Nightshade hosts for Canadian isolates of Globodera rostochiensis pathotype Ro1

Abstract

Two Canadian isolates of the potato cyst nematode (PCN), Globodera rostochiensis pathotype Ro1, were evaluated for their ability to replicate on several species of nightshade. Nightshade is commonly found in potato-growing fields and can potentially act as an alternative host, thereby interfering with eradication/control measures for PCN. Accessions of Solanum carolinense, S. americanum, S. dulcamara, S. ptychanthum, S. rostratum, S. sarrachoides, S. sisymbriifolium, S physalifolium, S. triflorum, S. villosum and S. nigrum, were evaluated. Of these, most accessions were resistant to PCN with the following exceptions. The Newfoundland (NFLD) isolate of G. rostochiensis could replicate on S. villosum and S. dulcamara. In contrast, the British Columbian (BC) G. rostochiensis isolate was found to replicate on S. dulcamara but not S. villosum. Furthermore, two of 17 S. nigrum accessions were hosts to PCN from NFLD but not BC; however, both of these accessions may not be true S. nigrum samples but S. nigrum/S. villosum hybrids.

Résumé

Deux isolats canadiens du nématode à kyste de la pomme de terre (NKP), Globodera rostochiensis pathovar Ro 1, ont été évalués quant à leur capacité à se répliquer sur différentes espèces de morelles. La morelle pousse fréquemment dans les champs de pommes de terre et peut servir de plante-hôte, entravant par conséquent l'éradication du NKP. Des accessions de Solanum carolinense, S. americanum, S. dulcamera, S. ptychanthum, S. rostratum, S. sarrachoides, S. sisymbriifolium, S. physalifolium, S. triflorum, S. villosum et S. nigrum ont été évaluées. Chez celles-ci, la plupart des accessions étaient résistantes au NKP à l'exception des suivantes. L'isolat de Terre-Neuve (T.-N.) G. rostochiensis pouvait se répliquer sur S. villosum et S. dulcamera. Par ailleurs, l'isolat de la Colombie-Britannique (C.-B.) G. rostochiensis se répliquait sur S. dulcamera, mais pas sur S. villosum. De plus, 2 des 17 accessions de S. nigrum étaient hôtes du NKP de T.-N., mais pas de celui de la C.-B. Toutefois, il est possible que ces 2 accessions ne soient pas de vrais échantillons de S. nigrum, mais plutôt des hybrides de S. nigrum et S. villosum.

Keywords:

• alternative host
• cyst
• Globodera
• host
• nematode
• potato
• Solanum

Mots clés:

• Globodera
• hôte
• kyste
• nématode
• pomme de terre
• plante-hôte
• Solanum

Introduction

The potato (Solanum tuberosum L.) is an important and popular source of food worldwide with a global production in 2008 of over 325 million metric tonnes (FAO, 2008). While Canada is only the 13th largest producer with almost 5 million tonnes, it is one of the world's largest exporters of certified seed potatoes, estimated at 120,000 tonnes (FAO, 2008). Solanum is a large and diverse genus which also includes other agriculturally important crops, such as tomato (S. lycopersicum L.) and eggplant (S. melongena L.). Nightshades, which also belong to the Solanum genus, are a common weed with widespread distribution but are best adapted to growing in tropical and subtropical America.

