木薯细菌性萎蔫病菌的检疫方法研究

本文对木薯细菌性萎蔫病菌的致病性测定、细菌的分离、细菌的培养条件和培养基选择、细菌的生理生化测定、分子生物学鉴定方法等方面进行了系统研究，确定了该病菌的菌落鉴定特征，建立了从木薯繁殖材料上进行病原菌检测的快速、灵敏、准确的PCR检测方法。     

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.     

两种植物病原黄单胞菌基因组中同义密码子使用的分析

 根据已释放的基因组序列,对野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv. campestris,Xcc)和地毯草黄单胞菌柑桔致病变种(X. axonopodis pv. citri,Xac)的密码子使用进行了分析。相对同义密码子使用值(relative synonymous codon usage,RSCU)的计算表明,它们具有高度相似的密码子使用模式。2个基因组密码子第3位的GC含量(GC3s)平均达0.806±0.077(Xcc)和0.791±0.075(Xac),倾向于使用GC含量较高的密码子。对有效密码子数量和密码子适应指数的分析表明,Xcc与Xac基因组中,高表达基因具有较高的GC含量,倾向于使用少数种类的密码子,而低表达基因具有较高的AT含量,倾向于随机地使用密码子。对密码子使用绝对次数进行的对应分析也证明了上述结论。同时,计算也证明了基因在基因组中的位置不影响密码子使用的模式。因此,基因组的GC含量、基因的表达水平和基因的种类与起源是影响这2个基因组密码子使用的主要因素。     

芒果细菌性黑斑病菌在不同场所的存活期

选择芒果细菌性黑斑病菌(Xanthomonas campestris pv.mangiferaeindicae,Xcm)抗利福平菌株RifXcm,通过人工模拟接种试验,采用半选择性平板分离、菌落PCR和常规PCR方法,研究该病原细菌在芒果叶片、土壤和水中的存活期,并对其能否作为侵染源进行了评价。结果表明,芒果细菌性黑斑病菌在芒果叶片病斑内可存活5~6个月,是此病发生最主要的侵染源。病原菌在土壤和自然水中的存活期有限,其中以含芒果残体土壤中的病原菌存活期最长(49~63d),但也没有超过3个月,因此年前存在于这些场所的病原菌均不可能成为第2年的初侵染源。     

Characterization,genetic diversity and distribution of Xanthomonas campestris pv. campestris races causing black rot disease in cruciferous crops of India

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a disease of crucifer crops. The objective of this study was to characterize races of Xcc, their distribution and genetic diversity in India. Two hundred and seventeen isolates of bacteria were obtained from 12 different black rot‐infected crucifer crops from 19 states of India; these were identified as Xcc based on morphology, hrpF gene and 16S rRNA gene based molecular markers and pathogenicity tests. Characterization of races was performed by using a set of seven differential crucifer hosts, comprising two cultivars of turnip (Brassica rapa var. rapa) and cultivars of Indian mustard (B. juncea), Ethiopian mustard (B. carinata), rapeseed mustard (B. napus), cauliflower (B. oleracea) and Savoy cabbage (B. oleracea var. sabauda). Races 1, 4 and 6 of Xcc were identified and, among these races, race 1 followed by race 4 dominated most of the states of India. Genetic diversity of the Indian isolates of Xcc was analysed using repetitive sequence‐based PCR (rep‐PCR) including primers for REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX (amplifying with BOX A1 R primer) repetitive elements. This method of fingerprinting grouped the isolates into 56 different DNA types (clusters) with a 75% similarity coefficient. Among these clusters, DNA types 22 and 53 contained two different races 1 and 4, whereas DNA type 12 contained races 1, 4 and 6. However, no clear relationship was observed between fingerprints and races, hosts or geographical origin.     

A study on the molecular mechanism of resistance to amicarthiazol in Xanthomonas campestris pv. citri

Three amicarthiazol-resistant mutants (Xuv10, Xuv20 and Xuv40) were obtained by UV induction and used in this study. Minimal inhibition concentrations (MICs) of amicarthiazol against the growth of mutants and wild-type isolate were 400 and 100 microg ml(-1) respectively. Inhibition by amicarthiazol of succinate dehydrogenase (SDH) activities of Xanthomonas campestris pv. citri (Hasse) Dye wild-type isolate (Xcc) and three resistant mutants derived from this isolate were assayed using triphenyltetrazolium chloride (TTC). The SDH activities of these mutants were significantly lower than that of Xcc. The complete nucleotide sequences of four subunits (SdhA, SdhB, SdhC and SdhD) of succinate-ubiquinone oxidoreductase (SQR) were cloned by polymerase chain reaction (PCR) amplification. An amino acid mutation (His229--> Leu229) in sdhB was found to confer resistance of X. campestris pv. citri to amicarthiazol. It is suggested that this mutation alters the SDH complex in some way that prevents binding of amicarthiazol.     

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.     

十字花科黑腐病菌应急反应相关基因spoT_(Xcc)对T3SS基因调控作用的分析

通过反向遗传学方法和半定量RT-PCR方法,研究了十字花科黑腐病菌中被注释的应急反应相关基因spoT_(Xcc)与Ⅲ型分泌系统(T3SS)基因的调控关系。基于自杀质粒pK18mob同源整合方法构建了十字花科黑腐病菌spoT_(Xcc)基因(XC_0956)的非极性整合突变体,检测了突变体的多种表型,发现spoT_(Xcc)突变体延迟了在非寄主植物辣椒ECW-10R上引发的过敏反应(HR)。利用GUS(β-葡糖苷酸酶)活性检测、原位组织染色和半定量RT-PCR的方法,发现T3SS相关基因在spoT_(Xcc)基因的非极性整合突变体中表达量降低,表明十字花科黑腐病菌中被注释的应急反应相关基因spoT_(Xcc)显著正调控T3SS相关基因的表达。     

辣椒细菌性疮痂病病原菌分类、检测及综合防治研究进展

辣椒细菌性疮痂病是一种世界性分布的细菌性病害,该病能引起辣椒严重的产量损失和品质下降。国外特别是美国对该病害研究较早且较深入,国内相关研究几乎是空白。本文主要围绕病原菌的分类、检测和病害综合防治等研究进展做一概述。     

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.     

安徽省水稻品种对水稻白叶枯病的抗性及白叶枯病小种鉴定

为明确安徽省白叶枯病菌小种组成及常用、备用品种对该病的抗性,用白叶枯病强毒性小种FuJ和YN24、中等致病力的安徽省优势小种AH以及弱致病小种YN7对安徽省常用及备用水稻品种进行人工接种鉴定;用鉴别品种IRBB5、IRBB13、IRBB3、IRBB14、IRBB2、R24对安徽的白叶枯病菌株进行鉴定.结果表明,有3.5％的品种抗FuJ,15.4％的品种抗YN24,29.8％的品种抗AH;安徽省白叶枯病菌小种有R2、R5和R8,其中R5为优势小种.抗AH的品种可以用于安徽的水稻生产;生产中应防止FuJ和YN24等毒性强的菌株传入.     

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

The present study developed a pathovar‐specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265‐bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant‐associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm‐specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.     

Polyphasic phenotypic and genetic analysis reveals clonal nature of Xanthomonas axonopodis pv. punicae causing pomegranate bacterial blight

Yellow-pigmented bacteria isolated from blight-affected pomegranate leaves and fruit across seven Indian states in epidemics during the years 2008–2016 were characterized and identified using phenotypic and genotypic tools. All bacterial isolates shared phenotypic traits such as colony morphology, NaCl and pH sensitivity and fuscan production, and caused typical lesions on pomegranate plants upon artificial inoculation. Analysis of 16S ribosomal DNA and 16S–23S rDNA intergenic spacer sequences confirmed their identity as Xanthomonas axonopodis pv. punicae. The new isolates collected after 2000 were compared with an old isolate from the 1950s using polyphasic taxonomic approaches including multilocus sequence analysis (MLSA). Nucleotide polymorphism in 24 isolates for nine genomic loci (dnaK, fyuA, gyrB (Young), gyrB (Almeida), rpoD, fusA, gapA, gltA and lepA) showed minor variations in loci fyuA and gyrB. Isolates were grouped into four nearly identical sequence types, ST1, ST2, ST3 and ST4, based on their allelic profiles, ST3 being widespread in Indian states. Molecular phylogenetic analysis of concatenated 5690 bp with other Xanthomonas pathovars revealed its close genetic similarity with the X. citri group. The blight outbreak in diverse geographical locations is attributed to a re-emerged clonal population of X. axonopodis pv. punicae on a genetically homogenous pomegranate cultivar. The latently infected vegetative planting material of elite pomegranate cultivars contributed to the dissemination of the bacterial inoculum. This study highlights and forewarns of the role played by the clonally propagated elite pomegranate cultivars in disseminating and sustaining clonal populations of this bacterial plant pathogen in many Indian states.     

Identification and Characterisation of Xanthomonas campestris pv. campestris Strains from Tanzania by Pathogenicity Tests, Biolog, rep-PCR and Fatty Acid Methyl Ester Analysis

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a major disease constraint to cabbage production by smallholder farmers in Africa. Variability exists within the pathogen, and yet differentiation of Xcc strains from other closely-related xanthomonads attacking crucifers is often difficult. The Biolog system, fatty acid methyl ester analysis using microbial identification system (MIS), rep-PCR and pathogenicity tests were used to identify and characterise Xcc strains from Tanzania. Great diversity was observed among Xcc strains in their Biolog and rep-PCR profiles. Specific rep-PCR genomic fingerprints were linked to some geographical areas in the country. Most of the Xcc strains were clustered in two groups based on their fatty acid profiles and symptom expression in cabbage although some deviant strains were found. Each of the methods allowed a degree of identification from species, pathovar to the strain level. Biolog and MIS identified all Xcc strains at least to the genus level. Additionally, Biolog identified 47% of Xcc strains to the pathovar and 43% to strain level, whereas MIS identified 43% of the strains to pathovar level. In the absence of a database, the utility of rep-PCR for routine diagnosis of strains was limited, although the procedure was good for delineation of Xcc to the strain level. These findings indicate the existence of Xcc strains in Tanzania that are distinct from those included in Biolog and MIS databases. The limitations noticed warrant continued improvement of databases and inclusion of pathogenicity testing, using universally susceptible cultivars, as an integral part of strain identification.     

东北水稻白叶枯病菌生理小种及水稻品种对9号小种的抗性

对东北地区水稻白叶枯病菌生理小种群体的构成及分布,以及水稻品种对白叶枯病菌9号小种的抗性进行了研究。结果表明,东北地区水稻白叶枯病菌生理小种群体由6个小种,即Race 1、Race 2、Race 3、Race 6、Race 8和Race 9构成。小种Race 1、Race 2和Race 9在东北三省吉林、辽宁和黑龙江都有分布,小种Race 3和Race 8只存在黑龙江省,Race 6只存在辽宁省。在30个水稻品种中,对9号生理小种表现抗病的有13个,出现频率为43.3%;中抗7个,频率23.3%;中感6个,频率20.0%;感病4个,频率13.3%。本研究为在东北粳稻地区挖掘白叶枯病的抗源,以及利用抗病品种控制白叶枯病危害提供依据。     

Races of Xanthomonas campestris pv. vesicatoria overcoming the gene Bs2 for bacterial spot resistance in pepper, prevalent on Capsicum chinense in Barbados and Grenada and weakly pathogenic on bell pepper and tomato in the field

A total of 404 isolates of Xanthomonas campestris pv. vesicatoria , obtained from Capsicum chinense cv. West Indian Red grown in Barbados and Grenada, were differentiated into pathogenic races, and of these, 96 were tested also for selected taxonomic group phenotypes. The response of C. chinense to infection by several X. campestris pv. vesicatoria races and the contribution of races isolated from this cultivar to severity of bacterial spot of bell pepper and tomato were also investigated. P4T2, P5T2 and P6T2 were the predominant races of X. campestris pv. vesicatoria isolated from C. chinense grown in Grenada, whereas nine races (T1, P4, P6, P0T2, P1T2, P4T1, P4T2, P6T1 and P6T2) were isolated in Barbados. Race P4T2 comprised 46.0 and 71.4% of the isolates from Barbados and Grenada, respectively. The 96 isolates, all of which overcame resistance conferred by the gene Bs2 , shared taxonomic group B strain characteristics, including the presence of the β-protein band, positive amylolytic activity and inability to oxidize cis -aconitate. The C. chinense cv. West Indian Red was susceptible only to races of X. campestris pv. vesicatoria that can overcome Bs2 gene resistance. Of six such races identified in Barbados, only P4T1, P4T2 and P6T1 affected bacterial spot-susceptible bell pepper or tomato in the field, and they amounted to only 1.5–2.1% of each sample of isolates from these plant species. Moreover, they were confined to the smallest bacterial spot lesions. Bell pepper was most severely affected by combinations of races T1 with P3T2 and T2 with P0T1, and tomato by race T1 only and combinations of races P0T1 with P0T2 and P1T1 with P1T0, all of which prevailed in the field despite selection against them by C. chinense cv. West Indian Red.     

Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris

Fifty-one Bacillus isolates were characterized by fatty acid methyl ester (FAME) analysis; universal primer polymerase chain reaction (UP-PCR) fingerprinting; production of secondary metabolites and antagonistic activity against Xanthomonas campestris pv. campestris (causal agent of black rot in cabbage) in vitro and in vivo . Based on FAME analysis and/or PCR fingerprinting, the isolates were clustered into three different groups, named as Bacillus amyloliquefaciens , B . subtilis and B . pumilus . Seed treatment with Bacillus spp. generally reduced germination of seeds and incidence of black rot, but no relationship was found between the results of in vitro and in vivo experiments. The B .  amyloliquefaciens group contained isolates that were generally the most effective at reducing attack of black rot in vivo . The metabolic profiles of these isolates suggested that they produced surfactin, iturin, bacillomycine and/or azalomycin F. Isolates belonging to the B . subtilis group were mostly able to synthesize surfactin and arthrobactin. Surfactin, amphomycin, arthrobactin and valinomycin were generally found in culture extracts of isolates belonging to the B . pumilus group. No effect on growth of the pathogen was detected when the activity of filtered culture extracts and selected metabolites produced by the three different Bacillus species was tested in vitro against X . c . pv. campestris . However, inhibition was seen when bacterial liquid cultures were used. When the ability to colonize cabbage endophytically was examined for seven selected isolates with different antagonistic potential against black rot, it was found that the ability was related to the species and not to the antagonistic activity of the isolates.     

Genetic, phenotypic and pathogenic diversity of Xanthomonas arboricola pv. corylina strains question the representative nature of the type strain

A collection of 31 Xanthomonas arboricola pv. corylina strains isolated from Corylus maxima and C. avellana of different countries were assessed by means of repetitive PCR using ERIC, BOX and REP primer sets and analysis of whole-cell protein extracts; pathogenicity tests to three hazelnut ( C. avellana ) cultivars; and some key biochemical tests. From these studies, the X. arboricola pv. corylina strains were clustered into five and three groups by repetitive PCR and protein analysis, respectively, and by using UPGMA cluster analysis, with two strains forming an outlier group to these. The groups showed a high degree of similarity. Strain membership between the groups designed by the two methods exhibited a high degree of congruence, and diversity between the groups was low. Surprisingly, the two strains originating from C. maxima , that include the type strain NCPPB 935, formed the most distinctive group. No relationship to geographic origin of the strains was evident. All strains proved pathogenic towards three different hazelnut cultivars, although the strains obtained from C. maxima did not incite any significant symptoms on buds and twigs. No other relationships between rep-PCR and whole-cell protein groups and pathogenicity were evident. The distinctiveness of the C. maxima strains was supported further by atypical negative gelatin liquefaction test and reduced quinate metabolism results.