两种植物粗提液对烟草青枯病菌致病力的影响

洋葱和大蒜对多种病原微生物具有较好地抑制作用,能有效降低病原菌的致病力。用洋葱和大蒜植物粗提液5种浓度对烟草青枯病菌做室内抑制测定,采用TTC培养基检测其抑制效果。结果表明,洋葱和大蒜粗提液均可以减弱烟草青枯病菌的致病力。在5种浓度下,随着植物粗提液浓度的升高,菌落在TTC培养基上表现的红斑范围越大,颜色越深。烟草青枯菌的致病力强弱与红斑大小、颜色深浅呈反比。因此,洋葱和大蒜粗提液可抑制烟草青枯病菌的致病力,是良好的植物源生物药剂。     

青枯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,室内平板喷雾法拮抗试验结果表明,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,Tmjd1 3和Aujd8 2 1两株菌具有较好控病效果,20 d后的相对防效分别为58.4％和97％。     

青枯菌生理分化及致病型测定研究进展

青枯病是一种毁灭性的植物病害。在烟草生产中,青枯病严重为害国内外烟草的产量和质量。青枯菌种以下分类较为复杂。本文综述了国内外青枯菌的生化型、生理小种和致病型的研究进展,以期为烟草青枯菌系的构成及烟草与青枯菌互作提供分析信息,并为烟草青枯病抗病品种选育提供依据,为烟草青枯菌生理分化及致病型进一步研究提供参考。     

不同渗透压及pH环境对烟草青枯病菌致病力的影响

为探究不同渗透压和pH环境对烟草青枯病菌Ralstonia solanacearum致病力的影响,用Biolog PM 9～10代谢板中96种渗透压和96种pH环境培养烟草青枯病菌,并采用穿刺法接种于烟草离体叶片,测定不同环境下烟草青枯病菌对烟草的致病情况。结果表明,烟草青枯病菌可致病的渗透压范围包括1%～2%氯化钠、2%～3%硫酸钠、5%～20%乙二醇、1%甲酸钠、2%尿素、1%乳酸钠、20～100 mmol/L磷酸钠、10～100 mmol/L硫酸铵、10～100 mmol/L硝酸钠及10～20 mmol/L亚硝酸钠。可致病pH范围为5.0～8.0;当pH 4.5时,烟草青枯病菌在分别与L-正缬氨酸和5-羟色氨酸共培养时均可致病,与其余33种氨基酸共培养时则均不能致病;当pH 9.5时,烟草青枯病菌在与所有35种供试氨基酸共培养时均不能致病;烟草青枯病菌在葡萄糖苷、辛酸盐、半乳糖苷等10种化合物培养下均可致病。表明渗透压和pH环境会严重影响烟草青枯病菌的生长和致病力。     

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1.59~16.68 μg/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。     

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%。     

青枯雷尔氏菌无致病力hrpB突变菌株的纯化分离及其异质性研究

获得纯度高的青枯雷尔氏菌无致病力菌株,是研发青枯病植物疫苗和防治青枯病害的一种新途径。作者以青枯雷尔氏菌强致病力菌株FJAT-91为出发菌株,通过对hrpB基因敲除,获得无致病力突变菌株FJAT-91ΔhrpB。高效离子交换色谱分离结果表明:FJAT-91和FJAT-91ΔhrpB色谱峰型不同,主要表现在峰的保留时间上,FJAT-91只有单一色谱峰,保留时间为6 min;FJAT-91ΔhrpB有P_1和P_2 2个色谱峰,保留时间分别为0.6 min和4.5 min。利用高效离子交换色谱对FJAT-91ΔhrpB进行纯化,获得只有P_1峰的高纯度菌株FJAT-91ΔhrpB-P。FJAT-91ΔhrpB和FJAT-91ΔhrpB-P与其出发菌株FJAT-91的菌落和菌体形态差异明显。致病力测定结果表明:FJAT-91接种4 d番茄植株开始发病,10 d发病率达100%;FJAT-91ΔhrpB和FJAT-91ΔhrpB-P接种20 d均未发病。防效试验结果表明:纯化后的菌株FJAT-91ΔhrpB-P对番茄青枯病的防效(81.64%)比未纯化FJAT-91ΔhrpB防效(61.04%)提高了33.75%。本研究获得一株高纯度的青枯雷尔氏菌无致病力突变菌株FJAT-91ΔhrpB-P具有良好的生防潜力。     

无致病力青枯菌株对番茄青枯病的防治效果

用紫外诱变法获得的青枯无致病力菌株ATm0 4 4和Asp0 6 1对致病菌没有直接的抑制作用 ;处理番茄后对青枯病产生抗性 ;两菌株可在番茄体内定殖并繁殖 ,移栽浸根是最佳的处理方法。盆栽试验结果表明 ,番茄经ATm0 4 4和Asp0 6 1处理后 ,分别较对照推迟 9和 7d发病 ,2 0d后的防效达 56 .7%和 53.4 %。田间小区试验结果表明 ,经ATm0 4 4和Asp0 6 1菌悬液浸根处理番茄 ,1 5d后对青枯病的防治效果分别为 53.8%和 37.7%。     

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。     

烟草感染青枯菌后的组织病理变化

病症观察表明,烟草感染青枯菌后168h叶片全部萎蔫下垂、褐变。茎部切片表明,青枯菌处理72h,个别导管内出现了染色较深的物质,髓部和皮层的部分薄壁细胞出现破损。120h后导管内染色较深的物质增多,导管堵塞程度加大。168h后局部区域的木质部和韧皮部分离。对感病植株的叶进行青枯病菌分离,结果表明:从叶中可分离出致病青枯菌,并可以导致复感植株得病继而死亡。     

