f u n g a l b i o l o g y 1 1 9 ( 2 0 1 5 ) 5 2 8 e5 3 7
journal homepage: www.elsevier.com/locate/funbio
Bioluminescence patterns among North American Armillaria species Jeanne D. MIHAIL* Division of Plant Sciences, 110 Waters Hall, University of Missouri, Columbia, MO 65211, USA
article info
abstract
Article history:
Bioluminescence is widely recognized among white-spored species of Basidiomycota. Most
Received 11 September 2014
reports of fungal bioluminescence are based upon visual light perception. When instru-
Received in revised form
ments such as photomultipliers have been used to measure fungal luminescence, more
31 December 2014
taxa have been discovered to produce light, albeit at a range of magnitudes. The present
Accepted 17 February 2015
studies were undertaken to determine the prevalence of bioluminescence among North
Available online 25 February 2015
American Armillaria species. Consistent, constitutive bioluminescence was detected for
Corresponding Editor:
the first time for mycelia of Armillaria calvescens, Armillaria cepistipes, Armillaria gemina, Ar-
Martin I. Bidartondo
millaria nabsnona, and Armillaria sinapina and confirmed for mycelia of Armillaria gallica, Ar-
Keywords:
representing all species had maximum intensity in the range 515e525 nm confirming
Diploid mycelium
that emitted light was the result of bioluminescence rather than chemiluminescence.
Emission spectrum
Time series analysis of 1000 consecutive luminescence measurements revealed a highly
Haploid mycelium
significant departure from random variation. Mycelial luminescence of eight species ex-
Mechanical disturbance
hibited significant, stable shifts in magnitude in response to a series of mechanical distur-
North American Biological Species
bance treatments, providing one mechanism for generating observed luminescence
millaria mellea, Armillaria ostoyae, and Armillaria tabescens. Emission spectra of mycelia
variation. ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction While the bioluminescence of basidiomata has been recognized for decades, the light produced by fungi in decaying wood has been recognized for centuries (Harvey 1952; Wassink 1978). Presently, 71 luminescent fungal species have been confirmed belonging to three distinct lineages, Omphalotus, Armillaria, and mycenoid (Desjardin et al. 2008, 2010). Confirmed reports of fungal bioluminescence are presently restricted to white-spored members of the Basidiomycota (Wassink 1978; Desjardin et al. 2008). A single biochemical light-producing system appears to be shared by all bioluminescent fungi (Oliveira et al. 2012). Fungal bioluminescence is
characterized by maximum light emission in the range 520e535 nm (e.g., Coblentz & Hughes 1926; Desjardin et al. 2008; Oliveira & Stevani 2009) irrespective of species. However, the majority of studies of fungal bioluminescence rely on human perception of light emission (Bermudes et al. 1992; Desjardin et al. 2008). When Bermudes and co-workers (1992) used a photomultiplier to measure the luminescence of several fungal genera, they were able to demonstrate detectable bioluminescence from basidiomata which were not luminescent to human observers. They inferred that there remain many fungal taxa with undiscovered luminescent properties. Among the widely-recognized luminescent taxa are four species of the white-rot wood-decay genus Armillaria (i.e.,
* Tel.: þ11 573 882 0574. E-mail address: [email protected] http://dx.doi.org/10.1016/j.funbio.2015.02.004 1878-6146/ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Bioluminescence patterns among Armillaria
529
Armillaria gallica, Armillaria mellea, Armillaria ostoyae, and Armillaria tabescens) (Wassink 1978; Rishbeth 1986; Mihail & Bruhn 2007; Desjardin et al. 2008). Armillaria is a genus with a wide host range and diverse interactions with host plants ranging from pathogenesis to necrotrophic wood decay to mutualism as a mycorrhizal partner with orchids (Fox 2000; Baumgartner et al. 2011). At least ten Armillaria species are currently recognized from North America (Volk & Burdsall 1995; Baumgartner et al. 2011; Ross-Davis et al. 2012). Although Armillaria mycelia are luminescent, light has not been documented in association with fruiting bodies (Harvey 1952; Wassink 1978). Previously, we reported constitutive luminescence of mycelia of several genets of A. gallica, A. mellea, and A. tabescens growing on agar and wood (Mihail & Bruhn 2007). Moreover, luminescence of A. gallica mycelia was consistently stimulated by mechanical disturbance. More recently, temporal luminescence patterns of the same Armillaria species were found to have quantifiable structure, representing significant departures from random variation (i.e., white noise processes) (Mihail 2013). During these studies, transient spikes of high-magnitude luminescence were episodically observed at random positions in the time series (Mihail 2013). The predominant form of most mushroom-producing members of the Basidiomycota is a dikaryon mycelium. The haploid mycelium resulting from germination and growth of basidiospores is of relatively short duration for these fungi. Armillaria spp. are unusual among the Basidiomycota in that the primary assimilative phase is a diploid mycelium (Baumgartner et al. 2011). Berliner (1963) reported that while diploid mycelia of Armillaria mellea were consistently luminescent, haploid mycelia derived from single basidiospores produced light inconsistently and for short periods although specific data were not provided. Bemudes et al. (1992) noted a similar phenomenon when comparing dikaryon and monokaryon mycelia of Panellus stipticus and Lampteromyces japonicas. In this latter study, light production by monokaryon mycelia was several orders of magnitude less than corresponding dikaryotic mycelia. The present research was undertaken to address four objectives: (i) to quantitatively compare luminescence of haploid and diploid mycelia of Armillaria; (ii) to explore the magnitude and temporal dynamics of light production by nine North American Armillaria species, of which five were not known to emit light; (iii) to characterize the emission spectra of newly
confirmed bioluminescent Armillaria species; and (iv) to quantitatively examine the role of mechanical disturbance in eliciting transient and/or stable changes in luminescence magnitude.
Materials and methods Strains used The investigation of luminescence of haploid and diploid mycelia was conducted with 21 isolates derived from fruiting bodies collected in connection with the Missouri Ozark Forest Ecosystem Project (MOFEP, Bruhn et al. 2000). Three fruiting bodies of Armillaria mellea collected from different locations in the upland Ozark forests were used to obtain cultures from three distinct genets (Table 1). One fruiting body of A. gallica was used to obtain diploid and haploid cultures (Table 1). Experiments focused on temporal luminescence patterns or luminescence in response to disturbance utilized three genets of each of nine North American Armillaria species obtained from the Forest Products Laboratory (FPL), U.S. Department of Agriculture, U.S. Forest Service, Madison, WI, USA (Table 2). All FPL isolates were monokaryons derived from single basidiospores. All isolates were maintained as active, working cultures on 2 % malt extract agar (2 M) during experimentation. When not in active use, isolates were maintained as archival cultures at 5 C in sterile distilled water or as archival cultures at 80 C as previously described (Mihail 2013).
