Fungal endophytes as priority colonizers initiating wood decomposition

Fungal endophytes as priority colonizers initiatingwood decompositionZewei Song1, Peter G. Kennedy2, Feng J. Liew3 and Jonathan S. Schilling*3,4,

1Department of Plant Pathology, University of Minnesota, St Paul, MN 55108, USA; 2Department of Plant Biology,University of Minnesota, St Paul, MN 55108, USA; 3Department of Bioproducts & Biosystems Engineering, University ofMinnesota, St Paul, MN 55108, USA; and 4Institute on the Environment, University of Minnesota, St Paul, MN 55108,USA

Summary

1. Priority effects among wood decomposers have been demonstrated by manipulating fungal

assembly history via inoculations in dead wood and then tracking community development

using DNA sequencing. Individual wood-degrading fungi have been shown, however, to initi-

ate decay after having colonized living trees as endophytes.

2. To track these ‘upstream’ colonizers across the endophyte–saprophyte transition, we cou-

pled high-throughput sequencing with wood physiochemical analyses in stem sections extracted

from healthy birch trees (Betula papyrifera; 4–7 cm dia.). We incubated wood in microcosms,

limiting communities as endophytes�only or challenging endophytes with Fomes fomentarius

or Piptoporus betulinus at high exogenous inoculum potential.

3. Initial fungal richness in birch stems averaged 143 OTUs and decreased nearly threefold

after five months of decomposition. Although F. fomentarius successfully colonized some stem

sections incubated at 25 °C, decayed wood was generally dominated by saprophytic fungi that

were present originally in lower abundances as endophytes.

4. Among saprophytes, fungi in the brown rot functional guild consistently dominated, match-

ing wood residues bearing the chemical hallmarks of brown rot. Despite this functionally

redundant outcome, the taxa that rose to dominate in individual sections varied. Surprisingly,

the brown rot taxa dominating wood decomposition were better known for lumber degrada-

tion rather than log decay in ground contact.

5. Given the isolation from colonizers in our design, this redundancy of brown rot as the out-

come suggests that these taxa and more generally brown rot fungi could have adapted to

decompose wood where there is lower competitive pressure. Competitive avoidance would

complement the diffuse depolymerization mechanisms of brown rot fungi, which are likely

more prone to sugar pilfering by other organisms than the processive depolymerization mecha-

nisms of white rot fungi.

6. Overall, this guild-level predictability of fungal endophyte development and consequence is

encouraging given the challenges of predicting wood decomposition, and it provides a base for

testing these dynamics under increasing natural complexity.

Key-words: brown rot, durability, endophytic, fungal community, historical contingency

latent, lignocellulose, white rot

Introduction

Temperate and boreal forests store an estimated 500 mega-

tons of carbon (C) in above-ground biomass (Myneni

et al. 2001), primarily as wood (Woodwell et al. 1978).

Predicting the rate at which this massive pool of C turns

over via decomposition, along with its fates above- and

below-ground, has become a priority for terrestrial ecosys-

tem modelling (Woodall et al. 2009; Keenan et al. 2013).

Unfortunately, it has proven more challenging to predict

turnover of dead wood relative to other plant litter types.

Climate explains little of the variability observed when

wood decays in the field (Bradford et al. 2014; Brischke &

Thelandersson 2014), and plant traits (e.g. nitrogen)*Correspondence author. E-mail: [email protected]

© 2016 The Authors. Functional Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License,which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial andno modifications or adaptations are made.

Functional Ecology 2016 doi: 10.1111/1365-2435.12735

http://creativecommons.org/licenses/by-nc-nd/4.0/

commonly used to explain decay rates in non-woody plant

litter often fail to explain decay rates in wood (Trofymow

et al. 2002; Cornwell et al. 2009; Weedon et al. 2009; van

Geffen et al. 2010; Freschet et al. 2012; Jackson, Peltzer &

Wardle 2013). Combined, these factors implicate biological

variability among wood decomposers as a major determi-

nant, including the saprotrophic fungi that dominate wood

decomposition in high latitude forests (Baldrian & Lindahl

2011).

There are several reasons why variability in fungal biol-

ogy, specifically nutritional mode, can steer the decay of

wood under the same environmental conditions towards

very different outcomes. First, many early colonizers target

‘easier’ non-structural carbohydrates (e.g. pectin, starch)

and then inhibit subsequent colonizers by defending terri-

tory (Zabel & Morrell 1992). Secondly, the fungi that

decay wood are not redundant in their strategies for

unlocking carbohydrates from lignocellulose, varying in

how much lignin they remove in order to access sugars

(Worrall, Anagnost & Zabel 1997; Schilling et al. 2015).

This variability in carbohydrate selectivity is no longer

viewed as binary (brown vs. white rot), rather as a spec-

trum of rot types that, at present, are poorly defined using

presence or abundance of lignocellulolytic genes (e.g. lignin

peroxidases) (Riley et al. 2014; Kaffenberger & Schilling

2015). Because lignin typically exceeds 20% of wood mass,

this biological variability in the amount of lignin mineral-

ized will affect carbon dioxide emission rates directly (Berg

& McClaugherty 2003). In addition, the residues produced

vary in their cation affinity, redox, sorption properties and

permeability, and these characters influence turnover in

other soil organic fractions (Ryp�a�cek & Ryp�a�ckov�a 1975;

Gilbertson 1980; Jurgensen et al. 1997; Filley et al. 2002;

Song et al. 2012).

