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Identification of transcription factor binding sites is one of the most important steps in understanding the function of transcription factors and regulatory networks in organisms. The assay for transposase accessible chromatin sequencing (ATAC-seq) is a simple protocol for detection of open chromatin that could be a powerful tool to advance studies of protein-DNA interactions. Although ATAC-seq has been used in systematic identification of cis-regulatory regions in animal and plant genomes, this method has been rarely applied in fungi. Here, we describe a valuable ATAC-seq resource in the genome of an economically important phytopathogen, the rice false smut fungus Ustilaginoidea virens. The ATAC-seq data of U. virens mycelia collected from potato sucrose broth (PSB) and PSB supplied with rice spikelet extract were both generated. This is the first genome-wide profiling of open chromatin and transcription factor binding sites in U. virens. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

False smut caused by Ustilaginoidea virens is an important rice fungal disease that significantly decreases its production. In the recent past, conventional methods have been developed for its detection that is time-consuming and need high-cost equipments. The research and development in nanotechnology have made it possible to assemble efficient recognition interfaces in biosensors. In this study, we present a simple, sensitive, and selective oxidized graphene-based geno-biosensor for the detection of rice false smut. The biosensor has been developed using a probe DNA as a biological recognition element on paper electrodes, and oxidized graphene to enhance the limit of detection and sensitivity of the sensor. Probe single-stranded DNA (ssDNA) and target ssDNA hybridization on the interface surface has been quantitatively measured with the electrochemical analysis tools namely, cyclic voltammetry, and linear sweep voltammetry. To confirm the selectivity of the device, probe hybridization with non-complementary ssDNA target has been studied. In our study, the developed sensor was able to detect up to 10 fM of target ssDNA. The paper electrodes were employed to produce an effective and cost-effective platform for the immobilization of the DNA and can be extended to design low-cost biosensors for the detection of the other plant pathogens.

Rice false smut (RFS) caused by Ustilaginoidea virens is one of the most important diseases in the majority of rice-growing areas worldwide. Rice false smut causes not only yield loss, but also threatens human or animal health by producing cyclopeptide mycotoxins. Cultivar resistance is the most economical, effective and environmentally friendly approach to control RFS. However, development of RFS-resistant rice cultivar still faces big challenges. In the field, disease severity of RFS is largely affected by rice growth period and variable weather conditions. To date, quite a few cultivars with stable resistance to RFS have been identified and could be used as resistant resource for disease-resistance breeding (Sun et al., 2020).

AIMS To characterize the geo-distinct isolates of Ustilaginoidea virens for morpho-molecular and mating-type locus diversity. METHODS AND RESULTS Sixty-one isolates of U. virens collected from Southern India exhibited significant diversity in mycelial width (3·45-5·50 µm), colony colour (yellow, pale yellow, and white), and growth pattern (thick leather mat, raised-fluffy, flat-fluffy, and raised). Field-borne chlamydospores of each isolate were significantly smaller in size (3·34-5·26 µm2 ) compared to those formed on culture media (18·6-100·89 µm2 ). The phylogenetic study based on internal transcribed sequences revealed two clusters; however, most isolates (n = 54) were grouped in cluster-I, indicating common ancestral origin. We also identified 42 haplotypes; among them, Hap_3 has the highest number of isolates (n = 19). Mating-type locus (MAT1) analysis revealed all sixty-one isolates as heterothallic, wherein 37 and 24 isolates belonging to MAT1-1-1 and MAT1-2-1 heterothallic mating types, respectively. The microsynteny analysis of MAT1 loci of one of the Indian strain (Uv-Gvt) along with Uv-8b (China) strain revealed synteny conservation at MAT1 locus, which is flanked by conserved genes SLA2 and a hypothetical protein in the upstream and APN2, COX12 and APC5 in the downstream of the locus. CONCLUSIONS Morpho-molecular study revealed the significant diversity among geo-distinct isolates, and MAT1 loci analysis indicated the distribution of heterothallic mating types in south Indian paddy fields. And also, complete synteny conservation between Indian and Chinese strain was observed at the MAT1 locus. SIGNIFICANCE AND IMPACT OF THE STUDY This is the first report describing the sexuality of Indian strains of the U. virens, which would help better understand the genetic diversity of the U. virens prevailing in Southern India and aid in developing resistant rice cultivars against this pathogen population.

