【作者】 郑青松；

【导师】 于汉寿； 陈怀谷；

【作者基本信息】 南京农业大学 ， 生物工程， 2014， 硕士

【摘要】 小麦全蚀病是由禾顶囊壳小麦变种(Gaeumannomyces graminis var.tritici)引起的真菌病害,在世界上许多小麦产区都有发生,为典型的小麦根部病害。小麦全蚀病首先在中国浙江省发现,随后在我国多个小麦产区发生,目前病害发生仍呈加重趋势。为筛选对小麦全蚀病有控制作用的生防菌,本研究从河南商丘全蚀病发生田块的小麦根际土,利用稀释分离法共获得648株细菌,通过平板生防测定初筛出对小麦全蚀病菌具有生防作用的细菌41株,再从中筛选出抑菌带大于0.3 cm或虽然抑菌带小于0.3 cm但长势强的细菌20株。根据20株细菌的16S rDNA和rpoB基因的序列对这些菌株进行了分子鉴定,并对这些菌株在温室盆钵试验中的防效进行了测定。进一步挑选出9株代表性细菌菌株(GP4,GP9,GP16,GP46,GP51,GP52,GP54,GP56,GP59)进行下一步研究。根据16S rDNA和rpoB的BLAST结果以及生理生化分析的结果表明这9株细菌均为假单胞菌。其中GP4,GP9,GP16,GP46,GP59为Pseudomonas poae,GP51和GP52为Pseudomonas chlororaphis,GP54和GP56为Pseudomonas putida。生理生化试验结果表明：9个菌株都产生蛋白酶；GP51.GP52.GP54和GP56可以产生嗜铁素；所有菌株均不产生几丁质酶。对9株细菌进行了生防物质产生相关基因的检测,这些菌株基因组中都不含2,4-二乙酰基间苯三酚和藤黄绿脓菌素产生相关基因；只有GP51,GP52含有吩嗪-1-羧酸和硝吡咯菌素产生相关基因。温室防效试验结果表明9株生防细菌对全蚀病都有一定的生防效果,其中GP51,GP52的效果最好,防效分别达到30.5%和33.3%。在明确9株生防菌对小麦全蚀病具有较好防治效果的基础上,进一步用平板生防试验测定了它们对其他一些小麦根部病害病原菌的抑制作用。结果表明9株细菌对小麦根腐病菌的抑制效果普遍较好,抑菌带宽度均在3.5 mm以上,只有GP51.GP52菌株对禾谷丝核菌有一定的抑制效果,对小麦赤霉病菌有抑制作用的菌株有GP9、GP16、GP46、GP51和GP59,但抑菌效果较差,这些生防菌对小麦茎基腐病菌不同化学型菌株的抑制效果存在差异。为了解9个生防菌在小麦根部的定殖情况,通过驯化筛选获得9株生防菌的利福平抗性菌株,用含菌玉米砂接种的方法分别测定了原始菌株与驯化菌株对小麦全蚀病的防效。这一步测定了6株细菌(2株Pseudomonas poae GP9和GP59,2株Pseudomonas putida GP54和GP56以及2株Pseudomonas chlororaphis GP5和GP52)在小麦根部定殖情况,结果表明这些菌在灭菌的土壤中的定殖量高于自然土的定殖量。其中GP51和GP52在4个处理中的定殖效果均较好,在一定程度上可解释GP51和GP52的具有较好生防效果的原因。本研究为小麦全蚀病的生物防治提供了重要的菌株材料,为后续高效生防菌的筛选、田间应用试验奠定了基础。更多还原

【Abstract】 The wheat take-all is a fungal disease caused by Gaeumannomyces graminis (Sacc.) Arx& Oliver var. tritici Walker. It is a typical wheat root disease and occurs in many wheat growing areas worldwide. The take-all of wheat was first discovered in Zhejiang province, and then the disease was discovered in other wheat growing regions in China. Currently, the occurrence of disease is becoming severer and.severer in Huanghui winter wheat growing areas.In order to obtain antagonistic bacteria against Gaeumannomyces graminis var. tritici, we isolated 648 strains of bacteria from the rhizosphere of wheat plants collected from Shangqiu City of Henan Province. We obtained 41 antagonistic bacterial strains against Gaeumannomyces graminis var. tritici through plate antagonistic determination experiment, and then we selected 20 strains of bacteria according to the antibacterial band width (not less than 0.3 cm or vigorous growth). They were identificated based on the sequences of 16S rDNA and rpoB gene, and the control effect of these strains was determined in greenhouse pots. We selected 9 representative bacterial strains for the further research and the nine antagonistic bacteria strains are GP4, GP9, GP16, GP46, GP51, GP52, GP54, GP56 and GP59 respectively.The molecular identification and physiological and biochemical test results showed that strain GP4, GP9, GP16, GP46, GP59 belong to Pseudomonas poae, and GP51 and GP52 were Pseudomonas chlororaphis, and GP54 and GP56 were Pseudomonas putida. The antifungal metabolites of 9 strains was analysed and the results indicated that all 9 strains produced proteinase, and GP51, GP52, GP54 and GP56 produced siderophore and lipopeptides, but they all can’t produce chitinase. The genes for antagonistic substances of the 9 strains were detected. None of them contained genes for 2,4-DAPG and Pyrrolnitrin production. GP51 and GP52 were only detected to contain genes forPhenazine-1-carboxylic acid, Pyoluteorin production. The disease control effect experiment in greenhouse showed that 9 strains can decrease disease index of wheat take-all, among which GP51 and GP52 were more effective, the relative control effect reached 30.5% and 33.3%.The antagonistic effect of 9 strains on other soil borne fungal pathogens was also tested. The results showed that all 9 bacteria strains had the high antagonistic effect on Bipolaris sorokinianum, and the inhibition zone were above 3.5 mm. Among 9 strains of bacteria, only GP51, GP52 strains had some antagonistic effect on Rhizoctonia cerealis, and strain GP9, GP16, GP46, GP51 and GP59 has weak antagonistic effect on Fusarium graminearum. These bacteria strains had different antagonistic effect on different chemical type pathogens of the wheat crown rot.In order to understand the colonization of these 9 bacteria strains in wheat rhizosphere, we developed rifampicin resistant strains. The colonization of the 6 bacteria strains including Pseudomonas poae (GP16 and GP59), Pseudomonas putida (GP54 and GP56) and Pseudomonas chlororaphis (GP51 and GP52) in wheat rhizosphere was determined, The results showed that the colonization of all 6 bacteria strains were better in the sterile soil than in the natural soil. The rhizosphere colonization of GP51 and GP52 were the best. It may explain the better biocontrol effect of GP51 and GP52 in a certain extent.The research provides strains for biological control of wheat take-all disease, and laid a foundation for selecting more effective bio-control strains and for control of wheat take-all in fields.更多还原