Triggering the biocontrol of Botrytis cinerea by Trichoderma harzianum through inhibition of pathogenicity and virulence related proteins

Abstract

This study reports a strain of Trichoderma harzianum CCTCC-SBW0162 with potential to enhance biocontrol activity against gray mold pathogen, Botrytis cinerea, and with a pivotal role in tomato (Solanum esculentum) plant growth enhancement. A total of 254 Trichoderma isolates were screened by in vitro antagonistic assay. Of these, 10 were selected for greenhouse experiments based on their greater inhibition of B. cinerea. The in vitro antagonistic assay and greenhouse experiments indicated that T. harzianum CCTCC-SBW0162 gave the highest inhibition rate (90.6%) and disease reduction (80.7%). Also, to study the possible mechanism associated with antifungal activity of CCTCC-SBW0162 against B. cinerea, molecular docking was used to assess the interactions between CCTCC-SBW0162-derived metabolites, and pathogencity and virulence related proteins of B. cinerea. The molecular docking results indicated that the combination of harzianopyridone, harzianolide and anthraquinone C derived from CCTCC-SBW0162 could synergistically improve antifungal activity against B. cinerea through the inhibition/modification of pathogenicity and virulence related proteins. However, this computerized modeling work emphasized the need for further study in the laboratory to confirm the effect T. harzianum-derived metabolites against the proteins of B. cinerea and their interactions.

Keywords

anthraquinone / Botrytis cinerea / harzianolide / harzianopyridone / molecular docking / Trichoderma harzianum

1 Introduction

Solanum esculentum, although technically a fruit, is one of the most economically important vegetable fruit cultivated worldwide. However, its productivity is substantially decreased by gray mold, and foliar diseases, root rots and wilts by several phytopathogens^[1–3]. The key phytopathogens belong to the genera Botrytis, Fusarium, Macrophomina, Phytophthora, Rhizoctonia and Sclerotinia^[4]. Of these, Botrytis is particular highly hazardous to the tomato plants, causing gray mold disease^[1]. Moreover, continued use of chemical fungicides is hazardous to the environment and can also lead to the resistance of the phytopathogens such as Botrytis cinerea and B. fabae^[5]. Therefore, there is a clear need for an effective biocontrol agent to reduce the impact of B. cinerea. In this regard, several biocontrol microorganisms, such as Bacillus and Pseudomonas and Trichoderma, are available to reduce the impact of phytopathogens^[6,7]. Trichoderma strains have potential to reduce gray mold rot^[8–10] and can improve plant growth through production of plant hormones, vitamins, and triggering plant immunity and nutrient uptake^[11]. Trichoderma harzianum has received global attention as an effective biocontrol agent for several plant pathogens^[12–15]. T. harzianum inhabits various ecological niches, such as soil, rhizosphere, lakes, forest sediments and coastal vegetation soil from agriculture or non-agriculture ecosystems^[16]. This fungus also occurs in close association with plants and has been isolated from different substrata of the plants^[17].

B. cinerea is ranked as the second most important phytopathogen in the world^[1], and it causes severe plant diseases that result in significant loss of yield in economically important crop plants including beans, berries, grapes and tomato^[18]. However, the underlying mechanism of disease incidence, virulence and pathogenicity of B. cinerea is not fully understood. The first and foremost process of interaction is initiated through contact of the fungal cell wall with the host plant. Hence, the fungal cell wall is essential for the penetration, colonization and infection of the plant tissues^[19] and the fungal cell wall proteins (glycoproteins) are involved in the host-pathogen interactions, virulence and pathogencity^[20,21]. The genome of B. cinerea exhibits over 100 putative GPI (glycosylphosphatidylinositol) proteins and these cell wall glycoproteins are involved in pathogencity, virulence and host interaction of B. cinerea^[21,22]. According to recent work, bcpmr1 from the B. cinerea is essential for protein glycosylation, cell wall structure and virulence of B. cinerea. In addition, examination of O-linked glycosylation pathways demonstrated that the PMT genes were crucial for the fungal pathogencity^[23].