Distribution of avirulence genes <Emphasis Type="Italic">avrA</Emphasis> and <Emphasis Type="Italic">popP1</Emphasis> in 22 Japanese phylotype I strains of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>

Sequence analysis of hrp loci and effector genes in the flanking regions showed significantly high similarities between two phylotype I strains of Ralstonia solanacearum, GMI1000 and Japanese strain OE1-1. Further sequence analysis of the distribution of avrA and popP1, known as determinants of a hypersensitive response (HR) induction on Nicotiana tabacum (tobacco), in 22 Japanese phylotype I strains revealed that all strains had one of the two distinct avrA alleles and that 10 strains had an identical popP1 but the other 12 did not. After infiltration of tobacco leaves, more than half of these 22 strains elicited HR. In combination with the ability to induce HR, avrA and popP1 are thus not likely to be the sole determinants of HR in Japanese phylotype I strains.     

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.     

Specific Detection of Biovars of Ralstonia solanacearum in Plant Tissues by Nested-PCR-RFLP

A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 10³cfu ml^–1, which corresponds to 1cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.     

我国长江流域和南方地区花生青枯菌遗传多样性分析

为明确不同青枯菌的遗传多样性和其在花生植株上的致病力差异,采用国际上新的青枯菌演化型分类模式,对从我国长江流域和南方地区9个花生种植区分离的95株花生青枯菌Ralstonia solanacearum菌株进行遗传多样性分析,基于内源葡聚糖酶基因egl对青枯菌进行系统发育研究,并对供试青枯菌的致病力进行测定。结果表明,所有95株菌株均属于青枯菌演化型I型,即亚洲分支类型。在序列变种分类上,所检测的9个花生种植区中有8个种植区的花生青枯菌菌株属于序列变种14,仅有1个种植区(广西壮族自治区贺州市)的花生青枯菌菌株属于序列变种48,表明我国长江流域和南方地区花生青枯菌群体遗传多样性水平较低。青枯菌致病力测定结果表明,来自赣州市的菌株GZ-1、贺州市的菌株HZ-2和宜昌市的菌株YC接种到花生植株14 d后,花生的病情指数分别为43.8、75.0和87.5,而来自其它6个花生种植区的菌株接种花生后,其病情指数均为100.0,表明菌株GZ-1和HZ-2的致病力较弱,而其它7个花生种植区代表性菌株的致病力均较强。     

丛枝菌根菌丝桥传递作用对烟草抗病性相关酶活性的影响

为明确供体与受体植株间菌丝桥传递抗病信号对受体植株生长以及抗病性相关酶活性的影响,利用丛枝菌根真菌在供体与受体烟苗植株间建立菌根菌丝桥,对供体植株接种青枯菌的方法进行研究。结果表明:在供体烟苗接种丛枝菌根真菌条件下,再接种青枯菌,比只有菌丝连接的受体烟苗叶片内的过氧化物酶(peroxidase,POD)、多酚氧化酶(polyphenol oxidase,PPO)、苯丙氨酸解氨酶(phenylalanine ammonialyas,PAL)的活性分别提高了21%、29%和14%,地上部干重和植株磷含量也有相同的趋势;而在不接种丛枝菌根真菌条件下,供体烟苗接种青枯菌与否对酶的活性、植株干重及氮、磷含量均无显著影响。表明供体烟苗产生的抗病信号可以通过菌丝桥传递给受体烟苗,增强受体烟苗的抗病性。     

Involvement of HLK effectors in Ralstonia solanacearum disease development in tomato

A multigene family encoding HLK effectors is well conserved in all Ralstonia solanacearum strains. The HLK family contains three paralogous members. We constructed single, double, and triple deletion mutants of hlk genes in R. solanacearum strain OE1-1 and characterized HLK effectors by comparing the phenotypes of the strains. HLK effectors exhibited some functional redundancy. While the triple deletion mutant was significantly impaired in virulence on tomato and multiplied less efficiently than the wild type in the leaves, the mutant was as virulent as the wild type on eggplant and tobacco.     

烟草青枯病菌与其拮抗菌解淀粉芽胞杆菌的代谢表型差异分析

为探究解淀粉芽胞杆菌X60作为烟草青枯病生防菌剂的潜力,采用Biolog代谢表型技术比较了2种细菌的不同代谢表型。结果表明,烟草青枯病菌和解淀粉芽胞杆菌分别能代谢19%、41%的碳源,43%、77%的氮源,95%、86%的磷源以及100%、69%的硫源,分别有94、91种生物合成途径,49、95种渗透压表型以及19、94种pH代谢表型;解淀粉芽胞杆菌比烟草青枯病菌代谢显著的碳源有L-果胶糖、D-甘露糖等34种,氮源有腺苷、胞苷等29种;烟草青枯病菌比解淀粉芽胞杆菌代谢显著的碳源有D-糖二酸、半乳糖醇等9种,氮源有缩二脲、葡萄糖苷酸等11种;解淀粉芽胞杆菌的渗透压和pH环境适应力比烟草青枯病菌强;解淀粉芽胞杆菌具有脱羧酶和脱胺酶的活性。研究表明,2种细菌的代谢表型间存在较大差异,解淀粉芽胞杆菌的碳源、氮源、渗透压及pH代谢表型较烟草青枯病菌的丰富,烟草青枯病菌的磷源、硫源和生物合成途径代谢表型较解淀粉芽胞杆菌的丰富。     

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.