New endophytic isolates of Muscodor albus, a volatile-antibiotic-producing fungus

David Ezra1, W. M. Hess2 and Gary A. Strobel1

1 Department of Plant Sciences, Montana State University, Bozeman, MT 59717, USA
2 Department of Integrated Biology, Brigham Young University, Provo, UT 84602, USA

Correspondence
Gary Strobel
email-uplgs{at}montana.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum, produces a mixture of volatile organic compounds (VOCs) in culture and its spectrum of antimicrobial activity is broad. Using the original isolate of M. albus as a selection tool, it has been possible to find other culturally and biochemically unique wild-type isolates of this organism existing as endophytes in a variety of other plant species, including Grevillea pterifolia (fern-leafed grevillea), Kennedia nigriscans (snake vine) and Terminalia prostrata (nanka bakarra) growing in the northern reaches of the Northern Territory of Australia. Interestingly, none of the new isolates had a culture morphology that was identical to the original isolate, nevertheless each possessed hyphal characteristics that resembled that isolate. Furthermore, their ITS-5·8S rDNA sequences were 96–99 % identical to that of M. albus and the isolates were considered M. albus on the basis of the DNA sequence data. However, the VOCs produced by these new isolates greatly differed in quality from the original strain by virtue of the production of naphthalene, naphthalene, 1,1'-oxybis-, and one or more other compounds. In bioassays with a range of test micro-organisms, including fungi and bacteria, each isolate possessed biological activity but the range of activity was great. Artificial mixtures of some of the VOCs mimicked the effects of the VOCs of the fungus. The value of these observations to the biology and practical uses of M. albus in agriculture and other applications is discussed.


Abbreviations: ITS, internal transcribed spacer; VOC, volatile organic compound


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Several years ago, an unusual endophytic fungus was isolated from Cinnamomum zeylanicum, growing in a rainforest in Honduras (Strobel et al., 2001; Worapong et al., 2001). The fungus lacked spores and spore-producing structures. All attempts to induce spore formation failed and thus initially, this organism could not be readily identified. Nevertheless, the fungus had distinctive whitish hyphae that were rope-like, interwoven and widely varying in diameter. Partial ITS-5·8S and 18S rDNA sequence data obtained on this organism indicated that it was a xylariaceous fungus with no complete molecular identity to any other fungus in the family Xylariaceae. The most notable property of this organism was its ability to produce a mixture of volatile organic compounds (VOCs) that were lethal to a wide variety of human- and plant-pathogenic fungi and bacteria. This mixture of gases consisted primarily of various alcohols, acids, esters, ketones and lipids. Artificial mixtures of the VOCs mimicked the biological effects of the fungal VOCs when tested against a wide range of fungal and bacterial pathogens. Based on the available molecular biological, chemical and structural information, this organism was given a novel genus and species designation – Muscodor albus.

While VOC-producing fungi have been isolated and studied chemically in the past 30–40 years, none have been found that have such a comprehensive spectrum of antimicrobial activity as that of M. albus (Strobel et al., 2001; McAfee & Taylor, 1999). Thus, it was of interest to learn whether other strains, variants or biotypes of this organism exist in nature and if they too possess biological activity via the production of VOCs. It was realized that in order to find other related Muscodor spp., the VOCs of M. albus could be used as a selection tool to effectively eliminate other competing endophytic micro-organisms and allow for only the growth of xylariaceous fungi including such organisms as xylaria, pestalotiopsis, daldinia and muscodor (Daisy et al., 2002a). By this method, other species of muscodor have been isolated from tropical trees and vines and characterized by chemical and molecular biological techniques including Muscodor roseus and Muscodor vitigenus (Worapong et al., 2002; Daisy et al., 2002a, b). More recently, a strain of M. albus was obtained from Myristica fragrans growing in Thailand and it very closely resembled the original culture of M. albus in most chemical, structural and molecular biological characteristics (Sopalun et al., 2003).

