Dynamics of bioluminescence by Armillaria gallica, A. mellea and A ...

Mycologia, 99(3), 2007, pp. 341–350. # 2007 by The Mycological Society of America, Lawrence, KS 66044-8897 Issued 20 August 2007

Dynamics of bioluminescence by Armillaria gallica, A. mellea and A. tabescens Jeanne D. Mihail1 Johann N. Bruhn

Panellus stipticus that required the presence of molecular oxygen, superoxide and a cationic surfactant for activity. Shimomura (1992) subsequently demonstrated that luminescence of Armillaria mellea increased as the superoxide dismutase (SOD) level declined. Because SOD is abundant throughout actively growing mycelium, Shimomura proposed that fungal luminescence occurs where SOD is locally absent or inhibited. The sensitivity of fungal bioluminescence to the nutritional status of the mycelium and to other culture conditions also has been demonstrated (Airth and Foerster 1965). The ecological context of fungal bioluminescence is still poorly understood, although many hypotheses have been offered (Hastings 1983, Wilson and Hastings 1998). Our studies focus on Armillaria, a genus of ca. 40 wood-decay species (Volk and Burdsall 1995, Pegler 2000). Both the diffuse mycelia and foraging rhizomorph systems of Armillaria spp. are typically slow growing (Rishbeth 1968). Armillaria spp. cause saprotrophic white-rot decay of dead woody portions of host plants, as well as root disease of living hosts (Gregory et al 1991, Fox 2000, Morrison 2004). The root disease-causing capabilities of different Armillaria spp. on a given host species can be thought of as points along a continuum with strict saprotrophism at one extreme and relatively virulent parasitism at the other (Fox 2000, Morrison 2004). Saprotrophic and parasitic Armillaria spp. are frequently sympatric. For example in the central USA (i.e. Missouri) A. gallica is often the more saprotrophic species while A. mellea and A. tabescens are more parasitic (Bruhn et al 2000). In Switzerland A. cepistipes is the saprotrophic species while A. ostoyae is parasitic (Prospero et al 2003a, b). The bioluminescence of Armillaria has been documented both anecdotally and experimentally for more than a century (Murrill 1915, Buller 1924, Harvey 1952). Although the mycelia and rhizomorphs of Armillaria are bioluminescent, the fruiting bodies apparently are not (Buller 1924, Harvey 1952, Wassink 1978). Bioluminescence of Armillaria mycelia incubated in complete darkness has been reported to exhibit diurnal periodicity with maxima observed at night (Berliner 1961, Calleja and Reynolds 1970). Similar diurnal bioluminescence patterns have been reported for mycelia of Panellus stipticus (Berliner 1961, Calleja and Reynolds 1970) and Mycena polygramma (Berliner 1961). The emission spectrum of actively bioluminescent A. mellea sensu lato grown on wood was 430–670 nm, with maximum emission at 520 nm (Coblentz and Hughes 1926). Similarly the

Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211

Abstract: Although fungal bioluminescence is well documented, the ecological significance is poorly understood. We examined bioluminescence by three sympatric species of Armillaria wood decay fungi, differing in parasitic ability. Luminescence by mycelia of four genets of A. gallica, A. mellea and A. tabescens was examined in response to environmental illumination or mechanical disturbance. Luminescence dynamics were assessed in a time series of measurements every 2 min for 72 h for mycelia growing on malt agar or on Cornus florida root wood. Luminescence by the necrotrophic species A. gallica was enhanced by environmental illumination and mechanical disturbance of mycelia. In contrast luminescence by the more parasitic A. mellea and A. tabescens was quenched by prolonged exposure to environmental illumination and less responsive to mechanical disturbance. With environmental illumination absent, all mycelia representing six genets of each Armillaria species were constitutively luminescent. The temporal dynamics of luminescence by all mycelia were complex with no evidence of the previously reported diurnal periodicity. Differences among Armillaria spp. in bioluminescence expression might reflect differences in ecological context as well. Key words: Armillaria gallica, Armillaria mellea, Armillaria tabescens, mycelial luminescence, temporal dynamics INTRODUCTION

