Many foliar endophytic fungi of Quercus gambelii are capable of psychrotolerant saprotrophic growth.

Many endophytic fungi have the potential to function as saprotrophs when living host tissues senesce and enter the litter pool. The consumption of plant litter by fungi obviously requires moisture but, in the arid, western USA, the native range of Quercus gambelii Nutt., most of the precipitation occurs during the coldest months of the year. Therefore, we hypothesized that the endophytic fungi of Q. gambelii have the potential to function as psychrotolerant saprotrophs, which we defined in this study as an organism capable of significant growth on leaf litter at 5°C. We further hypothesized that a tradeoff exists between growth of endophytic fungi at 5°C and at 17°C such that fungal isolates are either cold- or warm-temperature specialists. Consistent with our first hypothesis, we found that 36 of our 40 isolates consumed leaf litter at 5°C, but there was a surprisingly high degree of variability among isolates in this ability, even among isolates of a given species. Contrary to our second hypothesis, there was no tradeoff between saprotrophic growth at 5°C and saprotrophic growth at 17°C. Indeed, the isolates that grew poorly as saprotrophs at 5°C were generally those that grew poorly as saprotrophs at 17°C. By virtue of being endophytic, endophytic fungi have priority in litter over decomposer fungi that colonize plant tissues only after they enter the litter pool. Moreover, by virtue of being psychrotolerant, some endophytic fungi may function as saprotrophs during the cold months of the year when moisture is temporarily available. Therefore, we suggest that some endophytic fungi of Q. gambelii could play significant ecosystem roles in litter decomposition and nutrient cycling.

Introduction

In arid ecosystems, microbe-mediated litter decomposition and nutrient cycling can be limited by moisture [1], but this may depend on the time of year as some arid ecosystems are only seasonally dry. In the Great Basin desert of the western United States, for example, inadequate moisture frequently limits microbial decomposition of litter in the warmer months of the year, but it may not limit decomposition in the winter and early spring when evaporation is low and precipitation is relatively high [2]. Because moisture is least limiting in the winter and early spring, microbial decomposition of litter in the Great Basin of the western United States requires that some saprotrophic microorganisms have significant metabolic activity under cold conditions [3,4]. Such microorganisms are referred to as psychrotolerant, psychrotrophic or psychrophilic [5,6]. We hereafter refer to this phenomenon as psychrotolerance.

Endophytic fungi very commonly colonize living plant tissues [7]. In some cases the host plant benefits from their colonization, which may improve seed germination [8], vegetative growth [9] and tolerance to stresses such as drought [10,11], extreme temperature [12,13], salinity [13,14], heavy metals [15] and herbivory [16,17]. Moreover, upon senescence of the host tissues they occupy, many endophytic fungi are capable of obtaining nutrition saprotrophically [18-23]. Indeed, by colonizing living plant tissues, endophytic fungi are essentially pre-colonizing litter. Therefore, endophytic fungi are among the first with access to litter as a source of energy and nutrients [21,24]. Priority access to a resource frequently results in a competitive advantage [25-27]. Thus, in litter, endophytic fungi may have a competitive advantage over decomposer fungi that colonize the plant tissues only after they senesce [28].

If an endophytic fungus were strictly biotrophic, it would only have to maintain activity during the warmer growing season. However, if an endophytic fungus were to function as a saprotroph in the Great Basin, where moisture is least limiting in the winter and early spring, it would be advantageous to be psychrotolerant. Our first hypothesis, then, is that endophytic fungi isolated from leaves of Quercus gambelii Nutt. are psychrotolerant saprotrophs, and that there are significant differences among species in their ability to function as psychrotolerant saprotrophs. Organisms frequently exhibit functional tradeoffs. For example, tradeoffs have been observed in fungi between growth and enzymatic activity (Zheng et al., 2020), between growth and reproduction [29,30] and between growth and defense [31]. Still other tradeoffs occur because organisms frequently cannot excel along the entire length of an environmental gradient. For example, plants that are active in cool climates may adapt their physiology to lower temperatures such that they do not perform well at higher temperatures, while plants that are active in warm climates adapt their physiology to higher temperatures [32]. Therefore, our second hypothesis is that there is a tradeoff among the endophytic fungi isolated from Q. gambelii such that they specialize at either winter or summer temperatures.

