10_JPP1390 (Sagar)NOColore_321 - SIPaV

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Journal of Plant Pathology (2013), 95 (2), 321-328

Edizioni ETS Pisa, 2013

321

STATUS OF BACTERIAL WILT OF POTATO IN THE MALWA REGION OF MADHYA PRADESH IN INDIA V. Sagar1, A.K. Somani2, R.K. Arora3, S. Sharma1, S.K. Chakrabarti4, S.K. Tiwari5, R. Chaturvedi5 and B.P. Singh1 1Central

Potato Research Institute, Shimla 171 001 (Himachal Pradesh), India Potato Research Station, Gwalior 474 006 (Madhya Pradesh), India 3Central Potato Research Station, Jalandhar 144 003 (Punjab), India 4Central Tuber Crops Research Institute, Thiruvananthapuram (Kerala), India 5PepsiCo India Holdings Pvt Ltd Gurgaon 122 002 (Haryana), India 2Central

SUMMARY

Bacterial wilt caused by Ralstonia solanacearum, is one of the most important diseases of potato in tropical, subtropical and warm temperate regions of the world and is prevalent in many areas in India. The disease was only once reported from Indore and Jabalpur in Madhya Pradesh (India) in 1970, but was never a problem until 2007. From 2007, the disease suddenly emerged as a major constraint for potato cultivation in this region causing heavy crop losses; hence, warranting immediate steps for its management as the region is known to produce potatoes mainly for processing. Therefore, surveys were conducted to know the exact status of bacterial wilt and brown rot of potato and to collect plant and tuber samples from infested fields in several villages of Indore, Ujjain and Devas districts of the Malwa region of Madhya Pradesh during December 2009 and February 2010. The disease incidence ranged from traces to 80% depending upon the seed source. Wherever the farmers used healthy, certified seed, incidence of bacterial wilt and brown rot was negligible or very low. The identity of 21 of 22 bacterial isolates from infected potato plant/tuber samples from Indore, Ujjain and Devas districts of Malwa region was confirmed as R. solanacearum by colony characteristics on Kelman’s TZC agar medium, PCR with R. solanacearum specific primers YII and OLI 1and a pathogenicity test on potato. Using bio-chemical tests, based on the ability to utilize disaccharides and oxidise hexose alcohols, 21 isolates were categorized as biovar 2 which were further identified as phylotype II sequevar 1 as the characteristic race 3 biovar 2 specific 278 bp band was amplified from their DNA using the primer pair 630/631. All the 21 isolates proved to be pathogenic on potato, 18 on tomato, 15 on eggplant but none on pepper. To the best of our knowledge, this is the first report of occurrence of R. solanacearum race 3 biovar 2, phylotype II sequevar 1 in the Madhya Pradesh state of India.

Corresponding author: V. Sagar Fax: +91.177.2624460 E-mail: [email protected]

Key words: Biovar, brown rot, incidence, Ralstonia solanacearum.

INTRODUCTION

Bacterial wilt or brown rot, caused by Ralstonia solanacearum, limits potato production worldwide in Asia, Africa, and Central and South America, where it causes severe crop losses in tropical, subtropical, and warm temperate regions (Hayward, 1994; Elphinstone, 2005). In India, the disease is endemic in west coast from Thiruvananthpuram in Kerala to Khera in Gujarat, Karnataka, western Maharashtra, Madhya Pradesh, eastern plains of Assam, Orissa and West Bengal, and Andaman and Nicobar islands. The disease is also endemic in north-western Kumaon hills, eastern hills of West Bengal, Meghalya, Manipur, Tripura, Mizorum and Arunachal Pradesh and in Nilgiris, Annamalai and Palani hills of Tamil Nadu. However, the disease has not been found in the north-western high hills (excluding the Kumaon hills) and in the north-western and north-central plains (Shekhawat et al., 1992). The disease has become a limiting factor to potato cultivation in several parts of the country as yield losses of 30 to 70% have been reported in these areas (Somani et al., 2010). The disease causes damage at two stages: (i) wilting and killing standing plant (Fig. 1) and (ii) rotting of infected tubers in storage and transit (Fig. 2). Shekhawat et al. (1978) reported race 1 biovar 3 of R. solanacearum to be responsible for bacterial wilt of potato in the plains of India including Indore in Madhya Pradesh. Since then the disease was known to be endemic in Indore and other parts of Madhya Pradesh but was never a serious concern. However in 2007, the disease emerged as a major constraint for potato cultivation in Indore and adjoining districts of Madhya Pradesh causing heavy crop losses, therefore warranting immediate steps for its management as the region is producing potatoes mainly for processing (Somani et al., 2010). The present study was aimed at surveying the potato-growing areas of Indore, Ujjain and Devas districts of the Malwa region of Madhya Pradesh, in order to: (i) establish the exact status of bacterial wilt and brown


