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.

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.

Resin Acid Copper Salt, an Interesting Chemical Pesticide, Controls Rice Bacterial Leaf Blight by Regulating Bacterial Biofilm, Motility, and Extracellular Enzymes.

Bacterial virulence plays an important role in infection. Antibacterial virulence factors are effective for preventing crop bacterial diseases. Resin acid copper salt as an effective inhibitor exhibited excellent anti-Xanthomonas oryzae pv. oryzae (Xoo) activity with an EC50 of 50.0 µg mL-1. Resin acid copper salt (RACS) can reduce extracellular polysaccharides' (EPS's) biosynthesis by down-regulating gumB relative expression. RACS can also effectively inhibit the bio-mass of Xoo biofilm. It can reduce the activity of Xoo extracellular amylase at a concentration of 100 µg mL-1. Meanwhile, the results of virtual computing suggested that RACS is an enzyme inhibitor. RACS displayed good curative activity with a control effect of 38.5%. Furthermore, the result of the phytotoxicity assessment revealed that RACS exhibited slight toxicity compared with the control at a concentration of 200 µg mL-1. The curative effect was increased to 45.0% using an additional antimicrobial agent like orange peel essential oil. RACS markedly inhibited bacterial pathogenicity at a concentration of 100 µg mL-1 in vivo.

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.

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.

CRISPRi in Xanthomonas demonstrates functional convergence of transcription activator-like effectors in two divergent pathogens.

Functional analysis of large gene families in plant pathogens can be cumbersome using classical insertional mutagenesis. Additionally, Cas9 toxicity has limited the application of CRISPR-Cas9 for directed mutagenesis in bacteria. Here, we successfully applied a CRISPR interference strategy to investigate the cryptic role of the transcription activator-like effector (tale) multigene family in several plant-pathogenic Xanthomonas bacterial species, owing to their contribution to pathogen virulence. Single guide RNAs (sgRNAs) designed against Xanthomonas phaseoli pv manihotis tale conserved gene sequences efficiently silenced expression of all tales, with concomitant decrease in virulence and TALE-induced host gene expression. The system is readily translatable to other Xanthomonas species infecting rice, citrus, Brassica, and cassava, silencing up to 16 tales in a given strain using a single sgRNA. Complementation with plasmid-borne designer tales lacking the sgRNA-targeted sequence restored molecular and virulence phenotypes in all pathosystems. Our results evidenced that X. campestris pv campestris CN08 tales are relevant for symptom development in cauliflower. They also show that the MeSWEET10a sugar transporter is surprisingly targeted by the nonvascular cassava pathogen X. cassavae, highlighting a new example of TALE functional convergence between phylogenetically distant Xanthomonas. Overall, this novel technology provides a platform for discovery and rapid functional understanding of highly conserved gene families.

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.

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.

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.

First report in South Korea of a bacterial leaf blight of oats caused by Pseudomonas salomonii.

