Microbiological Research 235 (2020) 126445

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

Isolation of Trichoderma in the rhizosphere soil of Syringa oblata from Harbin T

and their biocontrol and growth promotion function
Bin Liua, Shida Jib,c, Huifang Zhangc, Yucheng Wangb,c, Zhihua Liua,c,*
a
State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, 26 Hexing Road, Harbin, 150040, China
b
Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, 830011, China
c
College of Forestry, Shenyang Agricultural University, 120 Dongling Road, Shenyang, 110866, China

ARTICLE INFO ABSTRACT

Keywords: For the effective biocontrol of Syringa powdery mildew (Mircosphaera syringejaponicae) and to promote seedling
Trichoderma growth, we identified 44 of the 181 Trichoderma isolates (T1-T181) isolated from the rhizosphere soil. Analysis
Syringa oblate identified 10 Trichoderma species, and T. pseudoharzianum T1 (TpseT1), T. afroharzianum T52 (TafrT52), and T.
Powdery mildew asperelloides T57 (TaspT57) were selected to make Trichoderma biofertilizer because of their fast growth and high
Biological control
spore production. Exposing Syringa oblata to Trichoderma biofertilizer showed that TafrT52 and TaspT57 could
induce abscisic acid (ABA) production, and promote the shedding of diseased leaves and the generation of new
leaves. Furthermore, TafrT52 increased the catalase (CAT) activity and reduced the H2O2 content. And the
disease incidence was reduced by 37.84 % by Tasp (highest) in 2017 year and by 13.84 % by TpseT1(lowest) in
2018 year. In addition, all Trichoderma strains we selected could promote the lateral root growth of S. oblata
seedlings; however, because of the downregulated gene expression at the late stage of chlorophyll synthesis, the
chlorophyll content decreased in the new leaves. Antagonism among different Trichoderma species led to low
biocontrol and growth promotion effects, thus the Trichoderma mixture cannot be use as biofertilizer. TafrT52,
with better biocontrol and growth promotion effects, could be used for biocontrol of M. syringejaponicae.

1. Introduction guava, and oak using Trichoderma selective medium (Anil and Lakshmi,
2010). Thirty-three Trichoderma species were isolated from 150 rhizo-
Powdery mildew disease are biotrophic pathogens that infect nu- sphere soil samples of chili (Nawaz et al., 2018).
merous plant species (Zou et al., 2017). For example, the leaves, fruit Trichoderma has been worldwidely researched for its biological
clusters, and shoots of Hazelnut (Corylus avellana) can be infected by ability against pathogenic fungi and growth-promotion ability to plants.
phypathogenic fungi Erysiphe corylacearum of powdery mildews disease, Trichoderma spp. can reduce the severity of plant diseases by inhibiting
causing great damage (Sezer et al., 2017). Powdery mildew is also one of plant pathogens through their highly potent antagonistic and myco-
the main diseases occurring in clonal nurseries of Eucalyptus spp. (Xia parasitic activity (Rosa et al., 2012). For example, the endophytic Tri-
et al., 2011a). Poplars are economically important fast growing trees that choderma V76-12 can inhibit mycelial growth in vitro, and reduce Cur-
are exposed to a broad range of fungal diseases, including powdery vularia oryzae symptoms in vivo and in natural fields. In addition, it is
mildew (Filiz and Vatansever, 2018). Furthermore, Syringa, as a city the most effective treatment tested in reducing leaf spot disease of oil
flower in Harbin, China, is often harmed by powdery mildew, which can palm seedlings (Sunpapao et al., 2018). Trichoderma harzianum ETS 323
detract from the attractiveness of the foliage while causing long-term effectively inhibited Botrytis cinerea hyphal growth, caused cytosolic
damage to the plant (Larsen et al., 2016). Chemical and physical methods vacuolization in the hyphae, and led to hyphal lysis (Chi-Hua et al.,
are often used to control the disease; however, there is an urgent need to 2012). The Trichoderma afroharzianum strain NAIMCC-F-01938 can be
find environmentally friendly biological control methods. Previous re- used with safe fungicides for enhanced control of powdery mildew in
search showed that Trichoderma is an important biocontrol fungus that vineyards (Sawant et al., 2017). In addition to antifungal ability of
exists widely in the rhizosphere soil of plants (Fu et al., 2019; Cummings Trichoderma itself, the metabolites of Trichoderma have also antifungal
et al., 2016). For example, fourteen strains of Trichoderma spp. were activity. The product of Trichoderma virens is involved in secondary
isolated from the forest tree rhizospheres of pinus, deodar, bamboo, metabolite biosynthesis, which can increase the antagonistic activity of

⁎
Corresponding author at: State Key Laboratory of Forest Genetics and Tree Breeding (Northeast Forestry University), 26 Hexing Road, Harbin 150040, China.
E-mail address: [email protected] (Z. Liu).

https://doi.org/10.1016/j.micres.2020.126445
Received 7 November 2019; Received in revised form 21 February 2020; Accepted 21 February 2020
Available online 22 February 2020
0944-5013/ © 2020 Elsevier GmbH. All rights reserved.
B. Liu, et al. Microbiological Research 235 (2020) 126445

