Microbispora clausenae sp. nov., an endophytic actinobacterium isolated from the surface-sterilized stem of a Thai medicinal plant, Clausena excavala Burm. f.

An endophytic actinobacterium, strain CLES2T, was discovered from the surface-sterilized stem of a Thai medicinal plant, Clausena excavala Burm. f., collected from the Phujong-Nayoa National Park, Ubon Ratchathani Province, Thailand. The results of a polyphasic taxonomic study identified this strain as a member of the genus Microbispora and a Gram-stain-positive, aerobic actinobacterium. It had well-developed substrate mycelia, which were non-motile and possessed paired spores. A phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this strain in the family Streptosporangiaceae, being most closely related to Microbispora bryophytorum NEAU-TX2-2T (99.4 %), Microbispora camponoti 2C-HV3T (99.2 %), Microbispora catharanthi CR1-09T (99.2 %) and Microbispora amethystogenes JCM 3021T and Microbispora fusca NEAU-HEGS1-5T (both at 99.1 %). The major cellular fatty acid of this strain was iso-C16 : 0 and major menaquinone was MK-9(H4). The polar lipid profile of strain CLES2T contained diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylinositol and phosphatidylinositol dimannosides. These chemotaxonomic data confirmed the affiliation of strain CLES2T to the genus Microbispora. The DNA G+C content of this strain was 70 mol%. Digital DNA-DNA hybridization and average nucleotide identity blast values between strain CLES2T and M. catharanthi CR1-09T were 62.4 and 94.0 %, respectively. The results of the polyphasic study allowed the genotypic and phenotypic differentiation of strain CLES2T from its closest species with valid names. The name proposed for the new species is Microbispora clausenae sp. nov. The type strain is CLES2T (=DSM 101759T=NRRL B-65340T).

Increased Carbazole Alkaloid Accumulation in Clausena harmandiana Callus Culture by Treatments of Biocontrol Agent, Trichoderma harzianum and Bacillus subtilis.

Clausena harmandiana (Pierre) Guillaumin is the Thai medicinal plant that possessed several pharmacological activities. The main active constituents of this plant are the carbazole alkaloids, isolated from wild plants. However, the in vitro culture for production of carbazole alkaloids from this plant has never been reported. Therefore, we aimed to develop callus culture of C. harmandiana elicited with two biotic elicitors, Trichoderma harzianum and Bacillus subtilis, as a sustainable source of carbazole alkaloids. The callus treated with living B. subtilis (BL) at 0.1 and 1% (v/v) for 3 days accumulated 5-fold increased level of clausine K. The highest level reached 309.37 ± 34.84 µg/g DW. This treatment also showed a significant increase in both total phenolic content and antioxidant capacity, which support the optimum usage of this elicitor. Moreover, only callus treated with 1% (v/v) Trichoderma culture filtrate (CF) showed a significant increase in the total phenolic contents and antioxidant capacity. The correlation analysis also revealed the significant correlation between antioxidant capacity and total phenolic level, total flavonoids, and clausine K but not 7-methoxymukonal. The results from our study suggested the use of C. harmandiana callus with the Bacillus elicitors for high-level production of clausine K.