Given that decay outcomes depend significantly on

which fungi become dominant, predicting community

assembly using fungal life-history strategies has emerged as

an important goal in microbial ecology (Crowther et al.

2014; van der Wal, Ottosson & de Boer 2015). One key

strategy among wood-degrading fungal colonizers is sim-

ply being the first to colonize. Wood decomposer commu-

nities have been used as a model system to demonstrate

priority effects using controlled inoculations and commu-

nity DNA-based profiling (Fukami et al. 2010; Dickie

et al. 2012; Hiscox et al. 2015; Song et al. 2015). The ulti-

mate priority, however, may begin with saprotrophic fungi

harboured in standing trees as wood endophytes (defined

broadly here as ‘organisms living inside living plants’; Car-

roll 1988; Griffith & Boddy 1991; Stone & Petrini 1997;

Crozier et al. 2006; Sieber 2007; Parfitt et al. 2010). These

fungi are often typified as stress-tolerant (S-selected)

(Boddy & Heilmann-Clausen 2008), weathering for long

time periods in tree xylem. Although endophytic fungi

have been shown in many culture-dependent studies to

persist after tree death and to initiate wood decay (e.g.

Chapela & Boddy 1988; Boddy 2001; Oses et al. 2008;

reviewed in Boddy & Griffith 1989), this transition from

endophytism to saprophytism, along with its influence on

wood C fates, has not yet been tracked using culture-inde-

pendent community assessments.

For this study, our goal was to characterize fungal wood

endophyte communities among multiple standing trees and

then track communities as they transitioned to saprotro-

phy after tree death. We specifically wanted to assess the

predictability of wood endophyte development, starting

here in a fixed environmental system and controlling all

exogenous colonization. To link these communities to their

functional consequences, we matched community sequenc-

ing with relevant outcomes of decay, including lignin min-

eralization. To do this, we incubated stem sections from

ten paper birch trees (Betula papyrifera) in isolation, using

microcosms to limit colonization events and to challenge

endophyte priority using high exogenous inoculum of

birch-associated fungi. In the absence of colonizers, we

expected that white rot-type endophytes would dominate

decay, as is true for many angiosperm wood species (Hib-

bett & Donoghue 2001), and that extensive wood lignin

losses would be the rule. We also expected, however, that

high inoculum potential of soil-inhabiting competitors

could override endophyte priority and alter patterns of lig-

nin mineralization (Holmer & Stenlid 1997; Song et al.

2015).

Materials and methods

WOOD SUBSTRATES

In October 2012, ten healthy B. papyrifera trees (targeting 9 cm

diameter at the base) were felled at the Cloquet Experimental For-

est, Cloquet, MN, USA. This size class is considered coarse

woody debris (CWD) and easily fits as stem rounds into our

microcosms. Tree stems were immediately transported whole to

our laboratory in Saint Paul, MN and sectioned into ~4 cm length

rounds while fresh. The saw blade used for the sectioning was

sprayed with 70% ethanol between every cut. Avoiding knots and

branch stubs, the stem rounds were matched in adjacent pairs,

with round one weighed fresh and retained for our decomposition

trial and the other weighed before and after oven-drying (100 °Cfor 48 h) to calculate moisture content (%). The latter allowed

back calculation of dry masses in the undried rounds. A third sin-

gle round from each of the ten trees was immediately frozen at

�80 °C and used for the fungal community sequencing analysis

(referred to as ‘time-zero’ samples). For the decomposition trial,

half of the rounds were twice autoclaved for 1 h, with a 24-h inter-

val, while the other half were stored at 4 °C (referred to as ‘de-

cayed’ samples).

FUNGAL ISOLATES FOR INOCULAT IONS

In the same plot where trees were extracted, the brown rot fungus

Piptoporus betulinus (Cloq001) and the white rot fungus Fomes

fomentarius (Cloq002) were isolated from sporophores on a single

B. papyrifera tree (Fig. S1, Supporting Information). P. betulinus

(brown rot; carbohydrate selective) and F. fomentarius (white rot;

lignin degrading) are both common dominant rot types in birch,

and both are common in standing trees where colonization by

other fungi is limited (Gilbertson 1980). Species were confirmed by

BLAST searching amplified ITS regions of rDNA. Both were

© 2016 The Authors. Functional Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.

2 Z. Song et al.

maintained on 2% malt extract agar (MEA) and remain publi-

cally available through the Forest Mycology Culture Collection

(University of Minnesota). The responses of elongation rate to

temperature were measured (cm day�1) under 20, 25, 28, 30 and

33 °C on 10 cm Petri dishes (2% MEA). Edge transfers (5 mm

dia. agar plug) from two-week-old colonies were used as inocu-

lant. P. betulinus and F. fomentarius reached their maximum

growth rate at 25 and 30 °C, respectively (Fig. S1). These tem-

peratures represent summer season mean temperatures at our

plots (Cloquet, MN, see Tjoelker et al. 2008) and have been used

to study competition of wood-degrading fungi in temperate

regions (Hiscox et al. 2016). Given that our goal, however, was

not to extrapolate a temperature relationship to field conditions,

but instead to simply give either fungus its best chance to over-

ride priority colonization, we included treatments at both temper-

atures.

SOIL M ICROCOSMS

We conducted our decomposition trial in soil-block microcosms,

modified from Song et al. (2012 and references within). Briefly,

300 g of soil mix (1 : 1 : 1 peat, vermiculite, fertilizer-free soil)

was added to ~240-mL glass jars and pressed to fill half of the jar.

