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Department of Biology, George Mason University, Fairfax, Virginia 22030-4422
Received for publication March 3, 1998. Accepted for publication July 9, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: chemical ecology • Fusarium • lichenicolous fungi • lichens • mycoparasites
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Rundel, 1978
; Lawrey, 1984
, 1986
). Nevertheless, certain obligate fungivorous animals (Lawrey, 1983
) and fungal parasites (Lawrey, 1995
) consume lichens, which suggests that tolerance to certain lichen compounds may play a role in the ecology of these organisms. Indeed, there is some evidence that lichen parasites are generally more tolerant of lichen compounds than nonlichenicolous fungi (Lawrey, 1997
). There is also evidence that the enzymes produced by lichenicolous fungi are more tolerant of certain lichen compounds than others, which may explain the host ecologies of these fungi (Torzilli and Lawrey, 1995
).
We recently discovered a new lichen parasite in the fungal genus Fusarium that attacks a variety of lichens and is tolerant of many lichen compounds in laboratory experiments. We were alerted to the existence of this new mycoparasite when we observed the familiar lichenicolous fungus Marchandiomyces corallinus (Roberge) Diederich & D.Hawksw. growing on lichens known to chemically suppress growth (Lawrey, 1993
). Lichens harboring M. corallinus were collected from a rock-inhabiting lichen community in the St. Mary's Wilderness in Virginia, and an unknown hyaline hyphomycete was isolated. Subsequent observations indicated that M. corallinus is found on certain lichens (including Lasallia spp.) only if this hyphomycete fungus is also present.
An analysis by Dr. Kerry O'Donnell (National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Peoria, Illinois) of 18S rDNA sequences obtained for the unknown fungus revealed that it is likely a new species in the genus Fusarium (NRRL 26803), closely related to the insect parasite F. larvarum larvarum Fuckel and nested within a small clade of entomogenous fungi. It, along with a Fusarium sister species (NRRL 26790) that also attacks lichens, has since been found in numerous lichen communities in the eastern United States. The experiments described in this paper concern only the isolate NRRL 26803, which has been found in numerous lichen communities in Virginia and Maryland. Degradation of lichens by this new Fusarium seems to expose them to attack by opportunistic fungi, such as M. corallinus, that are normally inhibited by lichen compounds. Since the behavior of lichenicolous fungi can be related to the enzymes they produce, we hypothesized that the degradative enzymes produced by this organism alter the chemistry of certain lichens, and that this is necessary for colonization of these lichens by M. corallinus. The present study describes results of experiments designed to test this hypothesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isolates of Fusarium sp. nov. (NRRL 26803) were obtained from thalli of the lichen Lasallia papulosa collected in 1996 in the St. Mary's Wilderness in Virginia. Surface sterilized conidia were plated onto SDA and subcultured monthly. Conidia formation declined in cultured mycelium of this fungus, but could be enhanced by growing the fungus on sterilized lichen tissues.
Lichen degradation experiments
The experimental approach used to determine degradative abilities of M. corallinus and Fusarium is similar to that used in previous studies (Lawrey, 1993
, 1997
; Lawrey, Rossman, and Lowen, 1994
). Mycelium of either Marchandiomyces corallinus or Fusarium sp. nov. was aseptically scraped from the surface of 2-wk-old agar plate cultures (incubated at 18°C) into a sterilized Waring blender with enough sterile water to cover the blades. This was blended for 30 s, and the homogenized inoculum was added to experimental dishes at a level of 0.3 mg of fungal biomass (dry mass) per dish.
Thalli of the lichens Lasallia papulosa and Flavoparmelia baltimorensis, collected in the St. Mary's Wilderness, Virginia, were chosen to be used as substrates for growth of the fungi. Fresh lichen tissues were cleaned of debris, washed in water, dried, and ground in a Wiley mill. A subsample of material for each species was washed several times in absolute acetone to remove all lichen phenolic compounds. Both acetone-washed and unwashed samples were then autoclaved and oven dried (100°C) for 4 h and stored in a desiccator. These materials were used as growth media for inocula of M. corallinus or Fusarium. Since lichen compounds are known to be unstable at high temperatures (Culberson, Culberson, and Johnson, 1977
), TLC and HPLC were used to monitor the breakdown of lichen compounds during sterilization in the autoclave.
