, Rice is one of the staple crops and constitutes a primary source of caloric intake for approximately half of the world’s population (Khush, 2005). The rice blast fungus, Magnaporthe oryzae, causes one of the most destructive diseases in rice by developing an appressorium, a specialized infection structure that can penetrate the plant leaf surface, resulting in a lot of crop losses of 10-30% (Martin-Urdiroz et al., 2016; Talbot, 2003). M. oryzae has two distinct reproductive phases. During asexual reproduction, conidia are produced by vegetative growth and subsequently infect into the plant leaf surface. In sexual reproduction, ascospores are formed by mating of vegetative hyphae, resulting in developing perithecia and ascus (de Jong et al., 1997; Wilson and Talbot, 2009; Zeigler, 1998). In order to efficiently manage rice blast disease caused by this fungal pathogen, its biological characteristics, genetic diversity, and adaptability to control approaches need to be well understood (Dean et al., 2012).
Volatile organic compounds (VOCs) are a mixture of low-molecular-weight compounds that exist in the gas phase and include alcohols, alkanes, aromatics, acids, esters, terpenes, thiols, and others (Kanchiswamy et al., 2015a; Korpi et al., 2009; Schulz and Dickschat, 2007). VOCs are produced by fungi, bacteria, plant, and other organisms (Hung et al., 2015), and are known to play roles in both inter- and intra-organismal interactions, such as fungi-fungi, fungi-bacteria, fungi-plant, bacteria-bacteria, and bacteria-plant interactions (Kanchiswamy et al., 2015a). In recent years, accumulating researches have focused on the use of VOCs as eco-friendly biological control agents against plant pathogens, including bacteria, fungi, nematodes, and insects (Kanchiswamy et al., 2015b; Razo-Belman and Ozuna, 2023; Tilocca et al., 2020).
In addition to the importance of VOCs, various aspects of fungal biology, including sporulation, primary and/or secondary metabolic pathways, stress responses, asexual and sexual developments, circadian clock, and pathogenicity, are regulated by light (Corrochano, 2011; Idnurm and Heitman, 2005; Rodriguez-Romero et al., 2010). Previous studies have reported that asexual development and spore release in M. oryzae are specifically regulated by blue light, whereas these aspects have not been well characterized under red light in the same fungus (Kim et al., 2011; Lee et al., 2006; Rodriguez-Romero et al., 2010; Yu and Fischer, 2019). Another study has revealed that asexual sporulation has been influenced by red light in Aspergillus nidulans (Purschwitz et al., 2008). Although many research groups have focused on elucidating and characterizing the functions of VOCs under light conditions in fungal-fungal and fungal-pathogen interactions, the underlying molecular mechanisms of the VOCs produced by the M. oryzae under light conditions remain poorly understood.
The importance of rice blast disease caused by M. oryzae, together with the potential role of VOCs in plant-fungal and fungal-fungal interactions, has prompted investigation into how VOCs produced by M. oryzae modulate fungal growth. In this study, we investigated whether VOCs produced by M. oryzae-mediated influenced intraspecific and interspecific interactions with other fungal pathogens, and identified VOCs that inhibit fungal growth under light conditions. Our results displayed that a total of eleven compounds were identified by the gas chromatography-mass spectrometry (GC-MS), among which four VOCs (1-hexanol, 2-ethylhexanoyl chloride, 1-octen-3-ol, and 3-octanol) exhibited strong self-inhibition effects. Further analysis revealed deletion of the MoCPKA and MoPMK1 genes reduced the self-inhibitory effects. In addition to self-inhibition effects, M. oryzae-mediated VOCs contributed to the interspecific antagonistic interactions with other fungal species (Aspergillus nidulans, Fusarium oxysporum f.sp. conglutinans, and Sclerotinia sclerotioum). These findings shed light on the functions of VOCs produced by M. oryzae under light conditions and suggest that they enhance growth inhibition both within a species (intraspecific) and among different species (interspecific).
Materials and Methods
Fungal strains and culture condition
Wild-type strains of Magnaporthe oryzae, including KJ201, CP987, Guy11, and 70-15, and knockout mutants, including ΔmagB and Δpmk1, were obtained from the Center for Fungal Genetic Resources (CFGR), South Korea (Supplementary Table 1). Additional knockout mutants, including Δmac1, ΔcpkA, Δplc1, and Δcrz1, were obtained from Sunchon National University, South Korea (Supplementary Table 1). All strains were grown on complete medium agar (CMA; 0.6% yeast extract (w/v), 0.6% casamino acid (w/v), 1% sucrose (w/v) and 1.5% agar powder (w/v)), oatmeal medium agar (OMA; 5% oatmeal (w/v) and 2.2% agar powder (w/v)), and V8 medium agar (V8A; 8% V8 juice (v/v), pH 6.79, and 1.5% agar powder (w/v)) under light or dark conditions at 25°C.
