Biocontrol activity of recombinant aspartic protease from Trichoderma harzianum against pathogenic fungi
Introduction
A major portion of postharvest losses in fruits and vegetables is due to rots caused by phytopathogenic fungi. Chemical fungicides are the most common means of controlling rot pathogens of fruits and vegetables. However, these chemicals are not environment-friendly, are harmful for human health, and lead to the development of pesticide-resistant fungi [1]. Promising approaches are available for developing new and potentially safer alternatives for controlling postharvest diseases. Eco-friendly antagonistic microorganisms can be used as biocontrol agents against soil-borne plant pathogens without any adverse effects on food security or human health [2]. The fungus Trichoderma is well-known for its antagonistic properties and has been widely used as chemical pesticide substitutes for controlling plant diseases [3]. Trichoderma spp. has been used to control a broad range of phytopathogenic fungi, such as Rhizoctonia solani, Sclerotinia sclerotiorum, and Botrytis cinerea [4]. In addition, Trichoderma spp. can also infect nematode egg shells [5]. Since these pathogenic plant fungi and plant nematodes cause mildew and rot of food, wine, and juice derived from crops, vegetables, and fruits, they are attractive targets for research aimed at increasing the shelf-life of food products.
Trichoderma spp. parasitizes phytopathogenic fungi (mycoparasitism) via the secretion of hydrolytic enzymes, such as proteases, chitinases, and glucanases [3,6,7]. These enzymes play critical roles in cell wall degradation of fungi [8] or shell degradation of nematode eggs [9]. Cloning and expression of genes encoding Trichoderma spp. enzymes are useful for exploring their antagonistic activities as well as for developing new potential alternatives for chemical pesticides. Several studies reported that enzymes from Trichoderma spp. could be used for controlling fungal pathogens in vitro [2,[10], [11], [12]]; however, the production yield of these enzymes was low. Most studies on biocontrol enzymes focus on phytopathogens because fungal pathogens of plants and animals are generally studied separately. Actually, several fungi such as Fusarium spp. cause diseases in both plants and animals, including humans [13]. Therefore, hydrolytic enzymes from Trichoderma spp. can be expected to be useful in the treatment of animal pathogens. Among the hydrolytic enzymes, proteases appear to regulate the expression or action of other hydrolytic enzymes involved in mycoparasitism. Reports show that knockout of a gene encoding a protease significantly down-regulated the expression of Trichoderma sp. chitinase [14], whereas overexpression of the papA protease (aspartic protease) increased the glucanase activity of Trichoderma harzianum by 30% [15].
To investigate the role of enzymes in mycoparasitism, the proteome and transcriptome of T. harzianum in the presence of crude fungal cell walls were analyzed. A protease, P6281, was identified to be the most prominent up-regulated enzyme during early mycoparasitism [16] and nematode egg-parasitism by T. harzianum [9]. Based on these results, the P6281 protease was suggested to be critical for cell wall or egg shell degradation during parasitism and may therefore find potential applications. However, the P6281 protease is a poorly characterized enzyme at the molecular and biological level as no systematic studies of its biochemical and enzymatic properties have been performed to demonstrate its direct role in mycoparasitism.
Fungal infections are common global problems that affect plants and humans. B. cinerea is an important fungal plant pathogen, and its broad host range and ability to cause disease in both pre- and postharvest fruits and vegetables causes immense economic loss (both in terms of yield loss and cost of infection control) [17]. Aspergillus flavus infects many important agricultural crops and it is also an opportunistic human and animal pathogen [18]. R. solani is a soil-borne, necrotrophic fungus causing damping off, root rot, and stem canker in many cultivated plants worldwide [19]. Mucor circinelloides is a fungal plant pathogen and conditional pathogen of humans. Aspergillus fumigatus is a fungus that causes spoilage and putrefaction of food and human aspergillosis [1]. Candida albicans infection is the most common fungal infection in critically ill or immune-compromised patients [20].
Here, the gene encoding P6281 was cloned and expressed in Pichia pastoris to identify the enzymatic characteristics of the P6281 protease from T. harzianum. The optimal pH and temperature required for the activity and stability of the purified recombinant P6281 (rP6281) were determined. Plant and animal pathogenic fungi such as B. cinerea, M. circinelloides, A. fumigatus, A. flavus, R. solani, and C. albicans were used for evaluating its antagonistic activity in vitro. The rate at which growth and spore germination of these fungi were inhibited was also determined. The cell wall of the protease-treated B. cinerea was observed by light microscopy and transmission electron microscopy (TEM). The applicability of rP6281 in inhibiting grey mold-mediated rot formation in apple, orange, and cucumber were evaluated.
Section snippets
Strains, plasmids, enzymes and reagents
T. harzianum strain GIM 3.442, B. cinerea strain GIM 3.47, M. circinelloides strain GIM 3.79, A. fumigatus strain GIM 3.20, and R. solani strain GIM 3.512 were purchased from the Guangdong Culture Collection Center. A. flavus GIM 3.493 was purchased from the Institute of Microbiology, Chinese Academy of Sciences. C. albicans was provided by Institute of Plant Protection, Guangdong Academy of Agricultural Sciences. The P. pastoris strain GS115 (his−, mut+) and pPIC9K vector were purchased from
Cloning and expression of rP6281
A 1107 bp p6281 cDNA fragment was cloned in the pMD18-T vector. This sequence was 97.7% identical to the sequence of p6281 with 25 silent mutations (GenBank accession no: AJ967001.1). The protein sequence of the P6281 protease encoded by our construct was identical to the sequence previously reported (GenBank accession no: CAI91181) [22]. A truncated form of p6281 without the sequence encoding the signal peptide was cloned downstream of the sequence encoding the α-factor signal in the pIC9BM
Discussion
Though many studies have been performed on the antagonistic activity of T. harzianum, its mechanism of action against plant pathogensis still unclear. A better understanding of the mechanism of action of T. harzianum is essential for developing appropriate commercial formulations and application methods, to maximize their potential use to manage fungal diseases. The production of lytic enzymes, such as proteases, is proposed as an important mode of action of T. harzianum. Here, we cloned and
Conclusions
Here, we cloned and expressed recombinant p6281 in P. pastoris, an aspartic protease of T. harzianum. The recombinant P6281 had high enzyme yield of 321.8 U/mL. rP6281 efficiently inhibited spore germination and growth of plant and animal fungal pathogens such as C. albicans, B. cinerea, M. circinelloides, A. fumigatus, A. flavus, and R. solani in vitro, suggesting that rP6281 could be a good biocontrol candidate against these pathogens. Microscopic and ultra-structural observation of
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
This work was supported by the Guangdong Province of China Science and Technology Projects (Project No. 2013B010404003, No. 2014A010107005, No. 2014A010107032); and Science and Technology Program of Guangzhou (grant No. 201604020135) to Dr. Xiaochun Luo.
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