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Chemical and Biological Technologies in Agriculture
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Changes in VOCs from a chestnut blight fungus Cryphonectria parasitica by a hypovirus infection: mycoviral infection alters fungal smell for insect vectors

Chemical and Biological Technologies in Agriculture volume 11, Article number: 123 (2024) Cite this article

Abstract

Background

The chestnut blight fungus, Cryphonectria parasitica, and its Cryphonectria hypovirus 1 (CHV1) comprise a useful model system to study fungus–virus interactions. CHV1 infection results in various phenotypic changes in the fungal host, including hypovirulence and other associated symptoms. Many studies have investigated the effects of hypoviral infection and how this infection affects physiological and biochemical states: however, no studies have examined volatile organic compounds (VOCs).

Results

This study characterized the global profile of VOCs released from C. parasitica, and by comparing CHV1-free with CHV1-infected isogenic strains, proved that CHV1 infection significantly affects the composition and levels of VOCs. We demonstrated that these VOC alterations were caused by viral regulation of the expression of fungal genes encoding enzymes responsible for the production of VOCs. The changed VOC profile resulting from CHV1 infection was more attractive to insects than the VOC profile from the virus-free strain, suggesting differences in the efficacy of fungal dissemination by the insect vector.

Conclusions

We characterized VOCs from C. parasitica and demonstrated that mycovirus (CHV1) infection affects fungal VOCs. We provided evidences that these alterations are caused by the modulation of the corresponding gene expression by CHV1 and resulted in changes in attractiveness to insects, suggesting the improved efficacy of hypovirulent C. parasitica for insect-borne dissemination.

Graphical Abstract

Background

Cryphonectria parasitica (Murrill) Barr, the causal agent of chestnut blight disease, destroyed American chestnut forests in the early twentieth century [1]. However, strains infected with the single-stranded ssRNA mycovirus Cryphonectria hypovirus 1 (CHV1) showed characteristic symptoms of attenuated virulence, a phenomenon known as hypovirulence [2,3,4]. This hypovirulence can be spread within the pathogen population via the transmission of CHV1 from an infected to a virus-free strain, ultimately resulting in protection of susceptible chestnut trees [5]. This type of virulence conversion is a naturally occurring example of biocontrol for a plant-pathogenic fungus mediated by mycoviruses, now referred to as a typical instance of “virocontrol” [6]. Specific hypovirulence-associated fungal symptoms include reduced sporulation and pigmentation, decreased oxalate accumulation and laccase production, and the suppression of female fertility [7,8,9,10,11]. Since the C. parasitica–CHV1 interaction is among the earliest successful biocontrol systems for plant pathogens mediated by mycoviruses, their interactions have been used as a model to study fungus–virus interactions [12]. Molecular genetic analysis, including recent systemic comparison of the transcripts and proteins of virus-free and virus-infected isogenic strains have proved that symptom development entirely depends on the fungal gene regulation by the hypovirus [13,14,15,16,17,18,19,20,21,22,23,24]. A smaller number of studies have investigated how metabolic changes influence C. parasitica behavior [25, 26], but few studies have analyzed the VOCs of this fungus and none have examined the alterations in the VOCs due to CHV1 infection.

VOCs are carbon-containing compounds with low molecular weights that easily evaporate at normal room temperature and pressure [27]. Although the importance of biogenic VOCs’ physiological and pathological effects has recently been recognized, the research on these aspects is scant [28, 29]. More than 300 fungal VOCs have been described, and the numbers of both fungal VOCs and fungal species continue to increase [30]. All fungal species release a mixture of a wide variety of VOCs and the same chemical species of VOC is produced by several fungi. Fungal VOCs behave as semiochemicals for many arthropods and hormones for fungal development [27]. They also act as flavors and aromas for food and living environments in addition to functioning as antibiotics and biostimulants to protect and promote plant growth [31,32,33,34]. In short, fungal VOCs are important in determining the interactions of fungi with host plants and other organisms.