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Bacterial wilt of potato in Malwa region


oping the typical irregular mucoid colonies were again streaked onto fresh TZC medium for further purification. Well separated typical wild type R. solanacerarum colonies were further transferred to a medium modified by exclusion of TZC for multiplication of inoculum and final check on purity. Two loopfuls of bacterial culture were then transferred in 2 ml of double distilled sterile water (pH 7.0) in vials and stored at 20±2oC.

Fig. 1. Standing crop of potato infected with bacterial wilt caused by R. solanacearum.

rot; (ii) collect diseased plant and tuber samples for isolation and identification of races/biovars and phylotype of R. solanacearum.

MATERIALS AND METHODS

Survey, bacterial isolates, media and growth conditions. Potato fields in several villages of the above specified areas were surveyed in December 2009 and February 2010 for recording the incidence of bacterial wilt. Stem pieces (5-6 cm long) and tubers of wilted plants were collected from each field, washed thoroughly, air dried and brought to laboratory for further studies. Samples were then surface disinfected by gently rubbing for 25-30 sec with a cotton swab dipped in 70% ethanol, peeled, subsampled and macerated in sterile distilled water. Macerates were streaked on Kelman’s (1954) triphenyltetrazolium chloride (TZC) agar medium: peptone, 10 g; glucose, 2.5 g; casamino acid, 1 g; agar, 18 g; TZC, 50 mg l-1; pH 7.0-7.1. Plates were incubated at 28±2oC for 48 to 72 h. Bacterial colonies devel-

Isolation of genomic DNA. Total genomic DNA was extracted as described by Chen and Kuo (1993). A bacterial colony was used to inoculate 1.5 ml of CPG broth (peptone, 10 g; glucose, 2.5 g; casamino acid, 1 g; distilled water 1 l; pH 7.0-7.1) in 2.0 ml Eppendorf tubes. The cultures were grown at 28±2oC for 48 h with vigorous shaking. Each culture (1.5 ml) was harvested with centrifugation for 3 min at 12,000 rpm. The pellet was re-suspended and lysed in 300 µl of lysis buffer (40 mM Tris-acetate pH 7.8, 20 mM sodium-acetate, 1 mM EDTA, 1% SDS, 20 µg RNase A) by vigorous pipetting and incubated for 30 min at 37oC. To remove most proteins and cell debris, 100 µl of 5 M NaCl solution were added and mixed well, and the viscous mixture was centrifuged at 12,000 rpm for 10 min at 4°C. After transferring the clear supernatant into a new vial, an equal volume of chilled chloroform was added, and the tube was gently inverted at least 50 times when a milky solution was completely formed. Following centrifugation at 12,000 rpm for 3 min, the extracted supernatant was transferred to another vial and the DNA was precipitated with 100% chilled ethanol, washed twice with 70% ethanol, dried in speed-vacuum, and re-dissolved in 50 µl of TE buffer. Confirmation of identity of R. solanacearum isolates.The identity of all the isolates was checked by PCR with R. solanacearum specific primers YII (5’-CCCACTGCTGCCTCCCGTAGGAGT-3’ and OLI1 (5’GGGGGTAGCTTGCTACCCTGCC-3’) (Seal et al., 1993). PCR amplification was done in a final reaction

Fig. 2. External symptoms on tubers (a); vascular browning of tubers (b); rotting of tubers in the field (c).