The search for new isolates of M. albus or other related organisms was centred in the monsoonal rainforest of the Northern Territory of Australia since this is where the second new species of muscodor was found, namely M. roseus (Worapong et al., 2002). This fungus differed from M. albus in its reddish mycelial coloration and the quality of its volatiles (Worapong et al., 2002). For a contrast in environmental conditions, plants in a temperate region of Australia were also sampled, in this case, the island of Tasmania. As a whole, it appears that the plants of Australia have an enormous diversity of endophytic micro-organisms that make bioactive metabolites (Strobel & Daisy, 2003). After collecting a number of specimens from woody plants in that area and using the VOCs of M. albus as a selection tool, at least seven widely differing isolates of an organism outwardly resembling muscodor were obtained. Each of these isolates was subjected to rigorous chemical analysis of its VOCs, subsequently analysed for its taxonomic position, and finally for its spectrum of biological activity. This report presents data describing these organisms, and how they compare to each other and to the original isolate of M. albus.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Collecting, isolating, culturing and storing M. albus.
The cultures of M. albus isolated and examined in this study were obtained as endophytes from small limbs of a number of tree and vine species in the Northern Territory of Australia. Three species, namely Grevillea pterifolia (fern-leafed grevillea), Kennedia nigriscans (snake vine) and Terminalia prostrata (nanka bakarra), are each native to the Northern Territory, and both the snakevine and nanka bakarra have medicinal uses by the Aborigines in this area, while the fern-leafed grevillea does not have any apparent medicinal applications. Many other plants around the Northern Territory, including Erythrophelum chlorostachys (ironwood), Bambusa arnhemica (native bamboo), Eucalyptus tetrodonta (stringy bark), Brachychiton diversifolus (kurrajong) were also sampled. In addition, many of the common forest trees of Tasmania were also sampled and some of the more notable ones included Acacia dealbata (silver wattle), Nothofagus gunnii (deciduous beech), Eucalyptus coccifera (Tasmanian snow gum) and Dacrydium franklinii (Huon pine).

A culture of M. albus was used as a selection tool to find and isolate other xylariaceous fungi, including the new isolates of M. albus. Potato dextrose agar (PDA) was poured into one quadrant of commercially available Petri plates that have the base half-plate separated into quadrants. Then, into that quadrant on each plate was placed a small plug of agar containing an actively growing culture of M. albus. The other three quadrants of each plate contained water agar. M. albus was incubated for 4 days at 23 °C before exposing the plant tissues being sampled to the VOCs of M. albus arising in the plates. The procedures used to isolate the original culture of M. albus were used to obtain the new isolates. The plant samples were treated with 70 % ethanol before excising the internal plant tissues and placing them onto the other three quadrants of each plate, containing water agar (Strobel et al., 2001; Worapong et al., 2001).

Endophytic fungi growing from the plant tissues, usually after 4–7 days, were then picked and recultured on PDA to determine culture purity. Pure fungi were tested at least three times by exposure to M. albus in order to exclude false positive growth in the initial screening.

The fungi obtained by these methods could best be stored by placing small pieces of agar supporting fungal growth on PDA in 15 % (v/v) glycerol and placing them at –70 °C. The fungi could also be stored after colonizing sterilized grain (including wheat, rye and barley), drying at room temperature and then placing them in sterile vials at –70 °C. The fungi, under these conditions, remained viable for over 2 years.

The cultures were each given a name designation, based on their plant source, as individual isolates of M. albus and deposited as living cultures at –70 °C in the Montana State University Mycological Collection (MONT) (Table 1).


