玉米根际细菌中PGPR的筛选及初步鉴定

通过离体拮抗试验和检测菌株产生铁载体的情况从玉米根际细菌中筛选出 8株拮抗菌和2 6株铁载体产生菌 ,其中 DC0 1、DC0 9、DC2 2、PY313既具有真菌抗性又能产生铁载体 ,并对部分菌株进行了初步鉴定     

拮抗菌DG1的鉴定及其对大白菜菌核病菌的抑制作用研究

为减少化学农药的使用,丰富生物防治菌株资源库,在分离大白菜菌核病菌的过程中,筛选到一株对其具有抑制作用的拮抗菌,命名为DG1.本研究通过形态观察、生理生化特征检测及16S rDNA序列分析,对拮抗菌DG1进行种属鉴定;通过测量菌落直径,研究DG1发酵液及挥发性物质对大白菜菌核病菌的抑制作用;通过平板对峙法测定拮抗菌DG...     

甘蔗黑穗病菌拮抗细菌的筛选及鉴定

从甘蔗根际土壤及甘蔗不同组织内分离到的928个细菌菌株中，对甘蔗黑穗病菌有拮抗作用的细菌菌株有301个．占32．4％．其中拮抗能力强(拮抗带大于10mm)的菌株有18个，占1．9％。经在KBA培养基上培养．发现具有拮抗作用的细菌主要是荧光菌和非荧光菌中的白色菌群。在18个拮抗性强的菌株中，13个菌株来自甘蔗的茎、芽(生长点)，占72％；5个菌株来自根际土壤，占28％；12个菌株为革兰氏阳性细菌中的芽孢杆菌属；6个菌株为革兰氏阴性细菌，其中4个菌株分别为假单胞杆菌属、不动杆菌属、伯克氏菌属及沙雷氏菌属，其余2个菌株有待进一步鉴定。     

一株百合内生细菌 Burkholderia sp. FJb-2的分离鉴定及其体外抑菌促生效应

从北京市房山区百合资源圃采集红芯百合(Fangio)植株样品,通过组织块分离法分离内生细菌;进一步以百合灰霉病菌—灰葡萄孢菌( Botrytis cinerea)ACCC36423、百合叶尖干枯病菌—葡萄座腔菌( Botryosphaeria dothidea)ACCC37263和百合枯萎病菌—尖孢镰刀菌( Fusarium oxysporum)F01139等几种主要百合病原真菌为指示菌,采用平板对峙法筛选具有拮抗活性的拮抗菌株;并对拮抗菌株进行产 1-氨基环丙烷 -1-羧酸(ACC)脱氨酶活性、产生长素(IAA)、溶磷、产铁载体等多种植物促生特性测定;最后通过形态观察及 16S rRNA基因序列分析,初步鉴定其种属。结果从百合茎部分离获得一株内生细菌菌株 FJb-2;该菌株对灰葡萄孢菌、葡萄座腔菌和尖孢镰刀菌均有较高拮抗活性,对病原真菌生长的抑制率范围为 60%～ 81%;该菌株产 ACC脱氨酶活性为     

苹果树腐烂病拮抗细菌鉴定及其抑菌作用效果测定

为了开发一种高效低毒的苹果树腐烂病生防制剂,通过对峙培养法、形态学及分子生物学的方法进行了苹果树腐烂病菌拮抗菌的分离筛选及鉴定,采用离体枝条法测定了拮抗菌对苹果树腐烂病的防效,并采用显微观察和液体培养法分别研究了拮抗菌对苹果树腐烂病菌的抑菌机理和无菌滤液对苹果树腐烂病菌生长的影响。分离筛选结果表明,从甘肃省各苹果产区果园土壤和苹果树枝条上分离得到23株细菌,2株对苹果树腐烂病菌具有较好拮抗作用,分别为LZ-1201和TS-1203,其对苹果树腐烂病菌菌丝生长抑制率分别为79.00%和85.00%。鉴定结果表明,菌株LZ-1201和TS-1203分别为枯草芽孢杆菌(Bacillus subtilis)和解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。离体枝条防效测定表明,拮抗菌无菌滤液对苹果树腐烂病的防效随着稀释倍数增大而降低,原液防效最高,分别为74.43%和77.07%。抑菌作用机理结果表明,两株拮抗菌均可导致苹果树腐烂病菌丝膨大畸形、原生质浓缩、释放及溶解。拮抗菌无菌滤液对腐烂病菌生长的影响测定结果表明,无菌滤液对腐烂病菌分生孢子萌发和菌丝生长量均有显著抑制作用(P0.05),其无菌滤液稀释40倍时对腐烂病菌分生孢子萌发和菌丝生长量的抑制率均高于60%,表明该拮抗菌具有很好的生防潜力。     

