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Article
Effects of Two Trichoderma Strains on Plant Growth,
Rhizosphere Soil Nutrients, and Fungal Community
of Pinus sylvestris var. mongolica Annual Seedlings
Saiyaremu Halifu 1, Xun Deng 2, Xiaoshuang Song 2and Ruiqing Song 1, *
1College of Forestry, Northeast Forestry University, Harbin 150040, China
2Institute of Forestry Protection, Heilongjiang Forestry Academy, Harbin 150040, China
*Correspondence: songrq1964@nefu.edu.cn; Tel.: +86-13804522836
Received: 16 June 2019; Accepted: 25 August 2019; Published: 2 September 2019
Abstract:
Trichoderma spp. are proposed as major plant growth-promoting fungi that widely exist
in the natural environment. These strains have the abilities of rapid growth and reproduction and
efficient transformation of soil nutrients. Moreover, they can change the plant rhizosphere soil
environment and promote plant growth. Pinus sylvestris var. mongolica has the characteristics of
strong drought resistance and fast growth and plays an important role in ecological construction and
environmental restoration. The effects on the growth of annual seedlings, root structure, rhizosphere
soil nutrients, enzyme activity, and fungal community structure of P. sylvestris var. mongolica were
studied after inoculation with Trichoderma harzianum E15 and Trichoderma virens ZT05, separately.
The results showed that after inoculation with T. harzianum E15 and T. virens ZT05, seedling biomass,
root structure index, soil nutrients, and soil enzyme activity were significantly increased compared
with the control (p<0.05). There were significant differences in the effects of T. harzianum E15 and
T. virens ZT05 inoculation on the growth and rhizosphere soil nutrient of P. sylvestris var. mongolica
(p<0.05). For the E15 treatment, the seedling height, ground diameter, and total biomass of seedlings
were higher than that those of the ZT05 treatment, and the rhizosphere soil nutrient content and
enzyme activity of the ZT05 treatment were higher than that of the E15 treatment. The results of
alpha and beta diversity analyses showed that the fungi community structure of rhizosphere soil
was significantly different (p<0.05) among the three treatments (inoculated with T. harzianum E15,
T. virens ZT05, and not inoculated with Trichoderma). Overall, Trichoderma inoculation was correlated
with the change of rhizosphere soil nutrient content.
Keywords:
Trichoderma spp.; growth promotion; Pinus sylvestris var. mongolica; soil microecological
environment; high-throughput sequencing
1. Introduction
Trichoderma species belong to Hyphomyceteales, Hyphomycetes, Deuteromycotina, and Eumycota.
Their existence in the natural ecological environment is widespread; for example, they are found in
plant seeds, the rhizosphere, the phyllosphere, corms, and soils. These species have plant growth
promotion and soil environment improvement abilities [
1
,
2
]. During the colonization in plant roots, the
mycelia of Trichoderma fungi twine around the plant roots to form an appressorium-like structure, then
penetrate the root epidermis layer and survive for a long time between the plant cells of the epidermis
and the cortex [
3
], having a direct promotional effect on the growth of seedlings [
4
–
6
], nutrient uptake in
the rhizosphere [
7
–
10
], and rhizosphere microbial community structure improvement [
11
–
14
]. Chang
and Baler [
15
] treated seeds or roots of pepper, Vinca, chrysanthemum, tomato, and cucumber with
a conidia suspension of Trichoderma harzianum T-203 and found that the germination rate of pepper
Forests 2019,10, 758; doi:10.3390/f10090758 www.mdpi.com/journal/forests
Forests 2019,10, 758 2 of 17
increased, the flowering period of Vinca came earlier, the number of chrysanthemum flowers increased,
the plant height and fresh weight of all these plants increased. Furthermore, the dry weight of tomato,
cucumber, and pepper fruits also increased significantly. During the interaction among Trichoderma
harzianum,Trichoderma virens, and Arabidopsis thaliana, the contents of the JA (jasmonic acid) and
(salicylic acid) SA and the number of lateral roots were all significantly increased [16,17].
Many nutrients in the soil exist in a sparingly soluble or insoluble state, which affects the circulation
of nutrients in the soil to some extent. Trichoderma species promote nutrient uptake by secreting organic
acids to dissolve minerals and activate nutrients in the soil, leading to the circulation and utilization of
nutrients in the soil. At the same time, due to the strong colonization ability of Trichoderma species, they
expand the contact area between the rhizosphere and soil and increase the secretion of extracellular
enzymes such as sucrase, urease, and phosphatase, as well as organic acids in the rhizosphere to
improve nutrient cycling and enzyme activity in the soil. Maeda [
18
] reported that Trichoderma species
can decompose nitrogen compounds into available nitrogen and release less NO
2
. Khan [
19
] and
Harman [
20
] found that Trichoderma species convert nutrients into effective nutrients to increase soil
nutrient circulation in the soil, enabling the reduction of the use of nitrogen fertilizer. Mbarki [
21
]
found that Trichoderma inoculation increased the effective nutrient content and the soil enzyme activity
to repair soil and promote plant growth. The imbalance of the soil microbial community structure
is the main cause of soil-borne diseases, and its diversity is an important indicator to measure soil
properties. Due to their advantages of fast growth and strong vitality, Trichoderma species rapidly
occupy the growth space and absorb the nutrients needed. The Trichoderma genus also has the feature
of hyperparasitism; it secretes cell wall-degrading enzymes such as chitinases, cellulases, xylanases,
glucanases, and proteinases. Trichoderma species absorb nutrients through degrading soil microbial
cells, leading to the change of the soil microbial community structure [
22
,
23
]. Wagner [
24
] and
Yadav [
25
] found that Trichoderma inoculation increases nutrient content and microbial biomass in
addition to improving the soil microbial community structure.
Mongolian pine (Pinus sylvestris var. mongolica), a geographical variety of Scots pine (P. sylvestris),
is naturally distributed in the Daxinganling mountains of China (50
◦
10
0
–53
◦
33
0
N, 121
◦
11
0
–127
◦
10
0
E),
in Honghuaerji of the Hulunbeier sandy plains of China (47
◦
35
0
–48
◦
36
0
N, 118
◦
58
0
–120
◦
32
0
E), and
in parts of Russia and Mongolia (46
◦
30
0
–53
◦
59
0
N, 118
◦
00
0
–130
◦
08
0
E) [
26
]. It is often planted as an
ornamental tree because of its height and greening characteristics. In addition, this tree is characterized
by cold hardiness, drought tolerance, strong adaptability, and rapid growth [
27
,
28
]. It is currently the
main coniferous tree species utilized in the“3-North Shelter Forest Program” and the “Sand-Control
Project” in China, and plays an important role in ecological construction and environmental restoration.
