进境菜豆种子中普通细菌性疫病菌的分离和鉴定

对经甘肃口岸进境的30批菜豆Phaseolus vulgaris种子进行了普通细菌性疫病菌的检测,利用选择性培养基MT从波兰进境菜豆种子上分离到1株细菌597,对该分离物进行菌落形态特征观察、致病性测定、16S rDNA及16S-23S rDNA ITS序列分析和特异性PCR检测。结果表明,该分离物在MT培养基上菌落呈黄色、圆形、黏稠、表面光滑向外隆起、菌落周围有水解圈。分离物597接种菜豆幼苗后导致叶片枯萎,接种点干枯。结合菌落形态、16S-23S rDNA ITS序列、特异性PCR检测结果,将分离物597鉴定为地毯草黄单胞杆菌菜豆致病变种Xanthomonas axonopodis pv.phaseoli。     

菜豆种子普通细菌性疫病菌检测

 由Xanthomonas axonopodis pv. phaseoli和Xanthomonas fuscans subsp. fuscans引起的菜豆普通细菌性疫病是严重影响菜豆生产的限制因子之一,可造成严重的产量和品种损失。染菌种子是病原菌传播的主要途径。本研究对5个菜豆主要产区的60份菜豆种子样品进行普通细菌性疫病菌检测。在MT选择性培养基上有36份种子样品浸提液检测到目标病原菌, 种子样品带菌量为2.49×10²~5.20×10⁷ CFU/粒。选择36个分离物接种感病品种“英国红”植株,所有分离物均引起接种植株发病。特异PCR检测结果表明,有20个分离物为X. fuscans subsp. fuscans,16个分离物为X. axonopodis pv. phaseoli。试验结果表明,我国一些菜豆主产区商业种植和研究用种子多数污染普通细菌性疫病菌,建议建立无菌种子生产区和加强种子管理,以有效控制病害发生。     

红掌细菌性疫病病原菌生物学特性研究

红掌细菌性疫病由地毯草黄单胞菌花叶万年青致病变种(Xanthomonas axonopodis pv.dieffenbachiae)引起。测定了病原菌在PDA、NA、SX、KB、TTC培养基上的生长状况,并初步研究该病的发生规律。试验表明,病菌典型菌株的胞外纤维素酶、淀粉酶、蛋白酶都具水解活性,但活性大小有所差异;在8种抗生素的筛选中,菌株在链霉素、利福平、氨苄青霉素及新露霉素上能生长,而在格丹霉素、卡那霉素、四环素和壮观霉素上不能生长。     

海南一品红细菌性叶斑病病原菌的分离与鉴定研究

 为明确海南省近几年发生的一品红细菌性叶部病害的病原菌特别是该病原菌的分子特征,为该病害的治理提供可行的依据,本文描述了2005年海南省部分花圃一品红植株上发生的细菌性叶斑病症状,并通过致病性测定、BIOLOG分析和16SrDNA序列比较将分离的病原菌鉴定为黄单胞菌属(Xanthomonas);部分碳源利用测定进一步显示2005年海南分离的菌株与2004年海南报道的细菌性疫病病原菌存在差异,但与杭州地区报道的一品红细菌性叶斑病菌基本一致。利用黄单胞菌模式种典型菌株的核糖体DNA内转录间隔区(internal transcribed spacer,ITS)序列构建系统发育树,结果显示3个海南菌株与巴豆黄单胞菌(Xanthomonas codiaei)和地毯草黄单胞菌(X.axonopodis)单独聚合成群,其中与巴豆黄单胞菌亲缘关系最近。     

芒果细菌性黑斑病病菌抗铜基因copA克隆与序列分析

芒果细菌性黑斑病是由野油菜黄单胞菌芒果致病变种(Xanthomonas campestris pv.mangiferaeindicae)引起的细菌性病害,是芒果生产中最为重要的病害之一。本试验通过对比已报道的黄单胞属的抗铜基因copA序列的分析、比较,设计简并引物Copa-F/Copa-R,采用同源克隆法从芒果细菌性黑斑病病菌中扩增出约1 800 bp的DNA片段。序列分析表明,该基因片段含有1个完整的copA同源基因(GenBank accession No.KJ890405.1),其核苷酸序列全长1 794 bp,编码597个氨基酸,其分子量约66.271 kDa,等电点(PI)为6.40。系统进化树结果显示,该基因编码的氨基酸序列与Xanthomonas属多个不同种的抗铜基因聚为一类。     