小麦赤霉病菌拮抗内生菌的抑菌活性及鉴定

为开发看麦娘内生菌生物防治资源,通过离体拮抗试验、生防试验和分子鉴定等方法,研究了野生日本看麦娘内生真菌KMN-1和KMN-11对小麦赤霉病菌的抑菌活性以及菌株的分类地位。离体生物测定结果表明,2株内生菌对小麦赤霉病菌的菌丝生长有明显拮抗作用,KMN-1和KMN-11菌株发酵液抑制小麦赤霉病菌菌丝生长的EC50值分别为6 572.97 mg·L-1、5 218.34 mg·L-1。进一步采用离体麦穗和田间小区系统研究KMN-1和KMN-11发酵产物对小麦赤霉病的防治效果,结果表明KMN-1和KMN-11对小麦赤霉病均表现一定的防治作用。离体麦穗上,KMN-1发酵产物对小麦赤霉病的保护和治疗作用防治效果分别为78.13%、76.52%;小区生防试验中,2株内生菌代谢产物均可降低小麦赤霉病的发病程度。经形态特征和分子学鉴定,初步确定KMN-1菌株为细极链格孢(Alternaria tenuissima),KMN-11菌株是变红镰孢(Fusarium incarnatum)。本研究表明从镰孢属和链格孢属内生菌中寻找生防菌株的前景广阔,为进一步研究开发有潜力的农用抗生素奠定了基础。     

大豆灰霉病和黑斑病拮抗菌株的筛选和鉴定

生物防治病虫害是当前农业可持续发展的重要方向之一。大豆灰霉病(Botrytis cinerea)和黑斑病(Alternaria tenuissima)是大豆上的主要病害,为了控制灰霉病和黑斑病对大豆的危害,通过生物防治的方法,采用土壤稀释法从江西省东乡野生稻根际土壤中分离获得64株拮抗菌,经反复筛选及平板对峙法测定菌株A2、A6、A10、F7和G6对大豆灰霉病菌和黑斑病菌有稳定拮抗作用。通过菌株发酵滤液和化学农药抑菌效果比较,菌株发酵液的抑菌率远超化学农药,其中拮抗菌的发酵滤液对灰霉病最佳抑制率达91. 7%,对黑斑病最佳抑制率在60. 9%～87. 0%之间;而灰霉病特效素和苯醚甲环唑对灰霉病和黑斑病的最佳抑制率仅为68. 0%和47. 3%。根据菌株的生理生化特征及16S r DNA分子鉴定,5株菌分别为米修链霉菌(Streptomyces misionensis)、绛红产色小单孢菌(Micromonospora purpureochromogenes)、圈卷产色链霉菌(Streptomyces ansochromogenes)、链霉菌(Streptomyces sp.)和类芽孢杆菌(Panenibacillus sp.)。本研究为大豆灰霉病和黑斑病的生防菌开发和利用提供了一定的支撑。     