The excessive and uncontrolled use of chemical fertilizers and pesticides have resulted in various
adverse effects such as serious diseases, soil environmental damage, and poor growth of seedlings [
29
].
The utilization of beneficial microorganisms, including Trichoderma spp. and microbial metabolites, is a
new environmentally friendly plant health management method compared with the use of chemical
pesticides. This approach has the advantages of being pollution-free, residue-free, safe for natural
enemies, difficult to produce resistance, and conducive to human and animal safety, as well as the
advantage of environmental protection [30,31].
In this study, T. harzianum E15 (introduced from the University of Edinburgh, UK) and T. virens
ZT05 (isolated from the Zhanggutai Experimental Forest Farm of Liaoning Province, China) were
used to study the effects of Trichoderma spp. on the growth and root structure of annual seedlings of
P. sylvestris var. mongolica, on the physical and chemical properties of rhizosphere soil, and on the
fungi community structure. A comparison was made of effects of the introduced and local isolates of
Trichoderma strains on seedling growth and the soil environment. The research objectives include the
assessment of the effects of (1) T. harzianum strain E15 and T. virens strain ZT05 on annual seedling
growth and root structure of P. sylvestris var. mongolica; (2) T. harzianum strain E15 and T. virens strain
ZT05 on rhizosphere soil physicochemical properties and enzyme activities, which in turn affect the
annual seedlings of P. sylvestris var. mongolica; and (3) T. harzianum strain E15 and T. virens strain ZT05
Forests 2019,10, 758 3 of 17
on the fungi community structure annual in the rhizosphere soil in which the P. sylvestris var. Mongolica
seedlings were planted.
2. Materials and Methods
2.1. Organisms and Growth Conditions
Two Trichoderma strains were used in this research. T. harzianum E15 was introduced from the
University of Edinburgh, UK to China. T. virens ZT05 was isolated from the rhizosphere soil of the
P. sylvestris var. mongolica forest of the Zhanggutai Experimental Forest Farm of Liaoning Province
(42
◦
43
0
–42
◦
51
0
N, 121
◦
53
0
–122
◦
22
0
E), China. These two strains were grown on a PDA medium
(potato extract 12 g/L, dextrose 20 g/L, agar 14 g/L; Haibo Biotechnology, China) at pH 6.0. The
Trichoderma species on PDA medium was cut with a sterile puncher (
∅
=5 mm) after culturing for
5 days. Suspension cultures of the two Trichoderma strains were obtained by transferring the mycelium
inoculum to liquid PD medium (PDA medium without agar) separately. Seven-day suspension cultures,
maintained in the dark at 25 ◦C under agitation (150 rpm), were used to inoculate the seedlings [32].
The experiment seeds of P. sylvestris var. mongolica (purchased from the Zhanggutai Experimental
Forest Farm in Zhangwu County, Liaoning Province, China) were surface-sterilized with potassium
permanganate (0.5%, v/v) for 30 min, then washed five times with sterile distilled water. They were
then germinated on sterile moistened gauze at 25
◦
C for 5 days. After germination, the seedlings were
transferred to plastic pot (15
×
15 cm, 20 seeds per pot) filled with a sterile culture substrate—namely, a
peat soil/vermiculite/sand (2:1:1, v/v/v) mixture that was sterilized in a high-temperature autoclave
for 2 h at 121
◦
C. The pots were kept under greenhouse conditions (day/night thermal regime of
22/30 ±3◦C
, and 14 h light/10 h dark photoperiod) and watered every 2 days for 1 month, after which
the seedlings were inoculated with the fungi [33,34].
2.2. Experimental Design and Seedling Inoculation
For all treatments, including the control, 20 pots (~20 seedlings per pot) were prepared, giving a
total of 400 seedlings per treatment. There were three treatments: (1) inoculation with PD blank culture
medium (CK); (2) single inoculation with T. virens ZT05; and (3) single inoculation with T. harzianum
E15. The inoculations were performed by transferring 100 mL of the fungal suspension culture into the
planting hole [
35
], where it was introduced at the root system level. The control plants were inoculated
with 100 mL PD blank culture medium. All treatments were arranged at random under the greenhouse
conditions given above.
2.3. Sampling and Analysis of Seedlings
The seedlings were harvested 3 months after inoculation. The harvest was conducted without
damaging the root system, which was carefully washed to remove the soil. A total of 50 seedlings
per treatment were randomly selected, the first 30 seedling were used to measure the biomass index.
For each seedling, the biomass index included calculations of the plant height, ground diameter, fresh
weight, and dry weight at harvest. Once the fresh weight had been measured, the seedlings were
oven-dried at 85 ◦C for 5 h to measure the dry weight.
2.4. Soil Properties Analysis
Soil samples were collected from 100 randomly selected seedlings per treatment. Rhizosphere
soil samples were collected from the root zone within 5 mm using a brush and passed through 1 mm
mesh screen. Soil samples used to determine the enzyme activity and physicochemical properties
were air-dried at 25
◦
C and collected into sterile sample bags, then kept in a 5
◦
C refrigerator until
further assays.
Organic matter (OM) was measured using the potassium dichromate oxidation heating method [
36
].
Total nitrogen (TN) was determined using the Kjeldahl method [
36
], total phosphorus (TP) was
Forests 2019,10, 758 4 of 17
determined using Mo–Sb colorimetry [
36
], available phosphorus (AP) was determined using the
antimony bismuth anti-colorimetric method with double acid leaching, rapidly available potassium
(AK) was measured using a NH4OAc leaching flame photometer [
36
], and total potassium was
determined using aflame photometer [
36
]. A pH meter [
36
] was used to determine soil pH (1:2.5).
The soil saccharase, catalase, acid phosphatase, and urease activities were measured using a kit
from Nanjing.
2.5. Fungal Diversity Analysis
The rhizosphere soil samples collected according to the method described above and 5.0 g soil
samples per biological repetition were placed in 50 mL sterile centrifugal tube and transported to
the laboratory in a cooler with an icepack. Soil samples used for high-throughput sequencing were
stored in a centrifuge tube at
−
80
◦
C [
37
–
39
] until soil DNA extraction. For the high-throughput
sequencing of soil microorganisms, the total genomic DNA was extracted from 0.5 g of soil using an
EZNA Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer ’s instructions.
