瓜类细菌性果斑病研究进展

瓜类细菌性果斑病是发生在甜瓜、西瓜等葫芦科植物上的一种严重的世界性病害,此病是典型的种传细菌性病害,病原为嗜酸菌属西瓜种(Acidovorax citrulli)。本文围绕瓜类细菌性果斑病菌的分离检测、致病机理、遗传多样性及防治等方面的研究进展作一概述,阐明了瓜类细菌性果斑病的研究现状。     

西瓜嗜酸菌趋化性的初步研究

瓜类细菌性果斑病是世界范围的检疫性细菌病害,病原菌为西瓜嗜酸菌,带菌种子为主要侵染源。病原细菌在寄主表面的定殖能力与其致病能力关系密切,而趋化性是决定定殖能力的关键因素之一,研究不同物质对西瓜嗜酸菌趋化性的影响对防治瓜类细菌性果斑病具有重要意义。本文采用毛细管法,研究了碳源、氨基酸、有机酸及其他物质对西瓜嗜酸菌趋化性的影响。结果表明,所测碳源中,麦芽糖、葡萄糖、乳糖、蔗糖和半乳糖均显著促进西瓜嗜酸菌的趋化性;所测氨基酸中,L-精氨酸、L-天冬氨酸、L-组氨酸、L-谷氨酸、L-亮氨酸、L-缬氨酸、L-谷氨酰胺和L-丙氨酸显著促进西瓜嗜酸菌的趋化性;所测有机酸中,琥珀酸、半乳糖醛酸和酒石酸显著促进西瓜嗜酸菌的趋化性;氯化钠、硫酸镁等对西瓜嗜酸菌的趋化性无显著影响。     

甜瓜细菌性果斑病菌致病性突变体筛选与hrcR基因的克隆

 细菌性果斑病(Bacterial Fruit Blotch,BFB)是西瓜和甜瓜的重要病害。本实验从发病甜瓜果实上分离得到致病菌株MH21,经鉴定为燕麦食酸菌西瓜亚种(Acidovorax avenae subsp. citrulli)。利用转座子Mini-Tn5构建MH21菌株的突变体库,Southern印迹杂交结果显示Mini-Tn5在菌株MH21染色体上可随机、单拷贝插入。通过浸种处理甜瓜种子进行突变体的致病性检查,筛选得到1株致病性完全丧失的突变体M543。对M543中转座子插入基因的克隆和测序表明其突变基因为燕麦食酸菌Ⅲ型分泌系统(TTSS)中的保守基因hrcR。推测菌株MH21中hrcR基因编码的蛋白定位于细菌内膜,是分泌通道主要成分之一。hrcR基因互补菌株的致病性检查结果显示其致病力恢复到野生型的84%。研究同时发现hrcR基因突变后MH21菌株丧失了在烟草上诱导过敏性坏死反应的能力。本研究从遗传学角度说明TTSS是西甜瓜果斑病菌致病性的重要因子。     

西瓜嗜酸菌LuxR家族基因luxR_(4229)的功能研究

瓜类细菌性果斑病是葫芦科作物上的一种严重的世界性病害,其病原菌为西瓜嗜酸菌(Acidovorax citrulli)。本研究以野生型菌株Ac-5为研究对象,通过同源重组双交换的方法,构建了LuxR家族功能基因luxR_(4229)的全基因缺失突变株,并对突变株进行致病性、生物膜形成能力和运动能力等生物学特性测定。同时,qRT-PCR定量检测了鞭毛基因fliR和fliC以及III型分泌系统功能基因hrpE和hrcN的表达水平。结果显示:与野生型相比,突变株致病力明显降低,运动能力减弱,而生物膜形成能力增强。fliR、fliC、hrpE和hrcN基因在突变株中的表达量下调,luxR4229对fliR、fliC、hrpE和hrcN基因均为正调控。基因luxR_(4229)在对西瓜嗜酸菌致病力、运动能力及生物膜的形成具有重要调控作用。     

