A novel Pik allele confers extended resistance to rice blast.

In the ongoing arms race between rice and Magnaporthe oryzae, the pathogen employs effectors to evade the immune response, while the host develops resistance genes to recognise these effectors and confer resistance. In this study, we identified a novel Pik allele, Pik-W25, from wild rice WR25 through bulked-segregant analysis, creating the Pik-W25 NIL (Near-isogenic Lines) named G9. Pik-W25 conferred resistance to isolates expressing AvrPik-C/D/E alleles. CRISPR-Cas9 editing was used to generate transgenic lines with a loss of function in Pik-W25-1 and Pik-W25-2, resulting in loss of resistance in G9 to isolates expressing the three alleles, confirming that Pik-W25-induced immunity required both Pik-W25-1 and Pik-W25-2. Yeast two-hybrid (Y2H) and split luciferase complementation assays showed interactions between Pik-W25-1 and the three alleles, while Pik-W25-2 could not interact with AvrPik-C, -D, and -E alleles with Y2H assay, indicating Pik-W25-1 acts as an adaptor and Pik-W25-2 transduces the signal to trigger resistance. The Pik-W25 NIL exhibited enhanced field resistance to leaf and panicle blast without significant changes in morphology or development compared to the parent variety CO39, suggesting its potential for resistance breeding. These findings advance our knowledge of rice blast resistance mechanisms and offer valuable resources for effective and sustainable control strategies.

A rapid, equipment-free method for detecting avirulence genes of Pyricularia oryzae using a lateral flow strip-based RPA assay.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive rice diseases worldwide. Using resistant rice varieties is the most cost-effective way to control rice blast. Consequently, it is critical to monitor the distribution frequency of avirulence genes in rice planting field to facilitate the breedings of resistant rice varieties. In this study, we established a rapid RPA-LFD detection system for the identification of AvrPik, Avr-Piz-t and Avr-Pi9. The optimized reaction temperature and duration were 37°C and 20 min, indicating that the reaction system could be initiated by body temperature without relying on any precision instruments. Specificity analysis showed that the primer and probe combinations targeting three Avr genes exhibited a remarkable specificity for at genus-level detection. Under the optimized condition, the lower detected thresholds of AvrPik, Avr-Piz-t and Avr-Pi9 were 10 fg/µl, 100 fg/µl and 10 pg/µl, respectively. Notably, the detection sensitivity of three Avr genes was much higher than that of PCR. In addition, we also successfully detected the presence of AvrPik, Avr-Piz-t and Avr-Pi9 in the leaf and panicle blast lesions with the RPA-LFD detection system. In particular, the genomic DNA was extracted using the simpler PEG-NaOH rapid extraction method. In summary, we developed the RPA detection system for AvrPik, Avr-Pi9 and Avr-Piz-t, combined with the PEG-NaOH rapid DNA extraction method. The innovative approach achieved rapid, real-time and accurate detection of three Avr genes in the field, which is helpful to understand the distribution frequency of the three Avr genes in the field and provide theoretical reference for the scientific layout of rice resistant varieties.

Autophagy-related protein UvAtg7 contributes to mycelial growth, virulence, asexual reproduction and cell stress response in rice false smut fungus Ustilaginoidea virens.

Autophagy is a conserved mechanism for nutrient and cytoplasmic components recycling in eukaryotic cell, in which E1-like enzyme Atg7 activates ubiquitin-like conjugation in the autophagy pathway. In plant pathogenic fungi Ustilaginoidea virens, UvAtg7, an ortholog of AAtg7 in baker's yeast was identified and functionally investigated. UvAtg7 was confirmed to be essential for autophagy, because the disruption of UvATG7 gene in U. virens completely blocked the fusion of autophagosome-like into vacuoles and catalytic degradation of GFP-UvAtg8 under N-starving condition. The fluorescent signal indicated UvAtg7 protein was dispersed in cytoplasma, but spatially coordinated with core autophagy protein UvAtg8 on occasion. Interestingly, disruption of UvATG7 in U. virens caused slightly reduction in mycelial growth, but resulted in a considerable decrease in virulence, conidia production in YT broth and chlamydospore formation on rice false smut balls. Moreover, the UvATG7 deletion mutants exhibited increased sensitivity to cell wall integrity stress caused by congo red and calcofluor white, meanwhile the UvATG7 deletion mutants showed decreased sensitivity to osmotic stress, cell membrane stress and reactiveoxygen stress caused by sorbitol, sodium dodecyl sulfate and H2O2, respectively. All of these defects in UvATG7 deletion mutants could be partially or completely restored by gene complementation. In general, our study indicates that UvAtg7 is essential in autophagy pathway and contributes to mycelial growth, virulence, asexual reproduction and cell stress response in U. virens.