DNA was eluted with 100
µ
L of elution solution from the kit. The DNA sample concentration and
quality (A260/A280 ratio) were measured using a NanoDrop2000 spectrophotometer (Thermo Scientific,
Walthan, MA, United States). Each treatment had three replicates in our experiment. High-throughput
sequencing analysis of the ITS region was performed to determine soil fungal communities. For each
treatment, three replicates were sequenced. The primers ITS1 (5
0
-CTTGGTCATTTAGAGGAAGTAA-3
0
)
and ITS2 (5
0
-GCTGCGTTCTTCATCGATGC-3
0
) were used to amplify the ITS1 region of the fungal
ITS [
10
–
13
]. PCR was performed in a 20
µ
L reaction system: 4
µ
L of 5
×
FastPfu buffer, 2
µ
L of 2.5 m
MdNTPs, 0.8
µ
L of each primer (5
µ
M), 0.4
µ
L of FastPfu polymerase, 0.2
µ
L of BSA, 10 ng of template
DNA, and 11.6
µ
L of double-distilled water [
37
,
38
]. The PCR conditions were as follows: 95
◦
C
for 3 min, 27 cycles of 30 s at 95
◦
C, 30 s at 55
◦
C, and 30 s at 72
◦
C, and with a final extension of
10 min at 72
◦
C. After PCR amplification, the obtained products were purified using an AxyPrep DNA
Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified with QuantiFluor-ST
(Promega, USA). Then, the purified amplicons were pooled in equimolar concentrations as a single
aliquot and employed for library construction, and sequencing was performed on an Illumina MiSeq
sequencer at Majorbio Biotechnology Co., Ltd. (Shanghai, China). Trimmomatic and FLASH software
were employed to quality-filter and merge raw fastqfu files [
39
], while UPARSE software (version
7.1, http://drive5.com/uparse/) was employed to further analyze the pyrosequencing data. The
sequences were then divided into operational taxonomic units (OTUs) with a 97% similarity cutoff,
after which chimeras were removed using UCHIME [
40
,
41
]. RDP Classifier (http://rdp.cme.msu.edu/)
was employed for the taxonomic annotation of each sequence within the confidence threshold of 0.7.
2.6. Data Analyses
Excel 2013 software was used for data processing. The differences of plant biomass, root structure
index, soil pH, chemical properties, and soil enzymes were determined by one-way analysis of variance
(ANOVA) in IBM SPSS 22.0 (IBM Corporation, New York, NY, USA). In addition, Pearson’s method was
performed for correlation analysis [
37
]. The differences were considered statistically significant at a 0.05
probability level in this study. Mothur software [
42
] was employed to analyze the alpha diversity index
and rarefaction, and the coverage index was used to represent the sequencing depth index. The Ace
(https://www.mothur.org/wiki/Ace) and Chao1 (https://www.mothur.org/wiki/Chao) indexes were used
to represent the community abundance, while the Shannon (https://www.mothur.org/wiki/Shannon)
and Simpson (https://www.mothur.org/wiki/Simpson) indexes were used to represent the species
richness and diversity of the fungal community [
43
,
44
]. Additionally, the similarities analysis (ANOSIM)
and heatmap analysis were calculated in the vegan package of R language [
45
,
46
] and the Unifrac
distance calculation was performed for fungal beta diversity comparisons. The figures were generated
using Origin 2019b (Origin Lab Corporation, Northampton, MA, USA).
Forests 2019,10, 758 5 of 17
3. Results and Analysis
3.1. Effects of Trichoderma Inoculation on Seedling Growth
3.1.1. Seedling Height
Trichoderma inoculation promoted the growth of seedlings, as indicated by the significantly
different heights between the control and treated groups (p<0.05) (Figure 1). Specifically, the seedling
height of the groups treated with ZT05 and E15 increased by 18.89% and 27.63%, respectively, relative
to the control. Additionally, the height of seedlings treated with E15 was 7.35% greater than the height
of those treated with ZT05.
Forests 2019, 10, x FOR PEER REVIEW 5 of 17
calculation was performed for fungal beta diversity comparisons. The figures were generated using
Origin 2019b (Origin Lab Corporation, Northampton, MA, USA).
3. Results and Analysis
3.1. Effects of Trichoderma Inoculation on Seedling Growth
3.1.1. Seedling Height
Trichoderma inoculation promoted the growth of seedlings, as indicated by the significantly
different heights between the control and treated groups (p < 0.05) (Figure 1). Specifically, the seedling
height of the groups treated with ZT05 and E15 increased by 18.89% and 27.63%, respectively, relative
to the control. Additionally, the height of seedlings treated with E15 was 7.35% greater than the height
of those treated with ZT05.
3.1.2. Seedling Diameter
As shown in Figure 1, the diameters of seedlings in the ZT05 and E15 treatment groups increased
by 27.50% and 68.50%, respectively, relative to the control. Moreover, the E15 treatment group
exhibited diameters increased 32.16% more than the ZT05 treatment group.
3.1.3. Seedling Biomass
Trichoderma inoculation increased seedling biomass (Figure 1). The fresh weight of seedlings
inoculated with ZT05 and E15 increased by 125.00% and 200.00%, respectively, compared to the
control, and the seedling fresh weight of the E15 treatment group increased 33.33% more compared
to the ZT05 treatment group. The dry weight of seedlings inoculated with ZT05 and E15 increased
by 28.57% and 44.44%, respectively, compared to the control, and the seedling dry weight of the E15
treatment group increased 22.22% more compared to the ZT05 treatment group.
Figure 1. Effects of Trichoderma inoculation on plant growth. CK: inoculation with PD blank culture
medium. ZT05: single inoculation with T. virens ZT05. E15: single inoculation with T. harzianum E15.
Figure 1.
Effects of Trichoderma inoculation on plant growth. CK: inoculation with PD blank culture
medium. ZT05: single inoculation with T. virens ZT05. E15: single inoculation with T. harzianum E15.
Different letters indicate significant difference at p<0.05, according to Duncan’s new multiple range
test. Vertical bars indicate standard error (SE).
3.1.2. Seedling Diameter
As shown in Figure 1, the diameters of seedlings in the ZT05 and E15 treatment groups increased
by 27.50% and 68.50%, respectively, relative to the control. Moreover, the E15 treatment group exhibited
diameters increased 32.16% more than the ZT05 treatment group.
3.1.3. Seedling Biomass
Trichoderma inoculation increased seedling biomass (Figure 1). The fresh weight of seedlings
inoculated with ZT05 and E15 increased by 125.00% and 200.00%, respectively, compared to the control,
and the seedling fresh weight of the E15 treatment group increased 33.33% more compared to the ZT05
treatment group. The dry weight of seedlings inoculated with ZT05 and E15 increased by 28.57% and
44.44%, respectively, compared to the control, and the seedling dry weight of the E15 treatment group
increased 22.22% more compared to the ZT05 treatment group.
