一株番茄青枯病生防菌的鉴定与防病、定殖能力

【目的】采用根系分泌物培养基筛选到一株番茄根际优势细菌YPP-9。本文分析测定该菌株对植物青枯病菌茄科雷尔氏菌的拮抗作用和控病能力,及其在番茄根际的定殖能力,并系统分析该菌株的分类学地位。【方法】以平板双重培养法和温室盆栽试验分别测定菌株对病原菌的拮抗能力和对番茄青枯病的控病能力;利用变性梯度凝胶电泳技术分析菌株在番茄根际的定殖能力;以形态学和生理生化特性以及16S rRNA基因序列分析确定菌株的分类地位。【结果】菌株YPP-9对茄科雷尔氏菌SSF-4的平板抑菌带宽为5mm,其盆栽控制番茄青枯病的效果达63.7%。菌株YPP-9在番茄根际具有较好的定殖能力。该菌株培养24h后菌落呈奶酪色,革兰氏染色阳性,菌体杆状、大小(1.8-4.1)μm×(0.9-1.1)μm,形成芽孢,芽孢中生或偏端生且为近似柱形,孢囊不膨大,无伴孢晶体,侧生鞭毛。菌株生长pH范围为pH5.5-8.5且最适生长pH为6.0,生长温度范围为20℃-45℃且最适生长温度为30℃。The BIOLOG GP2结果显示该菌为芽孢杆菌属。16SrRNA基因序列分析显示该菌株与Bacillus fumarioli的亲缘关系最近且序列相似性为97%,且其序列号为FJ231500。该菌株的G+C含量为41.9%,甲基萘醌主要类型为MK-7,细胞壁脂肪酸的主要种类为C14:0iso、C15:0iso和C16:0iso以及C16:1ω7 calcohol且含量分别为28.27%、19.59%、12.93%和10.88%。【结论】菌株YPP-9对茄科雷尔氏菌具有良好的拮抗作用和盆栽控病能力,且能良好的定殖于番茄根际。分类学上,该菌株归入芽胞杆菌属(Bacillus),并可能是一个新的种。     

植物根际促生菌的筛选及鉴定

【目的】植物根际促生菌(PGPR)和植物的互作关系往往不稳定,PGPR菌群有可能提高菌株对野外环境的适应性。为此,本文根据PGPR促生机制的多样性,从不同植物根际土壤进行了PGPR的筛选及鉴定。【方法】首先,按照固氮、解磷、解钾、拮抗6种常见病原真菌,同时能在植物根际定殖为基本初筛标准,然后在实验室条件下测定初筛菌株的多项促生能力(PGP),最后通过生理生化试验和16SrRNA基因序列分析对所筛菌株进行鉴定。【结果】从江苏扬州、盐城等地土壤样品筛选出14株PGPR,具有体外抑菌、产NH3、产IAA、产HCN、产嗜铁素、解磷、溶钾、固氮以及产抗生素等促生能力。分类鉴定结果显示:7株属于假单胞菌属(Pseudomonas)、3株属于类芽孢杆菌属(Paenibacillus)、2株为芽孢杆菌属(Bacillus)、1株为布克霍尔德氏菌属(Burkholderia)、1株为欧文氏菌属(Erwinia)。【结论】所筛细菌具有多种促生能力,且能在根际定殖,为进一步构建多功能PGPR广适菌群提供菌株资源。     

马铃薯立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测与应用

【背景】植物根际促生菌(plant growth-promoting rhizobacteria,PGPR)在根际的定殖是其发挥作用的基础,直观有效的跟踪技术和定量方法是研究PGPR在根际原位分布规律的重要工具。【目的】建立一种马铃薯黑痣病病原菌——立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测体系,并检测拮抗菌QHZ11在马铃薯根际的动态变化。【方法】根据GenBank中登录的类芽孢杆菌及近源菌株gyrB基因序列差异筛选特异性引物,优化反应条件;通过盆栽试验对马铃薯根际拮抗菌进行快速检测。盆栽试验设3个处理,T1:对照(无菌水,CK);T2:QHZ11菌悬液灌土(QHZ11);T3:将功能菌在有机肥中进行二次固体发酵制成生物有机肥(BOF11)。【结果】筛选出拮抗菌QHZ11的专用引物为gyrB-F/gyrB-R;建立的拮抗菌QHZ11实时荧光定量PCR检测方法特异性好、灵敏度高且重复性较好,线性相关系数为0.999 8,检测组内变异系数均在1%以内,扩增效率为0.9,可检测出1×103-1×1010copies/g-soil的拮抗菌,具有检出限低和扩增效率高的特点。盆栽试验...     

