OsPUB9 Gene Edited by CRISPR/Cas9 Enhanced Resistance to Bacterial Leaf Blight in Rice (Oryza sativa L.).

Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T0 plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene.

Introgression of blast and bacterial blight disease resistance genes in a rice genotype ADT43 through marker assisted back cross breeding.

Bacterial Leaf Blight (Xanthomonas oryzae pv. oryzae) and blast (Magnaporthe oryzae) are the major biotic stresses around the rice-growing zones of the world. The development of resistant varieties through Marker Assisted Backcross Breeding is the utmost economical and eco-friendly method for achieving stable yield. Amongst the resistance genes recognized, Xa21 and Pi54 possess broad-spectrum resistance to many Xoo and blast strains around the world. In the present study, we have effectively introgressed a Bacterial Blight resistance gene (Xa21) and a blast resistance gene (Pi54) into susceptible variety ADT43 from RP-Bio-Patho-2 coupled with phenotypic selection for agronomic, cooking quality and grain traits through MABC. MABC was sustained till BC2F2 generation with specific markers pTA248 for Xa21 and Pi54MAS for Pi54 resistance genes. A set of SSR markers for parental polymorphism were utilized for maximum regaining of recurrent parent genome in each backcrossing. "Positive plants" from BC2F1 were selfed to generate BC2F2 and the homozygous lines for bacterial leaf blight and blast resistance genes were identified for further assessment.

Constructed Rice Tracers Identify the Major Virulent Transcription Activator-Like Effectors of the Bacterial Leaf Blight Pathogen.

Xanthomonas oryzae pv. oryzae (Xoo) injects major transcription activator-like effectors (TALEs) into plant cells to activate susceptibility (S) genes for promoting bacterial leaf blight in rice. Numerous resistance (R) genes have been used to construct differential cultivars of rice to identify races of Xoo, but the S genes were rarely considered. Different edited lines of rice cv. Kitaake were constructed using CRISPR/Cas9 gene-editing, including single, double and triple edits in the effector-binding elements (EBEs) located in the promoters of rice S genes OsSWEET11a, OsSWEET13 and OsSWEET14. The near-isogenic lines (NILs) were used as tracers to detect major TALEs (PthXo1, PthXo2, PthXo3 and their variants) in 50 Xoo strains. The pathotypes produced on the tracers determined six major TALE types in the 50 Xoo strains. The presence of the major TALEs in Xoo strains was consistent with the expression of S genes in the tracers, and it was also by known genome sequences. The EBE editing had little effect on agronomic traits, which was conducive to balancing yield and resistance. The rice-tracers generated here provide a valuable tool to track major TALEs of Xoo in Asia which then shows what rice cultivars are needed to combat Xoo in the field.

Microbiome Analysis Revealed Acholeplasma as a Possible Factor Influencing the Susceptibility to Bacterial Leaf Blight Disease of Two Domestic Rice Cultivars in Vietnam.

The microbiomes of two important rice cultivars in Vietnam which differ by their susceptibility to the bacterial leaf blight (BLB) disease were analyzed through 16S rRNA amplicon technology. A higher number of operational taxonomic units and alpha-diversity indices were shown in the BLB-resistant LA cultivar than in the BLB-susceptible TB cultivar. The BLB pathogen Xanthomonas was scantly found (0.003%) in the LA cultivar, whereas was in a significantly higher ratio in the TB cultivar (1.82%), reflecting the susceptibility to BLB of these cultivars. Of special interest was the genus Acholeplasma presented in the BLB-resistant LA cultivar at a high relative abundance (22.32%), however, was minor in the BLB-sensitive TB cultivar (0.09%), raising a question about its roles in controlling the Xanthomonas low in the LA cultivar. It is proposed that Acholeplasma once entered the host plant would hamper other phytopathogens, i.e. Xanthomonas, by yet unknown mechanisms, of which the triggering of the host plants to produce secondary metabolites against pathogens could be a testable hypothesis.

