Introduction

Due to the changes in the pathogen population, the late blight disease of potatoes caused by the oomycete Phytophthora infestans, has become more difficult to manage. Economic losses caused by late blight result from both: the foliage and tuber susceptibility of grown cultivars. The number of P. infestans resistant cultivars used in potato production is not sufficient for reducing yield losses. The cultivars developed in the 19th and early 20th century provided a narrow genetic base. In Europe, the cultivated potato might have originated from only a few introductions (Ross 1986). Until the end of the 20th century, the germplasm available consisted of a small number of potato cultivars with field resistance to late blight (Colon 1994). By the 1980s, 77 % of European (including the Russian cultivars) and North American cultivars had introduced genes of resistance to P. infestans derived only from S. demissum.

The All Russian Institute of Plant Industry (VIR) potato collection was initiated with the accessions collected in 1925–1926 by Bukasov, Voronov and Juzepczuk in Mexico, Guatemala and Colombia, and in 1926–1928 by Juzepczuk in South and Central Peru, North Bolivia and Chile (Juzepczuk and Bukasov 1929, 1936). This wild potato germplasm was intensively evaluated and used in potato breeding in Russia over many years of research. However, the financial crisis of the 1990s caused difficulties in collection propagation and increased the risk of the accession losses.

In 1998–2000, the accessions from the VIR collection were reconstructed from old true seeds at IHAR-PIB, Młochów Research Center (Poland) with financial support of CEEM (Cornell-Eastern Europe-Mexico) Project on Late Blight Control. During those 3 years, the 305 accessions of wild potato species from VIR collection were sown in Młochów. Seed or tuber reproductions were obtained for 147 accessions. Reproduced seed samples were divided into three parts and distributed to the potato collections of VIR, IHAR-Młochów and PI, Sturgeon Bay (Wisconsin, USA). All of the grown accessions had been screened for resistance to Phytophthora infestans and a part of them - for resistance to Potato virus X (PVX) and Potato virus Y (PVY). This paper presents the results of this study.

Material and Methods

Plant Material

A total of 154 accessions of tuber-bearing Solanum species was screened for resistance to pathogens. Plant material used in this study is listed in Table 1. Plants obtained from the botanical seeds in the glasshouse were transplanted into the field (30 plants/acc.) in mid-May and in the screenhouse (10–30 pot-plants/acc.) in the beginning of June.

Table 1 The accessions of Solanum species in the VIR collection evaluated (V) for resistance to P. infestans (in leaflets = PhL, in tubers = PhT) PVX and PVY
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Leaflet and tuber tests were applied for evaluation of resistance to P. infestans. The resistance to viruses PVX and PVY was assessed in the laboratory tests.

Table 2 Leaflet resistance to P. infestans of wild potato species accessions from the VIR collection tested for 3 years 1998 (one test for field grown plants, 1999 (two tests for field and screenhouse grown plants) and 2000 (three tests: two for field and one for screenhouse grown plants)
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In the three years of testing for resistance to P. infestans 128 accessions of 48 species were evaluated for leaf resistance and 63 accessions of 35 species were evaluated for tuber resistance. For testing resistance to PVX and PVY artificial inoculations were used. The resistance to PVX was examined in 65 accessions of 20 species and 66 accessions of 20 species were tested for resistance to PVY.

Evaluation for Resistance to P. Infestans

Pathogen Material

The inoculation of detached leaflets was conducted using two highly pathogenic isolates of P. infestans: MP-322 (1.2.3.4.6.7.8.10.11.) in 1998 test and MP-324 (1.2.3.4.5.7.8.10.11.) in 1999 and 2000 tests. The virulence of isolates was examined using Black’s differentials carrying single R-genes from R1 to R11.

