Study Of Identification Of Pathogens And Molecular Detection Of Strawberry Root Rot

A high incidence of strawberry root rot which restricted development of strawberry planting, had been observed in production districts of Beijing in these years. In order to solve the problems in identification of pathogens of strawberry root rots and misuse of disease preventing and curing agent, identification of pathogens and molecular detection research of strawberry root rot had been studied in this paper.1. A Survey in strawberry planting areas Changping and Shunyi District of Beijing, from April2011to March2012, revealed that Strawberry root rot caused great economic losses had been one of the most important soil-borne diseases.105samples of the pathogens of strawberry root rot were isolated from disease tissue in affected plant which had gatherer random for field. Identificated the main species of the pathogens of strawberry root rot through the primary symptoms and microscopic observation.105isolated strains samples belong to Fusarium spp., Pythium spp., Rhizoctonia solani, Phytophthora spp., Pestalotiopsis clavispora and Sclerotinia sclerotiorum species. Among the six specie, A high incidence of Fusarium spp. and Pythium spp. indicated that the two species were dominant microflora in strawberry planting areas in Beijing.2. Pathogens of strawberry root rot were confirmed based on morphology and molecular biology method, through pathogenicity test.105isolated strains were inoculated the healthy beauty strawberry seedlings, only Fusarium spp. and Pythium spp. species had strong pathogenicity. So the pathogens of strawberry root rot in Beijing were primarily identified belong to theses two species.According to the Booth’s, Nelson’s Fusarium classification system and Yu’s Pythium classification system, the pathogens of strawberry root rot were confirmed belong to Fusarium oxysporum and Pythium ultimum through microscopic observation. ITS1/ITS4sequences of the isolations pathogens were amplified by PCR technique with universal primers of fungi, and the sequences were analyzed by comparing with those in GenBank with BLAST. The molecular identification indicated the same results as morphology identification.3. Gained a Real-time PCR detection system which used Fusarium oxysporum strain CM11032435as targets. Used strain CM11032435which had strong pathogenicity as targets, Constructed a expression vector with specific target sequence, expressed by E.coli expression system, the transformed plasmid of Fusarium oxysporum were formed. Gradient dilution method by plasmid was used to establish fluorescence quantitative standard curve. The detection limit of the systems was 0.37fg/μL,10CFU/g soils,The pathogens samples from the affected plant and soil were detected by Real-time PCR technology with all samples quantitatively assessed.4. Gained a Real-time PCR detection system which used Pythium ultimum strain CM11031012as targets. Constructed a expression vector with specific target sequence, expressed by E.coli expression system, the transformed plasmid of Pythium ultimum were formed. Gradient dilution method by plasmid was used to establish fluorescence quantitative standard curve. The detection limit of the systems was1.79fg/μL,100CFU/g soils;The pathogens samples from the affected plant and soil were detected by Real-time PCR technology with all samples quantitatively assessed.The species of pathogens of strawberry root rot were identificate firstly in this paper, and it is the first report for the detection of pathogens of strawberry root rot from the affected plant and soil by real-time PCR technique. This research will be helpful to confirm the species of the pathogens of diseases, and give an academic reference and practice methods on early warning, prevention and cure of diseases...