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.     

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.     

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.     

Pathogenic and genetic variability of Ralstonia solanacearum strains from the Philippines

In the Philippines, bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases affecting vegetables and banana. In this study, 89 strains of R. solanacearum isolated from various hosts were screened for their biovar, phylotype, pathogenicity, and genetic diversity. Foreign strains were included for comparison with these Philippine strains. Results of the biochemical and multiplex-PCR tests divided the Philippine strains into five biovars (1, 2, 3, 4, and N2) and three phylotypes (I, II, and IV). Three potato strains belonged to biovar N2/phylotype IV. Pathogenicity tests divided the strains into five pathogenicity types based on their virulence in tomato, potato, eggplant, sweet pepper, and tobacco. Strains classified as biovar N2 were weakly pathogenic to potato (pathogenicity type III) and almost all strains isolated from banana were not pathogenic to the test plants except potato (pathogenicity type V). The results of AFLP analysis divided the strains into four clusters. Cluster 1 was composed of strains isolated from solanaceous crops, ginger (Zingiber officinale), and Morus sp. from the Philippines and other Asian countries. Cluster 2 grouped the potato strains (biovar N2) from the Philippines and Japan and blood disease bacterium strains from Indonesia. Cluster 3 contained the local and foreign strains isolated from potato (biovar 2) and banana (biovar 1). Cluster 4 consisted only of the tomato strain from the USA.     

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.     

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.     

番茄青枯病内生拮抗细菌的筛选

 从广西一些市县采集番茄茎标本分离得到55个细菌菌株,分属为芽孢杆菌(Bacillus spp.)、黄单胞菌(Xanthomonas spp.)、假单胞菌(Pseudomonas spp.)和欧文氏菌(Erwinia spp.),其中芽孢杆菌为优势种群。经回接测试,有36个菌株为番茄植株内生菌。这些内生菌只有7个菌株对番茄青枯病菌有拮抗作用,芽孢杆菌B₄₇菌株对番茄青枯病菌拮抗作用较强,经室内和田间初步防治测定,它对番茄青枯病有较好的防治效果。     

Diversity and distribution of Ralstonia solanacearum strains in Guatemala and rare occurrence of tomato fruit infection

Fifty-nine Ralstonia solanacearum isolates from diverse crops and regions were collected and characterized to determine the distribution and diversity of this soilborne pathogen in Guatemala. Three distinct types were present: a phylotype I, sequevar 14 strain, probably originating from Asia, infecting tomatoes and aubergines at moderate elevations; a phylotype II, sequevar 6 strain of American origin causing Moko disease in lowland banana plantations; and a phylotype II, sequevar 1 (race 3 biovar 2) strain causing brown rot on potatoes, Southern wilt of Pelargonium spp. and bacterial wilt of greenhouse tomatoes at high elevations. These data on strain diversity will inform effective regional efforts to breed for wilt resistance. A sensitive enrichment method did not detect the pathogen in fruits from naturally infected commercial tomato plants in Guatemalan fields and greenhouses, although it was detected in 6% of fruits from a wilt-resistant hybrid. Low numbers of R. solanacearum cells were also infrequently detected in fruits from plants artificially inoculated in the growth chamber with either race 3 biovar 2 or a phylotype II tomato strain.     

Epidemiological evidence for the emergence of a new pathogenic variant of Ralstonia solanacearum in Martinique (French West Indies)

The emergence of a new genotype and pathogenic variant of Ralstonia solanacearum in Martinique is described. Bacterial wilt of solanaceous crops caused by phylotype‐I and ‐II strains (‘historical strains’), was reported in Martinique in the 1960s. From 1999, Anthurium and cucurbit production was strongly affected by strains described as a new pathogenic variant genotyped phylotype IIB/sequevar4NPB (phIIB/4NPB). The following questions concerning these strains were investigated: (i) were they introduced or endemic, (ii) was their distribution widespread in Martinique, and (iii) which factors could explain this emergence? This study examined 221 isolates collected from 1989 to 2003 after several surveys. The main survey (2002–03) included 115 vegetable and ornamental crop farms. From 1999 to 2001, these phIIB/4NPB strains were initially described as the ‘Anthurium‐cucurbit’ strain. In 2003, they made up one‐third of the isolates recovered from solanaceous hosts, particularly tomato. This pathogenic variant of R. solanacearum was consistently recovered from wild species and several weeds throughout Martinique, suggesting that these strains were well established in Martinique. Data reported are consistent with the emergence of a new population of R. solanacearum in Martinique, which has spread rapidly across the entire island and may overtake the previously established population, particularly on tomatoes. Evidence is presented which suggests that the emergence of these new strains is more frequent on vegetable crops when cucurbitaceous and musaceous plants are grown in succession.     