红掌细菌性疫病的病原菌初步鉴定

 在云南西双版纳的红掌上发现一种由细菌侵染引起的病害,从叶片的病组织上分离到具有致病性的杆状细菌,将分离的病原菌接种于健康的红掌上,表现出与田间一致的症状。感病初期在叶缘或叶脉间出现水渍状小点,后期病斑多呈棕褐色至黑色坏死,病健交界处多呈黄色。从接种发病的病斑与田间标样上分离的病原菌菌落形态完全相同。通过菌株的培养性状、常规生理生化特性及透射电镜下菌体形态观察结果,将病原菌初步鉴定为黄单胞菌属(Xanthomonas)。BIOLOG鉴定和致病性测定结果进一步鉴定病原菌为地毯草黄单胞菌花叶万年青致病变种Xanthomonas axonopodis pv. dieffenbachiae(Xad)(McCulloch & Pirone) Vauterin et al.     

产生ACC脱氨酶的PGPR种衣剂对黄瓜细菌性茎软腐病的防治效果

为防治近几年越冬茬黄瓜上的新病害——细菌性茎软腐病,从海边盐生植物根际土壤中富集培养、分离和鉴定了产1-氨基环丙烷-1-羧酸(ACC)脱氨酶的植物根际促生菌(PGPR),共鉴定出了嗜麦芽寡养单胞菌Stenotrophomonas maltophilia、土生拉乌尔菌Raoultella terrigena、土壤杆菌Agrobacterium tumefaciens、产酸克雷伯菌Klebsiella oxytoca、成团泛菌Pantoea agglomerans、产气肠杆菌Enterobacter aerogenes和克吕沃尔氏菌Kluyvera cryocrescens共7个种的PGPR。测定了ACC脱氨酶活性,其中嗜麦芽寡养单胞菌菌株CRG-2,产生ACC脱氨酶活性最高,为324.3μmol/(mg·h)。将菌株CRG-2和枯草芽胞杆菌复配制成丸化种衣剂后,测定了对黄瓜细菌性茎软腐病的田间防治效果。结果表明,嗜麦芽寡养单胞菌和枯草芽胞杆菌复配后形成的种衣剂对黄瓜细菌性茎软腐病的田间防治效果达到73.13%,高于中生菌素防治效果15.79%。由此可见该种衣剂有望在生产上用于防治黄瓜细菌性茎软腐病。     

CopAB基因突变导致PXO99对铜敏感和致病性降低

Cop基因簇是黄单胞菌属抗铜的主要基因位点,而水稻白叶枯病菌PXO99相对其他菌株对铜离子更加敏感。本研究发现抗铜基因Cop AB的6个氨基酸自然缺失突变是造成PXO99对铜离子敏感的主要因素。通过对Cop AB的缺失突变体和恢复抗铜能力的重组菌株的耐铜实验和接种实验,证实了Cop AB不仅参与黄单胞杆菌的抗铜功能,同时作为一种致病因子参与黄单胞杆菌与水稻的互作。为验证Cop AB基因的功能,从国内白叶枯病菌分离菌株中筛选到4株对铜离子敏感菌株,并证实其Cop AB基因也发生相似的突变。同时,恢复抗铜能力的PXO99互补菌株并不能克服xa13介导的抗性,而分离到的4株铜离子敏感的菌株中,3株不含Pth Xo1的菌株在IR24和IRBB13上致病性相当,说明PXO99对铜离子的敏感性与xa13基因介导的对PXO99专化抗性没有直接的关系。     

构树上一种新的丁香假单胞菌致病变种的研究

 构树细菌性疫病是一种荧光假单胞菌引起的新病害。主要症状有叶片角斑,嫩梢肿大和幼枝溃疡。从江苏一带分离获得的12个构树菌株和3个桑树对比菌株进行交互接种试验,发现构树菌株与桑树菌株之间不能交互侵染其寄主。细菌学特征和LOPAT试验及其它37项生理生化和营养特性试验表明,两种菌的表型特征基本相似,仅在7种化合物的利用上存在差异。两种菌的血清学反应无相关性。细胞全蛋白SDS一聚丙烯酰胺凝胶电泳图谱也略有不同。试验结果证明,构树细菌性疫病细菌属于假单胞菌属(Pseudomonas),丁香假单胞菌(P.syringae)的一个新致病变种,定名为Pseudomonas syrzngae pv.broussonetiae pv.nov.     

