[A preliminary study on origin of ligustrazine in Chuanxiong Rhizoma based on endogenetic Bacillus subtilis].

Ligustrazine is an important active ingredient of the traditional Chinese medicine Chuanxiong Rhizoma, but its content is a controversial topic. The endophytes of medicinal plants have the ability to produce the same active substances as the host, so this report focused on the endophytic Bacillus subtilis, to study the origin of ligustrazine in Chuanxiong Rhizoma preliminarily by inoculating the isolated endophytic B. subtilis to the Chuanxiong Rhizoma medium in vitro for solid state fermentation. Tissue grinding method was used to isolate the endogenetic B. subtilis. The morphological features, conventional physiological and biochemical reactions and 16S rRNA molecular techniques were combined to identify the endogenetic strains. Then, the strains that grew well in the medicinal matrix of Chuanxiong Rhizoma were screened out for further fermentation studies. The solid-state fermentation was performed at 37 °C for 30 d using Chuanxiong Rhizoma fermentation medium (40 g Chuanxiong Rhizoma powder, 100 mL sterile water, 121 °C, sterilization for 25 minutes). UPLC was used to detect the contents of ligustrazine, acetoin in the Chuanxiong Rhizoma fermentation medium and Chuanxiong Rhizoma. All the five strains were Gram-positive and had spores. Phylogenetic analysis of the 16S rRNA sequence showed that the endophytes were B. subtilis. The results of UPLC showed that ligustrazine was detected in the Chuanxiong Rhizoma fermentation medium inoculated with endogenetic B. subtilis LB3, LB3-2-1, LB4, LB5 and LB6-2, while not detected neither in blank Chuanxiong Rhizoma fermentation medium nor in Chuanxiong Rhizoma. This study showed that the endogenetic B. subtilis of Ligusticum chuanxiong Hort. can make use of Chuanxiong Rhizoma fermentation medium to produce ligustrazine. Endogenetic B. subtilis has a certain correlation with the accumulation of ligustrazine in Rhizoma Chuanxiong. We speculate that the ligustrazine may be derived from the catabolism of endogenetic B. subtilis in Ligusticum chuanxiong.

[Identification of Pathogens Causing Root Rot Disease on Ligusticum chuanxiong in Sichuan].

OBJECTIVE: To identify the pathogens causing root rot disease on Ligusticum chuanxiong from the main producing areas in Sichuan, and to provide gist of management of this disease in the future. METHODS: The diseased rhizomes of Ligusticurn chuanxiong with root rot symptom were sampled in two main producing areas in Sichuan. According to Koch's postulate, the pathogenicity on Ligusticum chuanxiong rhizomes of the isolates was confirmed. The representative isolates were identified based on their morphological characteristics and ribosomal DNA internal transcribed spacer(rDNA-ITS) sequences. RESULTS: The isolates(f2-16, 3-2, f4-19 and f5-7)which caused root rot disease held high homology reached 99%, 99%, 100% and 100% respectively, when compared with GenBank sequences GQ229075, JQ796755, JN232136 and FJ481024. CONCLUSION: The causing agents of root rot disease on Ligusticum chuanxiong belong to Fusarium solani, Fusarium oxysporum, Plectosphaerella cucumerina and Phoma glomerata. Pathogens from different regions are varied.

[Isolation and identification of endophytic fungi from Ligusticum].

OBJECTIVE: To investigate the correlation between entophytic fungal community, habitations and varieties of Ligusticum. METHOD: The solidified potato dextrose agar (PDA) of plates was applied for the isolation of the endophytic fungi, and the identification was completed by spot-planting method. RESULT: Fifty strains of the entophytic fungi were isolated from the rhizome of L. chuanxiong collected from 6 habitations. They were morphologically identified as belonging to 13 genera, 4 families, 3 orders and 1 class. CONCLUSION: There were some differences at quantity, species and distributing of the entophytic fungi in different habitats and varieties of Ligusticum, which was suggested that entophytic fungal community is related with specific habitat.