【作者】 李金云；

【导师】 王慧敏；

【作者基本信息】 中国农业大学 ， 植物病理学， 2004， 博士

【摘要】 葡萄根癌病是由葡萄土壤杆菌(Agrobacterium vitis)引起的一种在生产上造成了较大经济损失的世界性病害。无致病性的葡萄土壤杆菌E26菌株(A．vitis E26)是很有应用前景的根癌病生防菌株。本研究系统分析和研究了E26菌株的生防相关性状及其作用机理。 E26菌株的用量及应用时间对其防治作用有明显影响。在温室，以向日葵(Helianihus annus)为指示植物，当E26菌株先于葡萄根癌病菌K308菌株接种，或者，E26菌株的接种数量多于K308菌株时，其防治作用均比E26菌株与K308菌株以相同数量混合同时接种时的防治作用明显。在两个独立进行的田间实验中，当E26菌株与K308菌株以相同数量混合同时接种时，E26菌株均能够显著降低玫瑰香葡萄(Vitis vinifera cv Muscat humburg)的发病率，防治效果在70％以上。应用E26菌株可以有效预防葡萄根癌病的发生。 利用绿色荧光蛋白基因gfp标记示踪，检测了E26菌株浸沾硬枝扦插条后在葡萄根部环境的生存动态，分析了E26菌株与病原菌K308在葡萄植株伤口部位的相互作用。E26菌株可以在葡萄根表面和根际土壤中存活定殖，应用5个月后在根表面和根际土壤中均建立数量为10~3-10~4cfu／g根(土)的种群。E26与K308分别单独接种时均能以相似水平附着在伤口处；在E26与K308以相同数量混合同时接种时，附着在伤口细胞的K308的数量显著低于K308单独接种时附着在伤口部位的数量。扫描电镜观察证实E26能够和K308一样附着于葡萄根部伤口处。E26与K308能够竞争性地在伤口分泌液中共同生长，二者均可以利用葡萄组织中的主要营养物质。这些结果表明E26菌株是一个有效的伤口定殖菌，有可能在侵染位点与病菌竞争空间和营养而减少病菌的侵染。 从E26菌株发酵液中分离纯化了土壤杆菌素E26，物理和化学性质表明它可能是一种核苷类似物。土壤杆菌素E26对病菌的作用是通过干扰菌体DNA和RNA的合成实现的。比较了Tn5诱导的丧失产生土壤杆菌素能力的E26菌株的突变体ME005和ME084与野生型E26在温室和田间的防病效果。ME005和ME084都有一定的防治作用；但是，与野生型E26菌株相比，ME005和ME084的防病效果明显减弱。土壤杆菌素E26的产生是E26菌株生防机制中的重要因素，但是，不是唯一因素。 本研究结果表明E26菌株对葡萄根癌病的生防作用主要是预防作用，其机理复杂，有多种因子包含其中。产生土壤杆菌素、在伤口的定殖能力以及在侵染部位与病菌竞争空间和营养的能力都是E26菌株对葡萄根癌病的生防作用机制。更多还原

【Abstract】 Crown gall of grapevine, caused by pathogenic strains of Agrobacterium vitis, has been a problem with economical losses for extensive damage to grape health and quality all over the word. Since chemical pesticides are seldom effective and not environmentally friendly, biological control is receiving increased attention as a useful sustainable alternative. Nonpathogenic A. vitis strain E26 was naturally suppressive to crown gall bacteria both in vitro and in vivo tests, and was suggested to be a potential biocontrol agent of grapevine crown gall disease. The overall goal of this study was to determine mechanisms involved in the biological control process by strain E26.The effect of strain E26 dosage and applying time on suppression of tumor formation by a virulent strain of K308 on sunflower (Helianihus annus) was investigated in greenhouse. The suppression of tumor formation was more effective when E26 was inoculated either prior to strain K308 or at a higher concentration. In field tests conducted at two independent regions, E26 similarly exhibited strong effect of reducing the tumor index on grape (Vitis vinifera cv Muscat humburg) when co- inoculated with K308 at the same concentration. The controlling index was over 70% as compared with the check treatment.The dynamics of strain E26 were studied in grape root surface and rhizosphere under field conditions, using green fluorescence protein gene (gfp)-marked strains. Root colonization and survival in the rhizosphere of strain E26 was observed. Average populations of 104 cfu/g (fresh weight) of root and 104 cfu/g (dry weight) of soil of E26 were found 5 months after planting. The attachment and interaction of strain K308 and E26 directly on grapevine wound surfaces were also examined in vitro. E26 and K308 attached at a similar level to both stem and root explants. The attachment of strain K308 to grape cells was blocked by E26 in both systems. Scanning electronic microscope showed that strain E26 could attach to grape root wounded cells with the same manner as that of strain K308. Strain E26 and K308 were shown to grow competitively in grape stem wound exudates. There was also similarity in utilizing carbon and nitrogen nutrients reported to present in grape tissues in vitro between strain E26 and K308. These results indicated that E26 was an efficient colonizer of wound sites and suggested to exclude A.vitis from site essential for infection by competition for space and nutrients.Strain E26 was found to produce an agrocin that inhibited the growth of strain K308 on culture plate. This agrocin was isolated and purified from culture broth. Preliminary chemical analyses indicated that agrocin E26 was a nucleotide analogue. Agrocin E26 showed a broad host range of activity on tumorogenic bacteria. Mode of action of agrocin E26 was determined using strain K308.lt was shown to inhibit DNA and RNA syntheses.To determine the role of agrocin E26 in the control of crown gall, two Tn5- induced mutants, ME005 and ME084, that had lost their ability to inhibit A. vitis on plates were compared with the wild-type strain for their ability to control crown gall in greenhouse and fields. Although the two mutants grew both in culture medium and in grape stem explants at a rate similar to that of the wild-type strain and were active in suppressing crown gall, neither of them controlled crown gall as effective as strain E26 did. The data implied that agrocin production is an important factor of the biological control of grape crown gall by strain E26, but that another mechanism(s) is involved.The results obtained suggested that the biological control of grape crown gall by strain E26 is a complex process with a number of factors playing roles involved in. Agrocin production, efficient colonization on roots and wound sites, and competition for sites and nutrients are all one of these factors. The relative contribution of individual factors in the biocontrol situation is unknown at this time.更多还原