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Cryphonectria hypovirus 1 infection suppresses the pathogenicity but increases the mycotoxin deoxynivalenol production of Fusarium graminearum

Phytopathology Research volume 6, Article number: 54 (2024) Cite this article

Abstract

Fusarium graminearum is a prominent pathogen responsible for causing head blight disease in small grain cereals, leading to substantial agricultural damage. A recent study revealed the infectivity of a hypovirus, Cryphonectria hypovirus 1 (CHV1), in F. graminearum. However, the effects of CHV1 on the pathogenicity and development of the fungus remain largely unexplored. In this study, we investigated the effects of CHV1 infection on F. graminearum and made several interesting discoveries. First, we observed that CHV1 infection attenuated the pathogenicity of F. graminearum and reduced its conidiation. CHV1 efficiently spread through various transmission modes, such as hyphal fusion and spores, including conidiospores and ascospores. However, the accumulation of the virus was reduced following transmission through spores. Interestingly, despite causing hypovirulence, CHV1 infection enhanced the production of deoxynivalenol (DON), a mycotoxin known to play a role in F. graminearum’s virulence and induction of DON production by a DON-inducing medium elevated CHV1 accumulation. The transcriptional expression of key regulatory genes involved in DON biosynthesis or toxisome formation was upregulated in response to CHV1 infection. Furthermore, our study revealed that CHV1 infection suppressed the formation of lipid droplets. Overall, our study provides insights of how the infection of a hypovirulence-inducing mycovirus affects the physiological and metabolic processes in F. graminearum.

Background

Fungal diseases account for more than 70% of plant diseases, causing significant reductions in crop yield and quality (Giraldo et al., 2013; Li et al. 2023). Currently, the control of fungal diseases in crops mainly relies on chemical pesticides. However, the excessive use of these pesticides poses risks to food safety, environmental pollution, and resistance development among pathogens (Nicolopoulou-Stamati et al. 2016). In recent years, there has been growing interest in the use of mycoviruses for the biological control of pathogenic fungi. Mycoviruses multiply within fungal cells and can attenuate the virulence of phytopathogenic fungi; thus, they are referred to as hypovirulence-associated fungal viruses (Kotta-Loizou 2021). Generally, mycoviruses induce hypovirulence by significantly altering the fungal host’s morphology, development, and gene expression. Various effects of mycoviruses on fungal hosts have been observed. For instance, some mycoviruses can hinder fungal growth (Li et al. 2019), while others can reduce the secretion of secondary metabolites (Liu et al. 2022), and some can even transform host fungi into endophytes, promoting the growth and development of host plants (Zhang et al. 2020). Therefore, mycoviruses not only serve as potential biocontrol agents for fungal disease but also as probes for studying their fungal hosts (Myers et al. 2022).

Cryphonectria hypovirus 1 (CHV1) is the first mycovirus to be successfully used as a biological control agent to manage chestnut blight caused by Cryphonectria parasitica. It infects the chestnut blight fungus C. parasitica, causing hypovirulence with various phenotypic changes (Hillman et al. 1994; Sun et al. 2006). CHV1 is a prototype species in the genus Hypovirus in the family Hypoviridae. Like other hypoviruses, CHV1 lacks a rigid virion; instead, its genomic RNA is encapsulated within pleomorphic lipid vesicles derived from its fungal host (Dodds 1980). CHV1-EP713 is the first hypovirus to be sequenced. Its genome comprises a positive single-stranded RNA (+ssRNA) with a length of 12.7 kilobases and encodes two open reading frames (ORFs)-ORFA and ORFB (Shapira et al. 1991; Chiba et al. 2023). Notably, CHV1 is one of the most extensively studied mycoviruses. Reverse genetics techniques have been developed for several CHV1 strains, including CHV1-EP713 (Choi et al. 1992; Chen et al. 1999). A series of mutants have been constructed for analyzing the functions of viral factors in regulating host symptom induction, virulence attenuation, and viral replication (Craven et al. 1993; Suzuki et al. 2002; Suzuki et al. 2003). Several studies have extended CHV1’s host range to several other phytopathogenic fungi. Previously, CHV1 was artificially introduced into the phytopathogenic fungus Valsa mali, which is the causative agent of apple Valsa canker disease (Yang et al. 2021). CHV1 infection could reduce the hyphal growth, sporulation, and pathogenicity of V. mali. Previous studies also showed that CHV1 could be transfected into Fusarium graminearum by protoplast fusion. CHV1 infection also caused morphological changes in F. graminearum during its growth on potato dextrose agar (PDA) plates. Additionally, the cross-kingdom transmission of CHV1 between plant and fungus has been demonstrated (Bian et al. 2020). These studies indicate that CHV1 has a wide host range and the potential to be a biological control agent for a wide range of plant pathogenic fungi.

Apart from serving as a potential biological control agent, mycoviruses could be a valuable tool for studying fungal hosts, enabling researchers to investigate various aspects of fungal biology, pathogenicity, gene regulation, and defense mechanisms (Ghabrial et al. 2015; Myers et al. 2022). Mycoviruses can interact with their fungal hosts at the molecular level, influencing gene expression, protein production, and signaling pathways. Several studies have shown that CHV1 can alter the expression of a wide range of host genes involved in various biological processes (Allen et al. 2004; Deng et al. 2007; Andika et al. 2019; Chun et al. 2020). This alteration in gene expression is a key mechanism by which the virus affects fungal growth, metabolism, stress response, and pathogenicity (McCabe at al. 1999; Wu et al. 2017; Chun et al. 2020; Chun et al. 2022). Therefore, understanding the effects of CHV1 infection on gene expression in fungal hosts can provide valuable information about the regulation of key genes involved in pathogenicity or other biological processes.