Gas chromatography-mass spectrometry analysis for volatiles produced by M. oryzae
GC-MS analysis was performed to identify volatile compounds produced by M. oryzae that may contribute to growth inhibition. The M. oryzae strain KJ201 was cultured in 5-mL vials containing 1 mL of V8 agar for 1, 2, or 3 days. Volatile compounds accumulated in the headspace were collected using solid-phase microextraction fibers and analyzed by GC-MS. Peaks detected in the fungal samples but absent from the control samples were considered putative fungal-derived volatile compounds. The detected peaks were tentatively identified by comparison with spectra in the NIST library.
I-plate assay for volatiles
To examine the effect of fungal volatiles on mycelial growth, I-plate assays were performed using CMA, OMA, and V8A. A 5-mm-diameter mycelial plug was inoculated into one compartment of an I-plate and incubated at 25°C for 2 days. Thereafter, another 5-mm-diameter mycelial plug was inoculated into the other compartment of the same I-plate. This treatment was designated as the volatile-exposure treatment, referred to as volatile organic compounds-positive (VOCs+) treatment. For the non-volatile control, referred to as VOCs-, a 5-mm-diameter mycelial plug was inoculated into one compartment of a new I-plate without a pre-existing fungal colony. All inoculated I-plates were sealed and incubated at 25°C for 5 days. In the I-plate assay, the pre-existing fungal colony was designated as the sender, whereas the later-inoculated colony was designated the receiver. Colony diameters of the receiver colonies under VOCs- and VOCs+ conditions were measured, and colony areas were calculated using the formula for the area of a circle:
where A represents the colony area and r represents the colony radius. Relative growth was determined by normalizing the colony area under VOCs exposure to that of the VOCs- control. Growth inhibition was calculated using the following formula:
where AVOCs+ represents the colony area under volatile exposure and AVOCs- represents the colony area of the non-volatile control. Three plates were used for each strain, and the experiment was independently repeated three times.
I-plate assay using volatile compounds
To evaluate the effects of selected volatile compounds on fungal growth, a 5-mm-diameter mycelial plug was inoculated into one compartment of an I-plate. The volatile compounds tested in this study were 3-methyl-1-butanol, octane, 1-hexanol, p-xylene, 2-ethylhexanyl chloride, 1-octen-3-ol, 3-octanol, 1-octen-3-yl acetate, and 3-octyl acetate, all of which were purchased from Sigma-Aldrich (St. Louis, MO, USA). Each volatile compound was added to the other compartment at volumes of 100, 50, 25, or 5 μL, corresponding to final concentrations of 3.0, 1.5, 0.8, or 0.2 μL/mL, respectively, based on the internal headspace volume of the I-plate. The I-plates were then sealed and incubated at 25°C for 5 days. The four volatile compounds showing the strongest inhibitory effects were further examined using three plates per treatment in three independent experiments. Colony diameters were measured, and colony areas and growth inhibition percentages were calculated as described above, except that the non-treated control was used as the reference instead of the VOCs- control.
Statistical analysis
All experiments were independently repeated three times, with three plates per treatment in each replicate, unless otherwise stated. Colony area and growth inhibition data were analyzed using linear mixed-effects models or one-way analysis of variance, as appropriate. For I-plate assays comparing VOC- and VOC+ treatments, treatment was used as a fixed effect and biological replicate as a random effect. For comparisons among individual volatile compound treatments, Tukey’s honestly significant difference test was used for post hoc multiple comparisons. Data are shown as means ± standard errors. Statistical significance was defined as P < 0.05, and significance levels are indicated in the figure legends as follows: *P < 0.05, **P < 0.01, ***P < 0.001.
Results
Growth inhibitory effect by volatiles in M. oryzae
To determine any physiological changes of M. oryzae KJ201 growth by volatiles derived from M. oryzae, we conducted I-plate assay that allowed only exchange of VOCs between compartment and the fungus in one compartment of the I-plate cannot directly contact with the other side on various medium (Fig. 1). To confirm the effects of VOCs by this fungus, pre-inoculation was required on medium (left side of I-plate) and subsequently arriving inoculation was conducted in the same I-plate at 2 days after pre-inoculation (Fig. 1A). The tendency of self-inhibition by certain VOCs was consistently repeated in diverse medium (CM, oatmeal, and V8), where the strongest inhibition effect observed on V8 agar medium, indicating VOCs production in M. oryzae may be dependent on the growth medium (Fig. 1B). The quantification levels of fungal growth were approximately 28%, 30%, and 48% reduced in tested groups on CM, oatmeal, and V8 medium, respectively, as compared to controls (Fig. 1C). To assess any CO2 contamination of derived by the fungus, medium, and plastic plate, we measured CO2 levels during fungus inoculation. The CO2 levels were significantly lower in fungus inoculated plates than in dry ice (Supplementary Fig. 1). The self-inhibition effect of fungal growth could be promisingly proved by other strains of M. orzyae (i.e., Guy11, K1215, 70-15, and CP987 [Supplementary Fig. 2]). Taken together, our results indicate that certain VOCs play a role in suppression of fungal growth of late arriving colony.