phytopathogenic fungi and the capacity to promote plant growth (Ramã translation elongation factor 1-alpha gene (TEF1) were amplified using
Rez-Valdespino et al., 2017). All Trichoderma asperellum strains over- the EF1−728 F/TEF1LLErev primer pair (Druzhinina et al., 2005). The
grew the colonies of all Verticillium dahlia isolates to a similar extent, PCR reaction mix comprised 50 μl containing: 10 × PCR buffer, 5 μL;
and extracellular compounds from strains Bt3 and T25 showed higher 2 μmol/L dNTPs, 1 μL; Taq DNA polymerase, 0.5 μL; 20 μmol/L primer
anti-V. dahliae activities (Carrero-Carrón et al., 2016). In addition to its ITS1, 1 μL; 20 μmol/L primer ITS4, 1 μL; 100 ng/μL. DNA, 0.5 μL; and
good activity against fungal phytopathogen, Trichoderma can also pro- ddH2O, 41 μL. The PCR conditions were: 94 °C for 5 min; followed by 35
mote plant growth. For example, exposure to either Trichoderma virens cycles of 94 °C for 1 min, 50 °C for 30 s, and 72 °C for 1 min; 35 circles;
or Trichoderma atroviride increased the biomass production of wild-type and 72 °C for 7 min. The PCR products was detected using 1.0 %
Arabidopsis seedlings and stimulated lateral root development (Hexon agarose gel electrophoresis and purified using an AxyPrepDNA gel ex-
Angel et al., 2009a). Another study showed T. atroviride could also traction kit (Promega, Kit No. D5542-01). The sequencing was per-
regulate root architecture and promote plant growth (Garnica-Vergara formed by Shanghai Personal Biotechnology Co., Ltd on ABI 3730XL
et al., 2016). With respect to the control, T. atroviride promoted greater platform. The ITS sequences were aligned by “TrichoBLAST” tool at the
height of Capsicum annuum seedlings (Herrera-Parra, 2017). Tricho- International Subcommission on Trichoderma and Hypocrea (ISTH)
derma hamatum LU592 significantly promoted the growth of Pinus ra- website (http://isth.info/).
diata seedlings (increase their height by up to 16 %) and roots (in-
creased the dry weight by up to 31 %)(Gao, 2011). 2.2. Phylogenetic analysis of Trichoderma isolates
In the present study, in order to obtain the Trichoderma resource
against powdery mildew of S. oblata, 16 soil samples were collected from To study the relationships among the Trichoderma isolates, their ITS
the rhizosphere of S. oblata at eight districts of Harbin City, China. sequences were used to construct a phylogenetic tree together with other
Trichoderma strains were then isolated and identified. Furthermore, the Trichoderma sequences downloaded from the GenBank database at the
Trichoderma biofertilizer was made to research the biocontrol effect on NCBI (identified in the text by their accession numbers). The phyloge-
inhibiting powdery mildew and promoting growth of S. oblata seedlings. netic tree was constructed using the neighbor-joining method in the
To study the growth promotion effect of Trichoderma biofertilizer to MEGA 5.10 program. Our Trichoderma ITS sequences were submitted to
seedlings of S. oblata, the content of chlorophyll, the expression of GenBank database of NCBI (National Center for Biotechnology
chlorophyll synthesis genes and the changes in the lateral root number of Information) and received the accession numbers KX357824 to
S. oblata seedlings were determined. However, because Syringa oblata KX357867. Supplemental Table 1 illustrates the richness of Trichoderma
disease resistance genes are rarely reported, so we processed the seed- strains of the S. oblata rhizosphere from eight districts of Harbin City.
lings of S. oblata and sequenced the transcriptome, and then selected the
genes related to chlorophyll synthesis for fluorescence quantitative real- 2.3. Causative agent identification of S. oblata powdery mildew
time PCR, including Glutamyl tRNA reductase (HA78660), Mg chelatase
H subunit (CH80451), chlorophyll synthase (CG67251), and chlor- To determine the morphological characteristics and infection
ophyllide a oxygenase (CA67767). And to study the biological control method of S. oblata powdery mildew (M. syringejaponicae), leaves were
effect of Trichoderma on powdery mildew of S. oblata, the abscisic acid collected from outdoor infected S. oblata and glass slides were prepared
(ABA), H2O2, malondialdehyde (MDA) contents and catalase (CAT) ac- to observe the morphological characteristics of S. oblata powdery
tivity of infected leaves were determined. Our work aimed to evaluate mildew under a microscope.
the biological control effect of Trichoderma on powdery mildew and the
growth promotion effect on seedlings of S. oblata. 2.4. Confrontation culture of different Trichoderma species and preparation
of Trichoderma biofertilizer
2. Materials and methods
T. Pseudoharzianum T1 (TpseT1), T. afroharzianum T52 (TafrT52),
2.1. The isolation and identification of Trichoderma resources from 8 and T. asperelloides T57 (TaspT57) were cultured on PDA medium at
districts in Harbin 28 °C for 5 days in petri dishes. Then, the mycelia disk of 5 mm dia-
meter of two or three species were inoculated in the same petri dishe
Sixteen soil samples were collected from the S. oblata rhizosphere in with PDA medium. After co-cultured for 4 days, the colony radius were
eight districts of Harbin City, China, including Xiangfang measured to calculate the inhibition ratio among them. All the above
(45°39′∼45°46′N, 126°30′∼126°50′E), Daoli (45°32′-47′N, 126°08′-38′E), experiments were performed in triplicate.
Daowai (45°20′∼46°20′N, 126°15′∼127°30′E), Nangang(45°30′-45°40′N, T. pseudoharzianum T1 (TpseT1), T. afroharzianum T52 (TafrT52),
126°45′-126°43′E), Acheng(45°10′∼45°50′N, 126°40′∼127°39′E), Qunli and T. asperelloides T57 (TaspT57) are inoculate in PDA medium at
(44°04′∼46°40′N, 125°42′∼130°10′E), Shuangcheng (45°08′∼45°43′N, 28 °C for a week. The spores are collected and diluted into 108 ml−1
125°41′∼126°42′E), and Songbei(44°41′∼45°47′N, 125°16′∼125°37′E). spore suspension. Totally 10 ml spore suspension is added into 300 g
Put the soil sample into sterile water, mixed, and then diluted by 10−1, 10- solid fermentation medium (the thickness 1 cm–1.5 cm) and mix the
2
, 10-3, 10-4, and 10-5 times. The dilutions were cultured on Rose Bengal medium until blended, then the tray is sealed with preservative film.
Medium (Zhou et al., 2020). After 48 h, Trichoderma colonies were picked And they are cultured at 28 °C for a week as Trichoderma biofertilizer
and cultured on potato dextrose agar (PDA) medium. Putative Trichoderma (Liu et al., 2015).
colonies were purified by single spore isolation (Dou et al., 2019). The
single spore was transfered on PDA medium to obtain pure isolate. After 2.5. Antifungal ability Trichoderma against S. oblata powdery mildew
cultured for 48−72 h on PDA medium, the conidium, conidiophore and
phialide of Trichoderma were then observed under a scanning electron After treatment with Trichoderma biofertilizer, the diseased leaves
microscope(SEM; Quanta 200, FEI Company, USA) and photographed. were collected and stored at ↓80 °C. The CAT, H2O2, and MDA contents
Trichoderma strains were grown in 100 ml of liquid PD medium at of leaves were extracted using CAT (serial number: CAT-2-Y), H2O2
28 ± 0.5 °C in dark for 48 h (Zhou et al., 2020). The mycelia were (serial number: H2O2-2-Y), and MDA (serial number: MDA-2-Y) kits
collected through eight layers of gauze, rinsed three times with sterile from Suzhou Comin Biotechnology Co. Ltd. of China. The results were
distilled water, and stored at −80 °C. DNA extraction from Trichoderma determined using ultraviolet and visible light spectrophotometers. The
mycelia was performed using a DNA Kit (OMEGA Biotech, Norcross, ABA content of S. oblata leaves was determined by a biotechnology
GA, USA). The internal transcribed spacer regions were amplified by company using an ABA enzyme-linked immunosorbent assay (ELISA)
primer pair ITS1 and ITS4(White et al., 1990). And the partial kit (Mlbio ML-elisa-A11444, China). All data were analyzed and plotted

