一种快速鉴定玉米细菌性枯萎病菌的新技术——噬菌体株系鉴别法

利用８株玉米细菌性枯萎病菌噬菌体专化性的差异，结合氯化三苯基四氮唑（２，３，５－ｔｒｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍｃｈｌｏｒｉｄｅ）对不同致病力细菌显色反应的不同，组成了一种新的细菌快速鉴定技术，称为噬菌体株系鉴别法，可鉴定玉米细菌性枯萎病菌直至株系。用这个方法鉴定了从美国、日本、南斯拉夫、西德和罗马尼亚进口的玉米种子中截获的许多玉米细菌性枯萎病菌菌株，结果准确快速，并和血清学鉴定及致病性测定的结果相一致     

木薯细菌性萎蔫病菌的LAMP快速检测方法

建立了一种木薯细菌性萎蔫病菌的环介导恒温扩增快速检测方法,为木薯细菌性萎蔫病的快速检测提供有力的技术支持。针对木薯细菌性萎蔫病菌TAL效应器蛋白质(pthBXam)靶序列的6个位点设计4条特异性引物,并对反应温度和内引物浓度等参数进行了优化,设计的引物与试验中提供的其他黄单胞近缘种都没有扩增反应,表现了较好的特异性。LAMP方法对木薯细菌性萎蔫病菌菌株DNA的检测下限为1pg/μL,比常规PCR灵敏度高100倍。该方法采用SYBR Green I染料法对扩增产物闭管检测,裸眼观察颜色变化判断反应结果,能快速、准确地对田间样品进行检测,没有出现假阳性和假阴性。与其他检测方法相比,LAMP方法检测时间短,效率高,降低了设备投入,易于操作,适合木薯细菌性萎蔫病菌的现场检疫和大规模监测。     

PCR-DHPLC技术快速检测玉米细菌性枯萎病菌

本研究成功建立了玉米细菌性枯萎病菌的PCR检测方法.该方法根据细菌ITS序列的特异性,设计了对玉米细菌性枯萎病菌具有稳定性点突变的特异性引物及探针,并对5株玉米细菌性枯萎病菌及18种植物原性细菌的DNA进行了PCR、实时荧光PCR及PCR结合变性高效液相色谱技术(PCR-DHPLC)检测.结果表明,几种方法特异性强,检测灵敏度均为菌液浓度102 cfu/mL,PCR-DHPLC技术具有检测成本较低、高通量、自动化程度高、污染风险小及鉴定结果准确等特点,能够满足快速、准确诊断玉米细菌性枯萎病菌的要求.     

菠萝泛菌与水稻白叶枯病菌双重PCR检测方法的建立与应用

近年来水稻发生了一种由菠萝泛菌Pantoea ananatis引起的新型细菌病害，其发生时期与白叶枯病相近，病症与白叶枯病类似。为了实现该病害与白叶枯病的快速检测，本研究基于泛菌属看家基因acnA,通过序列比对，设计了22对特异性引物，分别与已知的白叶枯病菌检测引物XOO80进行配对并筛选，建立了一种双重PCR检测方法，可从菠萝泛菌和白叶枯病菌中分别扩增出910 bp和162 bp的特异性条带，而其他10种非目标细菌均未有扩增条带，25μL体系中可稳定地从至少1 pg/μL的基因组DNA模板中扩增出特异性条带。本研究建立的菠萝泛菌与水稻白叶枯病菌双重PCR检测方法具有良好的特异性和灵敏度，为水稻细菌病害病原鉴定及防治提供了技术支撑。     

玉米细菌性枯萎病菌TaqMan探针实时荧光PCR检测方法的建立

成功建立了玉米细菌性枯萎病菌快速检测鉴定的实时荧光PCR方法.该方法根据细菌16S rDNA序列的特异性,设计出对玉米细菌性枯萎病菌具有稳定性点突变特异性探针,并对10种细菌菌株和5种植原体进行了实时荧光PCR.结果表明,只有玉米细菌性枯萎病菌产生荧光信号,而其它参考菌不产生荧光信号,检测的绝对灵敏度是14.2 fg/μl质粒DNA,比常规的PCR电泳检测高约100倍.整个检测过程只需2h,完全闭管,降低了污染的机会,无须PCR后处理.     