Identification and effect of VOCs produced by M. oryzae
In order to specifically identify the VOCs produced by M. oryzae, we performed the GC-MS. A total of 11 VOCs such as 3-methyl-1-butanol, octane, 1-hexanol, p-xylene, 2-ethylhexanoyl chloride, (E)-2-hepten-1-ol, 1-octen-3-ol, 3-ocatnol, (Z)-2-octen-1-ol, 1-octen-3-yl-acetate, 3-octanol acetate, and CO2 were detected by the current protocols under our detection ranges of standard (Fig. 2A). Interestingly, most VOCs were detected at 2 days after fungal inoculation, a pattern that could be consistent with the strongest growth inhibitory effect observed in the I-plate assay at the same time point (data not shown).
Among 11 VOCs, nine VOCs were examined using a I-plate system to investigate how they modulated self-inhibition activity (supplementary Fig. 3). After pre-screening, four VOCs (1-hexanol, 2-ethylhexanoyl chloride, 1-octen-3-ol, and 3-ocatnol) were selected for further study. The inhibition morphologies were validated and their inhibition levels of all four VOCs increased in a dose-dependent manner (Fig. 2B, 2C). Therefore, the result indicated that four VOCs (1-hexanol, 2-ethylhexanoyl chloride, 1-octen-3-ol, and 3-ocatnol) may be responsible for self-inhibition effect in this system.
Growth inhibitory effect by VOCs under light and dark conditions
Accumulating evidence suggest that light affects the production of VOCs and secondary metabolites in fungi (Bayram et al., 2008; Rodriguez-Romero et al., 2010). To investigate growth inhibitory activity of M. oryzae by light, the I-plate assay was performed under light and dark conditions as shown in Fig. 3A. As similar to Fig. 1, the late arriving colonies were significantly smaller in the VOCs-producing compartment compared to the control compartment under light conditions. In contrast, this self-inhibition was remarkably diminished under dark conditions (Fig. 3B). These phenotypes were supported by a statistically significant 45% reduction in growth area under light conditions, whereas no such effect was quantitated un the dark (Fig. 3C).
Dependence of growth inhibitory effect on signaling pathways
Evidence support that MoCPKA and MoPMK1are required for turgor pressure generation and appressorium formation (Dean, 1997; Wilson and Talbot, 2009; Xu et al., 1996, 1997). To investigate whether VOCs influence and regulate growth inhibition through these signaling pathways, selected signaling mutants were analyzed as followed above part (Fig. 4A). Compared to control, growth levels of ΔcpkA and Δpmk1 under VOCs exposure conditions (AVOCs+) did not statistically decrease (Fig. 4B, 4C). Lack of CpkA and Pmk1may not affect the growth inhibition under light conditions. To further elucidate how these two genes CpkA and Pmk1 modulate self-inhibitory effect, we designed experiments in which the wild-type strain (70-15) was used as a sender and ΔcpkA and Δpmk1 mutants as receivers, and vice versa. As shown in Fig. 4D and 4E, regardless of the sender (wild-type or ΔcpkA), the level of growth inhibition in the receiver was not statistically different from that of control. The similar results were constantly observed by the wild-type or Δpmk1 (Fig. 4F, 4G). In contrast, Δplc1, Δcrz1, ΔmagB, and Δmac1 retained VOCs-mediated growth inhibition when tested either as self-paired mutants or in reciprocal sender-receiver combinations with the wild-type strain, indicating that these signaling components are not essential for VOCs production or perception under the conditions tested (Supplementary Fig. 4). These results demonstrate that disruption of the CpkA or Pmk1, but not Plc1, Crz1, MagB, or Mac1, compromises VOCs production, VOCs perception, or both. Taken together, the present study suggests that the CpkA and Pmk1 are involved in VOCs-mediated growth inhibitory activity, and that the VOCs produced may contribute to turgor pressure generation and appressorium formation.
