Molecular Screening of Rice Cultivated in Benin for the Identification of Xanthomonas oryzae Pv. oryzae and Bacterial Leaf Blight Resistance Genes

One of the most devastating diseases of rice worldwide is bacterial blight (BLB) caused by Xanthomonas oryzae pv. Oryzae (Xoo). In Benin, Xoo was first described in 2013 on wild rice Oryzae longistaminata. So far, no study has been done on Beninese Xoo strains. We do not know whether the pathogen has already passed into the rice varieties grown, or if they are exposed to other bacteria. Whereas the use of resistant varieties, carrying resistance genes, is the only highly effective and environmentally friendly way to control this disease, no information is available on these Xoo resistance genes in rice varieties grown in Benin apart from the one we recently. This study aims to identify Beninese Xoo strains, causing BLB and screen rice varieties grown in Benin for the main resistance genes. Diseased rice leaves showing typical symptoms of fire blight collected from different rice fields in the three phytogeographic areas of Benin were analyzed by PCR for Xoo-specific sequence identification. Furthermore, seventy-five collected rice accessions were screened to identify xa5, Xa7, xa13, and Xa21 resistance genes to Xoo. The results reveal that Xanthomonas oryzae was identified in two fields in Banikouara and one in Malanville. On the other hand, Sphingomonas sp. has been identified in several other rice fields in Benin. Forty-seven of seventy-five rice accessions examined (62.66%) carried Xoo resistance genes with 3 (4%) and 40 (53.33%) of xa5 and Xa21 respectively. None of the accessions had either Xa7 or xa13 resistance genes. Three accessions possess both xa5 and Xa21 genes. Isogenic lines IRBB60 and IRBB21, supposed to be a positive control, presented a Xoo sensitivity allele. These results indicate that Xoo has moved from the wild rice variety to the cultivated variety in northern Benin and varietal improvement programs must be implemented with varieties having several resistance genes for the efficient response against a possible BLB pandemic in Benin.

辣椒疮痂病菌(Xanthomonas vesicatoria)和水稻细菌性条斑病菌(X.oryzae pv.oryzicola)的质粒及其与耐链霉素和耐铜性关系

从不同省份收集到辣椒疮痂病菌Xanthomonasvesicatoria(XV) 7个菌株和水稻细菌性条斑病菌X .oryzaepv .oryzicola(XOZ) 14个菌株 ,进行了质粒微量制备。XV菌株除XV1外都检测到质粒 ,单个菌株拥有的质粒数为 2～ 5个 ,大小在 10～ 10 0kb,没有发现为所有菌株所共有的质粒 ,但XV2、XV3、XV4、XV5、XV6共享 1个大约 5 5kb的质粒。 14个XOZ菌株都含有 1个质粒 ,除XOZ5外 ,其余的 13个菌株都含 1个约 4 0kb的质粒。所有菌株都不耐铜。XOZ菌株仅XOZ7对链霉素表现耐性 ,4个XV菌株即XV4、XV5、XV6和XV7耐链霉素。用XV质粒转化无质粒菌株 ,发现XV5的 1个 76Kb质粒与耐链霉素有关。

水稻对白叶枯病Xanthomonas oryzae pv.Oryzae水平抗性的数量性状位点(QTL)定位

利用“Lemont/特青”重组自交系（RI）群体研究了水稻对白叶枯病致病菌株CR6的水平抗性。双亲和F1均为感病，重组自交系（RILs）的病斑长度（LL）为带有明显双向超亲的连续变异，显示出典型的多基因遗传特征。部分重组自交系（约占总数90％）对CR6表现高水平抗性（LL≤3cm）。利用由178个良好分离的RFLP标记构建的饱和连锁图，鉴定出11个数量形状位点（QTLs）和3对互作位点解释了RI群体的大部分病斑变异。抗性QTLs定位于水稻第2、3、4、8、9、10、11、12等8条染色体。在来自特青的Xa－4位点上检测到一个有很大加性效应的QTL。其余10个QTLs的抗性等位基因有7个来自特青，3个来自Lemont。研究结果表明多个数量性状位点和失效主基因（Xa－4）残效的累加效应构成了对白叶枯病水平抗性的遗传基础，是重要的抗性组成部分。可以预期在DNA标记的辅助下，这些数量性状位点与主效抗性基因的组合将使水稻品种具有持久抗病性。

