The diversity and antimicrobial activity of endophytic actinomycetes isolated from medicinal plants in Panxi Plateau, China. Authors: Ke Zhao, Petri Pentinen, Tongwei Guan, Jing Xiao, Qiang Chen, Jun Xu, Kristina Lindström, Lili Zhang, Xiaoping Zhang, and Gary A. Strobel This is a postprint of an article that originaly appeared in Curent Microbiology on 2010. The final publication is available at Springer via htp:/dx.doi.org/10.1007/s00284-010-9685-3. Zhao, K., Pentinen, P., Guan, T., Xiao, J., Chen, Q., Xu, J., Lindström, K., Zhang, L., Zhang, X., and Strobel, G. A. (2010). The Diversity and Anti-Microbial Activity of Endophytic Actinomycetes Isolated from Medicinal Plants in Panxi Plateau, China. Curent Microbiology, 62(1), 182–190. doi: 10.1007/s00284-010-9685-3 Made available through Montana State University’s ScholarWorks scholarworks.montana.edu The Diversity and Anti-Microbial Activity of Endophytic Actinomycetes Isolated from Medicinal Plants in Panxi Plateau, China Ke Zhao1, Petri Pentinen3, Tongwei Guan1,2, Jing Xiao4, Qiang Chen1, Jun Xu4, Kristina Lindström3, Lili Zhang2, Xiaoping Zhang1, and Gary A. Strobel Traditional Chinese medicinal plants are sour-ces of biologicaly active compounds, providing raw material for pharmaceutical, cosmetic and fragrance industries. The endophytes of medicinal plants participate in biochemical pathways and produce analogous or novel bioactive compounds. Panxi plateau in South-west Sichuan in China with its unique geographical and climatological characteristics is a habitat of a great variety of medicinal plants. In this study, 560 endophytic actinomycetes were isolated from 26 medicinal plant species in Panxi plateau. 60 isolates were selected for 16S rDNA-RFLP analysis and 14 representative strains were chosen for 16S rDNA sequencing. According to the phylogenetic analysis, seven isolates were Streptomyces sp., while the remainder belonged to genera Micromonospora, Oerskovia, No-nomuraea, Promicromonospora and Rhodococcus. Anti-microbial activity analysis combined with the results of amplifying genes coding for polyketide synthetase (PKS-I, PKS-I) and nonribosomal peptide synthetase (NRPS) showed that endophytic actinomycetes isolated from medicinal plants in Panxi plateau had broad- spectrum antimicrobial activity and potential natural product diver-sity, which further proved that endophytic actinomycetes are valuable reservoirs of novel bioactive compounds. Introduction Actinomycetes produce a diverse range of secondary metabolites including various antibiotics, antitumor and immunosuppressive agents and plant growth hormones [9, 26, 31] that play an important role in pharmaceutical industry. However, the dispersion of drug resistance in bacteria and the appearance of life-threatening viruses pro- mote the search for new and useful metabolites. Endophytes are microorganisms that for the whole or part of their life history live inside plant tissues by symbiotic, parasitic or mutualistic means without causing immediately overt neg- ative efects [28]. As a result of these long-held associations, endophytic microorganisms and plants have developed good information transfer [29]. Medicinal plants synthesize chemical substances, providing raw material for pharma- ceutical, cosmetic and fragrance industries [15]. Endophytes of medicinal plants probably participate in metabolic path- ways of medicinal plants and produce analogous or novel 1 Department of Microbiology, Colege of Resource and Environmental Sciences, Sichuan Agricultural University, 625000 Yaan, People’s Republic of China. e-mail: zhangxiaopingphd@126.com; zhaoke82@126.com 3 Department of Applied Chemistry and Microbiology, University of Helsinki, 00014 Helsinki, Finland 2 Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Corps, Tarim University, 843300 Alar, People’s Republic of