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Article

Uvpks1, Involved in Ustilaginoidin Biosynthesis, Contributes to the Metabolic Profile, Development, Stress Responses, and Pathogenicity in Villosiclava virens

by
Xuwen Hou
1,
Jie Dong
1,
Dan Xu
1,
Hao Liu
2,
Yu Li
1,
Yujun Huang
1,
Jiahang Sun
1,
Muhammad Abubakar Jakada
1,
Daowan Lai
1 and
Ligang Zhou
1,*
1
Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, China
2
The Key Lab of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China
*
Author to whom correspondence should be addressed.
J. Fungi 2026, 12(2), 111; https://doi.org/10.3390/jof12020111
Submission received: 4 January 2026 / Revised: 24 January 2026 / Accepted: 31 January 2026 / Published: 5 February 2026
(This article belongs to the Special Issue Pathogenic Fungi: Morphogenesis, Pathogenicity and Biosynthesis of Secondary Metabolites—Second Edition)
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Abstract

Villosiclava virens (anamorph: Ustilaginoidea virens) is a fungal pathogen that causes rice false smut, one of the most devastating diseases of rice. The Uvpks1 gene encoding polyketide synthase is responsible for the biosynthesis of ustilaginoidins, a major group of mycotoxins in V. virens. In this study, three strains, including the Uvpks1 deletion mutant ΔUvpks1, the complementation strain ΔUvpks1-C1, and the wild-type isogenic strain P1 of V. virens, were employed to investigate the role of Uvpks1 in shaping the metabolic profile and in the development, stress responses, and pathogenicity of V. virens. The deletion of Uvpks1 led to both the elimination of ustilaginoidin biosynthesis and the induction of many other secondary metabolite biosynthetic pathways. It decreased mycelial growth and sporulation, fungal tolerance to Congo red-induced cell wall damage stress, and susceptibility to the fungicides epoxiconazole and prochloraz. Meanwhile, it increased hyphal hydrophobicity, resistance to H2O2-induced oxidative stress and metal cation stress, and susceptibility to the fungicide azoxystrobin. Furthermore, the deletion of Uvpks1 resulted in reduced fungal pathogenicity toward rice plants. The findings reveal the functions of Uvpks1 in shaping the metabolic profile, development, stress responses, and pathogenicity of V. virens, which will be beneficial for developing new strategies to control rice false smut and ustilaginoidins.

1. Introduction

Rice false smut (RFS), caused by Villosiclava virens (anamorph: Ustilaginoidea virens), is a destructive disease in rice-producing areas of the world [1,2,3]. The RFS pathogen (V. virens) can produce toxic secondary metabolites (SMs), which are called mycotoxins. They are toxic to plants and animals and pose a serious threat to their health [4,5]. So far, three kinds of mycotoxins have been found in V. virens, which are ustilaginoidins [6,7,8], ustiloxins [5,9] and sorbicillinoids [10,11]. Among them, ustilaginoidins, belonging to bis-naphtho-pyrones polyketides, are the main mycotoxins in terms of their quantity and content in V. virens [7,8].
Ustilaginoidins exhibit a wide range of biological activities [12], such as phytotoxicity [7,8], cytotoxicity on cancer cells [7,13,14], hepatotoxicity [15,16], acute toxicity [16], teratogenicity [17], antibacterial activity [7,18], and inhibitory activity on adenosine triphosphate synthesis [19]. However, there are few reports on the physiological and ecological functions of ustilaginoidins in V. virens so far. In this study, the polyketide synthase (PKS)-encoding gene Uvpks1, which is responsible for ustilaginoidin biosynthesis, was deleted through CRISPR/Cas9-mediated homologous recombination [20] in order to reveal the functions of Uvpks1 through metabolic shunting (or genetic dereplication) [21] as well as to elucidate the roles of Uvpks1 deletion in contributing to the metabolic profile, development, stress responses, and pathogenicity of V. virens.

2. Materials and Methods

2.1. Fungal Strains, Plasmids, Gene Deletion and Complementation

The wild-type (WT) strain P1 of V. virens was kindly provided by Prof. Wenxian Sun from the Department of Plant Pathology, China Agricultural University. The vector pmCAS9-tRp-gRNA was used for the Uvpks1 gene deletion. The recombinant plasmid pCas9-tRp-Uvpks1 was constructed according to a previous method [20,22]. The PEG-mediated V. virens transformation was performed as described previously [23] using linear donor DNA fragments and the CRISPR construct pCas9-tRp-Uvpks1. The ΔUvpks1 strain was verified by PCR amplification and was unable to synthesize ustilaginoidins. To obtain the complemented strain, the vector pCBHT-RP27, which contained the Uvpks1 coding sequence (CDS), was transformed into the ΔUvpks1 protoplasts. The detailed construction and verification procedures of Uvpks1 deletion and complemented strains of V. virens are shown in the Supplementary Materials.
The complemented strains ΔUvpks1-C1, ΔUvpks1-C2, and ΔUvpks1-C3 were detected to restore the ability to synthesize ustilaginoidins. As all three complemented strains displayed very similar phenotypes, only ΔUvpks1-C1 was selected for this study.
The fungal strains were grown on PSA (potato sucrose agar) medium in Petri dishes in the incubator and in the dark at 28 °C. The strains ΔUvpks1, ΔUvpks1-C1 and P1 were stored in 20% glycerol at −80 °C in China Agricultural University. For the experiments using Petri dishes, 10 mL of solid medium was added to a 60 mm diameter Petri dish.

