Isolation And Identification And Molecular Study Of Rice False Smut Villosiclava Virens, And Infection And Karyotype Cytological Analysis Of Rhizcotonia Solani

The important fungal diseases of rice, such as rice blast、rice false smut and rice sheath blight, seriously affects the yield and quality of rice and causes great financial losses.So we study in-depth on these fungal diseases and cultivate the resistant rice. Predecessors have researched extensively and in-depth on rice blast, such as infection process of Magnaporthe oryzae, virulence genes, resistant genes, and cultivated some resistant varieties of rice. However, rice false smut and rice sheath blight are relatively few in these aspects of research. Rice false smut is a worldwide fungal disease in rice caused by the Villosiclava virens, which harmed the panicles of rice and formed rice false smut balls. The control method was less on the agricultural production, and the pathogen resistance was increasing year by year. So far, predecessors did a little research on pathogenic fungi, which was limited to study on the field investigation, infection process and biological characteristics in the past, but there were less deep reports on the pathogenic genes, genotype and other aspects of the molecules level.Therefore, based on the above research background, the first part of the thesis is carried out on separation identification and molecular studies of Villosiclava virens, such as morphology, biological characteristics and genome. Meanwhile, the second part content mainly incleds the infection process and chromosomal karyotype cytology of rice sheath blight fungus. These results lay the foundation for knowing the infection cytology and pathogenic mechanism, and the interaction mechanism between the pathogen and host. This research mainly obtain the following results:One, the isolation of rice false smut pathogens. A total of 291 samples were collected in a field at Sichuan, Yunnan, Fujian, Zhejiang, Anhui and other places. We obtained 206 isolates by suspending chlamydospores liquid of the method, and the success rate was 70.5%. A 26 sclerotium specimen was collected in four places of Sichuan, and obtained 26 strains by the isolating of sclerotium. The success rate of the method was 100%.Two, observation of the morphological characteristics of the rice false smut pathogens. We observed the germination ways of the chlamydospores and conidia by using optical microscope and found two germination ways of the chlamydospores:one is that spore grows hyphae in side and produced the conidia at the top of mycelium; another kind is that the spores germinate on both sides and produced conidia at the top of hyphae. The two approaches are common ways of the chlamydospore germination. The surface of chlamydospores can be clearly observed to spines by using scanning electron microscopy, which were pointed at the apex or irregularly curved and were approximately 250-550 nm long. But the surface of chlamydospores, which were directly produced on artificial medium, were clearly observed to the arc-shaped spines. Sclerotia appeared black horseshoe-shaped and irregular oblong or flat, and sizes ranged from 2 to 20 mm. The interior of each sclerotium was intertwined with compact hyphae.Three, research on biological characteristics of rice false smut pathogens. In our study, we examined the effect of the culture medium, carbon and nitrogen source, temperature, pH, water potential and light on mycelial growth and the effects of temperature and wetness duration on conidial germination, and identified the conidial lethal temperature. Potato sucrose agar (PSA) was the best medium for fast mycelial growth, and wakimoto toceshi (XBZ) and potato dextrose agar also favored mycelial growth, whereas Czapek agar was not suitable. Sucrose (2.6 mm d-1) and starch (2.4 mm d-1) were the best carbon sources and ammonium nitrate (2.1 mm d-1), ammonium sulfate (2.2 mm d-1) and ammonium chloride (2.2 mm d-1) were the most suitable nitrogen sources for mycelial growth. The fungus was able to grow at temperatures from 12 to 32℃, with the optimal mycelial growth occurring between 28 and 30℃. The average radial growth rate on PSA was 2.5 mm d-1 at 28℃ and 2.4 mm d-1 at 30℃. Active mycelial growth was observed at pH between 4.5 and 11, and optimal growth was observed at pH 7 to 8. The fungus was able to grow at a water potential as low as-6 Mpa but did not grow at-8 Mpa. The mycelial growth rate was significantly higher in the dark than in 12 h alternating cycles of dark and fluorescent light, and fluorescent light inhibited mycelial growth. Conidial germination occurred at temperatures from 12 to 34℃, and the optimal temperature for germination appeared to be 28 and 30℃ for which a minimum wetness period of 2 h was required. The lethal temperature of the conidia was 50℃. These results provided a better understanding of the biology and ecology of V. virens and the environmental conditions required for the infection