F. graminearum is an economically important fungal pathogen that causes various crop diseases. It is primarily known for causing Fusarium head blight (FHB) in grains such as wheat, barley, and corn (Pestka 2010; Zhang et al. 2021). FHB can lead to significant yield losses and reduce grain quality, thus posing a major threat to agricultural productivity and food safety (Machado et al. 2018). Apart from FHB, F. graminearum is also associated with other diseases such as Fusarium ear rot, stalk rot, and root rot in different crop species (Walter et al. 2010; Tang et al. 2021). F. graminearum infects plants through the flowers or wounds in the stems, leaves, or ears (Reid et al. 1994; Boenisch et al. 2011). Once inside the plant, the fungus produces mycotoxins, particularly deoxynivalenol (DON), which can have detrimental effects on both plants and animals (Chen et al. 2019; Hallaj Salahipour et al. 2019; Ma et al. 2021). DON plays a significant role in the virulence of the fungus, being involved in various pathogenic processes of F. graminearum and contributing to fungal colonization, spread, and survival during the plant’s infection (Voigt et al. 2005; Stephens et al. 2008). DON is a virulence factor known to be important for spread through rachis tissues (Bai et al. 2002; Bönnighausen et al. 2019). Currently, using the pesticide is the primary strategy for preventing and controlling FHB during the wheat flowering period. However, many fungicides have been observed to enhance DON production when used for disease control (Duan et al. 2020; Zhou et al. 2020). The biosynthesis of DON in F. graminearum involves a cluster of genes known as the Tri genes, which are responsible for the synthesis and regulation of DON, as well as other related trichothecene mycotoxins. The Tri gene cluster of F. graminearum consists of 12 genes, Tri1 to Tri16 (Kimura et al. 2007; Menke et al. 2013). Each gene within the cluster performs a specific function in the biosynthetic pathway of DON (Zhu et al. 2020). Understanding the relationship between DON synthesis and fungal virulence is essential for managing F. graminearum infections.

To date, a few mycoviruses have been identified as potential biological control agents for F. graminearum. For instance, Fusarium graminearum virus 1 (FgV1), a double-stranded RNA (dsRNA) mycovirus with hypovirulence-associated features, infects F. graminearum, reducing mycelial growth and pathogenicity (Chu et al. 2002). Fusarium graminearum gemytripvirus 1 (FgGMTV1), the first multi-component single-stranded DNA virus identified from fungi, can also cause F. graminearum to exhibit typical low-toxicity phenomena. Additionally, it has been developed as a virus-induced gene silencing (VIGS) vector suitable for F. graminearum (Li et al. 2020; Zhang et al. 2023).

We previously demonstrated the infection of F. graminearum by CHV1. In this study, we further investigate the effects of CHV1 infection on pathogenicity, the production of DON and lipid droplet formation of F. graminearum. We observed hypovirulence-inducing characteristic of CHV1 and an intricate relationship between CHV1 replication and DON biosynthesis in F. graminearum.

Results

CHV1 infection reduces the pathogenicity and sporulation of F. graminearum

In our previous study, we successfully introduced CHV1 into F. graminearum (PH-1 strain). CHV1 infection caused changes in morphology and a reduction in hyphal growth when compared to virus-free F. graminearum in Potato dextrose agar (PDA) medium (Bian et al. 2020). In this study, to investigate the impact of CHV1 infection on fungal pathogenicity, sporulation, and germination, we conducted the following experiments. To assess fungal pathogenicity, conidia obtained from CHV1-infected and virus-free F. graminearum were inoculated onto tomato fruits, wheat coleoptiles or wheat heads, following which the fungal lesion and disease index were measured. CHV1-infected F. graminearum produced significantly smaller lesions on tomato fruits and wheat coleoptiles than the virus-free F. graminearum strain (Fig. 1a–d). CHV1-infected F. graminearum produced milder head blight symptoms on inoculated wheat heads than the virus-free F. graminearum strain (Fig. 1e, f). Additionally, CHV1 infection also caused a significant reduction in asexual sporulation (Fig. 1g) but not in the morphology or germination of asexual spores (Fig. 1h). CHV1 infection did not affect the formation of perithecia (ascocarps) or the development of ascospores (Fig. 1i). The number of perithecia per cm2 was similar between CHV1-infected and virus-free F. graminearum (Fig. 1j). Therefore, in addition to inhibiting vegetative growth, CHV1 infection also reduces the pathogenicity and conidiation of F. graminearum.

