2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Allelic variation for broad‐spectrum resistance and susceptibility to bacterial pathogens identified in a rice MAGIC population

Quantitative trait loci (QTL) that confer broad‐spectrum resistance (BSR), or resistance that is effective against multiple and diverse plant pathogens, have been elusive targets of crop breeding programmes. Multiparent advanced generation intercross (MAGIC) populations, with their diverse genetic composition and high levels of recombination, are potential resources for the identification of QTL for BSR. In this study, a rice MAGIC population was used to map QTL conferring BSR to two major rice diseases, bacterial leaf streak (BLS) and bacterial blight (BB), caused by Xanthomonas oryzae pathovars (pv.) oryzicola (Xoc) and oryzae (Xoo), respectively. Controlling these diseases is particularly important in sub‐Saharan Africa, where no sources of BSR are currently available in deployed varieties. The MAGIC founders and lines were genotyped by sequencing and phenotyped in the greenhouse and field by inoculation with multiple strains of Xoc and Xoo. A combination of genomewide association studies (GWAS) and interval mapping analyses revealed 11 BSR QTL, effective against both diseases, and three pathovar‐specific QTL. The most promising BSR QTL (qXO‐2‐1, qXO‐4‐1 and qXO‐11‐2) conferred resistance to more than nine Xoc and Xoo strains. GWAS detected 369 significant SNP markers with distinguishable phenotypic effects, allowing the identification of alleles conferring disease resistance and susceptibility. The BSR and susceptibility QTL will improve our understanding of the mechanisms of both resistance and susceptibility in the long term and will be immediately useful resources for rice breeding programmes.     

Rice OsPAD4 functions differently from Arabidopsis AtPAD4 in host‐pathogen interactions

The extensively studied Arabidopsis phytoalexin deficient 4 (AtPAD4) gene plays an important role in Arabidopsis disease resistance; however, the function of its sequence ortholog in rice is unknown. Here, we show that rice OsPAD4 appears not to be the functional ortholog of AtPAD4 in host‐pathogen interactions, and that the OsPAD4 encodes a plasma membrane protein but that AtPAD4 encodes a cytoplasmic and nuclear protein. Suppression of OsPAD4 by RNA interference (RNAi) increased rice susceptibility to the biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo), which causes bacteria blight disease in local tissue. OsPAD4‐RNAi plants also show compromised wound‐induced systemic resistance to Xoo. The increased susceptibility to Xoo was associated with reduced accumulation of jasmonic acid (JA) and phytoalexin momilactone A (MOA). Exogenous application of JA complemented the phenotype of OsPAD4‐RNAi plants in response to Xoo. The following results suggest that OsPAD4 functions differently than AtPAD4 in response to pathogen infection. First, OsPAD4 plays an important role in wound‐induced systemic resistance, whereas AtPAD4 mediates systemic acquired resistance. Second, OsPAD4‐involved defense signaling against Xoo is JA‐dependent, but AtPAD4‐involved defense signaling against biotrophic pathogens is salicylic acid‐dependent. Finally, OsPAD4 is required for the accumulation of terpenoid‐type phytoalexin MOA in rice‐bacterium interactions, but AtPAD4‐mediated resistance is associated with the accumulation of indole‐type phytoalexin camalexin.     

Red‐Light‐Induced Resistance to Brown Spot Disease Caused by Bipolaris oryzae in Rice

In this study, the protective effect of red light against the brown spot disease caused by the fungus Bipolaris oryzae in rice was investigated. Lesion formation was significantly inhibited on detached leaves that were inoculated with B. oryzae and kept under red for 48 h, but it was not inhibited when the leaves were kept under natural light or in the dark. The protective effect was also observed in intact rice plants inoculated with B. oryzae; the plants survived under red light, but most of them were killed by infection under natural light or dark condition. Red light did not affect fungal infection in onion epidermis cells or heat‐shocked leaves of rice, and it did not affect cellulose digestion ability; this suggested that the protective effect is due to red‐light‐induced resistance. In addition, the degree of protection increased as the red light dosage increased, regardless of the order of the red light and natural light period, indicating that red‐light‐induced resistance is time dependent. Feeding of detached leaves with a tryptophan decarboxylase inhibitor, s‐α‐fluoromethyltryptophan (0.1 mm ), for 24 h inhibited the development of resistance in response to red light irradiation. Suppression of resistance was also observed in leaves treated with a phenylalanine ammonia‐lyase inhibitor, α‐aminooxy acetic acid (0.5 mm ). These results suggest that the tryptophan and phenylpropanoid pathways are involved in the red‐light‐induced resistance of rice to B. oryzae.     

Microbial secondary metabolite induction of abnormal appressoria formation mediates control of rice blast disease caused by Magnaporthe oryzae

Okinawa, the only subtropical area in Japan with numerous island ecosystems, is expected to have diverse microbial resources. Recently, we reported the construction of a culture filtrate library with microbes originally isolated from soils in Okinawa, including the Yaeyama Archipelago, and validated its phylogenetic diversity. In the present study, we investigated the inhibitory effect of the cell extract (CE) from microbial isolate 3–45 against Magnaporthe oryzae in rice (Oryza sativa). Abnormal appressorium formation by M. oryzae was induced in the presence of the CE from isolate 3–45. Additionally, melanization of appressoria was inhibited in the presence of CE from isolate 3–45. Sequence analysis of the 16S rDNA region of isolate 3–45 indicated that it shared similarities with Streptomyces erythrochromogenes. When rice leaves were inoculated with M. oryzae in the presence of CE from isolate 3–45, blast lesion formation was inhibited compared to leaves treated with M. oryzae in the absence of CE from isolate 3–45. In addition, M. oryzae infective activity was significantly inhibited in rice leaf sheaths treated with CE from isolate 3–45. Furthermore, abnormal appressorium formation was observed in the presence of heat‐treated CE from isolate 3–45. These results suggest that CE from isolate 3–45 can protect rice from blast disease caused by M. oryzae. Further studies are required to identify the active compounds present in 3–45‐CE and to clarify its mechanism of inhibition in full detail. The present study on isolate 3–45 might contribute to the development of a new fungicide for controlling rice blast disease caused by M. oryzae.     