The potato cyst nematodes (PCN) Globodera rostochiensis (Woll.) and G. pallida (Stone) are serious quarantine pests of potato grown worldwide and are thought to have co-evolved with their host in South America (Turner & Evans, 1998). They are highly specialized and successful obligatory parasites whose reproduction is dependent on the formation of a feeding site within the root of a susceptible host. There are five characterized pathotypes of G. rostochiensis (Ro1 to Ro5) (Nijboer & Parlevliet, 1990) determined based on the reproductive ability of PCN populations on select Solanum clones. In addition to potato, G. rostochiensis is also known to reproduce on some tomato, eggplant and nightshade species (Sullivan et al., 2007). Once a commercial field is infested, the cysts are difficult to eradicate, even in the absence of a suitable host, as the cysts can persist in the soil for up to 20 years (Turner, 1996). Globodera rostochiensis has only been found in a few sites in Canada, including on the west coast of British Columbia (in Central Saanich on Vancouver Island) and on the east coast near St. Johns, Newfoundland (Olsenand & Mulvey, 1962; Orchard, 1965). Both the Newfoundland (NFLD) and British Columbia (BC) G. rostochiensis isolates are of the Ro1 pathotype (Stone et al., 1977). In the Central Saanich area, a ban on the commercial production of potato, tomato and eggplant has been in effect since the early 1980s in an effort to eradicate the pest (Department of Justice, Canada, 1980, 1982 a, 1982 b). During a recent survey of quarantine fields in Central Saanich (Rott et al., 2010), it was noted that many of the fields sampled were also infested with the weed, nightshade. With over 30 nightshade species listed as potential hosts for PCN (Sullivan et al., 2007), several are known to be present in the Pacific Northwest, including bittersweet nightshade (S. dulcamara L.), cutleaf nightshade (S. trifolorum Nutt.), hairy nightshade (S. sarrachoides Sendt.), black nightshade (S. nigrum L.) and American black nightshade (S. americanum Mili.) (Hitchcock et al., 1959). Current regulatory acts in Central Saanich do not call for the control of nightshade in fields known to have been infested with PCN. As a result, nightshade species that could serve as a host for G. rostochiensis may have compromised eradication efforts. In particular, it has been stated that ‘Failure to ensure that weed hosts are controlled is likely to impede successful eradication’ (Sullivan et al., 2007).

Recent work has shown that the hairy nightshades, S. sarrachoides and S. physalifolium (Rusby), are suitable hosts for G. pallida in Idaho, whereas bittersweet nightshade, cutleaf nightshade and black nightshade are poor hosts (Boydston et al., 2010). A previous study also indicated that black nightshade is resistant to G. rostochiensis (Doncaster, 1953). However, other studies showed that the same isolate of G. rostochiensis could multiply on a particular type of black nightshade of German origin but not one of Dutch origin (Prummel, 1958). Most host range studies with G. rostochiensis were conducted over 40 years ago and there is considerable uncertainty regarding the results (Sullivan et al., 2007). In an effort to determine whether nightshade poses a significant risk in perpetuating PCN populations in quarantine areas, we undertook to evaluate several nightshade samples from Saanich, as well as known nightshade reference accessions, for their suitability as hosts for G. rostochiensis present in Canada.

Materials and methods

Inoculation procedure

Testing for resistance to G. rostochiensis was conducted following the standard EPPO protocol (EPPO, 2006). For each of the two G. rostochiensis isolates, five nightshade plants from each accession number, grown from seed, were inoculated with 10 cysts contained in a small nylon satchel, in pots with approximately 1 L of soil. A sixth plant was not inoculated and used as a negative control. An additional five potato ‘Desiree’ plants were each inoculated with one of the two G. rostochiensis isolates to serve as a positive control. Seeds were sown in two batches several months apart due to limitations in the amount of growing space, and were inoculated with cysts once the plant had reached approximately 10 cm in size. Plants were grown in growth chambers for 120 days with daily temperature regimes between 18 and 22 °C and 13 to 16 h light. At the end of the experiment, the soil was dried and the cysts recovered using a modified Fenwick can extraction procedure (Fenwick, 1940), and counted. Multiplication rates for each plant accession were determined by summing the total number of cysts from each of the five replicate plants (Pf) and dividing by the total number of cysts used for the inoculation of the five replicate plants (Pi). Relative susceptibility was expressed as a percentage according to the formula:

Scoring notation is based on relative susceptibility (5) with a score of 9 indicating maximum resistance and a score of 1 indicating a very high level of susceptibility (Table 1).

Table 1.  Standard susceptibility scoring notation for Solanum species inoculated with Globodera rostochiensis

Relative susceptibility (%)	Score
< 1	9
1.1–3	8
3.1–5	7
5.1–10	6
10.1–15	5
15.1–25	4
25.1–50	3
50.1–100	2
> 100	1
Note: Table reproduced from EPPO (2006). A score of 1 indicates a very high level of susceptibility whereas a score of 9 indicates the maximum level of resistance.

Coefficient of variance (CV), expressed as a per cent, was calculated by dividing the standard deviation by the average cyst count for the five replicates and multiplying by 100.

