侵染广东连州葫芦的黄瓜绿斑驳花叶病毒的分子特征 及致病性分析

【目的】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)是侵染瓜类作物的主要病毒之一,对瓜类产业造成巨大的危害。本研究旨在探明侵染广东省连州市葫芦的CGMMV分离物(CGMMV-GDLZ)分子特征及其在系统进化中的地位,并测定其对黄瓜、葫芦和西瓜的致病性,为CGMMV的防控提供理论依据。【方法】从广东省连州市葫芦种植基地采集2个疑似CGMMV侵染的病样以及1个无症状样品,提取总RNA,根据CGMMV参考序列（GenBank登录号：KX883801）设计引物进行RT-PCR检测,引物序列为F：CCACGAGTTGTTTCCTAATGCTG/R：TTTGCTAGGCGTGATCGGATTGT,退火温度53℃,扩增长度890 bp。将CGMMV全长序列分为前后两段,前半段1—3 511 nt,扩增引物序列为F：AAGTTCATTTCATTTGGAGAGGGTTTTAATTTTTATAA TTAAACAAA/R：AGTTCTGCATTAATTGCTATTTGGTAGGCACAGTGGTAG;后半段3 301—6 423 nt,扩增引物序列为F：GTGCGTGCTACCCCGACTCCAATAGGTTTGATTGCCCGTG/R：GGTGGAGATGCCATGCCGACCCTGGGCCCCTACCCGGGGAAAGG。将前后两段PCR产物通过同源重组的方法克隆到pCB301双元载体上,测序得到CGMMV-GDLZ分离物全长序列。利用CGMMV-GDLZ分离物全长序列在NCBI中进行Blast分析,然后通过MEGA7软件对CGMMV-GDLZ以及其他已经报道的CGMMV分离物进行系统进化树分析。将构建好的pCB301-CGMMV侵染性克隆注射接种本生烟验证其侵染性,然后再注射接种黄瓜、葫芦和西瓜的子叶,测定CGMMV-GDLZ分离物的致病性。【结果】RT-PCR结果证实,广东省连州市葫芦病样感染了CGMMV。CGMMV-GDLZ分离物全长序列为6 423 nt,编码4个蛋白,分别为129K复制酶（61—3 495 nt）、186K复制相关蛋白（61—5 007 nt）、运动蛋白MP（4 994—5 788 nt）和外壳蛋白CP（5 763—6 248 nt）。CGMMV-GDLZ核苷酸序列与CGMMV-eWT分离物（GenBank登录号：KY753928）同源性最高,为99.97%。系统进化树分析结果显示,CGMMV-GDLZ分离物与日本、韩国等东亚CGMMV分离物同属Group 1,在遗传距离上与山东、浙江和河南的CGMMV分离物最接近。pCB301-CGMMV侵染性克隆可以系统侵染本生烟,造成本生烟上部叶片出现皱缩、斑驳、凸起等症状,RT-PCR和Western blot进一步确认了侵染性克隆的侵染性。注射接种CGMMV-GDLZ分离物后15 dpi,葫芦和西瓜即可产生斑驳、花叶、突起、生长迟缓等症状,24 dpi时症状更明显。而15 dpi时,CGMMV-GDLZ分离物在黄瓜上的症状不明显,与未接种对照植株几乎没有区别;将植株从控温接种室移入网室中,30 dpi时,黄瓜植株上部叶片开始出现斑驳和花叶,40 dpi时,症状已经非常明显。RT-PCR和Western blot检测进一步确认了上述结果。【结论】侵染广东省连州市葫芦的CGMMV-GDLZ分离物与山东、浙江和河南的CGMMV分离物很可能具有相同的传染源;CGMMV-GDLZ分离物可以侵染本生烟、黄瓜、葫芦和西瓜等作物,但对这些作物的致病性存在差异。     

林麝肺源ST1249型铜绿假单胞菌的分离鉴定及其全基因组序列分析

【目的】对分离自林麝化脓肺脏的1株疑似铜绿假单胞菌(PA)进行鉴定,并分析其致病和耐药机制,为林麝PA感染化脓性疾病的防控奠定基础。【方法】将病原菌分离纯化后,依次进行生化试验、16S rRNA序列分析、药敏试验和小鼠致病性试验,并通过全基因组测序,对分离菌株进行群体进化和物种分型分析以及基因功能注释。【结果】分离自林麝化脓肺脏的1株疑似铜绿假单胞菌经鉴定与PA相符,命名为FMDP001。药敏试验显示其对阿莫西林、头孢曲松、氨曲南、多粘菌素B和林可霉素耐药;对小鼠半数致死量为9.4×10~5 CFU/mL。全基因组序列分析显示该菌基因组大小为6 955 100 bp,序列类型为ST1249,与B136-33株同源性最高,且两菌株基因组平均核苷酸一致性(ANI)值达98.93%;全基因组中共有357个序列编码FMDP001与致病性相关的基因,根据功能分为黏附、铁摄取、胞外毒性蛋白和调控系统;84个序列编码耐药基因,其中多药耐药外排泵为主要成分。【结论】从林麝化脓肺脏中分离到1株致病性较强的PA,并获得ST1249型林麝源PA的全基因组序列,序列显示该菌携带大量药物外排泵及生物膜形成相关基因,决定其具有多重耐药特性,哌拉西林等可作为该类型PA感染的临床用药。     

