Water Extract of Rice False Smut Balls Activates Nrf2/HO-1 and Apoptosis Pathways,Causing Liver Injury

Ustiloxins are vital cyclopeptide mycotoxins originally isolated from rice false smut balls that form in rice spikelets infected by the fungal pathogen Ustilaginoidea virens.The toxicity of the water extract of rice false smut balls(RBWE) remains to be investigated.Studies have shown that RBWE may be toxic to animals,but toxicological evidence is still lacking.In this study,we found that the IC50 values of RBWE to BNL CL.2 cells at 24 and 48 h were 40.02 and 30.11 μg/m L,respectively,with positive correlations with dose toxicity and time toxicity.After treatment with RBWE,the number of BNL CL.2 cells decreased significantly,and the morphology of BNL CL.2 cells showed atrophy and wall detachment.RBWE induced DNA presynthesis phase arrest of BNL CL.2 cells,increased the proportion of apoptotic cells and inhibited cell proliferation.RBWE up-regulated reactive oxygen species(ROS) levels and lowered mitochondrial membrane potentials.Additionally,Western blot and q RT-PCR results suggested that RBWE exerted the above effects by promoting the Nrf2/HO-1 and caspase-induced apoptosis pathways in vitro and in vivo.The contents of alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,and total bile acids in the serum of mice from Institute of Cancer were significantly up-regulated by RBWE.At the same time,RBWE can lead to increases in ROS and malondialdehyde contents,decreases in contents of oxidized glutathione,glutathione and reduced glutathione,as well as decrease in catalase and superoxide dismutase activities in mouse liver tissues,demonstrating that oxidative stress occurred in mice.Moreover,liver damage was further detected by haematoxylin-eosin staining and electron microscopy to verify the damage to the mice caused by RBWE.In general,RBWE may cause hepatotoxicity in vivo and in vitro via the apoptosis pathway,which provides a reference for hepatotoxicity and its mechanism of action.

A PCR-Based Method for Detecting Pathogen of Rice False Smut

Rice false smut is a destructive disease that affects rice grain badly.The disease seriously affects the yield and quality of rice in Heilongjiang Province.In this paper,a pair of specific primers was designed to detect the false smut pathogen rapidly and efficiently.The results showed that the pair of primers had strong specificity for false smut pathogen.In addition,the sensitivity of this primer to the genomic DNA of rice false smut pathogen in PCR reaction was 1 pg.By using these primers,the rice false smut pathogen could be detected within 48 h after inoculation,and a PCR reaction system with good specificity and high sensitivity was established.

Analysis on Quantitative Trait Loci for Resistance to Rice False Smut under Different Environmental Conditions

A recombinant inbred line （RILs） population with 157 lines derived from an inter-subspecies cross of Daguandao （japonica）/IR28 （indica） by the single seed de-scent method was used to detect quantitative trait loci （QTLs） conferring resistance to rice false smut caused by Ustilaginoidea virens（Cooke） Takahashi in Nanjing and Yangzhou. The disease rate index of the two parents and 157 RILs caused by rice false smut were scored and the QTLs for rice false smut resistance were detected accordingly by QTL Cartographer software. Eight QTLs control ing false smut resis-tance were detected on chromosomes 1, 2, 4, 8, 10, 11 and 12, respectively, with the phenotypic variance of 8.6%-22.5%. There were five QTLs detected in Nanjing and Yangzhou, respectively, and only two QTLs were found in both two years, the phenotypic variation was explained by individual QTL ranged from 18.0% to 18.9% for these two QTLs, and the additive effects of these two QTLs contributed to the 8.0%-14.6% decrease of disease index and therefore the disease resistance increased. The direction of the additive effects at six loci qFsr1, qFsr2, qFsr8, qFsr10a, qFsr11 and qFsr12 coincided with that predicted by phenotypes of the parents, and the IR28 al eles at these loci had positive effect against rice false smut while the negative effects were found in Daguandao al eles at qFsr4 and qFsr10b. Both qFsr10a and qFsr11 should be useful in rice breeding for resistance to rice false smut in marker-assisted selection （MAS） program.

