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.     

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.     

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.     

Validation and use of a qPCR protocol to quantify the spread of Ralstonia solanacearum in susceptible and resistant eucalypt plants

Bacterial wilt caused by Ralstonia solanacearum is a serious disease of eucalypt in humid and high temperature areas worldwide. Spreading of the bacterium in the field or to other nurseries occurs mainly by symptomless infected plant material. The use of pathogen-free propagating material as well as planting of resistant genotypes are currently the only strategies used for disease control. Therefore, a reliable and sensitive method for detection of low titres of R. solanacearum in infected plant tissue is essential for the success of management programmes. In this work, we adapted an efficient intercalating dye-based real-time PCR protocol to detect the bacterium in symptomless eucalypt plants as well as to investigate its movement in eucalypt clones CLR172 and CLR371, which exhibit resistant and susceptible phenotypes, respectively. We found that the bacterium translocates acropetally and basipetally in inoculated but symptomless cuttings of the resistant clone, as in cuttings of the susceptible clone displaying symptoms. Nevertheless, a smaller concentration of bacterial DNA was detected in tissues of the resistant clone. Mature biofilms occluding the xylem vessels were present in the susceptible clone whereas only single cells or small aggregates were observed in the resistant clone. This work contributes to improve our knowledge of the colonization process of R. solanacearum in eucalypt clones with different levels of susceptibility and to understand how the defence mechanisms against bacterial wilt in Eucalyptus work. Our findings could aid in the selection of the most resistant eucalypt clones to be used in wilt disease management programmes.     

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.     

Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds

Several seed extraction procedures, used for detection of Clavibacter michiganensis ssp. michiganensis ( Cmm ) in naturally infected and artificially infested tomato seed lots were evaluated. Extraction methods that included grinding the seeds were significantly better at detecting the pathogen in three different seed lots than methods that used only soaking. The detection threshold of Cmm in relation to seed sample size was determined by adding naturally infected seeds into samples of three different sizes. Cmm was detected by agar plating assay, on three media (CNS, mSCM, D₂ANX), and by direct PCR from seeds and Bio-PCR (bacteria cultured on agar media prior to PCR). In samples of 10 000 seeds containing one infected seed, Cmm could be detected only by Bio-PCR and in only one replicate out of five. In samples containing five or 10 infected seeds per 10 000 seeds, three of five and five of five replicates, respectively, were detected by the three detection methods. In samples of 5000 seeds, one infected seed could be detected in all five replicates only after adding a concentration step. A high correlation ( R ² = 0·9448) between artificially infested seeds and the disease incidence was found. Seed lots infested with less than 58 colony-forming units (CFU) per g did not cause disease under glasshouse conditions, whereas lots with about 1000 CFU g⁻¹ caused disease in 78 plants out of 2000.     

红掌青枯病的症状和病原菌鉴定

 描述海南儋州某红掌栽培基地红掌发生青枯病的症状,根据对该病原菌的形态、染色反应、培养性状、生理生化特性的测定结果,将病原菌鉴定为茄青枯拉尔氏菌[Ralstonia solanacearum (Smith) Yabuuchi et al.]。通过对病原菌的寄主范围测定,病原菌属茄青枯拉尔氏菌小种1,根据对6种糖、醇利用的测定结果,病原菌属于生物变种3。     

桑细菌性枯萎病病原菌的分离鉴定与全基因组序列分析

为明确华南蚕区桑树细菌性枯萎病主要病原菌的种类,本研究从广西、广东等地桑园采集了多份桑树细菌性枯萎病病样,采用分离培养、形态学鉴定和分子生物学鉴定的方法,对病原菌进行了分离鉴定,并通过药敏试验分析病原菌的耐药性。结果表明,结合柯赫氏法则,将分离到的桑枯萎病病原菌鉴定为产酸克雷伯氏菌Klebsiella oxytoca,编号AKKL-001(菌种保藏号GDMCC No.1.1602)。该病原菌属革兰氏阴性菌,基因组全长6 149 586 bp,含有5 792个基因,GC含量55.80%;药敏试验结果表明,病原菌对氨苄西林、头孢唑啉、头孢呋辛、头孢噻吩、链霉素、麦迪霉素等抗生素具有耐受性。本研究报道了桑细菌性枯萎病病样上分离的产酸克雷伯氏菌,为桑细菌性枯萎病病原菌的研究提供理论依据,全基因组测序的结果为桑细菌性枯萎病的相关致病机制研究奠定了基础。     

