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VOLUME 21 NUMBER 10 OCTOBER 2019 www.env-micro.com ISSN 1462-2912
environmental
microbiology
Unravelling the sulfur cycle of marine sediments
In search of microbial indicator taxa
Source and fate of H2 in Yellowstone hot springs
Geological gas-storage shapes deep life
Multi-omic analyses of exogenous nutrient bag
decomposition by the black morel Morchella importuna
reveal sustained carbon acquisition and transferring
Hao Tan ,
1,2
*Annegret Kohler,
3
Renyun Miao,
1,2
Tianhai Liu,
1,2
Qiang Zhang,
1,2
Bo Zhang,
1,2
Lin Jiang,
1,2
Yong Wang,
1,2
Liyuan Xie,
1,2
Jie Tang,
1,2
Xiaolin Li,
1,2
Lixu Liu,
1,2
Igor V. Grigoriev,
4,5
Chris Daum,
4,5
Kurt LaButti,
4,5
Anna Lipzen,
4,5
Alan Kuo,
4,5
Emmanuelle Morin,
3
Elodie Drula,
6,7,8
Bernard Henrissat,
6,7,8
Bo Wang,
1,2
Zhongqian Huang,
1,2
Bingcheng Gan,
1,2
Weihong Peng
1,2
and Francis M. Martin
3
*
1
National-Local Joint Engineering Laboratory of Breeding
and Cultivation of Edible and Medicinal Fungi, Mushroom
Research Center, Soil and Fertilizer Institute, Sichuan
Academy of Agricultural Sciences, Chengdu, China.
2
Scientific Observing and Experimental Station of Agro-
Microbial Resource and Utilization in Southwest China,
Ministry of Agriculture, Chengdu, China.
3
Université de Lorraine, Institut National de la Recherche
Agronomique, UMR Interactions
Arbres/Microorganismes, Centre INRA-GrandEst,
Champenoux, 54280, France.
4
US Department of Energy Joint Genome Institute,
Walnut Creek, CA, USA.
5
Department of Plant and Microbial Biology, University
of California Berkeley, Berkeley, CA, USA.
6
Architecture et Fonction des Macromolécules
Biologiques, CNRS, Aix-Marseille University, Marseille,
F-13288, France.
7
Institut National de la Recherche Agronomique,
USC1408 Architecture et Fonction des Macromolécules
Biologiques, Marseille, F-13288, France.
8
Department of Biological Sciences, King Abdulaziz
University, Jeddah, 21589, Saudi Arabia.
Summary
The black morel (Morchella importuna Kuo, O’Donnell
and Volk) was once an uncultivable wild mushroom,
until the development of exogenous nutrient bag
(ENB), making its agricultural production quite feasible
and stable. To date, how the nutritional acquisition of
the morel mycelium is fulfilled to trigger its fruiting
remains unknown. To investigate the mechanisms
involved in ENB decomposition, the genome of a culti-
vable morel strain (M.importuna SCYDJ1-A1) was
sequenced and the genes coding for the decay appara-
tus were identified. Expression of the encoded
carbohydrate-active enzymes (CAZymes) was then ana-
lyzed by metatranscriptomics and metaproteomics in
combination with biochemical assays. The results show
that a diverse set of hydrolytic and redox CAZymes
secreted by the morel mycelium is the main force driv-
ing the substrate decomposition. Plant polysaccharides
such as starch and cellulose present in ENB substrate
(wheat grains plus rice husks) were rapidly degraded,
whereas triglycerides were accumulated initially and
consumed later. ENB decomposition led to a rapid
increase in the organic carbon content in the surface
soil of the mushroom bed, which was thereafter con-
sumed during morel fruiting. In contrast to the high car-
bon consumption, no significant acquisition of nitrogen
was observed. Our findings contribute to an increas-
ingly detailed portrait of molecular features triggering
morel fruiting.
Introduction
Species in the fungal genus Morchella, commonly known
as morels, are important gourmet mushrooms. Morels
possess diverse ecological types including saprotrophic,
pyrophilic and ectomycorrhizal, and the boundary of eco-
logical types can be vague (Pilz et al., 2004). Commercial
demand for morel in world market is constantly growing,
despite their high prize (Tietel and Masaphy, 2018). Due
to limited production of wild morels, attempts to cultivate
morels artificially started over 130 years ago (Roze,
1882). Ascocarps (fruiting bodies) were once produced in
walk-in growth chambers (Ower et al., 1989), but further
development of this method ceased, as repeating Ower’s
success by others has proven difficult (Masaphy, 2010).
Although several kinds of morel cultivating techniques
Received 1 February, 2019; revised 12 July, 2019; accepted 13 July,
2019. *For correspondence. E-mail francis.martin@inra.fr; Tel. +33
383 39 40 80; Fax +33 383 39 40 69. E-mail h.tan@foxmail.com;
Tel. +86 28 84504867; Fax +86 28 84787971.
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.
Environmental Microbiology (2019) 21(10), 3909–3926 doi:10.1111/1462-2920.14741
emerged in the last two decades (Liu et al., 2018), morel
cultivation industry was boosted only after the breeding
of several black morel varieties with improved fruiting
yield and stability (Peng et al., 2016; Liu et al., 2018),
and more importantly, the development and widespread
application of an appropriate organic substrate contained
in the so-called exogenous nutrient bag (ENB), a special
type of culture substrate enriched in plant polysaccharides.
Thanks to the ENB technique, cultivation of black morel
expanded rapidly in China from 200 ha in 2011 to over
1200 ha in 2015 (Liu et al., 2016), which generated the
exportation of dried fruiting bodies from 180 to 900 tons
between 2011 and 2015 (Du et al., 2015). Despite its
widespread application, the decomposition mechanisms
taking place in ENB remain to be determined.
Use of ENB is the key technique that allowed large-
scale ascocarp formation from the Morchella elata clade
(O’Donnell et al., 2011). It was initially developed in 2000
as a prototype (Tan, 2016), improved later and evolved to
the present form. The most prevalent ENB formulation
today is a plastic bag filled with wheat grains plus rice
husks, and then autoclaved. After piercing or cutting its
bottom casing to allow colonization by morel mycelium
from the soil, ENBs are placed on the surface of soil inoc-
ulated with black morel, the so-called mushroom bed
(Fig. 1A and B). The mushroom bed is an outdoor soil
ecosystem containing natural microbial inhabitants, rather
than a quasi-sterile environment. The cultivation method
for black morel is unique, very different from the cultivation
of usual edible mushrooms such as Pleurotus ostreatus,
Lentinus edodes,Agaricus bisporus (Chang and Hayes,
2013) and Coprinus comatus (Stojkovi
cet al., 2013). For
unknown reasons, ENB is required for high-yield and sta-
ble fruiting of black morel. It is believed that ENB provides
key organic nutrients, including a sustained carbon
(C) source for morel mycelium and is considered as a spe-
cial type of mushroom culture substrate (Fig. 1C).
Wild morels are able to produce fruiting bodies on vari-
ous types of substrates, such as post-fire forest soils
(Larson et al., 2016), plant debris as well as living roots
(Pilz et al., 2004; 2007). In post-fire soils, wild morels are
unlikely to consume recent plant litter as primary C and
nitrogen (N) sources (Hobbie et al., 2016). Compared
with the contingent fruiting in the wild, ENB provides a
highly reproducible system which allows the black morel
to complete its life cycle in an artificial environment. It is
particularly helpful for studying physiological and bio-
chemical processes driving the fruiting of soil
saprotrophic mushrooms.
To investigate the mechanisms involved in ENB decom-
position, genome of Morchella importuna was sequenced
and genes coding for the decay apparatus were identified.
Expression of the encoded carbohydrate-active enzymes
(CAZymes) was then analyzed by metatranscriptomics
and metaproteomics in combination with bioassays.
Results
Genome features
The Illumina-sequenced haploid genome of M.importuna
SCYDJ1-A1, a cultivable strain from China, resulted in a
48.80 Mbp assembly, with an average read-depth cover-
age of 84×in 338 scaffolds (scaffold N50 = 27; Supporting
Information Table S1). By using the JGI Annotation Pipe-
line (Grigoriev et al., 2014), we identified 11 971 genes
(Supporting Information Table S2). The assembly size of
the haploid genome of an European wild strain, M.
importuna CCBAS932, was 48.21 Mbp (Supporting Infor-
mation Table S1), with a similar number of encoded genes
Fig. 1. A. ENB for morel cultivation.