Bioluminescence in fungi has been observed most often in species of the white-spored Agaricales (Basidiomycota) (Harvey 1952, Wassink 1978). Species with luminescent fruiting bodies often have luminescent mycelia although the reverse is not always true (Wassink 1978). The majority of reports of fungal bioluminescence document newly discovered luminescent taxa. The biochemistry of fungal luminescence has been characterized as the luciferinluciferase type (Airth and Foerster 1960, Wassink 1978). Shimomura (1991) identified a luciferin from Accepted for publication 2 February 2007. 1 Corresponding author. E-mail: [email protected]

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maximum emission of A. mellea s.l. mycelia growing on agar was 530 nm (Airth and Foerster 1960). In a comparison of mycelial bioluminescence of 10 basidiomycete species, A. mellea ranked fourth in intensity (Bermudes et al 1992). The sensitivity of A. mellea bioluminescence to temperature, pH and fluorescent light was investigated by Weitz et al (2001) with mycelia of one isolate growing on agar. Luminescence was greater at 22 C than that at 5, 15 or 30 C. Exposure of mycelia to 12 h or 24 h fluorescent light dramatically reduced bioluminescence. The studies of Weitz and co-workers (2001) also are significant as the only published records of the variation in bioluminescence among replicate mycelia. Variation among replicates was much less in their studies of temperature and pH effects (where the agar cultures were incubated completely in the dark) than in the study of light effects on bioluminescence. Ultraviolet irradiation also has been found to temporarily depress bioluminescence of A. mellea s.l. (Airth and Foerster 1960, Berliner 1963). Previous work on Armillaria bioluminescence has focused little attention on the ecological context of the phenomenon. Research with Armillaria published before 1980 also must be placed in the context of the fact that a suite of morphologically similar species were all included in A. mellea s.l. (Anderson and Ullrich 1979). At present at least 40 Armillaria spp. are recognized (Volk and Burdsall 1995, Pegler 2000). In the only previous comparison of bioluminescence among Armillaria spp., Rishbeth (1986) noted that the luminescence of A. mellea was visibly less than that of A. gallica (5A. bulbosa), A. ostoyae or A. tabescens. In the present report we have investigated the bioluminescence by three sympatric species, A. mellea, A. gallica and A. tabescens, which differ in parasitic ability. We asked whether there are consistent differences in the magnitude, amplitude or temporal dynamics of bioluminescence at the intra- and interspecific levels. We also investigated the separate influences on bioluminescence of environmental illumination and mechanical disturbance of mycelia. MATERIALS AND METHODS

Fungal isolates.— Preliminary studies of the effects of environmental illumination on mycelium luminescence used three field isolates: A. gallica Marx. & Romagn. genet C6, collected from a northern Michigan forest, U.S.A. (45u589280N, 88u219460W; Smith et al 1992); A. mellea (Vahl. : Fr.) P. Kumm. genet OZ550 collected from the upland Ozark forests of southern Missouri, U.S.A. (Site 5, Plot 49; Bruhn et al 2000); and A. tabescens (Scop.) Emel genet B1 collected from Carter County, in southern Missouri (Bruhn et al 2000).

