# Screening and Identification ofTrichoderma Strains isolated from Natural Habitats in China with Potential Agricultural Applications

Authors: Ming Xue; Rui Wang; Chongyuan Zhang; Weiwei Wang; Fengtao Zhang; Di Chen; Sen Ren; Zhang Manman; Jumei Hou; Tong Liu
Journal: BioMed Research International
 (2021)

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## Abstract

Trichoderma spp. are widely distributed in natural habitats and have been evaluated as a potential biocontrol agent (BCA) for disease control and plant growth promotion. In this study, 1308 Trichoderma strains were obtained from the plant rhizosphere soil, above-ground plants, and decaying wood from natural habitats in China. Among them, 49 Trichoderma strains showed a good inhibitory effect, especially against Botrytis cinerea, Fusarium oxysporum, and Colletotrichum gloeosporioides with inhibition rate above 85% in the dual culture test. Among these 49 strains, the 13 strains with broad-spectrum inhibitory effects also significantly promoted the seed germination of five crops (rice, cucumber, tomato, melon, and pakchoi) and root growth of four crop seedlings (watermelon, tomato, eggplant, and chili). Furthermore, these strains showed effective colonization in the rhizosphere and root of cucumber. Trichoderma strains SC012 and NX043 showed the highest chitinase and β-1,3-glucanase activity among all strains. Based on the morphological characterization and phylogenetic analysis of the nuclear ribosomal internal transcribed spacer (ITS) and translation elongation factor 1 (tef1), twelve Trichoderma strains were identified as Trichoderma asperellum and one as Trichoderma afroharzianum. This study suggests that the 13 Trichoderma strains are promising BCAs and could be developed as biofertilizers and biological pesticides for agricultural applications.


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## Body

## 1. Introduction

Soil-borne diseases significantly decrease the quality and quantity of cash crops including vegetables, fruits, and officinal plants [1]. Most of the causal agents are polyphagous ubiquitous fungi, such as F. oxysporum causing wilt [2, 3], C. gloeosporioides causing anthracnose [4, 5], and B. cinerea causing gray mold [6, 7]. Currently available strategies for managing soil-borne pathogens include cultivation practices, physical control, and chemical control through resistant plants [8]. Although these approaches can reduce pathogen-caused losses, they are time-consuming and nontargeting and have poor control effects in some cases; chemical control is effective, but there are potential risks to the environment, and pathogens are prone to drug resistance [9]. Biological control is one of the best options to overcome the above-mentioned hurdles to the management of soil-borne plant pathogens and is undoubtedly more suitable for the sustainable development of modern agriculture [10].Trichoderma spp. are recognized as important bioantagonists of phytopathogens in agricultural production and account for more than 60% of the registered biofungicides [11]. The biocontrol efficacy of Trichoderma is based on the activation of multiple mechanisms, including competing for nutrients and space, modifying the environmental conditions, and promoting plant growth and plant-defensive mechanisms, antibiosis, and mycoparasitism [12–15]. T. asperellum strain CCTCC-RW0014 exhibited antagonism activity to fusarium wilt disease due to its high hydrolytic activity of chitin, gelatin, carboxymethyl cellulose, and pachyman [16]. T. virens ZT05 showed a significant inhibitory effect on R. solani, and its mechanism of action was associated with hyperparasitism and antibiosis [17]; T. harzianum Ths97 can control F. oxysporum causing damping-off in Pinus massoniana seedlings by reducing reactive oxygen species, lowering lipid peroxidation and cell death, increasing osmolyte levels, stimulating the activities of antioxidant enzymes, and increasing soil fertility [18].Trichoderma is one of the most diverse genera of the biocontrol fungi having above 300 reported species [19]. However, only a few of them are used in agricultural production as BCAs or biostimulating agents including T. polysporum, T. hamatum, T. virens, and T. atroviride [20–22]. Therefore, exploring the greater diversity of Trichoderma and evaluating its biocontrol effect have greater advantages in the biocontrol of plant pathogens. In addition, the success of BCAs is dependent upon the complex interactions between beneficial microbes with pathogens and plants [23]. It is a prerequisite for their effective practical application to understand how the biocontrol agents exert their protective effects [24]. In this study, we isolated 1308 Trichoderma strains various natural habitats in China and screened for their antifungal effect against a range of plant pathogenic fungi. Those strains that showed prominent antifungal activity were further tested for their plant growth and germination effect on a variety of important cash crops. The selected strains were then evaluated for root colonization ability, chitinase, and β-1,3-glucanase activity and finally identified on morphological and molecular basis.


## 2. Materials and Methods

### 2.1. Strains and Media

The pathogenic fungi (B. cinerea WN-2, F. oxysporum CS-1, C. gloeosporioides Penz QH-5, Sclerotium rolfsii SDF-23, F. oxysporum f. sp. tracheiphilum MTX-6, Botryosphaeria dothidea CTG-7, Magnaporthe oryzae DWM-3, Botryodiplodia theobromae PERF-1, F. graminearum KDR-1, Rhizoctonia cerealis ROD-18, Stagonosporopsis cucurbitacearum SD-3, and R. solani MNV-6) were isolated, identified, and stored at the Key Laboratory of Green Prevention and Control of Tropical Diseases and Pests of Hainan University for the subsequent studies.Trichoderma selective medium (TSM) was used for the isolation of Trichoderma spp. [25]. Potato dextrose agar (PDA) medium was used to culture and preserve pathogenic fungi and Trichoderma. The β-1,3-glucanase synthetic medium (Yeast extract 30 g, NaNO3 3 g, MgSO4·H2O 0.5 g, K2HPO4 1 g, FeSO4·7H2O 0.01 g, KCl 0.5 g in 1 liter of distilled water, pH 6.0) and chitinase detection medium (chitin 5 g, MgSO4·7H2O 0.1 g, NH4NO3 3 g, KH2PO4 2 g in 1 liter of distilled water, pH 6.0) were used to ferment Trichoderma strains. Corn meal dextrose agar medium (CMD, 30 g corn flour, 18 g glucose, and 18 g agar; dilute the volume to 1 L with water) and synthetic low nutrient agar medium (SNA, 1.0 g KH2PO4, 10.5 g KCl, 1.0 g KNO3, 0.5 g MgSO4, 0.2 g glucose, 0.2 g sucrose, and 18 g agar; dilute it to 1 L with water) were used for the morphological identification of Trichoderma strains.


### 2.2. Collection of Samples and Isolation ofTrichoderma

A total of 1018 samples of the rhizosphere soil and plants of cultivated crops from different ecological habitats, and decaying woods were collected from China. All samples were placed into sterile plastic bags in an icebox, transported to the laboratory in an icebox, and stored at 4°C for further use. Add 10 g of soil into 90 mL sterile water, shook for 30 min (200 r min–1), and then dilute to 103-fold. Fresh plant samples and rotten woods were surface-disinfected by 70% alcohol for 30 s, ground in 1× PBS buffer, and diluted to 100-fold. The solutions (100 μL) were added to the surface of TSM for the isolation of Trichoderma spp. Each sample was performed in triplicate. Morphologically different colonies were selected, transferred onto PDA medium, and purified by a single spore isolation [26]. All purified strains were maintained on PDA slants at 4°C for further use.


### 2.3. Inhibitory Effect ofTrichoderma Strains on the Pathogenic Fungi

The antagonistic ability of allTrichoderma strains against pathogenic fungi was evaluated by a dual culture method. Five-millimeter diameter mycelial disks from Trichoderma strains and pathogenic fungi were transferred onto the border of PDA plate opposed to each other with a distance of 50 mm on Petri dishes (diameter, 90 mm) and incubated at 28°C for 7 days. The individual cultures of the pathogenic fungi were used as controls. The percentage of growth inhibition (PI) was calculated using the following equation: PI%=C−T/C×100, where C represents radial growth of control mycelial and T represents radial growth of the treatment mycelial. Each treatment was performed in triplicate.


### 2.4. Effect ofTrichoderma Strains on Seed Germination

Five crops (Supplementary TableS1), including rice, tomato, melon, cucumber, and pakchoi, were used to measure the effect of Trichoderma strains on seed germination. Uniform size 50 seeds of each crop were surface-disinfected by 75% ethanol for 1 min and 0.5% NaClO for 5 min and washed five times with sterile water. The seeds were soaked in Petri dishes containing 50 mL of Trichoderma strains spore suspension (105 spores mL–1) for 2 h, transferred into new petri dishes covered with a layer of cotton and a filter paper, and cultured for 7 days at 28°C under the conditions of 12/12 h light/dark. The seeds treated with an equivalent volume of sterile water were used as control. The seed germination rate was calculated using the following equation: seedgerminationrate%=numberofgerminatedseeds/numberoftotalseedstested×100. The experiment was performed in triplicate.


### 2.5. Determination of Growth Promoting Effect ofTrichoderma Strains

The root growth promotion experiment was performed at 28°C under the conditions of 12/12 h light/dark alternation with 80% relative humidity in a greenhouse. Uniform size seedlings of watermelon, chili, eggplant, and tomato were irrigated with a 100 mL spore suspension (105 spores mL–1) of the Trichoderma strains. The seedlings treated with water were used as control. After 15 days, the seedlings were harvested, and the roots and aboveground seedlings were separated with scissors. The root length of seedlings was measured. The experiment was carried out in triplicate.


