Journal of Integrative Agriculture 2021, 20(5): 1266–1276

Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Transcriptomic insights into growth promotion effect of Trichoderma

afroharzianum TM2-4 microbial agent on tomato plants

ZHAO Juan, LIU Ting, LIU Wei-cheng, ZHANG Dian-peng, DONG Dan, WU Hui-ling, ZHANG Tao-tao,
LIU De-wen

Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, P.R.China

Abstract
Plant growth promoting fungi are receiving increased attention as valuable beneficial microorganisms in crop cultivation due
to their capacity to produce bioactive substances, promote plant growth and enhance immune defense functions. In this
study, a novel Trichoderma isolate, designated as TM2-4, was screened from healthy tomato rhizosphere soil and identified
as Trichoderma afroharzianum. Culture filtrate of the isolate TM2-4 displayed obvious bioactive substance production and
an evident effect in promoting tomato seed germination, with hypocotyl length, radical length and vigor index increased
by 28.7, 19.4 and 62.1%, respectively, after a 100-fold dilution treatment. To assess the promotion effect and related
mechanism of isolate TM2-4, the plant biological indexes and gene expression profiles of tomato plants treated with or
without T. afroharzianum TM2-4 microbial agent were investigated by greenhouse pot experiment and RNA sequencing. The
results demonstrated that T. afroharzianum TM2-4 significantly promoted tomato plant growth in terms of plant height, dry
weight, number of leaves per plant and root activity, through efficient colonization in the rhizosphere and root system of the
plants. Transcriptome analyses identified a total of 984 differentially expressed genes in T. afroharzianum microbial agent
inoculated tomato roots, which were mainly engaged in the biological process of phytohormone homeostasis, antioxidant
activity, as well as metabolic pathways including phenylpropanoid biosynthesis and glutathione metabolism. These findings
provide useful information for understanding the mechanism of isolate TM2-4 for tomato plant growth promotion, which would
facilitate further development of T. afroharzianum TM2-4 microbial agent for use in vegetable crop production.

Keywords: Trichoderma afroharzianum, Solanum lycopersicum L., plant growth promotion, transcriptome analyses, qRT-
PCR

