Screening And Chemical Constituents Of Algicolous Fungi In The Sishili Bay Of Yantai

Marine-plant-derived fungi are now recognized as very promising sources of active compounds for pharmaceutical and pesticide development.Considerable progress has been made in exploring their diversity,species richness,and promise of biological applications.Marine algicolous fungi are believed to be good sources of structurally diverse and biologically active secondary metabolites due to the continuous pressure imposed on macroalgae by extreme marine environments.However,differences between the culture conditions of fungi in natural product studies and their real growth environment may lead to the silence of some gene clusters.Therefore,co-culture is gaining attention as an effective method to explore the potential of fungal secondary metabolites.To further understand the diversity composition and biological activity of marine algicolous fungi,endophytic and epiphytic fungal strains were isolated from macroalgae collected in the coastal zone of the Sishili bay of Yantai.The fungi were identified by morphological observation and ITS region sequence analysis,and the fungal isolates were screened for marine animal toxicity,anti-microalgal and antibacterial activities.A total of 77 epiphytic and 47 endophytic fungi were isolated from the macroalgae samples,totaling 124 strains.Of these,102 strains were identified into 17 genera.Using Artemia salina and Brachionus plicatilis as models for marine animal toxicity testing,extract of 13 strains showed 100% lethality to both marine animal species,at the concentration of 100 μg/m L.The anti-microalgal activity was tested on the models of Chattonella marina and Prorocentrum donghaiense.Extract of 21 strains showed more than 50% inhibition against the two species of marine red-tide microalgae,and 17 strains showed 100% inhibition,at the concentration of 100 μg/m L.Extract of 40 strains showed antibacterial activity against Pseudomonas citrate,4 strains showed antibacterial activity against Vibrio anguillarum,and 4 strains showed antibacterial activity against both tested bacteria in the antibacterial activity test,at the concentration of 100 μg/m L.47 strains of Trichoderma and 1 strain of Fusarium were selected from the isolated fungi,These Trichoderma molds were separately confronted against Fusarium.The results showed that the growth of Trichoderma was significantly dominant in the confrontation experiments,and the growth of Fusarium was inhibited to varying degrees.There are seven strains of Trichoderma are able to cover the colonies of Fusarium and grow on them,eventually leading to smaller colonies of Fusarium,where 1II1 F can even cover the colonies of Fusarium completely.Through the observation of mycelial morphology using microscopy,it was found that strains12A1 N,B41F and 1II1 F are able to hyperparasitize Fusarium in different ways,resulting in an inhibition of Fusarium.Combining the results of confrontation experiments and strain selection,T.flagellatum 12A1 N and F.graminearum 12II2 N were selected for secondary metabolite studies.Crude extracts from individual and co-cultures of 12A1 N and12II2N were analyzed using HPLC.The results of the analysis showed that some additional metabolites were produced during co-culture,while some metabolites present in separate cultures were absent during co-culture.Using various modern chromatographic techniques and other methods,the single constituents were separated,and then the structures of the compounds were identified using a combination of modern spectroscopic techniques.8 compounds were obtained from 12A1 N,12 compounds were isolated from 12II2 N,and 10 compounds were obtained from the co-culture of two fungal strains.Among them were 2 new compounds,including a cedramber derivative H-B-1 and a fusarin H-B-2.Analysis of secondary metabolites using HPLC showed that the metabolites of the fungus changed during co-cultivation: compounds H-B-6,H-B-7 and H-B-8 were not produced during co-culture,compound H-C-10 was a newly produced metabolite during co-culture,and there were also significant changes in the yield of some components.In this study,124 strains of algicolous fungi were isolated and screened for activity.The results indicated that the algal fungi along the coast of Yantai Sishili bay are rich in diversity.Some of them have biological activities,which portend a large number of potential natural active products and are valuable for further research.The confrontation experiment between Trichoderma and Fusarium proved the existence of hyperparasitoidism,indicating that Trichoderma has great potential in the role of biological protection.Structurally rich secondary metabolites were isolated from algicolous fungi,increasing the diversity of secondary metabolites of algicolous fungi.HPLC analysis of the crude extracts of 12A1 N and 12II2 N cultured individually and co-cultured,as well as the isolated monomeric compounds,showed that the metabolites of the fungi changed during the co-culture process,indicating that co-culture is an effective method to activate the silent gene cluster to make the fungi produce new metabolites.Through the research of algicolous fungi and their secondary metabolites in Sishili Bay,this study provides a theoretical basis and experimental basis for the development of marine drugs.