Application and Mechanism of Cryptolepine and Neocryptolepine Derivatives as T3SS Inhibitors for Control of Bacterial Leaf Blight on Rice.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv oryzae (Xoo) is extremely harmful to rice production. The traditional control approach is to use bactericides that target key bacterial growth factors, but the selection pressure on the pathogen makes resistant strains the dominant bacterial strains, leading to a decline in bactericidal efficacy. Type III secretion system (T3SS) is a conserved and critical virulence factor in most Gram-negative bacteria, and its expression or absence does not affect bacterial growth, rendering it an ideal target for creating drugs against Gram-negative pathogens. In this work, we synthesized a range of derivatives from cryptolepine and neocryptolepine. We found that compound Z-8 could inhibit the expression of Xoo T3SS-related genes without affecting the growth of bacteria. an in vivo bioassay showed that compound Z-8 could effectively reduce the hypersensitive response (HR) induced by Xoo in tobacco and reduce the pathogenicity of Xoo in rice. Furthermore, it exhibited synergy in control of bacterial leaf blight when combined with the quorum quenching bacterial F20.

Subtractive genomics study of Xanthomonas oryzae pv. Oryzae reveals repurposable drug candidate for the treatment of bacterial leaf blight in rice.

BACKGROUND: Xanthomonas oryzae pv. oryzae is a plant pathogen responsible for causing one of the most severe bacterial diseases in rice, known as bacterial leaf blight that poses a major threat to global rice production. Even though several experimental compounds and chemical agents have been tested against X. oryzae pv. oryzae, still no approved drug is available. In this study, a subtractive genomic approach was used to identify potential therapeutic targets and repurposible drug candidates that could control of bacterial leaf blight in rice plants. RESULTS: The entire proteome of the pathogen underwent an extensive filtering process which involved removal of the paralogous proteins, rice homologs, non-essential proteins. Out of the 4382 proteins present in Xoo proteome, five hub proteins such as dnaA, dnaN, recJ, ruvA, and recR were identified for the druggability analysis. This analysis led to the identification of dnaN-encoded Beta sliding clamp protein as a potential therapeutic target and one experimental drug named [(5R)-5-(2,3-dibromo-5-ethoxy-4hydroxybenzyl)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]acetic acid that can be repurposed against it. Molecular docking and 100 ns long molecular dynamics simulation suggested that the drug can form stable complexes with the target protein over time. CONCLUSION: Findings from our study indicated that the proposed drug showed potential effectiveness against bacterial leaf blight in rice caused by X. oryzae pv. oryzae. It is essential to keep in consideration that the procedure for developing novel drugs can be challenging and complicated. Even the most promising results from in silico studies should be validated through further in vitro and in vivo investigation before approval.

Alternative polyadenylation profiles of susceptible and resistant rice (Oryza sativa L.) in response to bacterial leaf blight using RNA-seq.

BACKGROUND: Alternative polyadenylation (APA) is an important pattern of post-transcriptional regulation of genes widely existing in eukaryotes, involving plant physiological and pathological processes. However, there is a dearth of studies investigating the role of APA profile in rice leaf blight. RESULTS: In this study, we compared the APA profile of leaf blight-susceptible varieties (CT 9737-613P-M) and resistant varieties (NSIC RC154) following bacterial blight infection. Through gene enrichment analysis, we found that the genes of two varieties typically exhibited distal poly(A) (PA) sites that play different roles in two kinds of rice, indicating differential APA regulatory mechanisms. In this process, many disease-resistance genes displayed multiple transcripts via APA. Moreover, we also found five polyadenylation factors of similar expression patterns of rice, highlighting the critical roles of these five factors in rice response to leaf blight about PA locus diversity. CONCLUSION: Notably, the present study provides the first dynamic changes of APA in rice in early response to biotic stresses and proposes a possible functional conjecture of APA in plant immune response, which lays the theoretical foundation for in-depth determination of the role of APA events in plant stress response and other life processes.

Introgression of bacterial leaf blight (BLB) resistant gene, Xa7 into MARDI elite variety, MR219 by marker assisted backcrossing (MABC) approach

Abstract Bacterial leaf blight (BLB) is one of the major rice diseases in Malaysia. This disease causes substantial yield loss as high as 70%. Development of rice varieties which inherited BLB resistant traits is a crucial approach to promote and sustain rice industry in Malaysia. Hence, this study aims were to enhance BLB disease resistant characters of high yielding commercial variety MR219 through backcross breeding approach with supporting tool of marker-assisted selection (MAS). Broad spectrum BLB resistance gene, Xa7 from donor parent IRBB7 were introgressed into the susceptible MR219 (recurrent parent) using two flanking markers ID7 and ID15. At BC3F4, we managed to generate 19 introgressed lines with homozygous Xa7 gene and showed resistant characteristics as donor parent when it was challenged with Xanthomonas oryzae pv. oryzae through artificial inoculation. Recurrent parent MR219 and control variety, MR263 were found to be severely infected by the disease. The improved lines exhibited similar morphological and yield performance characters as to the elite variety, MR219. Two lines, PB-2-107 and PB-2-34 were chosen to be potential lines because of their outstanding performances compared to parent, MR219. This study demonstrates a success story of MAS application in development of improved disease resistance lines of rice against BLB disease.