Introgression of Pi2 and Pi5 Genes for Blast (Magnaporthe oryzae) Resistance in Rice and Field Evaluation of Introgression Lines for Resistance and Yield Traits

Blast caused by Magnaporthe oryzae is the most devastating disease causing significant loss in rice production. The destructive nature of the disease is mainly due to the genetic plasticity of M. oryzae which complicates the breeding strategies. Blast can be effectively managed by the deployment of R genes. In this study, broad‐spectrum blast resistance genes Pi2 and Pi5 were introgressed independently into popular but blast susceptible rice variety, Samba Mahsuri (BPT5204) by applying marker‐assisted backcross breeding approach. Tightly linked markers AP5930 for Pi2 and 40N23r for Pi5 gene were used in foreground selection. Background selection helped to identify the lines with maximum recovery of recurrent parent genome (RPG). The RPG recovery in Pi2 introgression lines was up to 90.17 and 91.46% in Pi5 lines. Homozygous introgression lines in BC₃F₄ generation carrying Pi2 and Pi5 gene were field evaluated for blast resistance, yield per se and yield‐related traits. The lines showed resistance to leaf and neck blast in multilocation field evaluation. Improved BPT5204 lines with improvement for blast resistance were on par with original BPT5204 in terms of grain yield and grain features.     

Transgenic expression of the rice Xa21 pattern‐recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern‐recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21‐mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar ‘Gonja manjaya’ (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.     

Controlling and Defence‐related Mechanisms of Bacillus Strains Against Bacterial Leaf Blight of Rice

Bacillus strains are broadly studied for their beneficial role in plant growth and biological control of plant disease and pest; however, little is known about their underlying mechanisms. In this study, we assessed the controlling and defence‐related mechanisms of three Bacillus strains including rice seed‐associated strain B. subtilis A15, rhizobacterial strains B. amyloliquefaciens D29 and B. methylotrophicus H8, all of which are against bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae. Results indicated that all three strains showed strong biofilm formation ability. The culture filtrates of each strain significantly suppressed the growth and biofilm formation of X. oryzae, while changes in bacterial cell morphology such as cell swell and severe cell wall alterations were observed through the transmission electron microscopy images. PCR analysis revealed that all three strains harbour the antimicrobial‐associated genes that are responsible for biosynthesis of bacillomycin, fengycin, iturin and surfactin. Subsequent real‐time qPCR analysis revealed the upregulated expression of fenD and srfAA genes in D29 and H8, and fenD and ituC genes in A15 during their in vitro interaction with X. oryzae. It suggests that the antibacterial mechanisms of the three strains may be at least partially associated with their ability to secrete corresponding lipopeptides. Interestingly, the applications of the three strains in greenhouse conditions were found to be effective in controlling the BLB disease, which was achieved through the activation of inducing systemic resistance resulted from the enhanced activities of defence‐related enzymes. This is the first report of demonstration of the mode of antibacterial effect of Bacillus strains against X. oryzae. Overall, data from the current study provide valuable information for biological control of BLB disease in rice.     

The NB‐LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance

Plant resistance proteins of the class of nucleotide‐binding and leucine‐rich repeat domain proteins (NB‐LRRs) are immune sensors which recognize pathogen‐derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB‐LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR‐independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR‐Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo‐ and hetero‐complexes and interact through their coiled‐coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR‐Pia, neither RGA4 nor RGA5 is re‐localized to the nucleus. These results establish a model for the interaction of hetero‐pairs of NB‐LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.     

Activation of Systemic Resistance to Magnaporthe oryzae in Rice by Substances Produced by Fusarium solani Isolated from Soil

Of 70 micro‐organisms (fungi, bacteria and actinomycetes) isolated from soil using vegetable tissue baits, 16 produced substances in culture fluids capable of preventing the development of blast caused by Magnaporthe oryzae on rice leaves with little or no inhibitory effect on the conidial germination of the pathogen. Isolate KS‐F14, which secreted substances capable of activating resistance in untreated leaves, was selected and identified as Fusarium solani. The resistance‐inducing substances were effective at pH values ranging from 5 to 10 and were stable under high temperatures, maintaining approximately the same level of activity even after autoclaving for 20 min. After application, the activated resistance in rice leaves persisted for 14 days. The polar solvent extracts of freeze‐dried KS‐F14 secretions were effective in activating resistance against M. oryzae in rice plants. The non‐polar solvent extracts were also effective, albeit not as effective as the polar solvent extracts, indicating that although the majority of the secreted resistance‐inducing compounds are hydrophilic, some of the compounds are hydrophobic. Treating secretions with cation or anion exchange resins only partially reduced their resistance‐inducing ability, suggesting that the resistance‐inducing components include both charged and non‐charged compounds. The resistance‐inducing compounds produced by F. solani have the potential to be developed into a commercial product for the control of rice blast and possibly other plant diseases.