Rice Pathogen Research Articles

Xanthomonas oryzae Pv. oryzicola Response Regulator VemR Is Co-opted by the Sensor Kinase CheA for Phosphorylation of Multiple Pathogenicity-Related Targets

Two-component systems (TCSs) (cognate sensor histidine kinase/response regulator pair, HK/RR) play a crucial role in bacterial adaptation, survival, and productive colonization. An atypical orphan single-domain RR VemR was characterized by the non-vascular pathogen Xanthomonas oryzae pv. oryzicola (Xoc) is known to cause bacterial leaf streak (BLS) disease in rice. Xoc growth and pathogenicity in rice, motility, biosynthesis of extracellular polysaccharide (EPS), and the ability to trigger HR in non-host tobacco were severely compromised in the deletion mutant strain RΔvemR as compared to the wild-type strain RS105. Site-directed mutagenesis and phosphotransfer experiments revealed that the conserved aspartate (D56) residue within the stand-alone phosphoacceptor receiver (REC) domain is essential for phosphorelay and the regulatory activity of Xoc VemR. Yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) data identified CheA as the HK co-opting the RR VemR for phosphorylation. Affinity proteomics identified several downstream VemR-interacting proteins, such as 2-oxoglutarate dehydrogenase (OGDH), DNA-binding RR SirA, flagellar basal body P-ring formation protein FlgA, Type 4a pilus retraction ATPase PilT, stress-inducible sensor HK BaeS, septum site-determining protein MinD, cytoskeletal protein CcmA, and Type III and VI secretion system proteins HrpG and Hcp, respectively. Y2H and deletion mutant analyses corroborated that VemR interacted with OGDH, SirA, FlgA, and HrpG; thus, implicating multi-layered control of diverse cellular processes including carbon metabolism, motility, and pathogenicity in the rice. Physical interaction between VemR and HrpG suggested cross-talk interaction between CheA/VemR- and HpaS/HrpG-mediated signal transduction events orchestrating the hrp gene expression.

A secreted fungal effector suppresses rice immunity through host histone hypoacetylation.

Histone acetylation is a critical epigenetic modification that regulates plant immunity. Fungal pathogens secrete effectors that modulate host immunity and facilitate infection, but whether fungal pathogens have evolved effectors that directly target plant histone acetylation remains unknown. Here, we identified a secreted protein, UvSec117, from the rice false smut fungus, Ustilaginoidea virens, as a key effector that can target the rice histone deacetylase OsHDA701 and negatively regulates rice broad-spectrum resistance against rice pathogens. UvSec117 disrupts host immunity by recruiting OsHDA701 to the nucleus and enhancing OsHDA701-modulated deacetylation, thereby reducing histone H3K9 acetylation levels in rice plants and interfering with defense gene activation. Host-induced gene silencing of UvSec117 promotes rice resistance to U. virens, thus providing an alternative way for developing rice false smut-resistant plants. This is the first direct evidence demonstrating that a fungal effector targets a histone deacetylase to suppress plant immunity. Our data provided insight into a counter-defense mechanism in a plant pathogen that inactivates host defense responses at the epigenetic level.

Deep learning uncovers distinct behavior of rice network to pathogens response

SummaryRice, apart from abiotic stress, is prone to attack from multiple pathogens. Predominantly, the two rice pathogens, bacterial Xanthomonas oryzae (Xoo) and hemibiotrophic fungus, Magnaporthe oryzae, are extensively well explored for more than the last decade. However, because of lack of holistic studies, we design a deep learning-based rice network model (DLNet) that has explored the quantitative differences resulting in the distinct rice network architecture. Validation studies on rice in response to biotic stresses show that DLNet outperforms other machine learning methods. The current finding indicates the compactness of the rice PTI network and the rise of independent modules in the rice ETI network, resulting in similar patterns of the plant immune response. The results also show more independent network modules and minimum structural disorderness in rice-M. oryzae as compared to the rice-Xoo model revealing the different adaptation strategies of the rice plant to evade pathogen effectors.

