bioRxiv preprint doi: https://doi.org/10.1101/692814; this version posted July 4, 2019. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 1 Diversity and some bioactivities of soil actinomycetes 2 from southwestern China 3 4 Yi Jiang*，Guiding Li, Qinyuan Li, Kun Zhang, 5 Longqian Jiang, Xiu Chen and Chenglin Jiang* 6 7 Yunnan Institute of Microbiology, Yunnan University, 8 Cuihu Road 2#, Kunming, Yunnan, China 9 10 11 12 13 *Corresponding authors: Yi Jiang, e-mail:[email protected]; Tel: +86-871-65034073 14 Chenglin Jiang, e-mail:[email protected]; Tel +86-871-65034139 15 16 17 bioRxiv preprint doi: https://doi.org/10.1101/692814; this version posted July 4, 2019. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 18 Abstract: With the natural medicine exploring, the actinomycetes (actinobacteria) have gotten 19 more and more recognition. 815 soil samples were collected from six areas in the southwestern 20 China. 7063 purified strains of actinomycetes were isolated from these samples by using four 21 media. The 16S rRNA gene sequences of 1998 selected strains of the 7063 were determined, and 22 the phylogenetic analysis was carried out. The diversity of actinomycetes is analyzed. Total 33 23 genera of actinomycetes as the purified cultivation were identified from these soil samples. 14, 13, 24 5, 9 and 26 genera of actinobacteria were identified from E, A, B, D, C and F area respectively, 25 and the communities of actinomycetes are very different from each other. The diversity of 26 Xishuangbanna (F area) is the richest, and 26 genera were isolated. That of Emei and Qingcheng 27 Mountain (C area) is monotone, and only five genera were isolated. 158 of 1998 strains (7.9 %) 28 are possible new species. Antimicrobial activities of 1070 selected strains against 11 bacteria and 29 fungi were tested using agar well diffusion method, and biosynthetic genes of type I and II 30 polyketide synthases (PKS I, PKS II), nonribosomal peptide synthase (NRPS) and polygene 31 cytochrome P450 hydroxylase (CYP) of 1036 selected strains were detected by PCR. High rate of 32 antimicrobial activity and the four antibiotic biosynthetic genes existed in these actinomycetes. 33 Results of this study indicates that firstly more unknown actinomycetes can be obtained from soil 34 samples, specially primeval tropical forests. Second, isolation methods for actinomycetes must be 35 continually improved and improved. 36 Importance 37 First, discarding repeat of a mass of known microbes and compounds is very difficult. Second, 38 pharmaceuticals development can still not overtake the increase of resistance of pathogens to 39 antibiotics, and new diseases continuously and fleetly occur. Soil actinomycetes of these regions 40 are researched few by microbiologist yet. In order to get much more unknown actinomycetes for 41 discovery of new bioactive metabolites, the actinomycetes of forest soil in southwestern China 42 were isolated and identified. Anti-microbial activities and synthetic gene clusters’ of four kinds of 43 antibiotics of some selected strains were detected. 44 45 Key words: Actinomycete Diversity, Bioactivity, Biosynthetic genes, Southwestern China 46 47 bioRxiv preprint doi: https://doi.org/10.1101/692814; this version posted July 4, 2019. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 48 INTRODUCTION 49 Actinomycetes (Actinobacteria) has been paid a great attention owing to their production of 50 various natural drugs and other bioactive metabolites including antibiotics, enzyme inhibitors and 51 enzymes, and has special ecological function such as fixation of air nitrogen and degradation of 52 difficult disaggrega-tire substance in the nature world for maintaining a balance of ecosystem. 53 Over 22,000 bioactive secondary metabolites (including antibiotics) were published in the 54 scientific and patent literature, and about 11000 of 22000 metabolites were produced by 55 actinomycetes by the end of 2002. About 150 antibiotics have being applied in human therapy and 56 agriculture now. In the 150 antibiotics, bacteria produced 10-20, actinomycetes produced 100-120, 57 and fungi produced 30-35 (Berdy 2005). There are two difficult problems of natural 58 pharmaceutical development from microorganisms. First, discarding repeat of a mass of known 59 microbes and compounds is very difficult. So pharmaceutical development needs very long time, 60 tremendous investment, and manpower and material resources. Second, pharmaceuticals 