Phenotypes ofPhytophthora infestans in Nepal: mating types and metalaxyl sensitivity

Potato Research 44 (2001) 337-347

Phenotypes of Phytophthora infestans in Nepal: mating types and metalaxyl sensitivity

S.R. GHIMIRE, K.D. HYDE, I.J. HODGKISS and E.C.Y. LIEW

Centre for Research in Fungal Diversity, Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR, China

Accepted for publication: 23 November 2001

Additional keywords: fungicide resistance, phenylamide fungicides, potato late blight, Solanum tuberoston L.

Summary

Potato late blight has appeared in epidemic proportions in Nepal since the mid 1990s and fungicides have been reported to be decreasingly effective in managing the disease. Phytophthora infestans isolates were collected from potato crops during 1999-2000 and analysis of 371 isolates for mating types and 270 isolates for metalaxyl sensitivity revealed the presence of both new and old populations. This is the first report on the presence of metalaxyl-resistant isolates in Nepal. The frequencies of A1 and A2 were 83 and 17%, respectively. Metalaxyl- resistant, intermediate and sensitive isolates were recorded as 10%, 12% and 78% respectively. Metalaxyl resistance was distributed in both mating types. Sites with a history of meta'laxyl use had a significantly higher number of resistant and intermediate isolates (~-~=20, P<0.01) than sites where metalaxyl had not been used. This study confirms the changes in population structure of P. infestans in Nepal.

Introduction

Late blight, caused by the Oomycete Phytophthora infestans, is one of the most devastating diseases of potato (Solarium tuberosum) and tomato (Lycopersicon esculentum) worldwide (Hooker, 1981). Phytophthora infestans is a heterothallic organism with two distinct mating types, A1 and A2, and pairing between isolates of opposite mating types is required for sexual reproduction. In the absence of compatible mating types the organism reproduces asexually. Until the early 1980s both mating types were present only in Mexico and populations elsewhere were exclusively of A1 mating type (Fry & Spielman, 1991). Such geographical isolation of compatible mating types outside Mexico restricted the fungus to asexual reproduction. The first report on the presence of A2 mating type outside Mexico was from Switzerland (Hohl & Iselin, 1984). Subsequently it was also reported from Asia, Canada, Europe, Middle East, South America and USA (Spielman, 1991). In Asia the A2 mating type has been reported in Japan (Mosa et al., 1989), India (Singh et al., 1994), Korea (Koh et al., 1994), China (Zhang et al., 1996), Nepal (Shrestha et al., 1998), Indonesia, Thailand (Nishimura et al., 1999) and Pakistan (Mirza & Ahamad, 1999).

Fungicides have always been a major component in late blight management strategy. Since the discovery of phenylamide fungicides in 1977, metalaxyl has been used widely in late blight management due to its excellent protective, curative and

Potato Research 44 (2001) 337

S.R. GHIMIRE, K.D. HYDE, l.J. HODGKISS AND E.C.Y. LIEW

eradicative antifungal activities (Schwinn & Staub, 1987). However, metalaxyl- resistant isolates of P. infestans were recorded within a few seasons of metalaxyl application, and a declined level of disease control has been experienced. Metalaxyl- resistant isolates was first detected in field-grown potatoes in Ireland, The Netherlands and Switzerland in 1980, and since then resistant isolates have also been documented in various countries in Asia, Central America, Europe, Far East, Middle East and North America (Gisi & Cohen, 1996).

In Nepal, late blight is one of the most important biotic constraints to potato production. The disease has appeared in epidemic proportions since the mid 1990s, and a reduced effectiveness of fungicides has been observed. Such a change in epidemiological pattern and reduction in fungicide efficiency could be indicative of changes in the P. infestans populations. Ghimire et al. (2001) characterized 251 isolates for race composition among the isolates used in this study and revealed the presence of 30 different races with variable virulence factors ranging from none to seven per isolate. The population from the hills was the most diverse followed by terai (a part of the Indo-Gangetic plains) and the high hill populations. Gleason and Shannon diversity indices indicated a moderate level of diversity in the overall Nepalese P. infestans population, and the virulence complexities were minimal (Ghimire et al., 2001). However, it was not possible to make any inference regarding changes in pathogen population based on the race composition study. Since mating type and metalaxyl sensitivity tests have been very useful markers worldwide to provide first hand information on the changes in P. infestans populations, this study characterizes Nepalese isolates for these markers as a part of a larger investigation into understanding the population structure of P. infestans in Nepal.

