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Rice false smut, caused by Ustilaginoidea virens, threatens rice production by reducing yields and contaminating grains with harmful ustiloxins. However, studies on resistance genes are scarce. In this study, the resistance level of IR28 (resistant cultivar) to U. virens was validated through artificial inoculation. Notably, a reactivation of resistance genes after transient down-regulation during the first 3 to 5 dpi was observed in IR28 compared to WX98 (susceptible cultivar). Cluster results of a principal component analysis and hierarchical cluster analysis of differentially expressed genes (DEGs) in the transcriptome exhibited longer expression patterns in the early infection phase of IR28, consistent with its sustained resistance response. Results of GO and KEGG enrichment analyses highlighted the suppression of immune pathways when the hyphae first invade stamen filaments at 5 dpi, but sustained up-regulated DEGs were linked to the 'Plant–pathogen interaction' (osa04626) pathway, notably disease-resistant protein RPM1 (K13457, CNLs, coil-coiled NLR). An analysis of CNLs identified 245 proteins containing Rx-CC and NB-ARC domains in the Oryza sativa Indica genome. Partial candidate CNLs were shown to exhibit up-regulation at both 1 and 5 dpi in IR28. This study provides insights into CNLs' responses to U. virens in IR28, potentially informing resistance mechanisms and genetic breeding targets. [ABSTRACT FROM AUTHOR]

Main Conclusion: The Ustilaginoidea virens –rice pathosystem has been used as a model for flower-infecting fungal pathogens. The molecular biology of the interactions between U. virens and rice, with an emphasis on the attempt to get a deeper comprehension of the false smut fungus's genomes, proteome, host range, and pathogen biology, has been investigated. Meta-QTL analysis was performed to identify potential QTL hotspots for use in marker-assisted breeding. The Rice False Smut (RFS) caused by the fungus Ustilaginoidea virens currently threatens rice cultivators across the globe. RFS infects rice panicles, causing a significant reduction in grain yield. U. virens can also parasitize other hosts though they play only a minor role in its life cycle. Furthermore, because it produces mycotoxins in edible rice grains, it puts both humans and animals at risk of health problems. Although fungicides are used to control the disease, some fungicides have enabled the pathogen to develop resistance, making its management challenging. Several QTLs have been reported but stable gene(s) that confer RFS resistance have not been discovered yet. This review offers a comprehensive overview of the pathogen, its virulence mechanisms, the genome and proteome of U. virens, and its molecular interactions with rice. In addition, information has been compiled on reported resistance QTLs, facilitating the development of a consensus genetic map using meta-QTL analysis for identifying potential QTL hotspots. Finally, this review highlights current developments and trends in U. virens–rice pathosystem research while identifying opportunities for future investigations. [ABSTRACT FROM AUTHOR]

Rice false smut has become a serious threat to rice production. Durable chlamydospores of the fungus are the primary inoculum in paddy soil. Mixing converter slag into paddy soil can decrease disease incidence, but the mechanism is unknown. Here we first confirmed the suppressive effect of converter slag mixed into a paddy field: 3t/ha suppressed disease incidence by 75.6%. We then tested a converter slag solution (CSS), neutralized CSS (NCSS), and converter-slag-amended soil extract (CSSE), all at 3t slag/ha. In the CSS test, chlamydospores rapidly became transparent, then gradually collapsed, but NCSS slowed chlamydospore collapse. More germ tubes were abnormal in the CSS treatment than in the NCSS. The percentages of transparent, collapsed, and normally germinating chlamydospores in the CSSE treatment were intermediate to those obtained with CSS and NCSS. Hyphal invasion into rice roots in CSSE was also suppressed compared to that in control. These results suggest chlamydospores gradually collapse when exposed to cations and the pH is neutralized by soil, thus reducing hyphal production and infection of rice roots, contributing to disease suppression in fields. [ABSTRACT FROM AUTHOR]

The present experiment was conducted from June to November, 2017 at Crop Research Center, Chirodi, Sardar Vallabhbhai Patel University of Agriculture & Technology, Meerut (U.P.), India to assess the efficacy of six fungicides-propiconazole, strobin, flutolanil, copper oxychloride, azoxystrobin, flusilazol, and two bioagents viz., Trichoderma harzianum and Pseudomonas fluorescens-against false smut of rice under natural epiphytotic field conditions. Once panicle emergence reached 50%, each fungicide and bioagent was sprayed once. The findings of present investigation revealed that spray of propiconazole @ 0.1% reduced the false smut significantly and resulted in the lowest percentage of infected tillers (7.48%) and smutted grains (4.98%) along with maximum yield parameters like number of tillers (294), 10-ears weight (30.10 g), 1000-grain weight (24.50 g) and yield (4903 kg ha-1); it was followed by the spray application of either azoxystrobin @ 0.07% or strobin @ 0.06% with above respective parameters. Apart from the fungicides applied, the application of Trichoderma harzianum also reduced the false smut and increased the yield components significantly over the control plots. However, the lowest 10-ear weight (22.80 g), 1000-grain weight (17.50 g), and yield (3100 kg ha-1) along with the highest percentage of infected tillers (23.93%) and smutted grains (21.47%) were recorded in unprotected plots. Therefore, growers can be advised to use a foliar spray of one of three fungicides-propiconazole @ 0.1%, azoxystrobin @ 0.07%, or strobin @ 0.06%-at 50% panicle emergence stage to fungicidal control of false smut of rice. [ABSTRACT FROM AUTHOR]

Rice false smut, caused by Ustilaginoidea virens, is a devastating disease that greatly reduces rice yield and quality. However, controlling rice false smut disease is challenging due to the unique infection mode of U. virens. Therefore, there is a need for early diagnosis and monitoring techniques to prevent the spread of this disease. Lateral flow strip-based recombinase polymerase amplification (LF-RPA) overcomes the limitations of current U. virens detection technologies, which are time-consuming, require delicate equipment, and have a high false-positive rate. In this study, we used a comparative genomics approach to identify Uv_3611, a specific gene of U. virens, as the target for the LF-RPA assay. The designed primers and probe efffectively detected the genomic DNA (gDNA) of U. virens and demonstrated no cross-reactivity with related pathogens. Under optimal conditions, the LF-RPA assay demonstrated a sensitivity of 10 pg of U. virens gDNA. Additionally, by incorporating a simplified PEG-NaOH method for plant DNA extraction, the LF-RPA assay enabled the detection of U. virens in rice spikelets within 30 min, without the need for specialized equipment. Furthermore, the LF-RPA assay successfully detected U. virens in naturally infected rice and seed samples in the field. Therefore, the LF-RPA assay is sensitive, efficient, and convenient, and could be developed as a kit for monitoring rice false smut disease in the field.

