辣椒疫病生防菌的筛选及其抑菌机制初探

 观测了枯草芽孢杆菌BS、GF1和荧光假单胞菌LX1、BCA1 4株生防菌对7株来源不同的辣椒疫病病菌的拮抗作用和防治效果。结果表明:BS对辣椒疫病病菌菌丝的拮抗作用显著高于其它3株生防菌,抑制率达到33.2%~59.4%;温室防病试验中,BS喷雾处理和BCA1灌根处理对辣椒疫病具有明显的防治效果,防效分别达到56.83%和57.81%。BS抗菌粗提物对病菌菌丝生长具有明显的抑制作用,抑菌作用与抗菌粗提物稀释倍数呈负相关,稀释10倍时抑制率达到100%。BS抗菌粗提物处理使辣椒疫病病菌菌丝分枝增多,分枝间距明显缩短,分枝顶端原生质消解;同时可以显著抑制辣椒疫病病菌游动孢子的萌发速度和萌发率。对绿色荧光蛋白标记的BS菌株(gfp-BS)在辣椒幼苗各部位定殖情况的检测表明:该菌可以通过种子细菌化成功定殖于辣椒植株体内,出苗60 d后仍能检测到荧光标记的菌落,其定殖量维持在10³ cfu/g以上。     

辣椒疫霉生防菌的双重筛选

利用抑菌圈-定殖力双重测定法进行辣椒疫霉拮抗菌筛选。结果表明:有6株拮抗菌(28G14、03GY02②、F1、B2、95W06和31-4G02)在辣椒幼苗根茎部的定殖能力较强(定殖密度大于2.6×104cfu/g根),其中B2和F1的定殖密度为176.0和134.0,分别是致病菌辣椒疫霉(27.0×104cfu/g根)的6.5倍和5.0倍;这6株拮抗菌均具有较高的相对防效(81.2%~100%),不能定殖的菌株相对防效低(0~37.9%),说明抑菌圈-定殖力双重测定法是筛选具有双重功能生防菌的一种有效而简便的方法;6株有效菌在灭菌和未灭菌土壤中的相对平均防效分别为93.3%和87.0%;拮抗菌的相对防效与接种时间有关,有效菌株数按十叶期(44株)>开花期(38株)>结椒期(36株)的顺序排列。     

植物内生细菌在辣椒体内的定殖动态及对辣椒疫病的防治效果

为了探讨具生防作用的植物内生细菌在辣椒体内的定殖动态与其防治辣椒疫病的关系,采用对峙培养法和盆栽苗防效法筛选生防菌株,依据菌体形态、生理生化性质和16SrDNA序列鉴定菌种,用抗利福平标记研究菌株在辣椒苗中的定殖动态,在同时接入植物内生细菌和灌根接种辣椒疫霉菌的条件下分析生防菌株的定殖数量与防效的关系。结果表明,菌株G9、R15和J13对辣椒疫病防效最好,经鉴定均为荧光假单胞菌Pseudomonasfluorescens。菌株G9和R15在辣椒根部定殖量高于菌株J13;定殖周期均在30-40d,呈“先增后减”的变化趋势;菌株G9和R15在接种第15d时定殖量最高,菌株J13在根、茎和叶中定殖量达到最高的天数分别为第9、15和15-20d,定殖数量的变化为根〉茎〉叶。菌株G9定殖量达到9．73×10^5cfu·g-1时辣椒疫病的防效达到100％,保持该数量的时间约6d;菌株R15定殖量达到6．30×10^5cfu·g-1以上时对辣椒疫病的防效达到100％,保持该数量的时间约14d。研究结果展现了植物内生细菌在辣椒疫病生物防治上的应用潜力,为制定植物内生细菌防治辣椒疫病的施用技术提供了科学依据。     