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volume of 25 µl containing 1X buffer (M/S Applied Biosystems, USA), 100 µM dNTPs, 0.5 pmol of each primer, 1.5 mmol l-1 MgCl2, 1 U Taq DNA Polymerase (AmpliTaq of M/S. Applied Biosystems, USA) and 10 ng of template DNA. PCR amplifications were performed in a thermal cycler (Gen-Amp PCR System 9700 of M/S Applied Biosystems, USA). Reactions were heated at 96°C for 2 min, then 35 cycles of 94°C for 20 sec, 68°C for 20 sec and 72°C for 30 sec and final extension at 72°C for 10 min. The PCR product was subject to electrophoresis on 0.8% agarose gel stained with 0.5 µg ml-1 of ethidium bromide and visualised under a UVtrans illuminator. Biovar determination. Strains were classified to biovars using a variation of the physiological test developed by Hayward (1964). The basal medium used for biovar identification consisted of NH4H2PO4, 1.0 g; KCl,0.2 g; MgSO4.7H2O, 0.2 g; peptone, 1.0 g; 1% (wv-1) aqueous solution of bromothymol blue, 0.3 ml; agar, 1.5 g; distilled water, 1 litre. The pH of the medium was adjusted to 7.1 with 40% (wv-1) NaOH solution before addition of the agar. Five millilitres of a 10% (wv-1) pre-filtered (using syringe filters 0.22 µm, MF-Millipore MCF membrane) solution of the sugars (sucrose, lactose, maltose, cellobiose) and sugar alcohols (mannitol, sorbitol and dulcitol) were added to 45 ml of molten basal medium separately. Two hundred µl of these media were then dispensed into each of 8 wells in vertical row of 96-well micro-titre plates. Hayward’s medium without a carbon source and un-inoculated wells served as controls. Each well in a horizontal row was inoculated with 10 µl of a 2×109 CFU ml-1 cell suspension of an isolate prepared from overnight growth in CPG broth. The plates were incubated at 28±2°C for 3 weeks and the colour was recorded every 2 days. Positive cultures change the culture medium from green to yellow. Each test was replicated three times. Strains that were classified as biovar 2 were further tested using the PCR primer pair 630-F (5’-ATACAGAATTCGACCG-GCACG-3’) and 631-R (5’-AATCACATGCAATTCGCCTACG-3’), which specifically amplifies a 278 bp fragment from strains in phylotype II sequevar 1, historically known as race 3 biovar 2 (Fegan et al., 1998). Pathogenicity test. Pathogenicity of each isolate was tested on susceptible cultivars of potato (Solanum tuberosum cv. Kufri Jyoti), tomato (Solanum lycopersicum cv. Pusa Rubi), eggplant (Solanum melongena cv. Pusa purple long), and pepper (Capsicum annuum cv. Pachhad Yellow). The experiment was conducted in a glasshouse using 5 replications of one plant each. A suspension (1×108 CFU) of each isolate was prepared in sterile distilled water using a 48 h culture grown on CPG agar medium (Kelman, 1954). The plants were inoculated by stem stab method (Winstead and Kelman,

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1952) when they were 15-20 cm tall. The plants stabbed with sterilized distilled water only served as control. Inoculated plants were incubated at 28±8oC and were observed for wilt appearance till 40 days post inoculation. Re-isolations were made from section of stem about 2 cm above the inoculation point of symptomatic plants on TZC agar medium and observed for formation of typical R. solanacearum colonies.

RESULTS

Survey, collection of R. solanacearum isolates and their identification. Surveys involved 39 fields of 25 farmers in 16 villages of 10 Tehsils/Talukas in 3 districts in Malwa region of Madhya Pradesh (Table 1 and 2). The disease was present in 31 fields with disease incidence ranging from 0.5% to 80.0% (Fig. 1 and 2) depending on the source of seed used. The study revealed that wherever the farmers used certified seed or procured seed from Punjab (potato brown rot-free state of India), the incidence of bacterial wilt and brown rot was nil or negligible (Table 1 and 2). A total of 22 isolates were recovered from wilt-affected potato stems and tubers collected from different fields (Fig. 3). Out of these 22 isolates, 21 were confirmed as R. solanacearum as showing typical colony morphology on TZC agar, the expected single 280 bp fragment using the R. solanacearum specific primers YII and OLI1 (Fig. 4) and pathogenicity test on potato. Biovar determination. Using bio-chemical tests, based on the ability to utilize disaccharides and oxidise

Fig. 3. Typical R. solanacearum colonies on TZC agar medium.


