马铃薯黑痣病生防菌的筛选和鉴定及其生长条件的研究

从甘肃省景泰县条山农场不同连作年限的马铃薯地块中取根际土样品,分离筛选马铃薯黑痣病病原菌-立枯丝核菌(Rhizoctonia solani)的拮抗菌。采用土壤平板稀释、对峙法筛选拮抗菌;结合形态特征、生理生化及16Sr DNA分子鉴定;采用单因素试验设计对G1菌株的生长条件进行初步试验。通过初筛和复筛,从108株细菌中获得1株对立枯丝核菌拮抗效果稳定、拮抗能力较强的细菌G1。采用平板对峙法测定其拮抗作用,抑菌带宽度达6mm,抑菌圈直径2.5 cm,抑菌率为66.7%。结合形态特征、生理生化及16Sr DNA分子鉴定,G1为类芽孢杆菌(Paenibacillus sp.);采用单因素试验设计对G1菌株的生长条件进行初步试验。结果表明,菌株G1在4℃~45℃,p H 5~10,盐浓度0~50 g·L-1都能生长,碳源、氮源利用广泛,最适条件为:温度37℃,p H 7,蔗糖为碳源,蛋白胨为氮源,盐浓度为10 g·L-1。拮抗菌G1被鉴定为类芽孢杆菌,该菌株抑菌带较宽、抑菌效果稳定,生长条件与马铃薯生长的大田环境基本相符,具有作为一株优良生防菌的必要条件。     

枯草芽孢杆菌(Bacilllus subtilis)S9对植物病原真菌的溶菌作用

本研究共分离了976株细菌分离物,发现来自甘蔗根围的1株枯草芽孢杆菌(Bacillus subtilis)S9对立枯丝核菌(Rhizoctonia solani)、终极腐霉(Pythium ultimum)和西瓜枯萎病菌(Fusarium oxysporum f.sp.niveum)在PDA平板的对峙培养过程中不形成抑菌圈,但4 d后可使上述植物病原真菌的菌丝溶解.扫描电镜观察发现S9菌株在待测的立枯丝核菌表面形成了溶菌斑.S9菌株对立枯丝核菌的作用过程是通过吸附在病原真菌的菌丝上,并随菌丝生长而生长,而后产生溶菌物质消解菌丝体.液体共培养测定也证明了S9菌株具有上述作用.本研究还发现,S9菌株对植物病原真菌的拮抗真菌绿色木霉(Trichoderma viride)、鲜红毛壳菌(Chaetomium cupreum)和球毛壳菌(Chaetomiumglobosum)的生长没有影响.盆栽试验证明了S9菌株可有效地控制立枯丝核菌(R.solani)引起的番茄苗期病害.S9菌株与其它拮抗真菌混合具有促进防治植物病原真菌引起的植物病害的潜力.     

枯草芽孢杆菌（Bacillus subtilis）S9对植物病原真菌的溶菌作用

本研究共分离了976株细菌分离物，发现来自甘蔗根围的1株枯草芽孢杆菌(Bacillus subtilis)S9对立枯丝核菌(Rhizoctonia solani)、终极腐霉(Pythium ultimum)和西瓜枯萎病菌(Fusarium oxysporum f.sp.niveum)在PDA平板的对峙培养过程中不形成抑菌圈，但4d后可使上述植物病原真菌的菌丝溶解。扫描电镜观察发现S9菌株在待测的立枯丝核菌表面形成了溶菌斑。S9菌株对立枯丝核菌的作用过程是通过吸附在病原真菌的菌丝上，并随菌丝生长而生长，而后产生溶菌物质消解菌丝体。液体共培养测定也证明了S9菌株具有上述作用。本研究还发现，S9菌株对植物病原真菌的拮抗真菌绿色木霉(Trichoderma viride)、鲜红毛壳菌(Chaetomium cupreum)和球毛壳菌（Chaetomium globosum）的生长没有影响。盆栽试验证明了S9菌株可有效地控制立枯丝核菌(R．solani)引起的番茄苗期病害。S9菌株与其它拮抗真菌混合具有促进防治植物病原真菌引起的植物病害的潜力。     

芝麻茎点枯病菌两种细胞壁降解酶活性分析

为明确芝麻茎点枯病菌漆酶(Lac)和聚甲基半乳糖醛酸酶(PMG)活性,采用分光光度法测定了9株芝麻茎点枯病菌漆酶活性和聚甲基半乳糖醛酸酶活性。结果表明,芝麻茎点枯病菌胞内、胞外均能检测到漆酶,不同菌株之间漆酶活性差异显著;芝麻茎点枯病菌体外培养能持续产生聚甲基半乳糖醛酸酶,并且不同菌株之间聚甲基半乳糖醛酸酶活性差异显著。     

