Bacterial Panicle Blight Of Rice Research Articles

First Report of Burkholderia glumae Causing Bacterial Panicle Blight in Rice in Bangladesh.

Bacterial panicle blight (BPB) is one of the emerging diseases occurring in different Agro-Ecological Zones (AEZ) of Bangladesh and can cause up to 75% yield loss. In Bangladesh, the typical symptoms of BPB include sheath rot, panicle blight, grain spotting, and grain rot in both inbred and hybrid rice varieties, which resemble those reported by Zhou (2019). To confirm, 300 field samples of 20 panicles each with typical BPB symptoms from 20 districts (3 locations each district and 5 fields per location) were collected during mid-November 2022 for the causal pathogen(s) isolation. Nearly 70% of the panicles showed a dark brown chaffy appearance in the fields. For identification of the causal pathogen(s), 1 g of rice grains with typical BPB symptoms was surface sterilized by immersing for 15 seconds in 70% ethanol, 1 min in 3% sodium hypochlorite solution followed by rinsing the grains three times, and soaked in 1 mL sterile distilled water for 10 min (Mirghasempour et al. 2018). During grinding using mortar and pestle, 5 mL water was added (Islam et al. 2023) after which the suspension (20 μL) was then streaked onto the selective medium (S-PG) (Tsushima et al. 1986). Purple color colonies on the S-PG medium were selected and purified as candidate pathogens. For further confirmation, the genomic DNA of the bacterial isolates was extracted and amplified by PCR using 16SF (5'-AGAGTTTGATCCTGGCTCAG-3') and 16SR (5'-GGCTACCTTGTTACGACTT-3') (Nandakumar et al. 2009), and glu-FW (5'-GAAGTGTCGCCGATGGAG-3') and glu-RV (5'-CCTTCACCGACAGCACGCAT-3') primers (Maeda et al. 2006). The PCR products were visualized on 1% agarose gel resulting amplicons of 1494bp for 16S-rDNA and 529bp for gyrB. The PCR results revealed 529bp amplification for gyrB gene in one sample that was collected from a field in Natore (24°21'0.00" N 89°04'59.88" E) district cultivating Swarna (a local rice variety), primarily indicating the causal pathogen is Burkholderia glumae. The PCR products were sequenced using both primers and sequence data was analyzed by the BLAST nucleotide program. The obtained partial sequences of 16S rDNA and gyrB were deposited in Genbank (OR573691 and PP332812 respectively). The homology of 16S rDNA resulted over 98% with B. glumae (OK559611 and ON870618.1) and 100% with B. glumae (PP332812 and KX213523) for gyrB gene. To confirm B. glumae by pathogenicity test, 10 mL (108 UFC/ml) suspension of the representative strains, 0.5 mL was then injected into the panicles and sheaths of Horidhan (a susceptible local variety) in greenhouse condition and a control was inoculated with distilled water (Nandakumar et al. 2009). Typical BPB like symptoms were observed after 3 weeks post inoculation. The pathogen was again confirmed by reisolating from the infected spots as B. glumae to fulfill Koch's postulates. This report confirms the presence of B. glumae causing BPB of rice in Bangladesh. Future research for the investigation of BPB and the evolutionary origins of its causal bacteria is necessary to reduce the emergence of the disease and its management in Bangladesh.

Development of a Selective Medium for Surveillance of Fusarium Head Blight Disease.

