Etiology, Detection And Control Of Fusarium Wilt Of Chinese Water Chestnut

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.