Isolation and Sequence of a Gene, celD Xo Involved in Cellobiose Utilization in Xanthomonas oryzae pv. oryzae

A genomic library of Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174 was screened for 4-methylumbelliferyl β-D-glucoside-hydrolyzing (MUGase) activity. In subcloning of one of the MUGase-positive clones, an approximately 4.2-kb SacI-SphI fragment conferred not only MUGase activity but also 4-methylumbelliferyl β-D-cellobioside-hydrolyzing (MUCase) activity. Sequence analysis showed that the fragment contained an ORF of 2951 bp. The conceptual ORF product was significantly homologous with 1,4-β-D-glucan glucohydrolase D (CELD) from Pseudomonas fluorescens subsp. cellulosa, and was named CELD_Xo. Cell fractionation experiments suggested that CELD_Xo is localized in the cell-envelope fraction. We constructed a CELD_Xo-deficient mutant (74ΔCELD) from X. o. pv. oryzae. Little MUCase activity was detected in the cell-envelope fraction prepared from the mutant. The mutant 74ΔCELD did not grow in synthetic medium containing cellobiose as the sole sugar source. On the other hand, growth in rice leaves and pathogenicity of the mutant and the parental strain did not differ. These results suggested that CELD_Xo is involved in cellobiose utilization of X. o. pv. oryzae but that the gene is not required for bacterial growth in rice leaves. Received 16 February 2001/ Accepted in revised form 11 April 2001     

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     

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.     

在水稻悬浮细胞系中非亲和白叶枯病菌繁殖受到抑制的现象及其机理的研究

 研究了水稻白叶枯病菌在水稻悬浮细胞系中的繁殖动力学特征以及悬浮细胞胞外液中各类物质对白叶枯病菌繁殖的影响。结果表明,水稻-白叶枯病菌非亲和互作与亲和互作相比病原菌繁殖的数量明显减少。胞外液中粗提蛋白对白叶枯病菌繁殖不具有抑制作用。非亲和互作胞外液中二元酚含量显著增高,并且水稻细胞防卫反应基因的转录和翻译活性明显增强。表明非亲和互作中白叶枯病菌繁殖受到抑制与水稻细胞防卫反应的启动相关。     

hrp基因关键调控因子HrpG在水稻白叶枯病菌和条斑病菌中的交叉互补性研究

 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)和条斑病菌(X. oryzae pv. oryzicola, Xoc)在非寄主烟草上产生过敏反应(HR)和在寄主水稻上具有致病性,是由hrp致病岛决定的,其中HrpG为关键调控因子。Xoo和Xoc侵染水稻途径和在水稻上产生病害症状的不同,是否与hrpG基因有关,还不清楚。本研究利用反向遗传学方法获得了Xoo和Xoc的hrpG突变体PΔhrpG和RΔhrpG,并用hrpG_Xoo基因和hrpG_Xoc基因分别互补上述2个突变体,获得了相应的互补菌株。致病性测定结果显示,PΔhrpG和RΔhrpG突变体丧失了在水稻上的致病性和在非寄主烟草上产生HR的能力,而hrpG_Xoo基因和hrpG_Xoc基因可分别互补上述突变体至野生型水平。利用GUS报告基因检测基因启动子活性发现,hrpG_Xoo和hrpG_Xoc的启动子活性没有显著差别;RT-PCR和Western杂交结果显示,hrpG基因交叉互补菌株中HrpG调控的下游基因hrpX、hpaR、hrcT、hpa2和hrpD6的表达没有差异,且III型分泌系统分泌蛋白Hpa2的分泌性没有受到影响。这些结果表明,稻黄单胞菌hrpG基因可在Xoo和Xoc中交叉互置,位于其上游和下游的调控途径可能相似,而决定Xoo和Xoc在水稻上的侵染途径以及所致病害症状差异可能与hrpG基因位点无关。     