Each jar was then autoclaved twice for 1 h, with a 24-h rest

between runs to eliminate any source of external soil inoculum.

Experimental treatments included endophytes (presence, absence),

temperature (25, 30 °C) and external inoculum (one or two spe-

cies). In the latter treatment, we first added two B. papyrifera

wafers (15 9 50 mm; 1�5 mm thickness) pre-colonized by P. be-

tulinus, F. fomentarius or both to each microcosm for three weeks.

The stem rounds were then added, and all replicates were incu-

bated for 140 days in a dark dual-chamber incubator (Percival

Scientific, Perry, IA, USA) set at either 25 or 30 °C and a relative

humidity of 80%, to avoid soil and wood desiccation over the

5-month incubation (see Fig. S2 and Table S1). Both sets of these

stem rounds (i.e. autoclaved and not) were surface sterilized prior

to addition to microcosms by swabbing with 70% ethanol, in line

with recommendations of Burgdorf et al. (2014) for surface steril-

izing large samples.

WOOD HARVESTS

At harvest, individual stem rounds were bagged into sterile, pre-

weighed plastic bags, then weighed fresh inside a laminar flow

hood. After carefully removing the bark, each stem round was

then split inside the hood into two subsamples for molecular and

chemical analyses (Fig. S2). To account for possible position

effects within the wood (i.e. upper/lower portion of the stem

round), half of the upper and bottom subsamples for each stem

round were stored in �80 °C for molecular analysis. The remain-

ing subsamples were weighed separately and oven-dried for 48 h

to calculate average moisture content and then mass loss. Know-

ing that surface sterilization bears caveats (i.e. it may not com-

pletely eliminate fungal inoculum), bark removal and milling

relatively large intact wood samples were meant to reduce the

potential for ‘noise’ from surface inoculum.

WOOD CHARACTER IZAT ION

Mass loss (% dry weight) was calculated between initial and

decayed samples. Oven-dried subsamples were then pooled and

ground to 40 mesh (0�4 mm) in a Wiley mill (Arthur H. Thomas

Co., Philadelphia, PA, USA). Carbon fractions (w/w) were deter-

mined for cellulose (glucose), hemicellulose (arabinan, galactan,

glucan, mannan, xylan) and acid-insoluble lignin, as in Schilling,

Tewalt & Duncan (2009). The pH and dilute alkali solubility

(DAS) were determined following Shortle, Dudzik & Smith

(2010). Briefly, for pH, powder was added to 5 mM CaCl2 (0�1 mg

lL�1) and, for DAS, powder was autoclaved in 0�2 M NaOH, fil-

tered through tared fritted glass crucibles, and rinsed with distilled

water and 0�1 M HNO3. Per cent loss on extraction was calculated

by weighing extracted materials. In addition to providing basic

carbon utilization information, these chemical analyses can also

be used to quantify the dominant nutritional mode (rot type). Cal-

culating the carbohydrate selectivity using the ratio of lignin loss:-

density loss is a reliable rot-type measure (L : D; <0�8 when

brown rot dominates), accompanied in brown-rotted wood by

characteristically low pH and high DAS values (Worrall, Anag-

nost & Zabel 1997; Schilling et al. 2015).

DNA EXTRACT ION

Frozen subsamples were ground to fine powder in a modified bone

mill (Medtronic, St. Paul, MN, USA) after snap-freezing in liquid

nitrogen, as in Song et al. (2012). DNA was extracted following

Jasalavich, Ostrofsky & Jellison (2000), with slight modifications.

Powder (60–70 mg) in 800 lL 2X CTAB buffer with 2% PVP and

2% b-mercaptoethanol was incubated at 65 °C for 1 h, with initial

2 min vortex and mixing at 15-min intervals. Samples were then

extracted (upper phase) in a phenol:chloroform:isopropanol series

(800, 600, then 400 lL; vortex, centrifuge max speed 10 min each).

After the final extraction, 250 lL was added to 1250 lL binding

buffer PB (Qiagen, Valencia, CA, USA), mixed, and loaded into

mini-spin columns (Epoch Life Science, Sugar Land, TX, USA)

for two 1-min spins, with a 1 min wash in 750 lL washing buffer

PE (Qiagen). DNA was re-suspended in 100 lL EB buffer (Qia-

gen) and spun 1 min at max speed. After measuring DNA concen-

tration with Qubit HS dsDNA assay (Life Technologies,

Carlsbad, CA, USA), concentrations were adjusted to 1 ng uL�1

with nuclease-free water and stored at �20 °C.

HIGH-THROUGHPUT SEQUENCING

Fungal communities in the initial and decayed samples were iden-

tified using the Illumina compatible ITS1F-ITS2 primer pair in

Smith & Peay (2014). Each PCR consisted of 10 lL 2X Roche

FastStart PCR master mix (Roche, Indianapolis, IN, USA),

0�35 lL of each primer (10 lM), 1�2 lL MgCl2 (25 mM), 0�2 lLBSA (50 mg mL�1) and 2 lL template DNA (2 ng). For each

sample, triplicate PCRs were performed using thermocycling con-

ditions with 10-min initial denaturation (95 °C), thirty 30-s cycles

(95 °C), 20-s annealing (50, 53, or 55 °C) and 30-s extension

(72 °C), with 9-min final elongation (72 °C). Individual PCRs

were verified by gel electrophoresis, pooled by triplicate at equal

volume and purified with an Agencourt AMPure XP PCR purifi-

cation kit (Beckman Coulter Inc., Brea, CA, USA). For each,

20 lL of PCR product was mixed with 20 lL of AMPure XP

solution, instead of 38 lL as in the standard instructions, to better

remove large size primer dimers. Purified PCR products were

quantified with Qubit HS dsDNA assay (Life Technologies, Carls-

bad, CA, USA). 30 ng of DNA from each sample was combined

into a single pool and sent for Illumina MiSeq sequencing (250 bp

paired, V3 chemistry, 25% PhiX) at the University of Minnesota

Genomics Center (Saint Paul, MN, USA).