抗马铃薯晚疫病菌的粘细菌菌株X6-Ⅱ-1的分离鉴定、拮抗活性及发酵条件的优化

马铃薯晚疫病(potato late bright)是马铃薯(Solanum tuberosum)最为严重的病害,其病原菌为致病疫霉(Phytophthora infestans)。为了从土壤样品中筛选出对马铃薯晚疫病具有拮抗作用的粘细菌,本实验利用大肠杆菌(Escherichia coli)划线诱导法从土样中分离菌株,通过平板对峙法筛选具有抗马铃薯晚疫病菌活性的粘细菌,结合形态观察、生理生化特征和16S r RNA基因序列分析确定菌株的分类地位。采用称重法测定菌株生长曲线,滤纸片法确定活性物质的分布,并通过单因素分析与正交优化相结合的方法对菌株的发酵参数进行了研究。共从土壤样品中共分离得到5株粘细菌,其中有3株对致病疫霉表现出拮抗活性,菌株X6-Ⅱ-1的拮抗活性最强,致病疫霉菌丝生长边缘与菌株X6-Ⅱ-1菌饼最短距离可达到5 mm,经鉴定为叶柄粘球菌(Myxococcus stipitatus)。该菌株可以杀死并溶解大肠杆菌,且可以抑制枯草芽胞杆菌(Bacillus subtilis)的生长。拮抗致病疫霉的活性物质主要存在于细胞外,产活性物质的最适发酵条件为:接种量10%,摇床转速180 r/min,培养温度为30℃,发酵时间为7 d。粘细菌菌株X6-Ⅱ-1能够产生抗马铃薯晚疫病菌的活性物质,具有开发成抗马铃薯晚疫病生物农药的潜在价值。本研究为活性物质的分离鉴定以及抗马铃薯晚疫病农药的研发提供基础数据。     

防病促生枯萎病拮抗菌“98-Ⅰ”对4种枯萎病防治效果研究

经 8年系统试验研究从 330个细菌菌株中筛选出 1株防病促生枯萎病拮抗菌“98 Ⅰ” ,经鉴定属蜡质芽孢杆菌。该菌对黄瓜枯萎病、西瓜枯萎病、青椒枯萎病和番茄枯萎病 4种土传病害均有显著防治效果 ,其平皿孢子萌发抑制率分别为 79 2 %、75 1%、72 3%和 95 7% ,且该菌对多种蔬菜有促生和促进种子发芽功效。     

烟草根系分泌物酚酸类物质的鉴定及其对根际微生物的影响

【目的】烟草连作已导致土传病害发生、 烟株生长受抑制、 产量下降和品质恶化等问题。烟株对自身及土壤微生物产生的化感作用,是烟草产生连作障碍的一个重要原因,其中化感物质中的根系分泌物是烟株与土壤微生物间相互作用的重要物质,探索烟草根系分泌物对根际微生物生长的影响是生物防控烟草青枯病的理论依据。【方法】本文利用超高效液相色谱串联四级杆飞行时间质谱(UPLC-Q-TOF/MS)技术,分离、 鉴定烟草根系分泌物中主要酚酸类物质的种类和含量; 通过添加外源酚酸类物质,研究在液体培养基中烟草根系分泌物中的主要酚酸类物质对病原菌及拮抗菌的影响,并在土壤中添加鉴定出的主要酚酸类物质; 通过土壤培育试验,研究其对土壤微生物多样性和数量变化,特别是对烟草青枯病菌及其拮抗菌生长的影响。【结果】1)烟草根系分泌物粗提物对病原菌(茄科劳尔氏菌)生长的促进率为16.8％,对拮抗菌(短短芽孢杆菌)的生长抑制率达到29.4％; 2)UPLC-Q-TOF/MS检测根系分泌物中主要酚酸类物质为苯甲酸和3-苯丙酸,含量分别为0.25 μg/g干根重和1.15 μg/g干根重; 3)液体培养外源添加低浓度的苯甲酸(≤ 2 μg/L)和3-苯丙酸(≤ 3 μg/L)促进病原菌和拮抗菌的生长; 4 μg/L的苯甲酸对病原菌生长抑制作用不显著,对拮抗菌生长的抑制率达到90.2％,6 μg/L的 3-苯丙酸对病原菌的生长具有促进作用,对拮抗菌的生长抑制率达到81.1％,外源高浓度苯甲酸(≥4 μg/L)和3-苯丙酸(≥ 7 μg/L)抑制病原菌与拮抗菌的成长; 4)土壤中添加3 μg/kg土的苯甲酸时,土壤中病原菌的数量增加12.3％,而拮抗菌的数量减少21.0％,土壤细菌、 放线菌和真菌的数量分别降低37.5％,41.9％和55.6％; 3-苯丙酸浓度达到8 μg/kg土时,拮抗菌生长量减少14.5％,对病原菌没有显著影响,土壤细菌、 放线菌和真菌的数量分别降低69.9％,57.2％和80.7％; 5)土壤添加4 μg/kg的苯甲酸和7 μg/kg的3-苯丙酸后,土壤微生物Shannon指数、 Simpson指数、 McIntosh指数显著下降,分别仅为对照的57.7％、 94.1％、 88.1％和97.6％ 73.3％、 80.0％。【结论】烟草根系分泌物粗提物促进病原菌生长抑制拮抗菌生长,根系分泌物中酚酸类物质主要为苯甲酸和3-苯丙酸,液体培养中4 μg/L的苯甲酸或6 μg/L的 3-苯丙酸浓度是对病原菌生长抑制不明显但显著抑制拮抗菌生长的分界点,土壤外源添加3 μg/kg的苯甲酸或8 μg/kg的3-苯丙酸时,是土壤增加病原菌减少拮抗菌数量的分界点,同时土壤微生物功能多样性显著下降,病原菌对根系分泌物中苯甲酸和3-苯丙酸的利用优于拮抗菌,这也是烟草长期连作引起青枯病暴发流行的机理之一。     