苜蓿萎蔫病菌TaqMan探针实时荧光PCR检测方法的建立

苜蓿萎蔫病菌是我国对外检疫性二类有害生物，目前国内尚无发生6在出入境捡验检疫中主要是采用生物学和血清学方法进行检测，劳动强度大，耗费时间长。根据苜蓿萎蔫病菌与其它细菌菌株16SrDNA序列差异，设计出对苜蓿萎蔫病菌具有稳定点突交特异性探针，利用该探针对棒形杆菌属4个种及其它属细菌进行了实时荧光PCR检测实验。结果表明，只有苜蓿萎蔫病菌能检测到荧光信号，其它细菌没有荧光产生。该方法特异性强，灵敏度高，能检测到21．4fg质粒DNA，比常规PCR灵敏100倍，而且整个过程只需要2～3h。该方法可有效地应用于进出境病原菌检测之中。     

利用多重PCR技术快速检测4种水稻病原细菌

对水稻中多种病原细菌的检测,使用常规方法往往耗时耗力,而多重PCR可以更加高效地进行多种细菌的检测。根据水稻细菌性谷枯病菌gyrB基因,水稻细菌性叶鞘褐腐病菌PfsI/R quorum sensing 位点以及水稻细菌性条斑病菌和水稻白叶枯病菌含铁细胞接受因子基因设计引物,建立4种水稻病菌的多重PCR检测方法,对方法进行特异性和灵敏度测试,并对采自不同地区的水稻样本进行检测。结果显示,多重PCR方法能同步地快速检测出水稻细菌性谷枯病菌、水稻细菌性叶鞘褐腐病菌、水稻细菌性条斑病菌或水稻白叶枯病菌,检测灵敏度达到103 cfu/mL的菌液浓度,利用该方法对我国不同地区的58份水稻种子进行检测,其中17个样本检测出水稻细菌性条斑病菌或水稻白叶枯病菌,未检测到水稻细菌性谷枯病菌和水稻细菌性叶鞘褐腐病菌。     

杨树(细菌性)枯萎病菌快速检测方法研究

本文对杨树(细菌性)枯萎病菌的培养特征、生理生化特征、分子生物学特征等方面进行了研究与描述,设计并合成了特异性PCR引物,杨树(细菌性)枯萎病菌的PCR产物出现718 bp的特异性扩增条带,而其他近似种的细菌均未出现扩增条带,说明该对引物具有杨树(细菌性)枯萎病菌鉴定特异性,将病菌DNA模板做浓度梯度稀释,灵敏度可达10 pg/μL。该方法快速、灵敏、准确,可用于杨树枯萎病菌检测。     

梨果贮藏期病害拮抗细菌的筛选、鉴定和评价

梨轮纹病和炭疽病是梨果贮运过程中常见的两个重要病害,危害严重。本研究采集江苏省淮阴区、淮安区、盱眙县三地梨园病叶、病果、健叶、健果和土壤样本75份,分离纯化后得到365个细菌分离物。采用对峙生长法,测定细菌分离物对梨轮纹病菌和炭疽病菌菌丝生长的皿内抑制作用。采用梨果表面刺伤后接种菌块的方法,测定拮抗细菌对梨果轮纹病和炭疽病的控制作用。结果表明,4株拮抗能力较强的细菌对轮纹病菌菌丝生长的抑制率达73.45%~80.36%,对炭疽病菌菌丝生长的抑制率达72.02%~76.79%。菌株N106-2对黄冠梨和砀山酥梨接种轮纹病菌块后5 d的防治效果达68.58%和56.98%;对黄冠梨接种炭疽病菌块后5 d的防治效果为56.71%。对4株拮抗细菌进行16S r DNA种属鉴定,其中3株为芽胞杆菌属Bacillus,1株为假单胞菌属Pseudomonas。     

拮抗细菌Bs-208菌株鉴定及对几种植物病原菌的抑菌测定

从番茄田土壤中分离筛选获得的拮抗细菌Bs-208鉴定为枯草芽孢杆菌。采用不同方法测定了Bs-208菌株发酵液及分泌物滤液对番茄灰霉病菌、棉花黄萎病菌、棉花枯萎病菌及棉花立枯病菌的抑菌效果。结果表明，该菌株对供试植物病原菌具有很好的抑菌效果，并可在植物叶面定殖，有效排斥和干扰植物病原等杂菌在植物叶面上的定殖，达到抑菌防病效果。     

Development of a New Semiselective Medium for Isolating Xanthomonas campestris pv. manihotis from Plant Material and Soil

