【作者】 宋光桃；

【导师】 周国英；

【作者基本信息】 中南林业科技大学 ， 森林保护， 2012， 博士

【摘要】 油茶炭疽病(Camellia oleifera anthracnose)和根腐病(root rot disease)是我国油茶产区的主要病害。油茶炭疽病常引起落果、落蕾,落果率一般在20%-40%,严重时达80%以上；油茶根腐病使受害油茶根部腐烂,导致植株不能正常生长而死亡,它们给油茶生产带来巨大经济损失。当前主要通过化学防治来控制这些病害,但易引发病原菌产生抗药性和环境污染,直接或间接危害人体健康。本研究对这两种病原菌的分子快速检测、高效拮抗菌株分离筛选及鉴定、拮抗菌株对病原菌的抑菌机理、发酵工艺优化、菌剂配制与田间防治等技术进行了系统地研究,以期为油茶主要病害可持续控制提供理论依据和奠定应用基础。(1)从病原菌的形态特征、致病性测定、rDNA ITS序列测定、系统发育树构建等方面鉴定油茶炭疽病菌为胶孢炭疽菌(Colletotrichum gloeosporioides),油茶根腐病菌为层生镰刀菌(Fusarium proliferatum)。根据油茶炭疽病菌ITS的序列差异设计了特异性引物YT1和YT2,对胶孢炭疽菌进行检测,可扩增出约330bp的特异性条带,对炭疽菌属胶孢炭疽菌以外的16种炭疽菌进行检测,均未扩增出特异性条带。用引物YT1和YT2进行常规PCR方法扩增,可检测含量为1pg/25μL的胶孢炭疽菌,进行巢式PCR方法扩增,可检测含量为10ag/25μL的胶孢炭疽菌,检测灵敏度提高了100000倍；根据油茶根腐病菌ITS的序列差异设计了特异性引物GF1和GF2,对层生镰刀菌进行检测,可扩增出约400bp的特异性条带,对镰刀菌属层生镰刀菌以外的16种镰刀菌进行检测,均未扩增出特异性条带。用引物GFl和GF2进行常规PCR方法扩增,可检测含量为1pg/25μL的病原菌,进行巢式PCR方法扩增,可检测含量为100ag/25μL,检测灵敏度提高了10000倍。以YT1、YT2和GF1、GF2分别为油茶炭疽病菌和油茶根腐病菌的特异性引物,用巢式PCR方法并结合油茶组织总DNA的提取方法,可在病害发生潜育期,发病初期进行病原菌DNA的PCR扩增,快速、灵敏地检测油茶炭疽病菌和油茶根腐病菌。(2)采用稀释分离法从健康油茶组织中分离得到75株内生细菌,从不同来源的38份土壤中分离得到223株土壤细菌和194株土壤放线菌,以油茶炭疽病菌和根腐病菌为靶标,通过平板对峙试验以447株分离菌株进行初筛.共筛选出抑菌圈大于5mm的内生细菌6株,土壤细菌21株,抑菌圈大于10mm的放线菌14株,以8种植物病原菌为靶标复筛,筛选出对油茶主要病害有良好拮抗作用的3株拮抗细菌(菌株R6、菌株Z17、菌株Z26)和2株拮抗放线菌(菌株F10、菌株CF17)。通过形态特征、生理生化特征及16S rDNA序列分析,鉴定菌株R6为枯草芽孢杆菌(Bacillus subtilis),菌株Z17为科本类芽孢杆菌(Paenibcillus kribbens),菌株Z26为短短芽孢杆菌(Brevibacillus brevis),菌株F10为球孢链霉菌球孢亚种(Streptomvces globisporus subsp.globisporus),菌株CF17为小白链霉菌(Streptomyces albulus)。(3)菌株F10和菌株CF17能影响油茶炭疽菌和根腐病菌菌丝正常生长和分生孢子萌发,菌丝出现节间变短、变粗、扭曲变形、菌丝严重交联、细胞质聚集、外溢等现象,能产生抗菌活性物质,在常温下稳定,多次传代后对油茶炭疽病菌和根腐病菌仍有较强拮抗效应,与拮抗细菌菌株Z26无抑菌性,但不耐高温和抗紫外线,对强酸、强碱不够稳定：菌株F10能分泌蛋白酶和纤维素酶,能耐可杀得、代森锰锌等杀菌剂,对水稻、蚕豆、白菜种子萌发有促生作用。菌株CF17能分泌蛋白酶,能耐可杀得、敌克松等杀菌剂,对水稻种子萌发有促生作用。(4)综合考虑菌株CF17的菌体生长量(菌体干量)和对油茶炭疽病菌的抑菌率两个指标,研究不同发酵培养基、不同碳源、氮源等营养因子,以及接种量、装液量、初始pH值等非营养因子对菌株CF17发酵液生物活性的影响。结果表明：菌株CF17的优化培养液为：葡萄糖15g、酵母膏10g、NaCl1g、KH2P041g、CaCO33g、蒸馏水1000mL.pH7.0,在300mL三角瓶中装入发酵培养液100mL,接入种子发酵液的接种量为100%,在30℃、140r/min的条件下振荡培养7d。(5)以过80目细筛的米糠按1：2的料液比与菌株CF17的发酵液混合配成原粉,经抑菌活性测定,润湿性、分散性、悬浮性综合评价测定,菌剂最优配方为原粉40%,高岭土38%,木质素磺酸钠10%,十二烷基硫酸钠7%,腐植酸5%。此菌剂在盆栽防治中表明对油茶炭疽病防治效果很明显,500倍的菌剂稀释液的防治效果可达80%以上,优于对照药剂多菌灵的防治效果,对油茶根腐病也有较好的防治效果,而且可以促进油茶幼苗的生长。更多还原