进境西瓜种子中西瓜细菌性果斑病菌的检测鉴定

由嗜酸菌属西瓜种(Acidovorax citrulli)引起的西瓜细菌性果斑病是西瓜等葫芦科作物上的一种极具毁灭性的病害,该病原细菌可以由种子携带传播。本研究通过直接研磨种子,用双抗体夹心酶联免疫吸附测定法(DAS-ELISA)和免疫捕捉聚合酶链式反应法(IC-PCR)对进境的西瓜种子进行检测,并且对PCR产物进一步克隆测序。结果表明,在DAS-ELISA产生阳性结果的样品中,IC-PCR结果也产生阳性。序列分析表明,该序列与已知的该病菌16S rRNA基因的相应序列具有100%同源性。因此,2006年这批来自台湾的西瓜种子携带有嗜酸菌属西瓜种(Acidovorax citrulli)。     

瓜类果斑病菌胶体金试纸条的检测应用

瓜类果斑病是一种严重为害西甜瓜作物的细菌病害,胶体金试纸条是对其进行现场检测的主要手段.将中国检验检疫科学研究院研制的瓜类果斑病菌胶体金检测试纸条与市场上商品化的美国Agdia公司同类试纸条进行比较,发现两种试纸条的灵敏度和特异性相当,适用于检测出现疑似果斑病症状的叶片和果实;也可用于种子带菌检测,但易出现假阳性结果....     

西瓜噬酸菌效应蛋白AopK的功能研究

瓜类细菌性果斑病是西甜瓜产业中最重要病害之一，其病原菌西瓜噬酸菌的Ⅲ型分泌系统效应蛋白在致病过程中起重要作用。XopK为黄单胞菌效应蛋白，具有E3泛素连接酶活性，其在西瓜噬酸菌中的同源基因aopK鲜有研究。为鉴定效应蛋白AopK并探究其功能，本研究构建了西瓜噬酸菌AAC00-1菌株的aopK缺失突变株和互补菌株，发现基因aopK的缺失显著降低病原菌在西瓜幼苗上的致病力。通过AopK在本氏烟中的亚细胞定位实验，显示AopK定位于植物的细胞质膜。转录水平、外泌性及BAX共表达试验，证明aopK的表达受HrpX和HrpG正调控，其产物能够通过Ⅲ型分泌系统分泌到细胞外，并且能够抑制烟草叶片细胞中活性氧产生、胼胝质沉积以及细胞坏死产生，具有潜在抑制植物防卫反应的毒性功能。以上结果表明AopK为西瓜噬酸菌的Ⅲ型效应蛋白，研究结果为进一步探究其与寄主植物间的互作机理奠定了基础。     

不适温度条件诱导西瓜嗜酸菌进入VBNC状态的研究

由西瓜嗜酸菌(Acidovoraxcitrulli)引起的瓜类果斑病,是危害西瓜和甜瓜等葫芦科作物的一种典型的种传细菌性病害.已有文章报道西瓜嗜酸菌在铜离子诱导下可进入"有活力但不可培养"(viable but non-culturable,VBNC)状态,并在适当条件下复苏,成为生产中潜在的病害初侵染来源.本文结合实...     

LuxR家族调控因子 LuxR-2460 对西瓜嗜酸菌致病力及生物膜形成的调控

 LuxR家族的调控因子在革兰氏阴性细菌中起重要作用。对西瓜嗜酸菌一个LuxR家族的调控因子 LuxR-2460的调控作用进行研究。生物信息学分析表明该调控因子有2个保守结构域:N-末端为REC 结构域,C-末端为HTH 结构域。通过同源重组双交换的方法,构建了luxR-2460全基因缺失的突变株,该基因缺失突变株能够引起菌株致病力、运动能力及蹭行运动能力的下降及生物膜形成能力的增强。qRT-PCR 检测结果表明,该基因缺失导致鞭毛基因fliR在突变株中的表达量下调,影响病菌鞭毛的形成。说明luxR-2460基因对西瓜嗜酸菌的致病能力及相关因子具有重要调控作用,可以作为该病害防控的潜在靶标。     

