青枯菌致病机理及作物抗青枯病研究进展

青枯菌(Rdstonia solancearum)是引起植物青枯病的病原细菌.青枯菌通过T3S(Ⅲ型分泌系统)、T2S(Ⅱ型分泌系统)等分泌系统将多种毒性因子输送到胞外使寄主植物致病.转基因抗病、培育抗性品种和生物防治是防治青枯病的主要途径.     

一株番茄青枯菌拮抗菌的鉴定及抗病效果初探

从形态、生理生化、16S rDNA3个方面确定了番茄青枯菌拮抗菌株3-1-16的分类地位。光学显微镜下观察到菌体为杆状细胞,革兰氏染色均匀,并可见菌体染成蓝紫色。透射电镜进一步观察到细胞内有许多颗粒状物质,无伴胞晶体。Biolog鉴定,3-1-16与巨大芽孢杆菌(Bacillus megaterium)具有最高相似率为98%。16S rRNA分析,3-1-16与巨大芽孢杆菌MO31同源性最高为99.4%。聚类分析显示3-1-16与3株巨大芽孢杆菌聚成一支,支持度为100%。生理生化特征及培养特征测定结果表明,菌株3-1-16鉴定为巨大芽孢杆菌(Bacillus megaterium)。盆栽试验表明该菌株对番茄青枯病防病效果达到81.3%。     

烟草青枯雷尔氏菌噬菌体资源的发掘

由青枯雷尔氏菌(Ralstonia solanacearum)引起的烟草青枯病是烟草种植过程中一种重要的细菌性病害。为了开展利用噬菌体防控烟草青枯病的研究,本研究以烟田产区的7株烟草青枯菌为宿主,从烟田土壤中分离到了14个对烟草青枯菌具有侵染性的烈性噬菌体,说明烟田土壤中普遍存在烟草青枯菌噬菌体。选取噬菌体?PB2和?PB34,电镜观察确定其具有十二面体的头部和粗短的尾部,属于肌尾噬菌体科。噬菌体的宿主谱分析显示,筛选到的噬菌体对分别从烟草、西红柿和花生分离的青枯菌株系的侵染性有所差异。本研究分离发掘的14个对烟草青枯菌侵染的噬菌体,为利用噬菌体防控烟草青枯病的生防策略研究提供了噬菌体资源。     

番茄茎内生细菌的分离鉴定及青枯病拮抗菌的筛选

采用化学法进行表面灭菌处理,运用平板涂布法及平板划线法从番茄茎内得到17株内生细菌.通过形态观察和生理生化指标鉴定,17株内生细菌分属于6个属,即葡萄球菌属(Staphylococcus)、短芽孢杆菌属(Brevi-bacillus)、芽孢杆菌属(Bacillus)、棍状杆菌属(Clavibacter)、欧文氏菌属(Erwinia)和乳杆菌属(Lactobacillus).采用滤纸片法从17株内生菌中筛选出1株对青枯菌(Pseudomonas solanacearum)有拮抗作用的菌株,编号为TS-06,属于芽孢杆菌属(Bacillus).其抑菌圈半径为2.5 mm.     

广藿香内生真菌多样性及其对青枯菌的拮抗活性

【目的】对广藿香各部位内生真菌的类群结构及多样性进行系统分析,筛选出对青枯菌具拮抗活性的菌株。【方法】采用组织块分离法从广藿香健康植株的根、茎、叶中分离内生真菌,结合形态学和基于ITS-r DNA序列分析鉴定方法分析广藿香内生真菌的类群结构及多样性,并利用双层平板拮抗法筛选对青枯菌具有抑制活性的菌株。【结果】从广藿香中共分离获得313株内生真菌,隶属于30个属,其中链格孢属(占28.75%)、拟茎点霉属(占23.00%)和炭疽菌属(11.82%)为优势类群,此外还分离到炭皮菌属、弯孢聚壳属、Gibellulopsis、新萨托菌属、葡萄座腔菌属、篮状菌属等较为少见的类群。茎、叶内生真菌的定殖率和分离率明显高于根,而根部多样性指数为2.64,要高于茎(2.00)和叶(1.97);广藿香茎与叶、根与茎、根与叶之间的内生真菌相似性指数分别为0.35、0.20、0.19,均小于0.5,显示各部位之间的内生真菌组成不相似程度高。从313株广藿香内生真菌中通过拮抗实验筛选到16株对青枯菌有拮抗活性的菌株,其中GHXR07、GHXR27和GHXR29的拮抗活性尤为显著,分别被鉴定为Talaromyces sp.、Myrothecium roridum Tode和Talaromyces wortmannii(Kl?cker)Benjamin。【结论】广藿香内生真菌具有丰富的物种多样性且其类群分布具有一定的组织特异性,其中部分菌株对青枯菌具有明显的拮抗活性。     