Fusarium head blight (FHB), predominantly caused by Fusarium graminearum and F. asiaticum, is a significant fungal disease impacting small-grain cereals. The absence of highly resistant cultivars underscores the need for vigilant FHB surveillance to mitigate its detrimental effects. In 2023, a notable FHB outbreak occurred in the southern region of Korea. We assessed FHB disease severity by quantifying infected spikelets and grains. Isolating fungal pathogens from infected samples often encounters interference from various microorganisms. We developed a cost-effective, selective medium, named BGT (Burkholderia glumae Toxoflavin) medium, utilizing B. glumae, which is primarily known for causing bacterial panicle blight in rice. This medium exhibited selective growth properties, predominantly supporting Fusarium spp., while substantially inhibiting the growth of other fungi. Using the BGT medium, we isolated F. graminearum and F. asiaticum from infected wheat and barley samples across Korea. To further streamline the process, we used a direct PCR approach to amplify the translation elongation factor 1-α (TEF-1α) region without a separate genomic DNA extraction step. Phylogenetic analysis of the TEF-1α region revealed that the majority of the isolates were identified as F. asiaticum. Our results demonstrate that BGT medium is an effective tool for FHB diagnosis and Fusarium strain isolation.

First report of Burkholderia gladioli causing bacterial panicle blight of rice in Malaysia.

Rice (Oryza sativa) is a staple food for most of the world's populations, particularly in Asia (Gumma et al. 2011). The rice sector provides Malaysians with a food supply, food sufficiency, and income for growers (Man et al. 2009). From January to February 2022, panicle samples showing symptoms of bacterial panicle blight (BPB) disease, including reddish-brown, linear lesions with indistinct margins on flag-leaf sheaths and blighted, upright, grayish straw-colored florets with sterile and aborted grains on panicles were collected in granary areas in Sekinchan, Selangor, Malaysia with 90% disease incidence in fields. Surface-sterilization of infected leaf tissue was performed using 75% ethanol and 1% sodium hypochlorite, followed by rinsing three times in sterilized water. Leaf tissue was macerated in sterilized water and aliquots were spread on King's B agar medium, then cultured for 24 h to 48 h at 35 °C. All isolated bacteria were Gram-negative rods, positive for catalase and gelatinase but negative for indole, oxidase and hydrogen sulfide (H2S), and utilized sucrose, inositol, mannitol, glucose, and citrate. Colonies were circular and smooth-margined, producing a diffusible yellowish-green pigment on King's B agar medium, which are characteristics of Burkholderia species (Keith et al. 2005). Five representative isolates (UPMBG7, UPMBG8, UPMBG9, UPMBG15, UPMBG17) were selected for molecular and pathogenicity tests. PCR using specific primers targeting the gyrB gene for molecular characterization was performed, and the ∼470 bp amplicons were sequenced (Maeda et al. 2006) and deposited in GenBank (OM824438 to OM824442). A BLASTn analysis revealed that the five isolates were 99% identical to the B. gladioli reference strains MAFF 302533, GRBB15041, and LMG19584 in GenBank (AB190628, KX638432, and AB220898). A phylogenetic tree using Maximum-likelihood analysis of the gyrB gene sequences showed that the five isolates were 99% identical to B. gladioli reference strains (AB190628, KX638432, and AB220898). To verify the identification of these isolates, the 16S rDNA gene was amplified using 16SF/16SR primers (Ramachandran et al. 2021), producing ~1,400 bp amplicons. The resulting sequences of the five isolates (OM869953 to OM869957) were 98% identical to the reference strains of B. gladioli (NR113629 and NR117553). To confirm pathogenicity, 10 ml suspensions of the five isolates at of 108 CFU/ml were inoculated into the panicles and crowns of 75-day-old rice seedlings of local rice varieties MR269 and MR219 grown in a glasshouse with temperatures ranging from 37 °C to 41 °C (Nandakumar et al. 2009). Control rice seedlings were inoculated with sterilized water. All isolates produced BPB disease symptoms like those originally found in the rice fields at four weeks after inoculation. Control seedlings remained asymptomatic. To fulfill Koch's postulates, the bacteria were reisolated from symptomatic panicles and were confirmed as B. gladioli by sequence analysis of the gyrB and 16S rDNA genes. To our knowledge, this is the first report of B. gladioli causing BPB disease of rice in Malaysia. Since BPB disease causes a significant threat to the rice industry, it is crucial to investigate the diversity of this destructive pathogen for effective disease management strategies in Malaysia.