The M43 domain-containing metalloprotease RcMEP1 in Rhizoctonia cerealis is a pathogenicity factor during the fungus infection to wheat

Wheat(Triticum aestivum L.) is an important staple crop for global human. The necrotrophic fungus Rhizoctonia cerealis is the causal pathogen of sharp eyespot, a devastating disease of wheat. Herein, we identified RcMEP1, a zinc metalloproteaseencoding gene from R. cerealis genomic sequences, and characterized its pathogenesis function. RcMEP1 expressed at markedly-high levels during R. cerealis infection process to wheat. The predicted protein RcMEP1 comprises of 287 amino acid residues and contains a signal peptide and a M43 metalloprotease domain harboring the active site motif(HEVGHWLGLYH). The assays of Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana leaves indicated that RcMEP1 is an apoplastic elicitor of cell death, and that the predicted signal peptide functions and is required for secretion and cell death-induction. The purified RcMEP1 protein and its M43 domain peptide were individually able to induce plant cell death and H2 O2 accumulation, and to inhibit expression of host chitinases when infiltrated into wheat and N. benthamiana leaves, while the M43 domain-deleting peptide and negative control lacked the capacity. Moreover, compared with the control pretreatment, the purified RcMEP1 protein or its M43-domain peptide resulted in enhanced pathogenesis in the inoculated wheat, whereas the M43 domain-deleting peptide failed. These results suggest that RcMEP1 acted as an important pathogenicity factor during R. cerealis infection to wheat and that its signal peptide and M43 domain are required for the secretion and pathogenesis of RcMEP1. This study provides insights into pathogenesis role of M43 domain-containing metalloproteases during R. cerealis infection to wheat.     

Cherry picking by pseudomonads: After a century of research on canker,genomics provides insights into the evolution of pathogenicity towards stone fruits

Bacterial canker disease is a major limiting factor in the growing of cherry and other Prunus species worldwide. At least five distinct clades within the bacterial species complex Pseudomonas syringae are known to be causal agents of the disease. The different pathogens commonly coexist in the field. Reducing canker is a challenging prospect as the efficacy of chemical controls and host resistance may vary against each of the diverse clades involved. Genomic analysis has revealed that the pathogens use a variable repertoire of virulence factors to cause the disease. Significantly, strains of P. syringae pv. syringae possess more genes for toxin biosynthesis and fewer encoding type III effector proteins. There is also a shared pool of key effector genes present on mobile elements such as plasmids and prophages that may have roles in virulence. By contrast, there is evidence that absence or truncation of certain effector genes, such as hopAB, is characteristic of cherry pathogens. Here we highlight how recent research, underpinned by the earlier epidemiological studies, is allowing significant progress in our understanding of the canker pathogens. This fundamental knowledge, combined with emerging insights into host genetics, provides the groundwork for development of precise control measures and informed approaches to breed for disease resistance.     

Paraphoma root rot of alfalfa (Medicago sativa) in Inner Mongolia,China

Root rot symptoms were observed in fields of alfalfa in Chifeng city, Inner Mongolia, China in 2016. Disease incidences of seven alfalfa varieties planted in 2014 ranged from 56% to 95%, while incidence of Gongnong No. 1 planted in 2016 was 8%, 31% and 76% in 2016, 2017 and 2018, respectively. Paraphoma isolates were consistently recovered from black necrotic root tissues of diseased plants with a frequency of 77.1%. Based on morphological characters and phylogenetic analysis of rDNA internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and β-tubulin (TUB), this fungus was identified as Paraphoma radicina. Glasshouse pathogenicity experiments showed that P. radicina significantly reduced above- and below-ground biomass of alfalfa plants 2 months after inoculation. Paraphoma radicina infected 70% of the plants inoculated with a root dip in conidia, and these symptoms were consistent with the symptoms in the field. Paraphoma radicina was successfully reisolated from disease roots of the inoculated alfalfa plants. This is the first report of P. radicina as the causal agent of alfalfa root rot in China.     