Loop-Mediated Isothermal Amplification for Rapid Detection of Mating Types of Villosiclava virens.

Rice false smut (RFS), caused by Villosiclava virens, is an important fungal disease in panicles of rice. V. virens is a heterothallic ascomycete controlled by two opposite idiomorphs, MAT1-1 and MAT1-2. Previous study showed that sexual reproduction of V. virens plays an important role in the epidemic of RFS. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay to detect the mating type of V. virens easily and rapidly by using specific primers based on the mating type genes MAT1-1-2 and MAT1-2-1, respectively. The LAMP assay used only a water/dry bath and could recognize the mating type of V. virens in just 45 min. The LAMP assay was so sensitive that it could detect small amounts of V. virens genomic DNA (as low as 2.0 pg of MAT1-1 and 200.0 pg of MAT1-2) and was 10 times more sensitive than PCR. In addition, we demonstrated the application of mating type via LAMP assay by assessing the genomic DNA of V. virens isolated from rice fields. The high efficiency and specificity of this LAMP assay suggest that it can be used as a rapid testing tool in mating type recognition of V. virens isolates in the field.

Genome-Wide Prediction and Analysis of Oryza Species NRP Genes in Rice Blast Resistance.

Members of the N-rich proteins (NRPs) gene family play important roles in the plant endoplasmic reticulum stress in response, which can be triggered by plant pathogens' infection. Previous studies of the NRP gene family have been limited to only a few plants, such as soybean and Arabidopsis thaliana. Thus, their evolutionary characteristics in the Oryza species and biological functions in rice defense against the pathogenic fungus Magnaporthe oryzae have remained unexplored. In the present study, we demonstrated that the NRP genes family may have originated in the early stages of plant evolution, and that they have been strongly conserved during the evolution of the Oryza species. Domain organization of NRPs was found to be highly conserved within but not between subgroups. OsNRP1, an NRP gene in the Oryza sativa japonica group, was specifically up-regulated during the early stages of rice-M. oryzae interactions-inhibited M. oryzae infection. Predicted protein-protein interaction networks and transcription-factor binding sites revealed a candidate interactor, bZIP50, which may be involved in OsNRP1-mediated rice resistance against M. oryzae infection. Taken together, our results established a basis for future studies of the NRP gene family and provided molecular insights into rice immune responses to M. oryzae.

Pyricularia sp. jiangsuensis, a new cryptic rice panicle blast pathogen from rice fields in Jiangsu Province, China.

Pyricularia oryzae is a multi-host pathogen causing cereal disease, including the devastating rice blast. Panicle blast is a serious stage, leading to severe yield loss. Thirty-one isolates (average 4.1%) were collected from the rice panicle lesions at nine locations covering Jiangsu province from 2010 to 2017. These isolates were characterized as Pyricularia sp. jiangsuensis distinct from known Pyricularia species. The representative strain 18-2 can infect rice panicle, root and five kinds of grasses. Intriguingly, strain 18-2 can co-infect rice leaf with P. oryzae Guy11. The whole genome of P. sp. jiangsuensis 18-2 was sequenced. Nine effectors were distributed in translocation or inversion region, which may link to the rapid evolution of effectors. Twenty-one homologues of known blast-effectors were identified in strain 18-2, seven effectors including the homologues of SLP1, BAS2, BAS113, CDIP2/3, MoHEG16 and Avr-Pi54, were upregulated in the sample of inoculated panicle with strain 18-2 at 24 hpi compared with inoculation at 8 hpi. Our results provide evidences that P. sp. jiangsuensis represents an addition to the mycobiota of blast disease. This study advances our understanding of the pathogenicity of P. sp. jiangsuensis to hosts, which sheds new light on the adaptability in the co-evolution of pathogen and host.

