Ustilaginoidea Virens Research Articles

Effects of Carbon, Nitrogen, Ambient pH and Light on Mycelial Growth, Sporulation, Sorbicillinoid Biosynthesis and Related Gene Expression in Ustilaginoidea 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.

Genetic Diversity and Pathogenic Variation of the Rice False Smut Pathogen Ustilaginoidea virens from Different Rice Cultivars.

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 ten rice cultivars in Zhejiang, China. A total of 260 polymorphic loci and 27 haplotypes were identified based on the 2137-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.

Detection of Rice Fungal Spores Based on Micro- Hyperspectral and Microfluidic Techniques

As rice is one of the world's most important food crops, protecting it from fungal diseases is very important for agricultural production. At present, it is difficult to diagnose rice fungal diseases at an early stage using relevant technologies, and there are a lack of rapid detection methods. This study proposes a microfluidic chip-based method combined with microscopic hyperspectral detection of rice fungal disease spores. First, a microfluidic chip with a dual inlet and three-stage structure was designed to separate and enrich Magnaporthe grisea spores and Ustilaginoidea virens spores in air. Then, the microscopic hyperspectral instrument was used to collect the hyperspectral data of the fungal disease spores in the enrichment area, and the competitive adaptive reweighting algorithm (CARS) was used to screen the characteristic bands of the spectral data collected from the spores of the two fungal diseases. Finally, the support vector machine (SVM) and convolutional neural network (CNN) were used to build the full-band classification model and the CARS filtered characteristic wavelength classification model, respectively. The results showed that the actual enrichment efficiency of the microfluidic chip designed in this study on Magnaporthe grisea spores and Ustilaginoidea virens spores was 82.67% and 80.70%, respectively. In the established model, the CARS-CNN classification model is the best for the classification of Magnaporthe grisea spores and Ustilaginoidea virens spores, and its F1-core index can reach 0.960 and 0.949, respectively. This study can effectively isolate and enrich Magnaporthe grisea spores and Ustilaginoidea virens spores, providing new methods and ideas for early detection of rice fungal disease spores.

Development and application of recombinase polymerase amplification for rapid detection of rice false smut pathogen (Ustilaginoidea virens)

Ustilaginoidea virens (Cke.) Tak. is the causal pathogen of rice false smut, a grain disease which affects both grain quality and yield. In this study, a recombinase polymerase amplification (RPA) assay protocol was developed to detect U. virens directly from rice spikelet. Three pairs of primer were designed from the U. virens GTP binding protein beta subunit (UvG-β1) gene to function both in RPA and in polymerase chain reaction (PCR). The developed RPA assay efficiently detected U. virens from both genomic DNA and mycelial crude extract. A comparison of sensitivity of RPA with PCR and loop-mediated isothermal amplification (LAMP) revealed that the latter two methods failed to detect U. virens from crude extract. The specificity of the RPA primers was evaluated using DNA from other rice pathogens, as well as, by sequencing the RPA amplicons. For validation, the RPA assay was evaluated on 32 different U. virens isolates from eastern and north-eastern India, as well as, on field samples. The developed RPA assay was able to detect U. virens directly from crude extract prepared from infected rice spikelet. The developed RPA assay will be effective for field monitoring of U. virens.

Molecular characterization of a novel victorivirus (order Ghabrivirales, family Totiviridae) infecting Metarhizium anisopliae.

Here, we report the occurrence and complete genome sequence of a novel victorivirus infecting Metarhizium anisopliae, named "Metarhizium anisopliae victorivirus 1" (MaVV1). The genome is 5353 bp in length and contains two open reading frames (ORFs), encoding a coat protein and an RNA-dependent RNA polymerase (RdRp), that overlap at the octanucleotide sequence AUGAGUAA. These ORFs showed sequence similarity to the corresponding ORFs of Ustilaginoidea virens RNA virus L (68.23%) and Ustilaginoidea virens RNA virus 13 (58.11%), respectively, both of which belong to the family Totiviridae. Phylogenetic analysis based on RdRp sequences revealed that MaVV1 clustered with members of the genus Victorivirus. This is the first genome sequence reported for a virus belonging to the genus Victorivirus infecting the entomopathogenic fungus M. anisopliae.

