Studies on Biological Activity of Cyclic Imide Compounds

The structure-activity relationships of l-phenylpyrrolidine-2,5-dione derivatives were investigated on Sclerotinia sclerotiorum by the agar dilution method. In addition, several representative compounds were tested for antimicrobial spectra in vitro with 15 pathogenic microbes and for foliage protection activity in green house tests with rice blast, rice brown spot, rice sheath blight and kidney bean stem rot. It was found that 3,5-dihalo-substituents on the benzene moiety are essential to high antifungal activity against Sclerotinia sclerotiorum. Generally, l-(3′,5′-dihalophenyl)pyrrolidine-2,5-diones are active against Corticiaceae, Dematiaceae, Pleosporaceae and Sclerotiniaceae, especially active against Sclerotinia sclerotiorum and Botrytis cinerea (the conidia form of Sclerotinia fuckeliana). N-(3,5-Dichlorophenyl)itaconimide showed a peculiarly broad antimicrobial spectrum. In green house tests, these compounds showed high activity against rice brown spot, rice sheath blight and kidney bean stem rot. Results of green house tests on the above-mentioned diseases correlate fairly well with those of in vitro tests.     

Degradation of 3-(3′,5′ -Dichlorophenyl)-5,5-dimethyloxazolidine-2,4-dione by Plants,Soil and Light

Studies on uptake, tissue distribution, and metabolsim of 3-(3′,5′ -dichlorophenyl)-5,5-dimethyloxazolidine-2,4-dione (DDOD) by bean and grape plants, and degradation by soil and light were carried out using ¹⁴C- or ³H-DDOD. DDOD injected at stem or absorbed through roots of bean plants was transported mainly to leaf tissues. No downward movement of the label was observed. DDOD was decomposed in the nutrient solution to N-3,5-dichlorophenyl-N-α-hydroxyisobutyryl carbamic acid (DHCA) and α-hydroxyisobutyryl-3,5-dichloroanilide (HDA). Metabolism of DDOD in bean plants or on grape berries themselves occurred to only a small extent if at all. When injected into grape trees, DDOD underwent some degree of metabolization to HDA and, probably, 3-(3′,5′ -dichloro-4′ -hydroxyphenyl)-5,5-dimethyloxazolidine-2,4-dione. In soil, DDOD broke down to DHCA, HDA, and 3,5-dichloroaniline, but formation of tetrachloroazobenzene was not observed under the present experimental conditions. DDOD decomposed to some degree when irradiated with a xenon lamp.     

Studies on Biological Activity of Cyclic Imide Compounds

The structure-activity relationships of 3-phenyloxazolidine-2, 4-diones, 3-phenyl-4-imino-oxazolidine-2-ones and n-phenylcarbamates were investigated on Sclerotinia sclerotiorum by the agar medium dilution method. In addition, antimicrobial spectra of several compounds against other 15 pathogenic microbes were investigated by the same method. In each series, 3, 5-dihalo-substituents on benzene ring are essential to high antifungal activity against Sclerotinia sclerotiorum and in the case of n-phenylcarbamates, it is necessary that the α-position of alcohol moiety is substituted by such a group as cyano group, ethoxy-carbonyl group or carbamoyl group. α-Cyanoisopropyl N-(3, 5-dichlorophenyl) carbamate, 3-(3′, 5′-dichlorophenyl)-5, 5-dimethyl-4-iminooxazolidine-2-one and 3-(3′, 5′-dichlorophenyl)-5, 5-dimethyloxazolidine-2,4-dione were the most effective and completely inhibited the mycelial growth of Sclerotinia sclerotiorum at 3.2 γm (about l.0 ppm). In general, 3-(3′, 5′-dichlorophenyl) oxazolidine-2, 4-diones and related compounds are highly active against Sclerotinia sclerotiorum and Botrytis cinerea, and fairly active against Cochliobolus miyabeanus, Pellicularia filamentosa, Pellicularia sasakii and Alternaria kikuchiana.     