Genetic and pathogenic diversity analysis of Burkholderia glumae strains from Indian hot spot regions causing bacterial panicle blight of rice (Oryza sativa L.)

Genetic and pathogenic diversity analysis of Burkholderia glumae strains from Indian hot spot regions causing bacterial panicle blight of rice (Oryza sativa L.)

Development of Droplet Digital PCR Assay for Detection of Seed-Borne Burkholderia glumae and B. gladioli Causing Bacterial Panicle Blight Disease of Rice.

Bacterial panicle blight of rice or bacterial grain rot of rice is a worldwide rice disease. Burkholderia glumae and B. gladioli are the causal agents. The early and accurate detection of seed-borne B. glumae and B. gladioli is critical for domestic and international quarantine and effective control of the disease. Here, genomic analyses revealed that B. gladioli contains five phylogroups and the BG1 primer pair designed to target the 3’-end sequence of a gene encoding a Rhs family protein is specific to B. glumae and two phylogroups within B. gladioli. Using the BG1 primer pair, a 138-bp DNA fragment was amplified only from the tested panicle blight pathogens B. glumae and B. gladioli. An EvaGreen droplet digital PCR (dPCR) assay on detection and quantification of the two pathogens was developed from a SYBR Green real-time quantitative PCR (qPCR). The detection limits of the EvaGreen droplet dPCR on the two pathogens were identical at 2 × 103 colony forming units (CFU)∙mL−1 from bacterial suspensions and 2 × 102 CFU∙seed−1 from rice seeds. The EvaGreen droplet dPCR assay showed 10-fold detection sensitivity of the SYBR Green qPCR and could detect a single copy of the target gene in a 20-μL assay. Together, the SYBR Green qPCR assay allows for routine high-throughput detection of the panicle blight pathogens and the EvaGreen droplet dPCR assay provides a high-sensitive and high-accurate diagnostic method for quarantine of the pathogens.

Metabolomic characterization of Pseudomonas protegenssecretions for identification of biopesticides against Bacterial Panicle Blight of rice

Bacterial Panicle Blight (BPB) is a rice disease caused by Burkholderia glumae that has become a global concern in reducing rice yields. Currently, there are no effective chemical treatments or resistant cultivars to control BPB. We have observed that bacterial secreted fractions from the antagonistic microorganism Pseudomonas protegens PBL3 inhibits B. glumae infections in rice plants. However, the responsible antimicrobial molecules are unknown. Genome comparison between P. protegens PBL3 and the reference strain P. protegensCHA0, a well‐characterized microbial biological control agent, revealed the presence of 14 known biosynthetic gene clusters (BGCs) in P. protegensPBL3. From this genomic data, it is likely that P. protegensPBL3 secretes metabolites with activity against BPB that are not produced by P. protegensCHA0.Cultures of P. protegensPBL3 and the reference strain, P. protegensCHA0, were grown in M9 minimal media supplemented with 1% myo‐inositol. The metabolite secretome of P. protegens PBL3 and CHA0 were harvested in log phase and analyzed with a Waters Acquity H‐Class HPLC coupled to a Thermo Q‐Exactive HF‐X. In addition, active PBL3 fractions against B. glumaewere analyzed via LC‐MS to confirm the presence of bioactive compounds. The resulting data were searched against the Global Natural Products Social Molecular Networking (GNPS) spectral library. For potential antimicrobial peptide identification, data were searched against the Mascot database. To validate these results, known bioactive metabolites in P. protegensCHA0 were identified. By utilizing LC‐MS/MS based metabolomics, several compounds were uniquely identified in P. protegensPBL3 that could be used as biocontrol agents against BPB in rice.

Genome Sequence Resource of Burkholderia glumae UAPB13

Burkholderia glumae causes Bacterial Panicle Blight of rice. Here, we report the genomic sequence of B. glumae strain UAPB13 isolated from fields in Arkansas. The assembled genome consists of 123 scaffolds totaling 6,504,483 bp representing two chromosomes and two plasmids. The genomic complexity of B. glumae warrants the sequencing of additional strains. This additional genomic sequence will enable us to further understand this pathogen and the disease it causes.