Several workers have studied the pathogenesis and virulence of B. cinerea^[24–30]. One of the findings indicated that bcpmr1 encoded a P-type Ca²⁺/Mn²⁺-ATPase involved in protein glycosylation, cell wall structure and virulence of B. cinerea^[30]. The identification of the function of the monocarboxylate is, however, reported from the mammalian metabolism rather than fungi. The presence of this monocarboxylate transporter BcMctA has been reported as being essential for B. cinerea pathogenicity^[29]. Our study aimed to investigate the effects of T. harzianum and its metabolites on plant growth and enhanced biocontrol of B. cinerea through the modification/inhibition of the pathogenicity and virulence related proteins (bcpmr1 encoding a P-type Ca²⁺/Mn²⁺-ATPase, BcMctA) of B. cinerea.

2 Materials and methods

2.1 Microorganisms

Trichoderma strains were isolated from a coastal wetland ecosystem^[31], grown on modified potato dextrose agar (PDAm)^[32] and preserved in 20% glycerol at -80°C. A culture of B. cinerea isolated from infected tomato leaves was obtained from the Center of Trichoderma Culture Collection of Shanghai Jiao Tong University (CCTCCSJ), China.

2.2 In vitro screening

In vitro antagonism of 254 Trichoderma isolates (T1-T254) against B. cinerea was tested by the dual culture method, as described by Dennis and Webster^[33]. Percentage of mycelial growth was calculated by measuring the B. cinerea growth competing with Trichoderma isolates on PDAm in Petri dishes (9 cm).

2.3 Trichoderma enhanced plant growth

Seeds of tomato were surface-sterilized by the method of Huang et al.^[34], and germinated on sterile wet-paper at room temperature for 4 d. The pre-germinated tomato seedlings were planted in a greenhouse in pots containing the natural agriculture soil, sterilized at 180°C for 6 h to remove other microbes, insects and weeds. The conidial suspension of Trichoderma and B. cinerea were prepared according to the method of Vinale et al.^[35], and Nelson and Powelson^[36]. The microbial inoculation of Trichoderma/pathogens on tomato seedlings was applied by spraying^[37]. A 5 ml conidia suspension of Trichoderma (2.6 × 10⁵ conidia per mL) and/or a 5 mL conidia suspension of B. cinerea (5 × 10⁴ conidia per mL) were sprayed on tomato seedlings per treatment.

To analyze Trichoderma induced plant growth regulation, 12 treatments were applied: CK₁, uninoculated; CK₂, seedlings sprayed with B. cinerea; T1, seedlings sprayed with Trichoderma atroviride CCTCC-RW0008 and B. cinerea; T2, seedlings sprayed with Trichoderma asperellum CCTCC-RW0011 and B. cinerea; T3, seedlings sprayed with T. harzianum CCTCC-RW0006 and B. cinerea; T4, seedlings sprayed with T. harzianum CCTCC-SBW0162 and B. cinerea; T5, seedlings sprayed with T. atroviride CCTCC-RW0008 and B. cinerea; T6, seedlings sprayed with T. atroviride CCTCC-SBW0138 and B. cinerea; T7, seedlings sprayed with T. aureoviride CCTCC-SBW0122 and B. cinerea; T8, seedlings sprayed with T. atroviride CCTCC-SBW0074 and B. cinerea; T9, seedlings sprayed with T. atroviride CCTCC-SBW0068 and B. cinerea; and T10, seedlings sprayed with T. atroviride CCTCC-SBW0073 and B. cinerea. One month after microbial inoculation, the Trichoderma induced growth regulating indicators of tomato, including shoot length, root length, shoot biomass, root biomass and total biomass, were measured. Botrytis disease reduction was evaluated using the modified formula of Saravanakumar et al.^[31]. Each treatment had three replicates in a randomized design.