Resumo A mancha bacteriana das folhas (BLB) é uma das principais doenças do arroz na Malásia. Essa doença causa perdas substanciais de rendimento de até 70%. O desenvolvimento de variedades de arroz que herdaram características de resistência ao BLB é uma abordagem crucial para promover e sustentar a indústria do arroz na Malásia. Portanto, o objetivo deste estudo foi aumentar os caracteres BLB resistentes a doenças da variedade comercial MR219 de alto rendimento por meio de uma abordagem de cruzamento retrocruzamento com ferramenta de apoio de seleção assistida por marcador (MAS). O gene de resistência a BLB de amplo espectro, Xa7 do pai doador IRBB7, foi introgressado no MR219 suscetível (pai recorrente) usando dois marcadores flanqueadores ID7 e ID15. No BC3F4, conseguimos gerar 19 linhagens introgressadas com o gene Xa7 homozigoto e apresentamos características de resistência como genitor doador quando desafiado com Xanthomonas oryzae pv. oryzae por inoculação artificial. O pai recorrente MR219 e a variedade controle, MR263, estavam gravemente infectados pela doença. As linhas melhoradas exibiram características morfológicas e de desempenho de rendimento semelhantes às da variedade elite, MR219. Duas linhas, PB-2-107 e PB-2-34, foram escolhidas como linhas potenciais por causa de seus desempenhos excelentes em comparação com a mãe, MR219. Este estudo demonstra uma história de sucesso de aplicação de MAS no desenvolvimento de linhas de arroz melhoradas com resistência a doenças contra a doença BLB.

Introgression of bacterial leaf blight (BLB) resistant gene, Xa7 into MARDI elite variety, MR219 by marker assisted backcrossing (MABC) approach / Introdução do gene resistente à ferragem da folha bacteriana (BLB), xa7 na variedade MARDI elite, MR219 por abordagem de backcrossing assistida por marcador (MABC)

Abstract Bacterial leaf blight (BLB) is one of the major rice diseases in Malaysia. This disease causes substantial yield loss as high as 70%. Development of rice varieties which inherited BLB resistant traits is a crucial approach to promote and sustain rice industry in Malaysia. Hence, this study aims were to enhance BLB disease resistant characters of high yielding commercial variety MR219 through backcross breeding approach with supporting tool of marker-assisted selection (MAS). Broad spectrum BLB resistance gene, Xa7 from donor parent IRBB7 were introgressed into the susceptible MR219 (recurrent parent) using two flanking markers ID7 and ID15. At BC3F4, we managed to generate 19 introgressed lines with homozygous Xa7 gene and showed resistant characteristics as donor parent when it was challenged with Xanthomonas oryzae pv. oryzae through artificial inoculation. Recurrent parent MR219 and control variety, MR263 were found to be severely infected by the disease. The improved lines exhibited similar morphological and yield performance characters as to the elite variety, MR219. Two lines, PB-2-107 and PB-2-34 were chosen to be potential lines because of their outstanding performances compared to parent, MR219. This study demonstrates a success story of MAS application in development of improved disease resistance lines of rice against BLB disease.