2.2. Metabolic Profile Analysis of Fungal Strains

To perform chemical analyses, the WT strain, deletion mutant, and complemented strain were cultured on PSA plates at 28 °C for 4 weeks. The same plates with hyphae were extracted with ethyl acetate (EtOAc) three times. The combined EtOAc solution was concentrated using a rotary evaporator under reduced pressure to yield a tawny residue, referred to as the EtOAc extract. It was further dissolved in MeOH and filtered through a microporous filter (pore size, 0.22 μm) for HPLC-DAD analysis. HPLC-DAD analysis of the EtOAc extract was performed on a Shimadzu instrument equipped with a SPD-M20A photodiode array detector (LC-20A, Shimadzu Corp., Tokyo, Japan) using an analytical C18 column (250 mm × 4.6 mm i.d., 5 μm; Phenomenex Inc., Torrance, CA, USA). The column temperature was set at 30 °C. The mobile phase was composed of water containing 0.02% TFA (A) and MeOH (B). A gradient elution program from 10% to 100% MeOH was used, with a flow rate of 1.0 mL/min. The wavelength was set at 290 nm for the detection of ustilaginoidins [20].

2.3. Effects of Media with Different Carbon Sources on Mycelia Growth

To investigate the effects of different carbon sources on mycelial growth of V. virens, PDA (1000 mL medium containing 200 g of potato, 20 g of glucose, and 15 g of agar) was used as the basal medium, referred to as the glucose-containing potato agar medium. Glucose in PDA was then replaced with sucrose, cellulose, maltose, xylose, soluble starch, and galactose at the same concentration (20 g/L). They were called glucose-, sucrose-, cellulose-, maltose-, xylose-, soluble starch-, and galactose-containing potato agar media, respectively.
The experimental procedure was performed as described previously [24]. After the Petri dishes were filled with agar medium, 5 μL of a conidial suspension (2 × 106 conidia/mL) of V. virens was inoculated onto the solid medium in each Petri dish. The plates were incubated at 28 °C in the dark for 21 days. The diameters of the fungal colony extensions were then measured.

2.4. Effects of Ambient Temperature and pH on Mycelia Growth

To determine the effects of ambient temperature on mycelia growth of V. virens, each PSA plate was inoculated with 5 μL conidia suspension (2 × 106 conidia/mL). After the plates were cultivated in darkness at 23 °C, 28 °C, and 33 °C for 21 days, the fungal colony extension diameters were measured.
To understand the effects of ambient pH on mycelial growth of V. virens, the PSA plates were filled with medium whose pH was adjusted to 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 with 1 M HCl. Each PSA plate was inoculated with 5 μL of a conidia suspension (2 × 106 conidia/mL). After the plates were cultivated at 28 °C in darkness for 21 days, the fungal colony extension diameters were measured.

2.5. Effects of Uvpks1 Deletion on Sporulation

The high-nitrogen content medium YTD (1000 mL medium containing 1 g of yeast extract, 1 g of tryptone, and 10 g of glucose) was used for sporulation assessment of V. virens [25]. The aliquots of 1 mL conidia suspension (2 × 106 conidia/mL) of each V. virens strain were inoculated in 150 mL YTD medium. After the spores were germinated and cultured for 5 days at 28 °C, 180 rpm in darkness, the mycelia–spore mixtures were harvested and filtered through Miracloth. The spore suspension was centrifuged at 3500 rpm for 8 min. The centrifuged spores were resuspended in distilled water, and the volume was adjusted to 10 mL. The number of conidia was determined using a hemocytometer, with conidia observed and counted under a microscope. Sporulation was expressed as conidia per milliliter (conidia/mL) in liquid medium [24].

2.6. Hyphal Hydrophobicity Assay

To determine the hyphal hydrophobicity of fungal strains ΔUvpks1, ΔUvpks1-C1 and P1, 20 μL of either 2.5% bromophenol blue solution or ddH2O was dropped on the colony surface of each tested strain grown on PSA medium for 21 days according to the method previously described by Zhang et al. [22].

2.7. Effects of the Stresses on Mycelial Growth

To measure the susceptibility of the fungal strains to different stresses, 5 μL of spore suspension (2 × 106 spores/mL) of V. virens was inoculated in the center of the plate. Mycelial growth was assayed after incubation at 28 °C and pH 6.5 for 21 days on the plates of PSA treated with 0.25 M, 0.50 M, and 0.75 M of sodium chloride (NaCl); 0.50 M, 0.75 M, and 1.00 M of sorbitol (Sor); 0.01%, 0.02%, and 0.04% of hydrogen peroxide (H2O2); 0.25 mg/mL, 0.50 mg/mL, and 1.00 mg/mL of Congo red (CR); 0.1 mM, 0.5 mM, and 1.0 mM of copper chloride (CuCl2); and 0.025 M, 0.05 M, and 0.1 M of manganese chloride (MnCl2). The fungal colony diameters were measured, and the mycelial growth of the treated strains was compared with that of non-treated controls [26]. The inhibition rate was calculated using the following equation:
Inhibition rate (%) = [(average colony diameter of each strain on PSA without treatment − average colony diameter of each strain on PSA with treatment)/average colony diameter of each strain on PSA without treatment] × 100