Bioluminescence measurement and experimental design The comparison of bioluminescence of diploid and haploid mycelia used mycelia growing on 1.5 % malt extract agar (1.5 M) in 35-mm diameter plastic Petri dishes. Three replicate mycelia represented each of the 21 isolates included in the study. Bioluminescence was measured as relative light units per second (RLU$s1) using a Zylux FB-12 single tube luminometer (Zylux Corp., Oak Ridge, TN) which could accommodate Petri dishes of 35 mm diameter or less. Bioluminescence measured in one replicate consisted of 80 measurements of 1 s duration with a 14 s gap between consecutive measurements. All mycelia represented 7 d growth at the time of bioluminescence measurement owing to the
Table 1 e Armillaria isolates used in the comparison of bioluminescence among diploid and haploid mycelia. Species
Isolate
Ploidy
Locationa
A. gallica
OZ1502 OZ1502e5, OZ1502e10, OZ1502e13, OZ1502e14 OZ221 OZ221e1, OZ221e2, OZ221e4,OZ221e5, OZ221e6 OZ222 OZ222e1, OZ222e2, OZ222e3, OZ222e4 OZ1417 OZ1417e1, OZ1417e2, OZ1417e3, OZ1417e6
2N 1N 2N 1N 2N 1N 2N 1N
2e28 2e28 4e15 4e15 4e26 4e26 2e28 2e28
A. mellea
a All isolates were collected from the Ozark Mountains of Missouri, USA in conjunction with a long-term forest health study. Isolate locations are recorded in a ‘siteeplot’ format as indicated in Bruhn et al. 2000.
530
J. D. Mihail
Table 2 e Bioluminescence consistency among haploid mycelia of North American Armillaria species. Species
A. calvescens
A. cepistipes
A. gallica
A. gemina
A. mellea
A. nabsnona
A. ostoyae
A. sinapina
A. tabescens
Genet
FPL19 FPL20 FPL21 FPL37 FPL38 FPL39 FPL28 FPL29 FPL30 FPL16 FPL17 FPL18 FPL25 FPL26 FPL27 FPL31 FPL32 FPL33 FPL13 FPL14 FPL15 FPL22 FPL23 FPL24 FPL34 FPL35 FPL36
Original IDa
FFCe7, sse7 ILe7, sse4 TJVe94e114, sse1 TJVe93e167, sse6 HHBe14868, sse2 HHBe14867, sse7 MNe3, sse1 ILe30, sse3 133e2, sse1 JJWe233, sse1 Mielke, sse14 TJVe94e47, sse1 SHe7, sse10 ILe40, sse3 DAe2, sse2 HBe21, sse8 TJVe93e200, sse5 TJVe93e188, sse4 ONe19, sse4 FLe2, sse9 HHBe12879, sse7 HHBe14911, sse9 PRe7, sse9 ONe18, sse2 PORe100, sse8 De83, sse10 De85, sse1
No. luminescent at measurement interval (ms)b 100
200
400
800
1600
1 0 2 0 0 4 1 4 0 1 4 3 4 0 4 4 4 4 2 4 4 0 2 3 2 0 2
1 0 2 0 0 4 0 4 0 0 4 4 4 1 4 4 4 4 2 4 4 0 3 3 3 0 2
1 1 3 0 0 4 0 4 0 0 4 4 4 2 4 4 4 4 2 4 4 0 3 3 3 0 2
1 1 3 0 0 4 1 4 0 0 4 4 4 4 4 4 4 4 3 4 4 0 4 3 4 0 2
1 2 3 0 0 4 1 4 0 0 4 4 4 4 4 4 4 4 4 4 4 0 4 3 4 0 2
a All isolates were obtained from the Forest Products Laboratory, USDA, US Forest Service, Madison, WI, USA, with the original identifying designations as indicated. b Tabular values are the number of replicate mycelia (of four) from which light was consistently detected over 1000 measurements at the specified measurement interval (i.e., minimum luminescence > 0 RLU).
limitation of the size of Petri dish accommodated by the instrument. The bioluminescence of the nine North American Armillaria species was assessed using mycelia growing on 2 % malt extract agar (2 M) in 95emm diameter plastic Petri dishes. Four replicate mycelia represented each of the 27 isolates included in the study. Bioluminescence intensity was measured using a Sens-Tech photomultiplier tube detector, model P25232 (Sens-Tech, Ruislip, Middlesex, UK) which was not contained in a luminometer allowing for flexibility in the size of the Petri dish and the arrangement of sample and detector. For each replicate mycelium, a series of 1000 measurements was made of total light (RLU) emitted during a specified interval. The five measurement intervals used for each replicate mycelium were: 100, 200, 400, 800, and 1600 ms. Measurements were consecutive without intervening gaps. All mycelia were 21e28 d old at measurement. At the conclusion of each series of luminescence measurements, colony diameter was recorded. Emission spectra are typically measured using a spectrometer which parses unamplified incident light into user-defined bands of wavelengths. Preliminary studies of Armillaria mycelial emission spectra utilized an Ocean Optics USB2000þ Fiber Optic Spectrometer (Ocean Optics, Dunedin, FL, USA) and the iterative spectrum measurement protocol of Oba et al. (2013) for documenting low-intensity light emission. This approach was not sufficiently sensitive to produce reliable emission
spectra for the Armillaria mycelia. The Sens-Tech photomultiplier tube detector, noted above, represents the most sensitive method for measuring low light emission (Berthold et al. 2000). Bandpass filters allow only specific bands of wavelengths to be transmitted through them. A novel approach to emission spectrum measurement was developed by placing a series of bandpass filters between a luminescent mycelium and the photomultiplier tube detector. Sixteen Edmund Optics Hard Coated OD4 filters were utilized to measure light in the range 412.5e687.5 nm (Edmund Optics, Barrington, NJ, USA). Specifically, eight 25-nm-band width filters were used with central wavelengths of 425, 450, 475, 500, 600, 625, 650 and 675 nm and 90 % minimum transmittance. For example, the filter with a central wavelength of 425 nm permitted only light in the range 412.5e437.5 nm to be transmitted from the luminescent mycelium to the photomultipler tube detector. Eight 10nm-band width filters were used with central wavelengths of 510, 520, 532, 540, 550, 560, 568, and 580 nm and 85 % minimum transmittance. All bandpass filters were 25 mm in diameter with identical transmission areas. To characterize one emission spectrum, a series of 50 consecutive measurements of 1 s duration were made with each of the 16 bandpass filters. For each mycelium, bandpass filters were changed without disturbing the mycelium. A complete emission spectrum was measured in ca. 20 min so that bioluminescence intensity was temporally stationary.
