Diversity And Volatile Substances Of Endophytic Fungi From Litsea Cubeba (Lour.) Pers. (Lauraceae)

Endophytic fungi are abundant in plants.They are potential microbial resources,which have become a hot topic in the field of fungi in recent years.Endophytic fungi in plants have a long-term symbiotic relationship with their hosts in plant microecosystem,and their secondary metabolites have biological activities,such as anti-tumor,bactericidal,antioxidation,and plant growth,which is one of the effective ways to screen new bioactive components,and has potential development and application value in bio pharmaceutical and agricultural and forestry biological control.This study used tissue separation method to isolate endophytic fungi from Litsea cubeba health tissue and analyze the species diversity of endophytic fungi.The bacteriostasis strains were screened by plate confrontation test,and the morphological and molecular biological methods were used for classification and identification.At the same time,the active components were analyzed by active tracer.The main results are as follows:?1?970 strains of endophytic fungi were obtained from healthy tissues of Litsea cubeba,and the total isolation rate was 53.89%.There were 438 leaves?45.15%of the total number of bacteria?,241 stems?24.85%?,149 fruit?15.36%?and 142?14.64%?in the root,and the endophytic fungi were very rich in epiphyte.The number of endophytic fungi in Litsea cubeba was different,the low yield of oil was353?36.39%of the total number of bacteria?,the high yield oil rate was 336?34.64%?,and the middle oil rate was 281?28.97%?.?2?The endophytic fungi of Litsea cubeba were identified by morphological and molecular biological methods,belonging to 3 classes,9 orders and 17 genera of cysts,including 36 taxa,including 11 genera?67.57%?,3 genera?24.32%?,and 3 genera?8.11%?of cysts.The dominant flora of Endophytic Fungi of Litsea cubeba were Colletotrichum,Phomopsis,Diaporthe,Pestalotiopsis,Botryosphaeria and Fusarium,all of which belonged to common fungal groups.?3?Diversity analysis showed that the diversity index H'and Ds of endophytic fungi were 2.52 and 0.82 respectively,and the diversity index of endophytic fungi in the root was the highest?H'=2.74,Ds=0.90?,followed by stem?H'=2.56,Ds=0.90?,fruit?H'=1.99,Ds=0.76?,and leaves?H"?.The number of endophytic fungi in the root of Litsea cubeba was the most abundant,and the endophytic fungal flora of the leaf was unitary and unstable.The similarity coefficient analysis showed that the root and the stem endophytic fungi were the most similar,and the similarity index?CS?was 0.93.In addition,the difference between the root and fruit endophytic fungi was the largest,and the similarity coefficient was 0.58,followed by the second leaf.The similarity index with fruit was 0.80,root and leaf 0.79,stem and leaf 0.77,stem and fruit 0.64,and endophytic fungi among different tissues were significantly different.?4?Correlation analysis showed that the colonization rate and separation rate of endophytic fungi in different tissues were significantly different at the level of 0.01.Regression analysis showed that the endophytic colonization rate of Litsea cubeba was negatively correlated with low yield and medium oil production rate,and the endophytic colonization rate and oil production rate of Litsea cubeba were significantly positive.Correlation?p=0.018<0.05?.?5?Colletotrichum gloeosporioides,rice Rhizoctonia?Rhizoctonia solani?,rice evil seedling pathogen?Fusarium andiyazi?,shoot blight fungus?Ceratosphaeria phyllostachydis?,Phytophthora capsici,pear black spot pathogen?Alternaria alternata?,kiwifruit fruit rot pathogen?Phomopsis sp.?and other plant pathogenic indicator strains of inhibitory activity strains were screened by plate point confrontation method.The results showed that 69 endophytic fungi had at least 3 and more pathogenic fungi,of which SF14 and SF22 had strong inhibitory activity to 7pathogenic fungi.The strains SF14 and SF22 were identified as Penicillium minioluteum,Chaetomium globosum respectively by morphology and molecular biology.?6?The bacteriostasis activity of secondary metabolites and volatile substances of strain SF14 and SF22 were measured by filter paper diffusion method,mycelium growth rate method and plate clasp method.The results showed that the strain SF14and SF22 could produce antibacterial active substances,and the antibacterial activity was stronger.The volatile substances of strain SF12 and SF22 were determined by headspace solid phase microextraction?HS-SPME?and GC-MS.The results showed that the strain SF14 obtained 18 volatile components,the main active substances were twelve methyl ring six siloxane-C₁₂H₃₆O₆Si₆?Cyclohexasiloxane,dodecamethyl-?,1,3-two methyl benzene-C₈H₁₀?Benzene,?1,3-dimethyl-)and styrene-C₈H₈?Styrene?;strain SF22 obtained 16 volatile active components,the main active substances were2,3-butanediol-C₄H₁₀O₂?2,3-Butanediol?,p-xylene-C₈H₁₀?p-Xylene?and styrene-C₈H₈?Styrene?.In this study,the broad-spectrum antimicrobial active strains SF14 and SF22were screened from the endophytic fungi of Litsea cubeba,and their volatile substances had strong antibacterial activity to plant pathogenic fungi.The results provided a theoretical basis and scientific basis for the biological control of plant fungal diseases and the exploration of new active substances.