Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

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.     

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.     

Biological and molecular variability of Sarocladium oryzae, the sheath rot pathogen of rice (Oryza sativa L.)

Sheath rot disease of rice caused by Sarocladium oryzae (Sawada) (=Acrocylindrium oryzae, Sawada) has become an important production constraint in all rice-growing countries. Pathogenicity, phytotoxic metabolites, and random amplified polymorphic DNA (RAPD) markers were used to assess the level of genetic variability of S. oryzae derived from rice cultivars, CR1018, IR36, and IR50, of different locations in North East and South India. Variability in pathogenicity, phytotoxic metabolite production, and DNA polymorphisms was detected among S. oryzae isolates. Results indicated that S. oryzae isolates produced both cerulenin and helvolic acid at concentrations 0.3–0.62 and 0.9–4.8 μg mL⁻¹ of culture filtrate, respectively. Isolates that produce higher concentration of helvolic acid induced a high percent incidence of sheath rot disease. Oligonucleotide primers, GF and MR, generated either a simple (up to 2 bands) or complex (up to 6 bands) RAPD pattern. According to their level of similarity, S. oryzae isolates from North East and South India were grouped separately into two major clusters and 13 genotypes. Molecular- and pathogenicity-based classifications were not correlated, but a high level of genetic variability within S. oryzae isolates was identified. The molecular variability of S. oryzae isolates will be an important consideration in breeding programs to develop durable resistance for sheath rot disease.     

The XA21 binding protein XB25 is required for maintaining XA21‐mediated disease resistance

Plant genomes encode a large number of proteins that potentially function as immune receptors in the defense against pathogen invasion. As a well‐characterized receptor kinase consisting of 23 tandem leucine‐rich repeats, a transmembrane domain and a serine/threonine kinase, the rice (Oryza sativa) protein XA21 confers resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae (Xoo) races that cause bacterial blight disease. We report here that XA21 binding protein 25 (XB25) belongs to the plant‐specific ankyrin‐repeat (PANK) family. XB25 physically interacts, in vitro, with the transmembrane domain of XA21 through its N–terminal binding to transmembrane and positively charged domain (BTMP) repeats. In addition, XB25 associates with XA21 in planta. The downregulation of Xb25 results in reduced levels of XA21 and compromised XA21‐mediated disease resistance at the adult stage. Moreover, the accumulation of XB25 is induced by Xoo infection. Taken together, these results indicate that XB25 is required for maintaining XA21‐mediated disease resistance.     

Intercellular Production of Tamavidin 1, a Biotin-Binding Protein from Tamogitake Mushroom,Confers Resistance to the Blast Fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in Transgenic Rice

The blast fungus Magnaporthe oryzae, one of the most devastating rice pathogens in the world, shows biotin-dependent growth. We have developed a strategy for creating disease resistance to M. oryzae whereby intercellular production of tamavidin 1, a biotin-binding protein from Pleurotus cornucopiae occurs in transgenic rice plants. The gene that encodes tamavidin 1, fused to the sequence for a secretion signal peptide derived from rice chitinase gene, was connected to the Cauliflower mosaic virus 35S promoter, and the resultant construct was introduced into rice. The tamavidin 1 was accumulated at levels of 0.1–0.2% of total soluble leaf proteins in the transgenic rice and it was localized in the intercellular space of rice leaves. The tamavidin 1 purified from the transgenic rice was active, it bound to biotin and inhibited in vitro growth of M. oryzae by causing biotin deficiency. The transgenic rice plants showed a significant resistance to M. oryzae. This study shows the possibility of a new strategy to engineer disease resistance in higher plants by taking advantage of a pathogen’s auxotrophy.     

Accumulation of Indole‐3‐Acetic Acid in Rice sl Mutant Leaves Infected with Bipolaris oryzae

Rice leaves accumulate serotonin in response to infection by Bipolaris oryzae. The leaves of the sl mutant, which is deficient in the gene encoding tryptamine 5‐hydroxylase, accumulate tryptamine instead of serotonin upon infection by B. oryzae. Because tryptamine is a possible precursor of indole‐3‐acetic acid (IAA), we investigated the accumulation of IAA in sl leaves infected with B. oryzae. Liquid chromatography coupled with tandem mass spectrometry analysis indicated that IAA accumulated at approximately 1.5 μmol/gFW in the leaves of sl mutant. This accumulation was suppressed by 95% by the treatment with the tryptamine decarboxylase inhibitor, (S)‐α‐(fluoromethyl)tryptophan, at 100 μm , indicating that tryptamine served as the precursor of IAA. The accumulation of IAA was not reproduced by treatment with CuCl₂ or by exogenous feeding of tryptamine. Furthermore, inoculation of Magnaporthe grisea induced only a lower level of IAA accumulation. On the other hand, B. oryzae produced IAA in culture media containing tryptamine. These findings strongly suggested that the metabolism of tryptamine by B. oryzae was responsible for IAA accumulation in the leaves of the sl mutant. Serotonin added to the culture media was also converted into 5‐hydroxyindole‐3‐acetic acid (5HIAA) at a rate similar to that of tryptamine. Considering that wild‐type rice leaves accumulate serotonin for defensive purposes, reducing the concentration of serotonin by conversion into 5HIAA may be significant as a detoxification process in the interaction between B. oryzae and rice.     

