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MOLECULAR CHARACTERIZATION AND MYCOPARASITIC APTITUDE OF INDIGENOUS BIOCONTROL AGENT TRICHODERMA HARZIANUM.

Byline: G. Mustafa, S. Anwar, F. A. Joyia, M. B. Hayat, M. A. Zia, Ghazala and M. S. Khan

Keywords: Trichoderma harzianum, ITS, [beta]-1,3-glucanase, purification, characterization, SDS-PAGE.

INTRODUCTION

Biocontrol agents had always been preferred owing to the detrimental consequences of chemical fungicides used for fungal control. Trichoderma harzianum is an ancient and ecologically important mycoparasite that produces valuable hydrolytic enzymes. They are green-spored ascomycetes and are commonly found in rhizospheric soils (Harman et al., 2004). Such avirulent microbes acting as symbionts play their role as plant growth enhancers as well as antagonistic agents against many fungal pathogens i.e. Sclerotinia sclerotiorum, Rhizoctonia solani, Phytophthora megasperma, Gaeumannomyces gramminis, Cylindrocladium scoparium and Sclerotium rolfsii (Perez et al., 2007). Hence, they are of pivotal importance for plant growth as well to protect plants from fungal diseases. Trichoderma harzianum is an important mycoparasitic fungus used as a biocontrol agent to combat many devastating plant pathogens (Ganuza et al., 2019; Steyaert et al., 2004).

These biocontrol agents possess extraordinary competence of degrading phytopathogenic hyphae by virtue of numerous hydrolytic enzymes, most importantly chitinases, chitosinases, glucanases and proteases (Benhamou and Chet, 1996).

Glucanases are prominent fungal cell wall degrading enzymes which are found in higher plant tissues, yeast cells, insects' hemolymph and fish as well (Pan et al., 1989). They have been categorized into [beta]-1,3, [beta]-1,4 and [beta]-1,6-glucanases on the basis of glucosidic linkage(s) that they can cleave (Pitson et al., 1993). [beta]-1,3-glucanase is also vital for cell survival/integrity and plays an important role in cell division, movement of materials across the cell membranes via plasmodesmata, flowering as well as in the transport of [beta]-glucans (De La Cruz et al., 1995). Moreover, glucanases are key enzymes involved in mycoparasitism exhibited by Trichoderma sp. (Balasubramanian et al., 2012). Filtered culture of T. harzianum was found to contain [beta]-1,3-glucanase and chitinase. Further, the filtrate was employed to hydrolyze mycelium of a notorious plant pathogen named S. rolfsii (El-Katatny et al., 2000).

In addition to mycoparasitic impact, glucanases are valuable for their industrial applications especially in brewery industry in order to reduce viscosity, processing time and precipitation of [beta]-glucans in the beer. Recently, brewing industry has started using [beta]-glucanases to carry out better brewing process. Employing T. harzianum for industrial scale enzyme production is being practiced since ages (Blumenthal, 2004). Further, presence of [beta]-glucans in poultry and animal feed is a problem as it makes faeces sticky causing reduced animal performance (Jeroch et al., 1988). [beta]-1,3-glucanases are also being used to obtain depolymerized fragments with broader usage in pharmaceuticals, food and beverage industry (Kadi and Crouzet, 2008). They are also useful to produce gluco-oligosaccharides from lichen pustulan and algal paramylon, which may have role in prebiotics (nutraceuticals) and modifier of human immunity related activities (Giese et al., 2011).

They are also being employed in medication as are used for the treatment of allergies caused by foodstuff as well as to cure fungal diseases. [beta]-1,3-glucanases are quite useful for the preparation of protoplasts (Parrado et al., 1996) so can be employed in somatic hybridization or protoplast culture. So this group of enzymes has biotechnological, industrial as well as biomedical applications (McCleary et al., 1988). Keeping in view the widespread utility of Trichoderma derived glucanases, present study was conducted to isolate, purify and characterize extracellular [beta]-1,3-glucanase from indigenous Trichoderma harzianum isolates. Further, characteristics of the enzyme are an indicator of mycoparasitic ability of Trichoderma isolates depicting their efficiency to be employed as biocontrol agent.

MATERIALS AND METHODS

Preparation of potato dextrose agar broth media: Potato starch was prepared by boiling 500 g of potato infusions in 500 ml of distilled water until the volume remained half. Then it was filtered using muslin cloth to get liquid starch. In order to prepare 1000 ml of PDA, 20 g of agar and 20 g of sucrose were dissolved in an appropriate volume of distilled water and 250 ml of liquid starch was added. Then, volume was made up to 1000 ml and pH was adjusted to 5.5. Then media were autoclaved and one ml of filter sterilized chloramphenicol (1 M rexaphenicol) was added in order to check the bacterial growth.