Seed treatments to control Burkholderia glumae on rice seeds in the screenhouse

Burkholderia glumae is a rice seed-borne pathogen that cause severe yield which have impact to the food sustainability in the future. As none of control methods was effective, seed treatment is an alternative control technique to manage the disease. This study was aimed to determine the effective control method using liquid smoke, clove oil, citronella oil and copper hydroxide fungicide against B. glumae bacteria at the screen house, Faculty of Agriculture, Universitas Sumatera Utara, Medan, from December 2020 to May 2021. This study used a completely randomized design (CRD) non-factorial with 10 treatments, including positive control, negative control, 5% and 7% of liquid smoke, 2% of clove oil, 1% and 3% of citronella oil, 2% and 5% of copperhydroxide fungicides, with 3 replications. The results showed that the lowest percentage of disease incidence was the rice plants treated by 2% and 5% copperhydroxide fungicides (0.71%). The lowest percentage of disease severity was found in the copper hydroxide 2% and 5% of fungicide treatment (0.71%). The 5% copper hydroxide fungicide treatment had the lowest mean percentage of empty grain, (9.11%). The 5% of clove oil treatment gave the highest rice grain production, with an average of 57.50 g.

Biocontrol Potential of Arbuscular Mycorrhizal Fungi on Soil-borne Pathogens in Rice and Other Crops

Arbuscular mycorrhizal fungi (AMF) are soil-borne fungi that form mutualistic symbiosis with plant roots, and can signifi cantly improve plant nutrient uptake and increase resistance to several biotic and abiotic stresses. Though most rice cultivation systems involved submerged growing conditions, researchers are exploring the potential of AMF colonization and its benefi ts for rice. AMF has a potential of serving as a biocontrol agent in rice plants via direct mechanisms viz. nutrient improvement, competition, integration, morphological, biochemical, and physiological alteration and indirectly via induction of systemic induced resistance (ISR) in plants. Furthermore, other biotic factors (host genotype, age of the plant, species of AMF that is involved, degree of mycorrhization, presence of weed plants) and abiotic factors (soil temperature, moisture, nutrient content, and atmospheric gas composition, crop management systems) can be affected the process of biocontrol by AMF. These different mechanisms cannot be considered completely independent from one another. The potential for biocontrol by AMF depends mainly on all these mechanisms in conjunction with all of the other biotic and abiotic factors that affect mycorrhization. By considering such mechanisms, in this review we discussed the suitability of the application of AMF as a pathogen biocontrol agent in rice and the other crop plants, while reducing the application of chemical agents to control the diseases. However, for considering AMF as a better alternative for the synthetic agrochemicals, more research should be conducted by applying AMF and synthetic chemicals to compare the levels of pathogen control.

Transcriptome Analysis and SNP Identification Reveal That Heterologous Overexpression of Two Uncharacterized Genes Enhances the Tolerance of Magnaporthe oryzae to Manganese Toxicity.

ABSTRACTManganese is a crucial trace element that constitutes the cofactors of many enzymes. However, excessive Mn2+ can be toxic for both prokaryotes and eukaryotes. The mechanism of fungal genetics and metabolism in response to Mn2+ stress remains understudied, warranting further studies. Magnaporthe oryzae is well-established as the most destructive pathogen of rice. A field strain, YN2046, more sensitive to Mn2+ toxicity than other strains, was obtained from a previous study. Herein, we explored the genetic mechanisms of Mn2+ sensitivity in YN2046 through comparative transcriptomic analyses. We found that many genes previously reported to participate in Mn2+ stress were not regulated in YN2046. These non-responsive genes might cause Mn2+ sensitivity in YN2046. Weight gene correlation network analysis (WGCNA) was performed to characterize the expression profile in YN2046. Some overexpressed genes were only found in the Mn2+ tolerant isolate YN125. Among these, many single nucleotide polymorphism (SNP) were identified between YN125 and YN2046, which might disrupt the expression levels of Mn responsive genes. We cloned two uncharacterized genes, MGG_13347 and MGG_16609, from YN125 and transformed them to YN2046 with a strong promoter. Our results showed that the heterologous overexpression of two genes in YN2046 restored its sensitivity. Transcriptomic and biochemical analyses were performed to understand Mn tolerance mechanisms mediated by the two heterologous overexpressed genes. Our results showed that heterologous overexpression of these two genes activated downstream gene expression and metabolite production to restore M. oryzae sensitivity to Mn, implying that SNPs in responsive genes account for different phenotypes of the two strains under Mn stress.IMPORTANCE Heavy metals are used for fungicides as they target phytopathogen in multiple ways. Magnaporthe oryzae is the most destructive rice pathogen and is threatening global rice production. In the eukaryotes, the regulation mechanisms of Mn homeostasis often focus on the posttranslation, there were a few results about regulation at transcript level. The comparative transcriptome analysis showed that fewer genes were regulated in the Mn-sensitive strain. WGCNA and SNP analyses found that mutations in promoter and coding sequence regions might disrupt the expression of genes involved in Mn detoxification in the sensitive strain. We transferred two unannotated genes that were cloned from the Mn-tolerant strain into a sensitive strain with strong promoters, and the transformants exhibited an enhanced tolerance to Mn2+ toxicity. Transcriptome and biochemistry results indicated that heterologous overexpression of the two genes enhanced the tolerance to Mn toxicity by reactivation of downstream genes in M. oryzae.