Forests 2019,10, 758 6 of 17
3.2. Effect of Trichoderma Inoculation on the Root Structure of Seedlings
Root length and surface area are important parameters for measuring the distribution of roots,
while root average diameter, tip number, and branch number are important parameters for measuring
root absorption efficiency. As shown in Table 1and Figure 2,Trichoderma inoculation significantly
increased root system parameters such as root length, root surface area, average root diameter, and
number of root tips and branches (p<0.05).When compared with the control, treatment with ZT05
increased root length by 25.11%, root surface area by 98.19%, average root diameter by 5.66%, root
tip number by 45.89% and branch number by 74.42%.When compared with the control, treatment
with E15 increased these parameters by 3.43%, 18.21%, 3.77%, 22.10%, and 31.40%, respectively. When
compared with treatment with E15, these indexes were 20.96%, 67.66%, 1.82%, 19.48%, and 32.74%
higher, respectively, following treatment with ZT05.
Table 1.
Effects of Trichoderma inoculation on seedling root structure. CK: inoculation with PD blank
culture medium. ZT05: single inoculation with T. virens ZT05. E15: single inoculation with T. harzianum
E15. Different letters in the columns indicate significant differences (p<0.05), according to Duncan’s
new multiple range test.
Index CK ZT05 E15
Root length/cm 68.14 ±0.81 B 85.25 ±0.57 A 70.48 ±1.88 B
Surface area/cm211.59 ±0.10 C 22.97 ±0.72 A 13.70 ±0.31 B
Average diameter/mm 0.53 ±0.00 C 0.56 ±0.01 A 0.55 ±0.00 B
Apical number 100.90 ±2.13 C 147.20 ±0.99 A 123.20 ±2.09 B
Bifurcation number 17.20 ±0.36 C 30.00 ±0.63 A 22.60 ±0.83 B
Forests 2019, 10, x FOR PEER REVIEW 6 of 17
Different letters indicate significant difference at p < 0.05, according to Duncan’s new multiple range
test. Vertical bars indicate standard error (SE).
3.2. Effect of Trichoderma Inoculation on the Root Structure of Seedlings
Root length and surface area are important parameters for measuring the distribution of roots,
while root average diameter, tip number, and branch number are important parameters for
measuring root absorption efficiency. As shown in Table 1 and Figure 2, Trichoderma inoculation
significantly increased root system parameters such as root length, root surface area, average root
diameter, and number of root tips and branches (p < 0.05).When compared with the control, treatment
with ZT05 increased root length by 25.11%, root surface area by 98.19%, average root diameter by
5.66%, root tip number by 45.89% and branch number by 74.42%.When compared with the control,
treatment with E15 increased these parameters by 3.43%, 18.21%, 3.77%, 22.10%, and 31.40%,
respectively. When compared with treatment with E15, these indexes were 20.96%, 67.66%, 1.82%,
19.48%, and 32.74% higher, respectively, following treatment with ZT05.
Table 1. Effects of Trichoderma inoculation on seedling root structure. CK: inoculation with PD blank
culture medium. ZT05: single inoculation with T. virens ZT05. E15: single inoculation with T.
harzianum E15. Different letters in the columns indicate significant differences (p < 0.05), according to
Duncan’s new multiple range test.
Index CK ZT05 E15
Root length/cm 68.14 ± 0.81 B 85.25 ± 0.57 A 70.48 ± 1.88 B
Surface area/cm2 11.59 ± 0.10 C 22.97 ± 0.72 A 13.70 ± 0.31 B
Average diameter/mm 0.53 ± 0.00 C 0.56 ± 0.01 A 0.55 ± 0.00 B
Apical number 100.90 ± 2.13 C 147.20 ± 0.99 A 123.20 ± 2.09 B
Bifurcation number 17.20 ± 0.36 C 30.00 ± 0.63 A 22.60 ± 0.83 B
Figure 2. Effects of Trichoderma inoculation on seedling root structure of CK, ZT05, E15. CK:
inoculation with PD blank culture medium. ZT05: single inoculation with T. virens ZT05. E15: single
inoculation with T. harzianum E15.
Figure 2.
Effects of Trichoderma inoculation on seedling root structure of CK, ZT05, E15. CK: inoculation
with PD blank culture medium. ZT05: single inoculation with T. virens ZT05. E15: single inoculation
with T. harzianum E15.
Forests 2019,10, 758 7 of 17
3.3. Effects of Trichoderma Inoculation on Physicochemical Properties of Seedling Rhizosphere Soil
As shown in Table 2, significant differences were observed between the control and ZT05 and E15
treatment groups (p<0.05). Specifically, the organic content of the control group was higher than that
of the treatment groups inoculated with Trichoderma. This may have been due to the high capacity of
Trichoderma to transform soil nutrients. Specifically, Trichoderma species can rapidly degrade nutrients
produced by photosynthesis into a state in which they can be used for plant growth. The ability
of T. virens ZT05 to transform soil nutrients was higher than that of T. harzianum E15. Trichoderma
inoculation also significantly increased N and P nutrient contents in soil. This may be related to
the ability of Trichoderma to degrade soil macromolecular nutrients into an effective state for plant
utilization, thereby accelerating soil nutrient cycling and energy flow. However, organic matter refers
to compounds of different compositions with primary components of C, N, and P. Total potassium
levels showed no differences in CK, ZT05, and E15 groups (p<0.05), while the levels of available
potassium were organized in the order of CK >ZT05 >E15. These differences may have been a result
of diversified microbial communities, slower plant growth, and smaller root systems in the control
group than the two treatment groups. CO
2
released by root respiration as well as protons and organic
acids secreted during the growth of root tip cells can lead to a change in pH. In the present study,
the soil pH differed between the treated samples and the CK group (p<0.05), with the pH values of
samples treated with ZT05 and E15 increasing by 1.23% and 1.06%, respectively. This may be related to
Trichoderma promoting plant growth by reducing plant respiration.
Table 2.
Effects of Trichoderma inoculation on soil nutrients and soil enzyme activities of CK, ZT05, E15.
CK: inoculation with PD blank culture medium. ZT05: single inoculation with T. virens ZT05. E15:
single inoculation with T. harzianum E15. Different letters in the columns indicate significant differences
(p<0.05), according to Duncan’s new multiple range test.