Horizontal gene transfer of a syp homolog contributes to the virulence of Burkholderia glumae

Horizontal gene transfer (HGT) has been proved a major driving force in prokaryotic evolution. However, the molecular functions of these transferred genes in pathogenic bacteria especially plant pathogenic bacteria are still not fully investigated. In this study, the whole-genome in silico analysis was performed and found a syringopeptin synthetase (syp) homolog in Burkholderia glumae, which can cause bacterial panicle blight in rice, was predicted to be horizontally transferred from Pseudomonas ancestor with solid confidence by phylogenetic analysis. The comprehensive molecular experiments were performed to study the potential role of this gene in B. glumae. Inoculation of rice panicles with the syp mutant resulted in 60% lower disease index compared with the wild type (WT) parent strain, suggesting the requirement of syp for the full virulence of B. glumae. Chromatography analysis of exudates from B. glumae showed suppression of synthesis of metabolites analogous to syringopeptin in the mutants. All these data raise the possibility of HGT phenomenon in shaping the virulence and adaptation of B. glumae over evolutionary time.

Isolation and Characterization of Bacteriophages Infecting Burkholderia glumae, the Major Causal Agent of Bacterial Panicle Blight in Rice.

Bacterial panicle blight (BPB), caused by Burkholderia glumae, is one of the most severe seed-borne bacterial diseases of rice in the world, which can decrease rice production by ≤75%. Nevertheless, there are few effective measures to manage this disease. In an attempt to develop an alternative management tool for BPB, we isolated and characterized phages from soil and water that are effective to lyse several strains of B. glumae. After tests of host ranges, the phages NBP1-1, NBP4-7, and NBP4-8 were selected for further comprehensive characterization, all of which could lyse B. glumae BGLa14-8 (phage sensitive) but not B. glumae 336gr-1 (phage insensitive). This result indicates that the phages killing B. glumae cells have specific host ranges at the strain level within the bacterial species. In the greenhouse condition of this study, foliar application of the phage NBP4-7 reduced the severity of BPB caused by B. glumae BGLa14-8 ≤62% but did not cause any significant effect on the infection by B. glumae 336gr-1. Electron microscopy and whole-genome sequencing were also performed to characterize the three selected phages. Transmission electron microscopy revealed that the selected phages belong to the family Myoviridae. Furthermore, whole-genome sequence analysis indicated that the three phages belong to a same species and are closely related to the Burkholderia phage KL3, a member of the Myoviridae family.

Characterization and identification of Burkholderia glumae as the causal pathogen of bacterial panicle blight of rice (Oryza sativa L.) in Malaysian rice granaries

Bacterial panicle blight (BPB) like symptoms in rice plants were first detected in Sungai Ache, Penang, Malaysia in December 2017. The symptoms observed were straw-coloured panicles, discoloured florets with darker basal portion of the glumes. The panicles remained upright due to partially or aborted grain filling. The causal pathogen was confirmed as B. glumae based on phenotypic characterization, molecular identification, and phylogenetic analysis of 16S rRNA and gyrB genes. Koch’s postulates were fulfilled and pathogenic variations among the isolates were observed. This study is the first report of the occurrence of BPB caused by B. glumae in Malaysia.

