一株贝莱斯芽孢杆菌抑制禾谷镰刀菌的研究

为丰富生物防治菌株资源,本试验从感染赤霉病的麦穗上通过平板对峙法筛选到1株可高效抑制禾谷镰刀菌的细菌JS25R,在温室条件下该菌能有效降低小麦赤霉病的发病率、发病程度和病情指数。根据形态特征、生理生化特性和16S rDNA序列分析,将这株拮抗菌鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。进一步通过发酵液和挥发性物质抑制禾谷镰刀菌效果分析初步研究该菌抑制禾谷镰刀菌的机理。结果表明,JS25R的发酵液可抑制禾谷镰刀菌的生长,抑菌带宽度为0.58 cm。通过PCR扩增,确定该菌具有生防相关的5个脂肽合成基因。同时,JS25R可产生挥发性物质抑制谷镰刀菌的菌体生长(抑制率为27.7%)和孢子萌发(抑制率为26.7%),禾烷酮、2-壬酮和2-壬醇4种物质可完全抑制禾谷镰刀菌的生长。气相色谱-质谱法(GC-MS)分析结果表明,该菌可产生36种挥发性化合物,主要为酮类、烷类、苯类、醇类等,其中二甲基二硫醚、2-十一。本研究结果为禾谷镰刀菌生物防治提供了菌种材料。     

秸秆降解过程中拮抗链霉菌对禾谷镰刀菌消长动态的影响

由禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病是导致小麦产量和品质降低的主要因素，其在田间生长的基质主要为秸秆。基于各种拮抗菌的生物防治是控制小麦赤霉病的主要措施之一，但秸秆降解过程中拮抗菌的拮抗效应是否与平板对峙获取的拮抗效应一致尚不清楚。本研究首先通过平板对峙试验，筛选出对禾谷镰刀菌CGMCC3.4598有潜在拮抗作用的3株菌：吸水链霉菌ACCC41648 (Streptomyces hygroscopicus)、吸水链霉菌井冈变种ACCC40051 (S. hygroscopicus var. jingganggensis)和娄彻氏链霉菌ACCC41594(S.rochei)，其抑菌带平均宽度依次为2.67、1.67和1.12mm。基于室内70d培育试验，发现秸秆降解7、42和70 d后，吸水链霉菌ACCC41648及其井冈变种ACCC40051未表现抑制秸秆中禾谷镰刀菌生长的效应，而娄彻氏链霉菌ACCC41594在秸秆降解7 d和42 d后显著降低禾谷镰刀菌丰度分别达30.91%和69.28%。与对照相比，娄彻氏链霉菌ACCC41594显著促进秸秆降解...     

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。     

烟草青枯病菌拮抗菌的筛选、鉴定及生防特性研究

烟草青枯病危害严重,以拮抗菌进行防病的生物防治手段成为研究热点。从不同烟田分离纯化出238株细菌菌株,首先经牙签接种初筛,选取对青枯病菌抑制效果较好的菌株制备其抑菌物质的粗提物,以牛津杯法复筛,最终获得3株对烟草青枯病菌有明显抑制作用的拮抗细菌。全细胞脂肪酸、16S rDNA及gyrB基因测序等分析结果表明,菌株H19、Y6为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),菌株H34为甲基营养型芽孢杆菌(B.methylotrophicus)。3株拮抗菌经CAS检测平板法和Salkowski比色法,发现均具有产铁载体和吲哚-3-乙酸(IAA)的能力,以菌株H19能力最强。温室促生试验结果表明,3株拮抗菌能显著促进烟草株高、鲜重及干重等指标,与对照相比,平均增长率分别达到70%~115%、40%~49%和32%~42%。温室控病试验结果表明,菌株H19、H34和Y6明显降低烟草青枯病的发病率,防效达76.57%、60.98%和69.83%,稍逊于农用链霉素处理的78.66%。     