丛枝菌根真菌响应桉树茄科雷尔氏菌侵染进程基因的筛选与鉴定

【背景】桉树(Eucalyptus)青枯病危害严重,丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)与桉树共生影响桉树对青枯病的抗性,而AMF响应桉树青枯菌侵染的机制仍不清楚。【目的】探索AMF响应桉树茄科雷尔氏菌(Ralstonia solanacearum)的侵染机制。【方法】以非菌根化和异形根孢囊霉(Rhizophagus irregularis)菌根化巨桉(Eucalyptus grandis)分别受茄科雷尔氏菌侵染0、24、48和96 h接种后(hour post-inoculated, hpi)的根系组织为研究对象,基于转录组测序筛选和鉴定菌根化巨桉根系中异形根孢囊霉响应茄科雷尔氏菌侵染的基因信息。【结果】与对应非菌根化桉树受茄科雷尔氏菌侵染的时间点相比,菌根化桉树中异形根孢囊霉响应青枯菌侵染显著差异表达基因为3 382–5 989个,随青枯侵染时间进程的增加,异形根孢囊霉特异性响应茄科雷尔氏菌侵染差异表达基因数量逐渐增多。茄科雷尔氏菌侵染24 hpi时,异形根孢囊霉显著富集共生体生长、孢子形成和凋亡信号通路、铁载体等相关基因;茄科雷尔氏菌侵...     

关防风根腐病拮抗细菌筛选与鉴定

【背景】由木贼镰刀菌[Fusarium equiseti(Corda)Sacc.]引起的关防风根腐病,是近年来导致关防风产量及质量下降的主要土传病害之一。生物防治因其对环境安全及人畜无害等优势,成为目前植物病害防治的一种有效手段。【目的】挖掘关防风根际土壤中对木贼镰刀菌具有良好拮抗作用的生防菌株。【方法】采用稀释平板法分离根际土壤细菌;用滤纸片法和牛津杯法对拮抗细菌进行筛选和抑菌谱检测;用抗生素标记法标记拮抗细菌并测定其定殖能力;通过盆栽实验研究其对关防风根腐病的防效;通过形态学、生理生化特征及16S rRNA基因序列确定分类学地位。【结果】从健康关防风根际土壤中分离纯化了157株细菌,筛选获得对关防风根腐病菌具有显著抑菌作用的拮抗细菌SC-119,无菌滤液的抑菌率可达68.53%,而且兼具广谱抑菌能力和良好的定殖能力;盆栽实验表明,菌株SC-119对关防风根腐病的防治效果达到67.39%,其相对防效较接种哈茨木霉菌剂、枯草芽胞杆菌菌剂和代森锰锌分别提高了29.03%、32.26%和16.13%;对菌株SC-119进行分类学鉴定,确定其为萎缩芽胞杆菌(Bacillus atrophae...     

百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究

【目的】筛选对百合枯萎病具有抑菌活性的拮抗细菌,对其抑菌活性物质进行初步分离纯化分析。【方法】以强致病力的百合尖孢镰刀菌(Fusarium oxysporum)为靶菌,采用系列稀释法和平板对峙法初筛拮抗细菌,并通过产铁载体能力、水解酶活性、土壤定殖力等多种生防特性指标进行复筛,结合形态学特征、生理生化指标和16S rRNA基因序列比对鉴定其分类地位;利用百合尖孢镰刀菌作为靶菌进行活性追踪,结合酸沉淀、快速柱色谱、HPLC等分离纯化手段,对菌发酵液中的抑菌活性物质进行纯化分析。【结果】在64株百合根际细菌和386株海洋细菌中进行初筛,得到9株对百合镰刀菌具有较强拮抗活性的菌株,最后筛选了1株拮抗活性较强且产铁载体能力和水解酶活性、土壤定殖能力较高的菌株11B91,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其抑菌活性物质初步推测可能为iturin和fengycin脂肽类化合物。【结论】菌株11B91在百合枯萎病的生物防治中具有潜在的应用价值,证实海洋来源的微生物也具有防治陆地植物病原菌的潜力,为植物病害的防治拓宽了思路。     