拮抗链霉菌26B的筛选及其在不同含水量土壤中对白菜软腐病的防病效果

为挖掘防治白菜软腐病的生防菌,本研究采用牛津杯法从38株白菜根际放线菌中筛选到一株拮抗放线菌26B,通过形态学观察和16S rRNA基因序列分析将菌株26B初步鉴定为链霉菌。该菌除菌发酵滤液对供试的白菜软腐病菌Pectobacterium carotovorum subsp.brasiliensis BC1及3种马铃薯黑胫病菌均表现较高的抑菌活性,其中对白菜软腐病菌抑菌直径达到23.97 mm。萌发试验表明,白菜种子经26B除菌发酵滤液浸种后发芽势、发芽指数、根长及鲜重显著增加。采用浸根法测定菌株26B除菌发酵滤液对软腐病菌荧光标记菌株BC1-gfp在白菜根部定殖量的影响,浸根处理后48 h,白菜根上BC1-gfp的数量达到2.3×10⁹ CFU/g;未从26B除菌发酵滤液与BC1-gfp菌悬液混和处理（体积比10%）的白菜根上检测到病原菌。盆栽防病试验结果表明,在含水量为14%和20%的土壤中,菌株26B除菌发酵滤液对白菜软腐病的防效分别达96.0%和89.8%。上述结果表明,链霉菌26B是一株具有潜在开发应用价值的生防菌株,将链霉菌26B应用与土壤水分管理相结合可进一步提高其防病效果。     

生菜软腐和菌核病拮抗菌贝莱斯芽胞杆菌BPC6鉴定与防效

软腐病和菌核病是生菜生产中两大毁灭性病害。为有效控制这2种病害，利用平板稀释和对峙法，从生菜根际土壤中分离到一株拮抗菌BPC6，其菌悬液、无菌滤液对软腐病菌Pectobacterium carotovorum和菌核病菌Sclerotinia sclerotiorum的生长均具有抑制效果，该菌株也能抑制其他14种植物病原菌的生长。形态学、16S rDNA及基因组序列分析将其鉴定为贝莱斯芽胞杆菌Bacillus velezensis。BPC6能产生纤维素酶和蛋白酶，不能产生几丁质酶和嗜铁素。比浊法分析的生长曲线显示，BPC6能在1%～10% NaCl及pH 5.0～9.0条件下生长。通过分析不同浓度BPC6对软腐病菌和菌核病菌侵染生菜离体叶片的影响，明确了菌株BPC6适宜用量为1.4×10⁹～2.8×10⁹ CFU/mL。菌株BPC6对生菜软腐病和菌核病盆栽防效分别为44.09%和53.58%，对菌核病大田防效为77.41%。本研究表明BPC6是一株对生菜软腐病和菌核病具有拮抗作用的潜在生防菌。     

烟草青枯病生防菌混合接种对其定殖及防效的影响

本研究将枯草芽孢杆菌Bacillus subtilis B001、短短芽孢杆菌Brevibacillus brevis B011和侧孢芽孢杆菌Bacillus laterosporus 2-Q-9混合接种,探讨其协同防治烟草青枯病的有效性和可行性。平板拮抗试验表明,3株生防菌相互间无拮抗作用,且混合菌群的抑菌活性有所增强。定殖试验中,生防菌两两混接总体上可提升其定殖力,但3株混接对B011和2-Q-9有抑制作用;在烟株接种移栽后第60d,各混接组合的定殖总量均达到最大,其中B001＋B011和B011＋2-Q-9组合分别达7.28、7.23lg.cfu/g,远高于其它处理。小区试验表明,3株生防菌不论单用还是混用,其防治效果均优于72%农用链霉素;两菌混接优于对应的单菌处理,其中以B011＋2-Q-9组合防效最好,达83.39%,其次是B001＋B011组合,防效为78.34%;但三株混接的防效仅为45.49%,低于所有生防菌处理。     

拮抗细菌B11的鉴定及其分泌的拮抗蛋白抗菌谱

芽孢杆菌 (Bacillusspp .)是一群好氧兼性厌氧的产芽孢革兰氏阳性杆菌的总称 ,它不仅具有显著的生防潜力 ,还能产生耐热抗逆的芽孢 ,有利于生防菌剂的生产、剂型加工及在环境中存活、定殖与繁殖 ,是植物病害生防微生物的重要组成部分。广西地处亚热带 ,菌物资源丰富 ,生防资源 (基因库 )也多种多样 ,因此 ,在广西植物病害生物防治的研究具有较好的前景。黎起秦等[1] 从作物根围分离得到对西瓜枯萎病菌具有强烈抑制作用的拮抗细菌B11,本文对其进行了鉴定 ,并对其分泌的拮抗蛋白的抗菌谱进行了测定。现将结果报道如下。1　材料与方法1 .1　供…     

