Muscodor fengyangensis sp. nov. from southeast China: morphology, physiology and production of volatile compounds

The fungal genus Muscodor was erected on the basis of Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum. It produces a mixture of volatile organic compounds (VOCs) with antimicrobial activity that can be used as mycofumigants. The genus currently comprises five species. Here we describe the isolation and characterization of a new species of Muscodor on the basis of five endophytic fungal strains from leaves of Actinidia chinensis, Pseudotaxus chienii and an unidentified broad leaf tree in the Fengyangshan Nature Reserve, Zhejiang Province, Southeast of China. They exhibit white colonies on potato dextrose agar (PDA) media, rope-like mycelial strands, but did not sporulate. The optimum growth temperature is 25°C. The results of a phylogenetic analysis based on four loci (ITS1-5.8S-ITS2, 28S rRNA, rpb2 and tub1) are consistent with the hypothesis that these five strains belong to a single taxon. All five strains also produce volatile chemical components with antimicrobial activity in vitro, which were different from those previously described for other Muscodor species.     

Molecular and morphological evidence support four new species in the genus Muscodor from northern Thailand

The genus Muscodor comprises fungal endophytes which produce mixtures of volatile compounds (VOCs) with antimicrobial activities. In the present study, four novel species, Muscodor musae, M. oryzae, M. suthepensis and M. equiseti were isolated from Musa acuminata, Oryza rufipogon, Cinnamomum bejolghota and Equisetum debile, respectively; these are medicinal plants of northern Thailand. The new Muscodor species are distinguished based on morphological and physiological characteristics and on molecular analysis of ITS-rDNA. Volatile compound analysis showed that 2-methylpropanoic acid was the main VOCs produced by M. musae, M. suthepensis and M. equiseti. The mixed volatiles from each fungus showed in vitro antimicrobial activity. Muscodor suthepensis had the highest antifungal activity.     

Muscodor albus MOW12 an Endophyte of Piper nigrum L. (Piperaceae) Collected from North East India Produces Volatile Antimicrobials

Muscodor albus MOW12, an endophytic fungus isolated from Piper nigrum in Mawlong, Meghalaya, India, resembles some cultural and hyphal characteristics of previous isolates of Muscodor sp. In addition, it possesses about 99 % similarity in its ITS rDNA with other M. albus isolates and thus is nicely centered within the genetic tree to other Muscodor spp. This xylariaceae fungus effectively inhibits and kills certain plant pathogenic fungi by virtue of a mixture of volatile compounds that it produces. The majority of these compounds were identified by gas chromatography/mass spectrometry as small molecular weight esters, alcohols, and acids. The main ester components of this isolate of M. albus in its volatile mixture are acetic acid, ethyl ester; propanoic acid, 2-methyl-, methyl ester and acetic acid, 2-methylpropyl ester. This appears to be the first report of any M. albus strain from India.     

An Endophytic <Emphasis Type="Italic">Phomopsis</Emphasis> sp. Possessing Bioactivity and Fuel Potential with its Volatile Organic Compounds

An unusual Phomopsis sp. was isolated as endophyte of Odontoglossum sp. (Orchidaceae), associated with a cloud forest in Northern Ecuador. This fungus produces a unique mixture of volatile organic compounds (VOCs) including sabinene (a monoterpene with a peppery odor) only previously known from higher plants. In addition, some of the other more abundant VOCs recorded by GC/MS in this organism were 1-butanol, 3-methyl; benzeneethanol; 1-propanol, 2-methyl and 2-propanone. The gases of Phomopsis sp. possess antifungal properties and an artificial mixture of the VOCs mimicked the antibiotic effects of this organism with the greatest bioactivity against a wide range of plant pathogenic test fungi including: Pythium, Phytophthora, Sclerotinia, Rhizoctonia, Fusarium, Botrytis, Verticillium, and Colletotrichum. The IC₅₀ values for the artificial gas mixture of Phomopsis sp. varied between 8 and 25.65 μl/mL. Proton transfer reaction-mass spectrometry monitored the concentration of VOCs emitted by Phomopsis sp. and yielded a total VOC concentration of ca. 18 ppmv in the head space at the seventh day of incubation at 23°C on PDA. As with many VOC-producing endophytes, this Phomopsis sp. did survive and grow in the presence of the inhibitory gases of Muscodor albus. A discussion is presented on the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship.     