Inoculation

Detached leaflets collected from plants grown in the field were inoculated in mid-June to mid-July. For plants kept in the screenhouse, testing was done in mid-July. For inoculation, the samples of mycelia were multiplied on leaves of susceptible cv. Tarpan. Washed mycelium was used for preparation of the inocula, which comprised 50 sporangia per mm3. Inoculum was incubated at 4 °C for 1 h. Thirty plants from each accession were tested. Three leaflets detached from each plant in two replications were drop inoculated. For each accession from 10 to 30 plants were assessed. Incubation proceeded during 7 days at 23 °C (day) and 17 °C (night). Cultivars, which differ in resistance level were used as standards, namely Meduza (R), Bzura (MR), Irys (S) and Sokół (S). Each accession was tested once, twice or three times, however tests were performed six times in three-year period for field or screenhouse grown plants.

Disease Score

Leaflet disease rating was recorded using 1–9 grade scale, where 9 is the most resistant. General score criteria was a combination of a percent of affected leaf area and mycelia development intensity (Zarzycka 2001).

For evaluation of tuber resistance the method of decapitated tubers was applied (Zoteyeva and Zimnoch-Guzowska 2004). The decapitated tubers were drop inoculated with the inoculum comprising 50 sporangia/mm3. Tubers were incubated in the dark at 17°C during 10–14 days depending on the applied isolate aggressiveness. One or two tubers per plant, harvested from 6 up to 32 plants per accession were tested once at one of three tests performed. The number of plants and tubers per tested accession varied due to differences in tuberization among accessions.

Table 3 Tuber resistance to P. infestans of wild potato species accessions from the VIR collection
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Aerial mycelia growth on the decapitated surface was scored using scale 0–3, where 0 is the lack of mycelium growth and 3 is the most intensive growth. Lesion size on a longitudinal surface of cut tubers was scored using 1–9 scale, where 9 is the most resistant.

Evaluation for Resistance to PVX and PVY

The experiments were done in the greenhouse at Młochów. For evaluation of resistance to PVX, about 30 pot-plants from each accession were sap inoculated with the isolate of PVXO strain from potato cv. Osa in the presence of control cultivars (susceptible to PVX cvs Irys and Sokół and extremely resistant cv. Bzura). Four weeks after inoculation, ELISA was performed on inoculated plants with antibodies produced in the Gdańsk Laboratory of IHAR to test for the presence of virus (Zoteyeva et al. 2000).

For evaluation of resistance to PVY accessions were inoculated mechanically with three strains of PVY: PVYo (isolate PVYO LM), PVYNW (isolate PVYN WI) and PVYNTN (isolate 12/94) from the virus collection of the Młochów Research Center. For each accession 8 to 24 pot-plants were inoculated with each normal or necrotic strains using methods presented by Chrzanowska (2001).

The disease symptoms observed ranged from necrotic local lesions, systemic mosaic, top necrosis to systemic necrosis. Presence of the virus was tested 4 weeks after inoculation by ELISA with the cocktail mAb (Bioreba 112911) that recognizes all known isolates of all groups/subgroups of PVY and mAb PVYN specific (Bioreba 112712) that recognizes the necrotic strain group PVYN. Accessions were grouped into 5 groups: R - resistant – all plants were uninfected in greenhouse conditions, MR – moderately resistant, S – all plants were susceptible, and two groups R>S and R<S, which expressed different percentage of resistant/susceptible plants.

Results

Resistance to P. Infestans

Foliage Resistance

Four standard cultivars Irys (S), Sokół (S), Bzura (MR) and Meduza (R) performed as expected with the leaflet test applied (Table 2). In comparison with 1998 and 1999, the aggressiveness of the isolate applied in 2000 was significantly higher, which was accompanied by a higher infection of tested standards especially in the test of 18th July. Leaflets of cvs. Irys and Sokół were rated as susceptible in all tests (mean resistance scores varied form 1.0 to 3.4) indicating sufficient infection pressure for reliable characterization of resistance of assessed accessions.