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。     

Interaction of common bacterial blight bacteria with disease resistance quantitative trait loci in common bean

Common bacterial blight (CBB) of common bean (Phaseolus vulgaris L.) is caused by Xanthomonas campestris pv. phaseoli and X. fuscans subsp. fuscans, and is the most important bacterial disease of this crop in many regions of the world. In 2005 and 2006, dark red kidney bean fields in a major bean-growing region in central Wisconsin were surveyed for CBB incidence and representative symptomatic leaves collected. Xanthomonad-like bacteria were isolated from these leaves and characterized based upon phenotypic (colony) characteristics, pathogenicity on common bean, polymerase chain reaction (PCR) with X. campestris pv. phaseoli- and X. fuscans subsp. fuscans-specific primers, and repetitive-element PCR (rep-PCR) and 16S-28S ribosomal RNA spacer region sequence analyses. Of 348 isolates that were characterized, 293 were identified as common blight bacteria (i.e., pathogenic on common bean and positive in PCR tests with the X. campestris pv. phaseoli- and X. fuscans subsp. fuscans-specific primers), whereas the other isolates were nonpathogenic xanthomonads. Most (98%) of the pathogenic xanthomonads were X. campestris pv. phaseoli, consistent with the association of this bacterium with CBB in large-seeded bean cultivars of the Andean gene pool. Two types of X. campestris pv. phaseoli were involved with CBB in this region: typical X. campestris pv. phaseoli (P) isolates with yellow mucoid colonies, no brown pigment production, and a typical X. campestris pv. phaseoli rep-PCR fingerprint (60% of strains); and a new phenotype and genotype (Px) with an X. campestris pv. phaseoli-type fingerprint and less mucoid colonies that produced brown pigment (40% of strains). In addition, a small number of X. fuscans subsp. fuscans strains, representing a new genotype (FH), were isolated from two fields in 2005. Representative P and Px X. campestris pv. phaseoli strains, an FH X. fuscans subsp. fuscans strain, plus five previously characterized X. campestris pv. phaseoli and X. fuscans subsp. fuscans genotypes were inoculated onto 28 common bean genotypes having various combinations of known CBB resistance quantitative trait loci (QTL) and associated sequence-characterized amplified region markers. Different levels of virulence were observed for X. campestris pv. phaseoli strains, whereas X. fuscans subsp. fuscans strains were similar in virulence. The typical X. campestris pv. phaseoli strain from Wisconsin was most virulent, whereas X. campestris pv. phaseoli genotypes from East Africa were the least virulent. Host genotypes having the SU91 marker-associated resistance and one or more other QTL (i.e., pyramided resistance), such as the VAX lines, were highly resistant to all genotypes of common blight bacteria tested. This information will help in the development of CBB resistance-breeding strategies for different common bean market classes in different geographical regions, as well as the identification of appropriate pathogen genotypes for screening for resistance.     

Genetic Diversity and Pathogenic Variation of Common Blight Bacteria (Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans) Suggests Pathogen Coevolution with the Common Bean

ABSTRACT Common bacterial blight (CBB), caused by Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, is one of the most important diseases of common bean (Phaseolus vulgaris) in East Africa and other bean-growing regions. Xanthomonad-like bacteria associated with CBB in Malawi and Tanzania, East Africa, and in Wisconsin, U.S., were characterized based on brown pigment production, pathogenicity on common bean, detection with an X. campestris pv. phaseoli- or X. campestris pv. phaseoli var. fuscans-specific PCR primer pair, and repetitive element polymerase chain reaction (rep-PCR) and restriction fragment length polymorphism (RFLP) analyses. The common bean gene pool (Andean or Middle American) from which each strain was isolated also was determined. In Malawi, X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were isolated predominantly from Andean or Middle American beans, respectively. In Tanzania, X. campestris pv. phaseoli var. fuscans was most commonly isolated, irrespective of gene pool; whereas, in Wisconsin, only X. campestris pv. phaseoli was isolated from Andean red kidney beans. Three rep-PCR fingerprints were obtained for X. campestris pv. phaseoli strains; two were unique to East African strains, whereas the other was associated with strains collected from all other (mostly New World) locations. RFLP analyses with repetitive DNA probes revealed the same genetic diversity among X. campestris pv. phaseoli strains as did rep-PCR. These probes hybridized with only one or two fragments in the East African strains, but with multiple fragments in the other X. campestris pv. phaseoli strains. East African X. campestris pv. phaseoli strains were highly pathogenic on Andean beans, but were significantly less pathogenic on Middle American beans. In contrast, X. campestris pv. phaseoli strains from New World locations were highly pathogenic on beans of both gene pools. Together, these results indicate the existence of genetically and geographically distinct X. campestris pv. phaseoli genotypes. The rep-PCR fingerprints of X. campestris pv. phaseoli var. fuscans strains from East African and New World locations were indistinguishable, and were readily distinguished from those of X. campestris pv. phaseoli strains. Genetic diversity among X. campestris pv. phaseoli var. fuscans strains was revealed by RFLP analyses. East African and New World X. campestris pv. phaseoli var. fuscans strains were highly pathogenic on Andean and Middle American beans. Breeding for CBB resistance in East African beans should utilize X. campestris pv. phaseoli var. fuscans and New World X. campestris pv. phaseoli strains in order to identify germ plasm with the highest levels of resistance.     

