Fungal nutrition allocation enhances mutualism with fungus-growing termite

Fungal nodules and aged fungus gardens are products of termite fungiculture systems, and are the diets of termites. To understand the nutrition flow in fungiculture, we quantified the number and mass of fungal nodules produced along with fungus garden maturation and analysed the α-amino acid and fatty acid compositions of fungal nodules, fungus gardens, and termite tissues of a fungus-growing termite, Odontotermes formosanus. 1 g of fungus garden produced 5,148 fungal nodules (∼68.0 mg). Approximately 7.0% of α-amino acids were allocated to the fungal nodules and the rest (∼93.0%) remained in the fungus gardens. The compositions of α-amino acids or fatty acids in aged fungus gardens and fungal nodules were more similar to that of termite tissues than fresh fungus gardens, which supports the idea that termites nutritionally depend on the fungal products. Among the 18 α-amino acids, tryptophan was an essential amino acid and was the only one missing from fresh and aged fungus gardens, but found in fungal nodules at significantly higher concentrations. Hence, termites must consume fungal nodules to obtain tryptophan for survival. Furthermore, the fungus spores incorporated in nodules, were transferred when nodules were ingested by termites. We propose that allocating tryptophan in fungal nodules is crucial to enhance the mutualism between the fungus and termite.     

Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

Parallel evolution of termite‐egg mimicry by sclerotium‐forming fungi in distant termite groups

Among the great diversity of insect–fungus associations, fungal mimicry of termite eggs is a particularly fascinating consequence of evolution. Along with their eggs, Reticulitermes termites often harbour sclerotia of the fungus Fibularhizoctonia sp., called ‘termite balls’, giving the fungus competitor‐free habitat within termite nests. The fungus has evolved sophisticated morphological and chemical camouflage to mimic termite eggs. To date, this striking insect–fungus association has been found in eight temperate termite species, but is restricted to the lower termite genera Reticulitermes and Coptotermes. Here, we report the discovery of a novel type of termite ball (‘Z‐type’) in the subtropical termite, Nasutitermes takasagoensis. Phylogenetic analysis indicated that the Z‐type termite ball is an undescribed Trechisporoid fungus, Trechispora sp., that is phylogenetically distant from Fibularhizoctonia, indicating two independent origins of termite‐egg mimicry in sclerotium‐forming fungi. Egg protection bioassays using dummy eggs revealed that Reticulitermes speratus and N. takasagoensis differ in egg‐size preference. A comparative study of termite ball size and egg‐size preference of host termites showed that both fungi evolved a termite ball size that optimized the acceptance of termite balls as a unit investment. Termite‐egg mimicry by these fungi offers a model case of parallel evolution. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 531–537.     

Do the unique properties of nanometals affect leachability or efficacy against fungi and termites?

Nanotechnology has the potential to affect the field of wood preservation through the creation of new and unique metal biocides with improved properties. This study evaluated leachability and efficacy of southern yellow pine wood treated with copper, zinc, or boron nanoparticles against mould fungi, decay fungi, and Eastern subterranean termites. Results showed that nanocopper with and without surfactant, nanozinc, and nanozinc plus silver with surfactant resisted leaching compared with metal oxide controls. Nearly all nanoboron and boric acid was released from the treated wood specimens during leaching. Mould fungi were moderately inhibited by nanozinc oxide with surfactant, but the other nanometal preparations did not significantly inhibit mould fungi. Mass loss from Gloeophyllum trabeum was significantly inhibited by all copper preparations, while Antrodia sp. was not inhibited by nanometal treatments. Nanometals imparted high resistance in southern yellow pine to the white-rot fungus, Trametes versicolor. Unleached specimens treated with nanoboron or nanozinc plus surfactant caused 100% and 31% mortality, respectively. All specimens treated with nanozinc or nanozinc plus silver inhibited termite feeding, but the copper treatments were less effective against termites. Nanozinc possessed the most favorable properties: leach resistance, termite mortality, and inhibition of termite feeding and decay by the white-rot fungus.     

