Marine Fungal Diversity Analysis And Isolation In Deep Sea Samples By Ih Situ Enrichments Of South China Sea

In-situ enrichment culture is a method to study the attachment and enrichment of microorganisms by putting the equipment containing enriched substrates in the culture bin into the designated place in the deep sea for a period of time.The sediment and water samples collected from the northern South China Sea Basin after 13 months of operation were used for the isolation and identification of culturable fungi.Thirteen retrieved samples stored at-20 ? and 4 ? were sequenced respectively on an Illumina Mi Seq platform(Illumina,San Diego,USA)according to the standard protocols by Majorbio Bio-Pharm Technology Co.Ltd.(Shanghai,China),and culturable fungi were isolated and screened from 4 ? stored samples.Thirteen samples were divided into four groups: control group,petroleum enrichment sediment group,petroleum enrichment water group and macromolecular natural organic compounds enrichment water group.For the deepsea water sample stored at-20 ?(control group)of the South China Sea Experimental Station,a total of 50422 ITS r DNA high-throughput valid sequences(reads)were obtained by high-throughput sequencing.They were clustered into 334 operational taxonomic units(OTUs),belonging to 67 genera,the dominant group is unclassified_k__Fungi;A total of 71157 reads were obtained by high-throughput sequencing at for sample stored at 4 ?,and clustered into 648 OTUs,belonging to 118 genera,the dominant group at the plylum level was Ascomycota,and the dominant group at the genus level was unclassified_k__Fungi;A strain of genus Talaromyces was isolated by culturable method.It was the dominant genus,but not the dominant OTU in Illumina sequencing analysis.For the six petroleum hydrocarbon sediments stored at-20 ? sample,a total of 289 400 reads were obtained by ITS r DNA high-throughput sequencing,which clustered into 197 OTUs and belonged to 84 genera.Ascomycota was the dominant group and Cladosporium was the main fungus.For the six petroleum hydrocarbon sediments stored at 4 ? sample,a total of 334763 reads were obtained by ITS r DNA high-throughput sequencing,which clustered into 1237 OTUs and belonged to 186 genera.Ascomycota was the dominant group and unclassified_k__Fungi was the main fungus at genus level.Seventeen fungi were isolated by culturable method,12 belonged to ascomycetes and 5 belonged to basidiomycetes.The ITS sequences of strains of Soil03-F2,Soil36-B4,Soil36-F3 and Soil69-F6 match OTU detected by Illumina sequencing method.The matched OTU131 and OTU234 were the dominant OTU.For the petroleum hydrocarbon-enriched water sample stored at-20 ?,a total of 13624 reads were obtained by high-throughput sequencing of ITS r DNA and clustered into 25 OTUs,which belonged to 18 genera.Ascomycota was the dominant group,followed by Basidiomycota.Gibberella was the dominant group at the generic level,followed by Sistotrema,Philosophia and unclassified_Davidiellaceae.For the petroleum hydrocarbon-enriched water sample stored at 4 ?,a total of 68882 reads were obtained by high-throughput sequencing of ITS r DNA and clustered into 76 OTUs,which belonged to 45 genera.Ascomycota was the dominant group,followed by Basidiomycota.Alternaria was the dominant group at the generic level.A strain of Meyerozyma guilliermondii was isolated by culturable method,and a matching OTU was found by Illumina sequencing method of 4 ? samples.In the top fifty genera of total abundance,Acremonium?Aspergillus ? Beauveria ? Cladosporium ? Fusarium ? Mortierella ? Penicillium ?Phanerochaete?Stachybotrys?Talaromyces?Trichoderma reported to have petroleum degradation function.Alternaria was found to have potential petroleum degradation functions.Paraconiothyrium has been reported to produce laccase,which can remove toxicity of petroleum industrial wastewater.Fungi diversity analysis of-20 ? and 4 ? samples in macromolecular natural organic compounds enrichment water group shows that the community diversity index of-20 ? samples was higher than that for 4 ? samples,except in cellulose substrate enrichment samples is opposite and seeweed enrichment samples remain unchanged.Fungi diversity analysis of-20? and 4? sanmples in animal/plant tissues enrichment water group shows that the dominant groups are Ascomycota except Wfat sample were unclassified_k__Fungi.Candida and unclassified_k_Fungi,unclassified_p_Ascomycota were the genera with more than 5% abundance detected in samples stored at-20 ?.Candida,Penicillium and unclassified_k_Fungi are the genera whose abundance is more than 5% detected in samples stored at 4 ?.Eleven fungi were isolated by culturable method,ten belonged to Ascomycetes and one belonged to Basidiomycetes.The ITS sequences of strains of Wfat-F1-2?Wfiber2-F1?Wfiber-F2?Wfish-F1 and Wsw-F6 match OTU detected by Illumina sequencing method.The matched OTU1532 and OTU1622 were the dominant OTU.The dominant fungi of all samples were Ascomycota.The species with the widest niche at the family level was Trichocomaceae.In this study,the diversity of deep-sea fungi in 13 water and sediment samples enriched in situ in the northern basin of South China Sea was analyzed by means of unculturable method and culturable method,which is helpful to understand the composition of fungi community in situ enriched environment of South China Sea,deepen the understanding of fungi resources in situ environment of the South China Sea and response of fungi to different substrates in deep sea in situ environment,deep-sea fungi with special functions were excavated.