烟草赤星病菌拮抗菌解淀粉芽胞杆菌YW-2-6鉴定及生防效果

为获得有效拮抗烟草赤星病菌的生防资源,采用稀释分离法和平板对峙法从烟草根际土壤中分离筛选烟草赤星病菌拮抗菌株。通过形态学观察、生理生化特征和16S rDNA序列分析进行鉴定,并测定其对烟株的促生作用,验证菌株的产IAA能力,评价生防菌发酵液对烟草赤星病的田间防效。结果表明,从分离的65株细菌菌株中筛选到5株烟草赤星病拮抗细菌,其中菌株YW-2-6对烟草赤星病菌拮抗效果最好,抑制率为71.32%,初步鉴定菌株YW-2-6为解淀粉芽胞杆菌Bacillus amyloliquefaciens。菌株YW-2-6对小麦赤霉病菌、辣椒疫霉病菌、烟草靶斑病菌等病原菌具有显著的拮抗作用。YW-2-6发酵液对烟草赤星病防效为56.93%,且菌株YW-2-6对烟株有明显的促生作用。菌株YW-2-6具有良好的生防潜力和开发前景。     

不同配方存活基质对生防菌ANIT-8908A保存、防效和土壤定殖力的影响

研究了豆饼粉、泥碳土、木屑、KCl和尿素按不同配比制成的4种配方的存活基质对生防菌ANIT-8098A在室内的保存、田间防效和土壤定殖能力的影响。试验结果表明:用8.0%豆饼粉、46.0%泥炭土、40.0%木屑、5.0%KCl、1.0%尿素制成的生防菌制剂ANIT-3号存活基质对生防菌ANIT-8098A室内保存效果最好,经90 d后保存率仍高达83.33%;生防菌制剂ANIT-3号在田间对茄子青枯病的防效最高,达100.00%;生防菌制剂ANIT-3号对生防菌ANTI-8098A在土壤中定殖力的促进效果也最高,药后40 d定殖率为11.2%。     

烟草赤星病拮抗菌的鉴定、定殖和防病效果研究

为寻找可有效防控烟草赤星病的生防菌资源,利用平板对峙法从四川凉山的烟叶上分离并筛选得到6株对烟草赤星病菌（Alternaria alternata）有拮抗作用的菌株,其中以菌株SYL-3的拮抗作用最强。结合形态学特征、生理生化特性、16S r RNA及gyrA测序分析,鉴定SYL-3为贝莱斯芽孢杆菌（Bacillus velezensis）。进一步研究发现,SYL-3对烟草赤星病菌、马铃薯早疫病菌、小麦赤霉病菌等16种病原真菌均有较强拮抗作用,能成功定殖于烟草叶面并进入维管束组织,在田间施用后对烟草赤星病的防效优于对照药剂菌核净。     

Phenotypic and genetic characterization of Erwinia carotovora from mulberry (Morus spp.)

Phenotypic and genetic characteristics of nine bacterial strains isolated from mulberry ( Morus spp.), which were originally described as Erwinia carotovora ssp. carotovora (Ecc), were investigated. Based on the results of biochemical tests, these bacterial strains were divided into two different types, type 1 and type 2. Two strains of type 1 were similar to Ecc, whereas seven strains of type 2 were distinct from Ecc. A polyphasic study that included serological assay, specific PCR assay for E. carotovora ssp. atroseptica (Eca), PCR-RFLP of a pectate lyase ( pel ) gene and RAPD-PCR was performed on the type 2 strains, and the data were compared with those of related E. carotovora subspecies. The results of serological and specific PCR assays for Eca showed that the type 2 strains were distinct from Eca. In RFLP analysis of the pel gene using Sau 3AI, the type 2 strains showed a unique RFLP pattern. On the basis of RAPD analysis, similarity of RAPD patterns within the type 2 strains was very high. A unique RAPD fragment was isolated from the type 2 strains and used as a probe for Southern hybridization. This probe hybridized only with PCR products from the type 2 strains. Based on phenotypic, serological and genetic characteristics, the type 2 strains isolated from mulberry may belong to a distinct E. carotovora subspecies other than Eca or Ecc.     

大白菜软腐病菌游动性突变体在大白菜体内增殖扩展研究

 用培养皿滤纸吸附测定法和不伤根土壤拌菌处理及针刺接种法,测定了大白菜软腐病菌游动性突变体进入大白菜体内、并在其中侵染定殖和扩展的特性。结果表明,游动性丧失和增强的突变体都可以通过种子萌发和主动接触进入大白菜体内、并可以在体内有短期的繁殖,但菌量远低于野生菌。大白菜叶片接种实验说明,这两种突变体也都可以进行短距离扩展,但扩展距离和菌的繁殖量低于野生菌。     