棉花黄萎病生防菌脂肽类抑菌物质分离和鉴定

棉花黄萎病(Cotton Verticillium Wilt)是一种由大丽轮枝菌(Verticillium dahliae Kleb)引起的严重危害棉花生产的土传维管束病害[1]。因大丽轮枝菌菌丝体产生的大量微菌核在土壤中存活时间长以及病原菌变异快等特点,造成培育抗病品种、使用化学药剂和利用新的栽培技术等方法防病效果均不理想。近年来,以生物防治为主的综合防治措施日益受到人们重视,并取得了一定效果[2-4];该措施     

Control of cotton <Emphasis Type="Italic">Verticillium</Emphasis> wilt and fungal diversity of rhizosphere soils by bio-organic fertilizer

Cotton Verticillium wilt is a destructive soil-borne disease affecting cotton production. In this study, application of bio-organic fertilizer (BIO) at the beginning of nursery growth and/or at the beginning of transplanting was evaluated for its ability to control Verticillium dahliae Kleb. The most efficient control of cotton Verticillium wilt was achieved when the nursery application of BIO was combined with a second application in transplanted soil, resulting in a wilt disease incidence of only 4.4%, compared with 90.0% in the control. Denaturing gradient gel electrophoresis patterns showed that the consecutive applications of BIO at nursery and transplanting stage resulted in the presence of a unique group of fungi not found in any other treatments. Humicola sp., Metarhizium anisopliae, and Chaetomium sp., which were considered to be beneficial fungi, were found in the BIO treatment, whereas some harmful fungi, such as Alternaria alternate, Coniochaeta velutina, and Chaetothyriales sp. were detected in the control. After the consecutive applications of BIO at nursery and transplanting stage, the V. dahliae population in the rhizosphere soil in the budding period, flowering and boll-forming stage, boll-opening stage, and at harvest time were 8.5 × 10², 3.1 × 10², 4.6 × 10², and 1.7 × 10² colony-forming units per gram of soil (cfu g⁻¹), respectively, which were significantly lower than in the control (6.1 × 10³, 3.4 × 10³, 5.2 × 10³, and 7.0 × 10³ cfu g⁻¹, respectively). These results indicate that the suggested application mode of BIO could effectively control cotton Verticillium wilt by significantly changing the fungal community structure and reducing the V. dahliae population in the rhizosphere soil.     

