Australasian Plant Pathol. (2012) 41:453–461 DOI 10.1007/s13313-011-0098-y
Screening for resistance to potato cyst nematode in Australian potato cultivars and alternative solanaceous hosts R. Faggian & A. Powell & A. T. Slater
Received: 27 June 2011 / Accepted: 6 October 2011 / Published online: 10 November 2011 # Australasian Plant Pathology Society Inc. 2011
Abstract The potato cyst nematodes (PCN), Globodera rostochiensis (Woll.) and G. pallida (Stone), are major pests of ware and seed potato (Solanum tuberosum L.) crops worldwide and severely impact the movement of potatoes around the globe through quarantine restrictions. In Australia, only G. rostochiensis has been discovered, on four separate occasions between 1986 and 2008. The infested areas are the subject of strict regulation and quarantine procedures and while they are considered to be contained, managing nematode populations remains a priority. This study has identified the G. rostochiensis Ro1 resistance-status of potato cultivars currently grown by Australian potato growers, and new cultivars emerging
from the Australian Potato Breeding Program. Resistance was assessed by a simple and robust procedure carried out in a purpose-built quarantine facility. Of the 24 potato cultivars grown in the affected Koo Wee Rup district in 2004, 10 were resistant to nematode infestation, including the locally important cultivar Atlantic. Other cultivars important to the Victorian and Australian potato industry, such as Kennebec, Desiree, Sebago and Coliban, were classified as susceptible. Importantly, this study provided evidence that the Koo Wee Rup PCN population was able to complete its lifecycle on the native plant species, S. aviculare (kangaroo apple), potentially acting as an alternate host and spreading PCN among potato crops.
Electronic supplementary material The online version of this article (doi:10.1007/s13313-011-0098-y) contains supplementary material, which is available to authorized users.
Keywords PCN . Resistance . Globodera . Breeding . Potato . S. aviculare
This work was funded by the Victorian Department of Primary Industries and Horticulture Australia Ltd. R. Faggian (*) Department of Primary Industries, Future Farming Systems Research Division, Parkville Centre Victoria and University of Melbourne, PO Box 4166, Parkville, Vic 3052, Australia e-mail:
[email protected] A. Powell LaTrobe University, Bundoora, Vic 3086, Australia A. T. Slater Department of Primary Industries, Biosciences Research Division, Knoxfield Centre Victoria, 621 Burwood Highway, Knoxfield, Vic 3180, Australia Present Address: A. Powell Scottish Agricultural College, York, United Kingdom
Introduction The potato cyst nematodes (PCN), Globodera rostochiensis (Woll.) and G. pallida (Stone), are major pests of ware and seed potato (Solanum tuberosum L.) crops worldwide. They cause significant yield reductions and severely impact the movement of potatoes around the globe through quarantine restrictions. Both Globodera species cause serious disease problems in more than 61 countries (Dale and De Scurrah 1998), and pose considerable control problems (Whitehead and Turner 1998). Potato cyst nematodes attack plant roots and impact on the host plant’s ability to take up water and nutrients, in particular phosphorus, potassium, calcium and magnesium. Infective second stage juveniles (J2s) of Globodera spp. are preferentially attracted to root tips (Steinbach 1972 in Sheridan et al. 2004), which they explore by lip-rubbing and stylet-probing. When a suitable site is found in a
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susceptible host, cell-walls are penetrated by repeated stylet thrusts to provide access for the J2 (Wyss 2002). Once inside the root, the J2s migrate towards the vascular tissue by cutting through successive layers of cells, where they initiate a feeding site, or syncytium. Affected plants have less extensive root systems (Evans et al. 1977), up to half the root-shoot ratio, (Haverkort and Trudgill 1995) and suffer from chronic nutrient deficiencies (Trudgill 1980). In resistant hosts, the response to J2 invasion involves localised cell necrosis and browning, followed by the disorganisation and lysis of the syncytium (Sheridan et al. 2004). The lysis of the syncytium results in a food shortage for the nematode which causes the majority of nematodes to develop into males and therefore limit the development of cysts (Trudgill 1991). The impact of PCN on potato yield can be significant. Brown and Sykes (1983) recorded yield losses of up to 22 tonnes per hectare in field trials conducted over one growing season in the United States, and Greco and Moreno (1992) reported yield losses of up 70% of summer potato crops in Chile. In Australia, G. rostochiensis was first discovered in 1986 at Munster in Western Australia (Stanton 1986). Further infestations were detected in 1991 in Wandin and Gembrook on the outskirts of Melbourne, Victoria (Guy et al. 1992), and in 2004 in the Koo Wee Rup district of Victoria (Berg et al. 2004). The most recent detection was in 2008 at