Two thiadiazole compounds promote rice defence against Xanthomonas oryzae pv. oryzae by suppressing the bacterium's production of extracellular polysaccharides

Thiazole, isothiazole, thiadiazole, and their derivatives are used to control various human, animal and plant diseases. In addition to having direct anti‐microbial and anti‐fungal properties, these compounds are thought to induce host defences, but the mechanism of defence induction remains poorly understood. This article reports that the thiadiazoles of zinc thiazole and bismerthiazol induce H₂O₂ accumulation, up‐regulation of defence‐related genes, callose deposition and hypersensitive response‐like cell death in rice leaves infected with Xanthomonas oryaze pv. oryzae (Xoo) strain ZJ173, but not in non‐infected leaves. These defence responses in Xoo‐infected leaves were suppressed by the exogenous application of catalase, which reduces H₂O₂ accumulation. The application of extracellular polysaccharides (EPSs) extracted from strain ZJ173 significantly compromised rice defence against ZJ173 with or without thiadiazole treatment. The EPS‐deficient Xoo mutant ?gumH triggered a stronger defence than its parent strain ZJ173. The thiadiazole treatments reduced EPS production by strain ZJ173, but not by the thiadiazole‐resistant strain 2‐1‐1, which is thiadiazole resistant in vivo, but not in vitro; moreover, enhanced defence was not detected in thiadiazole‐treated rice inoculated with 2‐1‐1. Based on these data, we infer that zinc thiazole and bismerthiazol promote rice defence against Xoo by inhibiting the production of bacterial EPS.     

Rapid inhibition of protein histidine phosphorylation by UV-irradiation in Xanthomonas oryzae pv. oryzae

Abstract Exposure of Xanthomonas oryzae pv. oryzae cells to 254 nm UV radiation resulted in an alteration of protein phosphorylation. Labelling of the phosphohistidine-containing proteins with molecular masses of 81 and 32 kDa, named p81 and p32, was rapidly reduced following UV irradiation in the early exponential cells, but the decrease was not detected in mid-exponential cells. Mitomycin C, a DNA replication inhibitor, and rifampicin, a drug generally used to inhibit RNA synthesis and DNA replication, were also found to reduce the histidyl phosphorylation. However, this alteration of protein phosphorylation was not hindered by chloramphenicol treatment. A possible role for these histidyl phosphopfoteins in sensing UV light is proposed.     

Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

Involvement of Gluconeogenic Pathway in Virulence of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases of rice. A phosphoenolpyruvate synthase (ppsA)‐disrupted mutant OSPAM was generated by homologous suicide plasmid integration. The mutant was unable to grow in medium with pyruvate or C₄‐dicarboxylates as the sole carbon source, compared with the wild‐type, indicating a disruption in ppsA function. The mutant showed a reduction in virulence on rice but still induced a hypersensitive response in tobacco. When the mutant was complemented, the response was recovered to wild‐type. These results suggested that X. oryzae pv. oryzae possesses only PPSA route in gluconeogenesis, which is necessary for virulence.     

糖基转移酶基因rbfCxoo缺失突变导致水稻白叶枯病菌毒性表达增强

摘 要：【目的】阐明水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,简称Xoo)基因组中推导的脂多糖O抗原合成蛋白基因rbfCxoo (XOO2599) 的结构和生物学功能。【方法】通过基因克隆、序列分析、缺失突变和表型测定,对rbfCxoo的分子特征及其功能进行了鉴定。【结果】 用特异性引物进行PCR扩增,从野生型菌株PXO99A基因组DNA中成功地获得了与己测序菌株KACC10331序列完全一致的全长基因序列; RbfCxoo序列N端和C端分别具有一个糖基转移酶的保守结构域(Glycos_transf_2)。用标记置换法获得了基因缺失突变体△rbfCxoo。与PXO99A相比,△rbfCxoo 脂多糖O抗原合成能力并未发生变化,但鞭毛素糖基化能力有所降低。此外,△rbfCxoo鞭毛运动性、生物膜形成和胞外纤维素酶和木聚糖酶活性都无明显改变,但对水稻的致病性显著增强,毒性相关基因的表达也有所增加。【结论】RbfCxoo可能与细菌鞭毛素糖基化修饰以及毒性表达有关。     

水稻白叶枯病菌和水稻细菌性条斑病菌的实时荧光PCR快速检测鉴定

成功建立了水稻白叶枯菌与水稻细菌性条斑病菌快速检测鉴定的实时荧光PCR方法。根据含铁细胞接受子基因设计两菌的通用引物PSRGF/PSRGR（扩增一个152bpDNA片段）和特异性探针（Baiprobe和Tiaoprobe）,并对13种细菌和1种植原体进行实时荧光PCR。结果表明,两个特异性探针能分别特异性检测到目标病原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30.6fg/μL质粒DNA和103CFU/mL的菌悬浮液,相当于1个细菌细胞的基因,比常规PCR电泳检测高约100倍,相对灵敏度为105CFU/mL。整个检测过程只需2h,完全闭管,降低了污染的机会,无需PCR后处理。 用这两个特异性探针分别对自然感染白叶枯菌和条斑菌的叶片DNA提取液和种子浸泡液进行实时荧光PCR,结果均可特异性检测到目标菌的存在并完全可将两种病原细菌区分开来,且只需03g叶片和10g种子。     

Activity of a novel bactericide,zinc thiazole against Xanthomonas oryzae pv. oryzae in Anhui Province of China

Bacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv oryzae, is one of the most serious bacterial diseases in China. Presently, bismerthiazol has been the major bactericide for the control of BLB, however, bismerthiazol‐resistant strains of X. oryzae pv. oryzae have appeared in the field in China. Zinc thiazole is a novel bactericide with strong antibacterial activity against Xanthomonas spp. In this study, sensitivity of 109 X. oryzae pv. oryzae strains to zinc thiazole was determined. The EC₅₀ values for zinc thiazole in inhibiting bacterial growth of the 109 X. oryzae pv. oryzae strains were 0.53–9.62 µg mL^?1 with the average EC₅₀ value of 4.82 ± 1.86 µg/ml. The minimum inhibitory concentration (MIC) values of zinc thiazole against the 109 X. oryzae pv. oryzae strains were assessed and the results showed that the MIC values of zinc thiazole for completely inhibiting the growth of these 109 strains ranged from 5.0 to 40.0 µg mL^?1. In the evaluation of protective and curative activity test, zinc thiazole exhibited great activity against BLB and provided over 88% control efficacy (at 300 µg mL^?1) 1 and 3 days before or after inoculations, which was also higher that that of bismerthiazol in the corresponding treatments. Our field trials showed that zinc thiazole at 375 g.a.i ha^?1 provided over 70% control efficacy in 2012 and over 80% control efficacy in 2013 at both sites. Moreover, in all the four field trials, zinc thiazole at 250 g.a.i ha^?1 provided higher control efficacy than that of bismerthiazol at 250 g.a.i ha^?1. Taken together, zinc thiazole is therefore an alternative tool for the management of BLB.     

Pathotypic variation of Xanthomonas oryzae pv. oryzae in Bangladesh

Bacterial Blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo), a destructive disease of rice. Altogether, 96 isolates of Xoo were collected from 19 rice growing districts of Bangladesh in irrigated and rainfed seasons during 2014 to assess pathotypic variation. Pathotypic analyses on a set of 12 Near Isogenic Lines (NILs) of rice containing resistance genes viz. Xa1, Xa2, Xa3, Xa4, Xa5, Xa7, Xa8, Xa10, Xa11, Xa13, Xa14 and Xa21 and two check varieties IR24 and TN1 by leaf clip-inoculation technique. A total of 24 pathotypes were identified based on their virulence patterns on NILs tested. Among these, pathotypes VII, XII, and XIV considered as major, containing maximum number of isolates, (9.38% each) frequently distributed in North to Mid-Eastern districts of Bangladesh. Most virulent pathotype I recorded from Habiganj and Brahmanbaria. This pathotypic variation explained the pathogenic relatedness of X. oryzae pv. oryzae populations from diverse geographic areas in Bangladesh.     

High-quality mutant libraries of Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris generated by an efficient transposon mutagenesis system

A novel transposon mutagenesis system for the phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and X. campestris pv. campestris (Xcc) was developed using a Tn5-based transposome. A highly efficient transformation up to 10(6) transformants per microg transposon DNA was obtained. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses of Tn5 insertion sites suggested a random mode of transposition. The transposition was stable in the transformants for 20 subcultures. Eighteen thousand and 17000 transformants for Xoo and Xcc, respectively, were generated, corresponding to 4X ORF coverage of the genomes. The libraries will facilitate the identification of pathogenicity-related genes as well as functional genomic analysis in Xoo and Xcc.     

A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae,which confers an endo mode of activity,affects bacterial virulence,but not the induction of immune responses,in rice

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall‐degrading enzymes, including cellulases, xylanases and pectinases, to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo, but is also a potent inducer of innate immune responses of rice. In this study, we solved the crystal structure of the catalytic domain of the CbsA protein to a resolution of 1.86 Å. The core structure of CbsA shows a central distorted TIM barrel made up of eight β strands with N‐ and C‐terminal loops enclosing the active site, which is a characteristic structural feature of an exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in the catalysis and biological functions of CbsA. Intriguingly, the D131A CbsA mutant protein displayed the enzymatic activity of a typical endoglucanase. D131A CbsA was as proficient as wild‐type (Wt) CbsA in inducing rice immune responses, but was deficient in virulence‐promoting activity. This indicates that the specific exoglucanase activity of the Wt CbsA protein is required for this protein to promote the growth of Xoo in rice.     

水稻白叶枯病菌的母株与单细胞系的遗传与致病性差异的分析

由水稻白叶枯病菌(Xanthomonas oryxae pv.oryxae)引发的稻白叶枯病是水稻生产上的重要病害.水稻白叶枯菌自然群体是由包括基本无毒性的弱毒菌在内的不同致病型组成的混合群体,代表自然群体的原始菌株的致病力与其毒力结构紧密相关.     

中国、日本和菲律宾水稻白叶枯病菌遗传多样性比较分析

用IS-PCR和Rep-PCR对19个来自中国、日本和菲律宾的水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)群体进行遗传多样性分析.4个专化引物中的IS1113和ERIC能较好的区分三国水稻白叶枯病菌.UPGMA聚类结果表明,三国菌株主要呈现第2簇和第3簇遗传型;中国和菲律宾菌株在第2簇和笫3簇遗传型基础上有各自的特异性分化.病原菌的遗传分簇与致病群之间没有相关性.     

中国、日本和菲律宾水稻白叶枯病菌遗传多样性比较分析

用IS-PCR和Rep-PCR对19个来自中国、日本和菲律宾的水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae)群体进行遗传多样性分析。4个专化引物中的IS1113和ERIC能较好的区分三国水稻白叶枯病菌。UPGMA聚类结果表明, 三国菌株主要呈现第2簇和第3簇遗传型;中国和菲律宾菌株在第2簇和第3簇遗传型基础上有各自的特异性分化。病原菌的遗传分簇与致病群之间没有相关性。