Resistance to Taiwanese race 1 strains of Ralstonia solanacearum in wild tomato germplasm

A total of 252 wild Solanum accessions and one population of 49 introgression lines of LA716 were screened for resistance to a race 1/biovar 4/phylotype I strain Pss186 of Ralstonia solanacearum. Most wild tomato accessions were highly susceptible. However, five accessions of S. pennellii, i.e. LA1943, LA716, LA1656, LA1732 and TL01845 were resistant to strain Pss186. These accessions were then challenged against two other race 1/phylotpye I strains Pss4 and Pss190, which were more aggressive. All the five S. pennellii accessions were susceptible to Pss4, but displayed high to moderate resistance to Pss190 with a percentage of wilted plants ranging from 0% to 60%. Pss190 is an aggressive strain that made a resistant tomato line Hawaii 7996 susceptible. Thus, the results found in this study provide evidence of the presence of strain-specific resistance. LA3501, which has an introgression segment on chromosome 6, was found to be resistant to Pss186 among the screened introgression lines. This confirms the importance of resistance trait loci on chromosome 6 that have been identified by other studies. This is the first report of S. pennellii being resistant to bacterial wilt. These new resistant sources will provide breeders with more resources to breed for stable resistance to bacterial wilt of tomato.     

Ralstonia solanacearum virulence in tomato seedlings inoculated by leaf clipping

Ralstonia solanacearum is a phytopathogenic bacterium that colonizes the xylem vessels of host plants leading to a lethal wilt disease. Although several studies have investigated the virulence of R. solanacearum on adult host plants, infection studies of this pathogen on the seedling stages of hosts are less common. In a preliminary observation, inoculation of R. solanacearum F1C1 on 6‐ to 7‐day‐old tomato seedlings by a simple leaf‐clip strategy resulted in a lethal pathogenic condition in seedlings that eventually killed these seedlings within a week post‐inoculation. This prompted testing of the effect of this inoculation technique in seedlings from different cultivars of tomato and similar results were obtained. Colonization and spread of the bacteria throughout the infected seedlings was demonstrated using gus‐tagged R. solanacearum F1C1. The same method of inoculating tomato seedlings was used with R. solanacearum GMI1000 and independent mutants of R. solanacearum GMI1000, deficient in the virulence genes hrpB, hrpG, phcA and gspD. Wildtype R. solanacearum GMI1000 was found to be virulent on tomato seedlings, whereas the mutants were found to be non‐virulent. This leaf‐clip technique, for inoculation of tomato seedlings, has the potential to be a valuable approach, saving time, space, labour and costs.     

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.）。     

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.     

Current classification of Ralstonia solanacearum and genetic diversity of the strains in Japan

Ralstonia solanacearum is the causal organism of bacterial wilt of more than 200 species representing 50 families of plants in tropical, subtropical, and warm temperate regions in the world. Traditionally classified into five races based on differences in host range, R. solanacearum has also been grouped into six biovars on the basis of biochemical properties. With recent developments in molecular biology, various DNA-based analyses have been introduced and used to confirm that this binary system does not completely represent the diversity within R. solanacearum strains. Therefore, a new hierarchical classification scheme has been suggested, which defines R. solanacearum as a species complex and reorganized the concept of the species as a monophyletic cluster according to a phylogenetic analysis based on genomic sequence data. Here we discuss the current bacterial wilt situation and genetic relationships based on the recent classification system of Japanese R. solanacearum strains as well as worldwide strains. We also review the genetic, biochemical, and pathological characteristics of R. solanacearum strains, in particular, those affecting potato and Zingiberaceae plants as distinctly important pathogens in relation to continuously problematic and recent emergent diseases in Japan.     