In all subsequent experiments genets representing the three Armillaria spp. were selected as naturally co-occurring triplets collected from selected 0.2 ha. plots in the upland Ozark forests of southern Missouri (Bruhn et al 2000). Multiple isolates were used to let us draw general conclusions applicable to the three species. Specifically A. gallica, A. mellea and A. tabescens were represented by: Triplet A (isolates OZ97, OZ498, OZ865) from Site 2, Plot 69; Triplet B (OZ446, OZ253, OZ685) from Site 7, Plot 47; Triplet C (OZ182, OZ179, OZ795) from Site 7, Plot 55; Triplet E (OZ857, OZ604, OZ333) from Site 8, Plot 1; Triplet G (OZ94, OZ646, OZ650) from Site 3, Plot 70; and Triplet H (OZ316, OZ321, OZ320) from Site 6, Plot 22. All isolates were retrieved from water-agar culture disks stored longterm in 2 C sterile distilled water and maintained as working stock cultures on 2% malt-extract agar (2 M, malt extract 20 g L21, agar 20 g L21). Voucher cultures are deposited in the Center for Forest Mycology Research, USDA Forest Service, Forest Products Laboratory, Madison, Wisconsin. Bioluminescence measurement.—In all experiments bioluminescence was measured as relative light units per second (RLU s21) using a Zylux FB-12 single tube luminometer (Zylux Corp., Oak Ridge, Tennessee). The luminometer converts photons in the range of 370–630 nm emitted from the sample to electrons that are multiplied by a photomultiplier tube (Berthold et al 2000). Electrons then are counted when the amplification generates pulses above an internally set threshold, a process called photon counting. Luminometers convert photon pulse counts to RLU s21 units via an internal algorithm set by the manufacturer (Berthold et al 2000). Experiments evaluating mycelial bioluminescence on an agar substrate were conducted with 1.5%-malt extract agar (1.5 M, malt extract 15 g L21, agar 20 g L21) in 35 mm diam Petri dishes. Experimental mycelia were established with 5 mm diam disks cut from the margin of actively growing mycelia on 1.5 M. Background luminescence of uncolonized media was determined as the mean + se of at least four replicate Petri dishes. Except where mycelia were intentionally exposed to light as an experimental parameter, each Petri dish was placed in its own light-tight cardboard box for the duration of the experiment to avoid the confounding effects of exposure to environmental light as well as the possibility of any interaction among mycelia. Because our luminescence experiments required that most or all mycelia be maintained in complete darkness, it was not feasible to measure growth rates of the specific mycelia used in these experiments. However in a parallel study of mycelia grown similarly on 1.5 M we found that all three Armillaria spp. grew 2 mm radially in 7 d. At 12 d radial growth reached 4, 5.5 and 7 mm for A. gallica, A. mellea, and A. tabescens respectively. All bioluminescence experiments were conducted in a room maintained at 22–26 C. Weitz et al (2001) observed higher luminescence at 22 C compared with 15 or 30 C. All mycelia were undisturbed for at least 4 d before the initiation of luminescence measurements to let the mycelium recover from wounding associated with transfer.