【Abstract】 Camellia oleifera anthracnose and root rot disease are major diseases in the major producing areas of C. oleifera of China. C. oleifera anthracnose usually causes fruit drop and flower-bud abscission with the percentage of fruit drop20%-40%in normal case and even more than80%in serious case, while C. oleifera root rot disease can rot roots of the infected trees, which results into abnormal growth and death, leading to enormously economic losses. At present, chemical pestieides are employed to control the diseases, which can easily induce pathogen to produce drug resistance, pollute environment and damage human health both directly and indirectly. This paper has studied the rapid detection of molecular techniques, antagonistic stains screening, identification, inhibitory mechanism, optimization of fermentation, formula of inocula and field experiment, which hopes to provide theoretical basis for sustainable control and lay a foundation for application.(1)By morphological characteristics of pathogens, determination of the pathogenicity, ITS (internal transcribed space) sequencing, and construction of phylogenetic tree we identified that C. oleifera anthracnose and C. oleifera root rot disease was caused by Colletotrichum gloeosporioides and Fusarium proliferatum respectively. Based on differences in ITS sequences of C. oleifera anthracnose. a pair of species-specific primers(YT1and YT2) was synthesized, with which we could detecte C. gloeosporioides with about amplified330bp band. But for the other16strains, none of them could be amplified. The pathogen with content lpg/25μL could be dectected using general PCR amplification, but it was the only10ag/25μL content that was enough by the nest-PCR, the detection sensitivity had increased100000times. According to differences in ITS sequences of C. oleifera root rot, a pair of species-specific primers (GF1and GF2) was synthesized, with which we could detecte F. proliferatum with about amplified400bp band. But for the other16strains, none of them could be amplified. The pathogen with content lpg/25μL could be detected using general PCR amplification. but it was the only100ag/25μL content that was enough by the nest-PCR, the detection sensitivity had also increased10000times.Using YT1and YT2specific primers and GF1and GF2specific primers and the nest-PCR technique combined with the DNA extraction methods of C. oleifera tissue we can amplify pathogen DNA at the latent period or incipient stage, through which we can detect pathogens of C. oleifera anthracnose and root rot disease rapidly and sensitively.(2) By plate dilution method75endophytic bacterial strains were isolated from healthy C. oleifera and223bacterial strains and194actinomyces strains were isolated from38samples of different sources. Using C. oleifera anthracnose and root rot disease as the target, we screened the447strains primarily by pairing culture, and we obtained6endophytic bacterial strains and21bacterial strains whose inhibitory zone exceeded5mm and14actinomyces strains with inhibitory zone exceeding10mm. We also screened8phytopathogens secondarily and obtained3bacterial strains(strain R6, Z17, Z26) and2actinomyces strains (strain F10, CF17) with high efficient and broad-spectum antagonistic activity against main diseases of C. oleifera. By morphologic, physiological and biochemical characteristics and16S rDNA sequence analysis the strain R6, Z17, Z26. F10and CF17was identified as Bacillus subtilis, Paenibcillus kribbens, Brevibacillus brevis, Streptomyces globisporus subsp. globisporus and St. albulus, respectively.(3) The strain F10and CF17could affect the growth of mycelia and conidia germination of C. gloeosporioides and F. proliferatum, resulting into shortened and thick internode, twist deformation, serious cross linking of mycelia, cytoplasmic aggregation and the leak of cytoplasm. They both could secrete antifungal substance which was stable at room temperature but could not resist high temperature, ultraviolet, strong acid and strong base. They still retained antifungal effect on C. gloeosporioides and F. proliferatum after several passages without antagonistic effect on the strain Z26. The strain F10could secrete protease and cellulose which could resist fungicides such as Cupper hydroxide, mancozeb and promote seed germination of rice.(4)Based on comprehensive consideration of cell dry weight and inhibitory rate of CF17. we studied the effect of nutritional factors such as fermentation media. carbon and nitrogen and non-nutritional factors like inoculum size, medium volume and initial pH on bioactivity of fermentation broth. The results showed that the optimum fermentation medium of the strain CF17was as follows:glucose15g, yeast extract10g, NaCl lg.KH2PO4lg.CaCO33g. distilled water1000mL,pH7.0. the optimum inoculating quantity10%, flask content100mL/300mL,fermentation time7d, temperature30℃and rotation speed140r/min.(5)Raw powder was made up of the rice bran passed through a80mesh sieve and the fermentation broth of strain CF17at ratio of1:2. By determination of the antimicrobial activity and comprehensive assessment of wettability, dispersion and suspensibility we ultimately determine the optimal formula:the raw powder40%, kaolin38%, lignin sulfonate10%, SDS7%, humic acid5%. The potted experiment showed that the agent had good effect on the control of C. oleifera anthracnose, and the control effect of500times dilution could reach more than80%, which was superior to the control effect of carbendazim. In addition, it not only had good effect on the control of root rot disease, but also could promote the growth of C. oleifera seedlings.更多还原