The Genetic Mechanism of the Immune Response to the Rice False Smut (RFS) Fungus Ustilaginoidea virens

Rice false smut (RFS), which is caused by Ustilaginoidea virens (U. virens), has become one of the most devastating diseases in rice-growing regions worldwide. The disease results in a significant yield loss and poses health threats to humans and animals due to producing mycotoxins. In this review, we update the understanding of the symptoms and resistance genes of RFS, as well as the genomics and effectors in U. virens. We also highlight the genetic mechanism of the immune response to RFS. Finally, we analyse and explore the identification method for RFS, breeding for resistance against the disease, and interactions between the effector proteins and resistance (R) proteins, which would be involved in the development of rice disease resistance materials for breeding programmes.

The Effect of Trichoderma harzianum Hypovirus 1 (ThHV1) and Its Defective RNA ThHV1-S on the Antifungal Activity and Metabolome of Trichoderma koningiopsis T-51

Mycoviruses widely exist in filamentous fungi and sometimes cause phenotypic changes in hosts. Trichoderma harzianum hypovirus 1 (ThHV1) and its defective RNA ThHV1-S were found in T. harzianum and exhibited high transmissibility. In our previous study, ThHV1 and ThHV1-S were transferred to an excellent biological control agent T. koningiopsis T-51 to form a derivative strain 51-13. In this study, we assessed the metabolic changes in strain 51-13 and antifungal activity of its culture filtrate (CF) and volatile organic compounds (VOCs). The antifungal activity of CF and VOCs of T-51 and 51-13 was different. Compared with the CF of T-51, that of 51-13 exhibited high inhibitory activity against B. cinerea, Sclerotinia sclerotiorum, and Stagonosporopsis cucurbitacearum but low inhibitory activity against Leptosphaeria biglobosa and Villosiclava virens. The VOCs of 51-13 exhibited high inhibitory activity against F. oxysporum but low inhibitory activity against B. cinerea. The transcriptomes of T-51 and 51-13 were compared; 5531 differentially expressed genes (DEGs) were identified in 51-13 with 2904 up- and 2627 downregulated genes. In KEGG enrichment analysis, 1127 DEGs related to metabolic pathways (57.53%) and 396 DEGs related to biosynthesis of secondary metabolites (20.21%) were clearly enriched. From the CF of T-51 and 51-13, 134 differential secondary metabolites (DSMs) were detected between T-51 and 51-13 with 39 up- and 95 downregulated metabolites. From these, 13 upregulated metabolites were selected to test their antifungal activity against B. cinerea. Among them, indole-3-lactic acid and p-coumaric acid methyl ester (MeCA) exhibited strong antifungal activity. The IC50 of MeCA was 657.35 μM and four genes possibly related to the synthesis of MeCA exhibited higher expression in 51-13 than in T-51. This study revealed the mechanism underlying the increase in antifungal activity of T-51 because of the mycovirus and provided novel insights in fungal engineering to obtain bioactive metabolites via mycoviruses.

A natural allele of proteasome maturation factor improves rice resistance to multiple pathogens.

Crops with broad-spectrum resistance loci are highly desirable in agricultural production because these loci often confer resistance to most races of a pathogen or multiple pathogen species. Here we discover a natural allele of proteasome maturation factor in rice, UMP1R2115, that confers broad-spectrum resistance to Magnaporthe oryzae, Rhizoctonia solani, Ustilaginoidea virens and Xanthomonas oryzae pv. oryzae. Mechanistically, this allele increases proteasome abundance and activity to promote the degradation of reactive oxygen species-scavenging enzymes including peroxidase and catalase upon pathogen infection, leading to elevation of H2O2 accumulation for defence. In contrast, inhibition of proteasome function or overexpression of peroxidase/catalase-encoding genes compromises UMP1R2115-mediated resistance. More importantly, introduction of UMP1R2115 into a disease-susceptible rice variety does not penalize grain yield while promoting disease resistance. Our work thus uncovers a broad-spectrum resistance pathway integrating de-repression of plant immunity and provides a valuable genetic resource for breeding high-yield rice with multi-disease resistance.

High-Quality Genome Resource of Ustilaginoidea virens (UV2_4G), Causal Agent of an Emerging False Smut Disease in Rice.

