Sheath blight of rice is a global disease that significantly reduces rice yield. This study reports the antifungal activity of an active compound of essential oil, thymol, at different concentrations against Rhizoctonia solani (strain RS-Gvt). In vitro assay results indicated that thymol concentrations (0.5 mg mL-1 and 0.25 mg mL-1) completely inhibited (100%) the mycelial growth of RS-Gvt (p ≤ 0.01). Microscopic observations of thymol-treated mycelium of RS-Gvt at 0.0312 mg mL-1 and above concentrations, revealed a distorted mycelial morphology with deformed hyphae. Hyphae showed a bead-like appearance, reduction in size, and constriction of the hyphae at uneven points with increased hyphal density often entangling with each other. Further, an on-field experiment was conducted to study the field bio-efficacy of thymol for two consecutive Kharif seasons of 2022 and 2023 using a factorial RCBD design. The disease severity was measured as the percent disease index (PDI), and the results of two seasons were pooled. Pathogen (RS-Gvt) and thymol were inoculated in different combinations/methods as main treatments (M1-M3), and concentrations of thymol (0.0625-1.0 mg ML-1) as sub-treatments. The results indicated that all two factors significantly (P = 0.05) influenced the PDI and grain yield. The pooled data of two seasons indicated a significant difference between the main treatments (M1: RS-Gvt + thymol together; M2: thymol sprayed first followed by RS-Gvt; M3: RS-Gvt first followed by thymol spray) on PDI (53.39-59.67) and grain yield (4.16-4.75 t ha-1). M1 exhibited a lower PDI (53.39) and a higher grain yield (4.75 t ha-1) compared to M2 and M3, indicating a protective mode of action of thymol against sheath blight disease of rice. The sub-treatments have shown significant variation in PDI and grain yield. The PDI and grain yield ranged from 33.70 (at 1 mg mL-1) to 66.21 (at 0.0625 mg mL-1) and 4.18 (at 1 mg mL-1) to 5.26 (at 0.0625 mg mL-1) t ha-1, respectively, among the thymol concentrations. This indicates that increasing concentrations of thymol have negatively influenced the PDI and positively impacted the yield. Therefore, the spray of 1 mg mL-1 of thymol at the potential disease-infection stage is most effective in controlling the sheath blight disease of rice. This study provides an alternative green bioactive compound for controlling the sheath blight disease, and thymol can be included in developing eco-friendly integrated disease management practices. © 2024 Society of Chemical Industry.

BACKGROUND: Rhizoctonia solani Kühn is a pathogen causing rice sheath blight (ShB). Ammonium transporter 1 (AMT1) promotes resistance of rice to ShB by activating ethylene signaling. However, how AMT1 activates ethylene signaling remains unclear.
OBJECTIVE: In this study, the indeterminate domain 10 (IDD10)-NAC079 interaction model was used to investigate whether ethylene signaling is modulated downstream of ammonium signaling and modulates ammonium-mediated ShB resistance.
METHODS: RT-qPCR assay was used to identify the relative expression levels of nitrogen and ethylene related genes. Yeast two-hybrid assays, Bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) assay were conducted to verify the IDD10-NAC079-calcineurin B-like interacting protein kinase 31 (CIPK31) transcriptional complex. Yeast one-hybrid assay, Chromatin immunoprecipitation (ChIP) assay, and Electrophoretic mobility shift assay (EMSA) were used to verify whether ETR2 was activated by IDD10 and NAC079. Ethylene quantification assay was used to verify ethylene content in IDD10 transgenic plants. Genetic analysis is used to detect the response of IDD10, NAC079 and CIPK31 to ShB infestation.
RESULTS: IDD10-NAC079 forms a transcription complex that activates ETR2 to inhibit the ethylene signaling pathway to negatively regulating ShB resistance. CIPK31 interacts and phosphorylates NAC079 to enhance its transcriptional activation activity. In addition, AMT1-mediated ammonium absorption and subsequent N assimilation inhibit the expression of IDD10 and CIPK31 to activate the ethylene signaling pathway, which positively regulates ShB resistance.
CONCLUSIONS: The study identified the link between ammonium and ethylene signaling and improved the understanding of the rice resistance mechanism.