Corynespora cassiicola leaf spot of pawpaw (Carica papaya L.) in Nigeria

Leaf spot of pawpaw is hereby reported for the first time in Nigeria. The symptom is characterized by a papery center surrounded by a yellow halo. The causal organism is Corynespora cassiicola. Ripe fruits and abaxial surfaces of the leaves were significantly more susceptible to infection than unripe fruits and adaxial surfaces of leaves. Growth and sporulation of the fungus on several media was investigated. The organism grew faster on malt-extract agar (MEA) derived media and slowest on potato-dextrose agar (PDA) supplemented with thiamine. Sporulation was highest on Czapek-dox agar (CDA) plus biotin and lowest on PDA and PDA + thiamine. Reasons for increased susceptibility of ripe fruit are discussed.This revised version was published online in October 2005 with corrections to the Cover Date.     

Biocontrol of Rhizoctonia solani AG-2, the causal agent of damping-off by Muscodor cinnamomi CMU-Cib 461

Rhizoctonia solani is a damping-off pathogen that causes significant crop loss worldwide. In this study, the potential of Muscodor cinnamomi, a new species of endophytic fungus for controlling R. solani AG-2 damping-off disease of plant seedlings by biological fumigation was investigated. In vitro tests showed that M. cinnamomi volatile compounds inhibited mycelial growth of pathogens. Among nine solid media tested, rye grain was the best grain for inoculum production. An in vivo experiment of four seedlings, bird pepper, bush bean, garden pea and tomato were conducted. The results indicated that treatment with 30?g of M. cinnamomi inoculum was the minimum dose that caused complete control of damping-off symptoms of all seedlings after one month of planting. The R. solani-infested soil showed the lowest percentage of seed germination. In addition, M. cinnamomi did not cause any disease symptoms. From the results it is clear that M. cinnamomi is effective in controlling R. solani AG-2 both in vitro and in vivo.     

Biocontrol of a root rot of kale by <Emphasis Type="Italic">Muscodor albus</Emphasis> strain MFC2

Pythium ultimum is an oomycetous root rot pathogen that causes significant crop production losses on many crops including kale (Brassica oleracea), an economically important vegetable in Thailand. An endophytic fungus from Thailand designated Muscodor albus MFC2 controlled P. ultimum both in vitro and on kale seedlings grown under outdoor conditions via the production of volatile antibiotics. Ten-day old M. albus MFC2 PDA cultures killed P. ultimum in vitro. Thoroughly mixing three PDA plates of 10-day old M. albus MFC2 into a 500 g mixture of commercial soil and field soil did not adversely affect kale seed germination. The same amount of M. albus MFC2 could restore seedling emergence in P. ultimum inoculated soil to a level close to that of a non-infested control. In addition, M. albus MFC2 did not cause any disease symptoms, but rather seemed to promote the growth of kale in the presence or absence of P. ultimum for up to eight weeks after planting.     

Synergism among volatile organic compounds resulting in increased antibiosis in Oidium sp

Oidium sp. has been recovered as an endophyte in Terminalia catappa (tropical chestnut) in Costa Rica. The volatile organic compounds (VOCs) of this organism uniquely and primarily consist of esters of propanoic acid, 2-methyl-, butanoic acid, 2-methyl-, and butanoic acid, 3-methyl-. The VOCs of Oidium sp. are slightly inhibitory to many plant pathogenic fungi. Previous work on the VOCs of Muscodor albus demonstrated that besides esters of small organic acids, a small organic acid and a naphthalene derivative were needed to obtain maximum antibiotic activity. Thus, the addition of exogenous volatile compounds such as isobutyric acid and naphthalene, 1,1'-oxybis caused a dramatic synergistic increase in the antibiotic activity of the VOCs of Oidium sp. against Pythium ultimum. In fact, at elevated concentrations, there was not only 100% inhibition of P. ultimum but killing as well. In addition, a coculture of Muscodor vitigenus (making only naphthalene) and Oidium sp. plus isobutyric acid produced an additive antibiosis effect against P. ultimum. The biological implications of multiple volatile compounds acting to bring about antibiosis in nature are discussed.     

