一株西瓜枯萎病生防菌Bacillus methylotrophicus的筛选、鉴定及抑菌物质分析

【背景】西瓜专化型尖孢镰刀菌(Fusarium oxysporum f. sp. niveum, Fon)是西瓜枯萎病的致病菌,严重威胁连作西瓜的生产。【目的】筛选拮抗西瓜枯萎病菌的菌株并推测其无菌上清液(cell-free supernatant, CFS)的抑菌成分,为其在农业生产中的应用提供理论依据。【方法】利用平板对峙法筛选拮抗Fon的菌株,通过盆栽试验验证其抑制西瓜枯萎病的能力,并通过扫描电镜、共聚焦显微镜观察和抗氧化酶活性测定等方法研究Fon暴露在拮抗菌株次级代谢物下孢子形态的变化、膜的完整性和造成的氧化损伤,通过二代Illumina HiSeq联合三代PacBio Sequel测序平台进行全基因组测序、LC-MS非靶代谢组学、超高液相色谱系统和高分辨率质谱(UHPLC-ESI-MS/MS),明确拮抗菌株次级代谢物中的抑菌功能物质。【结果】从健康西瓜根际土筛选到拮抗Fon的一株优良菌株J4,盆栽试验表明其对西瓜枯萎病的防治效果为72.6%。经全基因组学鉴定为Bacillus methylotrophicus J4,该菌株可产蛋白酶、淀粉酶、葡聚糖酶和纤维素酶并有分泌铁载体的...     

拟康宁木霉T 51菌株对番茄枯萎病的生物防治及其机理研究

尖孢镰刀菌是番茄枯萎病的致病菌,该研究利用拟康宁木霉T 51菌株对尖孢镰刀菌展开对峙培养试验、防治最适温度、酸碱度筛选试验及生物防治效果试验,并测定防治试验中番茄幼苗叶片的生理生化指标和水杨酸、茉莉酸合成和信号途径关键基因表达量,探讨T 51菌株的生物防治机理。结果表明：(1)T 51菌株可显著抑制对峙培养中尖孢镰刀菌生长;随着环境温度和pH的增加,T 51菌株对尖孢镰刀菌的抑制率先升后降,并且在20 ℃、pH 7环境下效果最佳。(2)T 51菌株和尖孢镰刀菌混合孢子液处理番茄幼苗植株无明显的感病症状,植株生长状态与清水对照组无显著差异,枯萎病发病率和病情指数比单施尖孢镰刀菌孢子液处理显著大幅度降低,相对防效为87.5%。(3)与单施尖孢镰刀菌孢子液处理相比,混合孢子液处理番茄幼苗叶片的叶绿素荧光参数最大光化学效率、光化学淬灭系数和表观光合电子传递速率,以及抗氧化酶SOD、POD、CAT活性均显著增加,而非光化学淬灭系数和过氧化氢含量均显著降低。(4)与单施尖孢镰刀菌孢子液处理相比,混合孢子液处理番茄幼苗叶片的内源水杨酸(SA)显著降低,而茉莉酸(JA)含量显著升高,同时SA信号通路上的PR1和TGA2基因表达量均显著下调,JA合成基因LoxD的表达量显著上调。研究认为,拟康宁木霉T 51菌株对尖孢镰刀菌有显著的防治效果,并在20 ℃、pH 7环境下防治效果最佳;T 51菌株可能通过抑制尖孢镰刀菌侵染,增强番茄叶片抗氧化酶活性,提高叶片光合效率,以及调控水杨酸和茉莉酸合成和信号途径关键基因表达,调节内源水杨酸和茉莉酸含量,进而提高番茄植株的枯萎病抗病性。研究结果为番茄抗枯萎病提供了生防途径的理论依据。     

瓜菜枯萎病及生防根际微生物组研究进展

枯萎病是顽固性土传病害,称为瓜菜中的“癌症”,已成为制约我国瓜菜产业可持续健康发展的瓶颈问题。本文简要介绍瓜菜枯萎病危害,并从细胞壁降解酶、毒素、信号传导和致病基因等方面综述瓜菜枯萎病灾变机制,然后从根际微生物组自身与病原菌、土壤层面和植物层面等重点阐述了根际微生物组防治和抵御瓜菜枯萎病的机理,最后对枯萎病发生和抑制关键因子挖掘、核心微生物组构建及根际微生物组分子机制等进行了展望,期望生防微生物防治病害发生进入一个崭新且高效的阶段,为加快提升作物抗逆性机理研究提供一定思路。     

