Background: Rice blast is generally considered the most devastating rice disease worldwide. The development of resistant varieties has been proven to be the most economical strategy to control the disease. A cluster of resistant (R) genes on rice chromosome 12 including Pita, Pita2 and Ptr has been studies for decades. However, the relationship between these R genes has not been well established. Results: In this study, we compared the resistance spectra controlled by Pita2 and Pita by testing their monogenic lines (MLs) in four hotspots found in the Philippines and Burundi from 2014 to 2018. The reaction patterns were distinct in two countries and that Pita2-mediated field resistance was relatively prevalent. Pathogenicity tests using 328 single-spore isolates in greenhouse further verified that IRBLta2-Re for Pita2 conferred a relatively broader spectrum resistance than those of Pita. Rough and fine mapping of Pita2 were conducted using F 2 and F 3 populations derived from IRBLta2-Re [CO] and CO 39 consisting of 4344 progeny to delimit Pita2 in a genomic interval flanked by two markers 12 g18530 and 12 g18920 proximal to the centromere of chromosome 12. Alignment of the markers to the genomic sequence of IR64, which harbors Pita2 verified by genetic analysis, approximately delimited the candidate gene(s) within 313-kb genomic fragment. The two Pita2 suppressive mutants that contain mutations within Pita2 were verified and identified. Comparative sequence analysis in these two mutants further identified that each individual allele contains a single nucleotide substitution at a different position resulting in nonsense and missense mutations in the protein product of LOC_Os12g18729. On the contrary, no sequence mutation was detected in other candidate genes, indicating that mutations in LOC_ Os12g18729 were responsible for the loss of function of Pita2. Pita2 encodes a novel R protein unique from Pita, which is exactly identical to the previously cloned Ptr. Moreover, based on the resistance gene analysis of rice varieties and mutants containing Pita, it was found that Pita2 rather than Pita was responsible for the specificity to some differential isolates with AvrPita. The diagnosis and survey of Pita2 in IRRI released varieties showed relatively low frequency, implying a high value of its application for breeding resistant varieties against rice blast via marker assisted selection.

Avirulence (AVR) genes in Magnaporthe oryzae, the fungal pathogen that causes the devastating rice blast disease, have been documented to be major targets subject to mutations to avoid recognition by resistance (R) genes. In this study, an AVR-gene-based diagnosis tool for determining the virulence spectrum of a rice blast pathogen population was developed and validated. A set of 77 single-spore field isolates was subjected to pathotype analysis using differential lines, each containing a single R gene, and classified into 20 virulent pathotypes, except for 4 isolates that lost pathogenicity. In all, 10 differential lines showed low frequency (<24%) of resistance whereas 8 lines showed a high frequency (>95%), inferring the effectiveness of R genes present in the respective differential lines. In addition, the haplotypes of seven AVR genes were determined by polymerase chain reaction amplification and sequencing, if applicable. The calculated frequency of different AVR genes displayed significant variations in the population. AVRPiz-t and AVR-Pii were detected in 100 and 84.9% of the isolates, respectively. Five AVR genes such as AVR-Pik-D (20.5%) and AVR-Pik-E (1.4%), AVRPiz-t (2.7%), AVR-Pita (0%), AVR-Pia (0%), and AVR1-CO39 (0%) displayed low or even zero frequency. The frequency of AVR genes correlated almost perfectly with the resistance frequency of the cognate R genes in differential lines, except for International Rice Research Institute-bred blast-resistant lines IRBLzt-T, IRBLta-K1, and IRBLkp-K60. Both genetic analysis and molecular marker validation revealed an additional R gene, most likely Pi19 or its allele, in these three differential lines. This can explain the spuriously higher resistance frequency of each target R gene based on conventional pathotyping. This study demonstrates that AVR-gene-based diagnosis provides a precise, R-gene-specific, and differential line-free assessment method that can be used for determining the virulence spectrum of a rice blast pathogen population and for predicting the effectiveness of target R genes in rice varieties.

Methanosarcina species possess three operons (mtaCB1, mtaCB2, and mtaCB3) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (mtaA1 and mtaA2) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by constructing strains in which the mtaCB promoters were exchanged. When expressed from the strong PmtaC1 or PmtaC2 promoter, each of the MtaC and MtaB proteins supported growth and methane production at wild-type levels. In contrast, all mtaCB operons exhibited poorer growth and lower rates of methane production when PmtaC3 controlled their expression. Thus, previously observed phenotypic differences can be attributed largely to differences in promoter activity. Strains carrying various combinations of mtaC, mtaB, and mtaA expressed from the strong, tetracycline-regulated PmcrB(tetO1) promoter exhibited similar growth characteristics on methanol, showing that all combinations of MtaC, MtaB, and MtaA can form functional MT1/MT2 complexes. However, an in vitro assay of coupled MT1/MT2 activity showed significant variation between the strains. Surprisingly, these variations in activity correlated with differences in protein abundance, despite the fact that all the encoding genes were expressed from the same promoter. Quantitative reverse transcriptase PCR and reporter gene fusion data suggest that the mtaCBA transcripts show different stabilities, which are strongly influenced by the growth substrate.Methanosarcina species, such as M. acetivorans and M. barkeri, are strictly anaerobic archaea that derive their energy for growth via methanogenesis, the production of methane (CH 4 ) (35). These organisms utilize various substrates, including onecarbon (C 1 ) compounds (CO, CO 2, formate, methanol, and methylamines), acetate, and CO 2 , as terminal electron acceptors in an energy-conserving electron transport chain that ultimately results in the production of methane (3,7,22). Four distinct methanogenic pathways can be found in various methanogens, which alternatively allow the reduction of CO 2 , the reduction of methyl moieties from C 1 compounds, the splitting of acetate, and the disproportionation of C 1 compounds, known as methylotrophic methanogenesis.In the methylotrophic pathway for methanogenesis, methanol, methylamines, or methylsulfides are disproportionated into CO 2 and CH 4 , with the reducing equivalents from oxidation of one molecule of the substrate being used to reduce three additional molecules to CH 4 (reviewed in reference 14). Biochemical studies have shown that these methylated compounds are initially activated via methylation of 2-mercaptoethanesulfonic acid (coenzyme M [CoM]). The conversion of methanol to methyl-CoM is mediated by the concerted effort of at least three proteins that interact cooperatively ...