2.8. Fungicide Susceptibility Assay

To evaluate the susceptibility of fungal strains to various fungicides, the deletion mutant, complement strain, and WT strain were cultured on PSA medium. After 14 days of growth, 6 mm diameter mycelial plugs were taken from the edges of each colony and transferred to PSA plates containing a fungicide at different concentrations.
Four fungicides were used to evaluate the fungicide susceptibility of fungal strains. They included difenoconazole (98.4% purity) provided by Hangzhou Yulong Chemical Industry Co., Ltd. (Hangzhou, China); epoxiconazole (97.0% purity) provided by Hubei Bojie Biotechnology Co., Ltd. (Wuhan, Hubei, China); prochloraz (98.0% purity) provided by Jiangsu Huifeng Agrochemical Co., Ltd. (Nanjing, China); and azoxystrobin (98.0% purity) provided by Syngenta Biotechnology (China) Co., Ltd. (Shanghai, China).
The stock solutions of fungicides in DMSO at concentrations of 103 μg/mL and 102 μg/mL were prepared. For bioassay experiments, a series of concentration gradients of difenoconazole at 0.025, 0.05, 0.1, 0.4, and 0.8 μg/mL; epoxiconazole at 0.0125, 0.025, 0.05, 0.1, and 0.15 μg/mL; prochloraz at 0.025, 0.05, 0.1, 0.2, 0.4, and 0.8 μg/mL; and azoxystrobin at 0.025, 0.05, 0.1, 0.2, 0.4, and 0.6 μg/mL were employed. Then, 6 mm diameter mycelial plugs were transferred to PSA plates containing the fungicide at different concentrations. The plates were incubated at 28 °C for two weeks, after which the colony diameter in two perpendicular directions was measured for each plate. Three replicates for each concentration were used. The inhibition rate of mycelial growth was calculated based on the average colony diameter. The median inhibitory concentration (IC50) value (μg/mL) was calculated using a toxicity regression equation based on the logarithm of concentrations and the probability units of the inhibition rate [27].

2.9. Pathogenicity Assay of Fungal Strains

The pathogenicity assay was performed as described previously [25,28]. Briefly, the susceptible rice (Oryza sativa) cultivar ‘Wanxian98’ was planted in greenhouse pots during the summer for inoculation experiments. At the eighth stage of panicle development (i.e., before heading), the mixture of conidia suspension together with fragmented mycelia of each strain was diluted to 1 × 106 conidia/mL. Subsequently, 2 mL of the mixture was quickly injected into the sheath that wrapped the spikelet. The treated rice plants were placed in a greenhouse with relative humidity above 95% and a temperature of 25 ± 1 °C. At least eight rice spikelets were inoculated for each strain. The inoculated panicles were collected at 21 days post-inoculation (dpi) for statistical analysis of the rate of diseased panicles and the average number of false smut balls formed on each inoculated panicle. The experiment was repeated three times.

2.10. Statistical Analysis

All experiments were designed with three independent biological replicates. Three replicates were performed for each treatment. The treated samples were analyzed at the same growth stage. Extreme individual samples were excluded. All statistical analyses and charts were conducted using GraphPad Prism (version 8.0.2) software (GraphPad Software, Inc., San Diego, CA, USA) and Microsoft Excel 2016 (Microsoft Corporation, Washington, DC, USA). These comparisons were tested for statistical significance by Student’s t-test. The data were expressed as the mean ± standard deviation (SD). The differences at p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***) were considered statistically significant for the mutants compared with the WT strain under the same treatment.

3. Results

3.1. Deletion of Uvpks1 Resulted in Variation in the Metabolic Profile of the Fungal Strains

HPLC spectra of EtOAc extracts from the fungal strains WT, ∆Uvpks1, and ∆Uvpks1-C1 are shown in Figure 1. Compared with the extracts of ∆Uvpks1-C1 and WT strains, the ∆Uvpks1 mutant extract showed more peaks of various compounds with different polarities (retention times), though the intensity of each peak was relatively weak. The peaks observed between 30 and 40 min of retention time were identified as ustilaginoidins, consistent with previous reports [8,20]. Ustilagnoidins could not be produced in the strain of ∆Uvpks1 but could be produced in the strains of WT and ∆Uvpks1-C1 (Figure 1).