We used a customized bioinformatics pipeline to process the

sequencing data (https://github.com/ZeweiSong/FAST, see File S1

for the analysis pipeline). Briefly, all forward sequences were

renamed to add sample label and merged into a single FASTQ file

(FAST: add_labels.py). Primer sites, adapter and low quality ends

of reads were trimmed from the merged file using Trimmomatic

(Bolger, Lohse & Usadel 2014). Sequences with ambiguous bases

and homopolymers longer than 9 bp were removed (FAST:


Fungal endophytes and wood decay 3

https://github.com/ZeweiSong/FAST

filter_seqs.py). Sequences were then dereplicated (FAST: derepli-

cate.py) and clustered at 97% similarity using USEARCH v8.0

(Edgar 2013). The taxonomy of OTUs was assigned by BLAST

against the UNITE data base (Version No. 7, 2015-03-02 without

singletons; K~oljalg et al. 2013), resulting an unrarefied OTU table

that included taxonomic assignments. The abundances of each

OTU present in the negative control were subtracted from all sam-

ples. In order to remove artificial or non-fungal sequences, all

sequences representing <1% of total sequences or with a match

length or Pident (similarity level) ≤0�85 were removed. An abun-

dant plant OTU (matching Betula in BLAST searches) was also

removed.

QUANT ITAT IVE PCR

The abundances of P. betulinus, F. fomentarius and total fungi

in both the initial and decayed samples were measured using

quantitative real-time PCR. We designed species-specific ITS pri-

mers for P. betulinus and F. fomentarius (Table S2), while for

total fungal biomass, we followed Fierer & Jackson (2005).

Quantitative PCR was performed on an ABI 7900HT system

(Applied Biosystems, Foster City, CA, USA) in a 384 plate.

Each reaction contained 5 lL standard or sample DNA, 1 lLeach primer (10 mM) and 10 lL 2X SSoAdvanced SYBR Green

Supermix (Bio-Rad, Hercules, CA, USA) for 20 lL total vol-

ume. Standard DNA was diluted in a series (2500, 500, 250, 50,

25, 5, 2�5 and 0�5 pg). All reactions were performed in triplicate

and included three non-template controls. The cycling conditions

for P. betulinus and F. fomentarius were 3 min at 98 °C, and 40

one-min cycles at 60 °C. The cycling conditions for total fungi

were 3 min at 98 °C, followed by 40 cycles at 98 °C for 15 s,

55 °C for 30 s and 72 °C for 1 min. We performed dissociation

curve analyses on all plates using the default setting. Pure DNA

from P. betulinus and F. fomentarius cultures was used as the

corresponding standards. DNA of Saccharomyces cerevisiae cul-

tures was used as the standard for total fungi. We found that

all qPCR assays had efficiencies ranging from 87�2% to 95�1%and no inhibition (i.e. change in linearity) was detected in a

1 : 10 dilution series of standard and samples starting from

10 ng (2�5 ng was used in real reactions). We calculated final

abundances as the amount of DNA in measured samples (ng

mg�1), adjusted by the mass loss rate to yield volume-based

measures (ng cm�3).

DATA PROCESS ING AND STAT IST ICAL ANALYSES

To account for variation across individual rarefaction runs, we

took our unrarefied OTU table and, for each sample, conducted

1000 rarefactions at a depth of 5900 sequences (FAST: rar-

efy_otu_table.py). We then selected the sample (out of the 1000

runs) with median OTU richness to use in the final OTU table.

While this level of rarefaction removed a number of replicates

from multiple treatments (final n = 49), we found that it struck

the best balance between within-sample sequencing depth and

treatment replication (see File S2 for the change of the number of

sequences throughout the pipeline). To confirm this level of rar-

efaction did not bias our results, we also analysed the data at 1000

sequences per sample (which included 71 of the 80 samples) and

found the direction of treatment effects were all similar (Table S3).

Each OTU was first searched against the FUNGuild data base to

assign ecological functions (Nguyen et al. 2015; https://github.-

com/UMNFuN/FUNGuild). We then analysed the effects of tem-

perature (25 or 30 °C) and inoculation (endophytes only,

endophytes + P. betulinus, endophytes + F. fomentarius, endo-

phytes + both species) on fungal richness (observed), evenness

(Pielou) and diversity (Shannon and Simpson) of the decayed sam-

ples using two-way fixed factor ANOVA. To compare the change in

richness, evenness and diversity between the initial and decayed

samples, we used one-way ANOVA. To examine the effect of tem-

perature, inoculation and time (initial or decayed) on fungal com-

munity structure, we performed a permutational multivariate

analysis of variance using ADONIS. To visualize treatment

effects, we generated non-metric multidimensional scaling

(NMDS) plots using square root transformation. The community

structure analyses were conducted using the VEGAN package (Oksa-

nen et al. 2016) in R 3.2.2 (R Core Team 2013).