绿色木霉TV41对尖孢镰刀菌FW0在西瓜植株空间分布和枯萎病防控效果的影响

【目的】为进一步探究盆栽试验条件下绿色木霉TV41 (Trichoderma viride TV41)对尖孢镰刀菌FW0 (Fusarium oxysporum FW0)在西瓜植株空间分布的影响以及对西瓜枯萎病的防控效果。【方法】通过定期检测不同处理西瓜根际/根表尖孢镰刀菌的数量、西瓜植株根内/茎内尖孢镰刀菌的数量以及植株侧根被侵染比例和尖孢镰刀菌在植株内的侵染进程,进行多次盆栽试验并统计发病率。【结果】当绿色木霉和尖孢镰刀菌接种量均为5×105孢子/g基质时,绿色木霉TV41在西瓜根际/根表的定殖数量明显高于尖孢镰刀菌FW0的数量,接种了绿色木霉TV41的处理,根际/根表尖孢镰刀菌的数量(103/g基质)显著低于仅接种FW0的对照(104/g基质);绿色木霉TV41不仅能够有效减缓尖孢镰刀菌在西瓜植株内的侵染进程,而且能够有效降低西瓜植株根内、茎内尖孢镰刀菌的数量。与对照(只接种FW0)相比,接种绿色木霉后西瓜枯萎病的发病率从66%降低到27%。【结论】绿色木霉TV41能够通过影响尖孢镰刀菌FW0在西瓜植株的空间分布,从而有效防控西瓜枯萎病的发生,防控效果达到60%。     

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...     

石灰碳铵熏蒸与施用生物有机肥对连作黄瓜和西瓜枯萎病及生物量的影响

采用稀释涂布平板计数法,研究了石灰碳铵及碳铵熏蒸对黄瓜和西瓜连作土壤尖孢镰刀菌数量的影响,以及熏蒸后施用生物有机肥对黄瓜和西瓜枯萎病的防控效果及植株生长的影响.结果表明:与对照相比,石灰碳铵及碳铵熏蒸后,连作土壤中黄瓜尖孢镰刀菌的数量分别下降95.4%及71.4%,西瓜尖孢镰刀菌的数量分别下降87.2%及64.2%;多因素方差分析表明,熏蒸、施用有机肥及作物种类均对土壤中尖孢菌数量、枯萎病发病率、防控率及生物量有显著影响;与未熏蒸施用普通有机肥对照相比,石灰碳铵熏蒸后施用生物有机肥能显著减少后茬黄瓜或西瓜土壤中尖孢镰刀菌的数量并显著降低枯萎病发病率,防控率高达91.9%及92.5%,同时显著增加了植株的株高、茎粗、SPAD值及干质量.表明石灰碳铵熏蒸及施用生物有机肥能够降低土壤中尖孢镰刀菌数量,有效防控黄瓜和西瓜枯萎病的发生并促进其植株生长.     

低温茬口空闲期土壤强还原消毒对西瓜枯萎病的影响

为探明低温茬口空闲期土壤强还原消毒(RSD)对西瓜枯萎病的防控效果,设置对照(CK)、淹水对照(FCK)、淹水添加2％ (w/w)苜蓿粉(AL)、0.25％ (w/w)乙酸(AC)以及AL+AC的RSD处理进行西瓜盆栽试验,采用定量PCR和Illumina Miseq等测定真菌、尖孢镰刀菌数量及真菌群落组成,并统计发病...     

两株香蕉枯萎病拮抗菌在香蕉体内的定殖

本研究将2株香蕉枯萎病拮抗细菌0202和1112分别涂布于含有不同浓度的利福平培养基上,筛选出抗药性标记菌株0202-r和1112-r.将香蕉枯萎病菌Foc4接种于香蕉植株根部,3 d后再将抗药性标记菌株接种在同一部位或位点,于不同时间测定拮抗细菌在根际、根内、球茎和假茎的定殖情况.研究结果显示,将拮抗细菌接种香蕉根部1～3 d后,在植株根际土壤内存在大量拮抗细菌,而根内、球茎和假茎内存在少量拮抗细菌;接种7～14 d后,在植株根内、球茎和假茎内定殖的拮抗细菌增多,并达到最大量;在接种14～21 d后,在植株根内、球茎和假茎内定殖的拮抗细菌量急剧下降;在接种28～35 d后,在植株根内、球茎和假茎内定殖的拮抗细菌量稍有回升.     