3.2. Deletion of Uvpks1 Decreased Mycelial Growth

Saccharides (also called carbon sources) can affect fungal growth and metabolism. Secondary metabolism also affects fungi’s ability to utilize saccharides [29,30]. In PDA medium, an obvious decrease in mycelial radial extension was observed in the ∆Uvpks1 strain compared with the WT strain. The complemented strain (∆Uvpks1-C1) partially restored the growth phenotype of the WT strain. Glucose in PDA was replaced by sucrose, cellulose, maltose, xylose, soluble starch, and galactose with the same concentration (g/L). It was found that the potato agar media supplemented with 20 g/L of glucose, sucrose or soluble starch were beneficial for the growth of fungal strains. Compared with the WT and complemented strains, the ∆Uvpks1 strain showed a significant decrease in mycelial radial extension at 28 °C after 21 days of culture when the medium was separately supplemented with glucose, sucrose, maltose, soluble starch, or galactose. The growth (colony extension diameter) of the complemented strain (ΔUvpks1-C1) mycelia was between that of the ΔUvpks1 strain and the WT strain (Figure 2). Overall, deletion of Uvpks1 reduced mycelial growth when fungal strains were cultured in media supplemented with different carbon sources. PSA was selected as the basic medium for subsequent experimental research.
When the fungal strains WT, ∆Uvpks1, and ∆Uvpks1-C1 were inoculated on PSA media in darkness for 21 days at temperatures of 23 °C, 28 °C and 33 °C, the colony extension diameter of the ∆Uvpks1 strain was obviously smaller than that of the WT strain at the tested temperatures (23 °C, 28 °C and 32 °C), which indicated that the loss of ustilaginoidin production led to a decrease in fungal growth. The colony extension diameters of the complemented strain were partially (at 28 °C) or completely (at 23 °C and 33 °C) restored to the growth phenotype of the WT strain. The optimal growth temperature for the mycelia of fungal strains was 28 °C (Figure 3). In addition, mycelial growth at 23 °C was faster than at 33 °C. This may be due to the evolution of fungal strategies, such as the production of proteins and SMs at relatively low temperatures [31].
The pH values of PSA media were adjusted to 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 with 1 M HCl. When the fungal strains WT, ∆Uvpks1, and ∆Uvpks1-C1 were cultured for 21 days after inoculation at 28 °C with different pH values on PSA media, the colony extension diameters of the fungal strains were significantly different (Figure 4). The strain ∆Uvpks1 grew more slowly and produced less pigment than strain WT or ∆Uvpks1-C1 at a certain pH, which indicated that the loss of ustilaginoidin production decreased fungal growth. In addition, the optimal pH value for fungal growth of all strains was 6.5. The complementation strain partially restored mycelial growth at all test pH values in the media. Generally, low ambient pH (i.e., 5.0–6.5) is beneficial for fungal growth and the production of SMs. The results of this study are consistent with previous reports [24,32,33,34].

3.3. Deletion of Uvpks1 Decreased Sporulation

A high nitrogen content in the medium is generally beneficial for fungal sporulation [35,36]. YTD represented the high-nitrogen-content medium used for sporulation assessment in this study [25]. In order to investigate the effects of Uvpks1 on the sporulation of V. virens, the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 were cultured in YTD medium at 28 °C for 5 days. It was found that the conidia concentration of the ∆Uvpks1 strain was obviously lower compared with the WT strain in YTD media. The conidia concentration (4.7 × 107 conidia/mL) of the complemented strain ∆Uvpks1-C1 was restored to the level (4.6 × 107 conidia/mL) of the WT strain in YTD. The conidia concentration (0.35 × 107 conidia/mL) was approximately 13-fold lower in the ∆Uvpks1-C1 strain compared to that in strain WT or ∆Uvpks1-C1 (Figure 5).

3.4. Deletion of Uvpks1 Increased Hyphal Hydrophobicity

The hydrophobic property of the cell surface is a prominent feature of many fungal aerial hyphae [37]. The ∆Uvpks1 mutant formed raised colonies due to increased aerial hyphal growth, suggesting that the mutant might exhibit increased hydrophobicity on the hyphal surface (Figure 6A). As shown in Figure 6B, due to more aerial hyphae being present, both the 2.5% bromophenol blue solution and ddH2O maintained spherical droplets on the surface of the ΔUvpks1 colony without being absorbed or extended for more than 12 h, thereby demonstrating the strong hydrophobicity of the ΔUvpks1 hyphae. This indicates that Uvpks1 is involved in the growth and hydrophobicity of aerial mycelia in V. virens.

3.5. Effects of Uvpks1 Deletion on the Response to Various Stresses of V. virens

The roles of Uvpks1 in the response to various stresses of V. virens were investigated. These stresses included hyperosmotic stress, H2O2-induced oxidative stress, cell wall damage stress, metal cation stress, and fungicide stress.

3.5.1. Deletion of Uvpks1 Increased Fungal Tolerance to Hyperosmotic Stresses

To investigate whether Uvpks1 was involved in response to hyperosmotic stresses, the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 were cultured on the PSA medium treated with different concentrations of NaCl (Figure 7) and sorbitol (Figure 8).
The mycelial growth inhibition rate of strain ∆Uvpks1 under either NaCl or sorbitol stresses was significantly lower compared to the WT strain. It was plausible that the non-ustilaginoid metabolites produced by the deletion strain increased fungal tolerance to hyperosmotic stress. The results indicate that Uvpks1 plays a negative role in hyperosmotic responses in V. virens. However, the ∆Uvpks1-C1 strain could not restore the WT phenotype. This mechanism needs to be further studied.

3.5.2. Deletion of Uvpks1 Increased Fungal Resistance to H2O2-Induced Oxidative Stress

To investigate whether Uvpks1 was involved in response to oxidative stress induced by hydrogen peroxide (H2O2), the fungal strains WT, ∆Uvpks1, and ∆Uvpks1-C1 were cultured on the PSA medium containing 0.01–0.04% H2O2. When the concentrations of H2O2 in the medium were 0.01% and 0.02%, there were no obvious differences in their resistance to oxidative stress. As the concentration of H2O2 in the medium increased, the mycelial growth of the WT strain was more strongly inhibited than that of ∆Uvpks1 and ∆Uvpks1-C1 at 0.04% H2O2 (Figure 9). The non-ustilaginoidin metabolites synthesized in the deletion strain (∆Uvpks1) might have increased fungal tolerance to H2O2-induced oxidative stresses. However, the ∆Uvpks1-C1 strain could not restore the phenotype of the WT strain. The specific mechanism needs to be further studied.