To examine the overall effects of temperature, endophytes and

inoculation on wood characteristics, we conducted a three-way

fixed factor ANOVA. For each wood characteristic (mass loss, car-

bohydrates loss, lignin loss), we assessed specific differences across

the different endophyte and inoculation treatments using one-way

ANOVAs followed by Fisher’s LSD multicomparisons. This was

done for samples in the 25 and 30 °C treatments, separately. To

measure the performance of P. betulinus and F. fomentarius in the

inoculation treatments, the copy number ratios of inoculated fun-

gus to total fungi were similarly compared using one-way ANOVA

followed by Fisher’s LSD. Finally, to assess both wood character-

istics and copy number ratio in the same analysis, we conducted a

principal component analysis (PCA) and visualized the first two

axes. All analyses were conducted in R and considered significant

with P < 0�05.

Results

FUNGAL COMMUNIT IES

Endophyte fungal communities in the initial healthy B.

papyrifera sections had an average OTU richness of 143 � 7

(n = 10 trees, Fig. 1a). After five months of decay in the iso-

lation of microcosms, OTU richness decreased significantly,

to an average of 57 � 4 (n = 7 at 25 and 30 °C, Fig. 1a and

Table 1). In contrast to the overall trend of taxon loss, there

was a significant increase in the relative abundance of sapro-

trophs, from an average of 24�1% initially to an average of

96�1% after five months. These richness and abundance pat-

terns were similar at 25 and 30 °C. The addition of P. betuli-

nus and/or F. fomentarius had no significant influence on

OTU richness levels (Fig. 1a and Table 1), but their pres-

ence did vary the relative abundance of OTUs (Fig. 1b).

This variation was not linked to the dominance of either of

the added species, as only 16% of the samples had either

P. betulinus or F. fomentarius as the dominant OTU after

five months, but instead was due to an associated commu-

nity shift (File S3).

When the saprophytic fungal guild was categorized by rot

type, brown rot taxa were more common than white rot taxa

initially (Fig. 2a). This pattern of brown rot dominance

remained generally consistent across all the decayed sam-

ples, regardless of the presence of external inoculum

(Fig. 2b). For the endophyte-only treatment that was domi-

nated by brown rot taxa, in terms of relative abundance,

>90% of the total sequences were from four species in two

genera (Coniophora and Postia) (Fig. 3). Only in treatments

in which F. fomentarius was added were there cases in which

white rot became dominant (Fig. 2b; note, only at 25 °C).Similar to changes in OTU richness and abundance,

fungal community composition varied significantly

between the initial and decayed samples (Fig. 4 and


4 Z. Song et al.

https://github.com/UMNFuN/FUNGuild

https://github.com/UMNFuN/FUNGuild

Table 2). In decayed samples, both temperature and inocu-

lation each had a significant effect on community struc-

ture, but there was no interaction between these factors

(Table 2). Within this context, the relative abundances of

both P. betulinus and F. fomentarius (assessed via qPCR)

when inoculated externally depended on the presence of

endophytes. In general, both fungi were more abundant

inside wood when endophytes were absent (Fig. 5). This

result was consistent across temperatures, although the

effects of endophytes were stronger at 30 °C.

WOOD DECOMPOSIT ION

Comparing the sections without endophytes to those in

which only endophytes were present, the endophyte pres-

ence significantly increased both mass loss (average ~40%at 25 °C) and carbohydrate loss, but not lignin loss at both

temperatures (Fig. 6). The lignin loss calculated relative to

overall density loss was well below the 0�8 threshold, con-

firming brown rot. Supporting this redundant brown rot

outcome, endophytes significantly increased DAS and

decreased pH in wood residues without external

Fig. 1. Trophic mode of the OTUs by their richness (a) and abundance (b). The trophic mode was assigned using the FUNGuild data

base. The ten fresh wood samples are on top starting with ‘Initial’ and the sample labels (from A to J). Decayed wood samples are labelled

by their treatment and replicate numbers. From top to bottom are endophyte control at 25 and 30 °C; P. betulinus inoculated at 25 and

30 °C; F. fomentarius inoculated at 25 and 30 °C; and both fungi inoculated at 25 and 30 °C.

Table 1. Differences in fungal community diversity parameters

between initial and decayed samples as well as among decayed

samples in different treatments as determined by ANOVAs.

Observed number of OTUs (richness), evenness, Shannon and

Simpson indexes were used as response variables. Time was used

as explanatory factors between initial and decayed samples. Inocu-

lated fungus and temperature were used as explanatory factors

among decayed samples. The F statistics and their significant level

were reported in the table

Richness

Pielou’s

evenness Shannon Simpson

Initial and decayed samples

Time§ 201�80*** 80�80*** 112�31*** 53�5***Decayed samples

Inoculation† 1�36¶ 0�83 0�93 0�59Temperature‡ 0�23 0�61 0�60 1�45Temperature

9 Inoculation

0�46 0�26 0�21 0�43

†Pb, Ff, and Pb + Ff.‡25 and 30 °C.§Time zero and 5 month.¶Numbers represent F values.

