A simple pyrocosm for studying soil microbial response to fire reveals a rapid, massive response by Pyronema species.

We have designed a pyrocosm to enable fine-scale dissection of post-fire soil microbial communities. Using it we show that the peak soil temperature achieved at a given depth occurs hours after the fire is out, lingers near this peak for a significant time, and is accurately predicted by soil depth and the mass of charcoal burned. Flash fuels that produce no large coals were found to have a negligible soil heating effect. Coupling this system with Illumina MiSeq sequencing of the control and post-fire soil we show that we can stimulate a rapid, massive response by Pyronema, a well-known genus of pyrophilous fungus, within two weeks of a test fire. This specific stimulation occurs in a background of many other fungal taxa that do not change noticeably with the fire, although there is an overall reduction in richness and evenness. We introduce a thermo-chemical gradient model to summarize the way that heat, soil depth and altered soil chemistry interact to create a predictable, depth-structured habitat for microbes in post-fire soils. Coupling this model with the temperature relationships found in the pyrocosms, we predict that the width of a survivable "goldilocks zone", which achieves temperatures that select for postfire-adapted microbes, will stay relatively constant across a range of fuel loads. In addition we predict that a larger necromass zone, containing labile carbon and nutrients from recently heat-killed organisms, will increase in size rapidly with addition of fuel and then remain nearly constant in size over a broad range of fuel loads. The simplicity of this experimental system, coupled with the availability of a set of sequenced, assembled and annotated genomes of pyrophilous fungi, offers a powerful tool for dissecting the ecology of post-fire microbial communities.

Introduction

Fire is a natural part of many ecosystems, and organisms in systems with predictable fire regimes are often well adapted to survive or recolonize rapidly after fire. Plant adaptions are particularly well known with the ability to survive fire through thickened bark, serotinous pine cones, vegetative resprouting, or other traits [1]. Soil microbes also must survive or recolonize after fires, but much less is known about how they achieve this or what their roles are in the post-fire environment. Pressler et al. [2] conducted a meta-analysis on belowground effects of fire and reported that negative effects are commonly found on microbes including reductions in biomass, abundance, richness and evenness across taxonomic groups, and these effects were coupled with decade-long recovery times. Fungi in particular showed large reductions and slow recovery [2]. Dove and Hart [3] found similar effects in their meta-analysis of fungal communities, and both studies showed that the largest effects were associated with forest biomes. Post-fire changes in fungal communities were thought to be caused by both the direct killing effects of fire on the organisms, and by indirect effects on habitat, such as the loss of the organic layers and the reduction in living root biomass of mycorrhizal hosts.

Almost all studies considered in these meta-analyses focused on the reduction in post-fire microbial communities, but there is also evidence of microbes that respond positively to fire. In particular there is a set of “pyrophilous” fungi that fruit only in burned habitats and are abundant in the first weeks or months following fire. These predominantly saprophytic fungi have been known for over a hundred years [4], and the pattern of their fruiting in post-fire settings suggests a rapid successional sequence [5, 6]. Nevertheless, the rapid, post-fire, fungal succession has not been confirmed at the mycelial level with modern, molecular methods, nor is it known what these fungi degrade and live on in the chemically altered post-fire environment. How these fungi rapidly colonize post-fire soil is also unknown. Some fungi have heat-stimulated spores [7], or spores that can also be stimulated by post-fire chemicals [8]; these propagules may reside in the soil for decades between fire events [9]. Other post-fire fungi have been shown to colonize plants endophytically [10], but how this translates into colonizing the burnt soil habit is unresolved.

We propose that the post-fire microbial habitat is structured by a thermo-chemical gradient in which direct heating effects of fire and the production of temperature-specific soil chemicals change predictably with soil depth and fire intensity. The two main components of this conceptual model are: 1) a steep temperature gradient that causes differential mortality with soil depth; and 2) a gradient of chemically-altered substrates produced by this temperature gradient that structures resources available to recolonizing microbes. Fire heating of soils is reasonably well understood from a variety of models [11-13] and is the basis for component 1. These studies have shown that the heat capacity and water content of soil produce depth-stratified temperatures where high temperatures at the surface drop rapidly with depth. We assume that the temperatures achieved in soil will cause a gradient of death in which high temperatures near the surface kill all organisms down to some threshold depth, and below this depth there will be differential survival. The lethal temperature varies with the organism [14], and those organisms tolerant of higher temperatures will find themselves in a zone of reduced competition. We call this the “Goldilocks zone”, a depth layer where the not-too-hot, not-too-cool temperatures allow fire-specialized microbes such as the pyrophilous fungi to survive while killing most competitors. At least some pyrophilous fungi appear to have dormant spores or sclerotia in the soil, and these germinate following heating or chemical changes associated with fire [6, 15]. We would expect a rapid stimulation of such propagules within the Goldilocks zone.

The effects of fire on soil chemistry are known to be correlated with the temperature/depth gradient in general ways, and this knowledge is the basis of component 2 of our model. For example, extreme surface temperatures can completely combust much of the ligno-cellulosic biomass into CO2, while producing a cation-rich, high pH ash. Substantial amounts of biomass are also transformed into partially pyrolyzed (i.e., heat-modified), highly aromatic carbon sources [16]. Temperatures from 220–480 °C are known to convert biomass into a mixture of partially pyrolyzed organic compounds, while volatilized waxes and lipids typically condense at temperatures around 200°C in the soil below[16]. This process results in a hydrophobic zone that commonly occur in forest fires and can lead to heavy erosion by channelizing runoff [17-19]. Thus, the upper layers of soil will have highly modified, fire-specific carbon sources that may select for specific sets of organisms able to metabolism them. At lower depths, where peak temperature is below 200 °C, little pyrolysis occurs, but the high temperatures still kill most life. This creates a necromass zone, where dead organisms are likely to provide easily-mineralizable forms of carbon and nitrogen, creating a nutrient subsidy for any microbes that can rapidly recolonize. This zone is immediately above the Goldilocks zone and intergrades into it with the differential survival of heat tolerate microbes.

Experimental evidence of the effects of fire on microbial communities has been focused on sampling studies of wildfires and prescribed burns (see studies included in [2, 3]). Although generalities have been learned from these approaches, they have not been useful for connecting the detailed understanding of soil heating and chemistry to the structure of post-fire microbial communities. Post-fire successional studies of wildfires have generally been space-for-time comparisons with limited replication, and sampling was usually not conducted until at least a year or more post-fire [2,3]; thus the early responses would be missed. Prescribed burns have more potential for pre and post-fire sampling, replication, and fuel manipulations. Petersen’s [6] early work on successional patterns of pyrophilous fungi involved small experimental fires but were conducted prior to molecular identification methods and relied on fruiting. However, recent work by Reazin et al. [20] used replicated fuel manipulations, pre- and post-fire sampling, and high-throughput sequence analysis to show a strong response by pyrophilous fungi that varied with fire intensity. Nevertheless, all prescribed fires are necessarily limited to times when air temperatures, moisture, and other weather parameters make control manageable, and prescribed fires can only be manipulated in a relatively course way to achieve differences in soil temperatures. They also involve significant site preparation and costs that typically limit studies to small numbers of sites.

Our goal was to develop a more easily manipulated system to allow us to control soil heating and to dissect the fire effects on soil microbes at a finer scale. To that end we have developed a “pyrocosm” system where key fire effects on soil microbial communities can be controlled, monitored, and replicated in a few liters of soil. Here we show that we can use this system to control and reproduce soil heating effects by manipulating coarse fuels, and we use the results of these experiments to further develop our model of the Goldilocks and necromass zones. As a proof of concept we use forest soils to show that known pyrophilous fungi can be rapidly simulated to grow in these systems, and we discuss ways in which the growing genomic knowledge of these fungi can be used to further dissect post-fire, fungal ecology.

Materials and methods

Pyrocosm design

Our pyrocosms consisted of 10-quart (9.46 l) galvanized steel buckets, filled to a depth of 16cm with test soil or sand, and wired with K-type thermocouples at selected soil depths through small drill holes in the bucket sides. Total volume of soil in the buckets was 7 liters, which left a unfilled margin of approximately 8.5 cm at the top. Ten to 13 drill holes (0.6 cm diameter) were added to this unfilled margin to increase aeration for the fire. Assembled units were buried so that soil levels inside and outside the bucket were even. A small fire with weighed fuels consisting of pine needle litter, paper and charcoal briquettes was then burned on the top of it (Fig 1) and the test soil was watered the next day with to initiate microbial activity after it had completely cooled. Detailed notes on assembling these pyrocosms are given in the (S1 File)

Fig 1

Pyrocosm construction.

A) Pyrocosm in the lab filled with forest soil and wired with thermocouples; air holes around rim had not yet been drilled but are seen in remaining images; B) weighted pine needles, cardboard, tooth picks and newspaper constitute “flash fuels” used to ignite charcoals; C) early ignition phase; D) burned pyrocosm and unburned control in background.

Two pyrocosms were used to study post-fire fungal response and were filled with forest soil. Unburned test soil for these was collected from a Pinus ponderosa forest in Stanislaus National forest, CA, USA, 37.8140–120.0689, just outside the perimeter of the 2013 Rim Fire. The top 5 cm and the bottom 5–15 cm of soil were collected and kept as separate samples. Naturally burned test soil was collected from a nearby, fire-killed, Pinus ponderosa forest within the perimeter of the Rim fire in Stanislaus National forest, 37.8442–119.9402, located adjacent to previously studied plots [21], and was not depth stratified because it was derived from a disturbed pile. All soils were collected in Spring 2015, under Special Use Permit #GRO1087 from the USDA Forest Service, Stanislaus National Forest to TDB. Litter and F layers were collected from the same unburned site. The soils were sieved through a 2mm soil screen to homogenize them and remove rocks, roots and large aggregates, and then were air-dried in large, closed paper bags for 3 weeks in a fume hood to a moisture content of between 3.8% (bottom 5–15 cm) and 4.8% (top 5 cm), and then stored in sealed plastic bags at 5° C until needed. When assembling soil pyrocosms, bottom soil was used for the lower 5–16 cm and top soil was used for the upper 5 cm. In the second soil pyrocosm, burned forest soil was mixed with the bottom unburned soil in a 1:9 ratio with intent of adding inoculum of post-fire fungi to the experiment. Soil was allowed to settle evenly, but was not compressed. Bulk density was not measured, but was likely greater than in the field because sieving destroys structure that would otherwise produce larger air pockets.

