【作者】 张清华；

【导师】 李国庆；

【作者基本信息】 华中农业大学 ， 植物病理学， 2014， 博士

【摘要】 植物内生真菌有着存在的普遍性和丰富的多样性,同时有着多样的生态学作用,包括促进植物生长、增强植物的免疫性等。油菜是我国的重要油料作物,对于油菜体内的真菌和细菌类群及其防治病害的作用缺乏系统研究。鉴此,我们开展了油菜内生真菌的多样性分析,并对其防治植物真菌病害的潜力进行了研究。取得的主要研究结果如下：1.利用组织分离法分离纯化并鉴定了油菜内生真菌的种类,分析油菜的不同营养器官(根、茎、叶)中内生真菌的分布,不同生育期内生真菌的种类。利用数学统计分析的方法,计算了油菜内生真菌根茎叶不同部位以及整体的香农指数和辛普森指数。共获得97株油菜内生真菌,鉴定为24个目40个种,其中子囊菌是最多,占总数的84.6%。根、茎、叶中菌株的数量分别是35株,49株和13株。根茎叶不同部位的辛普森指数分别为0.936、0.962和0.731。综合油菜各部位来源的内生真菌的辛普森指数为0.959,表明油菜内生真菌存在着丰富的多样性。在所分离和鉴定的内生真菌中,Alternaria alternata和Chaetomium globosum在油菜根、茎、叶中都有分布；5种真菌(Macrophomina sp.、Aspergillus versicolor、Penicillium pinophilum、 Fusarium tricinctum和Fusarium oxysporum)只存在于根部。2.平板拮抗筛选试验表明：24株内生真菌能够抑制核盘菌生长的菌株,形成宽度为3-17mm的抑菌带,这些菌株属于链格孢属(Alternariasp)、曲霉属(Aspergillus)、毛壳菌属(Chaetomium)、粘帚霉Clonostachys)、Dothidea、附球菌属(Epicoccum)、镰刀菌属(Fusarium)、Leptosphaeria和Simplicillium。其中的4株真菌,即A.flavipesCanS-34A. C. globosum CanS-73、C. rosea CanS-43和L. biglobosa CanS-51的代谢产物在油菜叶片上表现出稳定的抑制核盘菌(Sclerotinia sclerotiorum)侵染的效果。此外,还筛选出6株真菌产生挥发性抗真菌物质。3.发现35株内生真菌可以产生吲哚乙酸(IAA),包括链格孢(Alternaria)、曲霉属(Aspergillus)、短梗霉属(Aureobasidium)、毛壳菌属(Chaetomium)附球菌属(Epicoccum)、镰刀菌属(Fusarium)、香灰菌属(Hypoxylon)、Macrophomina、黑孢菌属(Nigrospora)、Periconia、茎点霉属(Phoma)和锁掷孢酵母(Sporidiobolus)。其中的3株真菌对油菜的侧根数量有明显的促进作用,它们是Fusarium proliferatumCanR-9和两株酵母锁掷孢酵母(Sporidiobolus sp.)CanS-62和Dioszegia zsoltii CanS-69。通过两年的盆栽试验证明：A. alternata菌株CanL-18和F. tricinctum菌株CanR-70及CanR-71r显示出促进油菜生长的效果。4.发习见A. flavipes CanS-34A产生的抗菌物质可以抑制11个属15个种(亚种)的植物病原菌,尤其对核盘菌(S. sclerotiorum)及其近缘种(S. trifoliorum, S. minor和S. nivalis)抑制率(74.9%-100%)显著高于对其它植物病原真菌的抑制率(13.9%-71.9%)。试验还证明CanS-34A产生的抗真菌物质可以耐80℃C以下的温度处理。采用饱和硫酸铵不能盐析出CanS-34A产生的抗生物质。5.发现Fusarium oxysporum CanR-46产生挥发性物质(Volatile organic compounds, VOCs),这种混合物能够抑制多种植物病原真菌的生长。GC-MS鉴定出19种VOCs,其中的5-Hexenoic acid、(±)-Limonene、3,4-2H-dihydropyrana和Octanoicacid等成分具有抑制病原真菌生长的作用。发现CanR-46产生的VOCs抑制包括灰葡萄孢霉和大丽轮枝菌在内的16种植物病原菌的生长,延迟灰葡萄孢霉和大丽轮枝菌分生孢子萌发,抑制萌发后芽管的伸长。扫描电镜观察,VOCs可以破坏灰葡萄孢和大丽轮枝菌菌丝的完整性,导致菌丝干瘪。CanR-46产生VOCs可以在密闭条件下有效防治番茄果实灰霉病。通过与大丽轮枝菌分生孢子混合接种,CanR-46可以有效防治棉花黄萎病,并能够在棉花根部定殖。上述研究结果为进一步利用油菜内生真菌防治油菜病害奠定了基础。更多还原