水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)化学诱变hrp基因突变体及相关性状的研究

硫酸二乙酯 (diethyl sulfate,DES)化学诱变水稻白叶枯病菌 (Xanthomonas oryzae pv. oryzae,Xoo)日本系统 号小种 (JXO ) ,获得 16株 hrp-突变体 ,其在感病寄主水稻 IR2 6、汕优 6 3上失去致病性和在非寄主烟草上失去过敏反应。所得的 hrp-突变体并无黄色素缺失现象。16株 hrp-突变体的淀粉酶、果胶酶、蛋白酶、纤维素酶和脂酶等胞外酶活性基本与野生型一致。 JCM2 0 92、JCM2 2 11、JCM2 2 5 2和 JCM2 4 5 7这 4株 hrp-突变体为 型泌出通道突变体 ,JCM0 0 6 6等另 12株 hrp-突变体的超声波破碎液、离心上清液中无激发 HR的信号物质存在。细胞组分分析表明 ,激发烟草 HR的信号物质不是 L PS而是蛋白类物质。来自 JXO 基因文库 hrp基因克隆 p UHRX2 4 5中的 1.1kb Sac I-Kpn I片段 ,能够互补 hrp-突变体 JCM0 0 6 6和 JCM2 4 82 ,使其恢复在烟草上激发 HR的能力 ,但不能恢复其致病性。

非寄主抗病基因Rxo1介导的水稻对Xanthomonas oryzae pv.oryzicola的抗病反应

通过表型鉴定、反转录PCR和实时定量PCR方法,利用转基因和非转基因水稻植株,研究由Rxol基因介导的,水稻对细菌性条斑病菌的抗性反应。结果观察到3个涉及过敏性反应的基因由Rxol基因诱导表达,并对其进行了分析。这3个基因参与编码病程相关蛋白,在转基因水稻植株中呈上调表达,表明水杨酸信号转导途径在抗性反应中发挥重要的作用。

水稻品种次生物质与稻白叶枯病(Xanthomonas oryzae pv. oryzae)抗性的关系

应用高效液相色谱分析了对白叶枯病具有不同抗性水平的12个水稻品种中的19个(组)次生物质色谱峰(面积)的差异及其与白叶枯病抗性水平间关系。结果表明,水稻品种抗性水平与谱峰面积值之间相关极显著(R=0.992,p<0.01),被测的19个组分中,峰1、峰2、峰8、峰10、峰12、峰14、峰16和峰18是影响水稻对稻白叶枯病抗性水平的主要抗原次生物质。建立了水稻品种对白叶枯病抗性级别与以上次生物质含量谱峰面积之间的回归模型:Y=10.7603+0.1823X1-0.2287X2+0.2163X8-2.1975X10+0.0728X12-0.7438X14+1.1484X16-0.7795X18。研究结果表明水稻品种中起抗病作用的抗原次生物质不止一种,而是几种的组合,而且它们对水稻抗病性的贡献作用是不完全相同的,这与它们的性质与含量密切相关。提出了以抗原次生物质为标记的快速分析、鉴定、预测水稻品种对稻白叶枯病抗性水平的新途径、新方法。