Materials and methods

Study sites and leaf sampling

Q. gambelii is a small tree or shrub found mainly in Utah, Arizona, New Mexico and Colorado. In Utah it is widely distributed between approximately 1,200 and 2,400 meters in elevation [33]. In order to sample leaves from across the elevational range of the species in Utah, whole, unblemished leaves were collected on 24 September 2019 at Devil’s Kitchen (39.48’12.27”N, 111.41’17.96”W), elevation 2,553 meters, near Payson, Utah and on 8 October 2019 in Slate Canyon (40.13’30.59”N, 111.37’21.9”W), elevation 1,553 meters, in Provo, Utah. Air temperatures were monitored at the Devil’s Kitchen site from 24 Jun 2019 to 24 Sep 2019 using a temperature data logger (HOBO UA-001-64, Onset Computer Corporation, Bourne, MA, USA), set to log hourly. At Devil’s Kitchen, 15 leaves were collected from each of 7 trees and, in Slate Canyon, 10 leaves were collected from each of 6 trees, totaling 165 leaves. No sampling permits were required because of the small number of samples taken. In the field, leaves were stored in plastic bags and placed in a cooler on ice. Later on the same day, the leaves were transferred to an incubator at 5°C in the laboratory.

Fungal isolation

We isolated fungi from Q. gambelii leaves in a laminar flow hood to insure sterility. External leaf surfaces were sterilized by submerging leaves in 70% ethanol for 2 seconds, immediately submerging them in 3% sodium hypochlorite for 2 minutes, similar to Arnold et al. [7], rinsing away the hypochlorite by submerging leaves sequentially in three beakers of sterile water, then submerging, again, in 70% ethanol, after which they were laid to dry in a sterile petri dish. Each surface-sterilized leaf was subsampled once using a sterilized, 6 mm diameter, paper hole punch, taking care to re-sterilize the punch between leaves from individual trees with ethanol that was flamed off. Each leaf disk was placed on 2% malt extract agar in its own petri dish, sealed with parafilm and incubated initially at 10°C, a temperature that had previously been used to isolate psychrotolerant endophytic fungi [34]. After 3 weeks, fewer than 10 total fungal colonies grew out from the entire collection of leaf disks. These were isolated to their own dishes. The original dishes containing the leaf disks were then transferred to a 17°C incubator for an additional 3 weeks. Many new fungi growing from the leaf disks at 17°C were thus isolated. When there were multiple fungal colonies growing from a single leaf disk, each was isolated separately.

Sequencing and assigning taxonomy of fungal isolates

A total of six hundred seventy-five fungal isolates were sorted into morphological groups based on color, hyphal growth pattern and mycelial density. From these groups, 40 isolates were chosen for study, each of which was sequenced. Each isolate was subject to direct PCR [35-37] of the ITS region using APEX 2 Hotstart Master Mix (Genesee Scientific, El Cajon, CA, USA) with ITS1F and ITS4 primers [38]. The thermal cycling program included activation of the polymerase at 95°C for 15 minutes followed by 30 cycles of denaturation (95°C, 30 seconds), annealing (55°C, 30 seconds), and extension (72°C, 48 seconds), then ended with a final extension (72°C, 7 minutes). Primers and dNTPs were eliminated using exonuclease I and shrimp alkaline phosphatase (New England BioLabs, Ipswich, MA, USA) and sent to the Brigham Young University DNA Sequencing Center (https://biology.byu.edu/dnasc) for Sanger sequencing. Sequences were trimmed to exclude quality scores below 10 in CodonCode Aligner (CodonCode Corporation, Centerville, MA, USA, v. 9.0.1). The average trimmed sequence length was approximately 530 bp. Sequences were then searched against the UNITE database (https://unite.ut.ee/index.php) using BLASTn. We used a 99% identity criterion to match to a species and 97% identity to match to a genus. Five isolates could not be identified to a species or genus in the database. We, therefore, constructed a phylogenetic tree to assist in identifying these taxa. All sequences were aligned in MAFFT (https://mafft.cbrc.jp/alignment/server/) [39,40] and cleaned in Mesquite v. 3.70 [41]. The output from Mesquite was used to construct a tree in IQTree (http://iqtree.cibiv.univie.ac.at/) [42], which was visualized in FigTree v. 1.4.4. (http://tree.bio.ed.ac.uk/software/figtree/).