### 2.6. Determination of the Colonization Ability ofTrichoderma Strains

The spore suspension (107 spores mL–1) of the Trichoderma strains was added into sterilized composite soil containing vermiculite, matrix, and soil (1 : 3 : 7) and adjusted to a final concentration of 104 spores g–1. Sterilized distilled water was used as the control. The Trichoderma-inoculated and Trichoderma-uninoculated soils were placed into sterile glass test tubes (height, 25 cm; inner diameter, 2 cm), and then, a cucumber seed was planted inside. All tubes were inserted with a cotton ball, sealed with tin foil, and placed in a plant growth chamber at 28°C under the conditions of 12/12 h light/dark alternation. After 15 days, the Trichoderma populations in the rhizosphere soil and roots endoderm of cucumber were calculated by a dilution plate method [27] (thirty seedlings for each treatment). The experiment was performed in triplicate.


### 2.7. Determination ofβ-1,3-Glucanase and Chitinase Activity of Trichoderma Strains

For the determination ofβ-1,3-glucanase activity, the Trichoderma strain was incubated in synthetic medium and cultured at 28°C with shaking at 180 rpm for 3 days. Medium without Trichoderma spp. was used as control. Approximately 1 mL of the culture filtrate was used to assay activity of β-1,3-glucanase using the 3,5-dinitrosalicylic acid colorimetric method [28]. For the determination of chitinase activity, Trichoderma strain was incubated in chitinase detection medium and cultured at 28°C with shaking at 180 rpm for 3 days. Colloidal chitin was prepared according to the method described by Roberts and Selitrennikoff [29]. The assay was performed in 10 mL tubes containing 1 mL 1% colloidal chitin, 1.5 mL 50 mM acetate buffer pH =5.0, and 1 mL suspension, the mixture was incubated at 40°C for 1 h, and the chitinase activity was determined basing the standard curves of N-acetyl-D-glucosamine (GlcNAc) measured at 540 nm absorbance. Enzyme bioactivity was calculated as follows: enzymebioactivityU/mL=A1−A2+b1×d/K1×V1×T1, where A1 represents OD540 of samples, A2 represents OD540 of control, b1 represents the y-intercept of standard curves, d represents dilution rate of suspension, K1 represents the slope of standard curves, V1 represents the volume of mixture (mL), and T1 represents reaction time (h). Each treatment was performed in triplicate.


### 2.8. Identification ofTrichoderma Strains

The morphological characteristics of conidia, phialide, conidiophore, and chlamydospores and the colony growth patterns ofTrichoderma strains cultured on PDA, SNA, and CMD media were observed according to the description of Gams and Bissett [30]. For molecular identification, genomic DNAs of Trichoderma strains were extracted using the method of cetyl-trimethylammonium ammonium bromide (CTAB) as described by Ihrmark et al. [31]. The tef-1 gene was amplified with primers tef1-728F (5′-CATCGAGAAGTTCGAGAAGG-3′) and tef1-R (5′-GCCATCCTTGGGAGATACCAGC-3′), and ITS region was amplified with primers ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′). The PCR products were cloned into the Ti-19 vector and sequenced by the Sangon Biotechnology Co., Ltd. (Shanghai, China). All sequences from Trichoderma strains were aligned and spliced through “tef-1+ITS region” using MAFFT v7.215 [32]. The best substitution model (GTR+F+I+G4) was evaluated with IQ-TREE software using the ultrafast bootstrap method with 10,000 replicates. The phylogenetic tree was constructed based on tef-1 gene and ITS region for the molecular identification of the Trichoderma species.


### 2.9. Statistical Analyses

All experiments were repeated three times. The data were analyzed using IBM SPSS Statistics 21.0 software (IBM Corp., Armonk, NY, USA) using one-way analysis of variance (ANOVA). Means of treatment were compared using the least significant difference (LSD) test atP<0.05 and separated by superscript letters.


## 2.1. Strains and Media

The pathogenic fungi (B. cinerea WN-2, F. oxysporum CS-1, C. gloeosporioides Penz QH-5, Sclerotium rolfsii SDF-23, F. oxysporum f. sp. tracheiphilum MTX-6, Botryosphaeria dothidea CTG-7, Magnaporthe oryzae DWM-3, Botryodiplodia theobromae PERF-1, F. graminearum KDR-1, Rhizoctonia cerealis ROD-18, Stagonosporopsis cucurbitacearum SD-3, and R. solani MNV-6) were isolated, identified, and stored at the Key Laboratory of Green Prevention and Control of Tropical Diseases and Pests of Hainan University for the subsequent studies.Trichoderma selective medium (TSM) was used for the isolation of Trichoderma spp. [25]. Potato dextrose agar (PDA) medium was used to culture and preserve pathogenic fungi and Trichoderma. The β-1,3-glucanase synthetic medium (Yeast extract 30 g, NaNO3 3 g, MgSO4·H2O 0.5 g, K2HPO4 1 g, FeSO4·7H2O 0.01 g, KCl 0.5 g in 1 liter of distilled water, pH 6.0) and chitinase detection medium (chitin 5 g, MgSO4·7H2O 0.1 g, NH4NO3 3 g, KH2PO4 2 g in 1 liter of distilled water, pH 6.0) were used to ferment Trichoderma strains. Corn meal dextrose agar medium (CMD, 30 g corn flour, 18 g glucose, and 18 g agar; dilute the volume to 1 L with water) and synthetic low nutrient agar medium (SNA, 1.0 g KH2PO4, 10.5 g KCl, 1.0 g KNO3, 0.5 g MgSO4, 0.2 g glucose, 0.2 g sucrose, and 18 g agar; dilute it to 1 L with water) were used for the morphological identification of Trichoderma strains.


## 2.2. Collection of Samples and Isolation ofTrichoderma

A total of 1018 samples of the rhizosphere soil and plants of cultivated crops from different ecological habitats, and decaying woods were collected from China. All samples were placed into sterile plastic bags in an icebox, transported to the laboratory in an icebox, and stored at 4°C for further use. Add 10 g of soil into 90 mL sterile water, shook for 30 min (200 r min–1), and then dilute to 103-fold. Fresh plant samples and rotten woods were surface-disinfected by 70% alcohol for 30 s, ground in 1× PBS buffer, and diluted to 100-fold. The solutions (100 μL) were added to the surface of TSM for the isolation of Trichoderma spp. Each sample was performed in triplicate. Morphologically different colonies were selected, transferred onto PDA medium, and purified by a single spore isolation [26]. All purified strains were maintained on PDA slants at 4°C for further use.


## 2.3. Inhibitory Effect ofTrichoderma Strains on the Pathogenic Fungi

The antagonistic ability of allTrichoderma strains against pathogenic fungi was evaluated by a dual culture method. Five-millimeter diameter mycelial disks from Trichoderma strains and pathogenic fungi were transferred onto the border of PDA plate opposed to each other with a distance of 50 mm on Petri dishes (diameter, 90 mm) and incubated at 28°C for 7 days. The individual cultures of the pathogenic fungi were used as controls. The percentage of growth inhibition (PI) was calculated using the following equation: PI%=C−T/C×100, where C represents radial growth of control mycelial and T represents radial growth of the treatment mycelial. Each treatment was performed in triplicate.


## 2.4. Effect ofTrichoderma Strains on Seed Germination

Five crops (Supplementary TableS1), including rice, tomato, melon, cucumber, and pakchoi, were used to measure the effect of Trichoderma strains on seed germination. Uniform size 50 seeds of each crop were surface-disinfected by 75% ethanol for 1 min and 0.5% NaClO for 5 min and washed five times with sterile water. The seeds were soaked in Petri dishes containing 50 mL of Trichoderma strains spore suspension (105 spores mL–1) for 2 h, transferred into new petri dishes covered with a layer of cotton and a filter paper, and cultured for 7 days at 28°C under the conditions of 12/12 h light/dark. The seeds treated with an equivalent volume of sterile water were used as control. The seed germination rate was calculated using the following equation: seedgerminationrate%=numberofgerminatedseeds/numberoftotalseedstested×100. The experiment was performed in triplicate.


## 2.5. Determination of Growth Promoting Effect ofTrichoderma Strains

The root growth promotion experiment was performed at 28°C under the conditions of 12/12 h light/dark alternation with 80% relative humidity in a greenhouse. Uniform size seedlings of watermelon, chili, eggplant, and tomato were irrigated with a 100 mL spore suspension (105 spores mL–1) of the Trichoderma strains. The seedlings treated with water were used as control. After 15 days, the seedlings were harvested, and the roots and aboveground seedlings were separated with scissors. The root length of seedlings was measured. The experiment was carried out in triplicate.


## 2.6. Determination of the Colonization Ability ofTrichoderma Strains

The spore suspension (107 spores mL–1) of the Trichoderma strains was added into sterilized composite soil containing vermiculite, matrix, and soil (1 : 3 : 7) and adjusted to a final concentration of 104 spores g–1. Sterilized distilled water was used as the control. The Trichoderma-inoculated and Trichoderma-uninoculated soils were placed into sterile glass test tubes (height, 25 cm; inner diameter, 2 cm), and then, a cucumber seed was planted inside. All tubes were inserted with a cotton ball, sealed with tin foil, and placed in a plant growth chamber at 28°C under the conditions of 12/12 h light/dark alternation. After 15 days, the Trichoderma populations in the rhizosphere soil and roots endoderm of cucumber were calculated by a dilution plate method [27] (thirty seedlings for each treatment). The experiment was performed in triplicate.