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B. Liu, et al. Microbiological Research 235 (2020) 126445

using Excel and SigmaPlot 12.5 software. The comparison between

groups was performed using the paired T-test in SPSS11.0 (IBM Corp.,
Armonk, NY, USA) and the confidence level was determined.
The healthy and sick leaves treated with different Trichoderma bio-
fertilizers were collected and the back part of a leaf was removed and
made into a slide. The stomata were observed under the microscope and
statistical data were obtained. The morbidity of leaves were assessed by
Image J software (developed by National Institutes of Health)
(Weerakkody et al., 2017). The longitudinal length of guard cells on both
sides of the stoma was measured as the stomatal length (SL). The widest
area of shadow enclosed by the guard cells on both sides was measured
as the stomatal open level (SOL). SL/SOL of stomata were measured
using auto CAD and the images were created using SigmaPlot 12.5.

2.6. Growth promotion of S. oblata seedlings

TaspT57, TpseT1, and TafrT52 strains were selected to make

Trichoderma biofertilizers, and were cultured on PDA medium at
28 ± 0.5 °C in the dark for 5 d. A spore suspension was made and
transferred onto rice husk medium and culture for 5 d. Then, the roots
root of 5 month-old S. oblata seedlings were watered with 100 ml of
spore suspension (1 × 106 ml−1). After treatment, the new leaves of
seedlings were collected and stored at ↓80 °C and the roots of the
seedlings were photographed. The expression of chlorophyll-related
genes and the extraction of chlorophyll were performed to study the
growth promotion induced by Trichoderma biofertilizer.
The seedlings of S. oblata with cold, hot, salt, and alkali environ-
ment, toxins, and spores of Alternaria alternata to get more expressed
genes. And sequenced the transcriptome, then selected the 4 genes re-
lated to chlorophyll synthesis and 3 internal reference genes for RT-
qPCR detection. The sequence, Tm(°C) and size of product (bp) of Fig. 1. The species, number, and microscopic appearances of the Trichoderma
primer pair were illustrated in Supplementary table 2. The genes were isolates. a: The species and number of Trichoderma isolates. b: The microscopic
submitted to NCBI GenBank(Supplementary table 2). For the expression appearances of 10 Trichoderma species on PDA medium. From A to J: T. afro-
of chlorophyll-related genes, total RNA was extracted from new leaves harzianum, T. pseudoharzianum, T. velutinum, T. virens, T. viridescens, T. atro-
and cDNA was synthesized from the total RNA using an RNA Kit (Bio viride, T. hamatum, T. asperelloides, T. longibrachiatum, and T. brevicompactum. 1,
2 respectively represent the conidiophore and conidium.
Take RP3301) and a reverse transcription kit (Takara RR047A), re-
spectively. The cDNA was then used as a template for fluorescent
quantitative real-time PCR of chlorophyll genes. A 20-μL PCR system 3. Results
was used for the amplification: 2 × SYBR Green Realtime 10 μL, 10 μM
primer0.5 μL, 10×cDNA 2 μL, Rnase Free ddH2O 7 μL. The PCR con- 3.1. Isolation of Trichoderma in eight districts of Harbin
ditions were: 94 °C for 30 s; 35 cycles of 94 °C for 2 s, 59 °C for 30 s and
72 °C for 5 min. The gene expression levels were evaluated using RT- The total 181 Trichoderma strains (T1-T181) were isolated from 16
qPCR according to the 2−ΔΔCt method (Livak and Schmittgen, 2001). soil samples. According to the growth speed, conidiospore color, wheel
Tubulin and actin genes were used as internal references to normalize pattern and pigment secretion of colony on the PDA medium, the ex-
the amounts of total cDNA present in each reaction. PCR primers for the actly same Trichoderma isolates in each districts were divided into
amplification of S. oblata cDNA fragments were designed using Primer several categories: Hulan, 5 categories; Acheng 6 categories and etc.
6.0 (PREMIER Biosoft Co., Palo Alto, CA, USA). There were three RT- And one Trichoderma isolates was selected from each category, in-
qPCR replicates per cDNA sample. To extract the chlorophyll, 0.2 g of cluding total 44 isolates.Then, they were identified by morphological
leaves was weighed and put into mortar with a little SiO2 and CaCO3 (Fig. 1b) and molecular methods (Supplemental table 1) (Fig. 2), which
and 2–3 ml of 80 % CH3COCH3, and ground into a homogenate. The identified 10 Trichoderma species. The districts containing the highest
extract was filtered into a test tube and added with 25 ml of 80 % number of isolates (n = 41) and species (n = 5) of Trichoderma are
CH3COCH3. The optical density of the solution was then determined at Hulan and Nangang, respectively, whereas the districts containing least
645, 652, and 663 nm using UV756 ultraviolet and visible light spec- isolates (n = 11) and species (n = 2) were Songbei and Shuangcheng
trophotometers. The chlorophyll a and chlorophyll b were calculated districts (Supplementary table1). The dominant species in Harbin were
using the formula of Arnon (Arnon, 1949). The data for the RT-qPCR T. pseudoharzianum (46 strains) and T. afroharzianum (41 strains), T.
and chlorophyll content were analyzed and plotted by Excel and Sig- atroviride (n = 29), and T. asperelloides (n = 21). However, T. velutinum
maPlot12.5 software, respectively. (2 strains) and T. brevicompactum (1 strain) were the least abundant
Trichoderma species in Harbin (Fig. 1a).
2.7. Statistical analysis
3.2. Morphological characteristics
For each experiment, three replicates were included. Statistical
analyses of quantification data were carried out using SPSS11.0 soft- Based on the database of Trichoderma Taxonomy (http://isth.info/),
ware, and were subject to one-way analysis of variance (ANOVA) the 10 Trichoderma species are located in four sections and are involved
analysis; means were analyzed using Duncan’s multiple range tests at P in six clades. T. afroharzianum, T. pseudoharzianum, and T. velutinum
= 0.05. In the figures, the different superscript letters within the same belong to the Harzianum clade; and T. virens belongs to the Virens
column indicate statistically significant differences. clade. Both clades belong to the Pachybasium section. Species in this