Fig. 1
figure 1

Effects of CHV1 infection on the development and pathogenicity of F. graminearum. a, c, and e Infection assays were conducted on tomato fruits, coleoptiles, and wheat heads, using virus-free and CHV1-infected F. graminearum PH-1 (PH-1). Disease symptoms were photographed at 7 days and 14 days post inoculation (dpi). b, d, and f The lesion area of tomato fruits, lesion length of coleoptiles, and FHB disease progression were measured. g The impact of CHV1 infection on conidia was assessed. h CHV1 infection reduced the number of conidia but did not affect conidia morphology and germination after 6 hours of incubation. i and j The formation of perithecia and asci/ascospores was observed in virus-free and CHV1-infected strains after 15 days of culture in carrot medium at 25 and the number of perithecia was calculated. The data are expressed as mean ± SD (n=3 or n=10). ** indicates a significant difference at p<0.01, and *** indicates a significant difference at p<0.001 (Student’s t-test). Scale bars: 20 μm

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Vertical transmission reduces CHV1 accumulation

We also assessed the efficiency of both horizontal transmission through hyphal fusion and vertical transmission through conidia or ascospores of CHV1 in F. graminearum. To examine horizontal transmission, the virus-free strains were co-cultured with CHV1-infected strains (Fig. 2a). The resulting fusant isolates obtained from the virus-free side were then tested for viral infection through the extraction of viral dsRNA and subsequent RT-PCR analysis. All fusant isolates were found to contain the virus (Fig. 2b). Individual germlings generated from conidia or ascospores of CHV1-infected F. graminearum were examined for viral infection using an RT-PCR assay. The results revealed that the transmission efficiency of CHV1 was 94.7% and 80% for conidia and ascospores, respectively (Fig. 2c, d). Intriguingly, the inhibition of fungal growth caused by CHV1 infection in the fungal progenies derived from hyphal fusion was as severe as in the original infected strain, but CHV1 symptoms were significantly less severe in fungal progenies derived from conidia and even less in those derived from ascospores (Fig. 2e). qRT-PCR showed that CHV1 genomic RNA accumulation was, from lowest to highest levels, in the fungal progenies derived from ascospores, conidia, and fusants (Fig. 2f). These results showed that vertical transmission negatively affects CHV1 accumulation in the fungal progenies.

Fig. 2
figure 2

Efficiency of CHV1 transmission in F. graminearum. a Virus-free and CHV1-infected F. graminearum strains were co-cultured in PDA medium. After 5 days of hyphal contact, mycelia were collected from two specific locations on the recipient side (indicated with a red circle). b The resulting fusant mycelia were then grown in fresh PDA medium for RNA preparation. The extracted dsRNA, including samples from the donor, recipient, and fusant were electrophoresed on a 1.0% agarose gel in a 1xTAE buffer system and stained with ethidium bromide (top row). RT-PCR was used to detect CHV1 accumulation in fungal hosts by amplifying the CHV1 genomic RNA from 496 to 1239 nt or 2089 to 2275 nt using specific primers (bottom row). The number of CHV1-infected colonies versus the total number of tested fungal colonies is indicated below the gel. c, d CHV1-infected mycelia were cultured in CMC liquid medium for 3 days to induce conidiation, while carrot medium was used for 14 days to induce ascospore production. The resulting single conidia/ascospore spores were germinated and grown on a fresh PDA plate for RNA preparation. RT-PCR was used to detect CHV1 accumulation in these spores’ germlings using specific primers as described above. The resulting RT-PCR products were electrophoresed on a 1.0% agarose gel in a 1xTAE buffer system and stained with ethidium bromide. The number of CHV1-infected samples versus the total number of tested samples is indicated below the gel. e The phenotypes of fungal colonies with CHV1 through horizontal hyphal anastomosis and vertical conidial and ascospore transmission were observed. The efficiency of CHV1 transmission was calculated and is presented below the fungal phenotype images. f Quantification of CHV1 RNA accumulation levels in fungal strains was performed using qRT-PCR. The data are shown as mean ± SD (n=3). Different letters indicate a significant difference at p<0.05 (one-way ANOVA)

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CHV1 infection elevates DON production and toxisome formation in F. graminearum

As CHV1 infection reduced the pathogenicity of F. graminearum, we further investigated whether CHV1 infection affects the production and secretion of DON. The amount of DON produced by the virus-free and CHV1-infected F. graminearum strains cultured in DON-inducing medium was quantified. Interestingly, CHV1-infected strain showed a significant increase in DON production compared to the virus-free F. graminearum strain, regardless of whether they were cultured in Trichothecene biosynthesis inducing (TBI) (7 days), rice (28 days), or wheat head (15 days) medium (Fig. 3a). To further examine whether CHV1 infection enhances toxisome formation, CHV1 was introduced to a transgenic F. graminearum strain expressing a key DON synthetase protein, FgTri1, fused to GFP (FgTri1-GFP) driven by an endogenous FgTri1 promoter, which served as a reporter protein for toxisome formation (Tang et al. 2018), through hyphal fusion with a CHV1-infected fungal strain (Additional file 1: Figure S1). The virus-free and CHV1-infected FgTri1-GFP strains were grown in 24-well plates supplemented with TBI medium, and toxisome formation was observed using a confocal laser. After 48 h of culture, a higher number of GFP-labeled toxisomes was observed in the CHV1-infected strain than in the virus-free strain (Fig. 3b), indicating that CHV1 infection promotes toxisome formation. Consistent with the microscopic observation, the accumulation level of the Tri1-GFP transcript and protein detected by qRT-PCR and western blot assays was significantly higher in the CHV1-infected strain than in the CHV-free strain (Fig. 3c, d).