IBUN2755 Bacillus strain controls seedling root and bacterial panicle blight caused by Burkholderia glumae

Burkholderia glumae is the causal agent of seedling rot and bacterial panicle blight in rice. It has a worldwide distribution in most rice producer countries generating crop losses of up to 75%. Currently, there is no known effective control for this pathogen. In this study 74 Bacillus strains isolated from the rhizosphere of different plants were evaluated under in vitro conditions against three strains of B. glumae. Twenty-six strains of Bacillus were selected for their antagonistic activity and then were evaluated in plantae under the two different inoculation sources of B. glumae: the seeds, and the substrate, since these are the two main reservoirs of the pathogen in the field. Inoculated seeds with the selected Bacillus strains were planted in a previously inoculated substrate with B. glumae, finding that 11 strains showed a reduction of the infection up to 63.5%. When evaluating the biological control activity of those 11 Bacillus strains in B. glumae infested seeds, four strains (IBUN12A04, IBUN16A08, IBUN2755 and IBUN15A09), showed disease reduction of up to 62.4%. These four strains were again evaluated under greenhouse conditions with seeds infested with B. glumae to determine the presence of the pathogen in asymptomatic plants 10 and 30 days after germination. In this case, Bacillus strains reduced the population of B. glumae in the plant up to two orders of magnitude in relation to disease control, and some even promoted plant growth in relation to an uninoculated control. Finally, the IBUN 2755 strain, which showed the highest biocontrol activity in the greenhouse, was evaluated under field conditions, showing a significant reduction in the symptoms of the disease and an increase in crop yield of up to two tons per hectare with relation to the uninoculated control.

Harnessing Pseudomonas protegens to Control Bacterial Panicle Blight of Rice.

Bacterial panicle blight of rice is a seedborne disease caused by the bacterium Burkholderia glumae. This disease has affected rice production worldwide and its effects are likely to become more devastating with the continuous increase in global temperatures, especially during the growing season. The bacterium can cause disease symptoms in different tissues and at different developmental stages. In reproductive stages, the bacterium interferes with grain development in the panicles and, as a result, directly affects rice yield. Currently, there are no methods to control the disease because chemical control is not effective and completely resistant cultivars are not available. Thus, a promising approach is the use of antagonistic microorganisms. In this work, we identified one strain of Pseudomonas protegens and one strain of B. cepacia with antimicrobial activity against B. glumae in vitro and in planta. We further characterized the antimicrobial activity of P. protegens and found that this activity is associated with bacterial secretions. Cell-free secretions from P. protegens inhibited the growth of B. glumae in vitro and also prevented B. glumae from causing disease in rice. Although the specific molecules associated with these activities have not been identified, these findings suggest that the secreted fractions from P. protegens could be harnessed as biopesticides to control bacterial panicle blight of rice.

Type VI secretion systems of plant-pathogenic Burkholderia glumae BGR1 play a functionally distinct role in interspecies interactions and virulence.

In the environment, bacteria show close association, such as interspecies interaction, with other bacteria as well as host organisms. The type VI secretion system (T6SS) in gram‐negative bacteria is involved in bacterial competition or virulence. The plant pathogen Burkholderia glumae BGR1, causing bacterial panicle blight in rice, has four T6SS gene clusters. The presence of at least one T6SS gene cluster in an organism indicates its distinct role, like in the bacterial and eukaryotic cell targeting system. In this study, deletion mutants targeting four tssD genes, which encode the main component of T6SS needle formation, were constructed to functionally dissect the four T6SSs in B. glumae BGR1. We found that both T6SS group_4 and group_5, belonging to the eukaryotic targeting system, act independently as bacterial virulence factors toward host plants. In contrast, T6SS group_1 is involved in bacterial competition by exerting antibacterial effects. The Δ tssD1 mutant lost the antibacterial effect of T6SS group_1. The ΔtssD1 mutant showed similar virulence as the wild‐type BGR1 in rice because the ΔtssD1 mutant, like the wild‐type BGR1, still has key virulence factors such as toxin production towards rice. However, metagenomic analysis showed different bacterial communities in rice infected with the ΔtssD1 mutant compared to wild‐type BGR1. In particular, the T6SS group_1 controls endophytic plant‐associated bacteria such as Luteibacter and Dyella in rice plants and may have an advantage in competing with endophytic plant‐associated bacteria for settlement inside rice plants in the environment. Thus, B. glumae BGR1 causes disease using T6SSs with functionally distinct roles.