几丁质裂解微生物防治棉苗立枯病

研究了几丁质裂解微生物对立枯丝核菌引起的棉花立枯病的防治作用。用几丁质改良的Hutchinson培养基对几丁质裂解微生物进行分离,在42株几丁质裂解微生物中,有2株放线菌在培养基和土壤中都对立枯丝核菌有较强的抑制作用。选择上述两个菌株和几丁质配成10％生物拌种素,并用5％几丁质粉及20％几丁质酸溶物(20％拌种素),进行对棉花出苗的影响和对立枯丝核菌(立枯病)防效的田间试验。结果表明,10％生物拌种素对棉花出苗有明显促进作用,提高出苗率8.9％,出苗期提前5d以上。20％拌种素和5％几丁质对棉花出苗无明显不利影响;10％生物拌种素、5％几丁质和20％拌种素对棉花立枯病均有较好的防效,分别达68.2％、64.8％和51.9％。     

作物根际和叶围中产几丁质酶微生物的分布及其抑制真菌作用

从几十种作物的根际和叶围分离获得３５０株微生物，其中细菌和放线菌分别为２００株和１５０株。对所有这些分离物进行了抑制真菌试验和几丁质酶活性测定。在抑菌试验中，以棉花枯萎病菌、棉花黄萎病菌、黄瓜枯萎病菌，及西瓜枯萎病菌作为供试病原菌，有９６个分离物对其中的一种或一种以上的病原菌有抑制作用，其中１８个分离物对这４种病原菌都具有强烈的抑制作用。在几丁质酶活性方面，１０％的细菌和４０％的放线菌都能不同程度地产生几丁质酶。有些菌株几丁质酶活性强，抑菌作用也明显，有些菌株几丁质酶活性弱，抑菌作用弱或无作用，少数菌株几丁质酶活性强，但却无抑菌作用。用几丁质酶液处理病原菌孢子，其萌发率和芽管长度比对照显著降低     

西南地区玉米纹枯病病菌融合群鉴定和UP-PCR分析

为明确西南地区玉米纹枯病病菌组成结构,从西南地区(四川、贵州、云南和重庆)玉米纹枯病样上分离获得285个疑似丝核菌菌株,根据培养性状、菌丝形态和细胞核染色对其进行鉴定,通过载玻片配对培养法对鉴定出的菌株进行菌丝融合群鉴定,并采用UP-PCR方法从各融合群中选取代表性菌株进行遗传变异分析。结果显示,供试疑似菌株中共鉴定出253个丝核菌,分别为224株立枯丝核菌Rhizoctonia solani和29株玉蜀黍丝核菌R.zeae。立枯丝核菌包括5个融合群,其中AG1-ⅠA共201株,出现频率为79.4%;AG1-ⅠB共2株,出现频率为0.8%;AG2-2ⅢB共3株,出现频率为1.2%;AG4-HGⅠ共10株,出现频率为3.9%;AG-5共8株,出现频率为3.2%。29株玉蜀黍丝核菌融合群均为WAG-Z,出现频率为11.5%。遗传变异分析中,当相似系数为0.78时,按照出现频率从立枯丝核菌和玉蜀黍丝核菌选取的不同融合群的20株菌株被划分为6个类群,与菌丝融合分析结果完全吻合,其中AG2-2ⅢB融合群首次从西南地区玉米上分离到。     

辣椒溶杆菌NF87-2防治瓜类蔬菜蔓枯病的研究

由黑腐球壳菌Didymella bryoniae引起的瓜类蔬菜蔓枯病是一种重要真菌土传病害,造成瓜类蔬菜生产上重大的经济损失。本研究以蔓枯病菌DB-20为靶标菌,采用平板对峙培养法,测定了拮抗细菌及其代谢产物对蔓枯病菌菌丝生长和孢子萌发的的室内抑制作用,拮抗细菌和蔓枯病菌同时接种后,测定西瓜种子的发芽率和出苗率,采用盆栽试验研究拮抗细菌对黄瓜蔓枯病的防治效果。结果表明,辣椒溶杆菌Lysobacter capsici NF87-2对蔓枯病菌菌丝生长的室内抑制率为81.6%,菌株NF87-2及其次生代谢物对蔓枯病菌孢子萌发的抑制率分别为59.4%和67.2%。蔓枯病菌DB-20处理的京欣一号西瓜种子发芽率和出苗率分别为48%和38%,菌株NF87-2＋DB-20同时接种处理组的西瓜种子发芽率和出苗率分别为83%和82%。菌株NF87-2发酵液及其代谢产物对黄瓜蔓枯病的盆栽防治效果分别为81.6%和66.5%。辣椒溶杆菌NF87-2是一株具有较好生防应用潜力的拮抗菌株,有望开发成防治瓜类蔬菜蔓枯病的生物杀菌剂。     