Assessment of the pest status of Pratylenchus curvicauda and ultrastructural changes in roots of infected wheat and barley

Pratylenchus curvicauda, which was first described in metropolitan Perth in 1991, was recently identified in grain-growing areas in Western Australia. The biology of this root-lesion nematode, and especially its pest status, is unknown. We investigated its life cycle and interaction with host plants, because such information is essential for its management. The life cycle took 45 days to complete in a wheat cultivar maintained at 23°C. Over 10 weeks, the nematode multiplied in 26 of 61 genotypes; these host plants were all cereals and included widely grown cultivars of wheat and barley. Eighteen other cereal genotypes and 13 cultivars including canola, chickpea, ryegrass, lupin, soybean, and tomato, sustained the nematodes to different degrees without multiplication. Four cover crops were not suitable hosts. The patterns of attraction of the nematodes and penetration into roots of the host and tolerant plants were similar. The nonhosts attracted fewer nematodes, none of which penetrated the roots. Browning of infected roots was atypical—it occurred late in some roots, 55 days after inoculation, and in the presence of a fungus. The nematodes were confined to, and fed from, cortical cells. The ultrastructure of infected wheat and barley cells showed typical signs of damage caused by Pratylenchus spp. and included cell disorganization and lack of membrane integrity, disintegration of cytoplasm, hypertrophy of some nuclei, and deposition of tannin-like granules. This detailed characterization of P. curvicauda–host interaction indicates the nematode is likely to be a pest of major crops, and attention should be given to its management.     

植病生防菌株B1301的种类鉴定及其对生姜青枯病的作用机理和防治效果

从生姜田土分离的细菌B1 3 0 1鉴定为巨大芽孢杆菌。在盆栽条件下 ,B1 3 0 1处理种姜能够有效地防治由茄伯克氏菌引起的生姜细菌性青枯病 ,在种姜带菌率小于 5%的情况下 ,防治效果在75%以上 ;B1 3 0 1可在生姜块茎周围有效定殖 ,并能有效地降低青枯病菌的群体数量 ;也能从老块茎向新块茎转移。B1 0 3 1通过抗菌物质以及竞争作用达到防治病害的目的。     

茉莉酸甲酯对辣椒抗青枯病的诱导效应及抗氧化酶活性的影响

为探索茉莉酸甲酯(methyl jasmonate,MeJA)诱导辣椒抗青枯病的效应及与抗氧化酶活性的关系,以感青枯病品种粤红1号和抗青枯病品种辛香8号辣椒为试验材料,分别用浓度为0.025、0.05、0.1、0.5、1.0 mmol/L的MeJA浸种12 h和喷雾48 h处理后,接种浓度为107CFU/mL的青枯病菌Ralstonia solanacearum,测定MeJA对青枯病菌的抑制活性以及辣椒幼苗的病情指数、抗氧化酶活性和丙二醛含量。结果表明,0.025～1.0 mmol/L MeJA对青枯病菌无抑制作用;MeJA浸种处理对粤红1号和辛香8号辣椒幼苗的诱导效果最高,分别为21.38%和25.17%,而喷雾处理的诱导效果可依次提高至31.64%和37.30%,且诱导效果随MeJA浓度先升高后降低。与对照相比,MeJA处理后2个品种辣椒幼苗的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)及谷胱甘肽还原酶(GR)活性均随MeJA浓度先升高后降低,而丙二醛(MDA)含量则先降低后升高;CAT、POD、APX和GR活性最大值均为抗病品种辛香8号经MeJA喷雾处理后所达峰值,依次为13.51、49.27、11.58和5.28 U/g FW,MDA含量最低值为13.58μmol/g FW,而SOD活性最大值为感病品种粤红1号经MeJA喷雾处理所达峰值,为364.31 U/g FW。表明MeJA预处理可有效诱导辣椒抗青枯病,其诱导效应与其激活幼苗叶片抗氧化酶活性和降低丙二醛含量有关。     

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.     