To separate the effects of lichen secondary metabolites from those of other possible factors on fungal degradative activity, acetone-washed tissues of a single lichen (Lasallia papulosa) were spiked with acetone extracts of each of the test lichens at the same concentrations as naturally found in the test lichens. This yielded growth media that were identical in every way except secondary chemistry. Lasallia papulosa was chosen because all lichen parasites in our laboratory grow on tissues of this lichen if acetone-soluble compounds are removed. We also used a sample of purified lecanoric acid (LEC obtained from Dr. Chicita Culberson), a lichen compound known to inhibit fungal parasites in laboratory experiments (Lawrey, 1997
). This had been purified by repeated recrystallizations so that it contained mainly lecanoric acid (LEC) and only traces of orsellinic acid and an unidentified compound. It was added to washed L. papulosa tissues at a concentration of 5%, since LEC is frequently found at or near this concentration in many lichens. All washed tissues of L. papulosa treated with lichen extracts (or LEC) were dried in a hood and stored in a desiccator over anhydrous calcium sulfate for 1 wk prior to use in growth/degradation experiments.
Approximately 100 mg of lichen tissue were weighed to the nearest 0.01 mg, poured into 5-cm glass petri plates, autoclaved, and inoculated with 1 mL of fungal suspension containing 0.3 mg fungal tissue (dry mass). Plates were sealed in plastic film and placed in a growth chamber (12 h light/12 h dark cycle, 18°C) for 30 d. At the end of the experiment, the growth of the fungus was estimated by determining the net mass loss of the lichen tissue upon which it had been growing. Weights obtained at the end of each experiment included any fungal biomass that accumulated during the experiment since this could not be separated from the growth medium (Lawrey, Rossman, and Lowen, 1994
).
To test the hypothesis that previous degradation of lichen tissues by Fusarium is required before they can be used by M. corallinus, we prepared tissues of L. papulosa and F. baltimorensis as described above, inoculated these with Fusarium, and placed them in a growth chamber for 30 d. At the end of this period, tissues were dried and 100-mg samples placed in clean petri plates and autoclaved. These Fusarium-treated substrates were inoculated with M. corallinus and placed in the growth chamber for an additional 30 d. The controls for this Fusarium treatment were 100-mg samples placed in sterile water for 30 d, after which time they were also dried, autoclaved, and inoculated with M. corallinus and placed in the growth chamber. A comparison of the net mass loss of these previously altered tissues after 30 d was used to estimate the effect of Fusarium on subsequent degradative activity of M. corallinus.
HPLC experiments
Freshly collected and cleaned lichen tissues (around 50 mg) were extracted for 1 h at room temperature in 2 mL of acetone and centrifuged for 10 min (8000 rpm). The supernatant was collected and dried using a speedvac system (Savant AES 2000, Farmingdale, New York). The dried samples were reconstituted in 200 µL of methanol and analyzed using a Hewlett Packard 1100 HPLC system with a photo diode array detector set at a range of 200–450 nm; all peaks were analyzed at 254 nm. An analytical reverse phase C18 column (Prodigy, ODS, 150 x 4.6 mm, 3 µm; Phenomenex, Torrence, California) was used as the stationary phase. Mobile phase A contained 10% methanol and 90% water brought to a pH of 2.0 with phosphoric acid, and mobile phase B was 100% methanol. A linear gradient was applied over 30 min starting with 100% of mobile phase A at the start to 100% mobile phase B at the end. Chromatographs were analyzed by Hewlett Packard software; retention time and absorbance spectra were used to identify compounds. The concentration of LEC in the samples was determined by comparing with a concentration curve obtained by analysis of peak areas of a LEC standard at appropriate concentrations. Presence of LEC in the lichen extracts was confirmed by thin-layer chromatography (TLC) prior to HPLC analysis using a standardized method (Culberson, Culberson, and Johnson, 1981
).
Enzyme-mediated degradation experiments
A series of experiments was done to test the hypothesis that Fusarium enzymes degrade lecanoric acid, one of the inhibitory compounds produced by many lichens, including Lasallia species. A sample of purified lecanoric acid (LEC, same as in previous experiments) was used to make a standard solution (1 mg in 1 mL methanol), and 10 µL of this solution (10 µg LEC) were added to glass test tubes. The methanol was evaporated in a Savant vacuum centrifuge and the tubes treated with aqueous mixtures of fungal enzymes, as described below.
