Advertisement

GCB Bioenergy / Volume 15, Issue 9 / p. 1166-1185

RESEARCH ARTICLE Open Access  

Soil amendment with biochar and manure alters wood stake

decomposition and fungal community composition

Ruirui Zhao, Yong Liu , Michael J. Gundale 

First published: 24 July 2023

https://doi.org/10.1111/gcbb.13087
Citations: 1

Abstract
Biochar and manure can be used for sustainable land management. However,
little is known about how soil amendments might a"ect surface and
belowground microbial processes and subsequent wood decomposition. In a
split-split-split plot design, we amended soil with two rates of manure (whole
plot; 0 and 9 Mg ha−1) and biochar (split plot; 0 and 10 Mg ha−1). Wood stakes of
three species (hybrid poplar, triploid Populus tomentosa Carr.; aspen, Populus
tremuloides Michx.; and pine, Pinus taeda L.) were placed in two positions
Micr
(horizontally on the soil surface, and inserted vertically in the mineral soil), which
served as a substrate for fungal growth. In 3 years, the decomposition rate
(density loss), moisture content, and fungal community (via high-throughput
sequencing methods) of stakes were evaluated. Results indicated that biochar
and/or manure increased the wood stake decomposition rates, moisture
content, and operational taxonomic unit abundance. However, the richness and
diversity of fungi were dependent on wood stake position (surface > mineral),
species (pine > the two Populus), and sample dates. This study highlights that soil
amendment with biochar and/or manure can alter the fungal community, which
:
 in turn can enhance an important soil process (i.e., decomposition).

1 INTRODUCTION
Forest soils have a large role in the global carbon (C) cycle because they harbor a
substantial fraction of global soil C. Subsequently, key soil processes (e.g., organic C
formation, C mineralization, nutrient transformations, and organic matter
decomposition) in forests have implications for C sequestration and climate change
(Jiang et al., 2020; Nottingham et al., 2020; Yu et al., 2022). Soil organic matter (SOM)
is an important aspect of soil quality (Bhattacharyya et al., 2022; Jaroszewicz et
al., 2021) by increasing the water-holding capacity (Williams et al., 2016), reducing
erosion (Feng et al., 2016), limiting soil compaction (Somerville et al., 2020), and
serving as a substrate for microbial activity (Whalen et al., 2022). However, frequent
harvesting operations or land-use changes can cause C loss, resulting in nutrient
imbalances, drought, and reduced ecosystem services that soils provide (e.g., food
and #ber), especially in plantations (Coban et al., 2022; Mishra et al., 2022; Newbold
et al., 2015). Increasing SOM has proven to be an e"ective measure in restoring
fundamental physical, chemical, and biological soil properties (Krupek et al., 2022;
Page-Dumroese et al., 2021) and enhancing soil health.

Biochar, which can transform labile C into a more stable form (Lehmann et
al., 2012), has attracted worldwide attention for its capability to promote C
sequestration and storage (Chagas et al., 2022; Pingree et al., 2022), which can
serve to mitigate climate change (Hagenbo et al., 2022). In addition, biochar can
enhance soil health (Gundale et al., 2016) by improving soil microbiome structure
and function (Bahram et al., 2018; Xu et al., 2020), thus resulting in increased
underground C cycling and above-ground productivity. Organic fertilizer
amendment (e.g., manure and composts) is one of the most e"ective measures for
sustainable land management, both in agriculture and forestry (Reardon, 2014;
Williams, 1942; Yang et al., 2022), and its role in improving soil quality (e.g.,
physicochemical properties, structure, and microbiome functions), replenishing
SOM loss, and minimizing land degradation has been well accepted (Augarten et
al., 2023; Jin et al., 2022; Li et al., 2023).

In addition to C inputs from vegetation or organic amendments, microbial

decomposition is another key factor that controls soil C content (Jastrow et
al., 2007). The decomposition of organic matter in forests, both above and
belowground, is a function of soil properties (e.g., temperature, moisture, nutrient
:
availability, and microbial composition), substrate quality (e.g., nitrogen [N], lignin
content, and C:N; Middleton et al., 2021; Risch et al., 2022; Viotti et al., 2021), and
any soil condition changes at occur as a result of forest soil management practices
(Piaszczyk et al., 2022; Tie et al., 2022). Generally, high organic matter
decomposition rates are a sign of high soil quality and support higher forest
productivity (Wang et al., 2010), and thus decomposition can serve as a useful
response index of forest management activities. Biochar and/or organic
amendments could alter organic matter decomposition through positive or
negative priming e"ects (Joseph et al., 2021; Yu et al., 2020; Zheng et al., 2021), and
by altering the soil microbial community composition or abundance (Minamino et
al., 2019; Prayogo et al., 2013). However, the large variation in decomposition
substrate qualities investigated among published studies makes it di$cult to
distinguish to what extent such decomposition responses originate from the soil by
soil amendments or forest management practices.

The decomposition of “standardized” trembling aspen (Populus tremuloides Michx.)

and pine (Pinus taeda L.) wood stakes with di"erent qualities (e.g., C, N, lignin, and
lignin:N) can help hold organic matter quality constant, and thus allow for the
isolation of soil abiotic (e.g., moisture, temperature, and nutrient availability) and
biotic (e.g., microbial biomass, diversity, and function) changes caused by forest
management practices (Finér et al., 2016; Risch et al., 2022). Much work has been
done using the decay of wood stakes as an index of soil changes after land
management (e.g., organic matter removal, and soil compaction) (Adams et
al., 2021; Page-Dumroese, Jurgensen, et al., 2021). Especially, through the
degradation of hybrid poplar (triploid Populus tomentosa Carr.), aspen, and pine
stakes, our previous work indicated that biochar and/or manure amendment
treatments accelerated wood decomposition and altered nutrient %uxes within 6
months in a hybrid poplar plantation (Zhao et al., 2022). However, how these
amendments would a"ect wood decomposition and wood-inhabiting fungi over
longer time scales remains unknown, which is crucial for the development of
further forest soil management policies using biochar and/or organic fertilizers
(Yang et al., 2021).

As a result, we aimed to investigate wood stake decomposition (density loss) and

subsequent fungal community responses to soil manure and biochar amendments,
both on the soil surface and belowground, over a 3-year period. Wood stakes of
three species (hybrid poplar, trembling aspen, and pine) were placed horizontally
on the soil surface (hereinafter, surface stakes) and inserted vertically in the 0–20
:
cm mineral soil (hereinafter, mineral stakes) to serve as an index indicating soil
functioning and quality changes. These three species of wood stakes encompass a
range of lignin, C, and N concentrations, which could favor the development of
di"erent wood-decomposing microbial communities (Wang et al., 2020). We
hypothesized that biochar and/or manure would: (1) increase wood stake
decomposition both on the soil surface and in the mineral soil; (2) alter the fungal
community richness and abundance due to changed soil moisture; and further, (3)
wood stake species and position would also be important factors a"ecting
response to soil amendments.

2 MATERIALS AND METHODS

2.1 Study site and experimental design
This 3-year #eld experiment was conducted in Guan County, Liaocheng City,
Shandong Province, China (36°31′21″ N, 115°21′37″ E, 46 m above sea level), in the
%oodplain of the previous channel of the Yellow River left from the Western Han
Dynasty (202 BC–8 AD). Since 1980, this region has been the focus of management
activities aimed at preventing deserti#cation through a"orestation, and in the
decade prior to our study, the site was used for poplar seedling cultivation. The
climate at the study site is semiarid continental, and the average monthly
temperature and precipitation are in Figure 1. Soil (0–30 cm) in this study is sandy
loam (sand:silt:clay is 62.3%:35.0%:2.7%). Additional details on soil properties are
presented in Table 1.
:
 FIGURE 1 Open in !gure viewer  PowerPoint

Monthly average air temperature and precipitation in the study site. The data set is provided by

the Geographic remote sensing ecological network platform (www.gisrs.cn).

TABLE 1. Initial properties of sandy loam soil, maize straw biochar, pig manure, and
hybrid poplar, aspen, and pine stakes (Zhao et al., 2022).

Properties 0–30 cm Soil amendments Wood stakes

soil
Maize straw Pig Hybrid Aspen Pine
biochar manure poplar

pH 8.2 8.8 7.9 - - -

N (mg g−1) 2.20 10.70 14.64 1.40 1.10 0.80

P (mg g−1) 1.40 1.60 19.52 - - -

K (mg g−1) 8.30 2.60 26.84 - - -

:
 C (%) 1.10 47.56 - 46.75 46.40 48.62

H (%) - 2.80 - - - -

C:N 5.00 44.00 - 354.64 421.81 631.65

Speci#c surface area - 5.0 - - - -

(m2 g−1)

Pore volume (cm3 - 6.93 × 10−3 - - - -

g−1)

Pore size (nm) - 5.60 - - - -

Organic matter 7 - 450 - - -

content (g kg−1)

Lignin (%) - - - 20.92 20.64 31.09

Note: N, P, and K indicate total nitrogen, phosphorus, and potassium; C, carbon; H, hydrogen; -, not

determined.

