Fungal Volatiles Research Articles

Spontaneous vinification supports different microbiota, volatilome and leads to wines with different sensory attributes compared to vinifications inoculated with commercial and indigenous to vidiano cultivar Saccharomyces cerevisiae

Commercial Saccharomyces cerevisae yeasts are still the cornerstone of wine-making. However, use of indigenous S. cerevisae strains or spontaneous fermentations are gaining grounds. Still, we have limited understanding of the effects of S. cerevisiae strains on the composition and succession of vinification microbiota and its cascading effects on wine characteristics. We hypothesized that commercial or indigenous S. cerevisiae strains would lead to different vinification microbiota, volatiles, physicochemical and sensory attributes of wines. To verify this we employed vinifications inoculated with indigenous (W12, W13, W17) or commercial S. cerevisiae (GVS107, SH12) and one spontaneous (S). Amplicon sequencing and (GTG)5 analysis showed that fungal inocula dominated the inoculated vinifications, whereas the spontaneous supported a diverse community of Saccharomyces and non-Saccharomyces (NS) yeasts. The bacterial community didn't vary between vinifications, dominated by acetic acid bacteria (AAB), Burkholderia, Asinibacterium and Sphingomonas. The spontaneous fermentation showed a distinct volatilome compared to inoculated fermentations which didn't differ. The distinct fungal microbiota and volatiles in spontaneous vinification were reflected in the distinct oenological and sensory characteristics (nuts, herbaceous) of the wine compared to inoculated vinifications (fruits, blossoms). Saccharomyces were the key mediators of the volatilome in the inoculated fermentations, compared to spontaneous, where NS yeasts moderated volatiles.

Volatiles from nutritional fungal symbiont influence the attraction of Anisandrus maiche (Coleoptera: Curculionidae) to ethanol-baited traps.

Anisandrus maiche Stark (Coleoptera: Curculionidae: Scolytinae) is a non-native ambrosia beetle from central Asia that has been spreading throughout the eastern United States since 2005. Preferred hosts of A. maiche are not well characterized within its currently invaded range, but it is established in managed and natural forests throughout Indiana. Current monitoring and detection efforts for this beetle rely on ethanol-baited traps, but fungal volatiles may alter the attraction of A. maiche to ethanol. In this study, we conducted trapping experiments in Indiana to determine the extent to which a suite of common fungal alcohols influences the response of A. maiche to ethanol-baited traps. We then evaluated isoamyl and isobutyl alcohol as potential attractants for A. maiche and their ability to enhance attraction to ethanol. Lastly, we used SPME-GC-MS to identify volatiles from Ambrosiella cleistominuta (Mayers & Harr.), the fungal symbiont of A. maiche, grown for 7 and 14 days on malt extract agar. Benzyl alcohol, isobutyl alcohol, hexanol, methyl phenylacetate, phenethyl alcohol, and piperitone reduced the attraction of A. maiche to ethanol-baited traps in the field. Moreover, adding methyl benzoate and isoamyl alcohol individually to ethanol-baited traps did not further increase A. maiche capture. When paired with ethanol, isoamyl alcohol repelled beetles in the early flight period but did not significantly increase trap capture during the fall flight. These results represent a first step in understanding the role of fungal volatiles in the colonization behavior of A. maiche and may ultimately inform management strategies for this species.

Different genotypes and species of symbiotic fungi mediate the behavioral response of invasive Sirex noctilio fabricius (Hymenoptera: Siricidae).

In northeast China, the invasive woodwasp., Sirex noctilio, attacks Pinus sylvestris var. mongolica Litv and often shares habitat with native Sirex nitobei. Previous research showed that S. noctilio can utilize the volatiles from its symbiotic fungus (A. areolatum IGS-BD) to locate host trees. Consequently, symbiotic fungi (A. areolatum IGS-D and A. chailletii) carried by S. nitobei may influence the behavioral selection of S. noctilio. This study aimed to investigate the impact of fungal odor sources on S. noctilio's behavior in laboratory and field experiments. Our observations revealed that female woodwasps exhibited greater attraction toward the fungal volatiles of 14-day-old Amylostereum IGS-D in a "Y"-tube olfactometer and wind tunnel. When woodwasps were released into bolts inoculated separately with three strains in the field, females of S. noctilio exhibited a preference for those bolts pre-inoculated with A. areolatum IGS-BD. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the volatiles emitted by the two genotypes of A. areolatum were similar yet significantly distinct from those of Ampelopsis chailletii. Hence, we postulate that the existence of native A. areolatum IGS-D could potentially facilitate the colonization of S. noctilio in scenarios with minimal or no A. areolatum IGS-BD present in the host.