B. Large-scale morel cultivation
showing ENB laying on the mush-
room bed in a greenhouse. C. The
method of cultivation of black morels
using ENB differs from the cultivation
methods used for other commercial
mushrooms, such as the oyster mush-
room (P.ostreatus),wherefruitingbod-
ies are produced directly from the bag
containing a lignocellulosic substrate.
Red arrow means flow of organic
nutrients. [Color figure can be viewed
at wileyonlinelibrary.com]
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
3910 H. Tan et al.
(11 600; Murat et al., 2018). The completeness of genome
assemblies of the two strains are similar (Supporting Infor-
mation Table S1). Pairwise synteny of scaffolds between
the two strains was estimated by the vista synteny tool
(Martin et al., 2004) available at JGI genome portals of
Morchella. Almost every scaffold from the genome of
strain SCYDJ1-A1 had a highly syntenic scaffold hit from
the strain CCBAS932 (Supporting Information Fig. S1).
The two strains share 9783 common genes as determined
by BlastP Best Reciprocal Hit analysis (Supporting Infor-
mation Table S3). Strains SCYDJ1-A1 and CCBAS932
possessed 9891 and 9873 core-genes, respectively,
whereas strain-specific genes were 2080 and 1727
(Supporting Information Table S2). Most strain-specific
proteins are small proteins with unknown functions or
no PFAM domains. The results indicate that the two
M.importuna genomes are highly syntenic, whereas their
gene repertoires are substantially divergent. Genome
sequences, gene models and annotations of the two
M.importuna strains are publicly available from the JGI
MycoCosm database (Grigoriev et al., 2014). Their func-
tional portraits (GO, KEGG and KOG) are very similar
(available online from their JGI genome portals). The geno-
mic information indicates that M.importuna SCYDJ1-A1
has the capacity to secrete a large repertoire of CAZymes,
including glycoside hydrolases (GH), glycosyl transferases
(GT), carbohydrate esterases (CE), polysaccharide lyases
(PL) and several auxiliary activity enzymes (AA). Most of
the CAZymes are plant cell wall degrading enzymes
predicted to possess decomposition capabilities for plant
polysaccharides such as cellulose, hemicellulose and pec-
tins. Together, the results indicate the potential of M.
importuna to degrade a large set of substrates found in
decaying plant debris. To obtain experimental evidence for
the hydrolytic capabilities of M.importuna SCYDJ1-A1
against plant polysaccharides, profiling of transcripts and
proteins (see below) was performed on ENB extracts after
15, 45 and 75 days of growth.
ENB affects morel yield
M.importuna SCYDJ1-A1 was cultivated in a pre-
homogenized soil (Supporting Information Table S4),
which was used as the mushroom bed in this study. During
the entire cultivation course, the temperatures inside ENB
fluctuated between 6C and 13C (Supporting Information
Fig. S2). ENB weight showed no significant change over
the first 15 days (days 0–15) after contact with the mush-
room bed [p-value = 0.727, by one-way analysis of vari-
ance (ANOVA)] but shrank progressively from day 15 to
day 75 (day 45 < day 15, p-value = 8.50 ×10
−6
;day
75 < day 45, p-value = 9.15 ×10
−5
, by one-way ANOVA;
Fig. 2A). About 34% of the ENB dry weight was consumed
during days 15–45, with an additional 23% during days
45–75, indicating a sustained consumption of organic nutri-
ents. Indeed, total C content per ENB decreased at a
slow rate during days 0–15 (p-value = 0.037, by one-
way ANOVA), then at a higher rate during days 15–45
(p-value = 6.29 ×10
−4
, by one-way ANOVA), before
reaching a plateau during days 45–75 (p-value = 0.110,
by one-way ANOVA) (Fig. 3A; Supporting Information
Table S5), suggesting that ENB substrate was con-
sumed majorly in the middle stage. In response to ENB
decomposition, total organic C in the surface soil
(Fig. 2B) was increased significantly during days 15–45
(p-value = 3.41 ×10
−8
, by one-way ANOVA) and days
45–75 (p-value = 9.19 ×10
−3
, by one-way ANOVA).
After that, morel fruiting consumed a lot of the accumu-
lated organic C, by comparing day 75 with the comple-
tion of fruiting body harvest (p-value = 1.09 ×10
−7
,by
one-way ANOVA). After harvest, organic C in the sur-
face soil was still higher than the initial level before
morel sowing (6.04 0.05 g kg
−1
)(p-value = 3.91 ×10
−4
,
by one-way ANOVA; Fig. 2B).
Total N in ENB increased significantly during days
0–15 (p-value = 0.023, by one-way ANOVA; Fig. 3A;
Supporting Information Table S5), likely a result of ENB
colonization by the morel mycelium and other microbes. In
response, a temporary fall of inorganic ammonium N in
the surface soil took place during days 0–15 (p-values
between 4.06 ×10
−4
and 5.68 ×10
−4
,byone-way
ANOVA). After 15 days, total N content in ENB decreased
slowly until returned to its initial level (day 75 similar to day
0, p-value = 0.645, by one-way ANOVA). It suggests that
N was not substantially exported from ENB to the
surface soil.
Total C consumption in ENB was much higher than
total N (Fig. 3A) (p-value = 1.41 ×10
−6
,byttest), which
is supported by strikingly high activities of amylases and
lipases detected in ENB (Fig. 3B). The imbalance
between C and N consumptions resulted in a continuous
decrease in C:N ratio, from 36.9 to 19.3 (Fig. 3A). Total P
and total K were both consumed continuously from day
0 to day 75 (p-value = 8.31 ×10
−5
and 1.66 ×10
−7
,
respectively, by one-way ANOVA).
The duration of ENB contact with the mushroom bed
influenced fruiting body yield profoundly (Fig. 2A). Without
ENB, no fruiting took place, confirming that the nutrients
released by decaying ENB substrate are required for
fruiting. Removing ENB at day 15 or day 45 stopped the
increase in soil organic C (day 45 similar to day
15, p-value = 0.999; day 75 < day 45, p-value = 0.001, by
one-way ANOVA) and also lowered the fruiting body yield
significantly (p-value = 3.73 ×10
−10
and 4.46 ×10
−6
,
respectively, by one-way ANOVA). It indicates that the
consumed organic compounds from ENB were transferred
to the underlying soil, while future experiments with nets to
avoid mycelium colonization would be helpful to confirm
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
Decomposition mechanisms triggering morel fruiting 3911
the translocation through mycelial networks. Keeping ENB
until all fruiting bodies were harvested, the yield showed
no significant difference with removing ENB at day 75 (p-
value = 0.660, by one-way ANOVA; Fig. 2A). It means that
keeping ENB on the mushroom bed for at least 75 days is
essential to get as high yield as possible. Moreover, the
duration of ENB contact with the mushroom bed also
influenced the contents of total N, total proteins and free
amino acids in morel fruiting bodies (Supporting Informa-
tion Fig. S3).
Carbohydrate decomposition
A diverse array of CAZymes encoded by M.importuna
were identified in decaying ENB and their expression
strikingly varied along the time-course of decomposition
(Fig. 4). Consequently, carbohydrates as the major frac-
tion in ENB were degraded and consumed rapidly. About
70% of total carbohydrates were lost in 75 days. Amylo-
pectin, amylose and cellulose, consumed in large quan-
tity (Fig. 3A), were the most prominent C source for
metabolism. Over 90% of amylose was metabolized dur-
ing days 0–75, whereas amylopectin was metabolized by
72%. The consumed proportion of amylose was higher
than amylopectin (p-value = 8.73 ×10
−5
,byttest),
although the content of amylopectin in ENB was nearly
twice higher than amylose. The high degrading rates of
amylopectin and amylose are supported by the high
γ-amylase activity (Fig. 3B). A GH15 protein identified in
ENB (Fig. 4) seemed responsible for the γ-amylase activ-
ity. Its upregulated expression [fold-change = 5.79 during
days 15–45, p-value = 5.56 ×10
−4
,byttest with false
discovery rate (FDR) correction for multiple testing] was
similar with the growing trend of γ-amylase activity
observed during days 15–75 (day 45 > day 15, p-
value = 0.002; day 75 > day 45, p-value = 1.84 ×10
−5
,
by FDR-corrected ttest; Fig. 3B). In comparison, the
other amylases involved in starch hydrolysis showed
much lower activities (Fig. 3B). The GH13 proteins were
annotated as starch-hydrolysis-related enzymes, includ-
ing two α-amylases, an α-glucosidase, a branching
enzyme and a debranching enzyme (Fig. 4). Like the
γ-amylase, the upregulated expression of GH13_1 and
GH13_m42 proteins (GH13_1: fold-change = 5.42 during
days 15–45, p-value = 0.001; GH13_m42: fold-
change = 5.06 during days 15–45 and 2.70 during days
45–75, p-value = 5.35 ×10
−4
and 1.16 ×10
−4
, respec-
tively, by FDR-corrected ttest) supports the observed
increase in α-amylase activity (day 45 > day 15, p-
value = 2.30 ×10
−3
; day 75 > day 45, p-value = 0.004,
by FDR-corrected ttest; Fig. 3B). As M.importuna
genome lacks β-amylase gene (GH14), the observed
No ENB Remove ENB at da
y
15 Remove ENB at da
y
45 Remove ENB at da
y
75
0
25
50
75
100
0154575Entire
cultivation
course
0
50
100
150
200
0154575
thgiewyrdgniniameR
)g(BNErep
ENB lasting time (day)
Fruiting body yield
(g dry weight m-2)
ENB lasting time (day)
Day after ENB placing
niCcinagrolatoT
gkg(liosecafrus -1)
Day after ENB placing
Total N in surface
soil (mg kg-1)
Day after ENB placing
Ammonium N in
surface soil (mg kg-1)
5
10
15
20
0154575After
harvest
750
1000
1250
1500
0154575After
harvest
50
100
150
200
0 154575After
harvest
A
B
Fig. 2. A. The ENB substrate was consumed while the yield of morel fruiting body increased simultaneously. B. Time-course changes in total
organic C, total N and ammonium N in the surface soil. The coloured columns show mean of three biological replicates, with standard deviation
bars. Significant difference in multi-group comparison of an item at the three time-points was judged by one-way ANOVA. Significant difference in
pairwise comparison of two items at the same time-point, or during the same period, was judged by ttest. A full list of all p-values is provided in
Supporting Information Table S8.