关防风立枯病病原菌根际拮抗细菌筛选与鉴定

为筛选出对立枯丝核菌Rhizoctonia solani Kühn具有优良防效的拮抗菌株。本研究从关防风根际土壤中分离纯化出156株细菌,采用平板对峙法测定菌株SC-32和SC-127对立枯丝核菌均有良好拮抗效果,且兼具广谱抑菌能力;抗生素标记法测定菌株SC-32和SC-127接种35 d后在土壤中含菌量仍能保持2.93×10⁷和7.83×10⁶CFU/g;通过盆栽试验研究结果表明,菌株SC-32和SC-127对关防风立枯病的防治效果为60.53%和65.89%,高于菌剂和农药处理。通过形态学、生理生化特征、16S rDNA基因序列进行分类学鉴定确定菌株SC-32和SC-127均为贝莱斯芽胞杆菌Bacillus velezensis。     

水稻纹枯病生防细菌筛选及其与病原菌侵染垫形成的关系

为筛选对立枯丝核菌Rhizoctonia solani具有拮抗作用的生防细菌,利用稀释涂布平板法,从吉林省水稻纹枯病样中分离筛选出对立枯丝核菌AG-1A具有高拮抗活性的生防菌株,通过gyrB基因序列分析鉴定其分类地位,并采用离体侵染试验和温室防治试验测定筛选生防菌株对水稻纹枯病的防效,在显微镜下观察生防菌株预处理后接种病原菌的水稻叶片表面的菌丝生长情况,分析其生防机理。结果显示,从水稻纹枯病样中共分离获得35株菌株,其中菌株ND11对立枯丝核菌AG-1A的抑制率和抑菌圈直径最大,分别为74.12%和31.50 mm。基于gyrB基因序列分析最终将菌株 ND11鉴定为短小芽胞杆菌Bacillus pumilus;该菌株能够减缓立枯丝核菌AG-1A的侵染速度,抑制其侵染垫的形成,对水稻纹枯病的温室防效达70.69%以上。表明短小芽胞杆菌菌株ND11能够通过抑制立枯丝核菌侵染垫的形成来防治水稻纹枯病,且防效较好,具有开发为生防菌剂的潜力。     

水稻内生细菌的分离及其拮抗性与潜在致病性测定

从江苏省扬州、南通、常州和徐州等地水稻根、茎和种子分离获得内生细菌736个菌株,其中对稻瘟病菌、稻恶苗病菌、稻纹枯病菌和稻白叶枯病菌拮抗的菌株分别占20.7%、5.4%、3.1%和1.1%,且主要来自根和茎,并有24个和3个菌株分别对2种和3种病菌有拮抗活性。对稻瘟病菌和稻恶苗病菌拮抗的内生细菌转管培养20代后,多数菌株拮抗活性稳定,对其他两种病菌拮抗的菌株转管培养后则拮抗能力大都显著下降或丧失。经形态和生理生化鉴定,高拮抗菌株G87(对稻瘟病菌、稻恶苗病菌、稻白叶枯病菌拮抗)和J215(对稻瘟病菌、稻恶苗病菌拮抗)为枯草芽孢杆菌。针刺和剪叶接种试验表明,大多数水稻内生细菌不致病,少数(3.4%~4.8%)在人工接种条件下可有致病能力或潜在致病性。     

Application of Trichoderma and Gliocladium in alginate pellets for control of Rhizoctonia damping-off

Alginate pellets were prepared from wet fermentor biomass of 11 isolates of Trichoderma spp. and Gliocladium virens , with wheat bran as a food base carrier. Pellets with eight of the isolates reduced survival (34-78%) of Rhizoctonia solani in infested beet seed in soil. Pellets containing a T. harzianum isolate (Th-58) and a T. hamatum isolate (TRI-4) were the most effective. All isolates significantly reduced growth of the pathogen from infested beet seed into natural soil. Populations of isolates proliferated in soil to 10⁶−10¹¹ colony-forming units/g (cfu) from propagules within the pellets. Pellets with TRI-4 reduced pathogen survival and growth (>70%) in six different soils and were effective against six R. solani isolates in a natural loamy sand. Survival of R. solani in infested beet seed was not reduced when TRI-4 pellets were added to soil 1-6 weeks before the pathogen; however, saprophytic growth was prevented. Small amounts of biomass (3.0–7.5 g wet weight) in pellets were as effective as a large amount (300 g) in suppressing the pathogen. The storage of pellets for more than 6 weeks at 5 or 25C reduced their effectiveness against R. solani. Pellets prepared with four and three of the 11 isolates prevented damping-off of cotton and sugar beet in the greenhouse, respectively.     