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Table 1. Incidence of bacterial wilt of potato in the Malwa region of Madhya Pradesh (India) in December 2009. District

Tehsil/ Taluka

Village

Field (No.) 1

Deopalpur

Mahu

Incidence (%)

Home-saved Punjab

1.50 0.25

20.0 0.5

3 4 5 6

K. Lauvkar K. Chipsona-1 K. Jyoti K. Jyoti

Local Local Local Local

3.75 1.70 1.00 1.50

40.0 60.0 25.0 5.0

7

K. Laukar

Local

0.50

5.0

Navadapanth

1

K. Jyoti

Local

1.50

0.2

Harsola

1

K. Laukar

Local

0.30

25.0

1

K. Jyoti

Local

0.40

0.5

2

K. Laukar

Local

0.40

20.0

K. Chipsona-1

Local

0.30

40.0

2

K. Jyoti

Punjab

0.40

0

1 2

K. Jyoti K. Chipsona-1

Certified Local

1.50 12.0

0 0.5

3

K. Laukar

Local

4.50

0

1

K. Chipsona-1

Local

1.00

0

K. Chipsona-1

Home saved

2.00

30.0

K. Chipsona-1 K. Chipsona-1 K. Chipsona-1

Local Local Local

2.50 1.00 2.00

30.0 10.0 10.0

Mothala

Ambachandan

Ramgarh

Ujjain Ujjain

Area (ha)

K. Jyoti K. Jyoti

Teegaon

Ghatia

Seed source

2

Indore Indore

Cultivar

Ralayata Chandukheri

1

1 2 3 4

none on pepper (Table 3). The isolate number RS-4, however, did not produce any kind of symptoms on the crops tested. Re-isolations from symptomatic plants yielded typical R. solanacearum colonies.

hexose alcohols, 21 isolates were categorized as biovar 2 which corresponds to race 3 of the pathogen (Fig. 5). One isolate (RS-4), however, did not fall in any biovar category. The 21 isolates were further identified as phylotype II, sequevar I (race 3 biovar 2) as the characteristic race 3 biovar 2 specific 278 bp band was amplified from their DNA using the primer pair 630/631 (Fig. 6.)

DISCUSSION

Pathogenicity. Out of the 22 isolates, 21 proved to be pathogenic on potato, 18 on tomato, 15 on eggplant but

This study revealed a very high disease incidence (up to 80%) in some of fields surveyed whose incitant was

Fig. 4. PCR product banding pattern amplified from R. solanacearum strains with specific primers YII and OLI1 (lane M=ladder; lanes 1-22= R. solanacearum strains).


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Sagar et al.

Table 2. Incidence of brown rot of potato in the Malwa region of Madhya Pradesh (India) in February 2010. District

Tehsil/ Taluka Deopalpur

Indore

Field (No.) 1 2 3 4 5

Village Mothala

K. Jyoti K. Jyoti K. Lauvkar K. Chipsona-1 K. Jyoti

Seed Source of seed Home-saved Punjab Local Local Local

Cultivar

Incidence (%) 40.0 0.5 80.0 80.0 50.0

Navadapanth

1

K. Jyoti

Local

20.0

Shihana

1 2

K. Chipsona-1 K. Chipsona-3

Home-saved Home-saved

25.0 40.0

Kadua

1 2 3

K. Chipsona-1 Lady Rosetta K. Lauvkar

Punjab Punjab Punjab

0 0 0

0 2.0

Indore

Sagar

Malikheri Guran 1 2

K. Lauvkar K. Lauvkar

Punjab Home-saved

Devas

Devas

Sindhora

1

K. Chipsona-1

Local

25.0

Ujjain

Ujjain

Harsodan

1 2 3

K. Chipsona-1 K. Lauvkar K. Lauvkar

Home saved Home saved Home saved

20.0 05.0 10.0

Chandukhedi

1

K. Chipsona-1

Local

25.0

Table 3. Pathogenicity test of R. solanacearum isolates on potato, tomato, eggplant and pepper. Isolate No.

