Microbiological Research 239 (2020) 126520

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Colonization by Tuber melanosporum and Tuber indicum affects the growth of T

Pinus armandii and phoD alkaline phosphatase encoding bacterial
community in the rhizosphere
Xiaoping Zhanga,b,1, Xiaolin Lib,1,**, Lei Yeb, Yue Huanga,b, Zongjing Kanga,b, Bo Zhangb,
Xiaoping Zhanga,*
a
Department of Microbiology, College of Resources, Sichuan Agricultural University, Chengdu, China
b
Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences, Chengdu, China

A R T I C LE I N FO A B S T R A C T

Keywords: The synthesis of truffle ectomycorrhizae and the ecology of truffle-colonized seedlings in the early symbiotic
Truffle stage are important for the successful truffle cultivation. In this study, two black truffle species, Tuber mela-
Ectomycorrhizae nosporum and Tuber indicum, were selected to colonize Pinus armandii seedlings. 2, 4, 6 and 8 months after
phoD genes inoculation, the growth performance of the host and the rhizosphere soil properties were detected. The dynamic
Microbes
changes of two mating type genes in substrate were also monitored to assess the sexual distribution of truffles.
Mating type genes
Additionally, the variation of soil bacterial communities encoded by phoD alkaline phosphatase genes was in-
vestigated through next-generation sequencing. The results indicated that both T. melanosporum and T. indicum
colonization promoted the growth of P. armandii seedlings to some extent, including improving their biomass,
total root surface area, root superoxide dismutases and peroxidase activity. The organic matter and available
phosphorus in rhizosphere soil were also significantly enhanced by two truffles’ colonization. The phoD-har-
boring bacterial community structure was altered by both truffles, and T. melanosporum decreased their diversity
or richness on the 6th and 8th month after inoculation. Pseudomonas, Xanthomonas, and Sinorhizobium, a N2-fixer
with phoD genes, were found more abundant in truffle-colonized treatments. The mating type distribution of the
two truffles was uneven, with MAT1−1-1 gene occupying the majority. Overall, T. melanosporum and T. indicum
colonization affected the micro-ecology of truffle symbionts during the early symbiotic stage. These results could
give us a better understanding on the truffle-plant-soil-microbe interactions, which would be beneficial to the
subsequent truffle cultivation.

1. Introduction Murat, 2015; Li et al., 2019b). As a kind of ectomycorrhizal fungi

Tuber indicum and Tuber melanosporum are two black truffle species
belonging to Ascomycota, which have similar morphological char-
acteristics and close phylogenetical relationship (Murat et al., 2008).
Both T. indicum and T. melanosporum have high commercial values due
to their unique flavor for dishes and potential bioactive compounds for
medicinal function (Patel et al., 2017; Li et al., 2019b). T. melanosporum
is the most precious black truffle species mainly produced in some
native European countries like France, Italy, Spain, and some other
non-native regions like Australia, while T. indicum is the most common,
productive, and widely distributed species in China (Liu et al., 2011;
producing their fruiting bodies in the underground, truffles must co-
lonize the plant roots and live symbiotically with them to achieve the
whole life cycle (Mello et al., 2006; Benucci et al., 2012). The first
artificial synthesis of truffle ectomycorrhizae was performed at the end
of 1960s (Fontana, 1967; Fontana and Palenzona, 1969). The sub-
sequent planting of truffle-inoculated seedlings has always been one of
the hot spots in truffle research in order to realize their better com-
mercialization, and the truffle fruiting bodies have been successfully
obtained in some truffle plantations (Hu et al., 2005; Deng et al., 2014;
Murat, 2015). So the efficient synthesis of ectomycorrhizae with plants
is a fundamental and important step for the successful truffle artificial

⁎
Corresponding author at: Huimin Road, Chengdu, 611130, China.
⁎⁎
Corresponding author at: Shizishan Road, Chengdu, 610066, China.
E-mail addresses: kerrylee_tw@sina.com (X. Li), zhangxiaopingphd@126.com (X. Zhang).
1
Xiaoping Zhang and Xiaolin Li contributed equally to the work.

