Genetic Diversity of Xanthomonas oryzae pv. oryzae Strains from Sri Lanka

ABSTRACT Sixty strains of Xanthomonas oryzae pv. oryzae, collected from 29 locations in Sri Lanka in 1995, were analyzed by restriction fragment length polymorphism using either polymerase chain reaction-amplified 16S and 23S rDNA or the repetitive DNA element IS1112 from X. oryzae pv. oryzae as hybridization probes. Two different ribogroups were observed in the Sri Lankan strains using rDNA probes, whereas five clusters were identified by the IS1112 probe. Bootstrap analysis revealed that the five clusters defined by IS1112 were relatively robust. Our results suggest that the Sri Lankan strains are phylogenetically composed of five different groups. Each cluster was partially associated with climatic conditions (intermediate zone and wet zone) and was related to groups based on ribotyping. Based on virulence analysis using 12 rice cultivars, each containing a single resistance gene, 14 pathotypes were identified among the Sri Lankan strains. All strains were virulent to resistance genes Xa1, Xa2, Xa4, Xa10, Xa11, and Xa14. Only one strain (pathotype 1) was virulent to all major resistance genes including Xa21, while strains of the other pathotypes were all avirulent to Xa21. A partial relationship was found between the determined phylogenetic groups using the IS1112 probe and pathotypes for all but two clusters. The results of this study will facilitate the further understanding of the population structure of X. oryzae pv. oryzae in Sri Lanka.     

Distribution of Xanthomonas oryzae pv. oryzae Strains Virulent to Xa21 in Korea

ABSTRACT Strains of Xanthomonas oryzae pv. oryzae that are virulent to rice lines carrying the Xa21 resistance gene were widely distributed in Korea. A total of 105 strains collected during 1987 to 1996 in Korea was characterized by pathogenicity tests and restriction fragment length polymorphism analysis of the XorII methyltransferase (xorIIM) and avrXa10 genes. Although the lesion lengths on rice line IRBB21, which carries Xa21, decreased as plant age increased, resistance and susceptibility of the plants to 31 strains were clearly differentiated at the seedling (14, 21, and 28 days old), maximum tillering, and flag leaf stages. The resistance or susceptibility of seedlings was correlated with bacterial populations within an inoculated leaf. There was a significant change in the population structure of X. oryzae pv. oryzae with regard to virulence to Xa21 over the last 10 years; this change in population was confirmed by genome analysis. Lineage I, which is avirulent to Xa21 and does not have a genomic xorIIM homolog, was the predominant lineage found between 1987 and 1989, while lineage II, which is virulent to Xa21 and contains the xorIIM homolog, was predominant in strains collected between 1994 and 1995. Our results demonstrate that introduction of Xa21 into commercial rice should be based on the regional structure of X. oryzae pv. oryzae populations and suggest that Xa21 will not be useful in Korea.     

利用多重PCR诊断水稻白叶枯病和细菌性条斑病的复合发生

为准确检测水稻白叶枯病菌、细菌性条斑病菌及这两种病菌的复合发生,利用软件DNAStar分析比较这两种菌的部分核酸序列,设计了检测这两种病菌的特异性引物。引物Xoo F-Xoo R能特异性扩增出水稻白叶枯病菌中一条大小162 bp的条带;引物Xooc F1-Xooc R1和Xooc F2-Xooc R2能够分别特异性扩增出水稻细菌性条斑病菌中690 bp和945 bp的条带。通过优化PCR反应条件,成功建立了多重PCR技术,可以对不同国家的水稻白叶枯病菌和细菌性条斑病菌进行准确检测,对由这两种病菌引起的复合侵染实现了准确诊断。     

水稻细菌性条斑病菌拮抗细菌的筛选、评价与应用研究

从江苏省南京、泰州、扬州和宿迁等地区水稻田采集水稻病叶、健叶、根围土等样品90份,分离纯化得到1173个细菌分离物,对其进行水稻细菌性条斑病菌Xanthomonasoryzaepv．oryzicola皿内抑制试验,共获得12株拮抗能力较强的细菌,其中4株拮抗细菌抑菌圈直径大于27mm。盆栽试验结果表明,12株拮抗能力较强的细菌中有4株对水稻细菌性条斑病菌防效大于50％,其中菌株Lx-1l盆栽防效达62．5％。大面积示范试验结果表明菌株Lx．11田间防效达60．2％,显著高于化学药剂20％叶枯唑的防效（51．2％和45．8％）。形态特征、理化特性和分子鉴定分析确定菌株Lx．11为解淀粉芽孢杆菌Bacillusamyloliquefaciens。     