We employed four soil treatments set up in a split-split-split plot experimental

design. The #rst factor, manure (M), was applied at the whole plot level, with two
rates: 0 (M0) and 9 (M9) Mg ha−1. The second factor, biochar (B) was set up as a split-
plot treatment with two levels, 0 (B0) and 10 (B10) Mg ha−1. These manure and
biochar treatments resulted in four soil treatment combinations (M0B0, M0B10,
M9B0, and M9B10), each replicated three times, resulting in 12 plots in total, each
24 × 24 m squares separated with at least 8-m-wide bu"er strips. In addition to soil
amendment treatments, a third experimental factor was applied as a split-split plot
factor, which consisted of wood stakes that were installed in each of the two
positions within each plot (horizontally on the soil surface, and vertically within the
mineral soil). Furthermore, wood stakes were installed for each of three wood
species (hybrid poplar, aspen, and pine), which served as a split-split-split plot
factor.

2.2 Soil amendments and wood stakes

Our experiment utilized biochar made from maize via slow pyrolysis (450–500°C for
2 h in an oxygen-limited rotary furnace; Qin Feng Zhong Cheng Biomass New
Material Corporation), and pig manure used was supplied by Run Dong Fertilizer
:
Corporation. The properties of these soil amendments are described in Table 1.

Kiln-dried and knot-free sapwood was selected for stake production, in which two
surface (15 cm long) and two mineral (20 cm long) stakes of each species were cut
from 2.5 cm × 2.5 cm stakes of 40 and 50 cm lengths, respectively, with the middle
stake (10 cm) kept as a control (time = 0) to determine initial wood properties
(Table 1). Before installation, one end of each mineral stake was treated with a
wood sealer to avoid moisture loss (Jurgensen et al., 2006).

2.3 Soil amendment implementation, wood stake

installation, sampling, and calculations
After applying manure and biochar treatments to plot surfaces in April 2018, all soil
treatments (including the un-amended M0B0) were mixed into the top 20 cm of soil
using a rotary tiller (1GQN- 200; Weifang Sheng Xuan Machinery Corporation). After
that, plots were planted with 1-year-old hybrid poplar (triploid “Beilinxiongzhu1” [P.
alba × P. glandulosa] × [P. tomentosa × P. bolleana]) seedlings, with a 3 m × 4 m spacing;
however, we note that tree growth was not the focus of our current study.

Regarding wood stake treatments, in July 2018, after removing the debris from the
surface of forest land, 10 stakes of each species were placed on the soil surface
(each 30 cm apart) and secured with a stainless-steel landscape staple. Using a 2.5 ×
2.5 cm coring tool, we #rst created 10 holes per wood species (each 20 cm deep and
about 30 cm apart) which helped reduce compaction from stake installation,
mineral wood stakes were then inserted into the holes with the sealed end level
with the soil surface. For more details, see Wang et al. (2019). Totally, 720 wood
stakes were deployed (4 soil treatments × 3 replicates × 2 positions [surface and
mineral] × 3 tree species [hybrid poplar, aspen, and pine] × 10 individual stakes).

In July 2020 and 2021—2 and 3 years after stakes were installed—#ve surface and
#ve mineral stakes of each species were collected from each plot (a total of 720
stake samples, 4 soil treatments × 3 replicates × 2 stake positions × 3 tree species × 5
stakes × 2 sample dates). Wood stake samples were immediately weighed in the
#eld after adhering material was removed from their surfaces, and then kept on ice
as they were delivered to the laboratory.

Upon return from the #eld, wood shavings from each stake was collected for fungal
DNA analysis within 24 h. First, both ends of each stake were cleaned with a sterile
razor blade, then each end of the stake was drilled with sterile drill bits, and the two
:
shaving samples of each stake were composited into one individual sample. All
fungal samples were placed in 2 mL strip tubes and were frozen at −80°C until DNA
extraction.

Subsequently, 10 days after wood stakes were placed in a climate chamber (65%
relative humidity and 20°C), wood stake three dimensions (length × width × height)
were measured. Wood stake density loss was calculated by subtracting the #nal
from the initial wood density (g cm−3) and expressed as a persent (Cline et al., 2017;
Shorohova et al., 2008). After that, wood stakes were dried for 72 h at 105°C, and
weighed for moisture content calculation (Zhao et al., 2022).

2.4 DNA extraction, polymerase chain reaction ampli#cation,

and Illumina MiSeq sequencing
Microbial community genomic DNA was extracted using the FastDNA® Spin Kit for
soil (MP Biomedicals) according to the manufacturer's instructions. The extracted
DNA concentration and purity were determined using a UV–vis spectrophotometer
(NanoDrop 2000; Thermo Scienti#c), and the DNA quality was evaluated by 1%
agarose gel electrophoresis.

The primer pairs fITS7F (5′-GTGARTCATCGAATCTTTG-3′) and ITS4R (5′-

TCCTCCGCTTATTGATATGC-3′) were used to amplify the hypervariable ITS2 region,
and the ampli#cations were performed on an ABI GeneAmp® 9700 PCR
thermocycler (ABI) under the following programs: (1) initial denaturation at 95°C for
3 min, (2) 35 cycles of denaturing at 95°C for 30 s, (3) annealing at 55°C for 30 s, and
(4) extension at 72°C for 45 s, single extension at 72°C for 10 min, ending at 10°C.
polymerase chain reaction (PCR) reactions of each sample were performed in
triplicate. Furthermore, the PCR products were extracted from 2% agarose gel and
puri#ed using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) according to
manufacturer's instructions and quanti#ed using Quantus™ Fluorometer
(Promega).

Puri#ed amplicons were pooled in equimolar, and paired-end sequenced on an

Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina) according to the
standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. The raw reads were
deposited into the NCBI Sequence Read Archive database (Accession Number:
PRJNA899460).

2.5 Statistical analysis

:
For wood stake density loss and moisture content, we analyzed wood stake
position e"ects #rst, then data were analyzed separately by position to see the
main e"ects of manure, biochar, and wood species. Similarly, for wood stake fungal
composition, we analyzed wood stake position e"ects #rstly to see the overall
changes, and then the e"ects of manure and biochar soil treatment combination,
wood species, and/ or sample date were analyzed separately for each wood stake
position.

2.5.1 Wood stake decomposition and moisture content

After taking all factors (manure, biochar rates, wood stake position, and species)
into consideration, and using density loss and moisture content as dependent
variables for four-factor linear mixed e"ect (LME) models, three-factor LME models
were used with manure, biochar, and wood stake species as independent factors
and the density loss and moisture content as dependent variables. When the three-
factor analysis revealed signi#cant di"erences between treatment factors, we then
performed post hoc Tukey's pairwise comparisons. Subsequently, we adopted two-
factor LME models with manure and biochar as independent factors and the wood
stake density loss and moisture content of each species at each position as
dependent variables. When signi#cant interactions among manure and biochar
were detected, post hoc comparisons between the four soil treatments were
conducted.

All LME models were conducted using the lmerTest package (Kuznetsova et
al., 2017) in R version 4.1.1 (R Core Team, 2021), in which type III tests of #xed
e"ects were used to examine main e"ects and interactions. The post hoc Tukey's
pairwise comparisons were conducted using the emmeans package (Lenth et
al., 2022). Furthermore, Pearson correlation analyses were used to study the
correlations between wood stake density loss and moisture content for each
species and position with all soil treatments combined. Scatter and bar charts were
completed by Origin Pro 2022 (OriginLab).

2.5.2 Fungal data processing

The raw ITS2 gene sequencing reads were de-multiplexed, quality-#ltered by Fastp
(version 0.20.0, https://github.com/OpenGene/fastp) (Chen et al., 2018) and
merged by Flash (version 1.2.7, http://www.cbcb.umd.edu/software/!ash) (Magoc
& Salzberg, 2011) with the following criteria: (1) the reads were truncated to obtain
an average quality score <20, and shorter sequence reads (truncated length <50 bp)
were discarded; (2) raw reads containing ambiguous bases were discarded; and (3)
:
only overlapping sequences >10 bp were assembled according to the overlapped
sequences, and un-assembled reads were unwanted. The #ltered reads were
clustered into di"erent operational taxonomic units (OTUs) with 97% similarity
(Edgar, 2013; Stackebrandt & Goebel, 1994) by UPARSE (version 7.1,
http://drive5.com/uparse/) (Edgar, 2013). The taxonomy of each OTU
representative sequence was analyzed by RDP Classi#er version 2.2 (Wang et
al., 2007) against the Unite database 8.0 (http://unite.ut.ee/) using the con#dence
threshold of 0.70.