In March 2020, approximately 20% of leaves in a commercial oat field growing Avena sativa (cv. Samhan) in Jeongeup, Korea (35.3859°, 126.5607°), displayed leaf blight in the seedling stage of development. The lesions observed in 2020 were of a yellow discoloration that spread from the leaf tip downward with minimal brown delineation (Fig. S1A). These symptoms differed from the haloes delineated at the edges by extensive brown necrosis caused by Pseudomonas coronofaciens (Kim 2020a; Fig. S1C). To isolate the causal agent, 3-cm-long pieces of symptomatic leaves from ten different oat plants were disinfected by submersion in 70% ethanol for 5 min followed by immersion in 1% sodium hypochlorite for 5 min and rinsing extensively with sterile distilled water. The air-dried segments were transferred intact to nutrient agar, and only one colony type (yellow-colored, wet, with a shiny convex surface) was observed. After single colony isolation, three isolates from different 2020 field-grown diseased leaves, termed 2007, 2009, and 2011, were selected at random. The isolates produced fluorescent siderophores on King's medium B and triggered a hypersensitive response (HR) when infiltrated into tobacco (cv. Xanthi) leaves (Fig. S1D). Multilocus sequence typing analysis with four housekeeping genes was used for taxonomic identification of these isolates (Maiden et al. 1998). The 16S rRNA sequences were amplified with the 27F/1492R universal primers (Weisburg et al. 1991). The primers for three housekeeping genes were designed using genome sequence of P. coronaficiens X-1 causing halo blight disease in Korea (NZ_CP050260.1, Kim, 2020b). The products obtained had sizes of 640 bp for gltA (using primers F: 5'-CCT GGT AGC CAA GAT GCC GAC-3'; R: 5'-CAA AGA TCA CGG TGA ACA TGC TGG-3'), 710 bp for gyrB (with primers F: 5'-TCG GCA GCC GAG GTC ATC ATG AC-3'; R: 5'-TTG TCT TTG GTC TGC GAG CTG AA-3'), and 870 bp for rpoD ( with primers F: 5'-GTG AAG GCG AAA TCG AAA TCG-3'; R: 5'-CCG ATG TTG CCT TCC TGG ATC AG-3'). The concatenated sequences (2,353 bp/2,376 bp) had 99% identity with the gene sequences from P. salomonii type strain (AY091528.1, NZ_FNOX01000003, LC486864.1, LC486849.1), but lesser identity (90%) with P. coronafaciens (Fig. S2). Pathogenicity of the Korean isolates was confirmed by fulfilling Koch's postulates using leaves of 2 week-old greenhouse-grown 'Samhan' seedlings. Plants (n=50) were sprayed with 108 cfu/ml bacterial suspensions in water or with sterile water as controls. The plants were incubated for a week at 23 °C in 100% relative humidity under a 10 h light/14 h dark photoperiod. Five days after bacterial inoculation, yellow discoloration appeared at the leaf tips which progressed downward with time (Fig. S1B). Three bacterial isolates extracted from yellowed, inoculated leaves had 16S rRNA gene sequences identical to that of P. salomonii Korean isolates, 2007, 2009 and 2011, and they caused the anticipated symptoms when inoculated into oat leaves. These findings indicate that P. salomonii should be added to the potential pathogens of oats grown in Korea. Understanding whether spring weather conditions (warmth and humidity) boost this oat disease will help devise disease alert systems for farmers (Anderson 2004; Chakraborty 2005).

Diverse Virulence Attributes of Pantoea alfalfae sp. nov. CQ10 Responsible for Bacterial Leaf Blight in Alfalfa Revealed by Genomic Analysis.

Alfalfa is widely grown worldwide for its excellent nutritional value. Pantoea species living in alfalfa seeds can easily spread over great distances with frequent trade. However, the pathogenic properties of this dangerous hitchhiker on alfalfa have not been evaluated. Here, we identified the taxonomic status of Pantoea strain CQ10 isolated from the interior of alfalfa seeds based on the whole genome sequence. The diverse virulence attributes of strain CQ10 during host infection were characterized through pathogenicity assays and functional and genomic analyses. We report that strain CQ10 belongs to a novel species in the genus Pantoea, which was phylogenetically close to Pantoea vagans and Pantoea agglomerans. Strain CQ10 caused bacterial leaf blight of alfalfa after inoculation from the roots. We found that strain CQ10 possesses a large number of pathogenic genes involved in shaping the virulence properties during bacteria-host interactions, including motility, biofilm, type VI secretion system, and nutrient acquisition. Compared with P. vagans and P. agglomerans, the unique virulence factors of strain CQ10 were mainly involved in motility and biofilm, which were confirmed by in vitro experiments. Taken together, our results suggest that strain CQ10 is the first Pantoea species to infect alfalfa, and it possesses diverse virulence attributes among which motility and biofilm may be the best weapons.

The OsWRKY6 transcriptional cascade functions in basal defense and Xa1-mediated defense of rice against Xanthomonas oryzae pv. oryzae.

