Analisis Molekuler Burkholderia glumae Pada Varietas Padi Ciherang Di Sawah Tadah Hujan Lingkungan Universitas Wiralodra Indramayu

Deteksi patogen penyebab penyakit tumbuhan dapat dilakukan baik secara langsung maupun tidak langsung. Deteksi secara langsung umumnya dilakukan dengan menggunakan metode serologi dan molekuler. Deteksi dan identifikasi bakteri patogen dengan teknik molekuler dapat dilakukan dengan teknik polymerase chain reaction (PCR). Penyakit hawar malai yang disebabkan oleh Burkholderia glumae dilaporkan menginfeksi tanaman padi di Indonesia. B. glumae dapat terbawa benih sehingga berpotensi menyebar dengan cepat. Penelitian ini bertujuan untuk mengetahui wilayah sebar penyakit hawar malai di sawah tadah hujan lingkungan Universitas Wiralodra Indramayu. Metode yang dilakukan meliputi pengambilan sampel biji padi di sawah tadah hujan lingkungan Universitas Wiralodra Indramayu. Biji padi secara acak diambil langsung dari sawah tadah hujan serta dicatat varietas serta nama daerah pengambilan sampel, kemudian diuji secara molekuler. Hasil amplifikasi PCR terhadap 3 sampel biji padi menunjukkan ketiga sampel tersebut negatif terinfeksi B. glumae. Sampel biji padi yang negatif terdeteksi B. glumae dapat terjadi karena pada saat pengambilan sampel, wilayah Indramayu sedang memasuki musim penghujan, sehingga potensi berkembangnya penyakit hawar malai akan menurun.

T6SS Accessory Proteins, Including DUF2169 Domain-Containing Protein and Pentapeptide Repeats Protein, Contribute to Bacterial Virulence in T6SS Group_5 of Burkholderia glumae BGR1

Burkholderia glumae are bacteria pathogenic to rice plants that cause a disease called bacterial panicle blight (BPB) in rice panicles. BPB, induced by B. glumae, causes enormous economic losses to the rice agricultural industry. B. glumae also causes bacterial disease in other crops because it has various virulence factors, such as toxins, proteases, lipases, extracellular polysaccharides, bacterial motility, and bacterial secretion systems. In particular, B. glumae BGR1 harbors type VI secretion system (T6SS) with functionally distinct roles: the prokaryotic targeting system and the eukaryotic targeting system. The functional activity of T6SS requires 13 core components and T6SS accessory proteins, such as adapters containing DUF2169, DUF4123, and DUF1795 domains. There are two genes, bglu_1g23320 and bglu_2g07420, encoding the DUF2169 domain-containing protein in the genome of B. glumae BGR1. bglu_2g07420 belongs to the gene cluster of T6SS group_5 in B. glumae BGR1, whereas bglu_1g23320 does not belong to any T6SS gene cluster in B. glumae BGR1. T6SS group_5 of B. glumae BGR1 is involved in bacterial virulence in rice plants. The DUF2169 domain-containing protein with a single domain can function by itself; however, Δu1g23320 showed no attenuated virulence in rice plants. In contrast, Δu2g07420DUF2169 and Δu2g07420PPR did exhibit attenuated virulence in rice plants. These results suggest that the pentapeptide repeats region of the C-terminal additional domain, as well as the DUF2169 domain, is required for complete functioning of the DUF2169 domain-containing protein encoded by bglu_2g07420. bglu_2g07410, which encodes the pentapeptide repeats protein, composed of only the pentapeptide repeats region, is located downstream of bglu_2g07420. Δu2g07410 also shows attenuated virulence in rice plants. This finding suggests that the pentapeptide repeats protein, encoded by bglu_2g07410, is involved in bacterial virulence. This study is the first report that the DUF2169 domain-containing protein and pentapeptide repeats protein are involved in bacterial virulence to the rice plants as T6SS accessory proteins, encoded in the gene cluster of the T6SS group_5.

Applications of bacteriophages in controlling rice bacterial grain rot caused by Burkholderia glumae

The study on the procedure of applicating bacteriophage (or phage) to prevent rice bacterial grain rot caused by Burkholderia glumae was conducted in the greenhouse conditions. The first experiment investigated the effect of different phage titers (i.e. 105 PFU/mL, 106 PFU/mL, 107 PFU/mL, 108 PFU/mL) in controlling bacterial grain rot of rice. The results showed that all four titers gave disease reduction with different levels, among these were the titer of 108PFU/mL expressed highest efficacy in disease reduction with the lowest percentage of infected grains compared to the rest treatments. The second experiment examined the effect of phage application times (i.e. spraying phage at 2 hours before pathogen inoculation, 2 hours before and 5 days after pathogen inoculation, and 5 days after pathogen inoculation) in suppressing bacterial grain rot disease. The results found that two treatments (i.e. one time spraying at 2 hours before pathogen inoculation, and two times spraying at 2 hours before and 5 days after pathogen inoculation) expressed high efficacy in reduction of grain rot disease through percentage of infected grains and improved yield parameter regarding rate of filled grains.

