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.     

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。     

番茄青枯病生物防治研究进展

番茄青枯病是番茄生产上最严重的病害之一,化学防治效果不佳,且对环境不友好。本文概述了国内外应用无致病力青枯菌菌株、拮抗细菌和生物技术等方法对番茄青枯病进行生物防治的研究进展,并对生物防治存在的相关问题及应用前景进行了讨论。     

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...     

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.     

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.     

番茄青枯病植物疫苗胶悬菌剂的制备及其对病害的防治效果

研究了植物疫苗胶悬剂的制备工艺,确定胶悬剂制备的适宜参数为:琼脂终浓度为2.0‰、氯化钠终浓度为2.0%和pH为7.0。在此条件下,胶悬效果最好,质地均匀,无上下分层。试验了植物疫苗胶悬剂对番茄青枯病的防治效果,结果表明,该胶悬剂能较好地抑制番茄青枯病的发生,其防效(77.45%)与化学农药72%农用硫酸链霉素SP(77.23%)相当,配合生防菌剂20亿活芽孢/g蜡质芽孢杆菌WP施用,效果更佳,防效达81.81%。     

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.     

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.     

Susceptibility of Commercial Tomato Cultivars to Bacterial Wilt in Hydroponic System

Commercially available tomato cultivars were hydroponically cultured for inoculation, with Ralstonia solanacearum (K-101), which causes bacterial wilt, by pouring an inoculum suspension into the nutrient solution. Cultivar susceptibility to the bacteria was evaluated, based on the highest percentage of wilting. Because the length of time for wilt appearance varied among cultivars, some cultivars appeared to be suppressive to the translocation and/or multiplication of the invading pathogen. Thus, this hydroponic inoculation system is effective for examining levels of susceptibility in tomato cultivars to bacterial wilt. Received 13 December 2000/ Accepted in revised form 27 March 2001     

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.     

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.     

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.     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.     

烟草根际细菌铜绿假单胞菌swu31-2的定殖能力及其对烟草青枯病的防治作用

从重庆黔江烟草田间分离获得一株烟草青枯菌拮抗菌株Pseudomonas aeruginosa swu31 2（简称swu31 2）。采用逐步提高药物浓度的方法,筛选获得了抗链霉素300 μg/mL对烟草青枯病菌拮抗活性稳定的swu31 2突变菌株。采用灌根接种法,研究其在烟草根、茎和叶表面及内部的定殖能力及其对烟草青枯病的防治作用。结果表明,swu31 2能在烟草各组织的表面及内部定殖。该菌株在烟草各组织内部的数量均表现为“由增到减”的趋势。在接种后第8天定殖数量达到最高峰,随后有所下降;到第20天各组织内的数量仍然维持较高水平（105 cfu/g 以上）。同时,在接种20 d后,烟草的根、茎和叶的表面仍然可以检测到swu31 2的存在。盆栽试验结果表明,swu31 2的菌液和活性物质对烟草青枯病均有一定的防治效果。其中先施swu31 2菌液和活性物质粗提物的防效好于农用链霉素（51.25%）,分别为60.87%和 60.32%,而后施菌液和活性物质粗提物的防效也分别达39.50%和20.90%。     

青枯雷尔氏菌Tn5转座子无致病力突变株在番茄根部的定殖特性

为筛选防治番茄青枯病的优良生防菌株,本研究以弱化指数、胞外多糖含量和盆栽苗番茄发病率为指标确定20株经形态初步判定为无致病力的青枯雷尔氏菌Ralstonia solanacearum Tn5突变菌株的致病性,测定其在番茄根部的定殖数量,并于显微镜下观察其定殖特性。结果表明,供试的20株青枯雷尔氏菌无致病力突变菌株的弱化指数均大于0.75,胞外多糖含量介于1.59～16.68μg/mL之间,显著低于强致病力菌株FJAT-91,接种40 d番茄植株未出现青枯病症状;20株青枯雷尔氏菌无致病力突变菌株均能在番茄根部定殖,定殖数量呈先上升后下降的趋势,其中菌株T659的定殖数量最大,定殖时间最长,分别为2.86×10⁶CFU/g和35 d;透射电镜观察发现,青枯雷尔氏菌无致病力突变菌株T659从番茄植株根部表皮细胞中侵入,然后进入维管束厚壁细胞,并在维管束细胞中大量繁殖和定殖,但未引起番茄根部细胞结构病理变化。表明供试的青枯雷尔氏菌无致病力突变菌株T659的定殖能力最强,具有良好的生防潜力。