鸡粪高温蛋白分解菌的鉴定及发酵条件研究

从自然发酵鸡粪中分离筛选出2株(编号JS4、JS8)高温蛋白分解菌,能产较高活性的中性蛋白酶。对JS4、JS8菌株的形态、培养特征和生理生化特性进行鉴定,JS4、JS8菌株分别属于枯草芽孢杆菌和地衣芽孢杆菌。将2菌株复合接种发酵鸡粪,以中性蛋白酶活性作检测指标,研究鸡粪发酵条件。结果表明,最佳发酵条件为含水量为50%、接种量为1%、添加辅料为麸皮、pH值为7.0。     

固氮芽孢杆菌N3的筛选鉴定及其对二月兰的促生效果

从山东泰安农田土壤中筛选获得1株固氮能力强的菌株N3,通过形态观察、生理生化特征以及16S rDNA基因序列分析,确定为巨大芽孢杆菌属(Bacillus megaterium),该菌株固氮酶活性达C2H428.33 nmol/(h·ml)。温室条件下进行二月兰盆栽试验,设置不接菌对照(CK)、接种巨大芽孢杆菌N3、接种产吲哚乙酸(IAA)菌CDL29和复合接种菌株N3+CDL29四个处理,探讨了接种不同植物促生菌对二月兰的促生效果。结果表明:接种植物促生菌均促进二月兰的生长,且单一接种固氮菌株N3的促生效果最好,其显著提高二月兰地上部氮、磷和地下部氮、钾的含量,相比对照,增幅分别为33.03%、55.56%和29.87%、51.11%,其他处理也有提高二月兰养分含量的趋势,但并不显著;单一接种菌株N3,土壤有机质、全氮和速效养分含量显著提高;接种植物促生菌均提高土壤微生物生物量碳(MBC)和氮(MBN)的含量,单一接种固氮菌株N3提升效果最佳,接种产IAA菌株CDL29和复合接种次之。此外,接种固氮菌株N3能够显著提升土壤固氮酶活性,接种菌株CDL29对土壤固氮酶活性没有显著影响。因此,单一接种固氮菌株N3在促进二月兰生长、养分含量提高及土壤理化和生物学性质改良上都具有显著的效果。     

西瓜根际促生菌筛选及生物育苗基质研制

通过从西瓜根际分离筛选具根际定殖能力的植物根际促生菌,将其保活添加至普通育苗基质研制生物育苗基质,以确保功能菌株能够在苗期定殖根际,进而在移栽后发挥促生功能。结果表明,分离获得一株同时具有产吲哚乙酸(IAA)和NH_3,且对尖孢镰刀菌和茄科劳尔氏菌均有拮抗作用的植物根际促生菌(PGPR)菌株N23;在三季育苗试验中,与普通基质处理(CK)相比,添加菌株N23的生物育苗基质所育种苗,在多项苗期生长指标上均表现出稳定的促生作用;盆栽试验表明,除叶绿素相对含量测量值(SPAD)外,生物基质所育西瓜种苗的其他检测指标均显著高于对照(普通育苗基质所育种苗,下同);田间试验表明,生物基质所育种苗西瓜、黄瓜、辣椒和番茄种苗移苗后,在苗期植株株高和茎粗均显著优于对照,在产量上均增产10%以上。结合形态、生理生化特征和16S rDNA基因序列分析,初步鉴定菌株N23为芽孢杆菌属细菌(Bacillus sp.)。综上,利用芽孢杆菌N23研制的生物育苗基质能够有效促进所育不同作物种苗质量,增强移栽后作物的生长和田间产量。因此,本研究能够为根际有益微生物的应用提供新的思路,为生物育苗基质的研制提供理论支撑。     