Resumo A mancha bacteriana das folhas (BLB) é uma das principais doenças do arroz na Malásia. Essa doença causa perdas substanciais de rendimento de até 70%. O desenvolvimento de variedades de arroz que herdaram características de resistência ao BLB é uma abordagem crucial para promover e sustentar a indústria do arroz na Malásia. Portanto, o objetivo deste estudo foi aumentar os caracteres BLB resistentes a doenças da variedade comercial MR219 de alto rendimento por meio de uma abordagem de cruzamento retrocruzamento com ferramenta de apoio de seleção assistida por marcador (MAS). O gene de resistência a BLB de amplo espectro, Xa7 do pai doador IRBB7, foi introgressado no MR219 suscetível (pai recorrente) usando dois marcadores flanqueadores ID7 e ID15. No BC3F4, conseguimos gerar 19 linhagens introgressadas com o gene Xa7 homozigoto e apresentamos características de resistência como genitor doador quando desafiado com Xanthomonas oryzae pv. oryzae por inoculação artificial. O pai recorrente MR219 e a variedade controle, MR263, estavam gravemente infectados pela doença. As linhas melhoradas exibiram características morfológicas e de desempenho de rendimento semelhantes às da variedade elite, MR219. Duas linhas, PB-2-107 e PB-2-34, foram escolhidas como linhas potenciais por causa de seus desempenhos excelentes em comparação com a mãe, MR219. Este estudo demonstra uma história de sucesso de aplicação de MAS no desenvolvimento de linhas de arroz melhoradas com resistência a doenças contra a doença BLB.

Action mechanism of the potential biocontrol agent Brevibacillus laterosporus SN19-1 against Xanthomonas oryzae pv. oryzae causing rice bacterial leaf blight.

The causal agent of rice bacterial leaf blight (BLB) is Xanthomonas oryzae pv. oryzae (Xoo), which causes serious damage to rice, leading to yield reduction or even crop failure. Brevibacillus laterosporus SN19-1 is a biocontrol strain obtained by long-term screening in our laboratory, which has a good antagonistic effect on a variety of plant pathogenic bacteria. In this study, we investigated the efficacy and bacterial inhibition of B. laterosporus SN19-1 against BLB to lay the theoretical foundation and research technology for the development of SN19-1 as a biopesticide of BLB. It was found that SN19-1 has the ability to fix nitrogen, detoxify organic phosphorus, and produce cellulase, protease, and siderophores, as well as IAA. In a greenhouse pot experiment, the control efficiency of SN19-1 against BLB was as high as 90.92%. Further investigation of the inhibitory mechanism of SN19-1 on Xoo found that the biofilm formation ability of Xoo was inhibited and the pathogenicity was weakened after the action of SN19-1 sterile supernatant on Xoo. The activities of enzymes related to respiration and the energy metabolism of Xoo were significantly inhibited, while the level of intracellular reactive oxygen species was greatly increased. Scanning electron microscopy observations showed folds on the surface of Xoo. A significant increase in cell membrane permeability and outer membrane permeability and a decrease in cell membrane fluidity resulted in the extravasation of intracellular substances and cell death. The results of this study highlight the role of B. laterosporus SN19-1 against the pathogen of BLB and help elucidate the underlying molecular mechanisms.

XanFur, a novel Fur protein induced by H2O2, positively regulated by the global transcriptional regulator Clp and required for the full virulence of Xanthomonas oryzae pv. oryzae in rice.

IMPORTANCE: Although Xanthomonas oryzae pv. oryzae (Xoo) has been found to be a bacterial pathogen causing bacterial leaf blight in rice for many years, the molecular mechanisms of the rice-Xoo interaction has not been fully understood. In this study, we found that XanFur of Xoo is a novel ferric uptake regulator (Fur) protein conserved among major pathogenic Xanthomonas species. XanFur is required for the virulence of Xoo in rice, and likely involved in regulating the virulence determinants of Xoo. The expression of xanfur is induced by H2O2, and positively regulated by the global transcriptional regulator Clp. Our results reveal the function and regulation of the novel virulence-related Fur protein XanFur in Xoo, providing new insights into the interaction mechanisms of rice-Xoo.

Rice bacterial leaf blight drives rhizosphere microbial assembly and function adaptation.

IMPORTANCE: Our results suggest that rhizosphere bacteria are more sensitive to bacterial leaf blight (BLB) than fungi. BLB infection decreased the diversity of the rhizosphere bacterial community but increased the complexity and size of the rhizosphere microbial community co-occurrence networks. In addition, the relative abundance of the genera Streptomyces, Chitinophaga, Sphingomonas, and Bacillus increased significantly. Finally, these findings contribute to the understanding of plant-microbiome interactions by providing critical insight into the ecological mechanisms by which rhizosphere microbes respond to phyllosphere diseases. In addition, it also lays the foundation and provides data to support the use of plant microbes to promote plant health in sustainable agriculture, providing critical insight into ecological mechanisms.

Bio-functionalized nickel-silica nanoparticles suppress bacterial leaf blight disease in rice (Oryza sativa L.).

