The novel kasugamycin 2'-N-acetyltransferase gene aac(2')-IIa, carried by the IncP island, confers kasugamycin resistance to rice-pathogenic bacteria.
ABSTRACT: Kasugamycin (KSM), a unique aminoglycoside antibiotic, has been used in agriculture for many years to control not only rice blast caused by the fungus Magnaporthe grisea but also rice bacterial grain and seedling rot or rice bacterial brown stripe caused by Burkholderia glumae or Acidovorax avenae subsp. avenae, respectively. Since both bacterial pathogens are seed-borne and cause serious injury to rice seedlings, the emergence of KSM-resistant B. glumae and A. avenae isolates highlights the urgent need to understand the mechanism of resistance to KSM. Here, we identified a novel gene, aac(2')-IIa, encoding a KSM 2'-N-acetyltransferase from both KSM-resistant pathogens but not from KSM-sensitive bacteria. AAC(2')-IIa inactivates KSM, although it reveals no cross-resistance to other aminoglycosides. The aac(2')-IIa gene from B. glumae strain 5091 was identified within the IncP genomic island inserted into the bacterial chromosome, indicating the acquisition of this gene by horizontal gene transfer. Although excision activity of the IncP island and conjugational gene transfer was not detected under the conditions tested, circular intermediates containing the aac(2')-IIa gene were detected. These results indicate that the aac(2')-IIa gene had been integrated into the IncP island of a donor bacterial species. Molecular detection of the aac(2')-IIa gene could distinguish whether isolates are resistant or susceptible to KSM. This may contribute to the production of uninfected rice seeds and lead to the effective control of these pathogens by KSM.
Project description:A unique aminoglycoside antibiotic, kasugamycin (KSM), has been used to control many plant bacterial and fungal diseases in several countries. The emergence of KSM-resistant Acidovorax avenae ssp. avenae and Burkholderia glumae, which cause rice bacterial brown stripe and rice bacterial grain and seedling rot, respectively, is a serious threat for the effective control of these diseases. Previously, we have identified the aac(2')-IIa gene, encoding a KSM 2'-N-acetyltransferase, from both KSM-resistant pathogens. Although all KSM-resistant isolates from both species possess the aac(2')-IIa gene, only A.?avenae strain 83 showed higher resistance than other strains. In this research, kinetic analysis indicates that an amino acid substitution from serine to threonine at position 146 of AAC(2')-IIa in strain 83 is not involved in this increased resistance. Whole draft genome analysis of A.?avenae 83 shows that the aac(2')-IIa gene is carried by the novel IncP-1? plasmid pAAA83, whereas the genetic carrier of other strains, the IncP genomic island, is inserted into their chromosomes. The difference in the nucleotides of the promoter region of aac(2')-IIa between strain 83 and other strains indicates an additional transcription start site and results in the increased transcription of aac(2')-IIa in strain 83. Moreover, biological characterization of pAAA83 demonstrates that it can be transferred by conjugation and maintained in the host cells. These results demonstrate that acquisition of the aac(2')-IIa gene takes place in at least two ways and that the gene module, which includes aac(2')-IIa and the downstream gene, may be an important unit for the dissemination of antibiotic resistance.
Project description:Burkholderia glumae (bacterial grain rot), Xanthomonas oryzae pv. oryzae (bacterial leaf blight), and Acidovorax avenae subsp. avenae (bacterial brown stripe) are major seedborne pathogens of rice. Based on the 16S and 23S rDNA sequences for A. avenae subsp. avenae and B. glumae, and transposase A gene sequence for X. oryzae pv. oryzae, three sets of primers had been designed to produce 402 bp for B. glumae, 490 bp for X. oryzae, and 290 bp for A. avenae subsp. avenae with the 63°C as an optimum annealing temperature. Samples collected from naturally infected fields were detected with two bacteria, B. glumae and A. avenae subsp. avenae but X. oryzae pv. oryzae was not detected. This assay can be used to identify pathogens directly from infected seeds, and will be an effective tool for the identification of the three pathogens in rice plants.