Molecular validation of fungal isolates: Trichoderma strains were characterized using ITS-I (Internal Spacer Region-1) specific primers. Sequence information of the said gene was obtained from Genbank (http://www.ncbi.nlm.nih.gov/). Then Primer-3 online tool was employed to design primers (ITS-1 forward primer, 5'-TCCGTAGGTGAACCTGCGG-3' and ITS-1 reverse primer, 5'-GCTGCGTTCTTCTTCATCGATGC-3'). Amplification was carried out in total of 50 ul reaction volume comprising of, 5 ul of 10X MgCl2 free PCR buffer, 5 ul of 10X MgCl2, 0.25 ul of 2.5 units of Taq polymerase, 0.5 ul of 10 iM reverse and forward primers, 4 ul of 2.5 mM dNTPs and 1.5 ul of 100 ng/ul genomic DNA.

Thermal cycler profile was as follows: initial denaturation at 94AdegC for 5 minutes followed by 35 cycles of one minute denaturation at 94AdegC, one minute primer annealing and extension at 55AdegC and 72AdegC respectively and final extension at 72AdegC for 10 minutes. The resultant amplicons were examined by agarose gel electrophoresis 0.8% (w/v) with 10 mg/ml ethidium bromide.

Production of [beta]-1,3-glucanase: Mycelia of the indigenous Trichoderma isolates were inoculated in 500 ml of PDB containing sucrose as carbon source in Erlenmeyer flask. pH of the medium was adjusted to 5.5 and placed in incubator (Irmeco GmbH, Germany) at 28AdegC for 72 hours in dark at 180 rpm for growth. After filtration, the filtrate was centrifuged at 15000 rpm for 10 min. at 4AdegC in order to get cell free filtrate with maximum clarity. The supernatant (cell free filtrate) referred to as crude enzyme extract was taken into new falcon tube and further employed for isolation and purification of glucanase.

Determination of total protein contents: Total protein contents of the crude extract were quantified by Bradford method (Bradford, 1976) using Bovine Serum Albumin (BSA) as standard.

Isolation and purification of [beta]-1,3-glucanase

Protein precipitation using Ammonium sulfate: The crude enzyme was kept in ice for some time and saturated with ammonium sulfate up to 20%. Later on, it was incubated at 4AdegC overnight. Then, it was centrifuged at same temperature for fifteen minutes and at 15,000 rpm. The pellet was kept, and re-precipitation of supernatant was with different concentrations of ammonium sulfate (40, 60, 80, and 100%). For each concentration of ammonium sulfate, the process of slow dissolution, centrifugation and pellet separation was repeated. All pellets (hereafter termed as crude enzyme) were preserved at appropriate temperature.

Dialysis of crude enzyme: The pellets were re-suspended in 0.2 M phosphate buffer (pH 7.2). For dialysis, a piece of dialysis membrane of proper pore size was cut, filled with resuspended crude enzyme and dialyzed against distilled water (4 times at an interval of 5 hrs) to remove ammonium sulfate.

Sephadex G-75 column based gel filtration chromatography: The gel filtration chromatography of partially purified enzyme was carried out using a glass column of internal diameter 2 cm. It was packed with Sephadex G-75 (Sigma, USA) upto a height of 120 cm and was equilibrated with 0.2M phosphate buffer maintaining stable pH at 7.2. A total of 50 fractions were collected from the column with each fraction of 2 ml at a flow rate of 0.5 ml/min. Total protein contents and glucanase activity were determined for each fraction.

Glucanase assay: The activity of Glucanase was assessed by using 250 ul of (1%) carboxy methyl cellulase as substrate dissolved in potassium acetate buffer. Reaction mixture contained 0-125 ul glucanase enzyme. It was incubated at 37AdegC for 30 minutes. Then, 1.5 ml Dinitro Salicylic Acid (DNS) reagent was added to stop the reaction (Miller, 1959). The optical density (OD) was recorded using spectrophotometer at 550 nm wavelength to quantify reducing sugars in the samples as well as in standard samples (containing known concentration of glucose). The amount of enzyme needed to release 1 umole of glucose from substrate under given conditions was termed as one unit (IU) of glucanase activity.

Determination of various optimal conditions for purified [beta]-1,3-glucanase: Finally, the purified glucanase was assayed to evaluate its various optima by examining the effect of different pH values (4-9) using 0.2M phosphate buffer, effect of different incubation temperatures (0.0-50AdegC), various concentrations of substrate (0-11 ml of 0.5% CMC), enzymes (0-8 ml) and thirteen different incubation times were used in different reactions with the difference of six hrs each, to evaluate its impact on the activity of purified [beta]-1,3-glucanase produced from Trichoderma harzianum. Then enzyme activities for each case were determined under standard assay conditions using 0.5% CMC as substrate.