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Table 1. The M. albus isolates and their ITS-5·8S rDNA accession numbers in GenBank and their MSU culture collection deposit number

 
Scanning electron microscopy.
Once fungal isolates were obtained that outwardly resembled M. albus and possessed antimicrobial activity via VOCs, they were initially characterized by photographing and recording their cultural characteristics. Then, scanning electron microscopy was performed on each isolate suspected of being related to Muscodor spp. The fungal preparations were placed into 2 % glutaraldehyde in 0·1 M sodium cacodylate buffer (pH 7·2–7·4) with Triton X, a wetting agent, aspirated for 5 min and left overnight. Then the fixed carnation leaf pieces supporting fungal growth were placed into no. 1 Whatman filter paper packets, made by folding the filter paper over a piece of cork (1·5 cmx1·5 cm). The packets were tied with cotton string and two removable split shot sinkers (approx. 3·25 g each) were attached next to the packets to hold them under the surface of the dehydrating solutions and the liquid CO2 during critical-point drying. The next day they were washed in six 15 min changes of water-buffer, followed by a 15 min change in 10 % ethanol, a 15 min change in 30 % ethanol, a 15 min change in 50 % ethanol and five 15 min changes in 70 % ethanol, and were left overnight or longer in 70 % ethanol. They were then rinsed for 15 min in 95 % ethanol and then for three 15 min changes in 100 % ethanol, followed by three 15 min changes in acetone. The dehydration process was done slowly, to minimize hyphal shrivelling. Ultimately, for scanning electron microscopy the fungal material was critical-point dried, gold sputter-coated and images were recorded with an XL30 ESEM FEG in the high-vacuum mode using the Everhart–Thornley detector.

Fungal DNA isolation.
Each fungal test isolate was grown on PDA in a 9 cm Petri plate for 21 days at 25 °C. The mycelium was scraped directly from the surface of the agar culture and weighed. Nucleic acid (DNA) was extracted using the DNeasy Plant and Fungi Mini Kit (Qiagen) according to the manufacturer's directions. Agarose gel electrophoresis and a UV spectrophotographic system were used to record the data.

Amplification of internal transcribed spacer sequences (ITS) and 5·8S rDNA.
The ITS regions of the tested fungus were amplified using PCR and the universal ITS primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3'). PCR was performed in a 50 µl reaction vial containing 0·1 µg genomic DNA, 10 mM of each primer, 3 mM of the four dNTPs and 0·25 unit Taq polymerase (Novataq) in a 10x Taq buffer (Novataq) containing 500 mM potassium chloride, 15 mM magnesium chloride, 100 mM Tris/HCl (pH 8·8 at 25 °C) 1 % Triton X-100. The following cycle parameters were maintained: 95 °C for 5 min followed by 39 cycles of 45 s at 95 °C, 45 s at 60 °C and 45 s at 72 °C followed by 5 min at 72 °C.

The PCR products were purified and desalted using the QIAquick PCR purification kit (Qiagen). The PCR product was cloned into a pGEM-T easy vector (Promega) according to the manufacturer's instructions.

Transformation and DNA extraction.
Competent cells of E.coli strain DH5{alpha} were prepared by the CCNB80 method as described by Stinson et al. (2003a). Transformation of the cells with DNA was performed according to standard procedures. The transformed cells were plated on LB agar supplemented with 50 µg ampicillin ml–1 (Sigma), in the presence of IPTG and X-Gal for blue/white selection. White single colonies were grown in LB broth and DNA was extracted using a Perfectprep Plasmid Mini (Eppendorf) according to the manufacturer's instructions. Presence of the insert was confirmed by DNA digestion with EcoRI (Promega). Plasmid DNA extracted from the transformants, carrying the insert, was sequenced.

Cycle sequencing of the ITS regions and 5·8S rDNA.
The plasmid inserts were sequenced by the Plant-Microbe Genomics Facility at Ohio State University using an Applied Biosystems 3700 DNA Analyser and BigDye cycle sequencing terminator chemistry and the universal primers T7 and Sp6.

Test fungi and bacteria.
All plant-pathogenic fungi and other organisms used in the bioassay test system were obtained from Drs Don Mathre and Nina Zidack of the MSU Department of Plant Sciences or were a part of the Montana State University collection. All fungi and bacteria were grown on PDA and LB, respectively, at 23 °C prior to testing.