Out of the 128 accessions tested over 3 years, 30 were highly resistant, with weighted mean scores varying from 7.0 to 9.0 in 1–9 grade scale. This group included 6, extremely resistant accessions (with 100 % resistant plants assessed as 9.0), 16 accessions scored from 8.0 to 8.9 (with >80 % of resistant plants), and 8 accessions scored from 7.0 to 7.9 (with >50 % of resistant plants). On the other extreme, 60 accessions were susceptible, scored <5.0 (with up to 100 % of susceptible plants) and the other 38 accessions were moderately resistant, with scores from 5.0 to 6.9 (with variable number of resistant plants) (Table 2).

Among species from the Central America, the highest resistance to P. infestans was observed for those belonging to taxonomic series Demissa Buk. All 11 accessions of S. demissum were highly resistant with mean scores from 8.1 to 9.0. Among them six accessions possessed 100 % of resistant plants classified from 7.0 to 9.0 (k-3341, k-3342, 4-3345, k-3362, k-4445 and k-8462). It was also true for one accession of S. guerreroense (species closely related to S. demissum) k-18407. A large percentage of plants scored at 7.0–9.0 grade was found in the six out of ten tested accessions of S. stoloniferum (k-2492, k-3360, k-3533, k-3554, k-4226 and k-8416). The weighted mean scores ranged from 6.8 to 8.6. An accession of S. antipovichii, a species closely related to S. stoloniferum (Hawkes 1990), also showed a high foliage resistance with mean score of 7.8 and a significant predominance of resistant plants. The accession of S. neoantipovichii (k-8505), another relative of S. stoloniferum, expressed more than 80 % of resistant plants in three tests performed and its mean score was 7.6. The mean score of the three S. verrucosum accessions tested ranged from 5.8 to 7.3, reflecting a moderate resistance, however the plants scored at 9.0 in each test indicates probability of the presence of R genes. The percentage of resistant plants depended on the test and ranged from 42 % to 78 %. Out of five assessed S. papita accessions four possessed high mean scores: 6.3 (k-16889), 6.5 (k-8816), 6.7 (k-16888) and 9.0 (k-9145). The remaining one accession was susceptible with mean score 3.7. The weighted mean values of resistance in four accessions of S. polytrichon ranged from 5.6 (k-15258) to 8.1 (k-7423) with increased frequency of resistant plants (from 46 % to 80 % in individual tests). The tested four S. cardiophyllum accessions were characterized by high scores (from 7.6 to 8.3) and over 80 % of resistant plants. Within the six accessions of S. pinnatisectum, the highest level of foliage resistance was observed in k-15253 and k-19157 accessions (scored as 6.3 and 6.5, respectively). The accessions k-9174 and k-4455 (scored 5.3 and 5.7, respectively) were found to be relatively resistant. In the remaining accessions, approximately less than half of inoculated plants were resistant. It is important to note in all tested segregating populations the presence of plants scored as 9, a possible indication of the presence of major genes in evaluated accessions.

Among the South American species, a high score was found in the following accessions: S. berthaultii k-23047 (6.4), S. microdontum k-20320 (7.8), S. simplicifolium k-5400 (7.5) S. ruiz-ceballosii k-7370 (7.2), S. vernei k-20330 (6.1) and in one accession of S. chacoense k-3679 (6.4) out of 16 ones tested. In populations of S. chacoence resistance was clearly segregating, since the frequency of resistant plants fluctuated from 0 % up to 60 %. Generally, the South American species were characterized by a lower frequency of highly resistant plants within populations as compared to the Central American accessions.

Tuber Resistance

The tuber resistance reaction of two susceptible standards - cultivars Irys and Sokół indicated sufficient infection pressure in all tests performed. The resistance reaction of the set of applied standards was similar in all three tests, hence the results of evaluation of Solanum accessions from each test might be compared (Table 3). Mycelial development reflected the resistance levels of the standards: the least mycelium growth was observed on tubers of two resistant breeding forms: breeding line DG 94-15 and cv. Meduza while abundant mycelium growth was observed on tubers of susceptible cvs. Irys and Sokół. The most prolific mycelium growth was noted on decapitated tubers of susceptible cv. Irys in all tests.