A rapid diagnostic assay for detection and quantification of the causal agent of strawberry wilt from field samples

Verticillium dahliae Kleb, the cause of Verticillium wilt disease, is a destructive pathogen that leads to severe yield losses in strawberry fields and thus considerable economic damages. Although rapid identification and detection methods are becoming available more, pathogen quantification remains one of the main challenges in the disease management. In this study, a real-time polymerase chain reaction (rtPCR) assay was developed to quantitatively assess V. dahliae abundance directly from affected roots and soil collected from different areas in Estonia. A specific primer pair based on the ribosomal DNA (rDNA) internally transcribed spacer was designed for SYBR Green-based assay. Strawberry plant and soil samples were randomly collected from different areas in Estonia and analyzed for V. dahliae by soil plating technique and rtPCR assay. The assay was specific for V. dahliae so that the minimum detection limit was 0.93?pg?µl^?1 of pathogen DNA and the lowest amount of V. dahliae detected in soil was 10.48?pg?µl^?1 of target DNA corresponding to one microsclerotia per gram of soil. This technique allowed rapid detection and quantification of the pathogen DNA at the picogram level in soils and even in symptomless plants, facilitating the screening of the pathogen in diverse areas. This is the first study about the rtPCR technique being used successfully to assess populations of V. dahliae with high specificity and sensitivity in Estonia strawberry fields. Results of this research can be useful for growers and agricultural organizations to improve available disease management strategies against Verticillium wilt.     

土传棉花黄萎病拮抗菌的筛选及其生物效应

实验采用平板对峙法,从黄萎病发生严重的棉田中的健康植株根际土壤中分离筛选到11株对棉花黄萎病致病菌Verticillium dahliaeKleb(Vd)具有拮抗效果的菌株,抑菌率在51.8%和87.4%之间,经培养滤液抑菌率试验复筛,选择抑菌效果较好的3株菌株进行盆栽试验。结果表明:在施用拮抗菌摇床培养液(VS)、有机肥(VF)和两者结合(VFS)的3个处理中,VFS效果最显著,防病率达57%,植株生理性状显著改善,根际可培养微生物数量发生显著变化,细菌数量增加7.3~13.4倍、放线菌数量增加3.2~5.9倍,病原菌微菌核数量下降34%。结合生理生化和16S rDNA技术鉴定,初步确定供试的2株菌为死谷芽孢杆菌(Ba-cillus vallismortis),1株菌为枯草芽孢杆菌(Bacillus subtilis)。研究表明拮抗菌与有机肥共同施用不仅可以起到防病的作用,而且可以使棉花连作土壤微生物区系向健康、合理的方向发展。本文首次报道了死谷芽孢杆菌对棉花黄萎病有抑制作用。     

棉花内生解淀粉芽孢杆菌489-2-2对棉花黄萎病的防效研究

为更好地防治棉花黄萎病,在冀棉11根系中分离得到一株对大丽轮枝菌抗性明显的内生细菌,经分子鉴定为解淀粉芽孢杆菌489-2-2。本试验以489-2-2为材料,研究该菌株对棉花黄萎病的防效和机理。结果表明,解淀粉芽孢杆菌489-2-2能够抑制黄萎病菌Vd080的生长,可导致Vd080菌丝形态异常。用该菌株发酵液浸泡棉花种子,对棉花黄萎病的防效为54.99%,灌根法的防效为60.31%。解淀粉芽孢杆菌489-2-2能使植株产生防御酶,诱导的免疫反应较强,且该菌株能定殖到棉花幼苗根系内部。本研究结果为解淀粉芽孢杆菌489-2-2的利用提供了理论依据。     

Evaluation of Streptomyces spp. for the biological control of corky root of tomato and Verticillium wilt of eggplant

The effects of Streptomyces spp. isolates in the biological control of corky root of tomato and Verticillium wilt of eggplant was determined in in vitro, greenhouse and field trials. Twenty-six Streptomyces spp. isolates were obtained from the rhizospheres of different vegetable crops in southern Italy. In in vitro dual culture tests, mycelial radial growth of Pyrenochaeta lycopersici and Verticillium dahliae was reduced up to 18.6% and 30.1%, respectively. Radial growth of seven other fungal pathogens was variably reduced as well. The isolates StB-3, StB-6, StB-11 and StB-12 showed a good antagonistic effect against both P. lycopersici and V. dahliae, while the rest of isolates eventually showed antagonism against only one pathogen.In pot-experiments in the greenhouse three of the four above-mentioned Streptomyces spp. isolates significantly reduced corky root up to 64.9% (StB-11), and all four isolates reduced foliar symptoms of Verticillium wilt (AUDPC) up to 48.3%, but none of them reduced the severity of vascular browning. In naturally infested field trials, StB-11 significantly reduced corky root severity in tomato by 48.2%, StB-12 by 35% and StB-6 by 32.6%, but none of the isolates were effective in controlling Verticillium wilt of eggplant. The effectiveness of the streptomycete AtB-42, successfully used in previous researches, was here confirmed as it reduced corky root of tomato in the field by 33.6%. In conclusion, our research demonstrates that under field conditions corky root of tomato, but not Verticillium wilt of eggplant, can be effectively controlled by the Streptomyces spp. isolates used in this study.     

