【作者】 张雪；

【导师】 向文胜；

【作者基本信息】 东北农业大学 ， 微生物学， 2021， 硕士

【摘要】 番茄青枯病(Tomato bacterial wilt)是世界上危害最为严重的土传植物病害之一,危害性极大。在保护环境的前提下,生物防治是降低该病发病率的有效途径之一。放线菌作为生物农药资源菌,这几年倍受关注,其菌制剂的应用在农业上也发挥着越来越重要的作用。由于合成的杀菌剂等化学试剂在控制病害方面具有局限性和危害性,因此,利用环境中存在的拮抗微生物,开发环保型和高效型的微生物制剂来代替化学农药,根除这一类病害也是目前人们关注的话题。另一方面单一菌株有诸多缺点,比如持续时间较短,不经济等,导致生防效果差且不持久,因此本实验从多菌组合的角度出发,对生防菌株进行多菌复配,构建复合菌株,后期进行了盆栽实验,定殖实验,生物有机肥制备等一系列研究,并在此基础上,为复合菌肥在防治土传病害方面提供可行性的理论依据。本研究从山东省临沂市番茄根际土作为分离源,进行了放线菌的分离纯化,采用滤纸片的方法,以青枯病菌为指示性菌株筛选具有抗菌活性的菌株,6株具有较强抑菌活性的链霉菌,分别是NEAU-Z2、NEAU-D18、NEAU-88、NEAU-174、NEAU-C12和NEAU-66。并通过亲和试验,发现菌株NEAU-D18和NEAU-88具有良好的相容性,通过番茄苗期抗病试验、离体试验、盆栽试验等研究了复合菌(NEAU-D18+NEAU-88)对番茄青枯病的防治效果,另外并从营养竞争角度探讨复合菌株(NEAU-D18+NEAU-88)潜在的生物防治番茄青枯病的机制。后期以复合菌为研究对象,通过固体发酵方式制成复合生物肥,利用盆栽实验验证其生防效果,为尽快开发利用复合菌肥在实际生产中防治番茄青枯病提供参考。此外,对活性菌株NEAU-66进行了鉴定试验。主要实验结果如下:(1)选用六种分离培养基(CPA、DPA、SSA、HV、GS、AAG)对土壤中放线菌进行分离,经过形态观察和分子水平分析后合并,共筛选得到108株放线菌。共包括链霉菌属(Streptomyces),韩国生工菌属(Kribbella),诺卡氏菌属(Nocardia),小单胞菌属(Micromonospora),甲基杆菌属(Methylobacterium),其中链霉菌属(Streptomyces)为优势菌属。(2)对108株放线菌进行抗青枯病菌活性检测。共有4株菌株的孢子浸提液具有抑菌效果,分别是NEAU-88、NEAU-Z2、NEAU-C12和NEAU-66,它们对番茄青枯病具有较强的抑菌活性,抑菌区直径分别为25.16、15.32、17.78和13.00 mm。另外两株NEAU-174和NEAU-D18由实验室提供,抑菌直径为24.46 mm和24.51 mm。(3)将菌株NEAU-Z2、NEAU-D18、NEAU-88、NEAU-174、NEAU-C12和NEAU-66进行亲和性实验(NEAU-D18、NEAU-174为实验室提供),筛选出彼此之间无抑制作用的菌株。结果表明,NEAU-D18和NEAU-88之间彼此亲和,复合菌株的抑菌直径为28.98 mm。(4)在苗期试验和盆栽试验中,复合菌株(NEAU-D18+NEAU-88)的生防效果明显优于单一生防菌株(P<0.05),苗期实验中,当用浓度10~8CFU/m L且NEAU-D18:NEAU-88比例为1:1时,混合菌株孢子悬浮液的防效最佳,在盆栽实验中,混合菌株(NEAU-D18+NEAU-88)的生防效果(70.10%)显著高于单一菌株NEAU-D18(47.30%)和菌株NEAU-88(51.29%)。(5)营养竞争实验表明,NEAU-D18,NEAU-88以及彼此组成的复合菌株与青枯菌株QL-Rs1115具有营养相似性,测得NOI值分别0.71、0.82和0.88。并且表明混合菌株在理论上比单一菌株更具竞争力。(6)通过固体发酵基质的筛选,确定两株菌的最适固体发酵基质为蚯蚓粪和麦麸,且两者比例为2:1,后期对混合菌株进行固体发酵培养条件优化,得到最佳发酵条件,分别是接种量10%,含水量60%,温度28°C,并且在发酵7天时,复合菌株的孢子量达到最大。然后将发酵的产物制成的生物菌肥进行盆栽实验,生防效果为76.41%。(7)菌株NEAU-66的多相分类学鉴定。通过16S r RNA基因序列比对,生理生化特征、不同培养基的培养特征、形态特征以及化学成分分析,确定该菌株为链霉菌属内一株潜在新种。更多还原