稻白叶枯病菌不同毒力单细胞系互作关系及田间自然群体毒力组成初析

 稻白叶枯病菌不同毒力的单细胞系混合组配后的致病力随着不同毒力菌所占的比例、彼此之间及与寄主之间的相互作用而发生相应变化;同一病田不同病株分离菌以及同一叶片不同叶位段分离菌,均存在致病力的分化;表明稻白叶枯菌在同一块田、甚至同一病叶上都存在不同的毒力细胞。稻株发病程度,是在一定生态条件下,不同毒力的多细胞入侵和协同互作的结果。所以采用"段叶沙培切口取菌胶"法所得的分离株,是含不同毒力细胞的混合体。测定一定数量的此类菌株的致病型分化,即是对田间病菌自然群体毒力组成的表型反应。
来自同一田块的36株分离菌的致病型测定结果与从48县采集的208个菌株所测定的结果非常一致,因此,对一块稻田的病菌的毒力分化测定,可以作为对相似生态条件下的广大稻区病菌自然群体毒力组成监测的参考。     

Type-II secretion pathway structural gene xpsE, xylanase- and cellulase secretion and virulence in Xanthomonas oryzae pv. oryzae

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases in rice-growing areas worldwide. Four virulence-deficient mutants were identified from a transposon mutagenesis library of X. oryzae pv. oryzae . Sequence-analysis revealed that the transposon of the four mutants inserted at different sites in the same ORF, which is homologous to the xpsE gene encoding a component of the type-II secretion system in many bacterial pathogens. Extracellular enzymes, such as xylanase and cellulase, were not secreted to the extracellular space in the mutants. Analysis of the protein profile of the extracellular, periplasmic and intracellular fractions indicated that at least two secreted proteins accumulated in the periplasmic space in the mutants. After genetic complementation of these mutants with a functional xpsE gene, the xpsE gene could express normally and the pathogenicity of the mutants and their secretion of extracellular enzymes were restored. Western blot analysis with an anticellulase antiserum also showed that cellulase was secreted normally in the complemented strains. The results show that the type-II secretion pathway structural gene xpsE is required for xylanase and cellulase secretion and full virulence in X. oryzae pv. oryzae .     

Antagonistic Interactions Between Strains of Xanthomonas oryzae pv. oryzae

ABSTRACT The ability of some phytopathogenic bacterial strains to inhibit the growth of others in mixed infections has been well documented. Here we report that such antagonistic interactions occur between several wild-type strains of the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae. In mixed inoculations, a wild-type Philippine strain was found to inhibit the growth of a wild-type Korean strain. Furthermore, a nonpathogenic mutant of the Philippine strain maintained these antagonistic properties. Growth curve analysis indicated that both the wild-type Philippine strain and its nonpathogenic mutant inhibited the growth of the Korean strain 2 days after infection and prior to the onset of disease symptoms. When mixed with the nonpathogenic mutant, 10 out of 18 diverse wild-type X. oryzae pv. oryzae strains did not cause disease. Conversely, three of the strains that were not affected by the nonpathogenic mutant were found to inhibit the growth of both the wild-type and mutant Philippine strains, indicating that antagonism is widespread and strain specific. The observed growth inhibition occurred only in planta and did not correlate with bacteriocin activity in vitro. Antagonistic interactions also were found to affect resistance (R) gene-mediated resistance. The R gene Xa21 was capable of protecting rice plants coinoculated with nonantagonistic virulent and avirulent strains; however, when avirulent strains were coinoculated with virulent antagonistic strains, disease ensued. Taken together, these results indicate that X. oryzae pv. oryzae has evolved strategies to compete with rival strains in a fashion that allows virulent strains to evade R gene-mediated protection even when avirulent strains are present in the inoculum.     

Expression of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae hrp</Emphasis> Genes in XOM2, a Novel Synthetic Medium

To analyze the regulation of hrp expression and to detect and identify hrp-dependent secretion proteins of plant-pathogenic bacteria, an appropriate hrp-inducing medium is indispensable. In this study, two efficient hrp-inducing media for Xanthomonas oryzae pv. oryzae were designed by assaying the expression of a hrcU (the first gene of the hrpC operon) and a gus (β-glucuronidase) fusion gene. We modified XVM2, which is a hrp-inducing medium for X. campestris pv. vesicatoria, by adding 0.01% xylose in place of fructose and sucrose (0.18 and 0.34%, respectively) as a sugar source. The resulting medium induced approximately 15-fold more GUS activity from transformants containing a hrcU::gus gene than did XVM2. Moreover, a methionine-containing synthetic medium with 0.18% xylose as a sugar source was able to induce much stronger expression of HrcU::GUS, with GUS activity approximately 100-fold greater than that in XVM2. Induction depended on a regulator, HrpXo, and the PIP (plant-inducible-promoter) box, suggesting that HrcU::GUS was expressed in a hrp-dependent manner. The induction of operons hrpA to hrpF in XOM2 was also confirmed. These results suggest that both media, especially XOM2, are highly efficient hrp-inducing media for X. oryzae pv. oryzae. Received 7 October 2002/ Accepted in revised form 22 November 2002     

