Sensitivity to azoxystrobin in Didymella bryoniae isolates collected before and after field use of strobilurin fungicides

BACKGROUND: Isolates of Didymella bryoniae (Auersw.) Rehm, causal agent of gummy stem blight on cucurbits, developed insensitivity to azoxystrobin in the eastern United States 2 years after first commercial use in 1998. Baseline sensitivity of this fungus to azoxystrobin has never been reported. The objectives were to compare baseline sensitivities of D. bryoniae from South Carolina and other locations to sensitivities of isolates exposed to azoxystrobin for one or more seasons, and to compare sensitivity in vitro and in vivo. RESULTS: Sixty‐one isolates of D. bryoniae collected before 1998 were sensitive. Median EC₅₀ was 0.055 mg L^?1 azoxystrobin (range 0.005 to 0.81). Forty isolates collected after exposure during 1998 also were sensitive. Fifty‐three of 64 isolates collected in South and North Carolina between 2000 and 2006 were insensitive to 10 mg L^?1 azoxystrobin. Sensitive and insensitive isolates were distinguished by disease severity on Cucumis melo L. seedlings treated with azoxystrobin (20 or 200 mg L^?1). CONCLUSIONS: An azoxystrobin baseline sensitivity distribution was established in vitro for isolates of D. bryoniae never exposed to strobilurins. Baseline values were comparable with those of other ascomycetes. Insensitive isolates were found in fields with a history of strobilurin applications. An in vivo method distinguished sensitive and insensitive isolates. Copyright © 2009 Society of Chemical Industry     

甘肃省河西地区瓜类作物病毒病的病原鉴定

【目的】明确甘肃省河西地区瓜类作物病毒病害的主要病毒病原,为当地瓜类作物病毒病的防治提供参考。【方法】自甘肃省河西地区采集有病毒病症状的瓜类作物新叶或果实共37份,利用双抗体夹心酶联免疫吸附分析(DAS-ELISA)方法对样本进行检测,并采用RT-PCR、CP基因片段测序、序列比对等方法对DAS-ELISA检测呈阳性的代表性样本进行复检。【结果】在37份瓜类作物病毒病样品中,有30份感染了西瓜花叶病毒(Watermelon mosaic virus,WMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、番木瓜环斑病毒(Papaya ringspot virus,PRSV-W)和南瓜花叶病毒(Squash mosaic virus,SqMV),感染WMV、CMV、ZYMV、PRSV-W和SqMV的样品分别占样品总数的40.5%,32.4%,21.6%,21.6%和8.1%,其中27%的样品受2种以上病毒的复合侵染。【结论】该地区瓜类病毒病主要病原为WMV、CMV、ZYMV和PRSV-W,自然条件下常发生复合侵染。     

TaqMan探针法实时荧光定量PCR检测西瓜潜隐病毒

【目的】西瓜潜隐病毒(Citrullus lanatus cryptic virus,CiLCV)是近年来新发生的一种重要种传病害,研究旨在建立基于TaqMan探针法的实时荧光定量PCR(real-time fluorescent quantitative PCR with TaqMan probes,TaqMan-qPCR)检测技术,为开展种子、种苗带毒鉴定和病害防控提供技术支撑。【方法】通过基于小RNA高通量测序技术在北京西瓜产地发现了CiLCV,并克隆CiLCV的dsRNA1和dsRNA2全长序列,设计特异性引物建立RT-PCR和TaqMan-qPCR检测方法。以黄瓜绿斑驳花叶病毒(cucumber green mottled mosaic virus)、黄瓜花叶病毒(cucumber mosaic virus)、甜瓜内源病毒(Cucumis melo endornavirus)、瓜类褪绿病毒(cucurbit chlorotic yellows virus)、南瓜花叶病毒(squash mosaic virus)、番茄斑萎病毒(tomato spotted wilt virus...     