Mycorrhizal and pathogenic fungi as bio-elicitors for in vitro production of natural stilbenes in callus and cell suspension cultures of peanut (Arachis hypogaea L.)

Stilbenes, including resveratrol, are prominent bioactive compounds in peanuts widely recognized for their health benefits. However, limited research has focused on the production of resveratrol and its derivatives piceatannol and piceid from Arachis hypogaea. This research aims to study the production of these stilbenes using in vitro systems. Callus cultures have been established but only limited amounts of stilbenes were produced. Biotic elicitors, namely fungal culture filtrate (CF), mycelial extract (ME) and volatiles (VOCs) derived from three fungi (Serendipita indica, Fusarium solani and Alternaria alternata) were investigated in callus cultures. Based on Principal Components Analysis, 5 % v/v CF from A. alternata resulted in a significant increase in piceid production after 8 h. Similarly, 5 % v/v CF from S. indica after 16 h exhibited a notable enhancement in resveratrol and piceatannol levels. Enzyme activity of PAL and TAL was also promoted. Subsequent investigations involved establishing a cell suspension culture using CF from A. alternata and S. indica. In contrast to the callus culture, the levels of resveratrol remained comparatively lower. These results point towards a new method for piceid production using callus culture of peanuts, as well as a potential opportunity to obtain resveratrol and piceatannol via fungal elicitation.

An insect chemosensory protein facilitates locust avoidance to fungal pathogens via recognition of fungal volatiles

Locusts (Locusta migratoria) are one of the most destructive insect pests worldwide. Entomopathogenic fungi can infect and kill locusts, with Metarhizium acridum having evolved as a specialized acridid pathogen. However, locusts have evolved countermeasures to limit or avoid microbial pathogens, although the underlying molecular mechanisms behind these defenses remain obscure. Here, we demonstrate that L. migratoria exhibit avoidance behaviors towards M. acridum contaminated food via recognition of fungal volatiles, with locust perception of the volatile mediated by the LmigCSP60 chemosensory protein. RNAi-knockdown of LmigCSP60 lowered locust M. acridum avoidance behavior and increased infection and mortality. The fungal volatile, 2-phenylethanol (PEA), was identified to participate in locust behavioral avoidance. RNAi-knockdown of LmigCSP60 reduced antennal electrophysiological responses to PEA and impaired locust avoidance to the compound. Purified LmigCSP60 was able to bind a set of fungal volatiles including PEA. Furthermore, reduction of PEA emission by M. acridum via construction of a targeted gene knockout mutant of the alcohol dehydrogenase gene (ΔMaAdh strain) that contributes to PEA production reduced locust avoidance behavior towards the pathogen. These findings identify an olfactory circuit used by locusts to detect and avoid potential microbial pathogens before they are capable of initiating infection and highlight behavioral and olfactory adaptations affecting the co-evolution of host-pathogen interactions.

A predator breeding station for augmentative biological control of scolytine crop pests