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
3912 H. Tan et al.
A
B
-
0
2000
4000
6000
8000
day0 day15 day45 day75
free maltose free cellobiose
free trehalose free glucose
free fructose free galactose
free mannose free xylose
free arabinose
Content (mg) per ENB
Content (mg) per ENB
0
50000
100000
150000
day0 day15 day45 day75
total carbohydrates
total reducing sugars
amylose
amylopectin
cellulose
hemicellulose
protopectins
water-soluble pectins
0
1000
2000
3000
4000
day0 day15 day45 day75
crude fats triglycerides
free fatty acids
BN
E
re
p
)gm( t
ne
tn
oC
0
2500
5000
7500
10000
day0 day15 day45 day75
lignin total H units
total S units total G units
0
10
20
30
40
day0 day15 day45 day75
free H units free S units
free G units ferulic acid
Content (mg) per ENB
Content (mg) per ENB
Content (mg) per ENB
0
15000
30000
45000
day0 day15 day45 day75
total proteins soluble proteins
free amino acids
0
1500
3000
4500
day0 day15 day45 day75
total N organic N
ammonium N nitrate N
total P mineral P
total K
Content (mg) per ENB
0
40000
80000
120000
day0 day15 day45 day75
total C total N
BNE rep )gm( tnetnoC
36.9:1 27.6:1 21.3:1 19.3:1
C:N ratio NPK elements polysaccharides di- and mono- saccharides
lipids lignin lign
in metabolites proteins and amino acids
0
20
40
60
80
100
120
140
α-amylase
β-amylase
γ-amylase
α-glycosidase
endo-β-1,4-glucanase
endo-β-1,3-glucanase
exo-cellobiohydrolase
β-glycosidase
endo-β-1,4-xylanase
exo-β-1,4-xylosidase
β-mannosidase
α-L-arabinofuranosidase
endo-chitinase
exo-chitinase
pectin lyase
polygalacturonase
pectin esterase
pectate lyase
laccase
Mn peroxidase
versatile peroxidase
glyoxal oxidase
lipase
◇
total protease
◆
phosphatase
◆
day15 day45 day75
nim gm
(
ytivitca emyz
n
E-1g-1)BNE
th
giew yrd
0.00
0.25
0.50
0.75
1.00
1.25
α-amylase
β-amylase
γ-amylase
α-glycosidase
endo-β-1,4-glucanase
endo-β-1,3-glucanase
exo-cellobiohydrolase
β-glycosidase
endo-β-1,4-xylanase
exo-β-1,4-xylosidase
β-mannosidase
α-L-arabinofuranosidase
endo-chitinase
exo-chitinase
pectin lyase
polygalacturonase
pectin esterase
pectate lyase
laccase
Mn peroxidase
versatile peroxidase
glyoxal oxidase
lipase
◇
total protease
◆
phosphatase
◆
day15 day45 day75
Enzyme activity (mg min-1 g-1 dry weight ENB)
Fig. 3. Legend on next page.
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
Decomposition mechanisms triggering morel fruiting 3913
β-amylase activity was likely produced by other microbes
colonizing ENB.
The activity of endo-cellulase (endo-β-1,4-glucanase)
increased slightly during days 15–45 (p-value = 0.612, by
one-way ANOVA) and significantly during days 45–75 (p-
value = 1.53 ×10
−5
, by one-way ANOVA). The activities of
exo-cellulase (exo-β-1,4-glucanase) and β-glycosidase were
much lower than endo-cellulase (p-values between
5.03 ×10
−7
and 9.90 ×10
−7
, by one-way ANOVA; Fig. 3B).
It suggests that cellulose in ENB might be shredded into
short chains more readily than further hydrolysis into
β-D-glucose, yet the highly active γ-amylase was able
to produce β-D-glucose from starch. Hemicellulose-
hydrolyzing enzymes displayed a pattern similar to
cellulose-hydrolyzing enzymes, in that the enzymes for
shredding hemicellulose into short chains were also
more active than those detaching the short chains into
free monosaccharide units.
During days 0–45, the content of protopectins decreased
(p-value = 1.39 ×10
−7
, by one-way ANOVA), whereas
water-soluble pectins increased (p-value = 2.38 ×10
−7
,by
one-way ANOVA), and the sum of the two was similar (p-
values between 0.133 and 0.770, by one-way ANOVA). It
suggests that protopectins were solubilized but not eventu-
ally consumed during this period. Protopectins and water-
soluble pectins both decreased greatly after 45 days
(p-value = 2.35 ×10
−4
and 1.31 ×10
−6
, respectively, by
one-way ANOVA), which means that a substantial catabo-
lism of pectins took place. Four proteins of M.importuna
were identified as pectin lyases, which appeared to partici-
pate in the observed pectin degradation.
Free disaccharides and monosaccharides except fruc-
tose accumulated over the first 45 days and then
decreased (Fig. 3A; Supporting Information Table S5).
The temporary accumulation might be due to very active
shredding of polysaccharide chains while further catabo-
lism was not fast enough to consume the intermediates.
Lipid degradation
Lipase activity was the second highest in ENB (Fig. 3B),
suggesting that grain fats can act as a potential source
of C. As lipases encoded by non-CAZy genes were
absent from the metaproteomic profiles, the lipase activ-
ity detected in ENB was likely contributed by the CE5
proteins (Martinez et al., 1994; Nakamura et al., 2017)
of M.importuna (Fig. 4). However, lipids were not a
major C source in ENB, due to their low content. Crude
fats and triglycerides both accumulated during days
15–45 (Fig. 3A; p-value = 2.86 ×10
−6
and 3.93 ×10
−6
,
respectively, by one-way ANOVA), indicating that lipids
were synthesized and stored in ENB temporarily. After
45 days, net consumptions of crude fats and triglycerides
were both significant (p-value = 1.40 ×10
−5
and
5.10 ×10
−4
, respectively, by one-way ANOVA), suggesting
that excess of C nutrients could be converted to lipid stock
and consumed later. Noticeably, the triglyceride amount per
ENB at day 75 was still higher than the start (day 75 > day
0, p-value = 0.001, by one-way ANOVA).