## 2.7. Determination ofβ-1,3-Glucanase and Chitinase Activity of Trichoderma Strains

For the determination ofβ-1,3-glucanase activity, the Trichoderma strain was incubated in synthetic medium and cultured at 28°C with shaking at 180 rpm for 3 days. Medium without Trichoderma spp. was used as control. Approximately 1 mL of the culture filtrate was used to assay activity of β-1,3-glucanase using the 3,5-dinitrosalicylic acid colorimetric method [28]. For the determination of chitinase activity, Trichoderma strain was incubated in chitinase detection medium and cultured at 28°C with shaking at 180 rpm for 3 days. Colloidal chitin was prepared according to the method described by Roberts and Selitrennikoff [29]. The assay was performed in 10 mL tubes containing 1 mL 1% colloidal chitin, 1.5 mL 50 mM acetate buffer pH =5.0, and 1 mL suspension, the mixture was incubated at 40°C for 1 h, and the chitinase activity was determined basing the standard curves of N-acetyl-D-glucosamine (GlcNAc) measured at 540 nm absorbance. Enzyme bioactivity was calculated as follows: enzymebioactivityU/mL=A1−A2+b1×d/K1×V1×T1, where A1 represents OD540 of samples, A2 represents OD540 of control, b1 represents the y-intercept of standard curves, d represents dilution rate of suspension, K1 represents the slope of standard curves, V1 represents the volume of mixture (mL), and T1 represents reaction time (h). Each treatment was performed in triplicate.


## 2.8. Identification ofTrichoderma Strains

The morphological characteristics of conidia, phialide, conidiophore, and chlamydospores and the colony growth patterns ofTrichoderma strains cultured on PDA, SNA, and CMD media were observed according to the description of Gams and Bissett [30]. For molecular identification, genomic DNAs of Trichoderma strains were extracted using the method of cetyl-trimethylammonium ammonium bromide (CTAB) as described by Ihrmark et al. [31]. The tef-1 gene was amplified with primers tef1-728F (5′-CATCGAGAAGTTCGAGAAGG-3′) and tef1-R (5′-GCCATCCTTGGGAGATACCAGC-3′), and ITS region was amplified with primers ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′). The PCR products were cloned into the Ti-19 vector and sequenced by the Sangon Biotechnology Co., Ltd. (Shanghai, China). All sequences from Trichoderma strains were aligned and spliced through “tef-1+ITS region” using MAFFT v7.215 [32]. The best substitution model (GTR+F+I+G4) was evaluated with IQ-TREE software using the ultrafast bootstrap method with 10,000 replicates. The phylogenetic tree was constructed based on tef-1 gene and ITS region for the molecular identification of the Trichoderma species.


## 2.9. Statistical Analyses

All experiments were repeated three times. The data were analyzed using IBM SPSS Statistics 21.0 software (IBM Corp., Armonk, NY, USA) using one-way analysis of variance (ANOVA). Means of treatment were compared using the least significant difference (LSD) test atP<0.05 and separated by superscript letters.


## 3. Results

### 3.1. Screening the Antagonistic Activity ofTrichoderma Strains

A total of 1308Trichoderma strains were obtained by the dilution plate method and single spore isolation method, including 1018 strains from 1018 rhizosphere soil samples, 205 strains from 45 rotten wood samples, and 86 strains from 16 plant samples. The dual culture test showed that 49 Trichoderma strains had good inhibitory effects against F. oxysporum (FOC), C. gloeosporioides (CG), and B. cinerea (BC), and their PIs were all exceeded 85% (Table 1). These 49 strains used for further experiments.Table 1
The region of the isolates which were screened in dual culture and its PI to three pathogens.
Code
Region
PI (%)
FOC
CG
BC
HL003
Hegang, Heilongjiang
85.13
±
2.35
86.15
±
1.54
90.56
±
0.96
HN071
Ledong, Hainan
89.23
±
1.54
88.72
±
2.35
89.74
±
1.18
SC006
Panzhihua, Sichuan
86.15
±
6.71
97.95
±
0.89
89.33
±
6.11
SC017
Panzhihua, Sichuan
86.67
±
2.35
93.85
±
1.54
89.33
±
2.31
HN059
Ledong, Hainan
85.13
±
2.35
91.28
±
0.89
88.89
±
1.48
JX013
Pingxiang, Jiangxi
85.13
±
3.20
88.21
±
3.55
88.51
±
5.27
HL007
Hegang, Heilongjiang
86.15
±
1.54
91.28
±
1.78
88.33
±
0.22
HL101
Jiamusi, Heilongjiang
85.13
±
0.89
88.21
±
0.89
88.33
±
3.89
HN046
Danzhou, Hainan
86.67
±
0.89
89.74
±
0.89
88.03
±
1.48
HL008
Hailun, Heilongjiang
90.51
±
0.44
95.49
±
0.18
88.00
±
0.92
JX002
Pingxiang, Jiangxi
85.64
±
4.95
91.28
±
0.89
87.03
±
2.25
GX004
Guilin, Guangxi
88.21
±
0.89
93.33
±
0.89
87.03
±
5.94
HL137
Hailun, Heilongjiang
86.15
±
1.54
90.26
±
3.20
87.03
±
2.25
NM023
Chifeng, Neimeng
85.13
±
2.35
91.28
±
3.55
85.06
±
1.99
HL119
Yichun, Heilongjiang
85.13
±
3.87
85.13
±
0.89
86.67
±
2.31
FJ090
Ningde, Fujian
86.15
±
1.54
95.90
±
0.89
86.67
±
2.31
HL092
Daqing, Heilongjiang
90.82
±
0.09
93.33
±
0.89
86.67
±
2.31
ZJ019
Wenzhou, Zhejiang
88.72
±
3.87
88.21
±
3.20
86.50
±
2.34
HN027
Danzhou, Hainan
85.13
±
0.89
91.28
±
0.89
86.32
±
3.92
SX001
Changzhi, Shanxi
86.15
±
1.54
88.72
±
1.78
85.47
±
1.48
HL144
Hegang, Heilongjiang
87.69
±
1.54
92.82
±
2.35
85.73
±
4.49
HL018
Jiamusi, Heilongjiang
85.13
±
0.89
91.79
±
0.89
85.73
±
5.94
HL132
Hegang, Heilongjiang
86.67
±
4.70
89.74
±
1.78
85.73
±
5.94
HL131
Hegang, Heilongjiang
88.72
±
0.89
91.28
±
2.35
85.73
±
5.94
FJ065
Ningde, Fujian
87.18
±
3.87
91.28
±
2.35
85.73
±
4.49
SC011
Panzhihua, Sichuan
90.77
±
3.08
93.85
±
1.54
85.33
±
2.31
SC018
Panzhihua, Sichuan
86.15
±
1.54
96.41
±
3.55
85.33
±
2.31
SC013
Panzhihua, Sichuan
89.74
±
3.20
91.79
±
0.89
85.33
±
6.11
FJ058
Ningde, Fujian
85.13
±
0.89
94.36
±
1.78
85.33
±
2.31
HL015
Daqing, Heilongjiang
85.64
±
0.89
91.79
±
0.89
85.33
±
2.31
HL100
Hailun, Heilongjiang
90.26
±
3.20
95.90
±
0.89
85.33
±
4.62
FJ034
Fuzhou, Fujian
85.13
±
1.78
85.64
±
4.95
85.16
±
4.67
LN005
Dalian, Liaoning
86.67
±
1.78
87.18
±
0.89
85.06
±
3.98
XM002-21
Danzhou, Hainan
86.10
±
0.09
86.15
±
1.54
85.06
±
5.27
HL048
Daqing, Heilongjiang
92.82
±
0.89
96.41
±
0.89
85.20
±
2.08
SC010
Panzhihua, Sichuan
87.69
±
2.66
90.77
±
1.54
86.13
±
3.70
HL135
Hegang, Heilongjiang
90.77
±
1.54
90.26
±
3.20
85.20
±
2.12
SC012
Chengdu, Sichuan
90.21
±
0.98
94.87
±
3.20
85.33
±
6.11
HN018
Danzhou, Hainan
90.26
±
2.35
89.23
±
1.54
85.21
±
3.39
GZ070
Guiyang, Guizhou
90.15
±
2.32
92.31
±
1.54
86.50
±
4.67
FJ087
Fuzhou, Fujian
88.72
±
2.35
94.97
±
0.71
85.33
±
3.06
SC019
Panzhihua, Sichuan
89.74
±
0.89
96.92
±
2.66
85.33
±
7.02
JS016
Xuzhou, Jiangsu
85.08
±
0.80
89.74
±
2.35
91.11
±
1.92
BJ006
Haidian, Beijing
85.13
±
2.35
86.67
±
9.40
90.00
±
3.33
QH060
Haixi, Qinghai
87.79
±
0.18
87.69
±
1.54
85.13
±
2.35
SC101
Guangyuan, Sichuan
86.26
±
0.18
85.13
±
0.89
85.13
±
0.89
NX043
Qinghai, Ningxia
85.13
±
0.89
85.12
±
0.89
89.23
±
1.54
XJ035
Yili, Xinjiang
89.38
±
0.27
85.13
±
0.89
86.67
±
1.78
SC098
Guangyuan, Sichuan
87.74
±
0.09
85.17
±
2.35
86.15
±
0.27
XJ087
Kuerle, Xinjiang
86.15
±
0.27
85.13
±
0.89
85.13
±
0.89
Data represent themean±SD (n=3). FOC: Fusarium oxysporum; CG: Colletotrichum gloeosporioides; BC: Botrytis cinerea; PI (%): percentage of growth inhibition.