Analysis of Quantitative Trait Loci for Pathogenic Strain Pi-1 of Resistance to Rice False Smut in Rice(Oryza sativa)

In order to identify the resistant gene of rice false smut in rice, a recombi- nant inbred line （RILs） population with 157 lines derived from an inter-subspecies cross of Daguandao/IR28 by the single seed descent method was used to detect quantitative trait loci （QTLs） conferring resistance to strain Pi-1 of rice false smut caused by Usti/aginoiclea virens （Cooke） Takahashi in 2012 and 2013. The disease rate indexes of the two parents and 157 RILs caused by the strain Pi-1 of rice false smut were scored and the QTLs for rice false smut resistance were detected accordingly by QTL Cartographer software. Seven QTLs controlling false smut re- sistance were detected on chromosomes 2, 7, 8, 11 and 12, respectively, with the phenotypic variance of 8.5%-17.2%. There were four QTLs detected in 2012 and 2013, respectively, and only one QTL was found in both two years, the phenotypic variation explained by this individual QTL was 13.5% and 17.2%, and the additive effects of this QTL contributed to the 9.9% and 14.3% decrease of disease index and therefore the disease resistance increased. The direction of the additive effects at five loci qFsr2a, qFsr8a, qFsr8b, qFsr11 and qFsr12 coincided with that predicted by phenotypes of the parents, and the IR28 alleles at these loci had positive effect against rice false smut while the negative effects were found in Daguandao alleles at qFsr2b and qFsr7. The qFsr11 should be useful in rice breeding for resistance to rice false smut in marker-assisted selection （MAS） program.

Survey and examination of the potential alternative hosts of Villosiclava virens, the pathogen of rice false smut, in China

Rice false smut is caused by ascomycete Villosiclava virens, whose potential alternative hosts have been assumed previ- ously. Here its potential alternative hosts were surveyed and identified from 2008 to 2013 in the main rice-growing regions in China. Two common weeds in paddy fields, Digitaria sanguinalis Scop. and Echinochloa crusgalli （L.） Beauv., were found to present the similar symptoms to smut diseases in a few individuals in 2012 and 2013 in Zhejiang and Sichuan provinces of China, respectively. After the examinations of the spore morphology, their infection and extension mode in hosts, pathogen cell wall components, and molecular identification, the two pathogens were identified to be the Basidiomycetes, Ustilago syntherismae and Ustilago trichophora, respectively. So far there has been no alternative host of V. virens to be identified in China. These suggest that the alternative hosts of V. virens, if they do exist, are not possible to play an important role in the pathogen life cycle and the disease epidemics.

Rapid detection of the rice false smut fungus Ustilaginoidea virens by lateral flow strip-based recombinase polymerase amplification assay

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.

Interaction Between Ustilaginoidea virens and Rice and Its Sustainable Control

Ustilaginoidea virens is a common rice pathogen that can easily lead to a decline in rice quality and the production of toxins pose potential risks to human health.In this review,we present a comprehensive literature review of research since the discovery of rice false smut.We provide a comprehensive and,at times,critical overview of the main results and findings from related research,and propose future research directions.Firstly,we delve into the interaction between U.virens and rice,including the regulation of transcription factors,the process of U.virens infecting rice panicles,and the plant immune response caused by rice infection.Following that,we discuss the identification and characterization of mycotoxins produced by the pathogenic fungus,as well as strategies for disease management.We emphasize the importance of comprehensive agricultural prevention and control methods for the sustainable management of U.virens.This knowledge will update our understanding of the interaction between U.virens and rice plants,offering a valuable perspective for those interested in U.virens.