侧孢短芽孢杆菌AMCC 100017的分离鉴定及其生防潜力

【目的】从土壤中分离、筛选和鉴定具有抑制病原真菌活性等生防效果的菌株,为进一步开发利用具有良好定殖能力的生防菌株奠定基础。【方法】利用对峙试验筛选拮抗菌并评价其拮抗性能,根据其形态特征、生理生化特性、16S r RNA基因序列测定及Gen Bank序列相似性分析进行分离菌株的分类鉴定,并通过福林酚法测定该菌株的蛋白酶活力。【结果】从山东泰安各种类型土壤中分离得到侧孢短芽孢杆菌(Brevibacillus laterosporus),保藏编号为AMCC100017。平板对峙试验结果表明该菌株对多种植物病原真菌均有较强的拮抗作用,尤其是对镰刀菌属致病菌拮抗效果明显。另外,本试验还初步验证该菌能产生较高活性的胞外蛋白酶。【结论】侧孢短芽孢杆菌AMCC 100017在作物真菌病害生物防治方面,有较好的开发和利用潜力,并可望应用于线虫生物防治。     

青枯雷尔氏菌胞外多糖合成缺失突变株构建及其生物学特性

【目的】由青枯雷尔氏菌(Ralstonia solanacearum)引起的植物青枯病是一种毁灭性土传病害。胞外多糖(extracellular polysaccharides,EPS)是青枯雷尔氏菌关键的致病因子之一。通过构建胞外多糖缺失突变株,研究胞外多糖在青枯病致病中的作用。【方法】从青枯雷尔氏菌FJAT-91的基因组中克隆出胞外多糖合成结构基因epsD同源臂,克隆至自杀性质粒p K18mobsacB,再将庆大霉素抗性基因(Gm)插入同源臂中间,获得重组质粒p K18-epsD。将重组质粒转化至青枯雷尔氏菌FJAT-91感受态细胞中,通过同源重组敲除epsD基因,获得EPS合成缺失的突变株FJAT-91Δeps 。研究突变株与野生菌株在菌落形态、胞外多糖合成、运动能力、定殖能力的差异性。【结果】突变菌株FJAT-91ΔepsD与出发菌株FJAT-91相比:胞外多糖产量显著减少,生长较慢;泳动能力(swimming motility)和群集运动能力(swarming motility)显著降低;在番茄苗根部和茎部的定殖能力显著降低;弱化指数(AI)为0.905,鉴定为无致病力菌株。【结论】胞外多糖在青枯雷尔氏菌的致病中起着关键的作用,本课题研究成果为开发植物疫苗提供了优良的材料与研究基础。     

烤烟根际烟碱降解细菌的多样性及其促生特性分析

【背景】挖掘兼具烟碱降解和植物根际促生细菌(Plant Growth-Promoting Rhizobacteria,PGPR)功能的细菌资源,有助于保护土壤质量,实现绿色种植。【目的】分析烤烟根际细菌多样性,筛选可降解高浓度烟碱的PGPR。【方法】采用纯培养法在选择性培养基上分离烟碱降解细菌。通过BOXA1R-PCR分析技术、16SrRNA基因测序及系统发育树构建,对菌株的遗传多样性和分类学地位进行分析。进一步评价了菌株的吲哚乙酸(Indole-3-Acetic Acid,IAA)活性、溶磷能力、病原菌拮抗能力等PGPR指标,以筛选出高效PGPR,最后通过盆栽试验验证其促生效果。【结果】分离得到58株烟碱降解细菌,根据BOXA1R-PCR指纹图谱选取11株菌进行16S rRNA基因序列测定,结果表明,58株菌分别属于芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)、拉乌尔菌属(Raoultella)和短波单胞菌属(Brevundimonas)4个属,以芽孢杆菌属(Bacillus)为优势菌属。58株细菌中48.28%的菌株可产IAA,27.59%具备溶磷能力,37.93%具备纤维素降解能力,G2-13、G2-3及HT2-8因促生与抗病特性突出而被筛选为目标功能菌。盆栽试验结果表明,G2-13菌株对幼苗生长的促进作用明显,可使株高与地上部鲜重分别增加33.05%与53.32%。【结论】烤烟根际存在较为丰富多样的烟碱降解细菌,它们在种植业上具有潜在的应用价值。     

