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.     

Pythium yorkensis sp. nov., a potential soybean pathogen from southeastern Pennsylvania,USA

Biodiversity surveys of natural as well as agricultural ecosystems commonly reveal novel isolates. A new species, Pythium yorkensis sp. nov., is reported here, isolated from field soil during a survey of oomycete diversity in symptomless soybean across southeastern Pennsylvania. Molecular data from both mitochondrial and nuclear loci support a unique phylogenetic position for the isolates collected, and morphological features distinguish this new species from close relatives in Pythium clade J. Pathogenicity assays suggest that this new species may be a potential agricultural pathogen, as isolates were mildly aggressive on soybean. This study highlights the continued need for culture‐based surveys in collaboration with high‐throughput sequence‐based approaches for environmental sampling.     

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.     

Using image analysis for quantitative assessment of needle bladder rust disease of Norway spruce

High elevation spruce forests of the European Alps are frequently infected by the needle rust Chrysomyxa rhododendri, a pathogen causing remarkable defoliation, reduced tree growth and limited rejuvenation. Exact quantification of the disease severity on different spatial scales is crucial for monitoring, management and resistance breeding activities. Based on the distinct yellow discolouration of attacked needles, it was investigated whether image analysis of digital photographs can be used to quantify disease severity and to improve phenotyping compared to conventional assessment in terms of time, effort and application range. The developed protocol for preprocessing and analysis of digital RGB images enabled identification of disease symptoms and healthy needle areas on images obtained in ground surveys (total number of analysed images n = 62) and by the use of a semiprofessional quadcopter (n = 13). Obtained disease severities correlated linearly with results obtained by manual counting of healthy and diseased needles for all approaches, including images of individual branches with natural background (R² = 0.87) and with black background (R² = 0.95), juvenile plants (R² = 0.94), and top views and side views of entire tree crowns of adult trees (R² = 0.98 and 0.88, respectively). Results underline that a well‐defined signal related to needle bladder rust symptoms of Norway spruce can be extracted from images recorded by standard digital cameras and using drones. The presented protocol enables precise and time‐efficient quantification of disease symptoms caused by C. rhododendri and provides several advantages compared to conventional assessment by manual counting or visual estimations.     

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.     

内蒙古马铃薯黑胫病病原菌的分离和鉴定

 马铃薯是我国的第四大主粮。内蒙古是我国最大的马铃薯生产基地,马铃薯产业是当地农民收入的主要来源。2017年8月,内蒙古锡林郭勒盟部分马铃薯田块发生了一种细菌性黑胫病,发病面积约为200亩,发病率约为20%。发病初期,植株的茎基部变色、发黑,伴有臭味,严重时茎秆腐烂,植株死亡。为明确该地区马铃薯细菌性黑胫病的病原菌种类,本研究对该病原菌进行了分离,并利用生理生化测定,Biolog分析和分子生物学手段将该病原菌鉴定为胡萝卜软腐果胶杆菌巴西亚种(Pectobacterium carotovorum subsp. brasiliense)。据以往报道,我国马铃薯黑胫病主要由黑腐果胶杆菌(P. atrosepticum)和胡萝卜软腐果胶杆菌胡萝卜亚种(P. carotovorum subsp. carotovorum)两种病原菌引起,本研究是P. carotovorum subsp. brasiliense引起马铃薯黑胫病在国内的首次报道。     

广东南瓜细菌性叶枯病及其病原鉴定

 在广东省雷州市发生一种南瓜(Cucurbita moschata)叶枯病,病株叶片边缘开始出现水渍状病斑,逐步发展成大病斑,后期病斑焦枯;在叶片上也可形成近圆形水渍状病斑,伴有黄色晕圈,后期病斑联合形成不规则大枯斑;叶柄和匍匐茎被侵染后呈水渍状腐烂。从病斑上分离到一种细菌,在KB培养基上,菌落为椭圆形,乳白色,半透明,边缘参差不齐,紫外灯照射下产生荧光反应。致病性测定结果表明,该病原细菌可侵染6个南瓜品种引起与田间症状相同的叶枯病。生理生化试验结果表明,该病原细菌与丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)的特性一致。应用假单胞菌属特异引物Ps-for/Ps-rev和丁香假单胞丁香致病变种组群特异性引物Group III-F/Group III-R,可从该病原细菌中扩增出预期大小分别为1 018 bp和750 bp的目的片段。应用丁香致病变种syrB基因特异性引物B1/B2,可从该病原菌中扩增出预期大小为750 bp的丁香霉素基因片段。基于16S rDNA与gyrB基因序列系统进化分析均表明,南瓜叶枯病菌株与已报道的P. syringae pv. syringae菌株HS191(CP006256)亲缘关系最近,二者聚类在一起形成一个小分支。人工接种条件下,该病原细菌还可侵染西葫芦、丝瓜、茄子、番茄、菜豆、扁豆等植物。这些结果表明,引起广东省南瓜叶枯病的病原为丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)。这是首次在中国发现丁香假单胞丁香致病变种引起南瓜叶枯病。     