Interactions between Sarocladium oryzae and stem attacking fungal pathogens of rice

In laboratory tests Sarocladium oryzae, the sheath rot pathogen of rice was found to inhibit the mycelial growth of other stem-attacking rice pathogens. Among those inhibited, Sclerotium oryzae and Gaeumannomyces graminis var. graminis were most sensitive while Pyricularia oryzae and Rhizoctonia solani were less sensitive. Tissue-based tests made with rice culm segments established that Sarocladium oryzae inhibits mycelial growth and delays sclerotium formation in R. solani. Cerulenin, the toxin produced by Sarocladium oryzae showed a toxicity pattern towards rice pathogens similar to that of Sarocladium oryzae. The stem rot pathogen, Sclerotium oryzae was most sensitive to cerulenin. In two greenhouse experiments, IR58 rice plants inoculated with Sarocladium oryzae alone or together with Sclerotium oryzae, G. graminis var. graminis or R. solani were found to have reduced plant height and increased tiller number. Sheath rot severity increased when Sarocladium oryzae was inoculated as a single pathogen or together with others. Sheath rot inoculation reduced stem rot in rice plants by 76 and 58%, respectively, in Experiment 1 and 2. By its known antagonistic interaction towards stem rot and crown sheath rot pathogens which are sensitive to it and by other unknown interactions, sheath rot emerges as the dominant disease.     

Characterization of Pseudomonas fuscovaginae and Differentiation from Other Fluorescent Pseudomonads Occurring on Rice in Burundi

In various pathogenicity, serological, physiological and biochemical tests, performed in Belgium and Japan, the Pseudomonas fuscovaginae strains associated above 1,350 m elevation in Burundi with sheath brown rot of rice, rusty seed and black, stripes on seedlings, were found to be similar to reference strains of this pathogen from Japan. The same bacteria was detected on rice seeds imported from Asia to Burundi. Beside the serological characteristics, P. fuscovaginae can be differentiated from other oxidase and arginine dihydrolase positive non-pathogenic fluorescent pseudomonads, also isolated from lesions on rice seedlings, by the simultaneous occurrence of no production of 2-ketogluconate, acid production from trehalose, but not from inositol. Occasionally, other symptoms inducing, oxidase positive, fluorescent pseudomonads, different from any described species, were isolated from rice seedlings and sheath rot in Burundi.     

Rice oxalate oxidase gene driven by green tissue‐specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue‐specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence‐related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue‐specific manner for sheath blight resistance.     

Efficacy of formulation and storage on rice straw waste on the activation of bioagents against root-rot diseases of bean plants

Trichoderma harzianum and Trichoderma viride, which were grown on modified media of rice straw waste, were used as suitable natural media for determining the population of the bioagents and stimulating the production of antimicrobial substances, i.e. toxins, enzymes at different periods (1, 2, 3, 4, 5 and 6 weeks). Also, the evaluation of synergistic effect between biocontrol fungi and bacteria may play a key role in the natural process of biocontrol. Synergism can occur when different agents are applied together and cell wall degrading enzymes produced by fungi can increase the efficacy of bacteria. The evaluation of the bioagents formulated on rice straw waste has influenced the percentage of infection of root rot on bean plants under green house and field conditions. The formulation of the Trichoderma spp. to reduce the incidence of the diseases caused by soil-borne fungi in the field has great importance in the biocontrol of the diseases. This work was aimed to determine and improve the efficacy of application and formulation of Trichoderma spp. on the rice straw waste against root-rot disease of bean under green house and field conditions.     

一株水稻纹枯菌拮抗细菌的分离与鉴定

【目的】从土壤中分离并鉴定水稻纹枯菌拮抗细菌,测定其体外抑菌和温室防治效果。【方法】采用系列稀释法和平板对峙法筛选拮抗细菌,基于形态、生理特征及16S rDNA序列鉴定其分类地位,采用种子细菌化温室试验测定其防效。【结果】从蔬菜根际土壤中筛选出一株纹枯菌拮抗细菌,命名为kwkjT4。菌株具有明显的体外抑菌活性,对水稻纹枯病的温室防效与井冈霉素相当,初步鉴定为假紫色色杆菌(Chromobacterium pseudoviolaceum)。最适生长条件为pH 7.0,温度32°C,培养时间为36 h;抑菌活性物质产生的最适培养条件为pH 6.0,温度28°C,培养时间为48 h;表明两者并不一致。【结论】kwkjT4菌株在水稻纹枯病的生物防治中具有潜在的应用价值。这是C.pseudoviolaceum拮抗纹枯菌的首次报道。     