Transcription factors play critical roles in diverse biological processes in fungi. XlnR, identified as a transcriptional activator that regulates the expression of the extracellular xylanase genes in fungi, has not been extensively studied for its function in fungal development and pathogenicity in rice false smut fungus Ustilaginoidea virens. In this study, we characterized UvXlnR in U. virens and established that the full-length. N-terminal, and C-terminal forms have the ability to activate transcription. The study further demonstrated that UvXlnR plays crucial roles in various aspects of U. virens biology. Deletion of UvXlnR affected growth, conidiation, and stress response. UvXlnR mutants also exhibited reduced pathogenicity, which could be partially attributed to the reduced expression of xylanolytic genes and extracellular xylanase activity of U. virens during the infection process. Our results indicate that UvXlnR is involved in regulating growth, conidiation, stress response, and pathogenicity. [ABSTRACT FROM AUTHOR]

Rice false smut, which is caused by the soil-borne fungal pathogen Ustilaginoidea virens (U. virens), is one of the most threatening diseases in most of the ricegrowing countries including India that causes 0.5-75% yield loss, low seed germination, and a reduction in seed quality. The assessment of yield loss helps to understand the relevance of disease severity and facilitates the implementation of appropriate management strategies. This study aimed to mitigate biotic stress in rice by employing a rhizobacterial-based bioformulation, which possesses diverse capabilities as both a plant growth promoter and a biocontrol agent against U. virens. Rhizobacteria were isolated from the soil of the rice rhizospheres from the healthy plant of the false smut affected zone. Furthermore, they were identified as Bacillus strains: B. subtilis (BR_4), B. licheniformis (BU_7), B. licheniformis (BU_8), and B. vallismortis (KU_7) via sequencing. Isolates were screened for their biocontrol potential against U. virens under in vitro conditions. The antagonistic study revealed that B. vallismortis (KU_7) inhibited U. virens the most (44.6%), followed by B. subtilis BR_4 (41.4%), B. licheniformis BU_7 (39.8%), and B. licheniformis BU_8 (43.5%). Various biochemical and plant growth promoting attributes, such as phosphate and Zn solubilization, IAA, ammonium, siderophore, and chitinase production, were also investigated for all the selected isolates. Furthermore, the potential of the isolates was tested in both in vitro and field conditions by employing talc-based bioformulation through bio-priming and root treatment. The application of bioformulation revealed a 20% decrease in disease incidence in plants treated with B. vallismortis (KU_7), a 60.5% increase in the biological yield, and a 45% increase in the grain yield. This eco-friendly approach not only controlled the disease but also improved the grain quality and reduced the chaffiness. [ABSTRACT FROM AUTHOR]

Background: High efficiency, low toxicity, and environmentally safe pesticides are increasingly being sought and developed to ensure sustainable development and high-yield agricultural production. Chelerythrine has strong inhibitory effects against the mycelial growth of crop pathogens such as Fusarium oxysporum, Anthrax, and grey mold. Our previous studies have shown that chelerythrine inhibits fungus spore germination by up to 86.7% and induces the apoptosis of Ustilaginoidea virens cells. The application of chelerythrine as a green biological pesticide would not only enhance agricultural production but also enhance ecological protection. Materials and Methods: The formulation of a chelerythrine suspension concentrate was selected. According to the ratio of experimental design requirements to preparation sample, six groups were set up in the field assay; the first spraying was performed approximately 7 days before the rice rupture period, and the second spraying was performed after 10 days. The control effect was investigated at the milk-ripe stage. The present study preliminarily investigates the safety of chelerythrine as a pesticide using the oral acute toxicity test for mice. Results: In the present study, the effect of chelerythrine as the main ingredient in suspension concentrate formulations and its field effect on rice false smut (RFS) was investigated. The pesticide in the suspending agent was sprayed twice and had superior effects with regard to the prevention and control of RFS in the field. The control effect was 95.88% at an application rate of 192 mL/ha, with low residue, environmentally safe, and low resistance characteristics. An acute toxicity test following intragastric administration in mice showed that the LD50 of chelerythrine suspension was 1580 mg·kg−1, which was much lower than those of other low-toxicity commercial pesticides. Conclusion: Chelerythrine suspension was a low-toxicity agent and exhibited the characteristics of an environmentally friendly plant pesticide. The application of chelerythrine suspension could facilitate the achievement of the goal of pesticide reduction efficiency. [ABSTRACT FROM AUTHOR]

Oryza sativa (Rice) is one of the most cultivated staple food crops in India. The crop is affected by Rice False Smut (RFS) caused by Ustilaginoidea virens (UV), which leads to a substantial reduction in the crop yield, and the mycotoxins released pose a health risk. In the present study, bacterial strains were isolated from rhizosphere soil samples collected from the eastern regions of Uttar Pradesh, India. A total of 105 isolates were screened for their antagonistic activity against U. virens and the best four among them were selected and identified using 16S rRNA sequencing. Three isolates were identified as Bacillus subtilis, and one as Bacillus amyloliquefaciens. Additionally, plant growth promoting (PGP) traits such as siderophore production, IAA production, phosphate solubilization, HCN production etc. were assessed. To assess the biocontrol activity and PGP properties in rice a talc-based bioformulation was prepared using bacterial isolates. The control of UV infection was tested on rice variety HKR-126 involving 10 treatments with screened isolates. Enhanced germination was recorded in all bacterial treatments of UV infected seeds. Greenhouse experiments were also carried out to evaluate the biocontrol efficiency until the harvest stage, as well as plant growth in different stages. Collectively, the results suggested that biopriming with talc-based formulations consisting of B. subtilis and B. amyloliquefaciens can serve as an alternative to fungicides to control the severity of UV disease in susceptible rice varieties, along with improving overall plant growth and fitness. [ABSTRACT FROM AUTHOR]

The rice false smut disease, caused by Ustilaginoidea virens, has emerged as a significant global threat to rice production. The mechanism of carbon catabolite repression plays a crucial role in the efficient utilization of carbon nutrients and enzyme regulation in the presence of complex nutritional conditions. Although significant progress has been made in understanding carbon catabolite repression in fungi such as Aspergillus nidulans and Magnaporthe oryzae, its role in U. virens remains unclear. To address this knowledge gap, we identified UvCreA, a pivotal component of carbon catabolite repression, in U. virens. Our investigation revealed that UvCreA localized to the nucleus. Deletion of UvCreA resulted in decreased growth and pathogenicity in U. virens. Through RNA-seq analysis, it was found that the knockout of UvCreA led to the up-regulation of 514 genes and down-regulation of 640 genes. Moreover, UvCreA was found to be involved in the transcriptional regulation of pathogenic genes and genes associated with carbon metabolism in U. virens. In summary, our findings indicated that UvCreA is important in fungal development, virulence, and the utilization of carbon sources through transcriptional regulation, thus making it a critical element of carbon catabolite repression.

Abstract Ustilaginoidea virens causes rice false smut, one of the most devastating rice diseases. The pathogen produces various types of mycotoxins, such as ustilaginoidins and ustiloxins, which are harmful to both human and animal health. Histone deacetylases in fungi play an important role in regulating chromatin structure and gene expression. However, there is limited knowledge about how histone deacetylases control pathogenicity and mycotoxin biosynthesis in U. virens. Here, we characterize a putative class I histone deacetylase UvHOS2 in U. virens. The UvHos2-deletion mutants exhibit retarded vegetative growth, reduced conidial production and germination, and attenuated virulence. UvHOS2 positively regulates tolerance to various environmental stresses, including cell wall, cell membrane integrity, osmotic and oxidative stresses. UvHOS2 reduces the acetylation levels of histones at multiple Lys sites, including H3K9, H3K14, H3K27, and H3K56. ChIP-PCR assays revealed that UvHOS2-mediated H3K9 deacetylation regulates the expression of ustilaginoidin biosynthesis genes. Consistently, transcriptome analysis indicates that UvHOS2 regulates the expression of the genes involved in secondary metabolism, mycelial growth, conidiogenesis, and pathogenicity, thereby controlling U. virens virulence and mycotoxin (ustilaginoidins and sorbicillinoids) biosynthesis. This study provides a theoretical reference for revealing the epigenetic regulation of pathogenicity and mycotoxin biosynthesis in plant pathogenic fungi.