https://doi.org/10.1016/j.micres.2020.126520
Received 13 February 2020; Received in revised form 18 May 2020; Accepted 23 May 2020
Available online 03 June 2020
0944-5013/ © 2020 Published by Elsevier GmbH.
X. Zhang, et al.
cultivation. Pinus armandii, a kind of evergreen tree belonging to Pi-
naceae, is widely distributed in the southwest and northwest of China,
which has been confirmed to be able to form ectomycorrhizae with T.
indicum and T. melanosporum (Geng et al., 2009; Li et al., 2017).
However, rare reports focused on the physiological and molecular
mechanisms in response to the colonization of these two truffles and
made comparision between them (Bradshaw, 2005; Zhang et al., 2019).
There is no doubt that truffles have an important function in the
ecological environment. On the one hand, as an ectomycorrhizal fungi,
truffles can form the bridges with their mycelium in the underground,
which can move the nutrients in both directions between the plants and
soil. Many studies have shown that the formation of ectomycorrhizae is
beneficial for plants to absorb the nutrients and water from soil and also
contribute to the uptake of carbohydrates from plant roots to ectomy-
corrhizal fungi (Doidy et al., 2012; Hortal et al., 2017; Mello and
Balestrini, 2018; Gorka et al., 2019). The growth and development, and
the nutrients utilization status of the host plants can be improved by the
ectomycorrhizae to a certain extent, also, in some situation, ectomy-
corrhizal presence can alter the physiological activity of plant roots and
the stress resistance of the host (Turgeman et al., 2011; Jourand et al.,
2014; Guerrero-Galán et al., 2019; Zhang et al., 2019). During the early
symbiotic stage of truffles, the understanding of the host plant quality is
important, which can tell us the adaptation of different truffle species to
different hosts and justify whether it is fine for the artificial synthesis of
truffle ecomycorrhizae and the subsequent transplanting to plantations.
On the other hand, in the ecological environment, many soil mi-
crobes are involved in the life cycle of truffles, including bacteria and
other fungi. These soil microbes may play the key role in the formation
of truffle ectomycorrhizae, ascocarps and fragrance (Splivallo et al.,
2015; Chen et al., 2019). However, the specific roles or mechanisms of
these microbes are still unclear, because many authors have indicated
that there are complicated interactions between soil microbes, truffles,
truffle ectomycorrhizae and host plants, also with the changes of the
soil properties (Li et al., 2018; Mello and Balestrini, 2018). In the rhi-
zosphere soil associated with truffles, soil phosphorus (P) cycling plays
an important role, because the ectomycorrhizae can contribute to the
decomposition and release of P in soil for plants absorption (Köhler
et al., 2018; Liu et al., 2018). And in turn, soil P can also influence the
development of ectomycorrhizal fungi (Xue et al., 2008). So the in-
vestigation on soil microbes related to P cycling seems necessary in
truffle ecology. phoA, phoD and phoX are three kinds of genes encoding
alkaline phosphatase in bacteria, which can catalyze the hydrolysis of
phosphomonoesters and phosphodiesters, the dominate fraction of soil
organic P (Ragot et al., 2015). phoD alkaline phosphatase are pre-
dominantly membrane bound and are activated by Ca2+, which are
widespread in terrestrial ecosystems (Luo et al., 2009; Kageyama et al.,
2011; Ragot et al., 2015). So the bacterial community harboring phoD
genes were selected to assess in this study, which have not been re-
ported in the truffle-associated environment.
Since the genome sequencing project, T. melanosporum has been
found as a heterothallic fungus, and T. indicum is also verified to be
heterothallic in the subsequent researches (Martin et al., 2010; Rubini
et al., 2011b; Belfiori et al., 2013), which means that each strain of
Tuber only has single mating type gene. It has also been found that the
strains with MAT1−1-1 or MAT1−2-1 compete to colonize the root of
the host plants, and this competition results in the uneven sexual dis-
tribution in the ectomycorrhizae (Rubini et al., 2011a), which can be a
major factor that determine the production and yield of truffles. Few
studies addressed the distribution of Tuber mating types in soils com-
pared with the investigations on that in ectomycorrhizae (Rubini et al.,
2011a; Muratet al., 2013). Currently, the biological implications or the
mechanistic determinants of the nonrandom sexual distribution of
truffles in ectomycorrhizae or in soils are still uncertain. Therefore, the
more investigations on truffle mating type genes are of great sig-
nificance to know the development of truffles and will contribute to
their practical cultivation.
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Microbiological Research 239 (2020) 126520
In this study, we inoculated T. melanosporum and T. indicum on P.
armandii seedlings and assessed the truffle colonization level and the
host plant quality every two months after inoculation until the eighth
month. Additionally, the changes of soil properties, the phoD-harboring
bacterial community in rhizosphere, and the distribution of truffle
mating type genes in soil were also investigated, in order to enhance
our understanding on how the host plants and soil microbes with phoD
genes in response to the colonization of these two truffles during dif-
ferent periods in the early symbiotic stage, which can give us the insight
into the micro-ecology of truffle symbionts and the interaction between
truffles, host plants and phoD-harboring bacterial community.
2. Materials and methods
2.1. Cultivation of P. armandii ectomycorrhizal seedlings with T. indicum
and T. melanosporum
The seeds of P. armandii were purchased from Yunnan Academy of
Forestry Sciences. The ascocarps of T. indicum and T. melanosporum
respectively came from the Huidong County, China and Cahors, France.
The synthesis of ectomycorrhizal seedling included the P. armandii
seedlings cultivation and the truffle inoculation treatment in a green-
house, which were performed as our previous methods (Zhang et al.,
2019). In brief, the surface-sterilized P. armandii seeds were sowed in
the sterilized substrate (perlite: vermiculite: water = 1:1:1, v/v/v).
Two months later, each seedling was respectively transported to a pot
and filled with 1 L sterilized cultivation substrate (vermiculite: organic
soil: water = 1:1:0.5, v/v/v), then the truffle inoculation treatment was
followed up. The inoculum of T. melanosporum and T. indicum were
obtained by pulverizing their surface-disinfected ascocarps (Li et al.,
2018). Blend up their spore powder and inoculate 2 g of them closely to
the roots for each P. armandii seedling in the cultivation substrate. The
seedlings inoculated with T. melanosporum and T. indicum were assigned
as “mel. arm” and “ind. arm”, respectively. Meanwhile, the seedlings
without truffle inoculation treatment were assigned as “Control”. To-
tally, 180 P. armandii seedlings were cultivated, of which 120 were
inoculated with T. indicum or T. melanosporum, and 60 were not in-
oculated with truffles. Consequently, sixty seedlings were prepared for
each treatment. All the seedlings were put in a greenhouse and the
substrate was watered about every 3 days. The seedlings belonging to
different treatment were placed in different zones in the same green-
house.
2.2. Sampling strategy for analysis
After truffle inoculation, the seedlings and rhizophere soils were
sampled every two months until the eighth month. The samples col-
lected on the 2nd, 4th, 6th and 8th month after inoculation were re-
spectively denoted as M2, M4, M6 and M8. When collecting the seed-
ling samples, their root system were observed under a microscope to
determine whether the ectomycorrhizae formed based on their mor-
phology and structure, in other words, whether the seedlings were
successfully colonized by Tuber. Truffle colonization rates in mel. arm
treatment and ind. arm treatment were calculated by counting the
number of root segments with the presence of ectomycorrhizae and
randomly choosing thirty root segments in all in each seedling, which
was performed as previous method (Andres-Alpuente et al., 2014).
After that, the seedling samples were used for measuring the plant
growth, the root samples were used for the physiological assessment,
and the soil samples in the rhizosphere were utilized for the determi-
nation of soil properties, soil bacterial communities harbouring phoD
genes, and truffle mating type genes in soil. In this experiment, nine P.
armandii seedlings were analyzed in each treatment on each sampling
time. Root and rhizosphere soil samples from three P. armandii seed-
lings were mixed as one biological sample, respectively. Thus, three
biological replicates of root and rhizosphere soil samples were obtained
X. Zhang, et al.
from nine seedlings in each treatment on each sampling time, which
finally underwent corresponding analysis.
2.3. Measurement of plant growth, plant physiology and soil properties
The growth performance of P. armandii seedlings was obtained by
measuring their stem circumference, plant height, root length, total
root surface area, biomass and root-shoot ratio. A ruler or vernier ca-
liper was used to determine the indices of stem circumference, plant
height and root length. The total root surface area was assayed by
WINRHIZO root analysis scanner (Pornaro et al., 2017). The biomass
was obtained by assessing the dry weight of each seedling (Zhang et al.,
2019b). The dried seedlings were then cut into two sections according
to the aboveground and underground parts, and the root-shoot ratio of
the seedling could be got (underground dry weight / aboveground dry
weight).
The investigated physiological indices of P. armandii seedlings in-
cluded the root activity, root superoxide dismutases (SOD) activity and
root peroxidase (POD) activity. Root activity was determined by TTC
(2, 3, 5-triphenyl tetrazolium chloride) method and expressed as the
TTC reduction intensity (μg) of per gram of fresh roots within per hour
(Zhang et al., 2019b). The measurement of SOD activity in root used the
nitro-blue tetrazolium (NBT) method, based on the theory that the NBT
photochemical reduction could be inhibited by SOD (Shafi et al., 2015).
The POD activity in root was determined by guaiacol method, which
utilized the catalysis of POD during the oxidation of guaiacol under
H2O2 existence (Rácz et al., 2018).
The determination of soil properties, including pH, organic matter
(OM), total and available nitrogen (TN and AN), total and available
phosphorus (TP and AP), was carried out as previous description by Li