Course sand (~2mm grain) was used instead of soil in the series of 14 burning experiments to test the relationship between fuel and soil temperatures. Sand was selected because it was not altered by the fire, and therefore pyrocosm experiments could be repeated without reassembling the units between fires. Instead, the ash and charcoal formed were simply removed by hand and with the assistance of a small fan, and then a new fuel load and heating experiment were tested. Thus all 14 experiments were run with three sand pyrocosms. Controls for these temperature experiments consisted of leaving one of these units unburned. This gave us a reading of diurnal soil temperature changes unrelated to the fires.

Thermocouples were K-type, with an accuracy of 1°C. In the two soil pyrocosms, four thermocouples were installed through drilled holes in the side of the buckets at 0.5, 3.5, 6.5, and 9.5 cm from the surface (Fig 1A). These were inserted as the soil was added such that the tip of each thermocouple was located near the center of the bucket at the prescribed depths (see S1 File for detailed notes). The top two thermocouples were insulated K-type thermocouples that could withstand temperatures up to 1038°C (custom made by OMEGA.com, see Fig 1A). The lower two were plastic insulated wire thermocouples with temperature maximums of 200°C (Fig 1A). In later experiments, the number and arrangement of thermocouples per pyrocosm were modified as needed for specific uses. For experiments designed to determine the effect of fuel load on peak temperatures only two thermocouples were used and placed at 10.5 and 15.8 cm from the surface. These were the plastic-insulated types since temperatures were more moderate at these depths and the thinner wires were more accurately placed. These depths were selected to insure that the wire thermocouples did not experience temperatures above 200°C across the range of fuel loads tested. Temperatures were recorded using a data logger (Extech Instruments SDL200) at 10 sec or 30 sec intervals. The 30 sec intervals turned out to be more than sufficient because temperatures change fairly gradually at the depths measured.

Fuels, ignition and fire

Fuel in all experiments was weighed prior to ignition. Dried, weighed, forest litter and F-layer collected from the same unburned site as the soil were placed at the soil surface and gently tamped down to achieve an approximate depth of approximately 4 cm, which was similar to the depth of the organic layers at the site where the soil and litter were collected. Litter and F-layer samples was re-dried the night before in a drying oven at moderate heat (~110 C) to ensure that it burned readily and was consumed completely during the experiments. Charcoal was used as the course-fuel for these experiments, because it burns uniformly and completely once ignited. Differences in heating were achieved by simply varying the number of charcoal briquettes, which were weighed prior to burning, and were placed on top of the litter to ignite them (Fig 1). Our source of charcoal was a local grocery store.

The fire for the two soil pyrocosms contained approximately 300 g of litter and 1000 g of charcoal. The charcoal was piled in the middle of bucket, and was spread evenly after it was well lit. The temperatures at the four depths were monitored and the charcoal was then carefully removed with a small trowel when soil reached a target temperature. The removed charcoal was extinguished rapidly by dropping it in water, and was later dried and weighed and used to determine the amount that had been consumed. The temperature was monitored for six more hours and pyrocosms 1 and 2 ultimately reached temperatures of 146°C and 171°C, respectively, at a depth of 6.5 cm. The temperatures from these soil experiments were analyzed separately from the sand pyrocosms, since the heat capacity of soil and sand was assumed to differ.

In all sand experiments the amount of charcoal was reduced to 1 to 15 briquettes (~25 to 380 g) and was ignited by perching it on top of fine wood kindling (tooth picks), held in a 28–38 g cardboard ring that was filled with a single sheet of newspaper (Fig 1B). After the cardboard ring burned, and the ignited charcoal was moved toward the center, spread evenly, and allowed to burn completely. This complete combustion of a reduced amount of charcoal allowed us to determine the effect of charcoal quantity on peak soil temperatures more easily.

Fungal incubation, sampling, and controls

The two pyrocosms filled with forest soil were used to study fungal response to fire. The day following the experimental fires, after the soil temperatures had returned to normal, water was added to the pyrocosms in the form of weighed, crushed ice. This method for adding the water was meant to mimic snow, which is a typical way late fall precipitation occurs at mid elevation in the Sierra Nevada Mountains where the soil was collected. The slow melting also allowed the water to gently and uniformly percolate into the soil, even when the soil had become slightly hydrophobic from the fire. Pyrocosm 1 received 1000 g of ice on day 1, and 500 g on day 2, Pyrocosm 2 received 1500 g of ice on day one and 1,500g on day 3. This corresponds to 33 and 66 mm of rain for the two pyrocosms, respectively. The watering differences were meant to provide a broader range of post-fire conditions in these pilot studies with the intent that at least one of them might stimulate pyrophilous fungi. A control pyrocosm, filled with the same forest soil, setup in the same way and buried a meter from the test pyrocosm 1, was left unburned, but watered and incubated in the same way as the first pyrocosm (Fig 1D). After watering, pyrocosms were covered with foil to prevent additional precipitation and to limit aerial dispersal, and they were incubated in situ.

Soil within the pyrocosms was sampled from the soil surface to the bucket bottom with a single 2 cm diameter soil corer at each time point. Each soil core was vertically divided into four approximately even depth zones, and DNA was extracted from each zone sample via MoBio DNAeasy Power Soil kit (Qiagen, Carlsbad, CA, USA). Both experimental pyrocosms and the control were sampled this way at weeks 1 and 2 post-fire. In addition pyrocosm two was sampled at week 4.

Construction of Illumina libraries, and processing of Illumina data

We amplified ITS1 spacer, which is part of the Internal Transcribed Spacer region, the universal DNA barcode for fungi [22], with the ITS1F-ITS2 primer pair using Illumina sequencing primers designed by Smith and Peay [23], and we prepared libraries for Illumina MiSeq PE 2 x 250 sequencing as previously described [21]. These primers were used to enable direct comparison with earlier sequence studies that included these sites where the soil used was collected. Sequencing was performed at the Genome Center at the University of California, Davis, CA, USA. Bioinformatics was performed with UPARSE [24] usearch v7 and QIIME 1.8 [25] with the same methods as previously published [21]. All analyses are based on 97% sequence similarity for operational taxonomic units (OTUs) and taxonomy was assigned with the UNITE fungi database [26] accessed on 30 Dec 2014. Representative sequences for all OTU that represented 1% or more of the read abundance in either of the pyrocosms or the control were BLASTed [27] individually against the NCBI database, and the results were examined to improve the automated sequence-based identifications. Samples for a related study were run at the same time and OTUs were processed for both simultaneously to enable later comparison. As a result OTU identifying numbers reported here are greater than the 887 total found in this study. Sequences are available at the NCBI Sequence Read Archive (PRJNA559408), and representative sequences from each of the OTUs are deposited in NCBI MN724033-MN724919.

A mock community composed of equal quantities of DNA extractions of Coprinellus sp. Pholiota sp. Cyathus stercoreus, Penicillium citreonigrum, Aureobasidium pullulans, Rhodotorula mucilaginosa, Cladosporium sp., Suillus sp., Peyronellaea glomerata, and Peyronellaea glomerata was included to control for OTU processing, and a no DNA control was included to assay for spurious contamination and index jumping [28].

Statistical analyses

Plotting of temperature changes over time was initially done with Microsoft Excel, and then repeated in R along with all modeling and correlation analyses [29]. The response of individual OTUs was investigated by examining their changes in % sequence relative to the unburned control.

To determine overall abundance ranks of OTUs, the sequences across all depths in weeks one and two were separately summed for each OTU for each of the two pyrocosms and the control, and divided by total sequences of all OTUs from the same experimental unit and time sample (i.e., pyrocosm 1, 2 or control and weeks 1 or 2). The spreadsheet was then sorted by sequence abundance, first for the control and then for each of the two experimental pyrocosms. Ranks were assigned in descending order within each experimental unit with the most abundant OTU as 1.

OTU increases or decreases relative to fire were investigated by subtracting percent sequence abundance for each OTU from each pyrocosm from the percent sequence abundance in the control. The OTUs were then sorted by this metric to identify those that showed the largest changes.

Results

Soil heating characteristics in the pyrocosms

The heating experiments showed the following about the peak temperature that is reached at a given depth: 1) The reproducibility of the temperature profiles is excellent when fuel levels are constant (Fig 2A). 2) Peak temperatures at depth are reached hours after the fire has gone out and they linger near the peak for 40 minutes or more (Fig 2A). 3) Peak soil temperatures decrease by the natural log of the depth (Fig 2B) and can be predicted by the mass of course fuel (Fig 2C). 4). There are edge effects that result in progressively cooler temperatures away from the center (Fig 2D).

Fig 2

Thermal characteristics of pyrocosms.

A) temperature profiles from two depths of two fuel-replicated pyrocosms. Half-minute intervals are plotted over 800 minutes. Note that the temperature profiles are quite repeatable, and that temperature continues to rise after the fire is out (arrow); B) peak temperatures are predicted by the ln (or log10, not shown) of the depth, and C) by the mass of charcoal; D) peak temperature at three depths (7.5, 10.5, 15.8 cm) and three locations offset from center within a depth. Note that the center is hottest and the edge of the unit is cooler. Results shown in A,C, & D are from sand pyrocosms; B is from forest soil pyrocosm 1.