利用环介导等温扩增方法快速检测瓜类细菌性果斑病菌和番茄细菌性溃疡病菌

为建立简单、快速和灵敏地检测瓜类细菌性果斑病菌Acidovorax avenae subsp. citrulli(Aac)和番茄细菌性溃疡病菌Clavibacter michiganensis subsp. michiganensis(Cmm)的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,以Aac的ugpB基因和Cmm的micA基因为靶标,分别设计、合成和筛选特异性引物,摸索和优化各项反应条件和反应体系,成功建立了以钙黄绿素颜色为指示且只需要金属浴恒温反应30～60 min的LAMP扩增体系。特异性分析表明该LAMP方法可以快速检出5株不同的Aac菌株和2株不同的Cmm菌株,其它对照菌株如燕麦嗜酸菌燕麦亚种A. avenae subsp. avenae、丁香假单胞菌Pseudomonas syringae、水稻黄单胞菌水稻致病变种Xanthomonas oryzae pv. oryzae和茄科雷尔氏菌Ralstonia solanacearum则呈现阴性反应;引物比目前报道的LAMP引物有更高的DNA样品检测灵敏度,Aac和Cmm的灵敏度分别为1.72×10~2fg/μL和1.26×10~2fg/μL。研究结果表明,所设计的Aac和Cmm引物特异性好、灵敏度高,更有利于从源头上控制这2种检疫性细菌病害的流行和传播。     

哈蜜瓜种子带细菌性果斑病菌检测技术的研究

哈蜜瓜细菌性果斑病是世界性的检疫对象之一,主要以种子带菌进行远距离传播,本文通过温室育苗、室内分离和PCR(聚合酶链式反应)技术对哈蜜瓜种子带菌情况和主要带菌部位进行了检验,结果表明:3种方法都可以对种子带菌情况进行检测,分离检测和PCR检测还可以确定种子的带菌部位,PCR技术具有特异性强、灵敏度高、检测时间短等特点.另外检测结果也表明种子带菌以种皮为主,种仁的带菌量较少.     

Bacterial fruit blotch of melon: screens for disease tolerance and role of seed transmission in pathogenicity

Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax avenae subsp. citrulli, is a serious threat to the watermelon and melon industries. To date, there are no commercial cultivars of cucurbit crops resistant to the disease. Here we assessed the level of tolerance to bacterial fruit blotch of various commercial cultivars as well as breeding and wild lines of melon, using seed-transmission assays and seedling-inoculation experiments. Selected cultivars were also tested in a greenhouse experiment with mature plants. All tested cultivars/lines were found to be susceptible to the pathogen, and most of them showed different responses (relative tolerance vs. susceptibility) in the different assays; however, some consistent trends were found: cv. ADIR339 was relatively tolerant in all tested assays, and cv. 6407 and wild lines BLB-B and EAD-B were relatively tolerant in seed-transmission assays. We also provide evidence supporting a strong correlation between the level of susceptibility of a cultivar/line and the ability of the pathogen to adhere to or penetrate the seed. To the best of our knowledge, this is the first attempt to assess melon cultivars/lines for bacterial fruit blotch response.     

葡萄座腔菌(Botryosphaeria dothidea)全基因组分泌蛋白的预测及功能分析

葡萄座腔菌(Botryosphaeria dothidea)是引起多种果树和林木枝干溃疡病和果实腐烂病的重要病原菌.为明确葡萄座腔菌的致病机理,采用SignalP、WoLF PSORT、TMHMM、GPI-SOM等软件,对葡萄座腔菌全基因组14 998条蛋白序列进行了分泌蛋白预测和功能分析.结果表明B.dothidea...     

瓠瓜果斑病症状及其病原菌的鉴定

为有效防控瓠瓜果斑病,自浙江省象山县田间采集具有典型果斑病症状的瓠瓜样本,对其进行病原菌分离、形态观察、致病性测定及分子生物学鉴定,并利用特异性引物PL1/PL2 PCR扩增和基质辅助激光解吸电离飞行时间质谱（matrix assisted laser desorption ionization time-of-flight massspectrometry,MALDI-TOF-MS）技术对其病原菌进行亚群鉴定。结果表明：瓠瓜果斑病田间典型症状是发病叶片和果实上病斑由水渍状小斑点逐渐发展为伴有黄色晕圈的褐色不规则病斑,果实腐烂、有臭味。通过菌体形态和培养特性观察、PCR鉴定、16S rDNA序列分析和7个看家基因的系统发育分析将其病原菌鉴定为西瓜噬酸菌Acidovorax citrulli。从瓠瓜上分离的菌株均属于西瓜噬酸菌亚群I,从西瓜上分离的菌株均属于西瓜噬酸菌亚群II。     