3.5.3. Deletion of Uvpks1 Decreased Fungal Tolerance to Congo Red-Induced Cell Wall Damage Stress

Congo red (CR) is usually employed to induce fungal cell wall damage [38,39]. When the concentrations of CR in the medium were 0.25 mg/mL and 0.50 mg/mL, there were no obvious differences in their tolerance to CR-induced cell wall damage stress. As the concentration of CR in the medium increased to 1.00 mg/mL, the mycelial growth of the ∆Uvpks1 strain was more strongly inhibited than that of the WT and ∆Uvpks1-C1 strains. The strain ∆Uvpks1-C1 could restore the phenotype of the WT strain (Figure 10). This indicates that deletion of Uvpks1 decreases the tolerance of V. virens to the cell wall-damaging agent CR. The results of this study are consistent with previous reports [22,40].

3.5.4. Deletion of Uvpks1 Increased Fungal Resistance to Metal Cation Stresses

Metal ions are usually known to affect hyphal growth and secondary metabolism in fungi [41,42]. To investigate whether the deletion of Uvpks1 was involved in response to metal cation stresses, the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 were cultured on PSA amended with either 0.1, 0.5, 1.0, and 2.0 mM of CuCl2 (Figure 11) or 0.025, 0.05, and 0.1 M of MnCl2 (Figure 12). The mycelial growth of the ∆Uvpks1 strain under metal cation (Cu2+ and Mn2+) stresses was higher compared with the WT strain. These results showed that Uvpks1 played a negative role in response to metal cations Cu2+ and Mn2+ in V. virens.

3.5.5. Uvpks1 Involved in the Fungicide Stresses

At present, RFS management mainly relies on fungicides such as difenoconazole [43], though rice genetic resistance, biological control, and cultivation practices have also been used [44,45]. The susceptibility of the strains WT, ΔUvpks1, and ΔUvpks1-C1 to the fungicides difenoconazole, epoxiconazole, prochloraz, and azoxystrobin was determined by measuring fungal colony extension. The results with IC50 values showed that the ΔUvpks1 strain exhibited increased susceptibility to difenoconazole, epoxiconazole, and prochloraz (Table 1). However, when the strains were treated with azoxystrobin, the ΔUvpks1 strain showed greater resistance than the WT strain and the complemented strain ΔUvpks1-C. The complementation strain ∆Uvpks1-C1 could restore susceptibility to these two types of fungicides (i.e., sterol biosynthesis inhibitors and benzoquinone biosynthesis inhibitors). The results indicate that the deletion of Uvpks1 increases susceptibility to sterol biosynthesis inhibitor fungicides (i.e., difenoconazole, epoxiconazole, and prochloraz) and decreases susceptibility to benzoquinone biosynthesis inhibitor fungicides (i.e., azoxystrobin).

3.6. Uvpks1 Is Essential for Virulence and Pathogenicity of V. virens

To explore whether ustilaginoidins synthesized by Uvpks1 contribute to the virulence and pathogenicity of V. virens, the conidia suspensions prepared from the strains WT, ∆Uvpks1 and ∆Uvpks1–C1 were inoculated into the rice spikelets. Pathogenicity experiments showed that no false smut balls formed on rice spikelets infected with the ∆Uvpks1 strain. In contrast, the rates of diseased panicles of both the WT and ∆Uvpks1–C1 strains were 100% (Figure 13). These findings suggest that ustilaginoidins may serve as key virulence factors in the disease development process of V. virens, consistent with previous reports [46].

4. Discussion

4.1. Deletion of Uvpks1 Led to Metabolic Shunting and SM Diversity of V. virens

The gene Uvpks1 (Uv_2086), encoding a polyketide synthase, is responsible for the first step of ustilaginoidin biosynthesis in V. virens. Neither ustilaginoidins nor intermediates are produced when Uvpks1 is deleted [20,47]. The gene UvugsL (Uv_2091) encodes a laccase that dimerizes monomeric naphtha-γ-pyrones into ustilaginoidins in V. virens. If the gene UvugsL is deleted, the production of ustilaginoidins is also completely blocked [10].
Metabolic shunting regulation (also called genetic dereplication) can uncover new SMs from fungi [21,48] and has been considered one of the most efficient strategies for mining fungal SMs [49,50,51,52]. In this study, a genetic dereplication strain was created by deleting the ustilaginoidin synthase gene Uvpks1, thereby completely eliminating ustilaginoidin production in V. virens. Two new antibacterial sorbicillinids, ustisorbicillinols G and H, were previously isolated from the albino strain LN02 of V. virens [53], a natural genetic dereplication mutant with the white phenotype due to its inability to synthesize ustilaginoidins [54]. As shown in Figure 1, more non-ustilaginoidin metabolites were accumulated in the ∆Uvpks1 strain in comparison with the WT strain. These newly generated metabolites might play important compensatory roles in fungal development and pathogenicity, as well as other functions in response to environmental stresses. Subsequent work will aim to identify these metabolites and elucidate their biological activities and functions in V. virens.

4.2. Deletion of Uvpks1 Decreased Development and Pathogenicity of V. virens

Deletion of Uvpks1 impairs mycelial growth and sporulation, ultimately leading to attenuated pathogenicity. These phenotypic changes can be attributed to either the absence of ustilaginoidins or the production of other SMs synthesized through metabolic shunting. The laccase UvugsL is responsible for the dimerization of mono-naphtho-γ-pyrones to generate ustilaginoidins, which further act as effectors in the pathogenicity of V. virens [46]. Furthermore, ustilaginoidins have previously been shown to exhibit obvious phytotoxic activity on rice seedlings [7,8]. The ∆UvugsL strain was unable to cause false smut ball formation in rice spikelets. In addition, exogenous addition of ustilaginoidins during inoculation could partially restore its pathogenicity, indicating that ustilaginoidins function as phytotoxins during host colonization [46]. The other physiological and ecological functions of ustilaginoidins need to be further studied.
Uvpks1 is a PKS-encoding gene reported to be responsible for ustilaginoidin biosynthesis [20]. It is possible that supplementation with ustilaginoidins could restore mycelial growth, sporulation, and pathogenicity in the ΔUvpks1 mutant, which warrants further investigation through gene expression analysis, enzymology, protein–protein interactions, and metabolomics [22,46].