ABSTRACT An effective control for bacterial blight of cassava (Manihot esculenta), caused by Xanthomonas campestris pv. manihotis, requires the use of non-contaminated cuttings and seeds. Using classical agar plating techniques for screening planting material for contamination has not been very successful because of the lack of a reliable semiselective agar medium. The pathogen grows slowly on general plating media and is easily overgrown by saprophytic bacteria during isolation from diseased plants. In an effort to develop a semiselective medium, the utilization of several carbon and nitrogen sources was studied. Results of these tests provided information used to design a basal medium allowing good growth of the target organism while suppressing growth of several common saprophytes. Additional selectivity was achieved by incorporating three antibiotics into the basal medium. The new semiselective agar medium, designated cefazolin trehalose agar (CTA) medium, contained (per liter) 3.0 g of K(2)HPO(4), 1.0 g of NaH(2)PO(4), 0.3 g of MgSO(4).7H(2)O, 1.0 g of NH(4)Cl, 9.0 g of D(+)-trehalose, 1.0 D(+)-glucose, 1.0 g of yeast extract, 0.025 g of cefazolin, 0.0012 g of lincomycin, 0.0025 g of phosphomycin, 0.25 g of cycloheximide, and 14.0 g of agar. In comparison to a starch-based semiselective medium (SXM), plating efficiencies using pure cultures of 10 strains of X. campestris pv. manihotis were significantly higher on CTA, with an average of 85 and 50%, respectively. Likewise, isolation and recovery of X. campestris pv. manihotis from infected cassava leaves and contaminated soil were much higher on CTA than on SXM agar. When X. campestris pv. manihotis occurs in high concentrations in diseased tissue, the standard yeast trehalose glucose agar medium supplemented with 250 mug of cycloheximide per ml appears to be satisfactory. The newly developed CTA medium should prove useful for control strategies to identify and remove infected planting material of cassava, as well as for basic ecological studies of the pathogen.     

Development of a semiselective medium for isolating Xanthomonas campestris pv. musacearum from insect vectors, infected plant material and soil

A semiselective medium was developed for isolating Xanthomonas campestris pv. musacearum ( Xcm ) from infected banana plants, soil and insect vectors. The new medium was named cellobiose-cephalexin agar (CCA) and it contained (L⁻¹): 1 g yeast extract, 1 g glucose, 1 g peptone, 1 g NH₄Cl, 1 g MgSO₄·7H₂O, 3 g K₂HPO₄, 1 g beef extract, 10 g cellobiose, 14 g agar, 40 mg cephalexin, 10 mg 5-fluorouracil and 120 mg cycloheximide. The medium was evaluated for selectivity using 21 bacterial isolates and for plating efficiency using Xcm . The bacterial isolates included a soilborne Xanthomonas species and three pathogenic Xanthomonas strains that infect cassava, cabbage and beans. Although the plating efficiency of Xcm on CCA was lower (59%) than on non-selective yeast extract peptone glucose agar (YPGA), its selectivity was significantly higher, averaging 60 and 82%, when isolating from banana fruits and soil, respectively. CCA was also superior when isolating Xcm from insect vectors, with selectivity of 48–75%, compared with 8–17% on YPGA. Xanthomonas campestris pv. phaseoli did not grow on CCA, while X. campestris pv. campestris and X. axonopodis pv. manihotis grew, but their colonies were smaller than those of Xcm . Twenty-nine out of 33 suspected Xcm strains isolated from plants, soil and insects using CCA were pathogenic when inoculated onto banana plants, indicating that CCA can be a reliable tool in isolating Xcm populations. The medium should prove useful in studies on ecology, epidemiology and management of the banana bacterial wilt pathogen that is currently ravaging bananas in East and Central Africa.     

Molecular diagnostic procedures for production of pathogen-free propagation material

Production of disease-free propagation material is a major means of controlling most bacterial diseases of plants, particularly when neither resistant clones nor effective chemical treatments are available. For this purpose sensitive, specific and rapid detection methods are required. The advent of molecular biology and, in particular, the polymerase chain reaction (PCR) has opened new ways for the characterization and identification of plant pathogens and the development of disease-management strategies. PCR-based detection methods rely on the development of primers for the specific detection of the pathogen. The use of pathogenicity genes as targets for primer design is the preferred procedure for obtaining specific primers but other procedures may also be useful for this purpose. In the present review we describe four examples of procedures for detecting four important bacterial pathogens in Israel: Erwinia herbicola pv gypsophilae in gypsophila, Xanthomonas campestris pv pelargonii in geranium, Agrobacterium tumefaciens in asters and roses, and Xanthomonas campestris pv campestris in crucifers. Procedures for constructing specific PCR primers for each bacterium are illustrated and discussed as well as the combination of PCR with other methods.     