Use of omic approaches for characterizing microbiota from suppressive compost to control soil-borne plant pathogens

Abstract

Microbiomes composition, diversity, and variability into a collection of suppressive composts were investigated for effective biological control of soil-borne phytopathogens. Pyrosequencing resulted be a reliable and faster method for characterizing fungal and bacterial microbiomes into composts derived from a varied feedstock of different composition, origin and provenience. Differences in taxonomic structure assessed by bioinformatics analyses were related to feedstock origin. Green composts derived from agro-waste and agroindustrial co/byproducts provided the most varied microbiomes either related to suppression of Rhizoctonia damping-off in bean and Verticillium wilt in eggplant, either to control of Phytium damping-off in cucumber and Phytophthora root rot in tomato. On the other hand, composted municipal solid wastes and co-composted cow manure with household waste prevalently given a most specific microbiota related to suppression of Fusarium wilt in melon.     

Outbreak of Rhizopus Rot Caused by Rhizopus oryzae on Seedlings of Grafted Cucumber on Pumpkin Rootstock in South Korea

In 2013, an outbreak of Rhizopus rot caused by Rhizopus oryzae occurred in cucumber grafted onto pumpkin rootstock sampled from seedling farms in Changnyeong, South Korea. A water‐soaked appearance of the affected tissue was the first symptom of this soft fungal rot in the seedling stems of grafted cucumber. Lesions at the graft sites softened and rapidly, rotted, and turned brown or dark brown. Measurements and taxonomic characteristics were most similar to R. oryzae. DNA sequencing and phylogenetic analysis of the internal transcribed spacer rRNA gene region confirmed that the isolates were indeed R. oryzae. Koch's postulates were supported by pathogenicity tests conducted on healthy plants. Based on mycological characteristics, pathogenicity test, and molecular analysis, the causal fungus was identified as R. oryzae Went & Prinsen Geerligs. To our knowledge, this is the first report of Rhizopus rot caused by R. oryzae in seedlings of grafted cucumber on pumpkin rootstock in South Korea.     

Identification of major quantitative trait loci qSBR11-1 for sheath blight resistance in rice

Sheath blight caused by Rhizoctonia solani Kühn is one of the important diseases of rice, resulting in heavy yield loss in rice every year. No rice line resistant to sheath blight has been identified till date. However, in some rice lines a high degree of resistance to R. solani has been observed. An indica rice line, Tetep, is a well documented source of durable and broad spectrum resistance to rice blast as well as quantitative resistance to sheath blight. The present study identified genetic loci for quantitative resistance to sheath blight in rice line Tetep. A mapping population consisting of 127 recombinant inbred lines derived from a cross between rice cultivars HP2216 (susceptible) and Tetep (resistant to sheath blight) was evaluated for sheath blight resistance and other agronomic traits for 4 years across three locations. Based on sheath blight phenotypes and genetic map with 126 evenly distributed molecular markers, a quantitative trait loci (QTLs) contributing to sheath blight resistance was identified on long arm of chromosome 11. Two QTL mapping approaches i.e., single marker analysis and composite interval mapping in multi environments were used to identify QTLs for sheath blight resistance and agronomical traits. The QTL qSBR11-1 for sheath blight resistance was identified between the marker interval RM1233 (26.45 Mb) to sbq33 (28.35 Mb) on chromosome 11. This region was further narrowed down to marker interval K39516 to sbq33 (~0.85 Mb) and a total of 154 genes were predicted including 11 tandem repeats of chitinase genes which may be responsible for sheath blight resistance in rice line Tetep. A set of 96 varieties and a F₂ population were used for validation of markers linked to the QTL region. The results indicate that there is very high genetic variation among varieties at this locus, which can serve as a starting point for allele mining of sheath blight resistance.     

