromide) will destroy the pathogen, but only in the soil layers effectively penetrated by the fumes. However, this is a feasible method for use in greenhouses.

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Chapter 2

LITERATURE REVIEW The literature review on bacterial wilt presented in this rapport is mostly based on the literature review in the M.S.c. dissertation of Elizabeth Irmgard Maria Stander. The literature reviews on bacterial wilt in the M.S.c. dissertations of Nicolaas Johannes Jacobus Mienie and Wilma Van Broekhuizen are referred to on aspects which are not covered by Stander.

2.1

INTRODUCTION

More than a century ago Erwin F. Smith described a bacterial disease affecting potato, tomato and eggplant. The generic name for this solanaceous wilt was proposed as Bacillus. E.F. Smith probably did not realize that the disease he had described would become one of the most important plant diseases causing severe economic losses world-wide. Since his publication in 1896 (Smith, 1896 as reported by Kelman, 1953) a continuous stream of publications have been released internationally, each shedding some light on this unusually complex disease. Commonly known as bacterial wilt, other names include brown rot (used especially in Europe on potatoes), Granville wilt (on tobacco), southern bacterial blight (on tomato, eggplant and tobacco), and moko disease on bananas. Its ability to affect over 450 plant species (Prior et al., 1998), its world-wide distribution and its destructive ability has resulted in this disease to be ranked as the most important bacterial plant pathogen (Kelman, 1998). The economic impact can not only be measured in terms of crop losses, but must also be assessed indirectly in terms of soils becoming unsuitable for subsequent crop production . This is important to large commercial farms and to small family fields. The introduction of different strains to different geographic regions increases the risk of more crops becoming susceptible to wilt within a particular region. Bacterial wilt disease is considered difficult to control. Knowledge of the organism and the disease it causes are prerequisites for integrated management strategies.

2.2

THE CAUSATIVE AGENT

The causative agent of bacterial wilt is a gram-negative, non-spore-forming, non- capsulate bacterium (Kelman, 1953). It is an aerobic organism with optimum growth temperatures ranging from 27-37°C, depending on the strain. Maximum temperature for growth is about 39°C and the minimum between 10-15°C (Shekhawat et al., 1992). There are conflicting reports on the amount of flagella present in a single cell. According to Bergey's Manual of Determinative Bacteriology (Holt, et al., 1994) this bacterium has more than one flagellum. Shekhawat et al. (1992) however describes virulent isolates to be nonflagellate and avirulent ones as having 1-4 polar flagella. The shape and size of the causal organism was first described by E.F. Smith in 1896 as a small rod with rounded ends; its size varying according to growing conditions (Kelman, 1953). Bacteria isolated from infected tissues appeared as very short rods (0.3-0.6 x 0.4-1.2µ), those taken from young broths or cultures tend to be longer (ranging from 0.4-0.6 x 1.0-1.8µ), whereas those from old cultures have a short coccus-like form (Kelman, 1953). Cultural characteristics of the colonies on tetrazolium chloride medium (Kelman, 1953) are often used to identify the bacterium. Virulent isolates produce fluidal, slightly raised colonies that are creamy white with or without pink centres. Colonies are rarely round (Shekhawat et al., 1992). The pink centres often appear comma-like or half-moon in shape.

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Different generic names have been used to classify the causal agent of bacterial wilt. Smith first proposed the name Bacillus, as he believed the bacterium to have peritrichous flagella. In 1898, Chester subsequently changed it to Bacterium; a name later also adopted by Smith (Kelman, 1953). With the finding that the pathogen actually had a single polar flagellum, it was placed under the classification as either Bacterium solanacearum or Pseudomonas solanacearum. Bergey (1923) reclassified the bacterium as Phytomonas although most continued to use the name Bacterium. Studies and revision of the classification of gram-negative plant pathogens led to the provisional transferral of P. solanacearum to the new genus Xanthomonas. Since distinct cultural characteristics differed between P. solanacearum and the genus Xanthomonas, the pathogen was once again transferred back to the genus Pseudomonas. This agreed with the classification that was adopted in Bergey's Manual in 1948 (Kelman, 1953). The classification of P. solanacearum was adopted for the next 44 years. Studies conducted by Yabuuchi et al. (1992) led to the proposal that P. solanacearum be transferred to the new genus Burkholderia. The seven species that were placed into this new genus differed from the type species of Pseudomonas in their oxidation and assimilation capabilities of several polyalcohol’s and disaccharides. Yabuuchi et al. (1992) also disagreed with the description of P. solanacearum given in Bergey's Manual (Holt et al., 1994) in that the type strain they identified was non-motile and without any flagellum. Yabuuchi et al. (1995) reclassified Burkholderia solanacearum as Ralstonia solanacearum. This reclassification was based on studies involving phenotypic characterization, rRNA-DNA hybridization, phylogenic analysis o f 16SrDNA nucleotide sequences, and analysis of cellular lipids and fatty acids.

2.3

SUBSPECIFIC CLASSIFICATION

Several attempts have been made to find a suitable classification system for isolates of R. solanacearum as they often differ in host range, geographical distribution, pathogenicity, epidemiology and physiological properties. For almost thirty years two major approaches to differentiation were used, one based on hosts primarily affected resulting in the establishment of races, the other on selected biochemical properties conforming to distinct biovars. These classification systems do not abide by the Code of Nomenclature of Bacteria (Hayward, 1991a).

2.3.1

Classification according to race

Subspecific classification of strains into 5 races is achieved by determining the hosts that are primarily affected. Race 1: Strains affecting tobacco, tomato, a range of other solanaceous crops, some weeds and certain diploid but not triploid bananas Race 2: Those affecting triploid bananas, Heliconia spp. and other musaeous hosts Race 3: Strains affecting primarily potato and to lesser extent tomato. It is weakly pathogenic other solanaceous crops. Race 4: Strains affecting mainly ginger. Race 5: This race affects mainly mulberry

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2.3.2

Classification according to biovar

According to Hayward (1994b), five biovars can be identified based on ability to utilize three hexose alcohols, namely mannitol, sorbitol, dulcitol; and to produce acids from the three disaccharides, lactose, maltose and cellobiose (Table 2.3.2.1).

Table 2 . 3 . 2 . 1 . Identification o f biovars of R. solanacearum according to utilisation and/or oxidisation of certain carbohydrates (Hayward, 1994 b) Biovar/ Carbohydrate

Biovar 1

Biovar 2

Biopvar 3

Biovar 4

Biovar 5

Lactose

-

+

+

-

+

Maltose

-

+

+

-

+

Cellobiose

-

+

+

-

+

Mannitol

-

-

+

+

+

Sorbitol

-

-

+

+

-

Dulcitol

-

-

+

+

-

The distinction between race and biovar is not always: clear except in the case of race 3 and biovar 2. Generally it can be said that race 3 (potato race) is equivalent to biovar 2, although the reverse is not necessarily true. In other words not all biovar 2 strains belong to race 3 (Sequeira, 1993). biovar 2 strains that have been isolated in the Andean highlands seem to correspond to those of race 3 (Amat et al., 1978). Biovar 2 strains from the Amazon basin, however , differ in phenotypic properties regarding pathogenicity on various Solanum species and metabolic activities. These have been designated as biovar N2 (Gillings and Fahy, 1994). Since biovar 2 isolates were found on potato or tomato, the correspondence of rnce 3 and biovar 2 seems true. Biovar N2, however, is probably not equivalent to race 3, as it was isolated from other Solanum species such as the nightshades. Biovar N2 also does not occur in the region where potatoes originated. Biovar 2 has a more limited host range than biovar 3, and is known to contain some strains that are adapted to pathogenesis at lower temperatures. It appears that its ability to survive in fallow soil is less than for biovar 3. These generalisations about the epidemiology and control of biovar 2 (race 3) set it apart from other races and biovars (Hayward, 1991a). Strains of race 1 affecting potatoes can consist of biovars 1, 3 and 4 (He, et al., 1987). Within each of these races or biovars there are numerous subtypes that can be associated with certain geographical regions (He, 1983). This, together with the fact that R. solanacearum enjoys a world-wide distribution and that it is associated with indigenous plants in virgin soil supports the belief at this disease has been present in tropical soil for aeons (Sequeira, 1993).More recent attempts to classify R. solanacearum strains involve molecular methods such as restriction length polymorphism (RFLP) analysis. Two major divisions could be identified, division 1 consisting of race 1, biovars 3, 4 and 5; and division 2 of biovar 1 (race 1 and 2) and biovar 2 (race 3) (Cook and Sequeira, 1994). These authors recognise only three traditional races. Fegan, et al., (1998) does not specifically mention the separation of the races into the two divisions but, describes division 2 as consisting of biovars 1, 2 and N2.

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2.4 2.4.1

DISEASE DEVELOPMENT AND SYMPTOMOLOGY Mode of entry

Ralstonia solanacearum usually enters its hosts via wounds in the root system (Johnson and Schaal, 1952). Cultural practices such as interplanting prior to harvest often lead to increased root damage (Kelman, 1953). The presence of rootinvading parasitic fungi such as Phytophthora in the soil is believed to be another factor that may influence infection, although contradicting observations have been made in this regard (Kelman, 1953). The role of nematodes, especially the root-knot nematode (Meloidogyne spp.), in providing a wound for bacterial entry has been mentioned by several authors (Kelman, 1953; Buddenhagen and Kelman, 1964; Hayward, 1991a; Shekhawat, et al., 1992). Nematodes may also modify the host tissue making it more suitable for bacterial colonisation (Hayward, 1991a). Wilt resistant cultivars have been noted to become susceptible when attacked by nematodes. It is therefore not surprising that attempts were made to combine resistance to three of the Meloidogyne species and to R. solanacearum in potato plants (Gomez, et al., 1983). Root decay caused by unfavourable soil conditions is believed to provide further entrance sites for the pathogen. Invasion through insect wounds has been noted on peanut roots and on potato tubers. Even infection of aerial parts via wounds has been reported under field conditions (Kelman, 1953). Buddenhagen and Kelman (1964) reported transmission by several genera of insects visiting inflorescences of banana plants. Bacterial invasion does not occur directly through the flower, but occurs when open xylem vessels are exposed during natural dehiscence of bracts and male flowers. Although wounding of some kind was usually regarded as a prerequisite for infection, documentation exists where this is not the case (Kelman and Sequeira, 1965). It is postulated that the bacterium could enter the host through points of secondary root emergence (Kelman, 1953; Buddenhagen and Kelman, 1964; Kelman and Sequeira, 1965). Kelman and Sequeira (1965) observed that relatively large numbers of bacteria are needed to infect unwounded roots. They suggest that massing of bacterial cells at points of secondary root emergence is required for enzymatic digestion of the mucilaginous sheath or other barriers.

2.4.2

Histopathology of infected plants

The detailed study conducted by Wallis and Truter (1978) on the histopathology of tomato plants infected with Ralstonia solanacearum, shed considerable light on the spread of the pathogen within the host and the progressive destruction of its vascular tissue. Bacterial wilt was generally thought to be a vascular parasite confined initially to xylem vessels (Kelman, 1953; Buddenhagen and Kelman, 1964; Husain and Kelman, 1958a; Husain and Kelman, 1958b; Pegg and Sequeira, 1968). The Wallis and Truter (1978) study revealed, however, that initial colonisation of host tissue did not occur in the xylem vessels of the roots as expected, but in small diameter cells adjacent to large xylem vessels. Light microscopic examination could not identify whether these cells are tracheids, tracheid fibres or xylem parenchyma. Wallis and Truter (1978) observed that within 24 hours after inoculation additional small diameter cells had become invaded and filled with bacteria. The bacteria within these cells showed marked pleomorphism and appeared to contain granules of storage products such as poly-B-hydroxybutric acid or volutin, indicating metabolic activity. In some cases, the bacteria were in close contact with the host cell wall and seemed to orientate themselves toward the spaces between the bars of secondary thickening. In other cases they were contained in a bag-like structure, which effectively prevented them from affecting the primary wall. According to Wallis and Truter (1978) this concentration and orientation of bacteria towards bordered pits between vessels and adjacent cells during this stage of pathogenesis indicates that bacteria are either attracted by substances diffusing into the vessel from adjacent cells, or that they are drawn along an osmotic pressure gradient. This

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initial spread may be influenced by the method and the site of inoculation and may also indicate that at any particular stage of development of the host plant only certain cells are physiologically predisposed to invasion. Stimulation of tyloses formation was noted in invaded cells and less frequently in non-invaded cells about 24 hours after inoculation, an observation absent in healthy plants. This indicates that although infection stimulates the production of tyloses, the actual presence of bacteria is not required. Kelman (1953) reported similar findings. The formation of tyloses is believed to be stimulated by increased production of indole acetic acid (IAA) and other growth substances (Sequeira and Kelman, 1962; Buddenhagen and Kelman, 1964; Sequeira, 1965; Pegg and Sequeira, 1968). Wallis and Truter (1978) observed a slow migration of bacteria during the first 48 hours after inoculation and no bacteria could be detected at a distance greater than 3, 5 cm from the cut root-tip. No bacteria could be observed in the xylem vessels. During the next 24 hrs, however, disruption and collapse of tyloses had occurred, releasing the bacteria into the xylem vessels. Bacteria spread in root vessels above the region of tylose collapse. These bacteria increased steadily and in some cases the primary wall showed signs of degradation. Only at this stage of disease development were bacteria observed in the vessels of stems. During the first 72 hours after inoculation water uptake in inoculated plants had been 15 - 20% higher than in the control plants. Thereafter, however, the inoculated plants started to wilt and fluid uptake decreased relative to that in control plants. This observation correlates with the time when tyloses, after often obstructing vessels, collapsed, became disrupted and released bacteria into the xylem vessels (Wallis and Truter, 1978). After 144 hours the bacteria in the root vessel had reached such large numbers that they became compressed into irregular shapes. Longitudinal spread in the stem was now rapid but compression of bacterial cells did not occur to such an extent as in the root vessels, nor did bacteria reach such large numbers. Tissue collapse was observed after 168 hours and various plugging substances were noted in the vessels and cells of diseased plants. Complete wilting of all test plants occurred about 192 hours after inoculation. A dense, darkly staining material, possibly of cell wall origin, accumulated in many vessels. This material was found where vessel walls had been dissolved by bacterial enzyme action and in lysigenous cavities formed by bacterial degradation of adjacent parenchyma cell walls (Wallis and Truter, 1978). It has also been reported that in some instances these parenchyma cells seem to enlarge and divide, causing partial collapse of infected vessels (Kelman, 1953). According to Wallis and Truter (1978), the lack of damage to the walls of invaded root cells during the early stages of pathogenesis could possibly be ascribed to low levels of cellulolytic and pectinolytic enzyme activities as bacterial numbers are still relatively low. As bacterial numbers increased, degradation of wall material in the vessels was clearly visible. Kelman (1953) mentions that in solanaceous hosts, besides collapse of infected vessels, adjacent phloem areas can become infected and the cortex disorganized. The formation of a central cavity often results due to a breakdown of pith tissue. This general breakdown of walls and cells is, however, not found in stems of older plants that have well-developed secondary xylem.

2.4.3

Visual symptom expression

2.4.3.1 Aboveground symptoms Once the pathogen has entered the host, time elapses before visual symptoms appear. This incubation period varies greatly and depends on a variety of factors such as host species, environmental conditions, age of the host and level of resistance. The symptoms characteristic of bacterial wilt includes wilting, stunting and yellowing of the f o l i a g e , epinasty and v a s c u l a r b r o w n i n g in t h e stems (Kelman, 1953; Buddenhagen and Kelman, 1964; Shekhawat et al., 1992). It has also been noted that climatic conditions can affect the type of visual symptoms expressed. In hot dry weather, infected plants may show irregular scalded areas on their leaves, which then dry out and shatter at the edges (Kelman, 1953). Under moist conditions, however, the base of the stem or leaf petioles may completely rot and break off (Kelman, 1953; Shekhawat et al., 1992). In potatoes a slight yellowing of lower leaves is often noted as the first leaflets begin to droop (Kelman, 1953). The extent to which yellowing occurs often depends on whether the onset of disease is rapid, in which case no real change in colour would occur. In most cases, if the onset of disease is rapid, foliage of an entire hill may quickly

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droop and wilt without much change in colour (Smith, 1896 as quoted by Kelman, 1953). Dark narrow stripes beneath the epidermis, corresponding to the infected vascular strands, may be visible in the stems of young potato plants. Adventitious root formation on infected stems commonly occurs, especially if wilting is gradual (Kelman, 1953) or if an isolate of low virulence was responsible. A valuable diagnostic sign of the disease on any host is the appearance of slimy viscous ooze in the form of tiny, dirty -white to brownish glistening beads at the points on the cut stem where the vascular strands were severed (Smith, 1896 as quoted by Kelman,). If the two cut surfaces of a sectioned diseased stem are touched together and then drawn apart slowly, fine viscous bacterial strands may be seen between the stem ends. These strands will stretch a short distance before breaking according to Stevens and Sackett.(1903), as quoted by Kelman, 1953) .The presence of the pathogen can also be demonstrated by placing a longitudinal section containing vascular tissue from a diseased plant in a container of water. Within a few minutes fine milkywhite strands, composed of masses of bacteria, will stream from the margin of the tissue. This procedure is an aid in distinguishing this disease from certain fungal diseases which produce similar wilting symptoms and vascular discoloration in plants which are hosts of R. solanacearum (Ali, 1995). 2.4.3.2 Belowground symptoms Potato tubers from infected plants do not always show external symptoms. The presence of external greyish brown discoloration indicates an advanced stage of disease, which upon further development leads to a sticky bacterial exudate at the eyes or the stolon end of the tuber which mixes with the soil and causes the soil to adhere to the tuber surface (Grieve, (1936a) as quoted by Kelman, 1953)). A slight pressure on a cross section of a tuber will force the typical greyish-white bacterial slime out of the vascular ring. Cross sections of infected tubers often show distinct brown discoloration and decay in the vascular ring (Shekhawat et al., 1992). Tubers left in the soil continue to decay. Secondary organisms enter through breaks in the skin and complete the reduction of large portions to a slimy mass surrounded by a thin shell of outer tissue. Not all tubers produced on plants with aboveground symptoms are affected. However, diseased tubers are occasionally found on plants which show no apparent evidence of infection. The latter, however, usually carry latent infection (Eddins, 1941). Latent infection in plants can be considered as the best possibility for long-term survival of the pathogen. In the case of R. solanacearum long-term survival seems to be associated with physiological specialisation, race 1 having the widest host range, followed by race 2 and finally race 3 (Granada, 1987).

2.4.4

Symptom development and latency

A primary factor contributing to the persistence of bacterial wilt in the potato industry is that this disease can exist as symptomless (latent) infections. A number of variables can determine whether or not a bacterial wilt infection will be asymptomatic. Inoculum dosages at the time of infection and environmental conditions mainly affect expression of disease symptoms (Devi, et al., 1982). Additionally, the frequency of disease expression in a field may be so low that its detection in seed potato fields during field inspections is extremely difficult, if possible at all. Temperature has an influence on the virulence of R. solanacearum, as well as on the symptom expression of the disease. In general it may be accepted that symptoms are more intensely expressed with an increase in temperature. At lower temperatures a larger extent of latent infection occurs, whilst plants and tubers show no visual symptoms (Shekhawat, et al., 1992). This tendency holds great danger for the seed potato industry, seeing that the disease could easily be spread by tubers. It is clear that an indication of critical temperatures for latent infection will be of great value to seed potato growers and knowledge about the extent of symptom development at low temperatures will be valuable in cases where susceptible hosts are cultivated in infected soils.

Bacterial wilt disease on potato: The South African experience

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25

Although several researchers reported that the pathogen does not survive in tubers that were exposed to low storage temperatures (Eddins, 1941 and Ciampi et al., 1980), it cannot be assumed that the pathogen was eliminated. Seed potato storage in cold or country stores does not eliminate R. solanacearum and tuber infection can still result in wilting plants (Gadawar and Chakrabarti, 1995). Tubers from infested soil carried inoculums in lenticels and/or vascular tissues for varying periods. Surface inoculums was not detected after four months of cold storage (4-6°C), although it could be detected up to nine months in tubers stored at room temperature.

2.5

DISTRIBUTION

Bacterial wilt affects crops of economic importance in almost all the tropical, subtropical and warmer temperate regions of the world. Early reports of occurrence have often been incorrect, incomplete or not confirmed by proper identification techniques, making it difficult to truly assess its distribution. The distinct differences in geographical distributions of biovars suggest a separate evolutionary origin (Hayward, 1991a). Biovar 2 presumed to have originated in South America (presumed site of origin of the potato) now has a wide spread distribution, indicating the ease with which it can be transmitted as latent infections in potato seed tubers. In many countries of Southern Europe such as Portugal, biovar 2 is the sole biovar. This is also true for the Mediterranean area, Argentina, Chile and Uruguay (Hayward, 1991a). Biovars 1 and 2 are predominant in the Americas with biovar 3 being rare and biovar 4 not yet being identified with the exception of one case stated by Black and Sweetmore (1993). In Australia, however, biovar 3 predominates, biovars 2 and 4 occurring to a lesser extent. Biovars 2,3 and 4 also occur in India, Indonesia, Papua New Guinea, Sri Lanka and China (together with biovar 5). Only in the Philippines have all of biovars 1-4 been found and here as elsewhere in Asia, biovar 3 predominates in the lowland regions (Hayward, 1991a). The classification into divisions as suggested by Cook and Sequeira (1994) corresponds, with a few exceptions, to the geographic distribution of the strains. In DivisionI90% of the strains came from Asia and Australia, whereas 98% of those in Division 2 were from the Americas. This suggests that in early evolution R. solanacearum split into two groups which then evolved in geographical isolation to give rise to the strains typical of the Old World and the New World. All race 3 strains isolated in Africa, Asia and Australia belonged to the same RFLP groups which originated in the Andean region in South America. With the aid of RFLP techniques more data could be obtained to support the concept of geographic isolation in the pathogens evolution (Sequeira, 1993). Strains from Heliconia are in RFLP groups that are restricted to the Americas, whereas strains from mulberry and ginger form distinct groups that are again restricted to certain regions in Asia. Race 3 (potato race), although being widely distributed, consists of a very compact group originating in the Andean Region. Yet strains attacking bananas in the Asian continent seem to have evolved separately from those attacking bananas in the Americas (Sequeira, 1993). R. solanacearum has now been identified to be the cause of an old banana disease common in Asia, the so-called blood disease. RFLP patterns of these strains are so different from all other strains, that it was concluded that this group bears very little relationship to the American race 2 strains. So far only biovars 2 and 3 have been reported in South Africa with biovar 2 having a major impact on the potato industry. Since neighbouring countries such as Angola, Mozambique a n d Zimbabwe are reported to have biovar 1 of R. solanacearum (De Lourdes D'Oliveira, 1967), great care needs to be taken to prevent establishment of this biovar locally. In Table 2.5.1 the world-wide distribution of bacterial wilt is demonstrated with mention of the specific biovars and/ or races involved. In several instances literature sources only reveal the occurrence of R. solanacearum without further differentia1tion into biovars or races. This list may be incomplete and in some mentioned countries, such as Spain, Poland and Sweden, the disease is believed to be eradicated (Elphinstone, 1 9 9 6 ).

Bacterial wilt disease on potato: The South African experience

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26

Table 2.5.1 Distribution of bacterial wilt disease. COUNTRIES

BIOVARS

RACES

SOURCE

AMERICAS Argentina

2

3

Martin et al., 1982

Brazil

1.2,3

1,2,3

French et al. 1993

Bolivia

1.2

Smith el al. 1998

British Honduras (Belize)

1

Black and Sweetmore, 1993

Canada

1

Chile

2

Colombia

1,2

1,3

Martin et al., 1982

Costa Rica

1,2,3

1,3

Martin et al., 1982

1

Hayward, 1991a Hayward, 1991 a Ciampi-Panno, 1984

El Salvador

Kelman, 1953

Honduras

2

Woods, 1984

2

3

Fucikovsky, 1978

1

1

Fucikovsky, 1978

1

2

Fucikovsky and Santos, 1993; Fucikovsky, 1978

Panama

2

3

Martin et al., 1982

Peru

1,2,3

1,3

United States-

1

1

Martin et al., 1981 Martin et al., 1982

4

1

Black and Sweetmore, 1993

Uruguay

2

3

Hayward, 1991 a

Venezuela

2

3

Martin et al., 1982

Mexico

WEST INDIES Cuba

3

French West Indies

1,2,3

Grenada

1

Amat et al., 1978 2,3

Prior and Steva, 1990 Black and Sweetmore, 1993

Guadeloupe

Prior et al., 1993

Haiti

Kelman, 1953

Jamaica

Kelman, 1953

Martinique

2,3

1,3

Puerto Rica

1

1

Tobago Trinidad

Cook and Sequeira, 1994 Hosein and Phelps, 1997

1

1

Cook and Sequeira, 1994

Bacterial wilt disease on potato: The South African experience

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27

COUNTRIES

BIOVARS

RACES

SOURCE

EUROPE Albania

Wenneker et al., 1998

Austria

Kelman, 1953

Belgium

2

3.

Wenneker et al., 1998

Denmark France

Elphinstone, 1996

Elphinstone,1996, Hayward et al., 1998

2

3.

Greece

2

3

Walker, 1992

Netherlands

2

3

Janse et al., 1998

ltaly

2

3

Elphinstone, 1996

Portugal

2

3

De Lourdes D'Oliveira, 1967

3

Kelman, 1953 Elphinstone, 1996 Elphinstone, 1996

Rumania Spain

2

Sweden United Kingdom

Olsson, 1976 2

3

Elphinstone, 1996

U.S.S.R

Walker, 1992

Yugoslavia

Walker, 1992

ASIA China

2,3,4,5

1,3,4,5

India

2,3,4

1,2,3

2,3,4

1,3

Hayward, 1991; Machmud, 1986

2

3

Danesh and Bahar, 1984

3

Alvarez et al., 1993

Indonesia Iran Israel

He, 1983 Shekhawat et al.,1992; Sinha, 1986

Japan

1,2,3,4

1,3

Tsuchiya and Horita, 1998

Java

2,3,4

1,2,3

Cook and Sequeira, 1994

Malaysia

3,4

Nepal

2,3

Pakistan

2,3

Burney and Ahmad, 1997

Philippines

1,2,3,4

Valdez, 1986

Sri Lanka

Abdullah, 1993 1,3

Adhikari, 1993

2,3,4

1,3

Velupillai, 1986; Martin and Nydegger, 1982

Taiwan

3,2,4

l

Hsu, 1991

Thailand

3,4

Vietnam

Titatam, 1986 1

Hong and Mehan, 1993

Bacterial wilt disease on potato: The South African experience

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28

COUNTRIES

BIOVARS

RACES

SOURCE

AFRICA Kelman, 1953

Algeria Angola

1,2

3

De Lourdes D'Olivcira, 1967

Burundi

2

3

Berrios and Rubirigi, 1993

Egypt

2

3

Gillings and Fahy, 1994

Ethiopia Kenya

Kelman, 1953 2,3,4

1,3

Harris, D.C. 1976

Malawi

Black et al. 1998

Morocco

Kelman, 1953

Mozambique

l

De Lourdes D'Oliveira, 1967

Nigeria

2

3

Cook and Sequeira. 1994

Rwanda

2

3

Vander Zaag, 1986

Somalia South Africa

Kelman, 1953 2,3

1,3

Swanepoel, 1992

Tanzania

Black et al. 1998

Uganda

Tusiime et al., 1998

Zaire Zimbabwe

3

French et al., 1998 Robertson, 1998

1,2

AUSTRALIA Australia

2,3,4

1,3

Hayward , 1991b

INDIAN OCEAN Andaman and Nicobar Islands

Ramesh andBandyopadhyay, 1993

Madagascar

1

1

Lallmahomed et al., 1988

Mauritius

3

1

Saumtally et al., 1993

Reunion

1,2,3

1,3

Girard et al., 1993

PACIFIC OCEAN Fiji

Iqbal and Kumar, 1986

Hawaii

Alvarez et 4

1,2

New Zealand Papua New Guinea

al.,1993;

Hayward, 1986 Kelman, 1953

2,3,4

1,2

Tomlinson,1985; Tomlinson and Gunther, 1986; He, 1986

ATLANTIC OCEAN Madeira Island

2

3

De Lourdes D'Oliveira, 1967

Bacterial wilt disease on potato: The South African experience

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29

2.6

HOST RANGE

R. solanacearum is known to have a very extensive host range including not only economically important crop plants such as potatoes, tomatoes, tobacco and bananas, but also ornamental plants, trees and weeds. Species from more than 44 plant families have been identified by Hayward (1991a) and more hosts are being recognised and described. Some of the more recent reports include onion, Allium cepa, (Girard et al., 1993); custard apple, Annona spp., (Mayers and Hutton, 1987); florist geranium, Pelagornium hortorum, (Strider et al., 1981); strawberry, Fragaria L. spp., (Hsu, 1991) and radish, Raphanus sativus L., (Hsu, 1991). R. solanacearum, biovar 3, has recently also been noted on cashew in Indonesia and the Alexandra palm in Queensland, Australia (Hayward, 1991a). There appears to be some irregularity in the distribution of bacterial wilt in certain hosts. Cassava is cultivated in many countries where bacterial wilt is endemic, yet the disease on this host appears to be confined to Indonesia. Similarly bacterial wilt on sweet potato has only been reported in China (Hayward, 1991a). Eucalyptus was first reported as a host in Brazil and China. Eucalyptus in Australia appeared to be a non- host until recently, when biovar 3 was isolated from diseased plants (Hayward, 1994b). Specific strains pathogenic for certain hosts may have evolved only in certain parts of the world and are not found elsewhere. This theory is supported by recent RFLP studies. An alternative theory is that these hosts may only be susceptible where a number of environmental factors conducive to disease expression coincide, such as temperature regime, rainfall, soil type, inoculum potential, and other biological factors such as nematode populations (Hayward, 1991a; Hayward, 1994b). Hosts of R. solanacearum do not necessarily express symptoms but can serve as symptomless carriers. This is especially true for many of the weed hosts such as common purslane (Hayward, 199la), single leaved cleome (Shekhawat et al., 1992) and the apple of Peru (Olsson, 1976). The slow rate of colonisation and disease progress in symptomless hosts allows the bacteria to stay viable longer, serving as an inoculum source for susceptible crops or wild hosts. Studies conducted by Shekhawat et al. (1992) indicate that R. solanacearum can even survive symptomless in roots of weed-hosts and in plants considered to be non-hosts. Granada and Sequeira (1983a) have reported similar findings. Roots of bean and maize, both presumed non-hosts were invaded with bacteria. Infection was however localised and not all plants became infected. More than 450 species have been reported as hosts or symptomless carriers (Prior et al., 1998) of certain strains of R. solanacearum. In Table 2.6.1 host plants are listed with reference to the country of report and where possible the strain involved. The host range of individual strains of R. solanacearum differs considerably with race 1 (the solanaceous race) having the widest range. This race is more common in the sub- tropical and tropical climates where plant debris tends to decompose more rapidly thereby providing only temporary shelter to the bacteria. Al ternative hosts might therefore play an important role in the pathogen’s survival. Only about 30-40 species have been positively identified as hosts of biovar 2 (race 3).

Bacterial wilt disease on potato: The South African experience

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30

Table 2.6.1 Known hosts or symptomless carriers of Ralstonia solanacearum

Weed Hosts Scientific Name

Common Name

Biovar cited

Race cited

Country of Report

Literature Reference

Acanthospermum hispidum DC

Upright starbur

India

Shekhawat et al., 1992

Achyranthes aspera Cooke

Rough Chaff flower

India

Shekhawat et al., 1992

Aclypha boehmerioides Miq.

Kelman, 1953

Aclypha hispida

India

Shekhawat et al., 1992

Aclypha indica

India

Shekhawat et al., 1992 Machmud, 1986 Titatarn, 1986 Shekhawat, et al., 1992 Ramesh and Bandyopadhyay, 1993 Abdullah, 1993 Akiew et al. 1993

Ageratum conyzoides

White weed

Indonesia Thailand India Andaman and Nicobar Islands Malaysia Uganda

Ageratum houstoanianum Mill.

Blue billv-goat weed

Australia

Akiew et al., 1993

Amaranthus graecizans L.