MAIN CONCLUSION: The rice protein OsWRKY6 directly activates OsWRKY45 and OsWRKY47 expression, and also activates OsPR1a and OsPR1b through the two OsWRKYs, and this transcriptional module participates in Xa1-mediated defense against the pathogen Xanthomonas oryzae pv. oryzae. Biotic stress, the pathogen Xanthomonas oryzae pv. oryzae (Xoo) in particular, negatively impacts worldwide productivity and yield in the staple crop rice (Oryza sativa). OsWRKY transcription factors are involved in various biotic stress responses in rice, and OsWRKY6 specifically acts as an important defense regulator against Xoo. However, the relationship between OsWRKY6 and other OsWRKYs, as well as its role in resistance (R) gene-mediated defense, have yet to be studied in depth. Here, we characterized a transcriptional cascade triggered by OsWRKY6 that regulated defense against Xoo infection mediated by the NBS-LRR protein Xa1. OsWRKY45 and OsWRKY47 were identified as direct transcriptional targets of OsWRKY6, and their two gene products reciprocally activated their two genes. Furthermore, OsWRKY6 activated OsPR1a and OsPR1b via the OsWRKY45 and OsWRKY47. Two OsWRKY6 RNAi knockdown lines showed significantly reduced defense even against an incompatible Xoo infection, and the expression of OsWRKY6 was not regulated by OsWRKY51 and OsWRKY88. This study reveals that a novel downstream transcriptional pathway activated by OsWRKY6 is involved in Xa1-mediated defense against Xoo.

Osa-miR160a confers broad-spectrum resistance to fungal and bacterial pathogens in rice.

Rice production is threatened by multiple pathogens. Breeding cultivars with broad-spectrum disease resistance is necessary to maintain and improve crop production. Previously we found that overexpression of miR160a enhanced rice blast disease resistance. However, it is unclear whether miR160a also regulates resistance against other pathogens, and what the downstream signaling pathways are. Here, we demonstrate that miR160a positively regulates broad-spectrum resistance against the causative agents of blast, leaf blight and sheath blight in rice. Mutations of miR160a-targeted Auxin Response Factors result in different alteration of resistance conferred by miR160a. miR160a enhances disease resistance partially by suppressing ARF8, as mutation of ARF8 in MIM160 background partially restores the compromised resistance resulting from MIM160. ARF8 protein binds directly to the promoter and suppresses the expression of WRKY45, which acts as a positive regulator of rice immunity. Mutation of WRKY45 compromises the enhanced blast resistance and bacterial leaf blight resistance conferred by arf8 mutant. Overall, our results reveal that a microRNA coordinates rice broad-spectrum disease resistance by suppressing multiple target genes that play different roles in disease resistance, and uncover a new regulatory pathway mediated by the miR160a-ARF8 module. These findings provide new resources to potentially improve disease resistance for breeding in rice.

Photodynamic antibacterial and antibiofilm activity of riboflavin against Xanthomonas oryzae pv oryzae: an ecofriendly strategy to combat bacterial leaf blight (BLB) rice disease.

Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv oryzae (Xoo), is one of the most damaging rice diseases, causing severe production losses depending on the rice variety. The purpose of this study was to develop an antibacterial photodynamic treatment (aPDT) using riboflavin for the treatment of BLB disease. Combining light and riboflavin (RF) therapy significantly reduced bacterial planktonic cells compared to RF alone. Photoactivated riboflavin also decreased biofilm biomass by reducing the number of viable sessile cells and the production of extracellular polymeric substances (EPS). Reactive oxygen species (ROS) levels in Xoo cells treated with photoactivated riboflavin were found to be significantly higher than in cells treated with riboflavin and light individually. Malondialdehyde (MDA) increased greatly in photoactivated riboflavin treated cells, indicating that severe oxidative damage was induced. Subsequently, a reduction in lactate dehydrogenase (LDH) activity in photoactivated riboflavin treated Xoo cells indicates that oxidative stress has disrupted the respiratory system, leading to bacterial cell death. In an ex vivo aPDT assay, photoactivated riboflavin successfully eradicated Xoo on the surface of rice leaves. Photoactivated riboflavin had no side effects on rice seed germination in subsequent trials, indicating that it is safe for agricultural applications. Therefore, all these findings suggest that aPDT is a potential alternative management strategy for BLB disease.

Three semi-selective media for Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis.