首次报道一点红青枯病由茄青枯菌引起

一点红青枯病在广西种植区严重发生。通过柯赫氏法则证实病原菌为细菌。用16srDNA通用引物进行PCR鉴定,测序结果表明,PCR产物与茄青枯菌有99%的同源性。首次报道一点红青枯病由茄青枯菌引起。     

拮抗菌SB1的鉴定及其抗菌物质的分析

对番茄根系菌株SB1的抗菌活性进行测定,结果表明该菌株对多种植物病原真菌、细菌具有明显的抑制作用,表现出广谱抗菌活性。通过菌体形态、生理生化反应及16SrDNA序列分析,鉴定菌株SB1为枯草芽孢杆菌内生亚种。以青枯雷尔氏菌为指示菌,测定了菌株SB1抗菌物质的理化性质及组成。结果表明,其抗菌物质表现出良好的热稳定性、水溶性和醇溶性,且对紫外线照射和蛋白酶K处理不敏感。高效液相色谱分析结果进一步显示菌株SB1的抗菌物质中含有抗菌肽Surfactin。     

一株番茄青枯病菌拮抗细菌的筛选、发酵条件优化及田间小区防效

[背景]番茄青枯病是由青枯劳尔氏菌(Ralstonia solanacearum)引起的一种土传细菌性病害,该病原菌严重影响番茄的生产。[目的]筛选番茄青枯病的生防细菌,并将其用于病害防治。[方法]采用抑菌圈法、琼脂扩散法从湖南衡阳青枯病发病田的健康番茄根际土壤筛选对青枯劳尔氏菌具有较强拮抗能力的菌株,通过形态学观察、生理生化试验、16S rRNA基因和gyrA基因测序分析确定其分类地位;以单因素试验和正交试验对发酵条件进行优化;通过田间小区试验初探其防效。[结果]筛选的菌株TR-1被初步鉴定为贝莱斯芽孢杆菌(Bacillus velezensislezensis);菌株TR-1最佳培养基配方(g/L):可溶性淀粉20.0,大豆蛋白胨10.0,磷酸氢二钾5.0;最佳发酵条件:pH6.0-7.0,温度30-33℃,摇床转速160 r/min,发酵时长48 h,优化后TR-1无菌发酵上清液对青枯菌抑菌圈直径达2.95 cm,约为优化前的2倍;其田间小区防效为60.30%。[结论]通过对菌株TR-1发酵条件进行优化可大大提升其发酵液抑菌效果,而且菌株TR-1在田间小区试验中对番茄青枯病防效优...     

青枯菌特异性噬菌体的研究进展与应用

烟草等茄科植物青枯病的防治是一个世界性难题,传统的化学防治、合理轮作、抗病品种等措施无法有效控制该病的发生。噬菌体用于细菌性病害的防治已有很长历史,近年来利用噬菌体防治青枯菌引发的青枯病方面的研究越来越受重视。我们简要综述了青枯菌噬菌体的研究进展,并对青枯菌噬菌体生物防治的应用前景进行了展望。     

番茄青枯病拮抗菌筛选鉴定及其发酵条件初探

从健康番茄根系采样,筛选出4株对番茄青枯病有较强拮抗作用的菌株,在NA培养基上抑菌圈直径>9 mm。其中拮抗菌株YB6抑菌活性最强且拮抗效果稳定,通过形态学观察及部分生理生化特征测定,初步确定为节杆菌属。通过单因素试验进行了发酵条件初步研究,得到适宜的发酵条件为:发酵时间3 d,培养温度30°C,初始pH值9.0,接种量3%,转速100 r/min,碳源蔗糖,氮源酵母浸膏。通过初步优化后拮抗菌株抑菌活性明显增强,最终对青枯病菌SST-Y和G2M1.70抑菌圈直径与NB培养基相比增加了76.72%和81.14%,差异显著。     