Kaffir spinach

India

Shekhawat et al. 1992

Amaranthus hibridus L

Cape pig weed

RSA

Swanepoel, 1992

Malaysia

Abdullah, I993

Andaman and Nicobar Islands Uganda

Ramesh and Bandyopadhyay, 1993 Tusiime et al., l 998

3

3

1

Amaranthus sp.

Bayam

Ambrosia artemlsllfolia L.

Common ragweed

Kelman, 1953

Ambrosia trifida L.

Giant ragweed

Kelman, 1953

Anthirrhinum sp.

India

Kishore et al., 1993

Artemissia so.

India

Kishore et al., 1993

Arabidopsis thaliana

India

Kishore et al., 1993

Aritica dioca

India

Kishore et al., 1993

Asc/epias curassarica

Blood-flower

Honduras Australia

Berg, 1971 Akiew et al., 1993

Atrooa belladonna

Belladonna

Bidens bipinnata L.

Spanish blackjack

2

Kelman, 1953 3 3

Bidens pilosa L.

Common blackjack (Cobblers peg)

Brachiaria plantaginea

3

1

Brazil Uganda

Swanepoel. 1992 Shekhawat et al., 1992 Titatam, 1986 Shekbawat et al., 1992 Akiew and Trevorrow, 1994 Mariano, 1998 Tusiime et al., 1998

Brazil

Mariano, 1998

RSA India Thailand India Australia

Calendula officinalis L.

Calendula

India

Shekhawat et al..1992

Calopogonium mucunoides Desv.

Calopo

Australia

Akiew et al., 1993

Cannabis savita L. (s)

Indian Hemp

India

Shekhawat et al., 1992

Bacterial wilt disease on potato: The South African experience

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31

Weed Hosts Scientific Name

Common Name

Biovar Race cited cited

Country of Report

Literature Reference

Capsella bursa-pastoris

2

3

Netherlands

Van Beuningen et al., 1998

Cardamine spp.

2

3

Netherlands

Van Beuningen et al., 1998

Australia

Akiew et al., 1993

Honduras

Berg, 1971

India

Shekhawat et al., 1992

Netherlands

Van Beuningen et al, 1998

Nepals

Pradhanang, 1999

Netherlands

Van Beuningen et al, 1998

Cassia mimosoides L.

Five leaf cassia

Cecropia peltata Celosia argentea L.

2 Cockscomb

Cerastium fontanum

2

Cerastium glomeratum

2

Chenapodium album

2

Chenopodium ombrosoides L Chenopodium murals L.

3

3

India

Wormseed goosefoot Nettle-leaved goosefoot

India

Cichorium endivia

Shekhawat 1992 Shekhawat 1992

et

al.,

et

al.,

Brazil

Mariano, 1998

Australia

Akiew et al., 1993

1, 3

Kenya

Harris, 1976

1, 3

India

Shekhawa et al., 1992

Citrilus llanatus

Paddy melon

Cleome monopllylla L.

Single leaved cleome

Cleome soeciosissima

Cat's whisker

Malaysia

Abdullah, 1993

Commelina benghalensis L.

Common signal grass

India Brazil

Shekhawa et al., 1992 Mariani, 1998

Corchorus acutammlus L.

Native jute

Australia

Akiew et al., I993

Corchus olitarius

Corchorus

Malaysia

Abdullah, 1993

Cosmos caudatus

Ulam Raja

Malaysia

Abdullah, 1993

Andaman & Nicobar Islands

Ramesh and Bandyopadhyay, 1993

Andaman

Ansari, 1990

Thailand Indonesia Australia, Java Malaysia Indonesia Sri Lanka

Titatam, 1986 Machmud, 1987 Hayward, 1994 Hayward , 1986 Machmud, 1986 Velupillai, 1986 Shekbawat et al., 1992 Shekhawat et al., 1992 Swanepoel & Young, 1988

2

Cosmos sp. 1

Cosmos sulphureus 3 Crassocephalum erepidiodes 3 Croton hirlus

Croton

Croton speciflorus

India

Cyperus rotundus L.

Red nut grass

Datura ferox

Large thorn apple

Datura metel

3 2,3

India 1,3

RSA India

Bacterial wilt disease on potato: The South African experience

Schmiediche, 1986

Page

32

Weed Hosts Scientific Name

Common Name

Biovar Race cited cited

Kenya R.S.A India Peru

2, 3 Datura stramoniumL.

Common thorn apple

1, 2,3

2

Country of Report

3

Sweden Chile

Datura spp

4

Kenya

Drymaria cordata

2

Nepal

Dysophylla auricularia (L.) Blume Eclipta alba (L.)

3

White eclipta

lndia Australia

3

Thailand

Eleutheranthera ruderalis (Schw.)Sch.Bip. Erechtites hieracifolia Rafiin. Erigeron cannadensis

Harris, 1976 Swanepoel, 1992 Gishore et al., 1993 Marin & El-Nasbaar, 1993 Olsson, 1976 Fernandez, 1986 Martin and Nedegger, 1982 Pradhanang, 1999 Kelman, 1953

Eclipta prostrata

Eragrostis curvula

Literature Reference

Shekhawat et al., 1992 Akiew et al., 1993 Titatarn, 1986 Kelman, 1953

Weeping love grass

3

R.S.A.

Swanepoel, 1992

Malaysia Brazil

Abdullah, 1993 Mariano, 1998 Kelman, 1953

Horseweed

Erigeron floribundis (Conyza sumatrensis, C.albida)

Tall fleabane

Uganda

Tusiime et al., 1998

Erigeron hirta L.

Asthma plant

Australia

Akiew er al., 1993

Sweden India

Olsson, 1976 Shekhawat et 1992

Eupatorium cannabinum L.

Thailand Costa Rica

3 3

Eupatorium oderatum

Andaman & Nicobar Islands

Euphorbia geniculata L.

Painted Euphorbia

Euphorbia hirta L.

Red Euphorbia

India India Malaysia

Euphorbia maculate L. Euphorbia prunufolia

India 3

Euphorbia

Fagopyrum sagittatum

Titatarn, 1986 Black & Sweetmore, 1993 Ramesh & Bandyopadhyay, 1993

Shekhawat et 1992 Shekhawat et 1992 Addullah, 1993 Shekhawat et 1992

Malaysia

Hayward, 1986

Indonesia

Machmud, 1986

Galium aparine

India

Tusiime et al., 1998 Mariano, 1998 Kishore et al., 1993

Galphimia gracilis L.

Malaysia

Abdullah, 1993

Gomphrena sp

India

Shekhawat et al., 1992

Galansoga parviflora

Uganda Brazil

Small flowered quick weed

al.,

al., al.,

al.,

Heliconia acuminata

2

Costa Rica

Sequeira & Averre, 1961

Heliconia caribaea

2

Costa Rica

Sequeira & Averre, 1961

Bacterial wilt disease on potato: The South African experience

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33

WEED HOSTS Scientific Name

Common Name

Biovar Race cited cited

Country of Report

Heliconia imbricata

2

Costa Rica

Heliconia latispatha

2

Costa Rica

Literature Reference Sequeira & Averre, 1961 Sequeira & Averre, 1961

Hibiscus cannabinus

Malaysia

Abdullah, 1993

Hyptis capitata Jacq.

Malaysia

Abdullah, 1993

Hyptis suavealens

1

Hyptis suavealens

3

1

Sri Lanka Thailand Malaysia

Quezando-Soares & Lopes, 1994 Velupillai,1986 Titatarn, 1986 Abdullah, 1993

Maylasia

Abdullah, 1993

Andaman & Nicobar Islands Thailand Andaman & Nicobar Islands

Ramesh & Bandyopadhyay, 1993 Titatarn, 1986 Ramesh & Bandyopadhyay, 1993

Brazil

ipomea setose L. Ipomoea sp.

3 Jussiaea linifolia

Australia

Akiew et al., 1993

Lagosca mollis

India

Shekhawat et al., 1992

Lathyrus sp.

India

Kishore et al., 1993

Leonurus sibiricus L.

Malaysia

Abdullah, 1993

Leucas martinicensis

Uganda

Tusiime et al., 1998

Lablab purpureus

Lablab

Luzula campestris

2

3

Netherlands

Van Beuningen et al., 1998

Marsypianthes chamaedrys

1

1

Brazil

Mariano et al., 1998

Melampodium perfoliatum

1

1

Costa Rica

Black and Sweetmore, 1993

Merremia hastate (Desr.)Hall Merremia umbellate (Mey.) Hall

Kelman, 1953 Kelman, 1953 Kelman, 1953

Merremia vitifolia (l.) Hall 3

Milleria quinqueflora L. Nepat sp. Nicandra physaloides (L.)

2

Apple of Peru

Nicotiana alata Link & Otto Nicotiana glauca

Cuba

Amat et al., 1978

India

Kishore et al., 1993

Sweden India

Olsson, 1976 Shekhawat et al., 1992

Sweden

Kelman, 1953

Wild tobacco

Kelman, 1953 2

Peru Colombia

Martin & French, 1995 Thurston, 1963

India

Kishore et al., 1993

Uganda

Tusiime et al., 1998

Oxalis sp

India

Kishore et al., 1993

Oldenlandia corymbosa L.

India

Shekhawat et al., 1992

Parthenium hysterophorus L.

Cuba

Stefanova, 1998

Nicotiana glutinosa Oenothera rosea Oxalis latifolia

Red garden sorrel

3

Bacterial wilt disease on potato: The South African experience

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34

Weed hosts

3

Costa Rica

2

RSA

Literature Reference Black and Sweetmore, 1993 Swanepoel, 1992

Chile

Fernandez, 1986

Malaysia

Abdullah, 1993

India

Shekhawat et al., 1992

Colombia

Thurston, 1963

1

Australia

Akiew 1994

Piper auritum

2

Honduras

Berg, 1971

Piper peltatum

2

Honduras

Berg., 1971

Scientific Name Physalis angulata L

Common Name Wild gooseberry

Phvsalis floridana Phvsalis minima L.

Biovar Race cited cited

2

3

Bladder cherry

Phvsalis nirruri

1

Physalis peruviana Physalis spp.

Wild gooseberry

3

Country of Report

Polanisia viscosa (L) DC

andTrevorrow,

Kelman,1953

Polygala

India

Kishore et al., 1993

Polygonum hidropiper L.

India

Shekhawat et al., 1992

Polygonum napalense

Uganda

Tusiime et al., 1998

Polygonum sp.

India

Kishore et al., 1993

Kenya Thailand India Brazil Andaman and Nicobar Islands

Harris, 1976 Titatarn, 1986 Shekhawat et al., 1992 Mariano, 1998 Ramesh and Bandyopadhvav,1993

Australia

Li and Havward, 1993

Ranunculus sceleratus L

India

Shekhawat er al., 1992

Ricinus communis L

RSA Phillipines India Malaysia Brazil

Swanepoel. 1992 Persley et al 1986 Shekhawat et al., 1992 Abdullah, 1993 Mariano, 1998

Uganda

Tusiime et al., 1998

India Netherlands

Shekhawat et al., 1992 V a n Beuningen et al.,1998

Sweden

Olsson, 1976

1,3

Portulaca oleracea L.

Common purslane

3

Portulaca volarasia Pultenaea villosa

3

Casteroil plant

Rumex abyssinicum Rumex sp.

2

Salpiglossis sinuata R and P

3

Salvia privoides Benth. Scoparia dulcis L.

Kelman, 1953 Goutweed

Kelman, 1953

Scuttellaria scandens

lndia

Senecio sonchifolia Moench.

Kishore et al., 1993 Kelman, 1953

Sesbania exaltata

Indonesia

Machmud, 1986

Solanum capsicastrum

Sweden

Olsson, 1976

United States

Hayward 1991

Solanum carolinense L

Horse nettle

Bacterial wilt disease on potato: The South African experience

Page

35

Weed hosts Scientific Name

Common Name

Solanum cinereum Solanum dulcamara

Bitter nightshade

Biovar Race cited cited

Graham and Llovd, 1979

Sweden Netherlands France UK

Olsson, 1976 Janse et al., I998 Hayward et al., 1998 Elphinstone et al. 1998

Honduras

Berg, 1971

India

Shekhawat et al. 1992

3

Australia

Graham and Lloyd, 1979

1,3

Kenya

Harris, 1976

South Africa

Swanepoel, 1992

Honduras

Berg, 1971

2

Papua New Guinea

Tomlinson, 1985

3

Thailand

Titatarn, 1986

India

Shekhawat et al., 1992

Reunion

Girard et al., 1993

Andaman and Nicobar

Ramesh and

Islands

Bandyopadhyay, 1993

France

Hayward et al., I998

Sweden

Olsson, 1976

Peru

Martin andFrench, 1995

3

2

3

2

Solanum incanum I. 2

3 2

Black nightshade

2 1,2,3

3

Peru

2

3

Netherlands

Solanum capsicastrum Solanum carolinense L.

Literature Reference

Australia

2

Solanum hirtum

Solanum nigrum L.

Country of Report

Horse nettle

Solanum cinereum

Martin and El-Nashaar, 1993 Van Beuningen et al., 1998

Sweden

Olsson, 1976

United States

Hayward, 1991

Australia

Graham and Lloyd, 1979

3

Sweden Netherlands France UK

Olsson, 1976 Janse et al, 1998 Hayward, et al 1998 Elphinstone, et. al, 1998

2

Honduras

Berg, 1971

Solanum inocum L.

India

Shekhawat et al., 1992

Solanum jamaicense Miller

Philippines

Valdez, 1986

Indonesia

Machmud, 1987

Solanum khasianum Clarke

India

Shekhawat et al., 1992

Solanum luteum Mill.

Sweden

Olsson, 1976

Solanum muskiana L.

India

Shekhawat et al., 1992

Chile

Fernandez, 1986

2

Solanum dulcamare

Bitter night shade

Solanum hirtum

Solanum khasianum

Solanum sarrachoides

3

2

3

Bacterial wilt disease on potato: The South African experience

Page

36

Weed hosts Scientific Name Solanum sisymbriifolium

Common Name

Biovar cited

Race cited

Dense-thorned bitter apple

Country of Report

Literature Reference

Brazil

Mariano, 1998

Solanum umbellatum

2

Honduras

Berg, 1971

Solanum verbascifolium

2

Honduras

Berg, 1971

Solanum xanthocarpum

India

Shekhawat et al., 1992

Solanum indicum L.

India

Shekhawat et al., 1992

Sonchus arvensis

India

Shekhawat et al., 1992

India Uganda

Shekhawat et al., 1992 Tusiime et al., 1998

Indonesia

Machmud, 1986

Australia

Akiew et al., 1993

Uganda

Tusiime et al., 1998

Nepal Netherlands

Pradhanang, 1999 Van Beuningen et al., 1998

Malaysia

Abdullah, 1993

Australia Uganda India Philippes Nepal

Akiew et al., 1993 Tusiime et al., 1998 Shekhawat et al., 1992 Persley et al., 1986 Adhikari, 1993

Thaliana sp.

India

Kishore et al., 1993

Thalictum javanicum

India

Kishore et al., 1993

Trifolium sp.

India

Kishore et al., 1993

Tropaelum lobbianum Hort.

India

Kishore et al., 1993

RSA Sweden India Sweden

Swanepoel, 1992 Olsson, 1976 Shekhawat et al., 1992 Olsson, 1976

India

Kishore et al., 1993

Spergula arvensis L.

3

Corn spurry

Spigula anthelmia Stachytarphita jamaicensis L.

Snake weed

Stellaria senii 2 2

Stellaria media Synedrella nodiflora Gaertn.

3

Pig’s weed

Tagetes minuta Tagetes sp. 3

Tropaeolum majus L.

Garden nasturteum

Urtica dioeca L.

Bush stinging nettle

1

Valeriana hardwickii Verbesina alata L.

Kelman, 1953

Vernonia chinense Mill.

Kelman, 1953

Vernonia cinerea

3

Vicia sp. Xanthium chinense Mill. Xanthomonas roseum (s)

Thailand

Titatarn, 1986

India

Kishore et al., 1993 Kelman, 1953

Cocklebur 2

Honduras

Bacterial wilt disease on potato: The South African experience

Berg, 1971

Page

37

Tree and shrub hosts Scientific Name Aleurites Wild.

moluccana

(L.)

Biovar cited

Common Name

Race cited

Country of Report

Literature Reference

Candlenut

Kelman, 1953

Aleurites sp.

Tung oil tree

Kelman, 1953

Anacardium occidentale L.

Cashew

3 3

Annona L. spp.

Indonesia, Réunion 1

Australia

Custard apple Taiwan

Hayward, 1994 Mayers and Hutton, 1987 Hayward, 1986, Hayward, 1994

Archontophoenix alexandrae

Alexandra palm

3

Australia

Arnat et al., 1978

Azadirachta indica J.Juss.

Neem tree

3

Australia

Hayward, 1994

Carica papaya

Papaya

India

Shekhawat et al., 1992

Casuarina equisetifolia L.

Horsetail beefwood/casuarina

China India China, India, Mauritius Peru Peru Reuion

He, 1983 Shekhawat et al., 1992 Hayward, 1994 Martin and Nydegger, 1982 Martin and El-Nashaar, 1993 Girard et al., 1993

Cyphomandra betacea

3

2 1, 2, 3

Tree tomato

1

34

3

Diospyros digyna Jacq.

Black sapote

3

Australia

Hayward, 1994

Eucalyptus L’Her. Spp.

Eucalyptus

1 3 3

Brazil China Australia

Hayward, 1994 Dianese and Dristig, 1993 Hayward, 1994

Eucalyptus grandis

Brazil

Mariano, 1 998

Eucalyptus urophylla

Brazil

Mariano, 1998

Taiwan

Hayward, 1994

Indonesia

Machmud, 1986

Eugenia javanica Lam.

Java/wax apple

Manihot esculenta Crantz

Cassava

Manihot glaziovii M. Arg..

Ceara rubber

Moring oleifera Lam.

Horse radish tree

Morus alba L.

Mulberry

3,4

Kelman , 1953

5 3.5 1

Musa spp.

4 2 1 2

Banana 1 3

Myristica fragrans L.

Nutmeg

Olea europaea L.

Olive

3,4

Plantago sp.

Plantain

1

Pluchea indica Less.

Indonesian shrub

1

1

India

Estelitta et al., 1997

China India Honduras Philippines Costa Rica, Venezuela, Honduras India Malaysia Indonesia Brazil Australia Trinidad

He, 1983 Mathew et al., 1993 (b) Woods, 1984 Soguilon et al., 1994 French and Sequeira, 1970 French and Sequeira, 1970 Shekhawat et al., 1992 Abdullah, 1993 Black and: Sweetmore, 1993 Mariano, 1998 Akiew and Trevorrow,1994 Black and: Sweetmore, 1993

India

Mathew et al., 1993(a)

China

He, 1983

Costa Rica

Martin and Nydegger, 1982

India

Shekhawat et al., 1992

Bacterial wilt disease on potato: The South African experience

Page

38

Tree and shrub hosts Scientific Name

Common Name

Pogostemen patchouli L..

Patchouli

Schinus terebinthifolius

Pepper tree

Syzigium aromaticum Tectona grandis L.

Biovar cited

Race cited

Country of Report

Literature Reference

India

Mathew et al., 1994

3

Réunion

Havward, 1994

Clove tree

3

Indones ia

Machmud, 1987

Teak

3

Malaysia. Indonesia

Hayward, 1994

Leguminous Hosts Scientific Name

Common Name

Biovar cited

Race cited

Country of Report

Snapbean

3

Phillipines

Valdez, 1986

Stringbean

2

Phillipines

Valdez, 1986

Albizzia falcata Back

Kelman, 1953 3.4 3.4 .

Arachis hypogaea (L.)

Literature Reference

Groundnut 3

1

China Vietnam,Hawaii, Austratlia, Philippines, Papua New Guinea, Thailand, R.S.A, Reunion, Indonesia, Uganda etc India Malaysia Andaman and Nicobar Islands Brazil

Canavallia ensiformis DC.

Jack bean

Cassia tora L.

Cassia/Senna

India

Cyamopsis speciousus (sic)

He, 1983 Persley et al., 1986 Persley et al., 1986 Persley et al., 1986 Persley et al., 1986 Persley et al., 1986 Shekhavvat et al., 1992 Abdullah, 1993 Black and Sweetmore, 1993 Ramesh and Bandyopadhyay, 1993 Mariano, 1998 Kelman, 1953 Shekhawat, et al., 1992 Kelman, 1953

Desmodium diffusum

India

Shekhawat, et al., 1992

India India Vietnam Sweden

Dolichos lablab L.

Indian bean/ lablab

Glycine max L.

Soybean

Indigoferra arrecta Hochst

Natal indigo

Shekhawat, et al., 1992 Shekhawat, et al., 1992 Presley et al., 1986 Olsson, 1976 Kelman, 1953

Leucaena glauca

Leacaena

Kelman, 1953

Melilotus indica Mucuna sp. (capitata)

Shekhawat, et al., 1992 Mucana (vegetable)

Kelman, 1953

Phaseolus aureus Roxb.

Malaysia

Abdullal:t, 1993

Phaseolus coccineus L.

Scarlet runner bean

Kelman, 1953

Phaseolus mungo L.

Black gram

Kelman, 1953

Phaseolus vulgaris L.

Bean (French/kidney) Yellow wax bean

3, 4 3 1, 3

India Malaysia Uganda Réunion Brazil Sweden Colombia

Bacterial wilt disease on potato: The South African experience

Shekhawat et al., 1992 Hayward, 1994 Hayward, 1994 Girard et al., 1993 Melo and takatsu, 1997 Olsson, 1976 Thurston, 1963 Page

39

Scientific Name

Leguminous Hosts Biovar Race Common Name cited cited

Phaseolus vulgaris L. var. humulis

Bushbean

Pisum sativum. L.

Garden pea

Psophocarpus tetragonolobus

Country of Report Sri Lanka

Velupillai, 1986 Kelman, 1953

3 3

Winged bean

Sesbania bispinosa

Malaysia Philippines Sri Lanka India

Hayward, 1986 Valdez., 1986 Velupillai, 1986 Shekhawat et al., 1992

India

Shekhawat et al., 1992

Sesbania grandiflora Pers.

Kelman, 1953

Sesbania rostrata

4

Stylosanthes humilis HBK

Townsville luceme

Tephrosia vogellii Hook.

Vogel tephrossia

Vicia faba

Broad bean

Vignia sinensis Savi

Cowpea, Long bean

Malavsia

Havward , 1994

Australia

Havward, 1986 Kelman, 1953

Teraminus labialis Spreng.

1,2, 3

Vignia ungiculata

Voandzeia subeterranea Thou.

Literature Reference

India

Shekhawat et al., 1992

India Sweden Br.azil India Philippines Malaysia India Brazil

Persley et al.• 1986 Olsson, 1976 Melo and Takatsu, 1997 Hayward, 1994 Persley et al., 1986 Abdullah, 1998 Shekhawat et al., 1992 Mariano, 1998 Kelman. 1953

Earthpea

Ornamental Hosts Scientific Name

Common Name

Anthuthrium andreanum

Anthurium

Antirhinum sp.

Snapdragon

Argemone mexicana L

Mexican poppy

Aster chinensis L.

Aster pilosus Willd.

Aster

Canna glauca

Canna

Biovar cited

Race cited

3

1

3

I

Country of Report Sri Lanka Reunion, Australia Mauritius

Literature Reference

India Australia

Hayward, 1986 Hayward, 1994 Banymandhub-Munbodh et al., 1998 Shekhawat et al., 1992 Akiew and Trevorrow,1994

India

Shekhawat et al., 1992

India

Shekhawat et al., 1992 Kelman, 1953

India

Kelman, 1953

India

Shekhawat et al., 1992

India

Shekhawat et al., 1992

Crysanthemum coronarium

Philippines

Valdez., 1986

Crysanthemum indicum

India

Shekhawat et al., 1992

Canna indica Centaurea cyanus L.

Cornflower

Bacterial wilt disease on potato: The South African experience

Page

40

Ornamental Hosts Scientific Name Crysanthenum spp

Common Name

Biovar cited

Race cited

Crysanthemum

Country of Report

Literature Reference

India

Shekhawat et al., 1992

Dahlia cocinea L.

Cuba

Stefanova, 1998

Dahlia piñata

Brazil

Mariano. 1998

Dahlia rosea Cav.

Dahlia

India Malaysia

Shekhawat et al., 1992 Abdullah, 1993

Dahlia spp.

Dahlia

RSA

Swanepoel, 1992

India

Shekhawat et al., 1992

French West Indies

Prior and Steva, 1990

Réunion

Girard et al., 1993

Taiwan

Chao et al., 1995

Dahlia variabilis Ensete ventricosum

Ornamental banana

Euphorbia pulcherrima

Poinsettia

Eustomo grandiflora

Texas blue bell

Gerbera spp.

Barbarton daisy

Hed ychium Koenig spp.

Ornamental ginger

3

1

4

1

Kelman, 1953 Hawaii

Hayward, 1994

Cuba

Stefanova, 1998

Costa Rica, Columbia Australia, Hawaii

French and Sequeira, 1970 Akiew and Trevorrow, 1994 Havward, 1994

Helinconia caribaeo Lam.

Costa Rica

Hayward. 1994

Helinconia latispatha Benth.

Columbia

Hayward, 1994

India

Shekhawat et al., 1992

Brazil

Mariano, 1998

Réunion

Girard et al., 1993

l

Réunion

Girard et al., 1993

1

U.S.A Australia

Strider et al., 1981 Strider et al.• 1981

Heliychrysum bracteatum Andr. Helinconia

Impatiens balsamina

1 ,3

Helinconia

2 2,1

Garden balsam

Nasturtium officinale Pelagornium capitatum

Rose geranium

1

Pelagornium xasperum

Rose geranium

Pelargonium hortorum

Florist geranium

Petunia hybrida

Garden petunia

India

Shekhawat et al.• 1992

Petunia spp,

Petunia

Malaysia

Abdullah, 1993

Phlox drummondii Hook

Phlox

Malaysia

Abdullah, 1993

Salvia farinacea Benth.

Blue salvia

Malaysia

Abdullah, 1993

Strelitzia reginae Banks

Bird of Paradise

3

Hawaii Reunion,Hawai, Japan Taiwan, Australia

Hayward, 1994 Hayward, 1994 Hayward, 1994

Tagetea erecta L.

African marigold

1,3

India

Shekhawat et al., 1992

Vinca rosea L

Madagascar periwinkle

India

Shekhawat et al., 1992

India Malaysia

Shekhawat et al., 1992 Abdullah, 1993

India

Shekhawat et al., 1992

Philippes

Valdez, 1986

Zinnia elegans Jacq. Zinnia spp.

Zinnia Chinese cabbage

3

1

Bacterial wilt disease on potato: The South African experience

Page

41

Other Crop hosts Scientific Name

Common Name Sweet pepper

Biovar cited 2, 3, 4

Race cited 1

Country of Report

Literature Reference

Japan

Tsuchiya and Horita, 1998

Allium cepa

Onion

Réunion, Venezuela

Hayward, 1994

Avena savita L.

Oats

India

Shekhawat et al., 1992

Beta vulgaris L.

Beetroot

India Brazil Sweden India India Brazil India Brazil French West Indies Papua New Guinea China Philippines Thailand India RSA. Réunion Andaman and Nicobar Islands Brazil Peru

Shekhawat et al., 1992 Melo and Takatsu, 1997 Olsson, 1976 Shekhawat et al., 1992 Shekhawat et al., 1992 Melo and Takatsu, 1997 Shekhawat et al., 1992 Melo and Takatsu, 1997 Prior and steva, 1990 Tomlinson, 1985 He, 1983 Valdez, 1986 Titatarn, 1986 Shekhawat et al., 1992 Swanepoel, 1992 Girard et al., 1993 Ramesh and Bandyopadhyay, 1993 Mariano, 1998 Martion and French, 1995 Girard et al., 1993 Mariano, 1998 Thurston, 1963

Brassica napus L.v. napus Brassica oleraceae var. capitata Brassica oleraceae var. capitata

Capsicum annum L.

1, 3

Kohlrabi 3 1 3 1 3 3 3, 3,4

Cabbage Cabbage

Bell pepper, chillies

2, 3

1 1

1, 3 1, 3

Capsicum frutescens

Chillies (Bell pepper)

Capsicum pendulum (s)

Pepper

3

Réunion Brazil Colombia India Sweden

Capsicum sp.

Shekhawat et al., 1992

Brazil

Olsson, 1976 Black and Sweetmore, 1993 De Zoyza and Liyan, 1994 Melo and Takatsu, 1997

India

Shekhawat et al., 1992

Costa Rica Centella asiatica

Indian pennywort

Cichorium endivia

Chicory

Citrillus vulgaris Schrad.

Watermelon

Commelina nudiflora L.

Baby dewflower

Coriandrum sativum

Coriander

Brazil

Melo and Takatsu, 1997

Cucumis sativus

Cucumber

Japan

Hayward, 1984

Curcuma domestica

Tumeric

Sri Lanka

Velupillai, 1986

Curcuma longa L.

Tumeric

India Sri Lanka

Shekhawat et al., 1992 Hayward, 1994

Cucurbita maxima x C. moschata

Pumplkin

Japan

Tsuchiya and Horita, 1998

Cucurbita moschata Poit

Pumpkin

India

Mathew et al., 1994b

Cucurbita pep var. melopepo

Zucchini

1,2,3

Brazil

Melo and Takatsu, 1997

Daucus carota

Carrot

1.3

Brazil

Melo and Takatsu. 1997

Fragaria L. spp

Strawberry

3,4

Japan, Taiwan

Hayward , 1994

Go:ssipium sp.

Cotton

India

Shekhawat et al., 1992

3

1

Sri Lanka

1,3

Kelman, 1953 1

4

1

1

Bacterial wilt disease on potato: The South African experience

Page

42

Other Crop hosts Scientific Name

Common Name

Biovar cited

Race cited

Country of Report

Literature Reference

Helianthus annus L

Sunflower

India Malayia Cuba

Shekhawat et al., 1992 Abdullah, 1993 Stefanova, 1998

Hibiscus cannabinus

Indian hemp

Malaysia

Abdullah, 1993

Hibiscus esculentus L.

Okra

India

Shekhawat et al., 1992

Hibiscus sabdariffa L.

Roselle

Kaempferia galanta

Medicinal plant

4

Luffa cylindrica

Loofah

3

Kelman, 1953

1

Lycopersicum chilense 3,4

Lycopersicum esculentum

Tomato

3

1

4 1, 3, 4 3

1

1, 2

1, 3

1, 2, 3, 4

2 1, 2, 3

1 3

Maranta arundinaceae L.

3

Nicotiana tabacum

Tobacco

2, 3 1, 2, 3 3 4 1, 2, 3

1 2

China

He, 1986

Taiwan.

Pan et al., 1996

Sweden

Olsson, 1976

Papua New Guinea India, Vietnam,Fidji China Sri Lanka, Thailand etc. RSA

Tomlinson, 1985 Persley et al., 1986 Persley et al., 1986 Persley et al., 1986 Engelbrecht and Hattingh, 1989 He, 1983 Valdez, 1986 Adhikari, 1993 Abdullah, 1993 Girard et al., 1993

China Philippines Nepal Malaysia Réunion Andaman and Nicobar Islands Japan Brazil Sweden Peru Peru Colombia

Ramesh and Bandyopadhyay, 1993 Tsuchiya and Horita, 1998 Mariano, 1998 Olsson, 1976 Martin and French, 1995 Martin and El-Nashaar, 1993 Thurston, 1963

Malaysia

Abdullah, 1993

Thailand Vietnam Nepal Réunion Malaysia Andaman and Nicobar Islands Japan Brazil Australia RSA USA Peru

Titatarn, 1986 French and Sequeira, 1970 Adhikari, 1993 Girard et al., 1993 Abdullah, 1993 Ramesh and Bandyopadhyay, 1993 Tsuchiya and Horita, 1998 Mariano, 1998 Black and Sweetmore, 1993 Swanepoel and Young, 1988 Marin and El-Nashaar, 1993 Black and Sweetmore, 1993

Bacterial wilt disease on potato: The South African experience

Page

43

Other Host Crops Scientific Name

Common Name

Biovar cited

Race cited

3

Nicotiana tabacum

Tobacco

2, 3

1

1 1, 2, 3 3 4 1, 2, 3

2

3

Country of Report

Literature Reference

Thailand Vietnam Nepal Réunion Malaysia Andaman and Nicobar Islands Japan Brazil Colombia Australia RSA USA Peru

Titatarn, 1986 French and Sequeira, 1970 Adhikari, 1993 Girard et al., 1993 Abdullah, 1993 Ramesh and Bandyopadhyay, 1993 Tsuchiya and Horita, 1998 Mariano, 1998 Black and Sweetmore, 1993 Swanepoel and Young, 1988 Marin and El-Nashaar, 1993 Black and Sweetmore, 1993

Taiwan

Hayward, 1994

Japan

Tsuchiya and Horita, 1998

Perilla crispa

Perilla

Perilla ocymoides L.