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B. Liu, et al. Microbiological Research 235 (2020) 126445

Fig. 2. Phylogenetic relationships of ITS sequences from different Trichoderma isolates. Different colors represent different Trichoderma groups: Red for the
Pachybasium group, purple for the Trichoderma group, blue for the Longibrahiatum group, and green for the Lone Lineages group.

group have large conidiophores that are often arranged in tight clusters; was identified as T. velutinum, which was closely related to MF38363 (T.
branches and phialide are thick or swollen, relatively short, and ar- velutinum). T. afroharzianum, T. pseudoharzianum, and T. velutinum be-
ranged in tight rotations; some species have typical conidiophore con- long to the Harzianum clade. T32 was identified as T. virens, which has
tinuation (for example: T. virens), the sporogenous structure of many affinity with KU52185 (T. virens) and belongs to the Virens clade. All
strains is closely clustered, and adjacent conidiophores can fuse Trichoderma in group I belong to the Pachybasium section (Fig. 2). T72,
(Fig. 1A–D). T. viridescens and T. atroviride belong to the Viride clade, T143, T115, T141, T167, T83, T82, and T122 were identified as T.
and T. hamatum and T. asperelloides belong to the Hamatum subbranch. atroviride, and were located in the same group with the known T.
Both clades belong to the Trichoderma section. The morphological atroviride strain AF456920. T138, T157, T30, and T135 were identified
characteristics of this group are jagged conidiophore graciles, branches as T. viridescens belonging to the same clade (Viride) as T. atroviride and
are not compact branch, and it is rare for more than three branches to were also located in the same group with the known T. atroviride strain
come together in a rotate-like arrangement (Fig. 1E–H). T. long- AF456920. T67, T57, and T88 were identified as T. asperelloides, which
ibrachiatum belongs to the Longibrahiatum section, the typical char- showed affinity with T. asperelloides KU987251. T180 and T140 were
acteristic of this group is that the strains can produce a yellowish green identified as T. hamatum, and were closely related to KM054532 (T.
pigment, when observed through the bottom of the culture dish. This hamatum) and belong to the Hamatum clade. All Trichoderma in group II
group had few conidiophore branches and they were arranged irregu- belong to the Trichoderma section (Fig. 2). T128 was identified as T.
larly. There is also a phialide; however, vortex-like and wheel-like ar- brevicompactum (Fig. 2) and is closely related to MF669735 (T. brevi-
rangements are rare (Fig. 1I). T. brevicompactum belongs to the Lone compactum), which belongs to the Lone Lineages section and was lo-
Lineages section. The colony does not produce diffuse pigments and has cated in group III. T98 and T114 were identified as T. longibrachiatum
no odor. Spore producing clusters are hemispherical, flocculent, and (Fig. 2), which belongs to the Longibrahiatum section, and were located
bulk raw, with a bottle green, yellowish green, or celadon color in group IV. Therefore, the results of molecular identification were
(Fig. 1J). identical to those of the morphological identification.

3.3. Phylogenetic analysis of Trichoderma 3.4. The preparation of Trichoderma spores

The 44 identified strains (containing 10 species) were divided into TpseT1, TafrT52, and TaspT57 strains were selected to make
four groups based on the phylogenetic tree (Fig. 2), and the result was Trichoderma biofertilizers (Fig. 3) because of their high spore produc-
the same as morphological classification. According to the ITS se- tion and fast growth. After fermentation, the TpseT1, TafrT52, and
quences, T163, T127, T124, T170, T146, T155, T55, T42, T152, T97, TaspT57 cultures had 1.68 × 108, 1.7 × 108, and 1.71 × 108 spores/
and T52 were identified as T. afroharzianum; T145, T86, T87, T158, ml, respectively. Although they have advantages in fast growth and
T26, T30, T16, T84, and T129 were identified as T. pseudoharzianum; disease resistance, it is possible that antagonism could occur when they
both Trichoderma spp. have affinity with JX244281 (T. harzianum). T70 interact together. When two or three Trichoderma species were cultured

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B. Liu, et al. Microbiological Research 235 (2020) 126445