Lignin decomposition
Lignin decomposition took place rarely during days 0–15
(p-value = 0.963, by one-way ANOVA), slowly during
days 15–45 (p-value = 0.019, by one-way ANOVA) and
faster during days 45–75 (p-value = 4.98 ×10
−6
, by one-
way ANOVA; Fig. 3A). Over the 75 days, the ratios
among total p-hydroxyphenyl (H), total syringyl (S) and
total guaiacyl (G) units changed less than their free
monomers (Supporting Information Table S5). The ratio
of S:G (total units) decreased during days 0–45 and then
increased during days 45–75 (Supporting Information
Table S5). Free H and free G monomers as well as free
ferulic acid were rapidly consumed during days 0–15 (p-
value = 7.43 ×10
−12
, 1.28 ×10
−9
and 2.24 ×10
−12
,
respectively, by one-way ANOVA), unlike the significantly
accumulated free S monomer (p-value = 1.06 ×10
−10
,
by one-way ANOVA). During days 15–75, free S and free
G monomers both decreased (p-value = 4.76 ×10
−11
and 0.002, respectively, by one-way ANOVA)
(Supporting Information Table S5). The enzymes
involved in oxidative breakdown of lignin showed low
activities (Fig. 3B). Mn peroxidase (oxidizing Mn
2+
to Mn
3
+
) and versatile peroxidase (oxidizing veratryl alcohol)
had much lower activities than laccase (p-values
between 3.63 ×10
−6
and 8.56 ×10
−4
, by one-way
ANOVA).
Although the M.importuna SCYDJ1-A1 genome pos-
sesses several genes predicted as lignin-degrading
enzymes, only a laccase-like multicopper oxidase (LMCO)
Fig. 3. A. Content change of major chemicals in ENB at day 0, 15, 45 and 75. The values of the chemical contents are mean of three biological
replicates, with standard deviation bars. All the values, as well as pH and water content in ENB, are presented in the Supporting Information
Table S5. B. Enzymatic activities in ENB, measured at the pH and temperature of ENB at the sampling date. A subset of the figure with the verti-
cal axis zoomed in is shown to display low activity enzymes. Enzymatic activity which might be contributed by both CAZymes and non-CAZymes
is labelled with empty diamond, whereas the activity completely unrelated with CAZymes is labelled with solid diamond. The activity level is mean
of three biological replicates, with standard deviation bars. Significant difference in multi-group comparison of an item at the three time-points
was judged by one-way ANOVA. Significant difference in pairwise comparison of two items at the same time-point, or during the same period,
was judged by ttest. A full list of all p-values is provided in Supporting Information Table S8.
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
3914 H. Tan et al.
Color coding of expression patterns
(>: significantly up-regulated defined by fold-change > 2 plus p-value < 0.05; <: significantly down-regulated defined by fold-change < 0.5 plus p-value < 0.05; ≈: no significant up- or down- regulation)
,
Fig. 4. Major CAZymes of M.importuna SCYDJ1-A1 involved in ENB decomposition. A supplemental figure showing all 88 CAZymes
identified in ENB is provided in Fig. S5. Expression levels of transcripts and proteins were estimated by RNA-Seq and nanoLC-MS/MS
respectively. Steady-state transcript level (in RPKM) and protein relative abundance are the mean of three biological replicates. ND,
not detected. Functions of CAZymes were predicted according to their nearest analogues whose activities had been characterized in previ-
ous studies, as provided by the CAZy database. Fold-change in RPKM between time-points, together with p-value of pairwise comparison,
was calculated by the Baggerly’s proportion-based test (Baggerly et al., 2003) with a FDR correction for multiple testing (Benjamini
and Hochberg, 1995). Fold-change values of protein relative abundance between time-points, together with p-value of pairwise compari-
son, were calculated by ttest with FDR correction. Significant upregulation and downregulation were judged by fold-change > 2 and
fold-change < 0.5, respectively, whereas FDR-corrected p-value < 0.05. Fold-change values and p-values are provided in Supporting Infor-
mation Table S8. [Color figure can be viewed at wileyonlinelibrary.com]
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
Decomposition mechanisms triggering morel fruiting 3915
of AA1_3 family was detected in the metaproteomic pro-
files (Fig. 4), supporting the observed enzymatic activity.
The laccase activity of the AA1_3 LMCO protein has been
verified by biochemical characterization of the purified
enzyme (Zhang et al., 2019). The activity levels at
day 45 and day 75 were both higher than day 15 (p-
value = 6.52 ×10
−6
and 2.74 ×10
−7
, respectively, by
one-way ANOVA; Fig. 3B). This trend is consistent
with the significant lignin degradation taking place dur-
ing days 45–75 (p-value = 4.98 ×10
−6
, by one-way
ANOVA; Fig. 3A) and is also supported by increased
abundance of the AA1_3 LMCO protein in
ENB (Fig. 4).
N nutrition
Inorganic ammonium and nitrate represented a minor pro-
portion in the total N (Supporting Information Table S5),
most N being incorporated in organic compounds. The
amounts of soluble proteins and free amino acids at day
75 were all higher than the start (p-value = 2.59 ×10
−5
and 1.94 ×10
−7
, respectively, by one-way ANOVA). Total
proteins reached the highest content at day 15. Soluble
proteins showed a continuous increase during days 0–45
(days 0–15: p-value = 9.95 ×10
−4
;days15–45: p-
value = 0.001, by one-way ANOVA). Free amino acids
were initially consumed during days 0–15 (p-value = 0.001,
by one-way ANOVA) and then accumulated after 15 days
(day 45 > day 15, p-value = 6.84 ×10
−4
; day 75 > day
45, p-value = 2.59 ×10
−7
,byone-wayANOVA).These
results suggest that M.importuna mycelium colonizing
ENB, possibly together with other microbes, used some of
the free amino acids and borrowed some additional N from
the environment in the early period, which was likely used
to manufacture the large quantity of enzymes involved in
substrate decomposition. This contention was confirmed
by an elemental-tracing experiment with
15
N isotopic label-
ling of soil N, showing that ENB was indeed acquiring N
from the underneath soil over the first 15 days, and the
assimilated N was further enriched into the soluble pro-
teins in ENB (Supporting Information Fig. S4). In the late
period, lysis of dead microbial cells, as well as breakdown
of proteins, might release free amino acids as well as
ammonium into ENB substrate. Enzymes involved in N
scavenging from proteins and chitins showed low activities
in ENB (Fig. 3B), suggesting that degradation of proteins
and chitins seemed not very active.
Expression of CAZymes
ENB was colonized by M.importuna mycelium and a cor-
tege of environmental microorganisms. M.importuna,
together with 10 of the most abundant fungal genera (Mor-
tierella,Trichoderma,Monodictys,Peziza,Cladosporium,
Neonectria,Penicillium,Fusarium,Oliveonia and
Plectosphaerella), were defined as the major fungal taxa
in ENB. They represented 96.5% of the fungal community,
as determined by metabarcoding survey (see Changes in
the microbial community section).
Enzymes broadly characterized as hemicellulases and
pectinases (i.e., ß-xylosidases, endo-ß-1,6-glucanases,
polygalacturonases, pectin lyases and mannanases) were
among the most highly transcribed genes at day 15. Com-
plete breakdown of ENB substrate requires joint efforts
from multiple enzymes of GH, CE, PL and AA families.
At day 45, genes coding for α- and γ-amylases, GH13_8
branching enzyme and α-glucosidase/α-1,4-glucan lyase
were transcribed at a higher level. Cutinase/lipase, lytic
polysaccharide monooxygenases (LPMOs) and expansin-
related proteins showed a higher transcription level at the
later stage (Supporting Information Fig. S5).
A total of 1380 proteins belonging to M.importuna plus
the other 10 major fungal taxa in ENB were identified by
2D nanoLC-MS/MS, among which 60% (833) were from
M.importuna. The 833 proteins represented 7% of the
11 971 predicted genes in the morel genome. This set
included 88 CAZymes (24% of a total of 360 CAZy-genes
in the M.importuna SCYDJ1-A1 genome), and few were
encoded by other fungi (Supporting Information Table S6).
CAZymes expressed by M.importuna included 47 GH,
11 CE, 4 PL, 17 AA and 5 GT (Fig. 4). The diverse array of
CAZymes, including hydrolytic and redox enzymes, pointed
to the multiple pathways and degradative mechanisms
involved in ENB decomposition. During days 15–45,
168 out of the 833 morel proteins were upregulated,
whereas 5 were downregulated (Supporting Information
Fig. S6). Over a half of the 88 CAZy-proteins were
upregulated during this period, confirming a striking activa-
tion of the decay apparatus in ENB. Eighty eight out of the
833 morel proteins were downregulated during days 45–75.
It reflected the decline of M.importuna mycelium in ENB
during the late period, as evidenced by the fungal commu-
nity profiles (Fig. 5). However, an overwhelming majority
(86) of the 88 CAZy-proteins remained at a constant level
during days 45–75 (Supporting Information Fig. S6).