Bioluminescence patterns among Armillaria
531
The effect of mechanical disturbance on luminescence of the nine Armillaria species was assessed using mycelia 21e28 d old growing on 2 M. Three replicate mycelia representing each of the 27 isolates used in the study were assessed with a sequence of four series of 500 consecutive measurements of 500 ms duration each. During the first series of 500 measurements the mycelium was not disturbed so that a baseline luminescence magnitude was established. Subsequently, the mycelium was disturbed by application of a concentrated shock (200 N) using a chiropractic adjusting tool (JTECH Medical, Salt Lake City, UT, USA) within 15 s of the initiation of the series of 500 light measurements. Thus, a complete disturbance sequence for one replicate mycelium comprised a baseline luminescence series (500 measurements) followed by three disturbance-response series (500 measurements each). Less than 30 s elapsed between the conclusion of one component of the sequence and the initiation of the subsequent component. The Parafilm seal on each Petri dish was not disturbed or removed during or following disturbance treatments. Bioluminescence was measured using a Sens-Tech photomultiplier tube detector, as noted above. To avoid the confounding effects of environmental illumination, each Petri dish was placed in its own light-tight cardboard box between the initiation of the mycelium and the first luminescence measurement for an experiment. Petri dishes remained sealed with Parafilm (Pechiney Plastic Packaging, Chicago, IL, USA) during luminescence measurement. All bioluminescence measurements were obtained in a laboratory maintained at 22e26 C. Prior to each experiment, background luminescence of the agar medium was determined using at least 12 observations of the same duration as the experimental measurement interval. When band pass filters were used to measure emission spectra, the background luminescence was not altered by any of the 16 filters used. Each bioluminescence measurement taken was then reduced by a quantity representing the mean background luminescence designed to minimize the possibility of ascribing luminescence to an isolate which was not luminescent.
luminescence measurements. Time series analysis is used to detect cyclic patterns in a series of equally spaced, consecutive measurements (Fuller 1996; Warner 1998; Cryer & Chan 2008). Time series analysis is an appropriate analytical tool only if: (a) the data are normally distributed; (b) the mean value does not increase or decrease over the series (i.e., the series is stationary); and (c) the series variance is stable (Warner 1998; Cryer & Chan 2008). Time series analysis was applied to observations from the species comparison experiment using the 800 ms measurement interval where the analytical criteria were satisfied. Since all time series of 1000 observations were nonstationary, data were transformed by taking the ‘first difference’ in which an observation at time, t, is replaced by the difference between that observation and the preceding observation (Warner 1998; Cryer & Chan 2008). Differenced data were analysed using ordinary least squares regression to confirm the series’ stationarity by using PROC REG of SAS/ STAT. The ShapiroeWilk statistic, W, was calculated using PROC UNIVARIATE to confirm that data were normally distributed (Warner 1998; Cryer & Chan 2008). Time series analysis was conducted using PROC SPECTRA of SAS/STAT, in which a periodogram apportions total time series variance among a series of time intervals (i.e., periods). Departures from randomly fluctuating series were tested with Bartlett’s Kolmogorov-Smirnoff statistic (Fuller 1996). To assess the effect of mechanical disturbance upon luminescence magnitude, the mean luminescence magnitude of the baseline series (i.e., 500 measurements prior to disturbance) was compared with the mean magnitude of the final 450 values of the three series measured in response to disturbance shock. For each of the three response series, the first 50 luminescence values were removed as this portion of each series included pre-treatment luminescence levels as well as transient post-treatment luminescence spikes. Repeated measures ANOV were used to test the null hypothesis of no treatment effect, using the REPEATED option of PROC GLM in the SAS/STAT software. Specifically, the null hypothesis was tested as the contrast:
Data analysis
Lbase ¼ ½ðL1 þ L2 þ L3 Þ=3
Descriptive statistics measuring central tendency (i.e., mean, median, minimum, maximum) were calculated for all luminescence time series in each experiment. A series of luminescence measurements was considered to represent consistent luminescence only if the minimum value, after adjustment for background luminescence, was greater than zero. The relationship between luminescence intensity and colony diameter was assessed using PROC CORR of SAS/STAT analytical software (SAS/STAT System Release 9.3, Cary, NC, USA). Emission spectra were assembled from the mean luminescence intensity recorded for each of the 16 bandpass filters. The two groups of bandpass filters had identical transmission areas and similar minimum transmittances. Therefore, to account for the difference in light gathering capacity of 10 nm and 25 nm bandpass filters, the mean intensity measured using the 10 nm bandpass filters was increased by a factor of 2.5. In a previous study of Armillaria luminescence (Mihail 2013), the technique of time series analysis was useful in quantitatively characterizing the temporal dynamics of
where Lbase is the mean luminescence of the baseline series; L1, L2, and L3 are the mean luminescence magnitudes of the final 450 values of the three disturbance response luminescence series.
Results Luminescence of diploid vs monokaryotic mycelia Consistent bioluminescence was detected for all replicate mycelia of Armillaria mellea and Armillaria gallica irrespective of the haploid or diploid nuclear state (Fig 1). For all four genets examined, the greatest mean luminescence was expressed by one or more of the monokaryon single-spore strains, rather than the original diploid strain derived from the fruiting body (Fig 1). For A. mellea genet 222 and A. gallica genet 1502, three of four monokaryon strains expressed mean luminescence intensity at least as great as the original diploid strain (Fig 1).