Project description:The Hfq protein is a global small RNA chaperone that interacts with regulatory bacterial small RNAs (sRNA) and plays a role in the post-transcriptional regulation of gene expression. The roles of Hfq in the virulence and pathogenicity of several infectious bacteria have been reported. This study was conducted to elucidate the functions of two hfq genes in Burkholderia glumae, a causal agent of rice grain rot. Therefore, mutant strains of the rice-pathogenic B. glumae BGR1, targeting each of the two hfq genes, as well as the double defective mutant were constructed and tested for several phenotypic characteristics. Bacterial swarming motility, toxoflavin production, virulence in rice, siderophore production, sensitivity to H2O2, and lipase production assays were conducted to compare the mutant strains with the wild-type B. glumae BGR1 and complementation strains. The hfq1 gene showed more influence on bacterial motility and toxoflavin production than the hfq2 gene. Both genes were involved in the full virulence of B. glumae in rice plants. Other biochemical characteristics such as siderophore production and sensitivity to H2O2 induced oxidative stress were also found to be regulated by the hfq1 gene. However, lipase activity was shown to be unassociated with both tested genes. To the best of our knowledge, this is the first study to elucidate the functions of two hfq genes in B. glumae. Identification of virulence-related factors in B. glumae will facilitate the development of efficient control measures.
Project description:Burkholderia glumae is the causative agent of grain and seedling rot in rice and of bacterial wilt in many field crops. Here, we report the complete genome sequence of B. glumae BGR1 isolated from a diseased rice panicle in Korea.
Project description:Quorum sensing (QS) plays important roles in the pathogenicity of Burkholderia glumae, the causative agent of bacterial rice grain rot. We determined how QS is involved in catalase expression in B. glumae. The QS-defective mutant of B. glumae exhibited less catalase activity than wild-type B. glumae. A beta-glucuronidase assay of a katG::Tn3-gusA78 reporter fusion protein revealed that katG expression is under the control of QS. Furthermore, katG expression was upregulated by QsmR, a transcriptional activator for flagellar-gene expression that is regulated by QS. A gel mobility shift assay confirmed that QsmR directly activates katG expression. The katG mutant produced toxoflavin but exhibited less severe disease than BGR1 on rice panicles. Under visible light conditions and a photon flux density of 61.6 micromol(-1) m(-2), the survival rate of the katG mutant was 10(5)-fold lower than that of BGR1. This suggests that KatG is a major catalase that protects bacterial cells from visible light, which probably results in less severe disease caused by the katG mutant.
Project description:Rice world production is affected due to the growing impact of diseases such as bacterial panicle blight, produced by <i>Burkholderia glumae</i>. The pathogen-induced symptoms include seedling rot, grain rot and leaf-sheath browning in rice plants. It is currently recognized the entrance of this pathogen to the plant, from infected seeds and from environmental sources of the microorganism. However, it is still not fully elucidated the dynamics and permanence of the pathogen in the plant, from its entry until the development of disease symptoms in seedlings or panicles. In this work it was evaluated the infection of <i>B. glumae</i> rice plants, starting from inoculated seeds and substrates, and its subsequent monitoring after infection. Various organs of the plant during the vegetative stage and until the beginning of the reproductive stage, were evaluated. In both inoculation models, the bacteria was maintained in the plant as an endophyte between 1 × 10<sup>1</sup> and 1 × 10<sup>5</sup> cfu of <i>B. glumae</i>.g<sup>-1</sup> of plant throughout the vegetative stage. An increase of bacterial population towards initiation of the panicle was observed, and in the maturity of the grain, an endophyte population was identified in the flag leaf at 1 × 10<sup>6</sup> cfu of <i>B. glumae</i>.g<sup>-1</sup> fresh weight of rice plant, conducting towards the symptoms of bacterial panicle blight. The results found, suggest that <i>B. glumae</i> in rice plants developed from infected seeds or from the substrate, can colonize seedlings, establishing and maintaining a bacterial population over time, using rice plants as habitat to survive endophyticly until formation of bacterial panicle blight symptoms.