Nightshade accessions

Nightshade accessions (Table 2) were obtained as seed by request through the USDA Germplasm Resources Information Network (GRIN) and the Botanical and Experimental Garden from Radboub University, Nijmegen, the Netherlands. The two accessions of hairy nightshade, S. physalifolium, V206819 and V206818, were collected from infested fields and S. americanum, V206820, was collected near the Sidney laboratory. All three accessions are available through the Royal British Columbia Museum. A sample of S. dulcamara seed was obtained from a field in Ottawa, ON. Identity of the field samples was confirmed by taxonomists through the Royal British Columbian Museum herbarium.

Table 2.  Assessment of nightshade plant resistance to Globodera rostochiensis populations

		G. rostochiensis NFLD				G. rostochiensis BC
Solanum species	Accession	Pf/Pi *	Rel Suc %^†	Score	CV %	Pf/Pi	Rel Suc %	Score	CV %
S. carolinense	894750095	0	< 1	9	–	0	< 1	9	–
S. carolinense	914750002	0	< 1	9	–	0	< 1	9	–
S. carolinense	964750081	0	< 1	9	–	0	< 1	9	–
S. americanum	V206820	0	< 1	9	–	0	< 1	9	–
S. dulcamara	PI 645684	0.4	< 1	9	–	4.9	10.1	5	200
S. dulcamara	PI 643457	0.3	< 1	9	–	4.4	9.1	6	120
S. dulcamara	Ottawa	2.9	3.7	7	200	8	16.5	4	78
S. nigrum	PI 312110	0	< 1	9	–	0	< 1	9	–
S. nigrum	PI 381289	0	< 1	9	–	0	< 1	9	–
S. nigrum	954750338	0	< 1	9	–	0	< 1	9	–
S. nigrum	884750061	0	< 1	9	–	0	< 1	9	–
S. nigrum	904750188	0	< 1	9	–	0	< 1	9	–
S. nigrum	874750040	0	< 1	9	–	0	< 1	9	–
S. nigrum	884750070	0	< 1	9	–	0	< 1	9	–
S. nigrum	PI 312110	0	< 1	9	–	0	< 1	9	–
S. nigrum	884750071	0	< 1	9	–	0	< 1	9	–
S. nigrum	824750029	0	< 1	9	–	0	< 1	9	–
S. nigrum	A04750042	0	< 1	9	–	0	< 1	9	–
S. nigrum	964750044	0	< 1	9	–	0	< 1	9	–
S. nigrum	954750341	0	< 1	9	–	0	< 1	9	–
S. nigrum	954750358	0	< 1	9	–	0	< 1	9	–
S. nigrum	944750125	0	< 1	9	–	0	< 1	9	–
S. nigrum	954750355	0	< 1	9	–	0	< 1	9	–
S. nigrum	PI 381290	2.1	2.7	8	62	0	< 1	9	–
S. nigrum	PI 304600	23.9	30.4	3	47	0	< 1	9	–
S. physalifolium	V206818	N/D^‡	N/D	N/D	–	0	< 1	9	–
S. physalifolium	V206819	N/D	N/D	N/D	–	0	< 1	9	–
S. ptycanthum	A64750068	0	< 1	9	–	0	< 1	9	–
S. rostratum	PI 420997	0	< 1	9	–	0	< 1	9	–
S. sarrachoides	954750073	0	< 1	9	–	0	< 1	9	–
S. sarrachoides	A44750069	0	< 1	9	–	0	< 1	9	–
S. sarrachoides	954750170	0	< 1	9	–	0	< 1	9	–
S. sisymbriifolium	894750017	0	< 1	9	–	0	< 1	9	–
S. sisymbriifolium	894750018	0	< 1	9	–	0	< 1	9	–
S. tuberosum	Desiree	77.5	100	2	41	48.6	100	2	15
S. triflorum	924750074	0	< 1	9	–	0	< 1	9	–
S. triflorum	954750700	N/D	N/D	N/D	–	0	< 1	9	–
S. villosum	804750186	46.7	60.5	2	29	0	< 1	9	–
S. villosum	814750090	36.2	46.7	3	42	0.2	< 1	9	–
S. villosum	884750018	16.3	21.0	4	52	0	< 1	9	–
S. villosum	884750135	22.9	29.6	3	65	<1	< 1	9	–
S. villosum	884750151	9.5	12.3	5	34	0	< 1	9	–
S. villosum	914750047	19.3	24.9	4	44	0	< 1	9	–
Notes: *Pf/Pi = final number of cysts in each pot (Pf) divided by initial number of cysts used for the inoculation (Pi) for the five replicates.
^†Rel Suc % = Pf solanum weed/ Pf Desiree × 100%.
^‡N/D = not determined.

