Manure derived hydrochar reduced phosphorus loss risk via an alteration of phosphorus fractions and diversified microbial community in rice paddy soil.

Phosphorus (P) loss caused by the irrational use of manure organic fertilizer has become a worldwide environmental problem, which has caused a potential threat to water safety and intensified agricultural non-point source pollution. Hydrothermal carbonization is method with a low-energy consumption and high efficiency to deal with environmental problems. Application of pig manure-derived hydrochar (PMH) to soil exhibited potential of sustainable development compared with the pristine pig manure (PM). However, the effects of PMH on the distribution of P among the fractions/forms and the interaction between microorganisms and P forms and its relevance to the potential loss of P in paddy fields has not been clarified. Therefore, in this study, a soil column experiment was conducted using the untreated soil (control), and the PM, PMH1 (PMH derived at 180 °C), and PMH2 (PMH derived at 260 °C) treated soils (at the dose of 0.05 %) and rice was cultivated to investigate the effects of PM and PMH on the P fractions, mobilization, ad potential loss via the induced changes on soil microbial community after a complete growing season of rice. The trend of P utilization was evaluated by P speciation via continuous extraction and 31P NMR. The addition of PMH reduced the proportion of residual P in soil by 23.8-26.3 %, and increased the proportion of HCl-P and orthophosphate by 116.2-158.6 % and 6.1-6.8 % compared to PM. The abundance of gcd gene developed after the application of PMH2, which enhanced the mobile forms of soil P utilization via secreting gluconic acid. The network diagram analysis concluded that the changes in various P forms were mainly related to Proteobacteria, Bacteroides, Firmicutes and Acidobacteria. The results illustrated that PMH mitigate the potential risk of P loss more than PM by altering P fractions and affecting soil microbial community.

NIT24 and NIT29-mediated IAA synthesis of Xanthomonas oryzae pv. oryzicola suppresses immunity and boosts growth in rice.

Auxin plays a pivotal role in the co-evolution of plants and microorganisms. Xanthomonas oryzae pv. oryzicola (Xoc) stands as a significant factor that affects rice yield and quality. However, the current understanding of Xoc's capability for indole 3-acetic acid (IAA) synthesis and its mechanistic implications remains elusive. In this study, we performed a comprehensive genomic analysis of Xoc strain RS105, leading to the identification of two nitrilase enzyme family (NIT) genes, designated as AKO15524.1 and AKO15829.1, subsequently named NIT24 and NIT29, respectively. Our investigation unveiled that the deletion of NIT24 and NIT29 resulted in a notable reduction in IAA synthesis capacity within RS105, thereby impacting extracellular polysaccharide production. This deficiency was partially ameliorated through exogenous IAA supplementation. The study further substantiated that NIT24 and NIT29 have nitrilase activity and the ability to catalyse IAA production in vitro. The lesion length and bacterial population statistics experiments confirmed that NIT24 and NIT29 positively regulated the pathogenicity of RS105, suggesting that NIT24 and NIT29 may regulate Xoc invasion by affecting IAA synthesis. Furthermore, our analysis corroborated mutant strains, RS105_ΔNIT24 and RS105_ΔNIT29, which elicited the outbreak of reactive oxygen species, the deposition of callose and the upregulation of defence-related gene expression in rice. IAA exerted a significant dampening effect on the immune responses incited by these mutant strains in rice. In addition, the absence of NIT24 and NIT29 affected the growth-promoting effect of Xoc on rice. This implies that Xoc may promote rice growth by secreting IAA, thus providing a more suitable microenvironment for its own colonization. In summary, our study provides compelling evidence for the existence of a nitrilase-dependent IAA biosynthesis pathway in Xoc. IAA synthesis-related genes promote Xoc colonization by inhibiting rice immune defence response and affecting rice growth by increasing IAA content in Xoc.

Design, synthesis, and bioactivity studies of chalcone derivatives containing [1,2,4]-triazole-[4,3-a]-pyridine.

