| Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae is one of the most serious bacterial diseases of rice. Bismerthiazol and streptomycin are the most commonly used bactericide to control this disease and have been used above 30 years in China, so it is necessary to learn the status and mechanism of resistance of the two antibiotics. The objectives of this study were focused on 1) Characteristics and mechanisms of laboratory-induced streptomycin-resistant mutants of Xanthomonas oryzae pv. oryzae; 2) To screen for the streptomycin-resistance in the populations of X. oryzae pv. oryzae and X. oryzae pv. oryzicola from the south of China; 3) Mechanisms of streptomycin resistance in field isolates of Xanthomonas oryzae pv. oryzae and the character of resistance integron; 4) Characterization of bismerthiazol resistance of Xanthomonas oryzae pv. oryzae; 5) Monitoring bismerthiazol resistance of Xanthomonas oryzae pv. oryzae.1 characteristics and Mechanisms of Laboratory-Induced Streptomycin-Resistant Mutants of Xanthomonas oryzae pv. oryzaeSix streptomycin-resistant mutants of Xanthomonas oryzae pv. oryzae were obtained by ultraviolet light induction in the laboratory. The minimal inhibition concentrations (MICs) of streptomycin against wild-type strain ZJ173 and mutants were 0.10μg/mL and 600μg/mL, respectively. The medium effective concentration (EC50) of wild-type strain ZJ173 was 0.03μg/mL and the average EC50 of six mutants was 11.64 (3.15-23.75)μg/mL. The resistance factor (RF) of laboratory mutants relative to their parent strain ZJ173 was 388.PCR and nucleotide sequences were used to examine sensitive and resistant strains for differences that could account for streptomycin resistance, including mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA), and the presence of the strA gene (encoding aminoglycoside-3-phosphotransferase). Neither the strA gene nor mutations in the rrs were found. Four of five tested laboratory mutants had mutation at codon 43 (Lysâ†'Arg) and the other one at 88 (Lysâ†'Arg) in the rpsL gene.The resistant mutants maintained their resistance through 10 times transfers on NA plates. The pathogenicity and growth rate did not differ between resistant mutants and sensitive strain, while the competitiveness of resistant mutants is lower or slightly lower than sensitive strain.Considering the high spontaneous mutation frequency and one point mutation pattern, the risk of streptomycin-resistant mutants caused by rpsL mutation will be high.2) to screen for the streptomycin-resistance in the populations of X. oryzae pv. oryzae and X. oryzae pv. oryzicola from the south of ChinaRice leaves with symptoms of bacterial blight or bacterial leaf streak were collected in the south of China in 2007 and 2008. Five hundred and thirty four single-colony isolates of Xanthomonas oryzae pv. oryzae and 827 single-colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested for sensitivity to streptomycin, which has been used to control rice bacterial diseases. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (RF, the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was about 226. Besides resistant isolates, isolates of less sensitivity were also presented in the population of X. oryzae pv. oryzae from Province Yunnan. However, no streptomycin-sensitivity decreased isolates were obtained in the population of X. oryzae pv. oryzicola up to now.3) Mechanisms of streptomycin resistance in field isolates of Xanthomonas oryzae pv. oryzae and the character of resistance integronMutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of str A gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by PCR amplification and sequencing. Neither the presence of strA-strB gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.The sensitive and resistant isolates were examined for the presence of the three classes of integrons and for the aadA1 and aadA2 genes, which confer streptomycin resistance by PCR. The typeâ… integrase gene intâ… and the aminoglycoside adenylyltransferase gene aadAl were identified in all four resistant field isolates and not in 24 sensitive field isolates randomly selected. To our knowledge, this is the first report of a resistance integron in a phytopathogen.4041,3173,3801 and 3251 bp PCR products of integron amplified from YNA7-1, YNA10-2, YNA11-2 and YNA12-2, respectively were sequenced and analyzed and antibiotic sensitivity profile was examined by disk diffusion method and minimal inhibition concentration (MIC) method. The integron of YNA7-1 and YNA11-2 carried three gene cassettes in the order aac(6’) -â…¡-arr-3-aadA1, in which aac(6’) -â…¡conferred the resistance to tobramycin, kanamycin, gentamicin, and netilmicin, arr-3 conferred the resistance to