The rice seed market in Nepal is worth more than NRs. 600 million, with an annual demand of 20,000 tons. Rice seed production is threatened by False smut disease, caused by Ustilaginoidea virens. Although once considered minor, recent years have witnessed an alarming rise in false smut cases, causing substantial yield and economic losses globally. The disease can lead to the rejection of rice crops even at low levels, severely affecting seed producers. Depending on weather conditions and rice varieties, yield losses range from 3% to 70%. Moreover, this disease produces harmful mycotoxins, namely ustiloxins and ustilaginoidins, posing risks to human health. Initially introduced to Nepal in 1964 AD through Taiwanese varieties, localized outbreaks were observed over time, like the significant incidence in the Kanchan variety in Bhaktapur during the mid-1980s. Recent years have seen severe impacts on varieties such as Samba Mansuli, Chaite-5, Hardinath-1, and Ram Dhan in the Chitwan district, causing seed yield losses ranging from 80–90%. The disease severity is intensified by planting susceptible varieties, continuation of the same variety in specific fields, excessive use of nitrogenous fertilizers, and favorable weather conditions. Primarily the disease can be controlled by fungicide application at the booting stage. Commonly used fungicides include propiconazole, difenoconazole, and tebuconazole. However, the pivotal approach lies in cultivating resistant varieties, early planting, crop rotations, optimization of nitrogenous fertilizers and farmers’ knowledge are effective strategies for false smut management. These practices are crucial in mitigating the impact of false smut ensuring sustainable rice seed production.

ABSTRACTRice false smut disease is one of the most significant rice diseases worldwide. Ustilaginoidea virens is the causative agent of this disease. Although several developmental and pathogenic genes have been identified and functionally analyzed, the pathogenic molecular mechanisms of U. virens remain elusive. The velvet family regulatory proteins are involved in fungal development, conidiation, and pathogenicity. In this study, we demonstrated the function of the VelC homolog UvVELC in U. virens. We identified the velvet family protein UvVELC and characterized its functions using a target gene deletion-strategy. Deletion of UvVELC resulted in conidiation failure and pathogenicity. The UvVELC expression levels during infection suggested that this gene might be involved in the early infection process. UvVELC is also important in resistance to abiotic stresses, the utilization efficiency of glucose, stachyose, raffinose, and other sugars, and the expression of transport-related genes. Moreover, UvVELC could physically interact with UvVEA in yeast, and UvVELC/UvVEA double-knockout mutants also failed in conidiation and pathogenicity. These results indicate that UvVELC play a critical role in the conidiation and pathogenicity in U. virens. Functional analysis indicated that UvVELC-mediated conidiation and nutrient acquisition from rice regulates the pathogenicity of U. virens. Understanding the function of the UvVELC homolog could provide a potential molecular target for controlling rice false smut disease.

Ustilaginoidea virens causes rice false smut, one of the most devastating rice diseases. The pathogen produces various types of mycotoxins, such as ustilaginoidins and ustiloxins, which are harmful to both human and animal health. Histone deacetylases in fungi play an important role in regulating chromatin structure and gene expression. However, there is limited knowledge about how histone deacetylases control pathogenicity and mycotoxin biosynthesis in U. virens. Here, we characterize a putative class I histone deacetylase UvHOS2 in U. virens. The UvHos2-deletion mutants exhibit retarded vegetative growth, reduced conidial production and germination, and attenuated virulence. UvHOS2 positively regulates tolerance to various environmental stresses, including cell wall, cell membrane integrity, osmotic and oxidative stresses. UvHOS2 reduces the acetylation levels of histones at multiple Lys sites, including H3K9, H3K14, H3K27, and H3K56. ChIP-PCR assays revealed that UvHOS2-mediated H3K9 deacetylation regulates the expression of ustilaginoidin biosynthesis genes. Consistently, transcriptome analysis indicates that UvHOS2 regulates the expression of the genes involved in secondary metabolism, mycelial growth, conidiogenesis, and pathogenicity, thereby controlling U. virens virulence and mycotoxin (ustilaginoidins and sorbicillinoids) biosynthesis. This study provides a theoretical reference for revealing the epigenetic regulation of pathogenicity and mycotoxin biosynthesis in plant pathogenic fungi. [ABSTRACT FROM AUTHOR]

Ras GTPase‐activating proteins (Ras GAPs) act as negative regulators for Ras proteins and are involved in various signalling processes that influence cellular functions. Here, the function of four Ras GAPs, UvGap1 to UvGap4, was identified and analysed in Ustilaginoidea virens, the causal agent of rice false smut disease. Disruption of UvGAP1 or UvGAP2 resulted in reduced mycelial growth and an increased percentage of larger or dumbbell‐shaped conidia. Notably, the mutant ΔUvgap1 completely lost its pathogenicity. Compared to the wild‐type strain, the mutants ΔUvgap1, ΔUvgap2 and ΔUvgap3 exhibited reduced tolerance to H2O2 oxidative stress. In particular, the ΔUvgap1 mutant was barely able to grow on the H2O2 plate, and UvGAP1 was found to influence the expression level of genes involved in reactive oxygen species synthesis and scavenging. The intracellular cAMP level in the ΔUvgap1 mutant was elevated, as UvGap1 plays an important role in maintaining the intracellular cAMP level by affecting the expression of phosphodiesterases, which are linked to cAMP degradation in U. virens. In a yeast two‐hybrid assay, UvRas1 and UvRasGef (Ras guanyl nucleotide exchange factor) physically interacted with UvGap1. UvRas2 was identified as an interacting partner of UvGap1 through a bimolecular fluorescence complementation assay and affinity capture‐mass spectrometry analysis. Taken together, these findings suggest that the UvGAP1‐mediated Ras pathway is essential for the development and pathogenicity of U. virens. [ABSTRACT FROM AUTHOR]

The rice false smut disease, caused by Ustilaginoidea virens , has emerged as a significant global threat to rice production. The mechanism of carbon catabolite repression plays a crucial role in the efficient utilization of carbon nutrients and enzyme regulation in the presence of complex nutritional conditions. Although significant progress has been made in understanding carbon catabolite repression in fungi such as Aspergillus nidulans and Magnaporthe oryzae , its role in U. virens remains unclear. To address this knowledge gap, we identified UvCreA , a pivotal component of carbon catabolite repression, in U. virens. Our investigation revealed that UvCreA localized to the nucleus. Deletion of UvCreA resulted in decreased growth and pathogenicity in U. virens. Through RNA-seq analysis, it was found that the knockout of UvCreA led to the up-regulation of 514 genes and down-regulation of 640 genes. Moreover, UvCreA was found to be involved in the transcriptional regulation of pathogenic genes and genes associated with carbon metabolism in U. virens. In summary, our findings indicated that UvCreA is important in fungal development, virulence, and the utilization of carbon sources through transcriptional regulation, thus making it a critical element of carbon catabolite repression. [ABSTRACT FROM AUTHOR]

Ustilaginoidea virens is the causal agent of rice false smut, which has recently become one of the most important rice diseases worldwide. Ustilaginoidins, a major type of mycotoxins produced in false smut balls, greatly deteriorates grain quality. Histone acetylation and deacetylation are involved in regulating secondary metabolism in fungi. However, little is yet known on the functions of histone deacetylases (HDACs) in virulence and mycotoxin biosynthesis in U. virens. Here, we characterized the functions of the HDAC UvHOS3 in U. virens. The ΔUvhos3 deletion mutant exhibited the phenotypes of retarded growth, increased mycelial branches and reduced conidiation and virulence. The ΔUvhos3 mutants were more sensitive to sorbitol, sodium dodecyl sulphate and oxidative stress/H2O2. ΔUvhos3 generated significantly more ustilaginoidins. RNA‐Seq and metabolomics analyses also revealed that UvHOS3 is a key negative player in regulating secondary metabolism, especially mycotoxin biosynthesis. Notably, UvHOS3 mediates deacetylation of H3 and H4 at H3K9, H3K18, H3K27 and H4K8 residues. Chromatin immunoprecipitation assays indicated that UvHOS3 regulates mycotoxin biosynthesis, particularly for ustilaginoidin and sorbicillinoid production, by modulating the acetylation level of H3K18. Collectively, this study deepens the understanding of molecular mechanisms of the HDAC UvHOS3 in regulating virulence and mycotoxin biosynthesis in phytopathogenic fungi. [ABSTRACT FROM AUTHOR]