Rice false smut caused by Ustilaginoidea virens is one of the widespread rice diseases across the globe in recent years, however, we know little about its molecular mechanism of infection. The cAMP signaling pathway functions directly in the development and formation of infectious structures to regulate the infection process in many pathogenic fungi. In order to investigate the role of the cAMP signaling pathway in U. virens, UvCap1, a cyclase-associated-protein homologous to Saccharomyces cerevisiae Srv2 was identified. Three targeted deletion mutants of the UvCAP1 gene were obtained with gene replacement strategy assisted with CRISPR-Cas9 system. The UvCAP1 deletion mutants showed defects in mycelial growth and conidial production. Inoculation experiments demonstrated that ΔUvcap1 exhibited defects in pathogenicity. Compared with the wild-type strain, ΔUvcap1 showed decreased tolerance to sorbitol and H2O2, and increased tolerance to NaCl, CFW and SDS, and the intracellular cAMP level was significantly reduced in ΔUvcap1. Yeast two-hybrid assay identified the interactions of UvCap1 with UvAc1 (adenylase cyclase), two Ras proteins (UvRas1 and UvRas2) and UvSte50. Taken together, as a component of cAMP signaling pathway, UvCap1 plays important roles in the development and pathogenicity of U. virens.

Long non-coding RNAs (lncRNAs) play an important role in biological processes but regulation and function of lncRNAs remain largely unelucidated, especially in fungi. Ustilaginoidea virens is an economically important fungus causing a devastating disease of rice. By combining microscopic and RNA-seq analyses, we comprehensively characterized lncRNAs of this fungus in infection and developmental processes and defined four serial typical stages. RNA-seq analyses revealed 1724 lncRNAs in U. virens, including 1084 long intergenic non-coding RNAs (lincRNAs), 51 intronic RNAs (incRNAs), 566 natural antisense transcripts (lncNATs) and 23 sense transcripts. Gene Ontology enrichment of differentially expressed lincRNAs and lncNATs demonstrated that these were mainly involved in transport-related regulation. Functional studies of transport-related lncRNAs revealed that UvlncNAT-MFS, a cytoplasm localized lncNAT of a putative MFS transporter gene, UvMFS, could form an RNA duplex with UvMFS and was required for regulation of growth, conidiation and various stress responses. Our results were the first to elucidate the lncRNA profiles during infection and development of this important phytopathogen U. virens. The functional discovery of the novel lncRNA, UvlncNAT-MFS, revealed the potential of lncRNAs in regulation of life processes in fungi.

Lysine 2-hydroxyisobutyrylation (Khib ) is a newly identified post-translational modification (PTM) that plays important roles in transcription and cell proliferation in eukaryotes. However, its function remains unknown in phytopathogenic fungi. Here, we performed a comprehensive assessment of Khib in the rice false smut fungus Ustilaginoidea virens, using Tandem Mass Tag (TMT)-based quantitative proteomics approach. A total of 3 426 Khib sites were identified in 977 proteins, suggesting that Khib is a common and complex PTM in U. virens. Our data demonstrated that the 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Network analysis of the modified proteins revealed a highly interconnected protein network that included many well-studied virulence factors. We confirmed that the Zn-binding reduced potassium dependency3-type histone deacetylase (UvRpd3) is a major enzyme that removes 2-hydroxyisobutyrylation and acetylation in U. virens. Notably, mutations of Khib sites in the mitogen-activated protein kinase (MAPK) UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molecular dynamics simulations demonstrated that 2-hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent-accessible surface area and thereby affected binding between the UvSlt2 enzyme and its substrates. Our findings thus establish Khib as a major post-translational modification in U. virens and point to an important role for Khib in the virulence of this phytopathogenic fungus.