et al. (2016).
2.4. Soil DNA extraction
The total microbial DNA in the rhizosphere soil was extracted with a
FastDNA® SPIN Kit for Soil (Bio-Rad Co, USA) in accordance with the
instructions. After the detection of DNA quality and concentration
through 1% agarose gel electrophoresis and ultraviolet spectro-
photometry, the DNA samples were stored at −20 °C. All the DNA
samples had three replicates.
2.5. PCR amplification of phoD genes and Illumina MiSeq sequencing
ALPS-F730 (5′-CAGTGGGACGACCACGAGGT-3′) and ALPS-1101 (
5′-GAGGCCGATCGGCATGTCG-3′) primers were used to amplify the
phoD genes, including three technical replicates of each sample during
amplification (Luo et al., 2017). The reaction system consisted of 1 μL
forward and 1 μL reverse primers (10 μmol L−1), 2 μL dNTPs (2.5
mmol%L−1), 5 μL GC buffer, 5 μL reaction buffer, 2 μL DNA template
and 0.25 μL Q5 DNA polymerase, diluted to 25 μL with ddH2O. The
reaction programs contained 25–30 cycles, conducted according to
previous method (Zhang et al., 2019). The PCR products were mixed
and then detected, after that they were recovered by gel recovery kit
(Axygen, USA) and quantified using the Quant-iT PicoGreen dsDNA
Assay Kit on Microplate reader (BioTek, FLx800). The samples were
mixed in a corresponding proportion according to the fluorescence
quantitative results.
The sequencing library preparation was generated by TruSeq Nano
DNA LT Library Prep Kit (Illumina, USA) and their quality was in-
spected on Agilent Bioanalyzer. Illumina MiSeq sequencing platform
was used to perform the next-generation sequencing according to the
standard process, which was conducted by Personal Biotechnology, Co.,
Ltd. (Shanghai, China). The raw sequencing data were deposited in
NCBI SRA database with the accession number SRP242476/
PRJNA601834.
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Microbiological Research 239 (2020) 126520
2.6. Sequencing data analysis
The original paired-end sequencing data saved in FASTQ format
were screened by sliding the window and the FLASH software (v1.2.7)
was used to pair and connect the sequences according to the over-
lapping bases. The QIIME software (v1.8.0) and USEARCH (v5.2.236)
were utilized to eliminate the chimera sequences and the high quality
sequences of each sample were received (Caporaso et al., 2010).
Through QIIME and UCLUST, the obtained sequences with ≥97 % si-
milarity were merged as an operational taxonomic unit (OTU) (Edgar,
2010). The taxonomy information of each OTU were obtained using
RDP (Ribosomal Database Project) function gene database (Release
11.1) (Cole et al., 2009). The rarefaction curves and the alpha diversity
were calculated by QIIME, and the beta diversity which displayed the
differences between groups was conducted with non-metric multi-
dimensional scaling (NMDS) analysis through QIIME and R software.
2.7. Quantitative real-time PCR assay of truffle mating type genes
The mating type genes of T. melanosporum and T. indicum in rhizo-
sphere soil of P. armandii seedlings on four sampling times were abso-
lutely quantified by real-time PCR. With total DNA as the template, the
specific primers P19 and P20, and, P1 and P2 were used to respectively
amplify the MAT1−1-1 gene and MAT1−2-1 gene of T. melanosporum
(Rubini et al., 2011b) (Table S1). Meanwhile, the MAT1−1-1 and
MAT1−2-1 genes of T. indicum were amplified by primers i3 and i4,
and, i5 and i6, respectively (Belfiori et al., 2013) (Table S1). Each
sample had three technical replicates for amplification. The PCR reac-
tion system and reaction programs were shown in Table S2. The con-
struction of standard curves was performed using serial 10-fold dilu-
tions as previous method (Luo et al., 2017), and the copy numbers of
mating type genes were then calculated.
2.8. Statistical analysis
The means and standard deviations of the data including the growth
indices, physiological indices, soil properties, phoD gene diversities
(ACE, Chao1, Simpson and Shannon indices) and copy numbers of
mating type genes were calculated. The statistical analysis of these data
was performed by SPSS v22.0 (IBM, Armonk, NY, USA) with one-way
analysis of variance (ANOVA) using F test statistic, followed by least
significant difference (LSD) test when the ANOVA results were sig-
nificant at P < 0.05. P-values for multiple testing were then corrected
by Benjamini and Hochberg procedure, with a false discovery rate
(FDR) of 5% denoted as statistical significance. The differential taxa in
different treatments were obtained by STAMP software using ANOVA
statistical test followed by Tukey-Kramer post-hoc test, with sig-
nificance at P < 0.05 (Parks et al., 2014). Permutational multivariate
analysis of variance (PERMANOVA) was performed by QIIME and 999
replacement tests were also performed to identify the statistical sig-
nificance of the differences in taxonomic composition between groups.
The redundancy analysis (RDA) of environmental variables and the
bacterial communities harbouring phoD genes were conducted in R
statistical software package (version 2.15.0) using vegan packages, in
order to reveal the relationships between the two. The correlation be-
tween soil properties and truffle mating type gene abundances were
carried out by Pearson correlation analysis using SPSS v22.0.
3. Results
3.1. Ectomycorrhizal development and truffle colonization rate
Two months after inoculation, there was no presence of ectomy-
corrhizae in either mel. arm or ind. arm treatment. Four months after
inoculation, the ectomycorrhizae of T. melanosporum and T. indicum
were successfully observed, with the milky or yellowish color. As time
X. Zhang, et al.
Fig. 1. Ectomycorrhizae of P. armandii seedlings with T. melanosporum (a, b) and T. indicum (e, f); Roots of P. armandii seedlings that were not colonized by truffles (i,
j); Transversal sections of T. melanosporum ectomycorrhizae (c), T. indicum ectomycorrhizae (g) and ordinary root tips (k); Longitudinal sections of T. melanosporum
ectomycorrhizae (d), T. indicum ectomycorrhizae (h) and ordinary root tips (l).
went on, the ectomycorrhizae increased, showing the morphology of
monopodium, binary branches, coral-like or cluster-like, gradually
deepening the color to yellowish brown or dark brown (Fig. 1a, b, e, f),
which exhibited the obvious differences from the ordinary roots in
Control treatment (Fig. 1i, j). The structure of the Hartig net could be
clearly seen from the ectomycorrhizal slices in mel. arm and ind. arm
treatment (Fig. 1c, d, g, h), while the common roots didn’t have this
structure (Fig. 1k, l).
On the 4th and 6th months after inoculation, there were no sig-
nificant differences in colonization rate between the two truffles (Table
S3). However, on the 8th month after inoculation, the colonization rate
of T. indicum on P. armandii seedlings was 61.98 % ± 10 %, which was
significantly higher than that of T. melanosporum (47.94 % ± 8%)
(Independent t test, P = 0.046).
3.2. Effect of T. melanosporum and T. indicum colonization on the growth
and physiology of P. armandii
The quality of P. armandii seedlings were assessed by the growth
indices (Table 1), which were altered by T. melanosporum and T. indicum
colonization. The stem circumferences of seedlings were significantly
bigger in Control treatment on the second month (P = 0.048), however,
from the 4th month after inoculation, the stem circumferences of
truffle-colonized seedlings increased sharply, reaching the same level as
the Control. The plant height showed no significant differences between
three treatments under four sampling time. The main root length and
total root surface area increased with time. On the 6th and 8th month
after inoculation, the root length were significantly longer in Control
treatment (P = 0.005 on M6; P = 0.024 on M8), while the total root
surface area were significantly increased by the colonization of both
two truffles (P = 0.046 on M6; P = 0.04 on M8). There were no ob-
vious differences in these two indices between mel. arm and ind. arm
treatments.
The biomass of the seedlings also increased with time (Table 1).
Comparing with the Control, T. melanosporum and T. indicum coloni-
zation significantly increased the biomass of P. armandii on the 4th
month (P = 0.023) and 6th month (P = 0.039) after inoculation. The
4
Microbiological Research 239 (2020) 126520
root-shoot ratio of the seedlings gradually increased in the first 6
months, but decreased on the M8. The T. melanosporum-colonized
seedlings had significantly higher root-shoot ratio than Control under
four sampling time (P = 0.003, 0.002, 0.013, 0.033 on M2, M4, M6,
M8). However, T. indicum colonization only significantly increased the
root-shoot ratio of P. armandii on the 2nd month (P = 0.003) and 8th
month (P = 0.036) after inoculation. Although the root-shoot ratio in
mel. arm treatment were higher than that of ind. arm treatment, the
differences didn’t reach the significant level.
The physiological indices of P. armandii seedlings, including root
activity, root SOD and POD activity, were varied with time, and showed
a higher level in truffle inoculation treatments (Fig. 2). Detailedly, the
root activity reached the maximum on the 4th month after inoculation,
with significantly higher values in mel. arm (P = 0.008) and ind. arm
treatment (P = 0.006). The SOD activity in root showed no obvious
differences between the three treatments in the first 4 months, however,
it was significantly increased by T. indicum and T. melanosporum colo-
nization on the 6th month (P = 0.000) and 8th month (P = 0.045)
after inoculation. The root POD activity of the seedlings gradually in-
creased with time, which exhibited the significant differences between
Control treatment and two truffle inoculation treatments on the 4th
month (P = 0.009) and 6th month (P = 0.005) after inoculation, with
the minimum value in Control. No significant differences were observed
in root activity, root SOD and POD activity between T. melanosporum-
colonized seedlings and T. indium-colonized seedlings under four sam-
pling time.
3.3. Changes of soil properties in the rhizosphere
Some soil properties in the rhizosphere of P. armandii seedlings were
affected by T. melanosporum and T. indium colonization (Table 2). The
soil pH nearly didn’t show significant differences between the three
treatments under the four sampling time. The soil OM contents were
significantly more in the truffle-colonized treatments compared with
the Control on the four observed months (P = 0.03, 0.041, 0.042, 0.032
on M2, M4, M6, M8). Only on the 6th month after inoculation, there
were significant differences in OM between mel. arm and ind. arm
X. Zhang, et al. Microbiological Research 239 (2020) 126520