G. rostochiensis isolates

Experiments were performed with two Ro1 isolates of G. rostochiensis, one from Newfoundland (NFLD), and one from British Columbia (BC). Isolates were originally collected as field isolates and then multiplied as populations for several generations on S. tuberosum ‘Desiree’ in pots in a growth chamber. Cysts were kept at 4 °C in the dark for at least six months before use.

Results and discussion

Multiplication of PCN on the standard susceptible potato control was greater than 40-fold (P_f/P_i) with a coefficient of variation (CV) of 41% for NFLD and 15% for the BC isolates (Table 2). In general, it is preferable to have CV values of 35% or less for the standard susceptible control (EPPO, 2006); however, this was difficult to achieve with the NFLD isolate and may indicate some heterogeneity is present within this isolate. In Newfoundland, where the NFLD G. rostochiensis isolate was obtained, there are regions that contain mixed populations of G. rostochiensis and G. pallida (Stone et al., 1977). Although we could not detect the presence of G. pallida in our sample using molecular methods (data not shown), it is still possible that additional heterogeneity was present in this isolate. Solanum americanum was found to be highly resistant to both isolates of G. rostochiensis as were the two S. physalifolium accessions to G. rostochiensis BC, with no new cysts observed on any of the plants (susceptibility = 9, Table 1). These results would suggest that hairy nightshade as a potential host of G. rostochiensis, may not be a significant issue in the Central Saanich quarantine zone. By comparison, recent studies from Idaho have shown that S. physalifolium is a susceptible host for the closely related PCN, G. pallida (Boydston, 2010). While G. pallida is not present in Saanich, it does occur in mixed infection sites with G. rostochiensis in Newfoundland. With a few notable exceptions, nightshades tested were found to be highly resistant to both NFLD and BC G. rostochiensis isolates. On S. carolinese (L.), S. ptycanthum (Dun.), S. rostratum (Dun.), S. sisymbrifolium (Lam.), S. sarrachoides and S. triflorum, no new cysts were observed on any of the plants, with the exception of one cyst on one S. carolinese plant inoculated with the NFLD isolate (susceptibility = 9, Table 1). Solanum triflorum was also reported to be a poor host for the Idaho isolate of G. pallida, whereas S. sarrachoides was considered to be a suitable host (Boydston et al., 2010). Solanum physalifolium and S. sarrachoides are both present in North America and would be of concern anywhere G. pallida is found.

Seventeen accessions of S. nigrum were tested, 15 of which were fully resistant to G. rostochiensis. Solanum nigrum was also shown to be highly resistant to G. pallida (Boydston et al., 2010). Solanum nigrum accessions PI304600 and PI381290 were unusual in phenotype, with red instead of black berries typical of S. nigrum. Accession PI304600 was found to be susceptible to the NFLD G. rostochiensis isolate, with a total cyst count of 1195 from five plants, but not to the BC isolate with only 11 cysts from five plants (Table 2, susceptibility = 3). Accession PI381290 was fully resistant to the BC isolate, and only slightly susceptible to the NFLD isolate, with only 105 cysts total counted from five inoculated plants (susceptibility = 8). One-way ANOVA between accessions PI381290 and PI304600 inoculated with the NFLD isolate confirms a difference in susceptibility at P = 0.005 (data not shown). The berry colour for both these accessions was more similar to that of S. villosum. A closer examination of accessions PI381290 and PI304600 suggests that they may be hybrids between S. villosum and S. nigrum (data not shown). It has been suggested that S. nigrum is a natural hybrid between S. villosum and S. americanum (Lebecka, 2009). Species within the S. nigrum complex have been demonstrated to readily hybridize (Jacoby & Labuschagne, 2006); therefore, it may be possible for S. nigrum/S. villosum to hybridize and that the two accessions PI381290 and PI304600 were misidentified. In contrast, all six S. villosum accessions were found to be susceptible to the NFLD isolate (susceptibility of 2–5, CV of 29–64%), while at the same time resistant to the BC isolate. A total of 13 cysts were counted from the 30 S. villosum plants inoculated with the BC isolate and 7544 in total from the 30 S. villosum plants inoculated with the NFLD isolate. Considering the relatively high CV values for the S. villosum accessions inoculated with the NFLD isolate, a one-way ANOVA was used to demonstrate a significant difference between the S. villosum accessions at a confidence level of P = 0.001 (Table 3). While S. villosum is not known to occur in Saanich or anywhere else in Canada, it has been recorded in California, Florida, Maine, Massachusetts, New Hampshire and Pennsylvania (NRCS, 2010), and there is no reason to suggest that it could not survive in some parts of Canada as well.