In this study, 30 chalcone derivatives containing [1,2,4]-triazole-[4,3-a]-pyridine were designed and synthesized. The results of antibacterial activity showed that EC50 values of N26 against Xoo, Pcb was 36.41, 38.53 µg/mL, respectively, which were better than those of thiodiazole copper, whose EC50 values were 60.62, 106.75 µg/mL, respectively. The bacterial inhibitory activity of N26 against Xoo was verified by SEM. Antibacterial mechanism between N26 and Xoo was preliminarily explored, the experimental results showed that when the drug concentration was 100 mg/L, N26 had a good cell membrane permeability of Xoo, and it can inhibit the production of EPS content and extracellular enzyme content to disrupt the integrity of the Xoo biofilms achieving the effect of inhibiting Xoo. At 200 mg/L, N26 can protect and inhibit the lesions of post-harvested potatoes in vivo. The activities of N1-N30 against TMV were determined with half leaf dry spot method. The EC50 values of the curative and protective activity of N22 was 77.64 and 81.55 µg/mL, respectively, which were superior to those of NNM (294.27, 175.88 µg/mL, respectively). MST experiments demonstrated that N22 (Kd = 0.0076 ± 0.0007 µmol/L) had a stronger binding ability with TMV-CP, which was much higher than that of NNM (Kd = 0.7372 ± 0.2138 µmol/L). Molecular docking results showed that N22 had a significantly higher affinity with TMV-CP than NNM.

Phosphorus fertiliser application mitigates the negative effects of microplastic on soil microbes and rice growth.

Soil microplastics (MPs) have attracted widespread attention recently. Most studies have explored how soil MPs affect the soil's physicochemical parameters, matter circulation, and soil microbial community assembly. Similarly, a key concern in agricultural development has been the use of phosphorus (P) fertiliser, which is essential for plant health and development. However, the relationship between MPs and phosphate fertilisers and their effects on the soil environment and plant growth remains elusive. This study assessed the influence of adding low-density polyethylene MPs (1%) with different phosphate fertiliser application rates on microbial communities and rice biomass. Our results showed that MPs changed the structure of soil bacterial and phoD-harbouring microbial communities in the treatment with P fertiliser at the same level and suppressed the interactions of phoD-harbouring microorganisms. In addition, we found that MPs contamination inhibited rice growth; however, the inclusion of P fertiliser in MP-contaminated soils reduced the inhibitory action of MPs on rice growth, probably because the presence with P fertiliser promoted the uptake of NO3--N by rice in MP-contaminated soils. Our results provide further insights into guiding agricultural production, improving agricultural management, and rationally applying phosphate fertilisers in the context of widespread MPs pollution and global P resource constraints.

Biocontrol of bacterial seedling rot of rice plants using combination of Cytobacillus firmus JBRS159 and silicon.

Burkholderia glumae causes bacterial panicle blight (BPB) and bacterial seedling rot (BSR) which are difficult to control in rice plants. Seed disinfection using microbes and eco-friendly materials is an efficient alternative practice for managing BPB and BSR. In this study, we applied Cytobacillus firmus JBRS159 (JBRS159) in combination with silicon dioxide (SiO2) nanoparticle or potassium silicate (K2SiO3) solution to control BSR. JBRS159, SiO2 nanoparticle, and K2SiO3 independently suppressed the BSR disease and promoted growths of rice and Arabidopsis. Population of B. glumae in the treated rice seeds was suppressed by the application of JBRS159 via competitions for nutrients and niches. The mixture of JBRS159 and each Si compound (SiO2 nanoparticle or K2SiO3) was complementary for disease-suppressing and growth-promoting activities of individual treatment. The results of this study indicate that mixture of JBRS159 with each Si compound can be harnessed for disease control and growth promotion as efficient alternatives to chemical pesticides and synthetic fertilizers. The efficacy of JBRS159 and Si compounds in the control of BSR and BPB in the field remains to be evaluated.

Secondary Metabolites with Antifungal Activities from Mangrove Derived Fungus Monascus purpureus WMD2424.