Perilla

Petroselium crispum

Parsley

1, 3

Brazil

Melo and Takatsu, 1997

Piper hispidinervium

Long pepper

1

Brazil

Lopes et al., 1998

Pogostemon cablin

Medicinal plant

3

China

He, 1986

Pomoea batatus Lam.

Sweet potato

4

China

He, 1983

Raphanus sativa L.

Radish

Taiwan India China Sri Lanka Thailand India Andaman and Nicobar Islands

Hayward, 1994 Shekhawat et al., 1992 He, 1983 Velupillai, 1986 Titatarn, 1986 Shekhawat et al., 1992 Ramesh and Bandyopadhyay, 1993

1

1

3 Sesamum indicum

Sesame, gingelly

Setaria italica Beauv.

Indian millet

India

Shekhawat et al., 1992

Solanum auriculatum

Bringelier marron

Réunion

Girard et al., 1993

Sweden

Olsson, 1976

Brazil

Mariano, 1998

Australia RSA

Moffet et al., 1983, Engelbrecht and Hattingh, 1989 Tomlinson, 1985 He, 1983 Valdez, 1986 Titatam, 1986 Adhikari, 1993 Shekhawat et al., 1992 Abdullah, 1993 Girard et al., 1993 Ramesh and Bandyopadhyay, 1993 Tsuchiya and Horita, 1998 Mariano, 1998 Olsson, 1976 Marin and El-Nashaar, 1993

Solanum capsicastrum Solanum gilo

Nigerian vegetable 3 3 4 1,3,4

Solanum melangena

Eggplant

1

3

1 2,3,4 1,2,3

Papua New Guinea China Philippines Thailand, Nepal India Malaysia Reunion Andaman and Nicobar Islands Japan Brazil Sweden Peru

Bacterial wilt disease on potato: The South African experience

Page

44

Host crops Scientific Name

Common Name

Biovar cited

Race cited

2,3,4 2

Country of Report Philippines

Valdez, 1986

Nepal

Adhikari,.1993

Malaysia

Abdullah, 1993

Reunion

1,2

Andaman

Solanum tuberosum

Literature Reference

Girard et ol., 1993 and Nicobar

Ramesh and

Islands

Bandyop:adhyay, 1993

1,2

Bolivia

2,4

Japan Brazil

Smith et .a/., 1998 Tsucblya and Horita, 1998 Mariano, 1998

U.K., Netherlands, Italy

Hayward et al., 1998

France, Latin American

Hayward et ol., 1998

countries, Australia

Hayward et al., 1998

Sweden

Olsson, 1976

2,4

Papua New Guinea

Tomlinson, 1985

1,2, 3

Peru

Marin and El-Nashaar, 1993

2,3

South Africa

Swanepoel and Young, 1988

2,3

Australia

Black and :Sweetmore, 1993

1

USA

Black and :Sweetmore, 1993

1

Indonesia

Blaclc and !Sweetmore, 1993

Potato

Symohytum so.

Forage crop

3

China

He, 1986

TetroRonia exoansa

Spinach (N. '.Zealand)

1,3

Brazil

Melo and TalcatSu, 1997

Other host crops Scientific Name

Zingiber ojficinale

Common Name

Ginger

Biovar cited

Race cited

4 3 4 3,4 1,2,3 3,4 4 3

1

1,

3,4

Country of Report Costa Rica Mauritius China China Malaysia

Australia Hawaii, Philippines India Sri Lanka Thailand India

Literature Reference Martin and Nydegger, 1982 Hayward, 1986 He, 1983

Hayward, 1986 Hayward, 1986 Hayward, 1986 Hayward, 1986 Hayward. 1986 Velupillai, 1986 Hayward, 1994

Shelchawat et al., 1992

Andaman and Nicobar Islands

Ramesh and Bandvopadhvav, 1993

Squash

3

Philippines

Valdez, 1986

Fennel

4

Philippines

Valdez, 1986

Pechay

3

Philippines

Valdez, 1986

Honduras,Colombia,

French and Sequeira, 1970

2

Plantain

Peru, Costa Rica

Bacterial wilt disease on potato: The South African experience

Page

45

Other Plant Species Scientific Name

Common Name

Biovar cited

Race cited

Country of Report

Literature Reference

Hyoscyamus niger L. V.niger

Sweden

Olsson, 1976

Hyoscyamus niger L. V pallidus

Sweden

Olsson, 1976

USA

Valdez, 1986, Norman and Yuen, 1998

Australia

Havwardl, 1994

Blainvillea rhomboidea

Brazil

Mariano, 1998

Browallia speciosa Major

India

Shekhawat et al., 1992

Epipremun aureum

Pathos

1

Alpinia L. spp.

1

3

Corchortis capsularis

Jute

West Bengal

Hayward, 1986

Corchorus olitorius

Jute

West Bengal

Hayward, 1986

Corchorus spp..

India

Shekhawat et al., 1992

Corchorus trilocularis

India

Shekhawat et al., 1992

Coriandrum savitum

India

Shekhawat et al., 1992

Cosmos caudatus

Sarawak

Hayward, 1986

Cuinum cyminum

India

Shekhawat et al., 1992

Echinochloa crusgalli Beauv.

India

Shekhawat et al., 1992

Eleusine coracana Gaerlin.

India Japan

Tsuchiya and Horita, 1998

Foeniculum vulgare

India

Shekhawat et al., 1992

Galium aparine L..

India

Shekhawat et al., 1992

Heliotropium indicum

India

Shekhawat et al., 1992

Hyptis suaveolens (L) Poit.

India Brazil

Shekhawat et al., 1992 Mariano, 1998

lpomea obscura Ker.

India

Shekhawat et al., 1992

lpomoea setosa

Malaysia

Havward, 1986

Kalanchoe blossfeldiana Paellnitz v.

Réunion

Hayward, 1994

Kalanchoe tubiflora

Brazil

Mariano, 1998

Launea aspleniifolia DC

India

Shekhawat et al., 1992

Japan

Tsuchiva and Horita, 1998

Ludwiga suffruticosa

Brazil

Mariano, 1998

Marsypianthes chamaedrvs

Brazil

Mariano, 1998

Mimosa scabrella

Brazil

Mariano, 1998

Philippines Sri Lanka

Valdez, 1986 Velupillai, 1986

Eustoma russellianum

Russel prairie gentian

Liminium spp

Momordica charantia

3

Bitter Gourd

1,2,3

I

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Other host crops Scientific Name Obetia ficifolia

Common Name

Biovar cited

Race cited

Bois d'ortie

Country of Report

Literature Reference

Réunion

Girard et al., 1993

Parthenium hysterophorus

India

Shekhawat et al., 1992

Solanum americanum

Brazil

Mariano, 1998

Trachvspernum amni

India

Shekhawat et al., 1992

China

He. 1983

India

Shekhawat et al.• 1992

Urtica nivea

3

1

Verbena hybrida

2.7

SOURCES OF INOCULUM AND MODES OF DISPERSAL

Two major sources of inoculum exist, namely infected planting material and infested soil. Infected plants decaying in the soil can release masses of bacterial cells in a slime layer. These slime masses can adhere to soil particles and form pellets enhancing its survival (Shekhawat et al., 1992). The populations in the soil can then increase or decrease, depending on the presence of alternative hosts and cultural practices. The i n o c u l u m threshold for i n i t i a t i n g disease d e p e n d s highly o n predisposing factors. Devi et al. (1982) observed that the inoculum reached a threshold of about 10 7 cfu / g soil before infection started. Infected planting material such as potato tubers is the most effective source of inoculum and means of dispersal. Since the pathogen can be carried latently within tubers, controlling the transmission of this pathogen is complicated. Tubers can carry the bacteria in three manners, namely externally on tuber surfaces, in lenticels and in the vascular tissues (Shekhawat et al., 1992). Although surface carried bacteria can be eliminated by chemical treatments, internal infections remain a threat. A study conducted by Sunaina et al. (1989) showed that during storage, bacterial populations decreased rapidly on the tuber surface reaching a non-detectable limit within 30-60 days at 4°C and 60-90 days at room temperature. But in lenticels and vascular tissues R. solanacearum could still be detected after 240 days. Irrigation water, root contact and insects such as in the case of Moko disease of banana can also spread the disease. Mechanical dissemination occurs mainly by infested equipment both during sorting of seed tubers as well as on the field, and by movement of people and animals through infected fields. Insect dissemination plays an important role in the banana industry and has been reported in Honduras, Costa Rica and Colombia. Bees (Trigona spp.), wasps (Polybia spp.), fruit flies (Drosophila spp.), and flies of other genera have been identified as transmitters (Buddenhagen and Kelman, 1964). Reports where R. solanacearum was disseminated by chewing insects on potato (Colorado potato beetle, Leptinotarsa declimlineata Say.) and eggplants (green beetle, Diabrotica graminea Baly) have been made (Kelman, 1953). Negative results have however also been reported where insects were allowed to feed on infected plants and then on healthy ones (Kelman 1953). Some evidence suggests that R. solanacearum can have an epiphytic phase in its life cycle which contributes to its survival and provides another source of inoculum. Hayward and Moffet (1978) demonstrated that leaf spot disease of capsicum was caused by R. solanacearum. Further studies (Moffet et al, 1983) revealed that under conditions with relatively high humidity, epiphytic colonisation could occur, leading to the formation of lesions on leaves. Mist inoculation of eggplant, pepper and tomato cultivated in growth-chambers caused leaf spots and wilting (Kelman et al., 1994). Although leaf infection has been reported, there is no evidence that the pathogen can survive as an epiphyte on leaf and other plant

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surfaces (Kelman et al., 1994). Aerial transmission through rain splash dispersal on tobacco has been noted in Japan (Hayward, 1991a). Infected host debris is an important short-term shelter for R. solanacearum in soil (Graham et al., 1979) allowing survival between growing seasons. It also serves as a transmission agent. This is especially so for race 3 which has a limited alternative host range. Weeds serving as hosts are well-documented sources of inoculum and contribute greatly to the survival of R. solanacearum in the absence of a cultivated host. They may also serve as a source of infection when virgin lands are cleared for cultivation (Buddenhagen and Kelman, 1964, Martin et al., 1981).

2.8

PRESENCE OF RALSTONIA SOLANACEARUM IN VIRGIN SOILS

The occurrence of R. solanacearum in newly cleared lands or virgin soils has been cited in literature (Kelman, 1953; Sequeira and Averre, 1961; Martin et al., 1981) and has been attributed to the presence of wild hosts in the indigenous flora. In earlier reports proof was not always presented that bacterial wilt was truly a natural component of the soil microflora, and the possibility of contamination through planting material, drainage water or other means was not eliminated. Several authorities have studied the outbreak of bacterial wilt of bananas in newly planted areas in Costa Rica. A comprehensive study conducted by Sequeira and Averre (1961) involving 20 000 acres of virgin woodlands in Costa Rica revealed extensive infection of Heliconia latispatha, H. acuminata, H. imbricata with the banana strain (B strain) of R. solanacearum. Eupatorium oderatum was found to be infected with the weed strain of the pathogen (T strain). French et al. (1981) who summarised the findings of Buddenhagen (1960), Sequeira (1960), Sequeira and Averre (1961) and his own, refer to the strain causing rapid wilt of bananas as race 2, strain B and those causing slow wilt and distortion on helinconias as strain D of race 2. Repeated cutting back of heliconias caused the disease to be spread mechanically to other clusters of heliconias. When bananas were planted in cleared jungle, they developed a slow wilt and distortion not typical of the banana strain. In Costa Rica Heliconia were occasionally found diseased in virgin jungle showing distortion and slow wilt symptoms caused by D strains of race 2. Bananas planted in cleared jungle also developed distortion and slow wilt, symptoms not characteristic of the B strain wilt disease, which causes rapid wilting and was responsible for severe outbreaks. Continuous passage of the D strain through bananas resulted in a doubling of the disease index; leading to the conclusion that bacterial wilt of bananas arose by a selection pressure exerted by bananas upon strain D (French et al., 1981). Martin et al. (1981) found that biovar 1 (race 1) and biovar 2 (race 3) of the pathogen attacked potatoes grown in virgin soils in the Amazon basin. No potatoes or other wilt-susceptible crops had been planted before and infestation by contaminated water or by planting infected seed was excluded . This suggests that these strains were indigenous to the region. Sneviratne (1969) found biovar 2 to occur in virgin soils at elevations of 1891m in Sri Lanka. He believes it unlikely that the disease could have been introduced by European seed material. In their study on persistence of R. solanacearum in soil in Georgia, Dukes et al. (1965) had cleared land of timber and brush and planted tomatoes and found a high incidence of bacterial wilt indicating that the organism was indigenous.

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2.9. SURVIVAL OF RALSTONIA SOLANACEARUM IN SOIL Ralstonia solanacearum is a soil-borne pathogen that is found in various types of soils world-wide. Reports regarding its survival period are often conflicting. Bacterial wilt was found to survive in fallow soil for periods of 2 to 10 years, yet in a different soil poorer survival rates were reported despite the presence of host plants (Nesmith and Jenkins, 1985). Information on soil survival was often gathered indirectly or from glasshouse trials, partly because detection of the organism in field soils is difficult (Moffet and Wood, 1984). The survival of R. solanacearum in soil is affected by several factors such as the initial inoculum concentration, whether the land is left fallow or cropped to a non- susceptible host, as well as the biological, chemical and physical properties of the soil (Moffet et al., 1983). The temperature, moisture and oxygen status of the soil is further factors that influence the longevity of the pathogen. Survival of R. solanacearum in soil can be measured on the basis of two parameters, namely, the ability to withstand soil conditions to remain viable and the segregation into virulent and avirulent populations (Shekhawat and Perombelon, 1991). Several authors, amongst others Nesmith and Jenkins (1983), measure survival in terms of detection of fluidal cells on selective media. These cells are referred to as virulent. Denny et al. (1994) reason, however, that in soil Ralstonia normally exits in the "avirulent" (phenotype conversion) form, in which reduced production of extracellular proteins and extracellular polysaccharides occurs. In this form the bacteria can conserve energy and cellular resources thereby increasing its chances of surviving. Once host material is available, bacteria multiply and once sufficient cell density is obtained the extracellular virulence factors are produced. This hypothesis would greatly affect measurement of survival rate and might explain some of the discrepancies regarding the longevity of bacterial wilt in soil.

2.9.1

Influence of soil temperature

Temperature requirements for optimal growth are known to differ for the various strains. Biovar 2, race 3 isolates have a lower optimum growth temperature than strains of race 1 (Thurston, 1963). Disease development in terms of wilting and visible tuber infection, is known to occur at lower temperatures of 14/16°C with biovar 2 than with biovar 3 (race 1) (Swanepoel 1990). Katayama and Kimura (1984) also found that at lower as well as intermediate temperatures of 24°C, growth of biovar 2 (race 3) was better than that of race 1, biovar 4. Shekhawat and Perombelon (1991) studied the survival rates of biovar 3 (race 1) and biovar 2 (race 3) at various temperatures and confirmed that race 1 is better adapted to a wider range of temperature for growth than race 3. In their study it appears that population decline and loss of virulence of both races was slowest between 10-30°C, provided other soil factors were congenial. At 35°C, race 1 population declined to an undetectable limit within 10 weeks, whereas populations of race 3 could not be detected after 8 weeks. At low temperature of 5°C, population decline was the same for both races, reaching undetectable levels within 12 weeks. Granada and Sequeira (1983b) however reported that soil kept in plastic bags at 4°C maintained bacterial wilt populations for 673 days. This indicates that long-term survival in deeper soil levels at low temperatures is possible. Soil temperatures of over 40°C appear to be fatal to Ralstonia populations, depending on the period of exposure. Seneviratne (1988) found that the pathogen was able to survive in some of the soil samples kept at 40°C for seven days, but not in those kept at 43°C. Survival of races 1, 2, 3 in soil kept at 28°C and water potential of -2 bars varied with race 1 surviving longest. Race 2 and race 3 had shorter survival periods (Granada and Sequeira, 1983b).

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2.9.2

Influence of soil moisture

Soil moisture may influence at least four aspects of the bacterial wilt disease, namely the survival of the bacterium in its free state in soil, rate of infection, disease development after infection and spread through the soil. Native farmers in India have from an early date noted the relationship between soil moisture and bacterial wilt of tobacco and attributed the disease to high moisture levels (Shekhawat et al., 1992). Studies on the effect of soil moisture on the survival of R solanacearum h a v e provided conflicting evidence. This can partly be attributed to interactions with other soil factors. According to Shekhawat et al. (1992) soil moisture and temperature have a synergistic effect on disease development. High temperatures or high soil moisture alone will not induce symptoms. They found that in India, potato wilt was higher after onset of the monsoon, even though high temperatures prevailed earlier in the season. Wilt incidence declined when soil moisture dropped to 8-10% of water holding capacity (WHC). Tanaka and Noda (1973) found that growth rate of R. solanacearum in sterile soil is higher at high soil moistures (80-100% water content) than in soil at 40% of water holding capacity. Under identical conditions but in non-sterilised soil no increase in growth was observed, regardless of water content. Okabe (1971) found that R. solanacearum grew more actively in dry soil of 15-20% water content (WC) than in moist soil (40-50% WC) and reasoned that this pathogen had the specific nature to utilise small amounts of capillary water held among soil particles while growth of other micro-organisms was delayed. If, however, soil moisture were increased, re-growth of these micro-organisms would be stimulated leading to increased competitive colonisation of organic matter and antibiosis. This would reduce R. solanacearum populations. This theory could explain why Tanaka and Noda (1973) did not observe increase in growth in the non-sterilised soil at both moisture levels. Their moisture levels were higher than those investigated by Okabe (1971). Shekhawat and Perombelon (1991) reported that population decline was at its lowest in soil moisture at 60% of water holding capacity (WHC). Even after 13 weeks the pathogen could still be detected. A deviation in moisture led to an increased population decline. Greater population declines occurred at high moisture levels (80- 100% of WHC) than at low moistures (20-40% of WHC). At the high moisture levels a shift towards virulence was more pronounced when soil temperatures were low (5°C) (Shekhawat et al., 1992). In dry soils the populations decreased to an undetectable limit within 6 weeks. Hsu (1977) noted similar trends regarding high and low moisture contents. He reported a survival period of 30 days for dry soil (0.8-7.9% WC); 1 0 5 days for flooded soil; 150 days for very moist soil (43-47.4% WC); 225 days for moist soils (15.2-19.9% WC) and 390 days for moderately moist conditions (30.7-36.7% WC). Moffet et al. (1983) performed a study in which pressure potential rather than moisture content was used to measure soil moisture, since they regard this a more accurate measure of the availability of water to micro-organisms. Sensitivity t o degrees of drying varied with the biovar of the pathogen used and soil type, with biovar 2 decline being greater than biovar 3. Population decline for both biovars in the various soils (clay loam, sandy loam, and clay) was generally highest at -0,003 kPa. Sensitivity to dry conditions has been reported to be a contributing factor in reducing R. solanacearum populations in fallow land (Sequeira, 1962). Moffet et al. (1983) found, however, that the rapid decline of both biovars that occurred with drying was slowed if the pressure potential was maintained at a constant value. They did note that R. solanacearum decreased the most in the driest soils in the initial stages of the trials, but that in the end the driest soil contained higher numbers of viable pathogens than did the wetter soils.

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A rapid decline was observed in flooded or very dry soils, irrespective of soil type (Nesmith and Jenkins, 1985). Their study also revealed that antagonistic actinomycetes were most numerous in dry soil, whereas antagonistic bacteria were predominant in wet soils.

2.9.3

Influence of soil type

Relation between soil type and incidence of bacterial wilt is not clear and contrasting reports have been obtained. In Indonesia bacterial wilt of peanuts is most severe in heavy clay soils whereas in China it is prevalent in sandy, especially gritty soil, and not in heavy clay or loam (Hayward, 1991a). Nesmith and Jenkins (1985) found that soil type influenced soil moisture and antagonistic microbial populations, which in turn affected the Ralstonia populations. Soil bacteria are active primarily in soil of higher pressure potential and reduced activity is noted at -0.03 kPa and especially at -0.15 kPa (Cook and Papendick, 1970, as quoted by Moffet et al., 1983). The activity of the microbial population associated with organic matter varies with soil type and pressure potential. In a study on effect of moisture and soil type on survival of R. solanacearum in soil, Moffet et al. (1983) reported organic matter contents of 2.5% in clay loam, 1.7% in clay and 1.1% in sandy loam. They noted a greater R. solanacearum population decline in the clay loam than in clay or sandy loam at higher pressure potentials. The increased decline in the clay loam was attributed to the higher microbial activity associated w i t h the organic matter. R. solanacearum populations thus have to compete with the soil microbes for nutrition and be exposed to increased microbiostasis. The finding that population decline was less in the clay than in the sandy loam, even though the microbial activity is expected to be higher in clay, could be due to adsorption of Ralstonia onto clay particles, protecting them from microbiostasis (Moffet et al., 1983). The authors recorded greater decline of biovar 2 population on all of the three soil types in comparison to biovar 3 populations. This was ascribed to greater sensitivity of this biovar to microbiostasis or its poor competitive ability or to the intrinsic nature of the biovar 2 isolate. Tanaka (1976) previously reported the relation between organic matter, microbes and Ralstonia populations. In surface soil with high levels of organic matter and microbial activity, the pathogen population declined faster than in the subsoil with a lower content of these, and that addition of manure to the subsoil reduced the populations considerably. In a study conducted by Shekhawat and Perombelon (1991) population decline was slower in clay than in sand even under dry conditions. In dry clay, race 1 and race 3 were detectable after 5 weeks and 3 weeks, respectively, whereas in dry sand both races of the pathogen were undetectable within 3 weeks.

2.9.4

Influence of depth of soil layers

Results of several authors (Mc Carter et al., 1969, Okabe, 1 9 7 1 , Tanaka and Noda, 1973) suggest that R. solanacearum can survive in deeper layers of certain soils. Once the pathogen has entered the deeper layers it can survive in localised microsites (debris or "free soil") where microbial activity is likely to be low (Lloyd, 1978). Subsequent infection of a host plant would then depend on penetration o f the root system i n t o these mi c ros ites . The s u r v i v a l o f t h e pathogen i n these s i t e s would probably be limited to a few deep-rooted hosts such as potatoes and tobacco growing in favourable soil types such as loose sandy soils (Lloyd, 1978). Vertical distribution of the bacterial wilt pathogen in the soil varies, ranging from the presence in topsoil to almost a meter deep. Martin et al. (1981b) investigated the presence and distribution of R. solanacearum at three soil depths (0 30 cm, 30 - 60 cm, and 60 - 90 cm). In one location the bacterial population were highest in the 0-30 cm soil layer, Bacterial wilt disease on potato: The South African experience

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whereas in the other location the highest population was recorded in the 30 - 60 cm layer. The authors suggest that such varying results could be attributed to amount of rainfall, type of soil and differences in root development. Other authors (Okabe, 1971; Tanaka, 1976; Graham and Lloyd, 1979) reported highest concentrations of R. solanacearum at deeper soil layers. Tanaka (Tanaka, 1976) observed the distribution of the pathogen in the 0-80 cm layer of naturally infested sandy loam soil, and noted a high population at all depths even after one year of fallow. After two years, however, the pathogen had practically disappeared. Graham and Lloyd (1979) studied the distribution of R. solanacearum in soil at five sites in a naturally infested field. At three sites samples were collected at soil depths of 0-15 cm, 15-30 cm, 30 - 45 cm, 45 - 60 cm and 60 - 75 cm, at the other two only the 30 - 45 cm and 60 - 75 cm layers were sampled. The pathogen was detected at all five sites, but not at all depths. R. solanacearum could not be detected in any of the 0 - 15 cm samples and at one site the bacteria were only present in the 15 - 30 cm layer. Presence was detected in the 60 - 75 cm layer in four of the five sampling sites. The authors regard d e s i c c a t i o n o f R solanacearum cells due to dry weather prior to sampling as one of the reasons that the bacterium was absent from the 0-15 cm zone, but state that other factors could also be involved. Graham and Lloyd (1979) observed that their results contrasted with those obtained by Mc Carter et al. (1969) in a similar vertical distribution study. Mc Carter et al. (1969) recorded a high infestation in the top 30 cm layer, with markedly reduced or absent populations in the 30 - 45 cm zone. No Ralstonia populations could be detected deeper than 45-60 cm except for a low presence in one sample. Graham and Lloyd (1979) considered root development as one of the causes of variation. The work done by Mc Carter et al. (1969) involved soils infested by diseased tomato transplants whose roots are not likely to penetrate to great depths. Their study, however, involved soils infested with diseased potato plants. The study conducted by Sunaina et al. (1892) also supports the hypothesis that the depth of root systems of hosts might govern vertical distribution. They found that during the potato season population build-up was higher in the top 30 cm than in the deeper soil layers. During the non-cropping season the population declined much quicker in the top 30 cm as compared to deeper layers, and in the top 20 cm it decreased to an undetectable level. The pathogen survived at the 20-60 cm soil level even after the field had been kept fallow for 7 months. The longer survival of the pathogen in deeper soil layers is to be expected as the roots and bacterial exudates remain undisturbed.

2.9.5

Influence of anaerobiosis of soil

Longevity of R. solanacearum is also affected by the oxygen status of the soil. Anaerobic conditions cause a more rapid population decline with undetectable levels being reached within 7 weeks, whereas 11 weeks where required under aerobic conditions to reduce the population to undetectable limits. Anaerobic conditions also favoured a shift to avirulence (Shekhawat and Perombelon, 1991). R. solanacearum is an aerobic organism and conditions that reduce the availability of oxygen should affect its survival negatively. Flooding of soil has been reported to adversely affect the pathogen's survival (Kelman, 1953; Nesmith and Jenkins, 1985). Yet some reports indicate that flooding of rice fields for several weeks does not eradicate bacterial wilt (De S. Seneviratne, 1988; Shekhawat et al., 1992). In some cases bacterial wilt incidence was higher in a potato crop that followed after paddy rotation. The soil in flooded rice paddies appears to be more oxygen-rich than flooded fallow soils. Aerobic and anaerobic microsystems exist in soil of paddies. The diffusion of oxygen through the roots of rice creates an aerobic environment in the rhizosphere and rhizoplane, allowing R. solanacearum to survive ( Shekhawat et al., 1992).

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2.9.6

Influence of pH of the soil

Although the optimum pH for growth of Ralstonia solanacearum in vitro is about 6.8, bacterial wilt has been reported in both acidic and alkaline soils. In North Carolina a high incidence of potato wilt occurred in soil with a pH 4.5. Tobacco wilt in this state was more common in moderate acid soils (pH 5-5.5) (Kelman, 1953). Similarly, in Guadeloupe bacterial wilt was reported in soils of pH 5-5.5 (Prior et al., 1993) and in Mauritius it was not common in alkaline soils (Kelman, 1953). Soil conditions in which this bacterium occurred in Japan and Ceylon were often alkaline. In one instance a soil pH of 8.5 was recorded (Kelman, 1953). Shekhawat and Perombelon (1991) investigated the impact of different pH levels on race 3 (biovar 2) and race 1 (biovar 3) grown in broth culture. At pH 4.5, growth of both race 1 and race 3 decreased and virulence was completely lost. At pH 8.5 the virulence and growth of race 3 was reduced whereas race 1 grew well although virulence was reduced. At pH 5.5 and pH 7 race 3 grew well and maintained a high level of virulence. Race 1 also grew well but could maintain high virulence only at pH 7. It would therefore seem that race 3 is better adapted to retain virulence under low pH conditions.

2.10

CONTROL OF RALSTONIA SOLANACEARUM

2.10.1 Chemical Control Several chemical formulations have been evaluated for the control of bacterial wilt, with limited success. Disinfectants s u c h as potassium s u l p h i d e , copper acetate, potassium permanganate and formalin are not effective and often damage the crop and pose a threat to the environment (Kelman, 1953; Shekhawat et al., 1992). Control or eradication of bacterial wilt has been attempted with fumigants such as chloropicrin, ethylene dibromide and methyl bromide with varying results (Kelman, 1953; Enfinger et al., 1979; Engelbrecht et al., 1990; Melton and Powell, 1991; Chellemi et al., 1997). Chloropicrin has been applied since the 1940's and in most instances crop losses could be reduced, but complete control or eradication could not be obtained (Kelman, 1953). Enfinger et al. (1979) evaluated the application of an array of chemical formulations, amongst them fumigants such as chloropicrin, methyl bromide, DD- MENCS (a mixture of methyl isothiocyanate, dichloropropane and dichloropropene), and Metham. Chloropicrin was the only formulation that provided significant control throughout the season. DD-MENCS and a mixture of methyl bromide and chloropicrin (67-33%) were less effective than chloropicrin on its own, but reduced wilt more than methyl bromide. Methyl bromide was found to control wilt only until midseason. Metham applied as fumigant gave moderate to poor control early in the season. Applied as fumigant it was more effective than applied as drench or when incorporated. Dichloropropane and dichloropropene formulations have also been used since the 1940's to reduce the incidence of wilt. At high dosages where wilt was completely controlled, plants developed chlorine injury (Kelman, 1953). Engelbrecht et al. (1990) revealed that disease suppression was most effective if a mixture of ethylene bromide and chloropicrin (55:45 m/m) was applied at a rate of 120 L/ha. All chloropicrin treatments were found to reduce the disease incidence significantly. They also found at one of the trial sites that a chloropicrin/methyl bromide mixture was less effective than chloropicrin on its own, but that it did not differ significantly from methyl bromide application. In a trial where very high infestation levels were encountered, fumigation with ethylene bromide: chloropicrin 55:45 m/m at 120L/ha failed to suppress the disease for the entire growing season. Ethylene bromide has been banned in the United States since 1983 (Sittig, 1985). Melton and Powell (1991) were not successful in reducing the wilt index in tobacco fields by fumigating with 1, 3 dichloropropene and chloropicrin but yields were increased. Fortnum and Martin (1998) obtained both a reduction of the pathogen and an increased yield by fumigating with 1, 3-dichloropropene (78%): chloropicrin (17%) mixture with both in-

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row and broadcast applications. The authors suggest a waiting period of three weeks after application and warn that spring rains can interfere with the application program. A bactericidal formulation "Terlai" which has an active ingredient similar to 2,2- dichloro-N (2 hydroxy-l(hydroxy-methyl)2-(4-nitrophenyl) ethyl acetamide, significantly reduced the incidence of wilt, especially if applied in conjunction with P.fluorescens (Machmud and Machmud, 1994). Chlorosis on the leaves of tomato was noted, possibly due to high concentrations of "Terlai" applied. Chemical compounds have also been used to treat plants to offer protection against infection with R. solanacearum. Infection in tobacco seedlings could be delayed slightly by treating the roots and stems with compounds such as hydroxymercuricchlorophenol, zinc sulphate plus lime, Bordeaux mixture and sulphur. Aldicarb, a systemic insecticide, was found to hasten the development of wilt in tomato plants, by altering the quality and quantity of extracellular polysaccharide (Shekhawat et al., 1992). Rhizome treatment of ginger with Emisan 6 (an organomercurial) plus plantomycin for 30 minutes in addition to three spray applications resulted in 100% control of bacterial wilt. Plantomycin alone or in combination with Blitox was 95% effective (Shekhawat et al., 1992). Several studies on the use of antibiotics as control agents have been performed with varying success. Pre-treatment of potato tubers with antibiotic C-6 (similar to erythromycin) followed by two foliar sprays resulted in control and a threefold increase of yield. Dipping roots of eggplant seedlings into streptocycline prior to transplanting reduced the incidence of wilt (Shekhawat et al., 1992). Engelbrecht et al. (1990) could not control tobacco wilt effectively with frequent application of streptomycin sulphate.