Ustilaginoidea virens is the fungal pathogen causing an emerging false smut disease that affects crop yield as well as deteriorates quality of the grains by producing mycotoxins. A high quality genome of U. virens isolate UV2_4G was sequenced using Nanopore and Illumina HiSeq 2,000 sequencing platforms. The total assembled genome of Indian isolate UV2_4G was 35.9 Mb, which comprised 89 scaffolds with N50 of 700,296 bp. A total of 358,697 variants were identified in the genome, out of which 355,173 were SNPs and 3,524 were INDELS. Further, 7,390 SSRs belonging to different repeat types were also identified in the genome. Out of 7,444 proteins predicted, 7,206 were functionally annotated. A total of 1,307 CAZymes, 501 signal peptides, 1,876 effectors, and 2,709 genes involved in host-pathogen interactions were identified. Comparative analysis revealed isolate UV2_4G is distinct with 31 unique clusters and placed distantly in phylogenetic analysis. Taken together, this high-quality genome assembly and sequence annotation resource can give an improved insight for characterizing the biological and pathogenic mechanisms of U. virens.

BZIP Transcription Factor UvATF21 Mediates Vegetative Growth, Conidiation, Stress Tolerance and Is Required for Full Virulence of Rice False Smut Fungus Ustilaginoidea virens

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.

Impact of fungicides against false smut disease of rice caused by Ustilaginoidea virens

Impact of fungicides against false smut disease of rice caused by Ustilaginoidea virens

CHARACTERIZATION OF RICE FALSE SMUT PATHOGEN USTILAGINOIDEA VIRENS AND ITS USTILOXIN TOXIN IMPACT ON BRINE SHRIMP

Survey on rice false smut disease has been conducted at Erode, Tanjore, Perambalur and Coimbatore districts of Tamil Nadu, India. Among the areas surveyed, higher disease incidence was recorded in the paddy variety bhavani cultivated in Bhavanisagar, Erode district in which the rice false smut disease incidence was recorded as 40%. The pathogenicity test revealed the symptom development within 12-15 days of conidial suspension injection. In order to characterize the fungal isolates of Ustilaginoidea virens, a causal for rice false smut disease was cultured on Potato Sucrose Agar (PSA) medium. The pathogen was characterized morphologically which exhibits white mycelium that turns yellow and becomes olive green on maturity. Molecular confirmation was carried out among the collected isolates that were amplified at 380 bp region which is found specific for the pathogen Ustilaginoidea virens using the primers US 1-5 and US 3-3. The extraction and purification of ustiloxin was carried out using flash chromatography. On purification 100% water and 30% ethanol fraction was collected separately. The cytotoxicity assay of ustiloxin in brine shrimp resulted that the mortality at 1% concentration of ustiloxin was 96% and the lowest mortality (26%) was observed at 0.2% concentration. The lethal dosage 50(LD50) was confirmed at the concentration of 0.4% ustiloxin having the mortality rate of 52% using brine shrimp bioassay

Prevalence of Carbendazin Resistance in Field Populations of the Rice False Smut Pathogen Ustilaginoidea virens from Jiangsu, China, Molecular Mechanisms, and Fitness Stability.

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.

Eco-friendly management of false smut disease of rice incited by <i>Ustilaginoidea virens</i> through the application of <i>Trichoderma</i> spp

False smut disease of rice incited by Ustilaginoidea virens is an organ-specific pathogen that causes chalkiness of grain which leads to a reduction in 1000 grain test weight and yield. The dual culture assay results revealed that each Trichoderma isolates suppress the mean mycelial growth of U. virens under in-vitro conditions. Among the nine different Trichoderma isolates, 3 isolates were selected as effective isolates viz., TKM1, TKT9 and TTN5. Among these three effective isolates, maximum mycelial growth inhibition was recorded in the isolate TKM1 with 80.18 percentage reduction over control. The SEM photographs revealed that the hyphal round off in U. virens which is mainly due to the production of volatiles through direct antagonistic activity and competition through indirect antagonistic activity in which conidial adherence of T. harzianum over the surface of the mycelial mat of U. virens was observed. In 2020, the field experiment results revealed that the minimum disease severity was recorded when the Trichoderma isolate TKM1 was sprayed during booting stage with 4.61%, 50% PE stage with 17.91% and 100% PE with 21.86%. In 2021 the disease severity varied from 9.21% to 69.59%. The lowest disease severity was recorded in the plots sprayed with propiconazole fungicide with 9.21%. However, the disease severity recorded in fungicide treated plots were statistically on par with the Trichoderma isolate TKM1 treated plots at 50% PE spray with 10.60%. The disease severity recorded in the plots sprayed with TKM1 showed non-significant relationship with the fungicide treated plots which clearly revealed that the control efficacy of both TKM1 and Propiconazole treated plots were similar with each other. Among the Trichoderma treated plots the yield gain varied from 10.01% to17.20%. The yield gain was found to be 18.35% in fungicide treated plots. The yield and yield gain obtained by the effective isolate TKM1 (yield = 6405 kg/ha and yield gain = 17.20%) was statistically on par with propiconazole treated plots and significantly showed better yield and yield gain than the control plots. In 2021 among the Trichoderma treated plots the yield gain varied from 4.10% to 10.16%. The maximum yield gain was recorded in the fungicide treated plots (12.00%).