A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

HrcQ is necessary for Xanthomonas oryzae pv. oryzae HR-induction in non-host tobacco and pathogenicity in host rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae(Xoo), is one of the most destructive diseases of rice(Oryza sativa L.) worldwide. The type III secretion system(T3SS) of Xoo, encoded by the hrp(hypersensitive response and pathogenicity) genes, plays critical roles in conferring pathogenicity in host rice and triggering a hypersensitive response(HR) in non-host plants. To investigate the major genes conferring the pathogenicity and avirulence of Xoo, we previously constructed a random Tn5-insertion mutant library of Xoo strain PXO99A. We report here the isolation and characterization of a Tn5-insertion mutant PXM69. Tn5-insertion mutants were screened on indica rice JG30, which is highly susceptible to PXO99A, by leaf-cutting inoculation.Four mutants with reduced virulence were obtained after two rounds of screening. Among them, the mutant PXM69 had completely lost virulence to the rice host and ability to elicit HR in non-host tobacco. Southern blotting analysis showed a single copy of a Tn5-insertion in the genome of PXM69. PCR walking and sequencing analysis revealed that the Tn5 transposon was inserted at nucleotide position 70,192–70,201 in the genome of PXO99A, disrupting the type III hrc(hrp-conserved) gene hrcQ, the first gene in the D operon of the hrp cluster in Xoo. To confirm the relationship between the Tn5-insertion and the avirulence phenotype of PXM69, we used the marker exchange mutagenesis to create a PXO99Amutant, ΔhrcQ::KAN, in which the hrcQ was disrupted by a kanamycin-encoding gene cassette at the same site as that of the Tn5-insertion. ΔhrcQ::KAN showed the same phenotype as mutant PXM69. Reintroduction of the wild-type hrcQ gene partially complemented the pathogenic function of PXM69. RT-PCR and cellulase secretion assays showed that the Tn5-disruption of hrcQ did not affect transcription of downstream genes in the D operon and function of the type II secretion system. Our results provide new insights into the pathogenic functions of clustered hrp genes in Xoo.

An Inner Membrane Protein(Imp) of Xanthomonas oryzae pv. oryzicola Functions in Carbon Acquisition, EPS Production, Bacterial Motility and Virulence in Rice

Xanthomonas oryzae pv. oryzicola （Xoc） causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein （Imp）, showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant R？imp was constructed in the wild-type RS105. The R？imp mutant was signiifcantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufifciently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide （EPS） production and reduced motility. The deifciencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was signiifcantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also signiifcantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.

Molecular detection of Xanthomonas oryzae pv.oryzae, Xanthomonas oryzae pv. oryzicola, and Burkholderia glumae in infected rice seeds and leaves

The polymerase chain reaction(PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three important rice pathogens, Xanthomonas oryzae pv.oryzae, X. oryzae pv. oryzicola, and Burkholderia glumae. The unique PCR primer sets were designed from portions of a putative glycosyltransferase gene of X. oryzae pv. oryzae, an Avr Rxo gene of X. oryzae pv. oryzicola, and an internal transcribed spacer(ITS) sequence of B. glumae. Using a multiplex PCR assay, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected in one PCR reaction that contained the newly developed primer set mix. Using SYBR Green real-time PCR assays, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected at 1, 1, and 10 fg μL-1, respectively. These newly designed molecular assays are sensitive and could be reliable tools for pathogen detection and disease forecasting.

Exchangeability of Two hrp Gene Fragments from Xanthomonas oryzae pv.oryzae and pv.oryzicola for Hypersensitive Response on Tobacco and Pathogenicity on Rice

hrp mutants were produced from strain JXOIII of Xanthomonas oryzae pv. oryzae (Xoo) and strain RS105 of X.o. pv. oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che mical. All the hrp mutants lost their pathogenicity on a susceptible host plant, rice (Shanyou63), and elicitation of the hypersensitive response (HR) on a nonhost plant, tobacco (NC89). Extracellular enzyme (amy lase, pectate lyase, proteinase, cellulase and lipase) activities of all the hrp mutants were similar to those of the corresponding wild type strains. The response of tobacco to cell sonicated integrations of the wild type strains and the hrp mutants demonstrated that there existed an HR eliciting substance which was heat stable and sensitive to protease. No HR appeared on tobacco after infiltration of the lipopolysaccharide (LPS) of both the wild strains and hrp mutants into tobacco leaves. The ability of the Xooc hrp mutants to induce HR on tobacco and cause streak disease on rice was restored by complementation with pUHRX245 from JXOIII genomic DNA library and by pUHRS138 from RS105 genomic DNA library, respectively. Subcloning of a 38.6 kb hrp fragment insert in pUHRX245 and a 39.3 kb insert in pUHRS138 revealed that a 3.3 kb Sac Ⅰ fragment from pUHRX245 and a 4.5 kb Bam HⅠ Kpn Ⅰ fragment from pUHRS138 were the minimal functional portions required for restoration of the ability of Xooc hrp mutants to induce HR on tobacco and cause disease on rice. The disease symptom caused by the conjugant (M1005 plus 3.3 kb) on rice was similar to that caused by the wild type of Xooc. It suggests that the two fragments contain the same hrp gene(s) and are responsible reciprocally for HR induction on tobacco and pathogenicity on rice.