Baseline sensitivity of natural populations and resistance of mutants of Xanthomonas oryzae pv. oryzae to a novel bactericide,zinc thiazole

During 2009–2010, a total of 323 isolates of Xanthomonas oryzae pv. oryzae were obtained from rice with symptoms of bacterial leaf blight (BLB) in four provinces (Zhejiang, Jiangsu, Anhui and Hubei) in China. These isolates were tested for baseline sensitivity to zinc thiazole, a novel bactericide with strong antibacterial activity against Xanthomonas. The sampled pathogenic population had similar sensitivity to zinc thiazole (0·1–16·8 mg L^?1) in all four regions and over the whole two‐year study period. The baseline sensitivity was distributed as a unimodal curve with a mean EC₅₀ value of 6·79 ± 1·61 mg L^?1. The risk of mutation to resistance of zinc thiazole in X. oryzae pv. oryzae was further evaluated in vitro and in vivo. Twelve zinc thiazole‐resistant mutants were obtained through ultraviolet (UV) irradiation, culturing on zinc thiazole‐amended nutrient agar (NA) plates, and culturing on zinc thiazole‐treated rice plants. These zinc thiazole‐resistant mutants had resistance factors (RF = EC₅₀ value of a mutant / EC₅₀ value of the wildtype parent of this mutant) of 12·4 to 186·1 with a mean RF value of 44·1. Mutants obtained via UV irradiation, culturing on NA plates and culturing on rice plants had mean RF values of 51·8, 24·5 and 14·4, respectively. All mutants showed decreases in resistance to zinc thiazole after 20 successive transfers on bactericide‐free media or 10 successive inoculation–reisolations on bactericide‐free rice plants. No significant difference was found in bacterial growth and sensitivity to bismerthiazol between zinc thiazole‐resistant mutants and their parents. However, a significant decrease was observed in the pathogenicity of zinc thiazole‐resistant mutants compared with their parents, especially for mutants obtained via UV irradiation.     

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。     

Rep-PCR技术对中国水稻条斑病菌的遗传多样性初析

 采用Rep-PCR技术,对30个水稻细菌性条斑病菌株(Xanthomonas oryzae pv.oryzicola)进行遗传多样性分析,同时对李氏禾条斑病菌等其它10个参试菌株也进行了比较。Rep-PCR是利用一些基于细菌的短的重复序列引物(ERIC和BOX)的DNA扩增特性,2种引物组合的电泳图谱结合并分析,以水稻细菌性条斑病菌各自的指纹谱型在相似率80%时可分为6簇,初步表明我国水稻细菌性条斑病菌群体的遗传分化明显;发现自然界存在的弱或无毒性菌株与毒性菌株的Rep-PCR指纹图谱差异很大;毒性菌株的遗传分簇与其致病性具有一定的相关性。用ERIC扩增水稻条斑病菌基因组DNA的指纹比BOX更为多样,两者对菌株的分辨率不同。因此,Rep-PCR技术可有效地用于监测水稻细菌性条斑病菌的遗传变异,还可应用于菌株的鉴定和分类学研究。     

A single amino acid substitution in the SdhB protein of succinate dehydrogenase determines resistance to amicarthiazol in Xanthomonas oryzae pv. oryzae