The application of agricultural and forest residues can benefit the environment and the economy; however, they also generate a large amount of byproducts. In this study, bamboo tar (BT), a waste product of bamboo charcoal production, was dissolved in natural ethanol and the surfactant alkyl glucoside to manufacture a 50% (wt/wt) BT emulsifiable concentrate (BTEC) biopesticide. BTEC was screened for fungicidal activity against pathogens. The greatest activity was seen against Ustilaginoidea virens with a half-maximal effective concentration (EC50) value of 6 mg/liter. Four phytopathogenic fungi, Podosphaera xanthii, Rhizoctonia solani, Fusarium oxysporum, and Botrytis cinerea, showed EC50 values of 2- or 4-ethylphenol > 2- or 4-methylphenol > phenol > 4-ethylguaiacol > dimethoxyphenol > 4-methylguaiacol > 4-propenyl-2,6-dimethoxyphenol > 2,4-dimethylphenol. Some of the phenolic compounds identified from the tar might be fungicidally active components. BT is a biochar waste, which has potential as a biofungicide and has promise in organic agriculture. The value of this tar may not be because of any fundamental physical differences from other synthetic fungicides but rather caused by reduced production expenses and more efficient use of waste products.

False smut of rice caused by Ustilaginoidea virens are the most common diseasethat can devastating the yield of rice up to 5-85 per cent in India. In the recent years, the false smut was highly emerging disease and the climate changes, high yield varieties and high use of nitrogenous fertilizers are the most favourable conditions for the development of this disease. The present study was conducted to understand the activity of different chemical fungicides on growth of U. virens on the artificial media. Five fungicides Carbendazim (Bavistin 50 WP), Trifloxystrobin+Tebuconazole (Nativo 756WG), Propiconazole (Tilt 25 EC), Carbendazim (12%)+ Mancozeb (63%) and Tricyclazole 75% WP were evaluated at different five concentration 60ppm, 125ppm, 250ppm, 500ppm and 1000 ppm against U. virens using food poison technique on the potato dextrose yeast agar (PDYA) media.The percent zone of inhibition of the pathogens shows that Tilt 25EC (Propiconazol) and Nativo 75WG (Tryfloxystrobin + Tebuconazole) at all tested concentration followed by Baan 75WP (Tricyclazole) @250ppm show 100% zone of mycelium growth in vitro condition. Tilt 25EC (Propiconazol) and Nativo 75WG considered as best fungicide and remaining tested fungicide were less effective but superior over control.

Rice false smut (RFSm) disease caused by Ustilaginoidea virens (Cooke) Takah (teleomorph: Villosiclava virens) has become one of the most destructive rice diseases all over the world. The pathogen is known to produce secondary metabolites i.e. ustiloxins... which are known to be harmful to plant growth, human and domestic animals. This work had taken into consideration the effect of soaking seeds in either the pathogen spore suspension or rice false smut balls cell free extract (RFSBCFE) on seed germination, shoot length and root length of nine rice cultivars. Seed germination % ranged from 78.66 to 98.66 under soaking in spore suspension, however it ranged from 20 to 93.33 when seeds were soaked in RFSBCFE. Soaking rice grains of Sakha 104 in spore suspension, recorded the highest values in shoot and root length (5.23 and 5.80, respectively). While, Super 300, Sakha 108 and Sakha 104 cultivars showed the highest shoot and root length when their grains were soaked in RFSBCFE. Using LC-MS/MS method, five Ustiolxins (A, B, C, D and F) have been detected in RFSBCFE. Finally, toxic effect of using contaminated diets with these smut balls for feeding with five concentrations (0%, 1%, 5%, 10% and 25% (wt/wt)) has been studied on rats. RFSBs were found to pose risks of renal and hepato-toxicity in rats. Since biochemical parameters of liver and kidney functions (urea and creatinine concentration) were severely affected. Likewise, haematological indices were also influenced. On the other hand, the above mentioned lesions were relatively obscure in concentration 1%.