rifampim, aadA1 to streptomycin and spectinomycin; Compared to YNA7-1, YNA10-2 deleted aac(6’) -â…¡gene cassettes, carried arr-3-aadA1 gene cassettes; YNA10-2 deleted 550bp sequence including part of intâ… gene, but still carried aac(6’) -â…¡arr-3-aadAl gene cassettes. Isolates showed consistence resistance phenotype corresponded to their resistance gene cassettes, except YNA7-1 and YNA12-2 did not show rifampim resistance. The nucleotide sequence of integron of four resistance isolates is identical except deleted part. Sequence comparisons in GenBank revealed that no genecassettes are ranged as same as the order in integron of four resistant isolates in our research and the identical aadAl gene in four resistant isolates showed 99% identical to aadA1 gene known so far in GenBank database, with a substitution of a G for A at position 236, which resulted in amino acid substitution of Glycine (Gly) for glutamic acid (Glu) at codon 79.4) Characterization of bismerthiazol resistance of Xanthomonas oryzae pv. oryzaeNo in vitro resistant strains of Xanthomonas oryzae pv. oryzae against bismerthiazol were obtained through repeated ultraviolet light induction. Three in vivo resistant strains of X. oryzae pv. oryzae against bismerthiazol were obtained through bismerthiazol training. Bismerthiazol was effective in protection and treatment against X. oryzae pv. oryzae. To sensitive strain, the control effect of 100ug/ml bismerthiazol before 1d, after 1d,3d and 5d are 93.60,86.84,73.76 and 51.60%, respectively; To resistant strains, the best control effect of 1200ug/ml bismerthiazol is 50.14%.The resistant strains maintained their resistance through 10 times transfers on NA plates and one time transfer in vivo. In vitro, the inhibitory activity of bismerthiazol to in vovo resistant strains was not different to sensitive strain. The effect of different concentration of bismerthiazol to incidence of diseased leaves, recovery rate of colonies for leaves and resistance mutagenic rate was determined through two times of continuous spraying bismerthiazol and inoculation. With the increase in bismerthiazol concentration, the percentage of diseased leaves and recovery rate of colonies decreased. The recovery rate of colonies was 5.56% under the concentration of 600ug/ml. The colonies of X. oryzae pv. oryzae could be recovered from leaves not only with disease symptom but also without disease symptom when the concentration≤600ug/ml, although recovery rate from leaves without disease symptom was lower than from leaves with symptom. The percentage of resistant strains in recovery colonies was higher in the concentration of 100 and 300μg/mL of bismerthiazol and was the highest in 300μg/mL under which concentration the percentage of resistant strains was 31.90% and 28.57% in leaves with disease and without disease symptom, respectively. The offspring of sensitive strains could produce new resistant strains in the pressure of bismerthiazol, while the offspring of resistant strains would sustain resistance.The mechanism of bismerthiazol and its homologue 2-animo-1,3,4 thiadiazole (ADTA) were compared by niacinamide, which could reverse inhabitation of inosine 5’-monophosphate hydrogenase. In vitro, the EC50S of bismerthiazol and ADTA are 0.79 and 91.83μg/mL, respectively which showed the inhabitation of bismerthiazol was far higher than ADTA. The inhibitory activity of bismerthiazol against bacteria growth could be reversed by niacinamide, while the inhibitory activity of ADTA could not be reversed. Therefore, the mechanism of bismerthiazol and ADTA perhaps was different in vitro. The resistant strains of bismerthiazol showed cross-resistant to ADTA in vivo which showed that the mechanism of bismerthiazol and ADTA perhaps was identical in vivo.5) Monitoring bismerthiazol resistance of Xanthomonas oryzae pv. oryzaeIn 2007 and 2008,654 single-colony isolates of Xanthomonas oryzae pv. oryzae were were collected from 48 counties of seven provinces, including Yunnan, Jiangsu, Anhui, Hubei, Guangdong, Hainan, and Hunan Province in China, and 409 isolates of them were tested the sensitivity to bismerthiazol in vivo by inoculation. The resistance criterion for the in-vivo test was defined by the distribution frequency of relative inhabitation of field isolates treated by 300ug/ml bismerthiazol. The tested isolate was declared resistant when the inhabitation of it was less than 70% of the reference strain ZJ173. According to this discriminatory criterion,12.71% of 409 isolates monitored were resistant to bismerthiazol. Resistance frequency in the pathogen population differed among the provinces, ranged from 0 (Hubei and Hainan Province) to 19.21% (Jiansu Province). |
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