Testing hypothesis 1: Psychrotolerance

Each of the 40 isolates was grown in a 2 x 2 factorial experiment with two incubation temperatures (5 and 17°C) and two media (control and leaf litter). Our criterion for psychrotolerance was statistically significant growth at 5°C. See below for greater detail. The warmer temperature (17°C) represents the temperature during the growing season for Q. gambelii. The actual daily average temperature at Devil’s Kitchen during the majority of the growing season (24 Jun 2019 to 24 Sep 2019) was 16.9°C (SD, 6.2). The control medium contained starter glucose. The leaf litter medium contained starter glucose and leaf litter. There were 4 replicate 5 cm diameter dishes for each medium x temperature combination, 16 total dishes per isolate, for a total of 640 dishes. Each of the dishes was inoculated with the appropriate isolate using a 6 mm diameter piece of mycelium taken from the outer edge of the parent culture. Each liter of control medium contained 2.0 g glucose, 8.92 g agar, 0.46 g peptone, 1.0 ml of 300 g CaCl2 L-1, 10.0 ml of 30 g KH2PO4 L-1, and 1.0 ml of a solution containing 5 g MgSO4 L-1, 3.7 g FeSO4 L-1, 1.4 g MnSO4 L-1 and 3.7 g ZnSO4 L-1. Each liter of leaf litter medium additionally contained 7.18 g of Q. gambelii leaf litter, ground to pass a 2 mm screen in a Cyclone Mill (Retsch USA, Verder Scientific, Newtown, PA, USA). All media components were added prior to autoclaving. The litter had been collected from the forest floor of the Slate Canyon site in the summer of 2020.

The experiment was carried out in sets of 4 to 6 isolates. There were 8 sets total. Biomass growth rates were determined after 49–53 d of growth, depending on the set. To determine fungal biomass, the agar within each dish was melted within a stainless-steel tea ball (0.6 mm mesh) in boiling water for 10 min., the freed mycelium was dried on the lab bench at room temperature for 24 hours and weighed. It is unlikely that any of the fungal biomass was lost through the stainless-steel mesh of the tea ball because each mycelium consisted of a solid fungal mat. The biomass growth rate was calculated using the following formula: (DW2 –DW1) / elapsed time, for which we assumed DW1 = 0. In reality, the mycelium dry weight for any replicate at the beginning of the experiment was not zero, but was less than 0.0001 g, the lower limit of our balance.

To test hypothesis 1, we defined psychrotolerance as significant saprotrophic capacity at 5°C. We defined saprotrophic capacity as the difference between the biomass growth rate on leaf litter medium (containing starter glucose and leaf litter), and the biomass growth rate on control medium (containing starter glucose). At 5°C, there were 4 replicates for each of the media, but leaf litter replicates were not paired with corresponding control replicates for the purpose of calculating differences in growth rate (saprotrophic capacity). Therefore, we calculated the error associated with the difference by bootstrapping. First, differences were determined for the 16 possible combinations of leaf medium (4 replicates) and control medium (4 replicates). Then, 4 of the 16 differences were randomly sampled 1000 times with replacement in order to bootstrap a frequency distribution of the difference, and the mean, standard deviation and 95% confidence intervals were determined in R [43]. If this difference was significantly greater than zero for isolates grown at 5°C, according to the 95% confidence interval, we considered the isolate to be a psychrotolerant saprotroph. The R script for this procedure is given in the supporting information (S1 File).