Tuber resistance data for 63 accessions of 35 species are shown in Table 3. All of the five tested accessions of S. pinnatisectum were characterized by a very high tuber resistance to P. infestans: the mean score varied from 7.7 to 8.5, without mycelium growth. Among the other Central American species, the high frequency of resistant individuals was found in the five accessions of S. cardiophyllum with mean scores ranging from 6.5 to 8.1. A lack of aerial mycelium was observed in four of its accessions. Abundant mycelium growth was observed in individual tubers of accessions k-18086 and k-19986.

In tests performed in Młochów a high frequency of tuber resistant plants was noted within the following accessions: S. berthaultii k-23047 (6.9), S. fendleri k-5747 (7.9), S. kurtzianum k-12479 (6.8) and k-12488 (7), S. neoantipovichii k-8505 (6.5), S. papita k-16888 (6.4) and k-16889 (6.8), S. polytrichon k-15258 (7.5), S. ruiz-ceballosii k-7370 (7.1) and k-7381 (6.5), S. stoloniferum k-2536 (6.4), S. trifidum k-4451 (7.9) and S. verrucosum k-10556 (6.5). Two accessions of S. acaule were characterized by relatively high and uniform tuber resistance, their disease ratings were 5.9 and 6.7. The accessions S. arrac-papa k-9742, S. demissum k-3355, S. hougasii k-8818, S. polytrichon k-5682, S. subtilius k-2064 and S. verrucosum k-4220 were found to be relatively resistant.

The mycelium growth was highly variable within the species and within the accessions (Table 3). The high variation in intensity of mycelium growth was noted within the accessions of S. papita, S. polytrichon and S. stoloniferum. Tubers of S. berthaultii k-23047, S. cardiophyllum k-16828, S. ruiz-ceballosii k-7370 and of all tested accessions of S. pinnatisectum reacted to inoculation with local lesions.

Resistance to Viruses

In total, 70 accessions of 21 species were evaluated for resistance to PVX and three strains of PVY (Table 4).

The accessions of: S. acaule k-9786, k-10678, k-18007, k-18021, k-18522; S. albicans k-9813; S. berthaultii k-23047 and S. sparsipilum k-9808 expressed resistance to PVX in all tested plants (Table 4). Predominance of plants resistant to PVX was found in two S. acaule accessions. In 12 accessions of several Solanum species the segregation for PVX resistance with predominance of susceptible plants was noted. Among them were single accessions of S. acaule, S. chacoense S. guerreroense, S. kurtzianum, S. demissum, S. parodii and S. stoloniferum.

Table 4 Resistance of wild potato species accessions from the VIR collection to Potato virus X (PVX) and Potato virus Y (PVY)
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Resistance to three strains of PVY was found in 9 out of 11 tested accessions of S. stoloniferum: k-2492, k-2534, k-2536, k-3326, k-3360, k-3527, k-3533, k-3554 and k-4226. A single accession of S. neoantipovichii (k-8505) was also characterized by resistance to three strains of PVY. In S. polytrichon, resistance to three strains of PVY was expressed in accession k-5682, and for two strains (PVY N WI and PVY O) in accession k-5347. Two tested accessions of S. papita were susceptible to both viruses.

The majority of accessions of S. demissum expressed higher susceptibility to PVX than to PVY. Out of 9 tested accessions only k-3540 was relatively resistant to PVY strains. It is worth noting that this group of accessions was susceptible to PVYNTN. The accession of S. guerreroense species belonging to a taxonomic series Demissa Buk., did react to PVX and PVY similarly to some S. demissum accessions.

The accession k-4220 of S. verrucosum showed a variable reaction to PVY strains similar to some accessions of S. demissium and S. guerreroense.

