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.     

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。     

葡聚六糖诱导黄瓜抗霜霉病的研究

黄瓜经喷施人工合成的植物抗病性诱导剂葡聚六糖诱导后 ,黄瓜霜霉病的发病程度显著降低。葡聚六糖诱导黄瓜抗霜霉病的效果以质量浓度为10μg/L ,诱导间隔期为7d ,连续诱导 3次效果较好 ,其诱导持续期可达 7d。结果还表明葡聚六糖的诱导抗性表达不仅与它本身的作用机制有关 ,还与它的使用方法有关     

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.     

室内模拟塑料大棚黄瓜白粉病流行预测模型的建立

在室内人工气候箱内,通过人工接种和模拟高温高湿环境的方法研究了黄瓜白粉病的发生和流行趋势,采用SAS统计软件对试验数据进行相关和回归分析,建立了黄瓜白粉病始病期与流行程度的预测模型。始病期预测模型的RMSE值在2以下,流行程度预测模型的RMSE值在6以下,表明模型可对塑料大棚黄瓜白粉病始病期和流行程度进行定量预测。     

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.     

Structure and temporal shifts in virulence of Pseudoperonospora cubensis populations in the Czech Republic

The structure and temporal dynamics of the virulence of Pseudoperonospora cubensis (causal agent of cucurbit downy mildew) were studied in pathogen populations in the Czech Republic from 2001 to 2010. A total of 398 P. cubensis isolates collected from Cucumis (Cm.) sativus, Cm. melo, Cucurbita (Cr.) maxima, Cr. pepo, Cr. moschata and Citrullus lanatus were analysed for variation in virulence (pathotypes). Virulence was evaluated on a differential set of 12 genotypes of cucurbitaceous plants. All isolates of P. cubensis were characterized by their level of virulence (classified according the number of virulence factors, VF; low VF = 1–4, medium VF = 5–8, high VF = 9–12): high (75%), medium (24%) and low (1%). The structure and dynamics of virulence in the pathogen populations were expressed by pathotypes using tetrad numerical codes and a total of 67 different pathotypes of P. cubensis were determined. The most susceptible group of differentials was Cucumis spp., while the lowest frequency of virulence was recorded on Cr. pepo ssp. pepo, Ci. lanatus and Luffa cylindrica. A high proportion (c. 90%) of isolates was able to infect cucurbit species Benincasa hispida and Lagenaria siceraria, which are not commonly cultivated in the Czech Republic or elsewhere in central Europe. In the recent pathogen populations (2008–2010) there was prevailing frequency (70–100%) of isolates with high numbers (9–12) of virulence factors. ‘Super pathotype’ 15.15.15 was often observed in the study within the pathogen populations and was one of the four most frequently recorded pathotypes. Pseudoperonospora cubensis populations shifted to a higher virulence over time. From 2009 the pathogen population changed dramatically and new pathotypes appeared able to establish natural and serious infection of Cucurbita spp. and Ci. lanatus, which was not observed in 2001–2008. Generally, virulence structure and dynamics of P. cubensis populations are extremely variable in the Czech Republic.     

黄瓜-酸黄瓜渐渗系霜霉病抗性及感病前后几种酶活性的变化

 Using resistant and susceptible cultivars as controls, the resistant evaluation to Pseudoperonospora cubensis was carried out in 12 introgression lines from wide cross between cucumber(Cucumis sativus L.) and sour cucumber(Cucumis hystrix Chakr.). Three enzyme activities including POD, SOD and PAL were analyzed before and 7 d after inoculation, and the POD isozyme was detected by polyacrylamide electrophoresis. The inoculation results showed that, of the 12 accessions, 3 were identified as high resistant, 5 were moderately resistant and 4 were moderately susceptible to downy mildew. The enzyme activities of POD, SOD and PAL were greatly increased in resistant accessions after inoculation. PAL enzyme activities showed close correlation with disease rating before or after inoculation, which implicated that PAL enzyme activity might be used to estimate the resistance to downy mildew. POD isozyme electrophoresis showed that the number and intensity for the bands of resistant lines were significantly increased more than those of susceptible lines after inoculation.     