Fig. 3. The biofertilizer production and confrontation culture of three Trichoderma species. A: The production of Trichoderma spore powder. 1–3, TpseT1, TafrT52,
and TaspT57. B: the confrontation culture of Trichoderma. 1–3 represent TpseT1, TafrT52, and TaspT57 training alone, respectively. 4–6 respectively represent
confrontation cultures of TaspT57 and TafrT52, TaspT57 and TpseT1, and TafrT52 and TpseT1. 7,8 show confrontation cultures of TpseT1, and TafrT52 and
TaspT57. C: Inhibition Rate. The top of panel C shows the confrontation of two Trichoderma species, and below is the confrontation of three Trichoderma species. The
data were analyzed using a one-way analysis of variance (ANOVA), and the differences were compared using a Duncan’s multiple range test, The mean value ±
standard deviation (n = 3). Values with the same letter do not differ significantly (P < 0.05). There is no significant difference without *. Note that three *'s have
the highest significance, followed by two *'s, with one * having the lowest significance.

together, an obvious boundary was observed between the two colonies biofertilizers began to drop at 6 days (Fig. 5A2, B2); there were few
(Fig. 3B 4–8). When two Trichoderma species were cultivated together leaves left at the ninth day (Fig. 5A4, B3). Meanwhile, new leaves began
for 5 d, the inhibition rate of TafrT52 toward TaspT57 was 0.59; the to grow; by the twelfth day, many new, healthy leaves had appeared
inhibition rate of TaspT57 toward TpseT1 was 0.54 respectively; and (Fig. 5A6).To further understand this phenomenon of falling leaves, the
the inhibition rate of TpseT1 toward TafrT52 was 0.57 (Fig. 3C). When ABA content was determined. The results showed that the ABA content
all three Trichoderma species cultured together, the inhibition rates of increased with time. Meanwhile, in Syringa treated with TafrT52 and
TafrT52, TaspT57, and TpseT1 were 0.66, 0.74, and 0.61, respectively TaspT57 for 9 d, the ABA contents increase by 42.40 and 33.54 ng/ml,
(Fig. 3C). Therefore, for the selected Trichoderma species, the single respectively, higher than that of the ConD (by 30.80 ng/ml) (Fig. 5C).
species had advantages of high spore production, fast growth, and good In addition, the ABA content of Syringa treated with TafrT52 reached its
disease resistance; however, when they co-cultured, they exhibited peak on day 9 and was significantly higher than that of other treatments
antergic properties, which will be further studied in follow-up research. and ConD (Fig. 5C). However, the effect of TpseT1 on the ABA content
was not obvious.In conclusion, TafrT52 could promote the shedding of
3.5. The morphological characteristics of M. syringejaponicae powdery mildew infected leaves by increasing the ABA content.
To study the biocontrol function of Trichoderma to S. oblata powdery
The morphological characteristics of M. syringejaponicae, as the mildew in outdoor reared plants, the CAT activity and the H2O2 and
pathogen of S. oblata powdery mildew, were studied. Its asexual gen- MDA contents of leaves were determined. Both TpseT1 and TafrT52
eration belongs to the genus Oidium. The cleistothecium aggregates or could improve the antioxidant ability of Syringa, and the H2O2 content
disperses, and is spherical or oblate (Fig. 4A). The appendage branches showed a negative relationship with the CAT activity. The content of
form many sub-branches (Fig. 4B). There are also many asci (ovoid) in H2O2 in leaves treated with TpseT1 decreased with time (from 3.06 to
the cleistothecium (Fig. 4D). The ascospore is ovoid to oblong (Fig. 4D). 0.7 μmol/g) and was lower than that in the ConD group (4.03 μmol/g)
Although the disease can occur on both sides of the leaf, it mainly oc- at 6 days post interaction (dpi). The activity of CAT increased with
curs on the top (Fig. 4F). In the early stage of disease, scattered pink time, toy 115.12 nmol/min/g at 9 dpi, and was higher the that in the
spots appear on the diseased leaves, which gradually expand to connect ConD group (33.90 nmol/min/g)(Fig. 5D). The content of H2O2 in
with each other, covering the leaves. In the later stage of the disease, leaves treated with TafrT52 decreased by 1.89 μmol/g from 2.71 μmol/
the white powder layer becomes sparse and dust-like, with small white g (3 dpi) to 0.82 μmol/g (9 dpi), and was lower than the ConD group
particles appearing on it that eventually turn into black powder. This is (4.03 μmol/g). The activity of CAT increased by 203.2 nmol/min/g,
the sexual generation of the disease and the white powder layer is the from 47.5 nmol/min/g (3 dpi) to 250.7 nmol/min/g (9 dpi), which was
asexual generation (Fig. 4A). Compared with diseased leaves, the sur- significantly higher than that in the ConD group (33.90 nmol/min/g)
faces of the healthy leaves were obviously leathery, greener, and and in the other treatments (ranging from 27.12 nmol/min/g to
smoother (Fig. 4E). 63.28 nmol/min/g) (Fig. 5D). However, treatment with TaspT57 and
Tcom complexes had no obvious effect on improving the antioxidant
3.6. The biocontrol function of Trichoderma to powdery mildew of S. oblata ability of Syringa. The MDA content of leaves treated with TpseT1 and
in outdoor-cultivated plants TaspT57 decreased to 2.57 nmol/g and 1.81 nmol/g, respectively,
compared with the 7.22 nmol/g in the ConD group at 9 dpi. By contrast,
The diseased leaves that were treated with Trichoderma biofertilizers the MDA content of leaves treated with TafrT52 and Tcom showed no
would drop. Most of the leaves treated with all Trichoderma significant change compared with that in the control (Fig. 5D).

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B. Liu, et al. Microbiological Research 235 (2020) 126445

Fig. 4. The morphology and microscopic shape of M. syringejaponicae A: Cleistothecium, B: Appendage, C: Ascus produced by the ascocarp, D: Ascospore. E: Healthy
leaves, F: Diseased leaves.