SDS-PAGE analysis: Molecular size of the enzyme was determined using 12% SDS-PAGE following the protocols given by Laemmli (1970) with certain modifications. Protein samples were heated at 85AdegC for 3 minutes in sample buffer prior to separation on SDS-PAGE. Then protein samples were loaded in 12% polyacrylamide gel and run at constant voltage of 20 mA for 7-8 hours. Then, gel was stained with Coomassie brilliant blue R and destained with 40% methanol and 7% glacial acetic acid.

RESULTS

Molecular validation of fungal isolates: The selected Trichoderma strains were validated by ITS-1 specific PCR which is one of the most important markers in molecular systematics. Different PCR parameters were optimized to amplify target DNA fragments. A DNA fragment of around 300 bp was amplified (Figure 1), which was sequence characterized for the confirmation of of Trichoderma harzianum isolates.

Isolation and purification of [beta]-1,3-glucanase from Trichoderma harzianum: After 72 hrs of incubation, enzyme was isolated from the Trichoderma harzianum at log phase of growth. Ammonium sulfate precipitation, dialysis and sephadex G-75 column chromatography was used to purify the enzyme. We collected 50 fractions (2 ml each) in separate test tubes and subjected to spectrophotometric analysis for measuring optical density which resulted in the detection of proteins in first 25 fractions whereas no protein was detected in rest of the fractions. Among the isolated fractions, 9th fraction appeared to have the highest optical density (1880) (Figure 2).

Characterization of purified [beta]-1,3-glucanase

Impact of pH on enzyme activity: The purified enzyme was incubated at different levels of pH ranging from 4-8.5 using phosphate buffer of appropriate molarity. Enzyme assay was carried out 15 minutes after the incubation using 0.5% CMC as substrate. The absorbance (OD) was measured at 700 nm which revealed that the purified glucanase enzyme was active at a wide range of pH (5-8.5) but optimum activity was observed at a pH value of 5.5 (638.7567 U/ml) followed by 339.6306 U/ml and 443.6343 U/ml at pH 6 and 6.5 respectively (Figure 3A). The enzyme activity appeared to be decreased beyond pH range of 10.

Impact of temperature on enzyme activity: The enzyme with its substrate was incubated at a range of temperature to determine in order to assess its impact on the activity of isolated glucanase. The isolated enzyme showed maximum activity at 25AdegC with a value of 187.2263 U/ml followed by 170.8065 U/ml and 173.5261U/ml at 30AdegC and 35AdegC respectively (Figure 3B). Hence, isolated glucanase appeared to have maximum activity at 25AdegC.

Impact of enzyme concentration on its acticity: To determine the optimum glucanase concentration for maximum substrate degradation, numerous dilutions of the enzyme (upto eight-folds) were prepared and mixed with substrate solution of constant concentration. Enzyme-substrate interaction appeared to be increased with increase in enzyme concentration upto 4 ml but thereafter, it appeared to have consistent effects. The best measured enzyme activity was 45.88978 U/ml at an enzyme concentration of 4 ml per reaction (Figure 3).

Effect of substrate concentration on enzyme activity: To determine optimum substrate concentration for highest glucanase activity, eleven different concentrations of the substrate (carboxy methyl cellulose) were tested. The enzyme activity appeared to be increased with increase in substrate concentrations. The maximum values were determined as 646.4545 U/ml and 638.9549U/ml at 10 ml and 11 ml substrate concentrations respectively whereas further increase in substrate concentration did not show any profound effect on enhanced activity of the enzyme (Figure 3C).

Effect of incubation time on enzyme activity: Impact of incubation time on glucanase activity was determined by preparing enzyme-substrate reaction and incubating them for different time intervals. The maximum activity of enzyme was recorded at incubation time of 18 hrs (627.3255 U/ml). Thereafter, it appeared to be decreased but increase again at 42 hrs and 60 hrs (618.5705 U/ml) incubations (Figure 3D).

Determination of molecular size of enzyme through SDS-PAGE: Purified enzyme was electrophoresed on SDS-PAGE and appeared as single band when run on 12% SDS-PAGE, showing high purity level of protein having molecular mass of approximately 70 kDa (Figure 4). The gel was stained with coomassie brilliant blue and soaked in methanol, acetic acid and distilled water containing dye solution. Destaining of the gel was carried out using acetic acid solution (Meyer and Lamberts, 1965). The enzyme with specific activity of 618.5705U/ml was used for SDS-PAGE analysis.

