| Actinomycetes is an important resouse of microbial natural products. More than 22000 known biologically active compounds are obtained from microbes, and among them, 45% were produced by Actinobacteria. Unfortunately, as the result of conventional bioprospecting, the rate of new discoveries plummeted. As the development of seletivrly isolation menthods and genetic technology, the number antibiotics isolated from rare Actinomycetes increased significally. In this context, screening of biological resources that have not yet been explored for their potential natural products chould increase the likelihood of finding new strains, and the isolation of rare Actinomycetes could be crucial for modern drug-discovery programs to be successful. In order to take advantage of the microbial diversity in any habitat, it is necessary to use appropriate isolation techniques. In this study, we select seven plants, Lycium chinense Mill, Rehmannia glutinosa, Imperata cylindrica, Viola philippica Car, Bupleurum chinensis, Humulus scandens and Coptis chinensis Franch as the target. The endophytic and rhizospheric Actinomycetes diversity and the active compounds were studied by selectively isolation methods.(1) In this study, seven kinds of plants roots were dry heated and the rhizosphere soil were centrifuged for enrichment, and selective antimicrobial agents were used for isolation of actinomycetes. A total of 192 actinomycetes were isolated from the plant roots and rhizosphere soil, mostly on morphology of Streptomyces and Micormonospora, among them about 60-70% were Streptomyces, which was the dominance bacterium group. There were 70 endophytic strains and 122 rhizosphere actinomycetes strains. Despite the Lycium chinense Mill and Imperata cylindrica samples, the other plants have more rhizosphere actinomycetes than endophytic bacteria. The number of actinomycetes differentiate between the samples. More actinomycetes were isolated from rhizosphere soil of Viola philippica Car, Rehmannia glutinosa and Imperata cylindrica, and the strains have very different morphology. Even if the samples taken from the same region, the actinomycetes isolated different from each other. By 16 S r RNA gene sequencing of some strains, three specific strains st1, st4 and zh8 were finally select for polyphasic taxonomy and they were novel strains of Nonomuraea, Actinomycetospora and Micromonospora, respectively.(2) A total of 20 strains show activity against Bacillus subtilis or Micrococus luteus(diameter of inhibition zone more than 10mm), most of which were rhizosphere actinomycetes and only gq1, gq12 and sc6 were endophytic bacteria. Strain gt9, ht2 and and st18 showed strong activity against two bacteriea, with the diameter of inhibition zone more than 25 mm. A total of 16 fermentation of the strains show activity against two or more than two of the ten pathogenic fungi tested(diameter of inhibition zone more than 5mm), most of which were also rhizosphere actinomycetes except strains gq1, gq12 and hl7. Strain gq1 and gq12 have strong activity against Phytophthora sojae with the diameter of inhibition zone more than 30 mm, while strains st6, st14 and dht1 show activity against all ten pathogenic fungi tested. There were 14 strains show activity against both bacteriea and fungi, 6 strains(gt3, gt8, ht2, ht7, sc6 and st18) only against bacteriea and 2 strains(zt1 and hl7) only against fungi. The strains show activity in decussation method also have activity by confront culture method. All the strains have activity were Streptomyces likely and all the rare Actinomycetes show no activity. These may possibly because the other strains, such as Micromonospora have no aerial hypha, growing weakly in the medium, which were not conducive to the generation of antibiotics.(3) Strain st14 has a wide spectrum activity against 10 pathogenic fungi; the 16 S r RNA genesequence similarity analysis shows that strain st14 has a high similarity(99.66%) withStreptomyces scabies which cause potato common scab, so strain st14 was used for isolation ofnew compounds. The compound isolated from strain st14 has the same structure withConcanamycins A. The probability of isolation new antibiotics from Streptomyces isgreatlyreduced, and the isolation of rare actinomycetes still exist many problems. The isolation of activecompounds from rare actinomycetes is seriously restricted by their not easy to cultivate, and thestudy of rare actinomycetes is remote.(4) Characterization of strain st1 using a polyphasic approach: 16 S r RNA gene sequence similarity studies revealed that strain st1 belongs to the genus Nonomuraea, having the highest sequence similatity with Nonomuraea rosea GW12687T(98.91%), Nonomuraea solani NEAU-Z6T(98.44%), Nonomuraea rhizophila YIM67092T(98.24%) and Nonomuraea monospora PT708T(98.02%). The phylogenetic analysis also supported the position that strain st1 formed a distinct clade with its most closely related species. There are many different characteristics in spore arrangement, maximum Na Cl tolerance, some physiology and biochemistry characters and the major fatty acids between strain st1 and four type strains of Nonomuraea, and the DNA-DNA relatedness values between them are well below the threshold value of 70% recommended for the assignment of strains to the same species. Therefore, strain st1 represents a novel species of the genus Nonomuraea, for which the name Nonomuraea shaanxiensis sp. nov. is proposed. The type strain is NEAU-st1T(=CGMCC 4.7096 T =DSM 45877T).(5) Characterization of strain st4 using a polyphasic approach: 16 S r RNA gene sequence analysis showed that strain st4 was most closely related to Actinomycetospora rishiriensis RI109-Li102T(99.4%), Actinomycetospora corticicola 014-5T(99.1%), Actinomycetospora chiangmaiensis YIM 0006T(98.8%) and Actinomycetospora iriomotensis IR73-Li102T(98.2%). However, the low level of DNA-DNA relatedness differentiated strain st4 from its closest phylogenetic neighbours. The main chemotaxonomic properties of strain st4, such as the diamino acid of the peptidoglycan, the whole-cell hydrolysates, the predominant menaquinones and the phospholipid profile, supported its classification within the genus Actinomycetospora. Strain st4 formed sporangia directly on the substrate hyphae, which differentiated it from the other type strains of the genus Actinomycetospora. Strain st4 also had some phenotype characteristics such as maximum Na Cl tolerance and utilization of sole carbon sources. Consequently, strain st4 represents a novel species of the genus Actinomycetospora, for which the name Actinomycetospora atypica sp. nov. is proposed. The type strain is NEAU-st4T(=CGMCC 4.7093T=DSM 45873T).(6) Characterization of strain zh8 using a polyphasic approach: 16 S r RNA gene sequence similarity studies reveled that strain zh8 belonged to the genus Micromonospora, having the highest sequence similatity with Micromonospora chokoriensis 2-19(6)T(99.9%), Micromonospora saelicesensis Lupac 09T(99.3%) and Micromonospora lupini Lupac 14NT(99.0%). Study with gyr B gene indicated that strain zh8 should be assigned to the genus Micromonospora. The phospholipid profile of strain zh8 contained diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Whole-cell hydrolysates of strain zh8 were found to contain rhamnose, xylose, glucose and galactose. The cell-wall peptidoglycan of strain zh8 was consisted of meso-diaminopimelic acid and glycine. The major menaquinones of strain zh8 were MK-10(H4), MK-10(H2) and MK-10(H6). The major fatty acids were iso-C15:0, C16:0 and C17:0 10-methyl. A combination of DNA–DNA hybridization and some physiological and biochemical properties indicated that strain zh8 could be readily distinguished from the closest phylogenetic relatives. Therefore, it is proposed that strain zh8 represents a novel Micromonospora species, for which the name Micromonospora violae sp. nov. is proposed. The type strain is NEAU-zh8T(= CGMCC 4.7102 T = DSM 45888T). |
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