Introduction: Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastative diseases that threatens rice plants worldwide. Biosynthesized nanoparticle (NP) composite compounds have attracted attention as environmentally safe materials that possess antibacterial activity that could be used in managing plant diseases. Methods: During this study, a nanocomposite of two important elements, nickel and silicon, was biosynthesized using extraction of saffron stigmas (Crocus sativus L.). Characterization of obtained nickel-silicon dioxide (Ni-SiO2) nanocomposite was investigated using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Transmission/Scanning electron microscopy (TEM/SEM), and energy-dispersive spectrum (EDS). Antibacterial activities of the biosynthesized Ni-SiO2 nanocomposite against Xoo were tested by measuring bacterial growth, biofilm formation, and dead Xoo cells. Results and discussions: The bacterial growth (OD600) and biofilm formation (OD570) of Xoo treated with distilled water (control) was found to be 1.21 and 1.11, respectively. Treatment with Ni-SiO2 NPs composite, respectively, reduced the growth and biofilm formation by 89.07% and 80.40% at 200 µg/ml. The impact of obtained Ni-SiO2 nanocomposite at a concentration of 200 µg/ml was assayed on infected rice plants. Treatment of rice seedlings with Ni-SiO2 NPs composite only had a plant height of 64.8 cm while seedlings treated with distilled water reached a height of 45.20 cm. Notably, Xoo-infected seedlings treated with Ni-SiO2 NPs composite had a plant height of 57.10 cm. Furthermore, Ni-SiO2 NPs composite sprayed on inoculated seedlings had a decrease in disease leaf area from 43.83% in non-treated infected seedlings to 13.06% in treated seedlings. The FTIR spectra of biosynthesized Ni-SiO2 nanocomposite using saffron stigma extract showed different bands at 3,406, 1,643, 1,103, 600, and 470 cm-1. No impurities were found in the synthesized composite. Spherically shaped NPs were observed by using TEM and SEM. EDS revealed that Ni-SiO2 nanoparticles (NPs) have 13.26% Ni, 29.62% Si, and 57.11% O. Xoo treated with 200 µg/ml of Ni-SiO2 NPs composite drastically increased the apoptosis of bacterial cells to 99.61% in comparison with 2.23% recorded for the control. Conclusions: The application of Ni-SiO2 NPs significantly improved the vitality of rice plants and reduced the severity of BLB.

Pyramiding resistance genes for bacterial leaf blight (Xanthomonas oryzae pv. Oryzae) into the popular rice variety, Pratikshya through marker assisted backcrossing.

BACKGROUND: Bacterial leaf blight (BLB) is one of the major biotic stress in rice cultivation. Management techniques, such as the development of BLB-resistant cultivars, are required to lessen the severity of the disease attack and yield losses. Pratikshya was selected in the present investigation as the recipient parent, as it is one of the popular high-yielding rice varieties of Odisha, India, which is having excellent grain as well as cooking quality. However, Pratikshya is highly susceptible to BLB which is prevalent in Eastern Indian region. METHODS AND RESULTS: Three major BLB resistance genes xa5, xa13, and Xa21 from the donor source Swarna MAS (CR Dhan 800) were attempted to introduce into Pratikshya through a marker-assisted backcross breeding program. Those markers closely linked to the target genes were employed for foreground selection in the segregating generations till BC2F3. In each backcross generation, progenies containing all three targeted resistance genes and phenotypically more similar to the recipient parent, Pratikshya were selected and backcrossed. Screening of 1,598 plants of the BC2F2 population was conducted against BLB using Xoo inoculum and 35 resistant plants similar to Pratikshya were carried forward to the next generation. In the BC2F3 generation, 31 plants were found to possess all the three resistance genes. For background selection of plants carrying resistance genes 45 polymorphic SSR markers were employed. Evaluation of the pyramided lines at BC2F4 generation exhibited that, most pyramided lines were similar to Pratikshya in terms of morphological features and yield parameters, and some lines were superior to the recurrent parent in terms of morphological features and yield parameters. CONCLUSION: The three-gene pyramided lines showed a high level of resistance to BLB infection and are anticipated to offer a significant yield advantage over the recipient parent Pratikshya. The pyramided lines can further be used for multi-location trial, so as to be released as a variety or can be used as a potential donor for BLB resistance genes.

In vitro and in vivo antimicrobial activity of the fungal metabolite toluquinol against phytopathogenic bacteria.