### 3.2. Promotion Effect ofTrichoderma Strains on Seed Germination and Root Growth

The germination test showed that 13 strains (GZ070, HL100, HL119, HL135, HN059, JX013, SC012, XJ035, NX043, QH060, XJ087, SC098, and SC101) significantly promoted the seed germination of the five plants compared with the control. Among them, HL100 strain showed the best promotion effects on seed germination of rice, cucumber, and pakchoi, with increased germination rates of 12.17%, 13.23%, and 27.54%, respectively. The XM002-21 and XJ087 strains showed the highest promotion effects on seed germination of melon and tomato, with increased germination rates of 12.55% and 41.94%, respectively (Table2).Table 2
The germination rate of 5 kinds of seeds afterTrichoderma treatment.
Code
Seed germination rates (%)
Rice
Cucumber
Tomato
Melon
Pakchoi
GZ070
83.14
±
1.97
a
85.56
±
10.18
a
44.00
±
14.37
d
,
e
83.33
±
10.00
a
71.99
±
4.41
b
HL100
94.06
±
3.98
a
90.56
±
7.97
a
49.91
±
15.25
b
,
c
,
d
,
e
80.83
±
0.72
a
99.48
±
0.90
a
HL119
89.62
±
4.36
a
89.64
±
7.50
a
43.31
±
1.30
d
,
e
85.29
±
7.50
a
97.68
±
0.86
a
HL135
93.39
±
3.26
a
87.30
±
10.13
a
48.72
±
15.06
c
,
d
,
e
78.89
±
8.67
a
98.93
±
0.93
a
HN059
89.70
±
3.14
a
79.21
±
7.40
a
45.93
±
8.41
d
,
e
80.43
±
3.25
a
94.88
±
2.46
a
JX013
89.59
±
7.09
a
78.92
±
13.81
a
48.03
±
5.85
c
,
d
,
e
77.14
±
8.51
a
71.96
±
5.24
b
SC012
92.81
±
3.92
a
88.80
±
4.74
a
43.99
±
18.12
d
,
e
86.59
±
11.48
a
99.00
±
1.72
a
XJ035
83.78
±
19.05
a
82.22
±
16.44
a
72.00
±
16.00
a
,
b
,
c
76.81
±
9.77
a
78.67
±
13.32
b
NX043
82.33
±
15.53
a
80.00
±
15.28
a
74.67
±
2.15
a
,
b
82.22
±
10.09
a
81.11
±
7.37
b
QH060
87.00
±
7.55
a
85.56
±
6.94
a
88.00
±
2.00
a
77.78
±
13.47
a
83.33
±
3.06
b
XJ087
87.75
±
8.49
a
82.22
±
5.09
a
83.33
±
5.77
a
81.11
±
1.92
a
80.00
±
3.46
b
SC098
87.33
±
7.02
a
85.56
±
8.39
a
66.11
±
10.46
a
,
b
,
c
,
d
,
e
79.33
±
4.62
a
76.00
±
10.58
b
SC101
82.34
±
12.17
a
79.56
±
1.92
a
69.33
±
1.15
a
,
b
,
c
,
d
82.22
±
11.71
a
73.67
±
11.72
b
CK
81.91
±
17.41
a
77.33
±
8.03
a
41.39
±
12.41
e
76.34
±
4.93
a
71.94
±
7.25
b
Germination rates of seed were measured at 7 days, where seeds were treated by 105 spore mL–1 suspension in 2 hours. “CK” means to treat seeds with sterile water. Data represent the mean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test.The 13Trichoderma strains that significantly promoted the seed germination were further tested for their effect on root growth of watermelon, chili, eggplant, and tomato seedlings. In greenhouse experiment, all strains showed a promotion effect on root growth of chili seedlings, and among them, HL100 strain showed maximum increase 12.17% in root length compared to control. Three strains (GZ070, HL100, and HN059) significantly promoted the root growth of watermelon seedlings. HN059 strain showed maximum increase of 18.81% in root length compared to control. Trichoderma strains GZ070, HL100, HN059, JX013, XJ087, and NX043 strains and HL119, HN059, SC012, XJ035, SC098, and SC101 were found to promote the root growth of eggplant and tomato seedlings, respectively. The highest increase in root length of eggplant (40.99%) and tomato plants (34.68%) was recorded by GZ070 and SC098 strains, respectively (Table 3).Table 3
Root length of different crops treated with a spore suspension at 15 days.
Code
Length of roots (cm)
Watermelon
Chili
Eggplant
Tomato
GZ070
11.71
±
0.18
a
,
b
,
c
9.56
±
0.54
b
,
c
,
d
12.88
±
2.55
a
3.74
±
0.49
e
,
f
,
g
,
h
HL100
12.26
±
0.73
a
,
b
,
c
11.85
±
0.83
a
,
b
10.48
±
1.06
a
,
b
,
c
,
d
,
e
3.60
±
0.37
g
,
h
HL119
9.36
±
1.00
h
11.08
±
0.82
a
,
b
,
c
9.47
±
2.13
i
,
j
5.03
±
0.71
a
,
b
,
c
,
d
HL135
11.20
±
0.57
c
,
d
,
e
,
f
,
g
9.72
±
0.74
b
,
c
,
d
9.54
±
1.29
h
,
i
,
j
2.98
±
0.16
h
HN059
13.03
±
1.51
a
11.28
±
1.38
a
,
b
10.16
±
0.55
c
,
d
,
e
,
f
,
g
4.10
±
0.29
e
,
f
,
g
,
h
JX013
9.71
±
0.54
h
10.33
±
0.57
a
,
b
,
c
,
d
10.91
±
1.02
a
,
b
,
c
,
d
3.73
±
0.46
f
,
g
,
h
SC012
11.33
±
1.10
b
,
c
,
d
,
e
,
f
10.19
±
0.95
a
,
b
,
c
,
d
9.24
±
0.66
h
,
i
,
j
5.02
±
0.54
a
,
b
XJ035
10.69
±
0.49
c
,
d
,
e
,
f
,
g
10.96
±
0.90
a
,
b
,
c
,
d
8.24
±
0.70
h
,
i
,
j
4.76
±
0.54
b
,
c
,
d
NX043
11.65
±
1.66
b
,
c
,
d
,
e
,
f
,
g
10.51
±
0.68
a
,
b
,
c
,
d
9.39
±
1.58
e
,
f
,
g
,
h
,
i
,
j
3.37
±
0.38
h
QH060
11.50
±
0.99
c
,
d
,
e
,
f
,
g
9.20
±
0.51
a
,
b
,
c
,
d
8.45
±
0.57
h
,
i
,
j
3.43
±
0.61
f
,
g
,
h
XJ087
10.25
±
0.58
g
,
h
9.79
±
0.78
a
,
b
,
c
,
d
11.33
±
0.38
a
,
b
,
c
,
d
,
e
3.35
±
0.33
f
,
g
,
h
SC098
10.18
±
0.88
e
,
f
,
g
,
h
8.96
±
0.76
b
,
c
,
d
8.45
±
0.87
i
,
j
5.40
±
0.46
a
SC101
9.99
±
0.83
f
,
g
,
h
9.88
±
0.65
a
,
b
7.00
±
0.32
k
4.42
±
0.37
d
,
e
,
g
,
f
CK
11.26
±
0.18
a
,
b
9.34
±
0.38
d
9.63
±
0.15
f
,
g
,
h
,
i
,
j
3.93
±
0.14
f
,
g
,
h
The concentration of the conidial suspension was 105 spores mL–1. “CK” means to treat seeds with sterile water. Data represent the mean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test.


### 3.3. Colonization Ability ofTrichoderma Strains

The colonization of GZ070, HL100, and JX013 strains in rhizosphere soil was higher than that of other strains, which were8.30×104,1.50×105,and1.56×105 CFU g–1, respectively. In the root endoderm of cucumber, the colonization of GZ070, HL100, and QH060 strains was higher than that of other strains, showing 7.67×104,1.20×105,and8.00×104 CFU g–1, respectively (Figure 1).Figure 1
Determination of colonization ability ofTrichoderma on cucumber seedlings. Data represent the mean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test.


### 3.4. Activity of Chitinase andβ-1,3-Glucanase

The 13Trichoderma strains were used to measure the activities of chitinase and β-1,3-glucanase. The results showed that these strains possessed a wide range of activity of chitinase and β-1,3-glucanase (Table 4). SC012 strain exhibited the highest chitinase activity (0.154 U mL–1). NX043 and XJ087 strains showed the highest β-1,3-glucanase activity. The lowest chitinase activity (0.041 U mL–1) and β-1,3-glucanase activity (0.1765 U mL–1) were shown by SC098 and XJ035 strains, respectively.Table 4
Chitinase andβ-1,3-glucanase activity of Trichoderma isolates at 3 days in enzyme synthetic medium.
Code
Chitinase (U mL–1)
β-1,3-Glucanase (U mL–1)
GZ070
0.069
±
0.018
g
,
h
,
i
0.398
±
0.044
b
,
c
HL100
0.105
±
0.017
b
,
c
,
d
,
e
,
f
0.319
±
0.042
b
,
c
,
d
HL119
0.112
±
0.018
b
,
c
,
d
,
e
0.397
±
0.022
b
,
c
HL135
0.081
±
0.006
e
,
f
,
g
,
h
0.488
±
0002
b
HN059
0.068
±
0.016
g
,
h
,
i
0.340
±
0.002
b
,
c
,
d
JX013
0.082
±
0.001
e
,
f
,
g
,
h
0.489
±
0.05
b
SC012
0.154
±
0.013
a
0.3744
±
0.062
b
,
c
,
d
XJ035
0.087
±
0.009
d
,
e
,
f
,
g
,
h
0.1765
±
0.038
c
,
d
NX043
0.108
±
0.028
b
,
c
,
d
,
e
0.8162
±
0.172
a
QH060
0.126
±
0.009
b
0.2258
±
0.070
b
,
c
,
d
XJ087
0.104
±
0.039
b
,
c
,
d
,
e
,
f
0.7993
±
0.199
a
SC098
0.047
±
0.015
i
0.3814
±
0.048
b
,
c
,
d
SC101
0.095
±
0.006
c
,
d
,
e
,
f
,
g
0.3170
±
0.08
b
,
c
,
d
Data represent themean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test.