1. Introduction

Tomato (Solanum lycopersicum L.) constitutes one of the

Received 3 January, 2020 Accepted 16 September, 2020
ZHAO Juan, E-mail: zhaojuan119882@163.com; Correspondence
LIU Ting, Tel/Fax: +86-10-51503337, E-mail: lting@163.com
© 2021 CAAS. Published by Elsevier B.V. This is an open
access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
doi: 10.1016/S2095-3119(20)63415-3
most important vegetable crops in the world, the fruits of
which are a rich source of antioxidants including vitamins,
phenolics, flavonoids and folic acid (Hernández-Herrera
et al. 2014; Perveen et al. 2015). To meet the demands
of international markets, tomato growers use excessive
amounts of chemical fertilizers to ensure high yields,
 ZHAO Juan et al. Journal of Integrative Agriculture 2021, 20(5): 1266–1276
which are sometimes hazardous to human health and the
environment (Mengistie et al. 2017). The growing concern
regarding intensive use of chemical fertilizers has resulted
in an increasing attention towards more eco-friendly planting
strategies (Wang et al. 2016). Proper use of beneficial
microbial agents represents a viable strategy to maintain the
sustainable and healthy development of tomato production
(Cai et al. 2015).
Plant growth promoting microbes are widely accepted
as offering an environmentally-friendly green approach
in agricultural cultivation, due to their ability to stimulate
plant growth, defend against phytopathogen infection and
prevent plant disease occurrence (Cai et al. 2015; Ashmita
et al. 2018). The existing literature is mainly focused on the
plant growth promoting rhizobacteria including the genera
Bacillus and Pseudomonas (Otieno et al. 2015; Gamez et al.
2019). Some researchers have shown the plant growth
promoting fungi such as Aspergillus flavus, Penicillium
chrysogenum, as well as Trichoderma koningiopsis, which
also act as beneficial microorganisms in the promotion of
plant growth and yield through systemic resistance induction
and bioactive substance production (Adiyadolgor et al.
2020; Saad et al. 2020). According to previous studies,
Trichoderma spp. may serve as prominent fungal species in
promoting plant growth through increasing plant resistance
and enhancing nutrient uptake, in addition to their role as
biocontrol agents (Shukla et al. 2012; Pascale et al. 2017;
Contreras-Cornejo et al. 2018; Moya et al. 2020).
With the rapid development of high-throughput sequencing
technology, an RNA seq-based approach is often applied to
explore the mechanisms of plant growth promoting actions
of microbes on host plants (Sugimura and Saito 2017; Li
et al. 2018). There are some reports on the transcriptional
responses of Trichoderma-inoculated host plants such as
rice (Doni et al. 2019), tomato (Coppola et al. 2019; De
Palma et al. 2019) and cucumber (Yuan et al. 2019). The
Trichoderma treatment usually leads to upregulation of a
number of functional genes related to plant growth, stress
tolerance and disease resistance at the transcriptome level.
However, previous studies were mainly focused on the use
of Trichoderma spore suspensions. For further registration
and application of the beneficial fungus as a microbial
inoculation agent, a plant growth promoting Trichoderma
isolate designated as TM2-4 was screened and prepared
as a microbial agent after solid state fermentation. The
present study was conducted to (i) characterize the species
taxonomic identity of the Trichoderma isolate TM2-4 based
on morphological characters and molecular methods; (ii)
detect the beneficial biological activities, growth promotion
effect, and rhizosphere colonization ability of the isolate
TM2-4 on tomato plants; (iii) understand the gene expression
pattern of tomato roots in response to Trichoderma microbial
1267
agent inoculation at the transcriptional level.
2. Materials and methods
2.1. Isolation and screening of beneficial Trichoderma
isolates
Rhizosphere soil samples were collected from healthy tomato
plants in the Beijing suburb of Shunyi District in China via
the diagonal method. Trichoderma isolates were obtained
using the soil dilution plate technique and purified by the
single spore method on Trichoderma selective medium
(TSM) (Elad and Chet 1983). Biological characters including
indoleacetic acid production, siderophore production,
phosphate solubilization, and 1-aminocyclopropane-1-
carboxylate (ACC) deaminase production, as well as
seed germination promotion were assessed to screen
Trichoderma isolates with plant growth promoting activities.
For the quantitative determination of indoleacetic acid
(IAA) like substances, the obtained Trichoderma isolates
were cultured in Czapek liquid media supplemented with
500 mg L–1 of L-tryptophan for 5 days at 150 r min–1 and
25°C. The IAA concentration in the supernatant was
calibrated based on a standard curve after 1 mL of the
culture supernatant was added to 4 mL of Salkowski reagent
(Gordon and Weber 1951). Trichoderma isolates were also
incubated in Chrome azurol S (CAS) liquid media for the
detection of siderophore production according to previous
literature (Machuca and Milagres 2003). Quantitative
calculation of phosphate solubilization ability was performed
by growing the Trichoderma isolates in NBRIP liquid media
at 25°C and 150 r min–1 for 5 days (Nautiyal 1999; Achal
et al. 2007). ACC deaminase activity of the Trichoderma
isolates was evaluated quantitatively by measuring the
amount of a-ketobutyrate produced by the deamination of
ACC (Viterbo et al. 2010). The cell-free culture filtrate of
Trichoderma isolate and its 10-, 100-, 200-, 500-fold dilutions
with sterile water were prepared (Zhao et al. 2018) for seed
germination testing on tomato via the standard procedure
of the International Seed Testing Association (ISTA 2005).
2.2. Morphological and molecular identification of
the Trichoderma isolate
Morphological identification of the Trichoderma isolate
was completed based on colony and microscopic
characteristic observations according to the ISTH
(International Subcommission on Trichoderma and
Hypocera Taxonomy) after 5 days incubation at 25°C on
potato dextrose agar (PDA) media (Gams and Bisset 2002).
Molecular identification was conducted by multiple sequence
analyses of three different phylogenetic markers, including
1268 ZHAO Juan et al. Journal of Integrative Agriculture 2021, 20(5): 1266–1276
the internal transcribed spacer (ITS) region (White et al.
1990), translation elongation factor 1-alpha (tef1-α) gene
(Samuels et al. 2002) and RNA polymerase II second largest
subunit (rpb2) gene (Sawant et al. 2017). The sequences
of the rDNA-ITS, tef1-α and rpb2 genes of the Trichoderma
isolate were deposited in the NCBI GenBank (https://www.
ncbi.nlm.nih.gov/genbank/). Phylogenetic analysis of the
combined sequences of the rDNA-ITS, tef1-α and rpb2
genes was computed using the maximum likelihood (ML)
method in PAUP* version 4.0b10 (Swofford 2002).
2.3. Microbial agent preparation of the Trichoderma
isolate by solid state fermentation
The obtained Trichoderma isolate with biological activities
was used to prepare the growth promoting microbial agent
according to the revised method of Maurya et al. (2012).
Conidia suspension of the Trichoderma isolate (2.0×106
CFU mL–1) was inoculated in a 250 mL Erlenmeyer flask
containing 50 mL potato dextrose (PD) medium, and
incubated at 28°C on a rotary shaker at 150 r min–1 for
3 days in the dark. Afterwards, 8 mL of the Trichoderma
culture was transferred to a 500-mL beaker containing 100
g of solid fermentation medium (wheat bran, 70 g; corn cob
powder, 30 g; mineral salt solution, 60 mL). The beakers
were covered with six layers of sterile gauze and incubated
at 28°C for 7 days under 60% RH condition. Then the
fermentation product was dried by natural wind, pulverized
and screened through 80-mesh screens for further use as
Trichoderma inoculation microbial agent.
