青枯雷尔氏菌无致病力hrpB突变菌株的纯化分离及其异质性研究

获得纯度高的青枯雷尔氏菌无致病力菌株,是研发青枯病植物疫苗和防治青枯病害的一种新途径。作者以青枯雷尔氏菌强致病力菌株FJAT-91为出发菌株,通过对hrpB基因敲除,获得无致病力突变菌株FJAT-91ΔhrpB。高效离子交换色谱分离结果表明:FJAT-91和FJAT-91ΔhrpB色谱峰型不同,主要表现在峰的保留时间上,FJAT-91只有单一色谱峰,保留时间为6 min;FJAT-91ΔhrpB有P_1和P_2 2个色谱峰,保留时间分别为0.6 min和4.5 min。利用高效离子交换色谱对FJAT-91ΔhrpB进行纯化,获得只有P_1峰的高纯度菌株FJAT-91ΔhrpB-P。FJAT-91ΔhrpB和FJAT-91ΔhrpB-P与其出发菌株FJAT-91的菌落和菌体形态差异明显。致病力测定结果表明:FJAT-91接种4 d番茄植株开始发病,10 d发病率达100%;FJAT-91ΔhrpB和FJAT-91ΔhrpB-P接种20 d均未发病。防效试验结果表明:纯化后的菌株FJAT-91ΔhrpB-P对番茄青枯病的防效(81.64%)比未纯化FJAT-91ΔhrpB防效(61.04%)提高了33.75%。本研究获得一株高纯度的青枯雷尔氏菌无致病力突变菌株FJAT-91ΔhrpB-P具有良好的生防潜力。

Isolation,purification and heterogeneity analysis of avirulent hrpB mutant of Ralstonia solanacearum

Using an avirulent strain of Ralstonia solanacearum with high purity to produce ‘plant vaccine’ is a novel approach to control bacterial wilt disease. The hrpB gene of R. solanacearum virulent strain FJAT 91 was knocked out and avirulent mutant FJAT 91 ΔhrpB was obtained. Chromatographic behaviors of FJAT 91 and FJAT 91ΔhrpB were compared by using high performance ion exchange chromatography. The results showed that FJAT 91 formed single peak at the retention time of 6 min, and FJAT 91ΔhrpB had P₁ and P₂ two peaks at the retention time of 0.6 min and 4.5 min, respectively. Then, FJAT 91ΔhrpB was purified based on high performance ion exchange chromatography, and highly pure strain FJAT 91ΔhrpB P with only P₁ peak was obtained. FJAT 91ΔhrpB P and FJAT 91ΔhrpB significantly distinguished from FJAT 91 in terms of cell and colonies morphology. Pathogenicities of FJAT 91ΔhrpB P, FJAT 91ΔhrpB and FJAT 91 were detected with bioassay test using potted tomato in the greenhouse. The tomato plants began to wilt 4 d after inoculation of FJAT 91, and the disease incidence reached up to 100% at 10 d post inoculation. None of FJAT 91ΔhrpB P and FJAT 91ΔhrpB caused tomato plants wilting 20 d after inoculation. The control efficiency of FJAT 91ΔhrpB P reached to 81.64%, which was 33.75% higher than that of FJAT 91ΔhrpB. Therefore, the obtained high pure avirulent mutant FJAT 91ΔhrpB P had good potential for biocontrol against bacterial wilt disease.