高寒草地珠芽蓼内生拮抗固氮菌Z19的鉴定及其固氮功能

【目的】从东祁连山高寒草地珠芽蓼内生细菌中筛选和鉴定具有固氮能力和拮抗能力的内生细菌。【方法】采用16S rRNA基因序列同源性分析和生理生化指标测定方法对该菌株进行鉴定。【结果】从珠芽蓼中分离获得的21株内生细菌中6株内生菌具有固氮能力,76.2%具有抑菌能力,其中5株内生细菌对5种以上的病原菌有抑制作用;菌株Z19具有较强固氮能力和分解纤维素的能力,其纤维素溶解圈直径与菌落直径比达3.33,产生的纤维素酶活性为0.31 U,且对辣椒立枯丝核病菌(Rhizoctonia solani)、油菜菌核病菌(Sclerotinia sclerotiorum)、番茄灰霉病菌(Botrytis cinerea)、小麦根腐病菌(Bipolaris sorokiniana)和番茄早疫病菌(Alternaria solani)具有较强拮抗能力;经PCR扩增和测序,获得了菌株Z19的固氮基因(nifH)序列和16S rRNA基因序列,在GenBank中的登录号分别为EU693340和EU236746;菌株Z19呈革兰氏阳性,杆状,产芽孢。【结论】结合生理生化特征及16S rRNA基因序列同源性比较,鉴定其为枯草芽孢杆菌(Bacillus subtilis)。     

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...     

AM真菌对青枯菌和根际细菌群落结构的影响

利用传统的平板培养与DGGE相结合的技术手段,研究了接种AM真菌对番茄根际土壤中的青枯菌和细菌群落结构的影响。结果表明,菌根根际土壤中的细菌总量和总DNA量都高于非菌根根际土壤,其中前者的青枯菌种群数量比后者低60倍;DGGE图谱也证实了AM真菌对青枯菌的抑制效应,还揭示出接种AM真菌对根际土壤中细菌群落结构所产生的复杂的影响。文章对AM真菌抑制青枯菌的机制进行了探讨。     

烟草疫霉拮抗菌枯草芽孢杆菌21b菌株的分离鉴定及其生物学特性研究

【目的】为了控制烟草疫霉(Phytophthora nicotianae)引起的烟草黑胫病对烟草生产造成的危害。【方法】采用稀释平板法从贵州省毕节地区烟草根际土壤中分离筛选拮抗烟草疫霉的细菌菌株,然后经形态观察、Biolog鉴定及16S rRNA基因序列分析,对分离菌株进行鉴定,同时测定抗菌谱,单因子变量分析、优化生长条件。【结果】共分离得到44株拮抗烟草疫霉的细菌菌株,其中菌株21b对烟草黑胫病菌菌丝生长的抑制率达78.33%,鉴定为枯草芽孢杆菌(Bacillus subtilis)。该菌株对烟草青枯病菌(Ralstonia solanacearum)、烟草灰霉病菌(Botrytis cinerea)、烟草赤星病菌(Alternaria alternate)和烟草炭疽病菌(Colletotrichum destructivum)均具有拮抗作用,抑菌圈大小分别为19.5、18.2、14.6和13.4 mm,最佳的发酵条件为:温度30°C、p H 7.0–8.0、装液量12%、盐浓度0.5%。【结论】分离筛选到一株对烟草寄生疫霉有较强拮抗活性的细菌菌株,为进一步开发烟草黑胫病的生防菌剂提供了菌种资源。     