Introduction: Bacterial plant diseases cause tremendous economic losses worldwide. However, a few effective and sustainable control methods are currently available. To discover novel and effective management approaches, we screened marine fungi for their antibacterial activity against phytopathogenic bacteria in vitro and in vivo. Methods: We screened the culture broth of 55 fungal strains isolated from various marine sources (seawater, algae, and sediment) for their in vitro antibacterial activity using the broth microdilution method. Then, only the fungal strain (designated UL-Ce9) with higher antibacterial activity in vitro was tested in an in vivo experiment against tomato bacterial wilt. The active compounds of UL-Ce9 were extracted using ethyl acetate, purified by a series of chromatography, and the structure was elucidated by nuclear magnetic resonance spectroscopy. Pesticide formulations of toluquinol were prepared as soluble concentrates and wettable powder. The disease control efficacy of toluquinol formulations was evaluated against blight of rice and the bacterial wilt of tomato. Results and discussion: The culture broth of UL-Ce9 showed high antibacterial activity against Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas arboricola pv. pruni in vitro, and we selected UL-Ce9 for the in vivo test. The UL-Ce9 culture broth completely suppressed the bacterial wilt of tomato at a dilution of 1:5. The phylogenetic analysis identified UL-Ce9 as Penicillium griseofulvum, and the antibacterial metabolites were revealed as patulin, gentisyl alcohol, and toluquinol, all of which were associated with the biosynthetic pathway of the mycotoxin patulin. Patulin exhibited the highest antibacterial activity against 16 phytopathogenic bacteria in vitro, followed by toluquinol and gentisyl alcohol. As patulin is toxic, we selected toluquinol to investigate its potential use as a pesticide against bacterial plant diseases. Compared with the chemicals currently being applied in agriculture (streptomycin and oxytetracycline), toluquinol formulations exhibited similar and higher control efficacies against bacterial leaf blight of rice and bacterial wilt of tomato, respectively. To the best of our knowledge, this is the first report of the antibacterial activity of toluquinol against phytopathogenic bacteria. Our results suggest that toluquinol is a potential candidate for the development of novel and effective pesticides for the management of bacterial plant diseases.

First Report of Bacterial Leaf Blight caused by Enterobacter asburiae on Sorghum in Jiangsu Province, China.