Rice False Smut (RFS) caused by Ustilaginoidea virens is a major emerging disease of rice due to expanded area of hybrid rice cultivars, increasing use of nitrogenous fertilizers and change in climate. Due to the increasing incidences of this disease across the globe, there is a pressing need to develop techniques for false smut management. The application of fungicides with high efficiency, low toxicity, and low residue is currently the best option to control RFS. Therefore, current research was conducted to determine the effectiveness of fungicides to manage RFS. The experiments were conducted in a completely randomized block design with three replications of seven treatments at RFS-prone subtropical hills of Nepal in the main rice growing season, during 2020 and 2021. The fungicides include trifloxystrobin 25 % + tebuconazole 50 %, chlorothalonil 75 %, carbendazim 12 % + mancozeb 63 %, propiconazole 25 %, azoxystrobin 50 %, carbendazim 50 % and untreated control. Fungicides were applied as two foliar sprays, one at booting and the other at flowering. Fungicide spray significantly increased number of tillers per plant (P ≤ 0.01) and reduced the number of false smut-infected tillers per plant (P ≤ 0.05), false smut severity (P ≤ 0.05), and incidence (P ≤ 0.05). False smut incidence percentages were significantly reduced by all the fungicides except mancozeb + carbendazim compared to the non-treated control. The reduction in RFS incidence was 70 % in propiconazole, 71 % in trifloxystrobin + tebuconazole sprayed plots compared to the non-treated control plots. Thus, the application of suitable fungicide at the appropriate stage would give the satisfactory suppression of RFS in a farmers' field in Nepal.

Rice false smut, which is caused by the soil-borne fungal pathogen Ustilaginoidea virens (U. virens), is one of the most threatening diseases in most of the rice-growing countries including India that causes 0.5–75% yield loss, low seed germination, and a reduction in seed quality. The assessment of yield loss helps to understand the relevance of disease severity and facilitates the implementation of appropriate management strategies. This study aimed to mitigate biotic stress in rice by employing a rhizobacterial-based bioformulation, which possesses diverse capabilities as both a plant growth promoter and a biocontrol agent against U. virens. Rhizobacteria were isolated from the soil of the rice rhizospheres from the healthy plant of the false smut affected zone. Furthermore, they were identified as Bacillus strains: B. subtilis (BR_4), B. licheniformis (BU_7), B. licheniformis (BU_8), and B. vallismortis (KU_7) via sequencing. Isolates were screened for their biocontrol potential against U. virens under in vitro conditions. The antagonistic study revealed that B. vallismortis (KU_7) inhibited U. virens the most (44.6%), followed by B. subtilis BR_4 (41.4%), B. licheniformis BU_7 (39.8%), and B. licheniformis BU_8 (43.5%). Various biochemical and plant growth promoting attributes, such as phosphate and Zn solubilization, IAA, ammonium, siderophore, and chitinase production, were also investigated for all the selected isolates. Furthermore, the potential of the isolates was tested in both in vitro and field conditions by employing talc-based bioformulation through bio-priming and root treatment. The application of bioformulation revealed a 20% decrease in disease incidence in plants treated with B. vallismortis (KU_7), a 60.5% increase in the biological yield, and a 45% increase in the grain yield. This eco-friendly approach not only controlled the disease but also improved the grain quality and reduced the chaffiness.

Rice false smut, caused by Ustilaginoidea virens, threatens rice production by reducing yields and contaminating grains with harmful ustiloxins. However, studies on resistance genes are scarce. In this study, the resistance level of IR28 (resistant cultivar) to U. virens was validated through artificial inoculation. Notably, a reactivation of resistance genes after transient down-regulation during the first 3 to 5 dpi was observed in IR28 compared to WX98 (susceptible cultivar). Cluster results of a principal component analysis and hierarchical cluster analysis of differentially expressed genes (DEGs) in the transcriptome exhibited longer expression patterns in the early infection phase of IR28, consistent with its sustained resistance response. Results of GO and KEGG enrichment analyses highlighted the suppression of immune pathways when the hyphae first invade stamen filaments at 5 dpi, but sustained up-regulated DEGs were linked to the ‘Plant–pathogen interaction’ (osa04626) pathway, notably disease-resistant protein RPM1 (K13457, CNLs, coil-coiled NLR). An analysis of CNLs identified 245 proteins containing Rx-CC and NB-ARC domains in the Oryza sativa Indica genome. Partial candidate CNLs were shown to exhibit up-regulation at both 1 and 5 dpi in IR28. This study provides insights into CNLs’ responses to U. virens in IR28, potentially informing resistance mechanisms and genetic breeding targets.

False smut, caused by Villosiclava virens, is becoming increasingly serious in modern rice production systems, leading to yield losses and quality declines. Successful infection requires efficient acquisition of sucrose, abundant in rice panicles, as well as other sugars. Sugar transporters (STPs) may play an important role in this process. STPs belong to a major facilitator superfamily, which consists of large multigenic families necessary to partition sugars between fungal pathogens and their hosts. This study identified and characterized the STP family of V. viren, and further analyzed their gene functions to uncover their roles in interactions with rice. Through genome-wide and systematic bioinformatics analyses, 35 STPs were identified from V.virens and named from VvSTP1 to VvSTP35. Transmembrane domains, gene structures, and conserved motifs of VvSTPs have been identified and characterized through the bioinformatic analysis. In addition, a phylogenetic analysis revealed relationship between VvSTPs and STPs from the other three reference fungi. According to a qRT-PCR and RNA-sequencing analysis, VvSTP expression responded differently to different sole carbon sources and H2O2 treatments, and changed during the pathogenic process, suggesting that these proteins are involved in interactions with rice and potentially functional in pathogenesis. In total, 12 representative VvSTPs were knocked out through genetic recombination in order to analyze their roles in pathogenicity of V. virens. The knock-out mutants of VvSTPs showed little difference in mycelia growth and conidiation, indicating a single gene in this family cannot influence vegetative growth of V. virens. It is clear, however, that these mutants result in a change in infection efficiency in a different way, indicating that VvSTPs play an important role in the pathogenicity of virens. This study is expected to contribute to a better understanding of how host-derived sugars contribute to V. virens pathogenicity. [ABSTRACT FROM AUTHOR]

Chelerythrine (CHE) is a natural benzophenanthridine alkaloid with broad pharmacological activities. Herein, we investigated the responses to CHE of the fungal pathogen Ustilaginoidea virens using a proteomics approach. At 7.5 × 10–3 mg/mL, the U. virens inhibition rate reached 56.1%, significantly more potent than another two isoquinoline alkaloids and the commercial fungicide validamycin. The disease control effect on plants in the field was > 50% after spraying with 500 × liquid containing 20% CHE, significantly better than that of validamycin. A total of 4734 proteins were identified in CHE-treated U. virens using tandem mass tag (TMT)-based quantitative proteomics, and 823 were differentially expressed proteins (DEPs). Gene Ontology (GO) of DEPs yielded 107 biological process (BP) subcategories, 45 cellular component (CC) subcategories and 43 molecular function (MF) subcategories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were employed, and seven proteins were selected for subsequent parallel reaction monitoring (PRM) analysis. The results indicate that CHE might lead to apoptosis, but further studies are necessary. [ABSTRACT FROM AUTHOR]