Protection of tomato seedlings against infection by Pseudomonas syringae pv. tomato by using the plant growth-promoting bacterium Azospirillum brasilense

Pseudomonas syringae pv. tomato, the causal agent of bacterial speck of tomato, and the plant growth-promoting bacterium Azospirillum brasilense were inoculated onto tomato plants, either alone, as a mixed culture, or consecutively. The population dynamics in the rhizosphere and foliage, the development of bacterial speck disease, and their effects on plant growth were monitored. When inoculated onto separate plants, the A. brasilense population in the rhizosphere of tomato plants was 2 orders of magnitude greater than the population of P. syringae pv. tomato (10(7) versus 10(5) CFU/g [dry weight] of root). Under mist chamber conditions, the leaf population of P. syringae pv. tomato was 1 order of magnitude greater than that of A. brasilense (10(7) versus 10(6) CFU/g [dry weight] of leaf). Inoculation of seeds with a mixed culture of the two bacterial strains resulted in a reduction of the pathogen population in the rhizosphere, an increase in the A. brasilense population, the prevention of bacterial speck disease development, and improved plant growth. Inoculation of leaves with the mixed bacterial culture under mist conditions significantly reduced the P. syringae pv. tomato population and significantly decreased disease severity. Challenge with P. syringae pv. tomato after A. brasilense was established in the leaves further reduced both the population of P. syringae pv. tomato and disease severity and significantly enhanced plant development. Both bacteria maintained a large population in the rhizosphere for 45 days when each was inoculated separately onto tomato seeds (10(5) to 10(6) CFU/g [dry weight] of root). However, P. syringae pv. tomato did not survive in the rhizosphere in the presence of A. brasilense. Foliar inoculation of A. brasilense after P. syringae pv. tomato was established on the leaves did not alleviate bacterial speck disease, and A. brasilense did not survive well in the phyllosphere under these conditions, even in a mist chamber. Several applications of a low concentration of buffered malic acid significantly enhanced the leaf population of A. brasilense (>10(8) CFU/g [dry weight] of leaf), decreased the population of P. syringae pv. tomato to almost undetectable levels, almost eliminated disease development, and improved plant growth to the level of uninoculated healthy control plants. Based on our results, we propose that A. brasilense be used in prevention programs to combat the foliar bacterial speck disease caused by P. syringae pv. tomato.     

Static suppression of tomato bacterial wilt by bacterial coagulation using a new functional polymer that coagulates bacterial cells and is highly biodegradable

Tomato bacterial wilt by Ralstonia solanacearum was suppressed by coagulation of bacterial cells without disinfection using a copolymer of methyl methacrylate with N-benzyl-4-vinylpyridinium chloride in a molar ratio of 3:1 (PMMA-co-BVP) as a polymeric coagulant for bacterial cells. When 10 mg/kg of PMMA-co-BVP was added to soil before transplanting of tomato seedlings, and 2 mg/kg was supplemented once a week after transplanting, a 51% reduction of appearance and a 54% reduction of index of symptoms were observed. PMMA-co-BVP did not exhibit bactericidal activity against R. solanacearum, and coagulation of the bacterial cells appeared to reduce the opportunity for infectious contact of roots of tomato with cells of R. solanacearum, and resulted in disease suppression. PMMA-co-BVP was shown to be highly biodegradable, and the half-life was 5.1 d when treated with activated sludge in soil.     

A polyphasic approach for studying the interaction between Ralstonia solanacearum and potential control agents in the tomato phytosphere.