A regression between peak temperatures and ln of depth was linear with a R of 0.9977 (Fig 2B). The predictability of peak temperatures and total fuel is shown as a polynomial model with R2 values of 0.9682 (10.5 cm depth), and 0.9507 (15.8 cm depth), but a simpler logarithmic model works nearly as well with R2 values of 0.9659 and 0.9478, respectively.

Flash fuels, such as pine needles, tooth picks, cardboard, and newspaper, that produced few or no coals, had almost undetectable heating effects on the soil at depth. Burning of 172 g of flash fuels caused a 2° C rise in temperature at 10.5 cm below the surface, relative to the unburned control. This contrasts with approximately an 8° C rise at the same depth when a single 25.5 g charcoal was added. However, the flash fuels did cause a more rapid rise in temperature than that caused by solar heating in the control (S1 Fig), and using regression from the measured temperatures points (i.e., Fig 2B), the temperature 1 mm from the surface was predicted to have peaked at 108° C, and soil temperature was predicted to be heated to 70° C at a depth of 1.26 cm.

There is a significant edge effect on soil heating in the pyrocosms, and it is most pronounced at the shallower depths (Fig 2D). For example, at 7.5 cm the peak temperature varied from 91 to 61 °C, depending on whether the temperature was measured at the center or near the edge of the pyrocosm. Near the bottom of the unit at 15.8 cm, the temperature from center to edge only varied 5 ° from 48 to 43 ° C, which is a lesser absolute and relative difference than the variation recorded at 7.5 cm. Spreading out the coals more did not greatly reduce this edge effect (data not shown). The edge effects do not affect the results presented above with respect the relationships between depth and mass of charcoal on peak temperature, or the temperature-time profiles except to limit their accuracy to the center of the units. However, these edge effects do mean that peak temperature isoclines would be curved upward on the edges.

Fungal response to the pyrocosms

Table 1 summarizes the read depth, number of OTUs, and ranked abundance of the all OTUs in experimental and control samples that had sequence abundance of 1% or greater in control or treatment pyrocosms. Four of the 20 experimental samples yielded 58 or fewer sequences and were dropped. All other experimental samples yielded more than 3950 sequences and were retained. Because the dropped samples made the depth sampling difficult to compare across time, data from depth samples for single time points within an experimental unit were combined to give more robust measures of fungal communities within a time point for Table 1.

Table 1

Summary of samples, read depth, and OTUs.

	Unburned Control	Pyrocosm 1 Weeks 1 & 2	Pyrocosm 2 Weeks 1 & 2	Pyrocosm 2 week 4	Mock control	No DNA control
# samples retained (and lost)	8	7(1)	6 (2)	3(1)	2	4
Ave sequence depth/sample	54427	67308	25247	8495	21087	408
Range of sequence depth	9639–71080	37014–109855	6607–68272	3950–15637	16530–25643	90–1044
# OTUs > = 0.01% sequence	406	158	155	110	24	20
# OTUs with read depth > 5 seq	605	296	191	164	20	16
% reads of most abundant OTU	6.55	57.78	37.02	45.01	64.34	31.72

The fungal communities in the forest soil pyrocosms showed a fire response that is consistent with stimulation of Pyronema spp., a reduction in richness and evenness of the community, but little apparent change to underlying composition (Table 2). Non-metric multidimensional scaling analysis shows that the two pyrocosm communities are about as distinct from each other as they are from the unburned control and also shows much variation occurs between individual depth samples and time points (S2 Fig). However by sorting the OTU table by taxa that increase in the pyrocosms relative to the fire, three OTUs were found that exhibited dramatic increases in both pyrocosms relative to the control. All three were identified as Pyronema species (Table 2), a genus that is well known for its rapid fruiting following fire [4, 6].

Table 2

Most abundant OTUs summed across first two weeks.

		Control		Pyrocosm 1		Pyrocosm 2
OTU	Assigned Taxon1	Rank2	%seq3	Rank	%seq	Rank	% seq	Inferred Ecology4
7	Pyronema domesticum	30	0.85	1	57.78	2	10.85	pyrophilous fungi
5234	Pyronema omphalodes	165	0.05	2	6.81	1	37.02	pyrophilous fungi
4131	Pyronema aff. omphalodes	315	0.01	10	1.18	4	7.04	pyrophilous fungi
57	Sarocladium kiliense	––	0.00	––	0.00	6	5.66	Soil saprobe, opportunistic pathogen
149	Cortinarius sp.	37	0.24	26	0.25	11	1.09	ectomycorrhizal symbiont
37	Unknown Geminibasidiales	1	6.60	5	3.16	3	10.75	xerotolerant yeast (Basidiomycota)
61	Unknown Helotiatles	2	5.22	13	0.72	30	0.19	saprobe/ plant pathogen/ endophyte
42	Solicoccozyma sp.	3	4.25	3	4.59	12	1.02	yeast (Basidiomycota)
5444	Mortierella sp.	4	4.08	9	1.46	28	.27	soil saprobe/root endophyte
73	Russula acrifolia	5	3.21	4	4.58	5	6.19	ectomycorrhizal symbiont
89	Unknown Sporobolales	6	3.03	14	0.66	10	1.42	yeast (Basidiomycota)
5	Mortierella sp.	7	3.03	7	2.68	26	0.27	soil saprobe/root endophyte
161	Unknown Basidiomycota	8	2.86	81	0.03	132	0.01	Unknown
112	Hydropus sp.	9	2.30	NR	0.00	NR	0.00	Saprobe (Basidiomycota)
106	Wilcoxina aff. rehmii	10	2.08	19	0.33	34	0.14	Ectomycorrhizal symbiont
75	Pseudeurotium sp.	11	2.06	12	0.98	8	1.87	soil saprobe/root endophyte
102	Sebacina aff. dimitica	12	2.04	32	0.15	45	0.07	possible ectomycorrhizal symbiont
100	Solicoccozyma terricola	13	2.00	11	1.05	20	0.58	yeast (Basidiomycota)
114	Unknown fungus	14	1.91	NR	0.00	NR	0.00	unknown
115	Hyaloscypha sp.	15	1.87	39	0.09	131	0.01	saprobe/ endophyte
129	Ciliolarina sp.	16	1.86	30	0.16	130	0.01	saprobe/ plant pathogen/ endophyte
132	Unknown Helotiatles	17	1.83	––	0.00	NR	0.00	saprobe/ plant pathogen/ endophyte
195	Phialocephala sp.	18	1.77	22	0.32	81	0.02	dark septate root endophyte
190	Unknown Basidiomycota	19	1.73	NR	0.00	––	0.00	unknown
145	Unknown Ascomycota	20	1.68	67	0.04	NR	0.00	unknown
142	Unknown Pleosporales	21	1.52	NR	0.00	NR	0.00	saprobe/ plant pathogen/ endophyte
4306	Unknown Geminibasidiales	22	1.21	15	0.63	7	2.49	xerotolerant yeast (Basidiomycota)
52	Solicoccozyma terreus	23	1.15	6	2.92	9	1.85	yeast (Basidiomycota)
65	Unknown Basidiomycota	24	1.13	8	1.81	124	0.01	unknown
2145	Helotiales sp	25	1.02	33	0.14	67	0.03	saprobe/ plant pathogen/ endophyte

All taxa represented by at least 1% of the total sequence reads in the control or either experimental pyrocosm are listed.

1 Assigned taxa are based on automated assignments followed by individual Blast matches and evaluations; detailed information on the behavior of individual taxa in each sample is give in the deposited OTU table (doi:10.5061/dryad.45gd695).

2 ranked order of OTUs based on sequence abundance, where 1 is the OTU with the most sequence reads in that treatment.

3% of sequence reads assigned to the taxon in the control and each experimental pyrocosm within the first two weeks combined. First five taxa are those which are abundant in the pyrocosms, but not in the control, remaining 25 are ordered by their abundance in the control.

4 inferred ecology is based on knowledge of the taxon at the generic level. Data for Pyrocosm 2 is restricted to the first two weeks to allow comparison with the control and pyrocosm 1. NR = present but not ranked, below 0.01%.–– = not found.

The three Pyronema OTUs were highly abundant in the burned soils, but not the controls (Table 2). Pyronema domesticum (OTU7), was the most abundant taxon in Pyrocosm 1, accounting for 57.8% of the sequence in weeks 1 and 2 combined, and was the second most abundant sequence in Pyrocosm 2, accounting for 10.85% of the sequence. Pyronema omphalodes (OTU 5234) was the second most dominant taxon in Pyrocosm 1, accounting 6.81% of the total sequence, and it was the most dominant taxon in Pyrocosm 2 in which it accounted for 37.02% of the aggregate sequence in the first two weeks. A third Pyronema (OTU 4131) was ranked10th and 4th in abundance in Pyrocosms 1 and 2, respectively. All three Pyronema OTUs showed rapid rises, and declines in relative sequence abundance within the short time span of the experiment (Fig 3A). In addition, Pyronema domesticum (OTU7) fruited on the surface of the soil after 17 days in Pyrocosm 2, even though it was less abundant than Pyronema omphalodes (OTU 5234) in that experiment (Fig 3). All three Pyronena OTUs were detected in the control, but their read abundances were orders of magnitude lower and they did not increase with time as was the case in the burned pyrocosms (Fig 3A). Pyronema domesticum (OTU7) was the most abundant of the three in the unburned control, where it accounted for 1.69% in the first week but declined to 0.01% by week two. As discussed below it also appeared in the mock community control presumably via index switching [30] and accounted for 0.22% of the sequences (92 reads).

Fig 3

Pyronema species react rapidly to simulated fire in pyrocosms.