Sorghum (Sorghum bicolor [L.] Moench) is a major cereal crop in China, with a planting area of more than 674666 ha in 2021. In August 2022, bacterial leaf blight symptoms were observed on sorghum plants grown in a field in Huai'an (119.30437 ºE, 33.999644 ºN), in Jiangsu Province (Fig. 1). To determine the causal agent, four symptomatic leaves from different plants were surface sterilized with 75% (v/v) ethanol for 1 min and washed three times with ddH2O. The surface-sterilized plant tissues were cut into small pieces (4 × 4 mm in size) and cultured on Nutrient Agar (NA) plates at 28ºC for 24 h. To obtain pure cultures, these colonies were transferred to fresh NA plates by using the conventional streak plate method. The purified bacterial cells were rod-shaped, from 1.14 to 1.66 µm long, and from 0.61 to 0.86 µm wide (number of observations = 31) (Fig. 2). Three isolates were used for further characterization. The Gram stain test indicated that the three isolates were Gram negative. 16S rRNA (27F/1492R primers) and gyrB (UP1/Up2r) genes were amplified and sequenced (Marchesi et al. 1998; Yamamoto and Harayama 1995). The obtained 16S rRNA (0R143361-0R143363) and gyrB sequences (0R146993-0R146995) were submitted to GenBank. The 16S rRNA sequences of the three isolated strains showed over 98% identity (1447/1462, 1438/1462 and 1443/1460 bp) to the E. asburiae reference strains ENIPBJ CG1, CAV1043 and 1808 013 (CP014993.1, CP011591.1 and AP019632.1, respectively). Similarly, the gyrB sequences of the three strains showed 98% identity (1103/1129, 1105/1129 and 1108/1129 bp) to the same E. asburiae reference strains. Four-week-old sorghum plants were used in the pathogenicity tests. A phylogenetic tree was constructed with reference strains (Hoffmann et al., 2005). The healthy leaves were inoculated with bacterial suspensions of the three bacterial isolates (OD600 = 0.6~1.0) using the leaf cutting method (Kauffman et al. 1973). For the control group, sterilized ddH2O was used. Each isolate was inoculated in three healthy plants. Inoculated plants were incubated at 28ºC and 75% humidity with alternating 12-h light and 12-h dark cycles with a photon flux density of 200 mmol/m2/s. After 10 days, bacterial leaf blight symptoms were observed in all the inoculated leaves. The inoculated leaves showed severe browning near the inoculation site (1-2 cm), and advanced yellowing from 2 to 7 cm from the inoculation site, while no symptoms were found in control group. The pathogen was recovered from the infected leaves, and its identity was confirmed by 16S rRNA/gyrB sequencing and morphological analysis, fulfilling Koch's postulates (Fig 2). To our knowledge, this is the first report of E. asburiae causing bacterial leaf blight on sorghum worldwide. This species is a well-known pathogen of humans that can cause nosocomial infections (Markovska et al. 2019; Zhu et al. 2017). Recently, E. asburiae was identified as the causal agent of bacterial blight on rice and tuber rot on radish (Wang et al. 2023; Yu et al. 2021). The emergence E. asburiae as a plant pathogen may be produced by the numerous resistant strains reported during recent years. Pantoea ananatis has been reported as a common companion pathogen of E. asburiae (Xue et al. 2021). This report will help to better understand the host promiscuity of E. asburiae and reveals a new pathogen that affects sorghum production in China. This study also serves as a basis for future studies to develop management strategies and cultivation for the disease to prevent sorghum yield loss. As far as we know, no control method for the management of this new plant pathogen was reported to date, which highlights the potential hazard of this discovery. Reference Hoffmann, H., et al. 2005. Syst. Appl. Microbiol. 28:196. Kauffman, H. E., et al. 1973. Plant Dis. Rep. 57:537. Marchesi, J. R., et al. 1998. Appl. Environ. Microbiol. 64:795. Markovska, R., et al. 2019. Infect. Dis. 51:627. Wang, R., et al. 2023. Plant Dis. in press. https://doi.org/10.1094/PDIS-11-22-2650-PDN Xue, Y., et al. 2021. Plant Dis. 105:2078. Yamamoto, S., et al. 1995. Appl. Environ. Microbiol. 61:1104. Yu, L., et al. 2021. Plant Dis. 106:310. Zhu, B., et al. 2017. J. Glob. Antimicrob. Resist. 8:104.

Bacteriophage-mediated biosynthesis of MnO2NPs and MgONPs and their role in the protection of plants from bacterial pathogens.

Introduction: Xanthomonas oryzae pv. oryzae (Xoo) is the plant pathogen of Bacterial Leaf Blight (BLB), which causes yield loss in rice. Methods: In this study, the lysate of Xoo bacteriophage X3 was used to mediate the bio-synthesis of MgO and MnO2. The physiochemical features of MgONPs and MnO2NPs were observed via Ultraviolet - Visible spectroscopy (UV-Vis), X-ray diffraction (XRD), Transmission/Scanning electron microscopy (TEM/SEM), Energy dispersive spectrum (EDS), and Fourier-transform infrared spectrum (FTIR). The impact of nanoparticles on plant growth and bacterial leaf blight disease were evaluated. Chlorophyll fluorescence was used to determine whether the nanoparticles application were toxic to the plants. Results: An absorption peak of 215 and 230 nm for MgO and MnO2, respectively, confirmed nanoparticle formation via UV-Vis. The crystalline nature of the nanoparticles was detected by the analysis of XRD. Bacteriological tests indicated that MgONPs and MnO2NPs sized 12.5 and 9.8 nm, respectively, had strong in vitro antibacterial effects on rice bacterial blight pathogen, Xoo. MnO2NPs were found to have the most significant antagonist effect on nutrient agar plates, while MgONPs had the most significant impact on bacterial growth in nutrient broth and on cellular efflux. Furthermore, no toxicity to plants was observed for MgONPs and MnO2NPs, indeed, MgONPs at 200 µg/mL significantly increased the quantum efficiency of PSII photochemistry on the model plant, Arabidopsis, in light (ΦPSII) compared to other interactions. Additionally, significant suppression of BLB was noted in rice seedlings amended with the synthesized MgONPs and MnO2NPs. MnO2NPs showed promotion of plant growth in the presence of Xoo compared to MgONPs. Conclusion: An effective alternative for the biological production of MgONPs and MnO2NPs was reported, which serves as an effective substitute to control plant bacterial disease with no phytotoxic effect.