The mold Monascus, also called red yeast rice, anka, or koji, has been used as the natural food coloring agent and food additives for more than 1000 years in Asian countries. It has also been used in Chinese herbology and traditional Chinese medicine due to its easing digestion and antiseptic effects. However, under different culture conditions, the ingredients in Monascus-fermented products may be changed. Therefore, an in-depth understanding of the ingredients, as well as the bioactivities of Monascus-derived natural products, is important. Here, through the thorough investigation into the chemical constituents of M. purpureus wmd2424, five previously undescribed compounds, monascuspurins A-E (1-5), were isolated from the EtOAc extract of mangrove-derived fungus Monascus purpureus wmd2424 cultured in RGY medium. All the constituents were confirmed via HRESIMS and 1D- and 2D-NMR spectroscopy. Their antifungal activity was also evaluated. Our results showed that four constituents (compounds 3-5) possessed mild antifungal activity against Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae. It is worth mentioning that the chemical composition of the type strain Monascus purpureus wmd2424 has never been studied.

Integrated strength of osmotic potential and phosphorus to achieve grain yield of rice under water deficit by arbuscular mycorrhiza fungi.

Arbuscular mycorrhizal ecosystem provides sustainability to plant integrity under drought situations. However, host plants that survive in drought frequently lose yield. The potential of Funneliformis mosseae (F), Claroideoglomus etunicatum (C), and Acaulospora fovaeta (A) was assessed to evaluate in indica rice cv. Leum Pua during booting stage under 21-day water withholding. The effects of three inoculation types; (i) F, (ii) F + C (FC), and (iii) F + C + A (FCA), on physiological, biochemical, and yield traits were investigated. The three types showed an induced total chlorophyll content in the host as compared to uninoculated plants. Total soluble sugars and free proline were less regulated by FC and FCA inoculated plants than by F inoculated plants under water deficit conditions. However, the FC and FCA inoculations increased phosphorus content, particularly in the shoots of water-stressed plants. In the three inoculations, the FCA dramatically improved plant osmotic potential adaptability under water deficit stress. Furthermore, even when exposed to the water deficit condition, panicle weight, grain number, and grain maturity were maintained in FCA inoculated plants. According to the findings, the increased osmotic potential and phosphorus content of the FCA-inoculated rice plant provide a protection sign against drought stress and will benefit food security in the future.

Phosphorus and Serendipita indica synergism augments arsenic stress tolerance in rice by regulating secondary metabolism related enzymatic activity and root metabolic patterns.

The multifarious problems created by arsenic (As), for collective environment and human health, serve a cogent case for searching integrative agricultural approaches to attain food security. Rice (Oryza sativa L.) acts as a sponge for heavy metal(loid)s accretion, specifically As, due to anaerobic flooded growth conditions facilitating its uptake. Acclaimed for their positive impact on plant growth, development and phosphorus (P) nutrition, 'mycorrhizas' are able to promote stress tolerance. Albeit, the metabolic alterations underlying Serendipita indica (S. indica; S.i) symbiosis-mediated amelioration of As stress along with nutritional management of P are still understudied. By using biochemical, RT-qPCR and LC-MS/MS based untargeted metabolomics approach, rice roots of ZZY-1 and GD-6 colonized by S. indica, which were later treated with As (10 µM) and P (50 µM), were compared with non-colonized roots under the same treatments with a set of control plants. The responses of secondary metabolism related enzymes, especially polyphenol oxidase (PPO) activities in the foliage of ZZY-1 and GD-6 were enhanced 8.5 and 12-fold, respectively, compared to their respective control counterparts. The current study identified 360 cationic and 287 anionic metabolites in rice roots, and the commonly enriched pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was biosynthesis of phenylalanine, tyrosine and tryptophan, which validated the results of biochemical and gene expression analyses associated with secondary metabolic enzymes. Particularly under As+S.i+P comparison, both genotypes exhibited an upregulation of key detoxification and defense related metabolites, including fumaric acid, L-malic acid, choline, 3,4-dihydroxybenzoic acid, to name a few. The results of this study provided the novel insights into the promising role of exogenous P and S. indica in alleviating As stress.

The Perspective of Arbuscular Mycorrhizal Symbiosis in Rice Domestication and Breeding.