Fig. 3
figure 3

CHV1 infection increases the biosynthesis of DON. a DON production was determined in virus-free and CHV1-infected strains grown in TBI, rice medium, and wheat spikes. b The formation of toxisomes in the mycelia of virus-free and CHV1-infected Tri1-GFP strains grown in TBI for 48 hours was observed. The Tri1-GFP strain carries a reporter construct that expresses Tri1 fused with green fluorescence protein (GFP) under its native promoter. GFP fluorescence was visualized using confocal laser scanning microscopy (CLSM). c The total proteins were extracted from the mycelia and subjected to western blotting analysis using GFP-specific antibodies. d The total RNA was extracted from the mycelia and the transcript expression of Tri1-GFP was analyzed by qRT-PCR. e, f The transcript expression of key genes (Tri1, Tri4, Tri5, and Tri6) involved in the biosynthesis of DON was analyzed. The total ssRNA was isolated from the mycelia of virus-free and CHV1-infected strains grown in PDA or TBI, and qRT-PCR analysis was carried out. The data are presented as mean ± SD (n=3). ns indicates no differences, * indicates significant differences at p<0.05, ** indicates a significant difference at p<0.01, *** indicates significant differences at p<0.001 (Student’s t-test)

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Next, the effect of CHV1 infection on the transcript expression of Tri genes (FgTri1, FgTri4, FgTri5, and FgTri6), which are involved in the biosynthesis of DON in F. graminearum, was analyzed by qRT-PCR. When F. graminearum was grown in PDA medium (non-DON-inducing medium), the expression levels of Tri1, Tri4, and Tri6 (Xu et al. 2021), but not Tri5, were upregulated by CHV1 infection (Fig. 3e), while in TBI medium, CHV1 infection enhanced the transcript expression of all detected Tri genes, including the Tri5 gene, which showed a drastic increase (Fig. 3f). Tri5 (trichodiene synthase) catalyzes the first step in DON synthesis, facilitating the cyclization of farnesyl diphosphate (FPP) to produce the sesquiterpene trichodiene (Tijerino et al. 2011; Graziani et al. 2019). Taken together, these results show that CHV1 infection promotes DON biosynthesis in F. graminearum.

Induction of DON production enhances CHV1 accumulation in F. graminearum

As we observed that CHV1 infection promotes DON production in F. graminearum, we then questioned whether the presence of a higher amount of DON benefits CHV1 infection in F. graminearum. Therefore, we examined the effects of supplying exogenous DON on CHV1 symptoms and accumulation in F. graminearum. The virus-free and CHV1-infected strains were grown in PDA/PDB medium containing 50 µg/mL commercial DON for phenotypic observation and RNA preparation (Wang et al. 2018). Interestingly, the CHV1-infected strain exhibited significantly slower growth in DON-containing medium than in DON-free medium, whereas the addition of DON to the medium did not affect the growth of virus-free F. graminearum strains (Fig. 4a, b), showing that CHV1 induces more severe symptoms in the presence of exogenous DON. The CHV1 accumulation level was then analyzed using qRT-PCR. The results showed that exogenous application of DON did not significantly affect CHV1 RNA accumulation levels in F. graminearum cultured in Potato Dextrose Broth (PDB) medium, but CHV1 RNA accumulation was highly elevated in F. graminearum cultured in TBI medium (Fig. 4c). These results suggest that DON does not directly affect CHV1 replication but enhances CHV1 symptom induction in F. graminearum. Moreover, CHV1 infection enhances the process of DON biosynthesis, which, in turn, may promote virus replication.

Fig. 4
figure 4

The effect of the exogenous application of DON on CHV1 infection. a Phenotypic characteristics of virus-free and CHV1-infected strains were observed after 5 days of growth in PDA with or without the exogenous application of DON. b The growth areas of fungal strains were measured. c CHV1 accumulation was analyzed using qRT-PCR. Total ssRNAs were isolated from fungal mycelia cultured in PDB, PDB containing 40 μg/mL DON, or TBI medium and subjected to qRT-PCR. The data are presented as mean ± SD (n=3). The different letters indicate a significant difference at p<0.05 (one-way ANOVA)

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CHV1 infection interferes with the biosynthesis of lipid droplets (LDs) in F. graminearum

The regulation of DON biosynthesis is a complex process that involves various regulatory mechanisms, including pathway-specific and global regulators, signal transduction pathways, and epigenetic modifications (Chen et al. 2019). A previous study has reported that the deletion of FgVEA, encoding the velvet protein in F. graminearum can induce the aggregation of LDs and decrease DON production (Jiang et al. 2011). Both LDs and toxin-producing bodies originate from the endoplasmic reticulum (Chen et al. 2019; Chen et al. 2021), suggesting a close relationship between the production of DON and the aggregation of LDs in F. graminearum (Liu et al. 2019). To investigate whether CHV1 infection also affects LDs biosynthesis in F. graminearum, mycelia of CHV1-infected F. graminearum strains grown in PDB were then stained with BODIPY to monitor LDs (green dots) using Confocal Laser Scanning Microscope (CLSM) (Fig. 5a). As controls, virus-free strains were grown in PDB or PDB containing 40 μg/mL Triacsin C, a compound that reduces LDs formation (Dechandt et al. 2017). As expected, Triacsin C treatment markedly decreased LDs formation in the mycelial cells as compared to that in mycelial cells of non-treated virus-free strain (Fig. 5a, b), showing Triacsin C activities in reducing LDs biosynthesis in F. graminearum. Interestingly, CHV1 infection, similar to Triacsin C treatment, decreased LDs formation in F. graminearum compared to virus-free F. graminearum cultured in PDB (Fig. 5a, b). These results show that CHV1 infection not only increase DON production but also suppress LDs formation. Therefore, it is possible that CHV1 affects both biosynthesis of LDs and DON. Moreover, in contrast to the application of DON, the addition of Triacsin C increased CHV1 accumulation (Figs. 4c and 5c), suggesting that suppression of LDs biosynthesis is necessary to promote CHV1 infection in F. graminearum.