False smut of rice caused by Ustilaginoidea virens (Cooke) Takahashi (teleomorph Villosiclava virens) has emerged as a serious concern in rice production. While variation for resistance exists in rice germplasm, most of the cultivated varieties are susceptible to false smut. A total of 125 diverse lines from global rice diversity panel were screened for resistance to false smut along with susceptible checks GSR123 and PR116, under natural hot spot for 3 years at two different locations and artificial inoculation conditions in a fourth consecutive year. False smut resistance was recorded based on infected panicles per plants (IPP), number of smut balls per plant (NSBP) and a disease score (DS). Of 125, 21 germplasm lines showed high level of resistance in all the years under natural hot spot as well as artificial inoculation conditions whereas 31 germplasm lines showed moderate resistance. Genome wide association study for false smut related traits identified significant associations on chromosomes 2, 3, 6, 9 and 11 for IPP. Similarly, significant associations for NSBP were identified on chromosomes 3 and 8 and DS on chromosomes 3, 4 and 11. Most of these QTLs were found to be associated with all the traits. The study highlights the potential donors for resistance to false smut that could be utilized in rice breeding programme. The identified QTLs could be the potential candidate regions for further studying the false smut resistance.

Ustilaginoidea virens is an economically important fungus causing a devastating grain disease, rice false smut. An insertional mutagenesis screen was used to explore biological mechanisms underlying infection process of U. virens. T184, a new mutant was identified, with abnormal conidial morphology and deficient virulence. Analysis of the T-DNA inserted gene UvPal1 in the mutant confirmed it as a putative homologue of a cellular morphogenetic protein in yeast, Pal1, whose function has not been well characterized. Deletion of UvPal1 affected hyphal growth, cell morphology, stress adaptation and virulence. UvPal1 could interact with the endocytic proteins, UvEde1 and UvSla2, but was not required for receptor-mediated endocytosis. A yeast two-hybrid (Y2H) analysis was further carried out to screen the UvPal1-interacting proteins, resulting in the identification of 16 putative interacting proteins. Interestingly, UvPal1 interacted with a septin protein, UvCdc11 in vivo and in vitro, and also affected subcellular localization of UvCdc11 protein. Deletion of the four core septins impaired the growth, morphogenesis, stress response and virulence. Collectively, effects on cell morphology, oxidative stress response and virulence are similar to those of UvPal1, suggesting that UvPal1 physically interacts with UvCdc11 to mediate the septin complex to maintain the cellular morphology and virulence of U. virens.

Ustilaginoidea virens is the causal agent of rice false smut, which can be a highly destructive disease of rice. The plasma membrane phosphatase Psr1 proteins, which act as a regulator of the salinity stress response in yeast, are widely distributed across fungi, but their functional characterization is sketchy. In this study, we characterized the functions of Psr1 protein, UvPsr1, in U. virens. Analyses of the ΔUvpsr1 and its complementation strain showed that UvPsr1 is required for normal mycelial growth, conidiation and tolerance to oxidative, osmotic and cell wall stresses. When rice panicles were inoculated with the ΔUvpsr1 strains, no symptoms of false smut disease developed, showing that UvPSR1 also contributes to the pathogenicity of the fungus. The deletion mutant of UvPSR1 also appeared to produce a smaller titer of toxic compounds able to inhibit elongation of the germinated seeds. In conclusion, our results indicated that UvPsr1 is a new pathogenic factor of U. virens.