A) post-fire percent sequence read abundance of three Pyronema OTUs is shown in the control, and Pyrocosm 1 in weeks 1 and 2 and in Pyrocosm 2 in weeks 1, 2, and 4; B) mycelium and apothecia (ascocarps) of Pyromena domesticum fruiting on the surface of Pyrocosm 2, 17 days after the fire; C) closeup of same.

Only 79 OTUs had sequence abundance higher than 0.1% in at least one of the two experimental pyrocosms. Of these, 38 OTUs showed nominal increases in sequence abundance relative to the unburned control in at least one of the pyrocosms, but only 10 OTUs showed increases in both pyrocosms relative to the control. Most of these increases were quite small, and only four OTUs increased by more than 1% relative abundance in both pyrocosms. Three of these were the Pyronema OTUs just discussed, the fourth was a Russula species (OTU73), an ectomycorrhizal fungus, that increased in sequence abundance by 1.37% and 3.7% in Pyrocosms 1 and 2, respectively. The remaining six OTUs that increased in both pyrocosms, did so at much lower levels, and included two other ectomycorrhizal fungi (Rhizopogon arctostaphyli—OTU175, and an unknown Thelephoraceae-OTU879), two unidentified fungi (OTUs 304, 420), a Basidiomycota yeast (OTU631), and one additional Pyronema (OTU3739).

Evenness and richness were reduced in the burned pyrocosms relative to the control (Fig 4C), but composition differences, other than those related to Pyronema, were not striking (Table 2). The ranked abundance curves for the two experimental pyrocosms are very similar to each other, and distinct from that of the control (Fig 4). This pattern can also be seen in the level of dominance among component species. In unburned control the most abundant taxon, OTU 37 a xerotolerant yeast, represented 6.60% of the total sequence reads, and 25 OTUs each had more than 1% of the total reads. In contrast Pyrocosm 1 and 2 had only 11 and 12 OTUs, respectively, that each had more than 1% of the reads. Among the less abundant taxa, the control pyrocosm had 394 OTUs that were each represented by at least 0.01% of the sequence, versus 158 and 155 OTUs in Pyrocosms 1 and 2 respectively (Table 1, Fig 4).

Fig 4

Differences in diversity of fungal communities within pyrocosms and control.

A) Ranked abundance curves of pyrocosms and control based on % read abundance of for top 50 most abundant OTUs. B) Ranked abundance for all OTUs that are at least 0.01% of sequence. C) Richness estimates based on observed number of OTUs after rarifying samples to 3950 sequences; points show means for all samples; bars show SE.

The four no DNA controls revealed low levels of contamination as is typical of high throughput sequence studies. Of the 10 OTUs that were based on more than 5 sequences in these controls, 6 were identified as Morteriella sp.(Mucoromycota), four were identified as an unknown Ascomycota in the Dothideomycetes or Sordariomycetes, one was a Cortinarius sp. (Basidiomycota), and one was an unidentified fungus. Three of the Morteriella OTUs (64, 108, 794), and the unidentified fungal OTU (544), were found at similar or greater levels in the no DNA controls compared to the experiment. Of the remaining contaminating OTUs, three occurred at much lower levels compared to the experimental samples (OTUs 1,5,14) and three were found in the experimental samples at levels below the thresholds used for analyses (i.e., 0.01%, or 5 sequences) (OTUs 38,556,1021). None of these contaminants are relevant to the results or analyses discussed above.

All of the 10 knowns were recovered from the mock community controls, however, they yielded 16 OTUs with greater than 5 sequences each, and 20 OTUs with greater than 0.01% read abundance. This represents a 1.6 or 2-fold OTU inflation for these respective thresholds. This inflation was primarily due to minor amplicon variants of the known fungal species added, but also included some index switching from the experimental samples. The most obvious case of the latter involved Pyronema domesticum (OTU7), which was the most abundant sequence in the experimental samples (290,128 total reads), and it accounted for 0.22% (92 reads) in the mock community control, even though it was not included in the community.

Discussion

We have shown that these very simple pyrocosms provide an experimental system in which soil heating can be achieved in predictable ways by simply altering course fuel levels. Based on prior soil heating models [11-13], we would expect soils that differ in heat capacities and water contents to have different slopes (i.e. Fig 2B). However, the predictability within a given soil type should remain high, and we would expect the lognormal relationship between temperature and depth to remain. The strong correlation of peak temperature with natural log of depth (Fig 2B), means that one can accurately predict peak temperatures at any depth if temperatures at two depths are measured, although the edge effects will broaden the range of temperatures achieved within a give depth zone. Earlier models have shown that water content of the soil will have a large effect on soil heating, particularly as water content rises above 8% [13]. High water contents were specifically avoided in our studies because we assume that most large wildfires are likely to occur during very dry periods. We have also found that dry soil performed quite similarly to our sand pyrocosms, which helps with predicting peak temperatures in novel soils. However, if one were to model soil conditions of prescribed burns, higher water content of soils should be considered.

These results, and those of earlier modeling studies, have revealed features of the physical environment that are likely to be important for survival of propagules in the soil. The high heat capacity of soil increases with temperature [11], and means that soil heats slowly but retains heat for a long time. This is shown by the fact that peak temperatures at depth in the pyrocosms are achieved hours after the fire has gone out (Fig 2A). A study by Smith et al [31] found the same pattern of lingering soil temperatures under large fuel load in intact forest soils. Essentially the heated surface soils store heat and become the source for heating deeper soils. This means that at depths below a few centimeters, soil organisms would be “slow-cooked”, with temperatures hovering around the peak for tens of minutes. This is important because the spore germination of Pyronema domesticum and some other pyrophilous decrease as heat treatments persist for several minutes [7].

Our results show that coarse fuels (e.g. charcoal) transfer more heat to the surface and ultimately to the deeper soils and were the best predictors of peak temperatures (Fig 2C). To relate this to a forest fire setting we envision a pattern of spotty heating across the landscape driven by the uneven distribution of coarse fuels. This view is concordant with that of Smith et. al. [31], who showed that log piles generate much higher subsurface heating than that achieved from the finer fuels in the surrounding litter. In highly intense crown fires, radiant heating of the surface soils are likely to contribute to much more soil heating than the litter [32, 33] and would likely even out some of this patchiness. Nevertheless, the uneven distribution of larger course fuels is likely to contribute to heating heterogeneity. The less homogeneous nature of in situ forest soil compared to the sieved, dried, uniformity of the pyrocosm soil would add an additional source of spatial variation in peak soil temperatures [34].

To visualize the changes in peak temperatures caused by fuel differences and relate them back to soil organisms, we have used our results to model the depth and width of the Goldilocks and necromass zones (Fig 5). For the sake of argument we will assume the Goldilocks zone exists between 50 and 70 °C. This temperature range was selected because lethal temperature for many organisms falls within this range [14], but spores of some fire adapted fungi are stimulated to germinate at these temperatures [9, 35, 36]. Similarly, we define the temperature limits of the necromass zone from 70 to 200 °C, a temperature range that is lethal to almost all organisms, but is not hot enough to pyrolyzed carbon substrates. This should therefore be a zone that is rich in labile carbon and nitrogen sources that are derived from heat-killed organisms.

Fig 5

Modeled behavior of the “Goldilocks” and necromass zones as mass of charcoal is increased.

A) depth at which peak temperatures reach 50, 70, and 200 °C are modeled by using the ln relationships between mass of charcoal and peak temperature at two given depths (10.5 and 15.8), and then predicting the depths of the selected peak temperatures (i.e. 50, 70, 200) using a ln relationship between depth and peak temperatures (i.e., Fig 2B). The Goldilock and necromass zones are labeled between the hypothetical temperatures that define them B) Width of both zones over the same range of charcoal mass.

Our model shows that the depth of the Goldilocks zone increases with fuel load (Fig 5A), while the thickness of the zone increases rapidly with addition of charcoal up to a threshold. After this threshold is reached, the width of the zone decreases slowly with additional fuel, but retains a similar width (~3–5 cm, Fig 5B) over most of the modeled range. Note that the actual temperatures that define the Goldilocks zone would likely vary with specific organisms [14], and might be hotter or cooler than assumed here. Nevertheless, different defining temperatures would only cause the zone to move up or down in depth and modify the width, but the basic pattern of a thin selective zone that varies with local fuel load would still be valid. When we modeled the depth and width of the necromass zone in relation to coarse fuel, we found a similar pattern (Fig 5). The width of the zone increases rapidly up to a threshold fuel load, and then stays relatively constant (Fig 5B). Therefore our model predicts that in a forest setting there would almost always be a survivable zone for pyrophilous microbes as long as the soil is deep enough, and a larger open niche (i.e., the necromass zone) would occur directly above this zone when higher fuel loads occur.

Soils above the necromass zone would be expected to have unique post-fire soil chemistry that might provide substrates for fire adapted species. Soil temperatures in higher than 175 to 200 °C volatilize waxes and lipids that then condense at lower temperatures in the soil below and cause a hydrophobic layer that can facilitate erosion [37]. This should occur near the top of necromass zone. It is not known if these concentrated hydrophobic materials serve as energy-rich carbon sources for microbes, but it is known that the hydrophobic layer disappear with time [37], and we think it would be surprising if microbes were not involved in some way. Temperatures greater than 350 °C convert cellulose and lignin into highly aromatic forms of biochar [16] that are relatively recalcitrant to microbial degradation [38]. According to our regression-based model these temperatures would only occur within about a centimeter of the surface in the highest fuel loads tested, and would be non-existent in the lower fuel loads tested.