Two mating-type genes MAT1-1-1 and MAT1-1-2 with significant functions in conidiation, stress response, sexual development, and pathogenicity of rice false smut fungus Villosiclava virens.

Rice false smut caused by Villosiclava virens is one of the destructive diseases on panicles of rice. Sexual development of V. virens, controlled by mating-type locus, plays an important role in the prevalence of rice false smut and genetic diversity of the pathogen. However, how the mating-type genes mediate sexual development of the V. virens remains largely unknown. In this study, we characterized the two mating-type genes, MAT1-1-1 and MAT1-1-2, in V. virens. MAT1-1-1 knockout mutant showed defects in hyphal growth, conidia morphogenesis, sexual development, and increase in the tolerance to salt and osmotic stress. Targeted deletion of MAT1-1-2 not only impaired the sclerotia formation and pathogenicity of V. virens, but also reduced the production of conidia. The MAT1-1-2 mutant showed increases in tolerance to salt and hydrogen peroxide stress, but decreases in tolerance to osmotic stress. Yeast two-hybrid assay showed that MAT1-1-1 interacted with MAT1-1-2, indicating that those proteins might form a complex to regulate sexual development. In addition, MAT1-1-1 localized in the nucleus, and MAT1-1-2 localized in the cytoplasm. Collectively, our results demonstrate that MAT1-1-1 and MAT1-1-2 play important roles in the conidiation, stress response, sexual development, and pathogenicity of V. virens, thus providing new insights into the function of mating-type gene.

A Defect in the Twin-Arginine Translocation Pathway Decreases the Tolerance of Xanthomonas campestris pv. campestris to Phenazines.

Phenazine-1-carboxylic acid (PCA), a member of phenazines secreted by microorganisms, inhibits the growth of many bacteria and fungi. Xanthomonas campestris pv. campestris is the causal agent of black rot, the most important disease of cruciferous crops worldwide, and is more tolerant to PCA than other Xanthomonas species. Previous studies reported that reactive oxygen species (ROS) scavenging ability is involved in regulating the PCA tolerance of Xanthomonas species. Additionally, the cytochrome c maturation (CCM) system has been found to play a more important role in tolerance to phenazines than the ROS scavenging system. In this study, a highly PCA-sensitive insertion mutant of X. campestris pv. campestris, X-5, was identified and studied. The insertion site of X-5 was found to be in tatB gene (XC_4183), which encodes a subunit of the twin-arginine translocation (TAT) complex. Disruption of the three genes of TAT pathway resulted in decreased biological fitness and reduced tolerance to phenazines in comparison with the wild-type strain 8004. These results imply that the tolerance mechanism of the TAT pathway to phenazines is related to the CCM system, but not due to the ROS scavenging system. Furthermore, respiration-related characteristic tests and peptide analysis suggested that disruption of the TAT complex causes a defect in the cytochrome bc1 complex, which may be involved in the tolerance to phenazines. In summary, this study sheds new light on the critical role of the TAT pathway in influencing the fitness and phenazines tolerance of Xanthomonas species.

Effects of validamycin in controlling Fusarium head blight caused by Fusarium graminearum: Inhibition of DON biosynthesis and induction of host resistance.