2.10.2 Disinfection Cleaning and disinfection of storage and seed potato handling equipment each year to eliminate carryover of disease-causing organisms on equipment surfaces or in potato debris is an essential step in potato health management, even if disease has not been a problem in previous crops. All trash left from previous seasons, including tubers, vines, soil, broken boxes and old bags should be removed from the storage facilities and properly discarded and burned. Discarded potatoes should not be left near the stores or in cull piles, which could be a source of disease organisms. They should be burned, buried or completely removed from the farm (Secor and Gudmestad, 1993). After cleanup, storage bins, walls and floors should be thoroughly washed with a high-pressure washer using hot, soapy water. It should be remembered that the waste water could still contain viable pathogen cells and it is recommended that a suitable disinfectant is added to the hot soapy wash water. Thereafter, the surfaces should be rinsed effectively. After washing and rinsing a disinfectant should be applied according to the manufacturer’s instructions. It is critical to wash all surfaces before applying the disinfectant, as most disinfectants are ineffective on dirty surfaces, as they are rapidly inactivated by organic matter and soil. Disinfection of equipment is critical but workers should be provided with disinfectants/detergents and washing facilities to minimize spreading of the disease (Secor and Gudmestad, 1993). Important characteristics of a god disinfectant are high germicidal activity, raped action, and effectiveness in the presence of organic matter and in hard water, long shelf life, low human toxicity, non corrosiveness and ease of use. Several types of disinfectants are available, marketed under many brand names. Each type ha characteristics that affect its use (Secor and Gudmestad, 1993). Hypochlorite bleaches are extremely sensitive to inactivation by organic matter, and hence thorough prewashing is especially important when these compounds are used. Hypochlorite bleaches and iodine compounds are corrosive to metal. Alcoholic

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disinfectants are especially suitable for small tasks, such as sanitizing tissue culture tools, vessels and equipment (Secor and Gudmestad, 1993). One of the reasons for the widespread use of quaternary ammonium compounds (QACs) in especially the food industry is their compatibility with virtually any type of surface including soft metals, mild steel, wood and other porous materials. Thus QACs can be used on many types of equipment which would be corroded or damaged in some or other way by common disinfecting agents (Banner, 1995). Another desirable characteristic of QACs is their ability to coat and penetrate, making them suitable for disinfecting environmental, non-food contact areas such as floors, walls and external equipment surfaces (Secor and Gudmestad, 1993). Mienie and Theron (1999) recommended the use of 0.5% carbolic acid (Jeyes Fluid) for cleaning and disinfecting machinery. Disinfection of equipment/workers will form an integral part of an integrated control strategy. Table 2.10.2.1 indicates the types of disinfectants suggested for disinfection of potato-handling equipment and storage facilities.

Table 2.10.2.1

Active ingredient

Quaternary ammonium compounds

Types of disinfectants used for disinfecting potato-handling equipment and storage facilities (Secor and Gudmestad, 1993) Inactivated by organic matter

Slightly

Hypochlorites (including household bleach Yes and other chlorinebased disinfectants)

Inactivated by hard water

Corrosive to metal

Slightl y

No

Affected by iron only

Yes

Safety

Relatively; use caution (see labels)

Irritant; caustic

Recommended exposure time

Shelf life

Comments

Poisonous in concentrated form; relatively safe when diluted

10 min

1-2 y

10 min

5.25% bleach Quick acting, stable 3-4 mo at 21°C; once Inexpensive, diluted, it Caustic to skin and should be clothing, used within More effective at pH 7-8 hours Do not take internally

Iodine compounds

Yes

Affected by iron only

Yes

Relatively; use caution (see labels)

10 min

1-2 y

No longer effective when it loses yellowbrown colour Tamed iodophor compounds work best

Phenolic compounds

Alcohols

Moderat ely

slightly

Residual action No

No

No

Oral poison

No

Oral poison, flammable

10 min

10 min

Bacterial wilt disease on potato: The South African experience

1-2 y

1-2 y

Phenol listed as ingredient on label Best suited for small appliances Can be used to flame utensils

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2.10.3 Management through Cultural Practices 2.10.3.1 The use of disease free planting material One of the principles for cultural control is to avoid introducing the pathogen on planting material (Hartman and Elphinstone, 1994). Disease-free planting material is therefore especially important for potatoes, where dissemination of the pathogen by planting latently infected tubers allows for pathogen survival and transport and for the inoculum to come into direct contact with the host (Nyangeri et al., 1984). Certified seed potatoes grown in the RSA are produced under regulations mandating zero tolerance for bacterial wilt. Certified seed potatoes should only be purchased from reputable seed potato growers who grow them from pathogen tested seed stocks (Gudmestad and Secor, 1993). Shekhawat et al. (1992) reported that wilt incidence was high (1.6 to 5.6%) in crops grown from local latently infected seed tubers and that the use of disease free seed potatoes alone can reduce the disease incidence by 62.5 to 81.5%. The introduction of infected seed into cooler regions has often resulted in the production of latently infected seed on apparently disease-free fields. Such infected tubers have been known to cause severe outbreaks of the disease (French, 1994). In order to prevent the spread of the disease, cuttings from in vitro stocks were used on a large scale in Vietnam (Vander Zaag, 1986). Latent infections have also posed a major problem for plant breeders selecting for resistance to wilt. One example is the tolerant cultivar Cruza 148. Although plants do not show any external wilt symptoms or visible tuber infections, the bacterium is carried symptomless in the tuber (Hayward, 1991). Although such tolerance allows production of potatoes in infected regions, it assists in transmitting the disease. A zero tolerance for R. solanacearum is maintained in the South African seed potato industry (i.e. if this organism is detected, seed potato growers are immediately notified and their crop rejected). This ensures that no infected tubers can be sold as seed potatoes. However, once rejected as seed potatoes, farmers are still allowed to sell tubers as table potatoes. If these tubers should wrongfully be used as seed potatoes, the danger of spreading the disease still exists. . solanacearum can be transmitted by peanut seed (Machmud and Middleton, 1991). In China groundnut seed is preserved over the winter period and Yongxiang et al (1993) reason that application of dry preservation measures could prevent transmission. A subsequent study showed that peanut seed with water content of 10% or higher could transmit R. solanacearum (Dongfang et al., 1994). These authors also suggested that under normal conditions of preservation, the pathogen might not survive. Moffet et al. (1981) obtained infected plants from tomato and pepper seeds that had been inoculated artificially. In order to investigate seed transmission Shakya (1993) germinated tomato seeds and found that 21% of the seedlings developed water-soaked brown discoloration on their roots. After 8-9 days these seedlings collapsed. Isolated bacteria were identified as R. solanacearum. Singh (1994) was able to confirm the transmission of bacterial wilt through tomato seed as well as through eggplant seed. Occurrence and survival of R. solanacearum biovar 3 on eggplant seed was reported by Chatterjee et al. (1994). 2. 10.3.2 Use of whole seed versus cut seed material Many potato crops are grown from seed pieces cut from larger tubers, mainly to reduce the cost of planting material. Emergence from cut seed is usually more uniform across a field, since sprouting from whole seed within individual hills occurs over a longer period of time (Mosley and Chase, 1993). Cutting of seed tubers increases the risk of bacterial or fungal infections. During the cutting process the pathogen can be spread from a diseased tuber to healthy ones by contaminated blades. The cut surfaces also provide ideal entry points for soil-borne pathogens. In order to reduce fungal seed decay fungicides are usually applied. The impact of cutting seed material on the incidence of bacterial wilt has been noted by Shekhawat et al. (1988). Wilt increased up to 12 times in potato crops where cut seed had been used in comparison to uncut seed. Treatment of the cut pieces with the fungicide Dithane M-45 did not reduce the wilt.

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2.10.3.3 Hilling at planting Hilling is important to avoid sun damage of developing tubers, to protect tubers against potato tuber moth and to minimize late blight infection of tubers. In some production areas multiple hilling is performed as part of a weed control program, in others a single hilling is done after sprout emergence (Rowe and Secor, 1993). Shekhawat and Chakrabarti (1993) recommend that this procedure be completed at planting time since this minimizes injury to the potato plant. Wounds provide entry sites for bacterial wilt. Post-emergence ridging can increase injury by 10-15% (Shekhawat et al. 1992). Hilling three weeks after emergence does not appear to increase the incidence of wilt where low infestation levels were encountered. Where infestations were high, post-emergence hilling did increase the percentage of wilt (Kloos, 1986). 2.10.3.4 Control of nematodes and weeds The role of root-knot nematodes in the development of bacterial wilt by providing entry points and possibly assisting tissue colonization has been reported by several authors (Hayward, 1991a; Shekhawat et al., 1992). Practices for the control of nematodes include the planting of resistant varieties, using chemical soil treatments, fumigation and rotation with crops that are resistant to both nematodes and bacterial wilt (Akiew et al., 1993). Weed control is vital since several species serve as hosts and allow survival in the absence of a host crop (refer to Table 2.6.1). In one instance, however, the presence of susceptible weeds had no influence on the severity of wilt in the next season (Akiew et al., 1993). Most findings indicate that weeds promote the survival of Ralstonia in the soil, transmit the pathogen to the next crop, and reduce the success of rotation practices (Jackson and Conzales, 1981; Shekhawat and Perembelon 1991; Tusiime, et al., 1998). 2.10.3.5 Crop rotation and fallow Rotation with non-hosts as a means of reducing the disease incidence has been used successfully in several crops. The type of crop chosen for rotation varies greatly and depends on the region and the race of the pathogen involved. In India, a two-year rotation with wheat-lupin, wheat-maize or wheat-fallow was very effective in reducing wilt on potatoes. In other instances rotation with finger millet, horse gram, sorghum, wheat, cabbage, carrot, onion and garlic reduced wilt by more than 90% (Shekhawat et al., 1992). Soybean, cowpea, velvet bean, redtop grass, maize and cotton are recommended in two to three year rotation programs to control tobacco wilt in the United States, provided no root-knot nematode infestation is present (Akiew et al., 1993). In Australia forage sorghum, signal grass and Rhodes grass are often used in the rotation program (Akiew et al., 1993; Arthy and Akiew 1999). A seven-year rotation with signal grass reduced wilt of tobacco greatly, but when tobacco was cultivated for two consecutive years on these fields, wilt incidence increased again to 20%. Combining the use of resistant or moderately resistant tobacco cultivars with a two to three year rotation sequence of grass fallow appears to be effective (Akiew and Trevorrow, 1994). The potato strain of Ralstonia is reportedly brought under control with relative ease in comparison to other races. In the cool regions of Dorrigo, Australia, a 2¥2 -year pasture rotation was sufficient to eradicate race 3. Planting the tolerant variety Molinera or maize for several seasons after a six-month fallow eradicated the pathogen in the Peruvian highlands (French, 1994). Rotation with maize, oats or barley resulted in a 50-75% reduction in the incidence of potato wilt in India. Cowpea and cabbage are often included in rotation programs in potato cultivation (Shekhawat et al., 1988). The success of rotation in comparison to bare fallow programs varies. A rotation program using maize and bean intercrop or sweet-potato cultivation did not reduce the soil inoculum potential in Costa Rica. A five-month bare fallow with application of herbicide for weed control was more successful in reducing the incidence of wilt (Jackson and Gonzales, 1979). In another case, rice cultivation prior to a tomato crop was more successful in reducing wilt than fallow or other crops (French, 1994).

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Interestingly, Shekhawat et al. (1992) were able to isolate R. solanacearum from the root tissue of maize, wheat, paddy rice and bean. Populations of the wilt organism were however not high enough to exude from the root tissue into the soil. The lack of bacterial release could explain the experienced reduction in Ralstonia population after a rotation with these crops. Granada and Sequerira (1983a) also found that R. solanacearum could infect roots of presumed non-hosts such as maize. Infections remained localized in the roots and bacterial release into the soil was less from these plants than from true host plants. Infection rate of these presumed non-hosts was also lower in that not every individual plant of a given species became infected. These findings indicate why an overall reduction of wilt is experienced when applying rotation. They do however not explain why in some cases rotation is more effective than a bare-fallow treatment. The level of control achieved with crop rotation varies greatly and is dependent on factors affecting the survival of Ralstonia in the absence of a host (Akiew and Trevorrow, 1994). A short-term rotation appears in most instances, especially where race 3 is not involved, not to be effective in either eliminating or controlling wilt effectively. 2.10.3.6 Soil Amendmends and soil solarisation Several soil amendments have been studied in the hope of reducing wilt incidence. It was generally believed that alkalinity would favour the pathogen and acidity suppress it, but increasing soil acidity by adding potassium sulphate, nitric acid or sulphuric acid did not affect the incidence of wilt noticeably (Kelman, 1953). A range of fertilizers has also been evaluated, including superphosphate, calcium cyanamide and sodium nitrate, without much success. Urea applied at 1000kg/ha did reduce wilt significantly (Kelman 1953). Michel and Mew (1998) amended four soils with urea (200kg N/ha) and CaO (5000kg/ha) and reported that the success in reducing wilt varied with soil type. Differential reduction in wilt was also noted by Chellemi et al. (1992) in a trial with composted organic amendments. Site specific soil properties may be responsible for this phenomenon. A 99.9% reduction in wilt of tomato plants in glasshouses was reportedly achieved by an amendment developed by Sun and Huang (1985). The soil was amended with 1% S-H mixture which consisted of 4.4% bagasse, 8.4% rice husks, 4.25% oyster shell powder, 8.25% urea, 1.04% potassium nitrate, 13.16% calcium superphosphate and 60.5% mineral ash. Saumtally et al. (1993) stated that soil solarisation using clear plastic sheeting to cover moist soil for six weeks yielded only slight control. Unsolarised plots had 61% bacterial wilt compared with 54% in solarised plots. Modifications using a combination of transparent and dark plastic sheeting to achieve higher temperatures were being investigated. 2.10.3.7 Other cultural practices The removal of rogue plants, stubble and diseased plant material forms a vital part in disease management (Graham et al., 1979; Persley, 1986). Such plant material allows the pathogen to survive in the absence of a host crop and permit infection of a subsequent crop. The use of infected farming implements, lack of sanitation after handling diseased material contribute to the risk of transmitting the disease. Movement through infected fields should also be limited since soil attached to vehicles, shoes and animal hooves can spread the pathogen to adjacent fields (Swanepoel and Bosch, 1988). The pathogen can also be avoided by delaying the date of planting until temperatures are lower in summer- and autumnproduction regions. In winter- and spring-production regions, early planting and harvesting can reduce bacterial wilt and tuber rot. Short duration early maturing potato varieties would be ideal for this practice since the time for crop development is reduced to escape the disease (Shekhawat et al., 1992). However, this only applies for the production of table potatoes since the danger of latent infection still exists (Mienie and Theron, 1999).

2.10.4 Biological Control The concern over toxicity of chemical compounds combined with their relative inefficiency in controlling bacterial wilt has motivated the search for biological agents as part of an integrated management program. Trigalet et al. (1994) define Bacterial wilt disease on potato: The South African experience

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biological control in terms of direct microbial antagonism (competition, antibiosis) and indirect microbial antagonism (induced resistance in the host). Later the antagonistic effects of botanicals were included in this category (Trigalet and Urquhart, 1998). Several bacterial species have been reported as being antagonistic towards R. solanacearum, amongst them Pseudomonas fluorescens, P. glumae, P. cepacia, Bacillus polymyxa, other Bacillus spp. and Erwinia spp. (Trigalet et al., 1994; Shekhawat, et al., 1992). Avirulent mutants of R. solanacearum have also been identified as antagonists. These root colonizers antagonize the pathogen at the root infection site, resulting in reduced and delayed onset of wilt. Isolates of P.fluorescens reduced wilt by over 50 % and increased tuber yield. Similarly Bacillus spp. isolates have been reported to reduce wilt by almost 90% and tuber rot due to Ralstonia by more than 80% (Shekhawat et al., 1992). Biocontrol results obtained in the laboratory with these types of antagonists are often difficult to reproduce in field conditions. Antagonism observed on agar plates could have resulted from a different set of parameters than found under natural circumstances. In field conditions, the biocontrol organisms must compete with other soil microbes and must contend with both biological and physical factors (Trigalet et al., 1994). Since the plant is susceptible to infections for a long period of time, antagonists at the root infection site must be present continuously and without too many fluctuations in their population. The development of a bio-agent that acts as an endophytic antagonist has the advantage that once it is established in the plant, it can provide continuous protection. The agent should not cause disease and must be able to colonize roots, penetrate xylem vessels and multiply within the vascular tissue (Trigalet et al., 1994). For this reason several studies have been conducted on avirulent mutants of R. solanacearum (Trigalet et al., 1998; Smith et al., 1998). Problems that have been experienced with endophytic antagonism include limited systemic spread and population d e c l i n e , probably due to agglutination by plant lectins or by them being bound to host cell walls (Trigalet et al., 1994). Several botanicals have been evaluated for their ability to reduce bacterial wilt infections. Terblanche and de Villiers (1998) noted that French Marigolds (Tagetes patula L.) not only reduced Ralstonia populations in the soil, but also inhibited the development of wilt symptoms on tobacco plants. Two thiophenes were extracted from the roots, which proved inhibitory to R. solanacearum in in vitro tests. Similarly it was found that sugi bark (Crytomeria japonica D.Don), a substrate for horticultural crops in soilless culture, reduced the incidence of wilt on tomato plants (Yu et al. 1997). The inhibition was mainly attributed to volatile oils, phenolics and acidic substances. Injecting the volatile oils into rockwool (another soilless substrate) also suppressed the wilt incidence. The main components of the volatile oils were identified as isophyllodecene and ferruginol. Asafoetida (Ferula foetida) mixed with tumeric powder (Curcuma longa) at ratio 1.5g: 5g: 10 liter water and lg: 5g: 10 liter water, controlled wilt disease by 70.3% and 69% respectively (Mazumder, 1998). Soil drenching with a formulation of asafoetida and tumeric reduced the mortality of brinjals due to bacterial wilt, especially if three drenches were performed at 20, 50 and 80 days (Pun and Das, 1997)

2.10.5 Resistant Cultivars A practical and economic approach to managing bacterial wilt infested soils would be to plant resistant cultivars. According to Prior et al. (1998), the term resistance refers to "any measurable plant mechanism able to overcome completely or to limit the development of a pathogen or its effects". Tolerance is defined as "the overall ability for a plant to withstand development of a pathogen without major losses in yield". The use of resistant potato cultivars plays an important role in the integrated control of Bacterial wilt. Unfortunately the complexities of host-pathogen-environment interaction make breeding for resistance extremely difficult (Tung et al., 1990) and no immunity has as yet been identified in potato (Hayward, 1991). The effect of temperature on resistance to wilt caused by different strains and races of R. solanacearum have been investigated (Tung et al., 1990; Tung and Schmiediche, 1995). Resistance was found to be temperature sensitive as well as strain specific. A wide range of genes for resistance needs to be incorporated into a genotype to provide adequate resistance. Bacterial wilt disease on potato: The South African experience

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This should reduce strain specificity and improve the stability of resistance under changing environmental conditions (Tung et al., 1990). Since the the early 1970's Solanum phureja Juz. and Buk., a diploid cultivated species, has been used extensively in breeding programs by authorities such as the International Potato Centre in Peru, as the major source of resistance to bacterial wilt. It has, however, not been successful in all environments (Hayward, 1991). Its resistance is temperature sensitive and it is therefore best suited for higher elevations or cooler climates (Laferriere et al., 1998). Resistant genes from other diploid potato species have been identified and used in breeding programs. Some of these species are: S. chacoense Bitt., S. cilia tum Lam., S. jamesii Torr., S. multidissectum Hawkes, S. pinnatisectum Dun., S. raphanifolium Hawkes, S. sisymbrifolium Lam., S. sparsipilum Bitt., and S. stenotomum Juz. and Buk. (Madalageri and Patil,1995). Other Solanum spp. evaluated include: S. acaule Bitt. S. berthaultii Hawkes, S. blanco-galgosi Ochoa, S. boliviense Dun., S. brachycarpum Ochoa, S. chomatophilum Bitt., S. demissum Lindl., S. polytrichon Rydb., S. stoloniferum Schlechtd and S. sucrense Hawkes (Hartman and Elphinstone, 1994). According to Laferriere et al. (1998) another source of resistant genes was S. commersonii Dun., a diploid wild potato species from south-eastern South America. Attempts to incorporate this species (as with many other) into potato breeding programs were however thwarted by its sexual incompatibility with tetraploid S. tuberosum. To bypass this obstacle, protoplasts from the two species were electro fused where after the fused protoplasts multiplied to form callus tissue. This tissue differentiated to give rise to shoots that were used in further breeding programs. The stability of resistance of these plants under different field conditions and temperature regimes is still unknown and has to be evaluated. According to French and De Lindo (1982), resistance to bacterial wilt in potato is a partially dominant character and is more of a polygenic type. Tung and Schmiediche (1995) agreed on the dominant character of resistance and suggested that only a few genes control it although the number of genes involved is still unknown. Tung et al. (1990) and Tung and Schmiediche (1995) suggested that the genes for resistance in the host evolved independently from the pathogen and that a gene-for-gene relationship does not seem to be applicable to bacterial wilt. Whether or not resistance to R. solanacearum is controlled by minor or major genes is not considered to be a point of great concern in practical breeding (Tung et al., 1990). According to them, the inheritance and expression of resistance seem to be complex and new methods other than conventional breeding techniques should be evaluated to increase and stabilise resistance. Genetic engineering techniques are therefore being used to strengthen the basic composition of the host plant through the incorporation of suitable genes from other sources. Antimicrobial genes coding for lytic enzymes such as cecropins isolated from the lepidopteran Hyalophora were used for the control of bacterial diseases of plants and showed great potential against R. solanacearum (Montanelli et al., 1995). According to Hayward (1991), lysozyme and other potent antibacterial proteins derived from insects can also be introduced into potatoes to increase resistance.

2.11

SUMMARY

Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive and successful plant pathogens affecting several economically important crops. Although the disease is more common in the tropics, subtropics and the warmer temperate areas, it has also been reported in the cooler regions such as Sweden, Austria and the UK. During the last century, the pathogen has been reported in so many countries that its distribution can now be regarded as world-wide, though the strains encountered vary. The wilt organism is well adapted to survival, being able not only to infect a wide range of hosts but also being capable of remaining viable in the soil for several years. Reliable scientific data on soil survival under different circumstances is scarce. Its ability to survive in more than 450 different species has facilitated its survival even through harsh periods where soil conditions are not congenial for survival in its free state. Its success as a plant pathogen is favoured in the ready way it disseminates. Besides transmission through infected soil and infected plant material (often showing no symptoms), it can spread by mechanical contacts, contaminated water and in case of banana wilt even by insects. Bacterial wilt disease on potato: The South African experience

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Control of bacterial wilt lies in an integrated approach. Good agronomic practices such as using disease-free soils, disease-free and uncut planting material, effective weed and nematode control, incorporating good crop rotation systems and fallowing can assist greatly in reducing the incidence of wilt. Soil amendments and chemical control can be beneficial under specific circumstances. The potential of biological control as part of the management system is vast and the research input into this area has increased dramatically. Another important strategy in managing bacterial wilt infested soils lies in the use of resistant varieties. Although resistance in varieties have been globally reported over the years, most of these genotypes tend to be tolerant to bacterial populations, rather than being immune and often transmitted the disease to the progeny tubers without necessarily causing visual symptoms. Planting of such latently infected tubers could lead to infestation of previously disease-free fields or result in yield losses. Another concern for plant breeders is that the expression of resistance is strongly affected by environmental factors.

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Chapter 3

CHARACTERISATION AND DETECTION STUDIES ON RALSTONIA SOLANACEARUM 3.1

BACKGROUND

This chapter deals with the characterisation of R. solanacearum isolates and the detection of the pathogen in soil and plant material. This information constitutes an essential component of the effort to implement practical management practices in the control of bacterial wilt: 1. 2. 3. 4.

Characteristics of strains of R. solanacearum (A.E. Swanepoel and co-workers at Roodeplaat, late 1980s). Development of an alternative detection method for R. solanacearum in soil (W. van Broekhuizen and co-workers at the University of Pretoria, 1995 – 2002). Enzyme-linked immunosorbent assay for detection of R. solanacearum in potatoes (D. Bellstedt and co-workers at Stellenbosch University, late 1980s). Characterisation of South African R. solanacearum isolates using molecular techniques (W. van Broekhuizen and co-workers at the University of Pretoria, 1995 – 2002).

The sudden escalation of bacterial wilt outbreaks in the country in the late 1970s and the 1980s necessitated some basic research on R solanacearum, as very little was known about the disease in South Africa at the time. A decision was subsequently made to conduct a survey on the identification and distribution of the different biovars of South African strains of Pseudomonas solanacearum, as the organism was known at that time. Swanepoel (1988) found that with a single exception, the strains isolated from diseased potato plants were representative of biovar 2, and also that with a single exception, the strains isolated from diseased tomato and tobacco plants were representative of biovar 3. The tendency to isolate biovar 3 from tomatoes and biovar 2 from potatoes may reflect the practice of cultivating tomatoes in the warmer regions of the country and potatoes in the cooler regions, or during cooler periods in the warmer regions. In some areas, the two crops are grown together. According to Van Broekhuizen (2002), the use of indicator plants in fields suspected of being infected could be one possible way of overcoming the problem. However, it was found that this approach was not rapid enough for commercial use and therefore not suitable as a replacement for the existing ELISA test and other selective media diagnostic methods. Soil sampling in naturally infected fields remains a major problem with the current technique involving the warming of potato tubers before testing. In time, it became evident that there was a need for a sensitive and practical assay to detect the presence of Ralstonia solanacearum in all registered seed potato plantings. The contract for the development of a suitable test for this purpose was duly awarded to Prof. D.U. Bellstedt and his team at the Biochemistry Department of Stellenbosch University, resulting in the development of an ELISA test that was subsequently validated by the controlling laboratory, and which is currently being used at regional laboratories to test all registered seed potato plantings after the dying off of the crop. Any samples that test positive are retested by the controlling laboratory (Plantovita) by means of ELISA, as well as by other conventional pathological methods. A study aimed at characterising and evaluating possible variations between different R. solanacearum isolates, using different molecular techniques, was subsequently initiatedby Van Broekhuizen and co-workers. Although these techniques distinguished between biovar 2 and biovar 3 isolates, no correlation could be drawn between the different isolates and the regions from which they were isolated. Van Broekhuizen (2002) commented that although molecular techniques open up a whole new field for the classification of organisms, it should not be seen as the only criteria to be used for classification

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purposes. Traditional phenotypical and pathological observations should – and surely always will – play a fundamental role in classification.

3.2

CHARACTERISTICS OF SOUTH AFRICAN STRAINS OF R. SOLANACEARUM

South Africa is a country with climatic conditions varying from temperate to subtropical. The disease is endemic to the lowaltitude subtropical regions, such as the Lowveld areas of Mpumalanga, the coastal regions of KwaZulu-Natal, and the area known as the Cape Flats in the Western Cape, where the climate is characterised by hot, dry summers and cool, wet winters.

3.2.1

Materials and methods

3.2.1.1 Identification tests From 1984 to 1986, 45 strains of R. solanacearum were isolated from potatoes, tomatoes and tobacco in different locations around South Africa. These strains were isolated on TZC medium and identified by means of standard laboratory techniques, with every test being repeated five times. To determine whether differences existed among strains of the same biovar, tests were conducted to determine the ability of the strains to oxidise eight carbohydrates. 3.2.1.2 Virulence tests Virulence tests were conducted on potato, tomato, tobacco, eggplant, pepper, peanut, sunflower and D. ferox plants. A root inoculation technique was used to test plants measuring 100 to 150 mm in height, involving the application of 30 ml inoculum (with an absorbance value of 0.001) per plant. Five strains were selected for virulence tests: strains 1 and 5 were both isolated from potato plants, but originating from different parts of the country; strain 3 was isolated from a potato plant, but with distinct physiological differences compared to other potato isolates; strain 4 was isolated from a tomato plant; and strain 10 was isolated from a tobacco plant. Each isolate was inoculated into a total of at least 15 plants of each host. Plants were grown at temperatures of 28°C (day) and 25°C (night), while disease indices were recorded 40 days after inoculation on a scale of 1 to 5.

3.2.2

Results

3.2.2.1 Identification tests All strains were identified as rod-shaped and gram-negative strains, and all produced fluidal colonies with pink centres after 48 hours, with the exception of colonies of strain 3, which were red-pink and less fluidal. In fact, it was only strain 3 that was found to differ from other strains in some of the tests conducted. All strains were identified as R solanacearum. 3.2.2.2 Virulence tests Results of the virulence tests are given in Table 3.2.2. The strains differentiated as biovar 2 showed no virulence to sunflower, tobacco and peanut, whereas virulence to other hosts varied. Strain 3 was the only biovar 2 strain that showed virulence to Datura ferox. Biovar 3 strains were found to be virulent to all eight hosts.

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Table 3.2.2 Virulence ratinga of South African strains of Ralstonia solanacearum on eight hosts. (Swanepoel, 1988) Strain 1 3 5 4 10

Original Host Potato Potato Potato Tomato Tobacco

Biovar

Potato

Tomato

Eggplant

Pepper

Sunflower

Tobacco

Peanut

2 2 2 3 3

Hb H H M M

H H M H H

H M M H H

M L M H H

0 0 0 L L

0 0 0 L H

0 0 0 L L

Datura ferox 0 L 0 L L

Results based on average disease indices on a scale of 1 – 5, of 15 – 20 plants, 40 days after inoculation. H = High (4.1 – 5.0), M = Medium (2.6 – 4.0), L = Low (1.1 – 2.5), 0 = None (1.0) a

3.2.3

Discussion

With one exception, the strains isolated from diseased potato plants were representative of biovar 2, and also with one exception, the strains isolated from diseased tomato and tobacco plants were representative of biovar 3. The latter was mainly found in hot subtropical areas of the country, while biovar 2 was found mainly in the temperate regions. The tendency to isolate biovar 3 from tomatoes and biovar 2 from potatoes may reflect the practice of cultivating tomatoes in the warmer regions of the country and potatoes in the cooler regions, or during the cooler months in the warmer regions. In some areas the two crops are grown together. The isolation of biovar 3 (strain 8) from potatoes was an exceptional case, since the potatoes were grown on an old tomato field. Similarly, biovar 2 (strain 39) was isolated from diseased tomatoes grown on a field with a history of early summer potato production. No physiological differences were observed between strains isolated from tomato plants and those isolated from tobacco plants. With the exception of strain 3, all biovar 2 strains failed to oxidise trehalose and ribose. These sugars were indeed oxidised by all biovar 3 strains, however. Strain 3 was consistently identified as biovar 2, but it was similar to the biovar 3 strains in its ability to oxidise trehalose and ribose. It was the only strain capable of oxidising rhamnose, and differed from all the other strains in its ability to produce gas from nitrate and its inability to hydrolyse Tween 80. Since strain 3 was the only virulent strain with these particular characteristics, it could possibly be a mutant.

3.3

DETECTION TECHNIQUES FOR RALSTONIA SOLANACEARUM

3.3.1

Development of an alternative detection method for Ralstonia solanacearum in soil

3.3.1.1 Introduction The majority of detection techniques were primarily developed for the detection of the pathogen in infected plant material, although these same techniques can be applied in soil detection. Soil detection of the pathogen is subject to a number of complications and shortcomings, such as a low recovery rate. Most of the problems encountered can be attributed to the heterogeneous nature of soil and the subsequent difficulty of ensuring adequate sampling (Jenkins et al., 1967). The use of indicator plants was ultimately found to be a slow process, delivering inconsistent results. Various selective and semi-selective media, mostly developed on the basis of Kelman’s TZC medium (Kelman, 1954), and with the ability to detect problems such as background bacteria, can only detect the pathogen effectively in concentrations ranging from 104 cfu ml-1 soil sample (Jenkins et al., 1967; Nesmith and Jenkins, 1979). Serological techniques can also be used for this purpose, as in the case of the ELISA kit developed by Prof. D.U. Bellstedt of Stellenbosch University, which is currently being used by the South African seed potato industry to effectively detect pathogen concentrations of 103 cfu ml-1 of sample (Bellstedt and Van der Merwe, 1989). The efficacy and affordability of the trapping technique were evaluated in comparison to the selective media and ELISA techniques currently in use in South Africa. Bacterial wilt disease on potato: The South African experience

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3.3.1.2 Discussion Initially modified enrichment broths, based on commercial selective media, were used to enrich R. solanacearum concentrations. Although these enrichment broths were able to increase pathogen concentrations under sterile conditions, they proved unsuccessful when soil was added to the system, which was most likely due to the proliferation of soil microorganisms. Consequently, a decision was made to use only sterile distilled water for the preparation of soil suspensions. Concentrations of 101 cfu ml-1 could be detected, while concentrations of 103 cfu ml-1 (M-TZC and SMSA) and 104 cfu ml-1 (TZC) were detected with selective media. The ELISA proved effective from 104 cfu ml-1. The selective media proved most affordable, producing results within three days (as did the ELISA). The ELISA technique proved costly, requiring specialised equipment such as the multi-scan spectrophotometer. The trapping technique relevant to this study was based on the functioning of an indicator plant system. Tissue culture plants were successfully used to trap the pathogen in artificially inoculated soil suspensions, with the ability to detect pathogen concentrations as low as 101 cfu ml-1 soil suspension within ten days. This technique was ultimately found to be not rapid enough for commercial use, however, and is therefore not a suitable replacement for the existing ELISA and selective media diagnostic methods. None of the evaluated detection techniques proved capable of detecting R. solanacearum in soil samples taken from an infected potato field, which can most likely be ascribed to incorrect sampling methods, including sampling no deeper than 15 cm. Moreover, the field soil was extremely dry at the time of sampling, implying that the pathogen could have migrated to deeper soil layers. Soil sampling in naturally infected fields remains a major problem. The use of indicator plants in suspected fields is one possible way of overcoming this problem, although it is an extremely time-consuming process to obtain results regarding the presence of R. solanacearum in a field.