Materials and methods

Nepal consists of three agro-ecological regions: terai (100-300 m asl), hills (300-1600 m asl) and high hills (>1600 m asl). Based on the amount of annual precipitation, each region can be subdivided into three zones, i.e. low, medium and high rainfall areas. These factors play an important role in the potato production system in the country. A sampling frame of 3x3 matrix was devised and one potato-growing site was randomly selected from each grid for this study.

Sample collection and isolation. Infected potato leaf samples were collected from nine potato-growing sites across the country during the 1999-2000 growing seasons. Samples were collected from Bardiya, Chitwan and Morang for terai, Dhankuta, Hemja and Surkhet for the hills, and Baitadi, Ghandruk and Terathum for the high hills (Fig. 1). Potatoes are grown in summer in the high hills and as a winter crops in the hills and terai. Five to eight farms were sampled from each sampling site. Information on fungicides used against late blight was recorded for each site. Infected leaf pieces including a healthy portion were surface sterilized in 1% sodium hypochlorite solution for 1 min followed by 2-3 rinses with sterile distilled water. Leaf pieces were then plated on Rye A agar amended with 100 mg I "l vancomycin, 50 mg 1-1

338 Potato Research 44 (2001)

P HE NOT YPE S OF P H Y T O P H T H O R A I N F E S T A N S IN N E P A L

N

INDIA

Fig. 1. Map of Nepal showing sampling sites (o).

rifampicin and 100 mg 1-1 pimaricin. Inoculated plates were incubated in the dark at 18 ~ for a week. Fungal growth was then transferred to Rye A agar by sub-culturing single hyphal tips for genetic purity. Thirty-five to forty-five isolates per site were recovered to yield a total number of 371 isolates. These isolates were maintained on Rye A at 18 ~ under sterile distilled water by repeated revival every 4-6 months.

Mating type determination. Three hundred and seventy one isolates were tested for mating type. The number of isolates per site ranged from 35 to 45 for the test. P. infestans isolates, CBS 429.90 (A2) from the Centraal Bureau voor Schimmelcultures (CBS), The Netherlands and DKT 35 (A1) from Nepal were used as references (tester isolates) for mating type determination. Mating type of each isolate was determined by placing an agar strip of 15x5 mm containing mycelium on one half of a Rye A agar plate (Caten & Jinks, 1968) and a colony of the same size of the tester isolate on the other half of the plate 45-50 mm apart. All isolates were individually tested against each tester isolate in a separate plate, and were also tested for self- fertility by mating on its own. The test was conducted with 2 replications. Cultures were examined microscopically for the presence or absence of oospores after 10-16 days incubation at 18 ~ in the dark. Isolates forming oospores when paired with the A2 tester were designated as A1 mating type, and those forming oospores when paired with the A1 tester were designated as A2 mating type. Isolates forming oospores with either tester isolate as well as with themselves were designated self- fertile. Mating type data were analyzed using the chi-square test.

Metalaxyl sensitivity test. A radial growth assay on metalaxyl-amended Rye A agar was used to determine metalaxyl sensitivity levels since the method is operationally easier and known to provide equivalent results to the floating leaf disk method (Matuszak et al., 1994). Two hundred and seventy isolates, ranging from 28-31 isolates/site, were tested for metalaxyl sensitivity. Metalaxyl stock solution (100 mg ml l ) was prepared by dissolving metalaxyl in pure di-methylsulfoxide (DMSO). Rye


S.R. GHIMIRE, K.D. HYDE, I.J. HODGKISS AND E.C.Y. LIEW

A agar was amended with metalaxyl in DMSO to a final concentration of 5 or 100 mg 1 -l when autoclaved media cooled to 50 ~ The control was amended with DMSO without metalaxyl, and each treatment had the same amount of DMSO. Each isolate was plated on fungicide-amended and non-amended Rye A agar plates with 8 mm plugs taken from the edge of an actively growing colony. The experiment was replicated three times. Radial growth of the fungus in each plate was measured by taking the average of two perpendicular measurements of colony diameter after three weeks incubation at 18 ~ in the dark and the value was corrected by subtracting 8 mm for the diameter of the plug. The radial growth measurement on slow growing isolates was performed when colony diameter on the control treatment reached at least 30 mm. Relative radial growth (as percent of control) was determined for each isolate for both metalaxyl concentrations. Based on the relative growth responses, isolates were categorized as sensitive (relative growth <40% at 5 and 100 mg 1-1), intermediate (relative growth >40% at 5 mg 1 -l and <40% at 100 mg I" l) and resistant (relative growth >40% at 5 and 100 mg !1). This classification is similar to those described by Dagget et al. (1993) and Therrien et al. (1993). Chi- square analysis was performed to test the distribution pattern of metalaxyl sensitivity among agro-ecological regions and to test the independence of metalaxyl use on the number of isolates in the sensitivity classes.

