ประสิทธิภาพของ Bacillus subtilis S93 และ Bacillus siamensis RFCD306 ในการควบคุม โรคเหี่ยวฟิวซาเรียมของเบญจมาศ

Abstract

Fusarium wilt of chrysanthemum, caused by Fusarium spp., is a major disease causing severe damage to commercial chrysanthemums worldwide. The survey and sample collection found that 6 isolates of fungi were isolated including isolate CCR-01, CCR-02, CCR-03, CL-04, CL-05 and CL-06. The diseased sample was collected from Huay Nam Rin Royal Project Development Center. Fusarium oxysporum isolate COSP01 is the causal agent of chrysanthemum wilt, previously isolated from chrysanthemum which cultivated at Huay Luk Royal Project Development Center. Pathogenicity test on chrysanthemum of 7 fungal isolates, the isolate CCR-02 and F. oxysporum isolate COSP01 caused the most disease severity at 80.03 and 80.00 %, respectively. Afterward, the isolate CCR-02 was identified based on morphological characteristics, and molecular study of multiple DNA sequience of ITS and LSU, ITS with primer ITS1 and ITS4, and LSU-F and LSU-R, respectively. The result showed that isolate CCR-02 was identified as Fusarium equiseti (GenBank accession nos. OP247660 and OP247680). Efficiency of Bacillus subtilis S93 and Bacillus siamensis RFCD306 for in vitro inhibiting of F. equiseti CCR-02 and F. oxysporum COSP01 causing chrysanthemum wilt disease using dual culture method was investigated. The result showed that at 7 days of incubation of B. subtilis S93 and B. siamensis RFCD306 on PDA before challenged with F. equiseti CCR-02 showed the highest of percentage inhibition with 78.56 and 78.07 %, respectively. While, at 5 days of incubation antagonistic bacteria showed the highest inhibitory effect to F. oxysporum COSP01 with 71.25 and 73.75 %, respectively. Inhibition of fungal spore germination by cell-free culture filtrated (CF) of antagonistic bacteria (cultured in four liquid media including 3% TSB, TSB, MRS and AM2ab) was conducted by slide culture method. The results showed that after 4 hours of incubation, CF from B. siamensis RFCD306 in AM2ab broth had the highest inhibition of the spore germination up to 99.00 %. Moreover, the CF caused malformation and swelling germ tube, swollen mycelium of fungal pathogen. Besides, the fungal spore treated in CF from B. siamensis RFCD306 in AM2ab broth was tested for pathogenicity. The result showed that spore in CF of B. siamensis RFCD306 could reduce the severity of fungal the pathogen as 42.69 and 51.02 %, respectively. Greenhouse experiment was evaluated for ability of B. subtilis S93 and B. siamensis RFCD306 to control chrysanthemum wilt disease. The result showed that B. subtilis S93 could control wilt disease caused by F. equiseti CCR-02 and F. oxysporum COSP01 with 50.00 and 54.77 %, respectively. While, B. siamensis RFCD306 could control wilt disease with 60.81 and 65.37 %, respectively. For growth promotion efficiency, it was found that chrysanthemum plants treated with B. subtilis S93 and B. siamensis RFCD306 significantly increased height, fresh weight and root length of chrysanthemum plants. It is statistically significant compared to the control set at 95% confidence level.