Index CK ZT05 E15
OM g/kg 84.27 ±0.35 A 69.77 ±0.46 C 77.91 ±0.67 B
TN g/kg 2.20 ±0.01 C 2.61 ±0.00 A 2.40 ±0.01 B
AN mg/kg 206.26 ±0.03 B 219.59 ±0.59 A 191.41 ±0.36 C
TP g/kg 1.87 ±0.00 B 1.77 ±0.00 C 1.95 ±0.00 A
AP mg/kg 772.14 ±0.54 B 796.76 ±0.54 A 459.50 ±2.0 C
TK g/kg 7.08 ±0.07 A 7.13 ±0.09 A 7.06 ±0.12 A
AK mg/kg 235.86 ±0.26 A 161.41 ±0.48 B 135.38 ±0.07 C
pH value 5.68 ±0.00 B 5.75 ±0.01 A 5.74 ±0.01 A
Sucrase activity U/g 4.27 ±0.01 C 20.82 ±0.01 A 14.45 ±0.04 B
Catalase activity U/g 5.15 ±0.03 C 5.97 ±0.00 A 5.57 ±0.05 B
Acid phosphatase
activity U/g3.81 ±0.09 C 8.78 ±0.02 A 5.06 ±0.02 B
Urease activity U/g 938.26 ±0.08 C
1295.74
±
0.06 A
1094.43
±
2.42 B
3.4. Effects of Trichoderma Inoculation on Rhizosphere Soil Enzyme Activity
Soil enzymes, which comprise the most active organic component in the soil biochemical process,
are mainly derived from the secretions of soil microorganisms, plants, and animals. This component
plays an important role in soil organic matter circulation and energy conversion. As shown in Table 2,
Trichoderma inoculation led to a significant increase in the soil enzyme activity of the rhizosphere soil
of seedlings. When compared with the control group, the sucrase, catalase, acid phosphatase, and
urease activities of samples treated with ZT05 increased by 387.59%, 15.92%, 130.45%, and 38.02%,
respectively. Sucrase activity, catalase activity, acid phosphatase activity, and urease activity of samples
treated with E15 were increased by 238.41%, 8.16%, 32.81%, and 16.64%, respectively.
Sucrase activity, catalase activity, acid phosphatase activity, and urease activity in samples treated
with ZT05 showed increases that were 44.08%, 7.18%, 42.36%, and 15.54% greater, respectively, than
those seen in samples treated with E15. These results indicate that Trichoderma inoculation played an
Forests 2019,10, 758 8 of 17
important role in the circulation of nutrients and energy flow in soil, and strain ZT05 specifically had a
significant effect on promoting soil enzyme and nutrient cycle activity.
3.5. Effect on the Diversity of Rhizosphere Fungi of Seedlings
3.5.1. Soil Sample Sequencing Results and Sampling Depth Verification
A total of 541,936 fungal sequences was obtained from nine mixed soil samples in three treatments
using the Illumina MiSeqquome PE300 platform. Overall, 358 fungal OTUs were obtained upon OTU
clustering at 97% similarity after separation and elimination. The unique fungal OTUs of CK, ZT05,
and E15 groups amounted to 197, 17, and three, respectively. Overall, CK, ZT05, and E15 groups
shared 53 OTUs, CK and ZT05 groups shared 62 fungal OTUs, and CK and E15 groups shared 13
fungal OTUs. These findings indicate that the fungal communities of the CK and ZT05 groups were
more similar than the pairings of other groups (Figure 3).
Forests 2019, 10, x FOR PEER REVIEW 8 of 17
inoculation played an important role in the circulation of nutrients and energy flow in soil, and strain
ZT05 specifically had a significant effect on promoting soil enzyme and nutrient cycle activity.
3.5. Effect on the Diversity of Rhizosphere Fungi of Seedlings
3.5.1. Soil Sample Sequencing Results and Sampling Depth Verification
A total of 541,936 fungal sequences was obtained from nine mixed soil samples in three
treatments using the Illumina MiSeqquome PE300 platform. Overall, 358 fungal OTUs were obtained
upon OTU clustering at 97% similarity after separation and elimination. The unique fungal OTUs of
CK, ZT05, and E15 groups amounted to 197, 17, and three, respectively. Overall, CK, ZT05, and E15
groups shared 53 OTUs, CK and ZT05 groups shared 62 fungal OTUs, and CK and E15 groups shared
13 fungal OTUs. These findings indicate that the fungal communities of the CK and ZT05 groups
were more similar than the pairings of other groups (Figure 3).
Figure 3. Venn diagram showing the shared operational taxonomic units (OTUs) of CK, ZT05, E15
treatments. CK: inoculation with PD blank culture medium. ZT05: single inoculation with T. virens
ZT05. E15: single inoculation with T. harzianum E15.
3.5.2. Distribution of Soil Fungal Community
Categorical analysis of OTU representative sequences using a cutoff of 97% similarity revealed
a total of seven phyla, 19 classes, 56 orders, 89 families, 11 genera, and 197 species of soil fungi. As
shown in Figure 4, Ascomycota was the dominant fungi shared by the control, ZT05, and E15 groups
(relative abundance ≥10%). In addition, Ascomycota was dominant in the control group (relative
abundance 83.7%), while the relative abundances of Basidiomycota, Zygomycota, unclassified-k-
fungi, and Chytridiomycota were 7.2%, 4.57%, 4.3%, and 0.086%, respectively. In the ZT05 treatment,
Ascomycota was the dominant fungi (relative abundance 84.8%), while the relative abundances of
Zygomycota, Chytridiomycota, unclassified-k-fungi, and Basidiomycota were 7.17%, 6.4%, 1.6%, and
0.04%, respectively. For the ZT05 treatment, Ascomycota was the dominant fungi (relative abundance
99.05%), while the relative abundances of Chytridiomycota, Zygomycota, unclassified-k-fungi, and
Basidiomycota were 0.33%, 0.31%, 0.27%, and 0.03%, respectively. Significant differences were
observed in the relevance abundances of Basidiomycota and unclassified-k-Fungi between the
control and ZT05 and E15 treatments. The relevance abundance of Zygomycotain in the ZT05 group
was significantly different from those of the control and E15 groups.
Figure 3.
Venn diagram showing the shared operational taxonomic units (OTUs) of CK, ZT05, E15
treatments. CK: inoculation with PD blank culture medium. ZT05: single inoculation with T. virens
ZT05. E15: single inoculation with T. harzianum E15.
3.5.2. Distribution of Soil Fungal Community
Categorical analysis of OTU representative sequences using a cutoffof 97% similarity revealed
a total of seven phyla, 19 classes, 56 orders, 89 families, 11 genera, and 197 species of soil fungi. As
shown in Figure 4, Ascomycota was the dominant fungi shared by the control, ZT05, and E15 groups
(relative abundance
≥
10%). In addition, Ascomycota was dominant in the control group (relative
abundance 83.7%), while the relative abundances of Basidiomycota, Zygomycota, unclassified-k-fungi,
and Chytridiomycota were 7.2%, 4.57%, 4.3%, and 0.086%, respectively. In the ZT05 treatment,
Ascomycota was the dominant fungi (relative abundance 84.8%), while the relative abundances of
Zygomycota, Chytridiomycota, unclassified-k-fungi, and Basidiomycota were 7.17%, 6.4%, 1.6%, and
0.04%, respectively. For the ZT05 treatment, Ascomycota was the dominant fungi (relative abundance
99.05%), while the relative abundances of Chytridiomycota, Zygomycota, unclassified-k-fungi, and
Basidiomycota were 0.33%, 0.31%, 0.27%, and 0.03%, respectively. Significant differences were observed
in the relevance abundances of Basidiomycota and unclassified-k-Fungi between the control and ZT05
and E15 treatments. The relevance abundance of Zygomycotain in the ZT05 group was significantly
different from those of the control and E15 groups.