Molecular characterization of the incitant of cowpea bacterial blight and pustule, Xanthomonas campestris pv. vignicola

Strains of Xanthomonas campestris pv. vignicola (Xcv), isolated from cowpea leaves with blight or minute pustules and collected from various geographic areas, were selected on the basis of pathological and physiological features. All strains were analyzed for genotypic markers by two methods: ribotyping with EcoRI endonuclease, and RFLP analysis with a plasmid probe (pthB) containing a gene required for pathogenicity from Xanthomonas campestris pv. manihotis. Ribotyping revealed a unique pattern for all the strains that corresponded to the previously described ribotype rRNA7. Based on polymorphism detected by pthB among Xcv strains, nine haplotypes were defined. The observed genetic variation was independent of the geographic origin of the strains and of pathogenic variation. Some haplotypes were widely distributed, whereas others were localized. In some cases, we could differentiate strains isolated from blight symptoms and pustules according to haplotypic composition. However, in most cases, no significant differences were observed. Our results and the previous pathogenic and biochemical characterizations suggest that the strains isolated from leaves with blight symptoms or minute pustules belong to the same pathovar. We provide information on pathogen diversity that can be used to identify and characterize resistant germplasm.     

Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination

Xanthomonas campestris pv. vitians , the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.     

Methods to reduce the spread of the black rot pathogen,Xanthomonas campestris pv. campestris,in brassica transplants

Journal of Plant Diseases and Protection - Black rot of Brassica spp. caused by the bacterial pathogen Xanthomonas campestris pv. campestris (XCC), is a seedborne disease. Because the pathogen can...     

Measuring the Genetic Diversity of Xanthomonas axonopodis pv. manihotis Within Different Fields in Colombia

ABSTRACT Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis, is a widespread disease that affects cassava (Manihot esculenta). We collected 238 X. axonopodis pv. manihotis strains by intensively sampling single fields in four edaphoclimatic zones (ECZs) in Colombia. DNA polymorphism of different X. axonopodis pv. manihotis populations was assessed by restriction fragment length polymorphism (RFLP) analyses, repetitive sequence-based polymerase chain reaction (rep-PCR), and amplified fragment length polymorphism (AFLP) assays. Genetic diversity, phenetic relationships among strains, and the coefficient of genetic differentiation were determined. All strains were tested for aggressiveness on the susceptible cassava cv. MCOL 1522. Strains were also tested for virulence on cassava differentials adapted to the strains' respective ECZs. Our study showed that the Colombian X. axonopodis pv. manihotis population has a high degree of genetic diversity. The hierarchical analysis of diversity showed genotypic differentiation at all levels, among ECZs, among fields within ECZs, and among strains within fields planted to several cassava genotypes. New RFLP haplotypes were detected, leading to the characterization of a new pathotype. Dendrograms from AFLP were more robust than those from RFLP data. A close association between the strains' geographical origin and DNA polymorphism was obtained using RFLP and AFLP data. We suggest that the host played a role in causing pathogen differentiation.     

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.     

Use of phages for identifying the citrus canker bacterium Xanthomonas campestris pv. citri in Taiwan

Phages CP115 and CP122, which were isolated from canker lesions on grapefruit and Liucheng sweet orange, respectively, showed a high degree of specificity with respect to lysis of test bacterial strains. When used jointly, they lysed 135 (97·8%) out of 138 Xanthomonas campestris pv. citri strains isolated from the canker lesions on leaves, twigs, and fruits of various citrus species, cultivars, and hybrids grown throughout Taiwan, but they did not lyse other X. campestris pathovars and other phytopathogenic bacteria, nor other bacteria isolated from soil, clinical or environmental samples. Of 252 CP115/CP122-sensitive and 78 CP115/CP122-resistant bacterial strains with colony characteristics typical of or similar to those of X. campestris pv. citri , isolated from canker lesions of various citrus plants in diverse growing regions in Taiwan, 250 (99·2%) and 76 (97·4%) strains were pathogenic and non-pathogenic, respectively, when inoculated into Liucheng sweet orange or Mexican lime. Thus, phages CP115 and CP122, when used jointly, appear to be applicable for identifying X. campestris pv. citri in Taiwan.