3.3.2

Enzyme-linked immunosorbent assay detection of Ralstonia solanacearum in potatoes

The seed potato industry realised the need for a sensitive and practical assay to be developed to detect the presence of Ralstonia solanacearum in all registered seed potato plantings. The logistics involved in screening large statistical tuber samples by standard bacteriological identification methods were simply not practical, hence the need for a screening test capable of detecting the presence of the pathogen. Consequently, the Biochemistry Department of the University of Stellenbosch was contracted to develop a suitable test. The pathogen tends to be latent under low temperature conditions, but becomes pathogenic when temperatures rise, in which case infected soils can be rendered unsuitable for seed potato production for many years to come. An ELISA test that was developed in 1989 (Bellstedt and Van der Merwe, 1989) and subsequently subjected to extensive specificity and sensitivity testing, along with laboratory staff training, was ultimately implemented as part of the Potato Certification Scheme in 1996. This ELISA test is used as a first screen to detect infections, with subsequent confirmation by means of standard bacteriological identification methods. The cultivation of R. solanacearum, along with antibody production in rabbits and comprehensive procedures for conducting the assay, are discussed in the article published as a chapter in Methods in Molecular Biology: Plant Pathology 508. For the current purposes, this report on the ELISA test kit will suffice, with certain notes made by the developer.   

Ralstonia solanacearum biovar 2 (race 3) was isolated from contaminated tubers in the South African study, but any R. solanacearum race can be used for antibody production and be detected using this method. Ralstonia solanacearum biovar 3 (race 2) is very rarely isolated from potatoes. However, antibodies against R. solanacearum biovar 3 do cross-react with biovar 2 and can therefore also be detected by means of this ELISA test. During the initial phases of ELISA testing, microbiological trials were conducted to assess the number of bacteria present in latently infected tubers, i.e. those exhibiting no symptoms. These trials revealed that the vast majority of latently infected tubers contained significant numbers of bacteria. To illustrate: If sap from a latently infected tuber

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 

 

 

was diluted as much as 1:100, it still gave a significantly elevated ELISA reading indicative of the positive identification of the bacteria. A decision was therefore made to combine 100 tubers in a single sample. The ELISA is supplied in kit form to regional laboratories of South Africa. The inclusion of a dilution series of the positive control serves as an effective tool in monitoring assay performance. Over many years of testing with the ELISA, it became apparent that the following factors could lead to non-specific background readings: 1. Any tuber with signs of rotting; 2. Any tuber containing potato skin; 3. Any tuber with damaged skin caused during mechanical harvesting; 4. Tubers grown under waterlogged soil conditions during the three weeks prior to harvesting. The sensitivity of the ELISA was tested, with an absorbance reading of 0.15 being found in approximately 10 000 R. solanacearum cells. Several attempts were made (1993 – 1995) to increase the number of bacteria in the tubers by means of incubation at 37°C for up to two weeks prior to ELISA testing. The number of bacteria did increase, but so did the incidence of rotting tubers. The practice of subjecting tubers to warm incubation was consequently discontinued, except in cases where the presence of the pathogen was suspected. The incidence of infections detected by ELISA and subsequently confirmed is shown in Fig 3.3.2.1 It was found that even with the use of selective media, R. solanacearum bacteria could be easily overgrown by other bacteria. The inspection of bacterial growth at short intervals, the isolation of colonies, and the implementation of recultivation are therefore essential to ensure that symbiotic and/or contaminating bacteria do not overgrow the R. solanacearum bacteria. In many instances, the SMSA medium proved to be more selective than the TZC medium.

Number of confirmed infections

30 25 20 15 10 5 0 1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

Year

Fig 3.3.2.1 Number of Ralstonia solanacearum infections in South Africa during the period 1996 – 2006, detected by means of ELISA and subsequently confirmed through the use of selective media (Bellstedt, 2009).

3.4 CHARACTERISATION OF RALSTONIA SOLANACEARUM ISOLATES IN SOUTH AFRICA USING MOLECULAR TECHNIQUES 3.4.1

Introduction

R. solanacearum is a highly heterogeneous species (Fegan et al., 1998), due to factors such as differences in host range, geographical distribution, pathogenicity, epidemiological relationships and physiological properties (Hayward, 1991). Traditionally, strains were classified into five races based on differences in host range, and six biovars based on biochemical properties (Hayward, 1964; Hayward, 1991; Hayward, 2000; Poussier et al., 2000; Walker and Stead, 1993). These initial classifications focused on the physiological, biochemical and pathological properties of the pathogen, without incorporating the Bacterial wilt disease on potato: The South African experience

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molecular differences between isolates. Since proteins produced by different strains of bacteria can serve as a useful source of information for the identification and characterisation of different strains, scientists began to look at molecular properties as a means to subdivide isolates (Kerstes, 1990). With the development of molecular techniques such as polymerase chain reaction (PCR) and restriction fragment length polymorphism RFLP), it soon became evident that there are in fact clear differences between isolates that had previously been considered identical. Cook et al. (1989) used RFLP groupings to separate strains into two divisions that were genetically distinct from each other. Division 1 consisted of the metabolically more versatile biovars 3, 4 and 5, while division 2 consisted of the metabolically less versatile biovars 1, 2 and N2. This separation suggested an evolutionary divergence, with division-1 strains mainly originating from the Old World (Asia) and those in division 2 primarily from the New World (United States of America). A different approach was taken by some (Poussier et al., 1999; Poussier et al., 2000), involving the partial sequencing of the hrpB and endoglucanase genes, whereby isolates could be divided into three clusters, namely: Cluster 1, containing all isolates of biovars 3, 4 and 5, and equivalent to division 1; Cluster 2, containing isolates of biovars 1, 2 and N2 from Africa, the Antilles, the USA, and Central and South America; and Cluster 3, containing isolates of biovars 1 and N2 from Africa and the islands of Réunion and Madagascar. Once detected and isolated, the pathogen should be thoroughly characterised, which is also important in terms of determining the type of pathogen strain present in a certain area. Sufficient knowledge regarding the differences between strains plays an important role in the development of new control strategies. Different strains vary in terms of their ability to survive in soil (Elphinstone and Aley, 1993), and it is therefore important to know how strains differ from one another, especially where crop rotation is used as a control measure against bacterial wilt. Since different strains of the pathogen can infect different host plants (Hayward, 2000; Walker and Stead, 1993), identifying the particular strain that is present can play a role in choosing the best plants to use in crop rotation. The objective of this study was therefore to characterise and evaluate the possible variations between different R. solanacearum isolates, using different molecular techniques. As such, 44 isolates, which had previously been characterised primarily according to race and biovar, were collected from various potato production regions throughout South Africa. The enterobacterial repetitive intergenic consensus (ERIC) and ribosomal intergenic spacer analysis (RISA) PCR techniques, as well as RFLP with the enzyme Sau3A, were used to determine the homogeneity of the collection of R. solanacearum isolates.

3.4.2

Results and discussion

The ERIC- and RISA-PCR techniques were successfully used in this study to distinguish between biovar 2 and 3 isolates. Since these techniques take only a few hours to complete, results can be produced much faster than with carbon source utilisation and nitrate metabolism techniques. The sample should, however, be tested against biovars that are currently not found in South Africa (biovars 1, 4 and 5) in order to conclude whether or not it could successfully replace traditional identification methods. The RISA-PCR and RFLP with Sau3A techniques were used to characterise and evaluate possible variations amongst 44 R. solanacearum isolates collected throughout the potato-growing areas of South Africa. The dendogram subsequently constructed from the data showed two distinct groups (Fig. 3.4.2.1), with the first group comprising all four biovar 3 isolates, and the second group all biovar 2 isolates. Although these techniques served to distinguish between biovar 2 and biovar 3 isolates, no correlation could be drawn between the different isolates and the regions from which they were isolated. It is therefore clear that one has to be very careful in terms of choosing the correct molecular method and enzyme when seeking to answer specific questions. Although isolates from different areas did not group together in this study, considering a different region within the genome of R. solanacearum could have yielded different results. Van Broekhuizen (2002) commented that although molecular techniques open up an entirely new field for the classification of organisms, it should not be considered the only criterion to be used for

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classification purposes. Traditional phenotypical and pathological observations should – and always will – play a fundamental role in the classification process.

Figure 3.4.2.1 Dendogram showing the grouping of representative RFLP band profiles of Ralstonia solanacearum isolates using the unweighted pair group of arithmetic averages method (Van Broekhuizen, 2002)

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Chapter 4

HOST PLANT STUDIES 4.1

BACKGROUND

There is a constant need among potato producers to know which crops to include in a rotation system as part of an integrated management programme for bacterial wilt. It is also necessary to determine the susceptibility to bacterial wilt of all the weeds occurring in a particular potato field, since this information is important when considering specific management and control strategies. This chapter deals with studies on the susceptibility of crops and weeds to the pathogen Ralstonia solanacearum: 1. 2. 3. 4.

Survival of biovar 2 and biovar 3 strains of Ralstonia solanacearum in the roots and stems of weeds (A.E. Swanepoel and co-workers at Roodeplaat, early 1990s). The role of weeds in the perpetuation of bacterial wilt (E.I.M. Stander and co-workers at the University of Pretoria, 1994 – 2001). Screening of commercial crops as possible hosts for bacterial wilt (N.J.J. Mienie and co-workers at Roodeplaat, 1990s). Screening of herbal crops as possible hosts for bacterial wilt (W. von Broekhuizen and co-workers at the University of Pretoria, 1995 – 2002).

Swanepoel (1992) not only found Physalis angulata L. (wild gooseberry) to be a symptomless host for biovar 2, but also isolated biovar 3 from Eragrostis curvula (weeping love grass) without observing symptoms of wilt. Stander (2001) found biovar 3 to have a much wider host range, including 13 of the 25 weed species evaluated. This was the first time that Hibiscus trionum (bladder hibiscus) was found to be a host for bacterial wilt in South Africa. Stander also reported six other weed species to be hosts for biovar 3 for the first time in South Africa. Mienie (1998) identified several crops as being symptomless hosts for bacterial wilt, including turnips, radish, cotton and ginger. Van Broekhuizen (2002) reported that biovar 2 not only infected plants of the Solanacaeae family, but also that it could be isolated from herbal plants such as marjoram, nasturtium and parsley. In her study on crops as listed in Table 1 of the South African Seed Potato Certification Scheme, Espach (2008) confirmed the positive host status of all the crops concerned, but was unable to isolate the pathogen from certain plants that had developed wilt symptoms. It is clear that host studies should be extended and repeated in order to confirm the findings. It is also important to ensure that the methods used in these studies are standardised, and that the conditions under which these studies are conducted are favourable for disease development.

4.2 SURVIVAL OF BIOVAR 2 AND BIOVAR 3 STRAINS OF RALSTONIA SOLANACEARUM IN THE ROOTS AND STEMS OF WEEDS IN SOUTH AFRICA Worldwide, many weed species have been reported as hosts of the R. solanacearum pathogen (Kelman, 1953). Granada and Sequeira (1983) studied the pathogen’s ability to survive in the soil, rhizosphere and plant roots, finding that R. solanacearum can infect the roots of many plants previously considered non-hosts. Consequently, they concluded that the long-term survival of the pathogen appeared to be correlated with its ability to infect plant roots. Bacterial wilt disease on potato: The South African experience

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The purpose of the study at hand was to compare the ability of a biovar 2 isolate and a biovar 3 isolate to survive in certain weed species, generally found in tilled lands and in weeping love grass (Eragrostis curvula (Schrad.) Nees). This grass species had previously been recommended for use in crop rotation in soil infected with bacterial wilt. The investigation was restricted to these particular isolations, being the only biovars isolated thus far in South Africa (Engelbrecht and Hattingh, 1989; Swanepoel and Young, 1988).

4.2.1

Materials and methods

The seeds of weeping love grass plus nine weed species (Table 7.3.2) were sown in plastic pots and grown to 150 mm, then inoculated by pouring 30 ml inoculum (with an absorbance of 0.001 at 620 nm) onto the soil near the stem. Twenty seedlings of each species were inoculated with a biovar 2 isolate of R. solanacearum isolated from potatoes, while 20 seedlings were inoculated with a biovar 3 isolate, from tomatoes. Two non-inoculated control plants were included for each of the inoculum types for each species. The plants were grown in a glasshouse at temperatures of 28/25°C (day/night). Forty days after inoculation, the plants were scored on a wilting scale of 0 to 5, after which the results were transformed into percentage values (Kremer and Unterstenhöfer, 1967). Each plant that had survived inoculation was tested individually for the presence of the pathogen. After carefully removing the plants from the pots, the roots were washed in tap water and then surface sterilised in 1% sodium hypochlorite before being washed a second time. Each plant was then aseptically ground in 20 ml distilled water, and the resulting suspension was then used in a dilution series plated onto TZC medium (Kelman, 1954) and incubated at 30°C for 48 hours. Typical colonies were subsequently isolated and identified as R. solanacearum.

4.2.2

Results and discussion

The results are presented in Table 4.2.2.1. None of the plants developed wilt symptoms as a result of inoculation with the biovar 2 isolate, but the pathogen was isolated from Physalis angulate. Two of the weed species inoculated with the biovar 3 isolate developed wilt symptoms, but the pathogen was isolated from four plant species, including weeping love grass. These results confirm those of Granada and Sequeira (1983), who found that the pathogen is able to survive in presumed non-hosts. There is, however, an apparent difference in the variety of weeds in which the different biovars can survive, with biovar 3 having the ability to survive in weeds of different families. This tendency is in agreement with the natural host range of the biovars.

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Table 4.2.2.1 Mean percentage of wilting and percentage of plants infected with the pathogen 40 days after inoculation with islates of the biovar 2 or biovar 3 of Ralstonia solanacearum (Swanepoel, 1992). Mean % wiltinga

Plant species Gramineae Eragrostis curvula (weeping love grass)

% Plants infected

Biovar 2

Biovar 3

control

Biovar 2

Biovar 3

control

0

0

0

0

20

0

0

0

0

0

35

0

0

0

0

0

0

0

0

0

0

0

0

0

0 0

0

0

0

0

0

0

0

0

0

0

0 0 0 0

54 0 0 75

0 0 0 0

0 0 10 0

70 0 0 95

0 0 0 0

Amaranthaceae Amaranthus hybrids L. (cape pigweed) Chenopodiaceae Chenopodium album L. (white goosefoot) Compositae Bidens pilosa L. (common blackjack) Galinsoga parviflora Car. (small-flowered quick weed) Tagetis minuta L. (tall khaki weed) Solanaceae Datura stramonium L. (common thorn apple) Nicotiana glauca Graham (wild tobacco) Physalis angulata L. (wild gooseberry) Solanum nigrum L. (black nightshade) a

4.3

Mean percentage value determined according to the method of Kremer and Unterstenhöfer (1967)

ROLE OF WEEDS IN THE PERPETUATION OF BACTERIAL WILT

Stander (2001) investigated the possible host range by screening the weed species occurring on the site of the glasshouse trial in relation to pathogen survival, located at the University of Pretoria. Twenty-two weed species and three grass species occurring at the trial location were investigated for their susceptibility to infection with Ralstonia solanacearum biovar 2 and biovar 3.

4.3.1

Materials and results

A biovar 2 isolate and a biovar 3 isolate were cultured on Kelman’s tetrazolium medium (TZC) for a period of 48 hours (Kelman, 1953). A bacterial suspension was prepared in five litres of distilled water, after which a distilled series was prepared and plated on TZC. Colonies were counted after 48 hours, and the concentration was determined to be 2 x 106 cfu.ml-1. Vermiculite was soaked in the inoculum, and pots were then filled with 2 kg sterile vermiculite and soil (1:1 ratio) and inoculated by incorporating 40 g of soaked vermiculite. Ten seedlings from each weed species were transplanted into five pots inoculated with biovar 2, while 10 seedlings were transplanted into five pots inoculated with biovar 3, and two seedlings were transplanted into pots with non-inoculated soil to serve as controls. Once wilt had been established, isolations were performed to confirm the presence of Ralstonia solanacearum. After six weeks, isolations were performed on all plants. The stems and roots were washed thoroughly, after which all surfaces were sterilised in 1% sodium hypochlorite for 10 minutes and rinsed three times in sterile water. Each sample was blended or finely cut into sterile water, and the dilution series was then prepared, plated on TZC and incubated for 48 hours. Table 4.3.1.1 shows the individual host status of the 22 weed species and three grass species concerned. Bacterial wilt disease on potato: The South African experience

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Table 4.3.1.1

Evaluation of 22 weed species and 3 grass species for susceptibility to Ralstonia solanacearum biovar 3 (race 1) and biovar 2 (race 3) (Stander, 2001).

Plant species

% Plants wilting

Common name

% Plants infected

Biovar 2

Biovar 3

control

Biovar 2

Biovar 3

control

Amaranthus spp.

Pigweed

0

0

0

0

90

0

Bidens bipinnata

Spanish blackjack

0

50

0

0

90

0

Bidens pilosa

Common blackjack

0

0

0

0

0

0

Chamaesyce prostrata

Hairy creeping milkweed

0

20

0

0

100

0

Chenopodium album Chenopodium carinatum Chloris pycnotrix Commalina benghalensis Conyza albida

White goosefoot

0

0

0

0

70

0

Green goosefoot

0

0

0

0

100

0

Spiderweb chloris

0

0

0

0

0

0

Wandering Jew

0

0

0

0

0

0

Tall fleabane

0

0

0

0

0

0

Cyperus rotundus

Purple nutsedge

0

0

0

0

90

0

Datura ferox

Large thorn-apple

0

20

0

80

100

0

Datura stramonium

Common thorn-apple

0

60

0

100

100

0

Eragrostis curvula

Weeping love grass

0

0

0

0

30

0

Hibiscus trionum

Bladder hibiscus

0

0

0

10*

50

0

Hypochoeris radiacata

Hairy wild lettuce

0

0

0

0

0

0

Lepidium africanum

Pepperweed

0

0

0

0

0

0

Opuntia stricta

Australian pest-pear

0

0

0

0

0

0

Portulaca oleracea Pseudognophalium luteo-album Schkuria pinnata

Common purselane

20

40

0

70

100

0

Cudweed

0

0

0

0

10

0

Dwarf marigold

0

0

0

0

0

0

Sisymbrium thellungi

Wild mustard

0

0

0

0

0

0

Sonchus oleraceus

Sowthistle

0

0

0

0

40

0

Sporobolus africanus

Rat’s tail dropseed

0

0

0

0

0

0

Tagetes minuta

Khakiweed

0

0

0

0

10*

0

Tragopogon dubius

Yellow goat’s beard

0

0

0

0

30

0

*These findings must be re-evaluated to confirm infection of the host plant

4.3.2

Discussion

Datura ferox, D. stramonium, Portulaca oleracea and Hibiscus trionum were infected with the biovar 2 isolate, although wilting was observed only in some of the P. oleracea plants. The host status of Portulaca oleracea with respect to both races of Ralstonia solanacearum was established with plants expressing wilt symptoms. The host range for biovar 3 was much wider and included 13 of the 25 species evaluated. H. trionum has not yet (as at 2001) been reported as a host of bacterial wilt. Weeds reported for the first time as hosts of biovar 3 in South Africa include Sonchus oleraceus, Tragopogon dubius, Cyperus Bacterial wilt disease on potato: The South African experience

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rotundus, Bidens bipinnata and Chenopodium carinatum. Although Tagetes minuta is a host in both Australia (Akiew et al., 1993) and Uganda (Tusiime et al., 1998), the low percentage of plants infected with biovar 3 in this trial necessitates further investigation if it is to be declared a host of the local strain. Similarly, the low incidence of infection with biovar 3 found in Pseudognophalium luteo-album could be fortuitous and needs to be confirmed.

4.4 WILT

SCREENING OF COMMERCIALLY IMPORTANT CROPS AS POSSIBLE HOSTS FOR BACTERIAL

4.4.1

Materials and methods

Seeds were directly sown in plastic pots of 150 mm, while seedlings were directly transplanted into similar pots. After eight weeks, plants were inoculated with 10 ml inoculums (A620 = 0.01) applied to the soil near the stem base. Ten seedlings of each plant were inoculated with R. solanacearum biovar 2 (isolated from potatoes), while 10 seedlings were inoculated with biovar 3 (isolated from tomatoes). Five non-inoculated control plants were included for each inoculum type and crop species. The plants were kept in a glasshouse at day/night temperatures of 30/22°C and were watered with distilled water. Plants were evaluated and scored for wilt symptoms, 40 days after inoculation. The results were expressed as “percentage wilted plants” and then transformed into “percentage infected plants” according to the formula published by Kremer and Unterstenhöfer (1967). Each plant that survived inoculation was tested individually for the presence of the pathogen. Plants were blended aseptically and the suspension used for a dilution series plated onto tetrazolium chloride (Kelman, 1954) agar plates and incubated at 30°C for 48 hours. Plates were visually examined for typical R. solanacearum colonies, to be isolated and identified as R. solanacearum biovar 2 or biovar 3.

4.4.2

Results and discussion

The results are summarised in Table 4.4.2.1. The investigation identified cucumber, muskmelon, lettuce, beans, peas, lucerne, barley, maize, oats, sorghum, wheat, onions, beetroot, carrots, parsley, sweet potato and strawberry as non-hosts for R. solanacearum biovar 2 and 3, meaning that they can be recommended as rotational crops. It was also revealed that several crops can serve as symptomless hosts of R. solanacearum.

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Table 4.4.2.1 Mean percentage of wilting and percentage of plants infected with Ralstonia solanacearum biovar 2 or biovar 3 (Mienie, 1998)

Family

Crop species

Mean percentage wiltinga

Percentage plants infected

Biovar 2

Biovar 3

control

Biovar 2

biovar 3

control

Cucurbita pepo (Pumpkin)

0

30

0

0

50

0

Cucumis sativus (Cucumber)

0

0

0

0

0

0

Cucumis melo (Muskmelon)

0

0

0

0

0

0

Citrullis vulgaris (Watermelon)

0

17.5

0

0

40

0

Lactuca sativa (Lettuce)

0

0

0

0

0

0

Phaseolus vulgaris (Bean)

0

0

0

0

0

0

Pisum sativum (Pea)

0

0

0

0

0

0

Glycine max (Soybean)

0

55

0

0

70

0

Medicago sativa (Lucerne)

0

0

0

0

0

0

Hordeum vulgare (Barley)

0

0

0

0

0

0

Zea mays (Maize)

0

0

0

0

0

0

Avena sativa (Oat)

0

0

0

0

0

0

Sorghum spp. (Sorghum)

0

0

0

0

0

0

Triticum aestivum (Wheat)

0

0

0

0

0

0

Amaryllidaceae

Alium cepa (Onion)

0

0

0

0

0

0

Chenopodiaceae

Beta vulgaris (Beetroot) Brassica oleraceae var. capitata (Cabbage) Brassica oleraceae var. botrytis (Cauliflower) Brassica rapa (Turnip)

0

0

0

0

0

0

0

30

60

0

Cucurbitaceae

Compositae

Fabaceae

Gramineae

0 5

27.5

0

40

60

0

0

0

0

20**

80**

0

Raphanus sativus (Radish)

0

0

0

50**

80**

0

Daucus carota (Carrot)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Malvaceae

Petroselium crispum (Parsley) Ipomoea batatas (Sweet potato Gossypium hirsitum (Cotton)

0

0

0

0

30

0

Rosaceae

Fragaria spp. (Strawberry)

0

0

0

0

0

0

Zingiberaceae

Zingiber officinale (Ginger)

0

0

0

0

70

0

Cruciferae

Umbelliferae Convolvulaceae

a

Mean percentage value determined according to the method of Kremer and Unterstenhöfer (1967) ** Percentage plants with infected endorhizospheres

4.5

SCREENING OF HERBAL PLANTS AS POSSIBLE HOSTS FOR BACTERIAL WILT DISEASE

This investigation into the host status of herbal plants in respect of Ralstonia solanacearum formed part of the broader research project focusing mainly on the investigation of the possible suppression of the pathogen by herbal plant material.

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Seed of thirteen herbal plant species was germinated in seed trays and transplanted into two-litre pots containing 2 kg of a sterile clay-bark mixture. Sterile vermiculite was soaked in inoculums of either isolate 111 (biovar 2) or 117 (biovar 3) at 1 x 106 cfu ml-1 and added to the pot. Each plant was tested for the presence of the pathogen according to the technique described by Swanepoel (1992). None of the 13 herbal plant species selected developed typical wilt symptoms, but the pathogen was re-isolated from several species (Table 4.5.1).

Table 4.5.1

Host determination of herbal plant species (Van Broekhuizen, 2002) Herbal species

Host determination Biovar 2

Biovar 3

Ocimum basilicum L. (basil)

-

-

Borago officinalis L. (borage)

-

-

Apium graveolens L. (celery)

-

-

Matricaria recutita L. (chamomile)

-

-

Foeniculum vulgare Mill. (fennel)

-

-

Melissa officinalis L. (lemon balm)

-

-

Anethum graveolens Linn. (dill)

-

-

Allium tuberosum Rottler (chive)

-

+

Coriandrum sativum L. (coriander)

-

+

Origanum majorana L. (marjoram)

+

+

Brassica alba l. (mustard)

-

+

Tropaeolum majus L. (nasturtium)

+

+

Pertoselinum crispum L. (parsley)

+

+

Biovar 3 infected more of the 13 herbal species tested than did biovar 2. Since marjoram, nasturtium and parsley belong to the Labiatae, Tropaeolaceae and Umbelliferae families respectively, these results did not coincide with previous finding that biovar 2 (race 3) infects solanaceous plants only (Buddenhagen et al., 1962), and further investigation is thus required.

4.6 INVESTIGATION INTO HOSTS OF RALSTONIA SOLANACEARUM, BIOVAR 2 AND BIOVAR 3, AS LISTED IN TABLE 1 OF THE SOUTH AFRICAN SEED POTATO CERTIFICATION SCHEME 4.6.1. Introduction Common host plants – other than potato – for R. solanacearum (the bacterial wilt disease-causing organism) are listed in Table 1, as published by the South African Seed Potato Certification Scheme as per the Potato Plant Improvement Act, 1976 (Act 53 of 1976). Host plants of this organism are not limited to those listed in Table 1, however; a number of other crops and weed species also play a role. The implication of this table is that the crops listed cannot be included in a crop rotation programme for the growing of seed potatoes.

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The list of crops is as follows: Groundnut Arachis hypogea Cauliflower Brassica oleracea L. convar (L.) botrytis (L) Alef var. botrytis L. Cabbage Brassica oleracea L. convar (L.) capitata (L) Alef var. capitata L. Pepper Capsicum L. Watermelon Citrillus lanatus (Thunb) Matsum. et Nakai Pumpkin Cucurbita L. Soybean Glycine max (L.) Merrill Cotton Gossypium hirsutum L. Sunflower Helianthus annuus L. Tomato Lycopersicon lycopersicum L. Eggplant Solanum melongena L. Var. esculentum Nees Ginger Zingibor officinale Roscoe Tobacco Nicotinia tabacum L. Some questions were raised by seed potato growers regarding the feasibility of including soybeans as part of a crop rotation programme for the cultivation of seed potatoes. The Round-Up-Ready soybean cultivar proved to have an economic advantage over other crops when included in a crop rotation programme, especially in terms of weed control. At a meeting held on 8 November 2007, the ICCSP formulated a request for the investigation of soybeans in terms of the host status for R. solanacearum. The decision made in this regard was that once the results of the experiment had been evaluated and the relevant literature studied, the status of the soybean in terms of the crops included in the list as per Table 1 would be reviewed by the ICCSP. R. solanacearum affects over 450 plant species, making it the most significant bacterial disease worldwide. The species is highly complex and variable. Currently R. solanacearum is classified into five different races based on pathogenicity in host plants, and five different biovars based on the ability to produce acid from three hexose alcohols and three sugars. Race 1 (with a very broad host range) is pathogenic mainly for tobacco, tomatoes, potatoes, aubergines, diploid bananas, other solanaceous crops and weeds. Race 2 is pathogenic mainly for triploid bananas and Heliconia sp., while race 3 is pathogenic mainly for potatoes, tomatoes and other solanaceous crops, with two additional races affecting ginger and mulberries (fifth biovar) respectively, as reported in the Official Journal of the European Communities, Council Directive 98/57/EC on the Control of R. solanacearum (Smith) Yabuuchi et al. It is accepted that R. solanacearum race 3, biovar 2, as well as race 1, biovar 3, are the main causal agents for bacterial wilt in potatoes in South Africa. Internationally, the spread of the organism has been associated with dissemination via latently infected seed potatoes, implying that the most effective means of controlling the disease is through the use of pathogen-free seed tubers. Latent infection implies the colonisation of the vascular tissue of the tuber without any symptoms being caused, and this can occur at cooler temperatures when plants become infected, ultimately leading to latent infection in progeny tubers. The pathogen can survive in plant debris, as well as in the rhizosphere of potatoes, weeds and other crops. According to Miller (2003), Ralstonia solanacearum is not spread to any significant extent through air or splashing water. However, dissemination is easily achieved by means of sub-irrigation water, vegetative propagation and the handling of plants, as well as through soil adhering to shoes and cultivation tools. The pathogen is found in most tropical, sub-tropical and temperate regions of the world. Temperature is an important factor influencing the survival and host-pathogen interaction of R. solanacearum (Urquhart et al., 1998). Race 3, biovar 2, appears to have become better adapted to lower temperatures over time (thought to have originated in the highlands of Peru), while higher temperatures and soil moisture generally favour the survival and growth of the organism. Race 3, biovar 2, is a soil-borne

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pathogen that persists in wet, deep soil layers (> 75 cm) and reservoir plants, with irrigation water being an important source of dissemination for this sub-group. Extensive work has been done internationally on bacterial wilt and R. solanacearum with respect to different types of crops. A study conducted by Busolo-Bulafu (1998) found that in Uganda, where R. solanacearum occurs endemically, the disease can be seed-borne in groundnuts, and therefore the distribution of seed from planting areas where the disease is known to occur is discouraged. Schönfeld (2003) experimented with isolates from beans (Phasoelus vulgaris) to evaluate detection methods for R. solanacearum, while Terblanche and De Villiers (1998) evaluated Glycine max (soybean) as part of a crop rotation system in tobacco production. In Canada, the list of crop and weed hosts includes beans, beets and mustard plants. A study by Swanepoel (1988) on the characteristics of some South African strains of R. solanacearum (formerly known as Pseudomonas solanaceaum) found all the host plants included in that study (namely potato, tomato, eggplant, pepper, sunflower, tobacco and peanut) to be virulently infected by biovar 3 strains, while only potato, tomato, eggplant and pepper were found to be virulently infected by biovar 2 isolates. Also, strain variation was found to exist in virulence on hosts, as in the case of the weed species Datura ferox. An evaluation of root colonization in cultivated plants inoculated with R. solanacearum (Bringel et al., 2001) found soybeans to contain large populations of four isolates of the three biovars).