Project description:Panicle blight and seed rot disease caused mainly by Burkholderia glumae and Burkholderia gladioli is threatening rice cultivation worldwide. The bacteria have been reported as seed-borne pathogens from rice. Accurate detection of both pathogens on the seeds is very important for limiting the disease dissemination. Novel primer pairs targeting specific molecular markers were developed for the robust detection of B. glumae and B. gladioli. The designed primers were specific in detecting the target species with no apparent crossreactions with other related Burkholderia species at the expected product size. Both primer pairs displayed a high degree of sensitivity for detection of B. glumae and B. gladioli separately in monoplex PCR or simultaneously in duplex PCR from both extracted gDNA and directly preheated bacterial cell suspensions. Limit of detection was as low as 0.1 ng of gDNA of both species and 3.86 × 102 cells for B. glumae and 5.85 × 102 cells for B. gladioli. On inoculated rice seeds, the designed primers could separately or simultaneously detect B. glumae and B. gladioli with a detection limit as low as 1.86 × 103 cells per rice seed for B. glumae and 1.04 × 104 cells per rice seed of B. gladioli. The novel primers maybe valuable as a more sensitive, specific, and robust tool for the efficient simultaneous detection of B. glumae and B. gladioli on rice seeds, which is important in combating rice panicle blight and seed rot by early detection and confirmation of the dissemination of pathogen-free rice seeds.
Project description:In contrast to most Burkholderia species, which affect humans or animals, Burkholderia glumae is a bacterial pathogen of plants that causes panicle blight disease in rice seedlings, resulting in serious damage to rice cultivation. Attempts to combat this disease would benefit from research involving a phage known to attack this type of bacterium. Some Burkholderia phages have been isolated from soil or bacterial species in the order Burkholderiales, but so far there has been no report of a complete genome nucleotide sequence of a phage of B. glumae. In this study, a novel phage, FLC5, of the phytopathogen B. glumae was isolated from leaf compost, and its complete genome nucleotide sequence was determined. The genome consists of a 32,090-bp circular DNA element and exhibits a phylogenetic relationship to members of the genus Peduovirus, with closest similarity to B. multivorans phage KS14. In addition to B. glumae, FLC5 was also able to lyse B. plantarii, a pathogen causing rice bacterial damping-off disease. This is the first report of isolation of a P2-like phage from phytopathogenic Burkholderia, determination of its complete genomic sequence, and the finding of its potential to infect two Burkholderia species: B. glumae and B. plantarii.
Project description:Bacteria form biofilms as a means to adapt to environmental changes for survival. Pellicle is a floating biofilm formed at the air-liquid interface in static culture conditions; however, its functional roles have received relatively little attention compared to solid surface-associated biofilms in gram-negative bacteria. Here we show that the rice pathogen Burkholderia glumae BGR1 forms cellulase-sensitive pellicles in a bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)- and flagellum-dependent, but quorum sensing (QS)-independent, manner. Pellicle formation was more favorable at 28°C than at the optimum growth temperature (37°C), and was facilitated by constitutive expression of pelI, a diguanylate cyclase gene from B. glumae, or pleD, the GGDEF response regulator from Agrobacterium tumefaciens. Constitutive expression of pelI or pleD raised the levels of c-di-GMP, facilitated pellicle formation, and suppressed swarming motility in B. glumae. QS-defective mutants of B. glumae formed pellicles, while flagellum-defective mutants did not. Pellicles of B. glumae were sensitive to cellulase but not to proteinase K or DNase I. A gene cluster containing seven genes involved in bacterial cellulose biosynthesis, bcsD, bcsR, bcsQ, bcsA, bcsB, bcsZ, and bcsC, homologous to known genes involved in cellulose biosynthesis in other bacteria, was identified in B. glumae. Mutations in each gene abolished pellicle formation. These results revealed a positive correlation between cellulase-sensitive pellicles and putative cellulose biosynthetic genes. Pellicle-defective mutants did not colonize as successfully as the wild-type strain BGR1 in rice plants, which resulted in a significant reduction in virulence. Our findings show that cellulase-sensitive pellicles produced in a QS-independent manner play important roles in the interactions between rice plants and B. glumae.
Project description:Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B.?glumae strains showed pigmentation phenotypes producing at least two (yellow-green and purple) pigment compounds in casein-peptone-glucose agar medium. The B.?glumae strains LSUPB114 and LSUPB116 are pigment-deficient mutant derivatives of the virulent and pigment-proficient strain 411gr-6, having mini-Tn5gus insertions in aroA encoding 3-phosphoshikimate 1-carboxyvinyltransferase and aroB encoding 3-dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B.?glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.