Forests 2019,10, 758 9 of 17
Forests 2019, 10, x FOR PEER REVIEW 9 of 17
Figure 4. Relative abundance (%) of fungal phyla among CK, ZT05, E15 treatments, based on total
sequence reads. CK: inoculation with PD blank culture medium. ZT05: single inoculation with T.
virens ZT05. E15: single inoculation with T. harzianum E15.
The control group contained 134 genera of soil fungi, while the ZT05 group had 65 genera and
the E15 group had 33 genera. Figure 5 shows the community analysis of the top 10 fungi at the genus
level. In the control group, Fusarium, Phoma, and Gibberella were the dominant fungi genera (relative
abundance%), while the relative abundances of Trichoderma, Penicillium, Mortierella, Sphaerosporella,
Rhizophlyctis, unclassified-k-fungi, and Monographella were 1.09%, 5.16%, 1.72%, 7.31%, 0.08%, 4.34%,
and 3.99%, respectively. For the ZT05 group, Trichoderma was the dominant fungi (relative abundance
76.40%), while the relative abundances of Fusarium, Phoma, Gibberella, Penicillium, Mortierella,
Sphaerosporella, Rhizophlyctis, unclassified-k-fungi, and Monographella were 0.14%, 0.001%, 0.06%,
6.6%, 7.2%, 0.00%, 6.36%, 1.5%, and 0.0006%, respectively. For the E15 group, Trichoderma was the
dominant fungi (relative abundance 98.41%), while the abundances of all the other genera were less
than 0.33%.
Figure 4.
Relative abundance (%) of fungal phyla among CK, ZT05, E15 treatments, based on total
sequence reads. CK: inoculation with PD blank culture medium. ZT05: single inoculation with T. virens
ZT05. E15: single inoculation with T. harzianum E15.
The control group contained 134 genera of soil fungi, while the ZT05 group had 65 genera
and the E15 group had 33 genera. Figure 5shows the community analysis of the top 10 fungi at
the genus level. In the control group, Fusarium,Phoma, and Gibberella were the dominant fungi
genera (relative abundance%), while the relative abundances of Trichoderma,Penicillium,Mortierella,
Sphaerosporella,Rhizophlyctis, unclassified-k-fungi, and Monographella were 1.09%, 5.16%, 1.72%, 7.31%,
0.08%, 4.34%, and 3.99%, respectively. For the ZT05 group, Trichoderma was the dominant fungi
(relative abundance 76.40%), while the relative abundances of Fusarium,Phoma,Gibberella,Penicillium,
Mortierella,Sphaerosporella,Rhizophlyctis, unclassified-k-fungi, and Monographella were 0.14%, 0.001%,
0.06%, 6.6%, 7.2%, 0.00%, 6.36%, 1.5%, and 0.0006%, respectively. For the E15 group, Trichoderma was
the dominant fungi (relative abundance 98.41%), while the abundances of all the other genera were
less than 0.33%.
Forests 2019,10, 758 10 of 17
Forests 2019, 10, x FOR PEER REVIEW 10 of 17
Figure 5. Heat map showed the relative abundance of the top 10 genes at CK, ZT05, E15 treatments.
CK: inoculation with PD blank culture medium. ZT05: single inoculation with T. virens ZT05. E15:
single inoculation with T. harzianum E15.
3.5.3. Analysis of α Diversity Index
ANOVA of the ITS rDNA diversity index of the CK, ZT05, and E15 soil samples was conducted.
As shown in Table 3, the coverage indexes of the CK, ZT05, and E15 groups were close to 1, and they
did not differ significantly. These findings indicate that the sequencing results could accurately reflect
the actual situation of the tested soil samples. The Ace and Chao1 indexes of the soil samples were
found to be in the order of CK > ZT05 > E15, with obvious differences observed between CK and the
Figure 5.
Heat map showed the relative abundance of the top 10 genes at CK, ZT05, E15 treatments.
CK: inoculation with PD blank culture medium. ZT05: single inoculation with T. virens ZT05. E15:
single inoculation with T. harzianum E15.
3.5.3. Analysis of αDiversity Index
ANOVA of the ITS rDNA diversity index of the CK, ZT05, and E15 soil samples was conducted.
As shown in Table 3, the coverage indexes of the CK, ZT05, and E15 groups were close to 1, and they
did not differ significantly. These findings indicate that the sequencing results could accurately reflect
the actual situation of the tested soil samples. The Ace and Chao1 indexes of the soil samples were
found to be in the order of CK >ZT05 >E15, with obvious differences observed between CK and the
two treatment groups (p<0.05). No significant difference was observed between the ZT05 and E15
Forests 2019,10, 758 11 of 17
groups, indicating that the total number and the richness of fungi in CK soil were higher than those
in the two treatment groups. The total number and the richness of fungi communities in the ZT05
group were greater than those in the E15 group, but this difference was not significant. The Shannon
index was found to be in the order of CK >ZT05 >E15, with significant differences observed between
CK and the two treatment groups. No great difference was observed between the two treatments,
indicating that the fungal richness of CK soil was higher than those of the other two groups. The
Simpson index was found to be in the order of CK >E15 >ZT05, with significant differences observed
between CK and the two treatment groups, but not between ZT05 and E15. These findings indicate
that the complexity of CK soil was greater than that of the soil in ZT05 and E15 treatment groups;
furthermore, the complexity of ZT05 soil was greater than that of E15 soil, but not significantly.
Table 3.
Effects of Trichoderma inoculation on diversity indices of the soil fungal community in the CK,
ZT05, E15. CK: inoculation with PD blank culture medium; ZT05: single inoculation with T. virens
ZT05; E15: single inoculation with T. harzianum E15. Different letters in the columns indicate significant
differences (p<0.05), according to Duncan’s new multiple range test.