*P ≤ 0�05; **P ≤ 0�01; ***P ≤ 0�001.



inoculations (Fig. S3). In samples containing external

inoculum, however, mass and carbohydrate losses caused

by P. betulinus and/or F. fomentarius were significantly

higher in the absence of endophytes (Fig. 6a,b,d,e,

Table 3, Endophyte 9 Inoculation interaction). Lignin loss

also varied by endophyte presence, but only in the treat-

ments in which the white rot F. fomentarius isolate was

added (Fig. 6c,f). In those cases, lignin loss was signifi-

cantly decreased, with the exception at 30 °C when both

fungi were inoculated, simultaneously. When considered as

a main effect, increased temperature significantly slowed

decomposition (Fig. 6 and Table 3), but our treatment is

not necessarily a real-world proxy for temperature effects

(i.e. we applied constant rather than varying temperature)

and no other higher-order interaction effects were signifi-

cant.

The endophyte-present samples were separated from the

endophyte-absent samples on PCA ordinations using wood

characterizations and fungal ITS copy counts (Fig. 7). For

non-endophytic treatments, inoculated fungi had the lar-

gest effect in determining differences. There was no clear

effect of inoculated fungi on the endophytic treatment. The

effect of temperature was also not clear on PCA, although

all 30 °C treatment shifted away from their 25 °C corre-

spondents in the same direction.

Discussion

Allowing fungal wood endophytes to develop in the

absence of external colonizers demonstrated their potential

to both initiate and dominate decomposition, in this case

with a predictable functional consequence – brown rot.

The diverse fungal assemblages in these young birch trees

collectively caused significant wood mass loss (up to 40%)

over five months without external inoculation. This transi-

tion from endophytism to saprotrophy has been tracked

for individual fungi (e.g. Griffith & Boddy 1991), but using

community-scale high-throughput sequencing, we found a

redundant outcome that was better explained at a guild

level rather than a species level. Brown rot was the out-

come, but different brown rot taxa rose to dominate and

determine this outcome, and it could not be predicted by

Fig. 2. Decay type (trait) of the OTUs by their richness (a) and abundance (b). The decay type was assigned using the FUNGuild data

base. The ten fresh wood samples are on top starting with ‘Initial’ and the sample labels (from A to J). Decayed wood samples are labelled

by their treatment and replicate numbers. From top to bottom are endophyte control at 25 and 30 °C; P. betulinus inoculated at 25 and

30 °C; F. fomentarius inoculated at 25 and 30 °C; and both fungi inoculated at 25 and 30 °C.


6 Z. Song et al.

their relative abundances at the outset. This clear link

between community structure and function at the guild

level supports the logic for resolving function using traits

rather than per individual taxon (Crowther et al. 2014;

Nguyen et al. 2015; reviewed by Treseder & Lennon 2015).

The redundancy of brown rot on birch in these static

conditions is surprising for several reasons, particularly

given the taxa that rose to dominate decomposition. In

terms of the wood type, brown rot in birch does not fit the

generalized association of angiosperms and white rot fungi

(Hibbett & Donoghue 2001). Many brown rot fungi,

Fig. 3. The relative abundance of Top 20 OTUs in time-zero and decayed endophytes control under 25 °C in both treatments. (a) Top 20

OTUs in time-zero samples and their correspondents in 25 °C endophytes control. (b) Top 20 OTUs in 25 °C endophytes control and

their correspondents in time-zero samples. Letters in the parentheses following species name indicate fungal traits. B: brown rot, W: white

rot, S: saprotroph, P: Pathotroph and dash indicated unknown trait. Smaller figures in side A and B are relative abundance of OTUs at

the rank of 6–20.

Fig. 4. Non-metric multidimensional scaling plot of fresh wood,

and wood residue in the presence of endophytes under two tem-

perature and different soil fungi schemes. The soil fungi schemes

are endophyte control (circle), P. betulinus inoculated (triangle),

F. fomentarius inoculated (diamond) and both fungi inoculated

(star). All no-endophyte treatments were not included. The tem-

perature schemes are 25 °C (open) and 30 °C (shaded). The initial

wood samples were marked with uppercase letters.

Table 2. ADONIS analysis of differences in fungal composition


samples in different treatments. A Bray–Curtis dissimilarity matrix

was used as response variable using either square root transforma-

tion or presence/absence matrix. Time was used as explanatory

factors between initial and decayed samples. Inoculated fungus

and temperature were used as explanatory factors among decayed

samples. The F statistics and their significant level were reported

in the table

Square root† Presence/absence‡

Initial and decayed samples

Time§ 11�518***,¶ 23�293***Decayed samples

Inoculationk 1�97** 1�41*Temperature†† 4�71*** 2�15**Temperature 9 Inoculation 1�36 0�93

†Data were square root transformed before calculating the dissimi-

larity matrix.‡The abundance matrix was transformed to presence/absence

matrix before calculating the dissimilarity matrix.§Time zero and 5 month.¶Numbers represent F values.kPb, Ff, and Pb + Ff.††25 and 30 °C.*P ≤ 0�05; **P ≤ 0�01; ***P ≤ 0�001.



however, associate with angiosperms, including P. betuli-

nus on birch (Gilbertson 1980). Brown rot has also been

shown to peak at mid-decay stages (Rajala et al. 2015),

which would fit the decay stage in this study. More sur-

prising was that the four brown rot taxa with >90% of the

sequences in endophyte-only decayed wood were in genera

Coniophora and Postia (Fig. 3, 25 °C). Coniophora and

Postia genera include well-known slash-/lumber-degrading

fungi that are typically associated with conifer wood

(Buckland 1946; Gilbertson 1980; Ginns 1982), and the

most abundant taxon we observed in decayed wood was

the well-known building pest Coniophora puteana (>43%of sequences). While this was a surprising outcome, Cha-

pela & Boddy (1988)did find that C. puteana was an

endophyte commonly cultured from beech stems in Eur-

ope. It is possible that the fixed abiotic factors in our

experimental set-up, including relatively high moisture

levels (Boddy 1986), favoured the proliferation of these

particular fungi, or that the lack of exogenous competitors

favoured development of lumber-associated taxa.