Extraction and purification of ustiloxin A from rice false smut balls by a combination of macroporous resin and high-speed countercurrent chromatography

Rice false smut is an emerging plant disease worldwide.Ustiloxin A(UstA)is the major mycotoxin found in rice false smut balls,which are fungal colonies in rice florets.In this study,a new method consisting of macroporous resin column chromatography and high-speed countercurrent chromatography(HSCCC)was developed for UstA separation.UstA was extracted by a 3.81%HCOOH solution and adsorbed by XAD-4 resin.UstA was then eluted by a 40%methanol solution supplemented with 0.1%trifluoroacetic acid(TFA).Further purification was achieved by HSCCC using a two-phase solvent system consisting of n-butanol/TFA/H_(2)O(1/0.05/1,v/v/v).Under the optimized conditions,225 mg of UstA was obtained with a purity of 97.39%in a single run,with a final recovery of 65.2%.An inhibitory effect on seed germination of wheat and maize caused by UstA was observed in a preliminary phytotoxicity assay.

Carbon Catabolite Repressor UvCreA is Required for Development and Pathogenicity in Ustilaginoidea virens

The rice false smut disease, caused by Ustilaginoidea virens, has emerged as a significantglobal threat to rice production. The mechanism of carbon catabolite repression plays a crucial role in theefficient utilization of carbon nutrients and enzyme regulation in the presence of complex nutritionalconditions. Although significant progress has been made in understanding carbon catabolite repression infungi such as Aspergillus nidulans and Magnaporthe oryzae, its role in U. virens remains unclear. Toaddress 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 resultedin decreased growth and pathogenicity in U. virens. Through RNA-seq analysis, it was found that theknockout 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 genesassociated with carbon metabolism in U. virens. In summary, our findings indicated that UvCreA isimportant in fungal development, virulence, and the utilization of carbon sources through transcriptionalregulation, thus making it a critical element of carbon catabolite repression.

Genetic Diversity of Ustilaginoidea virens in Heilongjiang Province

In recent years, the occurrence of rice false smut has become increasingly serious and the area of damage has expanded year by year, so the false smut has become one of the diseases which have seriously affected rice yield and quality in Heilongjiang Province. However, the genetic diversity research on false smut in Heilongjiang Province was relatively weak. In the current investigation, seven polymorphic SSR markers were taken to analyze the genetic diversity among 89 strains of the pathogens of rice false smut collected from five main rice growing areas in Heilongjiang Province, China. The results showed that the amplified bands of each pair of primers were between 2 and 7. A total of 43 bands were obtained, and each primer was expanded for 4.8 bands. The genetic similarity coefficient(GSC) between strains by using SSR molecular marker analysis showed that the GSC of the strain of Ustilaginoidea virens was 0.613-0.955, with an average of 0.741. According to the results cluster analysis, when the genetic distance was 0.73, 89 strains were divided into three groups, and five strains of rice smut strains were distributed in three populations, and strains in Harbin City were mainly distributed in group Ⅲ. In Mudanjiang City, the strains were mainly distributed in groups Ⅰ and Ⅲ, and the strains in Suihua City were mainly distributed in groups Ⅰ and Ⅱ, and the strains in Jiamusi City were evenly distributed in groups Ⅰ, Ⅱ and Ⅲ, and the strains in Qiqihar City were mainly distributed in groups Ⅱ and Ⅲ. The results showed that the genetic background of Ustilaginoidea virens in Heilongjiang Province was relatively low in complexity, and the overall inheritance of the strain was relatively stable.

Biological Activity of Several Fungicides against Ustilaginoidea virens

[Objective] This study aims to screen for the high effective fungicides which could significantly decrease the disease incidence and disease index of rice false smut. [Method] The inhibitory activities of the fungicide against mycelial growth of Ustilaginoidea virens were measured to in vitro evaluate the ECho values. And 17 fungicides were sprayed to evaluate the efficacy and effect of the fungicides tested in the field trials on the rice characters, [Result] The results showed that epoxicona- zole, difenoconazole, propiconazole and procloraz exhibited high inhibitory activity against mycelial growth of Ustilaginoidea virens with the ECso values 0.04, 0.07, 0.12 and 0.11 pg/ml, respectively. The results of field trials showed that the efficacy of Wen- quning, and fungicides such as difenoconazole, prochloraz, propiconazole, epoxi- conazole and their mixtures in controlling rice false smut were all 70% or more. [Conclusion] The 17 tested fungicides behaved efficacy in controlling rice false smut and did not cause drug injury on leaves and grains of rice plants, sprayed when flag leaves of rice fully expanded.

