Temporal disease dynamics and relative importance of sexual and asexual reproduction of grape downy mildew (Plasmopara viticola) in an isolated vineyard in the North Georgia Mountains,USA

The North Georgia Mountains are the southernmost region along the United States East Coast where European wine grapes (Vitis vinifera) are grown commercially. Epidemics of downy mildew, caused by Plasmopara viticola, are frequent and severe, but little is known about the epidemiology and population biology of the pathogen in this region. Disease monitoring in an experimental vineyard from 2015 to 2017 indicated that times of disease onset and progress rates were highly variable across years and cultivars. Oospores were observed microscopically, and simulation with a process-based model indicated presence of conditions favourable for oospore germination in the spring and early summer each year. A total of 409 P. viticola isolates collected over three  years were genotyped with seven microsatellite markers, revealing very high genotypic diversity, which when combined with the observation of oospores is indicative of a sexually reproducing population. Among the 409 isolates, 225 multilocus genotypes (MLGs) were identified, of which 164 were detected only once and 61 were repeated (clonal). Eight MLGs (represented by 28 isolates) were detected across years, suggesting the possibility of asexual overwintering of P. viticola in this region. Across sampling dates, the percentage of isolates belonging to nonrepeated (unique) MLGs ranged from 27.3% to 63.2%. Even towards the end of the annual epidemic, the percentage of isolates in nonrepeated MLGs was still relatively high, around 30%. These MLGs may have originated from oospores germinating late during the growing season, although incomplete sampling at earlier dates and contribution by immigration cannot be fully excluded.     

二甲基二硫的生物活性评价及对土壤养分的影响

采用室内生物活性测定方法,评价二甲基二硫(dimethyl disulfide,DMDS)对土壤病原线虫和土传病原菌的毒力,比较不同浓度药剂处理对土壤理化性质和土壤呼吸的影响,为探究DMDS作为新型土壤熏蒸剂提供切实可行性的依据。结果表明:DMDS熏蒸对土传病原线虫和镰刀菌属的LD_(50)分别为4.743 mg/kg和1.513 mg/kg,可见DMDS对病原线虫和镰刀菌有良好的生物活性。对土壤理化性质进行数据分析发现:DMDS能显著增加土壤铵态氮含量,抑制硝化作用过程,减少NO~-_3-N的产生,提高植物可吸收态氮素水平。DMDS处理的土壤有机质含量和电导率均显著高于对照土壤,而土壤pH和速效钾含量较对照均有降低。此外,熏蒸土壤中有效磷含量较对照减少,但两者无显著差异。对DMDS熏蒸后土壤进行底物诱导呼吸试验,表明DMDS能够在试验初期抑制土壤微生物生物量。本试验结果可为指导DMDS的科学使用提供理论依据及对土壤微生物活性的影响作出科学评价。     

Isolation and characterisation of phages against Pseudomonas syringae pv. actinidiae

Purpose: Pseudomonas syringae pv. actinidiae causes bacterial canker of kiwifruit and is responsible for severe economic losses and emergence of drug-resistant bacteria. Bacteriophages are viruses that infect target bacterial hosts and may be the best strategy to prevent and control kiwifruit canker disease. The objective of this experiment was to monitor the prevalence of Pseudomonas syringae pv. actinidiae and provide insight for the use of phages in biological control.

Materials and methods: In this study, 52 strains of Pseudomonas syringae pv. actinidiae were isolated from 68 stem samples of kiwi plant (cv. Hongyang & Jinkui). Following polymerase chain reaction (PCR) analysis, 15 isolates belonging to biovar 3 were identified, one of which was named XWY0007 and used as the target strain to isolate the phages. Thirty-six phages were isolated and purified from a total of 51 surface water samples collected in Shanghai. All phages were identified by transmission electron microscopy (TEM) and their host ranges were evaluated. Three phages, designated φXWY0013, φXWY0014 and φXWY0026 were selected and further characterised using one-step growth curve and stability at different temperatures and pH.

Results and conclusions: The isolated phages are promising for use as antimicrobials against bacterial canker in kiwi. This report is regarding Pseudomonas syringae pv. actinidiae and its phages from major areas of kiwifruit cultivation.     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.     

星油藤青枯病病原菌鉴定

 星油藤(Plukenetia volubilis L.) 是一种重要的藤本油料植物,在我国华南地区广泛种植。青枯病是近两年在海南星油藤种植区发生的新病害,为探究星油藤青枯病菌的基本特性及种下分化情况,本研究对分离的6株代表菌株进行了相关分析。细菌学鉴定及致病性测定结果表明,该病害是由类茄科雷尔氏菌(Ralstonia pseudosolanacearum)侵染引起。同时,从传统分类及分子生物学不同层面分析了星油藤青枯病菌的遗传分化情况。生理小种及生化变种的测试结果表明,星油藤青枯病菌属于1号生理小种和生化变种Ⅲ;16S rDNA和egl基因部分序列聚类分析显示,星油藤青枯病菌属类茄科雷尔氏菌演化型Ⅰ即亚洲分支菌株,序列变种34。     

A highly sensitive,non‐invasive qPCR‐based strategy for direct quantification of Yersinia ruckeri in fish faeces