Results

Both mating types were present in isolates collected during the 1999-2000 growing seasons. The proportions of A1 and A2 types were 83% and 17%, respectively. Occurrence and distribution of mating types in various agro-ecological regions and study sites are presented in Table 1. Seven sites had only the A1 mating type, one site had only A2 and one site had both A1 and A2 mating types in approximately equal proportions. There was a considerable difference (X2=46, P<0.01) in the distribution of A1 and A2 among the agro-ecological regions. No A2 was recorded from the terai (Table 1) and none of the isolates tested was self-fertile. All A2 isolates exhibited a distinct colony morphology and lumpiness in Rye A agar as did those observed by Shattock et al. (1990). Such lumpiness was due to aggregation of dichotomously branched aerial hyphae.

The test isolates varied greatly in the degree of growth inhibition caused by metalaxyl (Figs 2 and 3), and all three sensitivity types, resistant, intermediate and sensitive isolates were recorded. The frequency of these isolates was 10%, 12% and 78%, respectively. This is the first report on the presence of metalaxyl-resistant P. infestans isolates in Nepal. About 36% test isolates had a relative growth rate of less than 20% of their control at 5 mg 1 1, whereas the frequency increased to 53% for the same relative growth rate (<20% of their control) in the higher concentration (100 mg l-l). None of the isolates had a relative growth rate of more than 80% of their control in both concentrations (5 mg 1 -l and 100 mg l-l). Metalaxyl resistance was distributed in both mating types. The frequency of A1/A2 mating types among the isolates tested for metalaxyl sensitivity was 81% and 19% respectively. Metalaxyl


P H E N O T Y P E S O F P H Y T O P I t T H O R A I N F E S T A N S IN N E P A L

Table 1. Distribution of P. infestans mating types in different agro-ecological regions and sites of Nepal (1999-2000).

Agro-ecological regions and sites Mating type frequency

A1 A2

Terai Bardiya 42 0 Chitwan 45 0 Morang 37 0

Sub total 124 0

Hills Dhankuta 35 0 Hemja 18 22 Surkhet 43 0

Sub total 96 22

High Hills Baitadi 45 0 Ghandruk 43 0 Terathum 0 41

Sub total 88 41

Total 308 63 Chi-square value ~t at 2dr: 45.6 Hs

Number of isolates tested

124

118

129

371

aValue to compare regional differences in mating type distribution.

70 60

50 0

--~ 40

~ 30 ,I0

~ 20 z 10

5 15

H J

25 35 45

Growth as percent of control

" ] , El, IS] 55 65 75

i

85 95

Fig. 2. Relative radial growth of 270 Nepalese isolates of Phytophthora infestans on agar with 5 mg 1 "j of metalaxyl expressed as percentage of growth on agar medium without metalaxyl. Histogram bar represent 0-10%, 11-20%, 21-30%, 31-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90% and 91-100% of control values.



90

80

70

, - 4 0

10

5 15

I-I I-I __ i J i i i i i i

25 35 45 55 65 75 85 95

Growth as percent of control

Fig. 3. Relative radial growth of 270 Nepalese isolates of Phytophthora infestans on agar with 100 mg 1-1 of metalaxyl expressed as percentage of growth on agar medium without metalaxyl. Histogram bar represent 0-10%, 11-20%, 21-30%, 31-40%, 41-50%, 51--60%, 61-70%, 71-80%, 81-90% and 91-100% of control values.

sensitivity distribution frequencies for two mating types were 16%/84% and 46%/54% for Alr /Als and A2r/A2s, respectively.

Five sites (Morang, Chitwan, Hemja, Bardiya and Terathum) had records of metalaxyl application whereas the other four sites (Baitadi, Dhankuta, Ghandruk and Surkhet) had no such records. Metalaxyl-resistant isolates (resistant and/or intermediate) were recorded from all sites except Ghandruk. The frequency of metalaxyl-resistant isolates per site ranged from 0% to 57% (Table 2). Sites having metalaxyl use usually had a higher number of metalaxyl resistant isolates than those sites without metalaxyl application (Table 3). The test of independence between sites with metalaxyl application and no metalaxyl application showed a significant (P<0.01) effect of metalaxyl on the number of isolates in sensitivity classes. The distribution pattern of metalaxyl sensitivity characteristics among agro-ecological regions was not statistically significant (P>0.05) (Table 2).