Selection of bacterial antagonists for the biological control of Rhizoctonia solani in oilseed rape (Brassica napus)

Spore-forming bacteria isolated from oilseed rape (OSR) roots were tested for biological control activity against Rhizoctonia solani damping-off of OSR, using a simple slurry coat formulation. From a total of 239 strains which were tested, Bacillus subtilis 205 was selected as the most promising biocontrol agent (BCA). A known isolate of B. subtilis (NCIMB 12376), which had previously shown activity against R. solani in cotton, was used as a standard comparison throughout. In an in vivo screening, pre-emergence damping-off disease control by B. subtilis 205 was excellent, with plant stands being almost as high as non-diseased controls. However, post-emergence survival was less good, although the surviving plants exhibited significantly reduced hypocotyl rot. Growth-room trials which assessed the effect of ambient temperature on the biological control efficacy of B. subtilis strains 12376 and 205 showed that isolate 205 was more effective at lower temperatures. Disease control by isolate 205 at 15°C/10°C (day/night) suggests that this bacterium could operate under autumn sowing field conditions. Further investigations also indicated that the addition of iron (III) to the BCA formulation may enhance disease control by both bacilli. The potential of bacterial BCAs for the control of seedling diseases is discussed.     

Characteristics and pathogenicity of isolates of Rhizoctonia spp. associated with damping-off of sugar beet

Rhizoctonia solani and R. cerealis were isolated from diseased sugar-beet seedlings in Ireland. Isolates of R. solani were assigned to anastomosis groups AG-2, AG-4, AG-5 and an unidentified group that did not anastomose with recognized tester isolates. Cultures of AG-2 were similar to those of AG-5 on oatmeal agar (OA) and potato-dextrose-marmite agar (PDMA). Cultures of AG-4, the unidentified group and R. cerealis were morphologically distinct from one another, AG-2 and AG-5. The optimum temperature for growth of AG-2 was 225 C, with optimum growth of AG-4, AG-5 and the unidentified group at 275-C. R. cerealis grew slower than all groups of R. solani, with optimum growth at 225°C. Hyphae of R. cerealis were significantly narrower than those of the groups of R. solani studied. In glasshouse pathogenicity tests, some AG-2 and all AG-4, AG-5 and isolates from the unidentified group caused damping-off of beet seedlings. In controlled environments of 10-25°C, an AG-2 isolate was the most aggressive at 10 C whilst AG-4, AG-5 and the unidentified group caused most disease at or above 15°C. R. cerealis was also pathogenic to beet seedlings, causing damping-off at 10 and 15 C.     

Biological control of Sclerotinia minor using a chitinolytic bacterium and actinomycetes

Isolates of 85 bacteria and 94 streptomycete and 35 nonstreptomycete actinomycetes were obtained from a lettuce-growing field in Al-Ain, United Arab Emirates, on colloidal chitin agar, and screened for their ability to produce chitinase. Twenty-three bacteria and 38 streptomycete and 15 nonstreptomycete actinomycete isolates produced high levels of chitinase and were examined in vitro for their ability to suppress the growth of Sclerotinia minor , a pathogen causing basal drop disease of lettuce. The three most suppressive isolates were examined further for their production of β-1,3-glucanase and antifungal activity as well as their ability to colonize the roots and rhizosphere of lettuce in vitro and in planta . The three isolates, Serratia marcescens, Streptomyces viridodiasticus and Micromonospora carbonacea , significantly reduced the growth of S. minor in vitro , and produced high levels of chitinase and β-1,3-glucanase. Streptomyces viridodiasticus also produced antifungal metabolite(s) that significantly reduced the growth of the pathogen in vitro . When the pathogen was presented as the sole carbon source, all three isolates caused extensive hyphal plasmolysis and cell wall lysis. Serratia marcescens and St. viridodiasticus were competent to varying degrees in colonizing the roots of lettuce seedlings after 8 days on agar plates and the rhizosphere within 14 days in pots, with their competency being superior to that of M. carbonacea . All three isolates, individually or in combination, were antagonistic to S. minor and significantly reduced incidence of disease under controlled glasshouse conditions.     