Oxalic acid-induced resistance to Rhizoctonia solani in rice is associated with induction of phenolics,peroxidase and pathogenesis-related proteins

Abstract

Oxalic acid (1 mM) when applied as a foliar spray to rice plants induced resistance to challenge infection with Rhizoctonia solani, the rice sheath blight pathogen. Maximum reduction in sheath blight incidence was observed when the plants were sprayed with oxalic acid three days before inoculation with the fungus. The biochemical alterations in rice plants treated with oxalic acid was also investigated. When rice plants were treated with oxalic acid, a two-fold increase in phenolic content in leaf sheaths was recorded three days after treatment. Phenylalanine ammonia-lyase and peroxidase activities increased significantly starting from two days after treatment. Peroxidase (PO) isozyme analysis indicated that PO-3 and PO-4 were induced two days after treatment with oxalic acid. Western blot analysis revealed that two chitinases (28 and 35 kDa) and two β-1,3-glucanases (30 and 32 kDa) were strongly induced in rice sheaths four to six days after treatment with oxalic acid. Immunoblot analysis of protein extracts from oxalic acid-treated plants demonstrated the induction of a 23 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antibody. These results suggest that the enhanced activities of defense enzymes and defense-related compounds in oxalic acid-treated rice plants may contribute to resistance against R. solani.     

Isolation and evaluation of the biocontrol potential of Talaromyces spp. against rice sheath blight guided by soil microbiome

Rice sheath blight caused by Rhizoctonia solani is the major disease of rice that seriously threatens food security worldwide. Efficient and eco-friendly biological approaches are urgently needed since no resistant cultivars are available. In this study, fallow and paddy soils were initially subjected to microbiome analyses, and the results showed that Talaromyces spp. were significantly more abundant in the paddy soil, while Trichoderma spp. were more abundant in the fallow soil, suggesting that Talaromyces spp. could live and survive better in the paddy soil. Five Talaromyces isolates, namely, TF-04, TF-03, TF-02, TF-01 and TA-02, were isolated from the paddy soil using sclerotia of R. solani as baits and were further evaluated for their activity against rice sheath blight. These isolates efficiently parasitized the hyphae and rotted the sclerotia even at higher water contents in the sterilized sand and the soil. Isolate TF-04 significantly promoted rice growth, reduced the severity of rice sheath blight and increased the rice yield under outdoor conditions. Defence-related genes were upregulated and enzyme activities were enhanced in rice treated with isolate TF-04. Our research supplies a microbiome-guided approach to screen biological control agents and provides Talaromyces isolates to biologically control rice sheath blight.     

Transgenic strategies for genetic improvement of Basmati rice

Transgenic approach offers an attractive alternative to conventional techniques for the genetic improvement of Basmati rice because they enable the introduction of one or more genes into a leading cultivar without affecting its genetic background. During the last ten years, a rapid progress has been made towards the development of transformation methods in rice. Several transformation methods including Agrobacterium, biolistic, and DNA uptake by protoplasts, have been employed to produce transgenic rice. An array of useful genes is now available and many of these have already been transferred in rice to improve the resistance against biotic and abiotic stresses. In Basmati rice, a beginning has already been made regarding the development of tissue culture protocols, transformation methods and production of useful transgenic plants. The application and future prospects of transformation technology to engineer the resistance against insect pests (stem borer, leaf folder, brown plant hopper, gall midge), fungal diseases (blast, bakanae/foot, rot), bacterial diseases (bacterial leaf blight, sheath blight), abiotic stresses (salinity and drought) and improved nutritional quality (accumulation of provitamin A and essential amino acids in endosperm) in Basmati rice, have been addressed.     

Comparison ofRhizoctonia solani isolates from web blight and basal canker of cowpea and from soil

Summary Isolates ofRhizoctonia solani from web blight and stem basal canker of cowpea and those obtained from soil had similar linear growth rates on potato dextrose agar (PDA) at various temperatures but differed in other features. The web blight isolates differed from the basal canker and the soil isolates in cultural appearance on PDA and on potato marmite agar (PMA). The web blight isolates readily formed discrete sclerotia on PDA, PMA and soil but the other isolates did not. In greenhouse tests, the former were generally the most virulent in inciting foliage and stem basal necrosis and damping-off of seven crop species. Of the plants tested, the legumes were the most susceptible toR. solani.     