The rice terpene synthase gene OsTPS19 functions as an (S)‐limonene synthase in planta,and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae

Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up‐regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)‐limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)‐limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)‐limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.     

Induction of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in rice by treatment with acibenzolar-S-methyl

The role of the plant defence activator, acibenzolar‐S‐methyl (ASM), in inducing resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) was studied. Application of ASM induced resistance in rice to infection by Xoo. When the pathogen was clip‐inoculated to the rice plants, it caused bacterial leaf blight symptoms in the untreated control. However, in the rice plants pretreated with ASM, infection was significantly reduced. Induced systemic resistance was found to persist for up to 3 days in the pretreated rice plants. Increased phenolic content and accumulation of pathogenesis‐related (PR) proteins, viz. chitinase, β‐1,3‐glucanase and thaumatin‐like protein (TLP; PR 5) were observed in rice plants pretreated with ASM followed by inoculation with Xoo. Immunoblot analysis using rice TLP and tobacco chitinase antiserum revealed rapid induction and over‐expression of 25 and 35 kDa TLP and chitinase, respectively, in rice in response to pretreatment with ASM followed by Xoo inoculation. Based on these experiments, it is evident that induction of disease resistance in rice was accelerated following treatment with ASM.     

Rice Resistance to Brown Spot Mediated by Nitrogen and Potassium

This study was undertaken to investigate the effects of both nitrogen (N) and potassium (K) rates on rice resistance to brown spot, caused by the fungus Bipolaris oryzae. Rice plants (cultivar ‘Metica 1’) were grown in soil corrected with 0, 25, 50, 75 and 100 mg of N / kg (as NH₄NO₃) of soil as well as with 25, 50, 75, 125 and 150 mg of K / kg (as KCl) of soil. Thirty‐three‐day‐old plants were inoculated with a suspension of Bipolaris oryzae conidia and the incubation period (IP), number of lesions (NL) per cm² of leaf area and disease severity was evaluated. Disease severity was scored at 24, 48, 72, 96, 120 and 144 h after inoculation and data were used to obtain the area under brown spot progress curve (AUBSPC). Soil plant analysis development (SPAD) index, plant dry weight and concentration of N and K in leaf tissues were also determined for both non‐inoculated (NI) and inoculated (IN) plants. Concentration of N in leaf tissue increased as the N rates in the soil increased. Concentration of K in leaf tissue increased sharply as the K rates in the soil increased for both NI and IN plants. Concentration of K in leaf tissue was not affected by N rates. The IP increased as the N rates increased, but was somewhat less impacted by increasing K rates. The NL decreased as the N rates increased. The NL dramatically declined at the highest K rates. The AUBSPC dramatically declined as the N and K rates in the soil increased. SPAD index values increased as the N and K rates in the soil increased for both NI and IN plants. Plant dry weight increased as the N and K rates in the soil increased for both NI and IN plants. Results from this study suggest that combining high N and K rates may contribute to reducing the intensity of brown spot in rice while improving plant development.     

Resistance-related physiological response of rice leaves to the compound stress of enhanced UV-B radiation and Magnaporthe oryzae

An enhanced UV-B radiation (5.0?kJ?m^?2) was supplied before, during, and after Magnaporthe oryzae infection. The effects of single and compound stress of the UV-B radiation and M. oryzae on the resistance physiology and gene expression of rice leaves were examined. Results revealed that UV-B radiation given before M. oryzae infection (UV-B?→?M.) significantly increased the pathogenesis-related proteins (PRs) activities of phenylalanine ammonialyase (PAL), lipoxygenase (LOX), chitinase (CHT), and β-1,3-glucanase, the resistance-related substances (flavonoids and total phenols) content, and resistance-related genes (OsPAL and OsCHT) expression, thereby improving the disease resistance of rice leaves. Simultaneous exposure to UV-B radiation and M. oryzae (UV-B/M.) significantly increased the OsLOX2 expression and the PRs activities. Exposure to UV-B radiation after M. oryzae infection (M.?→?UV-B) decreased the flavonoid content, did not improve the PRs activity, and increased OsLOX2 expression. Compound treatments of UV-B?→?M., UV-B/M., and M.?→?UV-B reduced the disease index by 62.3%, 40.2%, and 26.6%, respectively, indicating UV-B radiation inhibited the occurrence of M. oryzae disease, but its inhibitory effect weakened when it was provided after M. oryzae infection. Hence, rice responded to the compound stress of UV-B radiation and M. oryzae through a resistance-related physiological mechanism associated with the sequence of stress occurrence.