Study On The Disease And Seed-Borne Fungi Of Trifolium Pratense (cv. Minshan)

Trifolium pretense（cv.Minshan）is an important forage species that can be grown in alpine and cool regions.However,there is a lack of systematic studies on the diseases that affect this species.In this study,we investigated the main diseases and occurrence patterns of T.pretense（cv.Minshan）in Min County,Gansu Province,China.We assessed the loss caused by disease,explored the biological characteristics of the pathogens,and conducted indoor toxicity tests of fungicides on the main pathogens.Additionally,we studied the seed-borne fungi of T.pretense（cv.Minshan）using both conventional isolation methods and high-throughput sequencing.（1）Our findings showed that the main diseases affecting T.pretense（cv.Minshan）in the field were leaf spots caused by Stemphylium sarcniiforme,Leptosphaerulina trifolii,and Longiseptatispora melilotii.Of these,Stemphylium leaf spot was the most serious,with a highest incidence rate of 26.67%and a disease index of 8.20.The incidence rate of Leptosphaerulina leaf spot was 4.80%,with a disease index of 1.53.The incidence of Longiseptatispora leaf spot was lighter,and it could only be found on young leaves,with a maximum incidence rate of 4.60%and a disease index of 1.27.（2）Stemphylium leaf spot significantly affected the yield and quality of red clover,resulting in a 20.14%decrease in fresh weight and 20.93%decrease in chlorophyll content.The crude fiber,crude protein,crude fat and ash content of red clover plants inoculated with the pathogen decreased 1.53%-6.67%.Among the 20 amino acids measured,the isoleucine content of diseased plants was significantly reduced by 64.98%compared to the control（P<0.05）,and the methionine and tyrosine contents were significantly increased by 273.68%and 61.35%（P<0.05）.There was no significant change in the content of metal elements.（3）Using conventional methods,a total of 15 genera and 26 species of seed-borne fungi were isolated from the four species.A total of 9 genera and 14 species of seed-borne fungi were isolated and identified from T.pretense（cv.Minshan）,6 genera and 7 species from T.pretense（cv.Ruide）,8 genera and 8 species from T.pretense（cv.VNS）,and 8 genera and 12 species from T.pretense（cv.Honglong）.The top 5 isolation rate of genera were Cladosporium,Alternaria,Penicillium,Aspergillus and Stemphylium.A total of 30 genera were identified from the seed samples using high-throughput sequencing,mainly in Ascomycota and Basidiomycota at phylum level,consisting of the genera of Cladosporium,Alternaria,Penicillium,Aspergillus,Stemphylium,Sporobolomyces,Filobasidium and Udeniomyces.Theαandβdiversity analyses showed significant differences in community richness and diversity among the species,while the diversity of the seed-band fungi of T.pretense（cv.Minshan）was significantly higher than the other three species（P<0.05）.S.sarcniiforme isolated from seeds was the same as the causal agent of Stemphylium leaf spot in the field,and the seeds were the potential source of primary infestation of the disease.The eight fungicides tested against S.sarcniiforme showed that 0.3%tetramycin had the highest toxicity with an EC₅₀ of 0.0594 mg/L.In the toxicity test against L.meliloti,450 g/L mimosine was the most virulent with an EC₅₀ of 0.6424 mg/L.250 g/L pyrimethanil was the most virulent with an EC₅₀ of 0.3203×10^-7 mg/L for L.trifolii.（4）Among the 8 major fungi isolated rate>1%,A.pseudoglaucus,A.alternata,P.cyclopium and S.sarcniiforme significantly reduced the seed germination of red clover by 18.25%,10.00%,18.25%and 19.48%（P<0.05）.Except for A.ruber,all inoculated seeds of red clover with the fungus showed varying degrees of mold rot and root rot.The growth of seedlings was significantly inhibited by A.ruber,P.cyclopium and P.chrysogenum,which significantly reduced the length of seedlings by 42.01%,45.86%and 36.95%compared to the control（P<0.05）.（5）The optimum growth temperature of S.sarcniiforme was 25℃,and the other two pathogens was 20℃.The optimum sporulation temperature of S.sarcniiforme and L.melilotii were 20℃and 25℃.The sporulation of L.trifolii was not found.The three pathogens grew best at p H 7 or alkaline conditions.The optimum growth media for S.sarcniiforme and L.trifolii were CMA and OMA,and L.melilotii were PDA and PDA.The best caarbon source for these three pathogens was maltose,and the best nitrogen source was peptone.Light had no effect on the growth of the three pathogens,but the lowest spore production was achieved in 24 h light.