Changes in the microbial community
The bacterial and fungal communities in ENB at
15, 45 and 75 days were surveyed, respectively, through
metabarcoding of bacterial 16S ribosomal DNA (rDNA)
and fungal internal transcribed spacer (ITS). The bacterial
and fungal communities both showed a growing trend in
their taxonomic richness (i.e., the observed number of
operational taxonomic unit (OTU), ACE and Chao1) and
diversity (i.e., the Shannon–Wiener and Inverse Simpson’s
indices) during ENB decomposition (Table 1). Richness of
bacterial communities was higher than the fungal
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
3916 H. Tan et al.
Fig. 5. Legend on next page.
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
Decomposition mechanisms triggering morel fruiting 3917
communities at every time-point. Bacterial OTU richness
was similar at day 15 and day 45 but increased signifi-
cantly at day 75. The fungal OTU richness increased
mostly during days 15–45, suggesting that an increasing
number of fungal taxa colonized ENB at this stage.
PCA and hierarchical clustering showed that both bac-
terial and fungal communities were quite distinct between
different time-points (Fig. 5A and B). Community compo-
sition at genus (Fig. 5B) and phylum (Fig. 5C) levels were
both uneven in taxonomic abundance. Pseudomonas
was always the overwhelmingly major bacterial group. As
mentioned above, M.importuna and 10 other abundant
genera (belonging to 15 OTUs) represented 96.5% of the
fungal community in ENB. M.importuna was dominant at
day 15 and was greatly overturned by Trichoderma,Mor-
tierella and a few other taxa during days 45–75.
Discussion
Saprotrophic fungi can degrade soil polysaccharides
using a versatile arsenal of catabolic enzymes including
GH, CE, PL and AA, which are classified in the CAZy
database (http://www.cazy.org) (Lombard et al., 2014).
Compared with the genomes of other taxonomically
related Pezizomycetes (Fig. 6), the two strains of M.
importuna were characterized by an under-represented
set of CAZy-genes involved in lignin decomposition and
an over-represented set of CAZy-genes degrading pec-
tins. In comparison with commercially cultivated
Basidiomycota mushrooms L.edodes,P.ostreatus and
A.bisporus,M.importuna SCYDJ1-A1 genome encodes
over-represented sets of CAZy-genes involved in lipid
and pectin degradation and an under-represented set of
CAZy-genes involved in lignin decomposition (Supporting
Information Fig. S7A). Indeed, L.edodes (Gaitán-
Hernández et al., 2011; Cai et al., 2017) and P.ostreatus
(Isikhuemhen and Mikiashvilli, 2009) have been reported
to produce high levels of laccase and Mn peroxidase
activities thereby degrading lignin as one of their major C
sources. In contrast, M.importuna grows on deeply
decomposed plant biomass such as soil and plant-litter
compost. It is supported by the results of biochemical
assays, which revealed that M.importuna possesses
decomposition capabilities adapted to polysaccharides
over lignin.
In the genome of M.importuna SCYDJ1-A1, cellulose-
and hemicellulose-hydrolyzing enzymes are encoded by
over a dozen of GH genes, but the observed activities
were not as high as amylases. In comparison, the single
M.importuna GH15 protein contributed to a higher level
of γ-amylase activity. This finding suggests that gene
copy number and proteomic profiling should be com-
pleted by measurements of enzymatic activities to pro-
vide a comprehensive portrait of the decomposition
mechanisms. High amylase activity has been reported in
saprotrophic moulds such as Aspergillus and Mucor
(Saranraj and Stella, 2013; Gopinath et al., 2017) but
was rarely described in mushrooms. High activity levels of
enzymes hydrolyzing lipids and pectins were also observed
in ENB during the decomposition. Pectin solubilization could
disintegrate lignocellulosic complex and enhance accessibil-
ity to microbes and enzymes (Shirkavand et al., 2016).
The substantial expression of redox enzymes provides
additional insight into ENB degradative processes. Com-
pared with the Basidiomycota mushrooms P.ostreatus
and A.bisporus,M.importuna SCYDJ1-A1 displayed a
pattern of expressed CAZy-proteins with obvious short-
age in laccase (AA1), Mn peroxidase and versatile perox-
idase (AA2), as well as glyoxal oxidase (AA5) essential
for generating H
2
O
2
(Supporting Information Fig. S7B),
supporting the limited decomposition activities against lig-
nin. Two copies of AA1_3 LMCO were identified in the
M.importuna SCYDJ1-A1 genome, but only one was
expressed in ENB. Similar results were observed in the
sclerotium in mushroom spawn, the surface-soil myce-
lium before and after contact with ENB as well as in pri-
mordium and fruiting body of M.importuna SCYDJ1-A1
(Zhang et al., 2019). As laccase attacks mainly phenolic
units while Mn peroxidase and versatile peroxidase are
much more effective on non-phenolic units (Janusz et al.,
2017), the much higher laccase activity compared with
Mn peroxidase and versatile peroxidase suggests that
phenolic-unit components might be degraded faster than
non-phenolic components. ENB decomposition by M.
importuna caused the ratio of total S:G to increase initially
and then fall, differs with A.bisporus that induced a contin-
uous increase in the total S:G ratio during its entire vege-
tative growth in cultivation substrate (Kabel et al., 2017).
Concerning the other redox enzymes, Cu-dependent
LPMO has been shown to stimulate the performance of
endo- and exo-cellobiohydrolases (Vaaje-Kolstad et al.,
2010). The high transcription of benzoquinone reductase
(AA6) during days 15–45 is consistent with a role of
Fig. 5. Changes in the microbial communities colonizing ENB.
A. PCA analyses of bacterial and fungal communities in ENB at day 15 (empty squares), day 45 (empty triangles) and day 75 (circles), with three
replicates for each time-point coloured in red, green and blue respectively.
B. Relative abundance of bacterial and fungal genera, with hierarchical clustering tree constructed based on community similarity. Only the top
50 prominent genera are shown here.
C. Relative abundance of bacterial and fungal phyla in ENB at day 15, 45 and 75.
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
3918 H. Tan et al.
hydroxyl radicals in ENB decomposition (Cassagnes et al.,
2015). In addition to plant-polysaccharide degradation, chi-
tins constructing fungal cell walls are likely a substrate for
chitin deacetylases (CBM18-CE4). The high transcription
of chitin metabolism-related genes at day 15 might reflect
early colonization of ENB by morel mycelium. The
M.importuna SCYDJ1-A1 genome possesses two copies
of GH131 gene, a hallmark of plant-tissue-colonizing fungi
(Anasontzis et al., 2019), but none was likely expressed
as an enzymatic protein during ENB decomposition,
although their transcriptions were indeed observed.
The microbiota in ENB showed low OTU richness as
well as low diversity, as compared with soil environmen-
tal samples (Tan et al., 2013; Calderón et al., 2016),
composts (Wang et al., 2018), water (Thaler et al., 2017)
and guts (Griffinet al., 2017). It means that the decaying
ENB hosted a microbiota of limited complexity, compara-
ble to that of seed endophytic microbiota (Barret et al.,
2015), with a similar feature that their relative abun-
dances of microbial taxa were quite uneven. For
instance, the overwhelmingly high proportion of pseudo-
monads in the bacterial communities in ENB lasted for
the entire course of morel cultivation. Biofilms of soil-
borne pseudomonads around hyphae are known for sev-
eral mushrooms, such as P.ostreatus (Cho et al., 2003),
Laccaria bicolor (Deveau et al., 2007), A.bisporus and
Tuber borchii (Frey-Klett et al., 2011). Farming of
P.putida by M.crassipes has been reported (Pion et al.,
2013). M.importuna might tend to enrich pseudomonad
cohabitants as well.
Decline of M.importuna mycelium in ENB during days
45–75 was reflected by its relative abundance in the fun-
gal communities, but the CAZy-proteins produced by M.
importuna mycelium were durable enough to retain in
ENB until day 75, given that most of the expressed
CAZy-proteins were quantified similarly at day 45 and
day 75. This is also supported by the patterns of enzy-
matic activities, all of which not decreased from day
45 to day 75. The increase in surface soil total organic
C as well as increased fruiting body yield, during the
45 to 75 days period (Fig. 2A), indicated that ENB still
hadapositiveeffectonfruitinginthelatephase.Only
by retaining the ENBs to contact with the mushroom
bed for at least 75 days, can the dry weight of fruiting
body yield reach the average level in agricultural pro-
duction reported previously (Liu et al., 2018). The com-
pounds resulting from ENB decomposition are likely
exported to the mushroom bed via mycelial networks,
as well as free diffusion and running-off. Besides creat-
ing a surface soil with enhanced organic C content,
ENB caused N level in the mushroom bed to fall tem-
porarily during days 0–15 and restore later. Interest-
ingly, decaying plant litter had similar effects to
Table 1. Diversity of the bacterial and fungal communities in ENB at days 15, 45 and 75.