2.4. Growth promotion experiment of Trichoderma
microbial agent on tomato plants
The solid fermentation microbial agent of Trichoderma isolate
(108 cfu g–1 conidia content) was used for the evaluation
of its colonization ability and growth promotion effect on
tomato plants (cv. Jiafen 16) in pots in a greenhouse. The
experiment was conducted using a completely randomized
design, with ten plants per treatment and three replications.
Twenty-day-old plug seedlings were transplanted to pots with
diameter of 12 cm. Tomato plants transplanted in the pots
containing soil with Trichoderma agent added at a proportion
of 10 g kg–1 of soil were used as the Trichoderma agent
inoculation treatment, while plants potted in soil without
Trichoderma agent addition were taken as controls. Fifty-
day-old plants were then carefully uprooted and washed
under tap water. The capacities of Trichoderma isolate to
colonize and persist in the rhizosphere soil or root system
of tomato plants were evaluated using soil dilution plate
and tissue culture methods on TSM medium (Zhao et al.
2012). The plant growth promoting effect was determined
by measuring major growth parameters including plant
height, fresh weight, dry weight, number of leaves per plant,
diameter of leaf canopy, diameter of stem base, root activity
and leaf chlorophyll content (Wu et al. 2019).
2.5. Transcriptome analysis of tomato roots treated
with Trichoderma microbial agent
To assess tomato plant responses to the Trichoderma agent
inoculation at the transcriptome level, RNA-seq analysis
was conducted on the tomato roots treated with or without
Trichoderma microbial agent in the growth promotion assay.
2.6. RNA extraction, library construction and Illumina
sequencing
Root tissues from tomato plants treated with or without
Trichoderma microbial agent were harvested for RNA
extraction using QIAGEN RNeasy Plant Mini Kit (QIAGEN
Co., Ltd., Shanghai, China). After quality and purity
checking, the RNA samples were used for mRNA-seq library
construction by NEBNext Ultra RNA Library Prep Kit (NEB,
USA). Libraries were then indexed, pooled and sequenced
on an Illumina Hi SeqTM 2000 (San Diego, CA, USA) platform
with the pair-end method at Majorbio Bio-pharm Technology
Co., Ltd. (Shanghai, China).
2.7. Transcriptome de novo assembly and annotation
After discarding adaptor sequences, empty reads and low-
quality sequences, the remaining high-quality reads were
mapped to the Solanum lycopersicum reference genome
(Solanum lycopersicum, SL2.50: http://plants.ensembl.org/
Solanum_lycopersicum/Info/Index). The unigenes were
obtained by reference-based assembly and then used for
BLAST search and annotation in the Gene Ontology (GO),
Kyoto Encyclopedia of Genes and Genomes (KEGG),
Eukaryotic Ortholog Groups (KOG), non-redundant protein
sequences (NR), Swiss-prot protein, and Pfam database.
The RNA sequencing data were deposited in the NCBI
Sequence Read Archive (SRA) database.
2.8. Function annotation and enrichment analysis of
differentially expressed genes (DEGs)
The fragments per kilobase per million reads method
(FPKM) was used to determine the unigene expression
levels (Trapnell et al. 2010). The GO (http://www.
geneontology.org) database was adopted to predict the
functions of DEGs in relation to biological processes (BP),
 ZHAO Juan et al. Journal of Integrative Agriculture 2021, 20(5): 1266–1276
cellular component (CC) and molecular functions (MF) by
the GO seq R package (Young et al. 2010). The KEGG
(http://www.genome.jp/kegg/) database was employed to
determine the possible signal transduction or metabolic
pathways in which the genes could possibly be involved by
the KOBAS Software (Mao et al. 2005).
2.9. qRT-PCR analysis of gene expression levels
Quantitative real-time PCR (qRT-PCR) was carried out to
verify the expression patterns of ten genes that exhibited
significant differences via RNA-seq analysis. Elongation
factor 1-alpha (EF1α) gene was used as an internal
reference. The primers for the selected DEGs were designed
using the Primer Premier 5.0 Software. RNA samples from
tomato roots treated with or without Trichoderma microbial
agent were both extracted and purified as described above.
The cDNA was synthesized from 1 μg of purified RNA
samples using KR106 Fast Quant RT Kit (Tiangen Biotech
Co., Ltd., China) following the manufacturer’s instructions.
The qRT-PCR amplification was performed using CFX 96
Real-Time PCR Detection System (Bio-Rad, Hercules, CA,
United States) in a 20 μL system containing 2 μL of cDNA,
1 μL of each primer (10 μmol L–1), 10 μL of 2× TransStart
Top Green qPCR SuperMix (TransGen, Beijing, China), and
7 μL of RNase-free water. The qRT-PCR was initiated at
95°C for 3 min, followed by 40 cycles of 15 s at 95°C, 30 s
at 60°C, and 72°C for 30 s. Relative expression levels of
target genes were calculated by the 2−ΔΔCT method (Livak
and Schmittgen 2001).
2.10. Statistical analysis
Values were expressed as mean±standard deviation and
evaluated by analysis of variance (ANOVA) using SPSS
Statistics 17.0 Software (SPSS Inc., Chicago, United
States). The RNA-seq data were analyzed on the free online
platform of Majorbio Cloud Platform (www.majorbio.com).
3. Results
3.1. Biological characters of the obtained Trichoderma
isolate
A total of 14 Trichoderma isolates were obtained from the
rhizosphere soil samples of tomato plants in the Beijing
suburb of Shunyi district of China. One of them was a
novel isolate, designated as TM2-4, with apparent plant
growth promoting capabilities. A pink color was observed
after the culture filtrate of TM2-4 isolate was added to the
Salkowski reagent, indicative of production of indoleacetic
acid (IAA) like substances. An average of (6.45±0.72) µg
1269
mL–1 of IAA was detected for the isolate TM2-4. The isolate
TM2-4 also showed siderophore production (52.3±1.6)% in
Chrome azurol S (CAS) liquid medium and solubilization of
phosphate (32.4 μg mL–1) in NBRIP medium. The isolate
TM2-4 displayed no detectable 1-Aminocyclopropane-1-
carboxylate (ACC) deaminase production activity. Tomato
seeds treated with 500-fold dilution of culture filtrate of isolate
TM2-4 exhibited a germination rate of 93.3% compared to
the control (80%). The hypocotyl length, radical length and
vigor index were significantly increased compared to the
control by 28.7, 19.4 and 62.1%, respectively, under 100-
fold dilution treatment.
3.2. Morphological and molecular identification of
the Trichoderma isolate
The morphological observation showed that mycelia of
the isolate TM2-4 grew rapidly on PDA medium, covering
the entire plate on the 3th day of incubation. The aerial
hyphae were denser as white floss, which finally formed an
arachnoid and cottony colony with dark green conidia on
the surface. Conidiophores of the isolate TM2-4 were tree-
like, and composed of a main axis with several secondary
branches, which terminated in phialides. The phialides
of conidiogenous cells were ampullaceous or lagenate
in shape, (3.5–7.5)×(2.5–3.8) μm in size. Conidia were
ovoid to spherical, 1.3–2.8 μm in diameter. Based on
morphological and microscopic characteristics of the fungal
colonies on PDA plates (Fig. 1), the isolate TM2-4 was
confirmed as belonging to a Trichoderma species.
PCR amplification yielded fragments of approximately
582, 578 and 1087 bp, respectively, for the rDNA-ITS region,
tef1-α and rpb2 genes of the isolate TM2-4. According to
BLASTn results on NCBI, the ITS region of TM2-4 isolate
had 99% nucleotide identity with those of T. harzianum
(MG572200, KX632495) and T. afroharzianum (KP009262).
The tef1-α and rpb2 genes of isolate TM2-4 showed 99%
A B
Fig. 1 Culture and microscopic characteristics of isolate
TM2-4 on potato dextrose agar (PDA) medium. A, colony on
PDA medium. B, conidiophores, phialides and conidia under
microscopy (40×; scale bars=10 μm).
1270 ZHAO Juan et al. Journal of Integrative Agriculture 2021, 20(5): 1266–1276