China. e-mail: zhang63lyly@yahoo.com.cn 4 Key Laboratory of Marine Biogenetic Resources, The Third Institute of Oceanography, State Oceanic Administration, 361005 Xiamen, People’s Republic of China 5 Department of Plant Sciences, Montana State University, Bozeman, MT 59717, USA Abstract bioactive compounds, for example taxol [30]. Endophytic actinomycetes are considered as potential sources of novel bioactive compounds and various bioactive compounds have been isolated from them until now [4,11,32]. Species distribution and biological diversity are signif- icantly influenced by ecological environment [10,27]. In previous studies, the diversity and antibiotic activity of endophytic actinomycetes isolated from medicinal plants growing in tropical regions have been reported exclusively [2,17,23], while the medicinal plants from Panxi plateau, China, have not gained research atention before our study. The Panxi plateau is in the southwest of Sichuan province, located between the Qinghai-Tibet plateau, Yunnan-Guiz- hou plateau and Sichuan basin. The weather in this plateau is characterised with abundant rainfal and sunshine and with wet and dry seasons. Panxi plateau is the northernmost place with Southeast Asian tropical climate. Xerothermic climate is another characteristic of this area. The unique geographic position, favourable environment, complicated tectonic geographic condition, special climate and various agrotypes al contribute to the peculiar landscape and biodiversity in the area. About 2,400 species of wild medicinal plants are found in this area, with many of them having a long history of application by local people [14]. While the microbial diversity of Panxi plateau has been investigated by our laboratory in the past years [18], the actinomycetes and especialy the endophytic actinomycetes of this plateau have not been studied until now. In this study, we screened the endophytic actinomycetes of medicinal plants from Panxi plateau based on the medicinal function of the plants to identify their potential as biocontrol agents for phytopathogens and bacteria. Because polyketide synthase (PKS) and nonribosomal peptide syn- thetase (NRPS) pathways synthesize a large number of biologicaly active molecules and exist widely in the gen- omes of actinomycetes, we used degenerate primers of PKS-I, PKS-I and NRPS genes to analyse the potential capacity of the endophytic actinomycetes to synthesize secondary metabolites. The phylogenetic diversity of iso- lates was assessed using 16S rDNA RFLP analysis and sequencing. This is the were first report on the diversity and bioactivity of endophytic actinomycetes of medicinal plants from Panxi plateau where we hope to bioprospect endo- phytic actinomycetes resources possessing potential to be applied in pharmacy and agriculture field. Materials and Methods Sampling of Wild Medicinal Plants According to the ethnobotanical properties, healthy medicinal plant samples with antitumor, antibacterial and antiviral properties were colected from Panxi plateau in South-west Sichuan, China in July 2008. In order to ensure the endophytic nature of the isolates, the cut ends of root and stem samples were sealed with wax. Al the samples were screened for endophytic actinomycetes within 48 h. Surface Sterilization The procedure of surface sterilization was from Johannes et al.