Virulence of Xanthomonas oryzae pv. oryzae on Rice Near-Isogenic Lines with Single Resistance Gene and Pyramiding Lines in China

Ninety one isolates of Xanthomonas oryzae pv. oryzae were collected from different rice- growing regions in China and determined for their virulence on 24 rice near-isogenic lines containing single resistance gene and 2-4 genes: IRBB1 (Xa1), IRBB2 (Xa2), IRBB3 (Xa3), IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11), IRBB13 (xa13), IRBB14 (Xa14), IRBB21 (Xa21), IR24 (Xa18), IRBB50 (Xa4 + xa5), IRBB51 (Xa4 + xa13), IRBB52 (Xa4 + Xa21), IRBB53 (xa5 + xa13), IRBB54 (xa5 + Xa21), IRBB55 (xa13 + Xa21), IRBB56 (Xa4 + xa5 + xa13), IRBB57 (Xa4 + xa5 + Xa21), IRBB58 (Xa4 + xa13 + Xa21), IRBB59 (xa5 + xa13 + Xa21) and IRBB60 (Xa4 + xa5 + xa13 + Xa21). The results showed that most isolates were less virulent on lines with more than one genes pyramided than those with single resistance gene. The isolates tested were more virulent on IR24 and IRBB10, less virulent on IRBB5, IRBB7 and IRBB21. Based on interactions between isolates and rice near-isogenic lines, 7 cultivars with single gene (IRBB5, IRBB4, IRBB3, IRBB14, IRBB2, IRBB1 and IR24) were chosen as the differentials, and the tested isolates were classified into 7 virulence groups. The reaction patterns of the 7 groups in order were: RRRRRRR, RRRRRRS, RRRRRSS, RR/SRRSSS, RRRSSSS, RRSSSSS, RSSSSSS. The virulence frequencies were 7.69, 6.59, 14.29, 12.09, 14.29, 28.57 and 16.48% respectively. The elementary system for races identification has been established in China based on the results. It will be possible to compare with races in other countries, and the results will facilitate the development of rice resistance breeding to bacterial blight in China.

An improved protein expression system for T3SS genes regulation analysis in Xanthomonas oryzae pv. oryzae

Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.

Xoryp_08180 of Xanthomonas oryzae pv.oryzicola,Encoding a Hypothetical Protein,is Regulated by HrpG and HrpX and Required for Full Virulence in Rice

Xanthomonas oryzae pv.oryzicola（Xoc） causes a destructive bacterial leaf streak disease in rice.Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice.A mutant,Mxoc1679,screened from our previous Tn5-tagged mutant library for Xoc strain RS105,showed reduced virulence in rice.In this mutant,a gene named as Xoryp_08180 was disrupted by Tn5 insertion.Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp.Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice,a delayed hypersensitive response（HR） in non-host tobacco,and a decrease in extracellular protease activity.The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans.In addition,the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition.These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production,HR induction and full virulence of Xoc.

