BackgroundColletotrichum is one of the most widespread and important genus of plant pathogenic fungi worldwide. Various species of Colletotrichum are the causative agents of anthracnose disease in plants, which is a severe problem to agricultural crops particularly in Thailand. These phytopathogens are usually controlled using chemicals; however, the use of these agents can lead to environmental pollution. Potential non-chemical control strategies for anthracnose disease include the use of bacteria capable of producing anti-fungal compounds such as actinomycetes spp., that comprise a large group of filamentous, Gram positive bacteria from soil. The aim of this study was to isolate actinomycetes capable of inhibiting the growth of Colletotrichum spp, and to analyze the diversity of actinomycetes from plant rhizospheric soil.ResultsA total of 304 actinomycetes were isolated and tested for their inhibitory activity against Colletotrichum gloeosporioides strains DoA d0762 and DoA c1060 and Colletotrichum capsici strain DoA c1511 which cause anthracnose disease as well as the non-pathogenic Saccharomyces cerevisiae strain IFO 10217. Most isolates (222 out of 304, 73.0%) were active against at least one indicator fungus or yeast. Fifty four (17.8%) were active against three anthracnose fungi and 17 (5.6%) could inhibit the growth of all three fungi and S. cerevisiae used in the test. Detailed analysis on 30 selected isolates from an orchard at Chanthaburi using the comparison of 16S rRNA gene sequences revealed that most of the isolates (87%) belong to the genus Streptomyces sp., while one each belongs to Saccharopolyspora (strain SB-2) and Nocardiopsis (strain CM-2) and two to Nocardia (strains BP-3 and LK-1). Strains LC-1, LC-4, JF-1, SC-1 and MG-1 exerted high inhibitory activity against all three anthracnose fungi and yeast. In addition, the organic solvent extracts prepared from these five strains inhibited conidial growth of the three indicator fungi. Preliminary analysis of crude extracts by high performance liquid chromatography (HPLC) indicated that the sample from strain JF-1 may contain a novel compound. Phylogenetic analysis revealed that this strain is closely related to Streptomyces cavurensis NRRL 2740 with 99.8% DNA homology of 16S rRNA gene (500 bp).ConclusionThe present study suggests that rhizospheric soil is an attractive source for the discovery of a large number of actinomycetes with activity against Colletotrichum spp. An interesting strain (JF-1) with high inhibitory activity has the potential to produce a new compound that may be useful in the control of Colletotrichum spp.
Two new polyene macrolactone antibiotics, thailandins A, 1, and B, 2, were isolated from the fermentation broth of rhizosphere soil-associated Actinokineospora bangkokensis strain 44EHW(T). The new compounds from this strain were purified using semipreparative HPLC and Sephadex LH-20 gel filtration while following an antifungal activity guided fractionation. Their structures were elucidated through spectroscopic techniques including UV, HR-ESI-MS, and NMR. These compounds demonstrated broad spectrum antifungal activity against fungi causing anthracnose disease (Colletotrichum gloeosporioides DoA d0762, Colletotrichum gloeosporiodes DoA c1060, and Colletotrichum capsici DoA c1511) as well as pathogenic yeasts (Candida albicans MT 2013/1, Candida parasilopsis DKMU 434, and Cryptococcus neoformans MT 2013/2) with minimum inhibitory concentrations ranging between 16 and 32 μg/mL. This is the first report of polyene antibiotics produced by Actinokineospora species as bioactive compounds against anthracnose fungi and pathogenic yeast strains.
A novel actinomycete, strain 44EHW T , was isolated from rhizospheric soil under an Elephant ear plant (Colocasia esculenta) in Bangkok, Thailand. Strain 44EHW T produced long branching hyphae and abundant aerial mycelia with chains of rod-shaped spores. Whole-cell hydrolysates contained galactose, glucose, arabinose, ribose, mannose and rhamnose as diagnostic sugars. meso-Diaminopimelic acid was the diamino acid and glycine, alanine and glutamic acid were present in the cell-wall peptidoglycan with the acyl type of the peptidoglycan being acetyl. Phospholipids consisted of phosphatidylethanolamine, phosphatidylethanolamine with hydroxy fatty acids and diphosphatidylglycerol, as well as other unknown phospholipids; however, no mycolic acids were detected. The predominant menaquinone observed was MK-9(H 4 ) and major fatty acids were iso-C 16 : 0 and 2-OH iso-C 16 : 0 . The G+C content of genomic DNA was 74 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that this isolate was most similar to Actinokineospora enzanensis NBRC 16517 T . However, DNA-DNA hybridization revealed a low relatedness between this isolate and A. enzanensis NBRC 16517 T , indicating that this isolate represented a novel species in the genus Actinokineospora. On the basis of 16S rRNA gene sequence analysis, phenotypic characteristics and DNA-DNA hybridization data, we propose that strain 44EHW T represents a novel species in the genus Actinokineospora, Actinokineospora bangkokensis. The type strain is 44EHW T (5BCC 53155 T 5NBRC 108932 T ).The genus Actinokineospora was proposed by Hasegawa (1988) as a member of the family Pseudonocardiaceae and was later emended by Labeda et al. (2010), for non-motility in some species. Strains in the genus Actinokineospora contain meso-diaminopimelic acid, glycine, D-glutamic acid and L-alanine in their cell wall and have arabinose, galactose, mannose and rhamnose as the characteristic whole-cell sugars. The phospholipid pattern consists of phosphatidylethanolamine, including phosphatidylethanolamine containing hydroxylated fatty acids and the major menaquinone is MK-9(H 4 ) (Hasegawa, 1988;Labeda et al., 2010). Currently, there are 12 species in the genus During our search for new sources of bioactive compounds, many rare and slow-growing actinomycetes were isolated and identified. Strain 44EHW T was isolated from a soil sample collected under an Elephant ear (Colocasia esculenta) plant in Bangkok, Thailand. Air-dried soil samples were subjected to a pretreatment using dry heat at 120 u C for 1 h (Hayakawa et al., 1991). Serially diluted soil suspensions were spread on water-proline agar (proline 1.0 %, agar 1.2 %, tap water, pH 7.0) supplemented with 25 mg nalidixic acid ml 21 and 50 mg cycloheximide ml 21 and incubated for 4 weeks at 28 u C.
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