Table 1
The growth of P. armandii seedlings with or without T. melanosporum and T. indicum colonization under different sampling time.
Sampling time Treatments Stem circumferences (mm) Plant height (cm) Main root length (cm) Total root surface area (cm2) Biomass (g) Root-shoot
ratio

M2 Control2 0.97 ± 0.23a 13.63 ± 0.77a 5.77 ± 0.96a 10.52 ± 3.40a 0.95 ± 0.15a 0.13 ± 0.05a
ind.arm2 0.50 ± 0.12b 14.04 ± 1.51a 7.64 ± 2.80a 10.18 ± 4.42a 0.99 ± 0.11a 0.27 ± 0.04b
mel.arm2 0.51 ± 0.24b 13.10 ± 1.16a 7.38 ± 1.36a 12.21 ± 3.42a 1.21 ± 0.10a 0.29 ± 0.01b
M4 Control4 1.18 ± 0.41a 14.38 ± 0.60a 12.14 ± 2.56a 15.54 ± 1.08a 1.34 ± 0.05a 0.27 ± 0.02a
ind.arm4 0.60 ± 0.17a 14.23 ± 0.36a 10.95 ± 1.94a 20.16 ± 2.76a 1.50 ± 0.06b 0.32 ± 0.02ab
mel.arm4 0.80 ± 0.19a 13.38 ± 0.94a 11.37 ± 0.35a 20.11 ± 5.22a 1.48 ± 0.03b 0.39 ± 0.06b
M6 Control6 1.97 ± 0.07a 15.02 ± 1.34a 21.87 ± 1.01a 18.05 ± 5.13a 1.52 ± 0.04a 0.35 ± 0.01a
ind.arm6 2.02 ± 0.19a 14.89 ± 0.61a 12.96 ± 3.08b 28.40 ± 1.43b 1.67 ± 0.02b 0.40 ± 0.07ab
mel.arm6 2.00 ± 0.10a 14.37 ± 0.70a 14.10 ± 1.37b 26.78 ± 3.63b 1.68 ± 0.10b 0.47 ± 0.07b
M8 Control8 2.13 ± 0.16a 15.01 ± 1.41a 25.80 ± 2.32a 27.42 ± 4.65a 2.22 ± 0.04a 0.27 ± 0.04a
ind.arm8 2.18 ± 0.44a 14.70 ± 0.79a 16.32 ± 2.30b 40.88 ± 4.61b 2.29 ± 0.46a 0.35 ± 0.03b
mel.arm8 2.08 ± 0.16a 16.30 ± 1.01a 19.76 ± 4.06ab 41.25 ± 8.76b 1.81 ± 0.10a 0.37 ± 0.03b

Each value is the mean of nine replicates ( ± SD). ANOVA was performed using F test statistic, followed by LSD post-hoc test. Values followed by different lowercase
letters indicate significant differences (P < 0.05) between samples in the same sampling time. M2, M4, M6, and M8 represent P. armandii seedlings harvested on the
2nd, 4th, 6th, and 8th month after truffle inoculation, respectively. Control treatment, P. armandii seedlings without T. melanosporum and T. indicum colonization.
ind.arm treatment, P. armandii seedlings with T. indicum colonization. mel.arm treatment, P. armandii seedlings with T. melanosporum colonization.

treatment (P = 0.008), with significantly higher value in soil samples of
T. indium-colonized seedlings. Comparing with the Control, T. indium
colonization also significantly increased the soil TN under the 2nd
month (P = 0.009), 6th month (P = 0.003) and 8th month (P = 0.015)
after inoculation, while T. melanosporum colonization significantly in-
creased TN only on the 6th month after inoculation (P = 0.000). Soil
AN contents were significantly improved by T. indium colonization on
the 8th month after inoculation (P = 0.042), and they were not affected
by T. melanosporum colonization on the four observed months. Soil TP
showed obvious changes between different treatments from the 6th
month after inoculation, with significantly lower values in mel. arm
treatment (P = 0.002 on M6; P = 0.001 on M8). Both T. melanosporum
and T. indium inoculation significantly improved the AP contents in the
rhizosphere soil under the four sampling months (P = 0.000, 0.009,
0.002, 0.002 on M2, M4, M6, M8), however, the differences of AP be-
tween mel. arm and ind. arm treatments didn’t reach the significant
level.
by Control treatment (Fig. 3c, d).
In the first four month after inoculation, the richness and diversity
of bacterial community harbouring phoD genes showed no significant
differences in three different treatments, according to the ACE, Chao1,
Simpson and Shannon indices (Table 3). However, on the 6th month
after inoculation, Shannon and Simpson indices indicated that the soil
phoD-harbouring bacterial diversity was significantly decreased by T.
melanosporum colonization (P = 0.028; P = 0.033). And on the 8th
month after inoculation, the ACE and Chao1 values were also sig-
nificantly lower in mel. arm treatment (P = 0.045; P = 0.048), which
indicated that T. melanosporum inoculation significantly decreased the
richness of soil bacteria with phoD genes. Comparing with the Control
treatment, T. indium colonization didn’t affect the alpha diversity in-
dices of phoD-harbouring bacterial community.
3.5. Taxonomic composition and differences of phoD-harbouring bacterial
community