For enzyme induction, inoculum cultures (50 mL of Sabouraud's dextrose broth in capped 500-mL Erlenmeyer flasks) of either Fusarium sp. nov. or M. corallinus were homogenized for 30 s in a sterilized Waring blender and the homogenates centrifuged in sterilized centrifugation bottles. The pellets were resuspended and washed in sterile deionized water, centrifuged, and the pellets resuspended in deionized water. Aliquots from these mycelial homogenates (1.6 mg dry mass) were added to capped 500-mL Erlenmeyer flasks containing 200 mg of ground L. papulosa tissue (minus phenolic compounds), 200 mL of deionized water, and 200 µL of Tween 80 as a wetting agent. These cultures were incubated for 1 wk (for Fusarium sp.) or for 2 and 3 wk (for M. corallinus) without agitation at 18°C under a light regime of 12 h light/12 h dark. Cultures were harvested by centrifugation, and 0.5-mL aliquots of the supernatants were added to glass tubes containing 10 µg of LEC each. Chlorhexidine (0.002%) was added to all tubes to prevent bacterial contamination and autoclaved aliquots served as controls. All treatments were replicated four times. Tubes were incubated on a laboratory rotator at room temperature for 22.5 h, after which time the tube contents were taken to dryness using a Savant vacuum centrifuge. The dried contents of each tube were resuspended in 1 mL of methanol and filtered through a membrane filter (0.2 µm) before HPLC injection. The enzyme degradation of LEC was estimated by comparing areas under LEC peaks observed on HPLC chromatograms of treatment tubes (active enzymes) and control tubes (heat denatured enzymes).
Data analysis
Data sets were compared using one-way ANOVA, followed by a Duncan's multiple comparison test. All statistical programs were from NCSS-97 (NCSS, 1997
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) had shown that Marchandiomyces corallinus can easily degrade tissues of the lichen Flavoparmelia baltimorensis (from which it is frequently collected). However, it cannot degrade tissues of the lichen Lasallia papulosa unless they are first washed thoroughly in acetone to remove phenolic compounds. Therefore, when M. corallinus was discovered growing on this lichen in the St. Mary's Wilderness, we hypothesized that a tolerant ecotype of M. corallinus existed in this community. However, isolates of M. corallinus (ATCC 200797) from L. papulosa proved to behave exactly the same way as our original isolate (ATCC 200796) collected from F. baltimorensis. Both isolates of M. corallinus were significantly (ANOVA, P < 0.001) inhibited by the acetone-soluble compounds of L. papulosa. There was no significant difference in the degradation of washed vs. unwashed tissues of the preferred host, F. baltimorensis (Fig. 1).
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Results of 30-d degradation experiments (Fig. 4) indicated that LEC and extracts of Lasallia papulosa significantly (ANOVA, P < 0.01) inhibit M. corallinus. It is interesting that no significant inhibition was observed for extracts of F. baltimorensis, the preferred host of this parasite.
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HPLC results
HPLC chromatograms obtained for Lasallia papulosa showed the presence of lichen compounds typical of this species (Narui et al., 1997
). Gyrophoric (GYR) and lasallic (LAS) acids are the major compounds present, with lesser amounts of lecanoric (LEC) acid and other constituents. TLC analysis of autoclaved lichen tissues revealed no thermal breakdown of lichen compounds; however, small peaks present in HPLC chromatograms indicated that some breakdown of the major compounds took place in the autoclave. Inasmuch as autoclaved tissues were always used as growth media in degradation experiments, each experimental substrate contained the same compounds (the main constituents plus small quantities of breakdown products).