Blockboard with boron-treated veneers: laboratory decay and termite resistance tests

This study is evaluated by measuring the weight loss, and the ability of white- and brown-rot fungi and termites to attack untreated and boron-treated blockboard manufactured using untreated fir (Abies bornmulleriana M.) strips sandwiched between Ekaba (Tetraberlinia bifoliolata Harms.) veneers at final assembly. The veneers were treated with either boric acid or disodium octoborate tetrahydrate, or mixtures of these chemicals, and blockboard specimens were subjected to fungal decay resistance tests performed according to the Japanese Industrial Standard (JIS) A-9201 method using the brown-rot fungus Fomitopsis palustris (Berkeley et Curtis) Murrill and the white-rot fungus, Trametes versicolor (L. ex Fr.) Quel. Blockboard specimens were also tested against the subterranean termite Coptotermes formosanus Shiraki to determine termite resistance. Blockboard specimens with boron-treated veneers demonstrated increased durability against decay fungi and termite attack. However, sealing of untreated fir strips before decay resistance tests helped reduce significantly the weight losses in blockboard with either untreated or boron-treated veneers. Although blockboard is usually used in indoor applications, incorporation of boron-based biocides may be required for increasing resistance to fungal decay and termite attack and giving fire retardancy.     

Discovery of the termitophilous subfamily Termitoxeniinae (Diptera: Phoridae) in Japan,with description of a new genus and species

The subfamily Termitoxeniinae is discovered in Nansei‐shotô, Japan and the following species are recorded, described and keyed: Clitelloxenia formosana Shiraki 1925 , Horologiphora sinensis Disney 1997 , Pseudotermitoxenia nitobei Shiraki 1925 , and Selenophora shimadai Maruyama et Disney, gen. et sp. nov. Brevrostrophora Disney 1997 and B. fuscoterga Disney 1997 are synonymized with Pseudotermitoxenia Shiraki 1925 and P. nitobei, respectively. These species were collected from the fungus garden of the fungus growing termite Odontotermes formosanus Shiraki on the islands of Ishigaki‐jima and Iriomote‐jima of Nansei‐shotô, Japan.     

Comparison of gut-associated and nest-associated microbial communities of a fungus-growing termite (Odontotermes yunnanensis)

Abstract The digestion of cellulose by fungus-growing termites involves a complex of different organisms, such as the termites themselves, fungi and bacteria. To further investigate the symbiotic relationships of fungus-growing termites, the microbial communities of the termite gut and fungus combs of Odontotermes yunnanensis were examined. The major fungus species was identified as Termitomyces sp. To compare the micro-organism diversity between the digestive tract of termites and fungus combs, four polymerase chain reaction clone libraries were created (two fungus-targeted internal transcribed spacer [ITS]– ribosomal DNA [rDNA] libraries and two bacteria-targeted 16S rDNA libraries), and one library of each type was produced for the host termite gut and the symbiotic fungus comb. Results of the fungal clone libraries revealed that only Termitomyces sp. was detected on the fungus comb; no non-Termitomyces fungi were detected. Meanwhile, the same fungus was also found in the termite gut. The bacterial clone libraries showed higher numbers and greater diversity of bacteria in the termite gut than in the fungus comb. Both bacterial clone libraries from the insect gut included Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, Nitrospira, Deferribacteres, and Fibrobacteres, whereas the bacterial clone libraries from the fungal comb only contained Firmicutes, Bacteroidetes, Proteobacteria, and Acidobacteris.     

Evaluation of mold,decay and termite resistance of pine wood treated with zinc- and copper-based nanocompounds

In this work, the resistance of black pine wood (Pinus nigra L.) vacuum-treated with zinc oxide, zinc borate and copper oxide nanoparticles against mold and decay fungi and the subterranean termites was evaluated. Some of the nanocompounds tested were forced with acrylic emulsions to avoid leaching. Results showed that mold fungi were slightly inhibited by nanozinc borate, while the other nanometal preparations did not inhibit mold fungi. Mass loss from fungal attack by Trametes versicolor was significantly inhibited by the zinc-based preparations, while the brown-rot fungus, Tyromyces palustris was not inhibited by the nanometal treatments. Notably, nanozinc borate plus acrylic emulsion imparted very high resistance in pine wood to the white-rot fungus, T. versicolor with a mass loss of 1.8%. Following leaching, all pine specimens treated with nanozinc borate, with or without acrylic emulsion, strongly inhibited termite feeding, i.e. mass losses varying at 5.2–5.4%. In contrast, the copper-based treatments were much less effective against the subterranean termites, Coptotermes formosanus. In general, nanozinc borate possessed favorable properties, that is, inhibition of termite feeding and decay by T. versicolor.     