Rapd Pcr-based differentiation of Xanthomonas campestris pv. phaseoli and Xanthomonas campestris pv. phaseoli var. fuscans

A RAPD PCR-based method was used to differentiate between isolates of Xanthomonas campestris pv. phaseoli and Xanthomonas campestris pv. phaseoli var. fuscans. Using random primer OP-G11, a single, high intensity band of 820 bp was amplified from DNAs of all X. c. pv. phaseoli var. fuscans isolates, while multiple amplification products of varying sizes were generated from X. c. pv. phaseoli DNAs. Whereas RAPD PCR differentiation gave an unambiguous result in under 4 h, standard differentiation by recording the production of a brown pigment by X. c. pv. phaseoli var. fuscans isolates took up to 7 days and showed variation both between isolates and between media. The unequivocal nature of the RAPD PCR method was demonstrated when isolate 408, originally classified as X. c. pv. phaseoli var. fuscans, failed to produce the 820 bp band typical of X. c. pv. phaseoli var. fuscans isolates, and after also failing to produce a brown pigment, was re-classified as X. c. pv. phaseoli.     

Phenotypic and Genetic Diversity in Strains of Common Blight Bacteria (Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans) in a Secondary Center of Diversity of the Common Bean Host Suggests Multiple Introduction Events

ABSTRACT Common bacterial blight (CBB) disease of the common bean (Phaseolus vulgaris) is caused by Xanthomonas campestris pv. phaseoli and the brown-pigmented variant X. campestris pv. phaseoli var. fuscans. CBB first was described in Castilla y León County, Spain, in 1940, and is now a major constraint on common bean production. In this secondary center of diversity of the common bean, large-seeded Andean cultivars predominate, although medium-seeded Middle American cultivars also are grown. Xanthomonad-like bacteria associated with CBB in Castilla y León were characterized on the basis of carbohydrate metabolism, brown pigment production, genetic analyses (repetitive-element polymerase chain reaction [rep-PCR] and random amplified polymorphic DNA [RAPD]) and pathogenicity on cultivars representing the two common bean gene pools (Andean and Middle American). X. campestris pv. phaseoli was more prevalent (80%) than X. campestris pv. phaseoli var. fuscans (20%). Patterns of carbohydrate metabolism of Spanish CBB bacteria were similar to those of known strains; and only X. campestris pv. phaseoli var. fuscans strains utilized mannitol as a sole carbon source. rep-PCR and RAPD analyses revealed relatively little genetic diversity among Spanish X. campestris pv. phaseoli strains, and these strains were placed together with New World strains into a large cluster. Similar to other New World strains, representative Spanish X. campestris pv. phaseoli strains were highly pathogenic on bean cultivars of both gene pools, showing no gene pool specialization such as that found in certain East African strains. Genetic analyses and pathogenicity tests confirmed and extended previous results, indicating that these East African strains represent distinct xanthomonads that independently evolved to be pathogenic on common bean. X. campestris pv. phaseoli var. fuscans strains were more closely related and genetically distinct from X. campestris pv. phaseoli strains. However, two distinct clusters of X. campestris pv. phaseoli var. fuscans strains were identified, one having the most New World strains and the other having the most African strains. Spanish strains were placed in both clusters, but all strains tested were highly pathogenic on bean cultivars of both gene pools. Together, our results are consistent with multiple introductions of CBB bacteria into Spain. These findings are discussed in terms of breeding for CBB resistance and the overall understanding of the genetic diversity and evolution of CBB bacteria.     

Induction of superoxide dismutase,malate dehydrogenase and phenylalanine ammonia-lyase during enhancing resistance of common bean against Xanthomonas axonopodis pv. phaseoli by exogenous salicylic acid

Journal of Plant Diseases and Protection - Bacterial blight of common bean caused by Xanthomonas axonopodis pv. phaseoli (Xap) is one of the most devastating diseases and causes serious yield...     