Thorpdale, Victoria. All areas are the subject of strict regulation and quarantine procedures, and the infestations are considered to have been contained. In all cases, the only species detected was G. rostochiensis pathotype Ro1 (Berg et al. 2004; Quader et al. 2008). The costs to the Australian potato industry associated with the unrestricted spread of PCN (without action to prevent its spread or its entry to new areas), have been estimated at $20 m annually, and as much as $370 m over 20 years (Hodda and Cook 2009). Direct control of nematodes via eradication using nematicides is costly and extremely difficult, as some juveniles remain dormant for many years before hatching. Further, as few as two viable eggs per cyst can establish a new infestation (Brodie and Brucato 1993) so regardless of environmental conditions, an established field population can remain viable for a number of years (La Mondia and Brodie 1986). Also, many nematicides have been phased out over time due to their long environmental half-life and persistence, and because of the development of resistance in nematode populations. Indirect control, by managing the nematode populations and by limiting their spread, is a practically feasible and effective means of controlling the disease. Indirect control has been achieved using various management practices, such as by growing cultivars that can be sown and harvested earlier to avoid the higher summer temperatures
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that favour the nematode (Greco 1993), and by crop rotations with non-hosts (Evans 1993). Resistant cultivars are also important. Resistance to G. rostochiensis has been attributed to a number of genes, which confer partial (Gro1.2, Gro1.3, Gro1.4, Grp1) or near absolute (H1, Gro1, GroVI) resistance (Finkers-Tomczak et al. 2011). The H1 gene was introduced from S. tuberosum ssp. andigena (Toxopeus and Huijsman 1953) and has been widely introgressed into many commercial cultivars. This gene confers resistance against two of the five known pathotypes, Ro1 and Ro4 (Kort et al. 1977). Fortunately, Ro1 is the only pathotype known to occur in Australia (Hinch et al. 1998), and previous studies have shown that Ro1 resistant cultivars can significantly reduce yield losses and nematode populations. For instance, Brodie (1996) demonstrated that populations of G. rostochiensis can be reduced by up to 95% for each season that a resistant cultivar is grown, depending on initial inoculum density. Conversely, nematode population densities can increase 2–35 times when susceptible cultivars of potatoes are grown in an infected area. The aim of this study was to determine which potato cultivars currently grown in Australia and new lines emerging from the Australian Potato Breeding Program, exhibit resistance to the PCN detected in Australia, and also to investigate the possibility of an alternative host for PCN among other solanaceous species, including weeds and native plants.
Materials and methods Quarantine A quarantine facility was established to conduct all experiments using PCN. The facility consisted of a glasshouse modified to eliminate the risk of PCN escape, and quarantine procedures to ensure strict adherence to specific hygiene protocols, including the proper disposal of soil and other waste materials. Briefly, access to the glasshouse was restricted by lock and key and entry was via a separate anteroom with those entering the glasshouse-proper required to wear protective spray suits and watertight plastic boot covers. Before exiting the glasshouse, protective clothing was discarded, hands were washed with 1% chlorhexidine and footwear was treated in a Biogram (Ecolab, Castle Hill) footbath. In the glasshouse, 75-μmaperture mesh traps were installed in all drains to capture cysts in run-off water. All waste, including potting media, soil and plant material were double-autoclaved before disposal by deep burial. The quarantine facility and associated protocols were developed with, and approved by, Biosecurity Victoria, a government agency that delivers
Screening for resistance to potato cyst nematode in Australia
programs to preserve market access for Victoria’s livestock, plant, fisheries and forestry industries. Soil sampling and inoculum preparation PCN infested soil was sampled from infection ‘hotspots’ within the Koo Wee Rup PCN control area in Victoria, Australia. Soilsampling and hotspot-identification was carried out with Biosecurity Victoria staff. A total of 40 L of infested soil was sampled from an infested paddock in the summer of 2004/05 using a 10 m by 10 m grid sampling regime. Soil was sampled with a shovel in each grid section to a depth of approximately 30 cm after removing the upper pasture layer. Soil was placed in one of two 20 L plastic drums and transported directly to the laboratory for cyst isolation, identification and quantification. Cysts were extracted from 500 g sub-samples of soil using the Fenwick can flotation method (Fenwick 1940; Turner 1998), and counted. The concentration of cysts in the sub-sample was 525 cysts/500 