Six accessions of S. pinnatisectum were susceptible to PVX, though three of them (k-4455, k-4459 and k-19157) were found to be highly resistant to the three strains of PVY. All ten accessions of S. chacoense were susceptible to PVX and only one accession (k-4236) was resistant to three strains of PVY. Resistance to two PVY strains (PVY N WI and PVY O) was expressed by S. chacoense accessions: k-2731, k-2914, k-2917 and k-2926. The strong tendency to react with local necroses to PVY strains was noted in all S. chacosense accessions. The accessions of species: S. dolichostigma and S. gibberulosum, which are related to S. chacoense, expressed different levels of resistance to PVY and similarly to S. chacoense expressed hypersensitive responses to PVY isolates. The two accessions of S. dolichostigma k-7610 and k-7613 were extremely resistant to three PVY strains. The accession k-2937 of S. gibberulosum was found to be moderately resistant to PVYN WI and PVYNTN strains.

Discussion

The world germplasm collections are a great source of valuable traits for potato breeding. For a large number of wild potato accessions preserved in world gene banks, the data on resistance to pathogenes have been already obtained (Hanneman and Bamberg 1986; Zoteyeva 1986; Darsow and Hinze 1991a; Colon 1994; Bradshaw et al. 1995; Bamberg et al. 1996; Ramsay et al. 1999; Angeli et al. 2000). However, the resistance value of many collected accessions is still unknown.

Wild potatoes are known as reservoir of resistance genes to many diseases and pests. They are widely distributed in geographic zones differing in ecological and climatic conditions. The principal centers of widest species diversity are characterized either by large number of species and subspecies variation or by large number of major genes, including the genes coding for disease resistance. These centers are considered to be characterized by parallel distribution of specialized parasites co-evolving with the host plants (Vavilov 1926). Due to this theory, mutation rates and selection pressure lead to formation of resistance in local plant populations.

In many potato production regions significant losses from late blight are noted. Currently grown cultivars possess a narrow gene base for P. infestans resistance, and this coupled with an increase in the genetic variation of P. infestans populations creates increased risk of disease spread in potato crops. The use of new resistance sources would enrich the genetic background of cultivated potato.

The data obtained showed that stable high leaf resistance to P. infestans exists mostly in Central-American species. Using inoculum concentration of 50 sporangia/mm3, the high percentage of resistant plants (from 80–100 %) was registered in the accessions of S. antipovichii, S. ambosinum, S. demissum, S. guerreroense, S. neoantipovichii and several accessions of S. stoloniferum. In several accessions, namely S. guerreroense k-18407, S. papita k-9145 and seven accessions of S. demissum: k-3341, k-3342, k-3345, k-3355, k-3362, k-4445 and k-8462, the 100 % of tested plants expressed resistance.

Many wild potato species possess high variability for resistance to P. infestans. Variation for leaf resistance to late blight was found in the majority of 76 wild potato species from Central and South America evaluated in the field under epidemics conditions in Northwestern Russia and in laboratory studies conducted in the early 1980s (Zoteyeva 1986).

In this study a majority of the evaluated accessions were found segregating for leaflet resistance, while several accessions of S. demissum, and single accessions of S guerreroense, S. papita and S. stoloniferum were completely resistant. Accessions of Mexican species S. demissum, S. guerreroense and S. stoloniferum expressed full resistance also when inoculum concentration was 75 sporangia/mm3 (Zoteyeva 2000). The application of inoculum concentration of 50 sporangia/mm3 for resistance assessment could play an important role for the species with low and moderate foliar resistance (i.e. S. chacoense, S. kurtzianum, S. spegazzinii and many others) in the differentiation of their accessions for resistance to late blight.

