The Study On Detection And Biological Control To The Main Pathogens Of Camellia Oleifera

Camellia oleifera anthracnose and root rot disease are major diseases in the major producing areas of C. oleifera of China. C. oleifera anthracnose usually causes fruit drop and flower-bud abscission with the percentage of fruit drop20%-40%in normal case and even more than80%in serious case, while C. oleifera root rot disease can rot roots of the infected trees, which results into abnormal growth and death, leading to enormously economic losses. At present, chemical pestieides are employed to control the diseases, which can easily induce pathogen to produce drug resistance, pollute environment and damage human health both directly and indirectly. This paper has studied the rapid detection of molecular techniques, antagonistic stains screening, identification, inhibitory mechanism, optimization of fermentation, formula of inocula and field experiment, which hopes to provide theoretical basis for sustainable control and lay a foundation for application.(1)By morphological characteristics of pathogens, determination of the pathogenicity, ITS (internal transcribed space) sequencing, and construction of phylogenetic tree we identified that C. oleifera anthracnose and C. oleifera root rot disease was caused by Colletotrichum gloeosporioides and Fusarium proliferatum respectively. Based on differences in ITS sequences of C. oleifera anthracnose. a pair of species-specific primers(YT1and YT2) was synthesized, with which we could detecte C. gloeosporioides with about amplified330bp band. But for the other16strains, none of them could be amplified. The pathogen with content lpg/25μL could be dectected using general PCR amplification, but it was the only10ag/25μL content that was enough by the nest-PCR, the detection sensitivity had increased100000times. According to differences in ITS sequences of C. oleifera root rot, a pair of species-specific primers (GF1and GF2) was synthesized, with which we could detecte F. proliferatum with about amplified400bp band. But for the other16strains, none of them could be amplified. The pathogen with content lpg/25μL could be detected using general PCR amplification. but it was the only100ag/25μL content that was enough by the nest-PCR, the detection sensitivity had also increased10000times.Using YT1and YT2specific primers and GF1and GF2specific primers and the nest-PCR technique combined with the DNA extraction methods of C. oleifera tissue we can amplify pathogen DNA at the latent period or incipient stage, through which we can detect pathogens of C. oleifera anthracnose and root rot disease rapidly and sensitively.(2) By plate dilution method75endophytic bacterial strains were isolated from healthy C. oleifera and223bacterial strains and194actinomyces strains were isolated from38samples of different sources. Using C. oleifera anthracnose and root rot disease as the target, we screened the447strains primarily by pairing culture, and we obtained6endophytic bacterial strains and21bacterial strains whose inhibitory zone exceeded5mm and14actinomyces strains with inhibitory zone exceeding10mm. We also screened8phytopathogens secondarily and obtained3bacterial strains(strain R6, Z17, Z26) and2actinomyces strains (strain F10, CF17) with high efficient and broad-spectum antagonistic activity against main diseases of C. oleifera. By morphologic, physiological and biochemical characteristics and16S rDNA sequence analysis the strain R6, Z17, Z26. F10and CF17was identified as Bacillus subtilis, Paenibcillus kribbens, Brevibacillus brevis, Streptomyces globisporus subsp. globisporus and St. albulus, respectively.(3) The strain F10and CF17could affect the growth of mycelia and conidia germination of C. gloeosporioides and F. proliferatum, resulting into shortened and thick internode, twist deformation, serious cross linking of mycelia, cytoplasmic aggregation and the leak of cytoplasm. They both could secrete antifungal substance which was stable at room temperature but could not resist high temperature, ultraviolet, strong acid and strong base. They still retained antifungal effect on C. gloeosporioides and F. proliferatum after several passages without antagonistic effect on the strain Z26. The strain F10could secrete protease and cellulose which could resist fungicides such as Cupper hydroxide, mancozeb and promote seed germination of rice.(4)Based on comprehensive consideration of cell dry weight and inhibitory rate of CF17. we studied the effect of nutritional factors such as fermentation media. carbon and nitrogen and non-nutritional factors like inoculum size, medium volume and initial pH on bioactivity of fermentation broth. The results showed that the optimum fermentation medium of the strain CF17was as follows:glucose15g, yeast extract10g, NaCl lg.KH2PO4lg.CaCO33g. distilled water1000mL,pH7.0. the optimum inoculating quantity10%, flask content100mL/300mL,fermentation time7d, temperature30℃and rotation speed140r/min.(5)Raw powder was made up of the rice bran passed through a80mesh sieve and the fermentation broth of strain CF17at ratio of1:2. By determination of the antimicrobial activity and comprehensive assessment of wettability, dispersion and suspensibility we ultimately determine the optimal formula:the raw powder40%, kaolin38%, lignin sulfonate10%, SDS7%, humic acid5%. The potted experiment showed that the agent had good effect on the control of C. oleifera anthracnose, and the control effect of500times dilution could reach more than80%, which was superior to the control effect of carbendazim. In addition, it not only had good effect on the control of root rot disease, but also could promote the growth of C. oleifera seedlings.