The analysis of fungal data was completed by R version 4.1.1 (R Core Team, 2021),
which was mainly divided into the following parts. First, the MOTHUR program
(version 1.30.2) was employed to conduct the rarefaction analysis, and Venn
diagrams were used to demonstrate the number of unique OTUs in each group.
Then we obtained the alpha-diversity indices (Chao and Shannon) through UPARSE
(version 11) and utilized the Wilcoxon rank-sum test to check the di"erences
between groups. Subsequently, fungal community bar charts were employed to see
the overall structure at di"erent groups at the genus level, and the principal
coordinate analysis based on weighted-unifrac distance was applied to characterize
microbial beta diversity. Permutational multivariate ANOVA (PERMANOVA) was
performed by the Adonis function to show the di"erence between groups.
Furthermore, the Wilcoxon rank-sum and Kruskal–Wallis rank tests were conducted
to show the relative abundances of the top 15 genera di"erent between groups.
Finally, Pearson's correlations between the dominant genera and wood stake
density loss, and moisture content were displayed using heatmaps, in which the
third-year density loss and moisture were unpublished data.

3 RESULTS
3.1 Wood stake decomposition and moisture content
In our present study, the density loss and moisture content were investigated 2
years after wood stake installation. Overall, wood stakes decomposed faster in the
mineral soil (average, 52.33% density loss) than on the soil surface (average, 21.73%
density loss) with all other factors combined (F = 105.93, p < 0.001). On the soil
surface, biochar and wood species were the main factors a"ecting wood stake
density loss, and interaction e"ects among manure, biochar, and wood species also
existed (Table S1). Biochar application signi#cantly increased wood stake density
loss (average, 26.40%) with other factors combined (Figure 2a), and hybrid poplar
:
and aspen stakes decomposed faster as compared to pine (27.12%, 24.81%, and
9.08%, respectively, Figure 2b). As for each wood species, biochar had signi#cant
e"ects on the density loss of three species, while interactions among manure and
biochar were re%ected in hybrid poplar and aspen stakes (Table 2). As compared to
no biochar application, 10 Mg ha−1 biochar signi#cantly increased the decomposition
of hybrid poplar, aspen, and pine stakes (Figure 2c,e,g). In addition, biochar alone
or combined with manure (M0B10 and M9B10) signi#cantly increased the
decomposition of surface hybrid poplar and aspen stakes as compared to the
untreated M0B0 (Figure 2d,f).

FIGURE 2 Open in !gure viewer  PowerPoint

Surface wood stake density loss as a"ected by the main e"ect of biochar (a) and species (b);

surface hybrid poplar stake density loss as a"ected by the main e"ects of biochar (c), and the

interaction of manure and biochar (d); aspen stake density loss as a"ected by biochar (e), and the
:
 interactions of manure and biochar (f); pine stake density loss as a"ected by biochar (g); mineral

wood stake density loss as a"ected by wood species (h) with soil treatments combined; aspen (i)

and pine stake (j) density loss as a"ected by the main e"ect of biochar. Data are presented as

means ± SE, and di"erent letters indicate signi#cant di"erences at p ≤ 0.05.

TABLE 2. Surface and mineral hybrid poplar, aspen, and pine stake density loss and
moisture content responses to soil manure, biochar amendments, and their
interactions after 2 years of decomposition.

Position and Source of Density loss Moisture content

species variation
df F p Values df F p Values

Surface

Hybrid poplar Manure (M) 1 0.02 0.9002 1 6.75 0.9936

Biochar (B) 1 38.96 0.0000*** 1 0.00 0.0136*

M×B 1 6.19 0.0180* 1 2.99 0.0927

Aspen Manure (M) 1 0.01 0.9139 1 6.44 0.5542

Biochar (B) 1 95.04 0.0000*** 1 0.44 0.0157*

M×B 1 30.94 0.0000*** 1 3.16 0.0840

Pine Manure (M) 1 0.13 0.7402 1 0.33 0.6566

Biochar (B) 1 11.12 0.0021** 1 0.23 0.5722

M×B 1 0.68 0.4147 1 0.61 0.4397

Mineral

Hybrid poplar Manure (M) 1 0.01 0.9217 1 34.91 0.4444

Biochar (B) 1 3.57 0.0670 1 0.77 0.0000***

M×B 1 1.92 0.1750 1 2.19 0.1478

Note: *, **, and *** denote signi#cant e"ects at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001.
:
In the mineral soil, wood species instead of manure or biochar was the main factor
that a"ected wood stake decomposition (Table S1), in which signi#cantly higher
density loss was found in hybrid poplar and aspen stakes as compared to pine
stakes (65.50%, 66.30%, and 28.40%, respectively) (Figure 2h). For each species,
aspen and pine stake density loss was only a"ected by biochar amendment while
the density loss of hybrid poplar stakes was una"ected by soil amendments
(Table 2), in which biochar amendment signi#cantly increased aspen and pine stake
decomposition as compared to no biochar addition (Figure 2i,j).

Wood stake moisture content can re%ect the soil water conditions about 2 weeks
before sampling (Zhao et al., 2022). Generally, wood stakes had signi#cantly less
moisture on the soil surface (average, 46.67%) as compared to the mineral stakes
(average, 68.97%) with other factors combined (F = 65.55, p < 0.01). On the soil
surface, wood species instead of manure and biochar amendment was the main
factor a"ecting wood stake moisture content (Table S1), in which signi#cantly
higher moisture content was detected in pine (58.50%) as compared to hybrid
poplar (40.50%) and aspen stakes (41.00%) (Figure 3a). As for each wood species,
hybrid poplar and aspen stake moisture content was signi#cantly a"ected by
biochar with pine stake moisture content una"ected by soil amendments (Table 2),
in which biochar amendment signi#cantly increased the moisture content of hybrid
poplar and aspen stakes as compared to no biochar addition (Figure 3b,c).
Furthermore, the moisture content of surface hybrid poplar (r = 0.722, p < 0.0000),
aspen (r = 0.500, p = 0.0010), and pine (r = 0.493, p = 0.0012) was positively correlated
with the density loss with all the factors combined.
:
 FIGURE 3 Open in !gure viewer  PowerPoint

Surface wood stake moisture content as a"ected by the main e"ect of species (a); surface hybrid

poplar (b) and aspen (c) stake moisture as a"ected by the main e"ects of biochar; mineral stake

moisture content as a"ected by the main e"ects of biochar (d) and species (e) with soil

treatments combined; mineral aspen (f) and pine (g) stake moisture content as a"ected by the

main e"ects of biochar. Data are presented as means ± SE, and di"erent letters indicate

signi#cant di"erences at p ≤ 0.05.

In the mineral soil, biochar amendment, and wood species were the main factors
a"ecting wood stake moisture content (Table S1), in which higher wood stake
moisture content was detected in soil treated with biochar (Figure 3d), and the
highest moisture content was detected in aspen (79.20%) followed by hybrid poplar
(72.10%) and pine (55.60%) (Figure 3e). Similarly, for each species, hybrid poplar,
and aspen stake moisture content was notably a"ected by biochar amendment
while the moisture content of pine stakes was not a"ected (Table 2), in which
biochar addition signi#cantly increased the moisture content of hybrid poplar and
aspen stakes (Figure 3f,g). Similarly, positive correlations between moisture content
and density loss were detected in hybrid poplar (r = 0.505, p = 0.0009), aspen (r =
0.630, p = 0.0000), and pine (r = 0.570, p = 0.0001) stakes.

3.2 Wood stake fungal communities

:
3.2.1 The alpha-diversity responses
A total of 24,070,074 fungal sequences were recovered after quality #ltering
(average 90,489 per sample), representing 1881 fungal OTUs at 97% sequence
similarity. All of the fungal OTUs were classi#ed into 11 di"erent phyla, 39 classes,
102 orders, 234 families, 515 genera, and 874 species. Rarefaction curves for
Shannon and Chao indices approached a plateau for most samples (Figure S1),
which indicated that the sequences were adequate to represent the entire fungal
population in wood stakes, allowing meaningful comparison between treatments.

At the OTU level, 719 and 410 OTUs, representing 38.22% and 21.80% of the total
amount of OTUs, were exclusively represented in the mineral and surface stakes,
respectively (Figure S2a). For surface stakes, manure addition decreased
(Figure S2b) while biochar had little e"ect on the abundance of OTUs (Figure S2c).
Furthermore, stakes in soil treated with M0B10 had the most abundant and
exclusive OTUs as compared to the other three treatments (Figure S2d). As for
wood species, more OTUs and exclusive OTUs were found in pine stakes as
compared to hybrid poplar and aspen (Figure S2e).

However, in the mineral soil, both manure and biochar application increased the
OTU abundances of stakes (Figure S2f,g). Stakes in soil treated with 10 Mg ha−1
biochar (M0B10) had the most abundant OTUs while the most exclusive OTUs were
detected in the high rate of manure and biochar (M9B10) (Figure S2h). Similarly,
pine stakes had the most abundant and exclusive OTUs as compared to hybrid
poplar and aspen (Figure S2i). However, a similar abundance of OTUs was detected
after 2 and 3 years of decomposition (Figure S2j).