烟草青枯病拮抗内生细菌的分离、鉴定及其田间防效

从病区健康烟草植株茎杆内分离到一株对烟草青枯罗尔氏菌(Ralstonia solanacarum)有强拮抗作用的内生细菌,命名为B-001菌株.拮抗性研究表明,B-001菌株对多种革兰氏阳性细菌、革兰氏阴性细菌以及病原真菌均有较强的抑制作用.形态和生理生化特征初步表明菌株B-001为芽孢杆菌属(Bacillus)细菌.经扩增、测序得到B-001的16S rDNA序列,GenBank接收号为DQ444283.用ClustalX进行多重序列对比,并通过MEGA3方法构建16S rDNA系统发育树,表明:菌株B-001与Bacillus subtilis (DQ415893)的相似性为99.2%,并处于同一分支;结合形态和生理生化指标,将其鉴定为枯草芽孢杆菌(B. subtilis).2005和2006年在湖南省桂阳县、宁乡县进行了田间试验,防效在40.03%～78.14%,防治效果良好,且明显优于农用链霉素.     

Measurement of Horizontal and Vertical Movement of Ralstonia solanacearum in Soil

Two model systems were constructed to measure horizontal and vertical movement of bacteria in soil. These systems were applied to measuring movement of Ralstonia solanacearum (race 1, biovar 3), a causal agent of bacterial wilt of tomato, in andosol and sand at 28°C. The first system was used to measure horizontal movement of the bacteria in soil packed in a narrow horizontal frame. Suspension of the pathogen was applied to soil at one end of the frame, and bacterial number per gram of soil was measured over distance from the inoculation point after 4 days. Horizontal movement of R. solanacearum in supersaturated soil, but without flow, was possibly due to diffusion and the front advanced at 2.2 cm/day in andosol, and at 8.1 cm/day in sand. Using the same experimental system, but applying water inflow to one end of the frame only, the bacterium was detected at the front of water in andosol and sand. The front of the distribution advanced at 20.4 cm/h in andosol and 66.3 cm/h in sand. In the second experimental system, a cylinder of soil packed in a short tube was soaked with water, and soil at the top of the tube was inoculated with bacterial suspension. Immediately, soil cylinders were turned upward, and the bacterial number per gram of soil was measured along vertical distance from the inoculation point after 7 days. Using the system with andosol, the capillary water front rose to 32.5 cm over 7 days after inoculation, and R. solanacearum reached to 18.8 cm height. In sand, capillary water rose to 20.0 cm and the bacteria reached to 16.3 cm height.     

Evaluation of garlic intercropping for enhancing the biological control of Ralstonia solanacearum in flue-cured tobacco fields

Tobacco bacterial wilt, Ralstonia solanacearum, is an important disease affecting the root and stem. The disease causes extensive damage to flue-cured tobacco all over the word. Field trials were conducted in 2008 and 2009 at Longyan, Fujian Province, China, to evaluate garlic intercropping for enhancing the biological control of R. solanacearum in flue-cured tobacco fields. The results of the study demonstrate that tobacco bacterial wilt was clearly inhibited by intercropping garlic in 2008 and 2009. The appearance of the disease in intercropped fields was delayed for about 15 days. The total number of R. solanacearum in root system soils was significantly lower in intercropped fields than in monocultured fields in 2008. These numbers were between 138×10⁴ and 161×10⁴ cfu g^–1 dry soil in intercropped fields. The corresponding values in monocultured fields were 357×10⁴ cfu g^–1 dry soil. The monetary value of tobacco leaves was obviously higher in intercropped fields than in monocultured ones. The per cent increase in monetary values in the intercropped fields was between 14 and 34%. Consequently, intercropping tobacco with garlic might be very useful for enhancing biological control of R. solanacearum in flue-cured tobacco fields.     

Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains

Aims: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. Methods and Results: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single‐strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. Conclusions: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single‐strand DNA genome with an ORF encoding Zot‐like protein. Significance and Impact of the Study: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.     

