Investigation On Population And Resources Of Actinomycetes Associated With Lichen In Yunnan Province

Located in the southwest border of China,Yunnan province boasts the most diverse and abundant living form and natural gene pool,which is one of the areas with the richest lichen biodiversity worldwide.Lichens are specialized fungi consisting of an algae partner(photobiont)and a fungal partner(mycobiont)in a symbiotic relationship.Previous research indicated that diverse bacterial communities,including actinomycetes are dwelled in lichen symbiotic niches.Herein,we researched the diversity of lichen-associated Actinobacteria using culture-independent and culture-dependent methods.In addition,all cultivable actinomycetes were tested for antimicrobial activity and screened for genes associated with secondary metabolite production to evaluate their biosynthetic potential.Finally,one strain with high antimicrobial activity was further studied on its secondary metabolites.Detailed research progress is included:1.We studied the bacteria diversity in nine lichen samples collected from Yunnan province by Illumina MiSeq high-throughput sequencing.A total of 509134 bacteria V3 and V4 sequences of 16S rRNA genes were obtained and clustered into 11057 OTUs(with a threshold of 97%identity).These OTUs were distributed among 32 phylums.Among them,Proteobacteria,Cyanobacteria,Firmicutes and Actinobacteria were dominant in lichen samples.However,there are 7563 sequences can't be identified into any of the known phylums,indicating that these sequences may represented some new taxa.There are 49874 sequences(including 1831 OTUs)belonging to Actinobacteria,accounting for 9.24%of the total sequences in this study.Altitudes had great influence on the bacterial diversity of same kinds of lichens,the bacterial diversity decreased with the increasing of altitude.2.Excluding the spurious fraction of microorganisms present on the lichen thallus surfaces is important for studying lichen-associated microbial communities.In this study,we found that a two-step surface pretreatment:a 5-minute wash with running water,followed by three times of wash in sterile water(20 mL for each time)could effectively remov the external surface-borne microbes from lichen samples.We also found out that the selective Actinobacteria isolation media were amended with inhibitors(nystatin 150 mg/L,nalidixic acid 50 mg/L,potassium dichromate 50 mg/L)can greatly increase the rate of the Actinobacteria,in the meantime,significantly inhibit the growth of fungi and Gram-negative bacteria.3.In total,2415 colonies were isolated from the selective media of 68 lichen samples collected from Yunnan province,and 462 pure strains of actinomycetes were identified,which were distributed into 11 orders,23 families,and 47 genera:Actinomadura,A ctinophytocola,Actinoplanes,Agromyces,Amnibacterium,Amycolatopsis,Angustibacter,Arthrobacter,Blastococcus,Brevibacterium,Cellulomonas,Cellulosimicrobium,Curtobacterium,Flexivirga,Friedmanniella,Gordonia,Herbiconiux,Janibacter,Kibdelosporangium,Kineococcus,Kineosporia,Kitasatospora,Kocuria,Kribbella,Labedella,Lechevalieria,Leifsonia,Lentzea,Microbacterium,Micrococcus,Microlunatus,Micromonospora,Motilibacter,Mycobacterium,Nakamurella,Nocardia,Nocardioides,Nocardiopsis,Nonomuraea,Plantibacter,Pseudonocardia,Rhodococcus,Schumannella,Streptomyces,Streptosporangium,Umezawaea,and Williamsia.To the best of our knowledge,this is the first report to describe the isolation of 24 genera from lichens.Among the actinomycetes isolated from lichens,Streptomyces,Microbacterium,Rhodococcus,and Micromonospora were the dominant groups.4.At least 55 isolates(11.9%)are likely to represent novel actinomycetes taxa within 18 genera.Indeed,all of the Microlunatus,Nocardioides,Kibdelosporangium,Herbiconiux,Schumannella,Nakamurella isolates,and approximately 88.9%of the Pseudonocardia isolates appear as new species.5.All 462 isolates were tested for antimicrobial activities against Bacillus subtilis,Staphylococcus aureus,Escherichia coli,Mycobacterium tuberculosis,Aspergillus niger,and Candida albicans.One hundred and ten of the 462 isolates(23.81%)showed inhibitory activity against at least one of the indicator organisms.The high levels of activity was detected in the isolates of genera Streptomyces(35.36%),Amycolatopsis(36.36),Lentzea(20%),Nocardioides(20%),and Microlunatus(20%).All of the strains from six genera Cellulosimicrobium,Janibacter,Kibdelosporangium,Micrococcus,Motilibacter,and Umezawaea were found to have antibacterial activity.The 462 isolates were investigated for the presence of PKS ?,PKS ?,NRPS,APH,HMG-CoA and CYP gene sequences.More than half of all isolates(259 out of 462)screened gave a positive result.The highest rates were screened among members of the genera Streptomyces and Amycolatopsis.Approximately half of the isolates(233 strains)were closely related or identical to known strains producing bioactive compounds.More specifically,164 different compounds,including antibiotics,enzymes,enzyme inhibitors,immunomodulators,organic acids and amino acids,have been reported from species most closely related to the isolated actinomycetes according to queries against the natural products in the SciFinder(https://scifinder.cas.org)and REAXYS(http://www.reaxys.com)databases.The results confirmed the idea that species of actinomycetes isolated from lichens represents significant potential to produce biologically active compounds.6.Four new nanaomycins,4a?,10a?-dihydroxy-nanaomycin ?A(1),4a?,10a?-dihydroxy-nanaomycin ?A(2),4a?,10a?-dihydroxy-nanaomycin ?A(3),10?-hydroxy-nanaomycin aE(4),together with four known compounds,nanaomycin aA(5),nanaomycin ?A(6),3'-O-acetylthymidine(7),and 7-acetyl-3,6-dihydrox-8-methyl-tetralone(8)were isolated from a fermentation broth of Streptomyces hebeiensis YIM 130410 derived from lichen.Compounds 5 and 6 showed antibacterial activities against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with MIC values ranging from 3.13 to 100 ?g/mL.In a word,the present study enriched the understanding of actinomycetes associated with lichen symbiosis and facilitate the exploration and application of actinomycete resources from these unique niches.