Fig. 5. The growth status of diseased leaves treated with the Trichoderma biofertilizer and their ABA, CAT, H2O2 and MDA content. A. A1: healthy leaves, A2–A5: the
process of shedding diseased leaves to grow new ones; B. B1: healthy leaves, B2 and B3 spraying Trichoderma biofertilizer at day 6 and 9; C, D, E, F show the ABA
CAT, H2O2, and MDA contents of diseased leaves treated with Trichoderma biofertilizer. X-axis, time points; Y-axis, content. ConD, ConH, TpseT1, TafrT52, TaspT57
and the Tcom respectively represent diseased leaves, healthy leaves, T. pseudoharzianum biofertilizer, T. afroharzianum biofertilizer, T. asperelloides biofertilizer and
their combination biofertilizer treatment. The data were analyzed using one-way analysis of variance (ANOVA), and the differences were compared using a Duncan’s
multiple range test. The mean value ± standard deviation (n = 3) is shown. Values with the same letter do not differ significantly (P < 0.05). There is no significant
difference without *. Note that three *'s have the highest significance, followed by two *'s, with one * having the lowest significance.

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B. Liu, et al. Microbiological Research 235 (2020) 126445

Fig. 6. Leaf and leaf stomata of S. oblata in-

duced by Trichoderma. A: The leaves of S. oblata
in 2017. From left to right, successively, are
healthy leaves, Tasp treated leaves at 0, 3, 6, 9,
and 12 d. B: Stomata map corresponding to
panel A. C: The leaves of S. oblata in 2018. From
left to right, Successively, are healthy leaves,
diseased leaves, TpseT1, TafrT52, TaspT57, and
Tcom treated leaves at 6 d. D: Stomata map
corresponding to panel B. E: The morbidity of
leaves in panels A and C. F: SL/SOL of stomata
in panels B and D. ConH, ConD, TpseT1,
TafrT52, TaspT57, and Tcom respectively re-
present healthy leaves, diseased leaves treated
with water, T. pseudoharzianum biofertilizer, T.
afroharzianum biofertilizer, T. asperelloides bio-
fertilizer, and their combinations. The long-
itudinal length of guard cells on both sides of
the stoma is the stomatal length (SL). The
widest area of shadow enclosed by the guard
cells on both sides is the stomatal open level
(SOL). Data were analyzed using a one-way
analysis of variance (ANOVA), and the differ-
ences were compared using a Duncan’s multiple
range test. There is no significant difference
without *.

Continuous study for two years showed that Trichoderma bio- an obvious increase in number compared with that of the control
fertilizer could reduce the morbidity of S. oblata powdery mildew and (Fig. 7A3); however, TpseT1, TaspT57, and Tcom treatments did not
influence the morphological structure of stomata. The morbidity area of increase the number of lateral roots (Fig. 7A2, A4-A5). At the fifteenth
S. oblata powdery mildew was reduced by 4.69 % and 37.84 % after day, the numbers of lateral roots of seedlings treated with TpseT1 and
treatment with T. asperelloides biofertilizer for 3 d and 6 d in 2017 TafrT52 showed obvious increases compared with those of the control
(Fig. 6A, E). The morbidity of S. oblata powdery mildew was reduced by (Fig. 7B2-B3), and were higher than those of the seedlings treated for 7
7.91 %, 12.28 %, 13.84 %, and 8.72 % after treatment with TpseT1, d. At the thirtieth day, the numbers of lateral roots of seedlings treated
TafrT52, TaspT57, Tcom, respectively, for 6 d in 2018 (Fig. 6C and E). by the four Trichoderma biofertilizers showed obvious increases com-
The regenerated leaves of S. oblata treated with Trichoderma bio- pared with that of the control; however, the seedlings treated with
fertilizer were bigger and greener than those of the control (Fig. 6C). TpseT1 and TafrT52 had more lateral roots than the seedlings treated
The stomata of the diseased leaves had larger openings than those of with TaspT57 and Tcom (Fig. 7C1-C5). Thus, all the Trichoderma bio-
healthy leaves, and the degree of opening gradually increased up to the fertilizers could promote the growth of S. oblata seedlings by increasing
point where the leaves fell; however, the stomata of the regenerated the number of lateral roots, which would allow the plants to absorb
leaves had smaller openings (Fig. 6B and D). Following treatment of more water, inorganic salts, and small molecular compounds (Fig. 7).
Syringa with Trichoderma biofertilizer for 6 d, the SL/SOL ratio of the Following Syringa interaction with Trichoderma, the chlorophyll
stomata (4.77–4.80) was higher than that in the ConD group (4.41) in contents were determined. The chlorophyll contents of leaves treated
2017 (Fig. 6D). The SL/SOL (4.6) of leaves treated with TafrT52 was with TpseT1 and TafrT52 for 7 d were lower than those of the control,
significantly higher than that of the ConD group (3.9) and was even and the chlorophyll contents of leaves treated with TpseT1 and TafrT52
higher than that of the ConH group (4.1). However, the effects of other for 15 days showed no significant difference compared with those of the
Trichoderma treatments were not significant in 2018. These results control. In addition, TaspT57 and Tcom treatments had no obvious
suggested that Trichoderma could prevent and control the disease by effect on the chlorophyll content (Fig. 8). Therefore, to gain a better
reducing the morbidity of diseased leaves and increasing the SL/SOL understanding of why the chlorophyll content decreased after Syringa
ratio of the stomata to protect the guard cells. treated with Trichoderma biofertilizer, the expression levels of four
chlorophyll synthesis-related genes were detected using RT-qPCR. The
results show that the expression levels of HA78660 in all treatments
3.7. Promoting-growth function of Trichoderma to S. oblata seedling were significantly higher than those in the control: After treatment for 7
d, TaspT57 and the Tcom upregulated HA78660 expression by 24.46 and
To study the growth promotion effect of Trichoderma to S. oblata 24.6-fold, respectively (Fig. 8). TpseT1, TaspT57, and Tcom upregulated
seedlings, seedlings were watered with a suspension of 5 × 106 spores/ HA78660 expression by 23-fold or more at day 15 (Fig. 8). For the
ml. The results showed that in response to the spores, the roots of the CG67251 gene, all treatments upregulated its expression, especially
seedlings changed dramatically in a time dependent manner. At the TafrT52, reaching its maximum after 7 d (upregulated by 22-fold
seventh day, the lateral roots of seedlings treated with TafrT52 showed