The rapid, massive response of Pyronema species in both fire pyrocosms (Fig 3) demonstrates that this system is capable of simulating at least some known pyrophilous microbes, and it reproduces a response of similar magnitude reported from prescribed burning of log piles [20]. Pyronema’s response in our system is impressive in that three OTUs accounted for 68.7 to 86.7% of all fungal sequence reads in both pyrocosms within just two weeks of the fire (Fig 3), and the most dominant Pyromena OTU in each pyrocosm accounted for 55.1 to 58.4% of all reads in the two-week post-fire period, compared to less than 1% in the control (Table 2). For comparison the most dominant fungus in the unburned control was a xerotolerant yeast that accounted for less than 7% of the sequence (Table 2). This latter value is typical of dominants in other studies of soil fungi in which the most abundant species typically account for only few percent of the sequences, and all species with more than 0.1% are viewed as dominants [39]. Pyronema species achieved a dominance that is roughly 6 to 10-fold higher than is typical of the most dominant fungal soil fungi, and they did so in just one or two weeks.

Pyromena species have been known to be rapid responders to fire for over a century [4], but all of the earlier literature is based on the fruiting response of the species. Evidence of its in situ mycelial response was lacking until recently [20]. In vitro studies have shown that Pyronema species grow rapidly in situations where there are few competitors [7, 40]. Pyronema was also mentioned in Petersen’s (1970) experimental fire work, but its fruiting, though rapid, was not consistent enough for it to be considered in the successional patterns that Petersen discussed. Interestingly in our study, it only fruited in one of the two pyrocosms, even though it dominated in both. Thus, it may be much more common in post-fire soils then its fruiting record suggests. At least some pyrophilous fungi appear to have dormant spores or sclerotia in the soil, and these germinate following heating or chemical changes associated with fire [6, 15]. This mode of activating quiescent propagules is consistent with the rapid response we saw, but we did not specifically test for it by preventing dispersal. Nevertheless, the presence of all three dominant Pyronema OTUs in both pyrocosms shows that no inoculation was necessary for a rapid response.

The small response of Pyromena domesticum in week one in the unburned control is interesting (Fig 3), and corresponded to a total of 1.69% (3535 total reads) of the sequence in that sample. This is three orders of magnitude higher that the 92 sequence reads that contaminated the mock community, and therefore must be a real response of Pyronema within the unburned control soil. We interpret this as a short-term simulation following the wetting of the dried soil. One could imagine that such transient responses could take advantage of brief periods of low competition to renew soil inoculum between fire events. In any case, the prevalence of Pyromena domesticum in the control dropped to 0.006% by week two. The burned pyrocosms also show rapid increases and decreases in Pyronema within one-week periods (Fig 3). This rapid turnover shows that most of the Pyronema DNA in the soil does not linger long, and it suggests the occurrence of either autolysis or degradation by other components of the microbial community. In contrast the presence of ectomycorrhizal taxa such as Cortinarius and Russula spp. that occur in similar levels within the control and burned pyrocosms (Table 2) are likely due to survival of environmental DNA, perhaps as spores, as neither taxon would be expected to grow without a host.

It is less clear if fungi other than Pyronema specifically responded to the experimental fire in a positive way because abundance differences were not as great and replication was insufficient in this pilot study. However, the inclusion of ectomycorrhizal fungi in the set of fungi that appeared to increase after the fire at low levels (Table 2) shows that these apparent changes are within the level of stochastic sampling error, as growth of these ectomycorrhizal fungi could not have occurred without a host tree. For most other fungi we have no knowledge of their autecologies, so when they appear to respond at similar levels to the ectomycorrhizal fungi there is no strong evidence for or against their fire response. Longer incubations times and increased replication are necessary to resolve this.

The negative effects of fire on most fungi is clear from the reduced species richness and shape of the ranked abundance curves (Fig 4). This result is concordant with a meta-analysis of fungal response to fire [3], and makes intuitive sense based on the killing effect of soil heating. However, having taxa with extremely high read abundance (e.g. Pyronema) can distort perception of community structure because our ability to detect less abundant taxa is reduced as more of the sequence depth is consumed by the dominants [41]. However, if we remove the sequence reads from the top three Pyronema OTUs, and recalculate percent dominance, the rank abundance curves from the burned pyrocosms are still much less even than that of the control (S3 Fig). This shows that in addition to Pyronema other taxa are also driving the shape of the ranked abundance curves, and thus the reduction in richness and evenness are unlikely to be artifacts.

We propose that the pyrocosm system is an excellent model system to dissect the post-fire microbial community, and this can be done in a variety of ways. For example soil source, fire intensity, watering regime, access to external inoculum and incubation conditions can all be varied and replicated to study the process of post-fire community assembly. If pyrocosm experiments were incubated longer or under different conditions (e.g., water, soil type, temperature), other common, known pyrophilous fungi [6] might develop from the natural inoculum just as Pyronema did. However, even if they did not, most of them grow well in culture [7] and could be added into post-fire soils that lack them in varying orders and combinations to determine environmental versus biological interactions that underlie community assembly [42]. There are also indications of parallel postfire responses in bacteria and microfauna [2] that could be studied in similar ways with this system.

Finally, we propose that Pyronema is an excellent model organism for the study of fire fungal ecology. In addition to their ease of isolation and rapid growth rates, three genomes from Pyronema species have now been completely sequenced, assembled and annotated, and are available on Mycocosm (http://jgi.doe.gov/fungi), the genome portal for the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) [43;44]. Coupling these genomic resources with the rapid dominance of Pyronema in postfire soil can thus be used to explore the functional roles of this fungus in a realistic environment. For example, gene expression could be studied by incubating Pyronema on temperature defined soil layers removed from pyrocosms (S1 File) followed by extraction and sequencing of mRNA. This approach would help improve our understanding of what these fungi live on within the post-fire soil.

Furthermore, Pyronema is not unique within the post-fire community; genomes of 10 other pyrophilous fungi have now been sequenced, assembled, and annotated (Table 3). The list includes representatives of most of the common genera of pyrophilous fungi [6]. This is important because metatranscriptomic approaches with fungi are generally limited by the number and taxonomic coverage of sequenced and annotated genomes [45]. Due to its relative simplicity, the post-fire soil fungal community may now have the best genomic resources of any soil system, and these resources can be used in combination with the pyrocosms to dissect interacts with post-fire soil chemistry or with other organisms in this environment.

Table 3

Sequenced, assembled and annotated Genomes of pyrophilous fungi.

Phylum	Species	Website
Asco	Geopyxis carbonaria	https://genome.jgi.doe.gov/Geocar1/Geocar1.home.html
Asco	Morchella snyderi	https://genome.jgi.doe.gov/Morsny1/Morsny1.home.html
Asco	Peziza echinispora	https://genome.jgi.doe.gov/Pezech1/Pezech1.home.html
Asco	Pyronema omphalodes	https://genome.jgi.doe.gov/Pyrom1/Pyrom1.info.html
Asco	Pyronema domesticum	https://genome.jgi.doe.gov/Pyrdom1/Pyrdom1.home.html
Asco	Tricharina praecox (DOB1048)	https://genome.jgi.doe.gov/Pezsub1/Pezsub1.info.html
Asco	Tricharina praecox (DOB2270)	https://genome.jgi.doe.gov/Tripra1/Tripra1.home.html
Asco	Wilcoxina mikolae	https://genome.jgi.doe.gov/Wilmi1/Wilmi1.home.html
Basidio	Coprinellus angulatus	https://genome.jgi.doe.gov/Copang1/Copang1.home.html
Basidio	Crassisporium funariophilum	https://genome.jgi.doe.gov/Crafun1/Crafun1.home.html
Basidio	Lyophilum atratum	https://genome.jgi.doe.gov/Lyoat1/Lyoat1.info.html
Basidio	Pholiota molesta	https://genome.jgi.doe.gov/Phohig1/Phohig1.home.html

Conclusions

Pyrocosms now add to the growing list of experimental systems that can be used to study fungal community ecology [46], and they specifically relate to a natural environment that is growing in importance as fire size and intensity continue to rise [47]. Furthermore the community and the factors affecting this environment are simple enough that experimental and modeling approaches might be easily combined to achieve greater predictability [48], while the availability of genomic resources open avenues to dissect the functional roles of the component species in great detail.

Effect of flash fuels is subtle.

172 gm of flash fuels caused temperatures to rise slightly at 10.5 cm below the surface, but ultimately achieved the same peak temperature as solar heating in an unburned pyrocosm monitored simultaneously. Pyrocosms with the same flash fuel load, but one or two charcoal briquettes (25–51 g) heated more rapidly and achieve higher peak temperatures.

(TIF)

Click here for additional data file.

NMDS plots using Bray-Curtis dissimilarity.

Samples were rarified to 3950 reads/sample. Adonis R2 = 0.33 and 0.19 for Bray-Curtis (A) or Jaccard (B) respectively.

(TIF)

Click here for additional data file.

Ranked abundance curves with the three most abundant Pyronema OTUs dropped and percent read abundance recalculated without them.

Top 50 most abundant OTUs are shown.

(TIF)

Click here for additional data file.

Additional notes on pyrocosm assembly and use.

(DOCX)

Click here for additional data file.

24 Oct 2019

PONE-D-19-24954

A simple pyrocosm for studying soil microbial response to fire reveals a rapid, massive response by Pyronema species

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Reviewer #1: This manuscript was problematic for me. One the one hand, it clearly shows a methodology that could be used to study the effects of soil heating (by fire) on soil microbial communities. In the regard it is useful. On the other hand, the manuscript did not adequately test the proposed methodology. We are not shown how much variability occurs among multiple replicates, we do not know whether the conditions the system is able to produce in the soil actually mimic the conditions during a fire in the field, we are not shown how to alter fuel loads, etc. to tailor conditions to mimic a variety of field conditions during fire in the field, etc. etc. Therefore, this contribution is not nearly as useful as it might be. On balance, therefore, I think the manuscript could be publishable if the authors could remedy the deficiencies I have pointed out.

The introduction does a great job of introducing the reader to the effects of fire on soil chemistry, physics and biology. But there is little justification for the development of a “pyrocosm” system for experimentally producing heated soils. Are there no other systems that have been previously developed and used for similar purposes?