False smut or green smut, caused by Ustilaginoidea virens (Cooke) Takahashi is an important emerging disease of paddy causing 28-85 per cent yield losses in India and 18-36 per cent losses in some paddy growing districts of Uttar Pradesh. Thus, Integrated Disease Management approaches consisting of farmer's participatory on-farm trial for false smut management in two consecutive years in kharif 2019-20 and 2020-21 were evaluated. Technological gap between demonstrated technology and farmer's practices were studied based on survey and group discussion with farmers. Full gap was observed in case of use of seed/seedling treatment and a partial gap was in use of varieties, method of transplanting, number of hill/m2, use of fertilizers, weed management and plant protection measures, which definitely was the reason of not achieving potential yield. The average incidence of false smut in paddy fields, i.e. 7.05% was recorded in demonstrated practices while it was 12.35% in farmers' practice. On an average, disease reduction was 44.01 per cent noticed with the use of integrated disease management approach over farmer's practice, respectively. Average infected panicle/hill i.e., 1,87 and average infected panicle/m2 i.e., 3.35 was observed with use of integrated approach while it was noticed 2.82 and 4.92 in existing practices. The recommended rice cultivar yielded 48.60 q/ha yield during kharif 2019-20 while 47.80 q/ha was noticed in kharif 2020-21, it was 42.20 and 41.60 per cent more than farmer's practice. The average net returns, i.e. Rs. 77640.00/ha was received in demonstrated practices while farmers obtained Rs. 65530.00/ha by their own practices. On an average benefit-cost ratio 1.73 was obtained under demonstrated technologies while it was 1.45 in farmer's practices. The outcome of the trial inspired the farming communities to adopt area wise recommended varieties with judicious use of nitrogenous fertilizer and other technological options, including integrated disease management approach, which are being adopted in their cultivation. [ABSTRACT FROM AUTHOR]

Rice false smut, caused by the ascomycete fungus Ustilaginoidea virens, which infects rice florets before heading, severely threatens rice grain yield and quality worldwide. The U. virens genome encodes a number of glycoside hydrolase (GH) proteins. So far, the functions of these GHs in U. virens are largely unknown. In this study, we identified a GH42 protein secreted by U. virens, named UvGHF1, that exhibits β‐galactosidase activity. UvGHF1 not only functions as an essential virulence factor during U. virens infection, but also serves as a pathogen‐associated molecular pattern (PAMP) in Nicotiana benthamiana and rice. The PAMP activity of UvGHF1 is independent of its β‐galactosidase activity. Moreover, UvGHF1 triggers cell death in N. benthamiana in a BAK1‐dependent manner. Ectopic expression of UvGHF1 in rice induces pattern‐triggered immunity and enhances rice resistance to fungal and bacterial diseases. RNA‐seq analysis revealed that UvGHF1 expression in rice not only activates expression of many defence‐related genes encoding leucine‐rich repeat receptor‐like kinases and WRKY and ERF transcription factors, but also induces diterpenoid biosynthesis and phenylpropanoid biosynthesis pathways. Therefore, UvGHF1 contributes to U. virens virulence, but is also recognized by the rice surveillance system to trigger plant immunity. [ABSTRACT FROM AUTHOR]

It is perceived that sclerotia of the pathogen, in addition to chlamydospores, play an important role in the epidemiology of rice false smut disease. The propagule has been identified only in five rice growing countries of the world, and never recorded in Bangladesh. This study was undertaken to identify the sclerotia, quantify their concentration on smut balls and estimate the time of its formation. Intensive field-by-field monitoring was conducted during 2014, 2015 and 2016 at experimental farm of the Bangladesh Rice Research Institute, Gazipur. After rigorous search, sclerotia of rice false smut pathogen was identified on the 14th of December 2014. The was observed in the later part of crop maturity during the ‘T. Aman' season (early-November to mid-December) on olivaceous greenish-black smut balls, but not on orange smut ball. During the three years study, on average, 30.61±9.79% (± is 95%confidence interval) olivaceous greenish-black smut balls borne sclerotia, the average number being 1.63±0.09 per sclerotia-bearing ball. The ‘threshold temperature' for sclerotia formation was estimated as above 11°C difference in day-night temperature. Consistence presence and high concentration of sclerotia in smut balls signify its role in the epidemiology of the disease in the agroecology of Bangladesh. [Fundam Appl Agric 2023; 8(3.000): 580-589]

Abstract Rice false smut, caused by Ustilaginoidea virens, is one of the most important diseases in rice. The disease not only causes significant yield losses in China and worldwide but also produces multiple types of mycotoxins that pose a serious threat to the health of humans and animals. To effectively prevent and control the disease, the pathogenicity mechanisms of U. virens have been studied preliminarily, and some control strategies have been developed. This review focuses on recent progress in understanding the molecular mechanisms of U. virens pathogenicity, including virulence-related genes, transcriptional regulation of virulence genes, and effector-mediated interactions between rice and U. virens. Based on the molecular mechanisms underlying the rice-U. virens interactions, the possibilities of the pathogenicity genes in U. virens and host immune-related genes as potential targets for biological agents, host-induced gene silencing, and molecular design breeding are also discussed.

Dihydrochelerythrine (DHCHE) is an isoquinoline compound, which has distinct antifungal activity and can induce apoptosis. The antifungal activity of DHCHE against five rice pathogenic fungi was studied in vitro. At the concentration of 7.5 mg/L, DHCHE exhibited the highest efficacy among tested compounds in inhibiting mycelium growth, with an inhibition rate of 68.8% in Ustilaginoidea virens, which was approximately 2.4 times of that of validamycin (28.7%). After exposure to DHCHE, transmission electron micrographs revealed spores showed incomplete organelles, malformed cell walls and nuclear membranes, as well as irregular lipid spheres. Reactive oxygen species accumulation in treated spores was markedly higher than that in control spores. DHCHE induced cell damage increased in a dose-dependent manner, as indicated by the decrease in mitochondrial membrane potential and initiation of apoptosis. The differences of expression levels of Fip1, ACP1, PMS2 and COX13 that are important for oxidative phosphorylation and mismatch repair pathway were significant, which may be some of the reasons for the induction of apoptosis in DHCHE-treated U. virens. The protein levels of Fip1, ACP1, PMS2 and COX13 agreed with protein fold change ratio from parallel reaction monitoring Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway of differentially expressed proteins were further analyzed. These findings will help to elucidate the mechanisms associated with antifungal and pro-apoptotic effects of DHCHE on U. virens, thereby aiding the potential development of novel pesticides.