A breakthrough \"Green Revolution\" in rice enhanced lodging resistance by using gibberellin-deficient semi-dwarf varieties. However, the gibberellic acid (GA) signaling regulation on rice disease resistance remains unclear. The resistance test showed that a positive GA signaling regulator DWARF1 mutant d1 was more susceptible while a negative GA signaling regulator Slender rice 1 (SLR1) mutant was less susceptible to sheath blight (ShB), one of the major rice diseases, suggesting that GA signaling positively regulates ShB resistance. To isolate the regulator, which simultaneously regulates rice lodging and ShB resistance, SLR1 interactors were isolated. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and Co-IP assay results indicate that SLR1 interacts with Calcineurin B-like-interacting protein kinase 31 (CIPK31). cipk31 mutants exhibited normal plant height, but CIPK31 OXs showed semi-dwarfism. In addition, the SLR1 level was much higher in CIPK31 OXs than in the wild-type, suggesting that CIPK31 OX might accumulate SLR1 to inhibit GA signaling and thus regulate its semi-dwarfism. Recently, we demonstrated that CIPK31 interacts and inhibits Catalase C (CatC) to accumulate ROS, which promotes rice disease resistance. Interestingly, CIPK31 interacts with Vascular Plant One Zinc Finger 2 (VOZ2) in the nucleus, and expression of CIPK31 accumulated VOZ2. Inoculation of Rhizoctonia solani AG1-IA revealed that the voz2 mutant was more susceptible to ShB. Thus, these data prove that CIPK31 promotes lodging and ShB resistance by regulating GA signaling and VOZ2 in rice. This study provides a valuable reference for rice ShB-resistant breeding.

Sheath blight disease of rice caused by Rhizoctonia solani AG1-IA, is a major fungal disease responsible for huge loss to grain yield and quality. The major limitation of achieving persistent and reliable resistance against R. solani is the governance of disease resistance trait by many genes. Therefore, functional characterization of new genes involved in sheath blight resistance is necessary to understand the mechanism of resistance as well as evolving effective strategies to manage the disease through host-plant resistance. In this study, we performed RNA sequencing of six diverse rice genotypes (TN1, BPT5204, Vandana, N22, Tetep, and Pankaj) from sheath and leaf tissue of control and fungal infected samples. The approach for identification of candidate resistant genes led to identification of 352 differentially expressed genes commonly present in all the six genotypes. 23 genes were analyzed for RT-qPCR expression which helped identification of Oschib1 showing differences in expression level in a time-course manner between susceptible and resistant genotypes. The Oschib1 encoding classIII chitinase was cloned from resistant variety Tetep and over-expressed in susceptible variety Taipei 309. The over-expression lines showed resistance against R. solani, as analyzed by detached leaf and whole plant assays. Interestingly, the resistance response was correlated with the level of transgene expression suggesting that the enzyme functions in a dose dependent manner. We report here the classIIIb chitinase from chromosome10 of rice showing anti-R. solani activity to combat the dreaded sheath blight disease.