Fig. 5
figure 5

CHV1 reduces the quantity of lipid droplet (LD) and inhibiting LD biosynthesis enhances CHV1 replication. a The subcellular distribution of LDs in the hyphal cells of F. graminearum was examined using BODIPY staining (green fluorescence). The mycelia were cultured in PDB, PDB containing 5 μM/mL Triacsin C, and mycelia of the CHV1-infected strain were cultured in PDB medium. Confocal laser scanning microscopy was used to observe LDs. b The number of LDs in hyphal cells was measured in an area of 81 μm2. Data are presented as mean ± SD (n=10). c The accumulation of CHV1 in fungal strains was analyzed by qRT-PCR. The total ssRNA was extracted from the mycelia of CHV1-infected strains cultured in PDB and PDB Triacsin C (5 μM/mL) and subjected to qRT-PCR analysis. The data are presented as mean ± SD (n=3). The different letters indicate a significant difference at p<0.05 (one-way ANOVA). ** indicates a significant difference at p<0.01 (Student’s t-test)

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It had been demonstrated that target of rapamycin (TOR) pathway regulates LD synthesis in F. graminearum. The genes involved in TOR signaling pathway, such as FgNem1/FgCak1/FgSpo7 or FgPah1 play roles in the TOR-mediated regulation of LD biogenesis. Deletion of FgNem1/FgCak1/FgSpo7 or FgPah1 impairs the response to TOR signaling, and associates with decreased LD synthesis. Notably, absence of FgNem1/FgCak1/FgSpo7 or FgPah1 also leads to reduced levels of DON synthesis, hypha growth and pathogenicity in F. graminearum (Liu et al. 2019). To investigate whether CHV1 infection affects TOR pathway and LD synthesis, we examined the transcriptional changes of these genes with or without virus infection. CHV1 infection leads to reduced transcription levels of FgNem1/FgCak1/FgSpo7 or FgPah1 (Fig. 6a), accompanied with decreased LD formation (Fig. 6b, c) in F. graminearum growing on TBI medium. This observation suggests that CHV1 infection modulate the TOR signaling pathway and negatively affects the LD formation.

Fig. 6
figure 6

CHV1 reduced the transcription of FgNem1/FgCak1/FgSpo7 or FgPah1 and decreased LD formation. a Both virus-free and CHV1-infected strains of F. graminearum cultured in TBI medium. The total ssRNA was isolated from the mycelia and analyzed using qRT-PCR. The results are displayed as mean ± SD (n=3). b, c BODIPY staining (green fluorescence) was employed to investigate the LD formation in the hyphal cells of F. graminearum. LD observation was performed using confocal laser scanning microscopy, and the quantification of LDs in hyphal cells within an 81μm2 area is presented, as mean ± SD (n=10). *** indicates a significant difference at p < 0.001 (Student’s t-test)

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Discussion

Mycoviruses have attracted significant attention as potential biological control agents against pathogenic fungi. Researchers have investigated how mycoviruses affect the virulence and development of their fungal hosts, leading to a deeper understanding of the complex interactions between mycoviruses and fungi (Xie et al. 2014; Myers et al. 2022). The study of hypovirulence mechanisms, in particular, has unveiled the molecular and cellular processes involved in fungal pathogenesis. CHV1 is a well-known mycovirus that causes hypovirulence in its natural host, C. parasitica (Sun et al. 2008), as well as in a heterologous host, V. mali (Yang et al. 2021). CHV1 infection leads to milder or attenuated disease symptoms in both hosts. We previously introduced CHV1 into the phytopathogenic fungus F. graminearum, the causal agent of FHB worldwide (Bian et al. 2020). By utilizing the CHV1-F. graminearum virus-fungal host system, we investigated the effects of the virus on the fungal host’s biology and fitness. This knowledge can be useful in developing strategies to control fungal diseases.

We discovered that CHV1 infection significantly reduced the pathogenicity and conidiation of F. graminearum (Fig. 1), which is a typical feature of viral control agents of phytopathogenic fungi. The efficient transmission of a virus from one fungal host to another is crucial for the application of hypovirulence in biological control. Effective transmission ensures that the mycovirus can spread through the population of the target pathogen, exerting a substantial impact on its virulence. Therefore, we examined the transmissibility of CHV1 through hyphal fusion or spores (Fig. 2). Similar to CHV1 transmission efficacy in its fungal hosts, C. parasitica and V. mali, CHV1 exhibited a relatively high spread efficiency in F. graminearum. However, the virus load varied depending on the transmission pathway. The highest virus load was observed through hyphal fusion, while the lowest occurred via ascospores (Fig. 2f). This explains why CHV1 had less effect on the morphology of strains originating from ascospore germination (Fig. 2e). Indeed, it is commonly considered that ascospores are rarely involved in mycovirus transmission, and limited studies have been conducted on this subject (McFadden et al. 1983; Tuomivirta et al. 2009; Xie et al. 2014). One possible reason for the limited transmission of mycoviruses through ascospores is the potential physical and biochemical barriers that exist within the ascus, hindering the transfer and infection of mycoviruses (Xie et al. 2006; Son et al. 2013). Additionally, ascospore production in certain fungi might not coincide with active mycoviral infection or replication, further limiting the possibility of mycovirus transmission through this spore type (Gross et al. 2012; Khalifa et al. 2013). It is crucial to fully understand the mechanisms of mycovirus transmission, including the potential involvement of ascospores, to explore approaches that enhance the spread of mycoviruses in fungal populations for biological control applications.