532
J. D. Mihail
Fig 1 e Luminescence of diploid and haploid Armillaria mycelia. (A) A. mellea genet OZ221; (B) A. mellea genet OZ222; (C) A. mellea genet OZ1417; (D) A. gallica genet OZ1502. In each panel the first symbol represents the diploid mycelium; subsequent symbols represent haploid mycelia derived from single basidiospores. Each symbol with error bars represents the mean luminescence (RLU sL1) with standard deviation based on three replicate mycelia.
Luminescence of nine Armillaria species Luminescence was consistently detected for at least one of three genets for each of the nine North American Armillaria species (Table 2). Consistent bioluminescence was defined as detectable luminescence after adjustment for background for all 1000 observations in a series. Luminescence observations were made using five measurement intervals (i.e., 100e1600 ms). Only Armillaria nabsnona genets were consistently luminescent at all measurement intervals for all four replicate mycelia examined (Table 2). At the longest measurement interval (i.e., 1600 ms), consistent luminescence was detected from the three genets representing Armillaria calvescens, Armillaria mellea, Armillaria nabsnona and Armillaria ostoyae (Table 2). Only one of three Armillaria cepistipes genets was consistently luminescent (Table 2). The magnitude of luminescence varied among species, among genets within species, and among replicates within genets (Fig 2). The two luminescent genets of A. gallica, FPL28 and FPL29, had the lowest mean estimates of luminescence (102e103 RLU; Fig 2C) while the two luminescent genets of A. gemina, FPL17 and FPL18, had the highest mean estimates (107e108 RLU; Fig 2D). Mean luminescence estimates for the three A. nabsnona genets were the most similar (105e106 RLU; Fig 2F). For several genets, mean luminescence estimates varied by at least two orders of magnitude among the replicate mycelia (e.g., Fig 2F, A. nabsnona genet FPL32 and Fig 2H, A. sinapina genet FPL24). There was no significant relationship
between luminescence intensity and mycelium diameter for any genet or species. Emission spectra of luminescent mycelia of each of the nine Armillaria species showed maximum intensity using the bandpass filter with a central wavelength of 520 nm (Fig 3) which permitted detection of light in the range 515e525 nm. All emission spectra contained a notable positive skew (i.e., blue shift; Fig 3) and many contained a shoulder or decrease in intensity using the bandpass filters with central wavelengths of 550 or 560 nm (Fig 3). Luminescence time series of measurements at the 800 ms interval were examined to determine if evident variation was simply unstructured, random variation (Table 3). Of the 108 series available, 70 exhibited consistent luminescence and were analysed. Ninety percent of the consistently luminescent series met the criteria for time series analysis (Table 3). Where a series was not normally distributed, the reason was most often the presence of at least one exceptionally high or exceptionally low luminescence measurement (i.e., a transient spike or drop). Transient spikes were observed in luminescence series of A. calvescens (genets FPL20, FPL21), A. mellea (genet FPL26), A. ostoyae (genet FPL14), A. sinapina (genet FPL24) and A. tabsecens (genet FPL34). A transient drop was observed for one series of A. nabsnona (genet FPL34). For each of the nine species, at least 4 of the 12 series examined (i.e., three genets X four replicates) exhibited consistent luminescence (i.e., minimum > 0). All series which were normally distributed represented significant departures from random variation as
Bioluminescence patterns among Armillaria
533
Fig 2 e Mean luminescence of mycelia of nine North American Armillaria species. (A) A. calvescens genets FPL19, FPL20, FPL21; (B) A. cepistipes genets FPL37, FPL38, FPL39; (C) A. gallica genets FPL28, FPL29, FPL30; (D) A. gemina genets FPL16, FPL17, FPL18; (E) A. mellea genets FPL25, FPL26, FPL27; (F) A. nabsnona genets FPL31, FPL32, FPL33; (G) A. ostoyae genets FPL13, FPL14, FPL15; (H) A. sinapina genets FPL22, FPL23, FPL24; (I) A. tabescens genets FPL34, FPL35, FPL36. Each symbol with error bars represents the mean bioluminescence and standard deviations for 1000 consecutive 1.6 s observations using replicate mycelia which were consistently luminescent (e.g., minimum magnitude > 0 RLU).
determined by highly significant values of Bartlett’s KolmogoroveSmirnov statistic (Table 3).
Luminescence in response to mechanical disturbance The response of mycelial luminescence to a sequence of mechanical disturbance treatments revealed both transient and enduring responses to the disturbance (Fig 4). Transient responses were expressed as a short-duration spike in luminescence (e.g., Fig 4C, F, and K). Such luminescence spikes were observed for genets of A. cepistipes (genet FPL39), A. gallica (genet FPL29), A. gemina (genet FPL17), A. nabsnona (genets FPL31, FPL32, FPL33), A. ostoyae (genet FPL15), and A. sinapina (genets FPL23, FPL24). Enduring responses included a stable increase in luminescence magnitude (e.g., Fig 4B, E, K) or a stable decrease in magnitude (e.g., Fig 4D, I). Stable shifts in
luminescence magnitude in response to mechanical disturbance were documented for at least one genet of eight of the nine Armillaria species examined (Fig 4, Table 4). For seven of these species, both significant increases and decreases in magnitude were observed (Table 4). For example, luminescence magnitude of A. gemina mycelia was reduced by up to 41 % or increased by 318 % among the seven response sequences examined (Table 4). For each sequence of baseline and three response luminescence series, the null hypothesis that disturbance did not result in a shift in luminescence magnitude was examined using repeated measures ANOVA. For all sequences examined, the null hypothesis was rejected indicating significant shifts in mean luminescence in response to the mechanical disturbance treatment (Table 4). In this experiment, mycelia of A. tabescens did not produce consistent luminescence and thus were not included in the analyses.