Harnessing endophytes for industrial microbiology

Endophytic microorganisms exist within the living tissues of most plant species. They are most abundant in rainforest plants. Novel endophytes usually have associated with them novel secondary natural products and/or processes. Muscodor is a novel endophytic fungal genus that produces bioactive volatile organic compounds (VOCs). This fungus, as well as its VOCs, has enormous potential for uses in agriculture, industry and medicine. Muscodor albus produces a mixture of VOCs that act synergistically to kill a wide variety of plant and human pathogenic fungi and bacteria. This mixture of gases consists primarily of various alcohols, acids, esters, ketones and lipids. Artificial mixtures of the VOCs mimic the biological effects of the fungal VOCs when tested against a wide range of fungal and bacterial pathogens. Many practical applications for 'mycofumigation' by M. albus have been investigated and the fungus is now in the market place.     

Muscodor species- endophytes with biological promise

A novel fungal genus is described that produces extremely bioactive volatile organic compounds (VOC’s). The initial fungal isolate was discovered as an endophyte in Cinnamomum zeylanicum in a botanical garden in Honduras. This endophytic fungus was named Muscodor albus because of its odor and its white color. This fungus produces a mixture of VOC’s that are lethal to a wide variety of plant and human pathogenic fungi and bacteria. It also is effective against nematodes and certain insects. The mixture of VOC’s has been analyzed using GC/MS and consists primarily of various alcohols, acids, esters, ketones, and lipids. Final verification of the identity of the VOC’s was carried out by using artificial mixtures of the putatively identified compounds and showing that the artificial mixture possessed the identical retention times and mass spectral qualities as those of the fungal derived substances. Artificial mixtures of the available VOC’s mimicked some but not all of the biological effects of the fungal VOC’s when tested against a wide range of fungal and bacterial pathogens. Other species and isolates of this genus have been found in various tropical forests in Australia, Bolivia, Ecuador, and Thailand. The most recent discovery is Muscodor crispans whose VOCs are active against many plant and human pathogens. Potential applications for “mycofumigation” by members of the Muscodor genus are currently being investigated and include uses for treating plant diseases, buildings, soils, agricultural produce and many more. This report will describe how the fungus was discovered, identified, and found potentially useful to agriculture, medicine and industry.     

Effect of the antifungal activity of oxygenated aromatic essential oil compounds on the white-rot Trametes versicolor and the brown-rot Coniophora puteana

Essential oils and their constituents have a long history of applications as antimicrobial agents, but their use as wood preservatives has not yet been reported. In this study, 22 essential oil phenols, phenol ethers, and aromatic aldehydes have been tested for their antifungal activity against the wood-decaying fungi Trametes versicolor and Coniophora puteana. Minimal inhibitory concentrations of the selected compounds added to potato dextrose agar (PDA) in defined concentrations were determined by a screening test with the agar dilution method. A significant difference in the tolerance of the tested fungi towards the selected compounds was discovered and attributed to the differences in their metabolic characteristics. The influence of the chemical structure of the tested essential oil compounds on their antifungal activity is also discussed.     

Sclerotia formed by citric acid producing strains of Aspergillus niger: Induction and morphological analysis

Some strains of Aspergillus niger have been previously reported to produce sclerotia under certain conditions. Sclerotia are aggregations of hyphae which can act either as survival or as sexual structures in species related to A. niger. In this study, we were able to induce the formation of sclerotia in the progenitor of the industrial citric acid producing strains of A. niger, ATCC 1015, and in pyrG mutants derived from it. Sclerotia can be stably formed by ATCC 1015 on malt extract agar medium supplemented with raisins, showing a spatial differentiation of the fungus dependent on the addition and on the position of the fruits into the medium. On other media, including malt extract agar, pyrG auxotrophs also form abundant sclerotia, while the complementation of this gene reverses this phenotype. Additionally, a macro- and microscopical analysis of the sclerotia is reported. Our results show that the sclerotia formed by A. niger are similar to those formed by other fungi, not only in their morphology but also in their ability to germinate and regenerate the organism.     

