【作者】 朱志贤；

【导师】 黄俊斌；

【作者基本信息】 华中农业大学 ， 植物病理， 2014， 博士

【摘要】 荸荠枯萎病是中国荸荠生产上的最重要病害之一,主要为害植株的茎基部,最终导致植株倒伏或枯死,病株地下部结出发育差的浅白色球茎或不结球茎,或者球茎变成黑褐色腐烂,从而严重影响了荸荠的产量和品质。目前,对该病害只在国内有报道,且只进行了病原菌形态学鉴定、生物学特性和化学防治方面的初步研究,并没有进行系统的研究。本研究通过形态学、致病性测定和分子生物学手段对荸荠枯萎病菌进行鉴定,在此基础上对荸荠枯萎病菌遗传多样性、病原菌分子检测及病害田间发生动态进行研究并结合生产实际探讨了病害的防治技术。研究结果如下：1.为了鉴定荸荠枯萎病的病原菌,从湖北,浙江,福建,安徽,湖北,湖南6个不同省的发病荸荠植株上分离获得69株镰刀菌。根据形态学及分子生物学特征鉴定出了Fusarium commune (92.8%)是主要的分离物,广泛地分布在这6个省,在湖北省和浙江省还发现了藤仓赤霉复合种(GFSC)的新种(5.8%),同时在湖北省还发现了一种未知的镰刀菌(1.4%)。随机从不同省份选择了30株F. commune菌株和所有的GFSC菌株分析其线粒体小亚基rDNA (mtSSU),α-延伸因子(EF-1α)和核糖体基因间隔区(IGS)的序列。最大简约法和贝叶斯方法单独或联合分析这3个序列片段均显示这2种镰刀菌分别形成一个独立的分支。不同地方的菌株位于散布于系统发育树的不同分支上,显示与地理来源无联系。致病性测定结果表明这3种镰刀菌均可导致荸荠‘团风七号’品种枯萎,病原菌致病力大小显示与地理来源无相关性。这是在中国首次报道F. commune,藤仓赤霉复合种(GFSC)的新种,Fusarium sp是荸荠枯萎病的病原菌。2.建立了适合于F. commune的ISSR-PCR反应体系,对34条ISSR引物进行筛选,获得5条具有较高的多态性的ISSR引物。用这5条引物对供试的60株荸荠枯萎病菌F. commune进行ISSR扩增,对其遗传多样性分析显示,基因多样性指数(H)为0.22,Shannon信息指数(Ⅰ)为0.35,表明我国荸荠枯萎病菌具有丰富的遗传多样性。对不同地理种群遗传结构与分化分析结果表明,荸荠枯萎病菌在各群体间遗传多样性存在差异,群体内多样性大于群体间多样性,地理种群间存在着一定的基因流动,但各种群间的基因流动受到了一定的阻碍。6个省份11个不同地区子群体间遗传变异结果表明,湖北省团风县和福建省龙海市遗传相似性最低,遗传距离最大。湖北省团风县和湖北省孝感市遗传相似性最高,遗传距离最小。按照非加权算术平均(UPGMA)法聚类对种群聚类分析发现,11个不同种群可聚为四类,其中湖北省和安徽省群体亲缘关系最近。当相似系数设定为0.79时,用UPGMA聚类分析法可将60个供试菌株分为4个类群,其中类群Ⅱ包括了49个菌株,为我国荸荠枯萎病菌的一个优势类群。同时,聚类分析也说明菌株之间的遗传多态性与其地理来源之间无明显的相关性。3.根据荸荠枯萎病病原菌F. commune的mtSSU序列设计了一对引物(FO1/FO2)用于实时荧光定量PCR(qPCR)检测。参与引物特异性PCR检测的41个菌株,只有来自6个不同省份的的21株F. commune扩增出一条大小为178bp的特异性条带。用10倍梯度稀释的F. commune基因组DNA,根据扩增结果绘制qPCR标准曲线为：Y=-3.707x+31.85(R2=0.994,E=86.08％),检测灵敏度为1fg/μ1。加入10ng植物组织DNA至10倍梯度稀释的F. commune基因组DNA,根据扩增结果绘制标准曲线为：Y=-4.179x+33.84(R2=0.990,E:73.38％),检测灵敏度为1pg/μ1.由于植物组织DNA的加入影响了qPCR的扩增效率和标准曲线的相关系数,因此用加入植物DNA后获得的标准曲线计算植物组织中F. commune的DNA含量。以不同浓度分生孢子液接种土壤的总DNA为模板进行qPCR扩增,土壤中分生孢子数目的对数(X)和对应的土壤DNA在qPCR反应的CT值(Y)之间的线性关系为：Y=-3.039X+43.48(R2=0.990,E=113.30％),检测灵敏度为1,000个孢子/克土壤。荸荠茎秆中F. commune的DNA含量与枯萎病发病严重度呈显著正相关,不同发病级别植株下土壤中的F. commune的含量与枯萎病发病严重度无显著相关性。2012年荸荠枯萎病病情指数和F. commune在土壤中的含量呈显著正相关Y=2674X+24750,但是2011年荸荠枯萎病病情指数和F. commune在土壤中的含量无显著相关性。该qPCR方法可以快速和特异性地检测植物和土壤样品中的F.commune,这将有利于对病原菌进行监测,并有利于病害的防治。4.2010-2012年荸荠枯萎病田间发生动态调查数据表明,三年田间病害发展趋势基本一致,病害于8月中旬开始发生,由于温度较高,一直处于零星发生阶段。8下旬-9月上旬,温度适宜,病害开始缓慢增长。9月下旬-10月中旬为该病的高峰期,之后病害发生呈不断增长趋势至最后一次调查。三年的病情指数均与温度呈负相关,但各年份的病情指数与相对湿度和降雨量无显著相关性。通过12种药剂在PDA平板上对F. commune进行的抑菌试验,筛选出10%苯醚.甲环唑,25%多菌灵可湿性粉剂,40%氟硅唑,25%嘧菌酯4种药剂用于处理荸荠球茎。球茎药剂处理室内盆栽和田间试验结果均表明四种药剂处理均有利于荸荠出苗。球茎药剂处理假植到大田2个月后经多菌灵和氟硅唑处理的球茎对荸荠枯萎病仍有一定的防治效果,防效分别为22.3%,27.0%,嘧菌酯和苯醚.甲环唑均无防效。荸荠枯萎病抗病评价研究结果表明,沙洋荠、肇庆荠、韶关马坝荠、桂林-1在两年的田间试验中均表现出对枯萎病有较强的抗性。更多还原

【Abstract】 Fusarium wilt is one of the most important diseases of Eleocharis dulcis (Chinese water chestnut) in China. The stem base is the main infection area. The disease can eventually lead the whole plant to fall down or death. The underground of the diseased plants bear white corms, or not bear corms, or corms become dark brown rot. E. dulcis yield and quality can be significantly reduced. Thus far the disease has only been reported in China, and limited studies on morphological identification, biological characteristic and chemical control have been conducted. In this paper, the causal agents of Fusairum wilt were identified based on morphology, pathogenicity and molecular anlyses. And then, genetic divercity, molecular detection of the pathogen, occurence and control of Fusarium wilt disease were studied. The research results were summarized as