RS-1 RS-2 RS-3 RS-4 RS-5 RS-6 RS-7 RS-8 RS-9 RS-10 RS-11 RS-12 RS-13 RS-14 RS-15 RS-16 RS-17 RS-18 RS-19 RS-20 RS-21 RS-22 Control

Symptom appearance (21 dpi) (+/-)

Disease severity (21 dpi) (%)

Potato

Tomato

Eggplant

Pepper

Potato

Tomato

Eggplant

Pepper

+ + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + -

-

100 100 100 0 100 100 100 100 40 100 100 100 100 40 100 100 100 100 100 100 100 100 0

20 100 100 0 100 100 100 20 0 100 100 100 0 0 50 40 10 10 100 100 100 30 0

0 10 30 0 100 10 10 0 0 20 30 30 0 0 30 30 20 10 100 40 0 20 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

+Wilt symptoms appeared; -No wilt symptoms observed; dpi= Days post inoculation

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R. solanacearum. Though this disease was known to be endemic in Indore and adjoining areas since 1970, it was never a serious problem. Based on typical colony morphology on TZC agar medium, the expected single 280 bp fragment using the R. solanacearum specific primers YII and OLI and pathogenicity test on potato, 21 out of 22 isolates collected from Malwa region of Madhya Pradesh, India were identified R. solanacearum. Using bio-chemical tests, based on the ability to utilize disaccharides and oxidise hexose alcohols, 21 isolates were categorized as biovar 2 which were further identified as phylotype II sequevar 1 as the characteristic race


3 biovar 2 specific 278-bp band was amplified from their DNA using the primer pair 630/631(Fegan et al., 1998). Earlier, Shekhawat et al. (1978) had reported race 1 biovar 3 to be prevalent in plains of India including Madhya Pradesh. However in our study, all the isolates collected from various fields of Malwa region belonged to race 3 biovar 2. In the past, race 3 biovar 2 was known to persist in cool humid hilly areas only. However, seed from these areas was also used to raise winter crop in central India and it was only from 1970s onward that farmers started using disease free seed from north Indian plains (Shekhawat et al., 1992). Sunaina et

Fig. 5. Microtitre plates showing the results of biovar test with 22 R. solanacearum strains.


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Fig. 6. PCR amplification of 278 bp race 3 biovar 2 specific fragment using the primer pair 630/ 631 (lane M=1 kb ladder, lanes 1-14= R. solanacearum isolates belonging to biovar 2).

al. (1989) demonstrated that seed stock from mid hills i.e. Garud, the Garhwal hills, Mukteswar and Supi carried 54.5, 28.8, 25.0 and 17.1% latently infected tubers, respectively. The occurrence of race 3 biovar 2 in Malwa region can, therefore, be ascribed to its introduction through infected seed tubers from mid hills. Race 3 biovar 2 is generally considered to be better adapted to temperate or cool tropical highlands and the pathogen commonly cause latent (asymptomatic) infections, but when infected seed tubers are planted in warmer lowland fields, the resulting plants quickly wilt and die (Allen et al., 2001). All the 21 isolates, characterised as biovar 2, were pathogenic to potato, most to tomato and eggplant but none to pepper indicating that the isolates belong to race 3 biovar 2 of R. solanacearum i.e. potato race. Race 3 is described as pathogenic to potato and tomato but weakly virulent on other Solanaceae spp. (Buddenhagen et al., 1962) and biovar 2 coincides with race 3 (Martin and French, 1985). Also, the disease incidence was high in fields where farmers’ home saved seed or seed from local sources were used compared to fields where certified seed or seed from Punjab (brown rot free area) was used. The apparently healthy tubers may carry the pathogen in vascular tissues, on the tuber surface and in lenticels (Shekhawat, 1983). Storage of such tubers for use as seed in next crop will definitely aggravate the problem. The study also indicates that the disease could be managed in this region by using certified healthy seed or procuring seed from brown rot free areas like Punjab, Haryana and western Uttar Pradesh. Reason being, in plains of India, the summer temperature is very high (up to 42-43oC) with scanty vegetation and water deficit period for 40-45 days which adversely affect the survival of pathogen in soil (Shekhawat et al., 1992). Since the disease is known to be transmitted latently through seed tubers (Kelmin, 1953; Elphinstone, 2005), it is the seed tubers which serve as the primary source of inoculum in this region. In India, the north western high hills (2200 masl) and