Complete Genomic Data of Burkholderia glumae Strain GX Associated with Bacterial Panicle Blight of Rice in China.

Burkholderia glumae is a seedborne pathogen causing bacterial panicle blight of rice. Here, we report the complete genome of B. glumae strain GX, which represents the first whole-genome sequence of an isolate from China. The assembled genome consisted of five contigs, with two circular chromosomes of 3,712,850 and 2,750,046 bp and three plasmids of 201,571, 105,587, and 96,100 bp. This complete genome will provide a valuable resource for further studies on bacterial panicle blight worldwide.

First Report of Bacterial Panicle Blight of Rice Caused by Burkholderia glumae in Southern China

Rice (Oryza sativa L.) is the staple food for people in southern China. Bacterial panicle blight of rice (BPBR) caused by Burkholderia glumae, known as grain blight or seedling rot, leads to rice yield losses up to 75% and threatens rice production (Nandakumar et al. 2009). In 2007, B. glumae was isolated from symptomless rice seeds in Hainan, China (Luo et al. 2007). In 2017, 43 panicle samples showing typical symptoms of BPBR with reddish-brown, aborted or partially filled grains and linear lesions on flag-leaf sheaths were collected for bacterial isolation from fields in 15 towns of Guangxi and Guangdong provinces, China (cvs. Longfengyou139, Benyou9901, Baixiangyou 9978, Yaliangyou, Quanyousimiao, etc.). The diseased panicle incidence was about 6.5 to 20% in the field. The infected grains were sterilized with 70% ethanol for 30 to 60 s, rinsed with distilled water three times, cut and crushed, and soaked in 1 ml of distilled water for 10 min (Mirghasempour et al. 2018). The suspension was streaked and cultured on nutrient agar (NA) medium containing ampicillin (50 µg/ml) for 16 to 24 h at 37°C. The circular, smooth-margined, convex colonies were selected for further purification with serial dilution and subculturing on NA medium. The 29 isolated strains can all produce the diffusible yellow pigment on King’s B medium. Two isolated strains, GX1 and GX28, from Guangxi province were selected for the following molecular and pathogenicity assays. For molecular identification, B. glumae-specific polymerase chain reaction was first carried out using gyrase gene (GenBank D87080) primers BGgyrF (5′-GCAGCGGCAAGGAAGACG-3′) and BGgyrR (5′-GTCGTCGCCCGACGTCTC-3′). The amplified 317-bp fragments were sequenced (GenBank MN417293), and the sequence identity is up to 100% with other reported B. glumae strains 336gr-1 (GenBank assembly accession GCA_000503955), BGR1 (GenBank assembly accession GCA_000022645), and LMG2196 (GenBank assembly accession GCA_000300755) by NCBI BLASTn analysis. To further verify B. glumae, the 16S rRNA gene was also amplified with common 16s rRNA primers (5′-AGAGTTTGATCATGGCTCAG-3′ and 5′-AAGGAGGTGATCCAGCCGC-3′). Results showed that 1,525-bp 16S rRNA gene sequences from isolate GX1 (GenBank MN400210) and GX28 (GenBank MN400211) shared 99% sequence similarity with 336gr-1, BGR1, and LMG2196. For the pathogenicity test, bacterial suspension at 10⁸ CFU/ml was injected into the panicle bud at the early booting stage of rice (cvs. Zhongjiazao17, Zhongzao39, Tianyouhuazhan, and Yongyou12). The injected tissue showed the linear lesion at 2 to 3 days after inoculation; 20 to 30 days later, grains were reddish-brown, aborted and partially filled, with similar symptoms to those samples collected from rice fields of Guangxi and Guangdong provinces. The same B. glumae was reisolated from the inoculated rice plants, fulfilling Koch’s postulates. This is the first report of BPBR caused by B. glumae in southern China. The occurrence, spread, and damage of BPBR caused by B. glumae in China need to be determined for disease control in the future.