4.6.2

Materials and methods

4.6.2.1. Establishment of host plants All the crops as listed in Table 1, with the exception of ginger and tobacco, were planted for purposes of this study. Tomato was included as a positive control for the experimental inoculations. Seeds of the selected crops were obtained from local suppliers of commercial seed. Upon the emergence of seedlings, additional seeds were sown to correct emergence of less than 30 seeds per group. A potting-soil mixture obtained from a local nursery was steam-sterilised in an autoclave before being apportioned into disinfected containers suitable for the planting of seed from the selected hosts. These containers were then placed in the disinfected experimental facility, where more than 30 seeds were planted for each crop so as to ensure the emergence of approximately 30 seedlings from each. Controls of each crop were planted in small pots (approximately two seeds per crop) through a needle-puncture process in parallel to soil-soak inoculations. The seeds were watered using a fertiliser mixture as used in the fertiliser programme for in-house greenhouses. The seeds were not watered the following day (day before inoculation) so as to allow for the uptake of the pathogen by the roots after inoculation. 4.6.2.2 Preparation of inoculum In-house virulent isolates of R. solanacearum biovar 2 and biovar 3 from potato were grown on Tetrazoliumchloride (TZC) medium at 28°C for 48 hours to confirm purity of culture, as well as viability, and to facilitate subsequent confirmation of the identity of the organisms via biochemical methods. Both isolates were subjected to Gram-staining, 4% KOH disruption of cells, microscopic evaluation of cell morphology, colony morphology and colour on TZC and nutrient medium, as well as an oxidase test, nitrate reduction assay, Hugh-Leiffson oxidation/fermentation assay (aerobic or anaerobic utilisation of a carbon source in the form of glucose, and fluorescence on King’s B and Nile Blue media (intracellular accumulation of poli-βhydroxibutyric acid granules). To ascertain that both isolates were virulent, pure cultures were stab-inoculated into susceptible tomato plants (cv. Floradade) in the second true-leaf stage (approximately 14 days) via needle puncture of vascular tissue. The inoculum was drawn up into the needle and the plant tissue was punctured, after which a drop was deposited onto the puncture wound. The development of wilting symptoms was then monitored. Once wilting symptoms were observed, the isolates were multiplied on TZC medium to prepare the inoculum. The growth was removed from the solid medium and re-suspended into autoclaved distilled water. After suspension the number of viable cells was determined by means of dilution plating and counting with the aid of a Petroff-Hasser bacterial cell counting chamber.

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4.6.2.3 Soil-soak inoculation of host plants with R. solanacearum biovar 2 and biovar 3 isolates Approximately 150 ml of the cell suspension of both biovar 2 and biovar 3 were poured onto growth medium of unwounded plants (seeds) in the specific containers placed in areas (rows) where seeds were sown/planted on day three after planting. Inoculum density in the case of biovar 2 was 2.17 x 1012 cfu/ml, while for biovar 3 it was 7.9 x 1013 cfu/ml, giving final concentrations of 8.1 x 1011 cfu/g soil for biovar 2, and 3 x 102 cfu/g soil for biovar 3, for the first inoculation. Another inoculation was administered to the medium four weeks after planting. Inoculum was prepared and diluted to give the same order of cfu as the first inoculum, so as to ensure the presence of a viable inoculum in the planting medium for an extended time. The seeds were not watered on the day following soil-soak inoculation on day three, so as to allow for the uptake of the inoculum by the roots. The plants were then monitored for disease symptoms. Control plants aged between 14 and 21 days (depending on the cultivar) were inoculated through a process whereby the inoculum was drawn up into the needle and the plant tissue was punctured to allow a drop to be deposited into the puncture wound. Control plants were subsequently monitored for disease symptoms. 4.6.2.4 Symptom development in control plants and experimental inoculations All plants (controls and experimental inoculations) were monitored daily for the presence of symptoms, in an effort to establish disease development and to estimate when each experimental inoculation should be individually terminated by means of isolation of the pathogen. The plants were rated on a disease index scale of 0 to 4 (Table 4.6.3.1). The symptom development rating for all crops was based on the relative severity regarding progression (rapid progression of an individual plant also accounts for severe symptoms), and not necessarily the number of plants showing symptoms of infection. The disease rating was based on the number of plants that were affected/symptomatic/dead compared to the number of healthy plants. Upon termination of the experiment, the number of diseased plants in each group was counted and compared to the number of seemingly healthy plants of the same group. 4.6.2.5 Isolation of R. solanacearum from inoculated hosts Based on the host conditions and the symptom development observed, the pathogen was then isolated from the different crops. An individual plant was removed from the planting medium and the above-ground parts removed. Diseased plants and seemingly healthy plants were compared, and the crop was rated on the described disease index scale. Roots from the plants were divided into two equal groups. Roots from one group were surface sterilised, while those from the other group were merely rinsed in distilled water. The foliage representative of all plants from the particular container was pooled, while the roots of the two groups were pooled as divided. Six samples from each host (three samples for biovar 2 and three samples for biovar 3) were thus used to inoculate TZC and SMSA medium by spread-plating a dilution series of the samples. Pooled material was placed on one side of the sieve of a Bioreba extraction bag and then macerated. A 10-fold dilution series was prepared for each sample and the dilution series was plated out on TZC and SMSA medium. After 48 hours of incubation at 28°C, the solid medium was visually evaluated for the presence of typical R. solanacearum colonies. Typical colonies were isolated, purified and then subjected to the biochemical tests for verification of the identity of the particular isolate.

4.6.3

Results

Symptom development for all crops was based on the relative severity of progression (rapid progression on an individual plant also accounts for severe symptoms), and not necessarily on the number of plants showing infection. The disease rating was based on the number of plants that were affected/symptomatic/dead compared to the number of healthy plants. Symptom development is summarised in Table 4.6.3.1. In the case of soybeans, there was no rapid disease progression following inoculation with either biovar. However, up to 32% of the plants became affected/symptomatic at a later stage of the disease, with wilting and chlorosis being more evident for biovar 3 inoculations of PAN 1664 RR. Moreover, 38% of soybean Bacterial wilt disease on potato: The South African experience

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PAN 660 plants showed symptoms of disease at the termination of the experiment. This experiment revealed no clear differences between the cultivars in terms of disease progression.

Table 4.6.3.1.

Symptoms and disease development of crops soil-soak inoculated with R. solanacearum, and the isolation of R. solanacearum from plants under greenhouse conditions (Espach, 2008).

Crop Sunflower

Symptom development1 biovar 2 ++++

Symptom development1 biovar 3 ++++

Disease rating2 biovar 2

Disease rating2 biovar 3

4

4

Cotton

+

++++

1

2

Cabbage

++

++++

1

1

Cauliflower

+++

++++

1

2

Eggplant

+

+

1

1

Soybean PAN 1664 R

++

++

1

2

Soybean PAN 660

++

++

2

2

Pumpkin

+

++

1

2

Watermelon

+++

+++

2

1

Tomato

++++

+++++

4

4

Pepper

+

+

1

1

Groundnut

+

+

1

1

1

Symptom development: 0: no wilting 1: 1 % to 25 % wilted 2: 25 % to 50 % wilted 3: 50 % to 75 % wilted 4: 75 % to 100 % wilted

2

Disease rating was based on the number of plants that were affected/symptomatic/dead, compared to the number of healthy plants.

4.6.4. Summary The conclusion that can be drawn from these experimental infections is that the soybean can serve as a host for R. solanacearum. Studies to further clarify the virulence of different isolates of biovars on soybeans (and other crops that could be included in a suitable rotation programme for seed potato growing), as well as the colonisation of the plants and the survival of different population sizes in the rhizosphere, would be of use to assist growers in terms of considering means of reducing the R. solanacearum infection risk.

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Chapter 5

SURVIVAL IN SOIL AND THE INFLUENCE OF TEMPERATURE ON THE DEVELOPMENT OF BACTERIAL WILT 5.1

BACKGROUND

This chapter deals with several aspects of the ethiology of the bacterial wilt caused by Ralstonia solanacearum, of importance in the development of a strategy to manage bacterial wilt in the potato industry: 1. The effect of temperature on disease development and infection of progeny tubers during the production season (A.E. Swanepoel at Roodeplaat, late 1980s) 2. The effect of temperature, inoculation levels and storage period on spread of the pathogen in tubers and rot of tubers (N.J.J. Mienie and co-workers at Roodeplaat, 1990s) 3. Survival of R. solanacearum in soil (E.I.M. Stander and co-workers at University of Pretoria, 1994- 2001) Swanepoel (1990) found that wilting of infected plants might not occur at low temperatures, but that progeny tubers do become infected during the growing season. This led to the realisation by industry that reliable testing methods and rigorous testing of all seed potato plantings are of critical importance to limit the spread of R. solanacearum through seed potatoes. The concept of cold storage as a possible method of eliminating latent infections was investigated, also for purposes of determining the location of R. solanacearum cells in the tubers over time. Mienie (1998) confirmed the observations of Gadewar and Chakrabarti (1995), namely that low-temperature storage does not eliminate the pathogen from latent (symptomless) infected tubers and can still allow plants to wilt, meaning that the final concentration of viable counts in the tubers is therefore independent of the initial inoculum concentration and incubation temperature. Stander (2001) found that R. solancearum biovar 2 can survive in soil for a period of up to five years, which is much longer than previously believed. That field trial was partially continued after the five-year cropping sequence by Hammes (2013). After eight years, milky exudates were observed from potato stems from the potato monoculture plots. These results showed that although the wilt organism decreased over the eight years since the trial commenced, the organism still managed to survive, albeit in low numbers, in all four cultural practices. This emphasised the capacity of the organism to survive for long periods even under adverse (drought) conditions. This information assisted the South African potato industry in formulating regulations for the Seed Potato Certification Scheme. In 1999 the regulations stated that no seed potatoes may be cultivated on infected fields for a period of eight years in cases where biovar 2 has been isolated and never in cases where biovar 3 has been isolated.

5.2 EFFECT OF TEMPERATURE ON THE DEVELOPMENT OF WILTING AND ON PROGENY TUBER INFECTION OF POTATOES INOCULATED WITH SOUTH AFRICAN STRAINS OF BIOVAR 2 AND BIOVAR 3 OF RALSTONIA SOLANACEARUM 5.2.1

Introduction

Several authors have found that disease severity increased with an increase in temperature. It was also found that when resistant potato cultivars were grown in infested soil at high temperatures, only a few plants developed wilting symptoms. Nevertheless the pathogen was latently present in most plants and these produced both overtly and latently infected tubers. In South Africa it is in the cooler infested areas that latent tuber infection is a problem, as wilting symptoms are weak or absent and the harvested tubers appear healthy. Bacterial wilt disease on potato: The South African experience

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The purpose of the study by Swanepoel (1990) was to compare the ability of biovars 2 and 3 to induce wilting symptoms on plants grown at different temperatures and to investigate the infection of the progeny tubers of these plants. Results showed that at low temperatures (14/16°C) the mean percentage of wilting of plants inoculated with isolates of both biovars was low. It is therefore possible that symptoms on infected potato crops grown at low temperatures may not be evident during the production season, thus allowing infected progeny to transmit the disease.

5.2.2

Materials and methods

Disease-free tubers of the potato cultivar BP1 were planted in pots. When the plants were 15 to 200 mm tall, they were inoculated by the method of Winstead and Kelman (1952) using 10 ml inoculum (with an absorbance of 0.001) per plant of either biovar 2 or biovar 3, isolated from diseased potato and tomato plants respectively. After inoculation the plants were placed in growth chambers and set at the following temperatures (dark/light): 14/16, 16/18, 18/20, 20/22, 22/25 and 25/30°C. Each replicate consisted of 20 plants plus two non-inoculated controls. Sixty days after inoculation the plants were scored for wilting and the results transformed into percentage values (Kremer and Unterstenhöfer, 1967). Plants that did not develop wilt symptoms were kept until senescence and their progeny tubers harvested. If fewer than 20 tubers, they were scored for internal symptoms only. When more tubers were formed, the tubers were divided into two groups and scored for infection as follows: 1. Tubers were cut and examined for signs of infection. 2. Tubers were stored at 4°C for two months after which they were kept at 20 to 25°C to sprout and were then planted in pots and kept in a glasshouse at 25/28°C. Plants were examined weekly for symptoms up to 60 days. If wilting occurred, the pathogen was isolated and identified to confirm that it was R. solanacearum.

5.2.3

Results and discussion

The results are presented in Table 5.2.3.1. Plants inoculated with biovar 2 wilted when treated at temperatures of 14/16°C or higher, but plants inoculated with biovar 3 only wilted at temperatures of 18/20 °C or higher. After inoculation with biovar 2, plants died at temperatures of 18/20°C or higher, while after inoculation with biovar 3 they died only at the highest temperature tested, i.e. 25/30°C.

Table 5.2.3.1 Effect of temperature on the development of wilting symptoms, and progeny tuber infection in potatoes after inoculation with a South African strain of biovar 2 or biovar 3 of R. solanacearum (Swanepoel, 1990) Temperature treatment

Mean % wiltinga

Mean % internal symptoms on progeny tubersb

Mean % wilting of plants grown from progeny tubers

biovar 2

biovar 3

control

biovar 2

biovar 3

control

biovar 2

biovar 3

control

14/16°C

18.3

0

0

11.9

0

0

34.4

0

0

16/18°C

58.8*

0

0

26.3

0

0

57.0

7.2

0

18/20°C

100*

19.4*

0

NTHc

0

0

NTH

50

0

20/22°C

100*

43.2*

0

NTH

22.2

0

NTH

50

0

22/25°C

100*

58.2*

0

NTH

5.6

0

NTH

16.9

0

25/30°C

100

100

0

NTH

NTH

0

NTH

NTH

0

a Mean

% value determined according to the method of Kremer and Unterstenhöfer (1967) . analysis of variance was possible with the data in these columns due to plant mortality after inoculation. c NTH: No tubers harvested (all plants died after inoculation).  Significant (P≤ 0.05) mean separations between biovar 2 and biovar 3 (not the control plants) at the specified temperatures. LSD = 32.6. b No

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Browning or rotting of the vascular tissue was observed from 14/16°C when plants were inoculated with biovar 2, and from 20/22°C when inoculated with biovar 3. The lowest temperatures at which disease transmission by the progeny tubers could be detected were 14/16°C and 16/18°C after inoculation with biovar 2 or biovar 3 respectively. With each treatment the percentage wilting of plants grown from progeny tubers at 25/28°C was higher than the percentage internal symptoms on the progeny tubers. Latent infection may account for this tendency. Data on the internal symptoms occurring in progeny tubers and the wilting of plants grown from progeny tubers could not be subjected to variance analysis due to plant mortality after inoculation. The results indicate that at lower temperatures, the biovar 2 isolate has a greater capacity to cause disease than the biovar 3 isolate. Wilting, tuber symptoms and transmission by the progeny tubers occurred at lower temperatures after inoculation with biovar 2 than with biovar 3. At the lowest temperature (14/16°C) the mean percentage of wilting of plants inoculated with biovar 2 was only 18.2%, which did not differ significantly from those inoculated with biovar 3. It is therefore possible that symptoms on potato crops infected with biovar 2 and grown at low temperatures may not be evident, thus allowing infected progeny to transmit the disease. Ciampi and Sequeira (1980) reported differences in the virulence of different biovar 2 strains at different temperatures, identifying identified two biovar 2 strains able to cause wilting at 16°C. As these experiments were done with only one representative strain of each biovar, further research is necessary to determine whether there are similar differences between South African strains.

5.3 EFFECT OF INCUBATION TEMPERATURE, STORAGE PERIOD AND INOCULUM CONCENTRATION ON THE MULTIPLICATION AND SURVIVAL OF R. SOLANACEARUM IN POTATO TUBERS 5.3.1

Introduction

It was commonly believed that cold storage of tubers infected with R. solanacearum renders the organism non-viable. Several researchers have published results to the contrary, however. Ciampi et al. (1980) and Ciampi-Panno et al. (1981) reported that storage at 4°C did not eliminate the pathogen from inoculated tubers even after a 40-day incubation period. Gadewar and Chakrabarti (1995) also reported that the practice of seed storage in cold or country stores do not eliminate the pathogen and that latent tuber infection could still occur in wilting plants. The movement of R. solanacearum cells has been monitored in resistant potato plants, but not in potato tubers (Ciampi-Panno et al., 1991). Skoglund et al. (1993), following personal communication with El-Nashaar, reported that the location of the bacteria inside the tuber changes during storage. The concept of cold storage as a possible method of eliminating latent infections was investigated, also as a means to determine the location of R. solanacearum cells in the tubers over time.

5.3.2

Materials and methods

5.3.2.1 Preparation of inocula Inocula were prepared from 48-hour R. solanacearum biovar 2 cultures. Three concentrations were prepared, corresponding to viable counts of 1 x 105, 1 x 107 and 1 x 109 cfu ml-1 respectively. Distilled water was used as standard. Accurate viable counts and purity were confirmed with standard serial dilution and spread-plating of inocula on TZC (Kelman, 1954). 5.3.2.2 Inoculation of tubers In total, 270 tubers of cultivar BP1, approximately 50 mm in length, were selected and surface sterilised with 70% ethanol for three minutes. Ninety tubers were injected with each inoculum concentration respectively. Each tuber was inoculated to a depth of 5 mm at the stolon end with a micro-litre syringe (Hamilton No. 705). The number of cells injected into the tubers was Bacterial wilt disease on potato: The South African experience

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103, 105 and 107 cells per tuber, respectively. Thirty tubers from each inoculum group were incubated at 4°C, 18°C and 30°C respectively. 5.3.2.3 Sampling and testing of tubers Three tubers from each inoculum group and from each temperature treatment were withdrawn and tested for the presence of R. solanacearum at 3, 7, 14, 28, 42, 56, 70, 98, 126 and 154 days after inoculation. After peeling with a sterilised knife, three slices from the stolon end of each tuber were cut at depths of 0 to 10 mm, 10 to 20 mm and 20 to 30 mm. Each slice was weighed and cut into smaller pieces, then blended with double the quantity of sterile water (in ml) than the mass of the slice, after which the pulp was poured into a sterile McCartney bottle. The quantity of blending water was calculated in this manner to ensure the accuracy of the bacterial counts, since each tuber differed in size. Tenfold serial dilutions were prepared from each sample, and 0.1 ml quantities were spread onto TZC. Plates were incubated at 30°C for 48 hours and typical colonies were counted. The viable counts were transformed into log10 values and plotted against incubation time (days). 5.3.2.4 High-temperature incubation of tubers after cold storage After 10 weeks, all the tubers stored at 4°C were transferred to an incubator set at 30 °C in order to determine the effect of high-temperature incubation for an extended period of 28 days after cold storage. The sampling and testing of tubers then occurred as described above. 5.3.2.5 Data analysis Data analysis was done by means of Genstat 5 Release 2.2 copyright 1990, Lawes Agricultural Trust (Rothamstead Experimental Station).

5.3.3

Results

The tubers incubated at 18°C and 30°C started rotting after approximately 98 days and were lost by 126 days. Tubers placed at 30°C after ten weeks at 4°C could only be tested up to 98 days of storage, at which point after the experiment had to be terminated due to the rotting of tubers. Growth of R. solanacearum cells at different incubation temperatures Results of the 4°C incubation treatment are presented in Figure 5.3.3.1. There was a rapid decrease in the viable counts between zero and three days after incubation when the initial inoculum concentration was 103 cells per tuber. After 14 days the viable counts reached zero per gram tissue and remained so until 70 days, after which the remaining tubers were transferred to a 30°C incubator. The viable counts then increased to 1 x 108 cells per gram tissue within 28 days. Inoculation with 105 and 107 cells per tuber followed a similar trend during incubation and when transferred to a 30°C incubator. The viable counts at these concentrations did not drop as low as they did in the case of inoculation with 103 cells per tuber, at between 7 and 70 days. Results of the 18°C incubation treatment are presented in Figure 5.3.3.2. The three inoculation concentrations exhibited a similar trend regarding the viable counts. After an initial decrease in viable counts between zero and three days after incubation, a rapid increase in viable counts was experienced from 14 days after incubation until 28 days, when viable counts of 1 x 108 cells per tuber were attained and sustained until 70 days after incubation. At an incubation treatment of 30°C, a gradual increase in viable counts of the 10 3 cells per tuber treatment was observed until 42 days after incubation, when a viable count of 1 x 108 was reached and maintained until 92 days (Figure 5.3.3.3). The 105 cells per tuber treatment attained the same viable count at 14 days, while the 107 cells per tuber treatment reached this level seven days after incubation. Distribution of R. solanacearum cells in various tissue sections Bacterial wilt disease on potato: The South African experience

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Results of the distribution of cells in the inoculated tubers at 4°C, 18°C and 30°C incubation are presented in Figure 5.3.3.4, Figure 5.3.3.5 and Figure 5.3.3.6 respectively. At 4°C, the viable counts at different depths in the tuber followed the same trend over time, except that the viable counts for the deeper slice of the tuber (20-30 mm) were lower than for the two shallower slices. When incubated at 30°C from 70 days, the viable counts increased rapidly, reaching 1 x 10, 3.6 x 10 and 6.2 x 10 cells per gram tissue at depths of 0-10 mm, 10-20 mm and 20-30 mm respectively. At 30°C, the viable counts for the two shallower treatments reached 1 x 10 8 per gram tissue by seven days. The deeper treatment lagged behind until the seven-day mark, when viable counts increased rapidly and reached counts of 1 x 108 cells per gram after 28 days.

5.3.4

Discussion

The results of this study show that cold storage of infected tubers at 4°C for 10 weeks did not eliminate R. solanacearum, considering that after transferring the tubers to 30°C, the viable counts increased to 1 x 10 8 cells per gram tuber tissue. These results confirmed the observations of Gadewar and Chakrabarti (1995), namely that low-temperature storage does not eliminate the pathogen from latent (symptomless) infected tubers. It can be noted that even an initial low inoculum concentration (103 cells ml-1) of the pathogen at the onset of the trial subsequently increased under favourable temperatures to reach viable counts equivalent to those reached in tubers inoculated with a high initial inoculum concentration (10 7 cells ml-1). The final concentration of viable counts in the tubers is therefore independent of the initial inoculum concentration and incubation temperature.

Log of viable cells/g tissue

At the lowest incubation temperature (4°C), the highest concentration of viable R. solanacearum cells was found in the first 10 mm beneath the inoculation site. At 18°C, the bacteria moved deeper and the highest concentration of viable cells was found up to 20 mm deep. At 30°C, the number of viable cells was almost the same at all three depths at the conclusion of the trial. The movement or penetration of R. solanacearum cells deeper into artificially infected tubers is therefore temperature dependent. These results prove that the pathogen will multiply and spread inside latently infected tubers under favourable conditions. 8 7 6 5 4 3 2 1 0 0

3

7

14

28

42

70

98

Incubation time (days) Concentration 1

Concentration 2

Concentration 3

Fig 5.3.3.1 Multiplication of R. solanacearum cells at three concentrations in potato tubers incubated at 4°C for 70 days and thereafter at 30°C for 28 days (Mienie, 1998)

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Log of viable cells /g tissue

10 8 6 4 2 0 0

3

7

14

28

42

70

98

Incubation time (Days) Concentration 1

Concentration 2

Concentration 3

Fig 5.3.3.2 Multiplication of R. solanacearum cells at three concentrations in potato tubers incubated at 18°C (Mienie, 1998)

Log of viable cells/g tissue

10 8 6 4 2 0 3

7

14

28

42

70

98

Incubation time (Days) Concentration 1

Concentration 2

Concentration 3

Fig 5.3.3.3 Multiplication of R. solanacearum cells at three concentrations in potato tubers incubated at 30°C (Mienie, 1998)

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Log of viable cells/g tissue

8 6 4 2 0 3

7

14

28

42

70

98

Incubation time (days) Depth 0-1 cm

Depth 1-2 cm

Depth 2-3 cm

Fig 5.3.3.4 Multiplication of R. solanacearum cells at three depths in potato tubers incubated at 4°C for 70 days and thereafter at 30°C for 28 days (Mienie, 1998)

log of viable cells/g tissue

10 8 6 4 2 0 3

7

14

28

42

70

98

Incubation time (days) Depth 0-1 cm

Depth 1-2 cm

Depth 2-3 cm

Fig 5.3.3.5 Multiplication of R. solanacearum cells at three depths in potato tubers incubated at 18°C (Mienie, 1998)

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Log of viable cells/g tissue

10 8 6 4 2 0 3

7

14

28

42

70

98

Incubation time (Days) Depth 0-1 cm

Depth 1-2 cm

Depth 2-3 cm

Fig 5.3.3.6 Multiplication of R. solanacearum cells at three depths in potato tubers incubated at 30°C (Mienie, 1998)

5.4

SURVIVAL OF R. SOLANACEARUM IN ARTIFICIALLY INFESTED SOIL

5.4.1

Introduction

The occurrence, often inexplicable, of bacterial wilt in the fields and seed lots of a number of seed growers in the 1980s and 1990s emphasised the lack of knowledge regarding the survival of the causal organism under field conditions. At the time, biovar 3 was known to survive in soil for up to eight years, partly due to its wide host range. Biovar 2 was believed to be less adapted to soil survival, with eradication being possible within two years (Elphinstone, 1996). Rotations with pastures and maize have been reported to eradicate the pathogen after a few seasons (French, 1994). However, Martin et al. (1981) demonstrated that a bare fallow period of 140 days was insufficient to eliminate biovar 2 from any of the soil layers (at depths of 0-90 cm). Graham et al. (1979) reported that biovar 2 can survive in potato debris for at least 233 days. As little was known about the soil survival of biovar 2 in warmer regions, a long-term field trial with four cultural practices was initiated on the experimental farm of the University of Pretoria.

5.4.2

Materials and Methods

5.4.2.1 Infestation During the 1994/1995 season, a field of 20 x 48 m was selected on the experimental farm of the University of Pretoria and prepared for planting. The soil was a relatively well-drained clay-loam with a pH of 6.2. Ralstonia solanacearum biovar 2, race 3, was cultured on Kelman’s tetrazolium chloride (TZC) agar (Kelman, 1953) for 48 hours. A bacterial suspension was prepared in sterile distilled water and injected into a wound made in certified seed potato tubers. The tubers were dipped in a separate biovar 2 suspension and planted at a high density. Wilt symptoms were monitored throughout the growing season and the diseased crop was ploughed in to ensure uniform and severe infection levels. 5.4.2.1 Cultural practices The field was subdivided during the 1995/96 season into 12 plots representing three replicates of four treatments, namely maize monoculture, potato monoculture, bare fallow, and leaving the plots under weeds that could serve as alternative hosts. The field layout is depicted in Figure 5.4.2.1. In order to prevent cross-contamination between plots, metal plates were placed up to 1 m deep into the soil between plots. After rotovating the soil and applying herbicide, the potato and maize plots were planted and, together with the weed-fallowed plots, were monitored weekly for a period of 12 weeks for any wilt symptoms. Identifications were performed on weed and grass species. Isolations were performed from 1998 on roots and stems of various weeds from the weed-fallowed plots. Isolations from randomly selected maize plants were also made. During winter Bacterial wilt disease on potato: The South African experience

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months, plant growth was removed from the potato, maize and fallow treatments and the weeds were cut back. During the 1996/1997 season, half of each bare fallow plot was tilled to ensure greater exposure to the sun. These practices were conducted annually, and plots were irrigated during the crop season. Soil samples were drawn at different depths (down to 750 mm) at different times and analysed for the presence of R. solanacearum.

Repetition I

Repetition II

Repetition III

20 m Indicator plants 1999/2000

Indicator plants 1997/1998

Figure 5.4.2.1 Layout of the field infested with R. solanacearum (Stander, 2001)

Due to vast fluctuations in Ralstonia counts obtained from the soil samples, the use of indicator plants was introduced during the 1997/1998 season. A field area of five metres across all treatments was demarcated, and certified seed potatoes were planted in five rows of 10 plants each per treatment plot. To monitor wilting, milk-flow from cut stems of wilted plants was observed and bacterial isolations were performed from some infected stems to confirm bacterial wilt disease. All plant material was removed from this area after 12 weeks to minimise inoculum build-up. The area was separated from the rest of the field the following season by means of a wire fence to prevent movement through the area. This procedure was repeated during the 1999/2000 season in the demarcated region of five metres alongside the previous indicator plant site (Figure 5.4.2.1). 5.4.2.2 Plant samples Random samples of wilted potato plants were taken during each season and the stems analysed on TZC to confirm the presence of R. solanacearum. During the 1999/2000 season, 25 isolates from wilted indicator plants randomly selected across all plots were subjected to biovar identification tests to confirm the causal organism of the wilt as biovar 2. 5.4.2.3 Statistical analysis Wilt indices of potato plants in the potato monoculture plots over a five-year period were statistically analysed using the General Linear Models Procedure of the Statistical Analysis System (SAS®) 1 to determine variations between seasons. Wilt Bacterial wilt disease on potato: The South African experience

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indices of indicator plants in all treatment plots of both the 1997/1998 and 1999/2000 seasons were analysed to determine the effect of cultural practices. Turkey’s Studentized Range at the 5% significance level was used to identify significant differences.

5.4.3

Results and discussion

Ralstonia solanacearum populations in the soil were monitored after a three-year and a five-year cropping sequence by planting potato in a designated region across all treatment plots as indicators of wilt. Percentage wilt observed in the potato monoculture plots was 88% and 96% respectively (Figure 5.4.3.1). After both the three-year and five-year rotations, onset of wilt in bare-fallow plots was relatively slow, and the argument that survival of Ralstonia in the absence of a host is poor thus seemed to hold true. In the subsequent weeks, however, wilt increased rapidly, eventually surpassing wilt patterns in both maize and weed-fallow plots. The difference in the incidence of wilt between potato monoculture and bare fallow plots was not statistically meaningful after the three-year rotation period. Similar results were reported by Akiew and Trevorrow (1994), i.e. the incidence of tobacco wilt after two-year bare fallow was not significantly lower than after continuous tobacco culture. After five years, however, percentage wilt in the bare fallow plots was statistically lower than that observed in the potato plots. The desiccation of soil is considered a major factor in reducing soil populations of the wilt organism, and this is enhanced with bare fallow treatment. The disease pattern observed in both seasons indicates that either insufficient desiccation occurred, or R. solanacearum populations deeper in the soil profile remained less affected. It is also possible that the lack of plant material in the bare fallow plots resulted in a decrease in general soil organisms, thereby reducing competition and suppression. Maize has been used in rotation programmes since the early 1800s (Kelman, 1953), yet a relatively high percentage wilt (40%) was recorded in the maize plots after five years of monoculture. Granada and Sequeira 1983a) and Shekhawat et al. (1992) were able to isolate R. solanacearum from the root tissue of maize. Infections remained localised in the roots, and there was less bacterial release into the soil from these plants than from true host plants. The infection rate of the maize plants was also lower, since not every individual plant became infected. These findings may indicate why an overall reduction of wilt can still be experienced in a rotation with maize. Roughing of weeds is considered vital in the integrated management of bacterial wilt, since weeds can serve as hosts or shelter sites for long-term survival of the pathogen (French, 1994; Jackson and Conzales, 1979). However, potato plants in the weed-fallow plots exhibited the fewest symptoms of wilt, even though host species such as Datura stramonium and Portulaca were present. This may be an indication that certain weeds and grasses could have served to suppress the wilt organism either by harbouring antagonistic micro-organisms or by releasing inhibitory substances. The weeds in this case tended to be interspersed with the major cover being provided by grasses. Thirty species were identified in the weed-fallow plots over several seasons. In Australia, forage sorghum, signal grass and Rhodes grass are often used in rotation programmes (Akiew et al., 1993; Arthy and Akiew, 1999). The field study clearly demonstrated that Ralstonia solanacearum biovar 2 can survive much longer in soil than generally believed, irrespective of the cultural practice applied. This information should assist the South African potato industry in formulating regulations for the Seed Potato Certification Scheme. In 1999 the regulations stated that no seed potatoes may be cultivated on infected fields for a period of eight years in cases where biovar 2 has been isolated, and never in cases where biovar 3 has been isolated (Swanepoel and Theron, 1999). This was also confirmed by Nortje (2004). This study on the longevity of the bacterial wilt pathogen was, however, conducted in a clay-loam soil – a soil type not commonly found in most potato production regions. Since soil type is known to influence the survival period of R. solanacearum (Moffet et al., 1983; Shekhawat and Perombelom, 1991), the information obtained from this study cannot be extrapolated to situations where lighter soils are infected. The field trial was partially continued after the five-year cropping sequence. After eight years, milky exudates were observed from potato stems originating from the potato monoculture plots. Samples of 100 tubers per plot were drawn and submitted for analysis. Ralstonia was confirmed in five of the 12 samples, namely in two of the maize monoculture subplots, in two of the weed-fallow subplots, and in one of the three replicates of the bare fallow plots (Hammes, 2013). These results show that Bacterial wilt disease on potato: The South African experience

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although the incidence of the wilt organism decreased over the eight years since the start of the trial, the organism still managed to survive, albeit in low numbers, in all four cultural practices. This emphasises the capacity of the organism to survive for long periods even under adverse (drought) conditions.