534
J. D. Mihail
Fig 3 e Emission spectra of representative Armillaria mycelia. (A) A. calvescens genet FPL21; (B) A. cepistipes genet FPL39; (C) A. gallica genet FPL28; (D) A. gemina genet FPL16; (E) A. mellea genet FPL27; (F) A. nabsnona genet FPL31; (G) A. ostoyae genet FPL13; (H) A. sinapina genet FPL23; (I) A. tabescens genet FPL35. Each symbol represents the mean of 50 (1 s) measurements of light transmitted through a bandpass filter with the indicated central wavelength. All measurements were corrected for background luminescence of uncolonized agar with the band pass filters in place. Across the nine emission spectra, the variation in mean luminescence was inversely related to luminescence magnitude. The coefficients of variation associated with mean luminescence estimates were:
journal homepage: www.elsevier.com/locate/funbio
Bioluminescence patterns among North American Armillaria species Jeanne D. MIHAIL* Division of Plant Sciences, 110 Waters Hall, University of Missouri, Columbia, MO 65211, USA
article info
abstract
Article history:
Bioluminescence is widely recognized among white-spored species of Basidiomycota. Most
Received 11 September 2014
reports of fungal bioluminescence are based upon visual light perception. When instru-
Received in revised form
ments such as photomultipliers have been used to measure fungal luminescence, more
31 December 2014
taxa have been discovered to produce light, albeit at a range of magnitudes. The present
Accepted 17 February 2015
studies were undertaken to determine the prevalence of bioluminescence among North
Available online 25 February 2015
American Armillaria species. Consistent, constitutive bioluminescence was detected for
Corresponding Editor:
the first time for mycelia of Armillaria calvescens, Armillaria cepistipes, Armillaria gemina, Ar-
Martin I. Bidartondo
millaria nabsnona, and Armillaria sinapina and confirmed for mycelia of Armillaria gallica, Ar-
Keywords:
representing all species had maximum intensity in the range 515e525 nm confirming
Diploid mycelium
that emitted light was the result of bioluminescence rather than chemiluminescence.
Emission spectrum
Time series analysis of 1000 consecutive luminescence measurements revealed a highly
Haploid mycelium
significant departure from random variation. Mycelial luminescence of eight species ex-
Mechanical disturbance
hibited significant, stable shifts in magnitude in response to a series of mechanical distur-
North American Biological Species
bance treatments, providing one mechanism for generating observed luminescence
millaria mellea, Armillaria ostoyae, and Armillaria tabescens. Emission spectra of mycelia
variation. ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction While the bioluminescence of basidiomata has been recognized for decades, the light produced by fungi in decaying wood has been recognized for centuries (Harvey 1952; Wassink 1978). Presently, 71 luminescent fungal species have been confirmed belonging to three distinct lineages, Omphalotus, Armillaria, and mycenoid (Desjardin et al. 2008, 2010). Confirmed reports of fungal bioluminescence are presently restricted to white-spored members of the Basidiomycota (Wassink 1978; Desjardin et al. 2008). A single biochemical light-producing system appears to be shared by all bioluminescent fungi (Oliveira et al. 2012). Fungal bioluminescence is
characterized by maximum light emission in the range 520e535 nm (e.g., Coblentz & Hughes 1926; Desjardin et al. 2008; Oliveira & Stevani 2009) irrespective of species. However, the majority of studies of fungal bioluminescence rely on human perception of light emission (Bermudes et al. 1992; Desjardin et al. 2008). When Bermudes and co-workers (1992) used a photomultiplier to measure the luminescence of several fungal genera, they were able to demonstrate detectable bioluminescence from basidiomata which were not luminescent to human observers. They inferred that there remain many fungal taxa with undiscovered luminescent properties. Among the widely-recognized luminescent taxa are four species of the white-rot wood-decay genus Armillaria (i.e.,
* Tel.: þ11 573 882 0574. E-mail address: [email protected] http://dx.doi.org/10.1016/j.funbio.2015.02.004 1878-6146/ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Bioluminescence patterns among Armillaria
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Armillaria gallica, Armillaria mellea, Armillaria ostoyae, and Armillaria tabescens) (Wassink 1978; Rishbeth 1986; Mihail & Bruhn 2007; Desjardin et al. 2008). Armillaria is a genus with a wide host range and diverse interactions with host plants ranging from pathogenesis to necrotrophic wood decay to mutualism as a mycorrhizal partner with orchids (Fox 2000; Baumgartner et al. 2011). At least ten Armillaria species are currently recognized from North America (Volk & Burdsall 1995; Baumgartner et al. 2011; Ross-Davis et al. 2012). Although Armillaria mycelia are luminescent, light has not been documented in association with fruiting bodies (Harvey 1952; Wassink 1978). Previously, we reported constitutive luminescence of mycelia of several genets of A. gallica, A. mellea, and A. tabescens growing on agar and wood (Mihail & Bruhn 2007). Moreover, luminescence of A. gallica mycelia was consistently stimulated by mechanical disturbance. More recently, temporal luminescence patterns of the same Armillaria species were found to have quantifiable structure, representing significant departures from random variation (i.e., white noise processes) (Mihail 2013). During these studies, transient spikes of high-magnitude luminescence were episodically observed at random positions in the time series (Mihail 2013). The predominant form of most mushroom-producing members of the Basidiomycota is a dikaryon mycelium. The haploid mycelium resulting from germination and growth of basidiospores is of relatively short duration for these fungi. Armillaria spp. are unusual among the Basidiomycota in that the primary assimilative phase is a diploid mycelium (Baumgartner et al. 2011). Berliner (1963) reported that while diploid mycelia of Armillaria mellea were consistently luminescent, haploid mycelia derived from single basidiospores produced light inconsistently and for short periods although specific data were not provided. Bemudes et al. (1992) noted a similar phenomenon when comparing dikaryon and monokaryon mycelia of Panellus stipticus and Lampteromyces japonicas. In this latter study, light production by monokaryon mycelia was several orders of magnitude less than corresponding dikaryotic mycelia. The present research was undertaken to address four objectives: (i) to quantitatively compare luminescence of haploid and diploid mycelia of Armillaria; (ii) to explore the magnitude and temporal dynamics of light production by nine North American Armillaria species, of which five were not known to emit light; (iii) to characterize the emission spectra of newly
confirmed bioluminescent Armillaria species; and (iv) to quantitatively examine the role of mechanical disturbance in eliciting transient and/or stable changes in luminescence magnitude.
Materials and methods Strains used The investigation of luminescence of haploid and diploid mycelia was conducted with 21 isolates derived from fruiting bodies collected in connection with the Missouri Ozark Forest Ecosystem Project (MOFEP, Bruhn et al. 2000). Three fruiting bodies of Armillaria mellea collected from different locations in the upland Ozark forests were used to obtain cultures from three distinct genets (Table 1). One fruiting body of A. gallica was used to obtain diploid and haploid cultures (Table 1). Experiments focused on temporal luminescence patterns or luminescence in response to disturbance utilized three genets of each of nine North American Armillaria species obtained from the Forest Products Laboratory (FPL), U.S. Department of Agriculture, U.S. Forest Service, Madison, WI, USA (Table 2). All FPL isolates were monokaryons derived from single basidiospores. All isolates were maintained as active, working cultures on 2 % malt extract agar (2 M) during experimentation. When not in active use, isolates were maintained as archival cultures at 5 C in sterile distilled water or as archival cultures at 80 C as previously described (Mihail 2013).