DISCUSSION

Increased productivity of crops is direly needed owing to rapidly increasing population of the world which is expected to rise upto 10 billions by the year 2050. Various biotic stresses including fungal pathogens are significant in this perspective as plant diseases cause more than 12 percent yield losses in agriculture sector (Parvaiz et al., 2018; Fokunang et al., 2004). It further intensifies food shortage in a situation where more than 800 million people, around the globe, are already poorly fed.

Fungal diseases are currently being controlled by the application of toxic fungicides which get accumulated in soil, underground water and food. Such accumulations, on one hand, exhibit hazardous effects on human as well as animal health and environment. On the other hand, resistance development in fungal pathogens is also increasing due to abundant and persistent use of fungicides. In this situation, some bio-based alternatives are being explored and several biocontrol agents have been worked out. Besides other biocontrol agents, Trichoderma harzianum has shown consistent control of plant fungal pathogen (Ahmed and Omara, 2018). Trichoderma has evolved various nutritive adaptations including antagonism and mycoparasitism and this has made it a sturdy biocontrol agent. The phenomenon of mycoparasitism involves hydrolytic degradation of host cell wall which is carried out by production of extracellular hydrolytic enzymes i.e. glucanases and chitinases.

The degradation of pathogen cell wall permits parasitic hyphae (hyphae of Trichoderma) to penetrate the host cell (Elad, 1997). Such enzymes after purification from Trichoderma have been found highly effective against plant pathogenic fungi than enzymes isolated from other sources (Lorito, 1998). Many antibiotics and an array of hydrolytic enzymes including glucanases, chitinases, chitosinases and proteases are propsoed to endow the ability of mycoparasitism (Shoresh et al., 2010). Trichoderma-derived glucanases, chitinases and other hydrolytic enzymes are considered to be more effective to control fungal diseases, than the enzymes isolated from plants, bacteria or other fungi (Harman et al., 1993; Maximova et al., 2006). Such enzymes are known to elicit plant defence resposne, thus increasing disease tolerance (Kosambo-Ayoo et al., 2011). O'Kennedy et al. (2011) transformed pearl millet genome with T. atroviride [beta]-1,3-glucanase gene using gene gun.

Consequent transgenic lines showed 58% decrease in S. graminicola infection and the reason was found to be an elevated level of glucanase. The study under-discussion was carried out to explore [beta]-1,3-glucanase production from Trichoderma harzianum and its characterization for various optima. The optimal temperature for the growth of Trichoderma harzianum as well as for the induction of glucanase gene was found to be 28AdegC (De La Cruz et al., 1995). The similar temperature range was found to be optimum by Sexton et al. (2000). Similarly, Franco et al. (2004) also incubated Trichoderma harzianum strain T4 at 28AdegC to have optimum growth and enzymes production. As far as effect pH on enzyme activity is concerned, optimal range varies from species to species nevertheless, it is reported to be 3.0 to 6.0 in most of the cases (Niranjane et al., 2007). Glucanases exhibit maximum enzyme activity at 30AdegC to 55AdegC (Xu et al., 2006; Peng et al., 2009).

Crude exo-glucanase and endo-glucanase purified from Aspergillus niger showed optimum pH 3.5 and optimum temperature 30AdegC (Ghori et al., 2012). Likewise, B. subtilis-derived [beta]-glucanases showed 6.5-9.5 as optimal pH and 50AdegC as optimal temperature (Wichitra et al., 2006). In another study, [beta]-1,3-glucanase of around 36 kDa was isolated and purified from T. harzianum. The enzyme displayed optimum activity at pH 5.0 at 50AdegC (Marco and Felix, 2007). In our studies, [beta]-1,3-glucanase isolated from the T. harzianum was found to be highly active at 25AdegC while it was found stable up to 50AdegC. The enzyme was found to be functional at pH 5.0-8.5 however the best pH value with highest enzyme activity was found to be 5.5 (De La Cruz et al., 1995). In order to conclude, it is reported that the glucanase enzyme isolated in preset study is tolerant to a broad range of temperatures and a wide range of pH. It depicts its potential to be used as a valuable industrial compound.

Hence, Trichoderma strains produced stable [beta]-1,3-glucanase enzyme envisioning that these strains may be used as a biocontrol agent to control fatal fungal pathogens infecting valuable crops.

Authors' contributions: All of the authors contributed equally in this research

Conflict of interest: Authors have no conflict of interest for this research

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Author:G. Mustafa, S. Anwar, F. A. Joyia, M. B. Hayat, M. A. Zia, Ghazala and M. S. Khan
Publication:Journal of Animal and Plant Sciences
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Date:Dec 31, 2020
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