In nature, symbiosis with arbuscular mycorrhizal (AM) fungi contributes to sustainable acquisition of phosphorus and other elements in over 80% of plant species; improving interactions with AM symbionts may mitigate some of the environmental problems associated with fertilizer application in grain crops such as rice. Recent developments of high-throughput genome sequencing projects of thousands of rice cultivars and the discovery of the molecular mechanisms underlying AM symbiosis suggest that interactions with AM fungi might have been an overlooked critical trait in rice domestication and breeding. In this review, we discuss genetic variation in the ability of rice to form AM symbioses and how this might have affected rice domestication. Finally, we discuss potential applications of AM symbiosis in rice breeding for more sustainable agriculture.

Effects of rice-prawn (Macrobrachium nipponense) co-culture on the microbial community of soil.

In the Lixiahe region of China, co-culture has been rapidly promoted in flooded paddy fields owing to its ecological and economic benefits. Rice-prawn co-culture can reduce the damage of crab and shrimp to rice growth and paddy field and substantially change the soil microbial community and soil fertility. In this study, we compared changes in the soil microbial community and soil fertility in waterlogged paddies under conventional rice culture (CR), rice-prawn (Macrobrachium nipponense) co-culture (RP), and pond culture (PC). The microbial abundance in RP was significantly higher than that in CR. RP soil microbial diversity was significantly higher than PC soil microbial diversity. The dominant bacteria in RP soil were Proteobacteria, Chloroflexi, and Bacteroidetes. Compared with those in CR, total organic matter (TOM) and total nitrogen in RP were relatively stable, available potassium and available phosphorus (AP) decreased, and other indicators increased significantly. Soil fertility significantly benefited from co-culture, with total organic carbon (TOC) increasing. Interactive relationship analysis showed that TOM, TOC, AP, and NH4+-N were the main factors affecting the microbial community. Co-occurrence network analyses showed that network modularity increased with co-culture, indicating that a unique soil microbial community formed under co-culture, improving the adaptability and tolerance to co-culture. Thus, RP is a suitable culture method for this commercially important species. The results of this study can inform the practical operation of fertilizer use and sustainable development of rice-prawn aquaculture systems. KEY POINTS: • Microbial abundance and diversity increased under rice-prawn co-culture. • Co-culture significantly improved soil fertility, with an increase in TOC. • Rice-prawn co-culture is an ecologically suitable culture method for prawns.

Effects of elevated ozone on bacterial communities inhabiting the phyllo- and endo-spheres of rice plants.

To explore the effects of elevated ozone (O3) on microbial communities inhabiting phyllo- and endo-spheres of Japonica rice leaves, cultivars Nangeng 5055 (NG5055) and Wuyujing 27 (WYJ27) were grown in either charcoal-filtered air (CF) or elevated O3 (ambient O3 + 40 ppb, E-O3) in field open-top chambers (OTCs) during a growing season. E-O3 increased the values of the Shannon (43-80%) and Simpson (34-51%) indexes of the phyllo-and endo-spheric bacterial communities in NG5055. E-O3 also increased the values of the phyllosphere Simpson index by 58% and the endosphere Shannon index by 54% in WYJ27. Both diversity indexes positively correlated with the contents of nitrogen, phosphorus, magnesium, and soluble sugar, and negatively correlated with the contents of starch and condensed tannins. The leaf-associated bacterial community composition significantly changed in both rice cultivars under E-O3. Moreover, the leaf-associated bacterial communities in NG5055 were more sensitive to E-O3 than those in WYJ27. The chemical properties explained 70% and 98% of variations in the phyllosphere and endosphere bacterial communities, respectively, suggesting a predominant role of chemical status for the endospheric bacterial community. Most variation (57.3%) in the endosphere bacterial community assembly was explained by phosphorus. Gammaproteobacteria and Pantoea were found to be the most abundant class (63-76%) and genus (38-48%) in the phyllosphere and endosphere, respectively. E-O3 significantly increased the relative abundance of Bacteroidetes in the phyllosphere bacterial community and decreased the relative abundance of Gammaproteobacteria in the endophytic community. In conclusion, elevated O3 increased the diversity of bacterial communities of leaf phyllosphere and endosphere, and leaf chemical properties had a more pronounced effect on the endosphere bacterial community.

Antibacterial, anti-biofilm, and anti-virulence potential of tea tree oil against leaf blight pathogen Xanthomonas oryzae pv. oryzae instigates disease suppression.