Bioassay test for volatile antimicrobials.
A simple bioassay test system was devised that allows only for volatiles being the causative agents for any microbial inhibition being examined. Potato dextrose agar (PDA) was poured into all quadrants of commercially available Petri plates with the base half-plate separated into quadrants. Then, into one quadrant, a small plug of agar containing an actively growing culture of the tested isolate was placed. The plate was sealed and incubated for 4 days at 23 °C prior to exposure of the test fungi and bacteria to the VOCs of the M. albus isolate arising in the plates. Individual fungi were inoculated in the plate on a 3 mmx3 mmx3 mm plug of agar. Bacteria and Saccharomyces cerevisiae were streaked (1·5 cm streaks) on to the other quadrants of the plate, one organism in each quadrant. The dividing walls in the plate precluded the diffusion of any inhibitory soluble compounds emanating from the M. albus isolate to the organisms being tested. The plate was wrapped with two pieces of Parafilm and incubated at 23 °C. The growth of the yeast and bacteria was judged visually as described by Strobel et al. (2001). The linear growth of the filamentous fungi was measured from the edge of the agar inoculum plugs and compared with growth on a control plate. At the end of the assay, the viability of the each test fungus and bacterium was evaluated by transferring a portion of the original inoculum plug, or an area of the plate that had been streaked, on to a fresh plate of PDA and observing any growth developing within 2–3 days (Strobel et al., 2001). Each bacterium and fungus that was subjected to testing was obtained from a freshly growing culture. In each case, appropriate growth and viability of each organism was noted in the experimental set-up.

Quantitiative and qualitative analyses of VOCs.
The gases in the air space above the M. albus isolate mycelium growing in Petri plates were analysed as described previously (Strobel et al., 2001; Ezra & Strobel, 2003). A solid-phase micro-extraction syringe was used for trapping the fungal volatiles. The fibre material (Supelco) was 50/30 divinylbenzene/carburen on polydimethylsiloxane on a stable flex fibre. The syringe was placed through a small hole drilled in the side of the Petri plate and exposed to the vapour phase for 45 min. The syringe was then inserted into a gas chromatograph (Hewlett Packard 5890 Series II Plus) equipped with a mass-selective detector. A 30 mx0·25 mm i.d. ZB Wax capillary column with a film thickness of 0·50 mm was used for the separation of the volatiles. The column was temperature programmed as follows: 25 °C for 2 min, then increasing to 220 °C at 5 °C min–1. The carrier gas was helium (UltraHigh Purity; local distributor) and the initial column head pressure was 50 kPa. The helium pressure was ramped with the temperature ramp of the oven to maintain a constant carrier gas flow velocity during the course of the separation. Prior to trapping the volatiles, the fibre was conditioned at 240 °C for 20 min under a flow of helium gas. A 30 s injection time was used to introduce the sample fibre into the gas chromatograph. The chromatograph was interfaced to a VG 70E-HF double-focusing magnetic mass spectrometer operating at a mass resolution of 1500. The mass spectrum was scanned at a rate of 0·50 s per mass decade over a mass range of 35–360 Da. Data acquisition and data processing were performed on the VG SIOS/OPUS interface and software package. Initial identification of the unknowns produced by M. albus was made by library comparison using the NIST database.