g of soil. The number of eggs per cyst, and egg viability, were also determined. Eggs were counted by taking a sub-sample of 10 extracted cysts, soaking them in sterile distilled water for 24 h and breaking each open with a set of fine tweezers. Eggs were scraped out of the body of the cyst onto a glass slide containing a 3 mm grid where all eggs and hatched juvenile nematodes were counted. Egg-viability was assessed by soaking 10 cysts in a 0.01% Nile Blue stain for 48 h, breaking them open on to the 3 mm grid slide and counting stained (non-viable) and unstained (viable) eggs. Each cyst contained an average of 323.5 eggs and viability was 65%. The species of PCN was confirmed to be G. rostochiensis using a species-specific polymerase chain reaction (PCR) assay (Bulman and Marshall 1997). DNA was extracted from soil-extracted cysts that were sampled from potential infestation-sites, and also from cysts that developed on test cultivars in this study. DNA was extracted from the cysts according to the method outlined by Bulman and Marshall (1997) and also by using a FastDNA™ Spin kit for soil (Qbiogene, California, USA), as per the manufacturer’s instructions. The extracted DNA was subjected to PCR amplification using the specific primers PITSr3 and PITSp4 (Bulman and Marshall 1997) and the generic Internal Transcribed Spacer region primer ITS5 (White et al. 1990), in a Hybaid PCR Express ver. 2.0 thermocycler according to the conditions described by Bulman and Marshall (1997). Planting material and growth conditions Cultivars were selected from the Australian potato breeding germplasm collection of existing commercial cultivars and potential parental material used in the breeding program. All planting material consisted of small tubers
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produced by the DPI potato breeding program the previous summer. Selected potato cultivars were sown singly in 100 mmdiameter pots, in the aforementioned quarantine facility, during August 2005, 2007 and 2008 with three replicates of each cultivar. Pots were filled with approximately 100 g of a pine-based potting mix followed by a layer of infested soil. The volume of soil used was 45 mL, which was determined to achieve a final egg-per-pot concentration of approximately 9500 eggs. One small tuber of each cultivar was then partially imbedded in the soil and the pots were topped up with additional potting mix as required, with the average pot weight (soil and potting mix) of 250 g. Pots were placed on raised benches in a randomised block design and watered by hand. The tubers were fertilised initially with 10 g of Osmocote Plus and then periodically throughout the trial’s duration with Nitrosol. Positive control and negative control pots were also included; positive controls consisted of the susceptible cultivar Ilam Hardy inoculated as described above, and negative controls consisted of un-inoculated Ilam Hardy. Positive control pots were also harvested at regular intervals in order to monitor the development of cysts and to thereby allow prediction of the optimum time of harvest for the whole experiment. Assessment Harvest was carried out approximately 10 weeks after planting. Each plant was removed from the pot and its roots visually assessed for the presence of PCN cysts, and counted (Fig. 1). By this stage the plants were beginning to appear ‘pot-bound’ (that is, root growth filled the pot and were circling the root ball) and it was therefore easy to observe the root ball and also to replace them in the pots if further incubation was required. Plants with three or more cysts were considered susceptible and plants with no cysts were considered resistant (John Anderson, pers comm). A cultivar was classified as resistant or susceptible if all replicates were consistent. If an inconsistent result was observed, such as two resistant plants and one susceptible, the cultivar was reassessed. Cultivars that allowed development of greater numbers of cysts than the susceptible control, Ilam Hardy, were classified as very susceptible. Where possible, harvesting of plants was conducted several days earlier than optimum (as determined on Ilam Hardy plants) to avoid cysts becoming dark brown and therefore difficult to distinguish from the surrounding soil. Where white (presumably immature) cysts were encountered, the plants were replaced in their pots and left in the glasshouse for an additional week for reassessment, until such time as golden coloured cysts were observed. Once the data were collated, it was compared to the characteristics listed on the European Cultivated Potato
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Fig. 1 Comparison of the root ball and roots of a resistant cultivar, Atlantic (left) with a susceptible cultivar, Wilwash (right). Note the golden coloured cysts of G. rostochiensis on the roots of the susceptible cultivar (right)
Database to determine the likely pathotype of the PCN inoculum used in this study.
roots were sometimes discoloured and smaller than unaffected roots, and were sometimes excessively branched.