By infecting and replicating within fungal cells, viruses can disrupt or modify the expression of key genes involved in virulence and secondary metabolite biosynthesis. This interference can result in the attenuation or enhancement of virulence traits and the modulation of secondary metabolite production (Hurwitz et al. 2013). Fungal toxins, such as DON, are secondary metabolites produced by fungi under specific conditions (Chen et al. 2019). DON belongs to the B group of monotelomeric compounds and is widely distributed and influential as a fungal toxin worldwide. It is commonly referred to as a vomiting toxin and is primarily involved in fungal virulence (Desjardins et al. 1993). While viral infection can attenuate fungal virulence, the regulation of DON production can vary depending on the specific mycovirus-fungus interaction. For instance, a hypovirus, Fusarium graminearum hypovirus 2 (FgHV2), which also induces hypovirulence in F. graminearum, can reduce the accumulation of DON (Li et al. 2015). However, in our study, we noted that CHV1 reduced the phytopathogenicity of F. graminearum whereas increased DON production (Figs. 1 and 3). Moreover, genes related to DON biosynthesis were also upregulated upon CHV1 infection, particularly Tri5, which was significantly upregulated in TBI medium compared to PDA medium. Additionally, CHV1 infection induced a high accumulation of Tri-GFP-labeled toxisomes. While the wild-type strain required 72 h for toxisome induction, CHV1 infection achieved the same in just 48 h (Fig. 3). These results indicate that CHV1 upregulated the biosynthesis of DON production. In our study, we also observed that either DON production or the replication of CHV1 in TBI medium was significantly increased compared to that in PDB medium (Fig. 4). However, the addition of exogenous DON toxin did not actually increase the accumulation of CHV1 (Fig. 4). This indicates that the production process of DON is beneficial for the replication of CHV1, rather than the DON itself.

Previous studies have suggested that stressors such as H2O2 can stimulate DON synthesis (Ponts et al. 2006). Furthermore, CHV1 infection was shown to change stress enzyme activity in C. parasitica, and associated with the accumulation of H2O2 (Nuskern et al. 2017). This implies that CHV1 infection induces a stress condition during fungal growth, leading to increased DON accumulation. Given that CHV1 has a broad impact on host fungal metabolism (Brusini et al. 2017), it is likely that CHV1 affect diverse factors involved in F. graminearum pathogenicity beside DON biosynthesis. Further studies are needed to investigate the mechanism underlying CHV1’s contrasting effects in increasing DON production whereas diminishing the pathogenicity of F. graminearum.

LDs are widely present dynamic organelles that store and supply lipids in all eukaryotic cells and some prokaryotic cells for energy metabolism and lipid membrane synthesis (Olzmann et al. 2019), An increasing number of studies indicate that LDs are related to viral replication; for example, hepatitis C virus (HCV) relies on the host’s lipid metabolism pathway for replication, and its particle assembly occurs on LDs and endoplasmic reticulum membranes (Miyanari et al. 2007). LDs occur within the leaflets of the phospholipid bilayer in the endoplasmic reticulum (Kassan et al. 2013; Choudhary et al. 2015). A previous study by Liu et al. (2019) demonstrated the significant involvement of the TOR pathway in regulating LDs, DON biosynthesis, and phytopathogenicity in F. graminearum. FgNem1/FgCak1/FgSpo7 or FgPah1 are key players in TOR-mediated regulation of LD biogenesis. Deletion of FgNem1/FgCak1/FgSpo7 or FgPah1 disrupts the response to TOR signaling, resulting in decreased LD synthesis. The absence of these genes also reduced DON synthesis, as well as the pathogenicity in F. graminearum (Liu et al. 2019). Notably, in our observation, CHV1-infection downregulated the transcription of FgNem1/FgCak1/FgSpo7 or FgPah1 (Fig. 6a) and inhibited LD formation (Fig. 6b, c) during growth on TBI medium, suggesting the possibility that CHV1 diminishes LD formation through suppression of TOR pathway. Conversely, CHV1 infection upregulates the transcription levels of Tri genes (Fig. 3f) and enhances DON biosynthesis, thereby increasing DON production (Fig. 3). Further investigations are necessary to determine if CHV1 also influences other factors related to LD formation and DON synthesis.

Conclusion

In this study, we investigated the impact of CHV1 infection on F. graminearum. Our analysis revealed that CHV1 reduced the pathogenicity and conidiation of F. graminearum while enhancing the production of DON, a crucial factor in F. graminearum’s virulence. We observed that CHV1 triggered the biosynthesis and DON production, which in turn promoted virus replication. Additionally, our findings indicated that CHV1 infection disrupts the formation of lipid droplets while also leading to an increase in DON production.