[13] with some modifications. Samples were dried at room temperature for 48 h before being thoroughly washed with running tap water and graded by size and surface appearance. Al the visibly damaged material was exclu- ded. Samples were washed by sonification for 10 min at 150 w to dislodge soil and organic mater. The surface sterilization included the folowing steps: tissue pieces were rinsed in 0.1% Tween 20 for 30 s, sequentialy immersed in 75% ethanol for 5 min and in 2% sodium hypochlorite for 5 min and rinsed with 10% NaHCO3for 10 min to inhibit fungal growth. After each treatment, samples were rinsed three times in sterile water. Finaly, the samples were thoroughly dried in a laminar flow chamber. To confirm the success of the sterilization pro- cess, aliquots of the sterile distiled water from the final rinse were inoculated on ISP2medium plates. The surface- sterilized samples were asepticaly dissected into smal pieces, plated onto selective media and incubated for about 1 month at 28C. The actinomycetes isolates were stored on ISP4slope medium at 4C. Isolation Media The seven isolation media, al supplemented with 1.8% agar, were as folows: E1: Low-Nutrient Mineral Salts-agar (LNMS) [6]; E2: Type Water Yeast Extract agar (TWYE) [8]; E3: Oatmeal agar [24]; E4: Humic-vitamin agar (HV) [3]; E5: Histidine-Raffinose agar [37]; E6: Modified Gause No.1 [22]; E7: Chitin agar [21]. To inhibit the growth of nonactinomycetes, the isolation media were supplemented with nalidixic acid and K2Cr2O7, both to a final concen- tration of 50lg/ml. Identification of Actinomycetes The isolates were dereplicated by cultural and morpho- logical characteristics, including morphology and colour of aerial mycelium, characteristics of colonies on the plate, spore mass colour, colour of difusible pigments and spo- rophore and spore chain morphology. Visual observation using light microscopy and Gram-straining were performed for further identification [40]. 16S rRNA Gene Restriction Fragment Length Polymorphism Analysis (ARDRA) The total genomic DNA of isolates were extracted and amplified as described earlier [7]. The PCR products were, respectively, digested with restriction endonucleasesHae- II andRsa-I. The 10-ll reactions included 19bufer, 0.1- lg/ll BSA (Bovine Serum Albumin), 10-UHae-II/Rsa-I (TaKaRa, China) and 3-ll PCR product. The digestions were performed at 37C for 6 h. The digested fragments were separated by gel electrophoresis and visualised with a UV transiluminator. Isolates were grouped based on the combined amplified rDNA restriction analysis paterns using the approaches described [10]. 16S rDNA Sequencing and Analysis According to the results of 16S rRNA-RFLP, representa- tive isolates were chosen for 16S rRNA gene sequencing caried by Shengong Biotechnology Ltd. (Shanghai, China). Sequences were compared with GenBank database using BlastN for searching the closest match sequence. The sequences were pairwise aligned using Clustal X [34]. A phylogenetic tree was constructed under the Kimura two- parameter model and bootstrap analyses with 1,000 re- samplings were performed with MEGA 4.0. [33]. Evaluation of Antimicrobial Activity 11 indicator organisms (Verticilium dahliaeKleb. [SAUM 0110],Fusarium oxysporumf. sp. vasinfectum [SAUM 2312],Aspergilus niger[SAUM 1275],Fusarium oxy- sporumf. sp. Niveum [SAUM0674],Coletotrichum orbiculare[SAUM 0321],Fusarium graminearum[SAUM 2912],Exerohilum turcicum[SAUM 0218],Curvularia lunata[SAUM 1373],Botrytis cinerea[SAUM 1294], Staphylococcus aureus[ATCC 25923],Escherichia coli [ATCC 35218]) were used in the screening tests of anti- microbial activity as described earlier [35]. The antago- nism was detected by formation of an inhibition zone in 3 days. Detection and Analysis of PKS-I, PKS-I and NRPS Three sets of degenerate primers PKS-I-A: 50-GCS ATG GAY CCS CAR CAR CGS VT-30, PKS-I–B: 50-GTS CCS GTS CCR TGS SCY TCS AC-30[25]; PKS-I-A: 50-TSG CST GCT TGG AYG CSA TC-30, PKS-I-B: 50-TGG AAN CCG CCG AAB CCG CT-30[20]; NRPS-A: 50-GCS TAC SYS ATS TAC ACS TCS GG-30, NRPS-B: 50-SAS GTC VCC SGT SCG CTAS-30[1] targeting genes encoding polyketide synthases (PKS-I, PKS-I) and nonribosomal peptide synthetase (NRPS) were used to screen the biosynthetic potential of the isolates. Amplication and sequencing of the genes and sequence analysis were per- formed