甜瓜粉霉病菌效应蛋白编码基因的预测与分析

 粉霉病是一种严重影响甜瓜果实品质并造成经济损失的采后病害,效应蛋白在病原真菌致病机制中发挥着重要作用。为获得引起甜瓜粉霉病病原菌粉红单端孢(Trichothecium roseum)的效应蛋白,本研究利用一系列生物信息分析软件(SignalP, TMHMM, Protcomp, big-PI Predictor, TargetP和Blastp)对该菌进行效应蛋白预测与分析,并采用实时荧光定量PCR检测其中6个候选效应蛋白编码基因在离体诱导培养和接种果实时的表达模式。结果表明:从T. roseum的9 760个蛋白序列中预测得到154个候选效应蛋白,其中64个具有功能注释,其余为功能未知蛋白;候选效应蛋白的信号肽长度在14~30 aa之间,且大部分是由信号肽酶I型进行识别;信号肽中出现频率最高的氨基酸依次为丙氨酸、亮氨酸和丝氨酸;其中5个候选效应蛋白编码基因在离体诱导条件下和接种果实时表达量均显著升高,说明其可能为真正的效应蛋白。本研究预测获得了T. roseum中的效应蛋白,实时荧光定量PCR分析说明效应蛋白编码基因参与了T. roseum侵染甜瓜果实的致病过程,这些结果为进一步分析效应蛋白的功能与致病机制奠定了基础。     

Management of melon bacterial blotch by plant beneficial bacteria

Plant beneficial bacteria (PBB) have shown potential for disease control and are particularly important in the management of bacterial diseases, which are poorly controlled by conventional methods. In melon, bacterial fruit blotch caused by Acidovorax citrulli is a seedborne disease that is particularly destructive under certain conditions. PBB strains were screened for their ability to protect seeds and leaves from bacterial fruit blotch, and their antibiosis activity and plant colonization were studied. When Bacillus sp. RAB9 was applied to infected seeds, it reduced the area under the disease progress curve (AUDPC) by 47% and increased the incubation period (the time between inoculation and the first visible symptoms) by 35%. Three of the selected strains (JM339, MEN2 and PEP91) displayed antibiosis against A. citrulli. The RAB9^Rif-Nal mutant colonized seeds epiphytically and roots and stems endophytically. Paenibacillus lentimorbus MEN2 sprayed on melon seedlings protected leaves, and when challenged with A. citrulli, it reduced the AUDPC (by 88%), disease index (by 81%) and incidence (by 77%). Given that the production of both melon seedlings and commercially grown greenhouse melons is increasing, biocontrol strategies may well be integrated into bacterial blotch management programs.     

Cotyledons are the main source of secondary spread of Acidovorax citrulli in melon nurseries

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB) disease resulting in substantial economic damage to cucurbit crops worldwide. The plant parts and cultural practices (irrigation methods and bactericidal sprays) that affect the secondary spread of Ac in melon nurseries were investigated in this study. Overhead irrigation dispersed the pathogen from infected seedlings to 95% of the neighbouring healthy seedlings, with 80% of them displaying high disease severity. In contrast, when sub‐irrigation by floating was employed, the neighbouring plants of the infected ones did not display disease symptoms and were not colonized by Ac. Foliar treatment with Kocide after cotyledon emergence reduced disease incidence to 40%, with 37% of the plants displaying low disease severity. Assessment of Ac populations in different parts of the seedlings revealed that cotyledons were the most colonized part of the plant. Images of fluorescent binocular and confocal laser‐scanning microscopy of seedlings infected with a GFP‐labelled Ac strain showed that the pathogen forms abundant aggregates on the surface of cotyledons, colonizes the intercellular spaces of the parenchymatic tissues extensively, and moves through the vascular system of the hypocotyls, leading to infection of emerging leaves. Results of this study indicate that preventing secondary spread of Ac in melon nurseries by sub‐irrigation combined with a bactericidal spray at the cotyledon stage may provide an effective means for BFB control.