Methods

Fungal strains and culturing

The wild-type fungal strain F. graminearum PH-1 (PH-1 strain) was kindly provided by Prof. Huiquan Liu (Northwest A&F University, China) (Xin et al. 2023). The Tri1-GFP-expressing F. graminearum strain (FgTri1-GFP) (Tang et al. 2020) was kindly provided by Prof. Cong Jiang (Northwest A&F University, China). Fungal protoplast isolation and CHV1 transfection were performed following the methods described by Bian et al. 2020. All fungal strains used in this study were cultivated in PDA at 25℃ for 3–5 days for morphological observation and RNA preparation. All experiments were independently repeated at least three times.

Virus transmission assay

The horizontal transmission of CHV1 through hyphal fusion was carried out as previously described (Chiba et al. 2015). In brief, recipient strains of F. graminearum were co-cultured with CHV1-infected F. graminearum strains in PDA plates. After 5–7 days of co-culturing, mycelial plugs were collected from two different positions (1 and 2) on the recipient side. These mycelial plugs were then cultured on a new PDA plate layered with cellophane to extract RNAs for viral dsRNA analysis or perform reverse transcription-polymerase chain reaction (RT-PCR) detection. The vertical transmission of CHV1 was evaluated separately for asexual spores (conidia) and sexual spores (ascospores) in this study. Asexual spores were prepared in the liquid medium Carboxymethylcellulose (CMC) as described (Zhou et al. 2010). On the other hand, sexual spores were assayed in carrot solid medium (Kong et al. 2018). Both conidia and ascospores were collected and suspended in water before being placed on a PDA plate. Individual strains derived from a single conidium and ascospore were isolated and examined for CHV1 infection.

Pathogenesis assay

The pathogenesis of fungal colonies was assessed on coleoptiles, tomatoes and wheat heads following the methods as previously described (Zhang et al. 2012; Jiang et al. 2020; Ren et al. 2022). For each experiment, conidia were obtained from the tested fungal colonies grown in CMC medium for 5 days. These conidia were then resuspended in sterile water to a concentration of 106 spores/mL.

In the pathogenicity assay using spikelet, flowering wheat heads of the cultivar Xiaoyan 22 were inoculated with 10 μL conidia suspension (106 spores/mL) on the third or fourth spikelet from the base of the inflorescence. The wheat heads were enclosed in plastic bags for 48 h to maintain moisture. Scab symptoms were observed 14 days post-inoculation (dpi) (Ren et al. 2022). For the pathogenicity assay using coleoptiles, the top of the wheat coleoptile was excised, and 2 μL conidia suspension (106 spores/mL) was inoculated into the wounds for 7 days (Zhang et al. 2012). Pathogenicity assays on tomatoes were conducted as previously described (Jiang et al. 2020). The tomato fruits were wounded after surface disinfection and then inoculated with 10 μL conidia suspension (106 spores/mL). The inoculated tomatoes were incubated at 25℃ and 100% humidity for 7 days.

dsRNA and total RNA extraction, RT-PCR, quantitative RT-PCR (qRT-PCR)

dsRNA extraction from mycelia was conducted as described previously (Li et al. 2019). Total RNA was extracted from the mycelia as previously described (Suzuki et al. 2002). To eliminate fungal chromosomal DNA, the extracted total RNA was treated with RQ1 DNase I (Promega, USA, M6101) at 37℃ for 30 min, as previously described (Xiong et al. 2019). The first-strand cDNAs were synthesized using EasyScript Reverse Transcriptase (TransGen, China, AE101-02) and utilized for virus RT-PCR detection and qRT-PCR. The qRT-PCR reactions were performed with Green qPCR SuperMix (TransGen, China, AQ601-01-V2) on a CFX96TM Real-Time PCR Detection System. The 18S gene served as an internal control for normalization (Wei et al. 2019). All primers used in this study are listed in Additional file 2: Table S1. The relative expression levels of each gene were calculated with the 2–△△Ct method. Biological triplicates were included for each gene analyzed.

Analysis of DON production

Conidia were obtained from 3–5-day cultures of F. graminearum in liquid CMC. The conidia were then suspended and cultured in trichothecene biosynthesis induction (TBI) medium, and DON production was assessed as previously described (Ren et al. 2022) with some modifications. For TBI culturing, the conidia (20 μL, 106 spores/mL) were placed in 2 mL TBI medium in a 24-well plate, wrapped in tin foil, and incubated at 25℃ for 7 days. The resulting mycelia from the TBI medium were collected for RNA preparation. For wheat head culturing, a previously described method was followed (Jiang et al. 2020). The conidia (10 μL, 106 spores/mL) were inoculated onto wheat spikelets, as described above. In the case of rice medium, 6 g of rice was soaked in deionized water for 1 h and then sterilized at 121℃ for 1 h. The cooked rice was placed on a PDA plate and grown with fungal mycelia at 25℃ for 28 days (Dong et al. 2020). Afterward, the fungal mycelia in these culture media were collected and freeze-dried using a freeze dryer (LABCONCO, USA, Freezone PI J 2.5). The collected samples were ground into a homogeneous flour and subjected to mycotoxin extraction, as described previously (Ren et al. 2022). DON was quantified using gas chromatography-mass spectrometry (the GC-MS-QP2010 system with AOC-20i autoinjector (Jiang et al. 2020).