Growth inhibitory effect of other fungi by VOCs derived from M. oryzae
To investigate whether VOCs derived from M. oryzae modulate other fungal species, we performed I-plate bioassays. Consistent with the observed self-inhibitory effects, growth inhibition was generally detected, and the quantification levels were likewise reduced (Fig. 5). Among the tested fungal species, the growth inhibition of Aspergillus nidulans, Fusarium oxysporum f.sp. conglutinans, and Sclerotinia Sclerotioum, when used as receivers, exhibited statistically significant growth inhibition compared with the control (Fig. 5). Our results indicate that VOCs produced by M. oryzae can modulate the growth of other fungi. Overall, these findings suggest M. oryzae-derived VOCs not only affect self-inhibition but also directly mediate interspecific growth inhibition.
Discussion
Rice is one of the most important cereal crops (Khush, 2005); however, its production is severely threatened by the rice blast fungus, Magnaporthe oryzae, resulting in a lot of crop losses of 10-30% (Martin-Urdiroz et al., 2016; Talbot, 2003). To manipulate this disease, previous studies reports that VOCs produced by the antagonistic bacteria inhibit M. oryzae (Johnson et al., 2025; Patel et al., 2021; Sahu et al., 2021; Surovy et al., 2023). A study has demonstrated that the fungal VOC, helvolic acid, emitted by Pichia gulliermondii, exhibits strong antifungal activity by significantly inhibiting the spore germination of M. oryzae (Zhao et al., 2010). Although effective control of M. oryzae-mediated disease is critically important and several VOCs have functioned as potential biological control agents, the functional roles of VOCs produced by M. oryzae itself remain largely unknown. To fill the gap of current knowledge, we identified VOCs produced by M. oryzae and elucidated their roles in regulating both intra- and interspecific interactions. Our results intriguingly demonstrate that four VOCs (1-hexanol, 2-ethylhexanoyl chloride, 1-octen-3-ol, and 3-octanol) exhibited strong self-inhibition effects, and that deletion of the CpkA and Pmk1 genes reduced the self-inhibitory effects. Furthermore, M. oryzae-mediated VOCs contributed to interspecific antagonistic interactions with other fungal species, including Aspergillus nidulans, Fusarium oxysporum f.sp. conglutinans, and Sclerotinia Sclerotioum. Collectively, these new findings suggest that VOC production under light conditions enhances growth inhibition both within a species and among different species.
To confirm and verify the functions of VOCs, the I-plate assay is basically and widely utilized in many research groups, which only allows the exchange of gas components between two physically separated compartments (Farag et al., 2017). To date, the roles of VOCs as major components of fungal growth, plant immunity, and other biological aspects is no longer considered novel (Inamdar et al., 2020; Morath et al., 2012; Razo-Belmán et al., 2023). The reasons why VOCs produced by M. oryzae inhibit fungal growth both within the same species and across different species need to be addressed. First, a pre-existing colony (sender) may recognize a late coming colony (receiver) as a competitor even within the same species, thereby contributing to the preservation of its ecological niche under spatially restricted conditions. Second, M. oryzae may produce the VOCs as a potential mechanism to regulate overall population density, eventually preventing nutrient depletion and starvation. Third, as shown in Fig. 3, VOC activity is enhanced under light conditions, which may represent an evolutionary adaptation to regulate competition during spore dispersal. Collectively, the three hypotheses proposed in this study provide plausible explanations for the observed phenomena of self-inhibition and interspecies antagonism.
Among the identified VOCs, four VOCs, including 1-hexanol, 2-ethylhexanoyl chloride, 1-octen-3-ol, and 3-octanol, are shown to regulate self-inhibitory activity (Fig. 2). This VOCs-mediated physiological response in M. oryzae is consistent with previous findings. For examples, Sclerotinia sclerotiorum produces 3-methyl-1-butanol, which inhibits phytopathogen (Fialho et al., 2011). Similarly, 1-hexanol has shown to inhibit fungal growth while promoting plant growth at low concentrations, although it can suppress plant growth at higher concentrations (Splivallo et al., 2007). Furthermore, 1-Octen-3-ol, commonly referred to as mushroom alcohol, has been shown to inhibit various fungal pathogens (Herrera et al., 2015) and to induce plant defense responses, including the upregulation of defense-related genes and suppression of necrotic lesion expansion (Morath et al., 2012). Likewise, 3-Ocatnol also exhibits antifungal activity against various fungal pathogen (Herrera et al., 2015). Although we identify VOCs produced by M. oryzae, and elucidate their underly mechanisms of inhibitory effect, further analyses are still required to fully validate and extend the findings of the present study. First, the knock-out mutants of blue and/or red light receptors should to be analyzed to validate the possibility that specific wavelengths regulate growth inhibition. Second, the roles of VOCs produced by M. oryzae should be validated on plant surfaces. Upon obtaining these new data, we speculate that M. oryzae-derived VOCs may play distinct roles in antifungal activity against competing pathogens, as well as in modulating its own physiology and inducing responses in host plants.
PDF Links
PubReader
ePub Link
Full text via DOI
Download Citation
Supplement1
Print