2.4 Molecular interaction

Molecular interaction of T. harzianum-derived secondary metabolites and pathogenicity related proteins from B. cinerea were assessed with a computer-based molecular docking program. The presence of the known T. harzianum metabolites, including T22azaphilone, harzianopyridone, harzianolide, 1-hydroxy-3-methyl-anthraquinone and anthraquinone C^[38], in CCTCC-SBW0162 was confirmed by preliminary biochemical experiments (data not shown) and subsequent detailed chemical characterization of compounds. Therefore, these compounds were used for the molecular interaction study. The ligand structures (metabolites) were obtained from PubChem (NCBI-PubChem Compund), and the ligand was prepared by using the ACD/ChemSketch.

Earlier reports showed that bcpmr1 encodes a P-type Ca²⁺/Mn²⁺-ATPase, and BcMctA from B. cinerea is involved in pathogenicity and virulence^[29,30]. A BLAST analysis indicated that BcMctA is identical to MFS monocarboxylate transporter (CCD50452), and the bcpmr1 is identical to Ca²⁺/Mn²⁺-transporting P-type ATPase PMR1 (NP_011348) and hypothetical protein SS1G_09885 (EDN94018). Therefore, in the present study, bcpmr1 was considered identical to PMR1 (NP_011348) and SS1G_09885 (EDN94018), and BcMctA was identical to MFS (CCD50452)^[29,30]. Hence, available protein sequences of bcpmr1 and BcMctA for molecular interaction with T. harzianum metabolites were used, after retrieving the protein sequences from NCBI protein database and the protein structure were predictedusing SWISS-MODEL. Molecular docking was analyzed with ArgusLab 4.0.1, and interactions of the protein and ligand were visualized with BIOVIA Discovery Studio 2016 (Accelrys Software Inc., San Diego, CA, USA).

3 Results and discussion

3.1 In vitro antagonism

In vitro antagonistic experimental results indicated that mycelial growth of B. cinerea was significantly inhibited by Trichoderma isolates in dual culture. The percentage inhibition ranged from 1.56% to 90.6% with T. harzianum CCTCC-SBW0162 exhibiting the highest inhibition of B. cinerea (Table S1). Out of 254 Trichoderma isolates tested, the top 10 were selected for greenhouse experiments to assessing the Trichoderma induced enhanced growth in tomato (Fig. 1) based their high percentage of inhibition of Trichoderma. The selected isolates were T. harzianum RW0006 (81.3%), T. harzianum SBW0162 (90.6%), T. atroviride SBW0138 (76.6%), T. aureoviride SBW0122 (79.7%), T. atroviride SBW0074 (81.3%), T. atroviride SBW0073 (85.9%), T. atroviride SBW0068 (76.6%), T. atroviride SBW0008 (78.1%), T. asperellum RW0011 (84.4%) and T. atroviride RW0008 (75.0%).

Fig.1 In vitro antagonistic activity of Trichoderma against B. cinerea (BC) in dual culture: BC-CK, T. harzianum CCTCC-RW0006, T. atroviride CCTCC-RW0008, T. asperellum CCTCC-RW0011, T. atroviride CCTCC-SBW0008, T. atroviride CCTCC-SBW0068, T. atroviride CCTCC-SBW0073, T. atroviride CCTCC-SBW0074, T. aureoviride CCTCC-SBW0122, T. atroviride CCTCC-SBW0138, and T. harzianum CCTCC-SBW0162.