as described previously [12]. Results In total, we sampled 26 species healthy medicinal plants. No colonies emerged from the final rinses of the sterili- zation procedure showing that the surface sterilization was efective and the subsequent isolates were endophytes. We isolated 560 strains that showed morphological character- istics of actinomycetes after 1–1.5-month incubation. Most of the strains were isolated from root (58.2%), secondly from stem (27.8%) and least from leaves (14%). According to their colour on media and sporing structure, we selected 60 isolates for further analysis. We digested the 16S rRNA genes of 60 isolates by restriction endonucleasesHae-II andRsa-I and assigned the isolates to 12 groups at the similarity level of 90% (Table1). The isolates formed two dominant groups of 19 and 31 strains, named groups B and D, respectively. Each of the other ten groups contained one strain only. There was no direct corelation between the isolation sources and the RFLP grouping. For example, strains SAUK6024, SAUK6102 and SAUK6117, isolated fromPotentila dis- color,Pteris dactylinaHook andSenecio declouxiDunn, respectively, were al in RFLP group D, and strains SAUK6012 and SAUK6015, isolated fromAinsliaea hen- ryiDiels, were in groups F and H, respectively. Based on 16S rRNA-RFLP analysis and morphological characteris- tics, three strains were selected from RFLP group D and one from each of the other groups for sequence analyses. The 16S rRNA of those strains were compared with their closest relatives in GenBank and a phylogenetic tree of the 16S rRNA sequences were constructed by combining the sequences of the 14 representative strains with their closest relatives (Table1; Fig.1). The seven strains representing the 51 strains in RFLP-groups A–E were most similar to Streptomycesspecies. The other seven strains were mem- bers of the generaMicromonospora,Nonomuraea,Oer- skovia,PromicromonosporaandRhodococcus. In our study, 59 out of the 60 strains showed antago- nistic activity against at least one of the 11 indicator organisms (Table1). The 15 strains that inhibited the growth ofS. aureuswere considerable candidates for screening for new antibiotics (Table1). Over half of the strains, altogether 38, inhibited the growth of at least five indicator organisms (Table1). Al of them belonged to the genusStreptomyces, as assigned by morphological characteristics and DNA analyses. The rare actinomy- cetes strains were more limited in their antimicrobial scope. However, SAUK6015, a strain most similar to Ta ble 1 rD NA typ es, hos t p lan ts, sou rce pl ant or gan s, and is ola tio n med ia, pr ese nce of P KS- I, PK S-II a nd NR PS gen es an d t he ant im icr obi al act ivi tie s o f t he rep res ent ati ve str ain s o f end oph yti c a cti no myc ete s Rep res ent ati ve str ain s rD NA Typ ea Hos t p lan ts Org an Clo ses t s equ enc e i n Gen ban k ( si mil arit y) PK S- I PK S- II NR PS Ind ica tor or gan is m 1 2 3 4 5 6 7 8 9 10 11 SA UK 601 8 (FJ 842 556 ) A Im pat ien s c hin ens is L. Roo t Str ept om yce s a ura nti acu s (10 0% ) ?1 - ?2 ?? ? ? ? ? - ?? ? ? ? - - SA UK 602 3 (FJ 842 559 ) B Lys im ach ia for tu nei Max im . Roo t Str ept om yce s var iab ilis (10 0% ) ?3 ?4 ?5 - - - - - - - - - - - SA UK 615 9 B Rhi zo ma Ari sae mat is Roo t nd - ? - - ?? ? ?? ? - ? ?? ? ? - ?? ? ?? SA UK 616 9 B Dio sco rea op pos ita Ste m nd - ? - ?? - ?? - ? ?? ?? ? ? ? - SA UK 614 9 B Ste lle ra cha mae jas me L. Roo t nd ? ? - - - - - - ?? ?? ? - ?? ? - SA UK 613 8 B Sal via mi lti orr hiz aB ung e Ste m nd ? ? ? ? - ?? ? - - - ? - - - - SA UK 613 0 B Rhi zo ma Ari sae mat is Roo t nd ? ? ? ? - ?? ? - - - - ? ? ?? - SA UK 614 6 B Rhi zo ma Ari sae mat is Roo t nd ? ? ? - ? - - - - - ? ? - - SA UK 616 8 B Dro ser a p elt ata S mit h var .m ulti sep ala Y.Z .R uan Roo t nd ? ? - - - - - - - ?? - ? - - SA UK 616 1 B Rhi zo ma Ari sae mat is Roo t nd ? ? - ? ?? ? - ?? ? ?? ? ?? ? ?? - - SA UK 611 9 B Lys im ach ia for tu nei Max im . Ste m nd ? - - - - - - - - ?? ? - - - - SA UK 611 2 B Ste lle ra cha mae jas me L. Lea f nd ? ? - ? ?? ? - - - ?? ? - - - SA UK 612 7 B Ain sli aea he nry iD iel s Ste m nd ? - - - - ? - ? ? ?? ? ? ?? ? - SA UK 612 3 B Art emi sia an nua L. Ste m nd ? ? ? - ?? - - ? - ? - - - - SA UK 612 2 B Pte ris da cty lin aH ook . Ste m nd - ? - - - ? - - - ?? - - - - SA UK 614 5 B Pte ris da cty lin aH oo k. Roo t nd - ? - - - ?? ? - ? ? ?? ? - - - SA UK 616 7 B Lys im ach ia for tun ei Max im . Ste m nd - ? - ? ? ? - - - ? ? - - - SA UK 612 1 B Lys im ach ia for tun ei Max im . Roo t nd - ? - ? - - - - - - - - - - SA UK 61 48 B Lyc opo diu m j apo nic um Th unb . Lea f nd ? ? ? ? ?? ? ?? - ? - ?? ? - ?? ? - SA UK 602 7 B Pte ris da cty lin aH ook . Roo t nd - ? - ?? - ?? - ? ?? ? ?? - - - SA UK 602 2 (FJ 842 558 ) C Sen eci o d ecl oux ii Dun n Roo t Str ept om yce s gri seo car neu s( 99 %) ?6 ?7 ?8 - - - ?? ?? - ?? ? - - - - SA UK 602 4 (FJ 842 560 ) D Pot ent ill a d isc olo rB ge Roo t Str ept om yce s c hry seu s (10 0% ) ?9 ?1 0 ?1 1 - - - - ?? - - - ?? ? ? SA UK 602 0 (FJ 842 557 ) D Ach yra nth es asp era Lin n. Roo t Str ept om yce s v iri dis (99 %) ?1 2 ?1 3 ?1 4 ? - ? ?? - ? - - - - - SA UK 602 6 (FJ 842 561 D Cyn anc hu m a uri cul atu m R oyl e ex Wig ht. Ste m Str ept om yce s a lbo gri seo lus (10 0% ) - ?1 5 ?1 6 - - - - - ? ?? ? ? ? - - Ta ble 1 con tin ued Rep res ent ati ve str ain s rD NA Ty pe a Hos t p lan ts Org an Clo ses t s equ enc e i n Gen ban k (si mil arit y) PK S- I PK S- II NR PS Ind ica tor or gan is m 1 2 3 4 5 6 7 8 9 10 11 SA UK 602 8 D Gna pha liu m hyp ole ucu mD C. Lea f nd - - ? - ?? - - ? - ?? ? ?? - - - SA UK 602 9 D Ain sli aea he nry iD iel s Roo t nd - - ? ? - ?? ? - - ? ?? ?? ? - - SA UK 610 2 D Pte ris da cty lin aH oo k Roo t nd ? - - ? ? ?? ? - - ?? ?? ?? ?? ? - - SA UK 611 3 D Pra tia be gon ifo lia (W all. ) L ind l. Lea f nd - - - ? ? ? - ?? ? ? ?? ? ?? ? ? - - SA UK 611 4 D Dio sco rea op pos ita Ro ot nd - - - ? ? ? - ? ? ?? ? ?? ? - - SA UK 611 5 D Mos la dia nth era Ha m.) Max im Ste m nd - ? ? - ? - - ? ? ?? ? ? - - - SA UK 611 6 D Pru nel la vul gar is Lin n. Roo t nd ? ? ? ?? - ? - ? ? ?? ? ? ?? ?? ? - SA UK 61 17 D Sen eci o d ecl oux ii Dun n Roo t nd - ? - ? ? ? - ? ? ?? ? ?? ?? ? - - SA UK 611 8 D Im pat ien s c hin ens is L. Ste m nd ? ? - ? ?? - - ? ?? ?? ? ?? ? - SA UK 623 2 D Lys im ach ia for tun ei Max im . Ro ot nd ? ? ? ? ?? ?? ? - ? - ?? ? ?? ?? - - SA UK 623 3 D Mos la dia nth era (H am .) Max im Ro ot nd ? ? ? ?? ? ?? ? ?? ? ? ?? ? ?? ? ?? ? ? ?? ? ?? SA UK 623 4 D Vac cin iu m b rac tea tu m Thu nb Ro ot nd ? ? ? ?? ?? ?? ? - ? ?? ?? ? ?? ? ? ?? ? - SA UK 623 5 D Aco nit um ca rmi cha eli Deb x. Ste m nd ? ? ? - ? ? - ? ?? ? ? - - - - SA UK 623 6 D Gna pha liu m hyp ole ucu m D C. Roo t nd ? - ? ?? ? ? ?? ?? ? ? ?? ?? ? ?? ? ?? ? ? - SA UK 623 7 D Pot ent ill a d isc olo rB ge Roo t nd ? ? - ? - ?? - ?? ? ?? ? ? ? ?? - SA UK 623 8 D Im pat ien s c hin ens is L. Roo t nd - ? - ? - ?? - ?? ?? ?? ?? ? ?? - SA UK 623 9 D Gna ph ali um hyp ole ucu mD C. Roo t nd - ? - ? ? ?? - ? ?? ? ?? ?? - ? - SA UK 617 8 D Pte ris da cty lin aH ook . Ro ot nd - ? ? ? - ? - ? ?? ? ? ?? - - SA UK 617 9 D Dio sco rea op pos ita Roo t nd - ? - ?? ?? ? - ? - ?? ? ?? ?? ? ?? ? - SA UK 618 0 D Im pat ien s c hin ens is L. Roo t nd ? ? - ? - ?? - ? - ?? ?? ? ?? - SA UK 618 1 D Vac cin iu m b rac tea tu m Thu nb Ste m nd - ? ? ?? ? ? - ? ? ?? ?? - ? - - SA UK 618 2 D Aco nit um ca rmi cha eli Deb x. Roo t nd ? ? ? ? ?? ? ?? - - ? ?? ? ? - - - SA UK 618 3 D Ach yra nth es asp era Lin n. Roo t nd - ? ? ? ?? ? ? - ? - ?? ? - - - SA UK 618 4 D Cas syt ha filif or mis Roo t nd - ? - ? - ?? - ?? ? ?? ?? ? - - Ta