BACKGROUND: Xanthomonas oryzae pv. Oryzae Ishiyama, a causal agent of rice bacterial leaf blight, was found to be sensitive in vitro to the systemic fungicide amicarthiazol (2‐amino‐4‐methylthiazole ‐5‐carboxanilide), which is a potent inhibitor of succinate dehydrogenase (SDH, EC 1.3.99.1). This paper aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzae to amicarthiazol. RESULTS: UV‐induced resistant mutants of X. oryzae pv. oryzae to amicarthiazol were isolated. The activity of SDH in wild‐type X. oryzae pv. oryzae was strongly inhibited by amicarthiazol, while that in resistant mutants was insensitive, although their SDH activity was decreased compared with the wild‐type sensitive strain without amicarthiazol. A mutation of Histidine₂₂₉ (CAC) to Tyrosine₂₂₉ (TAC) was identified in sdhB, which encoded the iron–sulfur protein subunit of SDH. The sdhB from the mutant was ligated into a cosmid, pUFR034, to generate pUFR034RAX, which conferred resistance to amicarthiazol when transformed into the wild‐type sensitive strain. CONCLUSION: A mutation of His₂₂₉ (CAC) to Tyr₂₂₉ (TAC) in SdhB was responsible for determining amicarthiazol resistance. Copyright © 2010 Society of Chemical Industry     

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。     

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     

PCR-mediated Detection of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>by Amplification of the 16S–23S rDNA Spacer Region Sequence

A detection method specific for Xanthomonas oryzae pv. oryzae, the pathogen responsible for bacterial blight of rice, was based on the polymerase chain reaction (PCR) and designed by amplifying the 16S–23S rDNA spacer region from this bacterium. The nucleotide sequence of the spacer region between the 16S and 23S rDNA, consisting of approximately 580-bp, from X. oryzae pv. oryzae, X. campestris pv. alfalfae, X. campestris pv. campestris, X. campestris pv. cannabis, X. campestris pv. citri, X. campestris pv. cucurbitae, X. campestris pv. pisi, X. campestris pv. pruni and X. campestris pv. vitians, was determined. The determined sequences had more than 95% identity. Therefore, a pair of primers, XOR-F (5′-GCATGACGTCATCGTCCTGT-3′) and XOR-R2 (5′-CTCGGAGCTATATGCCGTGC-3′) was designed and found to specifically amplify a 470-bp fragment from all strains of X. oryzae pv. oryzae isolated from diverse regions in Japan. No PCR product was amplified from X. campestris pathovars alfalfae, campestris, cannabis, carotae, cucurbitae, dieffenbachiae, glycines, pisi, pruni, vitians or zantedeschiae, except for pathovars citri, incanae and zinniae. The method could also detect the pathogen in infected rice leaves within 3 hr, at a detection limit of 4×10¹ cfu/ml. Received 17 December 1999/ Accepted in revised form 10 April 2000     

Notes on the Occurrence of Pathogenic Races of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>Found in Hiroshima Prefecture in 1999

The pathogenic race of 59 cultures of Xanthomonas oryzae pv. oryzae, a pathogen of bacterial leaf blight of rice, isolated from six locations in the inland mountainous area of Hiroshima Prefecture in 1999, were determined by a set of traditional differentials. Four races—I, II, V and VII—were found across the area; however, we noticed the composition of the races as well as the dominant race in each location different. All races were avirulent on differential cultivar Te-tep. Races V and VII were new to Hiroshima. The rice cultivars infected with bacterial leaf blight in Hiroshima are thought to be grouped into the Kinmaze group, which does not have any resistance genes. Apparently, a variety of races occurred unexpectedly on the cultivars contrary to stabilizing selection theory. Received 25 February 2000/ Accepted in revised form 13 July 2000     

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.     

katExoo基因缺失突变对水稻白叶枯病菌H2O2抗性和致病性的影响

 为了揭示过氧化氢酶基因katExoo在水稻白叶枯病菌(Xanthomonasoryzaepv.oryzae,Xoo)过氧化氢(H₂O₂)抗性和致病性中的功能,本研究构建了基因缺失突变体ΔkatExoo,测定了突变体的H₂O₂抗性、过氧化氢酶(CAT)活性、在离体培养条件下的生长速率以及对水稻的致病性。用标记基因置换法获得了ΔkatExoo突变体,其保守的CAT结构域(GATase1_catalase和catalase_clade_2)被Gm^R片段所替换。katExoo基因缺失突变并不导致病菌的H₂O₂抗性和CAT活性降低或丧失,反而在一定程度上使之增强和升高。ΔkatExoo离体生长量显著降低,水稻接种叶片病斑明显缩短、在叶组织内的种群量下降。表明基因缺失突变显著地影响了病菌的生长、定殖和致病性。本研究结果为“KatExoo可能是Xoo的一个毒性因子”的假设提供了遗传学证据。