3.4. Alpha diversity of phoD-harbouring bacterial community
There were 1,272,571 high quality sequences obtained in all from
the 36 rhizosphere soil samples collected on the 2nd, 4th, 6th and 8th
month after truffle inoculation, which were clustered into 2330 OTUs
overall. The rarefaction curves of these OTUs in each sample are dis-
played in Fig. S1. The shared and unique OTUs number in the three
treatments under all the sampling time can be clearly seen from the
Venn diagrams (Fig. 3). The shared OTUs of mel. arm, ind. arm and
Control treatments gradually increased with the time going. On the 2nd
and 4th month after inoculation, there were more unique OTUs in
Control treatment (Fig. 3a, b), while on the 6th and 8th month after
inoculation, ind. arm treatments contained more unique OTUs, followed
A total of 5 phyla, 6 classes, 18 orders, 22 families and 32 genera
were identified in the 36 rhizosphere soil samples. At the phylum level,
Proteobacteria were dominant across all the samples, averagely ac-
counting for 71.86 %, followed by Actinobacteria (21.62 %) and
Gemmatimonadetes (6.30 %) (Fig. S2). The mel. arm and ind. arm
treatments contained more Proteobacteria under the four sampling
time. Contrary to Proteobacteria, Actinobacteria were significantly
more abundant in Control treatment compared with truffle-colonized
treatments on the 4th month after inoculation (P = 0.044). The
abundance of Gemmatimonadetes showed no significant differences
between the three treatments under all the sampling time.
At the class level, Alphaproteobacteria (34.85 %),
Gammaproteobacteria (23.27 %) and Betaproteobacteria (13.72 %)

Fig. 2. Physiology of P. armandii seedlings colonized or uncolonized by T. melanosporum or T. indicum. (a) root activity; (b) root SOD activity; (c) root POD activity.
M2, M4, M6, and M8 represent P. armandii seedlings harvested on the 2nd, 4th, 6th, and 8th month after truffle inoculation, respectively. Control treatment, P.
armandii seedlings without T. melanosporum and T. indicum colonization. ind.arm treatment, P. armandii seedlings with T. indicum colonization. mel.arm treatment, P.
armandii seedlings with T. melanosporum colonization.

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X. Zhang, et al. Microbiological Research 239 (2020) 126520

Table 2
The soil properties in the rhizosphere of P. armandii seedlings with or without T. melanosporum and T. indicum colonization under different sampling time.
Sampling time Treatments pH OM (g/kg) TN (g/kg) AN (mg/kg) TP (mg/kg) AP (mg/kg)

M2 Control2 8.78 ± 0.03a 28.89 ± 1.20a 0.86 ± 0.01a 38.50 ± 7.00a 1.83 ± 0.06a 4.00 ± 0.60a
mel.arm2 8.76 ± 0.04a 31.83 ± 1.45b 0.85 ± 0.00a 38.48 ± 0.98a 1.77 ± 0.07a 6.64 ± 0.28b
ind.arm2 8.64 ± 0.06b 32.08 ± 0.60b 0.95 ± 0.04b 40.83 ± 2.02a 1.75 ± 0.17a 6.32 ± 0.23b
M4 Control4 8.83 ± 0.04a 31.15 ± 1.19a 0.85 ± 0.02a 37.33 ± 5.35a 1.71 ± 0.10a 4.59 ± 0.67a
mel.arm4 8.87 ± 0.07a 32.76 ± 0.53b 0.89 ± 0.06a 36.17 ± 4.04a 1.77 ± 0.21a 8.34 ± 0.61b
ind.arm4 8.81 ± 0.08a 31.84 ± 1.18ab 0.99 ± 0.07a 37.33 ± 4.04a 1.75 ± 0.21a 8.81 ± 1.71b
M6 Control6 8.86 ± 0.08a 29.11 ± 0.73a 0.75 ± 0.06a 35.00 ± 3.50a 1.71 ± 0.14ab 4.24 ± 1.04a
mel.arm6 8.87 ± 0.08a 30.45 ± 0.47b 0.91 ± 0.02b 36.17 ± 2.02a 1.56 ± 0.02a 8.65 ± 0.89b
ind.arm6 8.85 ± 0.12a 32.48 ± 0.68c 0.99 ± 0.02c 44.33 ± 5.35a 1.82 ± 0.03b 8.63 ± 0.66b
M8 Control8 8.96 ± 0.03a 28.61 ± 0.55a 0.81 ± 0.09a 39.67 ± 2.02a 1.84 ± 0.03a 3.95 ± 0.57a
mel.arm8 8.86 ± 0.09a 30.32 ± 1.17b 0.91 ± 0.02ab 39.33 ± 1.89a 1.69 ± 0.02b 9.77 ± 1.58b
ind.arm8 8.83 ± 0.09a 30.17 ± 0.12b 1.01 ± 0.03b 44.33 ± 2.02b 1.81 ± 0.02a 9.03 ± 0.74b

OM, organic matter; TN, total nitrogen; TP, total phosphorus; AN, available nitrogen; AP, available phosphorus. Each value is the mean of three replicates ( ± SD).
ANOVA was performed using F test statistic, followed by LSD post-hoc test. Values followed by different lowercase letters indicate significant differences (P < 0.05)
between samples in the same sampling time. M2, M4, M6, and M8 represent rhizosphere soil samples of P. armandii seedlings harvested on the 2nd, 4th, 6th, and 8th
month after truffle inoculation, respectively. Control treatment, rhizosphere soil without T. melanosporum and T. indicum pattern. ind.arm treatment, rhizosphere soil
with T. indicum pattern. mel.arm treatment, rhizosphere soil samples with T. melanosporum pattern.

Fig. 3. Venn diagrams of shared and unique phoD-harbouring bacterial OTUs in different treatments under different sampling time. M2, M4, M6, and M8 represent
rhizosphere soil samples of P. armandii seedlings harvested on the 2nd, 4th, 6th, and 8th month after truffle inoculation, respectively. Control treatment, rhizosphere
soil without T. melanosporum and T. indicum pattern. ind.arm treatment, rhizosphere soil with T. indicum pattern. mel.arm treatment, rhizosphere soil samples with T.
melanosporum pattern.

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X. Zhang, et al. Microbiological Research 239 (2020) 126520

Table 3
The richness and diversity indices of phoD-harbouring bacterial community in the rhizosphere soil of P. armandii seedlings with or without T. melanosporum and T.
indicum colonization under different sampling time.
Sampling time Treatments Simpson Shannon Chao1 ACE

M2 Control2 0.96 ± 0.00a 6.39 ± 0.20a 583.42 ± 28.84a 589.88 ± 24.41a

mel.arm2 0.97 ± 0.01a 6.19 ± 0.14a 594.97 ± 75.47a 597.96 ± 68.27a
ind.arm2 0.92 ± 0.10a 5.71 ± 1.47a 580.22 ± 206.54a 599.31 ± 215.70a
M4 Control4 0.96 ± 0.00a 6.61 ± 0.18a 721.01 ± 24.55a 725.89 ± 19.15a
mel.arm4 0.89 ± 0.04a 5.41 ± 2.14a 530.79 ± 355.05a 534.02 ± 357.39a
ind.arm4 0.96 ± 0.02a 6.33 ± 0.34a 616.11 ± 61.43a 624.28 ± 41.15a
M6 Control6 0.96 ± 0.00ab 6.41 ± 0.24a 702.13 ± 87.62a 693.22 ± 82.21a
mel.arm6 0.94 ± 0.02b 5.92 ± 0.31b 643.4 ± 57.45a 666.67 ± 58.62a
ind.arm6 0.97 ± 0.01a 6.45 ± 0.13a 686.18 ± 79.43a 703.84 ± 65.37a
M8 Control8 0.97 ± 0.00a 6.78 ± 0.23a 731.38 ± 84.46ab 735.36 ± 83.90ab
mel.arm8 0.97 ± 0.01a 6.55 ± 0.23a 671.68 ± 28.14a 689.26 ± 46.44a
ind.arm8 0.97 ± 0.01a 6.61 ± 0.24a 775.93 ± 2.44b 803.95 ± 5.37b

