Soil Disinfestations And Molecular Biology On Identification Of Soilborne Fungi

The purpose of the research is to screen out some effective methyl bromide alternatives and develop a rapid and reliable molecular method to identify and quantify soilborne fungi.Some important soilborne fungi were isolated by traditional method. And the ITS regions of the isolated fungi were cloned and sequenced, species-specific primers based the rDNA ITS were designed. Then we used real-time PCR to quantify soilborne fungi in the soil samples which were treated with different dosage of sulfuryl fluoride. The following results were obtained:(1) Soil fumigation treated with different dosage of sulfuryl fluoride, the results of preliminary experiments showed that sulfuryl fluoride at the rate of 5.44 × 10^-2g/L⁴3.52×10^-2g/L,presented good control efficacy to Fusarium oxysporum f. sp. meloni. The results were identical with the research results which were done in the field by our laboratory. All the results show that sulfuryl fluoride is a promising soil fumigant.(2) Some important soilborne fungi including Phytophthora capsici, Phytophthora infestans, Fusarium oxysporum, Pythium aphanidermatum, Alternaria solani were isolated, and some other related pathogen fungi, including Phytophthora parasitica, Phytophthora boehmeriae, Fusarium Avenaceum, Fusarium Moniforme, Fusarium graminearum, Fusarium solani, Verticillium dahliae, Botrytis cinerea were collected.(3) Fungi's DNA were extracted by using improved CTAB method, the ITS regions of Fusarium solani, Fusarium spp., Phytophthora capsici, Phytophthora infestans, Verticillium dahliae, Pythium aphanidermatum were cloned and sequenced, the length of the fragments were 544bp, 544bp, 793bp, 884bp, 541bp, 866bp respectively. The results of sequences analysis show that Fusarium solani and Fusarium spp. are likely to belong to Fusarium oxysporum f. sp. meloni. Other fungi's sequences were all identical with their names.(4) Species-specific primers kfu1/kfu2, ljym1/ljym2, dllz1/dllz2 based the rDNA ITS of Fusarium oxysporum, Phytophthora capsici and Verticillium dahliae were designed and have been confirmed good specificity and sensitivity when the anneal temperature is 65℃.(5) Using real-time PCR and traditional method to quantify the soilborne fungi in the soil samples which were treated with different dosage of sulfuryl fluoride, the results showed that sulfuryl fluoride has good control effect to Fusarium oxysporum, an important pathogenic organism on strawberry in Mancheng County. And a rapid and reliable molecular method to identify and quantify soilborne fungi and evaluate the effect of soil fumigation was developed.Real-time PCR can quantify DNA templet quickly, accurately and sensitively. The using of Real-time PCR to evaluate the effect of sulfuryl fluoride can afford a new tool for the field of pesticide research.