The manuscript appears to be written hastily (see below).

Line 15. There is one too many “the”s in the sentence.

Line 17. This sentence indicates that “peak soil temperature” lingers near peak temperature, which is probably not what was intended.

Line 20. Should read “of charcoal”

Line 22. Is “massive” the best descriptor?

Line 25. How is it possible for there to be a reduction in richness while the other fungal taxa did not change significantly?

Line 29. Not clear what “it” refers to.

Line 33 uses the term “pyrophilous fungi”, while line 23 uses the term “postfire fungus”. Are these the same? If so, please use a single term.

Line 81. If they are “partially burned”, they can only be “partially pyrolyzed”, not “pyrolyzed”.

Line 81. Heat alone is not sufficient for pyrolysis. Oxygen must also be absent or very low.

Line 115. The written description here is inadequate. The photo helps, of course, but the written description ought to be accurate. “Filled to depth of 16 cm” does not tell how much space occurs above the soil within the bucket. And what does “buried to soil level” refer to, the soil in the bucket or the soil in the field? Which soil surface (in or outside the bucket) were the 10-13 holes positioned above?

Line 158. What is meant by the “first” soil pyrocosms? Were there more than one? How many? It appears that there were only two pyrocosms total ever used for testing. If that is true, the obvious difficulty is that we do not know how much variability exists among pyrocosms. If you only use 2, it is possible that by chance they were similar.

Line 168. Why 10.5 and 15.8 cm? Is that arbitrary?

Line 176. How were the samples dried?

Line 177. When tamping down, were field densities approximated?

Line 180-185. Why were charcoal briquettes used? Does their heat simulate that of burning wood? Is heating to a certain temperature in x amount of time equivalent to heating to the same temperature in 2x amount of time? Or is that unimportant?

Line 207. Again, how does a single replicate serve as a control?

Line 274. I do not think one can say much about reproducibility from n=2. True, the two look very much the same, but that could occur simply by chance. What if there were 4 or 5 or 10 replicates?

Line 275. The fact that peak temps are reached hours after the fire is out and linter for 40 minutes seems to be an artifact of the conditions. If there were a hotter fire, if there were thicker soil, if the buckets were a different size, etc. the results would be different. So how is it possible to know whether these conditions are relevant to those that occur in natural fires in the field?

Reviewer #2: The contribution describes a simple pyrocosm, in which soil communities and/or processes can be inexpensively studied in well replicated experimental settings. The value in this paper is not in its data or robust statistical analyses, but rather in the design of the experimental “fire bucket” and in a series of experiments that serve as a proof of concept for the experimental system. The manuscript demonstrates that the heat transfer into the soil profile - at least with the soils used - can be predicted by soil depth and mass of charcoal used in the experiments. Further, the acquired data suggest that pyrophilic organisms seem to respond in fire stimulation in the experimental system as inferred from MiSeq sequencing of the soil samples from the pyrocosm.

I see the value of describing and marketing this system as a valuable platform for fire studies without the excess complications from environmental noise. However, the absent replication minimizes the true inference of the organismal responses, and should be explicitly – and more concisely – be used as further proof for the system’s functionality. The meandering discussion on the poorly supported organismal responses is excessively lengthy and should be dramatically shortened. On the topic of presentation, the word microbes is used frequently, but in the body of the paper only fungi are discussed – are fungi microbes?

Also, the modeling of the potentially biologically interesting depths in the system is interesting but is described in excessive lengths. Although I do enjoy the modeling of the “Goldi Lock” zone, I think this could be more briefly described in the text and much of the detail moved to supplements.

I have a number of minor concerns that I list below.

MINOR COMMENTS:

Line 40: serotinous

Line 11: how is it possible to fit 7 liters of soil into a bucker that holds 3.8 liters?

Line 123: had

Line 128: is flash fuel tooth picks or pine needle litter? Compare to line 121

Line 135: Clarify what you mean by soils were collected separately

Line 196: I am uncertain how well does the single briquette transfer heat to a bucket of this size. The heat transfer is unlikely linear through the depth

Line 213: What is the depth of the core sample? What are the four even zones, based on depth?

Line 227: specify log (10, e, or something else)

Lines 301-303: I am not sure if the modeling correctly applies to flash fuels. I argue that heat transfer is a function of both duration and the temperature of the heat pulse. Therefore, extrapolating the models to flash fuels may be erroneous.

Line 315: effects

Line 15 on pager 21: Pyronema

Line 20 on page 21: It is curious that Pyronema would appear in the mocks. I have two questions: 1) were there any other taxa in the mocks; 2) is it possible that the mock is contaminated, because it was prepared in a lab that may maintain Pyronema cultures?

Lines 33-39 on page 22: While I do not question whether or not Pyronema spp. were stimulated by the fire treatments in pyrocosms, I am curious about the ectomycorrhizal fungi. I would ask the authors to elaborate on this - perhaps in the discussion. I would be particularly interested in discussion that would consider relative abundance increases when overall sequence pool changes (lower richness) and taxa (or their nucleic acid traces) may have been lost as a result of the fire treatment.

Line 69 page 23: why were the data rarified to 3950 sequences. There were a total of 6000 sequences for each of retained samples

Lines 121-122, Page 26: I believe this to be context dependent. While true for low severity fires, in wildfires that include whole log combustion the high temperatures last longer and heat is transferred deeper. See Smith et al (2016 - International Journal of Wildland Fire 25(11) 1202-1207).

Line 109, 110, 142 – In these lines, it was discussed that the soil was dried to mimic dry soil that would probably occur during a wildfire. Crushed ice was added the day after fire and the following days after to mimic snow fall. How realistic is this to dump ice onto a freshly burned plot to mimic slow snowfall? Would snowfall occur that close in time after the fire.

Line 176 Page 28: It is odd to return to definition of the necromass zone at this point. Perhaps the discussion points could be mane more concisely.

Line 202, Page 29: it may be an over statement that there was no prior evidence of mycelial in situ response for Pyronema. If my memory serves, Reazin et al (2016 - Forest Ecology and Management 377:118-227) included Pyronema observations.

Line 448: Please include the NCBI BioSample and representative sequence accession numbers.

Reviewer #3: Review of PONE-D-19-24954

This manuscript details a novel method of studying the response of fungi and other microbes to fire through the use of ‘pyrocosms.’ These in situ systems can be used to closely monitor the thermal dynamics of fire-frequented systems and study the effects of fire on microbes through inoculation. The authors use pyrocosms to describe temperature dynamics following fire in detail, and to stimulate the growth of native, pyrophilous fungi (Pyromena). Construction of the pyrocosms is well-documented. Given that the pyrocosms are cheap, easily reproducible, and can be used in a wide variety of applications, this work is a useful addition to the literature and suitable for publication in PLOS ONE.

The strengths of this paper are x and y, In revealing thermal dynamics in close detail and stimulating native, pyrophilous fungi, the authors demonstrate the clear utility of the pyrocosms, but the writing inhibits communication of this topic. For instance, I still have questions about how the experiments with the pyrocosms were conducted. So that readers can better visualize how they were used in these two experiments and in their own research, I recommend more thoroughly reporting how your two hypotheses/goals (thermal characteristics of the system and stimulating fungi) were tested in the Methods (see specific suggestions below). This being said, I found File S1 very helpful in understanding how the pyrocosms were created. In addition, if it is a key goal of the manuscript, your discussion of how a ‘Goldilocks zone’ was determined should be detailed here (along with a more explicit discussion of it in the Intro and Results) in place of solely the Discussion. I feel cautious about the number of replicate pyrocosms used in both experiments. From the writing, it is unclear how many sand pyrocosms were used in the heating experiment. Further, there were only 2 pyrocosm replicates for ‘soil’ portion of this study, and these 2 replicates contained different soils. With these 2 replicates, you can only soundly conclude that this system stimulated the growth of Pyromena. Because of this, there needs to be a more explicit statement that increased replication is needed to confirm the patterns of richness you observe, as well as an explanation for why you chose to use only 2 replicates in the Methods. Lastly, there are several potentially confusing wordings (see below for suggested copy edits). Clearing these confusions will ultimately create a tighter and more cohesive manuscript, and enable you to make a stronger case for the utility and applications of the pyrocosms.

Abstract/Introduction:

15 Should be “for studying”

27 Should be “wildfires”

37-51 Specify if you are considering wildfires only here, or fire in general (wildfires + low-intensity prescribed fires). While true of wildfires, some of these things, specifically high fungal mortality, can’t always be extended to prescribed fires.

39 Get rid of “either”

55 Consider changing “burnt” to “burned”

61-62 Although I recognize that we don’t know much about pyrophilous fungi in comparison to other groups, claiming that we don’t know what they are doing in post-fire environment seems slightly out-of-place, especially since you state earlier that we know they are saprotrophic.

64-71 From later portions of the manuscript, it does not seem like ectomycorrhizae are a central focus. Removing this paragraph would make for a better focus on your central goals.

95 Should be “wildfires”

100 This is not necessarily true for prescribed fires. Consider changing language to focus on wildfires.

100 should be “post-fire”

105-110 In reading the rest of the manuscript and from lines 90-92, I understand that you used the temperature experiment to investigate effects of fuel load and to determine a potential ‘Goldilocks zone.’ Mentioning these goals here will help readers track these ideas throughout the manuscript.

I assume that you aimed to use the pyrocosms in ways that mimic wildfires, rather than prescribed fires. However, for reader clarity, reiterate this when you state your hypotheses/goals. Also add that the pyrocosms directly answer to the need for increased sampling immediately following fire.

Methods:

126 Pictures are great for visualizing the pyrocosms!

132-146 Is there a specific reason why only 2 pyrocosms (and 1 per soil type treatment) were used to study fungal response? This is a very low number of replicates, and should be justified.