Ralstonia solanacearum biovar 2, the causative agent of brown rot in potato, has been responsible for large crop losses in Northwest Europe during the last decade. Knowledge on the ecological behaviour of R. solanacearum and its antagonists is required to develop sound procedures for its control and eradication in infested fields.A polyphasic approach was used to study the invasion of plants by a selected R. solanacearum biovar 2 strain, denoted 1609, either or not in combination with the antagonistic strains Pseudomonas corrugata IDV1 and P. fluorescens UA5-40. Thus, this study combined plating (spread and drop plate methods), reporter gene technology (gfp mutants) and serological (imunofluorescence colony staining [IFC]) and molecular techniques (fluorescent in situ hybridization [FISH], PCR with R. solanacearum specific primers and PCR-DGGE on plant DNA extracts). The behaviour of R. solanacearum 1609 and the two control strains was studied in bulk and (tomato) rhizosphere soil and the rhizoplane and stems of tomato plants.The results showed that an interaction between the pathogen and the control strains at the root surface was likely. In particular, R. solanacearum 1609 CFU numbers were significantly reduced on tomato roots treated with P. corrugata IDV1(chr:gfp1) cells as compared to those on untreated roots. Concomitant with the presence of P. corrugata IDV1(chr:gfp1), plant invasion by the pathogen was hampered, but not abolished.PCR-DGGE analyses of the tomato rhizoplane supported the evidence for antagonistic activity against the pathogen; as only weak R. solanacearum 1609 specific bands were detected in profiles derived from mixed systems versus strong bands in profiles from systems containing only the pathogen. Using FISH, a difference in root colonization was demonstrated between the pathogen and one of the two antagonists, i.e. P. corrugata IDV1(chr:gfp1); R. solanacearum strain 1609 was clearly detected in the vascular cylinder of tomato plants, whereas strain IDV1 was absent.R. solanacearum 1609 cells were also detected in stems of plants that had developed in soils treated with this strain, even in cases in which disease symptoms were absent, indicating the occurrence of symptomless infection. In contrast, strain 1609 cells were not found in stems of several plants treated with either one of the two antagonists.The polyphasic analysis is valuable for testing antagonistic strains for approval as biocontrol agents in agricultural practice.     

Survival of gfp-tagged antagonistic bacteria in the rhizosphere of tomato plants and their effects on the indigenous bacterial community

The survival and colonization patterns of Pseudomonas putida PRD16 and Enterobacter cowanii PRF116 in the rhizosphere of greenhouse-grown tomato plants and the effects of their inoculation on the indigenous bacterial community were followed by selective plating, molecular fingerprinting, and confocal laser scanning microscopy (CLSM) over 3 weeks. Both strains, which showed in vitro antagonistic activity against Ralstonia solanacearum, were previously tagged with gfp. Seed and root inoculation were compared. Although plate counts decreased for both gfp-tagged antagonists, PRD16 showed a better survival in the rhizosphere of tomato roots independent of the inoculation method. Analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and CLSM confirmed the decrease in the relative abundance of the inoculant strains. Pronounced differences in the Pseudomonas community patterns for plants inoculated with PRD16 compared to the control were detected 3 weeks after root inoculation, indicating a longer-lasting effect. Analysis by CLSM showed rather heterogeneous colonization patterns for both inoculant strains. In comparison with seed inoculation, root inoculation led to a much better colonization as evidenced by all three methods. The colonization patterns observed by CLSM provide important information on the sampling strategy required for monitoring inoculant strains in the rhizosphere.     

利迪链霉菌M01对番茄生长、青枯病发病率及根际细菌群落组成的影响

【背景】利迪链霉菌(Streptomyces lydicus)对多种作物均有较好的促生效果,且对病原真菌具有广谱抑制作用,但该菌对细菌性青枯病的防控研究较少。【目的】探究利迪链霉菌M01能否促进番茄生长并抑制番茄青枯病,以及M01对番茄生长的影响是否通过影响根际细菌群落结构实现。【方法】采用温室盆栽试验和扩增子高通量测序技术研究M01对番茄生长、青枯病发病率及根际细菌群落组成的影响。【结果】施用利迪链霉菌M01的番茄植株鲜重、干重、株高、用土壤与作物分析开发(soil and plant analyzer develotrnent, SPAD)方法测量的叶绿素浓度、根系活力和植株P含量比对照分别提高了22.7%、12.5%、16.0%、28.1%、18.4%和17.9%,其中对株高、SPAD值和植株磷含量影响显著(P<0.05)。M01处理延缓了番茄青枯病的发病时间,接种9周后发病率比对照降低了41.8%。此外,M01对番茄根际细菌群落无显著影响(门水平群落组成,P=0.4;属水平群落组成,P=0.4)。【结论】利迪链霉菌M01可促进番茄植株生长并抑制番茄青枯病,利迪链霉菌M01对番茄生长的影响并非通过调控根际细菌群落实现。     