Validamycin, known to interfere with fungal energy metabolism by inhibiting trehalase, has been extensively used to control plant diseases caused by Rhizoctonia spp. However, the effect of validamycin on controlling Fusarium graminearum has not been previously reported. In this study, when applied to F. graminearum in vitro, validamycin inhibited the synthesis of deoxynivalenol (DON), which is a mycotoxin and virulence factor, by decreasing trehalase activity and the production of glucose and pyruvate, which are precursors of DON biosynthesis. Because FgNTH encodes the main trehalase in F. graminearum, these effects were nullified in the FgNTH deletion mutant ΔFgNTH but restored in the complemented strain ΔFgNTHC. In addition, validamycin also increased the expression of pathogenesis-related genes (PRs) PR1, PR2, and PR5 in wheat, inducing resistance responses of wheat against F. graminearum. Therefore, validamycin exhibits dual efficacies on controlling Fusarium head blight (FHB) caused by F. graminearum: inhibition of DON biosynthesis and induction of host resistance. In addition, field trials further confirmed that validamycin increased FHB control and reduced DON contamination in grain. Control of FHB and DON contamination by validamycin increased when the antibiotic was applied with the triazole fungicide metconazole. Overall, this study is a successful case from foundational research to applied research, providing useful information for wheat protection programs against toxigenic fungi responsible for FHB and the consequent mycotoxin accumulation in grains.

The critical role of cytochrome c maturation (CCM) system in the tolerance of Xanthomonas campestris pv. campestris to phenazines.

Phenazine-1-carboxylic acid (PCA), a secondary metabolite produced by Pseudomonas spp., exhibits a high inhibitory effect in Xanthomonas oryzae pv. oryzae (Xoo), but less inhibitory effect in Xanthomonas oryzae pv. oryzicola (Xoc), and almost no inhibitory effect in Xanthomonas campestris pv. campestris (Xcc). In our previous study, reactive oxygen species (ROS) scavenging system was reported to be involved in PCA tolerance in Xanthomonas spp. However, the PCA tolerance mechanism of Xanthomonas spp. is unclear. In the current study, we constructed a Tn5-based transposon mutant library in Xcc and four highly PCA-sensitive insertion mutants were obtained. TAIL-PCR further confirmed that the Tn5 transposon was inserted in the cytochrome c maturation (CCM) system (XC_1893, XC_1897) of these mutants. Disruption of the CCM system significantly decreased the growth, motility and tolerance of Xcc to PCA and other phenazines, such as phenazine and 1-OH-phenazine. The CCM system is responsible for the covalent attachment of the apocytochrome and heme. Disruption of the transmembrane thioredox protein (Dsb) pathway (XC_0531), an essential process for the formation of mature apocytochrome, also exhibited a decreased tolerance to PCA, suggesting that the defect of cytochrome c caused decreased tolerance of Xcc to PCA. Meanwhile, disruption of the CCM system or Dsb pathway interfered with the functions of cytochrome c proteins, causing an increased sensitivity to H2O2. Collectively, we concluded that the CCM system and Dsb pathway, regulate the tolerance of Xcc to phenazines by influencing the functions of cytochrome c. Therefore, these results provide important references for revealing the action mechanism of PCA in Xanthomonas spp.

Characterization of propiconazole field-resistant isolates of Ustilaginoidea virens.

The plant-pathogenic fungus Ustilaginoidea virens (Cooke) Takah causes rice false smut (RFS), which is responsible for significant quantitative and qualitative losses in rice industry. Propiconazole is a triazole fungicide which belongs to Demethylation inhibitors (DMIs). It is used to control RFS in China. We previously screened 158 isolates of U. virens collected in the fields in 2015 in Jiangsu province of China, and found two of them were highly resistant to propiconazole (named 82 and 88, respectively). In this study, we have analyzed the physiological and biochemical characters of six field-sensitive isolates and the two field-resistant isolates, including mycelial growth and cell wall integrity. We found there was cross-resistance between different DMIs fungicides, but was no cross-resistance between DMIs and QoIs fungicides. We also analyzed the fitness, and found the pathogenicity in 88 was stronger than the field-sensitive isolates, but was completely lost in 82. Sequence analyses of CYP51 and the 1000-bp upstream of CYP51 coding region showed no mutation in 82 compared to the field-sensitive strains, but two more bases CC were identified at 154-bp upstream of the coding region in the field-resistant isolate 88. Moreover, the expression of CYP51 gene in all tested isolates was significantly induced by propiconazole. However, the up-regulation expression level in both 82 and 88 was much higher than that in the field-sensitive isolates. We also found propiconazole could inhibit the ergosterol biosynthesis in the field-sensitive isolates, but stimulated it in both field-resistant isolates 82 and 88. Given the high level of U. virens developing propiconazole resistance and the good fitness of the field-resistant isolate 88, the resistance of U. virens to DMIs must be monitored and managed in rice fields.