A Seed-Borne Bacterium of Rice, Pantoea dispersa BB1, Protects Rice from the Seedling Rot Caused by the Bacterial Pathogen Burkholderia glumae.

Seedling rot, caused by the bacterial pathogen Burkholderia glumae, is a major disease of rice. It originates from pathogen-contaminated seeds and is thus mainly controlled by pesticide treatments of seeds. We previously demonstrated that the seed-borne bacteria of rice may be a useful and sustainable alternative to pesticides to manage seedling rot, but they are limited in terms of variety. Here, we report that another seed-borne bacterium, Pantoea dispersa BB1, protects rice from B. glumae. We screened 72 bacterial isolates from rice seeds of three genetically different cultivars inoculated or non-inoculated with B. glumae. 16S rRNA gene sequencing revealed that pathogen inoculation affected the composition of culturable seed-borne bacterial communities and increased the presence of Pantoea and Paenibacillus species. Among three Pantoea and Paenibacillus isolates that exhibit tolerance to toxoflavin, a virulence factor of B. glumae, P. dispersa BB1 significantly mitigated the symptoms of rice seedling rot. The culture filtrate of BB1 inhibited the growth of B. glumae in vitro, suggesting that this isolate secretes antibacterial compounds. Seed treatment with BB1 suppressed pathogen propagation in plants, although seed treatment with the culture filtrate did not. Because BB1 did not show pathogenicity in rice, our findings demonstrate that BB1 is a promising biocontrol agent against seedling rot.

Streptomyces spinosus sp. nov. and Streptomyces shenzhenensis subsp. oryzicola subsp. nov. endophytic actinobacteria isolated from Jasmine rice and their genome mining for potential as antibiotic producers and plant growth promoters.

Two endophytic actinobacteria, strains SBTS01T and W18L9T, were isolated from leaf sheath and leaf tissue, respectively, of Jasmine rice (Oryza sativa KDML 105) grown in a rice paddy field in Roi EtProvince, Thailand. A polyphasic taxonomic study showed that both strains belong to the genus Streptomyces; they are aerobic, forming well-developed substrate mycelia and aerial mycelia with long chains of spores. Strain SBTS01T shares high 16S rRNA gene sequence similarity with Streptomyces rochei NRRL B-2410T (99.0%) and Streptomyces naganishii NRRL ISP-5282T (99.0%). Strain W18L9T shares high 16S rRNA gene sequence similarity with Streptomyces shenzhenensis DSM 42034T (99.7%). The genotypic and phenotypic properties of strains SBTS01T and W18L9T distinguish these two strains from the closely related species with validly published names. The genome analysis showed the dDDH, ANIb and ANIm values of the draft genome between strain SBTS01T and its close neighbour in the phylogenomic tree, Streptomyces corchorusii DSM 40340T to be 54.1, 92.6, and 94.3%, respectively; similarly for strain W18L9T and the closely related species S. shenzhenensis DSM 42034T values were 72.5, 95.1 and 97.0%. The name proposed for the new species represented by the type strain SBTS01T is Streptomyces spinosus (= NRRL B-65636T = TBRC 15052T). The name proposed for the novel subspecies of strain W18L9T is Streptomyces shenzhenensis subsp. oryzicola (= NRRL B-65635T = TBRC 15051T). Recognition of this subspecies also permits the description of Streptomyces shenzhenensis subsp. shenzhenensis. Strains SBTS01T and W18L9T can produce antibiotic against rice and human pathogens and showed plant growth promoting properties such as production of indole acetic acid, cytokinin, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, siderophores and cellulase. Genomic data mining of these two strains confirmed their potential as antibiotic producers and plant growth promoters. Their genomes contain multiple biosynthetic gene clusters including those for terpene, type 1, 2 and 3 polyketide synthase, Non-ribosomal peptide synthetase and lanthipeptides. Genes encoding plant growth promoting traits such; nitrogen fixation, ACC deaminase, siderophore production and stress-related adaption may have ecological significance.