The square-necked grain beetle, Cathartus quadricollis (Coleoptera: Silvanidae), is a predator of two significant crop pests in Hawaii: the coffee berry borer, Hypothenemus hampei, in coffee and the tropical nut borer, H. obscurus (Coleoptera: Curculionidae: Scolytinae) in macadamia nut. C. quadricollis is also a stored grain pest and is known to respond to its aggregation pheromone (i.e., quadrilure). Field tests were conducted in a macadamia nut orchard using sticky traps to identify the best lure dispenser and trap color based on captures of adult C. quadricollis. When quadrilure was released in different dispenser types, in combination with a fungal volatile blend released from a pouch, we found that a membrane-type quadrilure release dispenser (wafer) was superior to a red septa, gray septa, or open vial dispenser in attracting C. quadricollis. In subsequent tests, the combination of a quadrilure lure and a lure releasing a blend of fungal volatiles caught more C. quadricollis than the quadrilure lure alone. Also, we found that black sticky traps caught more C. quadricollis than did blue, green, red, yellow, white, or clear sticky traps. A predator breeding station consisting of a screened and sheltered enclosure containing food (250 g of cracked corn: cornmeal [4:1, w:w] mix) and the membrane-type quadrilure lure was developed to augment predator numbers in coffee and macadamia fields. In the laboratory, stocking a breeding station with 100 C. quadricollis resulted in production and dispersal of about 10,000 adults per station over a four-month period at 25 °C. In field tests, use of the quadrilure lure plus a fungal volatiles lure or the quadrilure lure alone were placed in breeding stations in a coffee field to attract naturally occurring wild adults for a five-week period, then returned to the laboratory where they both produced roughly 3,000 adults per station over a five-month period. The predator breeding station was an efficient way to multiply C. quadricollis numbers in the field and could be used to augment biological control of the coffee berry borer in coffee and tropical nut borer in macadamia nut.

Fungal volatiles from green manure-incorporated soils promote the growth of lettuce (Lactuca sativa) and mediate antifungal activity against Fusarium oxysporum in vitro

Fungal volatiles from green manure-incorporated soils promote the growth of lettuce (Lactuca sativa) and mediate antifungal activity against Fusarium oxysporum in vitro

Expression Pattern and Ligand Binding Characteristics Analysis of Chemosensory Protein SnitCSP2 from Sirex nitobei.

Sirex nitobei is an important wood-boring wasp to conifers native to Asia, causing considerable economic and ecological damage. However, the current control means cannot achieve better efficiency, and it is expected to clarify the molecular mechanism of protein-ligand binding for effective pest control. This study analyzed the expression pattern of CSP2 in S. nitobei (SnitCSP2) and its features of binding to the screened ligands using molecular docking and dynamic simulations. The results showed that SnitCSP2 was significantly expressed in female antennae. Molecular docking and dynamic simulations revealed that SnitCSP2 bound better to the host plant volatile (+)-α-pinene and symbiotic fungal volatiles terpene and (-)-globulol than other target ligands. By the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, the free binding energies of the three complexes were calculated as -44.813 ± 0.189 kJ/mol, -50.446 ± 0.396 kJ/mol, and -56.418 ± 0.368 kJ/mol, and the van der Waals energy was found to contribute significantly to the stability of the complexes. Some key amino acid residues were also identified: VAL13, GLY14, LYS61, MET65, and LYS68 were important for the stable binding of (+)-α-pinene by SnitCSP2, while for terpenes, ILE16, ALA25, TYR26, CYS29, GLU39, THR37, and GLY40 were vital for a stable binding system. We identified three potential ligands and analyzed the interaction patterns of the proteins with them to provide a favorable molecular basis for regulating insect behavioral interactions and developing new pest control strategies.

Oviposition substrate location by the invasive woodwasp Sirex noctilio: the combined effect of chemical cues emitted by its obligate symbiont Amylostereum areolatum and different host-tree species.

Sirex noctilio is an invasive forest wasp that affects pines and can result in severe economic losses. The use of semiochemicals offers an opportunity to develop sensitive and specific capturing systems to mitigatenegative impacts. Previous research showed that female S. noctilio would use volatiles emitted by its fungal symbiont, Amylostereum areolatum, but little is known about how these modulate behaviour when combined with pine-wood emissions. Our aim was to understand the relevance of fungal volatiles grown on artificial media and wood from two hosts trees, Pinus contorta and Pinus ponderosa, on behavioural and electroantennographic responses of wasp females. Because background odours can modify an insect's response towards resource-indicating semiochemicals, we propose that the behaviour towards the symbiont (resource) will be modulated by host pine emissions (background odours). Olfactometric assays showed that both host species with fungus were attractive when contrasted against air (P. contorta versus Air, χ2 = 12.19, P < 0.001; P. ponderosa versus Air, χ2 = 20.60, P < 0.001) and suggest a clear hierarchy in terms of female preferences towards the tested stimuli, with response highest towards the fungus grown on P. contorta (olfactory preference index: 5.5). Electrophysiological analyses indicate that females detect 62 volatile compounds from the tested sources. Results indicate a strong synergy between symbiont and host semiochemicals, suggesting that the pine species could play a fundamental role in the interaction. Further understanding of the chemical basis of this, could guide the development of specific and attractive lures, in order to maximize attraction of wasps in surveillance programmes. © 2023 Society of Chemical Industry.