MIHAIL AND BRUHN: ARMILLARIA BIOLUMINESCENCE DYNAMICS Bioluminescence in response to light.—Because it has been reported that fluorescent light quenches fungal bioluminescence (Bermudes et al 1990, Weitz et al 2001), two experiments were conducted to quantify the effects of fluorescent and incandescent light to set appropriate experimental conditions for further investigations. Experiment FL-1 examined the response of mycelial luminescence to fluorescent light supplied by Rapid Start bulbs (TL70, F32T8/TL741, Philips Lighting Co., Somerset, New Jersey), which provide a color rendering index of 78% of natural daylight, six emission peaks (410, 440, 485, 550, 590 and 610 nm), and a photon flux (mean 6 se) of 8.4 6 0.78 mmol m22 s21. Seven replicate mycelia of each of the three Armillaria spp. grown in darkness were exposed only to fluorescent light and only during the transfer of Petri dishes to the luminometer (i.e. brief exposure of 5–10 s). Eight replicate mycelia of each species grown in the dark were exposed only to low light emitted by a darkroom ‘‘safe light’’ fitted with an OC filter and only during transfer of Petri dishes to the luminometer. The filter reduced photon flux to ,1 mmol m22 s21. A single luminescence measurement was made for each mycelium between 1300 and 1600 daily on 3 successive d. Then the seven replicate mycelia of each Armillaria sp. in the ‘‘brief exposure’’ treatment were used in a ‘‘prolonged exposure’’ treatment of 10–12 h d21 fluorescent light (600–1800 daily), while the remaining eight replicate mycelia of each species were treated as before. After 4 d under the new light regime single bioluminescence measurements were made daily for each mycelium between 1200 and 1330 on 3 successive d. Experiment IL-2 examined the luminescence response when mycelia were exposed to incandescent light supplied by one cool white 60W bulb which provided a color rendering index of 100% (Osram Sylvania, Danvers, Massachusetts) and a photon flux (mean 6 se) of 13.3 6 1.33 mmol m22 s21. Separate sets comprising five replicate mycelia of the same genets representing the three Armillaria spp. were exposed either to incandescent light for 10 h d21 (i.e. ‘‘prolonged exposure’’), incandescent light only long enough for transfer of Petri dishes to the luminometer (i.e. ‘‘brief exposure’’) or only to ‘‘safe light’’ during transfer to the luminometer. Single luminescence measurements were made for each mycelium on 4 successive d. Variation in temporal dynamics of bioluminescence among species and genets.— Experiment TDA-3 addressed the variation among genets and between species in bioluminescence with mycelia growing on 1.5 M agar substrate as described above. Two replicate mycelia of each of four genets representing each Armillaria spp. (i.e. Triplets A, B, E and G) were included. Each of the 24 mycelia included in the test was initiated 6 d before luminescence measurement and maintained in its own individual light-tight cardboard box. Each Petri dish was transferred to the luminometer at the beginning of its measurement cycle with all sources of background light covered, including the LED display on the luminometer to avoid any confounding effects of environmental illumination. The luminescence by each mycelium was measured at 2 min intervals for 72 h. The order in

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which the six mycelia representing a Triplet were measured was determined by randomization, and Triplets were studied in the order, A, B, E and then G. Experiment TDW-4 addressed the variation in bioluminescence among genets and between species growing on sterile wood disks using a protocol similar to Experiment TDA-3. Two replicate mycelia of each of four genets representing each Armillaria sp. (i.e. Triplets A, C, E and H) were included. Root wood of Cornus florida L. was scrubbed gently under running tap water to remove soil. Disks (3–4 mm thick) were cut, autoclave sterilized for 1 h, then frozen until used. Each thawed disk was placed in a 35 mm diam Petri dish with sufficient 2% agar to slightly cover the disk. Mycelium was initiated from a 5 mm diam inoculum disk centered on the root wood disk. After 21 d growth in the dark the mycelial inoculum disk was removed and the Petri dish was placed in its own light-tight cardboard box until luminescence measurements were initiated 48 h later. All other conditions were as described for Experiment TDA-3. Four response variables were calculated for each mycelium included in Experiments TDA-3 and TDW-4: mean luminescence (RLU sec21); maximum luminescence (RLU sec21); luminescence amplitude (i.e. maximum minimum21); and the number of local maxima over the 72 h. A local maximum was defined as a luminescence measurement preceded and followed by a lower value. Bioluminescence in response to mechanical disturbance.— Experiment TDMD-5 examined the changes in luminescence in response to mechanical disturbance of mycelia grown on 1.5 M in the dark. Four replicate mycelia of each of two genets representing each Armillaria sp. (i.e. matched Triplets A and C) were included. Thirteen days after inoculation, luminescence of each mycelium was measured. Then two replicate mycelia of each genet were disturbed by sharply tapping the Petri dish on the lab bench. Luminescence was measured 0, 2 and 4 h after tapping. Luminescence of the two remaining replicate mycelia per genet initially was measured twice with gentle handling during measurements. This disturbance and measurement cycle was repeated the next day. On the following 5 d mycelia were not subject to disturbance or luminescence measurement. Then two additional measurement cycles followed during which disturbance treatments were reversed such that mycelia that were undisturbed during the first two measurement cycles received a disturbance treatment during the third and fourth measurement cycles and previously disturbed mycelia were handled gently. Statistical analysis.—For each combination of Armillaria isolate and measurement day in Experiment FL-1, treatment means were compared with the student’s t-test (Sokal and Rohlf 1995) with the TTEST procedure of the SAS 9.1 statistical software (#2003 SAS Institute Inc., Cary, North Carolina). Similarly for each combination of Armillaria isolate and measurement day in Experiment IL-2, treatment means were compared with one-way analysis of variance (Sokal and Rohlf 1995) with the GLM procedure of the SAS statistical software. In Experiments TDA-3 and TDW-4 response variable