Bacterial leaf blight (BLB) disease, caused by Xanthomonas oryzae pv. oryzae (Xoo), causes major annual economic losses around the world. Inorganic copper compounds and antibiotics are conventionally used to control BLB disease. They often cause environmental pollution, contributing to adverse effects on human health. Therefore, research is now leading to the search for alternative control methods. Tea tree oil (TTO) is obtained from a traditional medicinal plant, Melaleuca alternifolia, with antibacterial properties. In this study, we found that TTO showed antibacterial activity against Xoo with a minimum inhibitory concentration (MIC) of 18 mg/ml. These antagonistic activities were not limited only to planktonic cells, as further studies have shown that TTO effectively eradicated sessile cells of Xoo in both initial and mature biofilms. Furthermore, it was also observed that TTO reduced various key virulence properties of Xoo, such as swimming, swarming motility, and the production of extracellular polymeric substances, xanthomonadin, and exoenzymes. TTO triggered ROS generation with cell membrane damage as an antibacterial mode of action against Xoo. The in silico study revealed that 1,8-cineole of TTO was effectively bound to two essential proteins, phosphoglucomutase and peptide deformylase, responsible for the synthesis of EPS and bacterial survival, respectively. These antibacterial and anti-virulence activities of TTO against Xoo were further confirmed by an ex vivo virulence assay where TTO significantly reduced the lesion length caused by Xoo on rice leaves. All these data concluded that TTO could be a safe, environment-friendly alternative approach for the comprehensive management of BLB disease.

Discovery of Novel Dihydrolipoamide S-Succinyltransferase Inhibitors Based on Fragment Virtual Screening.

A series of new oxadiazole sulfone derivatives containing an amide moiety was synthesized based on fragment virtual screening to screen high-efficiency antibacterial agents for rice bacterial diseases. All target compounds showed greater bactericidal activity than commercial bactericides. 3-(4-fluorophenyl)-N-((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methyl)acrylamide (10) showed excellent antibacterial activity against Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, with EC50 values of 0.36 and 0.53 mg/L, respectively, which were superior to thiodiazole copper (113.38 and 131.54 mg/L) and bismerthiazol (83.07 and 105.90 mg/L). The protective activity of compound 10 against rice bacterial leaf blight and rice bacterial leaf streak was 43.2% and 53.6%, respectively, which was superior to that of JHXJZ (34.1% and 26.4%) and thiodiazole copper (33.0% and 30.2%). The curative activity of compound 10 against rice bacterial leaf blight and rice bacterial leaf streak was 44.5% and 51.7%, respectively, which was superior to that of JHXJZ (32.6% and 24.4%) and thiodiazole copper (27.1% and 28.6%). Moreover, compound 10 might inhibit the growth of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola by affecting the extracellular polysaccharides, destroying cell membranes, and inhibiting the enzyme activity of dihydrolipoamide S-succinyltransferase.

Bioactive Peptide F2d Isolated from Rice Residue Exerts Antioxidant Effects via Nrf2 Signaling Pathway.

Studies have shown that the peroxidation caused by oxygen free radicals is an important reason of vascular endothelial dysfunction and multiple diseases. In this study, active peptides (F2ds) were isolated from the fermentation product of rice dregs and its antioxidant effects were approved. Human umbilical vein endothelial cells (HUVECs) stimulated by H2O2 were used to evaluate the antioxidation effect and its molecular mechanism in the oxidative stress model. F2d protected H2O2-induced damage in HUVECs in a dosage-dependent manner. F2d can reduce the expression of Keap1, promote the expression of Nrf2, and activate the downstream target HO-1, NQO1, etc. It means F2d can modulate the Nrf2 signaling pathway. Using Nrf2 inhibitor ML385 to block the Nrf2 activation, the protective function of F2d is partially lost in the damage model. Our results indicated that F2d isolated from rice exerts antioxidant effects via the Nrf2 signaling pathway in H2O2-induced damage, and the work will benefit to develop functional foods.

Chemical or Genetic Alteration of Proton Motive Force Results in Loss of Virulence of Burkholderia glumae, the Cause of Rice Bacterial Panicle Blight.