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。     

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...     

Detection and Visualization of the Major Acidic Exopolysaccharide of Ralstonia solanacearum and its Role in Tomato Root Infection and Vascular Colonization

Exopolysaccharides play an important role in the pathogenicity of Ralstonia solanacearum. We compared in vitro and in planta exopolysaccharide production of the pathogenic strain AW1 with that of three related mutant strains impaired in both their exopolysaccharide production and aggressiveness on tomato. The distinction between the two hexosamine-rich exopolysaccharides, namely the N-acetyl-glucosaminorhamnan and the major N-acetyl-galactosamine-containing acidic polymer was emphasized. The major acidic polymer was identified specifically by electron microscopy using glutaraldehyde/ruthenium red/uranyl acetate staining, by immunofluorescence using specific monoclonal antibodies and correlated to an appropriate biochemical analysis. The two mutant strains AW1-1 and AW-19A were totally devoid of any production of the major exopolysaccharide in vitro or in planta whatever the technique used. Infection and vascular colonization of tomato roots by the pathogenic strain were also compared to those of the mutant strains by light microscopy. Pathogenicity on tomato was assessed by root infection without any artificial injury. Light microscopy showed that the two mutant strains AW1-1 and AW-19A were poorly infective and unable to invade xylem vessels, while they induced defence mechanisms in root tissues and appeared aggregated or degenerated within cortical infection pockets. These two mutant strains were non-pathogenic or weakly aggressive, respectively. In contrast, the pathogenic strain AW1 and the hypoaggressive AW1-41 strains, which produce large amounts of the major acidic exopolysaccharide in planta, were both infective and invasive, and tomato root tissues exhibited only limited defence reactions. Thus, the major acidic exopolysaccharide produced by Ralstonia solanacearum is involved in root infection and vascular colonization, though its precise role is still unknown.     

林木青枯病研究进展

青枯病是由青枯劳尔氏菌引起的一种严重的植物土传病害,我国南方多种树种被其侵染发病。本文介绍了我国林木青枯病的发生情况,概括了青枯病检测与防治方面的研究进展,并对其中的一些问题进行了探讨。     

Factors affecting the virulence of Ralstonia solanacearum and its colonization on tobacco roots

Tobacco bacterial wilt caused by Ralstonia solanacearum is a serious disease affecting tobacco cultivation in southwest China. The response surface methodology was employed to evaluate the optimal conditions of tobacco bacterial wilt, and green fluorescent protein gene (gfp) labelling was applied to monitor the location and survival dynamics of R. solanacearum (Rs::gfp) on tobacco roots and in soil under these optimal conditions. The results showed that the highest wilt incidence was 91.13%, which occurred when the population reached 6.6 × 10⁶ CFU/g soil, the temperature was 30.55 °C, and the humidity was >81.42%. The Rs::gfp densely colonized the root tips and root hairs, and cells of Rs::gfp were observed intermittently in the elongation zone or at the point of the emerging lateral roots. The Rs::gfp number in the rhizosphere soil was 10.75‐, 73.13‐ and 74.86‐times higher than that in the bulk soil at 10, 15 and 20 days after transplantation, respectively. Increased colonization by Rs::gfp was related to the population of the pathogen, the environmental temperature and the humidity in the soil. These three conditions determined whether R. solanacearum would induce tobacco wilt. This is the first study to investigate factors affecting the virulence of a tobacco wilt bacterial pathogen, which is important for conducting field diagnosis and biocontrol of tobacco bacterial wilt.     

马铃薯与玉米不同套种模式对马铃薯青枯病的防治作用

田间调查比较马铃薯与玉米不同套种模式对马铃薯青枯病的控制作用结果表明,马铃薯套种玉米可减少青枯病病菌在株行间的蔓延,抑制马铃薯青枯病的发生。套种田块的青枯病发生率、大中薯减产率和单株减产量均低于纯种田块。马铃薯与玉米二套二种植模式对马铃薯青枯病的防治作用最为明显,发病率仅为纯种模式的60%左右。