ble 1 con tin ued Rep res ent ati ve str ain s rD NA Typ ea Hos t p lan ts Org an Clo ses t s equ enc e i n Gen ban k (si mil arit y) PK S- I PK S- II NR PS In dic ato r o rga nis m 1 2 3 4 5 6 7 8 9 10 11 SA UK 618 5 D Pra tia be gon ifo lia (W all. ) L ind l. Lea f nd ? ? ? ? - ?? - - - ?? - - ?? ? SA UK 624 2 D Sen eci o d ecl oux ii Dun n Roo t nd - ? ? ? ? ?? - ? ? ? ?? ?? ? - SA UK 61 62 D Sal via mi lti orr hiz aB ung e Roo t nd - ? - ? ? ?? - ? ? ? ?? ? ?? ? - - SA UK 641 1 D Ain sli aea he nry iD iel s Roo t nd - ? - ?? ?? ? - ?? ?? ?? ?? - - - SA UK 601 1 (FJ 842 55 2) E Sal via mi lti orr hiz aB un ge Roo t Str ept om yce s o chr ace isc ler oti cus (10 0% ) ?1 7 ?1 8 ?1 9 - - - - - - ?? ? ? ? - - SA UK 601 2 (FJ 842 553 ) F Ain sli aea he nry iD iel s Roo t Mic ro mon osp ora pe uce tia (10 0% ) ?2 0 ?2 1 ?2 2 - - ? - - - ? ? - - SA UK 603 0 (FJ 842 562 ) G Ste lle ra cha mae jas me L. Roo t Mic ro mon osp ora ch oko rie nsi s (99 %) ?2 3 ?2 4 ?2 5 - - ? - - - ?? ? ? ? - - SA UK 601 5 (FJ 842 555 ) H Ain sli aea he nry iD iel s Roo t Non om ura ea ros eol a (10 0% ) - ?2 6 ?2 7 - - ? - - ?? ? ?? ? ? - - SA UK 604 2 (FJ 842 56 5) I Pra tia be gon ifo lia (W all. ) L ind l. Roo t Oer sko via tu rba ta (99 %) - - ?2 8 - - - ? - - ? - - - - SA UK 603 3 (FJ 842 563 ) J Jun cus eff usu sL. Va r. Dec ipi ens Buc hen . Roo t Pro mic ro mon osp ora cy mbo pog oni s ( 100 %) ?2 9 ?3 0 ?3 1 - - - - - - ?? - - - - SA UK 603 9 (FJ 842 564 ) K Vac cin iu m b rac tea tu m Roo t Oer sko via en ter oph ila (99 %) - - - - - - - - ? - - - - - SA UK 601 3 (FJ 842 554 ) M Rhi zo ma Ari sae mat is Roo t Rho doc occ us zop fii (99 %) - ?3 2 - - - - - - - ? ? - - - Ind ica tor or gan is m1 .V erti cill iu m d ahl iae Kle b., 2. Fus ari um o xys por um f. sp. v asi nfe ctu m, 3. Asp erg ill us nig er ,4 .F usa riu m o xys por um f. sp. ni veu m, 5. Col let otri ch um or bic ula re , 6. Fus ari um gr ami nea ru m, 7.E xer ohi lu m t urc icu m, 8.C urv ula ria lu nat a, 9.B otr ytis ci ner ea , 1 0.S tap hyl oco ccu s a ure us, 11 .Es che ric hia co li Gen Ban k No. fo r t he seq uen ced P KS 1, PK S2 and N PR S g ene s: 1. GQ 118 920 , 2 . GQ 118 954 , 3 . GQ 118 923 , 4 . GQ 118 938 , 5 . GQ 11 89 58, 6. G Q1 18 92 2, 7. GQ 11 89 36, 8. G Q1 18 95 7, 9. GQ 118 92 1, 10. G Q1 189 35, 11 . G Q11 895 5, 12. G Q11 891 8, 13. G Q11 892 9, 14. G Q11 894 9, 15. G Q11 893 4, 16. G Q11 895 1,, 17. G Q1 18 92 4, 18. G Q1 18 94 3, 19. G Q1 189 60, 20 . G Q1 18 92 8, 21. G Q11 894 0, 22. G Q11 894 6, 23. G Q11 892 6, 24. G Q1 189 39, 25 . G Q11 894 8, 26. G Q11 893 3, 27. G Q11 896 1, 28. G Q11 894 8, 29. G Q1 18 92 7, 30. G Q1 18 94 2, 31. G Q1 18 94 5, 32. G Q1 18 93 0 nd not de ter min ed, (- ) n o i nhi biti on, (? ) i nhi biti on zon e,? ?? wid th of gro wth in hib iti on zon e[ 10 mm ,? ? 5–1 0 mm ,? 1– 5 mm a rD NA typ es wer e d efi ned ba sed up on the re stri cti on pat ter ns of AR DR A d ige ste d wit hH ae -III an dR sa -I Nonomuraea roseola, was a very potent inhibitor of pathogensExerohilum turcicumandCurvularia lunata,as wel as SAUK6030, a strain most similar toMicromonos- pora chokoriensiswas a potent inhibitor ofCurvularia lunata. Among the tested endophytic strains, there were isolates from 18 plant species with known antibacterial activity (Supplemental material 2), and at least one of the isolates from 9 out of 18 plant species showed antibacterial activity (Table1). In this study, we assessed the biosynthetic potential of the 60 isolates by amplifying the genes encoding polyke- tide synthases (PKS-I, PKS-I) and nonribosomal peptide synthetase (NRPS) using three sets of degenerate primers. 