We analyzed the variation in psychrotolerant saprotrophy (saprotrophic capacity at 5°C) among isolates. Again, because leaf litter replicates were not paired with corresponding control replicates for the purpose of calculating differences in growth rate (= saprotrophic capacity), it was not possible to perform a standard analysis of variance of the difference in growth rate. Therefore, the analysis of variance was performed in R (see https://acetabulum.dk/) using the means and standard deviations of the differences in growth rate at 5°C obtained from the bootstrapping R script to analyze the variation among isolates in psychrotolerant saprotrophy (saprotrophic capacity at 5°C). To determine whether psychrotolerant saprotrophy differed among species, a conventional analysis of variance was performed with isolates as replicates within the four species comprising multiple isolates using Minitab v. 18 [44].

Testing hypothesis 2: Temperature specialization

To test hypothesis 2, we performed a linear correlation of saprotrophic capacities of isolates at 17°C and at 5°C. The P and r values were obtained through SigmaPlot for Windows, Version 11.0 [45].

Results

Based on the UNITE database, 35 of our 40 isolates were identified to the following taxa: Apiognomonia errabunda (Roberge ex Desm.) Höhn, Cladosporium herbarum (Pers.) Link Ex S. F. Gray, Ophiognomonia setacea (Pers.) Sogonov, Ophiognomonia sp., Pyronema omphalodes Bull. (Fuckel), Venturia sp., Coniochaeta polymorpha Z.U. Khan, J.P. Guarro & S. Ahmad, Antonie van Leeuwenhoek, Cladosporium allicinum (Fr.) Bensch, U. Braun & Crous, 2012, Tricharina cretea (Cooke) K.S. Thind & Waraitch, Parafenestella sp. The remaining 5 isolates were identified using the constructed phylogenetic tree: one isolate each in the Dothideales, Diaporthales, Coniochaetales, and two isolates in the Pezizomycetes.

Thirty-six of the 40 isolates had significant saprotrophic capacity at 5°C or, in other words, were psychrotolerant saprotrophs (Fig 1). In contrast, 4 isolates (QGsun2.2.A, Ophiognomonia setacea; QGsun5.7.A.4, Coniochaetales.; QGsun4.9.A.2.1, Pryonema omphalodes; QG6.6.A.1, Ophiognomonia sp.) did not exhibit significant psychrotolerant saprotrophy.

Fig 1

Psychrotolerant saprotrophy (saprotrophic capacity at 5°C) among the 40 isolates.

Error bars are 95% confidence intervals. The list of the fungal taxa for each of these isolates is given in the supporting information (S1 Table).

There was significant variation in psychrotolerant saprotrophy among fungal isolates (Fig 1, Table 1). Isolate was a significant factor (p = 6.42e-60), accounting for 95% of total variability in psychrotolerant saprotrophy, while the variability among replicates within an isolate accounted for only 5% (Table 1).

Table 1

ANOVA table for psychrotolerant saprotrophy (saprotrophic capacity at 5°C) among the 40 isolates.

Source	df	SS	MS	F	P
Among isolates	39	8.8673	0.22737	54.6571	6.42e-60
Within isolates	120	0.4992	0.00416
Total	159	9.3664

When we analyzed the four fungal species comprising more than a single isolate (Fig 2), species was a significant factor with respect to psychrotolerant saprotrophy (p = 0.007), with among species variation accounting for 39% of the total variability (Table 2). Ophiognomonia sp. had a saprotrophic psychrotolerance significantly lower than that of Apiognomonia errabunda and Ophiognomonia setacea (Table 3). However, within species variability was also large, accounting for 61% of total variability (Table 2).

Fig 2

Psychrotolerant saprotrophy (saprotrophic capacity at 5°C) among the 40 isolates, grouped by species.

The white bars represent taxa that were either species comprising a single isolate each or were not identified to species. Such taxa (white) were not included in the analysis of variance shown in Table 2. Error bars are 95% confidence intervals. The list of the fungal taxa for each of these isolates is given in the supporting information (S1 Table).

Table 2

ANOVA table for psychrotolerant saprotrophy (saprotrophic capacity at 5°C) among the four species each comprising multiple isolates.

Source	df	SS	MS	F	P
Among species	3	0.4306	0.14353	5.2	0.007
Within species	24	0.6628	0.02762
Total	27	1.0933

Table 3

Means and standard errors of the means (SEM) for psychrotolerant saprotrophy (saprotrophic capacity at 5°C) among the four species each comprising multiple isolates.

Different letters indicate a significant difference among species according to the Tukey Honestly Significant Difference Method.

Species	n	Mean (SEM)
Apiognomonia errabunda	4	0.3549 (0.0831) a
Ophiognomonia setacea	6	0.3194 (0.0678) a
Cladosporium herbarum	3	0.1637 (0.0959) ab
Ophiognomonia sp.	15	0.0668 (0.0429) b

The correlation between the saprotrophic capacity of isolates at 17°C and 5°C was positive and significant (p = 0.0003, r = 0.5434, Fig 3).

Fig 3

Plot of saprotrophic capacity of the 40 isolates at 17°C vs. 5°C.

Blue is Apiognomonia errabunda, red is Cladosporium herbarum, light green is Ophiognomonia setacea, dark green is Ophiognomonia sp., and white are taxa that were either not identified to species or species comprising a single isolate each.

Discussion

Because much of the microbially-mediated decomposition of Q. gambelii leaf litter must occur in the colder months of the year when moisture is available, we hypothesized that endophytic fungi colonizing Q. gambelii leaves were capable of saprotrophy at 5°C. Of the endophytic fungi we isolated, saprotrophic psychrotolerance was common but not universal, occurring in all but 4 of the 40 tested isolates. Others have reported psychrotolerant growth of endophytic fungi on glucose-based media [34,46]. However, this constitutes the first report of psychrotolerant saprotrophy of endophytic fungi using leaf litter as the carbon source.

We also hypothesized that there was significant variation among species in saprotrophic psychrotolerance. While the species designated Ophiognomonia sp. had a significantly lower saprotrophic psychrotolerance than the other species, there was a surprising level of variation within species as was seen for Aphiognomonia errabunda, Cladosporium herbarum and Ophiognomonia setacea. This result was unexpected because members of a given species are assumed to occupy the same niche and, therefore, to be functionally similar [47]. We note, however, that the unexpectedly high degree of variation in psychrotolerance within a species was expressed under artificial experimental conditions, including controlled temperatures and non-limiting availabilities of water and mineral nutrients. Under field conditions, where cold or insufficiency of water or mineral nutrients could limit saprotrophic activity, the ability to express variation in psychrotolerance may be more limited.

Among the 40 fungal isolates, we found no evidence of a tradeoff in saprotrophic capacity at 17°C with saprotrophic capacity at 5°C. In fact, the significant correlation between saprotrophic capacity at 17°C and at 5°C was positive. Therefore, these fungi appear not to specialize with respect to temperature. This result was somewhat unexpected given the existence of performance tradeoffs at different temperatures for important physiological processes such as photosynthesis [32]. The capacity to obtain nutrition saprotrophically under a wide range of temperatures, from 5°C to 17°C, could potentially allow endophytic fungi to obtain resources from leaf litter both during the colder months of late autumn, winter, and early spring, when moisture is most abundant, as well as during the late summer monsoon when limited rain falls [2]. We have not assessed the ability of endophytic fungi to compete against non-endophytic fungi in litter, and it is possible that endophytic fungi are generally not highly competitive. However, we previously showed that endophytic fungi do at least persist in leaf litter of Q. gambelii for weeks following leaf senescence [21]. The persistence of endophytic fungi in decomposing litter is not particularly surprising given that when leaves senesce and enter the litter pool, endophytic fungi are already present, have no need to colonize from the environment, and thus have priority [25] over non-endophytic fungi in capturing resources from leaf litter. Indeed, others have found that endophytic fungi can exert priority over decomposer fungi in litter [28].