Detection of fungal infestations of wood by ion mobility spectrometry

During their growth on wood many fungi produce characteristic volatile organic compounds as secondary metabolites. These microbial volatile organic compounds (MVOCs) can be used as indicators of fungal growth even when such growth is concealed. In order to investigate the formation of these volatile metabolites on building materials, specimens of pine sapwood on agar media colonized by the dry-rot fungus Serpula lacrymans and a mixture of six moulds were examined. MVOCs from this fungal growth were studied over a period of up to half a year by ion mobility spectrometry (IMS) headspace analysis using a sensitive, portable IMS mini-device. The IMS headspace spectra from the growing fungal specimens obtained during this time span are differed from non-incubated wood specimens and indicate the presence of a mixture of MVOCs. The composition and amount of volatile metabolites of the fungi changed during cultivation. This was confirmed by a principal component analysis (PCA). Identification of different MVOCs in the headspace according to drift time and the mobility of ionized gaseous species in reference to GC-MS investigations were proposed. It was concluded that IMS can be used as a rapid and sensitive on-site method to indicate actively growing fungi concealed within wood.     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Isolation and functional characteristics of the fungus Hypoxylon spp. Sj18 with biocontrol potential

A fungus with biocontrol potential was isolated from the roots of hickory trees. The strain named sj18 was classified as a member of the genus Hypoxylon (Hypoxylaceae) after multigene phylogenetic analysis (beta-tubulin gene, internal transcribed spacer, 28S large subunit ribosomal RNA gene, and RNA polymerase II subunit gene). The strain grew well on a PDA with an optimum temperature range between 32 and 34 °C. The fungus had obvious inhibitory effects on Botryosphaeria dothidea, Colletotrichum gloeosporioides, and Gibberella moniliformis in fumigation experiments on solid agar plates. In an inoculation experiment of Chinese cabbage, the fungus was also found to have an obvious repellent effect on cabbage caterpillars. In vitro experiments on Petri dishes showed that the fermentation broth of the sj18 strain could kill 100% of Bursaphelenchus xylophilus within 8 h even if the fermentation broth was diluted 8 times. The inoculation test of Arabidopsis thaliana showed that the fungus could promote the lateral root formation of plants and significantly increase their aboveground biomass. Through the analysis of solid phase microextraction (SPME), it was found that the main volatile components of the fermentation products were azulene 65.39% (61.77% + 3.62%), caryophyllene 7.41%, and eucalyptol 6.83% according to the peak area ratio. Therefore, sj18 can be used as a candidate for the further research and development of biocontrol agents.     

Characterization of lignosulfonate-induced phenol oxidase activity in the atypical white-rot fungus Polyporus dichrous

Polyporus dichrous, a white-rot fungus previously shown to lack phenol oxidase activity when grown on agar media in the presence of a variety of phenolic compounds, was found to exhibit phenol oxidase activity upon aging when grown on a lignosulfonate-containing agar medium. The phenol oxidase activity was compared with that of Trametes versicolor grown under the same conditions, in terms of substrate specificity, pH optimum, and temperature sensitivity. The phenol oxidase activity of P. dichrous was intracellular of tyrosinase type, with a pH optimum around 5.5, and was heat-sensitive, having a half-life of 10 min at 60°C.     

Volatile Emissions from an Epiphytic Fungus are Semiochemicals for Eusocial Wasps