Comparable analyses were conducted on Petri plates containing only PDA, and the compounds obtained therefrom, mostly styrene, were subtracted from the analyses done on plates containing the fungus. Final identification of the majority of the compounds was done by acquiring or making authentic samples of the tentatively identified compounds and then subjecting them to the same conditions of trapping, separation and mass spectroscopy as described above. However, a number of compounds were not available and thus their identification remains tentative. As a first approximation, the quantitative analysis of each compound found in fungal cultures is based on its relative peak area obtained after GC-MS analysis.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Isolation and identification of endophytic fungi
After exposing the stem tissues of various plant species to the VOCs of M. albus, a number of endophytic fungi appeared on the Petri plates from most plant samples after 4–7 days. These isolates were purified by picking hyphal tips and transferring them to PDA plates. Many of them were shown to be isolates of pestalotiopsis, xylaria and other related species (Daisy et al., 2002a). However, seven fungi appeared that produced odours and they survived repeated exposure to M. albus; these were kept for further examination. In addition, each of these fungi fitted the basic criterion of bearing no spores, like the original muscodor that was isolated (Worapang et al., 2001). Furthermore, each fungus produced odours that resembled those of the original culture of M. albus, which was used in a direct comparison. These sterile odoriferous fungi only appeared in plants obtained from the Northern Territory of Australia and not those from Tasmania. Furthermore, only Grevillea pterifolia (collected at 13° 07' 947'' S, 132° 27' 435'' E), Kennedia nigriscans (collected at 12° 57' 991'' S, 132° 48' 818'' E) and Terminalia prostrata (12° 12' 137'' S, 132° 47' 066'' E) yielded such sterile odoriferous fungi.

Each of the seven fungi was cultured on PDA and the growth patterns recorded (Fig. 1). Each pattern was distinctive from that of the original M. albus (CZ-620) on the basis of its cultural characteristics (Fig. 1). Each isolate developed a whitish mycelium with the exception of KN-27, which was tan, and GP-100, which appeared pinkish on PDA. The cultures also differed in growth rate, with CZ-620 and TP-21 being the most rapid after 10 days (Fig. 1), and in general culture morphology; e.g. GP-100 showed fuzziness of the mycelium, while GP-115, TP-21 and GP-100 produced a series of concentric rings that may be related to the response of the cultures to being in alternating cycles of light and darkness (Fig. 1).



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Fig. 1. The mycelial colonies of the new isolates of M. albus grown on PDA for 10 days. The original isolate of M. albus is also shown as CZ-620. The new isolates are each designated with a two-letter abbreviation (from the scientific name of their respective host plant) and a lab culture number.

 
Scanning electron microscopy of the mycelium of each of the cultures revealed all of the features that have been associated with this group of organisms, including right-angle branching, the intertwining of hyphae into rope-like strands, hyphal coiling, and sometimes submerged growth under the agar surface (Worapong et al., 2001) (Fig. 2a–d). One culture (KN-27), however, consistently preferred growing in a submerged state, while still developing hyphal coils, hyphal stranding and right-angle branching (Fig. 2d).



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Fig. 2. Scanning electron micrographs of the common hyphal characteristics of M. albus including (a) right-angle branching; (b) winding of hyphae into rope-like strands; (c) coiling of hyphae; and (d) hyphae growing submerged in PDA.

 
The partial ITS-5·8S rDNA sequences of the seven isolates were obtained (see Table 1 for GenBank accession numbers) and compared to sequences of all fungi in GenBank. There was 96–99 % sequence identity of these isolates to the original isolate of M. albus, CZ-620, (Table 2; sequence alignment available as supplementary data with the online version of the paper at http://mic.sgmjournals.org), making a very tight and clustered relationship among the ITS-5·8S rDNAs among all of the organisms whose sequences are now on deposit at the GenBank listed as M. albus (Strobel et al., 2001; Worapong et al., 2001; Sopalun et al., 2003) (Table 2). Extreme difficulty was encountered with isolate KN-205, in which five unsuccessful attempts were made to get total comparable partial ITS-5·8S rDNA sequence information. This unexpected result may be related to the presence of shorter sequences in isolate KN-205 that bear sequence similarity to the primers that were used, but only allow for partial reading of the ribosomal gene. Nevertheless, the very small partial sequences of this organism that were obtained showed similarity to the ITS-5·8S rDNA of M. albus (see the sequence alignment available as supplementary data).