Instead of manure, biochar, and soil treatments, wood stake position, species, and
sample date were the main factors a"ecting fungal richness and diversity
expressed by the Chao and Shannon index (Figure 4). In general, fungal
communities of surface stakes were signi#cantly more abundant than stakes in the
mineral soil (Figure 4a). Both on the soil surface and in the mineral soil, the highest
richness and diversity of OTUs were detected in pine followed by hybrid poplar and
aspen stakes, without signi#cant di"erences between hybrid poplar and aspen
stakes (Figure 4e,i). Furthermore, more fungal taxa were involved in the second
than the third year of mineral stake decomposition (Figure 4j).
:
 FIGURE 4 Open in !gure viewer  PowerPoint

Wood stake fungal diversity (Shannon, upper) and richness (Chao, lower) indices of di"erent

groups. (a) Represents wood stakes at di"erent positions; (b–e) represent surface stakes of

di"erent manure rates (b), biochar rates (c), biochar and manure combinations (d), and wood

stake species (e); and (f–j) represent mineral stakes of di"erent manure rates (f), biochar rates (g),

biochar and manure combinations (h), wood stake species (i), and sample dates (j). Wilcoxon

rank-sum test was performed. Data are presented as means ± SE, di"erent letters indicate

signi#cant di"erences at p ≤ 0.05, and bars without letters are not signi#cantly di"erent.

3.2.2 Fungal community structure and beta diversity responses

Overall, fungal communities at the genus level strongly clustered according to wood
stake positions (Figure 5a). For surface stakes, signi#cant di"erences were evident
with the manure treatment, but biochar had no e"ect on wood stake fungal
communities (Figure 5b,c), and all manure and biochar combinations did not
change the fungal communities signi#cantly as compared to the untreated M0B0
(Figure 5d). In addition, pine stake fungal communities were signi#cantly di"erent
from hybrid poplar and aspen (Figure 5e).
:
 FIGURE 5 Open in !gure viewer  PowerPoint

Principal coordinate analysis on genus level based on weighted-unifrac distance of all stakes at

di"erent groups. (a) Represents wood stakes at di"erent positions; (b–e) represent surface

stakes of di"erent manure rates (b), biochar rates (c), biochar and manure combinations (d), and

wood stake species (e); and (f–j) represent mineral stakes of di"erent manure rates (f), biochar

rates (g), biochar and manure combinations (h), wood stake species (i), and sample dates (j).

Permutational multivariate ANOVA (PERMANOVA) tests were performed by the Adonis function

to show the di"erence between groups.

However, in the mineral soil, manure and biochar application had no signi#cant
e"ects on the fungal communities of wood stakes (Figure 5f,g), while soil treated
with the high rate of both manure and biochar (M9B10) signi#cantly changed the
fungal communities of stakes as compared to the M0B0, M0B10, and M9B0
:
(Figure 5h). Similarly, mineral pine stake fungal communities were signi#cantly
di"erent from hybrid poplar and aspen (Figure 5i), but the sample date did not
change the fungal communities signi#cantly with other factors combined (Figure 5j).

Initial testing of genus-level e"ects for trial treatments (manure, biochar, soil
treatments, wood stake position, species, and sample date) was presented in
Figure S3, which provided identi#cation that represents>1% of the total community.
And the relative abundance di"erences of the top 15 genera among groups are
shown in Figures 6 and 7 and Figure S4. Wood stake position changed the fungal
taxa, in which only seven genera were shared for both surface and mineral stakes
(Figure S3a). Surface stakes harbored a higher abundance of Preussia,
unclassi!ed_c_Dothideomycetes, Coprinellus, and Coniochaeta than stakes in the
mineral soil, while the abundance of unclassi!ed_o_Sordariales was the opposite
(Figure S4).

FIGURE 6 Open in !gure viewer  PowerPoint

Relative abundances of the top 15 genera in surface wood stakes showed signi#cant di"erences

among manure (a), biochar (b), manure and biochar combination treatments (c), and wood stake

species (d). Comparisons between multiple and two groups were conducted using the Wilcoxon

rank-sum and Kruskal–Wallis rank test, respectively. In multiple comparisons, Welch's post hoc

tests were used. *, **, and *** indicate signi#cant di"erence at p < 0.05, p < 0.01, and p < 0.001.

FIGURE 7 Open in !gure viewer  PowerPoint

Relative abundances of the top 15 genera of mineral stakes showed signi#cant di"erences

among manure (a), biochar (b), manure and biochar combination treatments (c), species (d), and

sample date (e). Comparisons between multiple and two groups were conducted using the

Wilcoxon rank-sum and Kruskal–Wallis rank test, respectively. In multiple comparisons, Welch's

post hoc tests were used. *, **, and *** indicate signi#cant di"erence at p < 0.05, p < 0.01, and p <

0.001.

Small di"erences were observed between the manure treatments, in which only
:
unclassi!ed_f_Chaetomiaceae (5.55%) and unclassi!ed_f_Xylariaceae (4.05%) were
exclusive in soil treated with manure, and Trichoderma (4.05%) and Talaromyces
(3.01%) were exclusive in soil without manure application (Figure S3b). For surface
stakes, the abundance of all the top #ve genera was signi#cantly higher in soil
treated with manure (Figure 6a). However, in the mineral soil, manure amendment
signi#cantly increased the abundance of unclassi!ed_f_Chaetomiaceae, but less
Hydnophlebia, Exophiala, Trichoderma, and unclassi!ed_c_Dothideomycetes were
enriched as compared to no manure addition (Figure 7a).

Similarly, biochar had few e"ects on measured fungal community structure, in

which unclassi!ed_f_Chaetomiaceae (4.76%), Talaromyces (2.36%), and
Phaeoacremonium (1.90%) were exclusive in soil treated with biochar, and
Psathyrella (3.42%), unclassi!ed_f_Xylariaceae (2.62%), and Lasiodiplodia (2.62%) were
exclusively detected in soil without biochar (Figure S3c). For stakes on the soil
surface, the top #ve genera were less clustered in soil treated with biochar
(Figure 6b). In the mineral soil, however, biochar application decreased the
abundance of the top four genera but increased the abundance of
Phaeoacremonium (Figure 7b).

For the four soil treatments, a total of seven shared genera were detected in all
four treatments while the most unique taxa were found in M9B10 (Figure S3d). On
the soil surface, all soil treatments increased the abundance of
unclassi!ed_o_Pleosporales but decreased the abundance of unclassi!ed_o_Xylariales
as compared to the untreated M0B0. The highest abundance of Hypoxylon and
Coniochaeta was detected in M0B10, and the most abundant Paecilomyces was in
M9B0 (Figure 6c). In the mineral soil, all soil treatments increased the abundance of
Scytalidium but decreased the abundance of Exophiala. The abundance of
unclassi!ed_o_Sordariales and Hydnophlebia was decreased in M9B10 but increased
in M0B10 and M9B0 as compared to M0B0 (Figure 7c). Meanwhile, M9B0 increased
while M0B10 and M9B10 decreased the abundance of unclassi!ed_o_Hypocreales as
compared to the M0B0 (Figure 7c).

The fungal taxa composition of the hybrid poplar and aspen stakes was similar
(seven shared genera), while the most unique genera were found in pine stakes
(Figure S3e). On the soil surface, Preussia and Hypoxylon were most abundant in
hybrid poplar followed by aspen and pine stakes, and the abundance of Coprinellus
and unclassi!ed_c_Dothideomycetes was highest in aspen followed by hybrid poplar
and pine stakes. However, Coniochaeta was signi#cantly more abundant in pine
:
stakes (Figure 6d). In the mineral soil, unclassi!ed_o_Sordariales,
unclassi!ed_o_Hypocreales, and Exophiala were signi#cantly abundant in hybrid
poplar or aspen as compared to pine stakes, while the abundance of Hydnophlebia
was highest in pine followed by aspen and hybrid poplar stakes, and the most
abundant Scytalidium was detected in hybrid poplar followed by pine and aspen
stakes (Figure 7d).

Sample date also a"ected the top 15 abundant fungal genera composition of
mineral stakes, in which the top 3 abundant fungal genera were Hydnophlebia
(14.69%), unclassi!ed_o_Sordariales (8.24%), and Preussia (7.71%) 2 years after wood
stakes were installed, while in the third year, the top 3 genera were
unclassi!ed_o_Sordariales (14.50%), Hydnophlebia (12.15%), and
unclassi!ed_f_Chaetomiaceae (8.59%) (Figure S3f). Furthermore, signi#cantly more
abundant Hydnophlebia, Trichoderma, Psathyrella, and Preussia were detected after
2 years of decomposition, while in the third year, unclassi!ed_f_Chaetomiaceae was
more enriched (Figure 7e).

3.3 Relationships between wood stake decomposition,

moisture content, and fungal community
To better understand the relationship between wood stake decomposition and
fungi community, Pearson correlation analysis was conducted for three wood
species at each position (Figure 8). For surface stakes, both the density loss and
moisture content of hybrid poplar stakes were signi#cantly correlated with several
genera of Cystobasidiomycetes, Dothideomycetes, Eurotiomycetes, Sordariomycetes,
Tremellomycetes, and Agaricomycetes class, in which more positive correlations were
detected (Figure 8a). Similarly, aspen stake density loss and moisture content were
signi#cantly related to genera of Sordariomycetes, Dothideomycetes, Agaricomycetes,
and Eurotiomycetes class (Figure 8b). However, pine stake density loss was more
related to fungal taxa as compared to the moisture content, in which genera of
Agaricomycetes, Sordariomycetes, Dothideomycetes, Eurotiomycetes, and
unclassi!ed_p_Ascomycota class were involved (Figure 8c).