Microbes are ubiquitous on plant surfaces. However, interactions between epiphytic microbes and arthropods are rarely considered as a factor that affects arthropod behaviors. Here, volatile emissions from an epiphytic fungus were investigated as semiochemical attractants for two eusocial wasps. The fungus Aureobasidium pullulans was isolated from apples, and the volatile compounds emitted by fungal colonies were quantified. The attractiveness of fungal colonies and fungal volatiles to social wasps (Vespula spp.) were experimentally tested in the field. Three important findings emerged: (1) traps baited with A. pullulans caught 2750?% more wasps on average than unbaited control traps; (2) the major headspace volatiles emitted by A. pullulans were 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethyl alcohol; and (3) a synthetic blend of fungal volatiles attracted 4,933?% more wasps on average than unbaited controls. Wasps were most attracted to 2-methyl-1-butanol. The primary wasp species attracted to fungal volatiles were the western yellowjacket (Vespula pensylvanica) and the German yellowjacket (V. germanica), and both species externally vectored A. pullulans. This is the first study to link microbial volatile emissions with eusocial wasp behaviors, and these experiments indicate that volatile compounds emitted by an epiphytic fungus can be responsible for wasp attraction. This work implicates epiphytic microbes as important components in the community ecology of some eusocial hymenopterans, and fungal emissions may signal suitable nutrient sources to foraging wasps. Our experiments are suggestive of a potential symbiosis, but additional studies are needed to determine if eusocial wasp–fungal associations are widespread, and whether these associations are incidental, facultative, or obligate.     

Early discrimination of fungal species responsible of ochratoxin A contamination of wine and other grape products using an electronic nose

An electronic nose (e-nose) system using an array of metal oxide sensors (Fox 3000, Alpha MOS) was used to detect and discriminate two ochratoxigenic fungal species, Aspergillus carbonarius (Bain.) Thom and A. niger Van Tieghem, that are responsible for the contamination of wine and other wine grape products, using their volatile production patterns. Two well-known ochratoxigenic strains were used in this study: A. carbonarius A941 and A. niger A75. These strains were grown on three culture media, Czapek Dox modified (CDm) agar, yeast extract sucrose (YES) agar and white grape juice (WGJ) agar, and the volatile organic compounds produced in the headspace by these species were evaluated over periods of 48–120 h. The e-nose system was able to differentiate between the two species within 48 h of growth on YES and WGJ agar using principal component analysis (PCA), which accounted for 99.9% and 97.2% of the data respectively, in principal components 1 and 2, based on the qualitative volatile profiles. This differentiation was confirmed by cluster analysis of data. However, it was not possible to separate these species on CDm agar. Our results show that the two closely related ochratoxigenic species responsible for the contamination of wine and other wine grape products can be discriminated by the use of qualitative volatile fingerprints. This approach could have potential for rapid identification of A. carbonarius and A. niger on wine grape samples, thereby significantly reducing the time of detection of these ochratoxin A producing species.     

CmPEX6, a Gene Involved in Peroxisome Biogenesis,Is Essential for Parasitism and Conidiation by the Sclerotial Parasite Coniothyrium minitans

Coniothyrium minitans is a sclerotial parasite of the plant-pathogenic fungus Sclerotinia sclerotiorum, and conidial production and parasitism are two important aspects for commercialization of this biological control agent. To understand the mechanism of conidiation and parasitism at the molecular level, we constructed a transfer DNA (tDNA) insertional library with the wild-type strain ZS-1. A conidiation-deficient mutant, ZS-1TN22803, was uncovered through screening of this library. This mutant could produce pycnidia on potato dextrose agar (PDA), but most were immature and did not bear conidia. Moreover, this mutant lost the ability to parasitize or rot the sclerotia of S. sclerotiorum. Analysis of the tDNA flanking sequences revealed that a peroxisome biogenesis factor 6 (PEX6) homolog of Saccharomyces cerevisiae, named CmPEX6, was disrupted by the tDNA insertion in this mutant. Targeted gene replacement and gene complementation tests confirmed that a null mutation of CmPEX6 was responsible for the phenotype of ZS-1TN22803. Further analysis showed that both ZS-1TN22803 and the targeted replacement mutants could not grow on PDA medium containing oleic acid, and they produced much less nitric oxide (NO) and hydrogen peroxide (H₂O₂) than wild-type strain ZS-1. The conidiation of ZS-1TN22803 was partially restored by adding acetyl-CoA or glyoxylic acid to the growth media. Our results suggest that fatty acid β-oxidation, reactive oxygen and nitrogen species, and possibly other unknown pathways in peroxisomes are involved in conidiation and parasitism by C. minitans.