follows:1. In order to characterize the pathogens responsible,69Fusarium isolates were collected from diseased plants in E. dulcis production areas of the Chinese provinces Anhui, Fujian, Hubei, Hunan, Jiangsu and Zhejiang. These were then identified based on morphological and molecular characteristics. F. commune was the most common species (92.8%), and widely distributed in the six provinces, a novel species within the Gibberella fujikuroi Species Complex (GFSC) was found in Hubei and Zhejiang provinces (5.8%), and an unidentified Fusarium sp. was also found in Hubei province (1.4%). Thirty F. commune isolates from different provinces and four GFSC isolates were selected for sequence analyses of the translation elongation factor1-a (EF-la), the mitochondrial small subunit rDNA (mtSSU), and the nuclear ribosomal intergenic spacer region (IGS). Maximum parsimony and Bayesian analyses of the multilocus sequence data of these two species plus other taxa showed that the two species formed two distinct, well-supported clades among the three individual and combined gene genealogies. Isolates from different locations were scattered, with no evidence of geographic specialization. Pathogenicity assays showed that the two Fusarium species, including the unidentified Fusarium sp. were pathogenic to E. dulcis cv.’Tuanfeng seven’. There was no relationship between the source of isolates and their pathogenicity. This is the first description of F. commune, a novel species within the GFSC and an unidentified Fusarium sp. as causal agents of Fusarium wilt of E. dulcis in China.2. A suitable ISSR-PCR reaction system for F. commune was established. Five ISSR primers showed high polymorphism were screened out from34ISSR primers. The five primers were used to amplify60isolates of F. commune. The result of genetic diversity analyses showed that gene diversity index (H) and Shannon’s information index (I) were0.22and0.35, which indicated that the genetic diversity was considerably abundant among F. commune. The results of population genetic structure and differentiation of different geographical groups indicated that genetic variation within population was the main source of genetic variation. There was a certain gene flow between different geographical groups, however, it was obstructed. The results of genetic variation of11different geographical groups from six provinces showed that the genetic identity between Tuanfeng county, Hubei province and Longhai city, Fujian province was the lowest, and the gentetic distance was the most longest. The genetic identity between Tuanfeng county, Hubei province and Xiaogan city, Hubei province was the highest, and the gentetic distance was the closest. The11different geographical groups were clustered into four clusters by unweighted pair-group mean average (UPGMA) analysis, and the genetic relationship between population of Hubei province and Anhui province was the closest. The60tested isolates were cluster into four clusters at the genetic similar coefficient0.79based on UPGMA analysis. The cluster Ⅱ contained49isolates, including the majority of tested isolates, which indicated that cluster II was the dominant population of F. commune in China. There was no significant correlation between the genetic polymorphism of isolates and their geographical origins.3. A SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) assay was developed based on mtSSU sequences of F. commune. Assay specificity of FO1/FO2primers was tested on41fungal isolates. A single PCR band of approximately178bp DNA fragment was only amplified for21tested F. commune isolates collected from six provinces. Standard regression line for10-fold serial dilutions F. commune DNA was Y=-3.707X+31.85(R2=0.994, E=86.08%), with a detection limit of1fg/μl. When10ng DNA extracted from plant tissue was added to the10-fold serial dillutions of F. commune genomic DNA, the standard regression line was Y=-4.179X+33.84(R2=0.990, E=73.38%), with a detection limit of1pg/μl. Since the efficiency and correlation coefficient of the qPCR assay standard regression lines were influenced by presence of the host DNA, standard regression line of fungal genomic DNA added with the host DNA was used to estimate the amount of F. commune in plant tissues.When conidia were added to sterile soil, a linear relationship was obtained between the logarithm of inoculum concentrations (X) and the quantification cycle threshold values (Y):Y=-3.039X+43.48(R2=0.990, E=113.30%). The amount of target fungal DNA in stem tissues detected by qPCR was significantly correlated with the disease level, however, the qPCR assay showed no significant positive correlations between spore density in soil of different Fusarium wilt severity groups and disease level. The spore density of F. commune detected was positively correlated with disease index in the2012growing season, but not in2011. The qPCR method can be used for rapid and specific detection of F. commune in plant and soil samples, which will facilitate monitoring of this pathogen, and potentially improve plant disease management.4. The results of occurence study for Fusarium wilt of E. dulcis throughout2010to2012growing seasons showed that the disease development trend was nearly the same during the three years. Symptoms of the disease were initially occurred in the middle August. Due to the high temperature, the disease sporadically happened; Because of the appropriate temperature, late August to early September was suitable for the disease, and it began to grow slowly; Late September to middle October was the peak stage of the disease and disease index kept increase until the last survey. The disease index of the three years was negatively correlated with temperature, but has no significant correlation with relative humidity and rainfall.10%difenoconazole,25%carbendazim,40%flusilazole and25%azoxystrobin were screened out from12fungicides through inhibition of mycelial growth of F. commune and they were used for fungicidal corm treatments. The results of pot and field fungicidal corm treatments experiment indicated that all the four fungicides were good for E. dulcis corms to germinate. The results of field fungicidal corm treatments experiment indicated that two months after provisional planting, corms treated by carbendazim and flusilazole still have control effect, with control efficiency of22.3%and27.0%, respectively. Neither difenoconazole nor azoxystrobin had control efficiency. The evaluation of E. dulcis cultivar resistance showed that Shayang, Zhaoqing, Shaoguan maba and Guilin-1water chestnut have a relative strong resistance to Fusarium wilt in two year field trials.更多还原