FIGURE 8 Open in !gure viewer  PowerPoint

Pearson correlation heatmap of wood stake density loss, moisture content, and the top 50
:
 abundant genera of fungi in surface hybrid poplar (a), aspen (b), and pine (c), and mineral hybrid

poplar (d), aspen (e), and pine (f).

In the mineral soil, hybrid poplar and aspen stake density loss and moisture
content were only signi#cantly correlated with several genera of Sordariomycetes,
Dothideomycetes, Agaricomycetes, unclassi!ed_p_Ascomycota, and Eurotomycetes class,
while more genera of Sordariomycetes, unclassi!ed_k_Fungi, Eurotiomycetes,
Agaricostilbomycetes, Dothideomycetes, and Cystobasidiomycetes class were
signi#cantly related to the density loss and moisture of pine stakes (Figure 8d–f).

4 DISCUSSION
We hypothesized that biochar and manure would increase wood stake decay and
alter the richness and abundance of fungal communities, and that such e"ects
would depend upon wood species and positions. Biochar and/or manure indeed
accelerated the decomposition rates and increased the moisture content of wood
stakes both on the soil surface and in the mineral soil. Furthermore, soil
amendments increased the OTU abundance in wood stakes, especially in the
mineral soil, but the fungal richness and diversity were more a"ected by wood
stake position, wood species, and sampling dates.

4.1 Wood stake decomposition and moisture content

Biochar can enhance soil moisture content, especially in coarse-textured soils,
because of its high porosity (Razzaghi et al., 2020). Similarly, in our current study,
biochar at 10 Mg ha−1 signi#cantly increased the density loss and moisture content
of stakes both on the soil surface and in the mineral soil (Figures 2 and 3). However,
manure hardly a"ected wood stake decomposition and moisture content except
combined with biochar (Figures 2 and 3). This may be because the longer longevity
of biochar compared to manure may have caused longer-lasting positive e"ects
(Seyedsadr et al., 2022; Zhao et al., 2022). Furthermore, there may have been
synergistic positive e"ects of compost–biochar mixtures on the water-storage
capacity in the sandy, low-organic content soils (Liu et al., 2012; Seyedsadr et
al., 2022), and moisture uptake from the surrounding soil matrix may have served
as an important control on decomposition (Gora et al., 2019; Seyedsadr et
al., 2022). In addition, manure or other soil amendments can alter microbial
functions and subsequent wood decomposition by changing soil nutrient
availability (Pingree et al., 2022), however, in our previous study, we did not #nd soil
:
N availability in soil treated with manure (Zhao et al., 2022), which may explain why
we found no main e"ects of the manure treatment on decomposition in the
current study.

On the soil surface, soil amendments increased the density loss of three wood
species, but the increased moisture content was only detected in the Populus
species. Meanwhile, mineral pine stake density loss was increased by soil
amendments without moisture content changes. These inconsistent changes
indicate that soil biotic (e.g., fungal community) rather than abiotic (e.g.,
temperature and moisture content) changes caused by soil amendments were
responsible for the responses in wood decomposition we observed because
biochar can cause preferential adsorption of wood and litter-degrading fungi in
organic matter-poor sandy loam soil (Anasonye et al., 2018), which may cause
degradation changes.

4.2 Soil amendment, wood stake position, and species

e"ects on wood stake fungal community
4.2.1 Fungal alpha and beta diversity
Biochar and/or manure could indirectly increase the richness and diversity of fungi
related to wood degradation and litter decomposition through nutrient supply
(Pingree et al., 2022) or amelioration of soil pH (Han et al., 2022; Tarin et al., 2021).
Although soil manure, biochar, and their combinations had no signi#cant impacts
on the fungal richness and diversity of wood stakes at the OTU level (Figure 4),
fungal composition at the genera level indeed signi#cantly clustered according to
soil amendments (Figure 5). This #nding is consistent with data presented by Zhang
et al. (2021), where soil manure or biochar amendments caused soil micro-
environmental changes.

Higher species richness, in fungal ecology, is commonly thought to relate to higher

rates of nutrient cycling and decomposition via additive or synergistic bene#ts of
the fungal niche complementarity (van der Wal et al., 2013). However, consistent
with results reported by Wang et al. (2020), we found surface stakes had higher
fungal richness and diversity (Figure 4), but decomposed slower as compared to
mineral stakes (F = 105.93, p < 0.001). Furthermore, pine stakes decayed slower even
though had higher fungal richness and diversity as compared to the two Populus
stakes in our present study (Figures 2 and 4). These patterns may be due to that
poor soil conditions on the forest surface, and lower-quality pine stakes limit fungal
:
growth rates, which may prevent species with fast colonization and growth traits
from dominating substrates, and result in greater species coexistence with slower-
growing fungal species (Maynard et al., 2017).

4.2.2 Fungal composition and abundance

Wood-decaying fungi are divided into three main categories (brown-rot, white-rot,
and soft-rot fungi) depending on their enzyme capabilities to degrade lignocellulose
substrates. Soft-rot fungi (mostly Ascomycete) can degrade polysaccharides in the
surface layers of plants, brown-rot fungi (Basidiomycetes) mainly degrade cellulose
and hemicellulose and slightly modify lignin (Andlar et al., 2018; Sanchez, 2009;
Viotti et al., 2021), and white-rot fungi (especially basidiomycetes) are able to
decompose all the major polymers, for example, lignin, cellulose, and hemicellulose
(Tuor et al., 1995). Although soil temperature and humidity are thought to be the
main factors a"ecting saprotrophic fungi in speci#c forests, stakes placed in the
mineral soil did not always have a higher fungal abundance at the genus level
(Figure S4), suggesting other factors (e.g., oxygen, airborne spores, drought
tolerance of fungi) also serve as important control of fungal diversity (Jiang et
al., 2014; Li et al., 2022).

Soil manure amendment changed the abundance of the top 15 genera, which may
relate to the altered wood stake moisture content in the third year of
decomposition (F = 5.75, p < 0.01, not published data), or the improved
microenvironment of microorganisms (Zhang et al., 2022). But the di"erent
response directions of the two stake positions (positive for surface stakes, but
negative for mineral stakes; Figures 5 and 6) may have occurred because fungi
prioritized the utilization of labile manure C in the mineral soil rather than the more
complex wood substrate (Siedt et al., 2021). Biochar application, however,
decreased the abundance of the top 15 fungal genera in both surface and mineral
soil wood stakes (Figures 5 and 6), which may be due to the relatively high alkalinity
of the soil, biochar, and manure used in our study (pH = 8.2, 8.8, and 7.9
respectively), which was likely not conducive to the cellulose nonenzymatic
deconstruction process, and may have also disrupted lignocellulose degradation by
brown-rot fungi (Andlar et al., 2018).

Cellulose-degrading soft-rot fungi are believed to be the #rst decomposers of

woody debris, followed by brown- and white-rot fungi in the middle stages of the
wood decomposition (Rajala et al., 2015; Tarvainen et al., 2020). Furthermore,
Basidiomycete species are thought to dominate over Ascomycetes during the later
:
stages of decomposition (Duong et al., 2008) since they can synthesize the enzymes
required to degrade complex polymers such as lignin (Deacon, 1997; Kodsueb et
al., 2008). However, in our study, we found that Ascomycetes always dominated at
the two sampling dates (75.19% and 79.62%, respectively, after 2 and 3 years of
decomposition) with other factors combined, which may be because stakes were
still at an early stage of decomposition after 2 years of installation (average density
loss 27.30%), and the decay has not entered the later stages of decomposition,
even in the third year (density loss average 52.50%, unpublished data). In addition,
the most abundant 15 genera achieved their greatest abundance in mineral stakes
decomposed for 2 years (sampled in July 2020), as compared to 3 years after
installation (sampled in July 2021), which may relate to the low water content
(average 33.93%, unpublished data), or that the remaining C was in the form of
lignin, which is more recalcitrant (Fukasawa et al., 2009; Kurth et al., 2013).

5 CONCLUSIONS
Using standard decomposition substrates (i.e., wood stakes) as an index of soil
functioning, our study indicates that biochar alone or combined with manure
increased the density loss and moisture content of stakes both on the soil surface
and in the mineral soil. In addition, the application of biochar and/or manure
increased the OTU abundances in wood stakes, especially in the mineral soil. But
OTU richness and diversity were more strongly regulated by wood stake position,
wood species, and sample dates. We proposed that 10 Mg ha−1 biochar should be
taken into account by forest management operations aimed at enhancing or
sustaining soil quality in plantations with similar soil conditions, and additional
bene#ts can be achieved with the combined addition of 9 Mg ha−1 manure.
Furthermore, the degradation of standard substrates can provide scienti#c data
about soil process changes both on the soil surface and belowground, which can be
used to develop better forestry policy.

