姜瘟致病菌的分离鉴定及其生防芽胞杆菌的筛选

Isolation and identification of the causal organisms of ginger bacterial wilt and screening of Bacillusstrains for biocontrol

摘要:

【目的】探究湖南省郴州市姜瘟发病情况，鉴定病原菌，筛选出拮抗生防菌，为姜瘟的有效控制提供依据。【方法】系统调查湖南省郴州市生姜主产区姜瘟发生情况，采集具有典型症状的姜块及其根系土壤，分离其病原菌，通过形态特征和分子鉴定确定病原菌的种属，通过回接姜苗确定其致病性；基于内源葡聚糖酶基因egl对分离的青枯雷尔氏菌进行序列变种鉴定；以分离鉴定到的病原菌为靶标菌筛选生防芽胞杆菌，并测定其防效。【结果】田间姜瘟平均发病率8.52%；从姜瘟病块分离到2株病原菌(FJAT-15492和FJAT-15494)，姜瘟根系土壤分离到3株病原菌(FJAT-15495、FJAT-15496和FJAT-15497)；经鉴定FJAT-15492为桑树肠杆菌(Enterobacter mori)，而其他4株菌为青枯雷尔氏菌(Ralstonia solanacearum)；致病性测定结果表明，分离的桑树肠杆菌和青枯雷尔氏菌均会引起姜瘟病害。病原菌分布特性分析显示，青枯雷尔氏菌在病姜块及其根系土壤中均有分布，而桑树肠杆菌只在病姜块中分布，且分布数量(1.33×10³ CFU/g)显著低于青枯雷尔氏菌的分布数量(5.67×10³ CFU/g)。演化型和序列变种鉴定结果表明，分离到青枯雷尔氏菌属于演化I型，序列变种14；通过抑菌圈试验发现，短短芽胞杆菌(Bacillus brevis) FJAT-JK-2对青枯雷尔氏菌、贝莱斯芽胞杆菌(Bacillus velezensis) FJAT-54560对桑树肠杆菌有较强拮抗作用，抑菌圈直径分别为19.41 mm和16.11 mm，对姜瘟的室内防效分别为69.45%和61.11%。然而将2种生防菌发酵液按等体积混合对姜瘟的田间防效达52.57%。【结论】本研究鉴定出姜瘟的致病菌，并筛选出2株生防芽胞杆菌，为姜瘟的生物防治提供新的菌株资源。

Abstract:

[Objective] We investigated the occurrence of ginger bacterial wilt, identified the pathogens, and screened the antagonistic bacteria, aiming to provide a scientific basis for the control of the disease. [Methods] We systematically investigated the occurrence of bacterial wilt in the main ginger production areas in Chenzhou City, Hunan Province. The ginger tubers with typical symptoms of bacterial wilt and the rhizosphere soil were collected. Major pathogens were isolated and identified based on morphological characteristics and molecular evidence. The pathogenicity of the isolates was determined by inoculation of the isolates to ginger seedlings. Moreover, the sequevars of Ralstonia solanacearum isolates were identified based on the endogenous glucanase gene egl. Finally, the Bacillus strains for biocontrol of the pathogens were screened, and their biocontrol effects were measured. [Results] The average incidence of ginger bacterial wilt in the field was 8.52%. Two bacterial strains FJAT-15492 and FJAT-15494 were isolated from diseased ginger tubers, and three bacterial strains FJAT-15495, FJAT-15496, and FJAT-15497 were isolated from the rhizosphere soil of diseased ginger. The strain FJAT-15492 was identified as Enterobacter mori and the other four isolates were R. solanacearum. Both the isolates of E. mori and R. solanacearum could infect ginger seedlings and cause bacterial wilt. R. solanacearum strains existed in both diseased ginger tubers and rhizosphere soil, while E. mori only existed in diseased ginger tubers, with the count (1.33×10³CFU/g) lower than that (5.67×10³ CFU/g) of R. solanacearum.Furthermore, the R. Solanacearum isolates were identified as phylotype Ⅰ and sequevar 14. Brevibacillus brevis FJAT-JK-2 demonstrated inhibitory effect on R. solanacearum, and Bacillus velezensis FJAT-54560 on E. mori, with inhibition zone diameters of 19.41 mm and 16.11 mm and indoor control effects of 69.45% and 61.11%, respectively. Moreover, the fermentation mixture of the two biocontrol strains had the field control effect of 52.57%. [Conclusion] This work identified the pathogens of ginger bacterial wilt and provided two new biocontrol strains against the disease.