Genetic Structure and TALome Analysis Highlight a High Level of Diversity in Burkinabe Xanthomonas Oryzae pv. oryzae Populations.

Bacterial Leaf Blight of rice (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major threat for food security in many rice growing countries including Burkina Faso, where the disease was first reported in the 1980's. In line with the intensification of rice cultivation in West-Africa, BLB incidence has been rising for the last 15 years. West-African strains of Xoo differ from their Asian counterparts as they (i) are genetically distant, (ii) belong to new races and, (iii) contain reduced repertoires of Transcription Activator Like (TAL) effector genes. In order to investigate the evolutionary dynamics of Xoo populations in Burkina Faso, 177 strains were collected from 2003 to 2018 in three regions where BLB is occurring. Multilocus VNTR Analysis (MLVA-14) targeting 10 polymorphic loci discriminated 24 haplotypes and showed that Xoo populations were structured according to their geographical localization and year of collection. Considering their major role in Xoo pathogenicity, we assessed the TAL effector repertoires of the 177 strains upon RFLP-based profiling. Surprisingly, an important diversity was revealed with up to eight different RFLP patterns. Finally, comparing neutral vs. tal effector gene diversity allowed to suggest scenarios underlying the evolutionary dynamics of Xoo populations in Burkina Faso, which is key to rationally guide the deployment of durably resistant rice varieties against BLB in the country.

Genomic, LC-MS, and FTIR Analysis of Plant Probiotic Potential of Bacillus albus for Managing Xanthomonas oryzae via Different Modes of Application in Rice (Oryza sativa L.).

Xanthomonas oryzae causes tremendous damage in rice plants (Oryza sativa L). Therefore, this study is focused on siderophore-producing Bacillus albus (CWTS 10) for managing BLB disease caused by X. oryzae. Both B. albus and its crude siderophore (methanolic and diethyl ether) extracts inhibited X. oryzae (10-12 mm). Fourier transform infrared spectroscopy (FTIR) analysis of the extracts indicated the presence of catecholate siderophore functional groups. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed the presence of antimicrobial compounds such as 2-deoxystreptamine, miserotoxin, fumitremorgin C, pipercide, pipernonaline, gingerone A, and deoxyvasicinone. Complete genome sequencing revealed the gene clusters for antibiotic, siderophore, antibacterial, antifungal, and secondary metabolite production. An in vivo study revealed that bacteria (CWTS 10) and their siderophore extracts effectively inhibited X. oryzae. The mode of application of bacterial or siderophore extracts in terms of DI and DSI percentage was as follows: soak method > inoculation method > spray method. In addition to providing enhanced antagonistic activity, there was a significant increase in root and shoot length and weight (wet and dry) of treated plants compared to control plants challenged with X. oryzae. Thus, the results clearly indicate that siderophore-producing B. albus and its siderophore extracts strongly inhibited X. oryzae. However, further field experiments are required before being formulated to protect rice crops from X. oryzae.

Bulk segregant analysis coupled with transcriptomics and metabolomics revealed key regulators of bacterial leaf blight resistance in rice.

BACKGROUND: Bacterial leaf blight (BLB) is a highly destructive disease, causing significant yield losses in rice (Oryza sativa). Genetic variation is contemplated as the most effective measure for inducing resistance in plants. The mutant line T1247 derived from R3550 (BLB susceptible) was highly resistant to BLB. Therefore, by utilizing this valuable source, we employed bulk segregant analysis (BSA) and transcriptome profiling to identify the genetic basis of BLB resistance in T1247. RESULTS: The differential subtraction method in BSA identified a quantitative trait locus (QTL) on chromosome 11 spanning a 27-27.45 Mb region with 33 genes and 4 differentially expressed genes (DEGs). Four DEGs (P < 0.01) with three putative candidate genes, OsR498G1120557200, OsR498G1120555700, and OsR498G1120563600,0.01 in the QTL region were identified with specific regulation as a response to BLB inoculation. Moreover, transcriptome profiling identified 37 resistance analogs genes displaying differential regulation. CONCLUSIONS: Our study provides a substantial addition to the available information regarding QTLs associated with BLB, and further functional verification of identified candidate genes can broaden the scope of understanding the BLB resistance mechanism in rice.