水稻白叶枯病菌和细菌性条斑病菌的分子标记筛选及检测

 水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae）和细菌性条斑病菌(Xanthomonas oryzae pv. oryzicola)是水稻种子产地检疫中最重要的两种检疫对象,且同属于水稻黄单胞杆菌。本研究基于生物信息学技术构建比较基因组学算法对两种病原的全基因组序列比对分析,得到一系列能够区分两种病原的特异性PCR引物。结合简单的PCR技术及全自动DNA分析系统,我们选取了12对引物分别对23株水稻白叶枯病菌和5株水稻细菌性条斑病菌及其它相关菌株进行验证。结果获得了2对显性标记(Xoo-Hpa1和Xoc-ORF2)以及3对共显性分子标记(M568、M897和M1575)可以达到理想的区分检测两种病原的效果。分子标记的检测灵敏度从5×10⁴到5 × 10⁷cfu·mL^-1不等,且从水稻种子浸提液中也能成功地检测水稻白叶枯病菌和细菌性条斑病菌。本研究丰富了检测标记的靶位点,并有效的结合了高通量检测的手段对多位点联合分析,增强了检测的可靠性,有望在今后的植物检疫及病原鉴定中发挥着重要的作用。     

中生菌素对水稻白叶枯病的防治机制

对不同白叶枯病抗性水稻品种用200μg/ml中生菌素55℃温汤药液浸种，自然降温，秧田3—4叶期和移栽前5d各用30μg/ml中生菌素处理后，于成株期剪叶接种白叶枯病菌。结果表明中生菌素前期处理，于成株期接种白叶枯病菌时，高抗、中等抗性和感病品种中过氧化物酶、苯丙氨酸解氨酶和多酚氧化酶3种酶活性都有不同程度的提高，且以中等抗性品种酶活提高最多，接种24和48h，3种酶活性较清水对照分别增加26．92％、26．74％、24．06％和7．09％、1．31％、1．60％。盆栽试验表明，中生菌素对中抗品种的防治效果最好，达58．4％。说明中生菌素对水稻防御酶活性的诱激作用是其防治白叶枯病的机制之一。     

水稻白叶枯病菌单细胞系毒力分化研究

 以稀释倒平板法从0型菌086和IV型菌967-4和9620中分离到59个单细胞系;在12个近等基因系品种上,086和其单细胞系表现为弱毒力,2个IV型菌及其单细胞系能克服抗病基因Xa-1、2、3、8、10、11、14的抗性,不能克服Xa-21、4、5、7、13的抗性;带主效抗病基因的品种Asominori、XM5、M41、XM6和丰锦能把3个母株的59个单细胞系区分为12种数量差异或质量差异的不同致病型;将此5品种与近等基因系配合,适合作为病菌致病基因变异频度监测的寄主;采用"段叶沙培,切口取菌胶"法分离病菌,在中国致病型鉴定品种上划分的致病型,是田间病菌群体毒力结构的表型反应。     

Involvement of Phosphoglucose Isomerase in Pathogenicity of Xanthomonas oryzae pv. oryzae

ABSTRACT Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, was subjected to transposon mutagenesis to generate mutants defective in pathogenicity. A novel mutant 74M913 was attenuated in virulence but retained its ability to cause the hypersensitive response in leaf blight-resistant rice and tomato. Cloning and sequence analysis revealed that the transposon in 74M913 was inserted in a gene homologous to the phosphoglucose isomerase (pgi) gene of X. axonopodis pv. citri. Growth of the mutant in a synthetic medium containing fructose or xylose as a sole carbohydrate source was much reduced, indicating the transposon disrupted pgi function. The interaction between expression of pgi and hypersensitive response and pathogenicity (hrp) genes was investigated because we had demonstrated previously that expression of hrp genes of X. oryzae pv. oryzae is induced in a synthetic medium containing xylose. However, pgi and the hrp gene (hrcU) were expressed independently. This study suggests that PGI is involved in pathogenicity of X. oryzae pv. oryzae.     