similarity with those of T. afroharzianum strain LESF010
(KT279008) and strain TRS835 (KP009149), respectively.
Phylogenetic analysis of the combined data of rDNA-ITS
region, tef1-α and rpb2 genes showed that the isolate TM2-4
clustered together with the reference strain LESF010
(KT278854, KT279008 and KT278940) and strain TRS835
(KP009351, KP008787 and KP009149) of T. afroharzianum
with 100% bootstrap support (Fig. 2). The sequence data
of rDNA-ITS region, tef1-α and rpb2 genes of isolate TM2-4
were deposited in NCBI with GenBank accession numbers
of MK779176, MK779175 and MK779174, respectively.
Combining the molecular results with morphological
characters, the isolate TM2-4 was identified as Trichoderma
afroharzianum.
3.3. Growth promotion effect of T. afroharzianum
microbial agent on tomato plants
The microbial agent was obtained by solid state
fermentation of the obtained T. afroharzianum isolate TM2-4.
The isolate TM2-4 displayed competitive rhizosphere
colonization ability, since a T. afroharzianum colony was
recovered from the rhizosphere soil of tomato plants even
30 days post inoculation, with mean colony forming unit
(CFU) numbers remaining at 2.92×107 CFU g–1 soil. The
isolate TM2-4 was also detected from the root tissue of
tomato plants. The pot experiment indicated that the
T. afroharzianum TM2-4 microbial agent treatment gave
considerably higher values of plant height, dry weight,
number of leaves per plant and root activity, which were
increased by 36.1, 32.3, 56.3 and 26.2%, respectively,
compared with the control (P<0.05). The fresh weight,
diameter of stem base, diameter of leaf canopy and
chlorophyll content were also increased by 22.8, 14.8, 17.3
and 11.0% upon TM2-4 treatment (Fig. 3).
3.4. Unigene annotation and identification of DEGs
High-throughput sequencing was used for the systematic
analysis of gene expression profiles in tomato root treated
with or without T. afroharzianum TM2-4 microbial agent.
Approximately 47.24 and 45.23 million raw reads were
acquired from the control (Trichoderma agent free, TCK)
and the treatment (Trichoderma agent inoculation, TTM)
groups, respectively. Large proportions (98.74 and
98.66%) of clean reads (46.64 and 44.62 million for TCK
and TTM, respectively) were retained for assembly and
downstream analysis (Table 1). Sequencing data were
submitted to the NCBI Sequence Reads Archive database
with SRA accession number PRJNA561218. The clean
reads were mapped to the tomato reference genome,
with detailed mapping output summarized in Table 1.
Overall, the mapping ratios of the control and Trichoderma
treatment groups were 93.60 and 92.41% (average value
of uniquely mapped), respectively, suggesting high levels
of gene expression in both groups. A total of 31 631 unique
sequences were annotated based on blastx alignment
searches of six public databases including GO, KEGG, NR,