### 3.5. Detection of Antimicrobial Spectrum ofTrichoderma

The results showed that 13Trichoderma strains exhibited significant inhibitory effects on 9 kinds of pathogenic fungi (Table 5). The inhibition rates of 13 Trichoderma strains to FT and MO were all over 80% (80.81–88.37%). NX043 strain had the highest inhibition rate on FT (88.37%), and HL100 strain had the highest inhibition rate to MO (87.50%). However, the inhibition rate of 13 Trichoderma strains on BT was lower than that of other pathogenic fungi (59.49–70.26%).Table 5
Inhibition spectrum ofTrichoderma isolates, the PI of isolates against 9 pathogens in dual culture.
Code
PI (%)
SR
FT
BD
MO
BT
FG
RC
SC
RS
GZ070
78.97
±
1.78
a
87.50
±
1.33
a
83.61
±
4.09
a
,
b
83.62
±
1.49
a
67.69
±
1.54
a
83.08
±
1.54
a
83.97
±
1.11
c
78.09
±
1.65
a
,
b
85.29
±
3.23
a
,
b
HL100
75.38
±
4.07
b
87.50
±
2.80
a
86.34
±
2.3
7
a
,
b
87.50
±
1.98
a
62.50
±
2.35
a
81.03
±
0.89
a
,
b
85.58
±
0.96
b
,
c
79.17
±
0.62
a
,
b
76.31
±
1.41
b
HL119
79.49
±
0.89
a
88.08
±
0.50
a
90.44
±
4.73
a
86.64
±
0.75
a
68.72
±
0.89
a
82.05
±
0.89
a
,
b
85.90
±
1.11
b
,
c
79.17
±
2.49
a
,
b
85.70
±
4.64
a
HL135
78.46
±
2.66
a
86.34
±
0.50
c
78.14
±
4.73
b
,
c
84.05
±
1.98
a
65.64
±
1.78
a
84.36
±
1.18
a
85.26
±
1.11
b
,
c
78.09
±
0.62
a
,
b
84.89
±
3.08
a
,
b
HN059
78.56
±
0.18
a
86.63
±
0.50
a
87.70
±
4.10
a
,
b
86.21
±
3.73
a
65.64
±
9.01
a
81.28
±
1.94
a
,
b
81.41
±
1.11
b
,
c
78.45
±
2.85
a
,
b
82.84
±
4.42
a
,
b
JX013
80.00
±
5.33
a
86.45
±
1.31
a
81.56
±
2.05
a
,
b
,
c
85.78
±
2.59
a
64.10
±
0.89
a
83.85
±
2.77
a
83.33
±
1.06
c
82.04
±
5.42
a
,
b
85.70
±
3.54
a
SC012
78.97
±
3.55
a
86.05
±
0.87
a
84.29
±
1.18
a
,
b
86.64
±
1.49
a
70.26
±
8.75
a
82.15
±
2.51
a
,
b
87.82
±
1.11
a
,
b
79.89
±
3.29
a
,
b
83.66
±
3.54
a
,
b
XJ035
76.92
±
2.66
a
86.92
±
0.87
a
86.34
±
4.27
a
,
b
84.91
±
0.75
a
69.23
±
7.99
a
82.05
±
0.44
a
,
b
83.97
±
1.11
c
81.68
±
5.39
a
,
b
88.15
±
3.54
a
NX043
79.74
±
0.44
a
88.37
±
1.01
a
82.24
±
4.27
a
,
b
,
c
86.64
±
0.75
a
61.03
±
1.78
a
83.13
±
0.09
a
89.42
±
0.96
a
76.65
±
0.62
a
,
b
86.11
±
3.94
a
QH060
79.49
±
0.89
a
80.81
±
1.74
b
,
c
80.19
±
1.18
a
,
b
,
c
85.78
±
2.24
a
64.62
±
11.99
a
83.08
±
1.54
a
84.94
±
1.47
b
,
c
75.57
±
1.24
b
81.21
±
2.83
a
,
b
XJ087
78.46
±
1.54
a
84.88
±
1.82
b
81.56
±
2.05
a
,
b
,
c
84.05
±
1.49
a
69.23
±
6.15
a
82.05
±
1.78
a
,
b
83.97
±
1.11
c
78.45
±
3.73
a
,
b
87.75
±
2.45
a
SC098
76.92
±
4.07
a
88.08
±
1.33
a
72.68
±
6.26
c
82.76
±
1.49
a
66.67
±
6.22
a
78.97
±
0.89
b
79.49
±
2.94
d
73.42
±
1.24
a
,
b
87.13
±
0.87
a
SC101
76.41
±
5.40
b
84.59
±
3.30
b
89.07
±
6.26
a
83.19
±
1.29
a
59.49
±
4.44
a
82.31
±
1.33
a
,
b
84.29
±
1.47
c
85.27
±
5.53
a
81.62
±
6.13
a
,
b
Data represent themean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test. SR: Sclerotium rolfsii; FT: Fusarium oxysporum f.sp. tracheiphilum; BD: Botryosphaeria dothidea; MO: Magnaporthe oryzae; BT: B. theobromae; FG: F. graminearum; RC: Rhizoctonia cerealis; SC: Stagonosporopsis cucurbitacearum; RS: R. solani; PI: percentage of growth inhibition.


### 3.6. Identification ofTrichoderma Strains with Potential Agricultural Applications

All strains, except for SC098, showed the same morphological characteristics after growing on three kinds of medium for 7 days. These strains appeared as dark green colonies with a distinctive coconut odor on PDA plates, and they formed green conidial pustules on CMD medium and produced white, yellow, and green sorus, forming concentric zones on SNA plates (Figures2(a)–2(e) and 3(a)–(e)). Conidia and chlamydospores are smooth, watered, and subglobose, and the branches were pyramidal and verticillate or paired lateral arising. Phialides were tended to cluster on branches. SC098 strain produced a diffuse yellow pigment on both sides of PDA plates, and green and yellow cottony sori were observed on CMD medium at 48 h. The mycelia of the strains were wavy and grown floccose to arachnoid on SNA plates, and phialides were ampulliform (length, 5-6.5 μm; widest point, 2.9-3.2 μm), tending to cluster and to create a narrow neck at the top (Figures 2(f)–2(h) and 3(f)–(h)). A phylogenetic tree demonstrated that all the selected strains had two distinct clades. JX013, HN059, XJ097, QH060, HL100, HL119, HL135, SC012, NX043, SC101, GZ070, and XJ035 strains belonged to the Hamatum clade and had affinity to KP009011 (T. asperellum). SC098 strain was identified as T. afroharzianum, which was closely related to KP0699541 (T. afroharzianum) and belonged to the harzianum clade (Figure 4). Based on the morphological and molecular characteristics, the remaining 12 strains were identified as T. asperellum, and SC098 strain was identified as T. afroharzianum.Figure 2
Morphological identification of JX013 (Trichoderma asperellum). (a–c) Cultures of JX013 that were grown on PDA, CMD, and SNA medium. (d, e) Conidial pustules. (f) Conidia. (g, h) Conidiophore. (i) Chlamydospores. PDA: potato dextrose agar medium; CMD: corn meal dextrose agar medium; SNA: synthetic low nutrient agar medium.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)Figure 3
Morphological identification of SC098 (Trichoderma afroharzianum). (a–c) Cultures of JX013 which grew on PDA, CMD, and SNA medium. (d, e) Conidial pustules. (f) Conidia. (g, h) Conidiophore. (i) Chlamydospore. PDA: potato dextrose agar medium; CMD: corn meal dextrose agar medium; SNA: synthetic low nutrient agar medium.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)Figure 4
Phylogenetic analysis based on thetef-1 gene and ITS region from the strains screened. GenBank accession numbers of 13 Trichoderma strains shown in Supplementary Table 2.