Genome Re-Sequencing Reveals the Host-Specific Origin of Genetic Variation in Magnaporthe Species.

Rice blast is caused by Magnaporthe oryzae (M. oryzae), which is considered one of the most serious pathogens of rice around the globe. It causes severe losses owing to its proven capability to disrupt the host resistance. Recently, its invasion of new hosts like the Musa species or banana plants has been noticed. To understand the possible level of genetic variation, we sequenced the genomes of eight different isolates of the Magnaporthe species infecting rice, Digitaria (a weed), finger millet, Elusine indica, and banana plants. Comparative genomic analysis of these eight isolates with the previously well-characterized laboratory strain M. oryzae 70-15 was made. The infectivity of the newly isolated strain from Musa species suggested that there is no resistance level in the host plants. The sequence analysis revealed that despite genome similarities, both the banana and Digitaria isolates have relatively larger genome sizes (∼38.2 and 51.1 Mb, respectively) compared to those of the laboratory reference strain M. oryzae 70-15 (∼37 Mb). The gene contraction, expansion, and InDel analysis revealed that during evolution, a higher number of gene insertions and deletions occurred in the blast fungus infecting Digitaria and banana. Furthermore, each genome shared thousands of genes, which suggest their common evolution. Overall, our analysis indicates that higher levels of genes insertion or deletions and gain in the total genome size are important factors in disrupting the host immunity and change in host selection.

Biopriming for induction of disease resistance against pathogens in rice.

Rice is attacked by an armada of pathogens. Present review provides a critical evaluation of the potential of different biotic agents used to protect rice yield drop from pathogenicity and an account of unexplored areas, which might be taken into consideration to manage rice diseases. Rice (Oryza sativa L.), is the most important staple food of Asian countries. Rice production is significantly limited by a diversity of pathogens, leading to yield loss and deficit in current rice supply. Application of agrochemicals of diverse types has been considered as the only option to control pathogens and enhance rice production, thereby causing environmental concerns and making the pathogens resistant to the active ingredients. Increase in population and resistance of pathogen towards agrochemicals put pressure on the agronomists to search for safe, novel, eco-friendly alternative ways to manage rice pathogens. Inducing resistance in rice by using different biotic/abiotic agents provides an environmental friendly alternative way to effectively manage bacterial, fungal, and viral rice pathogens. In recent years, a number of protocols have been developed for inducing pathogen resistance by bio-priming of rice. However, a comprehensive evaluation of the potential of different biotic agents to protect rice crop loss from pathogens is hitherto lacking due to which the research on induction of defense against pathogens in rice is discontinuous. This review deals with the detailed analysis of the bacterial and fungal agents used to induce defense against rice pathogens, their mode of application, mechanism (physiological, biochemical, and molecular) of defense induction, and effect of defense induction on the yield of rice. It also provides an account of gaps in the research and the unexplored areas, which might be taken into consideration to effectively manage rice pathogens.

Characterization of the antagonism of the mycorrhizal fungus Waitea circinata against Magnaporthe oryzae, Cochliobolus miyabeanus, Monographella albescens and Sarocladium oryzae rice pathogens

Rice production is affected by important pathogens such as Magnaporthe oryzae, Cochliobolus miyabeanus, Monographella albescens and Sarocladium oryzae, and orchid mycorrhizal fungi can contribute to the biocontrol of these diseases. The aim of this study was to characterize the antagonism mechanisms of W. circinata against rice pathogens. The indoleacetic acid (IAA) and lytic enzymes of W. circinata were quantified. The effectiveness of the bioagent and its metabolites was assessed in vitro. Antagonism in vivo against rice blast was also investigated. Waitea circinata produced IAA (2.3 µg ml-1), cellulase and polyphenol oxidase in a qualitative test. Glucanase, chitinase and protease were also produced in culture with cell walls and in co-culture with pathogens to explain antibiosis. W. circinata acts on direct antagonism by antibiosis and volatile metabolites. Mycelial suspension reduced M. oryzae conidial germination, appressorium formation, and rice blast by 61, 82 and 84%, respectively. W. circinata, a unique bioagent, controls four rice pathogens from mechanisms of parasitism and antibiosis. The results could contribute to new formulations containing this fungus, enzymes as well as its volatile metabolites.