Trichoderma longibrachiatum S328 (JQ685875 JQ685867 KJ665291)

Trichoderma virens TRS110 (KP009298 KP008863 KP009100)
Trichoderma harzianum T22 (KX632495 KX632609 KX632552)
100
Trichoderma harzianum T9 (MG572200 KX572910 MG917680)
100
Trichoderma afroharzianum CBS466.94 (KP009262 KP008851 KP009150)
85 TM2-4(MK779176 MK779175 MK779174 )
95 Trichoderma afroharzianum TRS835 (KP009351 KP008787 KP009149)
Trichoderma afroharzianum LESF010 (KT278854 KT279008 KT278940)
100 Trichoderma afroharzianum GJS04-186 (FJ442265 FJ463301 FJ442691)
Trichoderma afroharzianum GJS04-197 (FJ442214 FJ463302 FJ442740)
Trichoderma asperellum CGMCC6422 (KF425754 KF425756 KF425755)
Trichoderma atroviride TRS18 (KJ786757 KJ786839 KP009061)
100 Trichoderma viride TRTRS575 (KP009372 KP008931 KP009081)
Trichoderma viride LELESF115 (KT278861 KT278989 KT278921)
90
Trichoderma koningii GJS90-18 (DQ323409 DQ289007 EU248600)
100 Trichoderma petersenii GJS04-164 (DQ323442 DQ289004 FJ442783)
10

Fig. 2 Maximum parsimonious tree obtained from the combined rDNA-ITS, tef1-α and rpb2 sequence data of isolate TM2-4.
Trichoderma longibrachiatum strain S328 was used as outgroup. Bootstrap support values >70% from 1 000 replications are
shown at the nodes. Trees were inferred using the heuristic search option with Tree–Bisection–Reconnection (TBR) branch
swapping. The numbers in the parentheses represent the GenBank accession numbers of rDNA-ITS, tef1-α and rpb2 genes of
the corresponding strains.
 ZHAO Juan et al. Journal of Integrative Agriculture 2021, 20(5): 1266–1276 1271

A B C D
20.00 2.00 1.00 * 3.00

Fresh weight (g/plant)

1.80

Dry weight (g/plant)

*
0.90

Diameter of stem
Plant height (cm)

16.00 1.60 0.80 2.50

1.40 0.70 2.00

base (cm)
12.00 1.20 0.60
1.00 0.50 1.50
8.00 0.80 0.40
0.60 0.30 1.00
4.00 0.40 0.20 0.50
0.20 0.10
0 0 0 0
TCK TTM TCK TTM TCK TTM TCK TTM

E F G H
20.00 16.00 * 2.50 0.30 *
18.00

Chlorophyll content
0.25
Diameter of leaf

16.00 12.00 2.00

Leaf number
canopy (cm)

Root activity
14.00

(mg g–1 h–1)

0.20
per plant

(mg g–1)
12.00 1.50
10.00 8.00 0.15
8.00 1.00
6.00 0.10
4.00 4.00 0.50
2.00 0.05
0 0 0 0
TCK TTM TCK TTM TCK TTM TCK TTM

Fig. 3 Growth promotion effect of Trichoderma afroharzianum TM2-4 microbial agent inoculation on tomato plants under greenhouse
conditions. Biological traits and physiological parameters were surveyed in fifty-day-old tomato plants. TCK and TTM, tomato
roots treated without and with T. afroharzianum TM2-4 microbial agent inoculation, respectively. Error bars represent the standard
deviations of three replicates. * denotes significant difference (P<0.05) in Trichoderma microbial agent treated samples compared
to the control.