Three semi-selective media, DTarTA, SPbc, and SPamt, were developed and tested to isolate Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca) from Raphanus sativus seeds. DTarTA contained D-tartaric acid as a carbon source and potassium tellurite, ampicillin sodium, and methyl violet as antibiotics. DTarTA suppressed growth in 19 of the 24 pathovars from the P. syringae complex, whereas Psm and Pca grew and formed gray to black colonies. SPamt contained sucrose and peptone as nutrient sources and was supplemented with bromothymol blue and the same antibiotics present in DTarTA and Psm and Pca formed yellowish to dark brown colonies on the SPamt medium. SPbc contained sucrose and peptone and was supplemented with cephalexin and boric acid as antibiotics and Psm and Pca formed semi-translucent to white colonies on the SPbc medium. SPamt and SPbc suppressed the growth of several plant-associated bacteria (except the P. syringae complex). The growth of saprophytic bacteria in seeds on the different media was compared with that on King's B medium, using five types of commercially available Raphanus sativus seeds. The suppression rate of DTarTA was 85-99% and was lower for seeds with more saprophytic bacteria. The suppression rates of SPamt and SPbc were 90-99%. In detection tests using 10,000 seed samples mixed with Pca or Psm-contaminated seeds, it was possible to selectively isolate Psm and Pca using SPamt and SPbc, even when the colony numbers of the target bacterium constituted less than 10% of the total colonies. KEY POINTS: • Bacterial leaf spot and blight pathogens were selectively isolated from seeds. • DTarTA medium distinguishes these pathogens from P. syringae complex pathovars. • SPamp and SPbc media have different selectivity for plant-associated bacteria.

Primers for specific detection and identification of Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis.

Bacterial leaf spot and bacterial leaf blight are global threats to the cultivation of cruciferous vegetables, and it is necessary to develop methods to easily detect, identify, and distinguish the causative pathogens Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca). Here, we used the sequence specificity of the exchangeable effector loci flanking the hrp gene cluster to design primers that can help detect and discriminate between Psm and Pca. Primers common to both bacteria (hrpK_fw1 and hrpK_fw2) were designed within hrpK at the end of the hrp gene cluster. Psm-specific primers (MAC_rv1 and MAC_rv2) were designed in hopPtoB1 and Pca-specific primers (ALS_rv1 and ALS_rv2) were designed in hopX1 adjacent to hrpK. PCR using hrpK_fw1 and MAC_rv1 or hrpK_fw2 and MAC_rv2 amplified DNA fragments of only Psm, P. syringae pv. tomato (causal agent of tomato bacterial speck), and P. syringae pv. spinaciae (causal agent of spinach bacterial leaf spot), among 76 strains of phytopathogenic bacteria. PCR using hrpK_fw1 and ALS_rv1 or hrpK_2 and ALS_rv2 amplified DNA fragments of only Pca. Multiplex PCR with these primers could easily distinguish Psm and Pca from bacterial colonies isolated on growth media and detect the pathogen in symptomatic leaves. Multiplex nested PCR with the primers detected contamination in one Psm- and/or one Pca-infected seeds in 1000 seeds. These results suggest that these PCR primers could help detect and discriminate Psm and Pca. KEY POINTS: • We investigated Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis. • Novel primers common to both bacteria were designed following genome comparison. • Multiplex PCR with new primers could discriminate Psm and Pca.

Temperature differentially modulates the transcriptome response in Oryza sativa to Xanthomonas oryzae pv. oryzae infection.

Bacterial blight is caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo). Genome scale integrative analysis on the interaction of high and low temperatures on the molecular response signature in rice during the Xoo infection has not been conducted yet. We have analysed a unique RNA-Seq dataset generated on the susceptible rice variety IR24 under combined exposure of Xoo with low 29/21 °C (day/night) and high 35/31 °C (day/night) temperatures. Differentially regulated key genes and pathways in rice plants during both the stress conditions were identified. Differential dynamics of the regulatory network topology showed that WRKY and ERF families of transcription factors play a crucial role during signal crosstalk events in rice plants while responding to combined exposure of Xoo with low temperature vs. Xoo with high temperatures. Our study suggests that upon onset of high temperature, rice plants tend to switch its focus from defence response towards growth and reproduction.

Biogenic silver nanoparticles as an antibacterial agent against bacterial leaf blight causing rice phytopathogen Xanthomonas oryzae pv. oryzae.