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B. Liu, et al. Microbiological Research 235 (2020) 126445

Fig. 7. Root growth condition of S. oblata seedlings interacting with three Trichoderma biofertilizers. Control, TpseT1, TafrT52, TaspT57, and Tcom, respectively,
represent water, T. pseudoharzianum biofertilizer, T. afroharzianum biofertilizer, T. asperelloides biofertilizer, and their combination biofertilizer treatment; A, B, and C
respectively display images of S. oblata seedlings treated with Trichoderma biofertilizer at 7, 15, and 30 d. The three images on the far right show the growth
conditions of whole S. oblata seedlings that interacted with TafrT57 at 7, 15, and 30 d.

(Fig. 8)). TaspT57 and Tcom upregulated CG67251 expression by 21.6 environments. Interestingly, the species and quantities of Trichoderma
and 21.35-fold, respectively, compared with the control (20.86-fold). varied according to the living environments. For example, nine Tri-
However, the expression of CA67767 was downregulated by all treat- choderma species (261 isolates) were identified from pine roots, in-
ments, except for TpseT1 on 7 d. For CH80451 gene, all treatments cluding T. spirale (n = 97), T. songyi (n = 56), T. hamatum (n = 52), T.
downregulated its expression compared with that of the control after 15 acrassum (n = 24), T. pyramidale (n = 5), T. polypori (n = 1) and other
d. Therefore, in general Trichoderma biofertilizers upregulated chlor- unknown species (n = 26)(Oh et al., 2018). In addition, three specific
ophyll synthesis genes at the early stage of new leaves chlorophyll groups of Trichoderma were observed in the roots of banana. Group one,
synthesis, but downregulated them at the middle and last stages. which made up the largest population, comprised T. asperellum, T. vi-
rens, and Hypocrea lixii, and was isolated from the inside and on the
surface of the banana roots. Group two comprised T. atroviride and T.
4. Discussion koningiopsis and existed on the surface only. Group three, comprising
only T. brevicompactum, was isolated from the inside of the roots (Xia
Trichoderma has been widely recognized as excellent biocontrol et al., 2011b). In addition, 20 Trichoderma isolates were obtained from
agent against plant pathogens, and can adapt to variety living en- the tomato rhizosphere, including T. harzianum (n = 8), T. virens
vironments (Saravanakumar et al., 2016), including some extreme

Fig. 8. Chlorophyll content and differential expression of four chlorophyll synthesis genes of S. oblata interacting with three Trichoderma species. X-axis, time points;
Y-axis, Chlorophyll content and fold change in expression. Expression levels were calculated according to the 2−ΔΔCt method. Values represent the mean of three
independent experiments ± standard deviation. HA, CH, CG, and CA represent glutamyl tRNA reductase, Mg chelatase H subunit, chlorophyll synthase, and
chlorophyllide a oxygenase, respectively. The data were analyzed using a one-way analysis of variance (ANOVA), and the differences were compared using a
Duncan’s multiple range test.

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B. Liu, et al. Microbiological Research 235 (2020) 126445

