| Polygala fallax is a Polygala medicinal plant of Polygalaceae,rich in active ingredients such as saponins,flavonoids and polysaccharides,and the endophytic fungi of medicinal plants are considered to be an important resource library for obtaining new medicinal active substances.Previous study had found that the diversity of endophytic fungi was relatively rich in P.fallax.Therefore,in order to better excavate and utilize the endophytic fungi,this study took the endophytic fungi of P.fallax as the research object,and used the plate confrontation method to evaluate the inhibitory activity of the endophytic fungi against 6 kinds of phytopathogenic fungi.The antioxidant activity of endophytic fungi was evaluated by DPPH free radical scavenging ability and total reducing ability index.And two highly active endophytic fungi HNLF-5 and HNLF-44 were further screened out,and then the two endophytic fungi were identified and belonged to.Then the growth rate method was used to determine the antifungal activity of the ethyl acetate crude extract and the 30-fold concentrated fermentation broth of the two strains,and the allelopathic effect of the fermentation broth stock solution and the 30-fold concentrated fermentation broth was detected by the seed germination method.Finally,the whole genome framework strain with important research value was sequenced,the sequencing results were annotated,and anti SMASH was used to predict secondary metabolite synthesis gene clusters and products.The main findings are as follows:1.HNLF-5 and HNLF-44 strains with significant antifungal activity were screened from 21 endophytic fungi of P.fallax by plate confrontation method.The inhibition rate of HNLF-5 and HNLF-44 against Fusarium oxysporum f.sp.cubense,Diaporthe citri,Fusarium solani,Phyllosticta zingiberi,Fusarium oxysporum,Mycosphaerella fijiensis was range from 50.3% to 91.4%.The antifungal rate of HNLF-5 against 6 pathogenic fungi ranged from 50.3% to 73.2%,and the antifungal effect of HNLF-5 against Diaporthe citri was the best,with a inhibition rate of 73.2%,followed by Phyllosticta zingiberi,with an anfungal rate of 70.3%.The inhibition rate of HNLF-44 was between 60.7% and 91.4%,and the antifungal effect of Fusarium oxysporum f.sp.cubense was the best,with an inhibition rate of 91.4%;followed by Diaporthe citri,with an inhibition rate of 67.7 %.This result indicates that endophytic fungi HNLF-5 and HNLF-44 have good antifungal activity.Morphological and molecular biological identification showed that HNLF-5 was a fungus of the genus Diaporthe sp.,and HNLF-44 was a fungus of the genus Alternaria burnsii.2.Antioxidant activity assay of 13 endophyte fungi showed that the DPPH clearance rate of endophytic fungal fermentation stock solution could almost reach 90%,and the absorbance of total reducing ability ranged from 0.279 2 to 0.748 8.The fermentation broth after ethanol treatment,the DPPH clearance rate ranged from 67.22% to 85.65%,and the absorbance of the total reducing ability ranged from 0.216 7 to 0.353 3.The fermentation broth after ethyl acetate treatment,the DPPH clearance rate ranged from31.13% to 45.23%,and the absorbance of the total reducing ability ranged from 0.132 1 to0.247 6.It shows that no matter the fermentation stock solution of endophytic fungi,alcohol precipitation solution or ethyl acetate extract have better DPPH removal ability and total reduction ability.3.The activity of the fermentation products of strains HNLF-5 and HNLF-44 was measured,and the results were as follows:（1）The antifungal activity of HNLF-5 ethyl acetate crude extract was enhanced with the increase of concentration.When the concentration was 10 mg/m L,the inhibition rates of Fusarium oxysporum f.sp.cubense,Mycosphaerella fijiensis,Diaporthe citri and Phyllosticta zingiberi were 43.3%,59.1%,60.1% and 49.47%,respectively.While the 30-fold concentrated fermentation broth had poor antifungal activity,with antifungal rates of 12.1%,20.8%,12.4%,and 32.4%,respectively.The ethyl acetate crude extract of HNLF-44 has poor antifungal activity against 6 pathogenic fungi,and the inhibition rate is all below 29.1%.While the 30-fold concentrated fermentation broth had 70.3% inhibition rate against Fusariun oxysporun f.sp.cubense,but the inhibitory activity against other pathogens was poor,and the antifungal rate was 18.9% to 31.5%.（2）HNLF-5 and HNLF-44 fermentation broth promoted the root length and shoot growth of rapeseed seeds during the germination period.Compared with the control group,the root length and shoot length of seeds treated with HNLF-5 increased by 0.6% and 1.1%,while those treated with HNLF-44 increased by 0.3% and 27.5%.The30-fold concentrated fermentation broth of HNLF-5 and HNLF-44 inhibited the germination rate,root length and shoot length of rape seeds.The inhibition rate of HNLF-5was 24.3%（germination rate）and 24.2%（root length）and 8.0%（bud length）,and HNLF-44 was 41.8%,86.0% and 17.4%.4.Whole-genome of HNLF-44 was sequenced by next-generation sequencing technology,and its functional annotation and prediction of secondary metabolite synthesis gene clusters were performed.The results showed that the genome size of HNLF-44 strain was 28671348 bp,the GC content was 51%.A total of 10692 coding genes were involved in the GO,KOG and KEGG annotation pathways,and 1,005 genes were annotated into the carbohydrate-active enzyme family（CAZy）.28 secondary metabolite synthesis gene clusters were predicted in the HNLF-44 genome,of which 8 known secondary metabolite synthesis pathways were associated with three pathways: T1PKS（type I polyketide synthase gene cluster）,NRPS（non-ribosomal peptide synthase）,and terpene（terpene synthesis pathway gene cluster）.Possible secondary metabolites are alternapyrone,alternariol,melanin,chichorine,abscisic acid,desmethyl TAN-1612,dimethylcoprogen,TAN-1612/1-（2,3,5,10-tetrahydroxy-7-methoxy-4-oxo-1,2,3,4-tetrahydroanthracen-2-yl）pe ntane-2,4-dione/desmethyl TAN-1612 and squalestatin S1.The study shows that the endophytic fungi of P.fallax had good antifungal activity and antioxidant activity,which provided a theoretical basis for the development and application of endophytic fungi in the future. |
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