## 3.1. Screening the Antagonistic Activity ofTrichoderma Strains

A total of 1308Trichoderma strains were obtained by the dilution plate method and single spore isolation method, including 1018 strains from 1018 rhizosphere soil samples, 205 strains from 45 rotten wood samples, and 86 strains from 16 plant samples. The dual culture test showed that 49 Trichoderma strains had good inhibitory effects against F. oxysporum (FOC), C. gloeosporioides (CG), and B. cinerea (BC), and their PIs were all exceeded 85% (Table 1). These 49 strains used for further experiments.Table 1
The region of the isolates which were screened in dual culture and its PI to three pathogens.
Code
Region
PI (%)
FOC
CG
BC
HL003
Hegang, Heilongjiang
85.13
±
2.35
86.15
±
1.54
90.56
±
0.96
HN071
Ledong, Hainan
89.23
±
1.54
88.72
±
2.35
89.74
±
1.18
SC006
Panzhihua, Sichuan
86.15
±
6.71
97.95
±
0.89
89.33
±
6.11
SC017
Panzhihua, Sichuan
86.67
±
2.35
93.85
±
1.54
89.33
±
2.31
HN059
Ledong, Hainan
85.13
±
2.35
91.28
±
0.89
88.89
±
1.48
JX013
Pingxiang, Jiangxi
85.13
±
3.20
88.21
±
3.55
88.51
±
5.27
HL007
Hegang, Heilongjiang
86.15
±
1.54
91.28
±
1.78
88.33
±
0.22
HL101
Jiamusi, Heilongjiang
85.13
±
0.89
88.21
±
0.89
88.33
±
3.89
HN046
Danzhou, Hainan
86.67
±
0.89
89.74
±
0.89
88.03
±
1.48
HL008
Hailun, Heilongjiang
90.51
±
0.44
95.49
±
0.18
88.00
±
0.92
JX002
Pingxiang, Jiangxi
85.64
±
4.95
91.28
±
0.89
87.03
±
2.25
GX004
Guilin, Guangxi
88.21
±
0.89
93.33
±
0.89
87.03
±
5.94
HL137
Hailun, Heilongjiang
86.15
±
1.54
90.26
±
3.20
87.03
±
2.25
NM023
Chifeng, Neimeng
85.13
±
2.35
91.28
±
3.55
85.06
±
1.99
HL119
Yichun, Heilongjiang
85.13
±
3.87
85.13
±
0.89
86.67
±
2.31
FJ090
Ningde, Fujian
86.15
±
1.54
95.90
±
0.89
86.67
±
2.31
HL092
Daqing, Heilongjiang
90.82
±
0.09
93.33
±
0.89
86.67
±
2.31
ZJ019
Wenzhou, Zhejiang
88.72
±
3.87
88.21
±
3.20
86.50
±
2.34
HN027
Danzhou, Hainan
85.13
±
0.89
91.28
±
0.89
86.32
±
3.92
SX001
Changzhi, Shanxi
86.15
±
1.54
88.72
±
1.78
85.47
±
1.48
HL144
Hegang, Heilongjiang
87.69
±
1.54
92.82
±
2.35
85.73
±
4.49
HL018
Jiamusi, Heilongjiang
85.13
±
0.89
91.79
±
0.89
85.73
±
5.94
HL132
Hegang, Heilongjiang
86.67
±
4.70
89.74
±
1.78
85.73
±
5.94
HL131
Hegang, Heilongjiang
88.72
±
0.89
91.28
±
2.35
85.73
±
5.94
FJ065
Ningde, Fujian
87.18
±
3.87
91.28
±
2.35
85.73
±
4.49
SC011
Panzhihua, Sichuan
90.77
±
3.08
93.85
±
1.54
85.33
±
2.31
SC018
Panzhihua, Sichuan
86.15
±
1.54
96.41
±
3.55
85.33
±
2.31
SC013
Panzhihua, Sichuan
89.74
±
3.20
91.79
±
0.89
85.33
±
6.11
FJ058
Ningde, Fujian
85.13
±
0.89
94.36
±
1.78
85.33
±
2.31
HL015
Daqing, Heilongjiang
85.64
±
0.89
91.79
±
0.89
85.33
±
2.31
HL100
Hailun, Heilongjiang
90.26
±
3.20
95.90
±
0.89
85.33
±
4.62
FJ034
Fuzhou, Fujian
85.13
±
1.78
85.64
±
4.95
85.16
±
4.67
LN005
Dalian, Liaoning
86.67
±
1.78
87.18
±
0.89
85.06
±
3.98
XM002-21
Danzhou, Hainan
86.10
±
0.09
86.15
±
1.54
85.06
±
5.27
HL048
Daqing, Heilongjiang
92.82
±
0.89
96.41
±
0.89
85.20
±
2.08
SC010
Panzhihua, Sichuan
87.69
±
2.66
90.77
±
1.54
86.13
±
3.70
HL135
Hegang, Heilongjiang
90.77
±
1.54
90.26
±
3.20
85.20
±
2.12
SC012
Chengdu, Sichuan
90.21
±
0.98
94.87
±
3.20
85.33
±
6.11
HN018
Danzhou, Hainan
90.26
±
2.35
89.23
±
1.54
85.21
±
3.39
GZ070
Guiyang, Guizhou
90.15
±
2.32
92.31
±
1.54
86.50
±
4.67
FJ087
Fuzhou, Fujian
88.72
±
2.35
94.97
±
0.71
85.33
±
3.06
SC019
Panzhihua, Sichuan
89.74
±
0.89
96.92
±
2.66
85.33
±
7.02
JS016
Xuzhou, Jiangsu
85.08
±
0.80
89.74
±
2.35
91.11
±
1.92
BJ006
Haidian, Beijing
85.13
±
2.35
86.67
±
9.40
90.00
±
3.33
QH060
Haixi, Qinghai
87.79
±
0.18
87.69
±
1.54
85.13
±
2.35
SC101
Guangyuan, Sichuan
86.26
±
0.18
85.13
±
0.89
85.13
±
0.89
NX043
Qinghai, Ningxia
85.13
±
0.89
85.12
±
0.89
89.23
±
1.54
XJ035
Yili, Xinjiang
89.38
±
0.27
85.13
±
0.89
86.67
±
1.78
SC098
Guangyuan, Sichuan
87.74
±
0.09
85.17
±
2.35
86.15
±
0.27
XJ087
Kuerle, Xinjiang
86.15
±
0.27
85.13
±
0.89
85.13
±
0.89
Data represent themean±SD (n=3). FOC: Fusarium oxysporum; CG: Colletotrichum gloeosporioides; BC: Botrytis cinerea; PI (%): percentage of growth inhibition.


## 3.2. Promotion Effect ofTrichoderma Strains on Seed Germination and Root Growth

The germination test showed that 13 strains (GZ070, HL100, HL119, HL135, HN059, JX013, SC012, XJ035, NX043, QH060, XJ087, SC098, and SC101) significantly promoted the seed germination of the five plants compared with the control. Among them, HL100 strain showed the best promotion effects on seed germination of rice, cucumber, and pakchoi, with increased germination rates of 12.17%, 13.23%, and 27.54%, respectively. The XM002-21 and XJ087 strains showed the highest promotion effects on seed germination of melon and tomato, with increased germination rates of 12.55% and 41.94%, respectively (Table2).Table 2
The germination rate of 5 kinds of seeds afterTrichoderma treatment.
Code
Seed germination rates (%)
Rice
Cucumber
Tomato
Melon
Pakchoi
GZ070
83.14
±
1.97
a
85.56
±
10.18
a
44.00
±
14.37
d
,
e
83.33
±
10.00
a
71.99
±
4.41
b
HL100
94.06
±
3.98
a
90.56
±
7.97
a
49.91
±
15.25
b
,
c
,
d
,
e
80.83
±
0.72
a
99.48
±
0.90
a
HL119
89.62
±
4.36
a
89.64
±
7.50
a
43.31
±
1.30
d
,
e
85.29
±
7.50
a
97.68
±
0.86
a
HL135
93.39
±
3.26
a
87.30
±
10.13
a
48.72
±
15.06
c
,
d
,
e
78.89
±
8.67
a
98.93
±
0.93
a
HN059
89.70
±
3.14
a
79.21
±
7.40
a
45.93
±
8.41
d
,
e
80.43
±
3.25
a
94.88
±
2.46
a
JX013
89.59
±
7.09
a
78.92
±
13.81
a
48.03
±
5.85
c
,
d
,
e
77.14
±
8.51
a
71.96
±
5.24
b
SC012
92.81
±
3.92
a
88.80
±
4.74
a
43.99
±
18.12
d
,
e
86.59
±
11.48
a
99.00
±
1.72
a
XJ035
83.78
±
19.05
a
82.22
±
16.44
a
72.00
±
16.00
a
,
b
,
c
76.81
±
9.77
a
78.67
±
13.32
b
NX043
82.33
±
15.53
a
80.00
±
15.28
a
74.67
±
2.15
a
,
b
82.22
±
10.09
a
81.11
±
7.37
b
QH060
87.00
±
7.55
a
85.56
±
6.94
a
88.00
±
2.00
a
77.78
±
13.47
a
83.33
±
3.06
b
XJ087
87.75
±
8.49
a
82.22
±
5.09
a
83.33
±
5.77
a
81.11
±
1.92
a
80.00
±
3.46
b
SC098
87.33
±
7.02
a
85.56
±
8.39
a
66.11
±
10.46
a
,
b
,
c
,
d
,
e
79.33
±
4.62
a
76.00
±
10.58
b
SC101
82.34
±
12.17
a
79.56
±
1.92
a
69.33
±
1.15
a
,
b
,
c
,
d
82.22
±
11.71
a
73.67
±
11.72
b
CK
81.91
±
17.41
a
77.33
±
8.03
a
41.39
±
12.41
e
76.34
±
4.93
a
71.94
±
7.25
b
Germination rates of seed were measured at 7 days, where seeds were treated by 105 spore mL–1 suspension in 2 hours. “CK” means to treat seeds with sterile water. Data represent the mean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test.The 13Trichoderma strains that significantly promoted the seed germination were further tested for their effect on root growth of watermelon, chili, eggplant, and tomato seedlings. In greenhouse experiment, all strains showed a promotion effect on root growth of chili seedlings, and among them, HL100 strain showed maximum increase 12.17% in root length compared to control. Three strains (GZ070, HL100, and HN059) significantly promoted the root growth of watermelon seedlings. HN059 strain showed maximum increase of 18.81% in root length compared to control. Trichoderma strains GZ070, HL100, HN059, JX013, XJ087, and NX043 strains and HL119, HN059, SC012, XJ035, SC098, and SC101 were found to promote the root growth of eggplant and tomato seedlings, respectively. The highest increase in root length of eggplant (40.99%) and tomato plants (34.68%) was recorded by GZ070 and SC098 strains, respectively (Table 3).Table 3
Root length of different crops treated with a spore suspension at 15 days.
Code
Length of roots (cm)
Watermelon
Chili
Eggplant
Tomato
GZ070
11.71
±
0.18
a
,
b
,
c
9.56
±
0.54
b
,
c
,
d
12.88
±
2.55
a
3.74
±
0.49
e
,
f
,
g
,
h
HL100
12.26
±
0.73
a
,
b
,
c
11.85
±
0.83
a
,
b
10.48
±
1.06
a
,
b
,
c
,
d
,
e
3.60
±
0.37
g
,
h
HL119
9.36
±
1.00
h
11.08
±
0.82
a
,
b
,
c
9.47
±
2.13
i
,
j
5.03
±
0.71
a
,
b
,
c
,
d
HL135
11.20
±
0.57
c
,
d
,
e
,
f
,
g
9.72
±
0.74
b
,
c
,
d
9.54
±
1.29
h
,
i
,
j
2.98
±
0.16
h
HN059
13.03
±
1.51
a
11.28
±
1.38
a
,
b
10.16
±
0.55
c
,
d
,
e
,
f
,
g
4.10
±
0.29
e
,
f
,
g
,
h
JX013
9.71
±
0.54
h
10.33
±
0.57
a
,
b
,
c
,
d
10.91
±
1.02
a
,
b
,
c
,
d
3.73
±
0.46
f
,
g
,
h
SC012
11.33
±
1.10
b
,
c
,
d
,
e
,
f
10.19
±
0.95
a
,
b
,
c
,
d
9.24
±
0.66
h
,
i
,
j
5.02
±
0.54
a
,
b
XJ035
10.69
±
0.49
c
,
d
,
e
,
f
,
g
10.96
±
0.90
a
,
b
,
c
,
d
8.24
±
0.70
h
,
i
,
j
4.76
±
0.54
b
,
c
,
d
NX043
11.65
±
1.66
b
,
c
,
d
,
e
,
f
,
g
10.51
±
0.68
a
,
b
,
c
,
d
9.39
±
1.58
e
,
f
,
g
,
h
,
i
,
j
3.37
±
0.38
h
QH060
11.50
±
0.99
c
,
d
,
e
,
f
,
g
9.20
±
0.51
a
,
b
,
c
,
d
8.45
±
0.57
h
,
i
,
j
3.43
±
0.61
f
,
g
,
h
XJ087
10.25
±
0.58
g
,
h
9.79
±
0.78
a
,
b
,
c
,
d
11.33
±
0.38
a
,
b
,
c
,
d
,
e
3.35
±
0.33
f
,
g
,
h
SC098
10.18
±
0.88
e
,
f
,
g
,
h
8.96
±
0.76
b
,
c
,
d
8.45
±
0.87
i
,
j
5.40
±
0.46
a
SC101
9.99
±
0.83
f
,
g
,
h
9.88
±
0.65
a
,
b
7.00
±
0.32
k
4.42
±
0.37
d
,
e
,
g
,
f
CK
11.26
±
0.18
a
,
b
9.34
±
0.38
d
9.63
±
0.15
f
,
g
,
h
,
i
,
j
3.93
±
0.14
f
,
g
,
h
The concentration of the conidial suspension was 105 spores mL–1. “CK” means to treat seeds with sterile water. Data represent the mean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test.