Cultural Characteristics and Molecular Confirmation of Ustilaginoidea virens the Incitant of False Smut of Rice

Aims: Ustilaginoidea virens, which causes false smut of rice (synonyms: green smut or pseudo smut), is an emerging grain disease around the world. The main aim of the study is to find out suitable culturable medium, carbon sources and pH for the growth of false smut pathogen and molecular confirmation of U. virens isolates.
 Place and Duration of Study: The in vitro studies were conducted in the Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, between December 2020 to April 2021.
 Methodology: To study the growth pattern of U.virens, five different carbon sources and mediums were used. Standardization of pH was done using a PSA medium amended with different pH levels. DNA extraction of all 13 isolates were done using the CTAB method. The phylogenetic tree was constructed using MEGA 12 software.
 Results: It is a slow-growing pathogen and grew well in the potato sucrose agar medium with a mycelial growth of about (54.00 mm) after 30 days and sucrose was found to be a good carbon source. The pH of 5 was found to be optimum. PCR reaction was carried out to further validate 13 isolates using three sets of primer pairs ITS 1/ITS 4, US1-5/US3-3, and US 2-5/US 4-3 with amplified products of 560 bp, 380 bp and 260 bp. Accession numbers were obtained for all the isolates.

Antifungal mechanisms of silver nanoparticles on mycotoxin producing rice false smut fungus

Ustilaginoidea virens, which causes rice false smut disease, is a destructive filamentous fungal pathogen, attracting more attention to search for effective fungicides against U. virens. Here, the results showed that the inhibition of 2nm AgNPs on U. virens growth and virulence displayed concentration-dependent manner. Abnormalities of fungal morphology were observed upon exposure to AgNPs. RNA-sequencing (RNA-seq) analysis revealed that AgNPs treatment up-regulated 1185 genes and down-regulated 937 genes, which significantly overlapped with the methyltransferase UvKmt6-regulated genes. Furthermore, we found that AgNPs reduced the UvKmt6-mediated H3K27me3 modification, resulting in the up-regulation of ustilaginoidin biosynthetic genes The decrease of H3K27me3 level was associated with the inhibition of mycelial growth by AgNPs treatment. These results suggested that AgNPs are an effective nano-fungicide for the control of rice false smut disease, but when using AgNPs, it needs to be combined with mycotoxin-reducing fungicides to reduce the risk of toxin pollution.

Gap-Free Nuclear and Mitochondrial Genomes of Ustilaginoidea virens JS60-2, a Fungal Pathogen Causing Rice False Smut

Rice false smut (RFS), caused by Ustilaginoidea virens, has become a major disease in recent years, and mycotoxins produced by U. virens often threaten food safety. To study fungal pathogenesis and identify potential targets for developing new fungicides, gap-free nuclear and complete mitochondrial genomes of U. virens JS60-2 were sequenced and assembled. Using the second and third generation sequencing data, we assembled a 38.02-Mb genome that consists of seven contigs with the contig N50 being 6.32-Mb. In total, 8,486 protein-coding genes were annotated in the genome, including 21 secondary metabolism gene clusters. We also assembled the complete mitochondrial genome, which is 102,498 bp, with 28% GC content. The JS60-2 genomes assembled in this study will facilitate research on U. virens and contribute to RFS control. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Spatial Distribution Patterns for Identifying Risk Areas Associated with False Smut Disease of Rice in Southern India

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.

Improving disease resistance to rice false smut without yield penalty by manipulating the expression of effector target

Rice grain production and quality are threatened by many devastating diseases. False smut caused by Ustilaginoidea virens is emerging as a major fungal disease of rice, leading to a severe loss of yield. U. virens invades only rice panicles at the late stage of booting and replaces grains with false smut balls containing mycotoxins, which endanger human and animal health (Sun et al., 2020). This successful colonization is accomplished through manipulating host immune responses by U. virens effectors.

The Toxicity of Salicylhydroxamic Acid and Its Effect on the Sensitivity of Ustilaginoidea virens to Azoxystrobin and Pyraclostrobin.

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.