基于外壳蛋白核酸序列的中国黄瓜绿斑驳花叶病毒分离物起源分析

以直接从中国进口源性葫芦种子中提取的总RNA为模板,经RT-PCR扩增获得种子上携带的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的外壳蛋白(CP)基因(LHP,Liaoning cucurbits seed protein ),将其克隆到pMD18-T载体上进行序列测定.结果表明该CP基因(GenBank登陆号:DQ997778)由483个核苷酸组成,编码161个氨基酸.对包括该分离物在内的19个CP基因核苷酸序列进行同源性比较和系统进化树构建,并结合寄主来源及地域来源进行LHP的起源分析,结果表明黄瓜绿斑驳花叶病毒不同分离物的序列分群和进化关系与上述两个因素无明显关系.     

Genetic differentiation in the French population of Erysiphe cichoracearum, a causal agent of powdery mildew of cucurbits

The relative incidence of Erysiphe cichoracearum and Sphaerotheca fuliginea , both agents of powdery mildew of cucurbits, was determined from 275 samples of mildewed leaves of cucurbits collected in 1994 from five regions of France. E. cichoracearum was identified in 9 to 39% of the mildewed leaf samples from four of the regions but was not detected in samples from the Mediterranean island of Corsica. The genetic structure of the French population of E. cichoracearum was examined using RFLPs of the ribosomal internal transcribed spacers amplified by PCR, random amplified polymorphic DNA (RAPD) markers, pathogenicity and mating-type tests. Forty-one isolates, including one from England, were analysed. Cluster analysis from 147 RAPD fragments using 16 primers revealed the existence of three distinct genetic lineages corresponding to three rDNA haplotypes (designated groups A, B and C). Bootstrap, genetic diversity, gametic disequilibrium and private allele analyses supported this differentiation. The genetic differentiation observed in the French population was not related to the geographical origin of the isolates. Group A isolates may be more specialized on melon as, with one exception, they were of race 1 (growth on four of the five melon cultivars tested) in comparison with group B and C isolates, which were of race 0 (growth on IranH only). Thus, the genetic differentiation observed may indicate a host-specialized subdivision within the French population of E. cichoracearum from cucurbits. Gametic disequilibrium analysis among RAPD loci and biological observations suggest that the sexual stage is of minor importance for epidemics of E. cichoracearum on cucurbits.     

Screening and identification of Cucurbita germplasm resistant to Xanthomonas cucurbitae,incitant of cucurbit bacterial spot

Bacterial spot of cucurbits, caused by Xanthomonas cucurbitae, is an emerging disease of cucurbits. This study was conducted to identify Cucurbita species that are resistant to X. cucurbitae. We developed a reliable method for inoculating cucurbit plants by spraying plants with X. cucurbitae suspensions containing carborundum. Symptoms of the disease developed within 7 days after inoculation. Subsequently, we evaluated the virulence of six isolates of X. cucurbitae in the greenhouse and observed that X. cucurbitae isolated from cucurbit fields in Illinois, Michigan, Kansas, Ohio, and Wisconsin were more virulent than the reference ATCC 23378 strain. Then, we screened 81 commercial cultivars of gourds, pumpkins, and squashes, as well as 300 Cucurbita spp. accessions, for their resistance to X. cucurbitae under greenhouse and field conditions. In the greenhouse study, all commercial cultivars and some of the accessions developed typical symptoms of bacterial spot disease, while some of the accessions developed fewer lesions. In the field studies, infection of leaves and fruits was caused by both natural inoculum and spray-inoculation of plants with a suspension of the X. cucurbitae isolate from Illinois. Among 300 accessions tested, 9 and 21 accessions were classified as resistant and less resistant, respectively. Resistant and less resistant accessions belong to the species Cucurbita maxima, C. maxima subsp. maxima, C. maxima subsp. andreana, and C. okeechobeensis subsp. martinezii. This is the first report of potential resistance to bacterial spot of cucurbits.     