Rice false smut is an emerging disease of rice caused by Ustilaginoidea virens (Cooke) Takashi. Along with yield loss, the pathogen affects the quality of the grains and produces mycotoxins also. Isolation of the pathogen is difficult due to its slow growing nature which often leads to the contamination. Hence, an improved methodology for the isolation was developed. For this, first the infected rice smut balls were taken and sterilized for 2 min in 1% (v/v) sodium hypochloride solution followed by washing three times (1 min each) with sterilized distilled water. Then, the smut balls were halved using a sterilized scalpel and spores from the inner surface of the ball were dusted onto the Petri plates containing ampicillin added potato sucrose agar (PSA). The plates were incubated at 27 °C ± 1 °C for 7 days after which white fungal colonies appeared were transferred in the PSA plates. The pathogen U. virens was further confirmed morphologically and through internal transcribed spacer (ITS) region amplification with 98–100% sequence homology. The developed methodology is comparatively faster with 80–85% success and will be helpful for the pathogen characterization, and understanding the biology of the pathogen.

False smut of rice, caused by Ustilaginoidea virens, has become one of the most important diseases in rice-growing regions worldwide. The disease causes a significant yield loss and imposes health threats to humans and animals by producing mycotoxins. In this review, we update our understanding of the pathogen, including the disease cycle and infection strategies, the decoding of the U. virens genome, comparative/functional genomics, and effector biology. Whereas the decoding of the U. virens genome unveils specific adaptations of the pathogen in successfully occupying rice flowers, progresses in comparative/functional genomics and effector biology have begun to uncover the molecular mechanisms underlying U. virens virulence and pathogenicity. We highlight the identification and characterization of the produced mycotoxins and their biosynthetic pathways in U. virens.The management strategies for this disease are also discussed. The flower-specific infection strategy makes the pathogen a unique tool to unveil novel mechanisms for the interactions between nonobligate biotrophic pathogens and their hosts.

False smut of rice caused by Ustilaginoidea virens is most destructive disease of rice causing 5-85 per cent yield losses in India. In the present investigations, six novel fungicides viz., Flusilazole 25% + Carbendazim 12.5% SE, Trifloxystrobin 25% + Tebuconazole 50% WG, Carbendazim 12% + Mancozeb 63% WP, Azoxystrobin 18.2% + Difenoconazole 11.4% SC, Azoxystrobin 11% + Tebuconazole 18.3% W/W and Copper hydroxide 50% WP were tested in vitro against the false smut of rice pathogen Ustilaginoidea virens. The maximum mycelial growth was observed in case of Copper hydroxide 50% WP (21.20 mm) as compare to control (60.70 mm) after 21 days incubation. All the fungicides significantly inhibited the fungal mycelial growth in all concentrations (10, 25, 50, 75 and 100 ppm). Among six fungicides evaluated under in vitro condition Trifloxystrobin 25% + Tebuconazole 50% WG showed highest mycelial growth inhibition (86.66%) at 100 ppm followed by Flusilazole 25% + Carbendazim 12.5% SE (78.91%) whereas least mycelial inhibition was recorded in case of Copper hydroxide 50% WP (65.07%) after 21 days of incubation.

Chelerythrine (CHE) possesses broad pharmacological activities. In this study, the extract of Chelidonium majus L. were characterized by high performance liquid chromatography (HPLC), infrared radiation (IR) spectroscopy and nuclear magnetic resonance (NMR). It was proved that the extract was CHE. The antifungal activity of CHE against five fungal pathogens of rice was researched in vitro, revealing that CHE inhibited Ustilaginoidea virens (U. virens) and Cochliobolus miyabeanus (C. miyabeanus) with 50% effective concentrations (EC50) of 6.53 × 10−3 mg/mL and 5.62 × 10−3 mg/mL, respectively. When the concentration of CHE was 7.5 × 10−3 mg/mL, the inhibition rate of U. virens reached 56.1%. Moreover, CHE (4 × 10−3 mg/mL) exhibited the greatest efficacy in inhibiting spore of U. virens growth with an inhibition rate as high as 86.7%. CHE displayed the best inhibitory activity against U. virens at the concentration of 7.5 × 10−3 mg/mL, compared with the other two isoquinoline alkaloids and commercial fungicide validamycin. After treating U. virens mycelia with CHE, twisted and atrophied mycelia were observed by optical microscopy. SEM results demonstrated narrow and locally fractured mycelium. TEM observations showed that the cell wall had become thin and broken, and most organelles were difficult to recognize. Furthermore, membrane of mycelia was destroyed and reactive oxygen species (ROS) of spores was accumulated, which induced apoptosis of pathogenic fungi. From these results, our understanding of the mechanisms of antifungal activity of CHE against U. virens was enriched and this research is relevant for developing novel pesticides.

In India, since 2001, false smut disease of rice has been observed in severe form among major rice growing states. This disease is difficult to tackle, because pathogens infect the plant during flowering stage, and the symptoms are only visible after emergence of the panicle. Thus, disease forecasting is necessary to predict the chance of disease in a certain set of conditions. We analysed 3 years of aerobiological data including pathogenic spore (Ustilaginoidea virens) concentration over the rice canopy in West Bengal, India. The crop growth stages in both rabi (winter or dry) and kharif (rainy) seasons during which rapid development of false smut disease takes place were also recorded. Multiple regression analysis of per cent disease index values was done using age of the plant, pathogen concentration and meteorological factors. In dry season, the false smut disease severity depends on age of the plant as well as pathogenic spore concentration, while in rainy season, the severity depends upon spore concentration, RH and minT. From all the analysed data, it can be predicted that spore concentration is the common factor for false smut disease incidence and severity in rice. If pathogen concentration is increased by one unit, there will be 0.981 unit increased in the expected value of disease severity. With odds ratio value, it can be assumed that with a change in spore concentration by 1 unit, the odds of “Severe” versus “High” or “Moderate” or “Low” or “Absent” combined will be 1.1031 times greater, when other variables held constant. The regression model made from the present study would be beneficial to the farmers for early prediction of false smut disease of rice by estimating the spore concentration over rice canopy in West Bengal, India.

Ustilaginoidea virens is an economically important biotrophic fungal pathogen that causes rice false smut worldwide. However, the regulatory mechanisms of smut ball formation under U. virens infection remain unclear. Here, we identified an important transcription factor, UvCom1, from this pathogen, which regulates the formation of smut balls on rice spikelets. UvCom1 contains two conserved internal repeat 1 (RPT) domains and is found only in fungi, with specific conservation in species of Basidiomycetes and Ascomycetes. UvCom1 protein N- or C-terminal fusion vectors both showed transactivation activity in yeast. Deletion of UvCom1 significantly affected the vegetative growth and conidiation of U. virens. UvCom1 negatively regulated the responses to oxidative, osmotic, and cell wall stresses. Remarkably, UvCom1 was found to be essential for the formation of rice smut balls, and UvCom1 deletion mutants lost the ability to stably utilize nutrients from the rice host. UvCom1 was also highly expressed at the mycelial expansion stage. Transcriptomic analysis and quantitative real-time PCR revealed that UvCom1 could affect the expression of genes significantly enriched in transmembrane transport. This study demonstrates that UvCom1 is a key transcription factor governing smut ball formation of this biotrophic fungus.