Alternative hosts
Cultivar resistance
In August 2006 a concurrent glasshouse trial was established to determine if PCN could survive on alternative hosts in the Koo Wee Rup area in the absence of potato crops. A range of Solanaceous horticultural crops, weeds and Australian native plants were obtained for the trial, which included nine types of tomato (Gardner’s Delight, Green grape, Grosse Lisse, Mini orange, Ox Heart, Roma, Tigerella, Tiny Tim, Yellow pear), six types of chilli (Anaheim, Hot pepper, Hungarian Hot Wax, Jalapeno, Long red cayenne, Small hot chillies), three types of capsicum (Corno di Torro, Marconi Rosso, Mixed capsicum), one type of eggplant (Black beauty), black nightshade (Solanum nigrum) from three locations and kangaroo apple (S. aviculare) from two locations. Seeds of these plants were germinated in pots containing the PCN infested soil and grown and assessed under the same conditions as the potato trial described above.
Average counts of cysts in replicate pots, and the resultant classification of cultivars (resistant, susceptible and very susceptible) for 302 cultivars revealed 89 resistant cultivars and 213 susceptible cultivars. For the sake of simplicity, Table 1 lists the cultivar classification only for the main cultivars currently grown commercially in Australia. Within this list, 17 were resistant to G. rostochiensis, 36 were susceptible, and five were very susceptible. Of these main commercial cultivars, 12 resistant cultivars are used in the fresh market, five in the crisping industry, while the main French fry cultivars are all susceptible.
Results Symptom expression PCN affected plants were generally stunted with yellowing leaves, and also showed signs of nutrient deficiency and water stress. Small golden cysts, ~500 μm in diameter were attached to the roots of susceptible plants. Heavily infested
PCN pathotype comparison The European Cultivated Potato Database (ECPD) lists the characteristics of a large number of cultivars, including, where determined, their resistance or susceptibility to G. rostochiensis and G. pallida and their pathotypes. We have compared the results listed on the ECPD with our results and found that our results are consistent with the results for G. rostochiensis Ro1 or Ro4 on the ECPD, but not the other pathotypes. For the 64 cultivars that were common between our study and the ECPD, 33 were resistant in this study and are listed as having a high or very high level of resistance to the Ro1 pathotype in the ECPD, while 31 were susceptible in this study and are listed as having very low or low resistance in the ECPD (data not shown). Moderately
Screening for resistance to potato cyst nematode in Australia Table 1 Main cultivars of potato grown in Australia (K. Blackmore pers. comm.), the mean number of cysts formed on their roots (averaged over three replicates) and their G. rostochiensis resistance classification (R=Resistant, S=Susceptible and VS=Very Susceptible)
C – Crisp processing, F – Fresh market, FF – French fry processing
a
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Cultivar
Main commercial usea
Mean No. of Cysts
PCN Resistance classification
Almera Argos Atlantic Bison Catani Celine Charlotte CMK Coliban Crop 13 Crop 17 Crop 4 Denali Desiree
F F C F C F F F F F F F C F
0 0 0 37 25 0 12 0 20 0 22 17 30 20
R R R S S R S R S R S S S S
Driver Exton FL1867 FL1953 FL2027 Ilam Hardy Inova Kennebec Kipfler Lady Christl Lustre Macrusset Malin McCain 1 McCain 4 Nadine Nicola Nooksack
F F C C C F FF F F F FF F FF FF F F FF
50 43 0 22 7 43 0 17 40 0 15 27 10 16 15 0 0 7
VS VS R S S VS R S S R S S S S S R R S
Onka Otway Red Pike Pink Eye Pontiac Ranger Russet Red Gem Red La Soda Red Rascal Royal Blue Ruby Lou Russet Burbank NG Russet Burbank Ruen Russet Burbank Tas. Sebago Sequoia Shepody
F F C F F FF F F F F F FF FF FF F F FF
28 20 0 23 42 35 5 43 20 0 27 27 27 14 29 37 25
S S R S S S S VS S R S S S S S S S
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R. Faggian et al. Table 1 (continued) Cultivar
Main commercial usea
Mean No. of Cysts
PCN Resistance classification
Simcoe Snowgem Spunta Trent Umatilla Valor White Lady Wilwash Wontscab
C F F C FF F F F C
0 8 21 27 13 0 0 57 0
R S S S S R R VS R
resistant cultivars in the ECPD were considered susceptible in this study. Our results disagree with the results for Ro2, where we found that Lady Rosetta, Liseta and Saturna were resistant, while the ECPD has them listed as low to moderate or moderately resistant. We disagree with Ro3, where we found that Lady Rosetta, Liseta, Mondial, Santana and Saturna were resistant, while the ECPD has them listed as low or moderately resistant. We also disagree with Ro5, where we found that Katahdin and Shepody were susceptible, while the ECPD has them listed as highly resistant. Not surprisingly, results from this study compared to the three G. pallida pathotypes disagreed across a number of cultivars (Table 2). Pathotype Ro4 is the only pathotype that cannot be distinguished from pathotype Ro1. Thus, these cultivars have allowed us to determine that the Australian pathotype is consistent with either Ro1 or Ro4. Alternative hosts Of the other Solanaceous horticultural crops, weeds and Australian native plants that were assessed as potential alternative hosts, low numbers of PCN cysts were observed on one tomato cultivar - Ox Heart (11 cysts), and three plants of Kangaroo Apple (four cysts). All remaining crops were resistant.