The Twin-Arginine Translocation (Tat) Pathway Is Essential for Virulence,Flagellation,and Chemotaxis in Xanthomonas oryzae pv. oryzicola Strain RsGD42

Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola, is an important disease of rice （Oryza sativa）. Genetic determinants （tatABC genes） of the twin-arginine translocation （Tat） pathway from X. oryzae pv. oryzicola strain RsGD42 were cloned and characterized, meanwhile, a tatC disruption mutant was generated. The tatC mutant lacked detectable flagella and was highly impaired in motility and chemotaxis. Furthermore, it was observed that the tatC mutant exhibited a reduced production of extracellular polysaccharide （EPS） and a significant reduction of virulence on adult rice plants compared to wild type strain. However, the tatC mutation in X. oryzae pv. oryzieola strain RsGD42 did not affect the growth rate and the ability to induce hypersensitive response （HR） in nonhost tobacco （Nicotiana tabacum L. cv. Samsun）. In conclusion, the data indicated that the Tat pathway significantly contributed to the virulence of X. oryzae pv. oryzicola.

Functional identification of phenazine biosynthesis genes in plant pathogenic bacteria Pseudomonas syringae pv. tomato and Xanthomonas oryzae pv. oryzae

Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens. In this study, we identified phenazine biosynthesis （phz） genes in two genome-completed plant pathogenic bacteria Pseudomonas syringae pv. tomato （Pst） DC3000 and Xanthomonas oryzae pv. oryzae （Xoo） PXO99A. Unlike the phz genes in typical phenazine-producing pseudomonads, phz homologs in Pst DC3000 and Xoo PXO99A consisted of phzC/D/E/F/G and phzC/E1/E2/F/G, respectively, and the both were not organized into an operon. Detection experiments demonstrated that phenazine-l-carboxylic acid （PCA） of Pst DC3000 accumulated to 13.4 IJg L-1, while that of Xoo PXO99A was almost undetectable. Moreover, Pst DC3000 was resistant to 1 mg mL-1 PCA, while Xoo PXO99A was sensitive to 50 IJg mL ~ PCA. Furthermore, mutation of phzF blocked the PCA production and significantly reduced the pathogenicity of Pst DC3000 in tomato, while the complementary strains restored these phenotypes. These results revealed that Pst DC3000 produces low level of and is resistant to phenazines and thus is unable to be biologically controlled by phenazines. Additionally, phz-mediated PCA production is required for full pathogenicity of Pst DC3000. To our knowledge, this is the first report of PCA production and its function in pathogenicity of a plant pathogenic P. syringae strain.

Differential Detection and Analysis of Pathotypes and Differentiation against Xanthomonas oryzae pv. oryzae in Southern China

Pathogenic isolates were collected from different rice-cropping regions in southern China to dissect the pathogenic disintegration and variation of Xanthornonas oryzae pv. oryzae （Xoo）. Two sets of rice-Xoo differential hosts, including Chinese system with such five euhivars as IR26, Java14, Nangeng15, Tetep and Jingang 30 and international system with a series of nearisogenic lines （NILs） including IRBB5, IRBB13, IRBB3, IRBB14, IRBB2 and IR24 carrying different known resistance genes,were used to detect pathogenic disintegration for the reaction between host and pathogen with leaf-clippingmethod at the rice booting stage. The results showed the type of the pathogen were divided intosix pathotypes, i.e. , Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ and Ⅸ based on the Chinese differential system, and seven pathogenicraces including RI, R2, R3, R4, R5, R8 and R10 based on the international differential system. The pathogenicity frequency of Xoo pathotypes V and IV and pathogenic races R8 and R5 were 27.40% , 19.30% and 44.67% , 15.34%, which were considered to be the prevailing races in southern China. Pathogenic rates of pathotypes Ⅳ, Ⅴ, Ⅸ and races R8, R5 against 500 varieties derived from southernChina were 96.40% , 95.00% , 50.40% , 62.00% , and 42.60%, respectively. Among which pathotype Ⅸ was the most virulent pathotype. The pathotype Ⅴ became preponderant pathotype and the new pathotype Ⅸ grew up quickly.