Summary Rice false smut has emerged as a serious grain disease in rice production worldwide. The disease is characterized by the transformation of individual rice florets into false smut balls, which is caused by the fungal pathogen Ustilaginoidea virens. To date, little is known about the host factors required for false smut ball formation by U. virens. In this study, we identified histological determinants for the formation of false smut balls by inoculating U. virens into rice floral mutants defective with respect to individual floral parts. The results showed that U. virens could form mature false smut balls in rice floral mutants with defective pistils, but failed to develop false smut balls in the superwoman mutant lacking stamens, identifying that U. virens requires rice stamens to complete its infection cycle. Comparative transcriptome analysis indicated a list of candidate host genes that may facilitate nutrient acquisition by U. virens from the rice stamens, such as SWEET11, SWEET14 and SUT5, and genes involved in the biosynthesis of trehalose and raffinose family sugars. These data pinpoint rice stamens as the key target organ of U. virens infection and provide a valuable starting point for dissecting the molecular mechanism of false smut ball formation.

In filamentous fungi, the conserved transcription factors play important roles in multiple cellular and developmental processes. The GATA proteins, a family of GATA-binding zinc finger transcription factors, play diverse functions in fungi. Ustilaginoidea virens is an economically important pathogen-causing rice false smut worldwide. To gain additional insight into the cellular and molecular mechanisms of this pathogen, in this study, we identified and functionally characterized seven GATA proteins from the U. virens genome (UvGATA). Sequences analysis indicated that these GATA proteins are divided into seven clades. The proteins in each clade contained conserved clade-specific sequences and structures, thus leading to the same motif serving different purposes in various contexts. The expression profiles of UvGATA genes at different infection stages and under H2O2 stress were detected. Results showed that the majority of UvGATA genes performed functions at both processes, thereby confirming the roles of these genes in pathogenicity and reactive oxygen species stress tolerance. This study provided an important starting point to further explore the biological functions of UvGATA genes and increased our understanding of their potential transcriptional regulatory mechanisms in U. virens.

Rice false smut caused by Ustilaginoidea virens is a serious disease of rice (Oryza sativa), severely reducing plant mass and yields worldwide. We performed genome-wide analysis of the CCHC-type zinc-finger transcription factor family in this pathogen. We identified and functionally characterized seven UvCCHC genes in U. virens. The deletion of various UvCCHC genes affected the stress responses, vegetative growth, conidiation, and virulence of U. virens. ∆UvCCHC5 mutants infected rice spikelets normally but could not form smut balls. Sugar utilization experiments showed that the ∆UvCCHC5 mutants were defective in the utilization of glucose, sucrose, lactose, stachyose, and trehalose. Deletion of UvCCHC5 did not affect the expression of rice genes associated with grain filling, as revealed by RT-qPCR. We propose that the ∆UvCCHC5 mutants are impaired in transmembrane transport, and the resulting nutrient deficiencies prevent them from using nutrients from rice to form smut balls. RNA-seq data analysis indicated that UvCCHC4 affects the expression of genes involved in mitochondrial biogenesis, ribosomes, transporters, and ribosome biogenesis. These findings improve our understanding of the molecular mechanism underlying smut ball formation in rice by U. virens.

False smut disease (FSD) of rice incited by Ustilaginoidea virens is an emerging threat to paddy cultivation worldwide. We investigated the spatial distribution of FSD in different paddy ecosystems of South Indian states, viz., Andhra Pradesh, Karnataka, Tamil Nadu, and Telangana, by considering the exploratory data from 111 sampling sites. Point pattern and surface interpolation analyses were carried out to identify the spatial patterns of FSD across the studied areas. The spatial clusters of FSD were confirmed by employing spatial autocorrelation and Ripley’s K function. Further, ordinary kriging (OK), indicator kriging (IK), and inverse distance weighting (IDW) were used to create spatial maps by predicting the values at unvisited locations. The agglomerative hierarchical cluster analysis using the average linkage method identified four main clusters of FSD. From the Local Moran’s I statistic, most of the areas of Andhra Pradesh and Tamil Nadu were clustered together (at I > 0), except the coastal and interior districts of Karnataka (at I < 0). Spatial patterns of FSD severity were determined by semi-variogram experimental models, and the spherical model was the best fit. Results from the interpolation technique, the potential FSD hot spots/risk areas were majorly identified in Tamil Nadu and a few traditional rice-growing ecosystems of Northern Karnataka. This is the first intensive study that attempted to understand the spatial patterns of FSD using geostatistical approaches in India. The findings from this study would help in setting up ecosystem-specific management strategies to reduce the spread of FSD in India.