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。     

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     

一体化智能孢子捕捉系统在黄瓜霜霉病和黄瓜白粉病预测上的应用

为探索国内研制的新型一体化智能孢子捕捉系统在黄瓜霜霉病和黄瓜白粉病预测预报上的应用,在田间自然发病情况下,通过对捕捉孢子的形态进行识别,优化一体化智能孢子捕捉系统主要工作参数如有/无空气切割头、空气采集口高度和空气采集时间;通过病害及孢子的动态监测分析大棚黄瓜霜霉病和黄瓜白粉病病情指数与孢子捕捉量的关系。结果表明,当不加装空气切割头、空气采集口高度为70 cm、孢子捕捉时间在10:00—10:30时段有利于孢子的捕捉。黄瓜霜霉病和黄瓜白粉病病情指数与连续7 d孢子捕捉总量具有强正相关性。连续多日监测到黄瓜霜霉病菌孢子囊且数量快速增加是黄瓜霜霉病发生或快速上升的一个预测指标。黄瓜白粉病发病之前没有监测到黄瓜白粉病菌分生孢子,且在病害盛发期分生孢子捕捉量仍较少。研究表明,一体化智能孢子捕捉系统适用于黄瓜霜霉病的预测,但在黄瓜白粉病的预测上尚存在一定问题。     

电解水对黄瓜生长、果实品质及黄瓜霜霉病和白粉病防效的影响

为明确KCl及NaCl电解水对黄瓜生长、果实品质及病害防效的影响,以3种电解水定期喷施大棚黄瓜,测定黄瓜生长指标及果实外观与营养品质,并测定其对黄瓜白粉病和霜霉病的防效。结果表明,除KCl微酸性电解水组的黄瓜叶面积显著高于农药组外,喷施电解水对黄瓜植株生长的影响均不显著;喷施KCl电解水可提高果实横径和果形指数,显著降低果实弓形高度,与自来水和农药对照组相比果实维生素C含量分别显著提高9.3%和37.6%,单果质量均提高11.0%,但总产量增加不明显;喷施NaCl电解水可显著增加果实糖酸比。NaCl和KCl电解水对黄瓜白粉病防效分别为72.2%和69.0%,与农药组防效相当;KCl微酸性电解水组防效稍低,为61.1%。NaCl和KCl电解水及KCl微酸性电解水对黄瓜霜霉病防效较差,分别为30.5%、26.4%和9.4%,显著低于农药组。研究表明,与NaCl电解水相比,喷施KCl电解水不但可替代农药防治黄瓜白粉病,还可显著提高黄瓜果实品质。     

两种候选甲氧基丙烯酸酯类杀菌剂对黄瓜霜霉病的作用方式

20%唑胺菌酯EC、20%SYP-3998 SC是甲氧基丙烯酸酯类候选杀菌剂,为探索两药剂对黄瓜霜霉病的作用方式,采用盆栽植株喷雾-叶碟法、叶碟喷雾法、盆栽植株喷雾-叶碟法分别测定两药剂对黄瓜霜霉病的保护、治疗及持效期,并采用离体叶片法、叶碟法测定二者对黄瓜霜霉病的铲除作用.结果表明:20%唑胺菌酯EC和20%SYP-3998 SC具有良好的保护、治疗及铲除作用.两药剂的铲除作用表现在能抑制黄瓜霜霉病菌病斑扩展、孢子囊产生及孢子囊再侵染,与孢子囊混合接种能显著降低孢子囊的致病性.此外,20%唑胺茵酯EC和20%SYP-3998 SC具有7～10天的持效期;20%唑胺茵酯EC预防、治疗、铲除效果及持效性均优于20%SYP-3998 SC及对照药剂25%嘧菌酯SC.     