Terpenes are representative VOCs that perform several functions in fungi [35,36,37]. Terpenes are derived from isopentenyl diphosphate (IPP, C5). IPP is produced through the mevalonate (MVE) pathway and it is then converted to geranyl-PP (C10) and farnesyl-PP (C15), which are precursors of monoterpene and sesquiterpene, respectively [38]. Geranyl-PP (GPP) and farnesyl-PP (FPP) become functional terpenes through interaction with terpene cyclase and tailoring enzymes. These GPP and FPP are further metabolized by several post-farnesyl pyrophosphate synthetase (FDS; encoded by the ERG20 gene in Saccharomyces cerevisiae) pathway genes, including ERG9 (farnesyl diphosphate farnesyl transferase; FDFT) and BTS1 (geranylgeranyl diphosphate synthase; GGPPS), which are involved in the production of squalene (sterols) and geranylgeranyl-pp (GGPP), using FPP and GPP as a precursor, respectively (Fig. S1). Studies on the genes involved in terpene metabolism in filamentous fungi are very limited [39, 40], but in silico analysis of the C. parasitica genome database (http://genome.jgi-psf.org/Crypa2/Crypa2.home.html) using a yeast homolog as a query revealed the presence of close homologs (CpERG20, CpERG9, and CpBTS1) in the C. parasitica genome. Genes encoding mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase, which are involved in the MVE pathway of IPP biosynthesis, have also been annotated in the C. parasitica genome (Fig. S1).

C. parasitica releases a characteristic “odor”. However, the nature of VOCs from C. parasitica has not been studied and their alterations due to CHV1 infection have not been characterized either. In this study, we conducted the first-time characterization of the VOCs from C. parasitica and analyzed their alteration resulting from CHV1 infection.

Methods

Fungal strains and growth condition

C. parasitica EP155/2 (ATCC 38755) and its isogenic hypovirulent strain UEP1 [41], containing Cryphonectria hypovirus 1 (CHV1-EP713), were used for this study. Fungal strains were cultured on potato-dextrose agar (PDA) supplemented with L-methionine (0.1 g/L) and biotin (1 mg/L) (PDAmb) under constant low light conditions at 25 °C as previously described [42]. To measure the VOCs, the agar block containing each strain of actively growing mycelium was placed in the center of the slant media consisting of 10 ml PDAmb in 20 ml-headspace vials (SU860097 Supleco, Sigma-Aldrich, St. Louis, MO, USA), then immediately sealed with stainless steel screw cap with PTFE fluorosilicone rubber septum (SU860101 Supelco, Sigma-Aldrich). Three biological replicates of each strain were sealed and incubated at 25 °C under constant low light for 5, 10, or 20 days.

Gas chromatography–mass spectrometry (GC–MS) analysis

The SPME fiber was preconditioned and cleaned through the heat-treatment at 270 °C for 30 min. Each vial containing EP155/2 and UEP1 was agitated at 50 °C at 350 rpm for 5 s with a 2-s break program for 3 min. Headspace SPME was used to collect VOCs from each vial, which were absorbed onto 50/30 µm DVB/CAR/PDMS fiber (57329-U Supelco, Sigma-Aldrich) at 40 °C for 30 min and penetrated at a rate of 20 mm/s at a depth of 40 mm. The compounds were separated and analyzed using gas chromatography tandem time-of-flight mass spectrometry (Pegasus BT 4D, LECO Corporation, St. Joseph, MI, USA) equipped with a multi-purpose sampler (Gerstel, Linthicum Heights, MD, USA). Sample introduction was performed in spitless mode with a 480-s desorption time at 270 °C. Other detailed instrumental parameters are listed in Table S1. Data were recorded in TIC mode. Agilent Chemstation software was adopted to process the mass spectra and chromatograms and their constituents were identified after comparing our results with the data in the GC–MS library available in the literature.