138 Why was this soil not depth stratified?

139 Confused about the litter and F layers.. these were collected from the unburned site, and then used in both pyrocosms?

149 How many sand pyrocosms were used? “14 experiments” seems vague.. does this mean pyrocosm replicates, number of times the pyrocosms were burned, or a combination of the two?

154 Here (and in line 167, and in Results) you imply that you used the sand pyrocosms to study thermal dynamics under different fuel loads. If this is the case, I would restate this more explicitly, as well as what the fuel loads were.

190 Does this mean that the soil pyrocosms were included in the temperature experiment?

193 For consistency, keep this in g only. In addition, why did the amount of charcoal added to the sand pyrocosms differ so much?

192-196 Why is this ignition protocol different from the one you describe for the soil?

203 Although I assume that the main point of adding ice to the pyrocosms was to stimulate fungal growth (mentioned line 123), it would helpful to remind readers why you chose to do this.

206 Why did the soil pyrocosms receive different amounts of ice?

208 So that readers do not get confused, move discussion of the 3rd forest soil pyrocosm (the control) to 132-146.

213-217 Does this mean that you took one soil core from each pyrocosm at each time point? Why was the 4-week sample taken only for the 2nd pyrocosm? Did you sample the entire depth of the pyrocosm (meaning 4in depth increments)?

221 Is there a specific reason why you chose to amplify ITS1 instead of ITS2 (ie is it more represented in UNITE)? If so, please cite here.

258 Provide full name for OTU (operational taxonomic unit)

Results

273-315 is this temperature data coming from the sand pyrocosms or the soil pyrocosms? The methods reflect that the sand pyrocosms were used for this. However, this section (especially the mention of 2 replicate pyrocosms, line 282, and Fig. 2A) makes it seem like this data is from the soil pyrocosms.

282 If this corresponds to pyrocosm 1 and 2, make sure that it reads this way in the figure (ie change A and B to 1 and 2).

292 Should be “0.94779 respectively”

295 To help readers better track ideas, the flash fuel/fuel load portion of the heating experiment should be mentioned in the Intro and Methods.

303 “to a depth of 1.26 cm”

310 “for the surface” is a little confusing

315 should be “effects”

Table 1: Should pyrocosm 2 week 3 be week 4 (see Methods line 214)?

320 “in control or treatment pyrocosms”

326 “Table 1”

Table 2: should be “Control”

5 (and on): capitalize pyrocosm 1 and 2 (ie “Pyrocosm 1”) to keep consistent with Methods

10 “ranked 10th the 4th in abundance” is confusing.

Discussion

141 If a key aspect of your study is finding exploring heating patterns and Goldilocks zones, it should also be discussed in the Intro, Methods, and Results.

173 What disappears with time? You might be missing a word here

246-259 Your statement that more replication is needed needs should be stronger and repeated in this paragraph. As mentioned above, only having 2 replicates calls these conclusions on richness into question.

274-294 This whole-genome sequencing section might be better combined in a sentence or 2 with the benefits of Pyromena as good natural inoculum. I believe that your manuscript has a nice focus on the benefits of pyrcosms and how they can stimulate pyrophilous fungi, and the level of detail on this sequencing portion is not necessarily needed.

297-303 I might add that they answer to the need for increased sampling immediately following wildfire.

**********

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23 Jan 2020

Here is the amended statement on funding that you requested:

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The work was funded by the Department of Energy grants DE-SC0016365 and DE-SC0020351 to TDB.”

And here is the updated data availability statement:

The underlying data for the work consisting of: temperature and fuel data for all pyrocosms and the complete OTU table representing fungal community response. These are deposited in Dryad: doi:10.5061/dryad.45gd695; Representative sequences for all the OTUs are deposited in NCBI MN724033-MN724919, and the raw sequence read data are deposited in the short read archive: PRJNA559408. R-scripts for all sequence analyses are deposited in Github: https://github.com/sydneyg/Pyrocosms

General comments about the revision: We thank the reviewers for their careful reading of the manuscript and their many useful edits, suggestions and comments. They have certainly helped us refine our message and the presentation of results in this revision.

Comments on content are addressed below in the line-by-line list by reviewer. Small edits (spelling, grammar, word use changes) suggested have been made and can be found in the marked up version of the revision but will not be covered below.

The most general criticism made was about the limited replication (two experiments) in the biological part of the study. We concede that this limits the organismal inferences that can be drawn, and we had acknowledged this in various parts of the first draft. However, it serves the purpose of demonstrating that our simple experimental system works to stimulate at some pyrophilous fungi, and reviewer 2 and 3 recognized this, but thought it should be clarified. Similarly at least two reviewers commented our description/presentation of the “Goldilocks zone”, and though found it interesting, thought that it was not well incorporated or was overly discussed.

In response to these comments we clarified our goals and role of the biological experiments in the introduction. We did this by outlining our thermo-chemical gradient model in the third and fourth paragraphs. This succinctly summarizes the way we theorize that heat, soil depth and altered soil chemistry interact to create a predictable, depth-structured habitat for microbes in post-fire soils. In the last paragraph of introduction, we make it clear that: 1) our primary goal was to develop an experimental system that allows the post-fire soil system to be manipulated at a fine scale; 2) the forest soil experiments were essentially a pilot study to show that our experimental system has biological relevance, and 3) the heating results are used to refine our concepts of the Goldilocks and necromass zones in our model.

Below we address the specific concerns of the three reviewers.

Reviewer 1

Thanks for your comments and edits. However, we are not completely sure that we understand exactly what your concern about the lack of replication is referring to. If it is the biological replication, we concede that two reps and one control is not enough to generalize, but as discussed above we have now clarified that they serve primarily as a proof of concept. If concern is referring to the heating experiments then this is incorrect. As we showed with regressions that soil heating is highly predicable from our data. Specifically peak temperatures at difference depths are related by the ln of the depth (Fig2b), and peak soil temperature at a selected depth are a function of the mass of fuel (Fig 2C). The R2 on all these regressions is quite high, and replication of specific fuel loads is not necessary to demonstrate this. Nevertheless, we show that replicated fuel loads result in nearly identical heating profiles (Fig 2A). This can also be seen in Fig 2C for fuel load just under 300g.

In terms of “showing readers how to alter fuel loads” we have made the methods clearer now, but we think that part of the problem may be that the reviewer apparently missed a supplementary file (S1) that goes into more detail on construction and uses of the pyrocosms.

We have now reworked the introduction to better justify the development of the pyrocosms. This is specifically addressed in the last paragraph of the introduction. Thank you for this suggestion.

Former line 22 - Yes, we think “massive” is an apt descriptor for a response that increases the sequence abundance of an organism from barely detectable levels to roughly 60% of all sequence. This is an order of magnitude more dominant than is typical of soil fungi in sequence studies.

Former line 25 - The reason reduction in richness can occur without large changes to the other fungal taxa is because we use a relative abundance measure (i.e., sequence %). Thus large increases in a very small subset of taxa (e.g. Pyronema) decreases the apparent abundance of other taxa. The taxa “lost” were those that had very small abundance in the pretreatment condition, but taxa that were reasonably abundant are retained at similar levels. This is covered more in the results and discussion.

Former line 29 - now reworded to make the antecedent of “it” clear.

“Pyrolyzed” is now qualified were it needs to be and defined more carefully. The reviewer’s comment about the need for low oxygen for pyrolysis may be technically correct, but in practice this is clearly not a limitation in soil systems, or even soil surfaces, where charred remains are common.

Former line 33 - Pyrophilous is now used uniformly

Former line 115 - We have revised the description of the pyrocosm setup to make it clearer and corrected the volume discrepancies that other reviewers caught as well. We also point to the supplemental notes (File S1) that have addition images and details.

Former line 158 and elsewhere the number and type of pyrocosm experiments is now clarified.

The selection of depths for the thermocouples was somewhat arbitrary but is explained on lines 310-313: “These depths were selected to insure that the wire thermocouples did not experience temperatures above 200ºC across the range of fuel loads tested.”

Former line 322; Method of drying litter is now explained

Former line 322 - F-layer and litter layer density was not measured; the tamping down was meant to approximate it, but we have no measure of how well we achieved that. However, it was completely incinerated, and we show that such flash fuels causes negligible soils heating, so we doubt it matters for the reproducibility or realism of the experiments.

Former line 180-185 The use of charcoal is now explained more fully. Essentially we use charcoal because they burn very predictably, transfer heat well to the soil, are probably not too different from natural substrates in forest fires, and are readily available.

Former line 275 - The finding that peak temperatures at depth are achieved hours after the fire goes out and linger for ~40 minutes is absolutely not artefactual; it is caused by the large heat capacity of soil. This makes soil resistant to temperature change, but allows it to absorb and hold heat. Thus the heat source for soil at depth is the soil above it, not the active combustion. The result occurred in all the sand pyrocosms and both the soil pyrocosms, and it is likely to be greater in nature where the volume of soil is larger and there are not unheated edges. You can see evidence of this same effect in Smith et al paper cited where they burned log piles in nature.

Reviewer 2

The first comments about the value of this work being in the system, and the value of the sequencing being in the demonstration of its biological value is exactly what we had hoped to communicate. After reading the reviews and realizing that this was not obvious to everyone, we reworked the introduction as discussed above. Thank you for this clear insight.

We agree that our limited biological replication limits inference. Our attempt to point out these limitations and to extract those results that transcend them was part of the reason the discussion was longer than the reviewer desired. We have now dropped at least two paragraphs of results and discussion about the biological response. What we retained was focused on Pyronema, and explaining why the mycorrhizal “response” indicates the noise level.