61 development can still not overtake the increase of resistance of pathogens to antibiotics, and new 62 diseases continuously and fleetly occur. But 90 % to 99 % of microorganisms in the nature world 63 are not cultured yet based on the research results of molecular technology (Chiao 2004; Hughes et 64 al. 2001; Joseph et al. 2003; Pachter 2007; Zengler et al. 2002). Making the uncultured 65 microorganisms to cultured microorganisms is one hope for getting new leader compounds for 66 development of new natural drug. Southwestern China including Yunnan, Guizhou, Sichuan and 67 Tibet is one of regions of the richest biodiversity in China. Soil actinomycetes of these regions are 68 researched few by microbiologist yet. In order to get much more unknown actinomycetes for 69 discovery of new bioactive metabolites, the actinomycetes of forest soil in southwestern China 70 were isolated and identified. Anti-microbial activities and synthetic gene clusters’ of four kinds of 71 antibiotics of some selected strains were detected. Some results are reported here. 72 73 MATERIALS AND METHODS 74 Collection and pretreatment of soil samples 75 280 soil samples were collected from primeval subtropical every-green broadleaf forest in 76 Zhangjiajie of Hunan and Fanjing Mountain in Guizhou, China. The two sampling spots belong to 77 Wuling Cordillera (A sampling area). 50 soil samples were from primeval subtropical every-green bioRxiv preprint doi: https://doi.org/10.1101/692814; this version posted July 4, 2019. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 78 broadleaf forest of Huangjing in Gulin (B area), south of Sichuan. 100 samples from secondary 79 every-green broadleaf forest in Emei and Qingcheng Mountains (C area), the western brim of 80 Sicuan Basin. 50 samples from primeval alpine taiga of Jiuzhaigou (D area), in the north of 81 Sichuan. 220 samples from primeval forests on various altitudes of Grand Shangri-La (E area) in 82 the common boundary of Sichuan, Yunnan and Tibet. 85 soil samples were collected from 83 primeval tropical rainy forest of Xishuangbanna in the south of Yunnan (Figure 1). Every soil 84 sample was collected from 3 to 5 holes with 10 to 20 cm depth and incorporated as one sample, 85 and put in one sterile plastic bag. Total 815 soil samples were preserving at 4 ºC before test. The 86 soil samples were dried in room temperature for 7 to 10 days, then pretreatment at 80 ºC for 1 87 hour. 88 89 Figure 1. Position of sampling areas in China 90 91 Isolation medium and method of actinomycetes 92 Plate dilution method was used for isolation of actinomycetes. Four media were used as following: 93 YIM 7 (HV medium) (Hayakawa and Nonomura 1987); YIM 171 [Improved Glycerol-asparagine 94 medium: Glycerol 10 g, asparagine 1 g, K2HPO4·H2O 1 g, MgSO4·7H2O 0.5 g, CaCO3 0.3 g, 95 Vitamin mixture (Hayakawa and Nonomura 1987) 3.7 mg, trace salts (Shirling and Gottlieb 1966) 96 1 ml, potassium dichromate (K2Cr2O7) 50mg, agar 15 g, distilled water 1000 ml, pH 7.7]; YIM 37 97 (Jiang and Xu 1997) (Improved Histidine-raffinose medium: Histidine 1 g, raffinose 10 g, 98 Na2HPO4 0.5 g, KCl 1.7 g, CaCO3 0.02 g, MgSO4·4H2O 0.05 g, FeSO4·7H2O 0.01 g; Vitamin bioRxiv preprint doi: https://doi.org/10.1101/692814; this version posted July 4, 2019. The copyright holder has placed this preprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, remix, or adapt this material for any purpose without crediting the original authors. 99 mixtures 3.7 mg, cycloheximide 50 mg, nystatin 50 mg, potassium dichromate 50 mg, agar 15 g, 100 distilled water 1000 ml, pH 7.2); YIM 212 (Jiang et al. 2006) (Mycose-proline medium: Mycose 5 101 g, proline 1 g, (NH4)2SO4 1 g, NaCl 1 g, CaCl2 2 g, K2HPO4 1 g, MgSO4·7H2O 1 g, vitamin 102 mixtures 3.7 mg; potassium dichromate 50 mg, agar 15 g, distilled water 1000 ml, pH 7.2). 103 104 Characterization of actinomycetes 105 Cultivation of cell, extraction of DNA, PCR and sequencing of 16S rRNA gene and phylogenetic 106 analysis of 1998 actinomycete strains were carried out with the methods described in previously 107 paper (Jiang et al. 2008). The strains were characterized at a genus and species level. 108 109 Determination of antimicrobial activity 110 920 actinomycete strains were fermented in YIM 61 broth (Soybean meal 20 g, glucose 10 g, 111 peptone 4 g, K2HPO4 1 g, MgSO4·7H2O 0.5 g, NaCl 1 g, CaCO3 2 g, distilled water 1000 ml, pH 112 7.8) on shaker at 28 ºC for 7 days.