Rice diterpenoid phytoalexins are involved in defence against parasitic nematodes and shape rhizosphere nematode communities.

Rice diterpenoid phytoalexins (DPs) are secondary metabolites with a well known role in resistance to foliar pathogens. As DPs are also known to be produced and exuded by rice roots, we hypothesised that they might play an important role in plant-nematode interactions, and particularly in defence against phytoparasitic nematodes. We used transcriptome analysis on rice roots to analyse the effect of infection by the root-knot nematode Meloidogyne graminicola or treatment with resistance-inducing chemical stimuli on DP biosynthesis genes, and assessed the susceptibility of mutant rice lines impaired in DP biosynthesis to M. graminicola. Moreover, we grew these mutants and their wild-type in field soil and used metabarcoding to assess the effect of impairment in DP biosynthesis on rhizosphere and root nematode communities. We show that M. graminicola suppresses DP biosynthesis genes early in its invasion process and, conversely, that resistance-inducing stimuli transiently induce the biosynthesis of DPs. Moreover, we show that loss of DPs increases susceptibility to M. graminicola. Metabarcoding on wild-type and DP-deficient plants grown in field soil reveals that DPs significantly alter the composition of rhizosphere and root nematode communities. Diterpenoid phytoalexins are important players in basal and inducible defence against nematode pathogens of rice and help shape rice-associated nematode communities.

Production of the plant hormone gibberellin by rhizobia increases host legume nodule size.

Plant-associated microbes have evolved the ability to independently produce gibberellin (GA) phytohormones as a mechanism to influence their host. Indeed, GA was first discovered as a metabolite from the fungal rice pathogen Gibberella fujikuroi, which uses it as a virulence factor. Though some bacterial plant pathogens similarly use GA to promote infection, symbiotic nitrogen-fixing bacteria (rhizobia), which inhabit the root nodules of legumes, also can produce GA, suggesting a role in symbiosis. The bacterial GA biosynthetic operon has been identified, but in rhizobia this typically no longer encodes the final metabolic gene (cyp115), so that these symbionts can only produce the penultimate intermediate GA9. Here, we demonstrate that soybean (Glycine max) expresses functional GA 3-oxidases (GA3ox) within its nodules, which have the capability to convert GA9 produced by the enclosed rhizobial symbiont Bradyrhizobium diazoefficiens to bioactive GA4. This rhizobia-derived GA is demonstrated to cause an increase in nodule size and decrease in the number of nodules. The increase in individual nodule size correlates to greater numbers of bacterial progeny within a nodule, thereby providing a selective advantage to rhizobia that produce GA during the rhizobia-legume symbiosis. The expression of GA3ox in nodules and resultant nodulation effects of the GA product suggests that soybean has co-opted control of bioactive GA production, and thus nodule size, for its own benefit. Thus, our results suggest rhizobial GA biosynthesis has coevolved with host plant metabolism for cooperative production of a phytohormone that influences nodulation in a mutually beneficial manner.

The Effects of Rhizobium Inoculation On The Growth Of Rice (Oryza Sativa L.) and White Radish (Raphanus Sativus L.)

The utilization of chemical fertilizers in agriculture production could cause harmful effects on human health and the environment, therefore the development and application of biofertilizers produced from beneficial microorganisms like plant growth-promoting rhizobacteria have been increasingly encouraged. The present study investigated the effects of the inoculation of the two Rhizobium pusense strains (R. pusense GCB108 and R. pusense GCB117) on the growth of the young rice plant (Oryza sativa L.) and white radish (Raphanus sativus L.). The results showed that the inoculation of the two R. pusense strains affected the growth of rice and white radish considerably. On day 7, the shoot length of Oryza sativa L. grown on the medium inoculated with the R. pusense GCB108 and GCB117 was 22.3 ± 0.42 cm and 21.6 ± 2.26 cm, respectively, while that recorded in the control sample (without bacterial inoculation) was only 18.25 ± 1.06 cm. On day 9, the shoot length of Raphanus sativus L. grown under the hydroponic condition and inoculated with the R. pusense GCB108 and GCB117 was 12.68 ± 1.22 cm and 13.69 ± 1.47 cm, respectively, while that recorded in the control sample was much lower (9.04 ± 1.86 cm). Moreover, the inoculation of R. pusense GCB108 and GCB117 also restricted the growth of the rice pathogen Pseudomonas oryzihabitans RL01 in the rice rhizosphere. Furthermore, the results from the biophysiological assay showed that both two strains R. pusense GCB108 and GCB117 could grow under a salinity environment (salt concentration of 3%) and a wide range of pH conditions (pH 5 – 11). The study demonstrates the importance of the R. pusense GCB108 and GCB117 inoculation in the growth of rice and white radish and suggests the potential application of those two strains in biofertilizer production to improve soil quality and stimulate plant growth, contributing to sustainable agriculture development.