Bioluminescence measurement and experimental design The comparison of bioluminescence of diploid and haploid mycelia used mycelia growing on 1.5 % malt extract agar (1.5 M) in 35-mm diameter plastic Petri dishes. Three replicate mycelia represented each of the 21 isolates included in the study. Bioluminescence was measured as relative light units per second (RLU$s1) using a Zylux FB-12 single tube luminometer (Zylux Corp., Oak Ridge, TN) which could accommodate Petri dishes of 35 mm diameter or less. Bioluminescence measured in one replicate consisted of 80 measurements of 1 s duration with a 14 s gap between consecutive measurements. All mycelia represented 7 d growth at the time of bioluminescence measurement owing to the
Table 1 e Armillaria isolates used in the comparison of bioluminescence among diploid and haploid mycelia. Species
Isolate
Ploidy
Locationa
A. gallica
OZ1502 OZ1502e5, OZ1502e10, OZ1502e13, OZ1502e14 OZ221 OZ221e1, OZ221e2, OZ221e4,OZ221e5, OZ221e6 OZ222 OZ222e1, OZ222e2, OZ222e3, OZ222e4 OZ1417 OZ1417e1, OZ1417e2, OZ1417e3, OZ1417e6
2N 1N 2N 1N 2N 1N 2N 1N
2e28 2e28 4e15 4e15 4e26 4e26 2e28 2e28
A. mellea
a All isolates were collected from the Ozark Mountains of Missouri, USA in conjunction with a long-term forest health study. Isolate locations are recorded in a ‘siteeplot’ format as indicated in Bruhn et al. 2000.
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Table 2 e Bioluminescence consistency among haploid mycelia of North American Armillaria species. Species
A. calvescens
A. cepistipes
A. gallica
A. gemina
A. mellea
A. nabsnona
A. ostoyae
A. sinapina
A. tabescens
Genet
FPL19 FPL20 FPL21 FPL37 FPL38 FPL39 FPL28 FPL29 FPL30 FPL16 FPL17 FPL18 FPL25 FPL26 FPL27 FPL31 FPL32 FPL33 FPL13 FPL14 FPL15 FPL22 FPL23 FPL24 FPL34 FPL35 FPL36
Original IDa
FFCe7, sse7 ILe7, sse4 TJVe94e114, sse1 TJVe93e167, sse6 HHBe14868, sse2 HHBe14867, sse7 MNe3, sse1 ILe30, sse3 133e2, sse1 JJWe233, sse1 Mielke, sse14 TJVe94e47, sse1 SHe7, sse10 ILe40, sse3 DAe2, sse2 HBe21, sse8 TJVe93e200, sse5 TJVe93e188, sse4 ONe19, sse4 FLe2, sse9 HHBe12879, sse7 HHBe14911, sse9 PRe7, sse9 ONe18, sse2 PORe100, sse8 De83, sse10 De85, sse1
No. luminescent at measurement interval (ms)b 100
200
400
800
1600
1 0 2 0 0 4 1 4 0 1 4 3 4 0 4 4 4 4 2 4 4 0 2 3 2 0 2
1 0 2 0 0 4 0 4 0 0 4 4 4 1 4 4 4 4 2 4 4 0 3 3 3 0 2
1 1 3 0 0 4 0 4 0 0 4 4 4 2 4 4 4 4 2 4 4 0 3 3 3 0 2
1 1 3 0 0 4 1 4 0 0 4 4 4 4 4 4 4 4 3 4 4 0 4 3 4 0 2
1 2 3 0 0 4 1 4 0 0 4 4 4 4 4 4 4 4 4 4 4 0 4 3 4 0 2
a All isolates were obtained from the Forest Products Laboratory, USDA, US Forest Service, Madison, WI, USA, with the original identifying designations as indicated. b Tabular values are the number of replicate mycelia (of four) from which light was consistently detected over 1000 measurements at the specified measurement interval (i.e., minimum luminescence > 0 RLU).
limitation of the size of Petri dish accommodated by the instrument. The bioluminescence of the nine North American Armillaria species was assessed using mycelia growing on 2 % malt extract agar (2 M) in 95emm diameter plastic Petri dishes. Four replicate mycelia represented each of the 27 isolates included in the study. Bioluminescence intensity was measured using a Sens-Tech photomultiplier tube detector, model P25232 (Sens-Tech, Ruislip, Middlesex, UK) which was not contained in a luminometer allowing for flexibility in the size of the Petri dish and the arrangement of sample and detector. For each replicate mycelium, a series of 1000 measurements was made of total light (RLU) emitted during a specified interval. The five measurement intervals used for each replicate mycelium were: 100, 200, 400, 800, and 1600 ms. Measurements were consecutive without intervening gaps. All mycelia were 21e28 d old at measurement. At the conclusion of each series of luminescence measurements, colony diameter was recorded. Emission spectra are typically measured using a spectrometer which parses unamplified incident light into user-defined bands of wavelengths. Preliminary studies of Armillaria mycelial emission spectra utilized an Ocean Optics USB2000þ Fiber Optic Spectrometer (Ocean Optics, Dunedin, FL, USA) and the iterative spectrum measurement protocol of Oba et al. (2013) for documenting low-intensity light emission. This approach was not sufficiently sensitive to produce reliable emission
spectra for the Armillaria mycelia. The Sens-Tech photomultiplier tube detector, noted above, represents the most sensitive method for measuring low light emission (Berthold et al. 2000). Bandpass filters allow only specific bands of wavelengths to be transmitted through them. A novel approach to emission spectrum measurement was developed by placing a series of bandpass filters between a luminescent mycelium and the photomultiplier tube detector. Sixteen Edmund Optics Hard Coated OD4 filters were utilized to measure light in the range 412.5e687.5 nm (Edmund Optics, Barrington, NJ, USA). Specifically, eight 25-nm-band width filters were used with central wavelengths of 425, 450, 475, 500, 600, 625, 650 and 675 nm and 90 % minimum transmittance. For example, the filter with a central wavelength of 425 nm permitted only light in the range 412.5e437.5 nm to be transmitted from the luminescent mycelium to the photomultipler tube detector. Eight 10nm-band width filters were used with central wavelengths of 510, 520, 532, 540, 550, 560, 568, and 580 nm and 85 % minimum transmittance. All bandpass filters were 25 mm in diameter with identical transmission areas. To characterize one emission spectrum, a series of 50 consecutive measurements of 1 s duration were made with each of the 16 bandpass filters. For each mycelium, bandpass filters were changed without disturbing the mycelium. A complete emission spectrum was measured in ca. 20 min so that bioluminescence intensity was temporally stationary.