Cloning of genes and quantitative analyses of transcript accumulation by RT-qPCR

An analysis of the C. parasitica genome sequence (http://genome.jgi-psf.org/Crypa2/Crypa2.home.html) was conducted to identify genes encoding members of the main metabolic pathways associated with the biosynthesis of mevalonate. In silico sequence analysis was performed to predict the full-length cDNA sequences of the genes for mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, farnesyl diphosphate synthase, farnesyl diphosphate farnesyl transferase, and geranylgeranyl diphosphate synthase, which are involved in the biosynthesis of terpenoids, and aryl-alcohol dehydrogenase, which is involved in the biosynthesis of phenylethyl alcohol. Genomic DNA and RNA was extracted as described previously [23, 24]. To quantify the expression levels of mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, farnesyl diphosphate synthase, farnesyl diphosphate farnesyl transferase, geranylgeranyl diphosphate synthase, and aryl-alcohol dehydrogenase genes, reverse transcription-quantitative PCR (RT-qPCR) was performed. Briefly, a total of 0.5 μg of RNA was treated with RQ1 RNase-free DNase1 (Promega, Madison, WI, USA), equal amounts of cDNA were synthesized with SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and oligo dT, referring to the manufacturer's protocol. RT-qPCR was performed using AmpiGene® qPCR Green Mix Lo-ROX (Enzo Biochem, NY, USA) as previously described and evaluated with the GeneAmp 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) [24]. Transcript accumulation levels were quantified by the 2−ΔΔCT method [43] and the transcript level of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was compared as the endogenously expressed gene [44]. The two-tailed Student’s t-test was conducted to statistically analyze the accumulation of transcripts of interest in the isogenic virus-free and hypovirulent UEP1 strains. A P-value of ≤ 0.05 was considered to be significant and standard deviation was denoted as an error bar. The primers used in the RT-qPCR analysis are listed in Table S2.

Behavior assays

To measure olfactory responses to VOCs released from C. parasitica, two-choice behavior assays were conducted using the adult fruit fly (Drosophila melanogaster) as the model insect, since it can produce many generations in a short time and has odor receptors to respond to VOCs [45, 46], and fall webworm moth larvae [Hyphantria cunea (Drury)] as generalist folivores on a chestnut tree [47]. To assess the insects’ olfactory behavior, 60 adult fruit flies were placed in a petri plate “release arena”, 10 cm in diameter, the bottom of which was connected to two 15-ml falcon tubes acting as pitfall traps. These pitfall traps were spaced 3.5 cm apart, and each pitfall trap contained a PDAmb agar block (15 mm in diameter) containing actively growing mycelia of a tested strain. Once in position, the apparatuses were placed under culture conditions, and the flies were given 12 h (six h of light and six h of darkness) to choose. At the end of each 12-h experiment, the number of flies in each falcon tube was recorded, then the flies were discarded. Olfactory preference was evaluated with three biological replicates at each time point (5, 10, or 20 days). In addition, mycelial invasive growth of strain on the chestnut stem was prepared as described previously [22]. When the stromal pustule eruptions of each strain were obvious after 20 days of inoculation, 60 adult fruit flies were placed in a petri plate “release arena”, 10 cm in diameter, the bottom of which was connected to two petri plates (9 × 2.5 cm) acting as pitfall traps. Once in position, the flies were given 12 h to choose as described above. For the larvae’s olfactory behavior assay, 10 larvae were placed in the center of a petri dish, which contained two PDAmb agar blocks (15 mm in diameter) containing actively growing mycelia of a tested strain in the two opposite zones (spaced 15 cm apart). The number of larvae less than 1 cm from the corresponding agar block was monitored every 10 min up to 180 min.