Table 1 Summary of trimming and read mapping results of the sequences obtained from tomato roots treated without or with
Trichoderma afroharzianum TM2-4 microbial agent inoculation
Sample1) Raw reads Clean reads Total mapped Multiple mapped Uniquely mapped
TCK1 48 613 434 47 983 752 46 243 986 (96.37%) 962 023 (2.00%) 45 281 963 (94.37%)
TCK2 48 225 004 47 675 052 45 957 634 (96.40%) 948 423 (1.99%) 45 009 211 (94.41%)
TCK3 44 874 730 44 262 370 41 603 122 (93.99%) 874 919 (1.98%) 40 728 203 (92.02%)
TTM1 44 703 550 44 041 048 40 980 852 (93.05%) 809 556 (1.84%) 40 171 296 (91.21%)
TTM2 47 318 126 46 722 380 44 181 363 (94.56%) 928 075 (1.99%) 43 253 288 (92.58%)
TTM3 43 664 422 43 103 098 41 108 532 (95.37%) 837 717 (1.94%) 40 270 815 (93.43%)
1)
TCK and TTM, tomato roots treated without and with T. afroharzianum TM2-4 microbial agent inoculation, respectively. Three
replicates of control (TCK1, 2 and 3) and treatment (TTM1, 2 and 3) groups were carried out in RNA-seq analysis.
Data in the parentheses indicated the percentage of reads mapped to the genome.

KOG, Swiss-prot and Pfam (Appendix A). We analyzed in Appendix E. To further clarify the definite functions of
the datasets derived from transcriptome of tomato roots the DEGs and based on GO enrichment analysis, 584 up-
treated with or without T. afroharzianum TM2-4 microbial regulated genes and 400 down-regulated genes were also
agent. Gene expression analysis showed that 984 genes assigned to the three categories. In the biological process
were significantly differentially expressed, including 584 (BP) category, the functions such as “hydrogen peroxide
up-regulated genes and 400 down-regulated genes. The metabolic process”, “detoxification”, and “secondary
list of differentially expressed genes (DEGs, FDR<0.05), metabolic process” were detected for the significant up-
including fold changes and annotation details, is presented regulated genes (Fig. 4-A); while “cellular response to
in Appendix B. The 984 DEGs were apparently enriched phosphate starvation”, “transmembrane transport”, and
in 44 terms of the three main GO categories (Appendix C), “cellular response to starvation” were detected for the
and in 206 pathways of the KEGG database (Appendix D). down-regulated genes (Fig. 4-B). The up-regulated genes
were also significantly enriched in the cellular component
3.5. GO function and KEGG pathway enrichment (CC) category, such as “extracellular region”, “intrinsic
analysis of the DEGs component of plasma membrane” and “integral component
of plasma membrane” (Fig. 4-A). For the molecular functions
In evaluating the DEGs in terms of their predicted functions, (MF), “peroxidase activity”, “oxidoreductase activity”, and
the top 30 significant GO enrichment terms are displayed “glutathione transferase activity” were moderately enriched
1272 ZHAO Juan et al. Journal of Integrative Agriculture 2021, 20(5): 1266–1276

for the up-regulated genes (Fig. 4-A). 4-coumarate-CoA ligase (EC: 6.2.1.12) and trans-cinnamate
We selected the twenty most abundant pathway A4-monooxygenase (EC: 1.14.14.91) are involved in the
entries of the 584 up-regulated and 400 down-regulated early stages of phenylpropanoid biosynthesis. In addition,
DEGs to display in the scatter plot graph (Fig. 5). For several higher expressed enzymes, namely cinnamoyl-CoA
the up-regulated DEGs, “phenylpropanoid biosynthesis” reductase (EC: 1.2.1.44), cinnamyl-alcohol dehydrogenase
and “glutathione metabolism” were the most enriched (EC: 1.1.1.195), Peroxidase (EC: 1.11.1.7), are concerned
KEGG pathways (Fig. 5-A), while “vitamin B6 metabolism” with the biosynthesis and modification of phenylpropanoids
was significantly enriched for the down-regulated DEGs (Appendix F). KEGG enrichment analysis of the glutathione
(Fig. 5-B). Biochemical synthetic and metabolic pathways, (GSH) metabolism showed that up-regulation of the
in which different genes were involved, were also determined glutathione peroxidase GPX (EC: 1.11.1.9), phospholipid-
through KEGG pathway enrichment analysis. The results hydroperoxide glutathione peroxidase GPX4 (EC: 1.11.1.12)
indicated that the gene products were obviously associated and glutathione reductase (EC: 1.8.1.7) were involved in the
with specific pathways, including phenylpropanoid conversion of GSH to GSH disulfide as well as the opposite
biosynthesis and glutathione metabolism. For example, reaction (Appendix G).

Biological process Cellular component Molecular function

A Hydrogen peroxide metabolic process

B
Detoxification Cellular response to phosphate starvation
Secondary metabolic process Transmembrane transport
Oxidation-reduction process Cellular response to starvation
Cellular oxidant detoxification Response to nutrient levels
Glutathione metabolic process Response to extracellular stimulus
Transmembrane transport Cellular response to nutrient levels
Defense response Chemical homeostasis
Secondary metabolite biosynthetic process Cellular response to extracellular stimulus
Plant-type cell wall organization or biogenesis Cellular response to external stimulus
Asparagine biosynthetic process Cell communication
Flavonoid biosynthetic process Inorganic anion transport
Cell wall organization Single-organism transport
Trehalose biosynthetic process Phosphate ion transport
Carbohydrate transmembrane transport
GO term

GO term

Extracellular region
Intrinsic component of plasma membrane
Integral component of plasma membrane
Plasma membrane part
Cell wall
External encapsulating structure
Peroxidase activity
Oxidoreductase activity, acting on peroxide as acceptor
Antioxidant activity
Glutathione transferase activity
Iron ion binding
Transporter activity
Transmembrane transporter activity
Glycerol channel activity
Glucosyltransferase activity
Asparagine synthase activity
0 2
Lipid biosynthetic process
Hormone metabolic process
Brassinosteroid homeostasis
Sulfolipid biosynthetic process
Brassinosteroid metabolic process
Phytosteroid biosynthetic process
Integral component of plasma membrane
Extracellular region
Integral component of membrane
Extrinsic component of thylakoid membrane
Intrinsic component of plasma membrane
Transporter activity
Substrate-specific transmembrane transporter activity
Transporter activity
Sugar transmembrane transporter activity
Secondary active transmembrane transporter activity
4 6 8 10
0 2 4 6
–Log10(P-value) –Log10(P-value)

Fig. 4 Most highly enriched GO functions related to biological process, cellular component, and molecular function among
Trichoderma afroharzianum TM2-4 microbial agent up-regulated (A) and down-regulated (B) differentially expressed genes (DEGs).

A B Number
Number Vitamin B6 metabolism
Phenylpropanoid biosynthesis
Glutathione metabolism 2 HIF-1 signaling pathway 1
Valine, leucine and isoleucine biosynthesis cAMP signaling pathway
Brassinosteroid biosynthesis 27 ErbB signaling pathway 4
Galactose metabolism Pentose phosphate pathway
Arginine and proline metabolism retinol metabolism
Nitrogen metabolism FDR Glycerolipid metabolism FDR
Beta-alanine metabolism 1.0 Carbon fixation in photosynthetic organisms 1.0
Starch and sucrose metabolism 0.8 Riboflavin metabolism 0.8
Alanine, aspartate and glutamate metabolism 0.6 0.6
0.4 Calcium signal pathway 0.4
Valine, leucine and isoleucine degradation 0.2 Alpha-linolenic acid metabolism 0.2
Cysteine and methionine metabolism 0 Pyruvate metabolism 0
Lndole alkaloid biosynthesis Glyoxylate and dicarboxylate metabolism
Cutin, suberine and wax biosynthesis Linoleic acid metabolism
Arginine biosynthesis
Pntothenate and CoA biosynthesis Diterpenoid biosynthesis
Biosynthesis of unsaturated fattyacids Steroid biosynthesis
Biotin metabolism Galactose metabolism
Plant-pathogen interaction Brassinosteroid biosynthesis
Tryptophan metabolism Arachidonic acid metabolism

0 0.05 0.10 0.15 0 0.05 0.10 0.15 0.20 0.25

Rich factor Rich factor

Fig. 5 The top 20 enriched KEGG pathways of the up-regulated (A) and down-regulated (B) differentially expressed genes (DEGs)
after the Trichoderma afroharzianum TM2-4 microbial agent inoculation. The vertical axis indicates different pathways, while the
horizontal axis indicates the enrichment factor. Red indicates highly expressed genes, blue indicates genes with low expression.
 ZHAO Juan et al. Journal of Integrative Agriculture 2021, 20(5): 1266–1276 1273

3.6. qRT-PCR validation of the gene expression levels High-throughput sequencing methods have revolutionized
of the DEGs genomic and transcriptomic research with advantages of
low cost and ultra-high data output. The RNA-seq method
To confirm the reliability of the RNA-seq data, the relative allowed us to undertake rapid gene expression analysis in
expression levels of ten DEGs related to phenylpropanoid plants interacting with rhizosphere microorganisms, which
biosynthesis (Solyc03g116910.2, Solyc12g005790.1), is especially applicable for plants with available reference
glutathione metabolism (Solyc01g099590.2, Solyc09g011630.2), genome sequences (Varshney et al. 2009; Camilios-
substance and energy metabolism (Solyc10g078550.1, Neto et al. 2014). Trichoderma spp. are recognized as
Solyc06g006080.2, Solyc04g080010.2), transporter activity beneficial fungi that can systemically alter and influence
(Solyc10g084120.1), and plant hormone signal transduction the expression profile of numerous plant functional genes
(Solyc04g080660.2, Solyc10g079640.1) pathways were (De Palma et al. 2019; Yuan et al. 2019). In this study, the
confirmed using qRT-PCR with specific primers (Appendix H). RNA-seq method was used to detect the gene expression
As shown in Fig. 6, the qRT-PCR results were highly levels of tomato roots treated by T. afroharzianum TM2-4
consistent with the RNA sequencing analysis, supporting microbial agent inoculation at the transcriptome level. The
the high quality of the RNA sequencing datasets. results indicated that the Trichoderma agent inoculation has
brought about up-regulation of numerous genes, which may
4. Discussion involve different signal transduction and metabolic process
of tomato plants.
This study demonstrated the obvious growth promotion According to the information obtained from GO function
ability of Trichoderma afroharzianum TM2-4 microbial agent and KEGG pathway enrichment analyses, the up-regulated
on tomato plants, which was closely related to successful differentially expressed genes (DEGs) were mainly related to
survival and colonization of the miroorganisms in the auxin signaling transduction, plant-type cell wall organization,
tomato rhizosphere. In addition, the isolate TM2-4 used for and photosynthesis pathways. Previous studies have
Trichoderma agent preparation and tomato plant inoculation verified the capacity of Trichoderma to release elicitors
was proven to produce a variety of aforementioned bioactive that may induce auxin signal transduction, such as salicylic
substances. These are consistent with previous studies acid (SA), jasmonic acid (JA) and ethylene (ET), which are
which declared that plant growth promoting characters often associated with induced systematic resistance within
of microbials commonly include the production of diverse the plants (Nawrocka and Malolepsza 2013). The cell wall
chemical substances such as phytohormones, siderophore, is also critical in the plant defense system considering its
phosphate solubilizing enzyme and ACC deaminase function of protecting plants from invasion of biotic or abiotic
(Rodriguez et al. 2008; Wani et al. 2015). stress (Underwood 2012; Malinovsky et al. 2014). The up-