Haradamyces foliicola anam. gen. et sp. nov., a cause of zonate leaf blight disease in Cornus florida in Japan

A fungus causing zonate leaf blight diseases in various evergreen and deciduous woody plant species in Japan was characterized by a discoid multicellular propagule arising from a hyaline sclerotium-like structure in the leaf tissue and dark-coloured microconidia produced enteroblastically from the terminal cells on the surface of the discoid propagules. Myrioconium-like microconidiophores also producing microconidia were occasionally produced in culture. No teleomorphic characteristics were observed on the fungus. Molecular analysis based on the partial nu-rDNA sequence data revealed that the fungus was phylogenetically related to the Sclerotiniaceae, Leotiomycetes, and Ascomycota. Because the morphology and sequence data of this fungus does not coincide with those of any known anamorphic fungi, Haradamyces foliicola is proposed here as a new anamorphic genus and species for this fungus.     

Low temperature enhances the ability of the termite-egg-mimicking fungus Athelia termitophila to compete against wood-decaying fungi

A unique symbiosis exists between subterranean termites and the sclerotium-forming fungus Athelia termitophila, which forms termite-egg-mimicking sclerotia called ‘termite balls’. While the sclerotia gain a competitor-free habitat by being harboured by termite eggs, A. termitophila mycelia have to compete with wood-decay fungi in the life stage without termites. To understand its relationship with termites, the factors that affect the ability of A. termitophila to compete with other wood-decay fungi must be clarified. Here, we show that A. termitophila is competitive against other wood-decay fungi at low temperatures. In Petri dish experiments to evaluate the effects of the physicochemical conditions, that A. termitophila experiences in termite nests, on its competitive ability, A. termitophila overcame surrounding fungi in the winter, when termites are less active. Further studies quantifying the effects of A. termitophila on termites in winter will help us to understand this relationship.     

The life cycle of Hymenoscyphus fraxineus on Manchurian ash,Fraxinus mandshurica,in Japan

Hymenoscyphus fraxineus causes a lethal disease known as “ash dieback” in the common ash, Fraxinus excelsior, in Europe. It is hypothesized that the fungus originated from East Asia. This fungus is found on the leaf litter of the Manchurian ash, Fraxinus mandshurica, in Japan and is reported to produce apothecia on pseudosclerotial plates formed mainly on decomposing rachises. However, dieback disease has not been reported in Japan, and little is known about the life cycle of H. fraxineus. This study was conducted to explore the behavior and life cycle of this fungus. It was revealed that, after infection by ascospores, H. fraxineus endophytically inhabits the living leaves of F. mandshurica. On fallen leaves, the fungus behaves saprophytically, producing apothecia on pseudosclerotial plates formed mainly on the decomposing rachises. Analysis by real-time quantitative polymerase chain reaction (qPCR) revealed that the amount of H. fraxineus DNA sharply increased in rachises, while such sharp increase of DNA was not found in leaflets.     

Taxonomy of an anamorphic xylariaceous fungus from a termite nest found together with <Emphasis Type="Italic">Xylaria angulosa</Emphasis>

Two xylariaceous fungi were isolated from a nest of a termite, Odontotermes formosanus, that was incubated in a laboratory after collecting from Iriomote Is., Okinawa Pref., in Japan. One of the two fungi was identified as Xylaria angulosa on the basis of the morphology of branched stroma produced on medium, tiny asci, and ascospores having a germ slit. Another fungus is an anamorphic fungus that produces synnemata up to 50 mm long from which dendritic conidiophores branch out. Unicellular conidia are holoblastically produced on a sympodially proliferating conidiogenous cell. Such morphological characters resemble those of the genus Geniculosporium. However, its distinctive synnema formation and dendritic conidiophores do not assign the fungus to Geniculosporium or other known genera and warrant establishment of a new genus. The phylogenetic tree based on the ITS regions of rDNA shows that the fungus is nested in the cluster of the genus Nemania (Xylariaceae), whose species have mainly Geniculosporium-like anamorphs. We describe here the present anamorphic fungus as Geniculisynnema termiticola gen. et sp. nov., and discuss its phylogenetic and ecological relationships to xylariaceous fungi, especially termiticolous species.     

Decay and termite resistance of medium density fiberboard (MDF) made from different wood species