AUTHOR CONTRIBUTIONS
Ruirui Zhao was involved in methodology, data curation, visualization, validation,
formal analysis, and writing—original draft. Yong Liu was involved in supervision
and funding acquisition. Michael J. Gundale was involved in writing review and
editing.
:
ACKNOWLEDGMENTS
The authors cordially thank Kai Wang at Beijing Forestry University for his
assistance in the #eld study. Ruirui Zhao was supported by the China Scholarship
Council (202206510017) as a visiting PhD student in Swedish University of
Agricultural Sciences, Department of Forest Ecology and Management, Forest
Vegetation Ecology group.

FUNDING INFORMATION
The study was funded by the National Key Research and Development Program of
China (2016YFD0600403).

CONFLICT OF INTEREST STATEMENT

We declare that we authors have no con%ict of interest.

Open Research 


DATA AVAILABILITY STATEMENT

The data are available from the corresponding author on reasonable request.

Supporting Information 


Filename Description

gcbb13087-sup-0001-DataS1.docxWord 2007 document , 11.7 MB Data S1.

Please note: The publisher is not responsible for the content or functionality of any
supporting information supplied by the authors. Any queries (other than missing
content) should be directed to the corresponding author for the article.
:
 REFERENCES 


Adams, M. B., Jurgensen, M., Palik, B., Miller, C., & Page-Dumroese, D. S. (2021). Wood
stake decomposition twenty years after organic matter removal at the Lake States LTSP
sites. Forest Ecology and Management, 496, 119456.
https://doi.org/10.1016/j.foreco.2021.119456

Anasonye, F., Tammeorg, P., Parshintsev, J., Riekkola, M.-L., & Tuomela, M. (2018). Role
of biochar and fungi on PAH sorption to soil rich in organic matter. Water, Air, & Soil
Pollution, 229(2), 1–14. https://doi.org/10.1007/s11270-018-3708-2

Andlar, M., Rezic, T., Mardetko, N., Kracher, D., Ludwig, R., & Šantek, B. (2018).
Lignocellulose degradation: An overview of fungi and fungal enzymes involved in
lignocellulose degradation. Engineering in Life Sciences, 18(11), 768–778.
https://doi.org/10.1002/elsc.201800039

Augarten, A. J., Malone, L. C., Richardson, G. S., Jackson, R. D., Wattiaux, M. A., Conley, S.
P., Radatz, A. M., Cooley, E. T., & Ruark, M. D. (2023). Cropping systems with perennial
vegetation and livestock integration promote soil health. Agricultural & Environmental
Letters, 8(1), e20100. https://doi.org/10.1002/ael2.20100

Bahram, M., Hildebrand, F., Forslund, S. K., Anderson, J. L., Soudzilovskaia, N. A.,
Bodegom, P. M., Bengtsson-Palme, J., Anslan, S., Coelho, L. P., Harend, H., Huerta-Cepas,
J., Medema, M. H., Maltz, M. R., Mundra, S., Olsson, P. A., Pent, M., Põlme, S., Sunagawa,
S., Ryberg, M., … Bork, P. (2018). Structure and function of the global topsoil
microbiome. Nature, 560(7717), 233–237. https://doi.org/10.1038/s41586-018-0386-6

Bhattacharyya, S. S., Ros, G. H., Furtak, K., Iqbal, H. M. N., & Parra-Saldívar, R. (2022). Soil
carbon sequestration – An interplay between soil microbial community and soil organic
matter dynamics. Science of the Total Environment, 815, 152928.
https://doi.org/10.1016/j.scitotenv.2022.152928
:
 Chagas, J. K. M., de Figueiredo, C. C., & Ramos, M. L. G. (2022). Biochar increases soil
carbon pools: Evidence from a global meta-analysis. Journal of Environmental
Management, 305, 114403. https://doi.org/10.1016/j.jenvman.2021.114403

Chen, S., Zhou, Y., Chen, Y., & Gu, J. (2018). fastp: An ultra-fast all-in-one FASTQ
preprocessor. Bioinformatics, 34(17), i884–i890. https://doi.org/10.1101/274100

Cline, L. C., Schilling, J. S., Menke, J., Groenhof, E., & Kennedy, P. G. (2017). Ecological and
functional e"ects of fungal endophytes on wood decomposition. Functional Ecology,
32(1), 181–191. https://doi.org/10.1111/1365-2435.12949

Coban, O., De Deyn, G. B., & van der Ploeg, M. (2022). Soil microbiota as game-changers
in restoration of degraded lands. Science, 375(6584), abe0725.
https://doi.org/10.1126/science.abe0725

Deacon, J. W. (1997). Modern mycology ( 3rd ed.). Blackwell Science.

Duong, L., McKenzie, E., Hyde, K., & Lumyong, S. (2008). Fungal succession on senescent
leaves of Castanopsis diversifolia in Doi Suthep-Pui National Park, Thailand. Fungal
Diversity, 30, 23–36.

Edgar, R. (2013). UPARSE: Highly accurate OTU sequences from microbial amplicon
reads. Nature Methods, 10(10), 996–998. https://doi.org/10.1038/nmeth.2604

Feng, Z., Leung, L. R., Hagos, S., Houze, R. A., Burleyson, C. D., & Balaguru, K. (2016).
More frequent intense and long-lived storms dominate the springtime trend in central
US rainfall. Nature Communications, 7, 13429. https://doi.org/10.1038/ncomms13429
:
 Finér, L., Jurgensen, M., Palviainen, M., Piirainen, S., & Page-Dumroese, D. S. (2016). Does
clear-cut harvesting accelerate initial wood decomposition? A #ve-year study with
standard wood material. Forest Ecology and Management, 372, 10–18.
https://doi.org/10.1016/j.foreco.2016.03.060

Fukasawa, Y., Osono, T., & Takeda, H. (2009). Microfungus communities of Japanese
beech logs at di"erent stages of decay in a cool temperate deciduous forest. Canadian
Journal of Forest Research, 39(8), 1606–1614. https://doi.org/10.1139/x09-080

Gora, E. M., Lucas, J. M., & Yanoviak, S. P. (2019). Microbial composition and wood
decomposition rates vary with microclimate from the ground to the canopy in a tropical
forest. Ecosystems, 22(6), 1206–1219. https://doi.org/10.1007/s10021-019-00359-9

Gundale, M. J., Nilsson, M.-C., Pluchon, N., & Wardle, D. A. (2016). The e"ect of biochar
management on soil and plant community properties in a boreal forest. GCB Bioenergy,
8(4), 777–789. https://doi.org/10.1111/gcbb.12274

Hagenbo, A., Anton-Fernandez, C., Bright, R. M., Rasse, D., & Astrup, R. (2022). Climate
change mitigation potential of biochar from forestry residues under boreal condition.
Science of the Total Environment, 807(Pt 3), 151044.
https://doi.org/10.1016/j.scitotenv.2021.151044

Han, Z. Q., Xu, P. S., Li, Z. T., Lin, H. Y., Zhu, C., Wang, J. Y., & Zou, J. W. (2022). Microbial
diversity and the abundance of keystone species drive the response of soil
multifunctionality to organic substitution and biochar amendment in a tea plantation.
GCB Bioenergy, 14(4), 481–495. https://doi.org/10.1111/gcbb.12926

Jaroszewicz, B., Cholewińska, O., Chećko, E., & Wrzosek, M. (2021). Predictors of diversity
of deadwood-dwelling macrofungi in a European natural forest. Forest Ecology and
Management, 490, 119123. https://doi.org/10.1016/j.foreco.2021.119123
:
 Jastrow, J. D., Amonette, J. E., & Bailey, V. L. (2007). Mechanisms controlling soil carbon
turnover and their potential application for enhancing carbon sequestration. Climatic
Change, 80(1–2), 5–23. https://doi.org/10.1007/s10584-006-9178-3

Jiang, M. K., Medlyn, B. E., Drake, J. E., Duursma, R. A., Anderson, I. C., Barton, C. V. M.,
Boer, M. M., Carrillo, Y., Castañeda-Gómez, L., Collins, L., Crous, K. Y., De Kauwe, M. G.,
Dos Santos, B. M., Emmerson, K. M., Facey, S. L., Gherlenda, A. N., Gimeno, T. E.,
Hasegawa, S., Johnson, S. N., … Ellsworth, D. S. (2020). The fate of carbon in a mature
forest under carbon dioxide enrichment. Nature, 580(7802), 227–231.
https://doi.org/10.1038/s41586-020-2128-9

Jiang, X. Y., Cao, L. X., Zhang, R. D., Yan, L. J., Mao, Y., & Yang, Y. W. (2014). E"ects of
nitrogen addition and litter properties on litter decomposition and enzyme activities of
individual fungi. Applied Soil Ecology, 80, 108–115.
https://doi.org/10.1016/j.apsoil.2014.04.002

Jin, L., Jin, N., Wang, S. Y., Li, J. W., Meng, X., Xie, Y. D., & Yu, J. H. (2022). Changes in the
microbial structure of the root soil and the yield of Chinese Baby Cabbage by chemical
fertilizer reduction with bio-organic fertilizer application. Microbiology Spectrum, 10(6), 1–
14. https://doi.org/10.1128/spectrum.01215-22

Joseph, S., Cowie, A. L., Van Zwieten, L., Bolan, N., Budai, A., Buss, W., & Lehmann, J.
(2021). How biochar works, and when it doesn't: A review of mechanisms controlling soil
and plant responses to biochar. GCB Bioenergy, 13(11), 1731–1764.
https://doi.org/10.1111/gcbb.12885

Jurgensen, M., Reed, D., Page-Dumroese, D. S., Laks, P., Collins, A., Mroz, G., & Degórski,
M. (2006). Wood strength loss as a measure of decomposition in northern forest
mineral soil. European Journal of Soil Biology, 42(1), 23–31.
https://doi.org/10.1016/j.ejsobi.2005.09.001
:
 Kodsueb, R., McKenzie, E. H. C., Lumyong, S., & Hyde, K. D. (2008). Fungal succession on
woody litter of Magnolia liliifera (Magnoliaceae). Fungal Diversity, 30, 55–72.