Community Sampling time Replicate Identified OTU OTU coverage ACE richness Chao1 richness Shannon-Wiener diversity Inverse Simpson’s diversity
Bacterial Day 15 1 110 0.9996 121 (114, 136) 120 (113, 140) 1.391 (1.379, 1.403) 2.464 (2.440, 2.488)
2 115 0.9996 132 (123, 152) 129 (120, 152) 1.373 (1.360, 1.385) 2.420 (2.397, 2.443)
3 103 0.9991 170 (144, 211) 143 (120, 195) 1.384 (1.369, 1.398) 2.444 (2.416, 2.473)
Day 45 1 106 0.9996 127 (115, 155) 127 (113, 168) 2.035 (2.021, 2.050) 4.248 (4.203, 4.296)
2 101 0.9996 114 (106, 135) 116 (106, 150) 1.995 (1.980, 2.011) 4.141 (4.093, 4.189)
3 105 0.9993 125 (114, 153) 121 (110, 151) 2.100 (2.083, 2.118) 4.468 (4.409, 4.531)
Day 75 1 212 0.9996 224 (217, 241) 233 (219, 277) 3.434 (3.419, 3.448) 13.263 (13.055, 13.477)
2 177 0.9996 182 (179, 193) 182 (178, 196) 3.237 (3.219, 3.255) 11.601 (11.377, 11.820)
3 144 0.9993 160 (151, 181) 160 (149, 190) 2.658 (2.639, 2.676) 7.246 (7.133, 7.369)
Fungal Day 15 1 36 0.9999 39 (37, 51) 38 (36, 50) 0.174 (0.165, 0.181) 1.050 (1.047, 1.053)
2 32 0.9999 37 (33, 53) 34 (32, 46) 0.224 (0.213, 0.235) 1.071 (1.067, 1.075)
3 34 0.9999 35 (34, 42) 36 (34, 49) 0.243 (0.233, 0.252) 1.077 (1.073, 1.081)
Day 45 1 51 1.0000 51 (51, 55) 51 (51, 51) 2.325 (2.314, 2.336) 6.489 (6.398, 6.579)
2 55 0.9999 57 (55, 65) 55 (55, 60) 2.414 (2.403, 2.425) 7.047 (6.954, 7.143)
3 50 0.9999 54 (51, 69) 52 (50, 62) 2.389 (2.378, 2.400) 7.117 (7.022, 7.215)
Day 75 1 54 0.9998 57 (55, 69) 59 (55, 83) 2.243 (2.230, 2.255) 6.180 (6.086, 6.277)
2 53 0.9999 56 (54, 69) 56 (54, 75) 2.185 (2.175, 2.196) 5.627 (5.556, 5.701)
3 55 0.9999 59 (56, 74) 60 (56, 84) 2.177 (2.167, 2.187) 5.577 (5.510, 5.647)
All samples had an OTU coverage above 0.999, showing that the sampling had sufficient scales. OTUs were clustered at 97% similarity. The 95% lower and upper confidence limits are presented
in parentheses.
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
Decomposition mechanisms triggering morel fruiting 3919
enhance soil organic C content and induce a migration
of soil N towards the decaying plant litter (Hori et al.,
2018). In this context, the man-made ENB in contact
with the mushroom bed plays a role that could mimic
the effects of plant litter, which is often abundant in
natural ecosystems such as forests and grasslands.
Interpretation of ENB decomposition by M.importuna
might provide insights into the mechanisms triggering
Fig. 6. Distribution and occurrence of genes coding for CAZymes involved in decomposition of plant polysaccharides, lignin and lipids. The
genomes of the two M.importuna strains SCYDJ1-A1 and CCBAS932 are compared with taxonomically related Pezizomycetes. The CAZy-
genes are sorted in categories according to their known targeted substrate. Significant over-representation and under-representation of CAZy-
genes in different categories of targeted substrates were estimated by Fisher’s exact test, with the statistical data shown in Supporting Informa-
tion Table S7. Pictures of the fungi are derived from the homepages of the species in the JGI genome portals.
Fig. 7. Schematic diagram of ENB decomposition by M.importuna SCYDJ1-A1.
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
3920 H. Tan et al.
wild saprotrophic mushrooms fruiting from soils, which
might advance potential attempts to domesticate more
species of wild saprotrophic mushrooms to artificial
cultivation.
Conclusions
During M.importuna cultivation, the vegetative mycelium
colonizing ENB substrate releases a complex set of deg-
radative CAZymes to efficiently decompose and metabo-
lize polysaccharides, such as starch and cellulose from
wheat grains and rice husks (Fig. 7). The metabolites
released by this decay mechanism are exported to the
adjacent surface soil of the mushroom bed, triggering
and sustaining fruiting of morels.
Experimental procedures
Morel strain
The cultivable black morel strain, M.importuna
SCYDJ1-A1, is a diploid strain as used in commercial
application. It was bred from an ancestor originally col-
lected in 2011, from the hilly terrain of Muerda village
(31.6N, 103.4E, altitude 2100 m), Lixian county, Sich-
uan province, China. The site belongs to the eastern part
of the Qinghai–Tibetan plateau, which has a cold climate
all through the year and had no forest fire for at least
10 years. The forest ecosystem from which the fruiting
body was collected had a vegetative cover composed of
mainly willow and shrub. The fruiting body grew in a
nearly-bare soil with very little coverage of plant litter. The
wild strain M.importuna CCBAS932 was collected from
an oak forest in France. The fruiting body grew directly
from a plant-litter compost without much soil. Haploid cul-
tures of monosporal isolates from the SCYDJ1-A1 and
CCBAS932 strains were used for genome sequencing.
Experimental treatments and morel cultivation
Morel cultivation in this study was carried out in a farm in
Tianjiaba Village (30.5N, 104.5E, Yangma town, Jianyang
city, Sichuan province, China). A total of 123 grids of nurs-
ery bed were built in a vegetable greenhouse. Each grid
was 1.5 m
2
in area, built with bricks and separated with
each other. A sandy loam soil was collected from a farm
nearby, thoroughly mixed to homogeneity, and evenly
loaded into all the grids. Physiochemical background of the
pre-homogenized soil was characterized (Supporting Infor-
mation Table S4).
Fifteen grids out of the 123 were randomly selected for
five treatments. In the five treatments, ENB contacted
with the mushroom bed for 0, 15, 45 and 75 days or for
the entire course (i.e., staying on the mushroom bed until
all fruiting bodies were harvested) respectively. Each
treatment included three individual grids as three biologi-
cal replicates. M.importuna strain SCYDJ1-A1 was culti-
vated in the 15 grids, using mushroom spawn produced
by Jindi-Tianlingjian company (Sichuan, China; see
Supporting Information). The mushroom spawn is free
from any bacterial or fungal contamination.
ENB was made by filling 350 g fresh weight of soaked
wheat grains and rice husks, with a dry weight ratio of
85:15, into polypropylene casing. ENB was autoclaved at
121C for 3 h, which inactivated potential decomposition
enzymes from cereal ingredients. Ten ENBs were placed
in each grid. The ENBs were pierced in the bottom cas-
ing and tightly pressed on the surface of the inoculated
soil (mushroom bed) 15 days after the morel sowing. The
15 grids consisted of five different treatments of ENB last-
ing time with three individual replicates for each treat-
ment. For the 0 day treatment, ENBs were not placed on
the soil. For the 15, 45 and 75 day treatments, ENBs
were removed and sampled at day 15, day 45 and day
75 after contact with the mushroom bed respectively.
Temperature inside ENB was measured with electronic
thermometer sensors, recorded every 30 min, and stored
automatically, throughout the entire cultivation course
(Supporting Information Fig. S2).
More details about the procedures for making mush-
room spawn and ENB, morel sowing and field manage-
ments are provided in the Supporting Information.
Sampling
For each experimental grid, 10 ENBs were sampled at
0, 15, 45 and 75 days after contact with mushroom bed,
snap frozen in liquid nitrogen, pooled and homogenized
(but not milled) to generate a replicate sample. Surface
soil of 0–2 cm depth was collected for chemical analysis.
Soil cores (2 cm ×2cm×2 cm) were collected using a
sterile blade, from 20 random points in each experimental
grid, and pooled as a replicate. Soils of 0, 15, 45 and
75 day treatments, as well as after completion of morel
harvest, were sampled respectively. For each treatment,
fruiting bodies were harvested when their size reached the
size-request (height, 5–8 cm; pileus length, 3–5 cm) for a
commercial product in the international trade.