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FIGS. 1–3. Luminescence by Armillaria mycelia in response to a brief exposure to fluorescent light (.) or darkroom ‘‘safe light’’ ( ) immediately before measurement in Experiment FL-1. 1. A. gallica genet C6. 2. A. mellea genet OZ550. 3. A. tabescens genet B1. Symbols are treatment means of seven or eight replicates for fluorescent and safe light treatments, respectively. Luminescence units, RLU sec21, expressed as multiples of base 10 exponents (e). * indicates means statistically different using the Student’s ttest, P , 0.05.

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FIGS. 4–6. Luminescence by Armillaria mycelia grown with prolonged exposures (10–12 h daily) to fluorescent light (.) or a brief exposure to darkroom ‘‘safe light’’ ( ) immediately before measurement. 4. A. gallica genet C6. 5. A. mellea genet OZ550. 6. A. tabescens genet B1. Symbols are treatment means of seven or eight replicates for fluorescent and safe light treatments, respectively. Luminescence units, RLU sec21, expressed as multiples of base 10 exponents (e). * indicates means statistically different using the Student’s ttest, P , 0.05.

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MIHAIL AND BRUHN: ARMILLARIA BIOLUMINESCENCE DYNAMICS

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means were compared among the three Armillaria spp. with the nonparametric Kruskal-Wallis test (Sokal and Rohlf 1995) as data were non-normally distributed. Analyses were conducted with the NPAR1WAY procedure of the SAS statistical software. RESULTS

Influence of environmental illumination.— Experiments FL-1 and IL-2 were conducted to quantify the effects of environmental illumination on luminescence expression. Brief exposure to fluorescent light resulted in significantly reduced bioluminescence for A. mellea as compared with exposure only to the OC filtered darkroom ‘‘safe light’ on the third measurement day, while the other two species were unaffected (FIG. 2 vs. FIGS. 1, 3). Prolonged exposure (10 h daily) of mycelia to fluorescent light significantly enhanced the luminescence of A. gallica but greatly reduced luminescence by A. mellea and A. tabescens (FIG. 4 vs. FIGS. 5–6). Prolonged exposure to incandescent light significantly enhanced the luminescence of A. gallica mycelia but did not consistently change the magnitude of luminescence by A. mellea and A. tabescens mycelia (FIG. 7 vs. FIGS. 8–9). Brief exposure to incandescent light had no statistically detectable effect on luminescence of any of the Armillaria spp (FIGS. 7–9).

FIGS. 7–9. Luminescence by Armillaria mycelia in response to three light regimes in Experiment IL-2: brief exposure to darkroom ‘‘safe light’’ immediately before measurement ( ); brief exposure to incandescent light immediately before measurement (.); or grown with prolonged exposures (10 h daily) to incandescent light (m). 7. A. gallica genet C6. 8. A. mellea genet OZ550. 9. A. tabescens genet B1. Symbols are treatment means (N 5 5). Luminescence units, RLU sec21, expressed as multiples of