(n = 4), T. viride (n = 3), T. koningii (n = 2) and T. asperellum (n = 2) Trichoderma are effective in preventing disease, the different Tricho-
(Rai et al., 2016). Therefore, we speculated that Trichoderma and plants derma strains have different ways of preventing disease.
shared the same living environment and formed a symbiotic system Furthermore, after Syringa seedlings were treated with Trichoderma
during the process of evolution. Therefore, to achieve better biocontrol biofertilizer, the number of lateral roots significantly increased, espe-
of Syringa powdery mildew, 16 soil samples were collected from the cially in the seedlings treated with TafrT52 (Fig. 7). A previous study
Syringa rhizosphere, and 10 Trichoderma species (from a total of 181 showed that wild-type Arabidopsis seedlings inoculated with either T.
strains) were isolated and identified, including T. pseudoharzianum virens or T. atroviride showed characteristic auxin-related phenotypes,
(n = 46), T. afroharzianum (n = 41), T. atroviride (n = 29), and T. as- including increased biomass production and stimulated lateral root
perelloides (n = 21) (Fig. 1a). Based on present study the taxonomy of development (Hexon Angel et al., 2009b). T. parareesei T6 exerted
the T. harzianum species complex is revised to include at least 14 species beneficial effects on tomato plants in terms of seedling lateral root
including T. guizhouense, T. harzianum, T. inhamatum, T. lentiforme, T. development, and in adult plants it improved defense against Botrytis
lixii, T. afarasin, T. afroharzianum, T. atrobrunneum, T. camerunense, T. cinerea and growth promotion (M Belén et al., 2014). The reason may
endophyticum, T. neotropicale, T. pyramidale, T. rifaii and T. simmonsii be that Trichoderma can produce or release a variety of compounds that
(Chaverri et al., 2015). Thus, the exact phylogenetic position of the induce localized or systemic resistance responses, and enhance root
majority of strains was not resolved and therefore attributed to a di- growth and development, and the uptake and use of nutrients (Harman
verse network of recombining strains conventionally called “pseudo- et al., 2004). The chlorophyll content of Miscanthus x giganteus in
harzianum matrix” (Druzhinina et al., 2010). Hence, we performed TEF September under PR6 treatment (including T. atrobrunneum and T.
molecules identification for T. harzianum. According to the growth harzianum) was 11 % higher than that of the control(Chirino-Valle
speed, conidiospore color, wheel pattern and pigment secretion of et al., 2016). However, interestingly, the chlorophyll content of plants
colony on the PDA medium, the 12 T. pseudoharzianum strains in 44 ITS treated with three kinds Trichoderma biofertilizer were lower than
identified Trichoderma isolates were divided into 8 categories and 10 T. control in the present study. The might be because the chlorophyll
afroharzianum strains of 44 ITS identified Trichoderma isolates into 7 synthesis gene expression of middle and late stage of chlorophyll
categories. Thus TEF identifications were performed for T1, T16, T30, synthesis is negatively regulated in newborn leaves (Fig. 8).
T26, T84, T86, T87 and T158 of T. pseudoharzianum and T42, T52, T55, Based on a previous study, the biocontrol effect of the Trichoderma
T124, T127, T146 and T152 of T. afroharzianum. The TEF identification mixture was better than that of a single strain. The combination of T1
results are identical with those of ITS. Furthermore, T. pseudoharzianum, (T. harzianum) and T2 (T. asperellum) isolates was more effective than
T. afroharzianum, and T. asperelloides produced more spores (Fig. 1a); the other treatments in controlling Fusarium, such that it reduced the
therefore, strains T. pseudoharzianum (TpseT1), T. afroharzianum disease severity from 20 to 44 % and increased the dry weight from 23
(TafrT52), and T. asperelloides (TaspT57) were selected as biocontrol to 52 % (Akrami et al., 2011). The combination of Trichoderma isolates
strains against Syringa powdery mildew. was applied at three time points (at 15 d before planting, at the second
Firstly, after treatment with TpseT1, TafrT52, and TaspT57, the month after planting, and at the fourth month after planting), resulting
morbidity of plants with S. oblata powdery mildew was reduced by in a significant reduction in banana Fusarium wilt disease and also in-
7.91, 12.28, and 13.84 % compared with that of the control (ConD), creased the bunch weight compared with that of untreated control
respectively (Fig. 6C, E). A previous study also showed that following plants (Thangavelu and Gopi, 2015). Interestingly, in our study, the
the treatment of coca leaves with T. asperellum ART-5, ART-6, and ART- effects of Trichoderma combinations (including TpseT1, TafrT52, and
8, the incidence of vascular streak dieback (VSD) disease decreased by TaspT57) were worse than those of the single treatments. After treat-
55.6, 38.9, and 32.9 %, respectively, compared with that of the positive ment, the ABA content of leaves treated with Tcom was lower than that
control (88.9 %). In addition, exposing cucumber roots to T. harzianum of plants treated with TafrT52 or TaspT57 (Fig. 5), the CAT activity was
T39 reduced the severity of gray mold (Botrytis cinerea) by 55 % same. As for promotion growth, the lateral roots number of Tcom
(Rosmana et al., 2015). Meanwhile, following exposure to Trichoderma, treatment were less than other treatment at 9 dpi. The reason for this
the leaves infected by Syringa powdery mildew fell from the Syringa tree phenomenon is that, when TafrT52, TaspT57 and TpseT1 were cultured
at 6 dpi, and the leaves regenerated at 9 dpi (Fig. 5). The reason may be together, the antagonism phenomenon of each other will arose
the ABA content (an important hormone regulating leaf shedding) of (Fig. 3C). Thus, T. afroharzianum T52, T. pseudoharzianum T1, and T.
leaves treated with TafrT52 (77 ng/ml) were higher than that of ConD asperelloides T57 strains cannot be used together, as the mixture may
(33.4 ng/ml; Fig. 5). A previous study showed that inoculation with T. not work well.
gamsii F18 significantly increased the ABA content of Arabidopsis Following the isolation and identification of Trichoderma, we found
thaliana leaves (28 % higher than that of control)(Zhou et al., 2018). that T. pseudoharzianum (n = 46), T. afroharzianum (n = 41), T. atro-
Therefore, we speculated that TafrT52 could promote the accumulation viride (n = 29), and T. asperelloides (n = 21) were the dominant species
of the ABA content in Syringa leaves, further accelerating the re- in the Syringa rhizosphere of Harbin city. Their excellent biocontrol and
generation of the leaves. Furthermore, Trichoderma could enhance the growth promotion function mean that T. afroharzianum T52 and T.
antioxidant ability of plants. Following inoculation with Trichoderma asperelloides T57 could be used as the preferred Trichoderma strain for
biofertilizer, the CAT activity of Syringa was markedly higher than that the biocontrol of S. oblata powdery mildew. However, because of the
of the control (33.9 μmol/g) at 9 dpi. In particular, the CAT activity of inhibition between different Trichoderma species, T. afroharzianum T52,
Syringa inoculated with TafrT52 and TpseT1, reached to 250.7 nmol/ T. pseudoharzianum T1, and T. asperelloides T57 cannot be used to-
min/g and 162.7 nmol/min/g, respectively (Fig. 5D). And, at 9 dpi, the gether. This study provided scientific guidance for the biocontrol of
H2O2 content of leaves inoculated with TpseT1 (0.59 μmol/g) and Syringa powdery mildew.
TafrT52 (0.51 μmol/g) was significantly lower than those of ConD
(5.62 μmol/g). However, TaspT57 had no significant influence on the
Ethical approval
H2O2 content of Syringa (Fig. 5E). In a study of the biocontrol of
pumpkin powdery mildew, the CAT activity of plants treated with T.
This article does not contain any studies with human participants or
harzianum (T1) and T. viridi (T2) increased by 175 and 160 (mMol-
animals performed by any of the authors.
H2O2FM/min*g), respectively, which was higher than that of the con-
trol (80 mMolH2O2FM/min*g)(Hafez et al., 2018). Pretreatment with
T. harzianum followed by Sclerotinia sclerotiorum inoculation, resulted in Declaration of Competing Interest
an increase in the CAT activity of soybean by 30 Umg−1plot compared
with that of the control (60 Umg-1 plot)(Zhang et al., 2016). Although The authors declare no conflicts of interests.

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B. Liu, et al. Microbiological Research 235 (2020) 126445

CRediT authorship contribution statement functioning. New Phytol. 209, 1496–1512.

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