Contribution of OxyR Towards Differential Sensitivity to Antioxidants in Xanthomonas oryzae pathovars oryzae and oryzicola.

OxyR and SoxR are two transcriptional regulators in response to oxidative stress in most bacteria, and SoxR has been reported to be activated by the endogenous redox-cycling compound phenazine in phenazine-producing organisms. However, which transcriptional regulator is activated in pathogens treated with the antibiotic phenazine-1-carboxylic acid (PCA) has not been determined. In this study, we found that PCA treatment activated OxyR rather than SoxR in the phytopathogenic bacteria Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola. We also found that X. oryzae pv. oryzae was much more sensitive to PCA and H2O2 and had a defective antioxidant system (i.e., less of total antioxidant capacity and total catalase activity than X. oryzae pv. oryzicola, although X. oryzae pvs. oryzae and oryzicola are very closely related). Based on KEGG sequences, OxyR differs in 10 amino acids in X. oryzae pv. oryzae versus X. oryzae pv. oryzicola. By exchanging OxyR between X. oryzae pvs. oryzae and oryzicola, we elucidated that OxyR contributed to the differences in antioxidant capacity, total catalase activity, and sensitivity to PCA and H2O2. We also found that OxyR affected X. oryzae pvs. oryzae and oryzicola growth in a nutrient-poor medium, virulence on host plants (rice), and the hypersensitive response on nonhost plants (Nicotiana benthamiana). Thus, OxyR is a critical regulator that relates to the differences in antioxidative stress between X. oryzae pvs. oryzae and oryzicola and contributes to the differences in survival of them against oxidative stress.

Ankyrin-Like Protein AnkB Interacts with CatB, Affects Catalase Activity, and Enhances Resistance of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola to Phenazine-1-Carboxylic Acid.

Xanthomonas oryzae pv. oryzae, which causes rice bacterial leaf blight, and Xanthomonas oryzae pv. oryzicola, which causes rice bacterial leaf streak, are important plant-pathogenic bacteria. A member of the adaptor protein family, ankyrin protein, has been investigated largely in humans but rarely in plant-pathogenic bacteria. In this study, a novel ankyrin-like protein, AnkB, was identified in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. The expression of ankB was significantly upregulated when these bacteria were treated with phenazine-1-carboxylic acid (PCA). ankB is located 58 bp downstream of the gene catB (which encodes a catalase) in both bacteria, and the gene expression of catB and catalase activity were reduced following ankB deletion in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Furthermore, we demonstrated that AnkB directly interacts with CatB by glutathione S-transferase (GST) pulldown assays. Deletion of ankB increased the sensitivity of X. oryzae pv. oryzae and X. oryzae pv. oryzicola to H2O2 and PCA, decreased bacterial biofilm formation, swimming ability, and exopolysaccharide (EPS) production, and also reduced virulence on rice. Together our results indicate that the ankyrin-like protein AnkB has important and conserved roles in antioxidant systems and pathogenicity in X. oryzae pv. oryzae and X. oryzae pv. oryzicola.IMPORTANCE This study demonstrates that the ankyrin protein AnkB directly interacts with catalase CatB in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola. Ankyrin protein AnkB can affect the gene expression of catB, catalase activity, and sensitivity to H2O2 In Xanthomonas spp., the locations of genes ankB and catB and the amino acid sequence of AnkB are highly conserved. It is suggested that in prokaryotes, AnkB plays a conserved role in the defense against oxidative stress.