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Variation in temporal dynamics among species and genets.—Experiments TDA-3 and TDW-4 addressed inter- and intragenet variation in luminescence over 72 h for the three Armillaria spp. grown on 1.5 M agar or sterilized wood disks. All 18 genets examined, regardless of species, were constitutively luminescent with measured mean luminescence of at least 104 RLU s21 (TABLE I) which was far greater than the background luminescence of the uncolonized agar media (i.e. 33 RLU s21). Significant differences among the three Armillaria species were detected with respect to four response variables. The mean and maximum luminescence of A. tabescens mycelia was significantly lower than that of A. mellea or A. gallica mycelia regardless of substrate (TABLE I). In contrast significant differences in luminescence amplitude were substrate dependent. Mycelia of A. gallica grown on agar had the lowest luminescence amplitude in Experiment TDA-3. However mycelia of A. gallica and A. tabescens had similar luminescence amplitude when grown on wood (TABLE I). Mycelia of A. tabescencs exhibited significantly greater fluctuation r base 10 exponents (e). * indicates means statistically different using one-way ANOV, P , 0.05.

346 TABLE I.

MYCOLOGIA Luminescence by mycelia of three Armillaria species grown on agar or wood disks in Experiments TDA-3 and TDW-4 Speciesa

Substrate Agar

Wood

Response variable Mean (RLU sec21) Maximum (RLU sec21) Amplitude No. local maxima Mean (RLU sec21) Maximum (RLU sec21) Amplitude No. local maxima

A. gallica 3.5 3 5.2 3 2.52 528 4.1 3 1.2 3 13.31 438

105 105

105 106

A. mellea 1.8 3 2.8 3 12.66 381 1.2 3 4.9 3 29.02 350

106 106

106 106

Kruskal-Wallis testb A. tabescens 1.2 3 3.2 3 18.15 652 1.2 3 3.7 3 12.32 669

104 104

104 104

x2

P

8.83 8.44 6.22 6.66 11.35 11.67 1.03 9.62

0.012 0.015 0.045 0.036 0.003 0.003 0.597 0.008

a

For each species tabular values are response variable means from luminescence measurements of two replicate mycelia for each of four genets (N 5 8). For each mycelium response variables were calculated from luminescence measurements (RLU sec21, relative light units) made every 2 min for 72 h. Amplitude 5 maximum minimum21. Luminescence local maxima were defined as luminescence values preceded and followed by lower values. Background luminescence of uncolonized 1.5 M was 33 RLU sec21. Background luminescence of uncolonized wood disks was 45 RLU sec21. b Response variable means were compared with the nonparametric Kruskal-Wallis test (Sokal and Rohlf 1995).

in luminescence than mycelia of A. mellea or A. gallica regardless of substrate as measured by the frequency of local maxima (TABLE I). Inspection of all 48 luminescence time series for mycelia in Experiments TDA-3 and TDW-4 did not provide any evidence of diurnal periodicity for any of the three Armillaria spp. (FIGS. 10–15, representative data). Temporal dynamics of luminescence were often complex, with little intraspecific similarity. Furthermore there was little similarity in the temporal dynamics of luminescence when comparing mycelia of the same genet grown on 1.5 M agar vs. on wood (FIGS. 10–15, representative data). The variation in temporal dynamics observed among species and genets extended to replicate mycelia (FIGS. 16–17, representative data). In all cases we measured only mycelial luminescence because rhizomorphs had not yet formed. In some instances replicate mycelia did exhibit similar temporal dynamics (data not shown). Bioluminescence and mechanical disturbance.—Experiment TDMD-5 focused on mechanical disturbance as one putative conditioning factor to explain complex temporal dynamics such as those observed in Experiments TDA-3 and TDW-4. Each mycelium was subjected to two measurement cycles, each comprising four luminescence measurements: immediately before a mechanical disturbance event and at 0, 2 and 4 h after the event. Each mycelium also was subjected to two similar measurement cycles lacking a disturbance event. For each measurement cycle, luminescence of each mycelium was normalized by the first measurement in the cycle (FIGS. 18–23). Thus a relative luminescence of 1 represents no change in luminescence relative to the first measurement. Both genets of A. gallica showed dramatically enhanced