Relationship between disease severity and escape of Pseudoperonospora cubensis sporangia from a cucumber canopy during downy mildew epidemics

Fundamental to the development of models to predict the spread of cucurbit downy mildew is the ability to determine the escape of Pseudoperonospora cubensis sporangia from infected fields. Aerial concentrations of sporangia, C (sporangia m^?3), were monitored using Rotorod samplers deployed at 0·5 to 3·0 m above a naturally infected cucumber canopy in two sites in central and eastern North Carolina in 2011, where disease severity ranged from 1 to 40%. Standing crop of sporangia was assessed each morning at 07·00 h EDT and ranged from 320 to 16 170 sporangia m^?2. Disease severity and height above the canopy significantly (P < 0·0001) affected C with mean concentration (C_m) being high at moderate disease. Values of C_m decreased rapidly with canopy height and at a height of 2·0 m, C_m was only 7% of values measured at 0·5 m when disease was moderate. Daily total flux (F_D) was dependent on disease severity and ranged from 5·9 to 2242·3 sporangia m^?2. The fraction of available sporangia that escaped the canopy increased from 0·028 to 0·171 as average wind speed above the canopy for periods of high C increased from 1·7 to 3·6 m s^?1. Variations of C_m and F_D with increasing disease were well described (P < 0·0001) by a log‐normal model with 15% as the threshold above which C_m and F_D decreased as disease severity increased. These results indicate that disease severity should be used to adjust sporangia escape in spore transport simulation models that are used to predict the risk of spread of cucurbit downy mildew.     

Detection and molecular characterization of viruses associated with tomato yellow leaf curl disease in cucurbit crops in Jordan

In this study, Tomato yellow leaf curl Sardinia virus (TYLCSV) and the strains Israel and Mild of Tomato yellow leaf curl virus (TYLCV-IL, TYLCV-Mld) were detected for the first time in four cucurbit crops in Jordan by nested polymerase chain reaction (nPCR). These viruses cause the tomato yellow leaf curl disease (TYLCD) in tomato. Cucumber, squash, melon and watermelon plants inoculated with TYLCV-IL[JO:Cuc], TYLCV-Mld, TYLCSV-IT[IT:Sar:88] and the Jordanian isolate of TYLCV (TYLCV-JV) did not show disease symptoms. However, virus-specific fragments were detected in uppermost leaves of symptomless plants by nPCR. A whitefly transmission test showed that Bemisia tabaci could transmit TYLCV-Mld from cucumber into tomato and jimsonweed plants. However, all infected tomato plants remained symptomless. In addition, results of semi-quantitative PCR (sqPCR) analysis showed that the relative amount of TYLCV-Mld DNA acquired by B. tabaci from cucumber plants was less than that acquired from tomato plants.     

Molecular,biological and serological variability of Zucchini yellow mosaic virus in Tunisia

A study was conducted to better understand the population structure of Zucchini yellow mosaic virus (ZYMV), a severe virus affecting cucurbit crops worldwide, in Tunisia and to estimate whether the use of resistant cultivars may provide durable control. Analysis of the polymerase and coat protein (NIb‐CP) partial sequences of 83 isolates collected in the three main cucurbit‐growing areas in Tunisia showed that ZYMV grouped into two distinct clusters within ZYMV molecular group A. An important variability was observed in the MREK motif of the P3 protein, a motif associated with tolerance breaking in ZYMV‐tolerant zucchini squash cultivars. Interestingly, significant differences were found in the distribution of the MREK variants in the two clusters defined by the partial NIb‐CP sequences, MREK and MKEK sequences being more common in cluster 1 and cluster 2, respectively. When combining NIb‐CP and P3 sequence information, ZYMV molecular variability was shown to be significantly higher in the Cap Bon region than in the Bizerte area. An important biological variability was observed in a subset of 23 isolates regarding symptomatology in susceptible or resistant cucurbits. Some isolates overcame ZYMV tolerance or resistance in zucchini squash and melon, but not in cucumber. Three serotypes were differentiated using a set of 13 monoclonal antibodies (MAbs). Seven parameters characterizing the 23 isolates, including molecular, serological and biological properties, were used for a multiple component analysis (MCA). This analysis revealed that symptom intensity of a given isolate was similar in different susceptible cucurbit hosts, suggesting similar degrees of aggressiveness in different hosts.