56 out of the 60 strains caried at least one of the biosyn- thetic enzyme genes (Table1). The number of strains with PKS-I, PKS-I and NRPS gene fragments matching the anticipated length were 32 (53%), 49 (82%) and 32 (53%), respectively. Sequence analysis of the amplified products of PKS-I, PKS-I and NRPS genes of the 14 representative isolates confirmed that al the partial sequences encoded parts of biosynthetic enzymes. SAUK6023 caried al the biosynthetical genes, yet it did not have antimicrobial activity against any of tested pathogens. Four strains SAUK6113, SAUK6114, SAUK6013 and SAUK6033 showed antimicrobial activity even though we were not able to amplify any of the PKS or NPRS genes from them (Table1). Discussion Plants growing in areas of great biodiversity are likely to house endophytes with equal or greater biodiversity. Plants with an ethnobotanical history and unusual longevity are expected to be more potent sources of endophytes pro- ducing active natural products than other plants [31]. 16S rRNA-RFLP technique is a powerful tool in grouping actinomycetes species and genera when studying diverse colections of microbial isolates [42]. However, there was considerable diversity within the RFLP groups B and D in respect of the presence of PKS-NPRS genes and antago- nistic activity of the strains. As assessed by 16S rRNA Streptomyces aurantiacusDSM40594T(AJ781383) SAUK6018 Streptomyces ederensisNBRC 15410T(AB184658) Streptomyces chryseusDSM40420T(AB184876) Streptomyces wedmorensisNRRL 3426T(DQ442557) SAUK6024 Streptomyces durhamensisNRRL B-3309T(AY999785) SAUK6020 SAUK6026 Streptomyces malaysiensisAT B - 1 1T(AF117304) SAUK6011 SAUK6022 SAUK6023 Streptomyces griseocarneusDSM40004T(X99943) Nonomuraea roseola ATCC33579T(U48980) Nonomuraea roseolaIFO 13155T(U48843) SAUK6015 Promicromonospora kroppenstedti DSM19349T (AM709608) SAUK6033 Oerskovia paurometabolaDSM 14281T(AJ314851) SAUK6039 SAUK6042 Rhodococcus zopfi ATCC 51349T(X81934) SAUK6013 Rhodococcus coprophilus ATCC29080T(X81928) Micromonospora peucetiaDSM 43363T (X92603) SAUK6012 SAUK6030 Bacilus pumilusDSM 27T(AY456263) 85 98 91 80 71 99 99 100 56 100 100 83 61 99 40 69 97 63 70 4288 0.02 Micromonospora chokoriensis JCM13247T(AB241454) Micromonospora olivasterospora DSM 43868T(X92613) Fig. 1Neighbor-joining phylogenetic tree analysis of 16S rDNA of endophytic actinomycete Thenumbersat the nodes indicate the levels of bootstrap support (%) based on 1000 resampled data sets. Only values above 50% are given. Thescale barcoresponds to 0.02 substitutions per nucleotide position. Numbers in parenthesesindicate accession numbers in Genbank.Bacilus pumilusDSM 27Twas used as an outgroup sequencing, the genus composition of endophytic actino- mycetes in our study was more diverse than that of acti- nomycetes isolated from rainforests [2,17], wheat root [5], herbaceous and woody plants [35] and Neem tree [36]. Stating that the dominance of the genus Streptomyces noticed in previous reports and in our study reflects the real world situation requires further culture-independent con- firmation [2,17,36]. We isolated actinomycetes of the generaPromicromonosporaandOerskovia, rare actino- mycetes that were first time found to be endophytic by Qin et al.[23] who also studied Chinese medicinal plants. Unlike Qin et al., our isolation methods were not designed to focus on the rare actinomycetes. Since similar isolation media and methods have been applied in studies on other plants, the findings of Qin et al.and the results of our study indicate that medicinal plants in general and the medicinal plants of Panxi plateau in particular host a great diversity of endophytic actinomycetes resources. However, to get more species information of endophytic actinomycetes in Panxi plateau, a wider range of isolation methods should be employed in further studies. Like in earlier studies, the strains we isolated possessed more antifungal than antibacterial properties [2,36]. In earlier studies with actinomycetes isolated from nonme- dicinal plant