Protection of Tomato Seedlings against Infection by Pseudomonas syringae pv. Tomato by Using the Plant Growth-Promoting Bacterium Azospirillum brasilense

Pseudomonas syringae pv. tomato, the causal agent of bacterial speck of tomato, and the plant growth-promoting bacterium Azospirillum brasilense were inoculated onto tomato plants, either alone, as a mixed culture, or consecutively. The population dynamics in the rhizosphere and foliage, the development of bacterial speck disease, and their effects on plant growth were monitored. When inoculated onto separate plants, the A. brasilense population in the rhizosphere of tomato plants was 2 orders of magnitude greater than the population of P. syringae pv. tomato (10⁷ versus 10⁵ CFU/g [dry weight] of root). Under mist chamber conditions, the leaf population of P. syringae pv. tomato was 1 order of magnitude greater than that of A. brasilense (10⁷ versus 10⁶ CFU/g [dry weight] of leaf). Inoculation of seeds with a mixed culture of the two bacterial strains resulted in a reduction of the pathogen population in the rhizosphere, an increase in the A. brasilense population, the prevention of bacterial speck disease development, and improved plant growth. Inoculation of leaves with the mixed bacterial culture under mist conditions significantly reduced the P. syringae pv. tomato population and significantly decreased disease severity. Challenge with P. syringae pv. tomato after A. brasilense was established in the leaves further reduced both the population of P. syringae pv. tomato and disease severity and significantly enhanced plant development. Both bacteria maintained a large population in the rhizosphere for 45 days when each was inoculated separately onto tomato seeds (10⁵ to 10⁶ CFU/g [dry weight] of root). However, P. syringae pv. tomato did not survive in the rhizosphere in the presence of A. brasilense. Foliar inoculation of A. brasilense after P. syringae pv. tomato was established on the leaves did not alleviate bacterial speck disease, and A. brasilense did not survive well in the phyllosphere under these conditions, even in a mist chamber. Several applications of a low concentration of buffered malic acid significantly enhanced the leaf population of A. brasilense (>10⁸ CFU/g [dry weight] of leaf), decreased the population of P. syringae pv. tomato to almost undetectable levels, almost eliminated disease development, and improved plant growth to the level of uninoculated healthy control plants. Based on our results, we propose that A. brasilense be used in prevention programs to combat the foliar bacterial speck disease caused by P. syringae pv. tomato.     

茄子青枯病拮抗放线菌XL-6的筛选、鉴定及发酵条件优化

【背景】茄子青枯病是一种毁灭性的土传病害,生产上化学农药无法对其有效防治。拮抗放线菌具有环保、无残留的优点,并已在植物多种病害上成功应用,这为茄子青枯病的生物防治提供了思路。【目的】从健康茄子根际分离获得对茄子青枯菌有显著拮抗作用的放线菌菌株。【方法】采用稀释涂布法分离放线菌;采用双层琼脂法、琼脂扩散法和平板对峙法筛选拮抗菌株;对目标菌株XL-6的形态、培养特征、生理生化特征及16S rRNA基因序列进行综合分析;通过单因素试验和正交设计试验优化目标菌株培养基组分及发酵条件。【结果】筛选得到一株对青枯菌有强抑制作用的放线菌菌株XL-6,它对其他3种病原菌均具有一定的抑制作用。菌株XL-6的形态和培养特征、生理生化特征与娄彻氏链霉菌相符,而且16S rRNA基因序列分析表明该菌株与娄彻氏链霉菌亲缘关系较近。该菌株最优发酵配方和培养条件分别为:玉米粉30.0 g/L、酵母粉5.0 g/L、K_2HPO_4 2.0 g/L、MgCl_2 2.0 g/L和NaCl 1.0 g/L;初始pH 7.0、培养基装瓶量70 mL/250 mL、摇床转速180 r/min、接种量6%,在28°C条件下培养6 d。【结论】菌株XL-6经鉴定为娄彻氏链霉菌,优化其发酵条件后对青枯菌具有更强的拮抗效果。