Samples Shannon
Index Simpson Index Chao1 Index ACE Index Coverage %
CK 2.85 ±0.44 A 0.151 ±0.07 A 260.04 ±16.39 A 257.51 ±17.23 A 0.99 ±0.00 A
ZT05 1.21 ±0.15 B 0.54 ±0.08 B 116.24 ±3.43 B 119.25 ±3.71 B 0.99 ±0.00 A
E15 0.58 ±0.02 B 0.71 ±0.01 B 82.72 ±5.71 B 100.71 ±12.45 B 0.99 ±0.00 A
p0.002 0.001 0.00 0.00 0.03
Pearson’s analysis was used to analyze the correlation between the
α
diversity index and the
physicochemical properties and enzyme activity (Table 4). The Ace, Chao1, and Shannon indexes
were positively correlated with organic matter, available nitrogen, available phosphorus, and available
potassium contents. In addition, theChao1 index was significantly positively correlated with the
available potassium (R
2
=1.00**, p<0.05, ** indicates a very significant difference), while the Ace,
Chao1, and Shannon indexes were negatively correlated with pH, total nitrogen, total phosphorus,
sucrase activity, catalase activity, urease activity, and acid phosphatase activity. These findings indicate
that Trichoderma has a crucial effect on soil nutrient cycling.
Table 4.
Correlation analysis of diversity indices and soil properties. The correlation coefficient
and significance were obtained using Pearson correlation analysis. Significant values are shown as:
** p<0.01.
pH
Value
Organic
Matter
Available
Nitrogen
Total
Nitrogen
Available
Phosphorus
Total
Phosphorus
Available
Potassium
Total
Potassium
Sucrase
Activity
Catalase
Activity
Urease
Activity
Acid
Phosphatase
Activity
Ace −0.97 0.76 0.29 −0.80 0.55 −0.04 1.00 −0.13 −0.88 −0.82 −0.76 −0.61
Chao1 −0.95 0.72 0.36 −0.75 0.60 −0.12 1.00** −0.64 −0.84 −0.77 −0.71 −0.56
Shannon −0.92 0.65 0.49 −0.69 0.68 −0.21 1.00 0.03 −0.79 −0.71 −0.64 −0.47
Simpson 0.90 −0.62 −0.48 0.66 −0.71 0.25 −0.99 −0.07 0.76 0.68 0.61 0.44
3.5.4. Analysis of βDiversity Index
The Bray–Curtis matrix was used to measure the heterogeneity of different sample communities
in the soils. As shown in Figure 6, CK, ZT05, and E15 groups were distributed in different quadrants
and the distribution distance was large, indicating that the composition of CK, ZT05, and E15 samples
differed greatly. Nonparametric results were subjected to ANOSIM, which revealed that the differences
between the fungal groups in the CK, ZT05, and E15 soil samples were greater than the within-group
differences (R=1), and that the differences were significant (p=0.003).
Forests 2019,10, 758 12 of 17
Forests 2019, 10, x FOR PEER REVIEW 12 of 17
Figure 6. PCoA(Principal co-ordinates analysis) ordination based on Bray–Curtis similarities of fungal
communities at CK, ZT05, E15 treatments. CK: inoculation with PD blank culture medium. ZT05:
single inoculation with T. virens ZT05. E15: single inoculation with T. harzianum E15.
4. Discussion
Trichoderma spp. are soil fungi widely distributed in the natural environment which form
symbioses with many plants [47]. The interaction between the plant rhizosphere and soil
microorganisms plays an important role in plant growth, yield, nutrient cycling, and energy
conversion in soil [48]. Plant root exudates promote the colonization of rhizosphere microorganisms,
while soil microorganisms improve plant growth and increase the amount of nutrients in the soil
environment by effectively utilizing plant photosynthates [49]. The beneficial effects of Trichoderma
spp. can be divided into direct and indirect beneficial effects. The direct beneficial effects include
promoting plant growth, promoting and improving plant root growth and structure [50], improving
seed vigor and growth [51,52], and decomposing, recycling, and utilizing soil nutrients [4,53,54]. Our
results show that inoculation with T. harzianum E15 and T. virens ZT05 could significantly promote
seedling growth and change root structure in annual seedlings of P. sylvestris var. mongolica. The
growth of seedlings inoculated with E15 was significantly higher than that of seedlings inoculated
with ZT05, and the growth and structure of seedling roots inoculated with ZT05 was higher than that
of seedling roots inoculated with E15.Many reports have shown that Trichoderma inoculation has
significant promotion effects on plants seedlings and crops yields, such as those of cotton [54], tomato
[55], and Leymus chinensis [8,30]. Harman [7] showed that the inoculation of Trichoderma spp. in maize
could significantly promote plant growth, change root structure, and increase root activity. Shen [56],
Fu [57], and Xiong [58] demonstrated that a Trichoderma agent stimulated banana root growth,
promoted plant growth, and increased fruit yield. Hung [59] and Zhang [30] showed that a mixture
of organic fertilizer and a Trichoderma agent could significantly improve plant growth and crop yield.
Trichoderma could be used as an organic fertilizer as a growth substrate to degrade soil nutrients and
improve the ability of plant photosynthesis, thereby improving plant growth. IAA is a molecule that
is synthesized by plants and a few microbes [60]. In plants, IAA plays a key role in root and shoot
development. The hormone moves from one part of the plant to another by specific transporter
systems that involve auxin importer (AUX1) and efflux (PIN1-7) proteins. IAA is a key regulator of
lateral root development and root hair development [61]. Studies have shown that all Trichoderma
Figure 6.
PCoA(Principal co-ordinates analysis) ordination based on Bray–Curtis similarities of fungal
communities at CK, ZT05, E15 treatments. CK: inoculation with PD blank culture medium. ZT05:
single inoculation with T. virens ZT05. E15: single inoculation with T. harzianum E15.
4. Discussion
Trichoderma spp. are soil fungi widely distributed in the natural environment which form symbioses
with many plants [
47
]. The interaction between the plant rhizosphere and soil microorganisms plays
an important role in plant growth, yield, nutrient cycling, and energy conversion in soil [
48
]. Plant
root exudates promote the colonization of rhizosphere microorganisms, while soil microorganisms
improve plant growth and increase the amount of nutrients in the soil environment by effectively
utilizing plant photosynthates [
49
]. The beneficial effects of Trichoderma spp. can be divided into direct
and indirect beneficial effects. The direct beneficial effects include promoting plant growth, promoting
and improving plant root growth and structure [
50
], improving seed vigor and growth [
51
,
52
], and
decomposing, recycling, and utilizing soil nutrients [
4
,
53
,
54
]. Our results show that inoculation
with T. harzianum E15 and T. virens ZT05 could significantly promote seedling growth and change
root structure in annual seedlings of P. sylvestris var. mongolica. The growth of seedlings inoculated
with E15 was significantly higher than that of seedlings inoculated with ZT05, and the growth and
structure of seedling roots inoculated with ZT05 was higher than that of seedling roots inoculated
with E15.Many reports have shown that Trichoderma inoculation has significant promotion effects on
plants seedlings and crops yields, such as those of cotton [
54
], tomato [
55
], and Leymus chinensis [
8
,
30
].