As with all laboratory studies, the ecological relevance of our results should be carefully weighed. This study was carried out under controlled temperatures (constant 17°C vs. constant 5°C), presumably without moisture or mineral nutrient limitation, and with every isolate grown separately, all conditions that are not likely to occur under field conditions. Because we do not know how these factors influence either saprotrophic capacity or psychrotolerance of a given isolate, the extent to which our results are relevant in the field is not clear. Nevertheless, our results suggest that a number of endophytic fungi of Q. gambelii leaves are potentially capable of saprotrophic growth under both cold and warm conditions and thus have the potential to influence litter decomposition and nutrient cycling in this arid ecosystem.

Isolate designation and species identity.

(DOCX)

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Bootstrapping R script.

(DOCX)

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Data set.

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26 Apr 2022

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Reviewer #1: The paper is well written, well organized, and absolutely clear about how the authors conducted the research. The study could be of certain interest to the target of Plos One.

Nevertheless, the overall quality of the manuscript needs to be slightly improved before publishing in Plos One, since minor issues are present.

I suggest the authors to better explain the second hypothesis in the material and method section since just a few words were spent about the goal of this second part of the paper.

The second hypothesis is given in the introduction. It is a straightforward hypothesis about an ecological tradeoff, and other examples of such tradeoffs are given in the introduction for clarification. Therefore, there doesn’t seem to be a need to further explain this hypothesis in the materials and methods section, which is devoted only to explaining the methods used to test the hypothesis.

Moreover, it is important to improve the clarity and quality of the tables provided in the text deriving from ANOVA analyses; it is not specified the number of ways (one way or two way) as well as the meaning of the acronyms.

The structure of the tables indicates that both anovas were one-way analyses. I am not sure what the reviewer means as far “acronyms” are concerned. The “df”, “SS”, “MS”, “F” and “P” are straightforward abbreviations of well-known anova terms (degrees of freedom, sums of squares, mean squares, F statistic and P value). If a reader does not recognize these terms, they will not be able to decipher an anova table anyway.

Furthermore, It would be useful to provide more details on the result of the statistical analysis reported in the tables within the text. Which variance gave greater significance? Between groups or within groups? This is important since It seems that these parameters differ a lot in terms of explained variance.

The variance is given by the sums of squares. The very reason we included the complete anova table was to allow the reader to make this assessment. Indeed, in the results section, we already make statements such as “with among species variation accounting for 39% of the total variability”.

Minor issues

The figures contains a lot of data, but are not properly legible. Is it possible to rotate the figures? I understand that could negatively affect the figure quality, but It surely could improve from a legibility point of view.

Yes, the figures could be rotated, but I do not see how that would increase how easily the figures are understood. Species names, some of which are very long, are not given in these figures to keep the figure as simple and easily interpreted as possible. S2 Table lists the species name for each of the isolates.

Reviewer #2: I think the authors satisfactorily replied to the reviewer 1 which raise some issues that go beyond the aim of the work. Moreover, some comments are inappropriate (e.g., since mid-2000s direct PCR is successfully applied to fungal material without any problem about the sequence quality). The aim of the work is clear and the biological hypotheses which the work is based on are appropriate and fully met. Experimental planning and procedures are detailed and they support appropriately what the authors intended to demonstrate although some parts of the M&M section can be improved (see the below comments).

Thank you for that confirmation.

However, it is not clear because you selected as “warm” temperature 17 °C that in many parts of the world is not so “warm”. I think this has to do with the climate where Q. gambelii grow. In this case the choice of this temperature level should be justified and an in-depth description of the study site should be reported (e.g., min and max temperatures of coldest and warmest months).