Medium density fiberboard (MDF) production worldwide is increasing due to the development of new manufacturing technologies. As a result, MDF products are increasingly utilized in traditional wood applications that require fungal and insect resistance. This study evaluated the ability of white and brown rot fungi and termites to decompose MDF consisting of different wood species by measuring weight loss. Furnish in the boards was prepared from heart and sapwood portions of pine (Pinus nigra Arnold var. pallasiana), beech (Fagus orientalis Lipsky), and European oak (Quercus robur L.) species. Fungal decay resistance tests were performed according to ASTM D 2017-81 standard method using two brown-rot fungi, Gloeophyllum trabeum (Pers. ex Fr.) Murr. (Mad 617), Postia placenta (Fries) M. Larsen et Lombard (Mad 698), and one white-rot fungus, Trametes versicolor (L. ex Ft.) Pilat (Mad 697). MDF and wood specimens were also bioassayed against the eastern subterranean termite, Reticulitermes flavipes (Kollar) in order to determine termite resistance of the specimens. MDF specimens containing oak and mixed furnish demonstrated increased durability against decay fungi. Only pine, oak, and mixed MDF specimens met the 25% or less weight loss limit to be classified resistant according to ASTM D 2017-81 standard method. Overall, MDF specimens made from oak showed better performance than oak solid wood specimens. Accelerated aging according to ASTM D 1037-96a standard method before fungal bioassay decreased fungal resistance of the specimens. In contrast to the fungal bioassay, MDF specimens made from beech and mixed furnish showed decreased weight losses from termite attack after 4 weeks. However, none of the MDF specimens were resistant to termite attack. In severe conditions, the MDFs may require the incorporation of chemical biocides prior to board production for increasing the resistance of MDF to termite attack.     

Distribution patterns of four Termitomyces species cultivated by a fungus-growing termite,Odontotermes formosanus,in Taiwan

Fungus-growing termites inoculate the obligate symbiotic fungus Termitomyces into the fungus comb in their colonies. In this study, Taiwanese Termitomyces species were determined by diagnostic PCR using the metagenome of the body of Odontotermes formosanus (the only host termite living in Taiwan) as a template. Phylogenetic analyses revealed that four different Termitomyces species are cultivated by O. formosanus in Taiwan. Three have previously been registered in the DNA database, but one was first recorded in Taiwan. Only Termitomyces sp. Type C was distributed in all areas investigated in Taiwan, whereas the other three species were distributed regionally. Field observations indicated that the flush period and the number of fruit bodies in each colony varied between species. The distribution patterns of Termitomyces spp. in Taiwan may be related to the Taiwanese climate and/or the fruiting pattern.     

Field Evaluations of Subterranean Termite Preference for Sap-Stain Inoculated Wood

Few studies have focused on interactions between subterranean termites and the ophiostomatoid fungal associates of pine bark beetles or root feeding weevils. Field stake tests were employed at four locations throughout Mississippi to determine the feeding preference of subterranean termites for blue-stained, unstained, and partially decayed southern pine sapwood stakes. This study also utilized wood decayed by Gloeophyllum trabeum, a fungus previously shown to elicit a positive subterranean termite feeding response, as a positive control. Stakes inoculated with G. trabeum received significantly more attacks than all other treatments after 16 weeks. Of the stakes attacked by subterranean termites, stakes inoculated with Ophiostoma minus were degraded faster than any other treatment. Subterranean termite preference for stakes treated with either of two Leptographium spp. and the untreated negative controls did not differ; however, each was fed upon less than all other treatments. The feeding rate on stakes inoculated with O. ips and G. trabeum being fed upon by subterranean termites was not significantly different. These results represent the first evidence of wood containing non-structurally degrading fungi (O. ips and O. minus) eliciting a feeding preference from subterranean termites greater than that of decayed wood. The implications of these results are particularly relevant to pine forest ecology, nutrient cycling, subterranean termite control, and the utilization of blue-stained southern pine building products in the southeastern U.S.     

Identifying the core microbial community in the gut of fungus‐growing termites

Gut microbes play a crucial role in decomposing lignocellulose to fuel termite societies, with protists in the lower termites and prokaryotes in the higher termites providing these services. However, a single basal subfamily of the higher termites, the Macrotermitinae, also domesticated a plant biomass‐degrading fungus (Termitomyces), and how this symbiont acquisition has affected the fungus‐growing termite gut microbiota has remained unclear. The objective of our study was to compare the intestinal bacterial communities of five genera (nine species) of fungus‐growing termites to establish whether or not an ancestral core microbiota has been maintained and characterizes extant lineages. Using 454‐pyrosequencing of the 16S rRNA gene, we show that gut communities have representatives of 26 bacterial phyla and are dominated by Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria and Synergistetes. A set of 42 genus‐level taxa was present in all termite species and accounted for 56–68% of the species‐specific reads. Gut communities of termites from the same genus were more similar than distantly related species, suggesting that phylogenetic ancestry matters, possibly in connection with specific termite genus‐level ecological niches. Finally, we show that gut communities of fungus‐growing termites are similar to cockroaches, both at the bacterial phylum level and in a comparison of the core Macrotermitinae taxa abundances with representative cockroach, lower termite and higher nonfungus‐growing termites. These results suggest that the obligate association with Termitomyces has forced the bacterial gut communities of the fungus‐growing termites towards a relatively uniform composition with higher similarity to their omnivorous relatives than to more closely related termites.     