Results

Identification of VOCs released from C. parasitica

The VOCs were identified through solid-phase microextraction followed by gas chromatography–mass spectrometry (SPME/GC–MS). VOCs that showed high similarity to those in the NIST Standard Reference Database and were detected from at least two replicates or accounted for more than 1% of the three repetitions were included for further analysis. In total, 65 predominant VOCs were identified (Table S3). Among these, 51 were released from the CHV1-free EP155/2 strain and 54 were from the CHV1-infected UEP1 strain. There were 40 commonly detected VOCs, 11 were specific to EP155/2, and 14 were specific to UEP1.

Analysis of VOCs from CHV1-free and CHV1-infected C. parasitica strains

At 5-day culture, 35 VOCs were detected from EP155/2 and 34 from UEP1. Of these, 26 were common to both (Fig. 1a). The predominant VOC from EP155/2 was phenylethyl alcohol (17.1%) followed by benzeneacetaldehyde (11.3%), (1R,5R)-1-isopropyl-8-methyl-4-methylenespiro[4.5]dec-7-ene (7.9%), and (1R,5S)-1,8-dimethyl-4-(propan-2-ylidene)spiro[4.5]dec-7-ene (7.7%). From UEP1, β-phellandrene (26.4%) was the most common, followed by α-phellandrene (11.5%), phenylethyl alcohol (9.19%), and 3-(methylthio)-1-propanol (9.16%). CHV1 infection affected the production of most of these 26 common VOCs, resulting in changes in order of prevalence. The dominant VOC from EP155/2, phenylethyl alcohol (comprising more than 17% of that strain’s total VOC output), was the third most produced VOC from UEP1 (9.19% of total VOC output), implying that CHV1 infection dramatically decreased phenylethyl alcohol production.

Fig. 1
figure 1

Number of VOCs released from each strain. CHV1-free EP155/2 and CHV1-infected UEP1 were the C. parasitica strains used. The numbers of strain-specific and common VOCs are represented by the Venn diagram. The proportions of VOCs are represented in the bar graph, and major VOCs are indicated at the bottom. Days of cultivation are: a 5 days, b 10 days, and c 20 days

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At 10-day culture, 36 VOCs were detected from both EP155/2 and UEP1, of which 28 were common (Fig. 1b). The predominant VOC from EP155/2 was phenylethyl alcohol (27.7%), followed by 3-(methylthio)-1-propanol (15.5%), β-phellandrene (11.1%), and benzeneacetaldehyde (6.7%). From UEP1, phenylethyl alcohol (32.7%) comprised the majority, followed by 4-methylene-1-(1-methylethyl)-bicyclo[3.1.0]hexane (16.2%), 3-(methylthio)-1-propanol (8.5%), 3-methyl-6-(1-methylethyl)- 2-cyclohexen-1-one (5.7%), and benzene acetaldehyde (5.07%). Among the common dominant VOCs, CHV1 infection affected the production of phenylethyl alcohol and 3-(methylthio)-1-propanol but no significant change was observed in the levels of benzeneacetaldehyde.

At 20-day culture, 31 VOCs were detected from EP155/2 and 36 from UEP1. Of these, 22 were common to both (Fig. 1c). The predominant VOC from EP155/2 remained phenylethyl alcohol (44.9%), followed by benzeneacetaldehyde (15.8%), and 3-(methylthio)-1-propanol (14.3%). From UEP1, phenylethyl alcohol (51.2%) was the most prevalent, then 3-methyl-6-(1-methylethyl)-2-cyclohexen-1-one (8.65%), benzeneacetaldehyde (8.27%), and 3-(methylthio)-1-propanol (5.19%). CHV1 infection affected the production of 15 VOCs of the 22 common VOCs, changing their order of prevalence.

We observed that the volatile metabolic fingerprints of C. parasitica altered significantly over the culture period (Fig. S2). Fifteen VOCs were consistently released from both strains, although the volumes differed over time. Among these 15 common VOCs, the volume of phenylethyl alcohol increased over time in both strains (Fig. S3a), while the volume of β-bisabolene decreased (Fig. S3b). Production of 3-(methylthio)-1-propanol increased in EP155/2 but decreased in UEP1 (Fig. S3c), demonstrating the impact of CHV1 infection on VOC production. Four VOCs were consistent and specific to EP155/2 and six were consistently specific to UEP1.