Table 3.  One-way ANOVA of S. villosum accessions inoculated with the NFLD isolate of G. rostochiensis

	Sum of squares	Degrees of freedom	Mean square	F-test	Significance
Between accessions	475,525	5	95,105	6.130	0.001
Within accessions	372,352	24	15,515
Total	847,877	29

Bittersweet nightshade (S. dulcamara) is widespread across North America and is found in almost every Canadian province and US state (NRCS, 2010). Two of the three S. dulcamara accessions were found to be resistant to the NFLD isolate while a third accession collected from eastern Canada was susceptible to the NFLD isolate. All three accessions were susceptible to the BC isolate (Table 2, susceptibility score of 4–6, CV of 77–200%). Coefficient of variance for S. dulcamara susceptibility to the BC isolate was high in all cases, making the data difficult to interpret. It should be noted that all Solanum species except for potato were seed-derived and the nematode populations were propagated from a population rather than from individuals. Therefore, it is expected that genetic variation would be present in both nematodes and host plants which could account for the high CV values observed. While it would be possible to use clonally propagated plants and to breed near-homozygous nematode lines, the results would not be reflective of normal diversity found in field situations. ANOVA was performed to determine whether differences were statistically significant between S. dulcamara accessions and the two G. rostochiensis isolates. No significant difference between the S. dulcamara accession inoculated with NFLD or BC isolates (P = 0.37 and 0.93, respectively), or between the NFLD or BC isolates (P = 0.35) could be detected.

It has been recommended that a standard isolate of G. rostochiensis, pathotype Ro1 and Ro5, be used for host resistance/susceptibility studies (EPPO, 2006). The present study suggests that using only two isolates could easily lead to incorrect conclusions on host range susceptibility. Both the NFLD and BC isolates used here have been identified as pathotype Ro1, yet they display marked differences in ability to reproduce on S. villosum. Solanum villosum is resistant to the BC isolate but readily susceptible to NFLD isolate. This underscores the heterogeneity among Ro1 pathotypes and the potential problems with generalized statements on PCN host range or even pathotype host range. The differences observed between the two isolates could explain some of the conflicting data on PCN host range in the literature, in which for example, one report would suggest S. nigrum to be a host for G. rostochiensis and another report would suggest the opposite (Sullivan et al., 2007).

There are two issues worth mentioning. First is the inadequate method used for PCN pathotyping. Pathotyping is based on the ability of the nematode to multiply on a very limited set of only seven Solanum clones (Nijboer & Parlevliet, 1990). It has been estimated that there are between 50 to 200 secreted proteins produced by PCN involved with pathogenicity (Davis et al., 2000), any of which could also be involved in pathotype determination. It is therefore unlikely that the current set of Solanum clones used for pathotyping is sufficient for accurate PCN pathotyping. In the future, a better molecular understanding of pathotype determinants along with new molecular markers, could lead to a better understanding of pathogenicity and host range prediction for PCN. Second, the cosmopolitan distribution, inter-species hybridization and the complex and evolving taxonomy of nightshade species can make it difficult to accurately identify local nightshade species or hybrids. More work is required to develop molecular markers for the analysis of both intra- and inter-species plant variation linked to PCN resistance genes. In the meantime, to ensure that local weeds are not hosts for PCN populations in a specific area and potentially compromise PCN eradication or control measures, it is important to test host range empirically using local PCN populations. This is often done when evaluating resistant potato cultivars to be planted into a new infested field for PCN management; our results suggest that this screening also be done with the local weed population, and that local weeds be monitored for infection on an ongoing basis.

Acknowledgements

We would like to thank Ken Marr from the Royal British Columbia Museum for help in the identification and creating new accession numbers for locally collected Solanum species.