Whole-Genome Sequence Resource of Fusarium oxysporum Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from Tetrastigma hemsleyanum

Whole-Genome Sequence Resource of <i>Fusarium oxysporum</i> Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from <i>Tetrastigma hemsleyanum</i>

An MKP-MAPK protein phosphorylation cascade controls vascular immunity in plants.

Global crop production is greatly reduced by vascular diseases. These diseases include bacterial blight of rice and crucifer black rot caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas campestris pv. campestris (Xcc). The molecular mechanisms that activate vascular defense against such pathogens remains underexplored. Here, we show that an Arabidopsis MAPK phosphatase 1 (MKP1) mutant has increased host susceptibility to the adapted pathogen Xcc and is compromised in nonhost resistance to the rice pathogen Xoo. MKP1 regulates MAPK-mediated phosphorylation of the transcription factor MYB4 that negatively regulates vascular lignification through inhibiting lignin biosynthesis. Induction of lignin biosynthesis is, therefore, an important part of vascular-specific immunity. The role of MKP-MAPK-MYB signaling in lignin biosynthesis and vascular resistance to Xoo is conserved in rice, indicating that these factors form a tissue-specific defense regulatory network. Our study likely reveals a major vascular immune mechanism that underlies tissue-specific disease resistance against bacterial pathogens in plants.

Effects of Marine Antagonistic Fungi against Plant Pathogens and Rice Growth Promotion Activity

Ten marine-derived fungi crude extracts, namely Emericella stellatus KUFA0208, Eupenicillium parvum KUFA0237, Neosartorya siamensis KUFA0514, N. spinosa KUFA 0528, Talaromyces flavus KUFA 0119, T. macrosporus KUFA 0135, T. trachyspermus KUFA0304, Trichoderma asperellum KUFA 0559, T. asperellum KUFA 0559 and T. harzianum KUFA 0631 were determined for their fungicidal activity against five rice pathogens in vitro. The results showed that the extracts of E. stellatus KUFA0208 and N. siamensis KUFA0514 exhibited the best antifungal activity, causing complete cessation of the mycelial growth of Alternaria padwickii, Bipalaris oryzae, Fusarium semitectum, Pyricularia oryzae and Rhizoctonia solani at 10 g/L. The N. siamensis KUFA0514 extract was fractioned and antifungal compounds were found in the fractions derived from petroleum-ether and chloroform (7: 3) evidenced by inhibition zones against the mycelial growth of A. padwickii around the disc containing each fraction. Moreover, in rice growth promotion tests, diluted cultural broth of T. asperellum KUFA 0559 and T. harzianum KUFA 0631 were found to strongly promote rice shoot and root elongation; however, higher concentrations of all marine fungal broths resulted in significantly reduced rice seedling growth rather than promotion. Meanwhile, Trichoderma showed great indole-3-acetic acid (IAA) production leading to the optimum IAA values of 45.38 and 52.30 µg/ml at 11 and 13 days after inoculation, respectively. The results of this study indicated that marine fungi are promising agents having antagonistic mechanisms involving antibiosis production and plant growth promotion and may be developed as novel biocontrol agents for rice disease management.

Isolation and Characterization of Bacillus Consortia for Plant Growth Promotion in Rice (Oryza sativa L.)

