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
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ABSTRACT |
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INTRODUCTION |
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While VOC-producing fungi have been isolated and studied chemically in the past 3040 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.
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METHODS |
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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 47 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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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 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 ml1 (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 23 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 min1. 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 35360 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.
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RESULTS AND DISCUSSION |
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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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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
3738 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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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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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
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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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, 37373741.[Medline]
Ezra, D. & Strobel, G. A. (2003). Effect of substrate on the biobioactivity of volatile antimicrobials produced by Muscodor albus. Plant Sci 165, 12291238.[CrossRef]
McAfee, B. J. & Taylor, A. (1999). A review of the volatile metabolites of fungi found on wood substrates. Nat Toxins 7, 283303.[CrossRef][Medline]
Mercier, J. & Jimenez, J. I. (2004). Control of decay of apples and peaches by the biofumigant fungus Muscodor albus. Postharvest Biol Technol 31, 18.[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, 239247.
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, 913922.[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, 13491354.
Strobel, G. A. & Daisy, B. (2003). Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67, 491502.
Strobel, G. A., Dirkse, E., Sears, J. & Markworth, C. (2001). Volatile antimicrobials from Muscodor albus, a novel endophytic fungus. Microbiology 147, 29432950.[Medline]
Worapong, J., Strobel, G. A., Ford, E. J., Li, J. Y., Baird, G. & Hess, W. M. (2001). Muscodor albus anam. nov., an endophyte from Cinnamomum zeylanicum. Mycotaxon 79, 6779.
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, 463475.
Received 14 May 2004;
revised 27 August 2004;
accepted 16 September 2004.
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