Rice is an important source of food for more than half of the world's population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. B. glumae synthesizes toxoflavin, an essential virulence factor that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis, named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae ΔdbcA strain. In addition to polymyxin sensitivity, the B. glumae ΔdbcA strain is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (∼15% of wild-type levels). Changes in membrane potential in the B. glumae ΔdbcA strain were reproduced in the wild-type strain by the addition of subinhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice, suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild-type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.

Growth Restriction of Rhizoctonia solani via Breakage of Intracellular Organelles Using Crude Extracts of Gallnut and Clove.

Plant diseases reduce crop yield and quality, hampering the development of agriculture. Fungicides, which restrict chemical synthesis in fungi, are the strongest controls for plant diseases. However, the harmful effects on the environment due to continued and uncontrolled utilization of fungicides have become a major challenge in recent years. Plant-sourced fungicides are a class of plant antibacterial substances or compounds that induce plant defenses. They can kill or inhibit the growth of target pathogens efficiently with no or low toxicity, they degrade readily, and do not prompt development of resistance, which has led to their widespread use. In this study, the growth inhibition effect of 24 plant-sourced ethanol extracts on rice sprigs was studied. Ethanol extract of gallnuts and cloves inhibited the growth of bacteria by up to 100%. Indoor toxicity measurement results showed that the gallnut and glove constituents inhibition reached 39.23 µg/mL and 18.82 µg/mL, respectively. Extract treated rice sprigs were dry and wrinkled. Gallnut caused intracellular swelling and breakage of mitochondria, disintegration of nuclei, aggregation of protoplasts, and complete degradation of organelles in hyphae and aggregation of cellular contents. Protection of Rhizoctonia solani viability reached 46.8% for gallnut and 37.88% for clove in water emulsions of 1000 µg/mL gallnut and clove in the presence of 0.1% Tween 80. The protection by gallnut was significantly stronger than that of clove. The data could inform the choice of plant-sourced fungicides for the comprehensive treatment of rice sprig disease. The studied extract effectively protected rice sprigs and could be a suitable alternative to commercially available chemical fungicides. Further optimized field trials are needed to effectively sterilize rice paddies.

Tandem DNA repeats contain cis-regulatory sequences that activate biotrophy-specific expression of Magnaporthe effector gene PWL2.

During plant infection, fungi secrete effector proteins in coordination with distinct infection stages. Thus, the success of plant infection is determined by precise control of effector gene expression. We analysed the PWL2 effector gene of the rice blast fungus Magnaporthe oryzae to understand how effector genes are activated specifically during the early biotrophic stages of rice infection. Here, we used confocal live-cell imaging of M. oryzae transformants with various PWL2 promoter fragments fused to sensitive green fluorescent protein reporter genes to determine the expression patterns of PWL2 at the cellular level, together with quantitative reverse transcription PCR analyses at the tissue level. We found PWL2 expression was coupled with sequential biotrophic invasion of rice cells. PWL2 expression was induced in the appressorium upon penetration into a living rice cell but greatly declined in the highly branched hyphae when the first-invaded rice cell was dead. PWL2 expression then increased again as the hyphae penetrate into living adjacent cells. The expression of PWL2 required fungal penetration into living plant cells of either host rice or nonhost onion. Deletion and mutagenesis experiments further revealed that the tandem repeats in the PWL2 promoter contain 12-base pair sequences required for expression. We conclude that PWL2 expression is (a) activated by an unknown signal commonly present in living plant cells, (b) specific to biotrophic stages of fungal infection, and (c) requires 12-base pair cis-regulatory sequences in the promoter.

Structural Alteration of Rice Pectin Affects Cell Wall Mechanical Strength and Pathogenicity of the Rice Blast Fungus Under Weak Light Conditions.