4.3. Deletion of Uvpks1 Changed Fungal Tolerances to Various Stresses

Deletion of Uvpks1 leads to an inability to synthesize ustilaginoidin. This genetic alteration also triggers the synthesis of other SMs in V. virens. Both ustilaginoidins and other SMs may alter the tolerance of V. virens to various environmental stresses. Therefore, it is important to elucidate the physiological and ecological functions of the metabolites in V. virens. Among these tolerances, fungicide stress is an important aspect. The fungicides difenoconazole, epoxiconazole, and prochloraz belong to the class of sterol biosynthesis inhibitors, which can disturb the formation of fungal cell walls [55,56,57], while azoxystrobin belongs to the class of benzoquinone biosynthesis inhibitors, which can inhibit fungal mitochondrial respiration [58,59,60]. The results of this study showed that deletion of Uvpks1 increased susceptibility to sterol biosynthesis inhibitor fungicides and decreased susceptibility to benzoquinone biosynthesis inhibitor fungicides, indicating that the ΔUvpks1 mutant enhances fungal mitochondrial adaptation to benzoquinone biosynthesis inhibitors and reduces tolerance of fungal cell wall formation to sterol biosynthesis inhibitors. The detailed mechanisms of action need to be further studied. In other stress response experiments, we observed that the biological phenotype of the ΔUvpks1-C1 strain was similar to that of the ΔUvpks1 mutant but did not match that of the WT strain. This might be attributed to the promoter used in the ΔUvpks1-C1 strain. We used the strong promoter Rp27 instead of the native promoter of Uvpks1, which led to the overproduction of ustilaginoidins in V. virens (Figure 1) and thereby interfered with its normal physiological processes. Furthermore, the overexpression of Uvpks1 driven by the Rp27 promoter in the complemented strain might have masked subtle phenotypic restoration effects [61].

5. Conclusions

In summary, this study revealed the multifaceted roles of Uvpks1 in V. virens. Our findings showed that the deletion of Uvpks1 directly led to the loss of ustilaginoid biosynthesis and the induction of many other SM biosynthetic pathways. Meanwhile, the deletion of Uvpks1 decreased mycelial growth and sporulation, fungal tolerance to Congo red-induced cell wall damage stress, susceptibility to the fungicides epoxiconazole and prochloraz, and pathogenicity. In contrast, the deletion of Uvpks1 increased hyphal hydrophobicity, resistance to H2O2-induced oxidative stress, resistance to metal cation stress, and susceptibility to the fungicide azoxystrobin. These results will help clarify the roles of ustilaginoidins in the metabolic profile, development, stress responses, susceptibility to fungicides, and pathogenicity in V. virens. Uvpks1 could be designated as a potential target for the development of novel fungicides, thereby providing avenues to manage ustilaginoid mycotoxins and RFS disease. However, the role of ustilaginoidins in the pathogenicity of V. virens and their precise mechanisms of action need to be studied in detail.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof12020111/s1, Construction of Uvpks1 gene deletion and complemented strains; Table S1: All the primers used in this study; Figure S1: Strategies for Uvpks1 knockout; Figure S2: Selection of ΔUvpks1 knockout transformants by PCR; Figure S3: Selection of ΔUvpks1-C complementary transformants by PCR. The references [10,20,22] cited in the Supplementary Materials are listed in the References Section of the text.

Author Contributions

L.Z. conceptualized and supervised the study. X.H., J.D., D.X., H.L., Y.L., Y.H., J.S. and M.A.J. performed the methodology, investigation, and data curation. X.H. and L.Z. prepared and wrote the manuscript. D.L. and D.X. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2023YFD1700703) and the National Natural Science Foundation of China (32202244, 32072373, and 31471729).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

We would like to acknowledge Jin-Rong Xu of the Department of Botany and Plant Pathology, Purdue University, for providing the vectors pmCAS9-tRp-gRNA and pCBHT; Pengfei Liu from the Department of Plant Pathology, China Agricultural University (CAU), for kindly providing the fungicides; Wenxian Sun from the Department of Plant Pathology, CAU, for providing the WT strain P1; and Vijai Bhadauria from the Department of Plant Pathology, CAU, for kindly editing the English of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CDScoding sequence
CRCongo red
CuCl2copper chloride
Dpidays post-inoculation
EtOAcethyl acetate
H2O2hydrogen peroxide
IC50median inhibitory concentration
MeOHmethanol
MnCl2manganese chloride
NaClsodium chloride
ORFopen reading frame
PDApotato dextrose (glucose) agar
PKSpolyketide synthase
PSApotato sucrose agar
PSBpotato sucrose broth
RFSrice false smut
SMssecondary metabolites
Sorsorbitol
TFAtrifluoroacetic acid
WTwild-type
YTDyeast–tryptone–dextrose medium
∆Uvpks1polyketide synthase gene knockout strain of V. virens
∆Uvpks1-Ccomplemented strain of ∆Uvpks1 mutant of V. virens

