Biological control of Magnaporthe oryzae using natively isolated Bacillus subtilis G5 from Oryza officinalis roots.

Rice blast, caused by Magnaporthe oryzae, is a major threat to global rice production causing significant crop losses and impacting grain quality. The annual loss of rice production due to this disease ranges from 10% to 30%. The use of biologically controlled strains, instead of chemical pesticides, to control plant diseases has become a research hotspot. In this study, an antagonistic endophytic bacterial strain was isolated from the roots of Oryza officinalis using the traditional isolation and culture methods. A phylogenetic tree based on 16S RNA and whole-genome sequencing identified isolate G5 as a strain of Bacillus subtilis. This isolate displayed strong antagonistic effects against different physiological strains of M. oryzae. After co-culture in LB medium for 7 days, the inhibition rates of the mycelial growth of four strains of M. oryzae, ZB15, WH97, Guy11, and T-39800E were 98.07 ± 0.0034%, 98.59 ± 0.0051%, 99.16 ± 0.0012%, and 98.69 ± 0.0065%, respectively. Isolate G5 significantly inhibited the formation of conidia of M. oryzae, with an inhibition rate of 97% at an OD600 of 2. Isolate G5 was able to provide 66.81% protection against rice blast under potted conditions. Whole-genome sequencing revealed that the genome size of isolate G5 was 4,065,878 bp, including 4,182 coding genes. Using the anti-SMASH software, 14 secondary metabolite synthesis gene clusters were predicted to encode antifungal substances, such as fengycin, surfactin, and bacilysin. The G5 isolate also contained genes related to plant growth promotion. These findings provide a theoretical basis for expounding the biocontrol mechanisms of this strain and suggest further development of biogenic agents that could effectively inhibit rice blast pathogen growth and reduce crop damage, while being environmentally friendly, conducive to ecological development, and a sustainable alternative to chemical pesticides. This study also enriches the relevant research on endophytes of wild rice, which proves that wild rice is a valuable microbial resource bank.

A new NLR disease resistance gene Xa47 confers durable and broad-spectrum resistance to bacterial blight in rice.

Bacterial blight (BB) induced by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating bacterial disease in rice. The use of disease resistance (R) genes is the most efficient method to control BB. Members of the nucleotide-binding domain and leucine-rich repeat containing protein (NLR) family have significant roles in plant defense. In this study, Xa47, a new bacterial blight R gene encoding a typical NLR, was isolated from G252 rice material, and XA47 was localized in the nucleus and cytoplasm. Among 180 rice materials tested, Xa47 was discovered in certain BB-resistant materials. Compared with the wild-type G252, the knockout mutants of Xa47 was more susceptible to Xoo. By contrast, overexpression of Xa47 in the susceptible rice material JG30 increased BB resistance. The findings indicate that Xa47 positively regulates the Xoo stress response. Consequently, Xa47 may have application potential in the genetic improvement of plant disease resistance. The molecular mechanism of Xa47 regulation merits additional examination.

Unveiling a Novel Source of Resistance to Bacterial Blight in Medicinal Wild Rice, Oryza officinalis.

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is among the oldest known bacterial diseases found for rice in Asia. It is the most serious bacterial disease in many rice growing regions of the world. A total of 47 resistance (R) genes (Xa1 to Xa47) have been identified. Nonetheless, these R genes could possibly be defeated to lose their qualitative nature and express intermediate phenotypes. The identification of sources of novel genetic loci regulating host plant resistance is crucial to develop an efficient control strategy. Wild ancestors of cultivated rice are a natural genetic resource contain a large number of excellent genes. Medicinal wild rice (Oryza officinalis) belongs to the CC genome and is a well-known wild rice in south China. In this study, O. officinalis was crossed with cultivated rice HY-8 and their hybrids were screened for BB resistance genes deployed through natural selection in wild rice germplasm. The molecular markers linked to R genes for BB were used to screen the genomic regions in wild parents and their recombinants. The gene coding and promoter regions of major R genes were inconsistently found in O. officinalis and its progenies. Oryza officinalis showed resistance to all thirty inoculated Xoo strains with non-availability of various known R genes. The results indicated the presence of novel genomic regions for BB resistance in O. officinalis. The present study not only provides a reference to investigate medicinal rice for R gene(s) identification against BB but also identified it as a new breeding material for BB resistance.

Identification and Fine-Mapping of a New Bacterial Blight Resistance Gene, Xa47(t), in G252, an Introgression Line of Yuanjiang Common Wild Rice (Oryza rufipogon).

Bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae is a common, widespread, and highly devastating disease that affects rice yield. Breeding resistant cultivars is considered the most effective measure for controlling this disease. The introgression line G252 derived from Yuanjiang common wild rice (Oryza rufipogon) was highly resistant to all tested strains, including C5, C9, PXO99, PB, T7147Y8, Hzhj19, YM1, YM187, YJdp-2, and YJws-2. To identify the BB resistance gene(s) of G252, we developed an F2 population from the cross between G252 and 02428. A linkage analysis was performed for the phenotype and genotype of the population. A segregation ratio of 3:1 was observed between the resistant and susceptible individuals in the F2 progeny, indicating a dominant resistance gene, Xa47(t), in G252. The resistance gene was mapped within an approximately 26.24-kb physical region on chromosome 11 between two InDel markers, R13I14 and 13rbq-71. Moreover, one InDel marker, Hxjy-1, co-segregated with Xa47(t). Three genes were predicted within the target region, including a promising candidate gene encoding a nucleotide-binding domain and leucine-rich repeat (NLR) protein (LOC_Os11g46200) by combining the structure and expression analysis. Physical mapping data suggested that Xa47(t) is a new broad-spectrum BB resistance gene without identified allelic genes.

Proteomic analysis of the rice (Oryza officinalis) provides clues on molecular tagging of proteins for brown planthopper resistance.

BACKGROUND: Among various pests, the brown planthopper (BPH) that damages rice is the major destructive pests. Understanding resistance mechanisms is a critical step toward effective control of BPH. This study investigates the proteomics of BPH interactions with three rice cultivars: the first resistant (PR) to BPH, the second susceptible (PS), and the third hybrid (HR) between the two, in order to understand mechanisms of BPH resistance in rice. RESULTS: Over 4900 proteins were identified from these three rice cultivars using iTRAQ proteomics study. A total of 414, 425 and 470 differentially expressed proteins (DEPs) were detected from PR, PS and HR, respectively, after BPH infestation. Identified DEPs are mainly enriched in categories related with biosynthesis of secondary metabolites, carbon metabolism, and glyoxylate and dicarboxylate metabolism. A two-component response regulator protein (ORR22) may participate in the early signal transduction after BPH infestation. In the case of the resistant rice cultivar (PR), 6 DEPs, i.e. two lipoxygenases (LOXs), a lipase, two dirigent proteins (DIRs) and an Ent-cassa-12,15-diene synthase (OsDTC1) are related to inheritable BPH resistance. A heat shock protein (HSP20) may take part in the physiological response to BPH infestation, making it a potential target for marker-assisted selection (MAS) of rice. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed eight genes encoding various metabolic proteins involved in BPH resistance. During grain development the expressions of these genes varied at the transcriptional and translational levels. CONCLUSIONS: This study provides comprehensive details of key proteins under compatible and incompatible interactions during BPH infestation, which will be useful for further investigation of the molecular basis of rice resistance to BPH and for breeding BPH-resistant rice cultivars.

Transcriptome analysis of WRKY gene family in Oryza officinalis Wall ex Watt and WRKY genes involved in responses to Xanthomonas oryzae pv. oryzae stress.

Oryza officinalis Wall ex Watt, a very important and special wild rice species, shows abundant genetic diversity and disease resistance features, especially high resistance to bacterial blight. The molecular mechanisms of bacterial blight resistance in O. officinalis have not yet been elucidated. The WRKY transcription factor family is one of the largest gene families involved in plant growth, development and stress response. However, little is known about the numbers, structure, molecular phylogenetics, and expression of the WRKY genes under Xanthomonas oryzae pv. oryzae (Xoo) stress in O. officinalis due to lacking of O. officinalis genome. Therefore, based on the RNA-sequencing data of O. officinalis, we performed a comprehensive study of WRKY genes in O. officinalis and identified 89 OoWRKY genes. Then 89 OoWRKY genes were classified into three groups based on the WRKY domains and zinc finger motifs. Phylogenetic analysis strongly supported that the evolution of OoWRKY genes were consistent with previous studies of WRKYs, and subgroup IIc OoWRKY genes were the original ancestors of some group II and group III OoWRKYs. Among the 89 OoWRKY genes, eight OoWRKYs displayed significantly different expression (>2-fold, p<0.01) in the O. officinalis transcriptome under Xoo strains PXO99 and C5 stress 48 h, suggesting these genes might play important role in PXO99 and C5 stress responses in O. officinalis. QRT-PCR analysis and confirmation of eight OoWRKYs expression patterns revealed that they responded strongly to PXO99 and C5 stress 24 h, 48 h, and 72 h, and the trends of these genes displaying marked changes were consistent with the 48 h RNA-sequencing data, demonstrated these genes played important roles in response to biotic stress and might even involved in the bacterial blight resistance. Tissue expression profiles of eight OoWRKY genes revealed that they were highly expressed in root, stem, leaf, and flower, especially in leaf (except OoWRKY71), suggesting these genes might be also important for plant growth and organ development. In this study, we analyzed the WRKY family of transcription factors in O.officinalis. Insight was gained into the classification, evolution, and function of the OoWRKY genes, revealing the putative roles of eight significantly different expression OoWRKYs in Xoo strains PXO99 and C5 stress responses in O.officinalis. This study provided a better understanding of the evolution and functions of O. officinalis WRKY genes, and suggested that manipulating eight significantly different expression OoWRKYs would enhance resistance to bacterial blight.