Each value is the mean of three replicates ( ± SD). ANOVA was performed using F test statistic, followed by LSD post-hoc test. Values followed by different lowercase
letters indicate significant differences (P < 0.05) between samples in the same sampling time. M2, M4, M6, and M8 represent rhizosphere soil samples of P. armandii
seedlings harvested on the 2nd, 4th, 6th, and 8th month after truffle inoculation, respectively. Control treatment, rhizosphere soil without T. melanosporum and T.
indicum pattern. ind.arm treatment, rhizosphere soil with T. indicum pattern. mel.arm treatment, rhizosphere soil samples with T. melanosporum pattern.

were the most abundant taxa in all the samples (Fig. S3). On the 4th
month after inoculation, the abundances of Alphaproteobacteria were
significantly increased by T. indicum colonization compared with mel.
arm treatment (P = 0.027). Gammaproteobacteria were more abundant
due to the colonization of T. melanosporum in the first 6 months. The
differences of Betaproteobacteria between three different treatments
under all the sampling time didn’t reach the significant level.
At the genus level, the ten most abundant bacteria with phoD genes
across all the samples were Bradyrhizobium (22.50 %), Pseudomonas
(17.01 %), Streptomyces (11.67 %), Saccharopolyspora (9.01 %),
Cupriavidus (7.13 %), Collimonas (6.56 %), Gemmatimonas (6.30 %),
Sinorhizobium (4.01 %), Pleomorphomonas (3.71 %), and Xanthomonas
(3.58 %) (Fig. 4). On the 4th month after inoculation, the abundances of
Bradyrhizobium were significantly lower in mel. arm treatments com-
pared with the ind. arm treatment (P = 0.012). Pseudomonas were
significantly richer in ind. arm treatment on the 8th month after in-
oculation (P = 0.042). Saccharopolyspora varied greatly over time, with
highest abundance on the 2th month after inoculation. T. melanosporum
and T. indicum colonization significantly decreased Saccharopolyspora
abundance on the 4th month after inoculation (P = 0.003). Sinorhizo-
bium were richer in truffle-colonized treatments under 2nd, 4th and 8th
month after inoculation, and were significantly increased by T. indicum

Fig. 4. Taxonomic composition of phoD-harbouring bacterial communities at the genus level. M2, M4, M6, and M8 represent rhizosphere soil samples of P. armandii
seedlings harvested on the 2nd, 4th, 6th, and 8th month after truffle inoculation, respectively. Control treatment, rhizosphere soil without T. melanosporum and T.
indicum pattern. ind.arm treatment, rhizosphere soil with T. indicum pattern. mel.arm treatment, rhizosphere soil samples with T. melanosporum pattern.