Biochemical assays
Chemical components in ENB substrate, fruiting body
and soil samples (Supporting Information Table S5) were
quantified with classical analytical methods based on
spectrophotometry or high-performance liquid chromatog-
raphy (HPLC). The activity of selected decomposition
enzymes was measured using crude soluble proteins
extracted from ENB. To investigate enzymes involved in
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
Decomposition mechanisms triggering morel fruiting 3921
substrate decomposition, mostly extracellular, proteins
were extracted by a soaking method (Zhu et al., 2016)
aimed to maximize extracellular enzyme sampling (see
Supporting Information), although potential contamination
from intracellular proteins cannot be ruled out. Activity of
enzymes listed in Fig. 3B was measured with colorimetric
or HPLC method (Supporting Information Table S9). Enzy-
matic activity was measured as the ability to catalyze sub-
strate conversion (in milligram) per minute by the protein
extracts from per gram (dry weight) of ENB. Phosphate
buffer for enzymatic reaction had the same pH of the cor-
respondent ENB sample, whereas assay temperature was
set at the average temperature at the sampling date.
Genome sequencing, assembling and gene annotation
The genome of a monosporal haploid culture from M.
importuna SCYDJ1-A1 was sequenced using a combina-
tion of Illumina fragment (270 bp insert size) and 4 kb
long mate-pair libraries, assembled using ALLPATHS-LG
(Gnerre et al., 2011) and annotated using the JGI Anno-
tation Pipeline (Grigoriev et al., 2014), as described by
Murat and colleagues (2018).
RNA-Seq
Total RNA extraction, cDNA library construction and
sequencing, RNA-Seq reads assembling, bioinformatic
procedures for transcript profiling as well as statistical
analyses in upregulation and downregulation of transcript
level were carried out as described by Morin and col-
leagues (2019). In brief, 1–3μg of total RNA was
extracted from the combined contents of all 10 ENBs
from each experimental grid, using the RNeasy Plant Mini
RNA Extraction Kit (Qiagen, Germany), and stored at
−80C until further analysis. cDNA library construction
and sequencing were performed at the sequencing facil-
ity of Beijing Genomics Institute (BGI, in Wuhan branch,
China) according to standard Illumina protocols. Raw
reads from paired-end sequencing were quality controlled,
trimmed and mapped to the M. importuna SCYDJ1-A1 ref-
erence transcripts (https://genome.jgi.doe.gov/Morimp1/Mor-
imp1.download.html, Folder: Annotation\Filtered Models
\Transcripts) to extract a M. importuna subset from the
metatranscriptome of the target fungal community
(Supporting Information Table S10), using the software
pipeline of the CLC Genomics Workbench 11 (Qiagen, Ger-
many). Low-quality reads with Phred-quality score < 20 or
length < 50 bp were discarded. Illumina-adapter strings
were removed. Alignment was performed with stringent
settings (similarity and length read mapping criteria at 98%
and 95%, respectively; maximum 10 hits for a read on differ-
ent genes). Details about the RNA-Seq libraries, including
the counts of mapped RNA-Seq reads in the ENB
metatranscriptomes at the three time-points, as well as
mapping rates were presented in Supporting Information
Table S11. The assembled metatranscriptomes were further
analyzed using the CLC Genomics Workbench. Total
mapped paired-end reads for each gene were calculated
and total read counts were normalized as reads per kilo-
base of gene model per million fragments mapped (RPKM;
Mortazavi et al., 2008), which was used to estimate tran-
script level of each gene. Fold-change values of the RPKM
of each transcript between different time-points, together
with p-value of pairwise comparison, were calculated by
the Baggerly’s proportion-based statistical test (Baggerly
et al., 2003) implemented in the CLC Genomic Work-
bench. It is a weighted t-type test designed for comparison
proportion of sequence counts, with a FDR correction for
multiple testing (Benjamini and Hochberg, 1995). Signifi-
cant upregulation and downregulation were judged by
fold-change > 2 and fold-change < 0.5, respectively, while
FDR-corrected p-value < 0.05.
Shotgun metaproteomics
ENB proteins were analyzed by two-dimensional nano-
liquid chromatography coupled with tandem mass tags
labelling mass spectrometry (2D nanoLC-MS/MS) on a
Q-Exactive system (Thermo Fisher Scientific) in Luming
Biotechnology, Shanghai, China. Crude protein extracts
were purified by trichloroacetic acid precipitation and ace-
tone washing, re-solubilized with urea and quantified as
previously described (Hori et al., 2018). The proteins were
digested with trypsin and labelled with isotopic tags as pre-
vious described by Wi
sniewski and colleagues (2009). Nine
different tags were assigned to the 3 ×3 individual replicate
samples. Peptide fragments were first separated by
reverse-phase HPLC using an Agilent Zorbax Extend C18
column on an Agilent 1100 HPLC system (Agilent Technol-
ogies) with a flow rate at 300 μlmin
−1
. Wavelength of UV-
detector was 210 and 280 nm. Phase A: acetonitrile-H
2
O
(2%:98%, v/v). Phase B: acetonitrile-H
2
O (90%:10%, v/v).
Gradient elution steps: 0–8min,98%A;8–8.01 min, 98%–
95% A; 8.01–38 min, 95%–75% A; 38–50 min, 75%–60%
A; 50–50.01 min, 60%–10% A; 50.01–60 min, 10% A;
60–60.01 min, 10%–98% A; 60.01–65 min, 98% A. The
eluted products during 8–50minwerecollectedwith1min
interval into centrifuge tubes until the end of the gradient.
Further separation was carried out using an Acclaim
Pepmap RSLC analytical column (C18, 2 μm, 100 Å,
75 μm×15 cm, Dionex) with a flow rate at 300 nl min
−1
.
Phase A: H
2
O-FA (99.9%:0.1%, v/v). Phase B: acetonitrile-
H
2
O-FA (80%:19.9%:0.1%, v/v/v). Gradient elution steps:
0–55 min, 8% B; 55–79 min, 30% B; 79–80 min, 50% B;
80–90 min, 100% B. The eluted fragments were scanned
in MS1 with resolution 70 000 and m/z range 300–1800,
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
3922 H. Tan et al.
followed by MS/MS fragmentation of 10 most intense pep-
tide fragments detected in the MS1. The MS/MS fragmen-
tation was scanned with resolution 17 500, dynamic
exclusion time 30 s.
Raw data were loaded into Proteome Discoverer software
v2.2 (Thermo Fisher Scientific) for protein identification and
quantification, with an FDR < 1%. A total of 6030 peptide
fragments were obtained in the MS/MS spectra, mapped to
the user-defined reference genomes (Supporting Informa-
tion Table S10). Protein hits belonging to wheat and rice,
which are ingredients of ENB substrate (wheat grains and
rice husks), were identified as substrate background to be
manually removed. The identified proteins must have a
Sequest HT score > 0 and unique peptide ≥1, as the
criteria previously adopted by Zhu and colleagues (2016)
and Cai and colleagues (2017). Fold-change values of pro-
tein relative abundance between time-points, together with
p-value of pairwise comparison, were calculated by ttest
with FDR correction. Significant upregulation and down-
regulation were judged by fold-change > 2 and fold-change
< 0.5, respectively, while FDR-corrected p-value < 0.05.
Microbiome metabarcoding
Metabarcoding survey on microbial diversity in ENB was
carried out using PCR amplicons of 16S rDNA V4-V5
region for bacterial community and ITS region for fungal
community. Total microbial DNA in ENB was isolated
with a CTAB extracting method (Tan et al., 2013). V4-V5
region of bacterial 16S rRNA gene fragment was amplified
with primers 515F (50-GTGCCAGCMGCCGCGG-30) and
907R (50-CCGTCAATTCMTTTRAGTTT-30; Jiang et al.,
2017). Fungal ITS region was amplified with primers ITS1-F
(50-CTTGGTCATTTAGAGGAAGTAA-30) and ITS2-R (50-
GCTGCGTTCTTCATCGATGC-30) (French et al., 2017).
Sequencing library was constructed from the PCR
amplicons, with index codes added, using NEB NextUltra
DNA Library Prep Kit for Illumina (NEB) following manufac-
turer’s recommendations. The libraries were sequenced on
an Illumina MiSeq platform at the sequencing facility of BGI
(Wuhan, China) according to standard Illumina protocols.
The paired-end reads were quality controlled, merged by
overlapping and analyzed with the QIIME pipeline
(Caporaso et al., 2010), as described in previous studies
(Barret et al., 2015; Awasthi et al., 2017). Bacterial and fun-
gal OTUs were clustered at 97% similarity threshold respec-
tively. Rarefaction curve of OTU was drawn to estimate
sequencing coverage (Supporting Information Fig. S7).