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Table 2. Percentage DNA identity of the ITS-5·8S rDNA sequence of the different new M. albus isolates to M. albus CZ-620, M. roseus and M. vitigenus

 
Although there are differences in colony morphology among the seven new isolates, their hyphal morphologies, with few exceptions, are quite comparable. In addition, they each produce VOCs and their partial ITS-5·8S rDNA sequences are similar to the original M. albus (designated CZ-620) and to each other (Table 2). We therefore designate them as new individual isolates of M. albus.

VOC production by the new isolates of M. albus
Each of the seven new isolates of M. albus, as well as the original isolate of this fungus (CZ-620), was subjected to analysis of the VOCs in a 10-day-old culture using the techniques as described. Final identification of the VOCs was done using authentic compounds obtained commercially and synthesized (Strobel et al., 2001; Daisy et al., 2002b). At least one compound that was not available has been given a tentative identification and this is indicated in Table 3. Quite surprisingly, none of the new isolates of M. albus produced any of the esters that are commonly known for the original M. albus isolate (CZ-620) or the relatively new isolate of M. albus from Myristica fragrans obtained in Thailand (Sopalun et al., 2003). However, with one exception, isolate TP-21, each new isolate of M. albus produced propanoic acid, 2-methyl- and the azulene derivative bulnecene. Also, all the new isolates produced naphthalene and an unknown compound having a retention at ~37–38 min (Table 3). Likewise, with the exception of KN-26 they each produced naphthlene, 1,1'-oxybis. Furthermore, with the exception of TP-21 and GP-100 each produced 1-butanol, 3-methyl-.


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Table 3. GC/MS analysis of the volatile compounds produced by various isolates of M. albus

Samples of gases were trapped from 10-day-old cultures and subjected to GC/MS as described in Methods.

 
It is noted that the original M. albus isolate (CZ-620) does not make naphthalene or naphthalene, 1,1'-oxybis- but does produce two different nonanones, ethanol and acetic acid, 2-phenylethyl ester and many esters which were not detected in the new M. albus isolates (Table 3). Also, in the most recent analysis of the original M. albus (CZ-620) VOCs, no bulnecene was detected, for unknown reasons, but the unknown compound of mass 204 Da appearing at ~37–38 min, was detectable in the VOCs of this isolate (Strobel et al., 2001) (Table 3). A number of other volatiles found in the original analysis of the gases of this fungus (Strobel et al., 2001) were not detected in this analysis (Table 3). The reasons for this may relate to the fungus having been continually transferred: bio-typical selection may have occurred; however this fungal isolate has retained its biological activity.

The isolates having similar major compounds appearing as VOCs are GP-206, KN-205, GP-115 and KN-27 (Table 2). Although the VOCs appear in differing amounts and ratios, the compound in greatest abundance in all of the new isolates of M. albus is naphthalene (Table 2). This is comparable to M. vitigenus, whose only detectable VOC is naphthalene (Daisy et al., 2002b).

Biological activities of the VOCs of the M. albus isolates
The noticeable variation in VOC production among the new isolates of M. albus, as contrasted to the original isolate, may suggest that the biological activities of these isolates would be different, given the broad spectrum of test micro-organisms used to assay them (Table 4). Overall, they have comparable biological activities against fungi and bacteria (Table 4). Those most alike in their spectrum of inhibition and being able to cause cell death were isolates KN-26 and KN-27 (Table 4). However, when evaluated on the same basis, isolate KN-205 was the most active followed by CZ-620 and TP-21. Most of the test organisms were, at the least, inhibited by the VOCs of the new isolates of M. albus (Table 4). However, isolate GP-115 had the lowest biological activity of all of the M. albus isolates tested followed closely by isolates GP-100 and GP-206 (Table 4). Since their qualitative VOC production is comparable, the differences in activity between isolates may relate to the quantitative production of the VOCs by these isolates. This cannot be readily measured by our GC/MS methods. This explanation seems reasonable, since one of the most active new isolates is TP-21 and it produces only three detectable VOCs, which were also detected in isolates GP-100 and GP-206.