Introduction: Rice false smut caused by Ustilaginoidea virens, is a destructive fungal disease encountered in many rice-producing areas worldwide. To determine the process by which U. virens infects rice spikelets in the field. Methods: The green fluorescent protein-labeled U. virens was used as an inoculum to conduct artificial inoculation on rice at the booting stage via non-destructive panicle sheath instillation inoculation. Results: The results showed that the conidia of U. virens germinated on the surface of rice glumes and produced hyphae, which clustered at the mouth of rice glumes and entered the glumes through the gap between the palea and lemma. The conidia of U. virens colonized in rice floral organs, which led to pollen abortion of rice. U. virens wrapped the whole rice floral organ, and the floral organ-hyphae complex gradually expanded to open the glumes to form a rice false smut ball, which was two to three times larger than that observed in normal rice. Discussion: Panicle sheath instillation inoculation was shown to be a nondestructive inoculation method that could simulate the natural infection of U. virens in the field. The entire infection process of U. virens was visualized, providing a theoretical reference for formulating strategies to control rice false smut in the field. [ABSTRACT FROM AUTHOR]

Rice false smut caused by Ustilaginoidea virens is one of the most devastating fungal diseases of rice (Oryza sativa) worldwide. Prochloraz and azoxystrobin belong to the groups of demethylation inhibitors and quinone outside inhibitors, respectively, and are commonly used for controlling this disease. In this study, we analyzed the sensitivities of 100 U. virens isolates from Yunnan, Sichuan, Chongqing, and Zhejiang in Southern China to prochloraz and azoxystrobin. The ranges of EC50 for prochloraz and azoxystrobin were 0.004−0.536 and 0.020−0.510 μg/mL, with means and standard errors of 0.062 ± 0.008 and 0.120 ± 0.007 μg/mL, respectively. However, the sensitivity frequency distributions of U. virens to prochloraz and azoxystrobin indicated the emergence of subpopulations with decreased sensitivity. Therefore, the mean EC50 values of 74% and 68% of the isolates at the main peak, 0.031 ± 0.001 and 0.078 ± 0.004 μg/mL, were used as the sensitivity baselines of U. virens to prochloraz and azoxystrobin, respectively. We found significant sensitivity differences to azoxystrobin among different geographical populations and no correlation between the sensitivities of U. virens to prochloraz and azoxystrobin. Among 887 U. virens isolates, the isolate 5-3-1 from Zhejiang showed moderate resistance to prochloraz, with a resistance factor of 22.45, while no nucleotide variation in the 1986-bp upstream or 1827-bp gene regions of CYP51 from 5-3-1 was detected. Overexpression of CYP51 is probably responsible for its resistance to prochloraz. Finally, artificial inoculation showed that 5-3-1 was highly pathogenic to rice, suggesting that the resistance of U. virens to prochloraz must be monitored and managed in Zhejiang. [ABSTRACT FROM AUTHOR]

Rice false smut, caused by the fungal pathogen Ustilaginoidea virens, is a worldwide rice fungal disease. However, the molecular mechanism of the pathogenicity of the fungus U. virens remains unclear. To understand the molecular mechanism of pathogenesis of the fungus U. virens, we performed an integrated analysis of the transcriptome and metabolome of strongly (S) and weakly (W) virulent strains both before and after the infection of panicles. A total of 7932 differential expressed genes (DEGs) were identified using transcriptome analysis. Gene ontology (GO) and metabolic pathway enrichment analysis indicated that amino acid metabolism, autophagy-yeast, MAPK signaling pathway-yeast, and starch and sucrose metabolism were closely related to the pathogenicity of U. virens. Genes related to pathogenicity were significantly upregulated in the strongly virulent strain, and were ATG, MAPK, STE, TPS, and NTH genes. However, genes involved in the negative regulation of pathogenesis were significantly downregulated and contained TOR kinase, TORC1, and autophagy-related protein genes. Metabolome analysis identified 698 differentially accumulated metabolites (DAMs), including 13 categories of organic acids and derivatives, lipids and lipid-like molecules, organoheterocyclic compounds. The significantly enriched pathways of DAMs mainly included amino acids and carbohydrates, and they accumulated after infection by the S strain. To understand the relevance of DEGs and DAMs in the pathogenicity of U. virens, transcriptomic and metabolomic data were integrated and analyzed. These results further confirmed that the pathogenesis of U. virens was regulated by DEGs and DAMs related to these four pathways, involving arginine and proline metabolism, lysine biosynthesis, alanine, aspartate and glutamate metabolism, and starch and sugar metabolism. Therefore, we speculate that the pathogenicity of U. virens is closely related to the accumulation of amino acids and carbohydrates, and to the changes in the expression of related genes. [ABSTRACT FROM AUTHOR]

False smut of rice (FSR) is an emerging disease posing a major threat to rice cultivation throughout the world. A comprehensive and systematic survey was conducted in the major rice growing regions of Karnataka. There was a large variation in disease severity over the rice growing seasons which ranged from 2.05 to 18.10 per cent. Fifteen isolates of Ustilaginoidea virens collected during the survey showed varied morphological characteristics and the isolate Uv-2 was highly virulent. Further, fifteen isolates were confirmed as Ustilaginoidea virens upon successful amplification with universal ITS1 and ITS4 primers as well as specific uvr-F and uvr-R primers. Approximately 500-620 bp amplicon was obtained with universal primers, whereas 280-320 bp amplicon was obtained for specific primers. Phylogenetic analysis based on ITS region among the isolates exhibited significant genetic variation especially isolate Uv-2 separated out from rest of cluster. [ABSTRACT FROM AUTHOR]

Kernel smut, caused by Tilletia horrida, is a disease characterized by the replacement of rice grains with black sooty masses of teliospores or chlamydospores. Kernel smut differs from rice false smut, caused by Ustilaginoidea virens, in the color of chlamydospores. False smut is characterized by globose, velvety spore balls ranging from orangish yellow to greenish black in color. Both kernel smut and false smut have been persistent but are considered minor diseases in many countries since they were discovered in the late 1870s to the 1980s due to their sporadic outbreaks and limited economic impacts. In recent years, however, kernel smut and false smut have emerged as two of the most economically important diseases in rice, including organic rice, in many countries, especially in the United States. The increased use of susceptible rice cultivars, especially hybrids, excessive use of nitrogen fertilizer, and short crop rotations have resulted in an increase in kernel smut and false smut, causing significant losses in grain yield and quality. In this article, we provide a review of the distribution and economic importance of kernel smut; our current understanding of the taxonomy, biology, and epidemiology of kernel smut; and the genomics of the kernel smut fungus as compared with false smut and its causal agent. We also provide an update on the current management strategies of pathogen exclusion, cultivar resistance, fungicides, biological control, and cultural practices for kernel smut and false smut of rice. [ABSTRACT FROM AUTHOR]

Rice false smut, caused by Ustilaginoidea virens, is one of the most important diseases in rice. The disease not only causes significant yield losses in China and worldwide but also produces multiple types of mycotoxins that pose a serious threat to the health of humans and animals. To effectively prevent and control the disease, the pathogenicity mechanisms of U. virens have been studied preliminarily, and some control strategies have been developed. This review focuses on recent progress in understanding the molecular mechanisms of U. virens pathogenicity, including virulence-related genes, transcriptional regulation of virulence genes, and effector-mediated interactions between rice and U. virens. Based on the molecular mechanisms underlying the rice-U. virens interactions, the possibilities of the pathogenicity genes in U. virens and host immune-related genes as potential targets for biological agents, host-induced gene silencing, and molecular design breeding are also discussed. [ABSTRACT FROM AUTHOR]