兼具水稻纹枯病拮抗功能的生物水稻育苗基质效果研究

水稻纹枯病是水稻上常见的土传病害之一,为了研发出可防治水稻纹枯病兼具促生效果的功能型生物水稻育苗基质,本研究选用前期筛选出具有固氮解磷功能的根际促生菌:LY5枯草芽孢杆菌(Bacillus subtilis)、LY11解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、X2摩拉维亚假单胞菌(Pseudomonas moraviensis)、X3沙芬西芽孢杆菌(Bacillus safensis)、X8绿针假单胞菌(Pseudomonas chlororaphis),首先探索该5株促生菌对水稻纹枯病病原菌的抑菌率,再将其与水稻纹枯病病原菌混合,在实验室已筛选出的最适配比基质基础上,研发功能型生物水稻育苗基质。在盆栽条件下,与市售水稻育苗基质对比,验证功能型生物水稻育苗基质对水稻纹枯病发病率、水稻幼苗生长和体内抗性相关酶类的影响。结果表明:(1)5株根际促生菌对水稻纹枯病病原菌均具有拮抗作用,抑菌率达到35.31%～40.20%。(2)5株拮抗促生菌能够降低水稻纹枯病的发病率,其中添加LY11菌株的处理较对照处理的每盘出苗数显著增加了110.41%,而发病率显著降低了89.32%。(3)在含有水稻纹枯病病原菌的育苗基质中添加拮抗促生菌后,水稻幼苗的茎粗、百株地下部干物质重、百株全株干物质重、根系活力、根总表面积、根平均直径、根总体积、根尖数等方面均有所提高,其中添加LY11菌株的处理比市售水稻育苗基质处理分别显著提高3.13%、4.21%、4.97%、68.02%、26.77%、16.67%、44.44%、7.82%。(4)添加拮抗促生菌显著促进了水稻幼苗对地上和地下部氮磷钾的吸收。(5)水稻幼苗体内超氧化物歧化酶、过氧化物酶、过氧化氢酶的活性以及丙二醛的含量均显著高于市售水稻育苗基质处理,提高幅度为27.08%～122.72%、16.09%～48.15%、53.33%～233.85%、110.24%～511.71%。因此,本研究研发出5种对水稻纹枯病具有较好的拮抗效果,同时可以显著促进水稻幼苗生长的功能型高效生物水稻育苗基质,其中添加了菌株LY11的育苗基质综合效果最优,为功能型高效生物水稻育苗基质的研制与应用奠定了理论与实践基础。     

烟草黑胫病拮抗菌的筛选及其生物效应

烟草黑胫病是烟草生产中危害最严重的土传病害之一,生物防治具有抗病和环保的作用,获得高效拮抗菌株是进行生物防治研究的基础。本研究采用平板对峙法,在烟草黑胫病发生严重的田块中选取健康烟株,从其根际土壤中分离筛选到12株对烟草黑胫病病原菌Phytophthora parasitica var.nicotianae具有拮抗效果的菌株,抑菌率59.4%～71.1%。选择抑菌率最高的C-5菌株进行试验。经鉴定C-5菌株为多粘类芽孢杆菌(Paenibacillus polymyxa)。抗菌谱试验结果表明:C-5菌株不仅对黑胫病病原菌有较强的拮抗作用,同时对甜瓜?黄瓜枯萎病病原菌和辣椒疫病病原菌也具有拮抗作用。苗期盆栽试验结果表明:利用C-5菌株发酵制备的微生物有机肥能抑制烟草黑胫病的发生,苗期防治率达80%。本文首次报道了多粘类芽胞杆菌菌株对烟草疫霉有拮抗作用。     