Fluorescent imaging and lipid droplets (LDs) staining

An FgTri1-GFP strain infected with CHV1 was obtained through hyphal fusion. Virus-free FgTri1-GFP and FgTri1-GFP with CHV1 were cultured in TBI medium at 25℃ for 5 days. To observe the expression of the FgTril-GFP fusion protein in the mycelium, confocal laser scanning microscopy was employed using an Olympus FV3000 instrument with a laser excitation/emission wavelength of 488/510–550 nm, as described previously (Niu et al. 2022). To monitor the lipid droplets (LDs) in the mycelium, the LD staining dyes BODIPY 493/503 (Sigma-Aldrich, USA, 790389) was used (Liu et al. 2019). Staining was performed following the manufacturers’ instructions.

Western blotting analysis

The western blotting analysis was carried out as previously described (Sun et al. 2006). The FgTril-GFP protein was detected by anti-GFP (1:5000; GFP Mouse Monoclonal Antibody, Beyotime, China, AF0159). For secondary antibody detection, a goat anti-rabbit immunoglobulin G-horseradish peroxidase (IgG-HRP, 1:5000; pRoteintech) was used.

DON and Triacsin C treatment

Fungus was cultured in a PDA medium containing 40 μg/mL DON (MedChemExpress, USA, HY-N6684) at 25℃ for 5 days (Wang et al. 2018). The conidia were harvested from CHV1-infected F. graminearum strains that were cultured in CMC medium at 25℃ for 5 days. The resulting conidia were suspended in 100 μL (106 spores/mL) and cultured in 20 mL medium, including PDB, PDB containing 40 μg/mL DON or 5 μM/mL Triacsin C (MedChemExpress, USA, HY-N6707) (Dechandt et al. 2017), and liquid TBI medium, at 25℃ for 3 days. The mycelia were collected for qRT-PCR assay and LD staining. All experiments were repeated at least three times.

Availability of data and materials

The authors declare that the data supporting the findings of this study are available within the paper, its supplementary information files.

Abbreviations

cDNAs:

Complementary DNAs

CHV1:

Cryphonectria hypovirus 1

CLSM:

Confocal Laser Scanning Microscope

CMC:

Carboxymethylcellulose

C. parasitica :

Cryphonectria parasitica

DON:

Deoxynivalenol

dpi:

days post-inoculation

dsRNA:

Double-stranded RNA

FgGMTV1:

Fusarium graminearum gemytripvirus 1

FgHV2:

Fusarium graminearum hypovirus 2

F. graminearum :

Fusarium graminearum

FgV1:

Fusarium graminearum virus 1

FHB:

Fusarium head blight

HCV:

Hepatitis C virus

H2O2 :

Hydrogen peroxide

LDs:

Lipid droplets

ORFs:

Open reading frames

PDA:

Potato dextrose agar

PDB:

Potato dextrose broth

qRT-PCR:

Quantitative real-time PCR

RT-PCR:

Reverse transcription-polymerase chain reaction

TBI:

Trichothecene biosynthesis induction

TOR:

Target of rapamycin

Tri:

Trichothecene

VIGS:

Virus-induced gene silencing

V. mali :

Valsa mali

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Acknowledgments

We thank the High-Performance Computing Center of Northwest A&F University for providing computing resources.

Funding

This research was supported by the National Natural Science Foundation of China (32170163) and the Natural Science Foundation of Xinjiang Uygur Autonomous Region (2021D01D12) to LS.

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Author notes

  1. Ruiling Bian and Huan Ren contributed equally to this study.

Authors and Affiliations

  1. State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, 712100, China

    Ruiling Bian, Huan Ren, Mengge Jiang, Zhihao Zhang, Shiyu Zhai, Tianxing Pang, Ida Bagus Andika & Liying Sun

Authors
  1. Ruiling Bian
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  2. Huan Ren
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  3. Mengge Jiang
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  4. Zhihao Zhang
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  6. Tianxing Pang
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  7. Ida Bagus Andika
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  8. Liying Sun
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Contributions

LS and RB designed research; RB, HR, MJ, ZZ, SZ, and TP performed research; RB, HR, and IBA analyzed data; RB and LS wrote the manuscript.

Corresponding author

Correspondence to Liying Sun.

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All authors of the manuscript have reviewed and consented to the publication of identifiable details, including photographs and any information within the text in the journal of Phytopathology Research.

Competing interests

Authors declare no conflict of interests.

Supplementary Information

Additional file 1: Figure S1.

Transfection of CHV1 into the FgTri1-GFP strain. a The FgTri1-GFP strain was co-cultured with CHV1-infected F. graminearum in PDA. CHV1 was transfected through hyphal anastomosis. b Detection of CHV1 infection in the FgTri1-GFP strain was achieved using dsRNA and RT-PCR analysis. c The FgTri1-GFP strains, with or without CHV1 infection, were cultured on PDA for 5 days and then photographed. Both virus-free and CHV1-infected F. graminearum PH-1 strains were cultured in parallelfor comparison.

Additional file 2.

 Table S1. A list of primers used in this study.

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Bian, R., Ren, H., Jiang, M. et al. Cryphonectria hypovirus 1 infection suppresses the pathogenicity but increases the mycotoxin deoxynivalenol production of Fusarium graminearum. Phytopathol Res 6, 54 (2024). https://doi.org/10.1186/s42483-024-00272-7

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Keywords

Phytopathology Research

ISSN: 2524-4167