In this study, soil samples from the rhizosphere of various uncultivated weeds were collected from fifteen different locations of Gujarat. Heat treatment was given at 65°C for 20 minutes prior to initial screening for spore-forming Bacillus spp. Among them, 20 nitrogen-fixing (NFB), 27 phosphate solubilising (PSB) and 15 potassium mobilizing (KMB) isolates were screened primarily. After molecular identification only Bacillus isolates were further selected and characterized. Three superior Bacillus isolates were selected from each category by secondary screening. All isolates belonging to different category were compatible in nature and showed significant ammonia production, ARA, phosphate solubilisation, potassium mobilization, siderophore production, IAA production and organic acid producers. The relative expression analysis of three genes NRT2.1 (Nitrate transporter), PT6 (Phosphorus transporter), and AKT1 (Potassium transporter) at transcriptional level were performed in the juvenile root tissues of Rice using qRT-PCR technique at 15th, 30th and 45th days after transplanting with Actin as a internal control. Rice transplants were inoculated with different NPK fixing Bacillus consortia, which significantly improved growth parameters as compared to control in field study. Various growth and yield attributing characters of rice viz., plant height (at 60 DAT, 90 DAT, and at harvesting), number of productive tillers, days to 50 % flowering, 100 seed weight, grain yield, straw yield, as well as dry biomass per hectare were significantly influenced by different treatments. An application of 100 % RDF with NPK consortia (T7) treatment of rice crop resulted in significantly higher plant height (49.25 cm at 60 DAT, 107.00 cm at 90 DAT, 129.50 cm at harvesting), days to 50% flowering (75.63), 100 seed weight (3.53 g), number of productive tillers per plant (12.13), grain yield (6889.00 kg ha-1), straw yield (8754.25 kg ha-1) and dry biomass (7889.25 kg ha-1) followed by the treatments T4 (T2 + N consortia), T5 (T2 + P consortia), T6 (T2 + K consortia), and T2 (RDF (NP) 100%). Nutrient content in grain and straw was differing significantly due to different treatments. However, significantly higher N, P, K, content in straw (1.28 %, 0.15 %, and 1.52 %) and grain (2.06 %, 0.23 %, and 0.25 %) respectively, were recorded under the treatment T7. The spore forming Bacillus consortia was able to survive at a wide range of temperature and pH fluctuations and found to be effective as N-fixers, P-solubilizers, K-mobilizers, siderophore producers, IAA producers with having antagonistic activity against rice pathogen Magnaportha oryzae.

Intracellular Ca2+ accumulation triggered by caffeine provokes resistance against a broad range of biotic stress in rice.

Chemical pesticides are still frequently overused to diminish such crop loss caused by biotic stress despite the threat to humans and the environment. Thus, it is urgent to find safer and more effective defense strategies. In this study, we report that caffeine, implanted through a transgenic approach, enhances resistance against variable biotic stresses in rice without fitness cost. Caffeine-producing rice (CPR) was generated by introducing three N-methyltransferase genes involved in the biosynthesis of caffeine in coffee plants. The CPR plants have no differences in morphology and growth compared to their wild-type counterparts, but they show strongly enhanced resistance to both bacterial leaf blight, rice blast, and attack of white-backed planthoppers. Caffeine acts as a repellent agent against rice pathogens. Moreover, caffeine triggers a series of Ca2+ signalling-like processes to synthesize salicylic acid (SA), a hormone associated with plant resistance. In CPR, phosphodiesterase was inhibited by caffeine, cAMP and cGMP increased, intracellular Ca2+ increased, phenylalanine lyase (PAL) was activated by OsCPK1, and SA synthesis was activated. This finding is a novel strategy to improve resistance against the biotic stresses of crops with a special type of defense inducer.

PAM-free loop-mediated isothermal amplification coupled with CRISPR/Cas12a cleavage (Cas-PfLAMP) for rapid detection of rice pathogens

Pathogenic disease is an important factor affecting rice growth, yield and quality, and the development and application of rapid diagnostic methods will contribute to the prevention and control of rice disease. Herein, we developed a novel protospacer adjacent motif (PAM)-free loop-mediated isothermal amplification (LAMP) assisted CRISPR/Cas12a cleavage (Cas-PfLAMP) assay for detection of three rice pathogens; Xanthomonas oryzae pv. Oryzae (XOO), rice stripe virus (RSV), and rice black-streaked dwarf virus (RBSDV). The Cas-PfLAMP assay showed high specificity due to doubly specific recognition of LAMP primer sets and FnCas12a/sgRNA, and high sensitivity down to 9 or 3 copies due to LAMP amplification and CRISPR/Cas12a trans cleavage activity. Furthermore, a visual on-spot Cas-PfLAMP platform was established for detection of rice pathogens by combining solid-phase nucleic acid extraction and a lateral flow strip (LFS) test. Analysis of rice leaf field samples confirmed the impressive performance of the Cas-PfLAMP platform, demonstrating its suitability for rapid (∼50 min) on-spot detection of rice diseases. The assay could also be extended to detection of other plant diseases, and other nucleic acid field tests.