Locally Isolated Trichoderma harzianum Species Have Broad Spectrum Biocontrol Activities against the Wood Rot Fungal Species through Both Volatile Inhibition and Mycoparasitism.

Pathogenic root/wood rot fungal species infect multiple urban tree species in Singapore. There is a need for sustainable and environmentally friendly mitigation. We report the local Trichoderma strains as potential biocontrol agents (BCAs) for pathogenic wood rot fungal species such as Phellinus noxius, Rigidoporus microporus, and Fulvifomes siamensis. Isolated Trichoderma strains were DNA-barcoded for their molecular identities and assessed for their potential as a BCA by their rate of growth in culture and effectiveness in inhibiting the pathogenic fungi in in vitro dual culture assays. Trichoderma harzianum strain CE92 was the most effective in inhibiting the growth of the pathogenic fungi tested. Preliminary results suggested both volatile organic compound (VOC) production and direct hyphal contact contributed to inhibition. SPME GC-MS identified known fungal inhibitory volatiles. Trichoderma harzianum strain CE92 hyphae were found to coil around Phellinus noxius and Lasiodiplodia theobromae upon contact in vitro and were possibly a part of the mycoparasitism. In summary, the work provides insight into Trichoderma inhibition of pathogenic fungi and identifies local strains with good potential for broad-spectrum BCAs against root/wood rot fungi in Singapore.

Fungal, host andnon‐hostvolatiles modify attraction of the walnut twig beetle,Pityophthorus juglandis, to pheromone lures

AbstractThousand Cankers Disease (TCD) of walnut trees is caused by the pathogenic fungusGeosmithia morbidavectored by the walnut twig beetle (WTB)Pityophthorus juglandis. Monitoring efforts for WTB rely on pheromone‐baited traps, but lures are likely effective at attracting beetles only over short distances. Fungal‐derived kairomones may increase the efficacy of current lures, while additional volatiles may repel beetles from valuable trees.The objective of this study was to determine the extent to which fungal, host and non‐host volatiles modify the attraction of WTB to pheromone‐baited traps. A trapping study that combined fungal, host‐associated and non‐host compounds with WTB‐pheromone lures was conducted over three years in black walnut plantations experiencing a TCD outbreak in Walla Walla, WA.Traps baited with pheromone andG. morbidavolatiles (i.e., isoamyl and isobutyl alcohol) consistently attracted more WTB, while other fungal volatiles inconsistently increased attraction compared to those baited with pheromone lure alone. This is the first field study that demonstrates fungal volatiles can increase the attraction of a bark beetle to its pheromone in a hardwood system.One fungal (benzyl alcohol) and two additional volatiles (limonene, piperitone) repelled WTB from pheromone‐baited traps. Although limonene is known to repel WTB, this is the first demonstration that benzyl alcohol and piperitone repel a bark beetle.Fungal volatiles may increase the efficacy of monitoring efforts and may play an important role in management tactics for WTB, especially in detecting the introduction and establishment of nascent populations and protecting trees from colonizing beetles.

Volatiles of fungal cultivars act as cues for host-selection in the fungus-farming ambrosia beetle Xylosandrus germanus.