RNA-Seq qRT-PCR
3
Solyc01g099590.2

Solyc06g006080.2

Solyc04g080010.2

Solyc04g080660.2

Solyc10g079640.1
Relative expression level (Log2fold change)

0
Solyc03g116910.2

Solyc12g005790.1

Solyc09g011630.2

Solyc10g078550.1

Solyc10g084120.1

–1

–2

–3

–4

Fig. 6 Comparison of the RNA-seq and qRT-PCR results validate the relative expression levels of differentially expressed genes
(DEGs). The graph shows the Log2(fold change) in expression of genes in Trichoderma afroharzianum TM2-4 microbial agent
inoculated tomato roots compared to the control. Error bars represent the standard deviations of three replicates.
1274 ZHAO Juan et al. Journal of Integrative Agriculture 2021, 20(5): 1266–1276
regulation of these genes related to plant defense activity
can be considered as an indication of better plant fitness
and a greater ability of the plant to resist external influences
(Doni et al. 2019). In addition, the up-regulation of genes
associated with chlorophyll biosynthesis and photosynthesis
also demonstrated an increase of the tomato plants’ capacity
to transport and accumulate photosynthetic products, which
has significant roles in plant growth and development (Doni
et al. 2019). We also found that the T. afroharzianum TM2-4
treatment could induce the up-regulation of phenylpropanoid
biosynthesis and glutathione metabolism related genes,
which usually play an important role in reinforcing plant cell
walls and improving plant defenses through modulation of
the plant secondary metabolism (Ben et al. 2017). Hence,
the growth promotion mechanism of T. afroharzianum
TM2-4 on tomato plants mainly manifests as growth and
metabolism enhancement, immune defense optimization,
and induced systematic resistance stimulation.
A decreased application rate of chemical fertilizers
combined with biological fertilizers, such as microbial
agents, can give the same levels of plant growth and yield
as those obtained with the full use rate of chemical fertilizers
(Cai et al. 2015). Therefore, plant growth promoting fungi
based microbial agents should be evaluated and used as
components of integrated management strategies in plant
cultivation. In this study, we demonstrated significant
stimulation of plant growth and the corresponding
mechanism at the transcriptomic level in tomato plants by
T. afroharzianum TM2-4 microbial agent inoculation. The
isolate TM2-4 was proven to have the potential of application
as plant growth promoting fungi (PGPF) in tomato plantation.
Further research will focus on the formulation development
and optimization of T. afroharzianum microbial agent
products, as well as the chemical fertilizer usage reduction
effect in field application.
5. Conclusion
In the present study, we described the tomato plant growth
promoting effect of Trichoderma afroharzianum isolate
TM2-4, a beneficial fungus previously obtained from
tomato rhizosphere soil. RNA-seq technology revealed the
transcriptome profiling of tomato plants by T. afroharzianum
TM2-4 microbial agent inoculation. The results indicated
that the growth promotion effect of isolate TM2-4 was
associated with up-regulated expression of hundreds
of genes which were implicated in nutrition and energy
metabolism, antioxidant activity, auxin signaling pathway,
and immune defense activity. The research provided an
in-depth understanding of the mechanism of action of the
rhizosphere competent T. afroharzianum isolate TM2-4 in
relation to tomato plant growth promotion.
Acknowledgements
This research was supported by the Youth Research Fund
of Beijing Academy of Agriculture and Forestry Sciences,
China (QNJJ201814), the National Key R&D Program of
China (2017YFD0201102), and the Beijing Key Laboratory
of Green Control of Fruit Tree Diseases and Pests in the
North China (BZ0432).
Declaration of competing interest
The authors declare that they have no conflict of interest.
Appendices associated with this paper are available on
http://www.ChinaAgriSci.com/V2/En/appendix.htm
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