HPLC analysis of lichen tissues before and after fungal degradation indicated that LEC was significantly reduced in concentration after exposure to Fusarium. Gyrophoric and lasallic acids were also degraded slightly. Since LEC is inhibitory to many fungi, including Marchandiomyces corallinus, we used HPLC to determine whether LEC can be degraded by extracellular enzymes produced by Fusarium. Partially purified LEC was exposed to culture supernatants containing enzymes produced by Fusarium. After only 24 h, most of the LEC exposed to these enzymes is degraded to a more water-soluble product, orsellinic acid (ORS); LEC exposed to autoclaved controls remains unchanged (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Clauzade, Diederich, and Roux, 1989
; Honegger, 1992
; Rambold and Triebel, 1992
; Gargas et al., 1995
). More than 1000 species within 300 genera are estimated to exist (Hawksworth, 1982
). Recent studies indicate that some of the fungi collected from lichens are not restricted to lichens but instead are generalized soil-borne saprobes (Petrini, Hake, and Dreyfuss, 1990
; Glenn, Gomez-Bolea, and Orsi, 1997
), some of which are able to exploit lichens weakened by pollution (Glenn, Gomez-Bolea, and Orsi, 1997
). Nevertheless, the high level of host specificity exhibited by many of the lichenicolous fungi (Hawksworth, 1982
) is evidence that they are uniquely adapted to lichen hosts, many of which produce antibiotic secondary metabolites. It is likely that many lichen parasites are derived from saprophytic fungi. If this is true, the transition to a lichenicolous habit would require the elaboration of enzymes that are specifically adapted to lichens and lichen secondary metabolites. Given the diversity of lichenicolous fungi, it would not be surprising to find that many produce enzymes tolerant of antibiotic lichen chemicals. The discovery of enzymes capable of destroying such chemicals is more interesting, however, and indicates the potential for a variety of ecological interactions among these fungi.
The existence of such hydrolytic enzymes in lichens has been known for many years (Culberson, 1969
). For example, Mosbach and Ehrensvärd (1966)
isolated enzymes from the lichen Lasallia pustulata and a culture of its Trebouxia photobiont that were capable of breaking down the depside evernic acid to simpler phenolic acid constituents. This esterase was also able to hydrolyze two tridepsides, including gyrophoric acid, which is the major compound in Lasallia papulosa and can also be found in some specimens of Flavoparmelia baltimorensis. We might expect to occasionally see such depside-degrading enzymes in nonlichenized fungi since the precursor phenolic acids of depsides are found in many nonlichenized fungi. The presence of these enzymes in the new Fusarium species we found supports this prediction. However, experiments with several other lichenicolous fungi that we maintain in our laboratory have yielded negative results so far, which may mean that this behavior is relatively rare in these fungi.
The Fusarium we worked with appears to be tolerant of lecanoric acid (LEC), and other lichen compounds as well, and depside degradation may be a necessary component of this tolerance. The nature of the inhibition of LEC on sensitive fungi is not known, nor is the cause of the tolerance exhibited by Fusarium. However, it appears that degradation of LEC to simpler, more water-soluble phenolic constituents renders this lichen substance less inhibitory to some lichenicolous fungi. Either orsellinic acid itself is less inhibitory or its greater solubility in water causes it to wash out of lichen tissues more rapidly and expose larger areas of lichen tissue to fungal attack. Since, in our experiments, the products of depside degradation remained in the glass plates after exposure to Fusarium and were a component of the substrate exposed to M. corallinus afterwards, it is likely that these compounds are themselves less inhibitory than LEC. This hypothesis needs to be tested experimentally, however.
The new Fusarium we discovered may play an important keystone role in lichen communities insofar as it increases opportunities for fungal parasites that would otherwise be excluded from lichen communities. For example, extracts of the lichen Punctelia rudecta (containing primarily LEC) are known from laboratory experiments to inhibit both M. corallinus (Lawrey, 1993
) and another common parasite Nectria parmeliae (Lawrey, Rossman, and Lowen, 1994
). Nevertheless, N. parmeliae grows commonly on this lichen in nature (we have never observed M. corallinus on this lichen). Once we realized that Fusarium may be involved in these interactions, we collected specimens of P. rudecta containing N. parmeliae and isolated the new Fusarium species from every specimen. We expect to see additional evidence of the involvement of Fusarium in other lichen–parasite interactions that we are beginning now to investigate.
The discovery of a lichen parasite in the genus Fusarium is somewhat surprising given the small numbers of this diverse group that appear to have evolved as lichen parasites (Hawksworth, 1979
). Since our new Fusarium appears to be derived from entomogenous fusaria, it is tempting to suggest that enzymes necessary to attack chitin-containing fungal tissues evolved earlier when these fungi were parasitic entirely on insects. However, a test of this hypothesis must await a complete understanding of the distribution of this new Fusarium, its host ecology, physiological ecology, and phylogenetic relationships with other fusaria. There is another new putative lichenicolous Fusarium species from Massachusetts (NRRL 26790) that is similar but slightly different in terms of SSU rDNA sequence (Dr. Kerry O'Donnell, personal communication, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois). It also appears to be more virulent than our isolate (D. Pfister and E. Kneiper, personal communication, Harvard University), which may have ecological significance in the lichen communities where it resides. This particular Fusarium has been noted in earlier published studies (Glenn, Gomez-Bolea, and Orsi, 1997
), and it is still not clear what its relationship is to our Fusarium, but a complete taxonomic description of the two will be undertaken in the near future.