Dihydrochelerythrine (DHCHE) is an isoquinoline compound, which has distinct antifungal activity and can induce apoptosis. The antifungal activity of DHCHE against five rice pathogenic fungi was studied in vitro. At the concentration of 7.5 mg/L, DHCHE exhibited the highest efficacy among tested compounds in inhibiting mycelium growth, with an inhibition rate of 68.8% in Ustilaginoidea virens , which was approximately 2.4 times of that of validamycin (28.7%). After exposure to DHCHE, transmission electron micrographs revealed spores showed incomplete organelles, malformed cell walls and nuclear membranes, as well as irregular lipid spheres. Reactive oxygen species accumulation in treated spores was markedly higher than that in control spores. DHCHE induced cell damage increased in a dose-dependent manner, as indicated by the decrease in mitochondrial membrane potential and initiation of apoptosis. The differences of expression levels of Fip1, ACP1, PMS2 and COX13 that are important for oxidative phosphorylation and mismatch repair pathway were significant, which may be some of the reasons for the induction of apoptosis in DHCHE-treated U. virens. The protein levels of Fip1, ACP1, PMS2 and COX13 agreed with protein fold change ratio from parallel reaction monitoring Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway of differentially expressed proteins were further analyzed. These findings will help to elucidate the mechanisms associated with antifungal and pro-apoptotic effects of DHCHE on U. virens , thereby aiding the potential development of novel pesticides. [ABSTRACT FROM AUTHOR]

bZIP proteins are widely distributed in eukaryotic organisms and regulate a diverse range of physiological processes. Several bZIP proteins have previously been identified in Ustilaginoidea virens. However, the biological roles of these bZIP proteins in this pathogen are still unknown. Here, one of these bZIP protein coding genes, UvATF21, was functionally characterized. Targeted deletion of UvATF21 resulted in reduced conidiation and pathogenicity despite of the increased vegetative growth. The deletion mutants also significantly decreased the sensitivity to osmotic and oxidative stresses. Interestingly, deletion of UvATF21 exhibited different performances to cell wall integrity stress. These results indicated that UvATF21 played crucial roles in vegetative growth, conidiation, stress response, and full virulence in U. virens.

Abstract Rice false smut, caused by Ustilaginoidea virens, is one of the most destructive diseases in major world rice-producing regions. Basic leucine zipper (bZIP) proteins, which belong to an evolutionarily conserved transcription factor family and play critical roles in various biological processes in eukaryotes, have been previously identified in U. virens; however, their functions still need to be further elucidated. Therefore, we aimed to analyze the biological roles of UvbZIP6, a member of the bZIP family in U. virens. In this study, we found that UvbZIP6 was highly up-regulated at 7 days post-inoculation. Deletion of UvbZIP6 in U. virens resulted in an increase in fungal growth and sensitivity to Congo red and calcofluor white, whereas a decrease in sensitivity to hyperosmotic, oxidative, and sodium dodecyl sulfate stresses. Conidiation capacity was reduced in UvbZIP6-knockout mutants, but conidial morphology and germination were not affected. Although UvbZIP6-knockout mutants caused infection in rice plants, they could not form false smut balls. Our study indicates that UvbZIP6 is required for fungal growth, conidiation, stress response, and false smut ball formation of U. virens.

Sorbicillinoids are a class of hexaketide metabolites produced by Ustilaginoidea virens (teleomorph: Villosiclava virens), an important fungal pathogen that causes a devastating rice disease. In this study, we investigated the effects of environmental factors, including carbon and nitrogen sources, ambient pH and light exposure, on mycelial growth, sporulation, as well as the accumulation of sorbicillinoids, and the expression of related genes involved in sorbicillinoid biosynthesis. It was found that the environmental factors had great influences on mycelial growth and sporulation of U. virens. Fructose and glucose, complex nitrogen sources, acidic conditions and light exposure were favorable for sorbicillinoid production. The relative transcript levels of sorbicillinoid biosynthesis genes were up-regulated when U. virens was separately treated with those environmental factors that favored sorbicillinoid production, indicating that sorbicillinoid biosynthesis was mainly regulated at the transcriptional level by different environmental factors. Two pathway-specific transcription factor genes, UvSorR1 and UvSorR2, were found to participate in the regulation of sorbicillinoid biosynthesis. These results will provide useful information to better understand the regulation mechanisms of sorbicillinoid biosynthesis, and be conducive to develop effective means for controlling sorbicillinoid production in U. virens. [ABSTRACT FROM AUTHOR]

Rice false smut, caused by Ustilaginoidea virens, has become one of the most devastating grain diseases of rice worldwide. Understanding the genetic diversity of U. virens is essential for efficient disease control and breeding for disease resistance. However, little is known about the genetic variation of U. virens from different rice cultivars. We investigated the genetic diversity and pathogenic variation of U. virens isolates from 10 rice cultivars in Zhejiang, China. A total of 260 polymorphic loci and 27 haplotypes were identified based on the 2,137-bp combined DNA fragments of all individuals; hap_4 was the most common haplotype, represented by 41 isolates. Phylogeny indicated that all isolates were divided into four genetic groups. Group I was the largest, with 98 isolates, distributed mainly in eight cultivar populations, whereas 90% of the isolates collected from a Changxiang cultivar were clustered in Group IV. Furthermore, the pairwise FST values exhibited significant genetic differentiation in 27 of the pairwise comparisons between populations, accounting for 23.21% of the total genetic variation. The genetic composition of the isolates of the CX population was distinguishable from that of the other nine populations, and genetic recombination was found in a few isolates. Finally, 27 haplotype representative isolates showed high variation in pathogenicity, and the isolates from the genetic subpopulation I were likely to be more virulent than those from genetic subpopulations II and III. Collectively, these findings suggest that differences in rice cultivars play an important role in the genetic variation of U. virens. [ABSTRACT FROM AUTHOR]

ABSTRACT Autophagy and apoptosis are evolutionarily conserved catabolic processes involved in regulating development and cellular homeostasis. Bax inhibitor 1 (BI-1) and autophagy protein 6 (ATG6) perform essential functions in these roles, such as cellular differentiation and virulence in various filamentous fungi. However, the functions of ATG6 and BI-1 proteins in development and virulence in the rice false smut fungus Ustilaginoidea virens are still poorly understood. In this study, UvATG6 was characterized in U. virens. The deletion of UvATG6 almost abolished autophagy in U. virens and reduced growth, conidial production and germination, and virulence. Stress tolerance assays showed that UvATG6 mutants were sensitive to hyperosmotic, salt, and cell wall integrity stresses but were insensitive to oxidative stress. Furthermore, we found that UvATG6 interacted with UvBI-1 or UvBI-1b and suppressed Bax-induced cell death. We previously found that UvBI-1 could suppress Bax-induced cell death and was a negative regulator of mycelial growth and conidiation. Unlike UvBI-1, UvBI-1b could not suppress cell death. UvBI-1b-deleted mutants exhibited decreased growth and conidiation, while the UvBI-1 and UvBI-1b double deletion reduced the phenotype, indicating that UvBI-1 and UvBI-1b antagonistically regulate mycelial growth and conidiation. In addition, the UvBI-1b and double mutants exhibited decreased virulence. Our results provide evidence of the cross talk of autophagy and apoptosis in U. virens and give clues for studying other phytopathogenic fungi. IMPORTANCE Ustilaginoidea virens causes destructive panicle disease in rice, significantly threatening agricultural production. UvATG6 is required for autophagy and contributes to growth, conidiation, and virulence in U. virens. Additionally, it interacts with the Bax inhibitor 1 proteins UvBI-1 and UvBI-1b. UvBI-1 suppresses cell death induced by Bax, unlike UvBI-1b. UvBI-1 negatively regulates growth and conidiation, while UvBI-1b is required for these phenotypes. These results indicate that UvBI-1 and UvBI-1b may antagonistically regulate growth and conidiation. In addition, both of them contribute to virulence. Additionally, our results suggest cross talk between autophagy and apoptosis, contributing to the development, adaptability, and virulence of U. virens.