of rice.Four, Electrophoretic karyotype analysis of false smut V. virens. We observed the nuclear numbers of both the conidia and hyphae using DAPI staining. The development of the germ tubes resulted in the top of hyphae becoming mononuclear conidia. The secondary conidia sprouted hyphae, giving rise to multinucleate apical. Because the fungus has small chromosomes and the numbers were not previously known, we analyzed the electrophoretic karyotype using a pulsed field gel electrophoresis (PFGE) technique. The results showed that V. virens has at least 10 chromosomes ranging in size from 0.6 kb to 6 Mb. The V. virens genome size is estimated to be 23.6 Mb. These results supply a foundation for further study of gene recombination, modified gene and inserted new gene of chromosomal location of this devastating pathogen.Five, transformation of GFP gene of rice false smut V. virens. The experiment transformed the dual-vector pCAMBIAl300GFP, which was carried gpd and sGFP gene, into conidia of V. virens by using Agrobacterium tumefaciens mediated transformation (ATMT). The experiment determined that the selection concentration of hygromycin was 200}ig/ml to transformant of V. virens. The results showed that there were two DNA bands of PCR products by using the positive transformant of PCR amplification. The bands were hygromycin B and GFP gene respectively by sequencing and comparing the sequence of gene, and the size was 600bp and720 bp, respectively. Therefore, the ATMT technology can build the mutant library of V. virens, and it supplied a foundation for screening functional genes.Six, identification mating-type of rice false smut V. virens. The primers for the MAT1 genes were designed based on the high similarity to amino acid sequences of the alpha box and HMG box in Clavicipitaceae. The experiment can amplify the MAT 1-1-1 and MAT1-2-1 genes in conidia and ascospore isolates by multiplex PCR.The length of the mating type genes MAT1-1-1 and MAT1-2-1 were 256 bp and 222 bp, respectively. These results suggested that V. virens is homothallic fungi and does not need the participation of a partner. The results laid the foundation of molecular for genetic analysis and pathogenic gene of research, and so on.Seven, preliminary analysis of genome of rice false smut V. virens. The experiment built a 180bp DNA library of V. virens genome, and assembled using velvet (VI.2.03) software. The results showed that the genomic library contained 21970 contigs and 17904 scaffolds. The genomic library included 3.6 G bases, and the correct recognition rate of between the bases was 95.39%. In addition, the GC content genomic library is 51.498%. Although this experiment only build a small library of the genome of V. virens, it provided a effective way for assessing the quality of genome, predicting the pathogenic genes of V. virens, etc.Rhizoctonia solani AG1 is pathogenic fungi of rice sheath blight and cause the whole rice dead in serious. R. solani AG1, which have a wide range of host, can infect grain crops, vegetables and fruit trees, such as rice, wheat, corn, soybean, chinese kale. In the midst of several subgroups of R. solani AG1, predecessors had done a lot of research on subgroup I A, but little research on IC. Therefore, this part mainly contained two contents which do IC research. The results are as follows:One, infection process observation of R. solani IC infecting soybean and chinese kale. Hyphae of R. solani AG-1 IC began to grow on leaves of Chinese kale and soybean at 8-12 h after inoculation and grown on Chinese kale was faster than that on soybean. The fungal mycelium completely covered the leaves surface of Chinese kale leaves in 30-48 h after inoculation. Disease severity index analysis revealed that the disease symptom of Chinese kale severity higher than soybean at the same time after inoculation. When disease development on leaves of Chinese kale and soybean was compared (96 h inoculated), the whole leaves of Chinese kale appeared tan and had water immersion-shaped symptoms, while soybean leaves only presented oval and irregular brown disease spots. Hyphal tips of IC produced lobate appressoria, which were swollen hyphal tips. In the next step, several of the swollen tips simultaneously formed infection pegs. These infection structures directly penetrated into the epidermal cell wall of host tissue or invaded host tissue through stomata.Two, karyological analysis of R. solani IC. Karyological analysis of DAPI-stained nuclei was performed on hyphal cells of R. solani AG-1 IC. The number of nuclei ranged from 1 to 17, but most of the hyphal cells contained between 4 and 6 nuclei. In this study, we investigated the number of chromosomes per nuclei by tabletting and staining with magenta. Chromosomes were small and linear shape. The nuclei ranged between 10 to 18 chromosomes per nucleus, with 12 to 13 nuclei per chromosomes most frequently observed. These results will be helpful in future molecular and genetic studies.