Isolation And Identification Of The Pathogen Of Gastrodia Elata Brown Rot, Study Of Biological Characteristics And Whole Genome Sequencing Analysi

Gastrodia elata which is a traditional and precious Chinese medicine is one of the major varieties of Chinese medicinal materials cultivated in Guizhou province.The planting area in the province exceeds 31,000 hectares.In recent years,G.elata industry has played a crucial role in increasing farmers’income,achieving targeted poverty alleviation,assisting in poverty alleviation and rural revitalization.However,with the annual expansion of cultivation area and the continuous increase of cultivation years,the brown rot disease of G.elata has become increasingly prominent,causing it to brown and rot in the nest,seriously affecting its yield and medicinal value,and even more seriously causing"rotten nest"and"empty nest"of G.elata,leading to a great of production.This study conducted the isolation,purification,and identification of pathogenic fungi from diseased samples with typical symptoms of G.elata brown rot,clarifying the classification status of the pathogenic fungi of this disease.Based on the result,research on the biological characteristics of the pathogenic fungi and indoor fungicides screening were performed to obtaining agents and methods that have effective inhibitory effects on the growth of the pathogenic fungi of brown rot,while conducting genome wide sequencing analysis of the brown rot fungi.The results will provide a good theoretical foundation for the efficient and sustainable control of G.elata brown rot and for the further study of the interaction between pathogen and host.The research results are summarized as follows:1.Identification of the pathogen of G.elata brown rot in Guizhou provinceA total of 96 samples with typical symptoms of G.elata brown rot disease were collected from the main production area of G.elata in Guizhou province.After isolation and purification,150 pure cultures were obtained,of which 142 were preliminarily identified as three categories of pathogenic fungi based on morphology.After further morphological and molecular biological identification（ITS,ACT polygenic phylogenetic analysis）and pathogenicity testing.It was determined that the pathogenic fungi causing the brown rot of G.elata in Guizhou province were Fusarium oxysporum,F.solani,and F.redolens.2.Biological characteristics of the brown rot pathogen of G.elata and indoor toxicity determination of fungicidesBased on the identification of pathogen,three representative virulent F.oxysporum strain GZQK8,F.solani strain SX13 and F.redolens strain QK1 were selected for biological characteristics research.The results showed that the strains GZQK8,SX13,and QK1 could grow at 5 to 37℃and p H 4 to 10.The suitable growth temperatures were 30℃,30℃,and 28℃,the p H was 9,7,and 5 respectively.NA,PDA and CDM medium were the optimal medium for the three strains,respectively.The most suitable carbon sources were fructose,mannitol,and sucrose respectively.beef extract,sodium nitrate and sodium nitrate were the best nitrogen sources for growth,and the best light conditions were dark alternating,dark alternating and light alternating respectively.Strain GZQK8 died at 40 minutes at 60℃water bath,strain SX13 could not survive after being heated in a water bath at 60℃for 40 minutes,and strain QK1 remained alive after 40 minutes at 65℃water bath.The indoor toxicity test results of 35 fungicides against indicated that four chemical fungicides,including trifloxystrobin·tebuconazole,tebuconazole,carbendazim·thiram,and imazalil,as well as two biological fungicides,including tetramycin,and eugenol,had strong toxicity against the strains GZQK8,SX13,and QK1.The EC₅₀ values for strain GZQK8 were 0.606μg/m L,0.671μg/m L,0.543μg/m L,2.043μg/m L,0.514μg/m L and 2.915μg/m L.The EC₅₀ values for strain SX13were 0.084μg/m L,0.155μg/m L,1.169μg/m L,3.277μg/m L,0.408μg/m L and 3.057μg/m L.The EC₅₀ values for strain QK1 were 0.803μg/m L,1.942μg/m L,0.551μg/m L,1.519μg/m L,0.572μg/m L and 2.658μg/m L.Among them,the most toxic fungicides for the test were trifloxystrobin·tebuconazole and tetramycin,the EC₅₀ value ranges were 0.084 to 0.803μg/m L and 0.408 to 0.572μg/m L.Fungicides such as chloroisobromine-cyanuric-acid and boscalid,have no toxic effects on the tested strains.3.whole-genome sequencing and analysisThrough Illumina and Pac Bio sequencing and assembly of three strains of G.elata brown rot pathogens,the genome size of F.oxysporum strain GZQK8,F.solani strains SX13 and F.redolens strain QK1 was 51,204,719 bp,55,171,989 bp and 50,423,669bp,and the content of GC was 47.21%,50.61%and 46.30%,encoded 16,650,15,917and 14,851 genes respectively.The phylogenetic analysis based on the construction of a single copy gene of the entire genome was consistent with morphological identification and polygenic phylogenetic analysis,confirming the taxonomic status of brown rot fungus.Through analysis of secretory proteins and gene function annotations,it was determined that strains GZQK8,SX13,and QK1 encoded 25,18,and 24CAZymes carbohydrate and PHI pathogenic genes,respectively.The research results laid a foundation for further research on pathogen host interaction and the screening of pathogen pesticide targets.In conclusion,the pathogen causing brown rot of G.elata in Guizhou were F.oxysporum,F.solani,and F.redolens.trifloxystrobin·tebuconazole（0.084 to 0.803μg/m L）and tetramycin（0.408 to 0.572μg/m L）had the strongest inhibitory effect on the growth of pathogen hyphae and can be used as a preferred fungicides for field control experiments,tebuconazole,carbendazim·thiram,imazalil,and eugenol can be used as alternative fungicides.By analyzing the genome of the pathogen causing the brown rot of G.elata,some pathogenic related genes were preliminarily screened.This study can provide theoretical basis for the comprehensive prevention and control of G.elata brown rot,and lay a solid foundation for the prevention and control of G.elata disease.