Rice false smut (RFS) caused by Ustilaginoidea virens has been one of the most severe rice diseases. Fungicide-based chemical control is a significant measure to control RFS. In the sensitivity determination of quinone outside inhibitor (QoI) fungicide in vitro, salicylhydroxamic acid (SHAM) has been commonly added to artificial culture media in order to inhibit alternative oxidase of phytopathogenic fungi. However, some studies showed that artificial media should not include SHAM due to its toxicity. Whether SHAM should be added in the assay of U. virens sensitivity to QoI fungicide remains unknown. In this study, two appropriate media, potato sucrose agar (PSA) and minimal medium (MM), were selected to test SHAM toxicity and sensitivity of U. virens to azoxystrobin and pyraclostrobin. The mycelial growth and sensitivity to azoxystrobin and pyraclostrobin had no significant difference between on PSA and MM. SHAM could significantly inhibit mycelial growth, conidial germination, peroxidase (POD) and esterase activity of U. virens. Average effective concentration for inhibiting 50% (EC50) values of SHAM against mycelial growth of ten U. virens were 27.41 and 12.75 μg/mL on PSA and MM, respectively. The EC50 values of SHAM against conidial germination of isolates HWD and JS60 were 70.36 and 44.69 μg/mL, respectively. SHAM at 30 μg/mL significantly inhibited POD and esterase activity of isolates HWD and JS60, and even SHAM at 10 μg/mL significantly inhibited POD activity of isolate HWD. In addition, SHAM significantly reduced EC50 values and EC90 values of azoxystrobin and pyraclostrobin on both PSA and MM. Even in the presence of SHAM at 10 μg/mL, average EC50 values of ten U. virens isolates for azoxystrobin decreased 1.7-fold on PSA and 4.8-fold on MM, and for pyraclostrobin that decreased 2.8-fold on PSA and 4.8-fold on MM. Therefore, these results suggest that SHAM should not be included in artificial media in the assay of U. virens sensitivity to QoI fungicides.

Rice false smut caused by Ustilaginoidea virens is a destructive disease in rice cropping areas of the world. The present study is focused on the morphology, pathogenicity, mating-type loci distribution, and genetic characterization of different isolates of U. virens. A total of 221 strains of U. virens were collected from 13 rice-growing regions in southwest China. The morphological features of these strains exhibited high diversity, and the pathogenicity of the smut fungus showed significant differentiation. There was no correlation between pathogenicity and sporulation. Mating-type locus (MAT) analysis revealed that all 221 isolates comprised heterothallic and homothallic forms, wherein 204 (92.31%) and 17 (7.69%) isolates belonged to heterothallic and homothallic mating types, respectively. Among 204 strains of heterothallic mating types, 62 (28.05%) contained MAT1-1-1 idiomorphs, and 142 isolates (64.25%) had the MAT1-2-1 idiomorph. Interestingly, strains isolated from the same fungus ball had different mating types. The genetic structure of the isolates was analyzed using simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNPs). All isolates were clustered into five genetic groups. The values of Nei’s gene diversity (H) and Shannon’s information index (I) indicated that all strains as a group had higher genetic diversity than strains from a single geographical population. The pairwise population fixation index (FST) values also indicated significant genetic differentiation among all compared geographical populations. The analysis of molecular variation (AMOVA) indicated greater genetic variation within individual populations and less genetic variation among populations. The results showed that most of the strains were not clustered according to their geographical origin, showing the rich genetic diversity and the complex and diverse genetic background of U. virens in southwest China. These results should help to better understand the biological and genetic diversity of U. virens in southwest China and provide a theoretical basis for building effective management strategies.