Finfish with asymptomatic Yersinia ruckeri infections pose a major risk as they can transmit the pathogen and cause clinical outbreaks in stock populations. Current tools have insufficient quantitative ability for accurately detecting the trace levels of Y. ruckeri typically associated with asymptomatic infection, necessitate invasive or lethal sampling, or require long processing times. This study presents a highly sensitive qPCR‐based method, targeting part of the Y. ruckeri 16S rRNA sequence, that is capable of detecting extremely low levels of Y. ruckeri in noninvasively collected faecal samples. Quantitative precision and accuracy of faecal sample analysis was consistent, despite the complexity of the faecal matrix. The assay demonstrated linearity over a six log‐wide dynamic range. Its limit of detection (LOD) and limit of quantification (LOQ) were 4 and 10 copies of the target sequence, respectively. Sensitivity of the assay was comparable to other qPCR‐based methods without requiring invasive or lethal sampling. Applicability as a screening strategy was tested using passively collected faecal samples. Asymptomatic Y. ruckeri infection was detected in all samples, although none of the fish exhibited overt infection. This method will be beneficial for finfish disease management if developed further as a noninvasive, screening tool against asymptomatic Y. ruckeri infection.     

Multilocus PCR assay reveals high diversity of vegetative compatibility types in populations of Cryphonectria parasitica in Croatia

The ascomycete fungus Cryphonectria parasitica, causal agent of chestnut blight, is probably one of the best known invasive fungal pathogens in forests of Europe and North America. Mycovirus that reduces virulence of C. parasitica can be used as a biocontrol agent of the chestnut blight. However, anastomosis‐mediated virus transmission is limited by a vegetative (in)compatibility (vc) system involving at least six known diallelic vic genetic loci. This study looked at vegetative compatibility (vc) diversity in two populations of C. parasitica in Croatia. For that purpose, a PCR assay was validated and implemented using already known/published and newly designed primers for amplification of six known vic loci. The vc genotypes determined by PCR for 158 C. parasitica isolates investigated in this study were in complete agreement with the vc genotypes determined by pairwise co‐culturing of the same isolates, revealing the specificity and accuracy of the PCR‐based molecular vic genotyping assay. Twenty‐six unique vc genotypes were found among 158 isolates, and 19 vc types per population, which makes Croatian C. parasitica populations among the most diverse in Europe regarding the number of vc types and genetic diversity. Low values of multilocus linkage disequilibrium suggest sexual reproduction as a major contributor to high C. parasitica genetic diversity in studied populations.     

What proteomic analysis of the apoplast tells us about plant–pathogen interactions

Plant pathogens have developed different strategies during their evolution to infect and colonize their hosts. In the same way, plants have evolved different mechanisms acting against potential pathogens trying to infect and colonize their tissues. Regulation of a wide variety of proteins is required in order to perceive the pathogen and to activate the plant defence mechanisms. The apoplast is the first compartment where these recognition phenomena occur in most plant–pathogen interactions, allowing the exchange of different molecules and facilitating inter‐ and intracellular communication in plant cells. Proteomic analysis of the apoplast in recent years has found the initial biochemical responses involved in pathogen recognition and early defence responses. However, this proteomic approach requires some specific experimental conditions to obtain an extract free of cytoplasmic proteins and nonprotein contaminants that affect the subsequent stages of separation and quantification. Obtaining the highest proportion of proteins from the apoplastic space in infected tissues requires different steps such as extraction of apoplastic washing fluids and preparation of total secreted proteins (protein precipitation, solubilization, separation and digestion). Protein identification using mass spectrometry techniques and bioinformatics tools identifying peptides for the extracellular exportation is required to confirm the apoplastic location. This review compiles the most commonly used techniques for proteomic studies, focusing on the early biochemical changes occurring in the apoplast of plants infected by a wide range of pathogens. The scope of this approach to discover the molecular mechanisms involved in the plant–pathogen interaction is discussed.     

直播稻田无人机全程防治病虫草害初探

针对不同设备设计不同喷液量,飞行参数验证防治效果,从而验证无人机在稻田全程应用的可能性,旨在探索2种小型多旋翼植保无人机低空喷洒在整个水稻生长时期病虫草害防治过程中的防效。结果表明:当喷洒水田封闭式除草剂的喷洒量为10650、15000 mL/hm²,其对杂草的株防效与鲜重防效与人工电动喷雾相近。在病虫害的防治试验中,使用推荐用量且喷洒量为10650、15000 mL/hm²时,以及使用70%推荐用量（减量30%）且喷洒量为15000 mL/hm²时,防治效果均与人工电动喷雾器无显著差异。通过验证,选择合适的药剂,配合科学的喷洒参数,可在保证药效的前提下减少农药使用量。多旋翼植保无人机在水稻田有进行大面积全程飞防植保应用有广阔的前景。     

金刺梨黑斑病病原菌的分离及鉴定

金刺梨黑斑病主要于开花期危害幼嫩的果梗,果梗受害后产生黑色干腐病斑,导致整个花序从果梗处脱落。对黑斑病病原菌进行了分离和鉴定,通过形态学特性、致病性测定、rDNA-ITS序列分析,初步确定该病主要由拟茎点霉属(Phomopsis Sacc.)的Phomopsis.capsici真菌引起。