Genome re-seqeunce and analysis of Burkholderia glumae strain AU6208 and evidence of toxoflavin: A potential bacterial toxin

Burkholderia glumae, the primary causative agent of bacterial panicle blight in rice, has been reported as an opportunistic pathogen in patients with chronic infections. This study aimed to re-sequence the clinical isolate B. glumae strain AU6208 and comparatively analyze its genome using B. glumae strain BGR1 from rice plant as the reference. Re-sequencing results revealed that the genome of strain AU6208 comprised 96 contigs corresponding to a 6.1 Mbp genome of the strain AU6208, with 5322 coding sequences and 68.2 % GC content; this is much larger compared to the genome previously sequenced by us and described by Seo et al (2015), which was reported to be 4.1 Mbp comprising >1200 contigs, 4361 coding sequences, and 67.31 % GC content. Moreover, this updated genome shares >80 % identity to the 7.2 Mbp genome of BGR1, which encodes 6491 coding sequences and has 68.3 % GC content. Further computational analysis revealed that the strain AU6208 encodes several bacteriocin biosynthesis genes, antibiotic, as well as virulent genes such as toxoflavin genes, which included 425 specialty genes and 12 toxoflavin genes. Upon further characterization, 12 toxoflavins (ToxA, B, C, D, E, F, G, H, I, J, TofI, and TofR) were found in AU6208 with 70–100 % sequence, family, and domain similarity with that of BGR1. Upon comparison with BGR1, the structural characterizations of selected toxoflavin genes (ToxB, ToxC, ToxG, H, and TofI) revealed variations in 2D and 3D structures such as differences in α-helix, β-sheets, loops, physiological properties of proteins, RMSD values, etc. These variations may play significant role in different mode of action in different hosts thereby indicating that in addition to their respective hosts, toxoflavins could also contribute to exploit other hosts across the kingdom. In addition to understanding the epidemiology of strain AU6208, this updated genomics data will also unfold the pathogenicity of bacteria in diversity of various hosts and anti-virulence.

Characterization and in Plant Detection of Bacteria That Cause Bacterial Panicle Blight of Rice

Burkholderia glumae presumably induces a grain rot symptom of rice that is threatening to rice production in most rice producing states of the USA. The present study was to identify the causal agent of bacteria panicle blight (BPB), virulence based on hypersensitive reactions and distribution of the pathogen within a plant. 178 rice panicles samples were analyzed with semi-selective media (CCNT), polymerase chain reaction (PCR) with bacterial DNA gyrase (gyrB) specific markers, and hypersensitive reactions on tobacco leaves. A total of 73 samples out of 178 produced a yellow bacterial colony with similar morphology on CCNT medium suggesting they were bacterial panicle diseases. However, with PCR reactions we only determined that 45 of 73 were due to B. glumae, and the causal agent for the remaining samples was undetermined. Within the 45 samples, 31 highly, 6 moderately, and 5 weakly virulent isolates were grouped based on lesion sizes of the hypersensitive reactions. Pathogenicity variability among the 45 B. glumae detected suggests that different degrees of pathogenicity exist. To determine the existence of bacteria in different plant tissues, naturally infected plant parts were examined with CCNT media and PCR analysis. B. glumae was again isolated from seeds followed by stems and sheaths from light yellow pigmented CCNT media. In contrast, roots and leaves show no visible yellow pigment on CCNT. Consistent PCR products were produced from the stem, sheath, and seed, but not from the root and leaves. These findings suggest that B. glumae is distributed in the stem, sheath, and seed, and not in the leaf and root. Together this study demonstrated the usefulness of artificial culture media, tobacco reactions, and DNA test with PCR for characterization of BPB, and distribution of bacteria in plants. These findings will help to understand the mechanism of bacteria translocation in plants.

Identification of new regulatory genes involved in the pathogenic functions of the rice-pathogenic bacterium Burkholderia glumae.