SILVA (Release 132) database of full-length sequences
and taxonomy references was used for bacterial OTU clus-
tering. UNITE v7.2 (Full UNITE+INSD dataset) was used for
fungal OTU clustering.
Accessibility of strain and data
Cultures from M.importuna SCYDJ1-A1 are available
(for non-commercial research only) on request to Jindi-
Tianlingjian company, Sichuan, China. The genome of
M.importuna SCYDJ1-A1 is available at the
corresponding MycoCosm genome portal at DOE Joint
Genome Institute (https://genome.jgi.doe.gov/Morimp1/
Morimp1.home.html) and also at NCBI BioProject
PRJNA334370 (Genbank accession number
SSHS00000000.1). RNA-Seq data: NCBI BioProject
PRJNA503787. Shotgun metaproteomic data: PRIDE
Archive identifier PXD012086. High-throughput sequenc-
ing of bacterial 16S rDNA V4-V5 and fungal ITS: NCBI
Sequence Read Archive SRP162892.
Acknowledgements
This research was mainly supported by the Sichuan Science
and Technology Program (Applied Fundamental Research
Project, 2018JY0637, HT), the Special Fund for Agro-
scientific Research in the Public Interest (201503137, ZH),
the Innovative Improvement Projects of Sichuan Province
(2016ZYPZ-028, WP; 2019LWJJ-009, HT; 2016LWJJ-007,
HT), the Key Breeding Project of Sichuan Province (BW),
and the Laboratory of Excellence ARBRE (ANR-11-LABX-
0002-01, FMM), Region Lorraine, European Regional Devel-
opment Fund. Isolation of the monosporal haploid culture
from M.importuna SCYDJ1-A1, preparation of genomic
DNA, as well as total mRNA for Expressed Sequence Tag
(EST) survey were supported by the SAAS International
Cooperation Fund 2015 (HT). Special thanks to Ms Lu Xiong
(internship MSc student from Sichuan Agricultural University)
for her participation in the extraction and purification of the
DNA and EST-RNA samples. Library construction, sequencing,
assembly, and annotation of the M.importuna SCYDJ1-A1
genome were performed within the framework of the 1000 Fun-
gal Genomes project by the U.S. Department of Energy Joint
Genome Institute, a DOE Office of Science User Facility, and
supported by the Office of Science of the U.S. Department of
Energy under Contract No. DE-AC02-05CH11231. We are
grateful to farmer Dafu Tian’s family for providing the field
experiment site and to all field technicians for their contribution.
Commercial use of M.importuna SCYDJ1-A1 is currently
under protection by Jindi-Tianlingjian company, Sichuan,
China.
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Supporting Information
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Appendix S1: Supporting information
Fig. S1. Pairwise synteny of scaffolds between the genomes
of M. importuna strains SCYDJ1-A1 (X-axis) and CCBAS932
(Y-axis). The VISTA program (Martin et al., 2004) integrated
in the JGI Annotation Pipeline was used for pairwise align-
ment of scaffolds as well as visualization of the alignment
results. Threshold of sequence length with continuous high
homology was set at 50 bp cut-off. The dot-plot figure was
extracted from the JGI MycoCosm genome portal of
M. importuna SCYDJ1-A1.
Fig. S2. ENB temperature measured by electronic thermom-
eter sensors inserted into three testing ENBs. Values are
mean of three replicates.
Fig. S3. Time-course changes in the content of organic com-
pounds and mineral elements in fruiting bodies. The
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
Decomposition mechanisms triggering morel fruiting 3925
coloured columns indicate mean of three biological repli-
cates, with standard deviations. Significance of difference
was judged by one-way ANOVA. A full list of all p-values is
provided in Table S8.
Fig. S4. Enrichment of
15
N in ENB which were placed on the
mushroom bed of a
15
N-labeled soil, indicating that decom-
position of ENB by M. importuna led to assimilation of N
from soil towards ENB, during the first 15 days. Significant
difference between day 0 and day 15 was judged by ttest.
Samples with a significantly increased level of
15
N relative
abundance are labeled with asterisks. The p-values are pro-
vided in Table S8.
Fig. S5. All the 88 CAZy-proteins of M. importuna SCYDJ1-A1
identified in ENB. Expression levels of the transcripts and pro-
teins were estimated by RNA-Seq and nanoLC-MS/MS,
respectively. Steady-state transcript level (in RPKM) and pro-
tein relative abundance are mean of three biological replicates.
ND: not detected. Functions of the CAZymes were predicted
according to their nearest analogs whose activities had been
characterized in previous studies, as provided by the CAZy
database. Significant up- or down-regulation was judged by
fold-change > 2 or fold-change < 0.5, respectively, while FDR-
corrected p-value < 0.05. Fold-change values and p-values are
provided in Table S8.
Fig. S6. Counts of proteins showing significant up-regulation
(fold-change > 2 and p-value < 0.05), significant down-
regulation (fold-change > 0.5 and p-value < 0.05) or no signifi-
cant shift, during days 15-45 or during days 45-75, respectively.
Proteins of the metaproteomes of the major fungal taxa in ENB
(M. importuna SCYDJ1-A1, Mortierella,Trichoderma,Mono-
dictys,Peziza,Cladosporium,Neonectria,Penicillium,Fusar-
ium,Oliveonia,Plectosphaerella), or those belonging to
M. importuna SCYDJ1-A1 only, were counted respectively.
Subset panels show expressing regulation in the CAZymes of
M. importuna SCYDJ1-A1. Areas of the circular sectors are all
proportional to gene counts.
Fig. S7. A. Distribution of occurrence of genes coding for
CAZymes involved in decomposition of plant polysaccharides,
lignin and lipids, compared between the genomes of the com-
mercially cultivated mushrooms M. importuna SCYDJ1-A1,
A. bisporus,P. ostreatus and L. edodes.B.Presenceof
CAZymes in the proteomic profiles of M. importuna SCYDJ1-A1,
P. ostreatus and A. bisporus. The CAZy-genes are sorted in cat-
egories according to their targeted substrates. Significant over-
representation and under-representation were judged by Fish-
er’s exact test shown in Table S7. The number of expressed
CAZy-proteins of P. ostreatus and A. bisporus are calculated
from available previous studies (Patyshakuliyeva et al., 2015;
Fernández-Fueyo et al., 2016), while M. importuna SCYDJ1-A1
is from this study.
Fig. S8. Rarefaction curves of bacterial 16S (a) and fungal ITS
(b) sequences. ENB at day 15, 45 and 75 are shown by solid
lines,dash lines and dot lines, respectively. The three repli-
cates of each time-point are coloured in red,green and blue.
Table S1. Genome completeness and assembly metrics of
M. importuna SCYDJ1-A1 and CCBAS932 strains.
Table S2. Comparison of gene-model characteristics
between M. importuna SCYDJ1-A1 (red) and CCBAS932
(blue) strains.
Table S3. 9783 common genes shared by the SCYDJ1-A1
and CCBAS932 strains of M.importuna, determined by
BlastP Best Reciprocal Hit analysis. The table is of big size,
and is therefore provided as an individual Excel file available
online: TableS3.xls.
Table S4. Initial state of physiochemical characteristics of
the pre-homogenized soil used as the mushroom bed for
morel cultivation.
Table S5. Content of chemicals in ENB at day 0, 15,
45 and 75.
Table S6. Proteins identified in the metaproteomes, with
relative abundance of the three replicates at day
15, 45 and 75. The table is of big size, and is therefore
provided as multiple working-sheets in an individual
Excel file available online: TableS6.xls.
Table S7. Crosstabs showing all the results of Fisher’s
exact test conducted in this study. Significant over-
representation is judged by adjusted residual value
> 1.96 (upper limit of 95% confidence of +1), and signifi-
cant under-representation by adjusted residual value <
−1.96 (lower limit of 95% confidence of −1), as the
criteria proposed by MacDonald and Gardner (2000). The
table is of big size, and is therefore provided as multiple
working-sheets in an individual Excel file available online:
TableS7.xls.
Table S8. p-values of all the statistical comparisons (except
for Fisher’s exact test) in this study. The table is of big size,
and is therefore provided as multiple working-sheets in an
individual Excel file available online: TableS8.xls.
Table S9. Methods for enzymatic activity estimation.
Table S10. User-defined reference metagenome.
Table S11. Mapping rate of RNA-Seq reads.
© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd., Environmental Microbiology,21, 3909–3926
3926 H. Tan et al.