Screening and characterization of Xanthomonas oryzae pv. oryzae strains with resistance to pheazine-1-carboxylic acid.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) and can be effectively controlled by phenazine-1-carboxylic acid (PCA), an antibiotic secreted by Pseudomonas spp. PCA resistance in Xoo was investigated in this research. Only four PCA-resistant strains were obtained by extensive screening, and the resistance was genetically stable in only one of them (P4). P4 was also resistant to phenazine and 1-hydroxyphezine but not to captan, bismerthiazol, or streptomycin. The following were reduced in P4 relative to the parental wild type: growth, virulence, EPS production, extracellular cellulase production and activity, biofilm formation, and swimming ability. ROS accumulation was reduced, resistance to exogenous H2O2 was increased, and expression of catalase genes and catalase activities were increased in P4, suggesting that PCA resistance in P4 results from a reduction in ROS production and/or an increased ability to metabolize ROS following PCA treatment. Given the low risk of Xoo developing PCA resistance and the reduced virulence and fitness of the resistant strain, PCA can be used in alternation with other common bactericides to control BLB in rice fields.

A new point mutation in ß2-tubulin confers resistance to carbendazim in Fusarium asiaticum.

Resistance to benzimidazole fungicides in many phytopathogenic fungi is caused by specific point mutations in the ß-tubulin gene (ß-tubulin). However, the mutated locus and genotype of ß-tubulin differ among phytopathogenic fungi. To validate the point mutation in Fusarium asiaticum ß2-tubulin that confers resistance to carbendazim and to analyze the molecular interaction between carbendazim and F. asiaticum ß2-tubulin. In this study, a new point mutation (GAG→GCG, E198A) at codon 198 of ß2-tubulin in a wild-type F. asiaticum strain was constructed by site-directed mutagenesis followed by a split marker strategy. The site-directed mutants were verified and exhibited a high level of resistance to carbendazim. In the absence of fungicide treatment, the biological characteristics did not differ between the site-directed mutants and the wild-type strain. Molecular docking between carbendazim and ß2-tubulin was carried out using the Surflex-Dock program in Sybyl X-2.0 version and the results indicated that the E198A mutation altered the configuration of ß2-tubulin, resulting in the change of the bonding sites and docking scores. We concluded that the point mutation of F. asiaticum ß2-tubulin conferring carbendazim resistance may not always be the bonding site for carbendazim.

Evaluation of Phenamacril and Ipconazole for Control of Rice Bakanae Disease Caused by Fusarium fujikuroi.

This study evaluated the use of phenamacril and ipconazole, alone and in mixtures, for the control of rice bakanae disease caused by Fusarium fujikuroi. Mixtures were studied with the goal of reducing the selection of fungicide-resistant field isolates of the fungus. When tested alone, both phenamacril and ipconazole exhibited high antifungal activity against F. fujikuroi mycelial growth; the average EC50 value for 19 field isolates was 0.1544 µg/ml for phenamacril and 0.0472 µg/ml for ipconazole. A 2:1 mixture of phenamacril and ipconazole caused a slightly synergistic (greater than additive) inhibition of mycelial growth. Inhibition of F. fujikuroi sporulation was highest for ipconazole alone, intermediate with the 2:1 mixture, and lowest for phenamacril alone. Inhibition by phenamacril and ipconazole alone or by the 2:1 mixture was substantially lower for spore germination than for mycelial growth or sporulation. When the total fungicide concentration was <24 g of a.i./100 kg of treated rice seeds, the fungicides, whether alone or in the 2:1 mixture, were not phytotoxic to seeds or seedlings of two rice cultivars. In a greenhouse experiment, the 2:1 mixture of phenamacril and ipconazole at 6 g of a.i./100 kg of treated seeds provided 100% control of rice bakanae disease on two cultivars. Overall, the results indicate that the use of a 2:1 mixture of phenamacril and ipconazole should control rice bakanae disease while reducing the occurrence of fungicide resistance in F. fujikuroi.

CatB is Critical for Total Catalase Activity and Reduces Bactericidal Effects of Phenazine-1-Carboxylic Acid on Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola.