Ability of Talaromyces flavus to occupy the rhizosphere and its interaction with Verticillium dahliae

The ability of Talaromyces flavus to occupy the rhizosphere of potato, cotton and eggplant and to reduce germinability of Verticillium dahliae microsclerotia (ms) was investigated. After 2 weeks the germination of microsclerotia of V. dahliae exposed to T. flavus-infested soil was 32–41% less than those exposed to soil that was not infested, depending on the soil type. With a new medium selective for T. flavus, TF medium, it was possible to monitor the populations of T. flavus in rhizosphere and non-rhizosphere soils. Significantly higher populations of T. flavus were found in rhizosphere soils than in non-rhizosphere soils of each of the test plants when T. flavus was applied as an ascospore drench to soil planted to potato, cotton or eggplant. The reduction of viable V. dahliae microsclerotia and the increase in T. flavus populations in rhizosphere soils may explain how T. flavus reduces Verticillium wilt of eggplant; further, it suggests the feasibility of testing the antagonist against V. dahliae on potato and cotton.     

拮抗菌和生物有机肥防治棉花黄萎病及其对土壤酶活性的影响

棉花黄萎病是世界上毁灭性病害之一,它由土传真菌病原菌大丽轮枝菌引起并可以通过特制的生物有机肥进行防治。将生物有机肥、有机肥和三种拮抗细菌S37、S44、S228分别施入土壤,研究其对棉花黄萎病的防治效果及对几种主要土壤酶活性的影响。试验结果显示:防病效果达到5.9%~38.3%,由高到低依次为S37>生物有机肥>S44>S228>灭菌的生物有机肥。两种肥料处理显著提高了土壤蔗糖酶、多酚氧化酶、碱性磷酸酶、蛋白酶和脲酶的活性。3种拮抗菌处理提高了多酚氧化酶、碱性磷酸酶和蛋白酶的活性。除过氧化氢酶之外,其他酶的活性两两之间均呈现极显著正相关,但是病情指数与各种酶活性相关性均不显著。     

Green manure effects on soil quality in relation to suppression of Verticillium wilt of potatoes

Verticillium dahliae is a major, persistent pathogen in soil but conventional control is becoming more difficult because of increasing costs and environmental impacts of pesticides. Organic amendments can naturally suppress plant diseases, but to be reliable, mechanisms and suppressive soil indicators need to be understood. Consequently, a 3 × 3 factorial experiment was conducted in two separate fields over 2 years with three green manure types (Austrian winter pea, Pisum sativum L.; broccoli Brassica oleracea L.; or Sudan grass, Sorghum vulgare), incorporated at three rates (6, 12, or 24 Mg ha⁻¹ dry biomass). The relationship between soil chemical and microbiological properties and suppression of Verticillium wilt of potato was investigated using correlation and stepwise multiple-linear-regression (MLR) analysis. V. dahliae inoculum density (ID) were positively correlated with relative area under the senescence progress curve (RAUSPC) in both 2002 and 2003. In 2002, in addition to ID, low soil pH, low Ca, high K, high Mg, high total soil C, and low arylsulfatase activity were associated with low RAUSPC. Soil pH, Ca, K, and Mg were not impacted by green manure treatments, but rather indicated a pre-existing soil gradient at the 2002 site. In 2003, in addition to ID, high values of NO₃ ⁻−N, total C, fluorescein diacetate hydrolysis (FDA), microbial respiration, and microbial biomass C were associated with low RAUSPC. These six factors were affected by green manure treatments. The best MLR model included terms for ID, FDA, and soil pH, and accounted for 70% of the variability in RAUSPC.