Evaluation of pathogencity and race classification of <i>Xanthomonas oryzae</i>pv. <i>oryzae</i>in guilan province-Iran

To evaluation of pathogenicity and race classification of Xanthomonas oryzae pv. oryzae agent bacterial leaf blight of rice, 153 isolates of X. oryzae pv. oryzae were collected from different rice-growing cities of Guilan province – Iran. All of isolates were inoculated to assess the differential characteristics of 26 near isogenic rice lines containing a single resistance gene or two to five genes. Inoculation was done 21 days after sowing in the greenhouse. Scoring of inoculated plants was done 18 days after inoculation. The level of infection was not so clear among pyramiding lines, expect IRBB53 and IRBB61. Therefore, the pyramiding lines can not be used as differentials for pathogenicity evaluation of X. oryzae pv. oryzae The 12 rice lines with a single resistance gene were used further to establish a system of races classification of X. oryzae pv. oryzae IRBB14, IRBB21and IRBB7 were resistance to the most isolates. Whereas, IRBB1, IRBB2, IRBB4 and IRBB10 were susceptible to all isolates. Based on the interactions between the isolates X. oryzae pv. oryzae and the 12 near-isogenic rice lines, seven singlegene rice lines were chosen as differentials, and the 153 tested isolates were classified into four races. Except for cultivar types, different terrain, climate, period of rice planting and other factors may be associated with the population diversity and virulent variation of X. oryzae pv. oryzae.

Xanthomonas oryzae pv.oryzae LAMP可视化检测方法的建立

由Xanthomonas oryzae pv.oryzae引起的水稻白叶枯病严重影响我国水稻安全生产,尤其在杂交稻上,损失高达40%~60%,在水稻种子市场监管过程中由于缺乏快速检测手段,造成带菌种子在市场中流通。利用SYBR Green I作为荧光指示剂,建立了水稻白叶枯病菌的环介导等温扩增(LAMP)可视化快速检测方法。以水稻白叶枯病菌为对象,通过Primer Explorer V4.0软件一共设计出四条特异性的LAMP引物以及两条环引物,优化并建立了水稻白叶枯病菌的可视化快速检测方法。结果表明:在65℃恒温条件下反应1 h,LAMP检测特异性和灵敏度,加入SYBR Green I荧光染料,水稻白叶枯病菌呈现荧光绿色,对照菌株则保持不变,依然呈橙色。LAMP反应检测灵敏度DNA的浓度大小为为100 fg/μL,菌悬液为3×103 cfu/mL。应用LAMP技术检测水稻白叶枯病菌Xanthomonas oryzae pv.oryzae具有特异性强、灵敏度高、操作简单快捷的特点,可用于市场监管过程中快速检测水稻白叶枯病菌。

Evaluation of the Pathotypes of Xanthomonas oryzae pv. oryzae and Preliminary Analysis of the Resistant Reactions of Main Japonica Rice in the Yunnan Plateau, China

The pathogenicity of 36 isolates of Xanthomonas oryzae pv. oryzae （Xoo）, which were collected from japonica rice varieties in the Yunnan Plateau, China, was evaluated. It was evaluated on 29 rice varieties including a set of seven varieties to identify pathogenicity, i.e., Haonuoyang, TN1, Kogyoku, Zhenzhu＇ai, IR26, Nanjing 33, and Kinmaze, which may be considered as a set of differential varieties for Xoo races from Yunnan japonica rice. The efficiency of the seven varieties was further confirmed. The results showed reversible and specific interactions between isolates and varieties. The isolates were classified into nine pathotypes from pathotyp Ⅰ to Ⅸ according to their pathogenic reactions on the seven rice varieties. The pathotype V was the epidemic, whereas pathogen Ⅶ was the most pathogenic. Most japonica varieties grown in the Yunnan Plateau were susceptible to Xoo. The rice lines IRBB21 （Xa-21）, Zhachanglong （Xa-22,, Xa- 24,）, and IR1545-339 （xa-5）, which were resistant to all the isolates tested, can be used as donors of resistant genes for bacterial blight in japonica rice breeding in the Yunnan Plateau.