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Figure 1. HPLC analyses of EtOAc extracts of V. virens strains WT, ∆Uvpks1, and ∆Uvpks1-C1 at 290 nm. The arrow indicates the peaks of ustilaginoidins.
Figure 1. HPLC analyses of EtOAc extracts of V. virens strains WT, ∆Uvpks1, and ∆Uvpks1-C1 at 290 nm. The arrow indicates the peaks of ustilaginoidins.
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Figure 2. Effects of different carbon sources on colony extension of V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured for 21 days at 28 °C on potato agar supplemented with 20 g/L of glucose, sucrose, cellulose, maltose, xylose, soluble starch, and galactose. (B) Effects of different carbon sources on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation (SD). The differences at p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
Figure 2. Effects of different carbon sources on colony extension of V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured for 21 days at 28 °C on potato agar supplemented with 20 g/L of glucose, sucrose, cellulose, maltose, xylose, soluble starch, and galactose. (B) Effects of different carbon sources on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation (SD). The differences at p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
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Figure 3. Effects of ambient temperatures on colony extension of V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA at 23 °C, 28 °C and 33 °C for 21 days. (B) Effects of ambient temperatures on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.05 (*) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
Figure 3. Effects of ambient temperatures on colony extension of V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA at 23 °C, 28 °C and 33 °C for 21 days. (B) Effects of ambient temperatures on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.05 (*) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
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Figure 4. Effects of ambient pH in medium on colony extension of V. virens strains. (A) The colony morphologies of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 were cultured at 28 °C for 21 days on PSA medium with pH values adjusted to 5.0–7.5. (B) Effects of ambient pH on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
Figure 4. Effects of ambient pH in medium on colony extension of V. virens strains. (A) The colony morphologies of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 were cultured at 28 °C for 21 days on PSA medium with pH values adjusted to 5.0–7.5. (B) Effects of ambient pH on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
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Figure 5. Effects of YTD medium on sporulation of V. virens strains. (A) (a–c) The cultured status of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 in YTD at 28 °C for 5 days. (B) The conidia concentrations of V. virens strains in YTD medium. Each datum is the average of three replicates ± standard deviation (SD). The difference at p ≤ 0.001 (***) was statistically significant for the strain ∆Uvpks1 compared with the WT strain.
Figure 5. Effects of YTD medium on sporulation of V. virens strains. (A) (a–c) The cultured status of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 in YTD at 28 °C for 5 days. (B) The conidia concentrations of V. virens strains in YTD medium. Each datum is the average of three replicates ± standard deviation (SD). The difference at p ≤ 0.001 (***) was statistically significant for the strain ∆Uvpks1 compared with the WT strain.
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Figure 6. Effects of Uvpks1 deletion on aerial hyphal hydrophobicity of V. virens strains. (A) Aerial hyphae of the ∆Uvpks1 mutant grew faster than those of the strains ∆Uvpks1-C1 and WT. All strains were cultured on PSA medium for 21 days at 28 °C. (B) Spherical water and bromophenol blue solution droplets (20 μL for each drop) were dropped on the colonies of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1, whereas the droplet dispersed on the colonies of the strains WT and ∆Uvpks1-C1.
Figure 6. Effects of Uvpks1 deletion on aerial hyphal hydrophobicity of V. virens strains. (A) Aerial hyphae of the ∆Uvpks1 mutant grew faster than those of the strains ∆Uvpks1-C1 and WT. All strains were cultured on PSA medium for 21 days at 28 °C. (B) Spherical water and bromophenol blue solution droplets (20 μL for each drop) were dropped on the colonies of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1, whereas the droplet dispersed on the colonies of the strains WT and ∆Uvpks1-C1.
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Figure 7. Deletion of Uvpks1 confers tolerance to NaCl-induced hyperosmotic stress in V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of NaCl for 21 days at 28 °C. (B) Inhibition of NaCl on the colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
Figure 7. Deletion of Uvpks1 confers tolerance to NaCl-induced hyperosmotic stress in V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of NaCl for 21 days at 28 °C. (B) Inhibition of NaCl on the colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
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Figure 8. Deletion of Uvpks1 confers sorbitol-induced tolerance to hyperosmotic stress in V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of sorbitol for 21 days at 28 °C. The CK (0 M Sor) photos of these strains were the same as those of CK (0 M NaCl) in Figure 7. (B) Inhibition of sorbitol on the colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.01 (**), and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
Figure 8. Deletion of Uvpks1 confers sorbitol-induced tolerance to hyperosmotic stress in V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of sorbitol for 21 days at 28 °C. The CK (0 M Sor) photos of these strains were the same as those of CK (0 M NaCl) in Figure 7. (B) Inhibition of sorbitol on the colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.01 (**), and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
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Figure 9. Deletion of Uvpks1 resulted in a decrease in tolerance to H2O2-induced oxidative stress in V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of H2O2 for 21 days at 28 °C. (B) Inhibition of H2O2 on the colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
Figure 9. Deletion of Uvpks1 resulted in a decrease in tolerance to H2O2-induced oxidative stress in V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of H2O2 for 21 days at 28 °C. (B) Inhibition of H2O2 on the colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
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Figure 10. Deletion of Uvpks1 contributes to the tolerance to Congo red (CR)-induced cell wall damage stress of V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1 and ∆Uvpks1-C1 cultured on PSA medium containing different concentrations of CR for 21 days at 28 °C. The CK (0 mg/mL CR) photos of these strains are the same as those of CK (0 M NaCl) in Figure 7. (B) Inhibitory effect of CR on the colony extension diameters of fungal strains. The difference at p ≤ 0.001 (***) was statistically significant for the strain ∆Uvpks1 compared with the WT strain under the same treatment.
Figure 10. Deletion of Uvpks1 contributes to the tolerance to Congo red (CR)-induced cell wall damage stress of V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1 and ∆Uvpks1-C1 cultured on PSA medium containing different concentrations of CR for 21 days at 28 °C. The CK (0 mg/mL CR) photos of these strains are the same as those of CK (0 M NaCl) in Figure 7. (B) Inhibitory effect of CR on the colony extension diameters of fungal strains. The difference at p ≤ 0.001 (***) was statistically significant for the strain ∆Uvpks1 compared with the WT strain under the same treatment.
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Figure 11. Deletion of Uvpks1 contributed to the tolerance to Cu2+ stress of V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of CuCl2 for 21 days at 28 °C. (B) Inhibition of CuCl2 on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.05 (*) and p ≤ 0.01 (**) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
Figure 11. Deletion of Uvpks1 contributed to the tolerance to Cu2+ stress of V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of CuCl2 for 21 days at 28 °C. (B) Inhibition of CuCl2 on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.05 (*) and p ≤ 0.01 (**) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
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Figure 12. Deletion of Uvpks1 contributed to the tolerance to Mn2+ stress in V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of MnCl2 for 21 days at 28 °C. The CK (0 M MnCl2) photos of these strains were the same as those of CK (0 mM CuCl2) in Figure 11. (B) Inhibition of MnCl2 on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
Figure 12. Deletion of Uvpks1 contributed to the tolerance to Mn2+ stress in V. virens strains. (A) Colony morphology of the strains WT, ∆Uvpks1, and ∆Uvpks1-C1 cultured on PSA medium treated with different concentrations of MnCl2 for 21 days at 28 °C. The CK (0 M MnCl2) photos of these strains were the same as those of CK (0 mM CuCl2) in Figure 11. (B) Inhibition of MnCl2 on colony extension diameters of fungal strains. Each datum is the average of three replicates ± standard deviation. The differences at p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***) were statistically significant for the strain ∆Uvpks1 or ∆Uvpks1-C1 compared with the WT strain under the same treatment.
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Figure 13. Pathogenicity of the strains ∆Uvpks1, ∆Uvpks1-C1 and WT on rice plants. (A) Rice spikelets were infected by the strains WT, ∆Uvpks1, and ∆Uvpks1-C. The arrows indicate the RFS balls. (B) Number of RFS balls per infected panicle for the strains. The difference at p ≤ 0.001 (***) was statistically significant for the strain ∆Uvpks1 compared with the WT strain. ND, not detected.
Figure 13. Pathogenicity of the strains ∆Uvpks1, ∆Uvpks1-C1 and WT on rice plants. (A) Rice spikelets were infected by the strains WT, ∆Uvpks1, and ∆Uvpks1-C. The arrows indicate the RFS balls. (B) Number of RFS balls per infected panicle for the strains. The difference at p ≤ 0.001 (***) was statistically significant for the strain ∆Uvpks1 compared with the WT strain. ND, not detected.
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Table 1. Susceptibility of V. virens strains to fungicides.
Table 1. Susceptibility of V. virens strains to fungicides.
Fungal
Strain		IC50 (μg/mL)
DifenoconazoleEpoxiconazoleProchlorazAzoxystrobin
WT0.20330.05770.25950.2071
ΔUvpks10.18040.04520.17590.4487
ΔUvpks1-C0.20100.05240.25840.2214
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MDPI and ACS Style