Application of image analysis in studies of quantitative disease resistance, exemplified using common bacterial blight-common bean pathosystem

The effectiveness of image analysis (IA) compared with an ordinal visual scale, for quantitative measurement of disease severity, its application in quantitative genetic studies, and its effect on the estimates of genetic parameters were investigated. Studies were performed using eight backcross-derived families of common bean (Phaseolus vulgaris) (n = 172) segregating for the molecular marker SU91, known to be associated with a quantitative trait locus (QTL) for resistance to common bacterial blight (CBB), caused by Xanthomonas campestris pv. phaseoli and X. fuscans subsp. fuscans. Even though both IA and visual assessments were highly repeatable, IA was more sensitive in detecting quantitative differences between bean genotypes. The CBB phenotypic difference between the two SU91 genotypic groups was consistently more than fivefold for IA assessments but generally only two- to threefold for visual assessments. Results suggest that the visual assessment results in overestimation of the effect of QTL in genetic studies. This may have been caused by lack of additivity and uneven intervals of the visual scale. Although visual assessment of disease severity is a useful tool for general selection in breeding programs, assessments using IA may be more suitable for phenotypic evaluations in quantitative genetic studies involving CBB resistance as well as other foliar diseases.     

Polyphasic characterization of xanthomonas strains from onion

ABSTRACT Xanthomonas leaf blight has become an increasingly important disease of onion, but the diversity among Xanthomonas strains isolated from onion is unknown, as is their relationship to other species and pathovars of Xanthomonas. Forty-nine Xanthomonas strains isolated from onion over 27 years from 10 diverse geographic regions were characterized by pathogenicity to onion and dry bean, fatty acid profiles, substrate utilization patterns (Biolog), bactericide resistance, repetitive sequence-based polymerase chain reaction fingerprinting, rDNA internally transcribed spacer (ITS) region, and hrp b6 gene sequencing. Multiplication of onion Xanthomonas strain R-O177 was not different from X. axonopodis pv. phaseoli in dry bean, but typical common bacterial blight disease symptoms were absent in dry bean. Populations from each geographical region were uniformly sensitive to 100 mug of CuSO(4), 100 mug of ZnSO(4), and 100 mug of streptomycin sulfate per ml. Biolog substrate utilization and fatty acid profiles revealed close phenoltypic relatedness between onion strains of Xanthomonas and X. axonopodis pv. dieffenbachiae (57% of strains) and X. arboricola pv. poinsettiicola (37% of strains), respectively. A logistic regression model based on fatty acid composition and substrate utilization classified 69% of strains into their geographical region of origin. Sequencing of a portion of the hrp B6 gene from 24 strains and ITS region from 25 strains revealed greater than 97% sequence similarity among strains. DNA fingerprinting revealed five genotype groups within onion strains of Xanthomonas and a high degree of genetic diversity among geographical regions of origin. Based on pathogenicity to onion, carbon substrate utilization, fatty acid profiles, rDNA genetic diversity, and genomic fingerprints, we conclude that the strains examined in this study are pathovar X. axonopodis pv. allii. Implications of genetic and phenotypic diversity within X. axonopodis pv. allii are discussed in relation to an integrated pest management program.     

The effect of populations of Xanthomonas campestris pv. phaseoli in bean reproductive tissues on seed infection of resistant and susceptible bean genotypes

Surface and internal populations of Xanthomonas campestris pv. phaseoli, causal agent of common bacterial blight of bean, on and in flower buds, blossoms and pods of seven bean (Phaseolus vulgaris) genotypes were studied. Bean plants were grown in the field and artificially inoculated at the seedling stage (18 days old). The pathogen was recovered in high numbers from flower buds, blossoms, pods and seed of both resistant and susceptible bean genotypes. Significant differences (P = 0.05) in population levels of X. c. pv. phaseoli between stages of reproductive tissue development were observed. Infected seed from resistant bean genotypes had no visible symptoms. Such seed may play an important role in the epidemiology of common bacterial blight because they are difficult to detect and may occur at low frequency in seed lots, as was the case in the current study.     

Identification of Resistance to Common Bacterial Blight Disease on Bean Genotypes Grown in Turkey

Common bacterial blight (CBB) in edible beans (Phaseolus vulgaris), incited Xanthomonas campestris pv. phaseoli, reduces bean yields and seed quality. The main objective of this study was to determine resistance to common bacterial blight in bean genotypes. Twenty-two bean genotypes grown in Turkey including common and snap bean cultivars/lines were collected from different parts of Turkey and tested for resistance against to Xanthomonas campestris pv. phaseoli strain MFD-11. All the common and snap bean lines/cultivars tested were moderately susceptible, susceptible or highly susceptible, except AG-7117 which was found resistant to Xanthomonas campestris pv. phaseoli. This is the first report of a resistance source in a common bean line (AG-7117) against Xanthomonas campestris pv. phaseoli.