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 of Ralstonia solanacearum (biovar 2A) in stems of symptomless plants before harvest of the potato crop using post-enrichment DAS-ELISA

The detection of Ralstonia solanacearum (biovar 2A) in stems of symptomless plants before harvest of the potato crop, instead of tubers, would not only save highly valued planting material but would be less time-consuming and would also enhance farmers' market decisions. Although pathogen detection in stems has been proven efficient for ring rot, this has never been investigated for bacterial wilt (BW). Therefore the possibility of detecting BW latent infection in stem pieces about three weeks before harvest was assessed in 57 fields of the Andean highlands of Peru. Two sensitive, specific and user-friendly serological methods were used to detect the pathogen in latently infected tubers and stems: double-antibody sandwich (DAS)-ELISA and indirect ELISA on nitrocellulose membrane (NCM) after enrichment of the plant extracts in a semi-specific broth. Optimum sample sizes of stems and tubers were evaluated for 37 potato crops showing between 0 and 0·1% BW incidence using a binomial distribution model to calculate the detection probabilities. Although results of detection using the two serological techniques had 100% concordance, detection probabilities were higher using DAS-ELISA, whatever the plant part tested. BW detection probabilities were higher for tubers than for stems; a 99% detection probability was obtained by analysing 400 stems sections or 250 tubers using DAS-ELISA. Detection of BW infection in symptomless plants 20 days before harvest using post-enrichment DAS-ELISA is a reliable and user-friendly technique that can easily be used by national plant protection services and seed programmes in developing countries.     

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%。     

山西省窖藏薯类块茎中2种短体线虫的种类鉴定

本文采用传统形态学与分子生物学相结合的方法,对采自山西省窖藏马铃薯和薯蓣块茎中的短体线虫进行了种类鉴定.结果 表明,从马铃薯块茎中分离出的短体线虫形态学特征与斯克里布纳短体线虫Pratylenchus scrib-neri一致,其SSU序列与P.scribneri美国群体相似性达99.8％.从薯蓣块茎中分离出的短体线虫...     

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.     

我国生物源农药标准制定现状及展望

随着农药减量化政策的实施,植物源农药因具有低毒、低残留等特点而越来越受到重视。柠檬烯是一种广泛存在于柑橘类精油中的天然单环萜烯,因其具有多种生物活性,在农业病虫草害防控中具有一定的潜能和应用前景。本文综述了近年来柠檬烯及其精油在杀虫、杀螨、除草、杀真菌等农业领域的研究与应用进展,并对其生物活性的作用机理进行了总结归纳。同时,介绍了柠檬烯制剂在我国的登记情况,以及柠檬烯纳米制剂在防治农业病虫害中的研究现状和应用,并对该领域的研究发展趋势和前景进行了展望,可为柠檬烯在农药减量化和病虫草害绿色防控中的应用提供科学依据。     

贝莱斯芽胞杆菌JK19发酵液稳定性及抑菌物质初步分析

本研究以具有自主知识产权的一株对番茄青枯病菌有较好抑菌效果的贝莱斯芽胞杆菌JK19为研究对象.通过TTC双层板平板对峙测定菌株JK19发酵上清液对温度、pH、酶及紫外照射等处理下抑菌效果及稳定性,进而对其拮抗活性物质进行了初步分析.结果表明菌株JK19发酵上清液抑菌圈直径在温度超过80℃时显著下降;不耐强酸强碱,适宜p...