luminescence 2 and 4 h after a mechanical disturbance event (FIGS. 18 and 19). The response of A. mellea and A. tabescens mycelial luminescence to mechanical disturbance was mixed. For example enhanced luminescence by A. mellea genet OZ179 was detected twice (FIG. 21) while no clear enhancement was detected for A. mellea genet OZ498 (FIG. 20). Enhanced luminescence similarly was detected for A. tabescens genet OZ865 (FIG. 22) but not for A. tabescens genet OZ795 (FIG. 23). None of the evaluated mycelia showed a response to gentle placement in the luminometer. DISCUSSION

The quenching effect of fluorescent light on A. mellea bioluminescence has been documented (Bermudes et al 1990, Weitz et al 2001). However we found differences among Armillaria spp. in their luminescence response to environmental illumination. Prolonged environmental illumination, whether fluorescent or incandescent, enhanced the luminescence of the more necrotrophic A. gallica. In contrast prolonged exposure to fluorescent illumination quenched the luminescence of the more parasitic A. mellea and A. tabescens while incandescent illumination had little effect. Our observations of strong influences of environmental illumination on bioluminescence prompted us to conduct all further tests with mycelia incubated in complete darkness and transferred to the luminometer in darkness with the LED instrument display covered to eliminate these influences on experimental measurements. All 18 genets representing the three Armillaria spp. included in our studies were consistently luminescent throughout 72 h observation periods. Furthermore


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FIGS. 10–15. Luminescence by Armillaria mycelia of Triplet E measured every 2 min for 72 h. FIGURES 10 and 11, 12 and 13 and 14 and 15 represent time series for two replicate mycelia of A. gallica genet OZ857, A. mellea genet OZ604 and A. tabescens genet OZ333, respectively. FIGURES 10, 12 and 14 represent mycelia grown on 1.5 M agar in Experiment TDA-3. FIGURES 11, 13 and 15 represent mycelia grown on wood in Experiment TDW-4. Each panel represents one randomly selected replicate mycelium (of two) for the combination for genet and substrate. Luminescence units, RLU sec21, expressed as multiples of base 10 exponents (e).

our studies demonstrated consistent and statistically significant differences in the luminescence responses of the three Armillaria spp. We observed significantly higher luminescence magnitude for A. gallica and A. mellea mycelia compared with those of A. tabescens. Weitz et al (2001) reported luminescence magnitude for A. mellea in the range 102 to 104 RLU (when correcting for the sensitivity factor they employed),

which was lower than our observations by a factor of at least 102 RLU. The discrepancy might be attributable both to differences among isolates used in the studies as well as differences between the two luminometers employed. Shimomura (1992) reported a coordinate increase in luminescence and mycelial weight for A. mellea and other fungi. In contrast we observed the lowest

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FIGS. 16–17. Luminescence time series for two replicate mycelia of Armillaria gallica genet OZ446 in Experiment TDA-3. Luminescence of each mycelium was measured every 2 min for 72 h. Luminescence units, RLU sec21, expressed as multiples of base 10 exponents (e).

luminescence intensity for A. tabescens, the species with the greatest linear extension rate. Thus differences in observed luminescence intensity are not attributable to differences in mycelial growth rate. The luminescence amplitude of the three species was responsive to substrate. In contrast, regardless of substrate, the luminescence of A. tabescens mycelia fluctuated at a rate nearly twice that of A. mellea mycelia as measured by a higher frequency of luminescence local maxima. While we were able to demonstrate significant and consistent differences among the Armillaria spp. in the overall luminescence response, the temporal dynamics of luminescence was varied and complex. There was no evidence of the diurnal periodicity reported previously (Berliner 1961, Calleja and Reynolds 1970). Given the sensitivity of Armillaria luminescence to environmental illumination and other cues, perhaps previously reported luminescence patterns actually were conditioned by unrecognized periodic environmental phenomena. The disparate bioluminescence time series patterns observed for replicate mycelia were surprising because all replicates of a particular genet–treatment combination within an experiment were prepared from a single mycelium to reduce variability attributable to inoculum source. Such variation suggests that the expression of bioluminescence is conditioned by subtle environmental cues. The issue of variability in luminescence was highlighted by Wassink (1978) who noted studies in which luminescent fungal species have both luminescent and nonluminescent strains. Similarly the results of Weitz et al (2001) demonstrated intrastrain variation in the luminescent response of one A. mellea isolate. Indeed it is the sensitivity of fungal bioluminescence to environmental context that prompted Weitz et al (2002) to develop a toxicity assay based on fungal mycelial luminescence.