Discussion

Mating type data show the presence of three distinct populations of P. infestans in Nepal. Seven sites had the A1 mating type, one site had the A2 mating type and one site had both types in approximately equal proportions. Therefore, this study shows that the pathogen reproduces mostly asexually in Nepal. In Hemja, where both mating types were present in equal proportions, there was a possibility of sexual reproduction. The presence of the A2 mating type in some study sites poses a high risk of moving compatible mating types among the potato growing areas in the country, which in turn may lead the pathogen to reproduce sexually.

The presence of the A2 mating type of P. infestans in Nepal was reported in 1998,


PHENOTYPES OF PHYTOPHTHORA INFESTANS IN N E P A L

Table 2. Occurrence and distribution of metalaxyl sensitivity of P. infestans isolates in different agro-ecologicai regions and sites in Nepal (1999-2000).

Agro-ecological regions & sites Sensitivity classes

Resistant Intermediate Sensitive

Terai Bardiya 7 7 16 Chitwan 0 5 25 Morang 5 0 26

Sub total 12 12 67

Hills Dhankuta 2 0 28 Hemja 1 6 23 Surkhet 2 5 24

Sub total 5 11 75

High Hills Baitadi 0 2 28 Ghandruk 0 0 28 Terathum 9 8 13

Sub total 9 10 69

Total 26 33 211 Chi-square value a 4dr: 3.42 Ns

Number of isolates tested

91

91

88

270

aValue to compare regional differences in metalaxyl sensitivity distribution.

Table 3. Metalaxyl-sensitivity distributions in P. infestans populations in Nepal between sites with and without metalaxyl application history (1999-2000).

History of metalaxyl use Response to metalaxyl Number of isolates tested

Resistant Intermediate Sensitive

With metalaxyl (5 sites) a 22 26 103 151 No metalaxyl (4 sites) b 4 7 108 119

Total: 26 33 211 270 Chi-square value at 2df: 20.0 Ils

aMorang, Chitwan, Hemja, Bardiya & Terathum. bBaitadi, Dhankuta, Ghandruk & Surkhet.

with 6% of isolations in 1996 and 42% in 1997 (Shrestha et al., 1998). The percentage of the A2 mating type (17%) in this study was between those observed in 1996 and 1997, but considerably lower than for 1997. The difference between these two studies was possibly due to differences in sampling. In the current study, sampling was made across the country, whereas in 1997 most of the samples (85%) were from central and



eastern Nepal (Nishimura et al., 1999). Such variations in the proportions of A2 types have been reported from several countries, e.g. Hawaii (Ho & Ko, 1999), Japan (Kato et al., 1998) and Poland (Sujkowski et al., 1994). Differences in the relative fitness of genotypes, the introduction of fitter genotypes and associated displacement of existing ones and the method of sampling could be responsible for variations in mating type frequency.

Regional differences were noted in the percentage of the A2 mating type with the highest frequency in the high hills (31%) followed by the hills (19%); A2 was absent in the terai. Shrestha et al. (1998) also reported A2 mostly from the high hills. A similar study conducted in India showed that all isolates from Indo-Gangetic plains were A1 (Singh et al., 1994). Shrestha et al. (1998) speculated that the A2 mating type might have migrated to Nepal with Andean potatoes (Solanum tuberosum var. andigena) recently introduced from Latin America. These potatoes are better adapted to cooler climates and hence their distribution in Nepal is limited to the hills and high hills. The distribution of A2 in Nepal seems to be dependent on host species distribution.

Chang & Ko (1990) demonstrated mating type conversion in P. infestans and P. parasitica on exposure to metalaxyl. In vitro induction of oospores and mating type change in P. infestans upon exposure to some commercial fungicides have recently been reported (Goves & Ristaino, 2000). Since in both of our study sites where A2 was reported have high metalaxyl use, 17 metalaxyl-resistant isolates of both mating types were tested in different metalaxyl concentrations for two months. However no evidence of fungicide-induced oospore formation in these isolates was found (unpublished data).