Many wood-boring insects use aggregation pheromones during mass colonization of host trees. Bark beetles (Curculionidae: Scolytinae) are a model system, but much less is known about the role of semiochemicals during host selection by ambrosia beetles. As an ecological clade within the bark beetles, ambrosia beetles are obligately dependent on fungal mutualists for their sole source of nutrition. Mass colonization of trees growing in horticultural settings by exotic ambrosia beetles can occur, but aggregation cues have remained enigmatic. To elucidate this mechanism, we first characterized the fungal associates of the exotic, mass-aggregating ambrosia beetle Xylosandrus germanus in Southern Germany. Still-air olfactometer bioassays documented the attraction of X. germanus to its primary nutritional mutualist Ambrosiella grosmanniae and to a lesser extent another common fungal isolate (Acremonium sp.). During two-choice bioassays, X. germanus was preferentially attracted to branch sections (i.e., bolts) that were either pre-colonized by conspecifics or pre-inoculated with A. grosmanniae. Subsequent analyses identified microbial volatile organic compounds (MVOCs) that could potentially function as aggregation pheromones for X. germanus. To our knowledge, this is the first evidence for fungal volatiles as attractive cues during host selection by X. germanus. Adaptive benefits of responding to fungal cues associated with an infestation of conspecifics could be a function of locating a suitable substrate for cultivating fungal symbionts and/or increasing the likelihood of mating opportunities with the flightless males. However, this requires solutions for evolutionary conflict arising due to potential mixing of vertically transmitted and horizontally acquired symbiont strains, which are discussed.

A new system to study directional volatile-mediated interactions reveals the ability of fungi to specifically react to other fungal volatiles

Microbes communicate with each other using a wide array of chemical compounds, including volatile organic compounds (VOCs). Usually, such volatile-mediated interactions are studied by growing two different microbes in a shared, confined environment and by subsequently collecting and analyzing the emitted VOCs by gas chromatography. This procedure has several drawbacks, including artificial volatile overaccumulation and potential oxygen limitation, as well as the impossibility to assign a producer to the compounds newly emitted during the interaction. To address these challenges, we have developed a novel system specifically designed to analyze volatile-mediated interactions allowing for sequential unidirectional exposure of a “receiver” microorganism to the VOCs of an “emitter” microorganism. Using hermetically sealed systems connected to an air compressor, a constant unidirectional airflow could be generated, driving emitted volatiles to be absorbed by a collection charcoal filter. Thus, our developed system avoids artificial overaccumulation of volatile compounds and lack of oxygen in the headspace and enables the univocal assignment of VOCs to their producers. As a proof of concept, we used this newly developed experimental setup to characterize the reaction of plant growth-promoting and biocontrol fungus (Trichoderma simmonsii) to the perception of VOCs emitted by two plant pathogens, namely Botrytis cinerea and Fusarium oxysporum. Our results show that the perception of each pathogen's volatilome triggered a specific response, resulting in significant changes in the VOCs emitted by Trichoderma. Trichoderma's volatilome modulation was overall stronger when exposed to the VOCs from Fusarium than to the VOCs from Botrytis, which correlated with increased siderophore production when co-incubated with this fungus. Our newly developed method will not only help to better understand volatile-mediated interactions in microbes but also to identify new molecules of interest that are induced by VOC exposure, as well as the putative-inducing signals themselves.

Semiochemicals produced by fungal bark beetle symbiont Endoconidiophora rufipennis and the discovery of an anti-attractant for Ips typographus.

Bark beetles vector symbiotic fungal species into their host trees during mass attacks. The symbiotic relationship with blue stain fungi of the Ascomycetes, including genera of Endoconidiophora (syn. = Ceratocystis), promotes successful establishment whereby the microbes help to overcome the host trees' defence and degrade toxic resins. This is the first study to evaluate both the volatile emissions from an insect-associated blue stain fungus over time and the insect response in a field trapping experiment. Volatile emissions from isolates of Endoconidiophora rufipennis (ER) were collected by solid-phase microextraction (SPME) and analysed by gas chromatography-mass spectroscopy (GC-MS) over a period of 30 days. This virulent North American fungus is closely related to E. polonica, a symbiotic fungus known from Eurasian spruce bark beetle Ips typographus.Nine volatiles were emitted by ER in substantial amounts: isoamyl acetate, sulcatone, 2-phenethyl acetate, geranyl acetone, geranyl acetate, citronellyl acetate, (R)- and (S)-sulcatol, and (R)-sulcatol acetate. A late peaking compound was geranyl acetone. In the field trapping experiment, three of the fungal volatiles (geranyl acetone, 2-phenethyl acetate and sulcatone) were tested in combination with a synthetic aggregation pheromone for I. typographus. Traps with geranyl acetone attracted lower numbers of I. typographus compared to traps with 2-phenethyl acetate, sulcatone or the pheromone alone as a control. The results showed that geranyl acetone acts as an anti-attractant and may act naturally on I. typographus as a cue from an associated fungus to signal an overexploited host.