7
X. Zhang, et al.
Fig. 5. Heatmap (a) and comparison analysis (b) of the significantly differential genera among the three different treatments. Control treatment, rhizosphere soil
without T. melanosporum and T. indicum pattern. ind.arm treatment, rhizosphere soil with T. indicum pattern. mel.arm treatment, rhizosphere soil samples with T.
melanosporum pattern.
colonization on the 6th month after inoculation (P = 0.012). Xantho-
monas were more abundant in mel. arm treatment under four sampling
time, which were significantly increased by T. melanosporum coloniza-
tion on the 6th month after inoculation compared with the other two
treatments (P = 0.015). The abundances of Streptomyces, Cupriavidus,
Collimonas, Gemmatimonas and Pleomorphomonas didn’t show the sig-
nificant differences between the three treatments under all sampling
time.
Overall, taking the samples in different time together, Pseudomonas,
Sinorhizobium, Xanthomonas, Lysobacter and Chromobacterium were the
significantly differential genera among the mel. arm treatment, ind. arm
treatment and Control treatment (Fig. 5a). Pseudomonas and Sinorhi-
zobium were more abundant in truffle-colonized treatments compared
with Control, especially in ind. arm treatment (P = 0.010; P = 0.026)
(Fig. 5b). Xanthomonas were richer in mel. arm treatment compared
with the other two treatments (P = 0.028) while Lysobacter were sig-
nificantly more abundant in Control treatment (P = 0.011) (Fig. 5b).
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Microbiological Research 239 (2020) 126520
3.6. phoD-harbouring bacterial community structure and their correlation
with soil properties
The structural differences in bacterial community harbouring phoD
genes among the samples were estimated by NMDS analysis (Fig. S4),
which indicated that T. indicum and T. melanosporum colonization al-
tered the structure of phoD-harbouring bacterial community (PERMA-
NOVA, P = 0.001). More specifically, on the 2nd month after in-
oculation, the phoD-harbouring bacterial community structure in ind.
arm treatment differed significantly from that in the other two treat-
ments (PERMANOVA, P = 0.009) (Fig. 6a). On the 4th month after
inoculation, mel. arm and ind. arm treatments contained significantly
different structure of phoD-harbouring bacterial community with the
Control (PERMANOVA, P = 0.002) (Fig. 6b). On the 6th and 8th month
after inoculation, the phoD-harbouring bacterial community structures
in three different treatments were significantly separate from each
other (PERMANOVA, P = 0.008 and P = 0.004) (Fig. 6c, d).
X. Zhang, et al.
Fig. 6. Non-metric multidimensional scaling (NMDS) analysis of phoD-harbouring bacterial communities in rhizosphere soil of P. armandii seedlings. M2, M4, M6,
and M8 represent rhizosphere soil samples of P. armandii seedlings harvested on the 2nd, 4th, 6th, and 8th month after truffle inoculation, respectively. Control
treatment, rhizosphere soil without T. melanosporum and T. indicum pattern. ind.arm treatment, rhizosphere soil with T. indicum pattern. mel.arm treatment, rhi-
zosphere soil samples with T. melanosporum pattern.
The relationship between phoD-harbouring bacterial community
and soil properties could be observed in redundancy analysis (RDA)
(Fig. S5). Soil pH, OM and AP contents showed significantly correlation
with the composition of phoD-harbouring bacterial community
(P < 0.05).
3.7. Distribution of truffle mating type genes in the rhizosphere soil and their
relationship with soil properties
The absolute quantification results of mating type genes of T. mel-
anosporum and T. indicum were presented with their copy numbers
(Table 4). However, the MAT1−2-1 genes of T. melanosporum were not
detected in all the rhizosphere soil samples under the four sampling
time. The amplification efficiencies for T. melanosporum MAT1−1-1
genes, T. indicum MAT1−1-1 genes, and T. indicum MAT1−2-1 genes
were 70.8 %, 84.2 % and 90.2 %, respectively. And the corresponding
R2 values of the regression curves were 0.998, 0.992 and 0.996. The
abundances of T. melanosporum MAT1−1-1 genes were significantly
increased on the 4th month after inoculation (P = 0.03), and main-
tained this level until the 8th month. The MAT1−1-1 genes of T. in-
dicum in the rhizosphere soil showed no significant differences between
different sampling time. The MAT1−2-1 genes of T. indicum were sig-
nificantly less on the 4th and 6th month after inoculation comparing
with the other two months (P = 0.041). For T. indicum, the MAT1−1-1
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Microbiological Research 239 (2020) 126520
genes were dominated in all the samples of ind. arm treatment, rather
than the MAT1−2-1 genes, which exhibited the uneven distribution.
The ratio of T. indicum mating type genes (MAT1−1-1/ MAT1−2-1)
were significantly increased on the 4th month after inoculation (P =
0.038), which indicated that the distribution of T. indicum mating type
genes altered with the time.
The mating type genes of both truffles showed no significant cor-
relation with the detected soil properties (Table S4). However, the
distribution of T. indicum mating type genes (MAT1−1-1/ MAT1−2-1)
exhibited the significantly positive correlation with soil pH (P = 0.002)
and AP (P = 0.004).
4. Discussion
The mutualistic symbiosis of fungi and host plants through the ec-
tomycorrhizae is an important and long-term stage during the life cycle
of truffles, which also play an important role in the ecological en-
vironment (Mello and Balestrini, 2018). In the early symbiotic stage,
the quality of truffle-colonized seedlings, including the truffle coloni-
zation level and the growth and physiology of host plants, is the im-
portant factor that determines the production of truffle fruiting bodies
in the plantations (Andres-Alpuente et al., 2014), which is crucial for
the successful truffle cultivation. Meanwhile, the truffle-associated
microbes are the third partner involved in the growth and development
X. Zhang, et al.
Table 4
Real-time absolute quantification of T. melanosporum and T. indicum mating type genes in the rhizosphere soil of P. armandii seedlings under different time after
inoculation.
Treatments Sampling time MAT1−1-1 Absolute RT-PCR (copies μL−1)
ind.arm M2 6436.76 ± 1120.36a
M4 9454.15 ± 1027.25a
M6 8459.89 ± 514.43a
M8 22300.14 ± 2203.87a
mel.arm M2 333.36 ± 151.79a
M4 1175.77 ± 312.34b
M6 996.75 ± 300.70b
M8 978.16 ± 289.66b
Each value is the mean of three replicates ( ± SD). ANOVA was performed using F test statistic, followed by LSD post-hoc test. Values followed by different lowercase
letters indicate significant differences (P < 0.05) between samples in the same treatment. M2, M4, M6, and M8 represent rhizosphere soil samples of P. armandii
seedlings harvested on the 2nd, 4th, 6th, and 8th month after truffle inoculation, respectively. ind.arm treatment, rhizosphere soil with T. indicum pattern. mel.arm
treatment, rhizosphere soil samples with T. melanosporum pattern.
of truffles, which also contribute to truffle formation (Benucci and
Bonito, 2016; Chen et al., 2019). So in this study, we collected the
seedling samples and rhizosphere soil samples associated with T. mel-
anosporum and T. indicum under four periods in the early symbiotic
stage to assess, and found that the micro-ecology of these two truffles
was significantly changed compared with control group.
Four month after inoculation, the ectomycorrhizae of T. melanos-
porum and T. indicum were successfully detected, with the presence of
Hartig net, a mycelium network occupying the space between root
cortical and epidermal cells (Daba et al., 2019). Although the coloni-
zation rates of these two truffle species on P. armandii were not very
high compared with our previous study (Zhang et al., 2019), T. indicum
colonization level was significantly higher than T. melanosporum’s,
which indicated that the ectomycorrhizal development of T. indicum
was better and T. indicum was more suitable than T. melanosporum to
select P. armandii as the host plant. Actually, P. armandii is not the
natural host range of T. melanosporum, however, from the results of this
experiment, the domestication of T. melanosporum with P. armandii can
be performed in some native areas in China in the future.
The truffle-host plant interactions were reflected in the substance
transportation through the ectomycorrhizae, as well as the metabolic
signals produced by truffles and hosts (Daba et al., 2019). Many studies
showed that the mycorrhizal symbiosis can improve the nutrients
transportation between host plants and mycorrhizal fungi, which brings
developmental and physiological benefits for the hosts (Liese et al.,
2017; Álvarez-Lafuente et al., 2018). Turgeman et al. (2011) reported
that the inoculation of desert truffle Terfezi boudieri improved the bio-
mass accumulation and shoot-root ratios of Helianthemum sessiliflorum,
and it also increased the drought resistance of the host. Domínguez
Núñez et al. (2008) proved that the mycorrhization of Pinus halepensis
seedlings with T. melanosporum improved the plant height, ground
diameter, biomass and nutrient absorption of the seedlings. In this
study, although the changes of various plant growth indices showed
different trends with time after inoculation, on the whole, T. indicum
and T. melanosporum colonization could promote the growth of the P.
armandii to a certain extent, like improving the biomass, root-shoot
ratios and total root surface area of the host. However, the plant height
and stem circumferences were not affected by truffle colonization.
Some studies reported that the morphology of host plant roots could be
regulated by the colonization of ectomycorrhizal fungi like truffles,
including the root shortening, the increase of lateral roots and bran-
ches, which were resulted by the metabolic signals released by fungi
(Felten et al., 2009; Splivallo et al., 2009). This maybe could explain
why the total root surface area of P. armandii increased while the main