一株黄曲霉拮抗菌的筛选、鉴定及特性研究

为获得拮抗黄曲霉和降解黄曲霉毒素的生物防治菌株,以烟台黄曲霉污染区域土壤为材料,采用稀释分离法和琼脂扩散法,筛选到一株对黄曲霉有抑制作用且可降解黄曲霉毒素的菌株Y-17-3。根据菌株的形态学特征、生理生化试验结合16S rDNA基因序列分析和系统发育树构建,确定菌株 Y-17-3 为空气芽孢杆菌(Bacillus aerius)。该菌株发酵液经硫酸铵沉淀的抗菌活性及稳定性试验,发现70%饱和硫酸铵沉淀的蛋白粗提物抑制黄曲霉活性最强,可降低黄曲霉孢子的萌发率,抑制菌丝的生长和菌丝球的形成,且该蛋白粗提物对温度、pH值和蛋白酶敏感。菌株Y-17-3能够降解黄曲霉毒素B₁,培养6 d时的降解率达90%。综上,所分离的空气芽孢杆菌Y-17-3菌株能够通过产生拮抗蛋白来抑制黄曲霉生长,且具有较强的黄曲霉毒素降解能力,在农业生物防治领域具有一定的应用前景。     

Fusarium graminearum TRI14 is required for high virulence and DON production on wheat but not for DON synthesis in vitro

Fusarium head blight (FHB) of wheat (Triticum aestivum L.), caused by the fungus Fusarium graminearum, is a major concern worldwide. FHB grain is reduced in yield, may fail to germinate, and is often contaminated with deoxynivalenol, a trichothecene mycotoxin linked to a variety of animal diseases and feed refusals. Annual losses in the tens of millions of dollars due to FHB underscore the need to develop improved methods of disease control and prevention. Previous research has identified deoxynivalenol biosynthesis as a virulence factor on wheat. Recently, we found that the TRI14 gene of F. sporotrichioides, closely related to F. graminearum, was not required for synthesis of a related trichothecene, T-2 toxin. TRI14 does not share similarity with any previously described genes in the databases. In this study, we examined the role that F. graminearum TRI14 may play in both deoxynivalenol synthesis and in virulence on wheat. TRI14 deletion mutants synthesize deoxynivalenol on cracked maize kernel medium and exhibit wild-type colony morphology and growth rate on complex and minimal agar media. However, FHB assays on greenhouse-grown wheat indicate that FgDeltaTri14 mutants cause 50-80% less disease than wild type and do not produce a detectable quantity of deoxynivalenol on plants. We discuss a number of possible roles that TRI14 may play in the disease process.     

不同喷药时期对甘肃陇南小麦赤霉病的防治效果

为明确小麦不同生长期喷施430 g/L戊唑醇悬浮剂对小麦赤霉病防治效果的影响,于2021年在甘肃省陇南市徽县栗川镇范寺村进行了田间试验。结果表明,在小麦抽穗期、扬花期、灌浆期喷雾,田间防效分别为93.23%、84.39%、36.51%,折合产量分别为9 168.0、8 949.0、8 376.0 kg/hm2,较喷清水分别增产10.76%、8.12%、1.19%。说明小麦抽穗期到扬花期是甘肃陇南防治小麦赤霉病的关键时期,在该阶段进行杀菌剂喷雾防治,可有效控制小麦赤霉病的发生流行。     

Impact of competitive fungi on Trichothecene production by Fusarium graminearum

Bioassays were used to determine the production of the trichothecene mycotoxin, deoxynivalenol (DON), by two isolates of Fusarium graminearum when grown in association with potentially competitive fungi and an antifungal chemical, 6-pentyl-alpha-pyrone (6PAP). The presence of 6PAP in the culture medium reduced DON production by as much as 80%, but this effect was reduced for the F. graminearum isolate that most efficiently metabolized the added 6PAP. A 6PAP-producing Trichoderma isolate grown in a competition assay system with the F. graminearum isolates was also able to substantially reduce DON production. When Fusarium isolates (F. crookwellense, F. culmorum, F. subglutinans, F. poae, F. equiseti, F. avenaceum, and F. sambucinum), which co-occur with F. graminearum in New Zealand maize plants (Zea mays), were grown in competition assays, the effect on DON production was variable. However, all isolates of F. subglutinans tested were shown to cause reductions in DON production (by 13-76%, mean = 62%). F. subglutinans frequently co-occurs with F. graminearum, but its presence can vary with location and time of the season. When the competitive fungus tested was also a trichothecene producer (e.g., of nivalenol), both toxins were produced in the assay medium. The results indicate that mycotoxin production by F. graminearum can be affected by the presence of particular competitive fungi. These results have implications for an ecological understanding of pathogenicity and of mycotoxin accumulation in plants. Early establishment of F. subglutinans, for example, may act as a biological control mechanism providing a temporary protection against invasion by more commonly toxigenic fusaria such as F. graminearum.     