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3.2 Plant growth enhancement induced by Trichoderma

The effects of Trichoderma on reduction of B. cinerea and enhancement of tomato growth under greenhouse conditions are shown in Table 1 and Fig. S1. Average shoot length was significantly affected by the treatments and ranged from 18.6±2.5 to 41.4±2.6 cm. Shoot length was increased significantly by about 1.22 times in T4 compared to the CK₂. Average root length varied with the treatments and it increased significantly 5.9 times in T3 when compared to CK₂. Average shoot biomass (18 times; P<0.05), average root biomass (7 times; P<0.05) and total biomass (12.5 times; P<0.05) increased significantly in T4 compared to CK₁. Disease reduction was significant between the treatments (P<0.05) with greatest reduction (80.7%) in T4 compared to (31.7%) in CK₂. Thus, treatment T4 showed that T. harzianum CCTCC-SBW0162 significantly reduce gray mold and improved tomato growth, which is consistent with other reports that Trichoderma spp. promote plant growth^[39].

Tab.1 Antagonistic effect of Trichoderma against B. cinerea on growth factors of tomato seedlings.

Treatment	Average shoot length/cm	Average root length/cm	Average shoot biomass/g	Average root biomass/g	Total biomass/g	Disease reduction/%
CK1	30.2±1.2 (0.62)	5.0±1.2 (0.56)	1.0±0.02 (4)	0.4±0.08 (1)	1.4±0.3 (2.5)	56. 7
CK2	18.6±2.5	3.2±0.8	0.2±0.01	0.2±0.09	0.4±0.1	31.7
T1	32.0±2.1 (0.72)	3.6±0.1 (0.125)	0.9±0.03 (3.5)	0.3±0.05 (0.5)	1.1±0.6 (0.75)	50.0
T2	29.7±1.5 (0.59)	4.2±0.6 (0.31)	0.2±0.02 (0)	0.2±0.06 (0)	0.4±0.8 (0)	40.0
T3	37.8±3.2 (1.03)	22.1±0.2 (5.90)	2.6±0.01 (12)	0.5±0.06 (1.5)	3.1±0.4 (6.75)	40.0
T4	41.4±2.6 (1.22)	11.5±0.4 (2.59)	3.8±0.03 (18)	1.6±0.04 (7)	5.4±0.5 (12.5)	80.7
T5	24.4±1.2 (0.31)	8.6±0.6 (1.68)	1.8±0.06 (8)	0.6±0.06 (2)	2.4±0.6 (5)	33.3
T6	32.4±0.6 (0.74)	6.5±1.2 (1.03)	3.4±0.06 (16)	0.2±0.04 (0)	3.6±0.2 (8)	56.7
T7	29.5±1.8 (0.58)	12.2±1.4 (3)	2.7±0.04 (2.5)	0.3±0.06 (0.5)	2.9±0.1 (1.5)	46.7
T8	20.0±1.6 (0.07)	3.5±0.6 (0.09)	1.2±0.06 (5)	0.5±0.01 (1.5)	1.7±0.2 (6.25)	33.3
T9	37.7±2.4 (1.02)	4.5±1.8 (0.40)	2.4±0.04 (11)	0.4±0.02 (1)	2.8±0.6 (6)	60.0
T10	32.6±1.6 (0.75)	7.0±1.6 (1.18)	1.9±0.09 (8.5)	1.1±0.03 (4.5)	3.0±0.1 (6.5)	56.7

Note: The values shown are means±SE (df= 60 seedlings per treatment) and one way ANOVA followed by multiple comparison using Duncan's test. The values in parentheses are the relative increase in treatment compared to negative control inoculated with B. cinerea and without Trichoderma treatment. CK1, uninoculated sterile soil; CK2, soil inoculated with B. cinerea; T1-T10, inoculated with different Trichoderma strains and B. cinerea as detailed in the materials and methods.

3.3 Generation of protein and metabolite structures

The predicted protein structures of bcpmr1 [PMR1 (NP_011348) and SS1G_09885 (EDN94018)] and BcMctA [MFS (CCD50452)] are shown in Fig. 2. The structure of T. harzianum metabolites (ligand) such as T22azaphilone, harzianopyridone, harzianolide, 1-hydroxy-3-methyl-anthraquinone, and 1, 8-dihydroxy-3-methyl-anthraquinone are shown in Fig. 3.