Bioluminescence patterns among Armillaria
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The effect of mechanical disturbance on luminescence of the nine Armillaria species was assessed using mycelia 21e28 d old growing on 2 M. Three replicate mycelia representing each of the 27 isolates used in the study were assessed with a sequence of four series of 500 consecutive measurements of 500 ms duration each. During the first series of 500 measurements the mycelium was not disturbed so that a baseline luminescence magnitude was established. Subsequently, the mycelium was disturbed by application of a concentrated shock (200 N) using a chiropractic adjusting tool (JTECH Medical, Salt Lake City, UT, USA) within 15 s of the initiation of the series of 500 light measurements. Thus, a complete disturbance sequence for one replicate mycelium comprised a baseline luminescence series (500 measurements) followed by three disturbance-response series (500 measurements each). Less than 30 s elapsed between the conclusion of one component of the sequence and the initiation of the subsequent component. The Parafilm seal on each Petri dish was not disturbed or removed during or following disturbance treatments. Bioluminescence was measured using a Sens-Tech photomultiplier tube detector, as noted above. To avoid the confounding effects of environmental illumination, each Petri dish was placed in its own light-tight cardboard box between the initiation of the mycelium and the first luminescence measurement for an experiment. Petri dishes remained sealed with Parafilm (Pechiney Plastic Packaging, Chicago, IL, USA) during luminescence measurement. All bioluminescence measurements were obtained in a laboratory maintained at 22e26 C. Prior to each experiment, background luminescence of the agar medium was determined using at least 12 observations of the same duration as the experimental measurement interval. When band pass filters were used to measure emission spectra, the background luminescence was not altered by any of the 16 filters used. Each bioluminescence measurement taken was then reduced by a quantity representing the mean background luminescence designed to minimize the possibility of ascribing luminescence to an isolate which was not luminescent.
luminescence measurements. Time series analysis is used to detect cyclic patterns in a series of equally spaced, consecutive measurements (Fuller 1996; Warner 1998; Cryer & Chan 2008). Time series analysis is an appropriate analytical tool only if: (a) the data are normally distributed; (b) the mean value does not increase or decrease over the series (i.e., the series is stationary); and (c) the series variance is stable (Warner 1998; Cryer & Chan 2008). Time series analysis was applied to observations from the species comparison experiment using the 800 ms measurement interval where the analytical criteria were satisfied. Since all time series of 1000 observations were nonstationary, data were transformed by taking the ‘first difference’ in which an observation at time, t, is replaced by the difference between that observation and the preceding observation (Warner 1998; Cryer & Chan 2008). Differenced data were analysed using ordinary least squares regression to confirm the series’ stationarity by using PROC REG of SAS/ STAT. The ShapiroeWilk statistic, W, was calculated using PROC UNIVARIATE to confirm that data were normally distributed (Warner 1998; Cryer & Chan 2008). Time series analysis was conducted using PROC SPECTRA of SAS/STAT, in which a periodogram apportions total time series variance among a series of time intervals (i.e., periods). Departures from randomly fluctuating series were tested with Bartlett’s Kolmogorov-Smirnoff statistic (Fuller 1996). To assess the effect of mechanical disturbance upon luminescence magnitude, the mean luminescence magnitude of the baseline series (i.e., 500 measurements prior to disturbance) was compared with the mean magnitude of the final 450 values of the three series measured in response to disturbance shock. For each of the three response series, the first 50 luminescence values were removed as this portion of each series included pre-treatment luminescence levels as well as transient post-treatment luminescence spikes. Repeated measures ANOV were used to test the null hypothesis of no treatment effect, using the REPEATED option of PROC GLM in the SAS/STAT software. Specifically, the null hypothesis was tested as the contrast:
Data analysis
Lbase ¼ ½ðL1 þ L2 þ L3 Þ=3
Descriptive statistics measuring central tendency (i.e., mean, median, minimum, maximum) were calculated for all luminescence time series in each experiment. A series of luminescence measurements was considered to represent consistent luminescence only if the minimum value, after adjustment for background luminescence, was greater than zero. The relationship between luminescence intensity and colony diameter was assessed using PROC CORR of SAS/STAT analytical software (SAS/STAT System Release 9.3, Cary, NC, USA). Emission spectra were assembled from the mean luminescence intensity recorded for each of the 16 bandpass filters. The two groups of bandpass filters had identical transmission areas and similar minimum transmittances. Therefore, to account for the difference in light gathering capacity of 10 nm and 25 nm bandpass filters, the mean intensity measured using the 10 nm bandpass filters was increased by a factor of 2.5. In a previous study of Armillaria luminescence (Mihail 2013), the technique of time series analysis was useful in quantitatively characterizing the temporal dynamics of
where Lbase is the mean luminescence of the baseline series; L1, L2, and L3 are the mean luminescence magnitudes of the final 450 values of the three disturbance response luminescence series.
Results Luminescence of diploid vs monokaryotic mycelia Consistent bioluminescence was detected for all replicate mycelia of Armillaria mellea and Armillaria gallica irrespective of the haploid or diploid nuclear state (Fig 1). For all four genets examined, the greatest mean luminescence was expressed by one or more of the monokaryon single-spore strains, rather than the original diploid strain derived from the fruiting body (Fig 1). For A. mellea genet 222 and A. gallica genet 1502, three of four monokaryon strains expressed mean luminescence intensity at least as great as the original diploid strain (Fig 1).