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FOOTNOTES |
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2 Author for correspondence [Tel: (703) 993-1059; FAX: (703) 993-1046;jlawrey{at}gmu.edu
].
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Culberson, C. F. 1969 Chemical and botanical guide to lichen products. University of North Carolina Press, Chapel Hill, NC.
———, W. L. Culberson, and A. Johnson. 1977 Thermally induced chemical artifacts in lichens. Phytochemistry 16: 127–130.[CrossRef][ISI]
———, ———, and ———. 1981 A standardized TLC analysis of ß-orcinol depsidones. Bryologist 84: 16–29.[CrossRef][ISI]
Gargas, A., P. T. DePriest, M. Grube, and A. Tehler. 1995 Multiple origins of lichen symbioses in fungi suggested by SSU rDNA phylogeny. Science 268: 1492–1495.
Glenn, M. G., A. Gomez-Bolea, and E. V. Orsi. 1997 Effects of thallus damage on interactions of lichens with non-lichenized fungi under natural and laboratory conditions. Lichenologist 29: 51–65.
Hawksworth, D. L. 1979 The lichenicolous Hyphomycetes. Bulletin of the British Museum of Natural History (Botany) 6: 183–300.
———. 1982 Secondary fungi in lichen symbioses: parasites, saprophytes and parasymbionts. Journal of the Hattori Botanical Laboratory 52: 357–366.
Honegger, R. 1992 Lichens: mycobiont-photobiont relationships. In W. Reisser [ed.], Algae and symbioses, 255–275. Biopress, Limited, Bristol, UK.
Lawrey, J. D. 1983 Lichen herbivore preference: a test of two hypotheses. American Journal of Botany 70: 1188–1194.[CrossRef][ISI]
———. 1984 Biology of lichenized fungi. Praeger, New York, NY.
———. 1986 Biological role of lichen substances. Bryologist 89: 111–122.[CrossRef][ISI]
———. 1993 Chemical ecology of Hobsonia christiansenii, a lichenicolous hyphomyete. American Journal of Botany 80: 1109–1113.[CrossRef][ISI]
———. 1995 The chemical ecology of lichen mycoparasites: a review. Canadian Journal of Botany 73(Supplement 1): S603–S608.
———. 1997 Isolation, culture, and degradative behavior of the lichen parasite Hobsonia santessonii. Symbiosis 23: 107–116.
———, A. Y. Rossman, and R. Lowen. 1994 Inhibition of selected hypocrealean fungi by lichen secondary metabolites. Mycologia 78: 842–846.[CrossRef]
Mosbach, K., and U. Ehrensvärd. 1966 Studies on lichen enzymes. Part I. Preparation and properties of a depside hydrolyzing esterase and of orsellinic acid decarboxylase. Biochemical and Biophysical Research Communications 22: 145–150.[CrossRef][ISI][Medline]
Narui, T., C. F. Culberson, W. L. Culberson, A. Johnson, and S. Shibata. 1996 A contribution to the chemistry of the lichen family Umbilicariaceae (Ascomycotina). Bryologist 99: 199–211.[CrossRef][ISI]
NCSS. 1997 NCSS User's Guide I and II. Kaysville, UT.
Petrini, O., U. Hake, and M. M. Dreyfuss. 1990 An analysis of fungal communities isolated from fruticose lichens. Mycologia 82: 444–451.[CrossRef][ISI]
Rambold, G., and D. Triebel. 1992 The inter-lecanoralean associations. Bibliotheca Lichenologia 48: 1–201.
Rundel, P. W. 1978 The ecological role of secondary lichen substances. Biochemical Systematics and Ecology 6: 157–170.
Torzilli, A. P., and J. D. Lawrey. 1995 Lichen metabolites inhibit cell wall-degrading enzymes produced by the lichen parasite Nectria parmeliae. Mycologia 87: 841–845.
Vartia, K. O. 1973 Antibiotics in lichens. In V. Ahmadjian and M. E. Hale, Jr. [eds.], The lichens, 547–561. Academic Press, New York, NY.
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