Discussion This study has identified the G. rostochiensis Ro1 resistancestatus of potato cultivars that are either currently grown by Australian potato growers, or that are emerging from the Australian Potato Breeding Program. Qualitative resistance to Ro1 is conferred from the H1, Gro1 and GroVI genes. While this study does not identify which of these genes are present in the Australian germplasm, it is likely that at least the H1 gene is present as it has been widely introgressed into
commercial cultivars (Finkers-Tomczak et al. 2011). The qualitative resistance conferred by these genes can be assessed by a simple and robust procedure (John Anderson, pers comm.) that allowed definitive classifications of the resistance-status of cultivars with few ambiguous results. The ‘window-of-opportunity’, where cysts were goldencoloured and therefore easy to see with the naked eye during visual assessments, was large enough that multiple assessments were generally not necessary. Low numbers of cysts (less than ten) were found on four cultivars (Table 1). These low numbers may indicate that these cultivars possess one of the quantitative resistance genes (Gro1.2, Gro1.3, Gro1.4, Grp1), which only confer partial resistance (Finkers-Tomczak et al. 2011). We have deliberately considered that these cultivars be regarded as susceptible for simplicity as they will still serve as a host for PCN and enable the nematode to reproduce, albeit in lower numbers. Although PCN has been recorded in Australia since 1986, it is still a relatively new and isolated disease problem for Australian potato growers. The first detection in Perth, Western Australia, was eradicated and the affected area converted from potato production to residential housing. Later, extensive soil testing could no longer detect cysts. Similarly, the second detection (Wandin/Gembrook, 1991–1993) was contained and remains under observation through routine soil testing. The 2004 detection, however, is in a major potato growing region (Koo Wee Rup, Victoria) such that eradication is not an economical or practical option. The detection affects a large number of growers and is a serious threat to interstate trade and to export markets. Therefore, containment and management of the nematode population, to ensure its decline, are important components of the overall effort to manage the affected area. But, one of the major limitations to adopting a containment and management approach in Australia is the limited data on which to judge resistance or susceptibility of locally grown cultivars. In the case of new breeding lines, the data are virtually non-existent. Similarly, there are
Screening for resistance to potato cyst nematode in Australia
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Table 2 Comparison of the results from our trials with the resistance ratings listed on the European Cultivated Potato Database (www.europotato. org) which can distinguish the Globodera spp. pathotypes. (L=Low, M=Moderate, H=High, V=Very) Cultivar Name
Australian PCN resistance
PCN Ro 1
Admiral Argos Atlantic Cabaret Celine Cunera Harborough Harvest Katahdin Lady Rosetta Liseta Maris Piper
R R R R R R R
VH VH H-VH VH VH H-VH H-VH
S R R R
VL-L H H H
Maxine Midas Mondial Nicola Santana Saturna Saxon Shepody Spey Valor White Lady
R R R R R R R S R R R
VH VH H H H H H L VH H VH
PCN Ro 2
PCN Ro 3
PCN Ro 4
PCN Ro 5
PCN Pa 1
PCN Pa 2
PCN Pa 3
L L-M VL-L VL-L L-M
L
L
L L L VL-L L-M VL-L L
L-M M-H
L L
L-M L-M L L
L-M M L L L
L L L L VL-L
L L L L VL-L
L VL-L L-M VL-L L
H-VH L-M M
L-M M H
L H L-M
few data relating to the potential host range of the pathogen in Australia. According to this study, of the 24 cultivars of potato that were grown in the Koo Wee Rup district in 2004, 10 are resistant to PCN infestation, including the locally important cultivar Atlantic. Before this study was conducted, it was thought that only seven were resistant, based on available information relating to cultivar traits. Of particular interest were two cultivars, Atlantic and FL1867, because they represent a large proportion of the seed-potatoes grown in Victoria. In 2004/2005, 407 ha (out of a total of 2167 ha) were planted with Atlantic (K. Blackmore, pers. comm.) and 136 ha of FL1867 under Victoria’s seed certification scheme. Confirmation that both are resistant to G. rostochiensis Ro1 will provide the Victorian potato industry with assurance that seed production schemes are unlikely to contribute to the spread of PCN, and that production with these cultivars may in fact assist with efforts to reduce the population of PCN. None of the other cultivars classified as resistant were grown in significant amounts commercially within the Koo Wee Rup district. However, other important cultivars, such as Kennebec, Desiree, Sebago and Coliban,