## 3.3. Colonization Ability ofTrichoderma Strains

The colonization of GZ070, HL100, and JX013 strains in rhizosphere soil was higher than that of other strains, which were8.30×104,1.50×105,and1.56×105 CFU g–1, respectively. In the root endoderm of cucumber, the colonization of GZ070, HL100, and QH060 strains was higher than that of other strains, showing 7.67×104,1.20×105,and8.00×104 CFU g–1, respectively (Figure 1).Figure 1
Determination of colonization ability ofTrichoderma on cucumber seedlings. Data represent the mean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test.


## 3.4. Activity of Chitinase andβ-1,3-Glucanase

The 13Trichoderma strains were used to measure the activities of chitinase and β-1,3-glucanase. The results showed that these strains possessed a wide range of activity of chitinase and β-1,3-glucanase (Table 4). SC012 strain exhibited the highest chitinase activity (0.154 U mL–1). NX043 and XJ087 strains showed the highest β-1,3-glucanase activity. The lowest chitinase activity (0.041 U mL–1) and β-1,3-glucanase activity (0.1765 U mL–1) were shown by SC098 and XJ035 strains, respectively.Table 4
Chitinase andβ-1,3-glucanase activity of Trichoderma isolates at 3 days in enzyme synthetic medium.
Code
Chitinase (U mL–1)
β-1,3-Glucanase (U mL–1)
GZ070
0.069
±
0.018
g
,
h
,
i
0.398
±
0.044
b
,
c
HL100
0.105
±
0.017
b
,
c
,
d
,
e
,
f
0.319
±
0.042
b
,
c
,
d
HL119
0.112
±
0.018
b
,
c
,
d
,
e
0.397
±
0.022
b
,
c
HL135
0.081
±
0.006
e
,
f
,
g
,
h
0.488
±
0002
b
HN059
0.068
±
0.016
g
,
h
,
i
0.340
±
0.002
b
,
c
,
d
JX013
0.082
±
0.001
e
,
f
,
g
,
h
0.489
±
0.05
b
SC012
0.154
±
0.013
a
0.3744
±
0.062
b
,
c
,
d
XJ035
0.087
±
0.009
d
,
e
,
f
,
g
,
h
0.1765
±
0.038
c
,
d
NX043
0.108
±
0.028
b
,
c
,
d
,
e
0.8162
±
0.172
a
QH060
0.126
±
0.009
b
0.2258
±
0.070
b
,
c
,
d
XJ087
0.104
±
0.039
b
,
c
,
d
,
e
,
f
0.7993
±
0.199
a
SC098
0.047
±
0.015
i
0.3814
±
0.048
b
,
c
,
d
SC101
0.095
±
0.006
c
,
d
,
e
,
f
,
g
0.3170
±
0.08
b
,
c
,
d
Data represent themean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test.


## 3.5. Detection of Antimicrobial Spectrum ofTrichoderma

The results showed that 13Trichoderma strains exhibited significant inhibitory effects on 9 kinds of pathogenic fungi (Table 5). The inhibition rates of 13 Trichoderma strains to FT and MO were all over 80% (80.81–88.37%). NX043 strain had the highest inhibition rate on FT (88.37%), and HL100 strain had the highest inhibition rate to MO (87.50%). However, the inhibition rate of 13 Trichoderma strains on BT was lower than that of other pathogenic fungi (59.49–70.26%).Table 5
Inhibition spectrum ofTrichoderma isolates, the PI of isolates against 9 pathogens in dual culture.
Code
PI (%)
SR
FT
BD
MO
BT
FG
RC
SC
RS
GZ070
78.97
±
1.78
a
87.50
±
1.33
a
83.61
±
4.09
a
,
b
83.62
±
1.49
a
67.69
±
1.54
a
83.08
±
1.54
a
83.97
±
1.11
c
78.09
±
1.65
a
,
b
85.29
±
3.23
a
,
b
HL100
75.38
±
4.07
b
87.50
±
2.80
a
86.34
±
2.3
7
a
,
b
87.50
±
1.98
a
62.50
±
2.35
a
81.03
±
0.89
a
,
b
85.58
±
0.96
b
,
c
79.17
±
0.62
a
,
b
76.31
±
1.41
b
HL119
79.49
±
0.89
a
88.08
±
0.50
a
90.44
±
4.73
a
86.64
±
0.75
a
68.72
±
0.89
a
82.05
±
0.89
a
,
b
85.90
±
1.11
b
,
c
79.17
±
2.49
a
,
b
85.70
±
4.64
a
HL135
78.46
±
2.66
a
86.34
±
0.50
c
78.14
±
4.73
b
,
c
84.05
±
1.98
a
65.64
±
1.78
a
84.36
±
1.18
a
85.26
±
1.11
b
,
c
78.09
±
0.62
a
,
b
84.89
±
3.08
a
,
b
HN059
78.56
±
0.18
a
86.63
±
0.50
a
87.70
±
4.10
a
,
b
86.21
±
3.73
a
65.64
±
9.01
a
81.28
±
1.94
a
,
b
81.41
±
1.11
b
,
c
78.45
±
2.85
a
,
b
82.84
±
4.42
a
,
b
JX013
80.00
±
5.33
a
86.45
±
1.31
a
81.56
±
2.05
a
,
b
,
c
85.78
±
2.59
a
64.10
±
0.89
a
83.85
±
2.77
a
83.33
±
1.06
c
82.04
±
5.42
a
,
b
85.70
±
3.54
a
SC012
78.97
±
3.55
a
86.05
±
0.87
a
84.29
±
1.18
a
,
b
86.64
±
1.49
a
70.26
±
8.75
a
82.15
±
2.51
a
,
b
87.82
±
1.11
a
,
b
79.89
±
3.29
a
,
b
83.66
±
3.54
a
,
b
XJ035
76.92
±
2.66
a
86.92
±
0.87
a
86.34
±
4.27
a
,
b
84.91
±
0.75
a
69.23
±
7.99
a
82.05
±
0.44
a
,
b
83.97
±
1.11
c
81.68
±
5.39
a
,
b
88.15
±
3.54
a
NX043
79.74
±
0.44
a
88.37
±
1.01
a
82.24
±
4.27
a
,
b
,
c
86.64
±
0.75
a
61.03
±
1.78
a
83.13
±
0.09
a
89.42
±
0.96
a
76.65
±
0.62
a
,
b
86.11
±
3.94
a
QH060
79.49
±
0.89
a
80.81
±
1.74
b
,
c
80.19
±
1.18
a
,
b
,
c
85.78
±
2.24
a
64.62
±
11.99
a
83.08
±
1.54
a
84.94
±
1.47
b
,
c
75.57
±
1.24
b
81.21
±
2.83
a
,
b
XJ087
78.46
±
1.54
a
84.88
±
1.82
b
81.56
±
2.05
a
,
b
,
c
84.05
±
1.49
a
69.23
±
6.15
a
82.05
±
1.78
a
,
b
83.97
±
1.11
c
78.45
±
3.73
a
,
b
87.75
±
2.45
a
SC098
76.92
±
4.07
a
88.08
±
1.33
a
72.68
±
6.26
c
82.76
±
1.49
a
66.67
±
6.22
a
78.97
±
0.89
b
79.49
±
2.94
d
73.42
±
1.24
a
,
b
87.13
±
0.87
a
SC101
76.41
±
5.40
b
84.59
±
3.30
b
89.07
±
6.26
a
83.19
±
1.29
a
59.49
±
4.44
a
82.31
±
1.33
a
,
b
84.29
±
1.47
c
85.27
±
5.53
a
81.62
±
6.13
a
,
b
Data represent themean±SD (n=3). Different letters indicate that the differences are significant (P<0.05) using Duncan’s multiple range test. SR: Sclerotium rolfsii; FT: Fusarium oxysporum f.sp. tracheiphilum; BD: Botryosphaeria dothidea; MO: Magnaporthe oryzae; BT: B. theobromae; FG: F. graminearum; RC: Rhizoctonia cerealis; SC: Stagonosporopsis cucurbitacearum; RS: R. solani; PI: percentage of growth inhibition.