% Potato Plants Tested positive (milk test)

Potatoes were planted over the entire trial field 10 years after the soil was initially infested. Samples consisting of 100 tubers were drawn from each plot and submitted for analysis. R. solanacearum could not be detected in any of the samples. This may imply that the organism had been totally eliminated during the dry and unfavourable conditions experienced since 2001. The possibility of some Ralstonia surviving in the deeper soil layers can, however, not be ruled out, as the test crop was grown under dry conditions and a shallow rooting system was observed (Hammes, 2013).

110 100 90 80 70 60 50 40 30 20 10 0 Weeds

Fallow 3 Years

Maize

Potatoes

5 years

Figure 5.4.3.1. Percentage wilting of indicator potato plants at three and five years after soil infestation (Stander, 2001)

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Chapter 6

ASPECTS OF THE MANAGEMENT OF BACTERIAL WILT 6.1

BACKGROUND

This chapter deals with several aspects of the management of bacterial wilt caused by Ralstonia solanacearum, namely: 1. Suppression of R. solanacearum in the rhizosphere by weeds (E.I.M. Stander and co-workers at University of Pretoria, 1994 – 2001). 2. Suppression of R. solanacearum in the rhizosphere by maize (E.I.M. Stander and co-workers at University of Pretoria, 1994 – 2001). 3. Suppression of R. solanacearum in the rhizosphere by herbal plant material (W. van Broekhuizen and co-workers, 1995 – 2002). 4. Evaluation of bacterial antagonists against bacterial wilt and their potential use as bio-control agents (N.J.J. Mienie and co-workers, 1994 – 1998). 5. Evaluation of commercially available disinfectants against Ralstonia solanacearum biovar 2 (N.J.J. Mienie and coworkers, 1994 – 1998). Stander (2001) recommended that further studies be conducted to determine whether high levels of antagonistic bacteria are present in the rhizosphere of the three grasses or the two weeds that occurred abundantly in the weed-fallowed plots. Further investigations should be conducted as to whether chemical compounds present in the grasses or weeds could be directly involved in the suppression of bacterial wilt. Varying results were obtained regarding the ability of maize to serve as a carrier or host of R. solanacearum. However, an antagonist tentatively identified as Chromobacterium violaceum, with the ability to suppress R. solanacearum, was found in the solution as well as in the maize plants. Several other saprophytic bacteria, mildly antagonistic, were also observed. Stander (2001) recommended further investigation of the hypothesis that certain bacteria (antagonistic and otherwise) present in the soil could be transmitted to seed and that some of these could have a protective effect on the developing maize plant. There are several possible reasons why no wilt symptoms developed and why all tissue isolations tested negative for the presence of R. solanacearum, such as low inoculum concentration, low soil temperatures, the dilution factor created by the soil, soil in pots being too dry, or loss of virulence. Van Broekhuizen (2002) recommended that bio-fumigation should be reevaluated under different glasshouse conditions to determine if herbal plant material could be used to suppress R. solanacearum in soil. Mienie (1998) concluded that further research is necessary to evaluate biological control under field conditions, but stated that bio-control clearly has the potential to be included as part of an integrated management strategy against bacterial wilt in potatoes in future. Unfortunately, none of the aforementioned studies were ever followed up. It is therefore evident that several options for the suppression (not exclusion) of bacterial wilt can be explored further in view of the management of bacterial wilt in soils endemically infected with R. solanacearum. At the time of this study, there were no registered chemicals available for use in sanitation practices against bacterial wilt. Chlorox, Jeyes Fluid, HTH and Sporekill were identified as bactericidal agents that can be used for the sanitation of implements.

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6.2

SUPPRESSION OF R. SOLANACEARUM IN THE RHIZOSPHERE BY WEEDS

6.2.1. Introduction Interest in the field of biological control as an alternative method for the control of weeds, insects and plant pathogens has grown due to the global move towards more natural and environmentally friendly farming systems (Lampkin, 1990; Lydon and Duke, 1989). According to Kirkegraad and Sarwar (1998), bio-fumigation refers to the suppression of soil-borne pests and pathogens by biocidal compounds released into the soil when certain plant material is broken down. During the decomposition of plant residues, substances such as glucosinolates can be hydrolysed to release a range of volatile compounds known as isothiocyanates, as well as other secondary compounds such as oxazolidinethiones, nitriles and thiocyanates (Akiew et al., 1996; Kirkegraad and Sarwar, 1998). The type of isothiocyanate released is specific to the type of glucosinolate present in the tissue. Isothiocyanates fall into the same class of chemical compounds as those produced by the decomposition of metham sodium (vapam), a commercial soil fumigant (Akiew et al., 1996). Since some of the substances released by bio-fumigation display broad biocidal activity, including insecticidal, nematicidal, fungicidal, antibiotic and phytotoxic effects, this practice can form part of an integrated control programme to suppress soil-borne pathogens (Kirkegraad and Sarwar, 1998).

6.2.2

Preliminary evaluation of various weeds for the suppression of R. solanacearum in the rhizosphere

6.2.2.2 Materials and methods Experiment 1 The rhizosphere soil from certain weeds used in the host range study was analysed to determine whether a reduction or increase in the R. solanacearum population had occurred in the six-week period. Soil was shaken from the rhizosphere of some plants of Amaranthus spp., Chamaesyce. prostata, Chloris pycnotrix, Eragrostis curvula and Datura ferox and allowed to dry for 72 hours. A 10 g sample of the soil was suspended in 100 ml sterile water and placed on a shaker for 30 minutes. A dilution series was prepared and plated on modified TZC without the addition of bacitracin. Colony counts were performed after 72 hours. Experiment 2 Due to the poor recovery of the pathogen from the soil, a second experiment was conducted to avoid the soil phase. Erlenmeyer flasks containing 90 ml sterile nutrient solution were inoculated with 10 ml R. solanacearum biovar 2 (6 x 105 cfu.ml-1) inocula. Six small plantlets of Sporobolus africanus, Eragrostis curvula and Chloris. pycnotrix each, were rinsed in distilled water, then transferred to the flasks and kept in a growth chamber for three weeks. Isolations were performed weekly on TZC from the solution of two flasks per specie and from two control flasks without any plants. Colony counts were performed after 48 hours. 6.2.2.3 Results and discussion Although rhizosphere populations of R. solanacearum have been determined successfully by several authors (Granada and Sequeira, 1983a; Terblanche and De Villiers, 1998), repeated efforts to obtain stable population counts from the rhizosphere soil failed in the case of this investigation. Growth of saprophytic organisms on the agar plates was high, possibly obscuring the presence of Ralstonia. The presence of bacterial antagonists can also inhibit the growth of R. solanacearum on agar plates (Elphinstone, 1996). The use of a hydroponic system to evaluate the effect of root exudates on pathogen populations has merit, if adapted and refined. It is not clear from the data obtained whether the plantlets cultured in inoculated nutrient solution had a direct effect on Ralstonia populations, or whether detection of Ralstonia was simply hampered by the overgrowth of saprophytes and/or the presence of antagonistic bacteria. The antagonistic effect that was observed when three bacterial isolates isolated from the grass exudates were streaked against R. solanacearum biovar 2 does not necessarily relate to antagonistic activity under natural conditions, but could be limited to the agar environment (Trigalet et al., 1994). To create an aseptic environment in which the chemical nature of suppression can be investigated, plants can be cultured in vitro or aseptically from seed before Bacterial wilt disease on potato: The South African experience

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being transferred to an inoculated hydroponic system. Surface sterilisation of roots would reduce microbial populations, but could affect normal root activity.

6.2.3

In vitro suppression of R. solanacearum with weed extracts

6.2.3.1 Materials and methods Experiment 1 Tagetes minuta, Conyza albida, Lepidium africanum and Opuntia stricta plants were rinsed to remove soil particles, surface sterilised in 1% sodium hypochlorite for 15 minutes, and rinsed three times in sterile water. The plant material was weighed and blended in sterile water at a ratio of 1 g plant material to 10 ml water. Supernatant of the extract was sterilised through microfiltration using a Millipore filter. The sterilised filter paper discs were impregnated with the sterile extract and allowed to dry in a sterile petri dish. Discs impregnated with a 1% sodium hypochlorite solution and sterile water were used as controls. Seeded TZC plates were prepared as follows: Standard TZC plates were poured and the medium was allowed to stall. Two additional bottles of 200 ml TZC medium each were prepared with only half the standard amount of agar. Once cooled to below 40°C, 20 ml R. solanacearum biovar 2 inoculum at concentration 3.1 x 105 cfu.ml-1 was added to one bottle and gently agitated, and a thin layer of cooled inoculated TZC medium was then poured onto standard TZC plates. To the other bottle, 20 ml of biovar 3 inoculum (2.0 x 105 cfu.ml-1) was added and poured onto standard TZV medium. Discs were placed in a random design on the seed TZC (10 replications) and incubated for 72 hours, after which inhibition zones were measured. This technique is similar to the one described by Korsten (1984). Experiment 2 Plants of Tragopogon dubius, Eragrostis curvula, Sporobulus africanus and Hypochoeris radicata were rinsed and soaked in sterile distilled water (ratio 1 g: 10 ml) for 16 hours without surface sterilisation. Suppression of R. solanacearum by these weeds was evaluated using three different techniques: Technique A: Paper disc The disc technique on seeded TZC, as described above, was used. Technique B: Poisoned medium Theron (1999) suggested the use of this technique, as described by Jones and Eheret (1976), after finding that the diffusibility of certain fungicides was not effective with the application of the disc technique. For this reason, filter-sterilised weed leaching samples (obtained as described above) were incorporated into cooled TZC medium at a ratio of 20 ml leaching to 500 ml TZC medium, prior to pouring. R. solanacearum biovar 2 inoculum (1.5 x 103 cfu.ml-1) was placed onto this medium and also as a control onto normal TZC medium where the leaching was replaced with sterile water. Technique C: Culture of R. solanacearum in leaching In this evaluation, 9 ml leaching was added to a sterile test tube with 1 ml R. solanacearum biovar 2 inoculum (2.5 x 106 cfu.ml-1), giving a final bacterial population of 2.5 x 105 cfu.ml-1. Sterile water and potato leaching were used in control tubes. Five repetitions of each treatment were incubated on a shaker for five days. A dilution series was prepared and plated onto TZC medium, and the number of colonies formed was compared. This technique takes into account the microbial activity associated with the weed.

6.2.3.2

Statistical analysis

The General Linear Models Procedure of the Statistical Analysis System (SAS®) 3 was applied to determine the effect of weed extracts on the growth of Ralstonia solanacearum. Tukey’s Studentized Range at the 5 % significance level was used to identify significant differences.

6.2.3.3.

Results and Discussion

The results obtained with the disc technique did not demonstrate any inhibitory interaction between the sterile weed extract or leaching and the colony growth of R. solanacearum on TZC medium. Similarly, inhibition was also not observed with the Bacterial wilt disease on potato: The South African experience

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poisoned medium technique. It is possible that the concentration of the extract or leaching was not sufficient to demonstrate inhibition. Neither the disc technique nor the poisoned medium technique permits the investigation of volatile substances. The apparent suppression of R. solanacearum biovar 2 observed in the inoculated leaching of E. curvula, S. africanus, T. dubius and H. radicata could probably be partly ascribed to the growth of other bacteria. This is highlighted by the fact that there was a substantial reduction in R. solanacearum colonies observed on TZC in the inoculated potato leaching. Elphinstone (1996) mentioned that cereal and grass crops are often recommended in crop rotation programmes, as they inhibit the development of weeds and allow the use of selective herbicides, along with the fact that natural bacterial populations in cereal rhizospheres are often antagonistic to bacterial wilt. Soil fumigation for the control of bacterial wilt is not recommended, since this practice does not affect R. solanacearum in the deeper soil layers, but does affect antagonistic populations in the upper layers (Elphinstone, 1996). Preliminary results obtained by Arthy and Akiew (1999) indicate that rotation with Rhodes grass (Chloris gayana) may be effective in reducing the incidence of wilt. Stander (2001) recommended further studies to determine whether high levels of antagonistic bacteria are present in the rhizosphere of the three grasses or the two weeds that occurred abundantly in the weed-fallowed plots. If so, it would partly explain the suppression observed in these plots. Further investigations should be conducted to determine whether chemical compounds present in the grasses or weeds could be directly involved in the suppression of bacterial wilt.

6.3

SUPPRESSION OF R. SOLANACEARUM IN THE RHIZOSPHERE BY MAIZE

6.3.1

Introduction

Several researchers have reported that rotation with crops immune to bacterial wilt could assist in disease control. Impressive results have already been achieved with maize (Zea mays L.), although the level of disease control achieved with maize rotations tends to vary. Stander (2001) conducted several experiments regarding the role of maize cultivation in terms of Ralstonia populations. This report concentrates on the findings of these investigations and the discussion thereof.

6.3.2. Effect of maize on Ralstonia populations – pot trial Ralstonia populations in soil from maize planted in pots were compared to those from fallow pots and from pots planted to potatoes. Pathogen populations declined faster in soil from maize pots than in fallow soil (Figure 6.3.2.1). A gradual decline was also observed in soil in which potato was grown. This data coincides with the findings of the field study and also supports the findings of Elphinstone and Aley (1993). The overall survival of R. solanacearum was, however, found to be poor in the pot trial. After six weeks no positive isolations could be obtained from the fallow and maize soil, while after eight weeks none could be obtained from the potato soil. This was surprising, since potatoes were still actively growing in those pots, although strong saprophytic growth could have hampered the detection of the pathogen. No Ralstonia could be isolated from the stems or roots of maize plants, which suggests that maize is not a host of the local biovar 2 strain.

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1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 2

4

6

8

10

Weeks after inoculation Potato

Fallow

Maize

Figure 6.3.2.1. Logarithmic presentation of R. solanacearum (biovar 2) populations in soil (cfu.g-1) from pots in which maize or potato was grown or which were kept fallow (Stander, 2001)

6.3.3

Effect of maize on Ralstonia populations in a soilless system

During the second phase, three consecutive trials were conducted in which maize was cultured in vitro in a nutrient solution. Varying results were obtained regarding the ability of maize to serve as a carrier or host of R. solanacearum. In the first two trials, no bacterial wilt could be isolated from the maize plants. R. solanacearum populations in the nutrient solution containing maize plants deteriorated rapidly to an undetectable level, whereas populations in the control flasks remained stable. In the first trial, an antagonist that was later tentatively identified as Chromobacterium violaceum was consistently found in the solution, as well as in the maize plants (Figure 6.3.3.1).

Fig 6.3.3.1. Ralstonia solanacearum biovar 2 cross-streaked against an antagonist tentatively identified as Chromobacterium violaceum, isolated from a maize plant on TZC medium (Kelman, 1953; Mienie, 1998)

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Further preliminary studies indicated that this bacterium is relatively successful in suppressing Ralstonia populations. Although this bacterium was not detected in the second trial, several other saprophytic bacteria were observed on the medium and some of these appeared mildly antagonistic. Once again, no R. solanacearum was isolated from maize plants. During the third in vitro trial, high Ralstonia populations were observed in some of the maize plants and in the corresponding nutrient solution. In plants not infected, either C. violaceum or other bacterial populations were observed. These results indicate that microbial populations present in the maize plants could play a role in the susceptibility of maize. Antagonistic bacteria associated with some maize plants or the maize rhizosphere could be partly responsible for the suppression of wilt, as reported. Elphinstone and Aley (1993) found high populations of Pseudomonas cepacia in maize roots that appear antagonistic to Ralstonia, reasoning that the presence of P. cepacia or similar bacteria may assist in explaining why Autrique and Potts (1987) reported a reduction in wilt disease when potatoes were intercropped with maize. Stander (2001) recommended the further investigation of the hypothesis that certain bacteria (antagonistic and otherwise) present in the soil could be transmitted to seed and that some of these could have a protective effect on the developing maize plant.

6.3.4

Summary

Bacterial wilt caused by Ralstonia solanacearum has plagued the South African potato industry since 1914. Little information is available on the longevity of R. solanacearum in soil under South African conditions and the way in which this is influenced by cultural practices. There is limited information available regarding local weeds that can potentially serve as alternative hosts. Potatoes were planted over the entire trial field ten years after the soil was initially infested. Samples consisting of 100 tubers were drawn from each plot and submitted for analysis. Ralstonia solanacearum could not be detected in any of the samples. This may imply that the organism had been totally eliminated during the dry and unfavourable conditions prevailing since 2001. The possibility of some Ralstonia surviving in the deeper soil layers can, however, not be ruled out, as the test crop was grown under dry conditions and with a shallow rooting system being observed (Hammes, 2013). Preliminary in vitro studies conducted to determine the suppressiveness of certain weeds/grasses found that microbial activity associated with some weeds could be involved in the suppression of the wilt organism. Further studies are required, however. The effect of maize on R. solanacearum populations was evaluated in a pot trial, as well as in hydroponic culture. Results indicate that microbial populations present in the maize plant could play a role in the susceptibility of maize to bacterial wilt infection. Antagonistic bacteria associated with some maize plants or with the maize rhizosphere could be partly responsible for the suppression of wilt.

6.4

SUPPRESSION OF R. SOLANACEARUM IN THE RHIZOSPHERE BY HERBAL PLANT MATERIAL

6.4.1

Introduction

Interest in the field of biological control as an alternative method for the control of weeds, insects and plant pathogens has grown due to the global move towards more natural and environmentally friendly farming systems (Lampkin, 1990; Lydon and Duke, 1989). According to Kirkegraad and Sarwar (1998), bio-fumigation refers to the suppression of soil-borne pests and pathogens by biocidal compounds released into the soil when certain plant material is broken down. During the decomposition of plant residues, substances such as glucosinolates can be hydrolysed to release a range of volatile compounds known as isothiocyanates, as well as other secondary compounds such as oxazolidinethiones, nitriles and thiocyanates (Akiew et al., 1996; Kirkegraad and Sarwar, 1998). The type of isothiocyanate released is specific to the type of glucosinolate present in the tissue. Isothiocyanates fall into the same class of chemical compounds as those produced by the decomposition of metham sodium (vapam), a commercial soil fumigant (Akiew et al., 1996). Since some of the substances released by bio-fumigation display broad biocidal activity, including insecticidal, nematicidal, fungicidal, antibiotic end

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phytotoxic effects, this practice can be used as part of an integrated control programme to suppress soil-borne pathogens (Kirkegraad and Sarwar, 1998).

6.4.2

Host determination

Seed of thirteen herbal plant species was germinated in seed trays and transplanted into two-litre pots containing 2 kg of sterile clay-bark mixture. Sterile vermiculite was soaked in inocula of either isolate 111(biovar 2) or isolate 117 (biovar 3) at 1 x 106 cfu ml-1 and then added to the pot. Each plant was tested for the presence of the pathogen according to the technique described by Swanepoel (1992). None of the 13 herbal plant species selected developed any typical wilt symptoms, but the pathogen was re-isolated from several species (Table 6.4.2.1). Table 6.4.2.1

Host determination of herbal plant species (Van Broekhuizen, 2002) Herbal species

Host determination biovar 2

biovar 3

Ocimum basilicum L. (basil)

-

-

Borago officinalis L. (borage)

-

-

Apium graveolens L. (celery)

-

-

Matricaria recutita L. (chamomile)

-

-

Foeniculum vulgare Mill. (fennel)

-

-

Melissa officinalis L. (lemon balm)

-

-

Anethum graveolens Linn. (dill)

-

-

Allium tuberosum Rottler (chive)

-

+

Coriandrum sativum L. (coriander)

-

+

Origanum majorana L. (marjoram)

+

+

Brassica alba l. (mustard)

-

+

Tropaeolum majus L. (nasturtium)

+

+

Pertoselinum crispum L. (parsley)

+

+

Biovar 3 was able to infect more of the 13 herbal species tested than biovar 2. Marjoram, nasturtium and parsley belong to the Labiatae, Tropaeolaceae and Umbelliferae families respectively. These results did not coincide with the previous finding that biovar 2 (race 3) would only infect solanaceous plants (Buddenhagen et al., 1962), and further investigation is therefore required.

6.4.3

Suppression of R. solanacearum with herbal plants

6.4.3.1. In vitro tests with crude herbal extracts Seven of the 13 herbal species were selected for in vitro testing of crude extracts based on their non-host characteristics. Extracts were prepared according to the method described by Terblanche and De Villiers (1998) using isolate 111 (biovar 2).

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Although some degree of suppression was noted around wells inoculated with non-filtered crude extracts, no clear inhibition zones developed. The limited suppression that was observed was likely due to endophytes developing in and around the wells. 6.4.3.2. Soil suppression trials Trial 1 Seed from the non-host herbal species was sown and raised as described in paragraph 4.3.2, and after four weeks was transplanted into 2-kg pots containing 2 kg of sterile clay-bark mixture. After eight weeks, plants from each species were removed, rinsed and cut into pieces of 1 to 2 cm. Potato seed tubers were planted into pots of the same size, in a 2:1 claybark mixture. For the first trial, vermiculite was soaked in isolate 111 (biovar 2) inoculum (refer to 6.4.2) and added together with 20 g herbal material to each pot and thoroughly mixed. For part 2, the vermiculite was omitted and only the herbal material was added. Controls were included where the herbal material was omitted. Pots were kept between temperatures of 10 to 12°C minimum and 20 to 22°C maximum. After six weeks, plants were removed and tissue isolations made on TZC medium from the bottom 10 cm of stems, in order to determine the presence of R. solanacearum. No wilt symptoms developed during the six weeks after emergence. All tissue isolations tested negative for the presence of R. solanacearum, and no internal symptoms could be observed within the vascular rings of cut tubers. To regain any possible loss of virulence, R. solanacearum isolate 111 was inoculated into potato plants and re-isolated according to the technique described by Kelman and Sequeira (1965). The potato stems were inoculated by piercing with a sterile needle. When the first symptoms of wilt appeared after 12 days, the plants were removed and tissue isolations prepared, after which the pathogen was re-isolated on TZC medium to be used in the second soil inoculation trial. Trial 2 Instead of soaked vermiculite, 50 ml isolate 111(1 x 106 cfu ml-1) was added to each pot after the tubers had been planted. Isolate 111 was again added to each pot after one week. Glasshouse temperatures ranged from 18 to 20°C minimum and 30 to 35°C maximum. The same procedure as described in Trial 1 was followed. No wilt symptoms developed and all tissue isolations tested negative for the presence of R. solanacearum. There could be several reasons why all tissue isolations tested negative for the presence of R. solanacearum, including low inoculum concentration, low soil temperatures, the dilution factor created by the soil, the soil in pots being too dry, or the loss of virulence. Van Broekhuizen discussed the hypothesis that R. solanacearum normally exists as the PC-type in soil (Denny et al., 1994), with suitable conditions enabling the pathogen to switch to its wild type, causing infection and inducing symptoms in the host. According to Schell (1996), phenotypic conversion occurs when some endogenous inducer, 3-OH PAME, exceeds a critical concentration. Van Broekhuizen (2002) recommended that bio-fumigation should be re-evaluated under different glasshouse conditions to determine whether herbal plant material could be used to suppress R. solanacearum in soil.

6.5 EVALUATION OF BACTERIAL ANTAGONISTS AGAINST BACTERIAL WILT AND THEIR POTENTIAL USE AS BIO-CONTROL AGENTS 6.5.1. Introduction Biological control strategies may be used as alternatives in the management of bacterial wilt, or may be integrated with other practices for practical field management (Shekhawat et al., 1992). The study conducted by Mienie (1998) had the following

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aims: 1) To evaluate the effectiveness of bacterial antagonists against R. solanacearum biovar 2 in vitro; and 2) to evaluate immobilisation matrixes as potential carriers for use in introducing antagonists into the soil.

6.5.2

Materials and methods

6.5.2.1. Evaluation of the effectiveness of bacterial antagonists against R. solanacearum biovar 2 in vitro. Maize was sown and grown to maturity in a greenhouse. The plants were harvested and the roots removed, after which the roots were surface sterilised in 5% NaOCL for five minutes and then rinsed in sterile water. The roots were then blended with sterile water, and serial dilutions were made and spread-plated onto nutrient agar (NA). The plates were incubated at 30°C for five days, and several bacterial colonies were picked from the plates and purified by streaking. The purified isolates were tested in vitro against R. solanacearum biovar 2 on King’s B medium, according to the crossstreaking method as described by Hartman et al. (1990). Identification of the isolates was done by means of biochemical tests and the Biolog MicroplateTM Identification System. 6.5.2.2 Evaluation of immobilisation matrixes in a greenhouse Two matrixes were used to introduce the antagonists to the potting soil, namely alginate beads and peat moss mix. 6.5.2.3. Alginate bead inoculum Each antagonist was streaked onto NA plates and incubated at 30°C for 48 hours. The cultured antagonists were scraped off the surface of the plates, and the cells were then weighed and mixed with 2% sodium alginate (BDH Limited Poole England) at a ratio of 1:10 (Bashan, 1986). Alginate beads were prepared for each antagonist and divided into equal portions (approximately 20 g) for each of the 20 pots, wit the beads being mixed into the potting soil. This procedure was followed for each of the three antagonists. Five non-inoculated control pots (not containing alginate bead inocula) were included. 6.5.2.4. Peat mix inoculum The growth of each antagonist was scraped off the NA plates and mixed with 400 ml sterile water. Viable counts of the suspensions indicated in excess of 109 viable cells ml-1. For each pot, 40 g sterile peat was mixed with 20 ml of the antagonist suspension and mixed into the potting soil. This was done for all three antagonist suspensions. Five non-inoculated control pots (not containing peat mix inocula) were included. Inoculation of plants with R. solanacearum: Twenty tubers per antagonist and five tubers per control were planted in separate plastic pots. After reaching a height of 150 cm, the plants were inoculated by pouring 10 ml R. solanacearum suspension (106 cells ml-1) onto the surface of the soil near the stem base. The plants were evaluated for wilt symptoms at seven-day intervals and scored according to the following disease scale: 0 = no wilting; 1 = wilting of one leaf; 2 = wilting of up to 50% of leaves; 3 = wilting of all leaves;and 4 = plant dead. The results were transformed into percentage values using the formula of Kremer and Unterstenhöfer (1967). Data analysis was done by means of Genstat 5 Release 2.2 Copyright 1990, Lawes Agricultural Trust (Rothamstead Experimental Station).

6.5.3

Results and discussion

Thirty-six bacterial isolates were obtained from the maize roots. Three of the isolates showed promising antagonism, with the antagonists being identified as Sporosarcina ureae, Stenotrophomonas maltophilia and Pseudomonas resinovorans. The in vitro inhibition of R. solanacearum biovar 2 by the three antagonists is shown in Figure 6.5.3.1.

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a

b

c

Fig 6.5.3.1 In vitro inhibition of Ralstonia solanacearum biovar 2 by a) Sporosarcina ureae, b) Stenotrophomonas maltophilia, and c) Pseudomonas resinovorans (Mienie, 1998)

Figure 6.5.3.2 and Fig 6.5.3.3 show the antagonistic effects of three antagonists immobilised in alginate beads and peat mix respectively against the pathogen, in vivo in greenhouse experiments. In both cases, the antagonist Pseudomonas resinovorans exhibited greater antagonism against the pathogen. P. resinovorans resulted in about 50 % wilt 35 days after inoculation, with no more plants having wilted until 49 days after inoculation. P. resinovorans seemed to be more effective in reducing the percentage wilt. The potential of immobilisation matrixes for introducing antagonists against bacterial wilt was indicated by these experiments.

Fig 6.5.3.2. Progression of bacterial wilt of potatoes inoculated with R. solanacearum biovar 2, both without and with three different antagonists in alginate beads (Mienie, 1998)

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Fig 6.5.3.3

Progression of bacterial wilt of potatoes inoculated with R. solanacearum biovar 2, both without and with three different antagonists in peat mix (Mienie, 1998)

In vitro evaluation of P. resinovorans showed total inhibition of R. solanacearum, which corresponds with the greenhouse trial where the same antagonist was again found to be most promising in terms of reducing wilt. Mienie (1998) speculated that since P. resinovorans was isolated from an endorhizosphere environment, it was to be expected that a rapid adaptation phase and immediate stability of the bacterial cells would have occurred during the greenhouse trial. Mienie (1998) recommended further research to evaluate biological control under field conditions, but keeping in mind that biocontrol clearly has the potential to be included as part of an integrated management strategy against bacterial wilt in potatoes in future.

6.6 EVALUATION OF THE EFFECTIVENESS OF COMMERCIALLY AVAILABLE DISINFECTANTS AGAINST RALSTONIA SOLANACEARUM BIOVAR 2 6.6.1. Introduction Disinfection during the handling of seed potatoes is an important aspect of bacterial wilt management. Several approaches can be taken to achieve the desired level of disinfection, including the use of hot water, steam, ultraviolet irradiation and chemical antimicrobial agents. Before any seed lot is handled, all containers, tools and implements (including cutters, planters, graders and diggers) should be thoroughly washed with detergent applied with a high-pressure washer, then rinsed in clean water and disinfected with a suitable disinfectant (Secor and Gudmestad, 1993). The goal in terms of bacterial wilt should be to exclude the pathogen from all phases of potato production. A strict sanitation programme to eliminate R. solanacearum from all production surfaces must be maintained (Secor and Gudmestad, 1993). The objective of this investigation was to evaluate commercially available disinfectants for possible use as part of an integrated management strategy against bacterial wilt.

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6.6.2. Materials and methods R. solanacearum biovar 2 was used, with the culture being streaked onto nutrient agar (NA) plates and incubated at 30°C for 24 hours. Bacterial growth was harvested from the plates and suspended in sterile distilled water. An inoculum suspension was prepared, corresponding to a viable count of 107 cfu ml-1. Each disinfectant used (Table 6.6.3.1) was diluted with sterile distilled water (pH 7.0) in sterile Erlenmeyer flasks to final concentrations of 0.2, 0.5, 1.0 and 2.0%. Water was used as control. A volume of 100 ml of each disinfectant concentration was inoculated with 10 ml R. solanacearum suspension and thoroughly mixed. After 30 seconds and then after 2, 5, 10, 30 and 60 minutes, 1 ml of the mixture was transferred to 9 ml sterile distilled water. Serial dilutions were made and plated onto TZC agar plates. After incubation for 4 days at 30°C, plates were examined for typical R. solanacearum colonies and counted. The viable counts were transformed to log10values and plotted against exposure time (minutes). Data analysis was done by means of Genstat 5 Release 2.2 Copyright 1990 Lawes Agricultural Trust (Rothamstead Experimental Station).

6.6.3

Results and discussion

The results indicated that the disinfectants differed in terms of their effectiveness against R. solanacearum biovar 2. Chlorox (15% NaOCL), Jeyes Fluid and Sporekill showed 100% control of the pathogen, after an exposure time of 0.5 minutes at the lowest concentration (0.2 %) and also the highest concentration (2%). The effectiveness of Terminator, Busan 1046 and HTH varied: Treatment with Terminator (0.2%, 0.5% and 1%) resulted in viable cell counts of 1 x 103, 4, 2 x 102 and 1 x 101 respectively, after five minutes of exposure. Exposure for between 10 and 60 minutes proved 100 % effective at all concentrations of Terminator. After two minutes of exposure, Busan 1046 provided 100% control against R. solanacearum biovar 2 cells at all concentrations. Treatment with Busan 1046 (0.2% and 0.5%) resulted in the survival of 2.8 x 10 5 and 1 x 104 cells respectively, after two minutes of exposure. In the case of treatment with HTH (0.2%), 2.3 x 10 2 cells survived after 0.5 minutes of exposure, but no cells were able to survive any of the treatments (0.2%, 0.5%, 1% and 2%) after an exposure time of between two and 60 minutes. It should, however, be kept in mind that soil adhering to the tuber surface and organic matter could have a detrimental effect on the sanitation process. Longer exposure times are therefore recommended, and it should be emphasised that waste water could still contain viable pathogen cells, making it important to use a suitable disinfectant concomitantly with the detergent. At the time of this study, no registered chemicals were available for use in sanitation practices against bacterial wilt. Chlorox, Jeyes Fluid, HTH and Sporekill were identified as bactericidal agents that could be recommended for use in sanitation.