Fungal volatiles have physiological properties

All fungi emit mixtures of volatile organic compounds (VOCs) during growth. The qualitative and quantitative composition of these volatile mixtures vary with the species of fungus, the age of the fungus, and the environmental parameters attending growth. In nature, fungal VOCs are found as combinations of alcohols, aldehydes, acids, ethers, esters, ketones, terpenes, thiols and their derivatives, and are responsible for the characteristic odors associated with molds, mushrooms and yeasts. One of the single most common fungal volatiles is 1-octen-3-ol also known as “mushroom alcohol” or "matsutake alcohol.” Many volatiles, including 1-octen-3-ol, serve as communication agents and display biological activity as germination inhibitors, plant growth retardants or promoters, and as semiochemicals (“infochemicals”) in interactions with arthropods. Volatiles are understudied and underappreciated elements of the chemical lives of fungi. This review gives a brief introduction to fungal volatiles in hopes of raising awareness of the physiological importance of these gas phase fungal metabolites to encourage mycologists and other biologists to stop “throwing away the head space.”

Mutational Analysis of Aspergillus fumigatus Volatile Oxylipins in a Drosophila Eclosion Assay.

Aspergillus fumigatus is a ubiquitous opportunistic pathogen. We have previously reported that volatile organic compounds (VOCs) produced by A. fumigatus cause delays in metamorphosis, morphological abnormalities, and death in a Drosophila melanogaster eclosion model. Here, we developed A. fumigatus deletion mutants with blocked oxylipin biosynthesis pathways (∆ppoABC) and then exposed the third instar larvae of D. melanogaster to a shared atmosphere with either A. fumigatus wild-type or oxylipin mutant cultures for 15 days. Fly larvae exposed to VOCs from wild-type A. fumigatus strains exhibited delays in metamorphosis and toxicity, while larvae exposed to VOCs from the ∆ppoABC mutant displayed fewer morphogenic delays and higher eclosion rates than the controls. In general, when fungi were pre-grown at 37 °C, the effects of the VOCs they produced were more pronounced than when they were pre-grown at 25 °C. GC-MS analysis revealed that the wild-type A. fumigatus Af293 produced more abundant VOCs at higher concentrations than the oxylipin-deficient strain Af293∆ppoABC did. The major VOCs detected from wild-type Af293 and its triple mutant included isopentyl alcohol, isobutyl alcohol, 2-methylbutanal, acetoin, and 1-octen-3-ol. Unexpectedly, compared to wild-type flies, the eclosion tests yielded far fewer differences in metamorphosis or viability when flies with immune-deficient genotypes were exposed to VOCs from either wild-type or ∆ppoABC oxylipin mutants. In particular, the toxigenic effects of Aspergillus VOCs were not observed in mutant flies deficient in the Toll (spz6) pathway. These data indicate that the innate immune system of Drosophila mediates the toxicity of fungal volatiles, especially via the Toll pathway.

Bark Beetles Utilize Ophiostomatoid Fungi to Circumvent Host Tree Defenses.