Fig.2 3D structure of target protein of B. cinerea. Bcpmr1 is identical to PMR1 (NP_011348) (a) and SS1G_09885 (EDN94018) (b), and BcMctA is identical to MFS (c)

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Fig.3 Structure of metabolites (ligand) of Trichoderma harzianum. (a) T22azaphilone; (b) harzianopyridone; (c) harzianolide; (d) 1-hydroxy-3-methyl-anthraquinone; (e) 1,8-dihydroxy-3-methyl-anthraquinone.

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3.4 Molecular interaction studies

A total of three target proteins were tested for interactions with T. harzianum-derived compounds and the results indicated that they can have a significant inhibitory effect against pathogenicity and virulence related proteins of B. cinerea (Table 2). Secondary metabolites produced from T. harzianum are known to inhibit the pathogens such as Gaeumannomyces graminis, Pythium ultimum and Rhizoctonia solani in vitro^[38]. Similarly the present work indicated that the T. harzianum-derived metabolites can inhibit the growth of B. cinerea through the inhibition/modification of pathogenicity and virulence proteins. The antifungal effect of T. harzianum-derived metabolites significantly varied between different types of phytopathogens^[35]. Similarly, the present study indicated that among the five tested compounds anthraquinone C can provide the greatest inhibition of bcpmr1 (-43.91 and -49.47 kJ·mol^-1 for PMR1 and SS1G, respectively) and BcMctA (-57.71 kJ·mol^-1) than the other compounds tested for docking energy.

Tab.2 Analysis of interactions between Trichoderma-derived compounds and pathogenicity related protein bcpmr1 of B. cinerea

S. No.	PubChem CID	T. harzianum derived Compound Name	Mol. Formula	Mol. Wt/(g·mol-1)	Docking Score/(kJ·mol-1)
					Bcpmr		BcMctA
					PMR1	SS1G	MFS
1	76326344	T22azaphilone	C18H18O6	330.336	-38.56	-42.57	-42.87
2	54697782	Harzianopyridone	C14H19NO5	281.308	-40.36	-35.34	-38.39
3	15719532	Harzianolide	C13H18O3	222.284	-40.27	-43.87	-45.17
4	164982	1-hydroxy-3-methyl-anthraquinone	C15H10O3	238.242	-38.22	-41.65	-48.13
5	641293	anthraquinone C	C20H17ClO4	356.802	-43.91	-49.47	-57.71

3.5 Molecular interaction of Bcpmr (PMR1 and SS1G_09885) with T. harzianum-derived metabolites

The examination of five candidate compounds of T. harzianum for molecular interaction indicated that they all had significant ability to inhibit and/or modify the pathogenicity and virulence related proteins Bcpmr (PMR1) of B. cinerea with strong docking scores from -38.22 to -43.91 kJ·mol^-1 (Table 2). Among the compounds, anthraquinone C showed the highest docking score of -43.91 kJ·mol^-1 with a strong interaction with hydrogen residues such as Arg182 and Asp703. Other residues were observed in the binding pockets such as Glu75, Gly241, Gly263, Ile76, Leu148 Phe262, Phe266, Thr702, Val704, Val682 and (Fig. 4). This interaction strongly indicated the potential of anthraquinone C to modify targeted protein structures and such modification could change protein function.

Fig.4 Docking complex showing the interaction between Trichoderma metabolites anthraquinone C with Bcpmr (PMR1) of B. cinerea

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The results indicated that all the tested compounds showed significant potential interactions with the SS1G_09885; and among the tested compounds anthraquinone C had the highest docking score of -49.47 kJ·mol^-1 with strong interactions with hydrophobic residues, such as Met447, Gln444 and Met520, and other residues, such as Glu524, Ile826, Ile830, Leu834, Leu495, Leu503, Leu443, Pro521, Thr441, Val523, Phe827, and Val499 (Fig. 5). This demonstrates that the fungal metabolites could have a significant ability to interact with and interfere with the functioning of Bcpmr-encoded protein.