Membrane Depolarization and Apoptosis-Like Cell Death in an Alkaline Environment in the Rice Pathogen Burkholderia glumae

The rice pathogen Burkholderia glumae uses amino acids as a principal carbon source and thus produces ammonia in amino acid-rich culture medium such as Luria–Bertani (LB) broth. To counteract ammonia-mediated environmental alkaline toxicity, the bacterium produces a public good, oxalate, in a quorum sensing (QS)-dependent manner. QS mutants of B. glumae experience alkaline toxicity and may undergo cell death at the stationary phase when grown in LB medium. Here, we show that the cell-death processes of QS mutants due to alkaline environmental conditions are similar to the apoptosis-like cell death reported in other bacteria. Staining QS mutants with bis-(1,3-dibutylbarbituric acid)-trimethine oxonol revealed membrane depolarization. CellROX™ staining showed excessive generation of reactive oxygen species (ROS) in QS mutants. The expression of genes encoding HNH endonuclease (BGLU_1G15690), oligoribonuclease (BGLU_1G09120), ribonuclease E (BGLU_1G09400), and Hu-beta (BGLU_1G13530) was significantly elevated in QS mutants compared to that in wild-type BGR1, consistent with the degradation of cellular materials as observed under transmission electron microscopy (TEM). A homeostatic neutral pH was not attainable by QS mutants grown in LB broth or by wild-type BGR1 grown in an artificially amended alkaline environment. At an artificially adjusted alkaline pH, wild-type BGR1 underwent apoptosis-like cell death similar to that observed in QS mutants. These results show that environmental alkaline stress interferes with homeostatic neutral cellular pH, induces membrane depolarization, and causes apoptosis-like cell death in B. glumae.

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.

Characterization of Streptomyces spp. from Rice Fields as a Potential Biocontrol Agent against Burkholderia glumae and Rice Plant Growth Promoter

The usage of plant growth-promoting (PGP) Streptomyces to improve plant growth is an alternative to existing strategies of chemical fertilizers that commonly caused environmental pollution. The aim of this study was to isolate and characterize PGP Streptomyces from Malaysian rice fields with antagonistic activity against Bacterial Panicle Blight disease in rice caused by Burkholderia glumae. A total of 50 bacterial isolates were recovered from the soil, rhizosphere, and endosphere; 22% showed antagonistic activity against B. glumae. Molecular identification using 16S rRNA amplification and phylogenetic tree analyses revealed that the antagonistic isolates belonged to the genus Streptomyces. Among the antagonistic isolates, Streptomyces sp. TBS5 (26.35 ± 0.14 mm) demonstrated the most antagonistic activity (p < 0.05) against B. glumae. Through API® ZYM analysis, the antagonistic isolates were revealed to have phosphoric hydrolase, ester hydrolase, peptide hydrolase, and glycosidase activities that play a crucial role in plant growth promotion. The antagonistic isolates demonstrated the highest (80%) PGP traits including able to fix nitrogen and solubilize phosphate, as well as produce siderophore and indole-3-acetic acid. Plant growth promotion assay under laboratory and greenhouse conditions were analyzed with the treatment of rice, Oryza sativa, seeds with the antagonistic isolates. Seeds treated with Streptomyces sp. TBS5, JAS2, R2-7, and TKR8 showed improvement in vigor index by more than 100% compared to water-treated control plants under both conditions. Augmentation of root length showed an overall increment of more than 101.5% under laboratory condition and 151.9% under greenhouse condition using Streptomyces sp. TBS5, JAS2, R2-7, and TKR8 treatments. Results suggest that Streptomyces sp. TBS5, JAS2, R2-7, and TKR8 are a good candidate to be developed as both biocontrol agent against B. glumae and PGP agent in rice.

Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae

The highly conserved ATP-dependent Lon protease plays important roles in diverse biological processes. The lon gene is usually nonessential for viability; however, lon mutants of several bacterial species, although viable, exhibit cellular defects. Here, we show that a lack of Lon protease causes pleiotropic effects in the rice pathogen Burkholderia glumae. The null mutation of lon produced three colony types, big (BLONB), normal (BLONN), and small (BLONS), in Luria–Bertani (LB) medium. Colonies of the BLONB and BLONN types were re-segregated upon subculture, while those of the BLONS type were too small to manipulate. The BLONN type was chosen for further studies, as only this type was fully genetically complemented. BLONN-type cells did not reach the maximum growth capacity, and their population decreased drastically after the stationary phase in LB medium. BLONN-type cells were defective in the biosynthesis of quorum sensing (QS) signals and exhibited reduced oxalate biosynthetic activity, causing environmental alkaline toxicity and population collapse. Addition of excessive N-octanoyl-homoserine lactone (C8-HSL) to BLONN-type cell cultures did not fully restore oxalate biosynthesis, suggesting that the decrease in oxalate biosynthesis in BLONN-type cells was not due to insufficient C8-HSL. Co-expression of lon and tofR in Escherichia coli suggested that Lon negatively affects the TofR level in a C8-HSL-dependent manner. Lon protease interacted with the oxalate biosynthetic enzymes, ObcA and ObcB, indicating potential roles for the oxalate biosynthetic activity. These results suggest that Lon protease influences colony morphology, growth, QS system, and oxalate biosynthesis in B. glumae.

Complete genome sequence data of four Burkholderia glumae strains isolated from rice fields in the United States

Bacterial panicle blight caused by Burkholderia glumae is a major disease in rice production worldwide. Currently, only a few whole-genome sequences of B. glumae strains isolated in the U.S. are available. Here, we report the complete genome sequence of four B. glumae strains, including three virulent strains (336gr-1, 411gr-6, and 957856-41-c) and the non-pathogenic strain B. glumae 257sh-1, which were isolated from rice fields in Louisiana (336gr-1, 957856-41-c, and 257sh-1) and Arkansas (411gr-6). The whole-genome sequence data of B. glumae strains will contribute to investigations of the molecular mechanism underlying bacterial pathogenicity and virulence to rice plants.