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Fig 1 e Luminescence of diploid and haploid Armillaria mycelia. (A) A. mellea genet OZ221; (B) A. mellea genet OZ222; (C) A. mellea genet OZ1417; (D) A. gallica genet OZ1502. In each panel the first symbol represents the diploid mycelium; subsequent symbols represent haploid mycelia derived from single basidiospores. Each symbol with error bars represents the mean luminescence (RLU sL1) with standard deviation based on three replicate mycelia.
Luminescence of nine Armillaria species Luminescence was consistently detected for at least one of three genets for each of the nine North American Armillaria species (Table 2). Consistent bioluminescence was defined as detectable luminescence after adjustment for background for all 1000 observations in a series. Luminescence observations were made using five measurement intervals (i.e., 100e1600 ms). Only Armillaria nabsnona genets were consistently luminescent at all measurement intervals for all four replicate mycelia examined (Table 2). At the longest measurement interval (i.e., 1600 ms), consistent luminescence was detected from the three genets representing Armillaria calvescens, Armillaria mellea, Armillaria nabsnona and Armillaria ostoyae (Table 2). Only one of three Armillaria cepistipes genets was consistently luminescent (Table 2). The magnitude of luminescence varied among species, among genets within species, and among replicates within genets (Fig 2). The two luminescent genets of A. gallica, FPL28 and FPL29, had the lowest mean estimates of luminescence (102e103 RLU; Fig 2C) while the two luminescent genets of A. gemina, FPL17 and FPL18, had the highest mean estimates (107e108 RLU; Fig 2D). Mean luminescence estimates for the three A. nabsnona genets were the most similar (105e106 RLU; Fig 2F). For several genets, mean luminescence estimates varied by at least two orders of magnitude among the replicate mycelia (e.g., Fig 2F, A. nabsnona genet FPL32 and Fig 2H, A. sinapina genet FPL24). There was no significant relationship
between luminescence intensity and mycelium diameter for any genet or species. Emission spectra of luminescent mycelia of each of the nine Armillaria species showed maximum intensity using the bandpass filter with a central wavelength of 520 nm (Fig 3) which permitted detection of light in the range 515e525 nm. All emission spectra contained a notable positive skew (i.e., blue shift; Fig 3) and many contained a shoulder or decrease in intensity using the bandpass filters with central wavelengths of 550 or 560 nm (Fig 3). Luminescence time series of measurements at the 800 ms interval were examined to determine if evident variation was simply unstructured, random variation (Table 3). Of the 108 series available, 70 exhibited consistent luminescence and were analysed. Ninety percent of the consistently luminescent series met the criteria for time series analysis (Table 3). Where a series was not normally distributed, the reason was most often the presence of at least one exceptionally high or exceptionally low luminescence measurement (i.e., a transient spike or drop). Transient spikes were observed in luminescence series of A. calvescens (genets FPL20, FPL21), A. mellea (genet FPL26), A. ostoyae (genet FPL14), A. sinapina (genet FPL24) and A. tabsecens (genet FPL34). A transient drop was observed for one series of A. nabsnona (genet FPL34). For each of the nine species, at least 4 of the 12 series examined (i.e., three genets X four replicates) exhibited consistent luminescence (i.e., minimum > 0). All series which were normally distributed represented significant departures from random variation as
Bioluminescence patterns among Armillaria
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Fig 2 e Mean luminescence of mycelia of nine North American Armillaria species. (A) A. calvescens genets FPL19, FPL20, FPL21; (B) A. cepistipes genets FPL37, FPL38, FPL39; (C) A. gallica genets FPL28, FPL29, FPL30; (D) A. gemina genets FPL16, FPL17, FPL18; (E) A. mellea genets FPL25, FPL26, FPL27; (F) A. nabsnona genets FPL31, FPL32, FPL33; (G) A. ostoyae genets FPL13, FPL14, FPL15; (H) A. sinapina genets FPL22, FPL23, FPL24; (I) A. tabescens genets FPL34, FPL35, FPL36. Each symbol with error bars represents the mean bioluminescence and standard deviations for 1000 consecutive 1.6 s observations using replicate mycelia which were consistently luminescent (e.g., minimum magnitude > 0 RLU).
determined by highly significant values of Bartlett’s KolmogoroveSmirnov statistic (Table 3).
Luminescence in response to mechanical disturbance The response of mycelial luminescence to a sequence of mechanical disturbance treatments revealed both transient and enduring responses to the disturbance (Fig 4). Transient responses were expressed as a short-duration spike in luminescence (e.g., Fig 4C, F, and K). Such luminescence spikes were observed for genets of A. cepistipes (genet FPL39), A. gallica (genet FPL29), A. gemina (genet FPL17), A. nabsnona (genets FPL31, FPL32, FPL33), A. ostoyae (genet FPL15), and A. sinapina (genets FPL23, FPL24). Enduring responses included a stable increase in luminescence magnitude (e.g., Fig 4B, E, K) or a stable decrease in magnitude (e.g., Fig 4D, I). Stable shifts in
luminescence magnitude in response to mechanical disturbance were documented for at least one genet of eight of the nine Armillaria species examined (Fig 4, Table 4). For seven of these species, both significant increases and decreases in magnitude were observed (Table 4). For example, luminescence magnitude of A. gemina mycelia was reduced by up to 41 % or increased by 318 % among the seven response sequences examined (Table 4). For each sequence of baseline and three response luminescence series, the null hypothesis that disturbance did not result in a shift in luminescence magnitude was examined using repeated measures ANOVA. For all sequences examined, the null hypothesis was rejected indicating significant shifts in mean luminescence in response to the mechanical disturbance treatment (Table 4). In this experiment, mycelia of A. tabescens did not produce consistent luminescence and thus were not included in the analyses.
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Fig 3 e Emission spectra of representative Armillaria mycelia. (A) A. calvescens genet FPL21; (B) A. cepistipes genet FPL39; (C) A. gallica genet FPL28; (D) A. gemina genet FPL16; (E) A. mellea genet FPL27; (F) A. nabsnona genet FPL31; (G) A. ostoyae genet FPL13; (H) A. sinapina genet FPL23; (I) A. tabescens genet FPL35. Each symbol represents the mean of 50 (1 s) measurements of light transmitted through a bandpass filter with the indicated central wavelength. All measurements were corrected for background luminescence of uncolonized agar with the band pass filters in place. Across the nine emission spectra, the variation in mean luminescence was inversely related to luminescence magnitude. The coefficients of variation associated with mean luminescence estimates were:
