高效离子交换色谱法分析青枯雷尔氏菌无致病力epsD突变菌株的异质性

Analysis of the heterogeneity of Ralstonia solanacearum avirulent espD-mutant by high performance ion-exchange chromatography

利用高效离子交换色谱(HPIEC)技术结合形态观察和胞外多糖(EPS)含量测定,比较分析青枯雷尔氏菌无致病力突变菌株FJAT-91⊿epsD与强致病力出发菌株FJAT-91及自然致弱的无致病力菌株FJAT-1458的异质性。结果表明,菌株FJAT-91⊿epsD与菌株FJAT-91在菌落和菌体形态上均有明显差异,与菌株FJAT-1458的形态相似。胞外多糖含量测定结果表明,菌株FJAT-91⊿epsD的EPS含量((12.64±1.46)μg/mL)显著低于菌株FJAT-91((30.49±2.97)μg/mL),与菌株FJAT-1458的EPS含量((11.30±1.38)μg/mL)相当。高效离子交换色谱分离结果表明,菌株FJAT-91、FJAT-91⊿epsD和FJAT-1458形成的色谱峰个数和保留时间不同,菌株FJAT-91只有单一色谱峰P3,保留时间为6.04 min;菌株FJAT-91⊿epsD有P1和P2两个色谱峰,保留时间分别为0.59 min和4.62 min;菌株FJAT-1458只有单一色谱峰P1,保留时间为0.59 min。对不同色谱峰回收培养,并对回收培养后的菌株进行致病力鉴定。结果表明,番茄植株接种菌株FJAT-91-P3后第4 d开始发病,第10 d发病率达100%;植株接种菌株FJAT-91⊿epsD-P2后第9 d开始发病,第20 d发病率达100%;植株接种菌株FJAT-1458-P1和菌株FJAT-91⊿epsD-P1后,在观察期内均未发病。研究结果可为利用菌株FJAT-91⊿epsD防治作物青枯病提供理论依据。

The heterogeneity of Ralstonia solanacearum avirulent espD-mutant,virulent wildtype strain FJAT-91 and spontaneous avirulent strain FJAT-1458 were compared and analyzed by using high performance ion-exchange chromatography(HPIEC). Their colony and cell morphologies were observed,and the contents of their extracellular polysaccharides(EPS)were determined. The results showed that the colony and cell morphologies of FJAT-91⊿ epsD were different from the original strain FJAT-91,but were similar to the spontaneous avirulent strain FJAT-1458. The EPS content of FJAT-91⊿ epsD was(12. 64 ± 1. 46)μg/mL,which was much lower than FJAT-91 with(30. 49±2. 97)μg/mL. The strain FJAT-91 were separated into single peak of P3 at the retention time of 6. 04 min,and FJAT-91⊿ epsD had two peaks(P1 and P2)at the retention times of 0. 59 min and 4. 62 min,respectively. FJAT-1458 formed single peak of P1 at the retention time of 0. 59 min. Then,the bacteria eluents from different chromatographic peaks were collected and their pathogenicities were detected with bioassay test using potted tomatoes in the greenhouse. The results showed that the tomato plants began to wilt 4 d after inoculation of strain FJAT-91-P3 eluted from P3,and the disease incidence reached 100% at 10 d post inoculation. The strain FJAT-91⊿ epsD-P2 eluted from P2 caused tomato plants wilting 9 d after inoculation,and the disease incidence reached 100% at 20 d. During 20 d of observation,none of tomato plants caused wilting while inoculation with FJAT-1458-P1 and FJAT-91⊿ epsD-P1 eluted from P1 of FJAT-1458 and FJAT-91⊿ epsD,respectively. In this paper,the morphologies,EPS contents and chromatographic behaviors of FJAT-91,FJAT-91⊿ epsD and FJAT-1458 were compared,which would provide theoretical basis for using FJAT-91⊿ epsD to control bacterial wilt disease.