Pectin, a component of the plant cell wall, is involved in cell adhesion and environmental adaptations. We generated OsPG-FOX rice lines with little pectin due to overexpression of the gene encoding a pectin-degrading enzyme [polygalacturonase (PG)]. Overexpression of OsPG2 in rice under weak light conditions increased the activity of PG, which increased the degradation of pectin in the cell wall, thereby reducing adhesion. Under weak light conditions, the overexpression of OsPG decreased the pectin content and cell adhesion, resulting in abnormally large intercellular gaps and facilitating invasion by the rice blast fungus. OsPG2-FOX plants had weaker mechanical properties and greater sensitivity to biotic stresses than wild-type (WT) plants. However, the expression levels of disease resistance genes in non-infected leaves of OsPG2-FOX were more than twice as high as those of the WT and the intensity of disease symptoms was reduced, compared with the WT. Under normal light conditions, overexpression of OsPG2 decreased the pectin content, but did not affect cell adhesion and sensitivity to biotic stresses. Therefore, PG plays a role in regulating intercellular adhesion and the response to biotic stresses in rice.

Gamma-irradiated fenugreek extracts mediates resistance to rice blast disease through modulating histochemical and biochemical changes.

The impact of gamma radiation on the activation of rice innate immunity to blast disease caused by Magnaporthe oryzae is described. In the present study, fenugreek seed extracts radiated with different doses of gamma rays viz. 5Gy, 10Gy, 15Gy, 20Gy and 25Gy were examined for their presence of biocompounds as well as for its ability to induce plant growth promotion and resistance against rice blast disease. The results of GC-MS analysis detected antimicrobial properties in methanolic extract. Enhanced germination (97%) and vigor (2718) was noticed in seeds pretreated with 20 Gy of gamma radiation in comparison with non-irradiated controls. Under greenhouse conditions, a significant disease protection of 56.7% on 3rd and 4th day after inoculation against rice blast was observed in 15Gy-irradiated rice plants challenge-inoculated with M. oryzae. Further, a significant increase in the hydrogen peroxide, phenol and lignin deposition was noticed in 20Gy-irradiated rice plants. Additionally, rice plants pretreated with 15Gy induced maximum activities of peroxidase (POX) and polyphenol oxidase (PPO) compared to untreated control plants. These findings revealed that rice plants-pretreated with gamma radiation elicit resistance against rice blast disease as well as strengthening the growth parameters by modulating cellular and biochemical defense system.

A novel aqueous extract from rice fermented with Aspergillus oryzae and Saccharomyces cerevisiae possesses an anti-influenza A virus activity.

Human influenza virus infections occur annually worldwide and are associated with high morbidity and mortality. Hence, development of novel anti-influenza drugs is urgently required. Rice Power® extract developed by the Yushin Brewer Co. Ltd. is a novel aqueous extract of rice obtained via saccharization and fermentation with various microorganisms, such as Aspergillus oryzae, yeast [such as Saccharomyces cerevisiae], and lactic acid bacteria, possessing various biological and pharmacological properties. In our previous experimental screening with thirty types of Rice Power® extracts, we observed that the 30th Rice Power® (Y30) extract promoted the survival of influenza A virus-infected Madin-Darby canine kidney (MDCK) cells. Therefore, to identify compounds for the development of novel anti-influenza drugs, we aimed to investigate whether the Y30 extract exhibits anti-influenza A virus activity. In the present study, we demonstrated that the Y30 extract strongly promoted the survival of influenza A H1N1 Puerto Rico 8/34 (A/PR/8/34), California 7/09, or H3N2 Aichi 2/68 (A/Aichi/2/68) viruses-infected MDCK cells and inhibited A/PR/8/34 or A/Aichi/2/68 viruses infection and growth in the co-treatment and pre-infection experiments. The pre-treatment of Y30 extract on MDCK cells did not induce anti-influenza activity in the cell. The Y30 extract did not significantly affect influenza A virus hemagglutination, and neuraminidase and RNA-dependent RNA polymerase activities. Interestingly, the electron microscopy experiment revealed that the Y30 extract disrupts the integrity of influenza A virus particles by permeabilizing the viral membrane envelope, suggesting that Y30 extract has a direct virucidal effect against influenza A virus. Furthermore, we observed that compared to the ethyl acetate (EtOAc) extract, the water extract of Y30 extract considerably promoted the survival of cells infected with A/PR/8/34 virus. These results indicated that more anti-influenza components were present in the water extract of Y30 extract than in the EtOAc extract. Our results highlight the potential of a rice extract fermented with A. oryzae and S. cerevisiae as an anti-influenza medicine and a drug source for the development of anti-influenza compounds.