Fig.5 Docking complex showing the interaction between Trichoderma metabolite, anthraquinone C, with Bcpmr (SS1G_09885) of B. cinerea

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3.6 Molecular interaction of BcMctA (MFS) with Trichoderma harzianum-derived compounds

BcMctA is significantly involved in the pathogenicity and virulence of B. cinerea and it was found that among the tested compounds, anthraquinone C could interact significantly with the BcMctA protein with a docking score of -57.71 kJ·mol^-1 and strong interaction with protein residues such as Leu111, Phe114, Ser110, Leu404, Phe401 and Phe117 (Fig. 6). All the docking scores of T. harzianum-derived compounds with pathogenicity and virulence related proteins in B. cinerea indicated the potential of T. harzianum to inhibit B. cinerea by targeting this protein. Several researchers have reported a potential reduction of protein function as evidenced by negative docking scores from the computational method using ArgusLab^[35,40–43] and the present results suggest that anthraquinone C could interact significantly with the BcMctA protein and inhibit its functioning.

Fig.6 Docking complex showing the interaction between Trichoderma metabolite, anthraquinone C, with BcMctA (MFS) of B. cinerea

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Harzianopyridone, harzianolide and anthraquinone C are known to reduce the gray mold disease in tomato caused by B. cinerea^[29,35]. The negative docking scores for these three compounds (Table 2) indicated that they could work synergistically against B. cinerea and inhibit its growth through targeting the pathogenicity and virulence related proteins. These molecular docking results indicate that some secondary metabolites from Trichoderma spp., could potentially interact strongly with phytopathogens. However further detailed laboratory and greenhouse studies are required to confirm this.

4 Conclusions

This work reports a potent Trichoderma strain with potentially useful biocontrol activity against the gray mold pathogen, B. cinerea, investigated by in vitro and greenhouse experiments. Significant potential molecular interactions between T. harzianum-derived metabolites and pathogenicity, virulence related proteins of B. cinerea indicated that the inhibition of the B. cinerea may not triggered by single metabolite, but is likely to be a synergistic effect of multiple metabolites from T. harzianum. Notably, the negative docking score for anthraquinone C indicated it could have the greatest ability to inhibit B. cinerea, and the combination of harzianopyridone, harzianolide and anthraquinone C may also increase the potential biocontrol activity of T. harzianum against B. cinerea. Taken together, these findings provide important new information about the molecular interactions of metabolites and pathogenicity-related virulence proteins. This contrasts with established methods which are generally based on trial and error testing, with a low probability of success in the laboratory experiments^[44]. Although this study needs to be confirmed by further study of the interaction of T. harzianum metabolites with B. cinerea proteins in a microbiology laboratory, this approach should lead to a greater likelihood of success.

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Supplementary materials

The online version of this article at https://doi.org/10.15302/J-FASE-2018214 contains supplementary materials (Table S1; Fig. S1).

This article does not contain any studies with human or animal subjects performed by any of the authors.

Acknowledgements

This study was supported by the National Key Research and Development Program of China (2017YFD0200400, 2017YFD0201108, SQ2017ZY06102), the National Natural Science Foundation of China (20090073110048), the National Modern Agriculture Industry Technique Systems (CARS-02), Special Project of Basic Work Project for Science and Technology (2014FY120900).

Compliance with ethics guidelines

Kandasamy Saravanakumar, Zhixiang Lu, Hai Xia, Meng Wang, Jianan Sun, Shaoqing Wang, Qiang-qiang Wang, Yaqian Li, and Jie Chen declare that they have no conflicts of interest or financial conflicts to disclose.

This article does not contain any studies with human or animal subjects performed by any of the authors.

RIGHTS & PERMISSIONS

The Author(s) 2018. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)