In research for late blight resistance, little attention has been paid to tuber resistance, although several authors recognize that it is an essential component of potato resistance to P. infestans (Darsow 1987; Świeżyński and Guzowska 2001). The opinion that cultivars with foliage resistance minimize the risk of tuber blight was the reason that interest in foliage resistance to late blight persisted. However, even slightly blighted foliage during the growing season can lead to a high proportion of infected tubers (Schwinn and Margot 1991). There are limited data for the assessment of Solanum species for tuber resistance. Among 128 tuber-bearing Solanum species studied by Darsow and Hinze (1991b) nearly 60 were found exhibiting foliage resistance. Within these species only 27 were found with tuber resistance.

For evaluation of the tuber resistance to P. infestans, the methods of whole tubers and tuber slice inoculation are usually used (Dorrance and Inglis 1998; Stewart et al. 1996). To evaluate the tuber resistance to P. infestans in wild potatoes characterized by small tuber size, we have developed a method of inoculation of decapitated tubers, which was applied in our study (Zoteyeva and Guzowska 2004). Among 35 species represented by 63 accessions, the accessions of S. cardiophyllum and S. pinnatisectum were those with the highest frequency of tuber resistant individuals. Variation for tuber resistance to P. infestans within accession was observed in all species tested with the exception of S. pinnatisectum. Accessions with resistant individuals were observed in S. acaule, S. berthaultii, S. kurtzianum, S. ruiz-ceballosii, S. fendleri and S. trifidum.

In potato, foliar and tuber susceptibility to late blight are not correlated. It is necessary to find new genetic sources of resistance either in leaves or in tubers (Kirk et al. 2001). Among the large number of accessions tested, several (S. berthaultii k-23047, S. cardiophyllum k-16828, S. ruiz-ceballosii k-7370, S. neoantipovichii k-8505 and S. pinnatisectum) contained individual clones with combined resistance expressed in leaves and in tubers. These accessions are promising breeding materials, possessing P. infestans resistance and good tuberization ability.

Evaluation of VIR’s collection for late blight and PVX and PVY resistance has resulted in the identification of accessions combining high resistance to P. infestans and to the viruses. The selected accessions of S. demissum, S. guerreroense, S. neoantipovichii, S. polytrichon and S. stoloniferum, are combining resistance to P. infestans and two or three strains of PVY and/or to PVX. These forms might be of special value for prebreeding purposes.

Testing the set of 66 accessions of 20 Solanum species for resistance to three strains of PVY is of particular value. Assessment of this gene pool for resistance to the recently found PVYNTN strains, which evoke Potato Tuber Necrotic Ringspot Disease on tubers in potato crops in Europe and other continents, has shown that several accessions resistant to older PVY strains (PVYO and PVYNW) are susceptible to PVYNTN. Among them were accessions of S. demissum, S. chacoense, S. guerreroense, S. polytrichon and S. verrucosum. It might be an indication of the virulence of this strain and possible breakdown of field resistance of currently grown cultivars. However, sources of resistance against PVYNTN were identified in accessions of S. stoloniferum, S. pinnatisectum and S. neoantipovichii and in selected accessions of S. chacoense, S. dolichostigma and S. polytrichon.

In the past decade, several accessions evaluated in this study from 1998 to 2000 have been exploited in genetic studies and prebreeding program conducted at Młochów Research Center, IHAR-PIB (Jakuczun and Wasilewicz – Flis 2004) and at the Swedish University of Agricultural Sciences (SLU) in collaboration with VIR’s Genetics Department (Zoteyeva and Carlson-Nilsson 2011). Recently, the accession k-5763 of S. michoacanum was the parental line for a population used in mapping the P. infestans resistance gene Rpi-mch1 on potato chromosome VII (Śliwka et al. 2012a). The Rpi- rzc1 gene was mapped to chromosome X by using accession k-7370 of S. ruiz-ceballosii as a parent of a mapping population. In addition, the first two maps of Solanum species with DArT markers were constructed (Śliwka et al. 2012b), providing proof of the ample possibilities of exploitation of the described pool of Solanum germplasm.