Displacement of the old populations by immigrant populations has been reported in P. infestans (Spielman, 1991; Koh et al., 1994; Sujkowski et al., 1994). New populations belonging to the A2 mating types were claimed to be more pathogenic and hence fitter than old ones (Gisi & Cohen, 1996). The virulence testing of Nepalese P. infestans isolates revealed the presence of higher numbers of virulence factors in A2 isolates compared with A1 isolates (Ghimire et al., 2001). A high proportion of A2 at Hemja and a monotypic A2 population at Terathum could be the result of a more virulent and fitter A2 than the native A1. Although previous data on mating type distribution for Hemja and Terathum are not available, it is likely that a gradual displacement of A1 by A2 is taking place at Hemja and such a displacement process could possibly have been completed at Terathum.

The major significance of co-presence of compatible mating types in P. infestans is sexual reproduction. Based on this study it is hard to establish whether the pathogen is reproducing sexually at Hemja, but it is apparent that sexual reproduction was not common at Terathum even during the displacement process. A small proportion of A1 isolates should otherwise have been detected at Terathum. The ploidy levels between A1 and A2 mating types in Nepal could be different (Tooley & Therrien, 1987), which would explain the low level of sexual reproduction, if at all present.

This study detected metalaxyl-resistant isolates (10%), and this is the first report on their presence in Nepal. Mancozeb and copper-based formulations have been in


PHENOTYPES OF PHYTOPHTItORA INFEST'INS IN N E P A L

extensive use in Nepal for many years to manage various plant diseases including potato late blight. Metalaxyl appeared in the Nepalese pesticide market in 1995 but became popular among potato growers in Nepal only after the nationwide blight epidemics of 1996. Due to the high cost of metalaxyl formulation (7- to 10-fold compared with mancozeb and copper fungicides) its use is still mainly restricted to commercial potato growers. A low proportion of resistant isolates in 1999-2000 is therefore not unexpected.

Metalaxyl-resistant and/or intermediate isolates were recorded from all sites but one, irrespective of metalaxyl use. It is not known whether metalaxyl-resistant isolates in these sites resulted from recent immigration of resistant genotypes or from the simultaneous occurrence of mutation due to selection pressure at different sites. However relationships were observed between the selection pressure and the appearance of metalaxyl-resistant isolates (Table 3). Since asexual reproduction is most probably the main form of reproduction, the number of spores and their rate of production are high enough to generate resistant isolates very rapidly.

The frequency of ALIA2 tested for metalaxyl sensitivity in this study was very close to that from Germany (Dagget et al., 1993), Poland (Therrien et al., 1993) and The Netherlands (Fry et al., 1991). However the metalaxyl-sensitivity distribution between mating types in Nepalese populations differed greatly from the average frequencies of these overseas populations (Gisi & Cohen, 1996). In many E1]ropean countries, with a high frequency of metalaxyl-resistant isolates, very few or no A2 isolates have been detected. By contrast, in Hawaii (Ho & Ko, 1999), Korea (Koh et al., 1994), Mexico (Deahl et al., 1995) and USA (Goodwin et al., 1996), with a high frequency of metalaxyl-resistant isolates, few or no A1 isolates have been detected. Based on mating type frequencies, the Nepalese P. infestans population is also similar to the European populations, although it is similar to non-European populations based on metalaxyl-sensitivity distribution between mating types. Further analysis of Nepalese isolates using molecular markers is required to establish their relationships with the isolates from Europe and the rest of Asia. It would also be interesting to trace the migration pathway of the pathogen in this region.

Analysis of isolates for mating type and metalaxyl sensitivity reveals the presence of new populations of P. infestans in Nepal. Co-presence of compatible mating types shows the possibility of this pathogen reproducing sexually in Nepal. Migration of compatible mating types among the potato-growing sites within and between the regions may provide the opportunity to undergo sexual reproduction. Such migration is more likely in Nepal due to the lack of domestic quarantine that restricts the movement of potato planting material. Hence the possibility of sexual reproduction in many locations in the near future should not be underestimated. Presence of metalaxyl-resistant isolates in most study sites indicates their wide distribution in Nepal. Although the current overall frequency of such isolates is low in Nepal it may increase within a short period if the current trend of metalaxyi use is continued. It would be wise to update fungicide use strategies against late blight based on the available information.


S.R. GHIMIRE, K.D. HYDE, I.J. HODGKISS AND E.C.Y. LIEW

Acknowledgements

This is part of a collaborative project between The University of Hong Kong and The Nepal Agricultural Research Council. Support from the Hong Kong Research Grant Council and Hill Agricultural Research Project, Nepal is gratefully acknowledged. Thanks are due to the farmers, extension workers and researchers who provided their precious support in the collection of isolates.

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Phenotypes ofPhytophthora infestans in Nepal: mating types and metalaxyl sensitivity