Isolation and properties of carboxylesterases of the termite gut-associated fungus,Xylaria nigripes. k., and their identity from the host termite,Odentotermes horni. w., mid-gut carboxylesterases

• 1.1. The termite, Odentoiermes horni. W., houses three fungal species, viz. Xylaria nigripes, Termitomyces microcorpus, and Trichoderma (species not identified), in its gut. X. nigripes was found to possess higher esterase activity levels than the other two.
• 2.2. Four esterase enzymes, viz. FE-I, -II, -III and -IV, with pI values 5.1, 5.25, 5.4 and 5.6, respectively, were identified, isolated and purified to apparent homogeneity from the fungus X. nigripes, their biochemical and enzymological properties were determined, and compared with those of the previously characterized host termite mid-gut enzymes, TE-I and -II.
• 3.3. The M_r, ofFE-I and -II was 85.1 kDa and those of FE-III and -IV was 87.5 kDa. However, TE-I and -II were relatively smaller (M_r ~ 78.5 kDa). Each of the fungal enzymes, viz. FE-I to -IV, was a homodimer with subunits associated non-covalently. The subunit M_r, were 42.6 kDa for FE-I and -II, and 43.7 kDa for FE-III and -IV. On the other hand, the termite mid-gut enzymes, TE-I and -II, were also homodimeric, but the subunits were associated covalently (subunit M, = 40 kDa). Immunologically the fungal esterase enzymes, viz. FE-I to -IV, were different from those of the host termite mid-gut esterases, viz. TE-I and -II.
• 4.4. The substrate specificity and inhibitor sensitivity studies classify these enzymes, i.e. FE-I to -IV, as carboxylesterases (EC 3.1.1.1). Steady-state product inhibition kinetics suggested; an ordered release of products, i.e. alcohol followed by acid, and a Uni-Bi kinetic reaction mechanism.
• 5.5. The two preliminary studies, i.e. the confinement of most esterase activity to the gut-tissue free from microorganisms and starvation of termites not leading to complete loss of esterase activity in the gut of the termites, suggested that there may not be any symbiotic relationship between termite, O. horni, and its gut associated microorganisms with regard to ester metabolism. Though the enzymes from the two sources were carboxylesterases, several of their properties were different and hence, they are different entities.

     

Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.     

Phragmidium satoanum,a new rust pathogen of Rosa hirtula in Japan

The causal fungus of a rust disease of Rosa hirtula, endemic to mountainous areas of Fuji-Hakone-Izu National Park, Japan, was thought to be a common species Phragmidium rosae-multiflorae. Continued field observations, morphological examination, and experimental inoculations proved that the fungus produced laterally three-angled aeciospores and urediniospores together with multi-cellular teliospores on the same R. hirtula trees. These morphological features were different from those of P. rosae-multiflorae. The fungus parasitized only R. hirtula. Experimental inoculations and field observations did not prove that R. banksiae, R. laevigata, and R. multiflora supported infection and sporulation of the fungus. Under the field observations, R. multiflora, the most common host of P. rosae-multiflorae, was not proven to harbor the R. hirtula fungus. Therefore, the fungus was concluded to be a species distinct from P. rosae-multiflorae; and a new name, P. satoanum, was proposed for it.     

Determination of volatile organic compounds in the stinkhorn fungus Pseudocolus fusiformis in different stages of fruiting body formation

A stinkhorn fungus was collected from the mountainous area of Yoshida campus, Yamaguchi University, Japan. Morphological characterization and similarity of large subunit ribosomal DNA sequences identified the fungus as Pseudocolus fusiformis. MonoTrap™ was combined with gas chromatography-mass spectrometry (GC-MS) to identify volatile organic compounds (VOCs) emitted from the fungus harvested at different stages of maturity. The main VOCs emitted from the mature fruiting body were 3-methyl-butanol, 4-methyl-phenol, and dimethyl tetrasulfide, while none of these compounds were detected in the egg-shaped state. Volatile sulfur-containing compounds, including dimethyl disulfide, trisulfide and tetrasulfide, which are commonly detected in stinkhorn fungi and truffles, were also emitted from this fungus. Furthermore, results elucidated that most VOCs occurred in the mature stage of Ps. fusiformis (fruiting body with arms fuse). This is the first study reporting VOC production of Ps. fusiformis.