Rice bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae, and rice bacterial leaf streak, caused by X. oryzae pv. oryzicola, are major diseases of rice. Phenazine-1-carboxylic acid (PCA) is a natural product that is isolated from Pseudomonas spp. and is used to control many important rice diseases in China. We previously reported that PCA disturbs the redox balance, which results in the accumulation of reactive oxygen species in X. oryzae pv. oryzae. In this study, we found that PCA significantly upregulated the transcript levels of catB and katE, which encode catalases, and that PCA sensitivity was reduced when X. oryzae pvs. oryzae and oryzicola were cultured with exogenous catalase. Furthermore, catB deletion mutants of X. oryzae pvs. oryzae and oryzicola showed dramatically decreased total catalase activity, increased sensitivity to PCA, and reduced virulence in rice. In contrast, deletion mutants of srpA and katG, which also encode catalases, exhibited little change in PCA sensitivity. The results indicate that catB in both X. oryzae pvs. oryzae and oryzicola encodes a catalase that helps protect the bacteria against PCA-induced stress.

Loop-Mediated Isothermal Amplification for the Rapid Detection of the F200Y Mutant Genotype of Carbendazim-Resistant Isolates of Sclerotinia sclerotiorum.

The point mutation at codon 200 (TTC→TAC, F200Y) confers moderate resistance to carbendazim in Sclerotinia sclerotiorum. This mutant genotype (F200Y) has been detected mainly by determining the minimum inhibitory concentration (MIC), which requires 3 to 5 days. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum. Specific LAMP primers were designed and concentrations of LAMP components were optimized. The optimal reaction conditions were 62 to 63°C for 45 min. The new LAMP assay requires no special equipment and is highly sensitive and specific (the i.e., it generated positive results with F200Y mutant genotype but generated negative results with other carbendazim-resistant mutants and with a variety of carbendazim-resistant mutants of Botrytis cinerea and Fusarium graminearum). Inclusion of the loop backward (LB) primer reduced the reaction time to 15 min. Results were identical with LAMP and MIC determinations. The advantages of the LB-accelerated LAMP assay for detection of the F200Y mutant genotype were demonstrated by assaying sclerotia produced on rape stems that were artificially inoculated in the field. The results indicated that the new LAMP assay represents an improved way to detect the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum.

Effects of phenazine-1-carboxylic acid on the biology of the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is the casual agent of bacterial blight, which is one of the most serious diseases of rice. The antibiotic phenazine-1-carboxylic acid (PCA), which is primarily produced by Pseudomonas spp., was found and previously reported very effective against Xoo. However, the biological effects of PCA on Xoo remain unclear. In this study, we found that PCA increased the accumulation of reactive oxygen species (ROS) and reduced the activities of catalase (CAT) and superoxide dismutase (SOD) in Xoo. Xoo was more sensitive to H2O2 than Xanthomonas oryzae pv. oryzicola (Xoc), and had a much lower expression of CAT genes. In addition, proteomic analysis indicated that PCA inhibited carbohydrate metabolism and nutrient uptake in Xoo, and analysis of carbon source utilization further confirmed that carbohydrate metabolism in Xoo was repressed by PCA. In conclusion, PCA acted as a redox-cycling agent that disturbed the redox balance in Xoo and reduced CAT and SOD activities, resulting in higher accumulation of ROS, altered carbohydrate metabolism, and lower energy production and nutrient uptake. Moreover, a deficient antioxidant system in Xoo made it very sensitive to PCA.

Proteome analysis of the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae.

The plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight, which is one of the most serious diseases of rice. Xoo has been studied for over one century, and much has been learned about it, but proteomic investigation has been neglected. In this study, proteome reference maps of Xoo were constructed by two-dimensional gel electrophoresis, and 628 spots in the gels representing 469 different protein species were identified with MALDI-TOF/TOF MS. The identified spots were assigned to 15 functional categories according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the annotations from the National Center for Biotechnology Information (NCBI) database. The data set has been deposited in the World-2DPAGE database (Database ID: 0044). In addition, comparative proteomic analysis revealed that proteins related to the TonB-dependent transportation system and energy metabolism are involved in the phenazine-1-carboxylic acid resistance in Xoo. In conclusion, we have established a proteome database for Xoo and have used this database in a comparative proteomic analysis that identified proteins potentially contributing to phenazine-1-carboxylic acid resistance in Xoo.