The term “microbe” is not a taxonomic term - it’s simply a common name for small organisms. So yes, we see microfungi as a subset of microbes. Using this term particularly in the introduction is useful, because bacteria, and fungi both have to deal with the same fire and post-fire conditions and substrates, and they would both be assessed through very similar high-throughput sequence approaches. However, we have now tried to clarify exactly when we are referring to only fungi, which is obviously most of the time.

We retained the modeling of the Goldilocks and necromass zones but as discussed above have now incorporated better into the focus of the paper as part of a broader thermo-chemical model.

Minor issues

Former line 11 - Yes, we had given the wrong size for the buckets - nice catch.

Former line128 - flash fuel is now defined more clearly.

Former line 135 - the soil separation is now spelled out better.

Comment on former line 196 - if you check the results (Fig S1) heat from one briquette was easily detectable, and had a greater effect than a much larger mass of flash fuels.

Former line 213 - the depth of sampling is now specified (all the way to the bottom of the bucket)

Former line 227 - natural log is now specified but the truth is that log10 is almost as good.

Comment on former lines 301-303: - I think there is confusion here on what is being regressed and we have now corrected that wording to clarify it. Basically there are there two main regressions used in this study. 1) temperature regression, which uses measured temperatures at 2 depths to predict temperatures at unrecorded depths (i.e, Fig 2b); and 2) course fuel related regression - where grams of charcoal are used to predict peak temperatures at particular depths (Fig 2C). We agree completely with the reviewer that flash fuels cannot be predicted by the second regression, but that is not what we did. Instead we are just using the measured peak temperatures achieved by the flash fuels at two depths to predict temperatures at other depths. This is valid because it is simply a function of the heat capacity of the soil - any heat source can be modeled this way.

Former Line 20 page 21 - Other contamination sources are always possible with PCR, and that is why no-DNA and mock community controls are so important. What we show is that the contamination level is quite low. However, it is very common for mock communities or other control results to be contaminated by abundant sequences from other samples. This is because of “index switching” where the primer tags from one sample are recombined with the amplicon from another sample during PCR is common (see the Carlson et al 2012 paper cited). The more abundant the amplicon is, the more likely that it will experience index switching and end up looking like it was in samples it did not actually occur in. Thus, we think this is the most likely source of contamination. To avoid this, recent libraries have used double indexing (tags on both primers).

Former lines 33-39 p 22. The nominal mycorrhizal “stimulation” was clearly artefactual, and that is (and was) mentioned in the discussion. We think that from the collective knowledge of Russula species we can say with some confidence that it did grow in a soil environment without a host. It is likely that the few percent increase seen in the two burned pyrocosms relative to the one control is due to soil heterogeneity/sampling issues that are compounded by low replication. We think the level of “mycorrhizal response" is a nice benchmark, in that we can’t infer anything about taxa that were apparently stimulated to similar or lower levels.

Former line 69, page 23; 3950 was correct, not 6000. We corrected that now - nice catch!

Line 121-122 larger, glowing, heat sources certainly heat the soil more and result in higher temperatures at depth, but heat transfer follows the same physics even when the heating is less - that’s what our first regression shows. Even the slight heating from flash fuels showed the slow rise and the lingering effect (Fig S1); the main difference is that the peak temperature achieved is much lower/g of fuel. However, whatever peak temperature is achieved is will linger near that temperature for some time, and organisms need to endure it; that’s the point.

Former Lines 109 etc, about ice: Water needs to be added. This is dry soil, so without it little activity would be expected. The various amounts and ways it could be added are clearly another variable that could be tested. The advantage of ice is that it melts slowly and wets the soil thoroughly. We were worried liquid water would channel through the hydrophobic upper layers and create uneven wetting, and would need to be added a little at time to avoid this problem. With a single pyrocosm that could be done, but with multiple pyrocosms the ice is much easier and provides a very uniformly timed wetting. Is a good mimic of snow? It’s clearly not perfect, but it’s probably good enough. The one big Sierra fire (Rim Fire 2013) we followed was initially wetted by a snow event that melted quickly at the elevation that these soils came from, and it spawned fungal activity similar to that we observed in the pyrocosms.

Former line 176 - we have incorporated the necromass zone discuss into the paper better now (see bit on introduction), and reordered it in the discussion

Former line202 p 29. We had missed the Reazin et al. paper before, but have now incorporated it. It’s an excellent addition as it shows a massive Pyromena response in the field. We think this is the only such published example, although we have unpublished results from the Rim fire that show this too.

All sequence samples now have accession numbers rather than the XXXXX seen in the original submission

Reviewer 3

As discussed above we have now focused the paper on the pyrocosm system and the thermal-chemical gradient model, and this was partially in response to reviewers’ clear summary of the manuscript - we thank you for that. We have also clarified how many pyrocosms were involved and addressed the replication issues.

Thank as well for the many edits suggested - these have been incorporated, but they will not be addressed line-by-line here.

Former lines 61-62. Knowing that most pyrophilous fungi are saprobes really only scratches the surface in addressing the question of what they do in this chemically altered environment. We have now clarified our point by stating: “…, nor is it known what these fungi degrade and live on in the chemically altered post-fire environment.”

Former line 64-71 Discussion of ectomycorrhizal response to fire is now removed as suggested.

Former line 100: The distinction between wildfires and prescribed fires is now clarified throughout.

Former line 105 - The Goldilocks zone is now incorporated better.

Former line 138 - the postfire soil added was not depth stratified because it was collected from a disturbed pile of soil in the fire zone. This is now mentioned.

Former line 139 - the origins, use, and treatment of the litter and F-layer is clarified now.

Former line 149 - As explained in the set-up they were reused by removing ash following one experiment before adding fuel for the next. That is why we said “14 experiments” rather than 14 pyrocoms. We have clarified this now.

Former line 154 - Soil pyrocosms were also used for temperature experiments to the extent that the Ln relationship between depth and peak temperature are shown with soil (Fig 2B). This is indicated in the legend.

Former lines 206 - The amounts of water differed because we had not standardized the amount of water yet. As explained above the biological side of experiment was a pilot study. If it had not succeeded in producing a biologically relevant result, the rest of the study would not have gone forward. So essentially we were trying different water and soil to see if anything worked.

Former line 192-196 The soil experiments were actually run prior to the sand experiments. We only learned later that could create the same temperatures profiles with less manipulation by simply letting fewer charcoals burn completely. In the more detailed instructions on assembling pyrocosm (File S1) the difference between the two burning methods is discussed more, but the bottom line is that either works.

Former line 203 - the ice is now explained more fully, and the explanation for the differences in ice are explained above.

Former lines 213-217 - The coring number and depth is now clarified.

Former lines 221 - our choice of ITS 1 was basically so we could compare it to our own earlier data. - This is now explained.

Line 258 - Operational Taxonomic Unit had already been spelled out at first use of the OTU on line 228 (now line 376)

Line 273-315,and line 182. Temperature results are primarily from the sand pyrocosm, and the specifics are now indicated in the legend to Fig 2

Former lines 274-294 - The whole-genome sequencing section is germane to the system as a whole, because it makes it more experimentally more powerful. We spent a short paragraph on it, and we feel this is important so that others that might want to use the system are aware of the resources available.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

6 Feb 2020

PONE-D-19-24954R1

A simple pyrocosm for studying soil microbial response to fire reveals a rapid, massive response by Pyronema species

PLOS ONE

Dear Dr. Bruns,

Thank you for submitting your manuscript to PLOS ONE. After careful review, your manuscript has been deemed suitable for publication. However, the reviewers did catch some minor grammatical errors and points for clarification. I would ask that you correct these small errors and resubmit a revised manuscript so as to permit formal acceptance.

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Reviewers' comments:

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1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

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Reviewer #3: (No Response)

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #2: Yes

Reviewer #3: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript has been substantially improved. With the exception of a few suggestions, below, I think it is ready to go.

Line 130. Probably should be “liters”.

Line 142. “10-quart” should be given in metric units.

Line 337. Probably should be “few or no coals”

Line 84, 113, 121 (Discussion) Probably should be “coarse”

Reviewer #2: I am reviewing this contribution for the second time. I see that the authors have been mindful and thorough about responding to previous reviews. They have also generously made all data, including sample-OTU tables and scripts available through appropriate depositories.

It is true that some of the reviewer comments are impossible to address without redoing the entire experiment – or repeating for additional blocks. That said, I fully agree with the authors, point of the contribution is not to describe an experiment to infer responses to treatment as much as it is to demonstrate the system and provide a proof of concept. I strongly feel that demonstrating heat penetration and organismal responses in a system that permits replicated fire studies is important. I only have a few editorial comments on few issues that may have slipped into the text during the revision.

Line 25: Typo – “We introduce a thermoschemical gradient model to summarize[s] the way that heat”

Line 28: The goldilocks zone is an orphan concept in the abstract. I agree this will be clarified in the forthcoming text, but might need to be omitted or better explained here.

Line 95: “Temperatures from 480-220” – why not “220-480?”

Line 248: Please provide an estimate of rainfall equivalence in mm.

Text below table 1: Pyronema spp. – “spp.” need not be italics

Line 8 in the second round of numbering: Grammar in “was ranked10th the 4th in abundance ”

Reviewer #3: (No Response)

**********

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Reviewer #3: No

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12 Feb 2020

all minor edits have been made.

this line is now added at line 168 "All soils were collected in Spring 2015, under Special Use Permit #GRO1087 from the USDA Forest Service, Stanislaus National Forest to TDB. "

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

14 Feb 2020

A simple pyrocosm for studying soil microbial response to fire reveals a rapid, massive response by Pyronema species

PONE-D-19-24954R2

Dear Dr. Bruns,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Reviewers' comments:

19 Feb 2020

PONE-D-19-24954R2

A simple pyrocosm for studying soil microbial response to fire reveals a rapid, massive response by Pyronema species

Dear Dr. Bruns:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

Dr. Garret Suen

Academic Editor

PLOS ONE