Sensitivity of Phytophthora infestans to flumorph: in vitro determination of baseline sensitivity and the risk of resistance

The sensitivity of 127 Phytophthora infestans isolates to flumorph was determined in 2003 and 2004. The isolates originated from two geographical regions and showed similar levels of sensitivity in both years. Baseline sensitivities were distributed as a unimodal curve with EC₅₀ values for growth of mycelia ranging from 0·1016 to 0·3228  µ g mL⁻¹, with a mean of 0·1813 (± 0·0405) µ g mL⁻¹. There was no cross-resistance between flumorph and metalaxyl. Laboratory studies were conducted to evaluate the risk of P. infestans developing resistance to flumorph. Mutants resistant to metalaxyl or flumorph were obtained by treating mycelium of wild-type isolates with ultraviolet radiation. Metalaxyl-resistant mutants were obtained with a high frequency and exhibited resistance factor values (EC₅₀ resistant/EC₅₀ sensitive phenotypes) of more than 100, while flumorph-resistant mutants were obtained at much lower frequencies and had very small resistance factors (1·5–3·2). There was cross-resistance between flumorph and dimethomorph, but not with azoxystrobin or cymoxanil. Most flumorph-resistant mutants showed decreases in hyphal growth in vitro and in sporulation both in vitro and on detached leaf tissues. These studies suggested that the risk of resistance developing was much lower for flumorph than metalaxyl. However, as P. infestans is a high-risk pathogen, appropriate precautions against resistance development should be taken.     

Assessing the risk of resistance in Pseudoperonospora cubensis to the fungicide flumorph in vitro

BACKGROUND: The oomycete fungicide flumorph is a recently introduced carboxylic acid amide (CAA) fungicide. In order to evaluate the risk of developing field resistance to flumorph, the authors compared it with dimethomorph and azoxystrobin with respect to the ease of obtaining resistant isolates to these fungicides, the level of resistance and their fitness in the laboratory. RESULTS: Mutants with a high level of resistance to azoxystrobin were isolated readily by adaptation and UV irradiation, and their fitness was as good as that of the parent isolates. Attempts to generate mutants of Pseudoperonospora cubensis (Burk. & MA Curtis) Rostovsev resistant to flumorph and dimethomorph by sporangia adaptation on fungicide-treated leaves were unsuccessful. However, moderately resistant mutants were isolated using UV mutagenesis, but their resistance level [maximum resistance factor (MRF) < 100] was much lower than that of the azoxystrobin-resistant mutant (MRF = 733). With the exception of stability of resistance, all mutants showed low pathogenicity and sporulation compared with wild-type isolates and azoxystrobin-resistant mutants. There is cross-resistance between flumorph and dimethomorph, suggesting that they have the same resistance mechanism. CONCLUSION: The above results suggest that the resistance risk of flumorph may be similar to that of dimethomorph but lower than that of azoxystrobin and can be classified as moderate. Thus, it can be managed by appropriate product use strategies.     

Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan

A total of 651 isolates of cucumber corynespora leaf spot fungus ( Corynespora cassiicola ) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC₅₀) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL⁻¹ and from 0·04 to 0·59 μg mL⁻¹, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL⁻¹. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC₅₀ values ranging from 1·1 to 6·3 μg mL⁻¹, and a very highly resistant (VHR) group consisting of 25 isolates with EC₅₀ values higher than 24·8 μg mL⁻¹. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.     

Resistance mechanism to carboxylic acid amide fungicides in the cucurbit downy mildew pathogen Pseudoperonospora cubensis

BACKGROUND: Pseudoperonospora cubensis, the causal oomycete agent of cucurbit downy mildew, is responsible for enormous crop losses in many species of Cucurbitaceae, particularly in cucumber and melon. Disease control is mainly achieved by combinations of host resistance and fungicide applications. However, since 2004, resistance to downy mildew in cucumber has been overcome by the pathogen, thus driving farmers to rely only on fungicide spray applications, including carboxylic acid amide (CAA) fungicides. Recently, CAA‐resistant isolates of P. cubensis were recovered, but the underlying mechanism of resistance was not revealed. The purpose of the present study was to identify the molecular mechanism controlling resistance to CAAs in P. cubensis. RESULTS: The four CesA (cellulose synthase) genes responsible for cellulose biosynthesis in P. cubensis were characterised. Resistant strains showed a mutation in the CesA3 gene, at position 1105, leading to an amino acid exchange from glycine to valine or tryptophan. Cross‐resistance tests with different CAAs indicated that these mutations lead to resistance against all tested CAAs. CONCLUSION: Point mutations in the CesA3 gene of P. cubensis lead to CAA resistance. Accurate monitoring of these mutations among P. cubensis populations may improve/facilitate adequate recommendation/deployment of fungicides in the field. Copyright © 2011 Society of Chemical Industry     

Biological and genetic characterization of Phytophthora capsici mutants resistant to flumorph

Baseline sensitivity to flumorph, a carboxylic acid amide (CAA) fungicide used to control some oomycetes, was examined using 83 Phytophthora capsici isolates, resulting in a unimodal distribution of effective concentration for 50% inhibition of mycelial growth ranging from 0·716 to 1·363, with a mean of 1·033 ± 0·129 μg mL^?1. To assess the potential risk of developing flumorph resistance, 13 flumorph‐resistant mutants of P. capsici were obtained using ultraviolet irradiation. Most of these mutants and their progeny had high levels of fitness, including mycelial growth, sporulation and virulence. The resistance to flumorph changed slightly, either increasing or decreasing, after 10 transfers on agar media. Cross‐resistance was found between flumorph and other CAA fungicides (dimethomorph and iprovalicarb), but not between flumorph and non‐CAA fungicides (cymoxanil, metalaxyl, azoxystrobin and cyazofamid). To investigate the genetics of the flumorph resistance, 619 progeny were obtained by self‐crossing and sexual hybridization. Segregation of sensitivity to fungicide was measured as a ratio of sensitive (S) to resistant (R) isolates. Segregation of the progeny, from self‐crossed isolate PCAS1 (flumorph resistant), was 1:15 in the first generation; and 0:1 or 1:15 in the second generation. In sexual hybridization, segregation of progeny was 0:1 and 1:7 for R × R hybridization; and 1:3 for R × S hybridization. Therefore, the resistance of P. capsici against flumorph was controlled by two dominant genes.     

Metalaxyl resistance in Phytophthora infestans on greenhouse tomatoes in Greece

In winter/spring 1984 resistance to metalaxyl developed in Phytophthora infestans within tomato greenhouses in Preveza, Greece. The appearance of resistant isolates followed continuous applications of metalaxyl and prevalence of weather conditions favourable for late blight. The majority of resistant isolates grew on tomato leaf discs floated on solutions containing 100 μg/ml metalaxyl whereas sensitive isolates were inhibited at concentrations of 0.1 μg/ml.
In trials within polyethylene tunnels metalaxyl had no effect on the development of the late blight induced by resistant strains but both a mixture of this fungicide with mancozeb and mancozeb alone, gave satisfactory control of the disease when applied in a 7-day schedule.     

六种杀菌剂对黄瓜霜霉病菌不同发育阶段的影响

用Calcofluor荧光染色和显微观察的方法研究了6种常用杀菌剂对黄瓜霜霉病菌Pseudoperonospora cubensis不同发育阶段的影响。结果表明:烯酰吗啉和氟吗啉对黄瓜霜霉病菌孢子萌发、芽管伸长、附着胞形成、菌丝扩展、孢囊梗和孢子囊的形成具有明显的抑制作用,其EC50值分别在0.07~5.98 μg/mL和0.10~3.50 μg/mL之间,而对游动孢子释放、游动孢子游动和休止孢形成没有抑制作用;霜脲氰对休止孢萌发和芽管伸长的EC50值分别为1.10和0.002 μg/mL,对其他阶段无明显的抑制效果;腈嘧菌酯对休止孢萌发、菌丝扩展和孢囊梗产生无影响,对游动孢子释放、芽管伸长和附着胞形成的EC50值分别为0.018、0.008和0.010 μg/mL;代森锰锌对游动孢子释放、休止孢萌发和芽管伸长的EC50值分别为2.40、2.88和1.53 μg/mL;甲霜灵对该菌的整个发育期均没有影响,这可能是因为供试菌株对甲霜灵已经产生了抗药性。     

黄瓜霜霉病菌抗氟吗啉菌株的适合度研究

 采用离体叶盘法和活体植株法,测定了从田间分离获得的对氟吗啉具有抗药性的黄瓜霜霉病菌菌株和通过室内紫外线诱变获得的对氟吗啉及对照药剂烯酰吗啉和嘧菌酯产生抗药性的突变菌株的生存适合度。试验结果表明,室内诱导获得的氟吗啉和烯酰吗啉抗药性突变菌株的适合度明显低于敏感菌株和嘧菌酯抗药性突变体及田产氟吗啉抗药性菌株,主要表现在菌株的侵染率、产孢能力和致病力的减弱。室内获得的氟吗啉和烯酰吗啉抗药性突变体和敏感菌株混合接种、继代培养后,其抗药菌株的检出率在混合群体中逐渐降低,其中抗性突变体FR4和敏感菌株以2:8的比例混合接种、培养7代后,其无性后代中检测不到抗药性菌株的存在,表明室内氟吗啉抗药性突变体竞争力弱于敏感菌株;而田间氟吗啉抗药性菌株和室内嘧菌酯抗药性突变体则表现出和敏感菌株相似或更强的竞争力,预示田间自然情况下抗药性菌株存在着可能形成优势菌群而导致药剂防治失败的潜在风险。     

山东省和河北省黄瓜霜霉病菌对烯酰吗啉及双炔酰菌胺的抗性监测

 为明确黄瓜霜霉病菌对烯酰吗啉及双炔酰菌胺的抗性时空动态及这两种药剂对黄瓜霜霉病的田间防效,采用叶盘漂浮法检测了2011~2016年采自河北省和山东省黄瓜主产区的霜霉病菌1 821个菌株对烯酰吗啉及双炔酰菌胺的敏感性,并采用茎叶喷雾法对这两种药剂的田间防效进行评估。结果表明:黄瓜霜霉病菌1 821个菌株对烯酰吗啉和双炔酰菌胺抗性频率分别为88.5%和34.3%,供试菌株中对烯酰吗啉的低抗菌株及对双炔酰菌胺的敏感菌株占优势,平均抗性倍数分别为8.92和2.44,抗性指数分别为0.42和0.27。黄瓜霜霉病菌对这两种药剂的抗性频率、抗性倍数及抗性指数随着监测年限及地域的不同而波动。在河北定兴和山东寿光日光温室进行的田间药效试验中,按照药剂田间推荐剂量喷施4次,50%烯酰吗啉可湿性粉剂和250 g·L^-1双炔酰菌胺悬浮剂对黄瓜霜霉病表现出良好的防效(85%以上),与两地黄瓜霜霉病菌对烯酰吗啉产生低抗、对双炔酰菌胺敏感的监测结果一致。由此推断,50%烯酰吗啉可湿性粉剂和250 g·L^-1双炔酰菌胺悬浮剂可在对甲霜灵(或精甲霜灵)、噁霜灵及嘧菌酯普遍产生抗性的地区,作为58%甲霜灵·代森锰锌WP、68%精甲霜灵·代森锰锌WG、64%噁霜灵·代森锰锌WP 和250 g·L^-1嘧菌酯SC的替代药剂单独使用或与不同作用机理的杀菌剂混用,防治黄瓜霜霉病。     

霜脲氰与不同杀菌剂混配对两种霜霉病菌的增效作用

采用叶盘法（定量喷药）测定霜脲氰与不同杀菌剂混配对黄瓜和葡萄霜霉病菌抗甲霜灵菌株的增效作用,证实了霜脲氰加代森锰锌或二者再加恶霜灵以不同比率混配对黄瓜、葡萄霜霉病菌均明显增效,发现了霜脲氰与碱式硫酸铜1：8混配对黄瓜霜霉病菌增交,代森锰锌（1：8）或再加恶霜灵（1：8：1）对葡萄霜霉病菌的增效作用随着供试菌株对甲霜灵抗性水平的提高而增强。霜脲氰与代森锰锌1：7混配以保护、治疗、铲除方式处理黄瓜叶盘,对黄瓜霜霉病菌均表现增效。     

Synergism in fungicide mixtures againstPseudoperonospora Cubensis

Mixtures of cymoxanil with mancozeb (Mancur) or with mancozeb plus oxadixyl (Pulsan, Sandocur-M) were effective in controlling either metalaxyl-sensitive (MS) or -resistant (MR) isolates ofPseudoperonospora cubensis on cucumbers (cv. ‘Elem’) in growth chambers. Metalaxyl+mancozeb (Ridomil-MZ) and oxadixyl+mancozeb (SAN-518) mixtures were effective against the metalaxyl-sensitive but less so against the metalaxyl-resistant isolate. Fungicide mixtures were far more effective in controlling either the sensitive or the resistant isolates than were the individual components applied alone, thereby representing significant levels of synergistic interactions. Increased efficacy of mixtures relative to their combined components (synergy factor) was calculated by the Wadley method. Mixtures containing cymoxanil showed high synergy factors against the resistant isolate, whereas Ridomil-MZ and SAN-518 did not evince this phenomenon. We concluded that cymoxanil mixtures might be suitable for the control of downy mildew incited by a metalaxyl-resistant isolate ofP. cubensis.     

Lack of cross-resistance to a novel succinate dehydrogenase inhibitor, fluopyram, in highly boscalid-resistant isolates of Corynespora cassiicola and Podosphaera xanthii

BACKGROUND: Recently in Japan, isolates resistant to boscalid, a succinate dehydrogenase inhibitor (SDHI), have been detected in Corynespora cassiicola (Burk. & Curt.) Wei and Podosphaera xanthii (Castaggne) Braun & Shishkoff, the pathogens causing Corynespora leaf spot and powdery mildew disease on cucumber, respectively. Resistant isolates of C. cassiicola are widely distributed and represent a serious problem in disease control at present. Novel SDHI fungicides, including fluopyram, are now under development. RESULTS: The growth of very highly boscalid‐resistant, highly resistant and sensitive isolates of C. cassiicola was strongly suppressed on fluopyram‐amended YBA agar medium. Although boscalid and another SDHI, penthiopyrad, hardly controlled Corynespora leaf spot and powdery mildew on cucumber plants when very highly or highly boscalid‐resistant isolates were employed for inoculation, fluopyram still exhibited excellent control efficacy against these resistant isolates as well as sensitive isolates of C. cassiicola and P. xanthii. CONCLUSION: Differential sensitivity to boscalid, penthiopyrad and fluopyram, clearly found in these two important pathogens of cucumber, may indicate involvement of a slightly distinct site of action for fluopyram from the two other SDHIs. This finding may lead to the discovery of unique SDHIs in the future. Copyright © 2011 Society of Chemical Industry     

番茄灰霉病菌对腐霉利的抗药性检测及生物学性状研究

采用区分剂量法,检测了来自福建省、上海市、辽宁省和内蒙古自治区不同采样点的番茄灰霉病菌Botrytis cinerea对腐霉利的抗性频率;选取未施药地区的敏感菌株,建立了灰霉病菌对腐霉利的敏感基线;比较了常年施药地区与未施药地区灰霉病菌的抗性频率差异,测定了施药10年以上地区番茄灰霉病菌的抗性指数;研究比较了抗、感菌株的生物学性状。结果表明:腐霉利对127株敏感菌株的平均EC50值为(0.31±0.08) μg/mL;供试4省市灰霉病菌对腐霉利均表现出了很高的抗性频率,其中内蒙古自治区和辽宁省采样点的抗性菌株频率高于90%;在施药历史超过10年的215株田间菌株中,抗性水平最高的菌株来自辽宁省,抗性指数为44.3;施药地区灰霉病菌的抗性频率均高于未施药地区;抗性菌株的抗药性可稳定遗传;供试11株抗性和敏感菌株在产孢量和孢子萌发率、菌丝生长速率、活体致病力等生物学性状方面的差异与其抗性水平之间无明显相关性。     

The occurrence of thiabendazole-resistant isolates of Polyscytalum pustulans and Helminthosporium solani on seed potato tubers in relation to fungicide treatment and storage

Tubers infected with thiabendazole-sensitive isolates of Polyscytalum pustulans and Helminthosporium solani were treated annually for 4 years with benomyl, thiabendazole or a formulated mixture of thiabendazole and imazalil, and grown at three farms in Scotland. The proportion of thiabendazole-resistant isolates of H. solani increased, and isolates producing black colonies became more common with successive annual applications of thiabendazole or benomyl. Silver scurf was not reduced after three annual applications. When these fungicides were applied once to untreated seed the incidence of resistant isolates of H. solani was much less in 1988 than in 1991 when tubers had been grown on farms for 3 years from untreated seed.
The proportion of resistant isolates of P. pustulans increased with the number of successive applications of thiabendazole or benomyl but at differing rates on each farm. At one farm, skin spot was not reduced by three annual applications of these fungicides whereas at the other farms it was reduced by 90–100% by four annual applications.
A smaller proportion of resistant isolates of P. pustulans and H. solani was obtained after applying the mixture of thiabendazole and imazalil than after benomyl or thiabendazole alone. Their occurrence was not related to the number of fungicide applications. The mixture also reduced both diseases by more than 75% over the 4 years.
At one farm where resistant isolates of H. solani were present, tubers were infected when stored on trays but not when stored in bags.     

Metalaxyl sensitivity of isolates of Phytophthora erythroseptica in Prince Edward Island

Isolates of Phytophthora erythroseptica , causal agent of pink rot of potato, were obtained from diseased tubers collected in Prince Edward Island (PEI), Canada. In an in vitro assay of field and single-zoospore isolates, all isolates of the pathogen from PEI were sensitive to metalaxyl (EC₅₀ < 0·5  µ g mL⁻¹), unlike moderately or highly resistant reference isolates obtained from Maine, USA. Allozyme-banding patterns at the glucose-6-phosphate isomerase ( Gpi ) locus were identical (91/91) for all isolates of P. erythroseptica examined from PEI and Maine, but could be used to distinguish isolates of P. erythroseptica from local isolates of Phytophthora infestans . Inoculation of potato tubers from plants treated with foliar applications of metalaxyl in the field indicated that compounds inhibitory to metalaxyl-sensitive isolates of P. erythroseptica were present in the periderm, even after 4 months' storage. By contrast, moderately or highly resistant isolates of the pathogen caused significantly ( P  ≤ 0·05) more disease than sensitive isolates in tubers from plants treated with metalaxyl. The effectiveness of metalaxyl in controlling pink rot in a particular region will depend on resistance levels of local populations of P. erythroseptica . Preventing the development and spread of resistant strains of P. erythroseptica will be critical in maintaining metalaxyl as an effective chemical for control of this pathogen.     

Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew

ABSTRACT Between 1998 and 1999, control failure of powdery mildew (Podosphaera fusca) and downy mildew (Pseudoperonospora cubensis) by the strobilurin fungicides azoxystrobin and kresoxim-methyl was observed in cucumber-growing areas of Japan. Results from inoculation tests carried out on intact cucumber plants and leaf disks clearly showed the distribution of pathogen isolates highly resistant to azoxystrobin and kresoximmethyl. Fragments of the fungicide-targeted mitochondrial cytochrome b gene were polymerase chain reaction amplified from total pathogen DNA and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochrome b gene, resulting in substitution of glycine by alanine at position 143, was found in resistant isolates of downy mildew. This substitution in cytochrome b seemed to result in high resistance to strobilurins in this pathogen. The same mutation was found in some but not all resistant isolates of powdery mildew. This study suggests that a mutation at position 143 in the target-encoding gene, resulting in an amino acid substitution, was probably a major cause of the rapid development of high strobilurin resistance in these two pathogens.     

基于SRAP标记的黄瓜霜霉菌遗传多样性分析

本研究对来自黑龙江、辽宁、河北、山东、江苏5个省12个黄瓜主产区的77个黄瓜霜霉菌菌株,采用SRAP分子标记进行了遗传多样性分析。从35对SRAP引物中筛选出10对引物,共产生9 554条扩增条带,其中9 132条表现多态性,占95.6%。基于SRAP分子标记,77个黄瓜霜霉菌菌株的遗传距离为0.60~1.00,表明黄瓜霜霉菌具有丰富的遗传多样性。通过聚类分析,77个黄瓜霜霉菌菌株聚类为8个类群,并且来自相同地区的菌株大多数聚集于同一类群中,表明黄瓜霜霉菌群体的遗传多样性与其地理来源密切相关。     

In vitro somatic growth and reproduction of phenylamide-resistant and -sensitive isolates of Phytophthora erythroseptica from infected potato tubers in Idaho

Pink rot of potato, most commonly caused by Phytophthora erythroseptica , is a major field and post-harvest problem in southern Idaho, USA, particularly since 1998 when isolates resistant to the phenylamide fungicide metalaxyl-M (mefenoxam) were detected. Isolates of P. erythroseptica were collected from infected tubers in 2001 and 2002 from six Idaho counties and tested for resistance to metalaxyl-M on amended agar. Metalaxyl-M resistant (MR) and metalaxyl-M-sensitive (MS) isolates were identified in six counties; 160 isolates were highly resistant, seven moderately resistant and 57 sensitive to metalaxyl-M with mean EC₅₀ values of 182, 23 and 0·5 mg L⁻¹ ai metalaxyl-M, respectively. Mycelial growth rates and oospore production in agar were assessed for 20 MS and 20 MR isolates at 10, 15, 20, 25 and 30°C. Growth rates of MR isolates were between 2·5 and 3·1 times greater ( P  < 0·05) than those of MS isolates at 10, 15, 20 and 25°C, and oospore production was between 6·8 and 20·5 times greater ( P  < 0·0001) for MR than for MS isolates at the same temperatures. Colony growth in V8 broth at 18°C was greater for MR than MS isolates ( P  < 0·0032). However, zoospore production at 18°C was greater for MS than for MR isolates ( P  < 0·0109), and zoospore production m m ⁻¹ of colony circumference was also greater for MS than for MR isolates, 14 191 and 9959, respectively ( P  = 0·0109). Sexual reproduction of MR isolates in nature may be greater than MS isolates, but MS isolates may be more asexually fit based on the fitness parameters studied.     

Baseline sensitivity of natural population and resistance of mutants in Phytophthora capsici to zoxamide

Laboratory experiments were conducted to determine the baseline sensitivity of Phytophthora capsici and its risk for developing resistance to zoxamide. In total, 158 P. capsici isolates were collected from China. All 158 isolates were sensitive to zoxamide, with effective concentrations for 50% inhibition of mycelial growth of 0.023 to 0.383 μg/ml and a mean of 0.114 μg/ml, which showed a skewed unimodal distribution. Zoxamide-resistant mutants of P. capsici were obtained by either treating mycelial culture and zoospores with ultraviolet irradiation or adapting a culture on zoxamide-amended plates. The frequency of resistance selection averaged 1.8 × 10(-7). Resistant isolates were also derived by selfing or crossing two sexually compatible isolates, resulting in a mean selection frequency of 0.47. The resistance factor (RF) for zoxamide was 25 to 100 in P. capsici mutants. Through 10 culture transfers, the mutants maintained high levels of RF (between 14 and 134) and had almost equal fitness as their wild-type parents in mycelial growth, sporulation, and virulence. There was no cross resistance between zoxamide and either flumorph, metalaxyl, azoxystrobin, or etridiazole. Based on the results above, P. capsici can develop resistance to zoxamide, and the risk is predicted to be moderate in nature.     

Molecular polymorphisms between populations of Pseudoperonospora cubensis from Greece and the Czech Republic and the phytopathological and phylogenetic implications

Molecular genetic polymorphisms within Pseudoperonospora cubensis isolates of different geographic origins were investigated to establish their phylogenetic relationships and to assess genetic variability between two distant pathogen populations. Thirty isolates originating from Greece (Crete; 15), the Czech Republic (13), the Netherlands (one) and France (one) were analysed by AFLP fingerprinting and ITS 5·8S rDNA sequence analysis. All isolates were obtained from cucumber ( Cucumis sativus ) plants showing typical downy mildew symptoms. Four AFLP primer combinations produced a total of 288 high-quality bands of which 45% were polymorphic, allowing isolates to be grouped into two separate clusters: one including the Central European (Czech Republic) and Western European (the Netherlands and France) and the other the Cretan isolates. Within each AFLP cluster there was some variation, which could be accounted for by geographic origin or pathogenicity. The two populations (Cretan vs. Central and Western European) exhibited a high degree of genetic isolation. There was no clear AFLP grouping of isolates on the basis of pathotypes. No variability was detected in the ITS1 region; however, ITS2 sequences grouped P. cubensis isolates in two subclusters: one with all investigated European and the other with Asian isolates. The two subclusters formed a larger P. cubensis cluster which was differentiated from the cluster of the neighbouring species Pseudoperonospora humuli . Within P. cubensis , AFLP fingerprints could resolve genetically isolated populations, even on small or medium geographic scales, while ITS2 sequence showed differences on a global scale, being only suitable for phylogenetic analyses.