细菌凝集素与菌体脂多糖在大白菜与软腐欧氏杆菌接触识别中的作用

 用3种试验方法测定了大白菜细菌凝集素(Agin-SD60)和软腐欧氏杆菌脂多精(LPS),在双方接触识别中的作用。在吸附抑制试验中,来自大白菜和马铃曾的Agin-SD60显示约98%的吸附抑制效应,另3种植物的Agin-SD60及大白菜外源凝集素(lectin)和细咆壁蛋白质(CWP)无明显作用;同时用不同种类的7个菌侏的LPS作测定,只有病菌的LPS吸附抑制作用明显(93.37%),其胞外多糖(EPS)也无作用。在菌体凝集试验中,也只有大白菜和马铃薯的Agin-SD60表现50%~100%的凝集活性。在琼脂双扩散试验中,寄主Agin-SD60可与病菌菌体及其LPS发生免疫沉淀。这些结果说明,Agin-SD60和菌体LPS在大白菜与软幅欧氏杆菌接触识别中分别作为植物识别子(cognor)和细菌识别子(cognon)起作用。     

软腐欧氏杆菌产细菌素菌株的筛选和细菌素类型的研究

采用Echandi方法,测定了从国内不同植物上分离,经鉴定的3个种和亚种的软腐欧氏杆菌(Erwiniacarotovora var.carotovora Dye,E.carotovora var.otroseptica Dye,E.chrysanthemi Burkholder.etal.)的224个菌株产生细菌素的能力。指示菌为代表3个不同种的菌株。测定结果表明:224个试验菌株中能产细菌素的有97个,占总数的43.3％。经测定产生细菌素的专化性有所不同,约各有1/3菌株分别对3种、2种、1种指示菌有抑制作用。有些广谱的细菌素甚至对其它属的植物病原细菌也有抑制作用。少数种内专化的产细菌素菌株可望用于软腐欧氏杆菌种鉴定的辅助手段。电镜观察初步表明,这些细菌素可能是分子大小不同的两种类型的细菌素:小分子细菌素在电镜下不可见,大分子细菌素具有短杆状结构,颇似无头壳的噬菌体的尾部。     

不同种类的细菌对大白菜软腐欧氏杆菌根表吸附的竞争作用

用吸附竞争试验,测定了8种细菌共10个菌株对大白菜软腐欧氏杆菌(Erwinia carotovorasubsp.carotovora)菌株 RL4在大白菜根表吸附的竞争作用。竞争菌的吸附竞争作用与它们对大白菜根表的吸附能力相一致,在等量混合接种时,只有能对大白菜根表发生吸附的7个菌株表现出程度不同的吸附竞争能力,其中以菌株 BCE 4(RL 4是从它筛选出的抗利福平菌株)竞争最强,它也是对寄主根表吸附能力最强的菌株,其吸附竞争作用最高可达98%,不表现1∶1的数量关系。RL4和 BCE4的热杀死菌体不发生吸附竞争。发生吸附竞争的有效时间为竞争菌对根表预吸附15分钟以后,或病菌(RL4)吸附15分钟以前。     

Detection of soft rot Erwinia spp. on seed potatoes: conductimetry in comparison with dilution plating, PCR and serological assays

Automated conductance measurements in polypectate medium were used for the detection of pathogenic soft rot Erwinia spp. in potato peel extracts. The detection threshold for Erwinia carotovora subsp. atroseptica (Eca) in inoculated peel extracts was ca. 10⁴ colony forming units (cfu) ml^-1 when samples were considered positive on the basis of a response within 48 h at 20 °C. Detection of E. chrysanthemi (Ech) was less sensitive, only 10⁵ cfu ml^-1 peel extract were detected within 36 h at 25 °C. The linear correlation between detection times in conductimetry and inoculum levels of Eca and Ech in peel extracts was used for a quantitative estimation of Eca and Ech in naturally contaminated peel extracts. Samples giving a positive conductimetric response had to be confirmed with an enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) for the presence of Eca and Ech, because E. carotovora subsp. carotovora (Ecc) also generated a conductance response. Conductimetry was sensitive and efficient for detection of contamination levels of Eca higher than 10⁴ cfu ml^-1 peel extract. For Ech, conductimetric detection was less sensitive and inefficient due to low contamination levels of Ech and the presence of high numbers of Ecc in many samples after enrichment, which interfered with the test. Immunofluorescence cell staining (IF) combined with enrichment and immunofluorescence colony staining (IFC) were suited to detect and quantify low numbers of Eca and Ech at less than 10⁴ cfu ml^-1 in peel extracts. However, since false positive and negative reactions in serology were observed, the use of PCR after enrichment, or in combination with IFC to confirm positive results, was required for accurate detection.     

