祁连山高山植物根际土放线菌生物多样性

从祁连山老虎沟不同海拔位点的15种植物根际土中培养得到78株特异表型放线菌,并结合菌体形态、生理代谢特征、抗菌活性及16S rDNA序列对其生理及系统发育多样性进行了研究。结果表明,分离菌株分属于链霉菌属(Streptomyces spp.)(73株)、诺卡氏菌属(Nocardia spp.)(4株),另有1株与GenBank中同源性最高的菌株Micromonospora saelicesensis相似性达92%,为1潜在新种。链霉菌属为主要类群,占分离菌株的93.6%,该属菌株在5个海拔位点的15种植物根际土中均有分布,但存在海拔位点、植物种类的差异性和特异性;诺卡氏菌属的菌株仅见于海拔2200 m的猪毛菜、海拔2800 m的钉柱萎陵菜和3800 m处的甘肃蚤缀根际土中;1潜在新种分离自海拔2200 m处的沙生针茅根际土。次级代谢物产生和拮抗性筛选研究结果表明:H2O2酶、脂酶2(Tween-40)、脲酶、蛋白酶、脂酶3(Tween-80)、淀粉酶、H2S、脂酶1(Tween-20)、可溶性色素及有机酸这10类次级代谢物产生菌分别占供试菌株的89.7%、82.1%、70.5%、62.8%、53.8%、52.6%、48.7%、44.9%、32.1%和17.9%,其中,淀粉酶、脂酶1、色素和有机酸仅由链霉菌产生;有29株放线菌对参试人类病原菌具有抑制作用,占供试菌株的37.2%,分布于5个海拔位点的12种植物根际土,其中,从药用植物甘肃黄芪和四裂红景天根际土中分离到的抗性菌株占拮抗性放线菌总数的60%。研究表明,高山地区植物根际土放线菌资源丰富,菌株生理功能多样,是新放线菌种和生物活性物质的重要资源库。

Phylogenetic and physiological diversity of actinomycetes isolated from plant rhizosphere soils in the Qilian Mountains

Actinomycetes are important producers of bio-active metabolites, including antibiotics, plant growth factors, enzyme inhibitors and many other beneficial compounds. Alpine extreme habitats that contain low temperature-, anoxia-, and stress-adapted plants, and rhizosphere soils may harbor novel actinomycete strains with unique metabolic characteristics and new secondary metabolites. The Laohugou Valley of the Qilian Mountains, in the northeastern of the Tibetan Plateau, is characterized by its high elevation (> 2000 m) and a distinct vertical distribution of vegetation types, ranging among desert steppe at 2200 m, mesophorbium at 2800 m, alpine shrub at 3350 m, alpine meadow at 3800 m, and alpine cold-desert at 4200 m. However, little is known about the diversity and distribution of actinomycetes in these habitats. Thus, we analyzed 15 rhizosphere soil samples from different vegetation types at increasing altitudes in Laohugou Valley to investigate the diversity and physiological characteristics of actinomycetes. We recovered 78 strains of actinomycetes with representative phenotype using glycerin arginine medium with potassium dichromate and penicillin. The morphology of colony hypha, small subunit 16S rRNA sequences, phylogenetic relationships, and physiological heterogeneity of these isolates were determined. The results revealed that 73 strains were closely related to Streptomyces spp. (93.6% of all isolates), which grouped into 21 species. Streptomyces spp. were found in all samples, where the abundance and diversity varied at different altitudes among rhizosphere soils. The highest diversity of Streptomyces spp. was found in the rhizosphere soil of Astragalus licentianu at 4200 m and the lowest in the rhizosphere soil of Poa annua at 3800 m. Four isolates (5.1% of all isolates) in the genus Nocardia were found in the rhizosphere soils of Salsola collina at 2200 m, Potentilla saudersiana at 2800 m and Arenaria kansuensis at 3800 m. We also isolated a strain from the rhizosphere soil of Stipa glareosa at 2200 m which shared 92% sequence similarity to its nearest match Micromonospora saelicesensis from database, indicating that it might be a new actinomyces species. The physiological analyses of the isolates indicated that strains producing catalase, lipase 2 (Tween-40), urease, proteinase, lipase 3 (Tween-80), amylase, H₂S, lipase 1 (Tween-20), diffusible pigment and organic acids accounted for 89.7%, 82.1%, 70.5%, 62.8%, 53.8%, 52.6%, 48.7%, 44.9%, 32.1% and 17.9% of the total actinomycetes isolates, respectively. Amylase, lipase 1, pigments and organic acid were produced only by Streptomyces isolates. The antimicrobial resistant patterns of the isolates were tested with Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Candida albicans (ATCC 66415), and a Pseudomonas aeruginosa strain isolated from clinical sputum. The results showed that 29 strains isolated from the rhizosphere soils of 12 plants at different altitudes exhibited antimicrobial activity, i.e., 37.2% of the total isolates. The isolates from the rhizosphere soils of two medicinal plants (Astragalus licentianu and Rhodiola quadrifida) comprised 60% of the total antimicrobial strains. The distinct antibiotic resistant patterns and physiological traits were detected among strains within the identical species. Our results suggested that the rhizosphere soils of alpine plants in the Qilian Mountain may be potential sources of new actinomycetes strains with novel bioactive compounds.