Inhibition of OsSWEET11 function in mesophyll cells improves resistance of rice to sheath blight disease

Pathogen–host interaction is a complicated process; pathogens mainly infect host plants to acquire nutrients, especially sugars. Rhizoctonia solani, the causative agent of sheath blight disease, is a major pathogen of rice. However, it is not known how this pathogen obtains sugar from rice plants. In this study, we found that the rice sugar transporter OsSWEET11 is involved in the pathogenesis of sheath blight disease. Quantitative real‐time polymerase chain reaction (qRT‐PCR) and β‐d ‐glucuronidase expression analyses showed that R. solani infection significantly enhanced OsSWEET11 expression in leaves amongst the clade III SWEET members. The analyses of transgenic plants revealed that Ossweet11 mutants were less susceptible, whereas plants overexpressing OsSWEET11 were more susceptible, to sheath blight compared with wild‐type controls, but the yield of OsSWEET11 mutants and overexpressors was reduced. SWEETs become active on oligomerization. Split‐ubiquitin yeast two‐hybrid, bimolecular fluorescence complementation and co‐immunoprecipitation assays showed that mutated OsSWEET11 interacted with normal OsSWEET11. In addition, expression of conserved residue mutated AtSWEET1 inhibited normal AtSWEET1 activity. To analyse whether inhibition of OsSWEET11 function in mesophyll cells is related to defence against this disease, mutated OsSWEET11 was expressed under the control of the Rubisco promoter, which is specific for green tissues. The resistance of transgenic plants to sheath blight disease, but not other disease, was improved, whereas yield production was not obviously affected. Overall, these results suggest that R. solani might acquire sugar from rice leaves by the activation of OsSWEET11 expression. The plants can be protected from infection by manipulation of the expression of OsSWEET11 without affecting the crop yield.     

Morphological,pathological and molecular characterisation of rice sheath blight disease causal organism Rhizoctonia solani AG-1 IA in Egypt

Rice sheath blight disease caused by Rhizoctonia solani is considered a distractive soil-borne disease of rice production worldwide. The study aimed to determine the causal organism of sheath blight symptoms in Egyptian rice fields. Sheath blight symptoms were first observed in a small area during 2013, 2014 and 2016 seasons, later in a wide area of rice fields in 2016 to 2018 seasons. Pathogen identification was carried out based on morphological traits and internal transcribed spacer sequencing. Thirty-six isolates were identified as R. solani fungus. The isolates exhibited a wide range of variability in their morphological traits and virulence patterns. Five isolates were sequenced and aligned with Chinese isolates with 75–100% identity. This is the first report of R. solani AG-1 IA that associated with rice sheath blight in Egypt. Initiate a breeding program for disease resistance and integrated disease management procedures are important to keep the disease under control.     

Activity and efficacy of Bacillus subtilis strain NJ-18 against rice sheath blight and Sclerotinia stem rot of rape

A strain of Bacillus subtilis (NJ-18) with broad antimicrobial activity was screened in the laboratory and in the field. NJ-18 inhibited the in vitro radial extension of hyphae of the phytopathogenic fungi Rhizoctonia solani and Sclerotinia sclerotiorum. The bacterium apparently produced antifungal metabolites that diffused through the agar and caused abnormal swelling of hyphae. The in vitro data and observations indicated that one of the mechanisms of inhibition by NJ-18 is antibiosis. In field experiments for control of sheath blight of rice, fermentation of NJ-18 at 5.0 × 10⁷ cfu ml⁻¹ significantly reduced disease incidence and severity; NJ-18 alone or combined with 50% kresoxim-methyl treatment at 225 g ai ha⁻¹ provided better control than 50% kresoxim-methyl at 225 g ai ha⁻¹ or Jinggangmycin at 120 g ai ha⁻¹, and control by NJ-18 alone was as high as 100.0%. In field experiments for control of Sclerotinia stem rot of rape, fermentation of NJ-18 at 1.0 × 10⁷ cfu ml⁻¹ again significantly reduced disease incidence and severity; control by NJ-18 was as high as 77.1% and was comparable with control by 46% dimethachlon and better than control by 50% carbendazim at 750 g ai ha⁻¹. We conclude that strain NJ-18 of B. subtilis is a promising biological control agent and should be further studied and tested for control of sheath blight of rice, Sclerotinia stem rot of rape, and other diseases.