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。     

影响引入微生物根部定殖的因素

从外界引入的各类有益微生物如生防菌（ＢＣＡ）和根际促生菌或增产菌（ＰＧＰＲ,ＹＩＢ）到种子表面随其生根发芽而蔓延或直接到根表沿根分布定殖。外来微生物在根际定殖的过程为与根尖接触,沿根分布,最后在根际建立自己的种群。定殖的位点以ＰＧＰＲ为例,是表皮细胞间隙,或侧根、根毛基部。外来微生物在根际定殖动态变化的原因,由于根际生物的和非生物的因素引起的。生物因子除去外来微生物本身的生理特性,还有根际土著微生     

绿豆尖镰孢枯萎病抗性鉴定方法

绿豆是我国的主要食用豆类之一。由尖镰孢引起的绿豆枯萎病是一种严重的土传病害,病原菌从根部侵入,引起植株矮化,叶片黄化、枯萎,根茎部维管束变褐,严重时导致植株死亡。防治枯萎病最经济、有效的方法是培育利用抗病品种。本研究在控制条件下以具有不同抗性表型绿豆品种为材料,分别对接种方法、植株生育期、接种体浓度、接种体处理时间及接种后植株培养温度等影响绿豆抗性表型的因素进行比较研究,以期建立一个快速、准确和高效的绿豆枯萎病抗性鉴定方法,为抗病资源的筛选和抗病育种提供技术支持。结果表明,绿豆枯萎病苗期抗性鉴定最适宜的接种方法为剪根浸根法,最适宜接种体浓度为105~106孢子/m L,接种最佳植株生育期为2叶期,最短有效接种体浸根时间为2 min,最适宜发病温度为25℃,接种后14 d调查病情。     

Isolation and characterization of endophytic streptomycete antagonists of Fusarium wilt pathogen from surface-sterilized banana roots

A total of 131 endophytic actinomycete strains were successfully isolated from surface-sterilized banana roots. These isolates belonged to Streptomyces (n=99), Streptoverticillium (n=28), and Streptosporangium (n=2) spp. The remaining 2 isolates were not identified. About 18.3% of the isolates inhibited the growth of pathogenic Fusarium oxysporum f. sp. cubense on banana tissue extract medium. The most frequently isolated Streptomyces sp. strain S96 was similar to Streptomyces griseorubiginosus. About 37.5% of the S. griseorubiginosus strains were antagonistic to F. oxysporum f. sp. cubense. The antagonism of strain S96 was lost when FeCl(3) was introduced into the inhibition zone. In vivo biocontrol assays showed that the disease severity index (DSI) was significantly (P=0.05) reduced and mean fresh weight increased (P=0.001) in plantlets treated with strain S96 compared to those grown in the absence of the biocontrol strain. These findings indicate the potential of developing siderophore-producing Streptomyces endophytes for the biological control of fusarium wilt disease of banana.     

First record of Fusarium vascular wilt on grapevine in Egypt

During the summer season of 2003 and 2004, wilt syndromes of grapevine leaves (Cv. crimson) and vascular discolouration of roots have been observed in 2-year-old grapevine plants in the field at two sides in Gharbeia Governorate, Egypt. First, symptoms of wilt began on bottom leaves borderline as chlorosis and then these turned to necrotic spots and the leaves died. Wilt symptoms were spread to apical associated with vascular discolouration of roots and stem basal. Routine isolations of discoloured root tissue from diseased plant yielded eight isolates of Fusarium oxysporum Schlechtend only where no other fungi were developed. Microscopic examination revealed the presence of three shapes of microconidia, first is avoid shape non-septate measuring 2.5–3.0 μm × 6–10 μm, second is cylindrical with one septa measuring 2.6 μm × 17.0 μm and third shape also cylindrical with two septate measuring 3.0 μm × 20.0 μm. Macroconidia was rarely with three septate measuring 3.5– 4.0 μm × 35.0–38.0 μm, and chlamydospores were found singly or in pairs or chains. F. oxysporum isolates attacked grapevine plants (Cv. crimson) causing vascular wilt (66.7%) and root-rot syndrome (33.3%). In vitro isolates of F. oxysporum causing wilt of grapevine (Cv. crimson) varied for producing lytic enzymes, i.e. polygalacturonase (PG) and cellulase. The reactions of several grapevines (Cvs.) with a virulent isolate of F. oxysporum indicated the presence of two different symptoms, i.e. vascular wilt only on grapevine plants (Cv. crimson) and root-rot on the other grapevine (Cvs.), i.e. superior, Thompson, King robi and flame seedless. All F. oxysporum isolates caused vascular wilt of grapevine Cv. crimson, successfully reisolated from symptomatic vascular infected tissue and complete identification on the basis of colony, conidia morphology and host range at formae speciales level as F. oxysporum f. sp. herbemontis (Tochetto) Gordan. This is the first report of Fusarium wilt on grapevine in Egypt.     