西瓜枯萎病生防细菌的筛选

从６个土壤标本中分离得１５个细菌菌株,其中Ｂ１１、Ｂ１４、Ｂ１９、Ｂ２４等４个菌株为芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｐ．）,它们对西瓜枯萎病菌有较强的抑制作用。经室内和田间初步防治试验测定,它们对西瓜枯萎病有较好的防治效果。     

Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels

Maize (Zea mays) and wheat (Triticum aestivum) collected in the foothills of the Nepal Himalaya Mountains were analyzed for Fusarium species and mycotoxins: fumonisins, nivalenol (NIV), and deoxynivalenol (DON). Predominant species were Gibberella fujikuroi mating population A (F. moniliforme) in maize and F. graminearum in maize and wheat; G. fujikuroi mating population D (F. proliferatum), F. acuminatum, F. avenaceum, F. chlamydosporum, F. equiseti, F. oxysporum, F. semitectum, and F. torulosum were also present. Strains of G. fujikuroi mating population A produced fumonisins, and strains of F. graminearum produced NIV or DON. By immunoassay or high-performance liquid chromatography, fumonisins were >1000 ng/g in 22% of 74 maize samples. By immunoassay or fluorometry, NIV and DON were >1000 ng/g in 16% of maize samples but were not detected in wheat. Fumonisins and DON were not eliminated by traditional fermentation for producing maize beer, but Nepalese rural and urban women were able to detoxify contaminated maize by hand-sorting visibly diseased kernels.     

Potential of Azotobacter spp. as biocontrol agents against Rhizoctonia solani and Fusarium oxysporum in cotton (Gossypium hirsutum), guar (Cyamopsis tetragonoloba) and tomato (Lycopersicum esculentum)

During the current investigation, 51 bacterial isolates/mutants of Azotobacter chroococcum, Azospirillum spp. and Gluconacetobacter diazotrophicus were tested for antifungal activity against three fungal pathogens, namely Rhizoctonia solani cotton, Rhizoctonia solani rice and Fusarium oxysporum tomato using a dual-culture technique under laboratory conditions. Ten isolates/mutants were found to be inhibitory against R. solani cotton, six against R. solani rice and twelve against F. oxysporum tomato. Nearly 50% of the isolates/mutants were siderophore positive. The antimicrobial substance was found to be extracellular and proteinaceous in nature, but in some strains it was found to be associated with other complex material also. Almost all the positive isolates/mutants showed production of antibiotic and antifungal compounds. On the basis of this study, in vitro results under laboratory conditions were reproduced in pot experiments in the greenhouse and chosen isolates/mutants of A. chroococcum were found to be effective biocontrol agents against R. solani cotton (disease index 16.7%) and R. solani rice (2.5%) in cotton crop, whereas in guar, the crop disease index was 2.5% against R. solani cotton, 13.3% against R. solani rice and 0% against F. oxysporum tomato in tomato crop compared with their respective checks.     