Some VOCs are pure hydrocarbons (consisting of only carbon and hydrogen), while others have functional groups containing nitrogen, sulfur, halogen, and/or oxygen, such as alcohols, aldehyde, esters, ethers, ketones, pyrazine, and furans (Fig. 2). Among the 39 hydrocarbon VOCs we identified, 34 were released from EP155/2 and 32 from UEP1. Seven were specific to EP155/2 while 5 were specific to UEP1 (Fig. 2a, b). Twenty VOCs with functional groups containing oxygen were released; 13 were common, four were EP155/2-specific, and nine were UEP1-specific (Fig. 2c, d). Two VOCs with functional groups containing sulfur were detected; 3-(methylthio)-1-propanol, which was common, and dihydro-2-methyl-3(2H)-thiophenone, which was UEP1-specific. 1,3,5-trichloro-2-methoxy-benzene and 3-ethyl-2,5-dimethyl-pyrazine were detected as common VOCs with a functional group containing halogen and nitrogen, respectively.

Fig. 2
figure 2

Numbers of classified VOCs depending on the hydrocarbon only (a, b) and functional groups (c, d). CHV1-free EP155/2 and CHV1-infected UEP1 were the C. parasitica strains used. Strain-specific and common classified VOCs are depicted by the Venn diagram. The numbers of each classified group are represented in the lower tables. Percentage of VOC constituents according to functional group are represented by the bar graph after 5-, 10-, and 20-day culture

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Terpenes are naturally occurring hydrocarbons with the formula (C5H8)n for n ≥ 2 and are further classified by their number of carbon atoms, for example, into monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20). Of the total 65 VOCs identified, 39 were terpenes: 25 were common to both strains, 10 were EP155/2-specific, and four were UEP1-specifc. The total levels of terpenes from both strains decreased as the culture aged (Fig. 3a). Sesquiterpenes were the major terpenes from EP155/2 throughout the culture period; however, UEP1 released more monoterpenes than sesquiterpenes except at 20-day culture (Fig. 3b). As the UEP1 culture aged, the level of sesquiterpenes increased while the level of monoterpenes decreased (Fig. 3b). These results indicated that CHV1 infection affects the terpenoid metabolic pathway modifying the proportion as well as the components of terpenes by modulating the expression of a gene encoding farnesyl diphosphate farnesyltransferase responsible for sesquiterpene biosynthesis and a gene encoding geranylgeranyl diphosphate geranylgeranyltransferase for monoterpene biosynthesis.

Fig. 3
figure 3

Analysis of terpene production. CHV1-free EP155/2 and CHV1-infected UEP1 were the C. parasitica strains used. a Proportion of terpenes in VOCs over time. b Ratio of monoterpenes and sesquiterpenes in total terpenes over time. The filled bar represents CHV1-free EP155/2, while the bar with slashes represents CHV1-infected UEP1

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Transcriptional regulation of VOCs by CHV1

The genetic sequence alignments of mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase, which are involved in the MVE pathway for IPP biosynthesis and phylogenetic analysis with closely related genes strongly suggest that these annotated genes are likely the corresponding genes in the MVE pathway (Fig. S4). Based on in silico sequence analysis of mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase, we performed RT-qPCR. The transcriptional accumulation of the three annotated genes responsible for each enzyme showed consolidated expression patterns, suggesting that although these genes were not topologically clustered, their expression was clustered for collinear regulation (Fig. 4a–c). The transcriptional accumulation of these three genes dramatically increased after 20-day cultivation of virus-free EP155/2, whereas in UEP1, the levels decreased. These results from EP155/2 are inconsistent with the results of the total amount of terpene measured in EP155/2.