【Abstract】 The term’endophytes’refers to any organisms (bacteria or fungi) occurring within plant tissues without causing apparent hurt to that plant or showing any signs of existence in that plant. Endophytic fungi are ubiquitous and have been found in all plant species examined to date. Endophytic fungi are hyperdiverse, also. Simultaneously, endophytic fungi have diverse functional roles. In this study, the endophytic fungi were isolated from oilseed rape (Brassica napus) and identified based on morphological chacterieristics and rDNA-ITS. Biocontrol efficacy of these fungal isolates was evaluated in vitro and in vivo. The main results were summarized below:1. The endophytic fungi were isolated from root, stem and leaf of healthy oilseed rape. The endophytic fungi were identified based on morphology together with ITS sequences. Distribution of endophytic fungi in different organs (root, stem, leaf) and at different growing stage (seedling, budding, flowering and pod-development) was analyzed. The Shannon index (H’) and Simpson’s diversity index (1-D) were calculated. Total of97endophytic fungal isolates were obtained from roots (35), stems (49) and leaves (13) of oilseed rape. Forty fungal species were identified and most species (80%) belong to Ascomycota. The Simpson’s diversity index of root, stem and leaf were0.936,0.962and0.731, respectively.The all species composition is highly diversified with Simpson’s diversity index reaching0.959. The result indicated the diversity of ednophytic fungi of oilseed rape. The specieses Alternaria alternate and Chaetomium globosum existed in root, stem and leaf. Five specieses Macrophomina sp., Aspergillus versicolor, Penicillium pinophilum, Fusarium tricinctum and Fusarium oxysporum exist in roots only.2. Twenty-four isolates exhibited antifungal activity against S. sclerotiorum in dual cultures on potato dextrose agar plate forming inhibition zones of3-17mm in width. These strains included Alternaria, Aspergillus, Chaetomium, Clonostachys, Dothidea, Epicoccum, Fusarium, Leptosphaeria, Simplicillium genera. The culture filtrates of Aspergillus flavipes CanS-34A, Chaetomium globosum CanS-73, Clonostachys rosea CanS-43and Leptosphaeria biglobosa CanS-51in potato dextrose broth exhibited consistent and effective suppression of oilseed rape leaf blight caused by S. sclerotiorum. In addition, six strains were found to be able to inhibit S. sclerotiorum growth by producing volatile organic compounds. 3. Thirty-five endophytic strains were found that could produce IAA. These fungi included Alternaria, Aspergillus, Aureobasidium, Chaetomium, Epicoccum, Fusarium, Hypoxylon, Macrophomina, Nigrospora, Periconia, Phoma and Sporidiobolus sp. The cultural filtrates of the strains Fusarium proliferatum CanR-9, Sporidiobolus sp. CanS-62and Dioszegia zsoltii CanS-69were found capable of promoting lateral root growth of oilseed rape. A. alternata CanL-18, Fusarium tricinctum CanR-70and CanR-71r, and L. biglobosa CanS-51exhibited growth-promoting effects on oilseed rape during the two-year pot experiment.4. The antifungal substances of Aspergillus flavipes CanS-34A have been studied. The cultural filtrates of A. flavipes CanS-34A inhibited15plant pathogenic fungi. The percentage of growth inhibition was higher for four Sclerotinia species (S. sclerotiorum, S. trifoliorum, S. minor and S. nivalis)(74.9%-100%) than for the other11fungi (13.9%-71.9%). The antifungal activity of cultural filtrates was found resistant to heat treatment below100℃.5. Nineteen compounds were identified in the VOCs emitted by non-pathogenic Fusarium oxysporum CanR-46via GC-MS. The Fo-VOCs inhibited growth of all the16tested fungal species, delayed conidial germination, and suppressed germ-tube elongation of Be and Vd. Damaged (shriveled and collapsed) mycelia of both Be and Vd caused by the Fo-VOCs were observed under scanning electron microscope. Four (5-hexenoic acid, limonene,3,4-2H-dihydropyran and octanoic acid) synthetic chemicals present in the Fo-VOC profile showed antifungal activity against Be and Vd. The VOCs emitted from Fo cultures significantly (P﹤0.05) reduced disease severity of Botrytis fruit rot of tomato compared to the treatment Be alone. Treatment of cotton roots with Fo provided effective control of Verticillium wilt of cotton compared to the treatment with Vd alone. Colonization of the epidermis and vascular tissues of cotton roots by a GFP-transformed strain of Fo was consistently observed under confocal laser scanning microscope.These results might be useful for utilization of the endophytic fungi to control diseases of oilseed rape in the future.更多还原