Burkholderia glumae is an emerging plant-pathogenic bacterium that causes disease in rice in several of the major rice-producing areas throughout the world. In the southern United States, B. glumae is the major causal agent of bacterial panicle blight of rice and has caused severe yield losses in recent decades. Despite its importance, few management options are available for diseases caused by B. glumae, and knowledge of how this pathogen causes disease is limited. In an effort to identify novel factors that contribute to the pathogenicity of B. glumae, random mutagenesis using the miniTn5gus transposon was performed on two strains of B. glumae. Resultant mutants were screened in the laboratory for altered phenotypes in various known or putative virulence factors, including toxoflavin, lipase and extracellular polysaccharides. Mutants that exhibited altered phenotypes compared to their parent strain were selected and subsequently characterized using a PCR-based method to identify the approximate location of the transposon insertion. Altogether, approximately 20 000 random mutants were screened and 51 different genes were identified as having potential involvement in the production of toxoflavin, lipase and/or extracellular polysaccharide. Especially, two regulatory genes, ntpR and tepR, encoding a LysR-type transcriptional regulator and a σ54-dependent response regulator, respectively, were discovered in this study as new negative regulatory factors for the production of toxoflavin, the major phytotoxin synthesized by B. glumae and involved in bacterial pathogenesis.

Infection Process of Burkholderia glumae in Rice Spikelets

AbstractBurkholderia glumae, which causes bacterial panicle blight of rice (BPBR), is a well‐known pathogen. The pathogen‐induced symptoms include seedling rot, grain rot and leaf‐sheath browning in rice plants. B. glumae can incubate in rice plants as endophytes before booting stage of rice. In this study, we constructed a gfp‐labelled system of B. glumae LMG 2196 and used SEM to clarify the colonization course of B. glumae at the heading stage. New locations of B. glumae were found. The pathogens initially distributed on the surface of the glumes and colonized in the glume hairs and cells of the edge of sterile lemma, palea and lemma. The base of glume hairs was the initial position for colonization. Bacterial population raised around glume hairs, penetrated into the inner surface of the palea and lemma, and spread on the gynoecium and stamens through contact. The spreading of B. glumae among the panicles mainly occurred through the contact or friction among glumes or leaf sheaths, but the inner spread of the stamens mainly occurred through the connective tissue of anther. We also detected the differences of bacterial content in stamens, gynoecia and glumes. The growing stage of B. glumae in spikelets could be divided into two sections. The biomass of all parts continued to increase to nearly 108 CFU/g at 10 DAI. This caused wilt symptoms and stopped the pollination. This work showed that glume hairs played an important role in the initial colonization of B. glumae, and provides a foundation for further studies of the infection manner of B. glumae and other pathogenic bacteria.

Infection Process of Burkholderia glumae Before Booting Stage of Rice

AbstractBurkholderia glumae is a well‐known pathogen for causing bacterial panicle blight of rice. In this study, the infection process of B. glumae in rice plants at different growing stages was tracked by means of real‐time fluorescence quantitative PCR. Burkholderia glumae tended to colonize at the growing point of rice plants, and the biomass of population was 104 to 108 CFU/g. The most intensive colonization was detected in the upmost leaf in the two‐leaf period. However, after the two‐leaf period, the population of pathogens decreased significantly, and they successfully recovered in the booting stage and broke out in panicles. We also illustrated the incubation location of B. glumae by presenting the infection pattern in the seedling and tillering stage of rice. Under fluorescent microscopy, the gfp‐labelled pathogens were first found in the vascular bundle of lateral roots, taproots and injured cells, then they were observed in the root hairs, epidermal cells and main root cap. The pathogens in the vascular bundle laterally dispersed towards the epidermal cells. By spray application of a bacterial suspension, the pathogens landed on the leaf sheaths and leaves, colonized in the epidermal hairs and leaf hairs, or invaded into the cells through the stomas. At the same time, the pathogens from the vascular bundle of the roots spread into the vascular bundle of leaf sheaths and leaves, which caused the leaves to curl and wilt, beginning from the tip.