L L-M
L-M
H H
L VL-L
which are grown in large quantities elsewhere in Victoria and Australia, were classified as susceptible and therefore should not be grown in affected areas. Coliban in particular is an important cultivar, as it makes up a significant proportion of the fresh produce potatoes found in Australian supermarkets. This situation may potentially impact on the sustainability of PCN-affected regions in the future, when market forces dictate a change in the predominant cultivars being grown. Of the other commercial cultivars, two resistant cultivars (Nadine and Riverina Russet) were planted in more than 10 ha in the certified seed program. Consequently, there are only two viable alternatives for large-scale growers should PCN becomes an issue outside of the Koo Wee Rup area. This study also provides evidence that the Koo Wee Rup PCN population is able to complete its life cycle on a native plant species, S. aviculare (commonly known as kangaroo apple). This is noteworthy because kangaroo apple is common in the Koo Wee Rup district, naturally occurring in remnant vegetation along roadsides and waterways, and also as a domestically cultivated garden plant. Solanum aviculare is listed as a potential host for both species of
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PCN according to the Crop Protection Compendium (CAB International 2010), and consequently in several other sources that cite the Compendium (for example, Sullivan et al. 2007). The original source for the establishment of this host-pathogen relationship seems to be Winslow (1954), in which all potential hosts were assembled into a ‘provisional’ list for various reasons, two of those being 1) the unconfirmed identification of the host plants involved and 2) the fact that “absolute purity” of the inoculum could not be guaranteed (since some experiments “used infested soil in the open field”). In the case of S. aviculare, its identity was confirmed only to genus level. In this study, seeds of S. aviculare were sourced from flowering and fruiting plants, so plant identity was easily confirmed. This study also used a robust assessment method, with confirmed G. rostochiensis inoculum, and the results determined that S. aviculare be classified as susceptible to G. rostochiensis. Therefore, failure to consider S. aviculare or other known hosts, such as tomato, may impede the success of PCN management and containment measures in Australia. Alternative hosts could interfere with PCN control efforts by providing feeding and reproduction sites for nematodes when potatoes are absent, and by allowing a population to become established, albeit at a low population density, in newly infested areas. Overall, the assessment method was effective and will prove a valuable tool for the selection of resistant cultivars in Australia. However, there does need to be further research into PCN population decline rates that can be achieved under Australian conditions using resistant cultivars, either in isolation or in rotation with other species/ cultivars. Quantifiable data will inform efforts at eradication within an affected region as well as contribute to substantiating any claims of PCN-freedom. Acknowledgements We would like to thank John Anderson, John Marshall (New Zealand Plant & Food Research) and Finlay Dale (Scottish Crops Research Institute) for discussions on the screening protocol; Pat Sharkey and David Eagling (Victorian Department of Primary Industries) for permission to develop a quarantine facility for the trial; Craig Murdoch (Victorian Department of Primary Industries) for access to PCN cysts; Lila Nambier and Motuil Quader (Victorian Department of Primary Industries) for assessment of cyst numbers and viability; Keith Blackmore (Victorian Certified Seed Authority) for access to data on cultivar planting area; and Phil Keane (La Trobe University) for plant pathology expertise. This work was funded by the Victorian Department of Primary Industries and Horticulture Australia Ltd.
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