Diazotrophic bacteria (syn. nitrogen-fixing bacteria, NFB) are plant growth-promoting rhizobacteria known to facilitate nitrogen availability for plant nutrition. Biochar may serve as a refuge for colonizing microbes influencing the mycorrhizal richness and activities in the rhizosphere, thereby affecting the mycorrhization helping bacteria. This study isolated, characterized, and identified diazotrophic bacteria from the cacao (Theobroma cacao L.) rhizosphere treated with arbuscular mycorrhizal fungi (AMF) and bamboo biochar (BB). Initial screening of the cacao rhizosphere yielded 1.28x106 CFU of diazotrophic bacteria g–1 soilgrown in nitrogen-free malate medium. A total of 12 isolates were further assayed for acetylene reduction and phosphate solubilization activities. All putative diazotrophic bacterial isolates successfully reduced acetylene to ethylene gas, ranging from 16.35 ± 1.68 to 60.71 ± 2.57 ppm. Of the 12 putative diazotrophic bacterial isolates, 11 have shown phosphate solubilization activity on Pikovskaya’s medium with the solubilization index (SI) ranging from 1.12 ± 0.08 to 3.74 ± 0.16 mm. Analysis of the 16S rRNA gene sequences revealed that these diazotrophic bacterial isolates belonged to the genera Bacillus, Burkholderia, Rhizobium, and Ralstonia. These results provide evidence of candidate plant growth-promoting rhizobacteria from cacao that are potential source of biofertilizers to improve the growth and yield of cacao crops, especially in the Philippines. As agricultural options, biochar and AMF pose beneficial influence on plant growth-promoting bacteria, which primarily provide nutrients for several crops.

Schistosomiasis is an acute and chronic parasitic disease caused by blood flukes of the genus Schistosoma. The Kato-Katz method is the standard for the diagnosis of intestinal schistosomiasis because of its specificity, simplicity, and low cost. However, Kato-Katz is less useful in light infections and other available detection methods are neither practical nor sensitive for routine or large-scale screening. Thus, improved tests are needed for the assessment of infection. The water buffalo is a natural host and reservoir of Schistosoma but is schistosomiasis-resistant, suggesting a robust immune repertoire, which can be tapped as a source of antibodies for diagnosis of schistosomiasis. Two schistosome antigens had been previously shown to be recognized by a phage display single-chain fragment of variation (scFv) library derived from a schistosome-infected water buffalo. However, specific antibodies have not been isolated using these antigens due to a lack of sufficient amounts and purified forms of the antigens. In this study, the schistosome antigens were cloned and overexpressed in vector with solubility tag, purified by immobilized metal affinity chromatography (IMAC), and used to interrogate the immune scFv library to isolate and identify specific schistosome antibody. An scFv specific to schistosome SAP domain-containing hypothetical protein was isolated but was only 105-bp long although certain regions align with the IgG VL, tetraglycylserine (Gly4Ser) linker, and IgG VH sequences of synthetic scFv constructs. Despite its length, scFv A63 can be potentially designed as a ligand in direct ELISA (enzyme-linked immunosorbent assay) by fusion with enzyme conjugates such as alkaline phosphatase to detect S. japonicum schistosomule and adult worm. The results in this study also suggest the need to construct a bigger and more diverse scFv library and to test a wider range of schistosome antigens, specifically those that will be present on the surface of the schistosome at all stages of S. japonicum life cycle.

Antimicrobial resistance (AMR) poses a global and developmental threat to humanity. The rapid emergence and spread of drug-resistant pathogens resulted in the ineffective use of currently available antibiotics. Therefore, there is a need to continue searching for additional sources of antibiotics, such as actinomycetes, which can potentially harbor unique and effective secondary metabolites. Furthermore, it is interesting to consider poorly explored environments like volcanoes, which can be potential sources of drug leads for medically important natural products. This study reports the antimicrobial activity of actinomycetes isolated from volcanic soil samples collected from Mount Mayon, Albay, Philippines. A total of 13 out of 30 morphologically distinct actinomycete isolates showed antagonistic activity against test microorganisms. Isolate A1-08, the focus of the study, exhibited a wide spectrum of antimicrobial activity against Salmonella enterica, Klebsiella pneumoniae, Staphylococcus aureus, methicillin-resistant S. aureus(MRSA), Candida albicans, Aspergillus niger, and Fusarium sp. Moreover, A1-08 was found to have anti-methicillin resistant S. aureus (MIC = 2.50 mg/mL) and anticancer activity against human colorectal cancer (HCT116) cell line (IC50 = 21.54 μg/mL). Whole-genome sequence-based phylogenetic analysis supported a novel species of Streptomyces closely related to S. olivaceus NRRL B-3009. A total of 48 biosynthetic gene clusters (BGCs) were identified that may be responsible for the biosynthesis of known and potentially novel secondary metabolites. This study concludes that Streptomyces sp. A1-08, a potentially novel species, is a good candidate to produce broad-spectrum antibiotics with anti-MRSA and anti-cancer activities and possibly novel secondary bioactive metabolites of medical and pharmaceutical importance.