Effect of temperature on the development of sclerotia in Villosiclava virens

The sclerotia of Villosiclava virens were found commonly in high altitude and the temperate regions, where the temperatures are relatively low in rice filling stage. To make sure if low temperature induce the sclerotial formation in V. virens, the inoculated rice panicles in laboratory and the diseased rice panicles cut from paddy fields were treated under different temperatures. The results showed that 3 days of night temperature at 15°C were enough to induce the sclerotial formation. The low temperature was much more effective for young balls with intact membranes. After appearance of chlamydospores on the ball surfaces, the sclerotium could not differentiate anymore. The sclerotia began to differentiate below the chlamydospore layer and gradually grew onto the ball surfaces. This suggests that low temperature in the early development stage of false smut balls is an important factor to induce the sclerotial differentiation, and rice cultivars with long growth periods are able to produce more sclerotium-bearing balls, which will produce mass of spores in paddy field in the coming year.

Putative Phosphatase UvPsr1 Is Required for Mycelial Growth, Conidiation, Stress Response and Pathogenicity in Ustilaginonidea 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 AUvpsr1 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 AUvpsr1 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.

UvWhi2 Is Required for Stress Response and Pathogenicity in Ustilaginoidea virens

Some stress response-related genes have been identified in Ustilaginoidea virens,but it is not clear whether and how defects of stress responses affect the pathogenesis processes of U.virens.In this study,we identified a general stress response factor UvWHI2 as a homolog of Saccharomyces cerevisiae Whi2 in U.virens.The relative expression level of Uv Whi2 was significantly up-regulated during infection,suggesting that UvWHI2 may be involved in pathogenesis.Furthermore,knockout of Uv Whi2 showed decreased mycelial growth,increased conidiation in the potato sucrose medium and a defect in pathogenicity.In addition,the RNA-Seq and phenotypic analysis showed that UvWHI2 was involved in response to oxidative,hyperosmotic,cell wall stress and nutrient limitation.Further studies revealed that the defects of stress responses of the?Uvwhi2 mutant affected the formation of secondary spores on the nutrient limited surface and the rice surface,resulting in a significant reduction of pathogenicity of U.virens.Our results suggest that UvWHI2 is necessary for fungal growth,stress responses and the formation of secondary spores in U.virens.In addition,the defects of stress responses can affect the formation of secondary spores on the rice surface,and then compromise the pathogenicity of U.virens.

Infection of Ustilaginoidea virens intercepts rice seed formation but activates grain-filling-related genes

Rice false smut has become an increasingly serious disease in rice （Oryza sativa L.） production worldwide. The typical feature of this disease is that the fungal pathogen Ustilaginoidea virens （Uv） specifical y infects rice flower and forms false smut bal , the ustiloxin-containing bal-like fungal colony, of which the size is usual y several times larger than that of a mature rice seed. However, the underlying mechanisms of Uv-rice interac-tion are poorly understood. Here, we applied time-course microscopic and transcriptional approaches to investigate rice responses to Uv infection. The results demonstrated that the flower-opening process and expression of associated transcription factors, including ARF6 and ARF8, were inhibited in Uv-infected spikelets. The ovaries in infected spikelets were interrupted in fertilization and thus were unable to set seeds. However, a number of grain-fil ing-related genes, including seed storage protein genes, starch anabolism genes and endosperm-specific transcription factors （RISBZ1 and RPBF）, were highly transcribed as if the ovaries were fertilized. In addition, critical defense-related genes like NPR1 and PR1 were downregulated by;Uv infection. Our data imply that Uv may hijack host nutrient reservoir by activation of the grain-fil ing network because of growth and formation of false smut bal s.