广东水稻白叶枯病菌遗传多样性和小种分化研究

 通过IS-PCR和rep-PCR指纹技术,分析了广东水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)群体遗传多样性。用2对特异性引物J3和ERIC对114个菌株基因组DNA进行了PCR扩增,分别呈现89和40种谱型,以彼此间的带位相似率达70%为界,J3扩增的谱型被分为11簇,ERIC的谱型被分为8簇。J3的簇1包含89个菌株,占总数的78.07%,ERIC的簇1包含52个菌株,占总数的45.61%,均为优势簇群。群体遗传多样性值J3为0.8919,ERIC为0.8278。上述结果表明,广东水稻白叶枯病菌的遗传多样性较高。全部参试菌株接种于含有不同抗性基因近等基因系及高感品种金刚30共6个鉴别品种,被划分为6个小种(X-gd1,X-gd2,X-gd3,X-gd4,X-gd5和X-gd6),X-gd4出现频率最高,为广东省的优势小种。     

Regulatory network of hrp gene expression in Xanthomonas oryzae pv. oryzae

Like other plant-pathogenic bacteria, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, has hrp genes that are indispensable for its virulence. The hrp genes are involved in the construction of the type III secretion (T3S) apparatus, through which dozens of virulence-related proteins, called effectors, are directly secreted into plant cells to suppress and disturb plant immune systems and/or induce plant susceptibility genes. The expression of hrp genes is strictly regulated and induced only in plants and in certain nutrient-poor media. Two proteins, HrpG and HrpX, are known as key regulators for hrp gene expression. Great efforts by many researchers have revealed unexpectedly that, besides HrpG and HrpX, many regulators are involved in this regulation, some of which also regulate the expression of virulence-related genes other than hrp. Moreover, it has been found that HrpG and HrpX regulate not only hrp genes and effector genes but also genes unrelated to the T3S system. These findings suggest that the expression of the hrp gene is orchestrally regulated with other virulence-related genes by a complicated, sophisticated regulatory network in X. oryzae pv. oryzae.     

Genotypic and Pathotypic Diversity in Xanthomonas oryzae pv. oryzae in Nepal

ABSTRACT Among the 171 strains of Xanthomonas oryzae pv. oryzae (the bacterial blight pathogen of rice) collected from eight rice-producing zones in Nepal, 31 molecular haplotypes were distinguished using two polymerase chain reaction-based assays. Six common haplotypes represented nearly 63% of the strains, and some haplotypes were geographically dispersed. Multiple correspondence analysis divided the collection into five putative genetic lineages. Lineages 1, 2, and 4 were the most frequently detected and occurred in diverse geographic populations. Twenty-six pathotypes (virulence phenotypes) of X. oryzae pv. oryzae were identified using 11 near-iso-genic rice lines, each containing a single gene for resistance. The 26 pathotypes grouped into five clusters, and cluster 1 contained wide virulence spectrum strains from all geographic populations. Although molecular variation was greatest between strains of different virulence phenotypes, some variation was observed among strains with identical virulence. There was a weak correlation (r = 0.52) between molecular haplotypes and virulence phenotypes. There are two major groups of X. oryzae pv. oryzae in Nepal. One group consists of strains with high molecular polymorphism and many pathotypes that are either virulent to the 11 major resistance genes or avirulent only to Xa21. Strains in the second group have low molecular polymorphism and are avirulent to Xa4, xa5, Xa7, and Xa21.     

水稻白叶枯病菌harpin基因的克隆与表达

 用PCR方法从水稻黄单胞菌白叶枯致病变种(Xanthomonas oryzae pv.oryzae,Xoo) JxoIII菌株中克隆到编码诱导植物过敏性反应激发子的基因hrfA。同源性比较发现其推测产物与水稻白叶枯病菌菲律宾小种Pxo86的Hpa1有97.2%的序列一致性,比Hpa1少了一个-GGG-GG-氨基酸残基序列重复。基因经NdeI和BamHI双酶切后连到含T7启动子的高表达载体pET 30a (+)上构建重组质粒pHRF4,并转化宿主菌BL21(DE3)产生表达菌株BLHR4。经过IPTG诱导之后,BLHR4产生一分子量为15.6 kD的蛋白质。研究表明,该蛋白在性质与功能上类似于其它已发现的harpins,即能够在烟草上引起典型的过敏性反应,富含甘氨酸、热稳定以及对蛋白酶敏感。因此,我们把该蛋白定名为harpin_Xoo。harpin_Xoo还具有诱导植物抗病性的功能。     

Growth of Glucose-uptake-deficient Mutant of Xanthomonas oryzae pv. oryzae in Rice Leaves