Krupek, F. S., Redfearn, D., Eskridge, K. M., & Basche, A. (2022). Ecological intensi#cation
with soil health practices demonstrates positive impacts on multiple soil properties: A
large-scale farmer-led experiment. Geoderma, 409, 115594.
https://doi.org/10.1016/j.geoderma.2021.115594

Kurth, V. J., Fransioli, N., Fulé, P. Z., Hart, S. C., & Gehring, C. A. (2013). Stand-replacing
wild#res alter the community structure of wood-inhabiting fungi in southwestern
ponderosa pine forests of the USA. Fungal Ecology, 6(3), 192–204.
https://doi.org/10.1016/j.funeco.2013.01.006

Kuznetsova, A., Brockho", P. B., & Christensen, R. H. B. (2017). lmerTest package: Tests
in linear mixed e"ects models. Journal of Statistical Software, 82(13), 1–26.
https://doi.org/10.18637/jss.v082.i13

Lehmann, J., Czimczik, C., Laird, D., & Sohi, S. (2012). Stability of biochar in soils. In J.
Lehmann & S. Joseph (Eds.), Biochar for environmental management: Science, technology
and implementation (pp. 215–238). Routledge.

Lenth, R. V., Buerkner, P., Herve, M., Love, J., Miguez, F., Riebl, H., & Singmann, H. (2022).
emmeans: Estimated marginal means, aka least-squares means (version 1.7.4-1). R package
version 1.7.5. https://github.com/rvlenth/emmeans

Li, L., He, L. X., Li, Y. Q., Wang, Y. F., Ashraf, U., Hamoud, Y. A., & Pan, S. G. (2023). Deep
fertilization combined with straw incorporation improved rice lodging resistance and
soil properties of paddy #elds. European Journal of Agronomy, 142, 126659.
https://doi.org/10.1016/j.eja.2022.126659
:
 Li, T., Cui, L. Z., Song, X. F., Cui, X. Y., Wei, Y. L., Tang, L., & Xu, Z. H. (2022). Wood decay
fungi: An analysis of worldwide research. Journal of Soils and Sediments, 22(6), 1688–
1702. https://doi.org/10.1007/s11368-022-03225-9

Liu, J., Schulz, H., Brandl, S., Miehtke, H., Huwe, B., & Glaser, B. (2012). Short-term e"ect
of biochar and compost on soil fertility and water status of a Dystric Cambisol in NE
Germany under #eld conditions. Journal of Plant Nutrition and Soil Science, 175, 698–707.
https://doi.org/10.1002/jpln.201100172

Magoc, T., & Salzberg, S. L. (2011). FLASH: Fast length adjustment of short reads to
improve genome assemblies. Bioinformatics, 27(21), 2957–2963.
https://doi.org/10.1093/bioinformatics/btr507

Maynard, D. S., Bradford, M. A., Lindner, D. L., van Diepen, L. T. A., Frey, S. D., Glaeser, J.
A., & Crowther, T. W. (2017). Diversity begets diversity in competition for space. Nature
Ecology & Evolution, 1(6), 156. https://doi.org/10.1038/s41559-017-0156

Middleton, T. E., McCombs, A. L., Gailans, S. R., Carlson, S., Karlen, D. L., Moore, K. J., &
McDaniel, M. D. (2021). Assessing biological soil health through decomposition of
inexpensive household items. Applied Soil Ecology, 168(2), 104699.
https://doi.org/10.1016/j.apsoil.2021.104099

Minamino, Y., Fujitake, N., Suzuki, T., Yoshitake, S., Koizumi, H., & Tomotsune, M. (2019).
E"ect of biochar addition on leaf-litter decomposition at soil surface during three years
in a warm-temperate secondary deciduous forest, Japan. Scienti!c Reports, 9(1), 16961.
https://doi.org/10.1038/s41598-019-53615-2

Mishra, A., Humpenöder, F., Churkina, G., Reyer, C. P. O., Beier, F., Bodirsky, B. L., &
Popp, A. (2022). Land use change and carbon emissions of a transformation to timber
:
 cities. Nature Communications, 13(1), 4889. https://doi.org/10.1038/s41467-022-32244-w

Newbold, T., Hudson, L. N., Hill, S. L. L., Contu, S., Lysenko, I., Senior, R. A., & Purvis, A.
(2015). Global e"ects of land use on local terrestrial biodiversity. Nature, 520(7545), 45–
50. https://doi.org/10.1038/nature14324

Nottingham, A. T., Meir, P., Velasquez, E., & Turner, B. L. (2020). Soil carbon loss by
experimental warming in a tropical forest. Nature, 584(7820), 234–237.
https://doi.org/10.1038/s41586-020-2566-4

Page-Dumroese, D. S., Jurgensen, M. F., Miller, C. A., Busse, M. D., Curran, M. P., Terry, T.
A., & Murray, M. (2021). Decomposition of wood stakes in the Paci#c Northwest after
soil compaction and organic matter removal. Forest Ecology and Management, 494,
119362. https://doi.org/10.1016/j.foreco.2021.119362

Page-Dumroese, D. S., Sanchez, F. G., Udawatta, R. P., Perry, C. H., & González, G. (2021).
Soil health assessment of forest soils. In D. Karlen, D. E. Stott, & M. M. Mikha (Eds.), Soil
health series (Vol. 1, pp. 100–138). John Wiley & Sons, Incorporation.

Piaszczyk, W., Lasota, J., Błońska, E., & Foremnik, K. (2022). How habitat moisture
condition a"ects the decomposition of #ne woody debris from di"erent species. Catena,
208, 105765. https://doi.org/10.1016/j.catena.2021.105765

Pingree, M. R. A., Kardol, P., Nilsson, M. C., Wardle, D. A., Maarou#, N. I., & Gundale, M. J.
(2022). No evidence that conifer biochar impacts soil functioning by serving as microbial
refugia in boreal soils. GCB Bioenergy, 14(8), 972–988.
https://doi.org/10.1111/gcbb.12978

Prayogo, C., Jones, J. E., Baeyens, J., & Bending, G. D. (2013). Impact of biochar on
mineralisation of C and N from soil and willow litter and its relationship with microbial
:
 community biomass and structure. Biology and Fertility of Soils, 50(4), 695–702.
https://doi.org/10.1007/s00374-013-0884-5

R Core Team. (2021). R: A language and environment for statistical computing (version
4.1.1). R Core Team. http://www.R-project.org/

Rajala, T., Tuomivirta, T., Pennanen, T., & Mäkipää, R. (2015). Habitat models of wood-
inhabiting fungi along a decay gradient of Norway spruce logs. Fungal Ecology, 18, 48–55.
https://doi.org/10.1016/j.funeco.2015.08.007

Razzaghi, F., Obour, P. B., & Arthur, E. (2020). Does biochar improve soil water
retention? A systematic review and meta-analysis. Geoderma, 361, 114055.
https://doi.org/10.1016/j.geoderma.2019.114055

Reardon, S. (2014). Manure fertilizer increases antibiotic resistance. Nature.

https://doi.org/10.1038/nature.2014.16081

Risch, A. C., Page-Dumroese, D. S., Schweiger, A. K., Beattie, J. R., Curran, M. P., Finér, L.,
& Jurgensen, M. F. (2022). Controls of initial wood decomposition on and in forest soils
using standard material. Frontiers in Forests and Global Change, 5, 829810.
https://doi.org/10.3389/"gc.2022.829810

Sanchez, C. (2009). Lignocellulosic residues: Biodegradation and bioconversion by fungi.