species, most of the strains have shown no antimicrobial activity and a smal percentage a narow antimicrobial activity [35]. In our study, only one out of the 60 strains was devoid of antagonistic activity.Streptomy- ces-like strain SAUK6233 isolated fromMosla dianthera Maxim, a plant with antibacterial activity [38], inhibited the growth of al tested indicator organisms, making it a good candidate for further studies. Also, strains SAUK6159, SAUK6024 and SAUK6185 isolated from Rhizoma arisaematis,Potentila discolorBge andPratia nummularia, respectively, with antioxidant, antitumor, antibacterial and antipyrotic activities [19,39,41], which had antimicrobial activity againstE. coliandStaphylo- coccus aureus, deserve further atention. The medicinal plants host numerousStreptomycesstrains expected to produce a wide variety of bioactive metabolites [17]. The antagonistic activity seen in our study further suggests that endophyticStreptomyceshosted by medicinal plants are a key source of bioactive compounds. In addition, the rare endophytic actinomycetes ofer a novel source for such compounds. Also, our results support the suggestion that the medicinal properties of medicinal plants could be at least partly due to the endophytes the plants host [31]. In recent years, scanning the genes encoding polyketide synthases and nonribosomal peptide synthetases that syn- thesize most of the biologicaly active polyketide and peptide compounds have been broadly applied in assessing the biosynthetic potential of culturable microorganisms and culture-independent samples [16,20]. However, the results from earlier studies and our results suggest that for the culturable actinomycetes, the antimicrobial potential may only be assessed by screening of antimicrobial activity against the desired indicator organisms. The percentages of strains with PKS-I, PKS-I and NPRS and the percentage of strains showing antimicrobial activity do not corelate, and there is an ample amount of examples of strains possessing the functional genes showing no antimicrobial activity and vice versa [17,23]. Here, the strain SAUK6023, most similar toStreptomyces aurantiacus, did not show any antimicrobial activity, yet we could amplify the PKS-NRPS genes from it. For this phenomenon there are several plausible explanations. Either the antimicrobials of SAUK6023 are efective against pathogens not tested by us or it produced them in quantities too low to inhibit patho- gens. It may also be due to the fact that the PKS-NRPS genes of SAUK6023 were silent or the PKS-NRPS gene clusters of the strain were incomplete. Also, four strains showed antimicrobial activity but none of the functional genes was detected in them. Possibly the code genes of antimicrobial products were not PKS or NRPS synthetical genes, or it may be due to the primers amplifying PKS- NRPS genes were not suitable to these strains. Interestingly, SAUK6033 belonged to genusPromicromonospora,in previous report [23] also noted that from the members of that genus the PKS-I, PKS-I and NRPS genes cannot be amplified, yet they possess antimicrobial activity. In conclusion, al but one of ourStreptomycesisolates tested displayed antimicrobial activity, with one-third of the strains showing antibacterial activity. In addition to Streptomyces, the medicinal plants caried rare actinomy- cetes al of which displayed antifungal activity. Our survey suggested that medicinal plants are a potent source of endophytic actinomycetes with wide biological activity against pathogenic fungi as wel as Gram-positive and Gram-negative bacteria. Acknowledgements This research was supported by the foundation of National Science Program of China (Project no. 30570062) and the fund of Sichuan provincal science and technology international cooperative project. 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