We isolated Xanthomonas oryzae pv. oryzae mutants deficient in the phosphoenolpyruvate : carbohydrate phos-photransferase system, a major glucose transport system in bacteria, using the glucose analogue 3-deoxy-3-fluoro-d-glucose (3FG). Glucose uptake by the mutants was decreased to 15–35% of the parental strain, and growth greatly decreased in synthetic media containing glucose as a sole sugar source. Growth of the mutants in rice leaves was, however, similar to the wild type. These findings suggest that glucose is not necessarily a major carbohydrate source for X. o. pv. oryzae in rice leaves. Received 11 August 2000/ Accepted in revised form 15 December 2000     

我国水稻白叶枯病菌致病力分化研究

综述了我国水稻白叶枯病菌致病力分化及其致病基因和致病机制的研究进展 ,查明稻白叶枯病菌致病力分化状况是进行水稻抗白叶枯病育种及其抗性品种利用的基础。     

云南水稻白叶枯病菌生理小种初析

从云南省 5个地州14个县市的水稻白叶枯病标本中分离水稻白叶枯病菌菌株 ,选择具有区域代表性的菌株 5 1株 ,利用已知抗病基因的近等基因系 ,在孕穗期应用剪叶法接种明确其生理小种。鉴定结果表明 ,云南省水稻白叶枯病菌小种复杂多样 ,共有 14种类型 ,暂定为YN1～YN14 ,优势生理小种为YN3、YN8、YN11,其中YN3分布在红河、德宏等 4个县市 ;YN8小种分布在大理等地区 ,小种的分布可能受地理区域的影响     

Genetic Diversity within the Population of Xanthomonas oryzae pv. oryzae in India

ABSTRACT Xanthomonas oryzae pathovar oryzae causes a serious disease of rice in India and is endemic in all of the major rice-growing areas of the country. Sixty-seven X. oryzae pv. oryzae strains, collected mostly in 1994 and 1995, from 18 locations in India were analyzed by DNA fingerprinting methods using two separate repeat element probes from the X. oryzae pv. oryzae genome. These results show that strains belonging to a single pathogen lineage can be isolated from 16 of the 18 locations sampled; many of these locations are separated from each other by hundreds of kilometers and represent ecologically diverse rice-growing areas. Pathotyping analysis indicated that the strains in this lineage belong to pathotype 1b of X. oryzae pv. oryzae.     

Variation for aggressiveness within and between lineages of Xanthomonas oryzae pv. oryzae

Isolates of Xanthomonas oryzae pv. oryzae (causal agent of bacterial blight of rice) from the Philippines representing two phylogenetic lineages, and five haplotypes within those two lineages, were evaluated for aggressiveness in two glasshouse trials. Aggressiveness was determined by clip-inoculating leaves of a rice cultivar lacking known, effective major genes for resistance and measuring the lengths of resulting lesions. Variance components analysis indicated that 55 and 46% of the lesion length variation were genetic in origin for the first and second trials, respectively. Variation of lesion length among isolates within haplotypes was highly significant in both trials ( P  = 0·002 and 0·027), but the effects of lineage and haplotype within lineage were not ( P  = 0·08 and 0·30 for lineage and P = 0·23 and 0·07 for haplotype). These results suggest that substantial heritable variation for aggressiveness exists within Philippine populations of X. oryzae pv. oryzae . This variation appears to be more prevalent within than among known phylogenetic groups, although mean differences among phylogenetic groups may still be of significant biological importance.     

水稻白叶枯病菌对噻枯唑的抗药性监测

采用活体寄主法和离体含药平板法 ,测定了 1999年安徽省滁州市田间水稻白叶枯病菌对噻枯唑的抗药性。结果表明 ,在水稻活体上存在噻枯唑抗药突变体 ,其比例为67.65%,比1997、1998年有较大程度升高 ,但是离体含药平板上未能监测到抗药突变体。     

中国水稻白叶枯病菌毒性变异研究

 利用13个含已知抗病基因的近等基因系材料,在水稻孕穗期采用剪叶接种法对我国285个水稻白叶枯菌株(其中108个采集于1970~1992年,177个采集于2003~2004年)的致病力变异进行研究。结果表明,病菌的致病力呈现多样性。通过试验系统比较了不同时期病菌的毒性和小种组成变化。在此基础上对病菌群体毒性变化的原因进行了分析。     

水稻品种对水稻细菌性条斑病的抗性鉴定

选用致病力强的水稻细菌性条斑病菌Rs105对206个水稻品种进行抗性鉴定，结果表明，供试品种对水稻细菌性条斑病的抗性存在明显差异，粳型水稻的抗性一般比籼型水稻好。品种抗侵入和抗扩展呈显著相关关系（r=0.7949)。