Another possible explanation for the redundant brown

rot outcome we observed is biotic, due in part to a lack of

combative colonizers in our microcosms. Forest dead wood

studies have shown dependable increases in species richness

over time (Rolstad et al. 2004; Rajala et al. 2015), yet here

we observed a significant loss of taxa when wood decay

developed in isolation. Taken in combination, this suggests

that the net increases observed on the forest floor result

Fig. 5. ITS copy number ratio between the inoculated fungus and total fungi. The data are means and SEs (n = 10, n = 1 for no endo-

phyte control). Different letters indicate significant differences (P < 0�05) among all treatments. Because the abundances of P. betulinus,

F. fomentarius and total fungi were calculated using different standard curves, we focus on the ratio between targeted fungi (i.e. P. betuli-

nus and F. fomentarius) and total fungi among treatments, using total fungi as a ‘reference gene’ for the fungal community.


8 Z. Song et al.

from significant species turnover. Immigration into forest

dead wood involves many cord-forming soil fungi with

combative life-history strategies (Boddy 1993), and relative

isolation may be more favourable for less-combative,

stress-tolerant endophytes. This might be the case for

brown rot fungi in standing dead trees, where the endo-

phyte–saprophyte transition could be a viable strategy that

suits the diffuse depolymerization mechanisms of brown rot

fungi, releasing sugars at relatively long distances from

hyphae in wood (Kerem, Bao & Hammel 1998). This is

notable in the light of Swift (1977), who observed that 40%

or more of branchwood density loss occurred before it fell

to the ground. We speculate that avoiding competitors that

might capitalize on the distant sugars they release relegates

brown rot taxa to lumber, slash and other stochastic envi-

ronments where the presence of competitors is limited.

The inability of F. fomentarius or P. betulinus to invade

our birch sections in most, but not all, cases may provide

more clues about the viability of an endophytic strategy.

Both of these fungi are considered to be S-selected (i.e.

stress-tolerant) and are known endophytes (Boddy & Heil-

mann-Clausen 2008). The general inability of these fungi

to invade matches studies where latent wood communities

inhibited colonization by Basidiomycete fungi (Chapela &

Boddy 1988). Although high exogenous inoculum potential

has been shown to override such priority effects (Holmer

& Stenlid 1997; Song et al. 2015), the non-combative nat-

ures of our ‘challengers’ may limit their ability to invade

occupied wood territory. This emphasizes the potential for

endophyte priority effects in wood, but it also suggests a

strategy for fungi colonizing wood in ground contact, hav-

ing been shown to differ from those colonizing above-

ground (Coates & Rayner 1985). It seems likely that com-

petitive (i.e. C-selected) strategies are more viable for cord-

forming soil fungi that encounter pre-colonized wood as

the rule (Boddy 1993), and testing various combative

Fig. 6. Mass, carbohydrates and lignin loss of wood samples under 25 and 30 °C in the absence and presence of endophytes. The data are

means and SEs (n = 10, n = 1 for no endophyte control). Different letters indicate significant differences (P < 0�05) among all treatments.



colonizers might help explain the low colonization suc-

cesses of the S-selected F. fomentarius and P. betulinus,

and also their common occurrences as latent propagules

associated with trunk rots in standing trees (Boddy & Heil-

mann-Clausen 2008).

The instances where the external colonizer did manage

to invade may also be telling of endophyte lifestyles and

competitive strategies. Although P. betulinus had a strong

influence on community shifts (seen in Fig. 4 NMDS

plots), the success of only F. fomentarius in gaining signifi-

cant ingress and altering the chemical signatures in wood

residues may indicate a discrepancy in competitive abili-

ties. The head-to-head interactions we observed in steril-

ized wood did not reflect any such discrepancy, but

F. fomentarius may be a better competitor within a com-

munity and notably is the less host-restricted of these two

strains (Ryvarden & Gilbertson 1993; McCormick et al.

2013). Also notable is that F. fomentarius succeeded in

invading only at the lower temperature (25 °C), which

may reflect the growth efficiencies of the endophytes

developing within wood, rather than the performance of

F. fomentarius (File S3). Endophytes were given no ‘head

start’ for growth when placed atop lawns of hyphae, and

the latent propagules of endophytes have been shown to

compartmentalize in vessels, etc. (Boddy & Rayner 1983),

leading to spatial heterogeneity. At lower temperatures,

albeit relative here given that 25 °C is quite high, territory

captured by endophytes prior to invasion may have been

lower than at the higher temperature, allowing F. fomen-

tarius more time to access non-degraded wood. In all

cases, both as exceptions (white rot dominance) and as the

rule (brown rot dominance), these dynamics between endo-

phyte community development and exogenous coloniza-

tion suggest that the endophyte life-history strategy may

work best when allowed to develop in relative isolation,

again as one might find in standing dead trees or in

lumber.