root length of them decreased in this study. As for the physiological
changes in this paper, the root activity, root SOD and POD activity of P.
armandii showed the enhancement in different time after T. melanos-
porum and T. indicum colonization. SOD and POD are free radical sca-
vengers widely existing in the organisms, which play the important
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Microbiological Research 239 (2020) 126520
MAT1−2-1 Absolute RT-PCR (copies μL−1) MAT1−1-1/MAT1−2-1
4265.13 ± 482.62a 1.52 ± 0.29a
1873.70 ± 150.42b 5.06 ± 0.51b
1816.42 ± 138.53b 4.86 ± 0.39b
4498.64 ± 2110.97a 4.79 ± 3.41ab
Not detected /
physiological functions in anti-oxidation, anti-stress, respiration and
photosynthesis (Bela et al., 2015; Luis et al., 2018). In our previous
studies, T. indicum inoculation improved the SOD activity of Quercus
acutissima roots on the 6th month after inoculation but had no obvious
effects on the host plant POD activity (Zhang et al., 2019b). In terms of
the comparision between these two truffle species, the effects of T.
melanosporum on the growth and physiology of P. armandii seedlings
were similar as that of T. indicum.
Soil properties are closely related to the truffle growth and their
production, also in turn, truffle colonization can have a feedback on the
soil properties (Ponce et al., 2014; Li et al., 2018). In our study, both T.
indicum and T. melanosporum colonization significantly improve the
organic matter and available P in the rhizosphere soil of P. armandii
seedlings on four sampling periods, especially available P, averagely
increased by 97 % in truffle-colonized treatments. T. indicum also en-
hanced the soil total and available N to some extent, however, the effect
of T. melanosporum on the improvement of soil N was not obvious as
that of T. indicum. Some previous studies showed that the colonization
of T. panzhihuanense and T. borchii on Corylus avellana seedlings could
significantly increase the soil available P (Li et al., 2019a; Yang et al.,
2019), which were similar as our results. However, the organic matter
was not altered obviously by T. borchii and T. panzhihuanense. Li et al.
(2018) reported that the organic matter, total and available N in the
rhizosphere of Quercus aliena could be enhanced by T. indicum mycor-
rhization. These changes of soil properties may contribute to truffle
ectomycorrhizal synthesis.
In the environment associated with Tuber, some soil microbes are
likely involved in the molecular mechanisms that control the formation
of truffle ascocarps (Siebyła and Hilszczańska, 2017; Chen et al., 2019).
Many studies have explored the truffle-associated microbes, including
the microbes in the brûlé areas, truffle ascocarps, truffle ectomycor-
rhizae and rhizosphere soil, and some bacteria or fungi have been found
to have the relationship with truffles (Mello et al., 2013; Vahdatzadeh
et al., 2015; Deveau et al., 2016; Chen et al., 2019). However, rare
reports have focused on a combination of the microbial community and
bacterial functional genes during the growth of truffles (Barbieri et al.,
2010; Chen et al., 2019), which may be beneficial to explore the spe-
cific roles of these truffle-associated microbes. In this study, due to the
significant changes of the soil available P after T. indicum and T. mel-
anosporum colonization and the importance of P cycling in the ecto-
mycorrhizal soil, phoD-harbouring bacterial community were detected.
phoD alkaline phosphatase was responsible for hydrolyzing a range of
organic phosphoesters, which could contribute to the release of avail-
able P (Ragot et al., 2015). However, the results found that the richness
or diversity of phoD-harbouring bacterial communities was decreased
by T. melanosporum colonization on the 6th and 8th month after in-
oculation. This may indicate that T. melanosporum may improve the
available P through other ways rather than phoD-harbouring bacteria,
e.g., through acid phosphatase (Fraser et al., 2017). Some reports also
X. Zhang, et al.
showed that truffles could produce some metabolites that inhibited the
growth of some other organism, which lead the microbial diversity
decreased in truffle brûlé or in the rhizosphere soil of truffle-colonized
seedlings (Streiblova et al., 2012; Mello et al., 2013; Zhang et al.,
2019b). phoD alkaline phosphatase was distributed across twenty bac-
terial phyla and was mainly found in Actinobacteria, Cyanobacteria,
Planctomycetes and Proteobacteria according to other researches
(Ragot et al., 2016; Luo et al., 2017). In this paper, Proteobacteria was
the most dominant taxa across all the samples, followed by Actino-
bacteria. Proteobacteria was increased while Actinobacteria was de-
creased with the colonization of truffles. At the genus level, a symbiotic
and free-living N2-fixer, Bradyrhizobium, was most abundant in all the
samples, which was also discovered in some truffle ascocarps, like T.
magnatum and T. indicum (Barbieri et al., 2010; Chen et al., 2019). Si-
norhizobium, also a N2-fixer with phoD genes, was more abundant in
truffle-colonized treatments, indicating that this taxa may play an im-
portant role in coupling N and P cycle in the truffle-associated en-
vironment. Pseudomonas was richer with the colonization of T. indicm in
this study. Previous reports showed that some species belonging to
Pseudomonas were beneficial to truffle ectomycorrhizal synthesis, like P.
fluorescens, known as the “mycorrhizal helper bacteria” (Dominguez
et al., 2012). Although some taxa with phoD genes exhibited no obvious
differences between different groups in this study, the structure of
phoD-harbouring bacterial communities were significantly altered by T.
melanosporum and T. indicum colonization under the four periods in the
early symbiotic stage, which suggested that the bacteria with phoD
genes played an important role in truffle growth. Some researches have
reported that soil pH and OM could drive the structure of phoD-har-
bouring bacterial communities in cropping systems or in orchard soil
(Cui et al., 2015; Ragot et al., 2016). In our study, the composition of
phoD-harbouring bacterial communities was found correlated with soil
pH, AP and OM. And OM and AP contents were affected by truffle
colonization in this study. So the phoD-harbouring bacterial community
structure had the feedback on these soil properties and truffle activities.
Due to the heterothallic patterns of T. melanosporum and T. indicum,
the coincidence probability of spatiotemporal distribution of strains
with different mating type genes may affect the fructification and yield
of truffles (Rubini et al., 2014). In this paper, the results showed that
the MAT1−1-1 genes of both T. melanosporum and T. indicum occupied
the dominant position in the rhizosphere soil, and the MAT1−2-1 genes
were not detected in the rhizosphere soil associated with T. melanos-
porum, suggesting that MAT1−1-1 genes seemed to be more adapted to
this condition than MAT1−2-1 genes. In truffle orchards, MAT1−1-1
was found more abundant than MAT1−2-1 in T. melanosporum ecto-
mycorrhizae in some previous studies (Linde and Selmes, 2012; Murat
et al., 2013; Taschen et al. (2016); De la Varga et al., 2017). Some
reports also found the occurrence of only one mating type gene in
truffle ectomycorrhizae, indicating a competition on the host roots
between the strains with opposite mating genes (Rubini et al., 2011a;
Murat et al., 2013). In terms of the sexual distribution of truffles in soil,
Murat et al. (2013) showed that, in Montemartano, all soil samples
contained single mating type, either MAT1−1-1 or MAT1−2-1. In
Rollainville, although many soil samples included both mating type
genes, some soils only contained MAT1−1-1 or MAT1−2-1. Rubini
et al. (2011a) obtained the similar results in Borgo Cerreto, which was
that the majority of soil samples exhibited single mating type, and this
corresponded to the mating type genes in the nearby ectomycorrhizae
of T. melanosporum. From these results, the two mating type genes could
co-exist in soil, however, the detection of the only one of them was
consistent with the dominant mating type in the ectomycorrhizae. The
distribution of T. indicum mating type genes in our study was altered
with the time, and the values of MAT1−1-1/MAT1−2-1 significantly
increased on the 4th month after inoculation, exhibiting an unbalanced
distribution from this time. The correlation analysis between mating
type genes and soil properties told us that the pH and available P may
play an important role in the distribution of T. indicum mating type
11
Microbiological Research 239 (2020) 126520
genes. So in truffle plantations, the management of soil pH and avail-
able P needs to be emphasized. Furthermore, the distribution and dy-
namic changes of mating type genes in truffle ectomycorrhizae and soils
after establishment in the field require further investigation.
In conclusion, after the dynamic monitoring, both T. melanosporum
and T. indicum colonization could promote the growth of P. armandii
seedlings to some extent, and T. indicum was more suitable for P. ar-
mandii than T. melanosporum according to the colonization rate. The
colonization of these two truffles also had a feedback on the soil, with
the changes of soil properties and soil phoD-harbouring bacterial com-
munities in the rhizosphere. The structure of phoD-harbouring bacteria
and the distribution of T. indicum mating type genes were found cor-
related with some soil properties. During the early stage of symbiosis,
these truffle-plant-soil-microbe interactions results could give us a
better understanding on truffle growth, which would be beneficial to
the subsequent cultivation of truffles.
Funding
This work was supported by the National Naltural Science
Foundation of China (No. 31900079).
Declaration of Competing Interest
The authors declare that they have no potential conflict of interest.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.micres.2020.126520.
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