Development of ELISA for the detection of Ralstonia solanacearum in tomato: its application in seed health testing

Bacterial wilt caused by Ralstonia (formerly Pseudomonas) solanacearum is worldwide in distribution. It is one of the most destructive bacterial diseases of economically important crops. The serological assays so far developed for the detection of R. solanacearum were able to provide information as to the presence or absence of the pathogen in soil and plant materials. However, they could not discriminate between virulent and avirulent strains of the pathogen and were not specific to strains and races. In the present investigation, virulent bacterial cells (encapsulated with mucin) from tomato seeds were used as antigen and polyclonal antisera were developed in rabbit using a classical immunization protocol. Antisera thus developed were examined for the antibody titre, sensitivity, specificity, rapidity and the efficacy of the antibody in identifying the virulent and avirulent strains of the pathogen and the potential for application of this assay to the screening of infected plant materials and seeds. Our results indicate that the anti-tomato R. solanacearum: (i) has a good antibody titre of 1:10,000; (ii) can detect as few as 100 bacterial cells/ml; (iii) is tomato-specific (it reacted with tomato R. solanacearum, and not with isolates from chilli or eggplant); (iv) is reactive to all isolates of R. solanacearum from tomato; (v) is not cross-reactive with non-pseudomonads; (vi) is virulent strain-specific as it recognizes the virulent exopolysaccharide component as an antigenic determinant; (vii) reactivity could be correlated well with the degree of infection in tomato seeds and plant materials. The enzyme linked immunosorbent assay developed is sensitive, specific and rapid, therefore suitable for the detection of R. solanacearum isolates from tomato seeds during routine assays.     

Study on intraspecific diversity of Ralstonia solanacearum strains in West Malaysia using whole cell fatty acid analysis

Abstract

Surveys were conducted between the years of 2005 and 2006 at several locations in the northern, central and southern parts of West Malaysia to study the polymorphism of Ralstonia solanacearum strains. These sites included vegetables and farms with known hosts of the pathogen, such as banana, tomato, eggplant, chili and tobacco. Samples were collected from the suspected wilted plants and weeds, including soil and water samples, in selected areas. The bacterium was isolated in all samples using semi-selective tetrazolium chloride medium (TZC). The bacteria strains were detected by using the BIOLOG identification system and were confirmed by nested-PCR. Fatty Acid Methyl Esters (FAME) profiling was performed to determine polymorphism among 58 bacterial isolates. The results showed that the fatty acid composition varied for all R. solanacearum isolates. Grouping of R. solanacearum isolates by fatty acid composition suggested that the existence of distinct groups that were significantly related to host of bacteria but low correlation between fatty acid profiles and biovar or sampling site was detected. A unique FAME profile was found among the strains that have been isolated from banana.     

一株西瓜枯萎病生防菌的鉴定与田间防效

[背景]西瓜枯萎病是由尖孢镰孢菌西瓜专化型(Fusarium oxysproum f.sp.niveum)引起的土传病害,是西瓜最为严重的病害之一.利用拮抗微生物抑制病原菌繁殖,降低其危害程度,已成为西瓜枯萎病防控领域的研究热点.[目的]筛选对西瓜枯萎病病原菌具有拮抗作用的菌株,并探究其抑菌作用,为有效防控西瓜枯萎病提...     

Genetic and pathogenic diversity of Ralstonia solanacearum species complex strains isolated in Burkina Faso

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is a disease that negatively affects the cultivation of Solanaceae crops in Burkina Faso. Knowledge of the pathogen diversity is essential to deploy locally adapted control methods. In this study, diseased plants showing typical bacterial wilt symptoms were collected in the three main agroclimatic zones of Burkina Faso for the detection of RSSC isolates. Strain characterization was achieved through a phylogenetic and pathogenicity diversity assessment. A total of 102 isolates were sampled, and Phylotype I (Ralstonia pseudosolanacearum) was predominant (n = 101; sequevars 14, 31, 34, and 46). The remaining isolate was characterized as Phylotype IIA-35 (Ralstonia solanacearum). Phylotypes I-31 and I-46 were predominant and both characterized as the most the aggressive group of strains amongst a subset of 33 representative isolates. Our findings provide valuable information as regard RSSC diversity that breeders and resistance programme should target in order to fight this pathogen in Burkina Faso and around the world.