Using microcosms to observe wood endophyte commu-

nities developed in isolation offers insight by deliberately

removing many variables present in a natural forest sys-

tem. By design, this restricts the focus and harbours vari-

ous important caveats. Without sequencing bacterial

communities, for example, we may have overlooked inter-

play between dominant sugar-releasing mechanisms and

guilds of microbes that might either enable wood degrada-

tion (iron acquisition, Herv�e et al. 2016) or inhibit it

(sugar-intercepting ‘cheaters’, Allison 2005) in this system.

Also, extrapolations remain limited when working with a

single wood type from a single plot, and introducing wood

modifications (e.g. hemicellulose side chain loss) in those

treatments using autoclave sterilization. Furthermore, the

surface sterilization bears caveats that some surface inocu-

lum on the end grain may persist to invade the wood or

that propagules on the bark may have similarly colonized

prior to the bark removal at harvest.

Despite a restricted design, our study showed a surpris-

ing and predictable outcome of brown rot that was driven

by relatively few fungi present at low initial abundances.

This is welcome encouragement to those trying to predict

wood decomposition. If we assume that biological variabil-

ity is the key to predicting wood decomposition, the histor-

ical contingencies of priority and inoculum potential might

seem a daunting prospect for modellers aiming to predict

carbon cycling from wood. If, however, we accept these

contingencies and imagine that they have shaped the

strategies of wood decomposers, it leads us ‘upstream’ to

the endophytes that are initiators of wood decay. Active

host active responses will restrict community diversity,

Table 3. Effects of endophyte, inoculation and temperature on

wood characteristics as determined by a fully factorial three-way

ANOVA. The wood characteristics (mass loss, carbohydrates loss

and lignin loss) were used as response variables. Endophyte (pres-

ence or absence), inoculated fungi and temperature were used as

explanatory factors among decayed samples. The F statistics and

their significant level were reported in the table

Mass loss Carbohydrates loss Lignin loss

Endophyte† 9�14**,¶ 1�37 6�21*Inoculation‡ 19�11*** 20�71*** 21�28***Temperature§ 5�05* 9�55** 0�04Endophyte

9 Inoculation

14�96*** 13�62*** 3�76*

Endophyte

9 Temperature

0�42 1�82 0�32

Temperature

9 Inoculation

1�27 2�28 0�51

Temperature

9 Endophyte

9 Soil fungi

0�33 0�03 0�96

†Endophyte absent (autoclaved) and endophyte present (non-auto-

claved).‡Pb, Ff, and Pb + Ff.§25 and 30 °C.¶Numbers represent F values.

*P ≤ 0�05; **P ≤ 0�01; ***P ≤ 0�001.

Fig. 7. Principle component analysis plot of all treatments (25 and

30 °C, and in the absence and presence of endophytes) using wood

characteristic and fungal biomass as variables.


10 Z. Song et al.

likely explaining why wood endophytes tend to be more

host specific than soil-inhabiting wood-degrading fungi

(Boddy & Heilmann-Clausen 2008). This suggests that

endophyte development might, at least initially, be pre-

dicted by tree host. Our study thus provides motivation

and a solid starting point for testing these dynamics under

increasing natural complexity.

Acknowledgements

This research was made possible through the generous support of the Con-

servation and the Environment grants programme of The Andrew W. Mel-

lon Foundation (New York, NY). A doctoral dissertation fellowship of the

University of Minnesota, awarded to Zewei Song, also provided generous

support for this research, along with the Minnesota Agricultural Experi-

ment Station funding #MIN-12-087 for Schilling.

Conflict of interest

We have not circulated this manuscript for pre-review, and we cannot iden-

tify any direct competitors that would be conflicts here. We similarly do

not identify groups with a history of dispute with the authors, and we claim

no financial interest in the outcome of this work.

Data accessibility

Raw sequence data and associated metadata were deposited in Data Repos-

itory for University of Minnesota (DRUM) under project number 181161

(http://doi.org/10.13020/D69880).

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ern United States. Forest Ecology and Management, 257, 1434–1444.Worrall, J.J., Anagnost, S.E. & Zabel, R.A. (1997) Comparison of wood

decay among diverse lignicolous fungi. Mycologia, 89, 199.

Zabel, R.A. & Morrell, J.J. (1992) Wood Microbiology: Decay and its

Prevention. Academic Press Inc, San Diego, CA, USA.

Received 21 April 2016; accepted 29 July 2016

Handling Editor: Alison Bennett

Supporting Information

Additional Supporting Information may be found online in the

supporting information tab for this article:

Fig. S1. Fruiting body of the two inoculum fungi in field and their

growth response to temperature.

Fig. S2. Flow chart of the experimental design.

Fig. S3. Diluted alkaline solubility and pH of wood samples under

25 and 30 °C in the absence and presence of endophytes.

Fig. S4. Nonmetric Multidimensional Scaling Plot of fresh wood,

and wood residue in the presence of endophytes under two tem-

perature and different soil fungi schemes.

Table S1. Experiment treatment structure.

Table S2. Species specific primer sets for P. betulinus and F. fo-

mentarius in qPCR.

Table S3. ADONIS analysis of differences in fungal composition


samples in different treatments, using an OTU table rarefied to the

depth of 1000 sequences.

File S1. High throughput sequencing analysis pipeline.txt.

File S2. Sample QC report.xlsx.

File S3. OTU taxonomic information.xlsx.

File S4. Rarefied OTU table with functional informatin.xlsx.

File S5. Diversity indexes of all samples.xlsx.


12 Z. Song et al.

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