Phylotype and sequevar variability of Ralstonia solanacearum in Brazil,an ancient centre of diversity of the pathogen

The β‐proteobacterium Ralstonia solanacearum causes bacterial wilt of many plant species. Knowledge of phylotype and sequevar variability in populations of this microorganism is useful for implementing control measures, particularly host resistance. To this end, 301 isolates of R. solanacearum were collected from different geographic regions and hosts in Brazil. Their phylotype and sequevar characterization was used to determine the amount and distribution of phenetic and phylogenetic variability. Isolates were classified into phylotypes I (n = 48), clade 1; and phylotype II, clades 2–5. Phylotype II was divided into subclusters IIA (n = 112) and IIB (n = 141). Phylotype II was widely distributed, whereas phylotype I isolates were found in Central, Northern, and Northeastern regions of Brazil. There were 108 haplotypes identified among endoglucanase (egl) gene sequences from 301 isolates and 32 haplotypes among DNA repair (mutS) gene regions from 176 isolates. The egl and mutS sequence analyses identified eight known (1, 4, 7, 18, 27, 28, 41 and 50) and four new (54, 55, 56 and 57) sequevars. Phylotype IIB showed high diversity in sequevars and host range. Multiplex PCR, using primers specific to the Moko ecotype, characterized banana and long pepper isolates as sequevar 4 and 4/NPB, respectively. This constitutes the first report of the emergent ecotype IIB/4NPB in a new host, long pepper. The majority of sequevars were associated with geographic regions. This high variability of R. solanacearum in Brazil suggests use of host resistance to control bacterial wilt should be mainly focused by region.     

福建及贵州等地烟草青枯菌系统发育分析

[目的]探寻烟草上青枯菌的系统发育.[方法]采用演化型分类框架对福建及贵州等地的62个烟草青枯病菌株进行鉴定分析.[结果]基于内切葡聚糖酶基因系统发育学的分析结果表明:所有参试菌株均归属于青枯菌亚洲分支的4个序列变种,分别为序列变种15、17、34和44;尚未发现归属于美洲或非洲分支的烟草青枯病菌株.其中序列变种15和17为优势菌系,序列变种34的菌株都来自福建省,只发现3个菌株属于序列变种44.基于avrA基因的氨基酸序列比对结果表明4个序列变种的avrA基因都属于RS1000类型.[结论]本研究表明福建及贵州等地烟草上的青枯菌存在一定的遗传分化.     

Host specificity and genetic diversity of race 4 strains of Ralstonia solanacearum

Ralstonia solanacearum race 4 isolates were obtained from Zingiberaceae plants in India during bacterial wilt outbreaks. Polyphasic phenotypic and genotypic analysis revealed intraracial diversity and dominance of biovar 3 over biovar 4. Biovar 3 strains were isolated from very severely wilted Zingiberaceae plants in the field and found to be present across diverse geographical, host and seasonal boundaries. It was hypothesized that these isolates belong to a single, ‘fast wilting’, lineage. Using one ‘fast wilting’ isolate in controlled inoculations, rapid wilt was observed in ginger within 5–7 days. Wilting was also observed in several other closely and distantly related hosts such as turmeric (Curcuma longa), aromatic turmeric (Curcuma aromatica), black turmeric (Curcuma caesia), sand ginger (Kaempferia galanga), white turmeric (Curcuma zeodaria), awapuhi (Zingiber zerumbet), greater galangal (Alpinia galanga), globba (Globba sp.), small cardamom (Elettaria cardamomum) and large cardamom (Ammomum subulatum) of the Zingiberaceae family, and in tomato (Solanum lycopersicum). Molecular analysis, including multiplex PCR‐based phylotyping, sequence analysis of 16S rDNA, 16–23S intergenic spacer and the recN gene, and multilocus sequence typing, revealed minimal differences between fast wilting isolates, confirming that almost all belong to the same lineage. Biovar 4 was isolated from plants showing slow wilt progression and self‐limiting wilting in restricted geographical locations instead, and was identified to be genetically distinct from the fast wilting biovar 3 isolates. To the authors' knowledge, this is the first report of host range and genetic analysis of R. solanacearum race 4 in India.     

Wide genetic diversity of Ralstonia solanacearum strains affecting tomato in Trinidad,West Indies

The genetic diversity of Ralstonia solanacearum causing bacterial wilt of tomato in Trinidad was assessed using the hierarchical phylotyping scheme and rep‐PCR DNA fingerprinting. Seventy‐one isolates were collected in 2003 on infected tomato crops in the four main vegetable cropping areas of Trinidad (North, Central, South‐East and South). Two phylotypes were present, with phylotype II being much more prevalent (66%) than phylotype I (34%). Phylotype II strains consisted mainly of sequevar 7 in Central and South‐East, and sequevar 35 in North, South‐East and South. This is the first report of sequevar 7 outside south‐eastern USA. In contrast, no ‘brown rot’ (phylotype IIB/1, race 3 biovar 2) or emerging strains of phylotype IIB/4NPB were identified. Rep‐PCR data were used to assess population genetic structure. No significant clustering by geographical distance was found, suggesting regular gene flow among cropping areas (via waterways, plant or soil). However, the population from Central was significantly differentiated from the others, containing only phylotype II/seq 7 strains, with a high degree of clonality, suggesting a possible recent introduction from abroad. The South population was less aggressive and more genetically diverse, suggesting horizontal gene transfers within the population, even among isolates of different phylotypes. Phylotype I and phylotype II populations differed slightly in clonality levels, with indications of more frequent recombination events within phylotype I populations. Possible factors influencing genetic diversity and distribution within the island are discussed.     

Phylogenetic and pathogenic variability of strains of Ralstonia solanacearum causing moko disease in Colombia

Moko disease, caused by the bacterium Ralstonia solanacearum, is one of the most devastating diseases of Musa spp. in Colombia, where banana and plantain are major crops. The disease epidemiology is poorly understood and little is known about the diversity of the bacterial populations associated with this disease. This study assessed the diversity, phylogenetic relationship and pathogenicity of R. solanacearum strains associated with moko disease in Colombia. For this, the genetic diversity of 65 isolates obtained from four banana/plantain-growing regions was evaluated by using multiplex PCR and analysing the partial sequences of the mutS, rplB and egl genes. These analyses revealed that all the strains belonged to the R. solanacearum phylotype II, sequevars 4 and 6. In addition, the phylogenetic analysis assorted the strains into three subgroups, which matched the region of isolation: (i) central region (i.e. Eastern plains and Andes, IIB/4); (ii) northwest (i.e. Urabá and a few strains from Magdalena, IIB/4); and (iii) north coast (Magdalena and a few strains from Urabá, IIA/6). In addition, this evolutionary pattern was associated with pathogenicity, as 63 of the 65 isolates caused wilting of banana and plantain plants under greenhouse conditions, whilst only 32, those isolated from the central region, caused such symptoms in tomato plants. In conclusion, this study shows that banana and plantain crops in Colombia foster genetically diverse strains of R. solanacearum that belong to at least three different genetic groups, which show biogeographic and host range association.     

Implication of C-terminal mutation of PopA of Ralstonia solanacearum strain OE1-1 in suppression of bacterial wilt

Ralstonia solanacearum strain OE1-1 causes bacterial wilt on tobacco plants. The popA -mutant 31b, derived from OE1-1 by insertion of transposon Tn 4431 , did not cause wilt on tobacco plants inoculated through the roots. However, when 31b was directly inoculated into xylem vessels, the tobacco plants wilted, similarly to those inoculated with OE1-1. 31b retained its exopolysaccharide productivity and its type-III secretion function. Furthermore, 31b grew in intercellular spaces and systemically infected tobacco plants, similarly to OE1-1. popA consists of an operon with popB and popC , and suppression of popB and popC expression resulting from polar mutation by transposon insertion did not affect the virulence of 31b. The mutated popA ( popA31b ) was composed of 960 nucleotides, including 39 derived from Tn 4431. A recombinant mutant from OE1-1, where popA31b was introduced by marker exchange, showed the same phenotype as 31b. PopA31b protein was extracellularly secreted by 31b co-cultured with Arabidopsis thaliana . These results suggest that PopA31b extracellularly secreted by 31b in intercellular spaces may be implicated in suppression of disease development, leading to inability of the bacteria to induce wilt on plants. Taken together, interactions between host plants and R. solanacearum existing in intercellular spaces immediately after invasion may be involved in disease development.     

烟草青枯病菌拮抗细菌的筛选、鉴定和抑菌机制研究

青枯雷尔氏菌Ralstonia solanacearum造成的烟草青枯病是烟草主要毁灭性病害之一.本研究采用牛津杯法从运城盐湖湖岸土壤中筛选获得一株对烟草青枯病菌具有较好拮抗效果的菌株FY-C;并进一步分析了菌株FY-C的抑菌谱、对青枯病菌的潜在生防效果.结果 显示,经菌株FY-C无菌滤液处理24 h后的青枯病菌细胞壁...     

青枯雷尔氏菌胞外蛋白指纹多态性分析

 采用SDS-PAGE技术对70株不同来源及致病力青枯雷尔氏菌（Ralstonia solanacearum）进行胞外蛋白指纹多态性分析,研究结果表明,供试青枯雷尔氏菌菌株呈现丰富的胞外蛋白指纹多态性,多态性比率为100%。不同来源青枯雷尔氏菌分泌的胞外蛋白不同,EZ-Tn5TM插入诱变菌株电泳出20条不同大小蛋白条带,分子量集中在20~97 kD ,且菌株间蛋白条带相似或完全相同;60Co辐射诱变菌株共电泳出16条不同大小的蛋白条带,多数蛋白分子量44.3 kD;野生型菌株电泳的蛋白条带最多,共获得26条不同大小的蛋白条带。进一步对37株不同致病力的野生型菌株进行胞外蛋白含量测定,结果表明,不同致病力菌株胞外蛋白含量差异大,强致病力菌株分泌的胞外蛋白含量较高,为1.026～5.963μg/mL,无致病力菌株胞外蛋白含量较低,为0.083～0.761 μg/mL。     

Identification and use of a wild plant with antimicrobial activity against Ralstonia solanacearum, the cause of bacterial wilt of potato

Fresh aerial tissue and roots of 14 wild plants in Okinawa prefecture were investigated for their antimicrobial activity against Ralstonia solanacearum , which causes bacterial wilt of potato. A 70% aqueous ethanol extract of fresh aerial tissue of Geranium carolinianum L. showed strong antimicrobial activity against R. solanacearum . This extract also showed antimicrobial activity against the pathogens causing common scab of potato and soil rot of sweet potato. The antimicrobial substance could be extracted with hot water, and was effective against R. solanacearum in soil. In the field test, a treatment combining incorporation of dried aerial tissue into the soil and solarization was highly effective for control of bacterial wilt of potato. These findings suggest that G. carolinianum L. could be used as a biological agent for the control of bacterial wilt of potato.     

青枯雷尔氏菌无致病力突变菌株的构建及其防效评价模型分析

 利用青枯雷尔氏菌(Ralstonia solanacearum)无致病力菌株防治番茄青枯病具有很好的应用潜力。作者通过分离筛选自然弱毒株、⁶⁰Co辐射诱变和EZ-Tn5插入诱变,分别获得3、12和40株青枯雷尔氏菌无致病力突变菌株。经盆栽番茄苗致病性检测,15 d后均未发病,证实均为无致病力青枯雷尔氏菌。进一步对番茄青枯病的防治试验表明,从番茄青枯病发病田块分离的无致病力突变菌株FJAT1458的防治效果最好,防效达100%。该菌株能定殖番茄植株根系土壤、根部和茎部,定殖数量均表现为“先增后减”的趋势,并且接种浓度越大、苗龄越小,定殖数量越大。从构建的防效模型可以看出,不同接种浓度条件下,植株发病率随时间变化符合的回归方程不同,相关系数R值也不同,接种浓度越大,R值越小。本研究获得的青枯雷尔氏菌无致病力突变菌株FJAT1458对番茄青枯病具有很好的防病效果。     

Phylogenetic relationships and population structure of Ralstonia solanacearum isolated from diverse origins in Taiwan

Bacterial wilt caused by Ralstonia solanacearum is a destructive disease for many crops. The aim of this study was to investigate the phylogenetic relationships and genetic structure of an R. solanacearum population from diverse origins in Taiwan. All 58 tested isolates belonged to phylotype I, except the two biovar 2 isolates from potato. These belonged to phylotype IIB sequevar 1 and were identical to known potato brown rot strains, which were probably introduced. Phylotype I isolates were grouped into 10 sequevars. Sequevar 15 was predominant (34 out of 56 isolates). Its distribution covered the whole island and it was largely associated with solanaceous crops such as tomato, and with tomato field soil. A total of 14 haplotypes were identified based on a partial endoglucanase gene sequence. Parsimony network analysis revealed that haplotype A was the oldest haplotype in the local population. It encompassed all but one of the sequevar 15 isolates. Large variation in virulence on tomato was observed among the 58 isolates, and seven pathotypes were identified. Significant genetic differentiation was detected among pathotypes. Moreover, genetic differentiation was detected between biovar 3 and biovar 4 subgroups and between the strains associated with solanaceous and non‐solanaceous species, but none was detected between strains from different geographic origins. The results suggest that the phylotype I population in Taiwan is homogeneous, while mutation and local adaptation to specific ecological niches keep shaping the population.     

壳聚糖对青枯劳尔氏菌生长及其生物膜形成的影响

为建立快速检测壳聚糖抑菌效果的方法,研究其对青枯菌生物膜形成的影响并探索其抑菌机理,利用酶标仪检测壳聚糖与菌悬液混合培养物的吸光值,计算抑菌率,同时利用结晶紫染色法对壳聚糖处理后青枯菌生物膜形成能力进行评价,并进行透射电镜观察.结果表明,在壳聚糖浓度低于0.15 mg/mL时,随浓度增加其抑菌效果亦显著增加,其浓度超过0.15 mg/mL之后,其抑菌效果达到同一水平,最高抑菌率为74.3％;而壳聚糖浓度对青枯菌生物膜形成能力未产生显著影响;透射电镜观察结果显示:壳聚糖通过破坏青枯菌细胞膜抑制细菌生长.     

我国植物青枯菌菌株的遗传多样性和组群划分

 采用15条随机引物对我国11个省(市、区)6种不同寄主植物的43个青枯菌代表性菌株和4个国外青枯菌菌株,进行了PCR扩增.引物OPB11、OPA15、OPE1和OPZ10对上述所有菌株扩增获得了相似的产物电泳图谱,分别具1~5条谱带不等;引物OPB7、OPA10和OPF1对马铃薯菌株获得了相同的产物图谱,但对其它寄主菌株的产物间有明显差别;引物OPA14、OPC,6、OPG14、OPF5、OPK14、OPK20和OPK17对于不同菌株的扩增产物多态性很强.供试菌株被聚类为2个组群,即组群A和组群B.组群A中又可分为7个亚组(A1、A2、A3、A4、A5、A6、A7),其中A1含有2个类型(A1-1、A1-2);组群B中也可分为2个亚组(B1、B2),其中B1含有3个类型(B1-1、B1-2、B1-3),B2也含有3个类型(B2-1、B2-2、B2-3).RAPD组群A中包含了27个来自我国不同地区的马铃薯菌株,主要是3号小种、生化变种2;组群B中含有20个来自不同地区、不同寄主的菌株,分属于其它不同的小种和生化变种.研究结果表明,我国青枯菌菌株RAPD组群的划分与菌株的地理来源关系不大,而与寄主来源有明显相关性.此外,通过对我国青枯菌菌株组群进行同源性PCR分析表明,来源自马铃薯的3号小种菌株属于美洲分支"Americanmm",而来自其它寄主的青枯菌1号、5号小种菌株属于亚洲分支"Asiaticum",与本研究RAPD组群A和组群B的划分是一致的.     

广东茄科青枯菌致病力分化及其DNA多态性分析

 分别采自广州、增城、东莞、花都、三水、清远、电白、高要8个菜区的番茄、茄子和辣椒上的31个青枯病菌株,经人工接种于10个鉴别寄主植物上,结果表明,它们的致病力存在明显的差异。聚类分析这31个菌株,可以聚为3个组:第I组菌株主要来自种植番茄和茄子历史较长的广州、东莞、增城老菜区,其致病力较强;第Ⅱ组菌株主要分离自茄子和辣椒上,其致病力中等;第Ⅲ组菌株主要来自近年来新发展的三水市各番茄产区,它们的致病力较弱。从200个随机引物中筛选出17个引物用于上述31个菌株DNA的RAPD分析,共扩增出523条带,其中468条为多态性带,占89.5%。聚类分析这31个菌株,又可聚为4个簇群:第I簇群主要分离自已推广种植多年的丰顺、金丰等抗病番茄品种上;第Ⅱ簇群分离自抗病的番茄新品种新星、年丰和石碣紫红茄上;第IV簇群主要分离自辣椒和茄子;而第Ⅲ簇群来源包括番茄、茄子和辣椒这3种寄主植物。上述试验结果说明,广东茄科青枯菌的致病力存在明显的分化现象,其DNA存在较高的遗传多样性。     

Ralstonia solanacearum preferential colonization in the shoot apical meristem explains its pathogenicity pattern in tomato seedlings

Ralstonia solanacearum causes a lethal bacterial wilt disease in many plants by colonizing the vascular tissues of the hosts. Upon inoculation of tomato seedlings through either leaf or root, the wilting symptoms occur first at the apical region and then proceed downward along the shoot. The systemic order of the disease initiation and progression in the host, independent of the site of pathogen inoculation, is yet to be investigated. To understand the disease progression more clearly, we have carried out a systematic study of the pathogen localization by GUS staining of inoculated tomato seedlings, at 24-hour intervals from 0 days post-inoculation (dpi) to 5 dpi. In both inoculation methods, pathogen colonization was observed at 1 dpi at the apical meristem as well as the cotyledon leaves, where the disease initiates. As the disease progressed, colonization by the pathogen towards the lower region of the shoot was observed. Disease consistency and pathogenicity magnitude were observed to be higher using the leaf inoculation method than the root inoculation method. Several R. solanacearum transposon-induced mutants that were reduced in virulence by root inoculation but virulent by leaf inoculation were obtained. Using GUS staining, it was observed that these mutants were unable to localize in the shoot region when inoculated in the root. Our study indicates that the apical meristem and the cotyledon leaves are the first regions to be colonized in inoculated tomato seedlings, which might explain the disease initiation from this region.     

An improved enrichment broth for the sensitive detection of Ralstonia solanacearum (biovars 1 and 2A) in soil using DAS–ELISA

A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g⁻¹ inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6 (SE 14·8) and 100·9 (SE 22·6) CFU g⁻¹ were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.     

青枯雷尔氏菌温度相关致病基因yqhE功能研究

不同温度下青枯雷尔氏菌表达谱PPI网络分析发现,yqhE可能是青枯雷尔氏菌新的温度相关致病基因。克隆青枯雷尔氏菌CBM613的yqh E基因,结果发现该基因长度为831 bp,共编码276个氨基酸。基因敲除结果显示,28℃培养的yqhE突变株较野生型菌株致病力降低,病程延长,但并未彻底丧失致病力。20℃和28℃培养的yqh E突变株维生素C的生物合成皆被抑制;与28℃相比,20℃培养下野生型菌株维生素C的合成能力降低。突变株在20℃和28℃培养下甲基乙二醛（MG）和丙二醛（MDA）明显积累,其中20℃积累更多;20℃培养的野生型菌株MG和MDA比28℃积累更多。维生素C的生物合成被抑制导致青枯雷尔氏菌自由基清除能力减弱与氧化应激反应增强,MG和MDA积累产生细胞毒性,上述结果可能与28℃培养的突变株致病力减弱,20℃培养的野生型菌株致病力丧失有关。综上结果表明yqhE是青枯雷尔氏菌温度相关致病基因。     

Genetic diversity of Ralstonia solanacearum species complex strains obtained from Guangxi,China and their pathogenicity on plants in the Cucurbitaceae family and other botanical families

Bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC), is a destructive plant disease in Guangxi, China. However, the diversity of RSSC populations in the area is unknown. To this end, we performed an extensive bacterial wilt survey from 2015 to 2018. Using phylotype-specific multiplex PCR (Pmx-PCR) and an egl-based tree, 189 strains collected from 20 plant species were identified as R. pseudosolanacearum phylotype I, which included 14 sequevars (12, 13, 14, 15, 16, 17, 18, 30, 34, 44, 48, 54, 70, and 71); two strains isolated from potato plants belonged to R. solanacearum phylotype II, sequevar 1. Sequevars 13, 17, and 44 were prevalent in Guangxi, and sequevar 13 dominated the RSSC sequevars of four Cucurbitaceae plants. The susceptibility of different Cucurbitaceae species to bacterial wilt and the host range of 16 representative strains were further tested. Members of the Cucurbita, Momordica, and Luffa genera were susceptible to bacterial wilt, with wilt incidence ranging from 73% to 100%. Most strains were pathogenic to solanaceous plants, mulberry, and ginger plants but not to melon crops; however, the strains from kidney bean, pepper, and Cucurbitaceae plants were highly virulent to melon crops. This is the first comprehensive report on the genetic and host range diversity of the RSSC in Guangxi and the susceptibility of different Cucurbitaceae species to bacterial wilt, which can provide valuable information for the development of bacterial wilt control strategies.     

青枯菌无致病力菌株对烟草青枯病的控病作用初步研究

从茄子、番茄、辣椒、烟草青枯病株中分离出116株无致病力青枯菌,室内平板喷雾法拈抗试验结果表明,有21株菌在NA培养基上可明显抑制青枯菌TbRs的生长;烟草MSK326品种温室盆栽控病试验表明,Tnljdl-3和Aujd8—2—1两株菌具有较好控病效果,20d后的相对防效分别为58．4％和97％。     

福建青枯雷尔氏菌的遗传多样性及其生防放线菌的筛选

为了明确福建青枯雷尔氏菌（简称青枯菌）的遗传多样性,综合菌株的演化型、生化型及基于内源葡聚糖酶基因egl的序列变种鉴定,对福建省8个地区的番茄、辣椒和茄子寄主分离的56株青枯菌进行分析。结果表明：供试的56株青枯菌均属于演化型Ⅰ;53株为生化型Ⅲ（占94.64%）,1株为生化型Ⅱ,2株为非标准生化型;从序列变种来看,4株来自茄子的青枯菌均属序列变种15,24株来自辣椒的青枯菌中,23株属于序列变种14,1株为序列变种16,28株番茄青枯菌鉴定出7个序列变种。进一步,选择上述鉴定的生化型Ⅲ和生化型Ⅱ的代表菌株为靶标菌进行生防菌筛选。结果表明,供试14株放线菌中,筛选到1株对生化Ⅲ青枯菌有拮抗作用的放线菌FJAT-31535。基于菌落形态特征和16S rRNA基因序列相似性分析,菌株FJAT-31535属于链霉菌属（Streptomyces sp.）。