Biotechnology Advances, 27(2), 185–194.
https://doi.org/10.1016/j.biotechadv.2008.11.001

Seyedsadr, S., Sipek, V., Jacka, L., Snehota, M., Beesley, L., Pohorely, M., & Trakal, L.
(2022). Biochar considerably increases the easily available water and nutrient content in
low-organic soils amended with compost and manure. Chemosphere, 293, 133586.
https://doi.org/10.1016/j.chemosphere.2022.133586
:
 Shorohova, E., Kapitsa, E., & Vanha-Majamaa, I. (2008). Decomposition of stumps in a
chronosequence after clear-felling vs. clear-felling with prescribed burning in a southern
boreal forest in Finland. Forest Ecology and Management, 255(10), 3606–3612.
https://doi.org/10.1016/j.foreco.2008.02.042

Siedt, M., Schä"er, A., Smith, K. E. C., Nabel, M., Roß-Nickoll, M., & van-Dongen, J. T.
(2021). Comparing straw, compost, and biochar regarding their suitability as agricultural
soil amendments to a"ect soil structure, nutrient leaching, microbial communities, and
the fate of pesticides. Science of the Total Environment, 751, 141607.
https://doi.org/10.1016/j.scitotenv.2020.141607

Somerville, P. D., Farrell, C., May, P. B., & Livesley, S. J. (2020). Biochar and compost
equally improve urban soil physical and biological properties and tree growth, with no
added bene#t in combination. Science of the Total Environment, 706, 135736.
https://doi.org/10.1016/j.scitotenv.2019.135736

Stackebrandt, E., & Goebel, B. M. (1994). Taxonomic note: A place for DNA-DNA
reassociation and 16S rRNA sequence analysis in the present species de#nition in
bacteriology. International Journal of Systematic Bacteriology, 44(4), 846–849.
https://doi.org/10.1099/00207713-44-4-846

Tarin, M. W. K., Fan, L. L., Xie, D. J., Tayyab, M., Rong, J. D., Chen, L. Y., & Zheng, Y. S.
(2021). Response of soil fungal diversity and community composition to varying levels of
bamboo biochar in red soils. Microorganisms, 9(7), 1385.
https://doi.org/10.3390/microorganisms9071385

Tarvainen, O., Saravesi, K., Pennanen, T., Markkola, A.-M., Suokas, M., & Fritze, H. (2020).
Fungal communities in decomposing wood along an energy wood harvest gradient.
Forest Ecology and Management, 465, 118070.
https://doi.org/10.1016/j.foreco.2020.118070
:
 Tie, L. H., Hu, J. X., Peñuelas, J., Sardans, J., Wei, S. Z., Liu, X., & Huang, C. D. (2022). The
amounts and ratio of nitrogen and phosphorus addition drive the rate of litter
decomposition in a subtropical forest. Science of the Total Environment, 833, 155163.
https://doi.org/10.1016/j.scitotenv.2022.155163

Tuor, U., Winterhalter, K., & Fiechter, A. (1995). Enzymes of white-rot fungi involved in
lignin degradation and ecological determinants for wood decay. Journal of Biotechnology,
41, 1–17. https://doi.org/10.1016/0168-1656(95)00042-O

van der Wal, A., Geydan, T. D., Kuyper, T. W., & de Boer, W. (2013). A thready a"air:
Linking fungal diversity and community dynamics to terrestrial decomposition
processes. FEMS Microbiology Reviews, 37(4), 477–494.
https://doi.org/10.1111/1574-6976.12001

Viotti, C., Bach, C., Maillard, F., Ziegler-Devin, I., Mieszkin, S., & Buee, M. (2021). Sapwood
and heartwood a"ect di"erentially bacterial and fungal community structure and
successional dynamics during Quercus petraea decomposition. Environmental
Microbiology, 23(10), 6177–6193. https://doi.org/10.1111/1462-2920.15522

Wang, Q., Garrity, G. M., Tiedje, J. M., & Cole, J. R. (2007). Naive Bayesian classi#er for
rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and
Environmental Microbiology, 73(16), 5261–5267. https://doi.org/10.1128/AEM.00062-07

Wang, S. L., Zhang, W. D., & Sanchez, F. (2010). Relating net primary productivity to soil
organic matter decomposition rates in pure and mixed Chinese #r plantations. Plant
and Soil, 334, 501–510. https://doi.org/10.1007/s11104-010-0400-8

Wang, W. W., Lindner, D. L., Jusino, M. A., Page-Dumroese, D. S., Palmer, J. M., Banik, M.
T., Jurgensen, M., Kymberly, D., & Liu, Y. (2020). Wood-colonizing fungal community
:
 response to forest restoration thinnings in a Pinus tabuliformis plantation in northern
China. Forest Ecology and Management, 476, 118459.
https://doi.org/10.1016/j.foreco.2020.118459

Wang, W. W., Page-Dumroese, D. S., Jurgensen, M., Miller, C., Walitalo, J., Chen, X., & Liu,
Y. (2019). Restoration thinning impacts surface and belowground wood decomposition.
Forest Ecology and Management, 449, 117451.
https://doi.org/10.1016/j.foreco.2019.117451

Whalen, E. D., Grandy, A. S., Sokol, N. W., Keiluweit, M., Ernakovich, J., Smith, R. G., &
Frey, S. D. (2022). Clarifying the evidence for microbial- and plant-derived soil organic
matter, and the path toward a more quantitative understanding. Global Change Biology,
28(4), 7167–7185. https://doi.org/10.1111/gcb.16413

Williams, A., Hunter, M. C., Kammerer, M., Kane, D. A., Jordan, N. R., Mortensen, D. A., &
Davis, A. S. (2016). Soil water holding capacity mitigates downside risk and volatility in
US rainfed maize: Time to invest in soil organic matter? PLoS One, 11(8), e0160974.
https://doi.org/10.1371/journal.pone.0160974

Williams, G. B. (1942). Manufacture of fertilizer from urban wastes. Nature, 150(3801),

299. https://doi.org/10.1038/150299a0

Xu, W. H., Whitman, W. B., Gundale, M. J., Chien, C. C., & Chiu, C. Y. (2020). Functional
response of the soil microbial community to biochar applications. GCB Bioenergy, 13(1),
269–281. https://doi.org/10.1111/gcbb.12773

Yang, Q., Zhou, H. W., Bartocci, P., Fantozzi, F., Mašek, O., Agblevor, F. A., & McElroy, M.
B. (2021). Prospective contributions of biomass pyrolysis to China's 2050 carbon
reduction and renewable energy goals. Nature Communications, 12(1), 1–12.
https://doi.org/10.1038/s41467-021-21868-z
:
 Yang, Y. H., Li, M. J., Wu, J. C., Pan, X. Y., Gao, C. M., & Tang, D. W. S. (2022). Impact of
combining long-term subsoiling and organic fertilizer on soil microbial biomass carbon
and nitrogen, soil enzyme activity, and water use of winter wheat. Frontiers in Plant
Science, 12, 788651. https://doi.org/10.3389/fpls.2021.788651

Yu, Z., Ciais, P., Piao, S. L., Houghton, R. A., Lu, C. Q., Tian, H. Q., & Zhou, G. Y. (2022).
Forest expansion dominates China's land carbon sink since 1980. Nature
Communications, 13(1), 5374. https://doi.org/10.1038/s41467-022-32961-2

Yu, Z. Y., Ling, L., Singh, B. P., Luo, Y., & Xu, J. M. (2020). Gain in carbon: Deciphering the
abiotic and biotic mechanisms of biochar-induced negative priming e"ects in
contrasting soils. Science of the Total Environment, 746, 141057.
https://doi.org/10.1016/j.scitotenv.2020.141057

Zhang, M. Y., Zhang, L., Riaz, M., Xia, H., & Jiang, C. C. (2021). Biochar amendment
improved fruit quality and soil properties and microbial communities at di"erent
depths in citrus production. Journal of Cleaner Production, 292, 126062.
https://doi.org/10.1016/j.jclepro.2021.126062

Zhang, X., Zhang, Q. Q., Zhan, L. P., Xu, X. T., Bi, R. Y., & Xiong, Z. Q. (2022). Biochar
addition stabilized soil carbon sequestration by reducing temperature sensitivity of
mineralization and altering the microbial community in a greenhouse vegetable #eld.
Journal of Environmental Management, 313, 114972.
https://doi.org/10.1016/j.jenvman.2022.114972

Zhao, R. R., Liu, Y., Page-Dumroese, D. S., Dumroese, R. K., & Wang, K. (2022). Enhancing
soil quality of short rotation forest operations using biochar and manure. Forests,
13(12), 2090. https://doi.org/10.3390/f13122090

Zheng, T. H., Zhang, J., Tang, C. J., Liao, K. T., & Guo, L. P. (2021). Positive and negative
priming e"ects in an Ultisol in relation to aggregate size class and biochar level. Soil &
:
 Tillage Research, 208, 104874. https://doi.org/10.1016/j.still.2020.104874

Citing Literature 


Download PDF

ABOUT WILEY ONLINE LIBRARY

Privacy Policy
Terms of Use
About Cookies
Manage Cookies
Accessibility
Wiley Research DE&I Statement and Publishing Policies
Developing World Access

HELP & SUPPORT

Contact Us
Training and Support
DMCA & Reporting Piracy

OPPORTUNITIES

Subscription Agents
Advertisers & Corporate Partners

CONNECT WITH WILEY

The Wiley Network

Wiley Press Room

Copyright © 1999-2023 John Wiley & Sons, Inc. All rights reserved
:
: