Amino acid substitutions in GyrA of Burkholderia glumae are implicated in not only oxolinic acid resistance but also fitness on rice plants

Abstract

Oxolinic acid (OA) resistance in field isolates of Burkholderia glumae, a causal agent of bacterial grain rot, is dependent on an amino acid substitution at position 83 in GyrA (GyrA83). In the present study, among spontaneous in vitro mutants from the OA-sensitive B. glumae strain Pg-10, we selected OA-resistant mutants that emerged at a rate of 5.7 x 10(-10). Nucleotide sequence analysis of the quinolone resistance-determining region in GyrA showed that Gly81Cys, Gly81Asp, Asp82Gly, Ser83Arg, Asp87Gly, and Asp87Asn are observed in these OA-resistant mutants. The introduction of each amino acid substitution into Pg-10 resulted in OA resistance, similar to what was observed for mutants with the responsible amino acid substitution. In vitro growth of recombinants with Asp82Gly was delayed significantly compared to that of Pg-10; however, that of the other recombinants did not differ significantly. The inoculation of each recombinant into rice spikelets did not result in disease. In inoculated rice spikelets, recombinants with Ser83Arg grew less than Pg-10 during flowering, and growth of the other recombinants was reduced significantly. On the other hand, the reduced growth of recombinants with Ser83Arg in spikelets was compensated for under OA treatment, resulting in disease. These results suggest that amino acid substitutions in GyrA of B. glumae are implicated in not only OA resistance but also fitness on rice plants. Therefore, GyrA83 substitution is thought to be responsible for OA resistance in B. glumae field isolates.

FIG. 1.

MIC of OA in spontaneous in vitro mutants from B. glumae strain Pg-10 and their deduced amino acid sequences of the QRDR in GyrA. aa, amino acids.

FIG. 2.

In vitro growth of B. glumae recombinants with amino acid substitutions in GyrA. Values represent the means ± standard deviations for three separate experiments. OD₅₈₀, optical density at 580 nm.

FIG. 3.

Populations of B. glumae recombinants with amino acid substitutions in GyrA on rice spikelets at 5 days after flowering. Rice spikelets were inoculated with the recombinants at 10 days before flowering. Values represent the means ± standard deviations for three separate experiments.