| Plant endophytic fungi are an important part of fungal diversity in nature and play an important role in the ecosystem.There may exist or produce active substances that are the same or like the secondary metabolites of their host plants.Cupressaceae plants are rich in essential oils,terpenes,phenols,flavonoids,and other compounds with different biological activities.Thuja sutchuenensis essential oil and its plant body also contain a variety of similar secondary metabolites.Among them,the most critical terpenoids have good antibacterial effects and contain certain pharmacological effects,such as heat-clearing,detoxification,anti-tumor,anti-inflammatory and so on.It’s a rare and endangered gymnosperm and a Cretaceous relic plant in China.It is also a very small population of wild protected plants in Chongqing.It is mainly distributed in dangerous areas such as ridges and cliffs at an altitude of 900-2200 m.The population and individual number of the wild Thuja sutchuenensis is small,the distribution range is narrow,the habitat is dangerous,and the living condition is harsh.It has been pointed out that the tolerance of host plants to biological stress is related to the natural products of fungi.Therefore,it is speculated that Thuja sutchuenensis endophytic fungi are rich in novel secondary metabolites with diverse structures and medicinal activities.This research takes the endangered plant Thuja sutchuenensis endophytic fungi as the research object.On the basis of morphological observation and molecular biological identification,explore its community composition,division rules and genetic diversity,and evaluated its antibacterial activity through antibacterial experiments on plant pathogenic fungi,animal pathogenic bacteria and fermentation broth extracts,and screened out those with strong broad-spectrum antibacterial activity strains,and carried out subsequent experiments such as optimization of fermentation conditions,and terpenoid metabolomics analysis of fermentation products,the main conclusions are as follows:(1)Using conventional tissue isolation methods,the roots,stems and leaves of Thuja sutchuenensis root,stem and leaves were used for different disinfection time to isolate 112 endophytic fungi.After molecular biology combined with morphological identification,Thuja sutchuenensis can cultivate endophytic fungi mainly composed of ascomycetes(74.1 %)and Basidiomycetes(25.0%),belonging to 5 classes,13 orders,18 families,and 29 genera,showing a rich diversity of species,and the number,distribution and population structure of roots,stems,and leaf organs There are obvious differences,showing obvious tissue specificity;Among them,28 strains were isolated from the root,the dominant genus was Fusarium;39 strains were isolated from the stem,and the dominant genus was Pestalotiopsis;45 strains were isolated from the leaf,and the dominant genus was Nigrospora.(2)The antibacterial activity of endophytic fungi showed that 27.7% of endophytic fungi had different antibacterial effects on at least one plant pathogenic fungi,and the number of active strains accounted for 25.0%;Most of the active strains showed antibacterial activity against more than three plant pathogenic fungi,and the best antibacterial activity was against Sclerotinia sclerotiorum.44 strains of endophytic fungi had antibacterial activity against animal pathogenic bacteria,and the number of active strains accounted for 39.29%;However,the overall broad-spectrum bacteriostasis was poor,and most of the active strains only had bacteriostasis on one or two kinds of animal pathogens.Through the re-screening of the antibacterial activity of the ethyl acetate extract of the fermentation broth,it was found that the overall antibacterial effect of the strain TSY-27 was strong;Strain TSY-27 was identified as Nigrospora sphaerica.(3)The single factor experiment results show that the optimal basic medium for the PDB culture medium,the concentration of the best carbon source for corn flour,corn flour for the 25 g/L,the best nitrogen source for tryptone,tryptone best concentration of 14 g/L,the best fermentation medium initial p H value of 7,the best fermentation time is 9 d,best inoculation amount is 12%,the best temperature for28 ℃,the best table speed of 140 r/min,the best fluid volume is 130 ml..Plackett-Burman experimental design and analysis show that temperature is a significant factor,p H and liquid volume are significant factors,and other factors are not significant.The results of response surface test showed that the optimal levels of each factor were initial p H6.44,temperature 29.72 ℃ and liquid loading 136.68 m L,respectively.Using the optimized medium formula,the inhibition zone diameter of strain TSY-27 against Bacillus subtilis is 20.03 mm,which is 27.74% larger than the original fermentation medium,which is close to the predicted value Y=20.85 mm of the regression model.(4)The terpenoid metabolomics of the fermentation products of strain TSY-27 was analyzed by ultra-performance liquid liquid-triple quadrupole mass spectrometry(UPLC-MS/MS),and a total of 10 terpenoids were detected,namely Hinokitiol and Decahydrotrimethylnaphthofuranone((-)-drimenin),heptaketide,conocenolide A,abscisic acid,betulin,ursonic acid,betulinic acid,pomolic acid,3,24-Dihydroxy-17,21-(13)oleanolic fruit.Isolation and identification of culturable endophytic fungi showed that Thuja sutchuenensis contains abundant endophytic fungi resources.The screening results of antibacterial activity showed that endophytic fungi of Thuja sutchuenensis had certain antibacterial activity,which provided candidate strains for the screening of antibacterial substances.In addition,the results of studies on terpenoid secondary metabolites of the broad-spectrum active strain Nigrospora sphaerica TSY-27 show that the endophytic fungus TSY-27 is rich in terpenoids,which provides valuable fungal sustainability for natural active drugs.Resources provide a basis for follow-up research. |
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