Characterization of an antibacterial substance produced by <Emphasis Type="Italic">Erwinia carotovora</Emphasis> subsp. <Emphasis Type="Italic">carotovora</Emphasis> Ecc 32

A total of 88 strains of Erwinia carotovora subsp. carotovora (Ecc) isolated from various host plants in several geographic regions were screened for production of antibacterial substances using the same strains as indicators. Of the 88 strains, 72 produced antibacterial substances. One of these 72 strains, a Brazilian strain Ecc 32, produced an antibacterial substance active against all tested Ecc strains on TSA medium. The antibacterial spectrum of the compound from Ecc 32 strain was limited to closely related strains of soft-rot Erwinia species. Such a narrow spectrum of activity is typical of bacteriocins. The compound produced by Ecc 32 strain, however, was resistant to some enzymes and detergents. Moreover, the compound was heat-stable and active over a wide pH range. The physical characteristics of the compound were not in agreement with those of bacteriocin or carotovoricin.     

绿针假单胞菌YL-1抗细菌活性相关基因的克隆和分析

绿针假单胞菌YL-1对植物多种重要病原细菌和病原真菌有较强的抑制作用。采用转座子插入突变技术电转化绿针假单胞菌YL-1,构建随机插入突变体库。通过平板抑菌试验从突变体库中筛选对3种病原细菌荚壳伯克霍尔德菌Burkholderia glumae、解淀粉欧文氏菌Erwinia amylovora和胡萝卜果胶杆菌Pectobacterium carotovora抑菌效果显著下降的突变体,并采用PCR和Southern Blot验证转座子是否成功插入到YL-1染色体基因组上。利用质粒拯救技术克隆转座子插入位点基因、测序和生物信息学分析。结果表明:本研究成功构建了绿针假单胞菌YL-1随机插入突变体库,筛选出7株对3种病原细菌抑制效果明显下降的突变体,但其对立枯丝核菌的抑制效果与野生型菌株YL-1相同;PCR和Southern Blot试验结果表明绿针假单胞菌YL-1的7株突变体均是单拷贝;克隆到7个突变体插入位点的基因序列并测序,生物信息学分析结果表明其中6个突变位点是嗜铁素合成基因簇,1个突变位点是蛋白分泌基因sec Y。     

Resistance enhancement of transgenic tomato to bacterial pathogens by the heterologous expression of sweet pepper ferredoxin-I protein

ABSTRACT Expression of a foreign gene to enhance plant disease resistance to bacterial pathogens is a favorable strategy. It has been demonstrated that expressing sweet pepper ferredoxin-I protein (PFLP) in transgenic plants can enhance disease resistance to bacterial pathogens that infect leaf tissue. In this study, PFLP was applied to protect tomato (Lycopersicon esculentum cv. cherry Cln1558a) from the root-infecting pathogen, Ralstonia solanacearum. Independent R. solanacearum resistant T(1) lines were selected and bred to produce homozygous T(2) generations. Selected T(2) transgenic lines 24-18-7 and 26-2-1a, which showed high expression levels of PFLP in root tissue, were resistant to disease caused by R. solanacearum. In contrast, the transgenic line 23-17-1b and nontransgenic tomato, which showed low expression levels of PFLP in root tissue, were not resistant to R. solanacearum infection. The expansion of R. solanacearum populations in stem tissue of transgenic tomato line 24-18-7 was limited compared with the nontransgenic tomato Cln1558a. Using a detached leaf assay, transgenic line 24-18-7 was also resistant to maceration caused by E. carotovora subsp. carotovora; however, resistance to E. carotovora subsp. carotovora was less apparent in transgenic lines 26-2-1a and 23-17-1b. These results demonstrate that PFLP is able to enhance disease resistance at different levels to bacterial pathogens in individual tissue of transgenic tomato.     

抑制植物病原细菌的植物筛选

以3种重要的植物病原细菌—辣椒青枯病菌(Ralstonia solanacearum)、柑橘溃疡病菌(Xanthomonas citri)和大白菜软腐病菌(Erwinia carotovorapv.carotovora)为供试菌,对43种植物甲醇提取物进行离体抑菌活性测定。研究结果表明,漆树对辣椒青枯病菌抑菌活性最强,其次是金秀清明茶;抑制柑橘溃疡病菌活性最高的植物有漆树和十大功劳;对大白菜软腐病菌没有筛选到抑菌活性较好的植物;垫状卷柏、七叶一枝花、少花龙葵、水半夏和豆瓣菜粗提物对3种植物病原细菌均无抑制作用。