Identification of potential native chitinase-producing Trichoderma spp. and its efficacy against damping-off in onion

Chitinase-producing Trichoderma species have been recognized long ago against the phytopathogenic fungi. In this study, we evaluated the production of chitinase enzyme for seventeen isolates of Trichoderma isolated from onion growing districts of Punjab and assessed their bio-efficacy against damping-off in onion. In vitro, these Trichoderma isolates were screened for their antagonistic activity against the damping-off pathogen Fusarium oxysporum f.sp. cepae by dual culture assay. These isolates were also screened for their chitinase enzyme activity; it was found that isolates T5 and T8 are showing higher antagonistic activity on Fusarium oxysporum f.sp. cepae and also produced large amounts of chitinase enzymes in the presence of commercial colloidal chitin. The selected chitinolytic isolates were used in field studies to confirm the feasibility of their biological control efficacy against onion damping-off. In the field experiment, the seed+soil treatment of chitinolytic isolate (T8) showed a critical decrease of damping-off in onion by 88.75% over control.

     

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。     

Biological control by Trichoderma harzianum of damping-off of lettuce caused by Rhizoctonia solani1

Ten isolates of Trichoderma harzianum were tested for their ability to control lettuce seedling damping-off caused by introduced Rhizoctonia solani. T. harzianum isolates TRC 9 and 28 both reduced damping-off. Dual culture experiments were used to select isolates for the study of antibiotic production and mycoparasitism. T. harzianum isolate TRC 12 produced volatile and non-volatile antibiotics, whilst TRC 33 produced only non-volatile antibiotics. T. harzianum isolates 018-2/Y and TRC 9,15 and 28 mycoparasitized R. solani by coiling around and lysing the host hyphae. It appeared that mycoparasitism was more important than antibiosis in the biological control of damping-off.     

Control ofRhizoctonia solani andSclerotium rolfsii in the greenhouse using endophyticBacillus spp.

Isolates of different endophytic bacteria were recovered from surface-disinfected seeds obtained from commercial companies, plants in the field and tissue culture. The bacteria were isolated from seeds after stringent surfacedisinfection.Pseudomonas fluorescens (isolate no. 14) from bean inhibited growth of all fungi tested and was fluorescent on King B medium.Bacillus cereus fromSinapis (isolate no. 65) inhibited growth ofRhizoctonia solani, Pythium ultimum andSclerotium rolfsii and also exhibited chitinase activity.Bacillus subtilis from onion tissue culture (isolate no. 72) inhibitedR. solani andP. ultimum growth.B. cereus from cauliflower (isolate no. 78) inhibited growth ofR. solani. B. pumilus from sunflower (isolate no. 85) inhibited growth ofR. solani andS. rolfsii. B. cereus (isolate no. 65) was introduced into cotton, and by using radioactive labelling we found that it was present for 16 days in the root-stem junction. It is most likely that these bacteria were still found 72 days after their introduction in the root and stem, at levels of 2.8·10⁵ and 5·10⁴ cfu g^–1 fresh weight, respectively, when selective medium was used. There was no difference between control and treated plants in their height or in the fresh weight of roots, stems and leaves.When cotton seedlings were inoculated withB. cereus (isolate no. 65),B. subtilis (isolate no. 72) orB. pumilus (isolate no. 85), disease incidence caused byRhizoctonia solani was reduced in the greenhouse by 51%, 46% and 56%, respectively. In bean seedlings inoculated withB. subtilis (isolate no. 72),B. cereus (isolate no. 78) orB. pumilus (isolate no. 65), disease incidence caused bySclerotium rolfsii was reduced by 72%, 79% and 26%, respectively, as compared to control. In both cotton and bean seedlings, these endophytes reduced the disease index more than 50%. These results indicate that endophytic bacteria can survive inside cotton plants and are efficient agents for biological control against plant pathogens under greenhouse conditions.     

拮抗细菌B20-006对玉米根系内生细菌种群的影响

 Treatment of corn seeds with Bacillus subtilis strain B20-006, a potential biocontrol agent, had no effect on type of endophytic bacteria on corn. However, it stimulated endophytic bacterial reproduction in the plants. B20-006 did not affect the original population of endophytic bacteria in corn roots in early stage. In late stage, population of endophytic bacteria in corn roots treated with the bacteria was significantly higher than CK. Colonization of the bacteria in corn roots would benefit the micro-ecological resistance of cultivars against the diseases. The results suggested that strain B20006 could enhance the percentage of antagonistic endophytic bacteria against Rhizoctonia solani. The percentage in the resistant cultivar, Lianyu 11 was higher than in the susceptible one, Lianyu 13.