【Abstract】 Tomato bacterial wilt is one of the most serious soil-borne plant diseases in the world and has quite a bit of damage.Under the premise of environmental protection,biocontrol is one of the effective ways to reduce the incidence of the disease and yield loss.Actinomycetes as BCAs attention resources,the application of the BCAs also play an increasingly important role in agriculture,and chemical reagents such as synthetic fungicides have limitations and harmfulness in biocontrol.Therefore,using antagonistic microorganisms present in the environment,development of environmental protection and efficient type of probiotics as an alternative to chemical pesticides,so this disease is a subject of great concern at present.Because of a single strain has many shortcomings,such as shorter duration,not economic,etc,therefore this study from the perspective of the mixed strains,opposite to carbendazim resistant strain distribution,build mixed strain.In the later stage,a series of studies were carried out,such as pot experiment,colonization experiment and preparation of biological organic fertilizer.On the basis of studying the preparation,provide composite researches in the aspects of controlling soil-borne disease provide theoretical basis for the feasibility.In this study,actinomycetes were isolated and purified from tomato rhizosphere soil in Linyi city,Shandong Province.The antagonistic strains were screened by using filter paper with Ralstonia solanacearum as the indicator.Six strains of Streptomytes NEAU-Z2,NEAU-D18,NEAU-88,NEAU-174,NEAU-C12 and NEAU-66 had strong antibacterial activity.Through compatibility test,the strains NEAU-D18 and NEAU-88 had good compatibility,and the biocontrol effect of mixed strain(NEAU-D18+NEAU-88)on tomato bacterial wilt was studied through seedling disease resistance test,in vitro test and pot experiment,and the mechanism of biological control of mixed strain(NEAU-D18+NEAU-88)was discussed from the perspective of nutritional competition.In the later stage,the mixed strains were taken as the research object and cultivated by solid fermentation to make the compound bioorganic fertilizer,so as to clarify its stability and mechanism and effect of biocontrol action,and to provide reference for the development and utilization of the compound bioorganic fertilizer in the actual production of tomato bacterial wilt as soon as possible.In addition,the biocontrol bacteria strain NEAU-66 was identified.The main experimental results are as follows:(1)Six separate media(CPA,DPA,SSA,HV,GS,AAG)were used to isolate actinomycetes from soil.After morphological observation and molecular level analysis,the bacteria were combined,and a total of 108 actinomycetes were screened.Streptomyces,Kribbella,Nocardia,Micromonospora and Methylobacterium are included in the total.Streptomyces is the dominant bacterium.(2)The activity of 108 actinomycetes against bacterial wilt was detected.NEAU-88,NEAU-Z2,NEAU-C12 and NEAU-66 had strong antimicrobial activity against R.Solanacearum QL-Rs1115,with diameter of 25.16,15.32,17.78 and 13.00 mm,respectively.The other two strains,NEAU-174 and NEAU-D18,were provided by the laboratory with antibacterial diameters of 24.46 mm and 24.51 mm.(3)The compatibility of NEAU-Z2,NEAU-D18,NEAU-88,NEAU-174,NEAU-C12 and NEAU-66 strains were tested to screen out the non-inhibitory strains.The results show that NEAU-D18 and NEAU-88 are mutually compatible.The antibacterial diameter of the compound strain was 28.98 mm.(4)In the seedling stage experiment and pot experiment,the biocontrol effect of the mixed strain(NEAU-D18+NEAU-88)was significantly better than that of the single biocontrol strain(P<0.05).In the seedling stage experiment,when the concentration of 10~8 CFU/m L and the ratio of NEAU-D18:NEAU-88 was 1:1,the spore suspension of the mixed strain had the best control effect.In the pot experiment,the control effect of the mixed strains(NEAU-D18+NEAU-88)(70.10%)was significantly higher than that of single strains NEAU-D18(47.30%)and NEAU-88(51.29%).(5)Nutrition competition experiments showed that NEAU-D18,NEAU-88 and the mixed strain had nutritional similarities with R.Solanacearum QL-Rs1115,with NOI values of 0.71,0.82and 0.88,respectively.And it showed that the mixed strains are more competitive than the single strain in theory.(6)Through solid fermentation matrix of screening,to determine the optimal solid fermentation of two strains of bacteria matrix for vermicompost and wheat bran,and the ratio between the two late to solid mixed strains fermentation culture conditions optimization,get the best fermentation conditions,respectively is the quantity of 10%,60%water content,temperature28°C and 7 days in fermentation,mixed strains reached the maximum amount of spores.Then the biological fertilizer made from the fermentation products was potted,and the biological control effect was 76.41%.(7)Polyphasic taxonomic identification of strain NEAU-66.According to 16S r RNA gene sequence alignment,physiological and biochemical characteristics,culture characteristics of different media,morphological characteristics and chemical composition analysis,the strain was identified as a potential new species in Streptomyces.更多还原