BACKGROUND: The development of sheath blight (ShB) resistance varieties has been a challenge for scientists for long time in rice. Activation tagging is an efficient gain-of-function mutation approach to create novel phenotypes and to identify their underlying genes. In this study, a mutant population was developed employing activation tagging in the recalcitrant indica rice (Oryza sativa L.) cv. BPT 5204 (Samba Mahsuri) through activation tagging.
RESULTS: In this study, we have generated more than 1000 activation tagged lines in indica rice, from these mutant population 38 (GFP- RFP+) stable Ds plants were generated through germinal transposition at T2 generation based on molecular analysis and seeds selected on hygromycin (50 mg/L) containing medium segregation analyses confirmed that the transgene inherited as mendelian segregation ratio of 3:1 (3 resistant: 1 susceptible). Of them, five stable activation tagged Ds lines (M-Ds-1, M-Ds-2, M-Ds-3, M-Ds-4 and M-Ds-5) were selected based on phenotypic observation through screening for sheath blight (ShB) resistance caused by fungal pathogen Rhizoctonia solani (R. solani),. Among them, M-Ds-3 and M-Ds-5 lines showed significant resistance for ShB over other tagged lines and wild type (WT) plants. Furthermore, analysed for launch pad insertion through TAIL-PCR results and mapped on corresponding rice chromosomes. Flanking sequence and gene expression analysis revealed that the upregulation of glycoside hydrolase-OsGH or similar to Class III chitinase homologue (LOC_Os08g40680) in M-Ds-3 and a hypothetical protein gene (LOC_Os01g55000) in M-Ds-5 are potential candidate genes for sheath blight resistance in rice.
CONCLUSIONS: In the present study, we developed Ac-Ds based ShB resistance gain-of-functional mutants through activation tagging in rice. These activation tagged mutant lines can be excellent sources for the development of ShB resistant cultivars in rice.

Sheath blight (ShB) disease, caused by Rhizoctonia solani Kühn, is one of the most serious rice diseases. Rice breeding against ShB has been severely hindered because no major resistance genes or germplasms are available in rice. Here, we report that introduction of Gastrodia antifungal protein (GAFP) genes from Gastrodia elata B1 into rice significantly enhances resistance to rice ShB. Four GAFP genes were cloned from G. elata B1, and all displayed a strong ability to inhibit R. solani growth in plate assays. Two versions, with or without a signal peptide, for each of the four GAFP genes were introduced into XD3 and R6547 rice cultivars, and all transgenic lines displayed stronger ShB resistance than the corresponding wild-type control in both greenhouse and field conditions. Importantly, GAFP2 showed the highest ShB resistance; GAFPs with and without its signal peptide showed no significant differences in enhancing ShB resistance. We also evaluated the agronomic traits of these transgenic rice and found that ectopic expression of GAFPs in rice at appropriate levels did not affect agronomic traits other than enhancing ShB resistance. Together, these results indicate that GAFP genes, especially GAFP2, have great potential in rice breeding against ShB disease.

Nitrogen fertilization can promote rice yield but decrease resistance to sheath blight (ShB). In this study, the nitrate transporter 1.1b (nrt1.1b) mutant that exhibited less susceptibility to ShB but without compromising yield under NH4+ fertilization was screened. NRT1.1B\'s regulation of ShB resistance was independent of the total nitrogen concentration in rice under NH4+ conditions. In nrt1.1b mutant plants, the NH4+ application modulated auxin signaling, chlorophyll content, and phosphate signaling to promote ShB resistance. Furthermore, the findings indicated that NRT1.1B negatively regulated ShB resistance by positively modulating the expression of H+-ATPase gene OSA3 and phosphate transport gene PT8. The mutation of OSA3 and PT8 promoted ShB resistance by increasing the apoplastic pH in rice. Our study identified the ShB resistance mutant nrt1.1b, which maintained normal nitrogen use efficiency without compromising yield.