Fig. 4
figure 4

Transcriptional analysis of the genes involved in the (MVE) pathway for IPP biosynthesis (a–c) and the post-IPP pathway for terpene biosynthesis (d–f). CHV1-free EP155/2 and CHV1-infected UEP1 were the C. parasitica strains used. Transcript accumulation of genes for a mevalonate kinase (CpERG12), b phosphomevalonate kinase (CpERG8), c diphosphomevalonate decarboxylase (CpERG19), d farnesyl diphosphate synthase (CpERG20), e farnesyl diphosphate farnesyl transferase (CpERG9), f geranylgeranyl diphosphate synthase (CpBTS1). CHV1-free EP155/2 and CHV1-infected UEP1 were the C. parasitica strains used. The filled bar represents EP155/2, while the bar with slashes represents UEP1

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Sequence alignment and phylogenetic analysis of farnesyl pyrophosphate synthetase, farnesyl diphosphate farnesyl transferase, and geranylgeranyl diphosphate synthase homologs from C. parasitica with other fungal genes, including genes from S. cerevisiae, strongly suggest that these gene products are involved in the post-IPP pathway (Fig. S5). Transcription analysis of CpERG20, CpERG9, and CpBTS1 genes confirmed that the transcript encoding of all three increased in the virus-free EP155/2 as the culture proceeded (Fig. 4d–f). However, different expression patterns were observed in CHV1-infcted UEP1; the expression of CpERG20 and CpERG9 decreased while the expression of CpBTS1 did not significantly changed (Fig. 4d–f). Pairwise comparison of CpERG9 transcript accumulation, the gene involved in squalene biosynthesis, indicated that the transcript accumulation was significantly higher in UEP1 than EP155/2 at 5-day culture.

Olfactory behavioral assay

Because phenylethyl alcohol and β-phellandrene, two major VOCs from C. parasitica, are known to function as insect attractants [48, 49], we tested the insects’ olfactory behavior in response to VOCs from C. parasitica (Fig. 5). In the two-choice chemotaxis behavioral assay for adult fruit flies, 10-day and 20-day cultured virus-infected UEP1 were more attractive than virus-free EP155/2 (Fig. 5a). In addition, when we tested the infected sterile pieces of chestnut stems resembling more of natural ecosystem, chestnut stems infected with virus-infected UEP1 were more attractive than those with virus-free EP155/2 (Fig. 5b, Video S1). Hyphantria cunea (Drury) larvae also found UEP1 significantly more attractive, and this was accentuated after 20 days of inoculation (Fig. 6). We were unable to identify the specific attractive components from the total VOCs, but we found that CHV1-infected C. parasitica underwent dramatic changes in its VOC output. These dramatic VOC changes resulted in changes in the attractiveness to the insects, which became most significant after 20 days. Insects are important potential disseminators of C. parasitica [50]. Larvae of the fall webworm H. cunea used in this study are generalist folivores that grow on chestnut trees [47]. Our results showed a difference in attractiveness between CHV1-free and CHV1-infected strains, implying that there may be a difference in the insect-borne dissemination of this fungus depending on infection with CHV1.

Fig. 5
figure 5

Two-choice chemotaxis behavioral assay of EP155/2 and UEP1 using adult D. melanogaster. CHV1-free EP155/2 and CHV1-infected UEP1 were the C. parasitica strains used. Behavioral assay using adult fruit flies responding to agar plugs of actively growing mycelia (a) and chestnut stems infected with each strain (b). A representative picture of the behavior test of fruit flies using pathogenic growth is shown below the graph. * indicates the significant difference using Tukey’s t-test. The red arrows denote the fruit flies in the figure