红三叶根际促生菌中具生防效果菌株筛选、鉴定及特性研究

【目的】 从岷山红三叶根际分离的109株促生菌,鉴定、筛选了具有生防能力的菌种资源并研究其生防特性,为生产应用提供服务。 【方法】 采用平板对峙法筛选优良生防菌株,对其产铁载体能力进行定性、定量测定和16S rRNA基因序列分析鉴定。 【结果】 平板对峙试验显示,109株促生菌中有8株促生菌对3种病原真菌 (立枯丝核菌、黄瓜枯萎菌、西瓜尖镰孢菌) 具有拮抗效果,分别是菌株MHS3、MHS7、MHS9、MHS27、MHS31、MHS38、MHS39和MHS48。其中,菌株MHS27、MHS31发酵液对立枯丝核菌抑菌率分别为40.1%、47.1%;菌株MHS7和MHS48发酵液对黄瓜枯萎菌抑菌率分别为26.1%、22.8%;菌株MHS27和MHS31发酵液对西瓜尖镰孢菌抑制率分别为34.0%、30.5%;产嗜铁素是生防菌 (MHS7、MHS27、MHS31、MHS48) 拮抗3种病原真菌的主要途径之一。经16S rRNA基因序列分析初步鉴定,菌株MHS7为特基拉芽孢杆菌 (Bacillus tequilensis),菌株MHS27为溶蛋白芽孢杆菌 (Bacillus proteolyticus),菌株MHS31为Pseudomonas paralactis,菌株MHS48为克雷伯氏菌 (Klebsiella oxytoca)。 【结论】 从岷山红三叶根际分离筛选出4株具有优良生防作用的PGPR菌株,为今后生物菌肥应用推广提供了菌种资源支持。      

Seed priming with Pseudomonas putida isolated from rhizosphere triggers innate resistance against Fusarium wilt in tomato through pathogenesis-related protein activation and phenylpropanoid pathway

Pathogenesis-related (PR) proteins are one of the major and preliminary proteins accumulated as a defense against biotic stress. This defense response can be induced by using beneficial rhizobacteria, which has been studied in various host-pathogen interactions. In the present study, eleven Pseudomonas isolates were assessed for their potential to ferment sorbitol, reduce nitrate, and produce mycolytic enzymes, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, phenazine antibiotics, and N-acyl homoserine lactones (AHLs). All isolates were tested against the host-specific pathogen Fusarium oxysporum MTCC1755 in tomato under greenhouse conditions, and shortlisted isolates were tested for their rhizosphere competence. In-vitro test results showed that the isolates were able to produce mycolytic enzymes, including protease, lipase, chitinase, cellulase, and amylase, and the antibiotic phenazine and were negative for pyoluteorin. All the isolates except two were positive for ACC deaminase production. Greenhouse results showed that the isolates M80, M96, and T109 significantly reduced symptoms of Fusarium wilt. Extended greenhouse tests under autoclaved and unautoclaved soil conditions showed that M80, M96, and T109 were excellent rhizosphere competitors and were identified as Pseudomonas putida. In brief, the defense-specific biochemical variations in the host could describe the improved defense against Fusarium wilt occurring in the primed plants. These three Pseudomonas strains could be used as potential biocontrol agents, along with their rhizosphere competence.     

Purification and identification of barley (Hordeum vulgare L.) proteins that inhibit the alkaline serine proteinases of Fusarium culmorum

It has been proposed that microbial proteinase inhibitors, which are present in abundance in cereal grains, protect the seed against plant pathogens. So far, however, very little is known about the interactions of those inhibitors with the proteinases of phytopathogenic microbes. The increased alkaline proteinase activities of Fusarium head blight (FHB) diseased wheat and barley grain imply that the Fusarium fungi synthesize those enzymes during the colonization of the kernel. To study which barley proteins can inhibit Fusarium proteinases, and hence, possibly protect the seed from FHB, the proteins of a grain extract have been separated and tested for their abilities to inhibit two alkaline serine proteinases that we previously isolated from F. culmorum. The proteins were separated by size exclusion, ion exchange, and reversed-phase-HPLC chromatographies. The purified inhibitors were identified by their molecular masses and N-terminal amino acid sequences. The proteins that inhibited the subtilisin-like Fusarium proteinase were the chymotrypsin/subtilisin (CI) inhibitors 1A, 1B, and 2A and the barley alpha-amylase/subtilisin inhibitor (BASI). Only one of the purified proteins inhibited the trypsin-like proteinase, the barley Bowman-Birk inhibitor (BBBI). No novel inhibitors were detected.