## 3.6. Identification ofTrichoderma Strains with Potential Agricultural Applications

All strains, except for SC098, showed the same morphological characteristics after growing on three kinds of medium for 7 days. These strains appeared as dark green colonies with a distinctive coconut odor on PDA plates, and they formed green conidial pustules on CMD medium and produced white, yellow, and green sorus, forming concentric zones on SNA plates (Figures2(a)–2(e) and 3(a)–(e)). Conidia and chlamydospores are smooth, watered, and subglobose, and the branches were pyramidal and verticillate or paired lateral arising. Phialides were tended to cluster on branches. SC098 strain produced a diffuse yellow pigment on both sides of PDA plates, and green and yellow cottony sori were observed on CMD medium at 48 h. The mycelia of the strains were wavy and grown floccose to arachnoid on SNA plates, and phialides were ampulliform (length, 5-6.5 μm; widest point, 2.9-3.2 μm), tending to cluster and to create a narrow neck at the top (Figures 2(f)–2(h) and 3(f)–(h)). A phylogenetic tree demonstrated that all the selected strains had two distinct clades. JX013, HN059, XJ097, QH060, HL100, HL119, HL135, SC012, NX043, SC101, GZ070, and XJ035 strains belonged to the Hamatum clade and had affinity to KP009011 (T. asperellum). SC098 strain was identified as T. afroharzianum, which was closely related to KP0699541 (T. afroharzianum) and belonged to the harzianum clade (Figure 4). Based on the morphological and molecular characteristics, the remaining 12 strains were identified as T. asperellum, and SC098 strain was identified as T. afroharzianum.Figure 2
Morphological identification of JX013 (Trichoderma asperellum). (a–c) Cultures of JX013 that were grown on PDA, CMD, and SNA medium. (d, e) Conidial pustules. (f) Conidia. (g, h) Conidiophore. (i) Chlamydospores. PDA: potato dextrose agar medium; CMD: corn meal dextrose agar medium; SNA: synthetic low nutrient agar medium.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)Figure 3
Morphological identification of SC098 (Trichoderma afroharzianum). (a–c) Cultures of JX013 which grew on PDA, CMD, and SNA medium. (d, e) Conidial pustules. (f) Conidia. (g, h) Conidiophore. (i) Chlamydospore. PDA: potato dextrose agar medium; CMD: corn meal dextrose agar medium; SNA: synthetic low nutrient agar medium.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)Figure 4
Phylogenetic analysis based on thetef-1 gene and ITS region from the strains screened. GenBank accession numbers of 13 Trichoderma strains shown in Supplementary Table 2.


## 4. Discussion

Trichoderma grows rapidly and can quickly occupy the growth space of pathogenic fungi, which is one of the important mechanisms of their antimicrobial effect [19]. The competitive effect of Trichoderma has been widely reported. For example, T. asperellum (T3, T4, T15, and T19) and T. harzianum T6 showed effective inhibition on graminicola in the dual culture assay [33]; T. atroviride, T. harzianum, and T. viride showed strong inhibitory effects on Phytophthora, Botrytis cinerea, and Rhizoctonia solanacearum in competitive bioassay tests [34]. In this study, we found that the growth rate of Trichoderma was significantly faster than that of pathogenic fungi. It could be clearly observed that Trichoderma covered most parts of the plates, and some can even continue to grow on the colony of pathogenic fungi and then covered the entire colony, showing obvious competition.The production of cell wall-degrading enzymes (CWDEs) and volatile antibiotics is key parameters forTrichoderma as a biocontrol agent [35]. Saravanakumar et al. proved that there was a positive correlation between the CWDs of Trichoderma strains and its antagonism to pathogens [15]. We found that 13 Trichoderma strains (PI>85%) with excellent antagonism to pathogens could secrete chitinase and β-1,3-glucanase, which were closely related to the cell wall composition of pathogens. Therefore, Trichoderma can not only compete with pathogens for space and nutrition but also degrade the cell walls of pathogens, deform, or even digest the hyphae, and inhibit the growth of pathogens. It has also been reported that the cell wall degrading enzymes such as chitinase and glucanase secreted by Trichoderma also play a vital role in the hyperparasitism process of Trichoderma on pathogens [36]. In addition, we also found that some Trichoderma strains had a distinctive coconut odor, which may be related to the production of the volatile compound namely 6-pentyl-2H-pyran-2-one (6PP) [37]. Wonglom et al. reported that T. asperellum T1 could produce 22 volatile compounds including 6PP, which could inhibit the growth of two leaf spot fungal pathogens, Corynespora cassiicola and Curvularia aeria [38].This study confirmed that 13Trichoderma strains had a good growth-promoting effect on many crops. Trichoderma promotes plant growth by enhancing nutrient absorption efficiency and the secretion of growth-promoting metabolites in host plant [39]. Previous studies have shown that T. virens promotes the growth of Arabidopsis thaliana by mediating the activation of the classical auxin response pathway [40]. Additionally, diverse VOCs from T. asperellum also contribute immensely to plant growth [37, 38]. Trichoderma T-22 could increase the efficiency of nitrogen application in maize, and under the same growth conditions, it could reduce the nitrogen application rate of corn by 40% [41]. Besides, Trichoderma can also induce plant hormone synthesis to promote plant growth by upregulating plant genes for hormone biosynthesis or downregulating hormone catabolism-related genes [42]. For instance, some reports suggested an indirect altered balance of the auxin hormone and cytokinin by Trichoderma, through the synthesis induction of these hormones by the plant, thereby achieving the purpose of promoting plant growth [43, 44].As a mycorrhizal fungus,Trichoderma can successfully colonize in soil and plant roots, which is the premise of acting its biocontrol effect (including competition, growth promotion, antagonism, induced resistance) [45]. Trichoderma is precisely regulated in the process of colonizing plant roots. To successfully colonize roots, Trichoderma can instantly reduce the activity of root defense-system and promote its colonization in the root cortex [46]. Some reports suggested that root colonization by Trichoderma strains could increase levels of defense-related plant enzymes, including various peroxidases, chitinases, β-1,3-glucanases, and the lipoxygenase-hydroperoxide lyase pathway [47, 48]. In cucumber, root colonization by strain T-203 causes an increase in phenolic glucoside levels in leaves; their aglycones (which are phenolic glucosides with the carbohydrate moieties removed) are strongly inhibitory to a range of bacteria and fungi. Root colonization by these fungi therefore induces significant changes in the plant metabolic machinery [19]. In this study, we also evaluated the colonization ability of 13 Trichoderma strains on cucumber and found that all of them could colonize in the rhizosphere soil of cucumber, and the corresponding Trichoderma strain could also be isolated from other parts of cucumber, proving that they have strong colonization ability. However, the biocontrol effect of Trichoderma is the result of multiple mechanisms, and the interaction mechanism between Trichoderma, host plants, and pathogens needs to be further explored.


## 5. Conclusion

In this study, thirteenTrichoderma strains that showed high antagonistic potential against B. cinerea, F. oxysporum, and C. gloeosporioides were screened. At the same time, they could increase the germination rate of a variety of crop seeds, promote root growth, efficiently colonize in cucumber root soil, and show high β-1,3-glucanase and chitinase activities. These 13 strains of Trichoderma were identified as T. asperellum (12 strains) and T. afroharzianum (1 strain). These strains have good biological control potential, which provides a feasibility basis for the next evaluation of their field control effects and their application in production.



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*Source: 7913950-2021-12-21.xml*