广州地区黄瓜霜霉病流行速率的预测模型

通过对广州地区黄瓜霜霉病 4点 4年的系统调查 ,初步明确了该地区黄瓜霜霉病的发生规律 ,对该病的流行速率及其相关因子进行逐步回归分析 ,组建了广州地区黄瓜霜霉病流行速率的预测模型 ,利用历史数据对该模型进行检验 ,其预测 5d和10d的病情指数的准确度分别为88.16%和68.11% ;用该模型预测2000年黄瓜霜霉病的病情指数 ,其预测5d和10d的病情指数的准确度分别为94.47%和69.43%。     

Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid

A total of 618 isolates of corynespora leaf spot fungus (Corynespora cassiicola) collected from 24 commercial cucumber greenhouses in 12 cities in Ibaraki Prefecture, Japan, were tested for their sensitivity to boscalid. Boscalid‐resistant isolates were detected in 17 out of 19 greenhouses with a history of use of this fungicide and detection frequencies of the resistant isolates exceeded 47% in nine greenhouses. Frequencies of very highly resistant (VHR) isolates with 50% effective concentration (EC₅₀) values of boscalid exceeding 30 μg mL^?1 were higher than those of moderately resistant (MR) isolates with EC₅₀ ranging from 2·0 to 5·9 μg mL^?1 in 11 greenhouses. Additionally, highly resistant (HR) isolates with EC₅₀ from 8·9 to 10·7 μg mL^?1 were first detected. Furthermore, molecular characterization of genes encoding succinate dehydrogenase (SDH) subunits (SdhA, SdhB, SdhC and SdhD) was carried out to elucidate the amino acid substitution responsible for the resistance to boscalid. All 23 VHR isolates had the same mutation from CAC to TAC in the SdhB gene leading to the substitution of histidine with tyrosine at amino acid position 278 (B‐H278Y). At the same position, the substitution to arginine conferred by a mutation to CGC (B‐H278R) was detected in all four HR isolates. Some MR isolates showed a substitution from serine to proline at position 73 in SdhC (C‐S73P), from serine to proline or from glycine to valine at position 89 (D‐S89P) and 109 (D‐G109V), respectively, in SdhD. There was no common mutation in SDH genes of all MR isolates.     

A new pathotype of<Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> causing downy mildew in cucurbits in Israel

Downy mildew, incited by the comycetePseudoperonospora cubensis (Berk.et Curt.) Rost., was recorded in Israel during the years 1979–2001 on cucumber (Cucumis sativus) and melon (Cucumis melo) and classified as pathotype 3. In July 2002 severe outbreaks of downy mildew occurred on pumpkin (Cucurbita moschata) and summer squash (Cucurbita pepo subsp.pepo). Host range pathogenicity studies revealed high compatibility of the new population with cucumber, melon, pumpkin and summer squash but low compatibility with watermelon. This new population was therefore designated as pathotype 6. The possible origin of this new pathotype in Israel is discussed. http://www.phytoparasitica.org posting Oct. 9, 2003.     

四氟醚唑对黄瓜的安全性及其对黄瓜白粉病的防治效果

为明确四氟醚唑对黄瓜植株的安全性,采用浸种及茎叶喷雾处理的方法测定其对不同生长时期黄瓜植株的影响,采用子叶保湿培养法测定白粉病菌对其敏感性,并在田间进行防治白粉病的药效试验,综合评价了四氟醚唑对黄瓜白粉病的效果。浸种处理后,黄瓜子叶上白粉病发病率明显降低,对黄瓜株高和根长的抑制率、茎周增长率及叶绿素含量增加率与己唑醇相比均较低;发芽期及幼苗期的黄瓜植株使用四氟醚唑处理,新生节间均出现轻微的抑制伸长现象;4％四氟醚唑水乳剂对黄瓜白粉病菌的EC50为0．8146mg／L,敏感毒力高于50％醚菌酯水分散粒剂;田间间隔期10天喷雾,在末次药后7天对黄瓜白粉病的防治效果为76．02％～85．77％,与5％己唑醇水乳剂防效相当,明显高于50％醚菌酯水分散粒剂的防效。表明四氟醚唑对黄瓜生长安全且用药间隔期长,是防治白粉病的高效轮换药剂。