north western plains comprising Punjab, Haryana, western Uttar Pradesh and Rajasthan States are known to be free of brown rot pathogen (Shekhawat et al., 1992). The north western plains of India have been identified suitable for healthy seed tuber production of potato by Central Potato Research Institute (ICAR), Shimla, India. This region is largely responsible for supply of healthy seed tuber to rest of India. Since the bacterial wilt problem in Malwa region is largely because of latently infected seed tubers (as indicated in our study), the problem can be overcome to a great extent by using healthy certified seed or procuring seed from north western plains of India. REFERENCES Allen C., Kelman A., French E.R., 2001. Brown rot of potatoes. In: Stevenson W.R., Loria R., Franc G.D., Weingartner D.P. (eds). Compendium of Potato Diseases, pp. 1113. APS Press, St. Paul, MN, USA. Buddenhagen I.W., Sequeira L., Kelman A., 1962. Designation of races in Pseudomonas solanacearum. Phytopathology 52: 726. Chen W.P., Kuo T.T., 1993. A simple and rapid method for the preparation of Gram-negative bacterial genomic DNA. Nucleic Acids Research 21: 2260. Elphinstone J.G., 2005. The current bacterial wilt situation: a global overview. In: Allen C., Prior P., Hayward A.C. (eds). Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex, pp. 9-28. APS Press, St. Paul, MN, USA. Fegan M., Taghavi M., Sly L.I., Hayward A.C., 1998. Phylogeny, diversity and molecular diagnostics of Ralstonia solanacearum. In: Prior P., Allen C., Elphinstone J. (eds). Bacterial Wilt Disease: Molecular and Ecological Aspects, pp. 19-33. INRA Editions, Paris, France. Hayward A.C., 1964. Characteristics of Pseudomonas solanacearum. Journal of Applied Bacteriology 27: 265-277. Hayward A.C., 1994. The hosts of Pseudomonas solanacearum. In: Hayward A.C., Hartman G.L. (eds). Bacterial Wilt: The Disease and its Causative Agent, Pseudomonas solanacearum, pp. 9-24. CAB International, Wallingford, UK.


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Kelman A., 1953. The bacterial wilt caused by Pseudomonas solanacearum. A literature review and bibliography. North Carolina Agricultural Experiment Station, Technical Bulletin No. 99. Kelman A., 1954. The relationship of pathogenicity of Pseudomonas solanacearum to colony appearance in a tetrazolium medium. Phytopathology 44: 693-695. Martin C., French E.R., 1985. Bacterial wilt of potato, Pseudomonas solanacearum. International Potato Centre Lima, Technical Bulletin No. 13. Seal S.E., Jackson L.A., Young J.P.W., Deniel M.J., 1993. Differentiation of Peudomonas solanacearum, P. syzygii, P. picketii and the blood disease bacterium by partial 16S RNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction. Journal of General Microbiology 139: 435-440. Shekhawat G.S., Singh R., Kishore V., 1978. Distribution of bacterial wilt and races and biotypes of the pathogens in

Received September 3, 2012 Accepted November 8, 2012

Journal of Plant Pathology (2013), 95 (2), 321-328 India. Journal of Indian Potato Association 5: 155-165. Shekhawat G.S., 1983. Bacterial Diseases. In: Nagaich B.B. (ed.). Potato Production, Storage and Utilization. pp. 400411. Central Potato Research Institute (ICAR), Shimla, India. Shekhawat G.S., Chakrabarti S.K., Gadevar A.V., 1992. Potato bacterial wilt in India. Central Potato Research Institute Shimla, Technical Bulletin No. 38. Somani A.K., Chakrabarti S.K., Pandey S.K., 2010. Spread of bacterial wilt and brown rot of potato in Indore region of Madhya Pradesh. CPRI News Letter 42: 16-17. Sunaina V., Kishore V., Shekhawat G.S., 1989. Latent survival of Pseudomonas solanacearum in potato tubers and weeds. Journal of Plant Disease Protection 96: 361-364. Winstead N.N., Kelman A., 1952. Inoculation techniques for evaluating resistance to Pseudomonas solanacearum. Phytopathology 42: 628-634.