Hou, X.; Dong, J.; Xu, D.; Liu, H.; Li, Y.; Huang, Y.; Sun, J.; Jakada, M.A.; Lai, D.; Zhou, L. Uvpks1, Involved in Ustilaginoidin Biosynthesis, Contributes to the Metabolic Profile, Development, Stress Responses, and Pathogenicity in Villosiclava virens. J. Fungi 2026, 12, 111. https://doi.org/10.3390/jof12020111

AMA Style

Hou X, Dong J, Xu D, Liu H, Li Y, Huang Y, Sun J, Jakada MA, Lai D, Zhou L. Uvpks1, Involved in Ustilaginoidin Biosynthesis, Contributes to the Metabolic Profile, Development, Stress Responses, and Pathogenicity in Villosiclava virens. Journal of Fungi. 2026; 12(2):111. https://doi.org/10.3390/jof12020111

Chicago/Turabian Style

Hou, Xuwen, Jie Dong, Dan Xu, Hao Liu, Yu Li, Yujun Huang, Jiahang Sun, Muhammad Abubakar Jakada, Daowan Lai, and Ligang Zhou. 2026. "Uvpks1, Involved in Ustilaginoidin Biosynthesis, Contributes to the Metabolic Profile, Development, Stress Responses, and Pathogenicity in Villosiclava virens" Journal of Fungi 12, no. 2: 111. https://doi.org/10.3390/jof12020111

APA Style

Hou, X., Dong, J., Xu, D., Liu, H., Li, Y., Huang, Y., Sun, J., Jakada, M. A., Lai, D., & Zhou, L. (2026). Uvpks1, Involved in Ustilaginoidin Biosynthesis, Contributes to the Metabolic Profile, Development, Stress Responses, and Pathogenicity in Villosiclava virens. Journal of Fungi, 12(2), 111. https://doi.org/10.3390/jof12020111

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