False smut, caused by Villosiclava virens, is becoming increasingly serious in modern rice production systems, leading to yield losses and quality declines. Successful infection requires efficient acquisition of sucrose, abundant in rice panicles, as well as other sugars. Sugar transporters (STPs) may play an important role in this process. STPs belong to a major facilitator superfamily, which consists of large multigenic families necessary to partition sugars between fungal pathogens and their hosts. This study identified and characterized the STP family of V. viren, and further analyzed their gene functions to uncover their roles in interactions with rice. Through genome-wide and systematic bioinformatics analyses, 35 STPs were identified from V.virens and named from VvSTP1 to VvSTP35. Transmembrane domains, gene structures, and conserved motifs of VvSTPs have been identified and characterized through the bioinformatic analysis. In addition, a phylogenetic analysis revealed relationship between VvSTPs and STPs from the other three reference fungi. According to a qRT-PCR and RNA-sequencing analysis, VvSTP expression responded differently to different sole carbon sources and H2O2 treatments, and changed during the pathogenic process, suggesting that these proteins are involved in interactions with rice and potentially functional in pathogenesis. In total, 12 representative VvSTPs were knocked out through genetic recombination in order to analyze their roles in pathogenicity of V. virens. The knock-out mutants of VvSTPs showed little difference in mycelia growth and conidiation, indicating a single gene in this family cannot influence vegetative growth of V. virens. It is clear, however, that these mutants result in a change in infection efficiency in a different way, indicating that VvSTPs play an important role in the pathogenicity of virens. This study is expected to contribute to a better understanding of how host-derived sugars contribute to V. virens pathogenicity.

ABSTRACT Magnaporthe oryzae and Ustilaginoidea virens are two filamentous fungal pathogens that threaten rice production worldwide. Genetic tools that permit fast gene deletion and silencing are of great interest for functional genomics of fungal pathogens. As a revolutionary genome editing tool, clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) enable many innovative applications. Here, we developed a CRISPR interference (CRISPRi) toolkit using nuclease activity dead Cas9 (dCas9) to silence genes of interest in M. oryzae and U. virens. We optimized the components of CRISPRi vectors, including transcriptional repression domains, dCas9 promoters, and guide RNA (gRNA) promoters. The CRISPRi tool was tested using nine gRNAs to target the promoters of MoATG3, MoATG7, and UvPal1. The results indicated that a single gRNA could direct the dCas9-fused transcriptional repression domain to efficiently silence the target gene in M. oryzae and U. virens. In both fungi, the target genes were repressed >100-fold, and desired phenotypes were observed in CRISPRi strains. Importantly, we showed that multiple genes could be easily silenced using polycistronic tRNA-gRNA in CRISPRi. Furthermore, gRNAs that bind different promoter regions displayed variable repression levels of target genes, highlighting the importance of gRNA design for CRISPRi efficiency. Together, this study provides an efficient and robust CRISPRi tool for targeted gene silencing in M. oryzae and U. virens. Owing to its simplicity and multiplexity, CRISPRi will be a useful tool for gene function discovery in fungal pathogens. IMPORTANCE Many devastating plant diseases are caused by fungal pathogens that evolve rapidly to adapt to host resistance and environmental changes. Therefore, genetic tools that enable fast gene function discovery are needed to study the pathogenicity and stress adaptation of fungal pathogens. In this study, we adopted the CRISPR/Cas9 system to silence genes in Magnaporthe oryzae and Ustilaginoidea virens, which are two dominant fungal pathogens that threaten rice production worldwide. We present a versatile and robust CRISPRi toolkit that represses target gene expression >100-fold using a single gRNA. We also demonstrated that CRISPRi could simultaneously silence multiple genes using the tRNA-gRNA strategy. The CRISPRi technologies described in this study would accelerate the functional genomics of fungal pathogens.

bZIP proteins are widely distributed in eukaryotic organisms and regulate a diverse range of physiological processes. Several bZIP proteins have previously been identified in Ustilaginoidea virens. However, the biological roles of these bZIP proteins in this pathogen are still unknown. Here, one of these bZIP protein coding genes, UvATF21 , was functionally characterized. Targeted deletion of UvATF21 resulted in reduced conidiation and pathogenicity despite of the increased vegetative growth. The deletion mutants also significantly decreased the sensitivity to osmotic and oxidative stresses. Interestingly, deletion of UvATF21 exhibited different performances to cell wall integrity stress. These results indicated that UvATF21 played crucial roles in vegetative growth, conidiation, stress response, and full virulence in U. virens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rice false smut (RFS), caused by Ustilaginoidea virens, is an important fungal disease of rice. In China, Methyl Benzimidazole Carbamates (MBCs), including carbendazim, are common fungicides used to control RFS and other rice diseases. In this study, resistance of U. virens to carbendazim was monitored for three consecutive years during 2018 to 2020. A total of 321 U. virens isolates collected from Jiangsu Province of China were tested for their sensitivity to carbendazim on PSA. The concentration at which mycelial growth is inhibited by 50% (EC50) of the carbendazim-sensitive isolates was 0.11 to 1.38 µg/mL, with a mean EC50 value of 0.66 μg/mL. High level of resistance to carbendazim was detected in 14 out of 321 isolates. The resistance was stable but associated with a fitness penalty. There was a statistically significant and moderate negative correlation (r= −0.74, p < 0.001) in sensitivity between carbendazim and diethofencarb. Analysis of the U. virens genome revealed two potential MBC targets, Uvβ1Tub and Uvβ2Tub, that putatively encode β-tubulin gene. The two β-tubulin genes in U. virens share 78% amino acid sequence identity, but their function in MBC sensitivity has been unclear. Both genes were identified and sequenced from U. virens sensitive and resistant isolates. It is known that mutations in the β2-tubulin gene have been shown to confer resistance to carbendazim in other fungi. However, no mutation was found in the Uvβ2Tub gene in either resistant or sensitive isolates. Variations including point mutations, non-sense mutations, codon mutations, and frameshift mutations were found in the Uvβ1Tub gene from the 14 carbendazim-resistant isolates, which have not been reported in other fungi before. Thus, these results indicated that variations of Uvβ1Tub result in the resistance to carbendazim in field isolates of Ustilaginoidea virens. [ABSTRACT FROM AUTHOR]

Summary: Rice false smut caused by Ustilaginoidea virens is becoming one of the most recalcitrant rice diseases worldwide. However, the molecular mechanisms underlying rice immunity against U. virens remain unknown.Using genetic, biochemical and disease resistance assays, we demonstrated that the xb24 knockout lines generated in non‐Xa21 rice background exhibit an enhanced susceptibility to the fungal pathogens U. virens and Magnaporthe oryzae. Consistently, flg22‐ and chitin‐induced oxidative burst and expression of pathogenesis‐related genes in the xb24 knockout lines were greatly attenuated.As a central mediator of energy signaling, SnRK1A interacts with and phosphorylates XB24 at Thr83 residue to promote ATPase activity. SnRK1A is activated by pathogen‐associated molecular patterns and positively regulates plant immune responses and disease resistance. Furthermore, the virulence effector SCRE1 in U. virens targets host ATPase XB24. The interaction inhibits ATPase activity of XB24 by blocking ATP binding to XB24. Meanwhile, SCRE1 outcompetes SnRK1A for XB24 binding, and thereby suppresses SnRK1A‐mediated phosphorylation and ATPase activity of XB24.Our results indicate that the conserved SnRK1A‐XB24 module in multiple crop plants positively contributes to plant immunity and uncover an unidentified molecular strategy to promote infection in U. virens and a novel host target in fungal pathogenesis. [ABSTRACT FROM AUTHOR]