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Fig. 6
figure 6

Two-choice chemotaxis behavioral assay of EP155/2 and UEP1 using H. cunea larvae. CHV1-free EP155/2 and CHV1-infected UEP1 were the C. parasitica strains used. Behavioral assay using fall webworm moth larvae. An actual picture of the behavior test of H. cunea larvae is shown below the graph. The agar block with EP155/2 was placed on the top of the plate, and the agar block with UEP1 was placed on the bottom of the plate. * indicates the significant difference using Tukey’s t-test

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Discussion

Fungal VOCs have been studied in many disciplines, including fragrance chemistry, chemical ecology, industrial hygiene, medical mycology, and plant pathology [27, 29, 31, 51,52,53,54]. In addition to their implications in agriculture as off-odors for fungal spoilage, plant growth effectors (stimulant or retardant), and semiochemicals, their antimicrobial activity, such as antibiosis and mycofumigation, makes fungal VOCs more attractive as an effective biocontrol potential [27]. However, their remarkable heterogeneity renders them difficult to study, and research regarding the total complement of compounds at a given time from a fungal pathogen (fungal volatome) are very limited. No previous studies of the VOCs from C. parasitica have been conducted, though many studies using multi-omics, including metabolomics, have used this model.

We observed that the VOCs released from C. parasitica are chemically diverse. Many of the VOCs have been previously found in natural essential oils and other fungi, suggesting fragrant and antimicrobial functions [34]. However, the VOC fingerprint of C. parasitica was dissimilar from any other specific fungus, including the well-known fungal biocontrol agent Trichoderma spp. [55, 56]. These results indicate that the VOCs from C. parasitica are specific to this fungal species. Although there were several consistently detected VOCs, temporal changes of emitted VOCs were also observed implying that the VOC profiles changes dynamically with fungal growth stages, i.e., its physiological status.

Analysis of the VOCs fingerprints of virus-free and virus-infected isogenic strains revealed significant differences depending on CHV1 infection. We identified strain-specific VOCs: four that are EP155/2-specific and that six are UEP1-specific. These VOCs indicate whether a strain is infected with CHV1.

We demonstrated that the VOC changes resulted from modification in the expression of genes responsible for VOC production. We compared the production of the most commonly detected VOC, phenylethyl alcohol, with the expression of the gene responsible for phenylethyl alcohol synthesis (Fig. 7). As shown in Fig. S3a, the production of phenylethyl alcohol increased as both the EP155/2 and UEP1 cultures aged. Aryl-alcohol dehydrogenase is responsible for phenylethyl alcohol production, so transcriptional analysis of the aryl-alcohol dehydrogenase gene was conducted (Fig. 7b). Transcriptional accumulation of the mRNA encoding the aryl-alcohol dehydrogenase showed a significant increase after 20 days of inoculation, which was similar to the pattern of the increasing proportions of phenylethyl alcohol, in that transcript accumulation increased as the culture proceeded. These results indicated that VOC changes in the CHV1-infected strain occurred through alterations in the expression of the corresponding genes by CHV1. Our transcriptional analysis of more complicated terpene biosynthesis pathway genes, which aligns with our previous RNA-Seq results [21], further supports VOC changes due to fungal gene regulation by CHV1. It has been known that various symptomatic changes in virus-infected strains occurred because a specific set of fungal genes were affected by CHV1 infection [14, 21, 23, 57, 58]. Our results clearly indicated changes in the transcription of VOC-related genes and changes in the compound species and relative amounts of VOCs. However, further studies are required to reveal the correlation between the analyzed genes and the changes in VOCs. Our results of changes in the expression of genes related to VOCs and VOC profiles after CHV1 infection strongly suggest that VOC changes can be attributed to the CHV1-infected strain that occurred through alterations in the expression of the corresponding genes by CHV1. Major fungal taxa are infected with viruses, and many more are yet to be identified [59, 60]. Among these, the C. parasitica–CHV1 interaction is a pioneering model used to study fungus–virus interactions. The findings of our study suggest that mycoviral infection affects fungal volatomes, resulting in significant changes in fungal ecology.