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Table 6.6.3.1

Technical information regarding the disinfectants included in this study, as supplied by the manufacturers (Mienie, 1998) Disinfectant (Trade name)

Characteristics Busan 1046

Jeyes Fluid

HTH

Sporekill

Terminator

Active ingredient

Metham sodium

Carbolic acid

Calcium hypochlorite

Synergised quaternary compound

Dimethyldidecylammoniumc hloride (quaternary ammonium compound)

Incompatibility

Oxidising agents

None

None

None

None

Irritations

Eyes and skin

Eyes and skin

Eyes and skin

Eyes and skin

Eyes and skin

Stability

Stable

Stable

Stable

Stable

Stable

Teflon, polyethylene, nylon, silicone, rubber, PVC, stainless steel

No Information

No information

No information

No information

Toxic

Toxic

Toxic

Non-toxic at recommended rates

Low mammal toxicity

None

None

None

None

Potassium hydroxide, tarry acids and creosote Adcock-Ingram Pharmaceuticals

Contact with acids liberates toxic gas Olin Pool Products

None

None

Hygrotech Seed

Zeneca Agrochemicals

Compatibility

Toxicity

Corrosive action

Hazardous degradation products Supplier/ manufacturer

Avoid contact of concentrated product with aluminium Hydrochloric acid and methyl chloride Buckman Laboratories

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and ecological aspects. Reports of the second international bacterial wilt symposium, Gosier, Guadeloupe, France, 22-27 June 1997, pp. 337342. SOGUILON, C.E., MAGNAYE, L.V. and NATUREL, M.P. 1994. Bugtok disease of cooking bananas in the Philippines. Bacterial Newsletter 10: 5-7. SOHI, H.S., RAO, M.V.B., RAWAL, R.D. and KISHUN, R. 1981. Effect of crop rotations on bacterial wilt of tomato and eggplant. Indian Journal of Agricultural Science. 8: 572-573. STANIER, R.Y., PALLERONI, N.J. and DOUDOROFF, M., 1966. The aerobic pseudomonads: A taxonomic study. Journal Gen. Microbiology 43: 179. STEFANOVA, M. 1998. Current situation of bacterial wilt (Ralstonia solanacearum Smith) in Cuba. . In: Prior, P.H., Allen, C. and Elphinstone, J. (eds.). Bacterial wilt disease: Molecular and ecological aspects. Reports of the second international bacterial wilt symposium, Gosier, Guadeloupe, France, 22-27 June 1997, pp. 364-368. STEVENS, F.L. and SACKETT, W.G. 1903. The Granville tobacco wilt, a preliminary bulletin. North Carolina Agricultural Experimental Station Bulletin 188: 77-96. STEYN, P.J. 1999. Die herkoms en groeistadiums van die aartappelplant. Pages 2-6 in: Steyn, p.j. (ed.). Handleiding vir aartappelverbouing in Suid-Afrika. Landbounavorsingsraad, Roodeplaat. STRIDER, D.L. JONES, R.K. and HAYGOOD, R.A. 1981. Southern bacterial wilt on geranium caused by Pseudomonas solanacearum. Plant Disease 65: 52-53. SUN, S.K. and HUANG, J.W. 1985. Formulated soil amendment for controlling Fusarium wilt and other soil-borne diseases. Plant Disease 69:917-920. SUN, S.K. and HUANG, J.W. 1985. Formulated soil amendment for controlling Fusarium wilt and other soil-borne diseases. Plant Disease 69:917-920. SUNAINA, V., KISHORE, V. and SHEKHAWAT, G.S. 1989. Latent survival of Pseudomonas solanacearum in potato tubers and weeds. Journal of Plant Disease and Protection 96: 361-364. SWANEPOEL, A., (1988). Characteristics of South African strains of Pseudomonas solanacearum. Plant Disease 72: 403-405. SWANEPOEL, A.E. 1990. The effect of temperature on the development of wilting and on progeny tuber infection of potatoes inoculated with South African strains of biovar 2 and 3 of Pseudomonas solanacearum. Potato Research 33: 287-290. SWANEPOEL, A.E. 1992. Survival of South African strains of biovar 2 and biovar 3 of Pseudomonas solanacearum in the roots and stems of weeds. Potato Research 35 35(3): 329-332. SWANEPOEL, A.E. and BOSCH, S.E. 1988. Beheer verwelksiekte so. SA Groenteboer Maart/April 1988: 14-15. SWANEPOEL, A.E. and THERON, D.J. 1999. Control measures for bacterial wilt, caused by Ralstonia solanacearum, as applied by the South African potato certification scheme. In: Leone, A., Foti, S., Ranalli, P., Sonnino, A., Vecchio, V., Zoina, A., Monti, L. and Frusciante, L. (eds) 14th Triennial Conference of the European Association for Potato Research. Abstracts of Conference Papers, Posters and Demonstrations, Sorrento, Italy, 2-7 May 1999, pp 241-242. SWANEPOEL, A.E. and YOUNG, B.W. 1988. Characteristics of South African strains of Pseudomonas solanacearum. Plant Disease 72: 403405. TANAKA, H. 1985. Induced resistance in tobacco against bacterial wilt and its possible mechanism. Bulletin of the Utsuhomiya Tobacco Experimental Station 21: 1-66. TANAKA, H., NEGISHI, H. and MAEDA, H. 1990. Control of tobacco bacterial wilt by an avirulent strain of Pseudomonas solanacearum M4S and its bacteriophage. Annals of the Phytopathological Society of Japan 56: 243-246. TANAKA, Y. and NODA, N. 1973. A study of factors governing the survival of tobacco wilt disease bacteria. Bulletin of Okayama Tobacco Experimental Station 32: 81-93.

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TANAKA, Y. 1976. Factors affecting survival of Pseudomonas solanacearum. In: Proceedings of the first International Planning Conference and Workshop on the Ecology and Control of Bacterial wilt caused by Pseudomonas solanacearum, North Carolina State University, Raleigh, pp. 122. TERBLANCHE, J. and DE VILLIERS, D.A. 1998. The suppression of Ralstonia solanacearum by marigolds. In: Prior, P.H., Allen, C. and Elphinstone, J. (eds.). Bacterial wilt disease: Molecular and ecological aspects. Reports of the second international bacterial wilt symposium, Gosier, Guadeloupe, France, 22-27 June 1997, pp. 325-331. THERON, D.J., 1999. Fusarium dry rot of potatoes: etiology, epidemiology, toxicity and control. PhD thesis, University of the Orange Free State, Bloemfontein. THORNTON, R.E., STEVENS, R.G. and HAMMOND, M.W. 1993. Selecting the site and preparing it for planting. In: Potato Health Management (R.C., ed.). pp. 11-18. THURSTON, H.D. 1963. Bacterial wilt of potatoes in Colombia. American Potato Journal 40: 381-391. TITATARN, V. 1986. Bacterial wilt in Thailand. In: Persley, G.J. (ed.). Bacterial Wilt Disease in Asia and the South Pacific. Proceedings of an international workshop, PCARRD, Los Baños, Philippines, 8-10 October 1985, pp. 65-67. TOMLINSON, D.L. and GUNTHER, M.T. 1986. Bacterial wilt in Papua New Guinea. In: Persley, G.J. (ed.). Bacterial Wilt Disease in Asia and the South Pacific. Proceedings of an international workshop, PCARRD, Los Baños, Philippines, 8-10 October 1985, pp. 35-39. TOMLINSON, D.L. 1985. A preliminary study of the distribution of biovars of Pseudomonas solanacearum in Papua New Guinea. Australian Plant Pathology Vol 14(1): 8-10. TRIGALET, A. and URQUHART, L. 1998. Chair’s perspectives on biological control and epidemiology. In: Prior, P.H., Allen, C. and Elphinstone, J. (eds.). Bacterial wilt disease: Molecular and ecological aspects. Reports of the second international bacterial wilt symposium, Gosier, Guadeloupe, France, 22-27 June 1997, pp. 323. TRIGALET, A. and TRIGALET-DEMERY, D.!990. Use of avirulent mutants of Pseudomonas solanacearum for the biological control of bacterial wilt of tomato plants. Physiological and Molecular Plant Pathology 36:27-38. TRIGALET, A., FREY, P. and TRIGALET-DEMERY, D. 1994. Biological control of bacterial wilt caused by Pseudomonas solanacearum. State of the art and understanding. In: Bacterial Wilt: The disease and its causative agent, Pseudomonas solanacearum (A.C. Hayward and G.L. Hartman, eds.). CAB International. pp. 225-233. TRIGALET, A., FREY, P. and TRIGALET-DEMERY, D.1994. Biological control of bacterial wilt caused by Pseudomonas solanacearum. State of the art and understanding. In: Bacterial Wilt: The disease and its causative agent, Pseudomonas solanacearum (A.C. Hayward and G.L. Hartman, eds.). CAB International. pp. 225-233. TRIGALET, A., TRIGALET-DEMERY, D. and PRIOR, P. 1998. Elements of biocontrol of tomato bacterial wilt. In: Prior, P.H., Allen, C. and Elphinstone, J. (eds.). Bacterial wilt disease: Molecular and ecological aspects. Reports of the second international bacterial wilt symposium, Gosier, Guadeloupe, France, 22-27 June 1997, pp.332-342. TSUCHIYA, K. and HORITA, M. 1998. Genetic diversity of Ralstonia solanacearum in Japan. In: Prior, P.H., Allen, C. and Elphinstone, J. (eds.). Bacterial wilt disease: Molecular and ecological aspects. Reports of the second international bacterial wilt symposium, Gosier, Guadeloupe, France, 22-27 June 1997, pp. 61-73. TUNG, P.X. and SCHMIEDICHE, P. 1995. Breeding potato for resistance to bacterial wilt (Pseudomonas solanacearum): Looking for stable resistance? In: Hardy, B. and French, E.R. (eds.). Integrated management of bacterial wilt. Proceedings of an international workshop held in New Delhi, India, October 11-16, 1993. Lima, Peru, International Potato Centre. pp. 173-178. TUNG, P.X., HERMSEN, J.D.T.H., VANDER ZAAG, P. and SCHMIEDICHE, P. 1992a. Effects of resistance genes, heat tolerance genes and cytoplasms on expression of resistance to Pseudomonas solanacearum (E.F. Smith) in potato. Euphytica 60: 127-138. TUNG, P.X., HERMSEN, J.D.T.H., VANDER ZAAG, P. and SCHMIEDICHE, P. 1992b. Effects of heat tolerance on expression of resistance to Pseudomonas solanacearum E.F. Smith in potato. Potato Research 35: 321-328. TUNG, P.X., RASCO, E.T.J.R., VANDER ZAAG, P. and SCHMIEDICHE, P. 1990. Resistance to Pseudomonas solanacearum in the potato: II Aspects of host-pathogen-environment interaction. Euphytica 45: 211-215.

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TUSIIMI, G., ADIPALA, E. OPIO, F. and BHAGSARI, A.S. 1998. Weeds as latent hosts of Ralstonia solanacearum in Highland Uganda: implications to development of an integrated control package for bacterial wilt. In: Prior, P.H., Allen, C. and Elphinstone, J. (eds.). Bacterial wilt disease: Molecular and ecological aspects. Reports of the second international bacterial wilt symposium, Gosier, Guadeloupe, France, 22-27 June 1997, pp. 413-419. URQUHART, L., MIENIE, N.J.J. and STEYN, P.L., (1998). The effect of temperature, storage period and inoculum concentration on symptom development and survival of R. solanacearum in inoculated tubers. Reports of the second bacterial wilt symposium In Bacterial wilt disease, Ecological and molecular aspects. Eds Prior PH, Allen C., Elphinsone J. G., (1998). Springer. VALDEZ, R.B. 1986. Bacterial wilt in the Philippines. In: Persley, G.J. (ed.). Bacterial Wilt Disease in Asia and the South Pacific. Proceedings of an international workshop, PCARRD, Los Baños, Philippines, 8-10 October 1985, pp. 49-56. VAN BEUNINGEN, A.R., DERKS, J.H.J., GORKINK, R, RONDA, B.H.N.A.M. and JANSE J.D. 1998. Plantenziektenkundige Dienst Wageningen, Annual Report Diagnostic Centre 1998: 44-45.

Verslagen en mededelingen

VAN ELSAS, J.D., KASTELEIN, P., VAN BEKKUM, P., VAN DER WOLF, J.M., DE VRIES, P.M. and VAN OVERBEEK, L.S. (2000). Survival of Ralstonia solanacearum Biovar 2, the causative agent of Potato Brown rot, in field and microcosm soils in temperate climates. Phytopathology 90 (12) : 1358-1366. VAN DER PLANCK, J.E., 1957/58. Annual Report of the Secretary for Agriculture for the year ended 30 June 1956, 60 – 62. VANDER ZAAG, P. 1986. Potato production under Pseudomonas solanacearum conditions: sources and management of planting materials. In: Persley, G.J. (ed.). Bacterial Wilt Disease in Asia and the South Pacific. Proceedings of an international workshop, PCARRD, Los Baños, Philippines, 8-10 October 1985, pp. 35-38. VASSE, J., FREY, P. and TRIGALET, A. 1995. Microscopic studies of intercellular infection and protoxylem invasion of tomato roots by Pseudomonas solanaceaum. Molecular Plant-Microbe Interaction 8 (2), 241 – 251. VELUPILLAI, M. 1986. Bacterial wilt in Sri Lanka. In: Persley, G.J. (ed.). Bacterial Wilt Disease in Asia and the South Pacific. Proceedings of an international workshop, PCARRD, Los Baños, Philippines, 8-10 October 1985, pp. 57-64. VERWOERD, L., 1929. A preliminary checklist of diseases of cultivated plants in the winter rainfall area of the Cape Province. Scientific Bulletin of the Department of Agriculture, South Africa. 88: 1 – 128. VISSER, A.F. 1999. Aartappelkultivarkeuse en kultivar eienskappe. Pages 25-61 6 in: Steyn, P.J. (ed.). Handleiding vir aartappelverbouing in Suid-Afrika. Landbounavorsingsraad, Roodeplaat. WAGER, V.A., 1972. Technical Communication of the Department of Agricultural Technical Services, South Africa 100: 1 - 14. WAKIMOTO, S. 1987. Biological control of bacterial wilt of tomato by non-pathogenic strains of Pseudomonas glumae. Korean Journal of Plant Pathology 3: 300-303. WALKER, D. 1992. Potato brown rot Pseudomonas solanacearum. Central Science Laboratory, Plant Disease Notice, No 73, U.K. WALKER, D.R.I. and STEAD, D.E. 1993. Potato brown rot: A new threat to potato production in the EC. 1993 BCPC Monograph No 54: Plant Health and the European Single Market. WALLIS, F.M. and TRUTER, S.J. 1978. Histopathology of tomato plants infected with Pseudomonas solanacearum with emphasis on ultra structure. Physiological Plant Pathology 13: 307-317. WANG, J.S., HOU, X.Y. and HU. B.J. 1983. Studies on the control of bacterial wilt of peanut. Acta Phytophylactica Sinica 10: 79-84. WENNEKER, M. VERDEL, M.S.W. and JANSE, J.D. 1998. Verslagen en mededelingen Plantenziektenkundige Dienst Wageningen, Annual Report Diagnostic Centre 1998: 53. WINSTEAD, N.N. and KELMAN, A., 1952. Inoculation techniques for evaluating resistance to Pseudomonas solanacearum. Phytopathology 42: 628 – 634. WOODS, A.C. 1984. Moko disease: atypical symptoms induced by afluidal variants of Pseudomonas solanacearum in banana plants. Phytopathology 74: 972-976.

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YABUUCHI, E., KOSAKO, Y., OYAIZU, H., YANO, I., HOTTA, H., HASHIMOTO, Y., EZAKI, T. and ARAKAWA, M. 1992. Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas Homology Group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes, 1981) comb. Nov. Microbiology and Immunology 36 (12): 1251-1275. YABUUCHI, E., KOSAKO, Y., YANO, I., HOTTA, H. and NISHIUCHI, Y. 1995. Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: Proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. Nov., Ralstonia solanacearum (Smith 1896) comb. Nov. and Ralstonia eutropha (Davis 1969) comb. Nov. Microbiology and Immunology 39 (11): 897-904. YONGZIANG, Z. JINGYUE, H. and LIYUAN, H. 1993. Effect of infected groundnut seeds on transmission of Pseudomonas solanacearum. Bacterial Wilt Newsletter 9: 9-10. YU, J.Q., KOMADA, H., YOKOYAMA, H. YAMAMOTO, M, TERADA, T. and MATSUI, Y. 1997. Sugi (Cryptomeria japonica D.Don) bark, a potential growth substrate for soilless culture with bioactivity against some soil-borne diseases. Journal of Horticultural Science 72(6): 989-996. ZANDSTRA, H. 1997. Potatoes in the developing world: A thirty-year perspective. In: World Potato Congress. 2-7 march 1997. Durban, South Africa. ZUCKERMAN, L. 1998. The potato. How the humble spud rescued the world. Faber and Faber. Boston.

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OUTPUTS SCIENTIFIC PUBLICATIONS EMANATING FROM THE RESEARCH REPORTED ON BELLSTEDT, D.U., 2008. Enzyme-linked Immunosorbent Assay Detection of Ralstonia solanacearum in Potatoes: The South African Experience. Chapter 5. Methods in Molecular Biology. Plant Pathology 508. SWANEPOEL, A.E. 1990. The effect of temperature on the development of wilting and on progeny tuber infection of potatoes inoculated with South African strains of biovar 2 and 3 of Pseudomonas solanacearum. Potato Research 33: 287-290. SWANEPOEL, A.E. 1992. Survival of South African strains of biovar 2 and biovar 3 of Pseudomonas solanacearum in the roots and stems of weeds. Potato Research 35 35(3): 329-332. SWANEPOEL, A.E. and YOUNG, B.W. 1988. Characteristics of South African strains of Pseudomonas solanacearum. Plant Disease. 72: 403-405. URQUHART, L., MIENIE, N.J.J. and STEYN, P.J., 1998. The effect of temperature, storage period and inoculum concentration on symptom development and survival of Ralstonia solanacearumin inoculated tubers. Bacterial wilt disease. Molecular and ecological aspects. Eds.: P. Prior, C. Allen and J.G. Elphinstone. Publisher: Springer.

PAPERS READ BELLSTEDT, D.U. and VAN DER MERWE, K.J., 1989. The Development of ELISA kits for the Detection of Pseudomonas solanacearum Bacterial Wilt in Potatoes. Proceedings of the First South African Potato Research Symposium, Warmbaths, South Africa, pp 64-69. SWANEPOEL, A.E. and THERON, D.J. 1999. Control measures for bacterial wilt, caused by Ralstonia solanacearum, as applied by the South African potato certification scheme. In: Leone, A., Foti, S., Ranalli, P., Sonnino, A., Vecchio, V., Zoina, A., Monti, L. and Frusciante, L. (eds) 14th Triennial Conference of the European Association for Potato Research. Abstracts of Conference Papers, Posters and Demonstrations, Sorrento, Italy, 2-7 May 1999, pp 241-242. TRIGALET, A. and URQUHART, L. 1998. Chair’s perspectives on biological control and epidemiology. In: Prior, P.H., Allen, C. and Elphinstone, J. (eds.). Bacterial wilt disease: Molecular and ecological aspects. Reports of the second international bacterial wilt symposium, Gosier, Guadeloupe, France, 22-27 June 1997, pp. 323.

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POPULAR PUBLICATIONS CARLSON, R., 2012. Bakteriese verwelk – die mees verwoestende plantsiekte ter wêreld. Chips January/February pp 20-23. HAMMES, P.S., 2006. Die bestuur van bakteriese verwelksiekte. Chips 20 No 2. March/April. HAMMES, P.S.2013. Survival of bacterial wilt organisms in soil. Chips Sept/Oct pp. 40-42 MIENIE, N.J.J. 1997. Integrated control of bacterial wilt. Pages 92-98 in: Potato short course. Potato production in SA with the emphasis on KwaZulu-Natal, Agricultural Research Council, Roodeplaat. MIENIE, N.J.J. 1998. An integrated management approach to the control of bacterial wilt in potatoes. MSc Thesis. University of Pretoria, Pretoria. MIENIE, N.J.J. and THERON, D.J. 1999. Voorkoms en beheer van bakteriese siektes. Pages 109-116 In: Steyn, PJ. (ed.). Handleiding vir aartappelverbouing in Suid-Afrika. Landbounavorsingsraad, Roodeplaat. MIENIE, N.J.J., 2003. Bakteriese verwelk maak weer sy opwagting. Chips 41, May/June pp 41-42. NORTJE, P.F. 2004. Bacterial wilt. 6th Seed Potato Growers Forum. Chips Sept/Oct pp. 27-29. SWANEPOEL, A.E. and BOSCH, S.E. 1988. Beheer verwelksiekte so. SA Groenteboer Maart/April 1988: 14-15.

OTHER SCIENTIFIC PUBLICATIONS ON BACTERIAL WILT BY SOUTH AFRICANS ENGELBRECHT, M.C., 1994. Modification of a semi-selective medium for the isolation and quantification of Pseudomonas solanacearum. Bacterial Wilt Newsletter 10: 3 -5. ENGELBRECHT, M.C. and HATTINGH, M.J. 1989. Numerical analysis of phenotypic features of Pseudomonas solanacearum strains isolated from tobacco and other hosts in South Africa. Plant Disease 73: 893-898. ENGELBRECHT, M.C., VAN WYK, R.J., ENGELBRECHT, S.L.A.G. and JANSE VAN RENSBURG, J.N. 1990. Control of bacterial wilt of tobacco with Chloropicrin in South Africa. Phytophylactica 22:269-271. TERBLANCHE, J. and DE VILLIERS, D.A. 1998. The suppression of Ralstonia solanacearum by marigolds. In: Prior, P.H., Allen, C. and Elphinstone, J. (eds.). Bacterial wilt disease: Molecular and ecological aspects. Reports of the second international bacterial wilt symposium, Gosier, Guadeloupe, France, 22-27 June 1997, pp. 325-331.

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ANNEXURE 1

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ANNEXURE 2

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ANNEXURE 3

Procedure: Testing and confirmation of the presence of Bacterial wilt with ELISA and conventional plating out.

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ANNEXURE 4

Diagrammatic presentation of actions followed after finding a suspicious plant or tuber. PCS = Potato Certification Service PSA = Potatoes South Africa

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ANNEXURE 5 GUIDELINES FOR THE BACTERIAL WILT COMMITTEE GENERAL It is the responsibility of the Bacterial Wilt Committee to continuously evaluate and monitor the risk which bacterial wilt presents to the potato industry. The Committee is also responsible to continuously keep the relevant authorities within the industry, such as the Independent Certification Council for Seed Potatoes (ICCSP), Potatoes South Africa (PSA) and the Department of Agriculture (DA), well informed. MEETINGS 1

2

3 4

The Committee convenes at least twice per annum, preferably before scheduled meetings of the ICCSP, and the National Council of Potatoes South Africa in order to be able to give feedback. Notification of the meetings of the Bacterial Wilt Committee, as well as the agendas, minutes of the previous meeting and supporting documents, must be made available at least fourteen days before the scheduled meeting. The convener of the Committee will act as secretary or the secretary will be designated by the convener. The Committee has the right to co-optation to general meetings.

OBJECTIVE AND PROCEDURE OF COMMITTEE INVESTIGATIONS INTO CASES OF BACTERIAL WILT The objective of the investigation is to determine the risk related to seed potato production on farms where the occurrence of bacterial wilt has been confirmed and to establish the conditions in terms of which seed potato production may be continued on such farm. 1

When can an investigation be conducted? 1.1 Upon the request of the local office of Potato Certification Service. 1.2 Upon the request of the seed grower. 1.3 When the Authority is of the opinion that an investigation is justified for specific reasons. 1.4 When the occurrence of bacterial wilt was already confirmed during the growing stage of the crop or when a latent infection was detected through a laboratory test.

2

Nature of the investigation 2.1 The Committee must consider future plantings where the occurrence of bacterial wilt on the farm was already confirmed on a previous occasion. 2.1.1 The investigation must provide the seed grower with the necessary advice and recommendations with regard to containing the disease. 2.1.2 The committee must acquaint itself with all the local circumstances and conditions during the investigation in order to be able to inform the ICCSP comprehensively and to take responsible decisions 2.1.3 The Committee must advise the seed grower in writing with regard to actions which the seed grower should take before the Authority will be willing to consider continued seed production on the specific farm. Bacterial wilt disease on potato: The South African experience

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2.1.4

By implication the recommendations of the Committee will serve as requirements with which the grower has to comply.

The following aspects, amongst others, should be considered by the Committee: 1 2 3 4 5 6 7 8 9

10

All infested and presumably infested units. Distances between units (clean and unifested). The topography of fields and especially slopes between units. Irrigation and water sources (e.g. dams, streams and rivers). Implements, sorting equipment and stores, as well as sanitary measures. The possible influence on farms in the immediate vicinity. The sharing of implements and renting of fields by different growers. The source of the planting material. The isolation and sanitary measures that have already been taken in respect of the infected unit and infected material in order to prevent the dissemination and re-infestation of the organism. Crop rotation practices (specific crops and rotation).

The PCS must, as convener of the committee, conduct a comprehensive investigation with regard to the source of the seed potatoes and localities where the same seed source has been planted before the visit of the Committee takes place. If the seed potatoes could possibly be regarded as infected in terms of the results of the preinvestigation, PCS must endeavour to prevent the further planting of seed potatoes of the same source until the investigation has been finalised. Investigations of the Committee should be conducted timeously and be given priority. 3

Composition of the Bacterial Wilt Committee -

4

Chairperson – Representative of Department of Agriculture Manager: Research and Development – Potatoes South Africa Chief Executive Officer – PCS (Convener) One independent expert on bacterial wilt The regional certification officer

Procedure of the Investigation 4.1 The seed grower or the regional certification office requests the Authority to launch the investigation. 4.2 The convener (CEO of PCS) requests the regional office of PCs to prepare the case study in writing in cooperation with the grower in order to provide all the relevant information (guidelines 1 – 10 must be consulted). 4.3 The authority determines whether the proposed investigation is justified. 4.4 The convener makes the case study available to the committee members. 4.5 The convener arranges the visit to the grower. 4.6 The committee discusses the case/cases with the regional certification officer in camera before the investigation commences in order to clarify any questions and uncertainties of committee members. 4.7 The case is investigated comprehensively on the farm/farms of the grower. 4.8 The investigation is conducted in private with the seed grower. Bacterial wilt disease on potato: The South African experience

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4.9 The report and recommendations is compiled by the convener and signed by all the committee members and the regional certification officer. The convener submits the report and recommendations to the Executive Committee of the ICCSP. 4.10 The Executive Committee of ICCSP must approve the report and recommendations in writing. 4.11 The convener makes the approved report and recommendations available to the seed grower, regional certification office, members of the Committee and the Department of Agriculture. 4.12 The Department of Agriculture issues a mandate in respect of the field concerned and provides PCS with confirmation of the issuing of the mandate. 4.13 The regional certification office monitors the implementation of the recommendations and regularly reports to the head office of PCS and the regional office of the Department of Agriculture. The convener of the Committee keeps the head office of the Department of Agriculture well-informed regarding the case. 4.14 The report must include the following: - Particulars of the grower, farm name, field number, unit registration number and date of visit - Discussion of the findings - Recommendations and conditions agreed upon - Present plantings and planning of future plantings 5 Expenses of the Committee Responsibility of Potato Certification Service All travel and subsistence costs of: -

The Department representative Convener Regional certification officer

Responsibility of Potatoes South Africa All travel and subsistence costs of: -

Independent expert

-

Manager: Research and Development – Potatoes South Africa

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ANNEXURE 6 Isolation requirements (4) The crop plants specified in Table 1 (Table 1.3.4.1) of the Annexure shall not be cultivated or irrigated together with plants established on a registered unit. Prohibited organisms 7(1) A grower shall notify the authority forthwith of the occurrence or presumed occurrence of prohibited organisms on –

(2)

(a)

a unit;

(b)

land adjacent to a unit;

(c)

land within 50 metres from the area of land specified in paragraph (b); and

(d)

land under his or her control upon which crops are being cultivated or are going to be cultivated.

A unit shall be regarded as a presumably infected unit if – (a)

it is situated on property upon which a prohibited organism occurs or had occurred;

(b)

it is situated on property adjacent to or within 50 metres of a property upon which a prohibited organism occurs or had occurred;

(c)

it is situated on property where livestock occurs and such livestock had access previously to land upon which a prohibited organism occurs or had occurred and the authority regards such livestock as carriers of the prohibited organism;

(d)

Seed potatoes on the unit concerned originated from an origin which is or was infected with a prohibited organism;

(e)

water that flows over the unit originates from land upon which a prohibited organism occurs or had occurred;

(f)

the unit is irrigated with water which flows off land upon which a prohibited organism occurs or had occurred;

(g)

the plants that occur on the unit may be infected with a prohibited organism; or

(h)

equipment that has previously been used for the cultivation of land upon which a prohibited organism occurs or had occurred, is used on the unit concerned, without decontamination thereof.

(a)

If, in the case of an uncovered plot of land, a prohibited organism occurs on the property or an adjacent property, the unit shall be surrounded by an isolation area in which no host plants specified in Table 1 of the Annexure or plants of the spp. Solanum tuberosum L. shall occur.

(b)

the isolation area shall be at least 50 metres wide or as wide as the authority may determine after inspection.

(3)

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(4)

Equipment used in soil that is infected with a prohibited organism shall not be used again for the cultivation of seed potatoes unless it has been effectively decontaminated.

Field samples 18 (2) In the case of a field sample to determine the presence of the organisms that cause bacterial wilt disease – (a)

the sample shall be taken as late as possible during the growing season or after the foliage has died off;

(b)

one sample shall be taken in accordance with the provisions of the protocol from plantings that are cultivated and irrigated together;

(c)

only one tuber per plant shall be taken; and

(d)

unless otherwise determined by the authority, the size of the sample shall, in the case of – (i)

G0 seed potatoes, be 4 tubers per 100 plants or a portion thereof;

(ii)

G1 seed potatoes, be 1 tuber every 10 metres in each row to a maximum of 4 605 tubers over the whole planting;

(iii) G2 to G8 seed potatoes, be 4 605 tubers taken in accordance with the provisions of the protocol in plantings that are cultivated and irrigated together; and (iv) G2 to G8 seed potatoes on units of 1 hectare or smaller, be 1 tuber every 10 metres in each row to a maximum of 4 605 tubers over the whole planting. (3) In the case where the growth stages of seed potatoes on different units that have been cultivated and irrigated together, overlap, certification of such seed potatoes shall only take place once field samples of all the units concerned have been taken and the provisions of section 23 of the Scheme have been complied with.

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Table 1.3.4.1

(TABLE 1). Common host plants of bacterial wilt disease other than Solanum tuberosum L. Scientific name

Common name Crops

Arachis hypogaea L.

Groundnut

Brassica oleraceae L.*

Cabbage crops*

Capsicum L.

Pepper

Citrillus lanatus (Thunb.) Matsum. Et Nakai

Watermelon

Cucubita L.

Pumpkins

Gossypium hirsitum L.

Cotton

Helianthus annuus L.

Sunflower

Lycopersicon lycopersicon L. (=L. Esculentum, Solanum lycopersicon)) Nicotiana tabacum L.

Tomato

Solanum melongena L. var. esculentum Nees

Eggfruit

Tobacco

Weeds Amaranthus deflexus L.

Pigweed

Bidens bipinnata L.

Spanish blackjack

Datura ferox L.

Large thorn apple

Datura stramonium L.

Common thorn apple

Nicandra physalodes L. Gaertn.

Apple of Peru

Nicotiana glauca R. C. Grah.

Wild tobacco

Physalis angulate L.

Wild gooseberry

Ricinus communis L.

Castor-oil plant

Solanum nigrum L.

Black nightshade

Samples originating from units where crops indicated with an * has been planted in rotation with potatoes must be warmed up before testing for bacterial wilt.

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