Potential host range of four Phytophthora interspecific hybrids from Clade 8a

In recent years several interspecific hybrids have been reported in the plant pathogenic oomycete genus Phytophthora. Due to the large genotypic and phenotypic changes, these hybrids might have broader or more limited host ranges compared with their parental species. It is crucial to understand the host range of Phytophthora hybrids to minimize the economic losses caused by their infection. The potential host range of four hybrids belonging to Clade 8a of the Phytophthora phylogenetic tree was investigated in this study. Thirty species of herbaceous plants as well as eight species of woody plants were inoculated and monitored for any symptom of infection. In addition, the detached twigs of 32 tree species, fruits of six plant species, tubers of potato, and roots of carrot and sugar beet were investigated for susceptibility to these hybrids. Almost all hybrids caused severe rot on all tested fruits, tubers, and roots, although different isolates showed different pathogenicity on detached tree twigs. All hybrids tested had a different host range compared with their parental species: they were able to infect plants outside the host range of their parents, infect hosts of both parental species, although these parents did not have overlapping hosts, or, in some cases, they were not able to infect hosts infected by the parents.     

云南、广西三七黑斑病病原链格孢菌的鉴定

正黑斑病是三七栽培生产中常见的一大病害,叶片受害产生近圆形或不规则水浸状病斑,常导致成株落叶、幼苗生长点及茎秆顶端腐烂枯死。其病原一般认为是链格孢属真菌人参链格Alternaria panax Whetzel~([1,2]),也有相关研究证明黑斑病病原为细链格孢Alternaria tenuis Nees~([3]),后定名为链格孢Alternaria alternata Keissl~([4])。本研究利用ITS序列和histone 3部分编码序列的PCR鉴定,结合形态学鉴定,分析三七主产区黑斑病菌的组成和分布情况及几种病原菌的致病力差异,以期为三七黑斑病防治提供理论依据。     

芝麻棒孢叶斑病病原菌鉴定与致病力比较

 为明确芝麻叶斑病病原菌及其致病力,本研究以河南不同地区芝麻上分离所得叶斑病分离物为研究对象,通过形态观察和分子鉴定探讨这些分离物的分类属性,并且进行离体叶片和活体茎杆接菌,进一步探究了不同菌株的致病力差异。结果显示分离所得的6株芝麻叶斑病分离物的菌落形态、生长速率和产孢量存在差异,分生孢子多数呈倒棍棒状,具隔膜,6个菌株的ITS序列与GenBank中Corynespora cassiicola的一致性达99%以上,结合形态特征与ITS序列将其鉴定为山扁豆生棒孢。室内人工接菌芝麻发现,这6个菌株的致病力存在显著的差异;在芝麻叶片上,20180909-03属强致病力菌株,20180821-01、20180909-05和20180824-01属中等致病力菌株,20180904-02-02和SF1-1属弱致病力菌株;在芝麻茎杆上,20180824-01致病力最强,其次是20180821-01、20180909-03和20180909-05,SF1-1和20180904-02-02致病力最弱。研究结果证明山扁豆生棒孢不同菌株在芝麻上的致病力存在明显的分化。     

Phaeosphaeria sp.HD-06菌株对马唐的致病性分析及初步鉴定

为了得到能够有效抑制马唐Digitaria sanguinalis生长的生防微生物,在农田采集马唐种子,进行萌发试验,观察种子的萌发状况,从发病的种子和马唐幼苗基部分离致病菌,通过形态学观察以及ITS序列分析对致病菌进行鉴定。结果从罹病的马唐幼苗基部分离得到一株致病菌HD-06,该菌株的发酵液用水稀释至60%浓度时,对马唐种子的萌发抑制率和幼苗发病率均达到100%,这说明菌株HD-06对马唐有较好的生物防治作用。根据其形态学特点及ITS序列分析结果,鉴定菌株HD-06为暗球腔菌属Phaeosphaeria的真菌。这些结果为深入研究其对马唐的防治作用奠定了基础。     

刺葡萄棒状拟盘多毛孢叶斑病病原菌的分离鉴定及其防治药剂筛选

葡萄叶斑病是一种严重影响葡萄产量的病害。为了明确引起刺葡萄叶斑病的病原菌种类及筛选有效防治药剂,本研究从福建农林大学实践教学基地采集刺葡萄‘高山二号’10片典型叶斑病病叶片,对分离得到的菌株进行形态学和分子鉴定。结果表明,通过形态学特征结合rDNA-ITS(ITS)、β-tubulin和EF-1α序列分析确定分离到的菌株均为棒状拟盘多毛孢Pestalotiopsis clavispora。防治药剂筛选的结果显示,不同的防治药剂对炭疽病菌的抑制效果不同,60%唑醚·代森联水分散粒剂和25%嘧菌酯悬乳剂对该病原菌菌丝生长的抑制效果最好,其EC_(50)分别为0.119 mg/L和0.178 mg/L;其次为80%波尔多液可湿性粉剂,其EC_(50)为1.766 mg/L;抑制效果最差的药剂为50%多菌灵可湿性粉剂,仅表现出轻微的抑制作用。防治棒状拟盘多毛孢菌丝生长和孢子萌发的最佳药剂是60%唑醚·代森联水分散粒剂和25%嘧菌酯悬乳剂,可作为备选药剂进行田间刺葡萄叶斑病防控试验。