Fig. 7
figure 7

Pairwise comparison of production and gene expression of phenylethyl alcohol. CHV1-free EP155/2 and CHV1-infected UEP1 were the C. parasitica strains used. a Proportion of phenylethyl alcohol released from EP155/2 and UEP1 over time. b Relative quantification of transcript accumulation encoding aryl-alcohol dehydrogenase from EP155/2 and UEP1. The filled bar represents EP155/2, while the bar with slashes represents UEP1

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VOCs are involved in interkingdom communication [61, 62]. Our behavioral assay indicated that the attractiveness of C. parasitica to insects was altered by CHV1 infection. CHV1 infection modifies the fungal VOC profile, rendering the CHV1-infected strain more attractive to insects. Considering the quantitative and qualitative changes in the VOC fingerprint caused by CHV1-infection and the interaction of blended VOCs [49], we did not define which component is responsible for the changes in attractiveness. In forest ecosystem, the life cycle of C. parasitica includes asexual and sexual stages, the spores of which are disseminated via different mechanisms. Asexual conidia are believed to be dispersed over short distances by water splashes and over long distances by vectors, such as animals and insects [47, 63, 64]. The matured sexual ascospores, which are thought to be ejected from ascocarp structures into the air, are dispersed by the wind [64,65,66]. Hypovirulent strains are characterized by reduced virulence, which is represented by the formation of superficial cankers on the bark of chestnut trees. As a result, asexual and sexual sporulation is not as abundant as in the virus-free virulent strain and is even rare, often absent, because of superficial cankers. However, a high disease incidence and low disease severity are essential for hypovirulence to preserve chestnut forests. Thus, deciphering the interactions between the tripartite chestnut/C. parasitica/CHV1 pathosystem is important to understand how the CHV1-infected hypovirulent strains with reduced conidiation and female fertility prevail in chestnut forests over time. Dissemination methods for virus-free C. parastica are relatively well known, but how the hypovirulent strain disseminates has not yet been fully elucidated [67, 68]. Our finding that insects are more attracted to the CHV1-infected strain than the CHV1-free strain suggests that the hypovirulent strain would have a greater chance of being exposed to the insect to collect conidia and mycelia than the virus-free strain, and that the attracted insect acted as an efficient vector by simply moving around for the dissemination of the hypovirulent strain. These findings help explain how hypovirulent strains are efficiently disseminated in chestnut forests, even when sporulation is reduced. This finding is expected to significantly affect the disease cycle of the hypovirulent strains.

Availability of data and materials 

All data generated or analyzed during this study are included in this published article and its supplemental information files.

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Funding

This work was supported by the National Research Foundation of Korea (NRF) grant [NRF-2022R1A2C3005906]. This research was supported by “Research Base Construction Fund Support Program” funded by Jeonbuk National University in 2022.

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  1. Yo-Han Ko and Jeesun Chun contributed equally to this work.

Authors and Affiliations

  1. Institute for Molecular Biology and Genetics, Jeonbuk National University, Jeonju, 54896, Jeonbuk, Korea

    Yo-Han Ko, Jeesun Chun & Dae-Hyuk Kim

  2. Korea Basic Science Institute (KBSI) Jeonju Center, Jeonju, 54907, Jeonbuk, Korea

    Sei-Jin Lee

  3. Department of Bioactive Material Sciences, Department of Molecular Biology, Jeonbuk National University, Jeonju, 54896, Jeonbuk, Korea

    Dae-Hyuk Kim

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D.H.K. wrote the manuscript; Y.H.K. and S.J.L. performed the experiments; D.H.K. and J.C. analyzed the data.

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Ko, YH., Chun, J., Lee, SJ. et al. Changes in VOCs from a chestnut blight fungus Cryphonectria parasitica by a hypovirus infection: mycoviral infection alters fungal smell for insect vectors. Chem. Biol. Technol. Agric. 11, 123 (2024). https://doi.org/10.1186/s40538-024-00657-y

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