We now indicate in the materials and methods section that “air temperatures were monitored at the Devil’s Kitchen site from 24 Jun 2019 to 24 Sep 2019 using a temperature data logger (HOBO UA-001-64, Onset Computer Corporation, Bourne, MA, USA), set to log hourly.” Furthermore, in the section “Testing hypothesis 1: psychrotolerance”, we added “Each of the 40 isolates was grown in a 2 x 2 factorial experiment with two incubation temperatures (5 and 17 ºC) and two media (control and leaf litter). Our criterion for psychrotolerance was statistically significant growth at 5 ºC. The warmer temperature (17 ºC) represents the temperature during the growing season for Q. gambelii. The actual daily average temperature at Devil’s Kitchen during the majority of the growing season (24 Jun 2019 to 24 Sep 2019) was 16.9 �  C (SD, 6.2).”

The results about strain identification (ln 114-120) should be moved at the beginning of the result section and the phylogenetic tree generated to identify the unknown isolates should be shown. This part is a result and not a methodology and, in addition, this shift would compensate the gap of length between M&M and Result sections.

As suggested, the identity of the isolates has been moved to the results section. However, we consider the generation of the phylogenetic tree to be part of the methods section because it was used to identify five of the unknown isolates. Therefore, the section title for this part of the materials and methods has been changed to “Sequencing and assigning taxonomy of fungal isolates”. Because the tree was used as a method and was not a result, we do not feel it should be included as a result.

Accession numbers and site of collection of each isolate should be added in the supplementary Table 1

The UNITE ID numbers and collection sites have been added to S2 Table.

Specific comments

Ln 40, a brief description of the differences between psychrotolerant, psychrotrophic and psychrophilic should be provided to ensure that the reader understand why you chose the first term.

Unfortunately, these terms have been used interchangeably depending on the author. Therefore, any of the terms could have been used. We chose psychrotolerant because the condition of a psychrotolerant organism is easily described as “psychrotolerance”, whereas the conditions describing a psychrotrophic or psychrophilic organism are awkward.

Ln 85, delete “2000”

Done.

Ln 88, how many sub-samples (disks) from each leaf?

We now clarify that “Each surface-sterilized leaf was subsampled once…”

Ln 92, “very few” is too subjective, “less than xx” would be better

The statement now reads “After 3 weeks, fewer than 10 total fungal colonies grew out from the entire collection of leaf disks.”

Ln 92-96, I suppose the plates were inspected every day to isolate strains before R selected fungi (e.g. Penicillium, Trichoderma,…) form and release conidia

Yes.

Ln 103, “ITS is considered a universal barcode for fungi” delete this sentence because superfluous

Deleted

Ln 116, 119,…, “sp.” must not be italicized; What is “Ophiognomonia 1”? perhaps “Ophiognomonia sp.”?

These have been corrected throughout the manuscript.

Ln 132, for a total of 640 plates, is it correct?

Yes, this fact has been added.

Ln 133-136, it is not clear which is the size of the plates.

We now specify that the plates were 5 cm diameter.

I do not think that 200 ml of medium was added to each plate. Moreover the amount of each component of the medium should be reported as weight (g, mg,…) per L.

The components of the media are now given per liter.

Ln 137, was the leaf litter autoclaved?

Yes, the leaf litter was added to the medium prior to autoclaving. We now write “All media components were added prior to autoclaving.”

Ln 143, how did you separate mycelium biomass from residues of litter?

Surprisingly, the vast majority of the ground leaf litter was easily separated from the mycelium during the boiling process.

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Reviewer #1: No

Reviewer #2: No

Submitted filename: Response to reviewers.docx

Click here for additional data file.

26 Sep 2022

Many foliar endophytic fungi of Quercus gambelii are capable of psychrotolerant saprotrophic growth

PONE-D-22-12165R1

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3 Oct 2022

PONE-D-22-12165R1

Many foliar endophytic fungi of Quercus gambelii are capable of psychrotolerant saprotrophic growth

Dear Dr. Koide:

I'm 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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