六种重要玉米病害病原名称的厘定

真菌、细菌、病毒等引起的病害是玉米生产的重要威胁。在中国,玉米种植地域广,种植区生态类型多,病害种类复杂,常见病害有30余种。中国的玉米病害研究历史不足百年,从事研究的专业人员也较少,导致玉米病害知识的传播受到一定影响,其表现之一就是我国在一些玉米病害病原名称的采用上存在明显混乱,已被弃用的旧病原名称还在普遍应用。这种现状影响了玉米病害科学研究信息在国内和国际上的正常交流。本文梳理了瘤黑粉病、丝黑穗病、红叶病、北方炭疽病、圆斑病、黑束病6种玉米病害病原学名的历史变迁,重点结合近年在形态学和分子系统分类学等方面的研究进展,明确了这6种病害病原在目前应该采用的正确名称。(1)玉米瘤黑粉病病原无论是形态学和分子生物学都有别于Ustilago属物种的特征,已恢复其名称为1912年命名的Mycosarcoma maydis(DC.)Bref.(玉蜀黍瘿黑粉菌),曾被广为采用的Ustilago maydis(DC.)Corda(玉蜀黍黑粉菌)成为异名;(2)基于Sporisorium属与Sphacelotheca在寄主科选择上的差异以及对玉米丝黑穗病致病菌的形态学、寄主病害特征和多基因序列分析的结果,Sporisorium reilianum(J.G.Kühn)LangdonFullerton.(丝孢堆黑粉菌)被确定为玉米丝黑穗病病原的正确名称,而Sphacelotheca reiliana(J.G.Kühn)Clinton成为异名之一。由于Sporisorium reilianum种内存在对玉米和高粱的致病性分化,玉米致病菌又可称为Sporisoriumreilianum f.sp.zeae(丝孢堆黑粉菌玉米专化型);(3)普遍认为大麦黄矮病毒(Barley yellow dwarf virus,BYDV)引起玉米红叶病,但近年通过对致病病毒的测序,明确了多种病毒和株系是该病的病原。在中国,引起玉米红叶病的为马铃薯卷叶病毒属(Polerovirus)的小麦黄矮病毒-GPV(Wheat yellow dwarf virus-GPV)和玉米黄矮病毒-RMV(Maize yellow dwarf virus-RMV);(4)尽管在形态学方面有许多相似性,但通过多基因序列的比较研究,证明Kabatiella属完全有别于Aureobasidium属,因此,玉米北方炭疽病病原的学名应该采用Kabatiella zeae Narita et Hiratsuka(玉蜀黍球梗孢),Aureobasidium zeae(Narita et Hiratsuka)Dingley作为异名处理;(5)分生孢子呈蠕孢状的真菌经历了数次属的变化,但形态学、基因序列信息、生活史、次生代谢物等特征的研究表明,引起玉米圆斑病的病原Bipolaris zeicola(G.L.Stout)Shoemaker(玉米生平脐蠕孢)是一个独立的物种,其无性态名称的保留得到了国际真菌命名委员会的同意;(6)对物种名称Cephalosporium acremonium的长期混乱使用使得其已经失去了纯粹种的含义,而Acremonium拥有150余个种并对应多个有性态属,已经成为了一个庞杂属。大量的分子生物学特征研究揭示了Acremonium属的异质性,以分子特征和形态学结合的方法,重新划分和新建了相关的属,Sarocladium strictum(W.Gams)Summerbell(直帚枝杆孢)成为了玉米黑束病病菌的新种名,而Cephalosporium acremonium Corda和Acremonium strictum Gams是其异名。     

Cronobacter spp., foodborne pathogens threatening neonates and infants

Cronobacter spp. (formerly Enterobacter sakazakii) are special foodborne pathogens. Cronobacter infection can cause necrotizing enterocolitis, sepsis and meningitis in all age groups, especially neonates and infants, with a high fatality of up to 80%, although the infection is rare. Outbreaks of Cronobacter infection are epidemiologically proven to be associated with contaminated powdered infant formula (PIF). Cronobacter spp. can resist dry environments and survive for a long period in food with low water activity. Therefore, Cronobacter spp. have become serious pathogens of neonates and infants, as well as in the dairy industry. In this review, we present the taxonomy, pathogenesis, resistance, detection and control of Cronobacter spp.