UNASSIGNED: Sheath blight caused by Rhizoctonia solani is one of the major diseases of rice, causing widespread crop losses. The use of semi-dwarf rice varieties in the ongoing nutrient-intensive rice cultivation system has further accentuated the incidence of the disease. An ideal solution to this problem would be identifying a stable sheath blight-tolerant genotype.
UNASSIGNED: A multi-environment evaluation of 32 rice genotypes against sheath blight infection was conducted over six seasons across two locations (Agricultural Research Farm, Institute of Agricultural Sciences, Banaras Hindu University (28.18° N, 38.03° E, and 75.5 masl), for four years during the wet seasons (kharif) from 2015 to 2018 and two seasons at the National Rice Research Institute (20°27\'09\" N, 85°55\'57\" E, 26 masl), Cuttack, Odisha, during the dry season (rabi) of 2019 and the kharif of 2019, including susceptible and resistant check. Percent disease index data were collected over 4 weeks (on the 7th, 14th, 21st, and 28th day after infection), along with data on other morphological and physiological traits.
UNASSIGNED: The resistant genotypes across seasons were the ones with a higher hemicellulose content (13.93-14.64) and lower nitrogen content (1.10- 1.31) compared with the susceptible check Tapaswini (G32) (hemicellulose 12.96, nitrogen 1.38), which might explain the resistant reaction. Three different stability models-additive main effect and multiplicative interaction (AMMI), genotype + genotype x environment (GGE) biplot, and multi-trait stability index (MTSI)-were then used to identify the stable resistant genotypes across six seasons. The results obtained with all three models had common genotypes highlighted as stable and having a low area under the disease progress curve (AUDPC) values. The ideal stable genotypes with low disease incidence were IC 283139 (G19), Tetep (G28), IC 260917 (G4), and IC 277274 (G10), with AUDPC values of 658.91, 607.46, 479.69, and 547.94, respectively. Weather parameters such as temperature, rainfall, sunshine hours, and relative humidity were also noted daily. Relative humidity was positively correlated with the percent disease index.

Rice sheath blight (ShB) is a devastating disease that severely threatens rice production worldwide. Induction of cell death represents a key step during infection by the ShB pathogen Rhizoctonia solani. Nonetheless, the underlying mechanisms remain largely unclear. In the present study, we identified a rice transcription factor, OsERF65, that negatively regulates resistance to ShB by suppressing cell death. OsERF65 was significantly upregulated by R. solani infection in susceptible cultivar Lemont and was highly expressed in the leaf sheath. Overexpression of OsERF65 (OsERF65OE) decreased rice resistance, while the knockout mutant (oserf65) exhibited significantly increased resistance against ShB. The transcriptome assay revealed that OsERF65 repressed the expression of peroxidase genes after R. solani infection. The antioxidative enzyme activity was significantly increased in oserf65 plants but reduced in OsERF65OE plants. Consistently, hydrogen peroxide content was apparently reduced in oserf65 plants but accumulated in OsERF65OE plants. OsERF65 directly bound to the GCC box in the promoter regions of four peroxidase genes and suppressed their transcription, reducing the ability to scavenge reactive oxygen species (ROS). The oserf65 mutant exhibited a slight decrease in plant height but increased grain yield. Overall, our results revealed an undocumented role of OsERF65 that acts as a crucial regulator of rice resistance to R. solani and a potential target for improving both ShB resistance and rice yield.

Beauveria bassiana, an entomopathogenic fungus, has recently drawn attention worldwide not only as a potential biocontrol agent against insect pests but also for its other beneficial roles as plant disease antagonist, endophyte, plant growth promoter, and beneficial rhizosphere colonizer. In the present study, 53 native isolates of B. bassiana were screened for antifungal ability against Rhizoctonia solani, the causal agent of sheath blight of rice. Also, the mechanisms underlying such interaction and the responsible antimicrobial traits involved were studied. Following this, potential B. bassiana isolates were assayed against the reduction of sheath blight of rice under field conditions. The results showed that B. bassiana exhibited antagonistic behavior against R. solani with a percent mycelial inhibition recorded maximum of up to 71.15%. Mechanisms behind antagonism were the production of cell-wall-degrading enzymes, mycoparasitism, and the release of secondary metabolites. The study also deciphered several antimicrobial traits and the presence of virulent genes in B. bassiana as a determinant of potential plant disease antagonists. Under field conditions, combined application of the B. bassiana microbial consortium as a seed treatment, seedling root dip, and foliar sprays showed reduced sheath blight disease incidence and severity up to 69.26 and 60.50%, respectively, along with enhanced plant-growth-promoting attributes. This is one of the few studies investigating the antagonistic abilities of the entomopathogenic fungus B. bassiana against phytopathogen R. solani and the underlying mechanisms involved.