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Table 4. The VOC bioassay of the various isolates of M. albus as compared to the original isolate, CZ-620

 
Saccharomyces cerevisiae was the most resistant test organism used in our assays. It survived all M. albus VOCs even though it was 100 % inhibited by CZ-620 (Table 4). The other resistant micro-organism was the spore-forming bacterium Bacillus subtilis which was not killed by any isolate of M. albus; however, it was inhibited by CZ-620 and KN-205 (Table 4). Another notable survivor of M. albus VOCs was Fusarium culmorum, which was more or less inhibited by each isolate but not killed by any isolate under the conditions of this test. Likewise, those isolates that totally inhibited Pythium ultimum also killed it. P. ultimum survived under conditions in which it was only partially inhibited in its growth e.g. in response to GP-100, GP-115 and GP-206 (Table 4). Phytophthora cinnamomi behaved in an almost identical manner (Table 4).

Artificial mixtures of some of the main compounds, or derivatives thereof, that were present in the VOCs of the M. albus isolates were tested against several plant-pathogenic fungi. The artificial VOCs were optimized in quantity and quality for their biological activity. The plant pathogens were used in a Petri plate test according to established procedures in which a small plastic vial containing the test solution was placed in the plate along with the test fungus (Strobel et al., 2001). Many individual compounds, as well as mixtures of various compounds, were examined in the plate test. The most biologically active mixture contained naphthalene, propanoic acid and butanol, 3-methyl, at a ratio of 9 : 45·5 : 45·5 (w/v/v), respectively. Pythium ultimum responded with 100 % inhibition, after a 6 day exposure, at a minimum inhibitory concentration (MIC100) of 15 µl in the test vial. The test organisms also died after exposure to this mixture of VOCs. Butanol, 3-methyl-, naphthalene and propanonic acid alone each had MIC100 values of 100 µl or greater. Other test fungi including Rhizoctonia solani and Sclerotia sclerotiorum responded to the artificial mixture in nearly the same manner as P. ultimum.

Conclusions
Clearly, all the isolates of the sterile, VOC-producing xylariaceous fungus M. albus make compounds that are inhibitory, and in many cases lethal, to various test organisms including plant-pathogenic fungi, including yeasts and bacteria. Previously, it had been established that the main VOCs responsible for the inhibitory activity of M. albus isolate CZ-620 were esters, alcohols and acids (Strobel et al., 2001). This work shows that the new isolates of M. albus make other VOCs such as naphthalene and an alcohol, an acid, and/or other naphthalene/azulene derivatives that possess biological activity both in vivo and in artificial mixtures. Interestingly, no individual compound by itself possessed major antifungal activity, but a combination of compounds was required, as was previously determined for M. albus isolate CZ-620 (Strobel et al., 2001). It was also previously noted that naphthalene, the sole distinguishing VOC of M. vitigenus, was produced in great enough quantities to cause modifications in insect behaviour (Daisy et al., 2002b). It may be the case that the new isolates of M. albus have comparable anti-insect activities; if they do, it becomes reasonable to hypothesize a new biological role of M. albus. That is, as an endophyte in plants, it may deter insects that would otherwise inhabit and destroy plant tissues. Certainly this concept is worthy of further testing.