西瓜枯萎病病株空间分布格局及其抽样技术

研究了西瓜枯萎病病株田间空间分格局及其抽样技术 ,通过频次分布和聚集度指数的测定以及 m* - m回归和 Taylor幂法则分析 ,结果表明 :西瓜枯萎病病株田间分布趋向于聚集分布。m* - m回归分析表明病株空间分布的基本成分是个体群 ,病株个体间相互吸引 ,病株在大田中存在明显的发病中心 ,个体群在田间呈均匀分布格局 ,即分布的基本成分个体群之间趋于均匀分布 ,而个体群内的个体与核心分布相吻合。Taylor幂法则分析表明 ,西瓜枯萎病病株个体的空间格局随着病株密度的提高越趋均匀分布。用 Iwao最适理论抽样模型 n=t2a/D2 [(α 1) /X β- 1],计算出不同病情指数情况下所需的最适抽样数 ,随着病情指数的增加 ,所需抽样数递减。序贯抽样模型为 T0 ( N) =0 .9871N± 1.2 347N ,调查株数 N株时 ,若累计病情指数超过上界可定为防治对象田 ,若累计病情指数未达到下界时 ,可定为不防治田 ,若累计病情指数在上下界之间 ,则应继续调查 ,直到最大样本数 m0 =0 .9871时 ,也即病情指数 2 5 % ,所需抽样数 130株止。     

蔓割病不同抗性甘薯品种的茎部细胞结构观察

蔓割病是我国南方薯区甘薯主要病害之一,本研究采用直接观察、显微和超微结构观测等方法,对高抗、中感和高感蔓割病的3个甘薯品种(高抗品种:金山57,中感品种:热薯1号,高感品种:新种花)接种蔓割病菌28 d植株充分发病后其茎基部细胞的侵染结构进行了观察。结果表明,在用清水作对照处理时,3个品种茎部组织的细胞形态和结构正常且完整,细胞代谢强。高抗品种金山57无论是接种组还是对照组,均未发现病原菌丝的存在,其茎下部、中部和上部细胞结构相对完整。中感品种热薯1号,蔓割病菌从其茎基部侵入后导致茎基部细胞破损坏死,而中部寄主-病原互作较为活跃和典型,造成养分运输受阻,茎基部接种后病原菌丝会沿着寄主茎部的维管束和其他组织一直向寄主的茎部末端蔓延,遭受侵染后的部位其细胞反应与高感品种新种花类似。高感品种新种花遭受蔓割病原菌侵染后,病原菌菌丝从茎基部新鲜剪口侵入后进入表皮细胞、皮层、维管束并在甘薯茎基部蔓延至中部、上部,直至寄主整株枯死,甘薯茎的木质部导管出现侵填体,质壁分离;与此同时,细胞壁的沉积物及乳突在病原菌的入侵处形成,各种无定型物质或纤丝构成的织网迅速包围入侵菌丝。     

Stability of transformed antagonistic Fusarium oxysporum strains in vitro and in soil microcosms

The stability of a genetically modified strain of Fusarium oxysporum used as antagonist against phytopathogenic formae speciales of F. oxysporum was evaluated both in vitro and in microcosm assays. The Escherichia coli hygromycin B phosphotransferase gene (hph), conferring hygromycin B resistance, was introduced by genetic transformation into a recipient strain marked by benomyl resistance and a dark red pigmentation. Hybridization with the complete plasmid suggested that the integration had generally occurred in a multiple-tandem array at multiple sites. Among nine independent transformants tested, only three of them were mitotically stable after four rounds of vegetative growth with no selective pressure, while six showed various changes in the integration pattern. One transformant had lost the ability to grow in the presence of hygromycin B. In soil microcosms all the transformants maintained the hygromycin B resistant phenotype, but six of them showed rearrangement of transforming DNA. Only one strain (coded T26.40) underwent no obvious rearrangement both after in vitro growth and after recovery from the soil microcosm. The nine transformants were used in three biological control experiments against Fusarium wilt of carnation in comparison to two untransformed reference strains and to the recipient mutant. A high degree of variability in the biocontrol activity was observed throughout the experiments and only transformant T26.40 consistently controlled the incidence of disease. The results are discussed in relation to risk assessment of the release of transgenic antagonistic fungi.