Among the bioluminescent marine organisms, dinoflagellates (Dinoflagellata) are of interest because they emit light flashes in response to a variety of stimuli including mechanical disturbance (Harvey 1952, Hastings 1978). When we examined the luminescence of the three Armillaria spp. in response to mechanical disturbance we observed that luminescence of A. gallica genets was greatly enhanced by a single disturbance event. The effect lasted at least 4 h in some instances. While mechanical disturbance did enhance the luminescence of one genet each of A. mellea and A. tabescens, the effect was much less consistent than that observed for A. gallica. Shimomura (1991, 1992) demonstrated that luminescence of several species increased during active mycelial growth, which generates superoxide. Shimomura (1992) proposed that luminescence intensity is inversely related to local concentrations of superoxide dismutase (SOD). One avenue for future investigation of Armillaria luminescence should be the local short-term accumulation/degeneration of SOD in response to mechanical disturbance. Our studies have revealed consistent differences among the three sympatric Armillaria spp. in the expression of bioluminescence. The necrotrophic A. gallica exhibited bioluminescence that was enhanced by both environmental illumination and mechanical disturbance. In contrast the more parasitic A. mellea and A. tabescens exhibited bioluminescence quenched by environmental illumination and only occasionally responsive to mechanical disturbance. Intragenet variability in luminescence magnitude was least for A. tabescens which has the most stringent requirements for the production of foraging rhizomorphs (Mihail et al 2002). Nevertheless intragenet variability was remarkable, considering that replicate mycelia were initiated as disks taken from the actively growing margin of the same mycelium.


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FIGS. 18–23. Luminescence of Armillaria spp. in response to mechanical disturbance over four measurement cycles in Experiment TDMD-5. 18. A. gallica genet OZ97. 19. A. gallica genet OZ182. 20. A. mellea genet OZ498. 21. A. mellea genet OZ179. 22. A. tabescens genet OZ865. 23. A. tabescens genet OZ795. For each genet four replicate mycelia were subjected to two measurement cycles with disturbance and two measurement cycles without disturbance. A disturbance treatment comprised luminescence measurements immediately before mechanical disturbance and at 0, 2 and 4 h after disturbance. Mycelia not receiving a disturbance treatment were measured twice at the beginning of the measurement cycle and subsequently after 2 and 4 h. For each mycelium luminescence at 0, 2, and 4 h post-treatment was normalized by the first (pretreatment) measurement in the cycle. Each symbol represents one post-treatment observation. Symbols ( ) and (%) indicate isolates disturbed in measurement cycles 1 and 2 or 3 and 4, respectively.

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While the phenomenon of bioluminescence is common among these three Armillaria spp., the differences in its expression may reflect a diversity of selection pressures acting upon it. Bioluminescence might have a distinct ecological context for each of these fungal species.

ACKNOWLEDGMENTS

This research was supported by Missouri Agricultural Experiment Station Project PSSL0112. The authors gratefully acknowledge the Missouri Department of Conservation Missouri Ozark Forest Ecosystem Project (http://mofep. conservation.state.mo.us) as the source of all Armillaria

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MYCOLOGIA

isolates except A. gallica C6. Contribution from the Missouri Agricultural Experiment Station. LITERATURE CITED

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