Using the approach of finding and isolating fungi that are tolerant of the VOCs of M. albus as a selection tool, it has been possible to find not only these new isolates of this organism, but others as well, including M. vitigenus and M. roseus (Daisy et al., 2002b; Worapong et al., 2001). Most recently, at least eleven new isolates of VOC-producing fungi with a white sterile mycelium have appeared from a number of plants obtained in the tropical zones of Venezuela. These each possess volatile antibiotic properties, but their morphological and molecular biological properties have not been defined. A pattern is beginning to emerge that indicates the preferred habitat of the Muscodor spp. They have only been isolated as endophytes in tropical or monsoonal rainforests, e.g. Thailand, Hondouras, Peru, Venezuela and Australia. Attempts to find them in temperate rainforests, using the original M. albus isolate as a selection tool, such as those in Tasmania or southwestern Australia, and the temperate zones of Chile have failed. Also, attempts to find them in tropical, but more seasonally dry climates such as the island of Socotra, Yemen, also have failed. However, an antibiotic VOC-producing Gliocladium sp. was isolated from a temperate rainforest in central Chile using M. albus VOCs as the selection technique (Stinson et al., 2003a). Initially it was thought that M. albus was a unique organism confined to only one locale, but as more studies are done, it appears that it and related organisms are common inhabitants of the world's tropical rainforests. Their life cycle remains a mystery.

Potential applications for M. albus and its VOCs are currently being investigated. These include uses for treating various seeds, fruits and cut flowers, to reduce or eliminate harmful or disease-causing micro-organisms (Mercier & Jimenez, 2004). Another promising option is as an alternative to methyl bromide fumigation of soil to control soil-borne plant pathogens (Stinson et al., 2003b).


   ACKNOWLEDGEMENTS
 
This research was supported by Vaadia-BARD Postdoctoral Award no. FI-321-2001 from BARD, The United States–Israel Binational Agricultural Research and Development Fund. Funding was also supplied by the National Science Foundation, the R&C Board of the State of Montana, the USDA, and the Montana Agricultural Experiment Station. Dr Joe Sears of the MSU Department of Chemistry acquired mass spectral data using GC/MS methods and Ms Sigal Ezra did bioassay testing. Dr Uvi Castillo collected plant materials from Venezuela and did fungal isolation work on them. Mr and Mrs Djalu Gurruwiwi of Yirrkala, Gove Pennisula, NT, Australia, provided valuable assistance in plant collection.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Daisy, B., Strobel, G., Ezra, D., Castillo, U., Baird, G. & Hess, W. M. (2002a). Muscodor vitigenus sp. nov., an endophyte from Paullinia paullinoides. Mycotaxon 84, 39–50.

Daisy, B. H., Strobel, G. A., Castillo, U., Ezra, D., Sears, J., Weaver, D. K. & Runyon, J. B. (2002b). Naphthalene, an insect repellent, is produced by Muscodor vitigenus, a novel endophytic fungus. Microbiology 148, 3737–3741.[Medline]

Ezra, D. & Strobel, G. A. (2003). Effect of substrate on the biobioactivity of volatile antimicrobials produced by Muscodor albus. Plant Sci 165, 1229–1238.[CrossRef]

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Mercier, J. & Jimenez, J. I. (2004). Control of decay of apples and peaches by the biofumigant fungus Muscodor albus. Postharvest Biol Technol 31, 1–8.[CrossRef]

Sopalun, K., Strobel, G. A., Hess, W. M. & Worapong, J. (2003). A record of Muscodor albus, an endophyte from Myristica fragrans, in Thailand. Mycotaxon 88, 239–247.

Stinson, M., Ezra, D., Hess, W. M., Sears, J. & Strobel, G. A. (2003a). An endophytic Gliocladium sp. of Eucryphia cordifolia producing selective volatile antimicrobial compounds. Plant Sci 165, 913–922.[CrossRef]

Stinson, A. M., Zidack, N. K., Strobel, G. A. & Jacobsen, B. J. (2003b). Effect of mycofumigation with Muscodor albus and Muscodor roseus on seedling diseases of sugarbeet and Verticillium wilt of eggplant. Plant Dis 87, 1349–1354.

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Worapong, J., Strobel, G. A., Daisy, B., Castillo, U., Baird, G. & Hess, W. M. (2002). Muscodor roseus anna. nov., an endophyte from Grevillea pteridifolia. Mycotaxon 81, 463–475.

Received 14 May 2004; revised 27 August 2004; accepted 16 September 2004.



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