Bark beetles maintain symbiotic associations with a diversity of microbial organisms, including ophiostomatoid fungi. Studies have frequently reported the role of ophiostomatoid fungi in bark beetle biology, but how fungal symbionts interact with host chemical defenses over time is needed. We first investigated how inoculations by three fungal symbionts of mountain pine beetle affect the terpene chemistry of live lodgepole pine trees. We then conducted a complimentary laboratory experiment specifically measuring the host metabolite degradation by fungi and collected the fungal organic volatiles following inoculations with the same fungal species on lodgepole pine logs. In both experiments, we analyzed the infected tissues for their terpene chemistry. Additionally, we conducted an olfactometer assay to determine whether adult beetles respond to the volatile organic chemicals emitted from each of the three fungal species. We found that all fungi upregulated terpenes as early as two weeks after inoculations. Similarly, oxygenated monoterpene concentrations also increased by several folds (only in logs). A large majority of beetles tested showed a strong attraction to two fungal species, whereas the other fungus repelled the beetles. Together this study shows that fungal symbionts can alter host defense chemistry, assist beetles in overcoming metabolite toxicity, and provide possible chemical cues for bark beetle attraction.

The Potential of Ectomycorrhizal Fungi to Modulate below and Aboveground Communities May Be Mediated by 1-Octen-3-ol

It is known that ectomycorrhizal (ECM) fungi can modulate below and aboveground communities. They are a key part of belowground communication as they produce a vast array of metabolites, including volatile organic compounds (VOCs) such as 1-octen-3-ol. Here, we tested if the VOC 1-octen-3-ol may be involved in the ECM fungal mechanisms that modulate below and aboveground communities. For that, we conducted three in vitro assays with ECM fungi and the 1-octen-3-ol volatile to (i) explore the effects of mycelium growth of three ECM species, (ii) investigate the impact on the germination of six host Cistaceae species, and (iii) study the impact on host plant traits. The effects of 1-octen-3-ol on mycelium growth of the three ECM species depended on the dose and species: Boletus reticulatus was the most sensitive species to the low (VOC) dose, while T. leptoderma was the most tolerant. In general, the presence of the ECM fungi resulted in higher seed germination, while 1-octen-3-ol resulted in lower seed germination. The combined application of the ECM fungus and the volatile further inhibited seed germination, possibly due to the accumulation of 1-octen-3-ol above the plant species' threshold. Seed germination and plant development of Cistaceae species were influenced by ECM fungal volatiles, suggesting that 1-octen-3-ol may mediate changes in below and aboveground communities.

Expression Pattern, Molecular Docking and Dynamics Simulation Analysis of CSP4 from Sirex nitobei Provides Molecular Basis of CSP Bound to Scent Molecules

Insects stimulate specific behaviors by correctly recognizing scent molecules in the external environment. Sirex nitobei, a wood-boring wasp species native to Asia with a distribution area that includes the Palaearctic and Oriental regions, is a significant pest of conifers. Focusing on the molecular mechanism of protein-ligand binding, this study resolved the tissue expression profile of CSP4 from S. nitobei (SnitCSP4) and probed its binding properties with target ligands using molecular docking and dynamics simulations to verify the odor recognition function of this protein. The open reading frame (ORF) of SnitCSP4 was 396 bp, encoding 131 amino acids. Tissue expression analysis revealed that SnitCSP4 was significantly expressed in female antennae and docking showed that all ligands were bound in hydrophobic cavities and close to many hydrophobic amino acid residues. GLN68 and LEU49 were important amino acid residues for SnitCSP4 to bind various odors, and THR9 was the key ligand-binding site in identifying (-)-globulol in the SnitCSP4. Molecular dynamics verified the docking results, confirming that SnitCSP4 bound well to two sex pheromone molecules, three host plant volatiles, and three symbiotic fungal volatiles, with (Z)-7-heptacosene, (Z)-9-nonacosene, and (-)-globulol binding being the most highly stable. These results mean that SnitCSP4 is critical for insects recognizing scent molecules, providing a favorable molecular basis for regulating the behavioral interactions between S. nitobei and the environment, and offering the possibility of developing new strategies for more environmentally friendly and effective control.

Tris(methylthio)methane produced by Mortierella hyalina affects sulfur homeostasis in Arabidopsis

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC–MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Incorporation of the sulfur from the fungal volatile into plant metabolism was shown by 34S labeling experiments. Under sulfur deficiency, TMTM down-regulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.