Harman [
7
] showed that the inoculation of Trichoderma spp. in maize could significantly promote
plant growth, change root structure, and increase root activity. Shen [
56
], Fu [
57
], and Xiong [
58
]
demonstrated that a Trichoderma agent stimulated banana root growth, promoted plant growth, and
increased fruit yield. Hung [
59
] and Zhang [
30
] showed that a mixture of organic fertilizer and a
Trichoderma agent could significantly improve plant growth and crop yield. Trichoderma could be
used as an organic fertilizer as a growth substrate to degrade soil nutrients and improve the ability
of plant photosynthesis, thereby improving plant growth. IAA is a molecule that is synthesized by
plants and a few microbes [
60
]. In plants, IAA plays a key role in root and shoot development. The
hormone moves from one part of the plant to another by specific transporter systems that involve
auxin importer (AUX1) and efflux (PIN1-7) proteins. IAA is a key regulator of lateral root development
Forests 2019,10, 758 13 of 17
and root hair development [
61
]. Studies have shown that all Trichoderma spp. isolated from different
geographical areas can secrete IAA and promote the growth of cucumber, bottle gourd, and bitter
gourd [
61
]. Contreras-Cornejo [
5
] showed that IAA, a mycelial secretion of Trichoderma spp., could
significantly improve plant and lateral root growth. The volatile and non-volatile secondary metabolites
of Trichoderma spp.—including 6-n-pentyl-6H-pyran-2-one (6PP), gliotoxin, viridin, harzianopyridone,
harziandione, and peptaibols [
62
,
63
]—have a significant growth-promoting effect on plants [
7
,
64
].
Vinale [
65
] showed that secondary metabolites of T. harzianum commercial strains T22 and T39, T.
atroviride P1, and T. harzianum A6 also had significant growth-promoting effects on plant growth.
In this study, Trichoderma inoculation was found to significantly increase the nutrient content and
soil enzyme activity in rhizosphere soil of P. sylvestris var. mongolica seedlings. T. virens ZT05 had a
more significant effect on soil nutrient and enzyme activity in the rhizosphere of seedlings compared
to T. harzianum E15. Many nutrient elements in soil exist in a slightly soluble or insoluble state, which
limits the normal circulation of nutrients in soil. Trichoderma spp. can change the pH value of plants in
the rhizosphere soil and secrete organic acids to degrade minerals such as large amounts of elements (P)
and trace elements (Fe, Mn, and Zn), and activate soil nutrients, thus promoting the uptake of nutrients
by plants as well as the recycling and utilization of nutrients in the soil environment [
50
,
66
,
67
]. At the
same time, Trichoderma spp. have a strong colonization ability which, with the growth and extension
of the root system, can increase the contact area between root and soil and increase the secretion of
extracellular enzymes such as sucrase, urease, phosphatase, and organic acids in the rhizosphere, so
as to improve the nutrient cycle and enzyme activity in the soil [
6
,
68
,
69
]. Yedidia [
70
] showed that
under a hydroponic system, T. harzianum T203 could significantly increase the nutrient conversion
and absorption of P, Fe, Mn, Zn, Cu, and Na, thus promoting cucumber growth and yield. Khan [
19
]
showed that Trichoderma spp. can significantly improve the degradation and absorption of P, K, Ca, Mg,
Cu, Fe, Mn, and Zn in fertilizers. Li [
71
] showed that the inoculation of tomato plants with Trichoderma
asperellum CHF78 could significantly increase the soil’s available nutrient content and plant nutrient
uptake ability. Zhai showed that T. asperellum ACCC30536 improved the yield of A. annua, while the
moisture, pH stability, organic matter content, and availability of nitrogen, phosphorus, and potassium
in inoculated soil were also significantly improved [
72
]. El-Katatny found that Trichoderma spp. are
important soil microorganisms that can have a significant effect on soil phosphorus, potassium, and
nitrogen fixation, as well as on the restoration of degraded soil environments [67].
In this study, the Illumina Miseqquome PE300 high-throughput sequencing results show that
Ascomycota was the dominant group in the rhizosphere soil of all treatments—CK, ZT05, and E15—and
the relative abundance of Ascomycota in the E15 treatment rhizosphere soil was 99.05%. At the genus
level, Fusarium,Phoma, and Gibberella were dominant in the CK treatment, while Trichoderma was the
dominant genus in the ZT05 and E15 treatments. The results of the alpha and beta diversity analyses
showed that Trichoderma spp. inoculation had a significant effect on the community structure of fungi
in the rhizosphere soil of seedlings; this is consistent with the research results of Yu [
73
], Shen [
60
],
Zhang [
30
]. Trichoderma spp. have the advantages of fast growth and vigorous vitality; thus, they can
occupy the growing space quickly and absorb the required nutrients. At the same time, Trichoderma
spp. can secrete cell wall-degrading enzymes including chitinases, cellulases, xylanases, glucanases,
and proteinases, which can degrade microbial cells in the soil environment to absorb nutrients, thus
changing the structure of the microbial community [
22
,
23
]. Stefania [
74
] showed that lemon plant soil
microbial biomass increased by 46% after Trichoderma inoculation. Likewise, Mclean [
75
] showed that
Trichoderma inoculation could change soil nutrient content and the soil microbial community structure
of grassland soil.
5. Conclusions
1. Trichoderma inoculation increased the total biomass, seedling height, ground diameter, root
length, root area, root diameter, number of root tips, and number of branches of P. sylvestris var.
mongolica seedlings, thereby increasing the absorption area and growth potential of seedlings. The
Forests 2019,10, 758 14 of 17
contribution of T. harzianum E15 to seedling height, ground diameter, and total biomass was more
significant. ZT05 had greater effects on root length, root area, root diameter, number of root tips, and
bifurcation of seedlings.
2. Trichoderma inoculation increased soil nutrient content and soil enzyme activity in the rhizosphere
soil of P. sylvestris var. mongolica annual seedlings. Specifically, Trichoderma inoculation had a significant
effect on the community structure of fungi in the rhizosphere soil of seedlings, especially at the
genus level.
Author Contributions:
S.H., X.D., and R.S. conceived and designed the study. S.H., X.D., and Y.A. performed the
experiments. S.H., X.D. and X.S. contributed to the sample measurement and data analysis. S.H. and X.D. wrote
the paper.
Funding:
This research was funded by [National Key Research and Development Program] grant number
(2017YFD0600101), [National Natural Science Foundation of China] grant number (31670649, 31700564, 31170597,
31200484).
Conflicts of Interest: The authors declare no conflict of interest.
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