Silver nanoparticles (Ag NP) were produced utilizing leaf extract of rice cultivar Taichung native-1. Various factors like leaf extract, silver nitrate concentrations, and duration of autoclaving were standardized during synthesis. Nanoparticles were analyzed with UV-visible absorption spectroscopy (UV-vis), dynamic light scattering, zeta potential, X-ray diffraction and transmission electron microscopy techniques. The synthesis was noted at 0.4% extract, 0.6 mM silver nitrate, 30 min of autoclaving and NP formation was confirmed from 424 nm peak in UV-vis. NP showed zeta potential of - 27 mV, face-centered cubic (fcc) crystal nature and sized around 16.5 ± 5.9 nm. Biogenic NP synthesized from susceptible rice variety were used as an antibacterial agent against phytopathogen Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight (BLB) disease in rice. Antibacterial effect of Ag NP was evaluated using in vitro assays and in vivo efficacy under greenhouse conditions. Results confirmed effective inhibition of Xoo growth and colony formation by Ag NP and found to be the more powerful antibacterial agent. Besides, Ag NP treatment (10 µg/mL) caused an enhancement in seedling vigor index. Pots treated with Ag NP (15 µg/mL) in vivo in greenhouse showed disease severity of 26.6% and disease decrease over control of 49.2%, at a much lower NP concentration than earlier reported studies. Thus, the current report implies using the leaf extract synthesized Ag NP to control and BLB disease management in field conditions.

Flavonone 3-hydroxylase Relieves Bacterial Leaf Blight Stress in Rice via Overaccumulation of Antioxidant Flavonoids and Induction of Defense Genes and Hormones.

Efficient accumulation of flavonoids is important for increased tolerance to biotic stress. Although several plant defense mechanisms are known, the roles of many pathways, proteins, and secondary metabolites in stress tolerance are unknown. We generated a flavanone 3-hydroxylase (F3H) overexpressor rice line and inoculated Xanthomonas Oryzae pv. oryzae and compared the control and wildtype inoculated plants. In addition to promoting plant growth and developmental maintenance, the overexpression of F3H increased the accumulation of flavonoids and increased tolerance to bacterial leaf blight (BLB) stress. Moreover, leaf lesion length was higher in the infected wildtype plants compared with infected transgenics. Kaempferol and quercetin, which scavenge reactive oxygen species, overaccumulated in transgenic lines compared with wildtypes in response to pathogenic infection, detected by scanning electron microscopy and spectrophotometry. The induction of F3H altered the antioxidant system and reduced the levels of glutathione peroxidase activity and malondialdehyde (MDA) contents in the transgenic lines compared with the wildtypes. Downstream gene regulation analysis showed that the expression of F3H increased the regulation of flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and slender rice mutant (SLR1) during BLB stress. The analysis of SA and JA signaling revealed an antagonistic interaction between both hormones and that F3H induction significantly promoted SA and inhibited JA accumulation in the transgenic lines. SA-dependent nonexpressor pathogenesis-related (NPR1) and Xa1 showed significant upregulation in the infected transgenic lines compared with the infected control and wildtype lines. Thus, the overexpression of F3H was essential for increasing BLB stress tolerance.

Identification of a Chitooligosaccharide Mechanism against Bacterial Leaf Blight on Rice by In Vitro and In Silico Studies.

This study focuses on a commercial plant elicitor based on chitooligosaccharides (BIG®), which aids in rice plant growth and disease resistance to bacterial leaf blight (BLB). When the pathogen (Xoo) vigorously attacks rice that has suffered yield losses, it can cause damage in up to 20% of the plant. Furthermore, Xoo is a seed-borne pathogen that can survive in rice seeds for an extended period. In this study, when rice seeds were soaked and sprayed with BIG®, there was a significant increase in shoot and root length, as well as plant biomass. Furthermore, BIG®-treated rice plants showed a significant reduction in BLB severity of more than 33%. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) analysis was used to characterize BIG®'s mechanism in the chemical structure of rice leaves. The SR-FTIR results at 1650, 1735, and 1114 cm-1 indicated changes in biochemical components such as pectins, lignins, proteins, and celluloses. These findings demonstrated that commercial BIG® not only increased rice growth but also induced resistance to BLB. The drug's target enzyme, Xoo 1075 from Xanthomonas oryzae (PDB ID: 5CY8), was analyzed for its interactions with polymer ingredients, specifically chitooligosaccharides, to gain molecular insights down to the atomic level. The results are intriguing, with a strong binding of the chitooligosaccharide polymer with the drug target, revealing 10 hydrogen bonds between the protein and polymer. Overall, the computational analysis supported the experimentally demonstrated strong binding of chitooligosaccharides to the drug target.