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

Abstract

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.