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BioTechnologia vol. 101 (2) C pp. 117–133 C 2020 Journal of Biotechnology, Computational Biology and Bionanotechnology RESEARCH PAPERS . http://doi.org/10.5114/bta.2020.94771 CC S. = BY NC ND Screening and identification of novel cellulolytic Trichoderma species from Egyptian habitats OMAR A. HEWEDY *, A. M. EL-ZANATY, A. I. FAHMI Department of Genetics, Faculty of Agriculture, Menoufia University, Shibin, El-Kom, Egypt Abstract The present study aimed to isolate indigenous Trichoderma fungi possessing cellulolytic activities from different geographical locations in Egypt. Such strains can be used to convert crop residues to bioproducts, which presently is one of the most important issues in developing countries. The isolates were identified using molecular ap- proaches and morphological characteristics. Favorable growth conditions for Trichoderma isolates (media & tem- perature) were assessed in vitro. The growth rate of Trichoderma isolates was assessed on two media, namely Potato Dextrose Agar (PDA) and Synthetic Nutrient Agar (SNA), and morphological parameters, i.e., shape, pig- ment, and edge were observed. A comparison of sequences encoding for internal transcribed spacer (ITS) and translation elongation factor 1-α (tef1 ) was conducted to determine the species. Cellulase assay was performed using different cellulose substrates (Swollen, CMC (low viscosity carboxymethylcellulose), and Avicel). Among the isolated fungi, Trichoderma viride EGY-T27 had the highest endoglucanase activity, while Trichoderma harzianum EGY-T30 showed the highest exoglucanase activity. A total of 27 strains of Trichoderma were identified using ITS, and the tef1 gene was sequenced for 23 strains. The tested strains were identified with acceptable quality based on ITS sequences, which ranged from 98 to 100%, except for strain MH908499 (85%). This research constitutes the most comprehensive study on screening for fungal strains with the highest biodegradable potential isolated from various locations in Egypt. Key words: Trichoderma, cellulase activity, biodegradation, ITS, tef1 Introduction such as wood, soil, crop residues, and some plant habitat Trichoderma species are well known for their ability niches as endophytes (Samuels et al., 2006; Bailey and to colonize different substrates and diverse environ- Melnick, 2013; Chaverri and Samuels, 2013). They are mental habitats. They inhabit the rhizosphere layer of currently widely used in industrial applications because the soil with a rapidly growing rate, producing a large of their ability to produce extracellular hydrolases in number of spores and degrading enzymes for their nutri- large amounts (Mach and Zeilinger, 2003; Samuels tion. Additionally, Trichoderma fungi are considered to et al., 2006). Their ability to produce numerous diges- be one of the most widely distributed fungi in nature and tive enzymes such as cellulose, hemicellulose, lignin, can grow in a wide range of climates (Seidl, 2006; Topo- and chitin that break down various biopolymers have lovec-Pintarić, 2019). Some Trichoderma strains are been utilized as a strategy for degrading various agri- endophytes that promote plant health and serve as bio- cultural wastes and biomass, with biofuel as the most va- control agents via mycoparasitism; they are also used as luable product of this hydrolysis process (Druzhinina biopesticides instead of synthetic pesticides and as bio- et al., 2018c). The development of alternative energy fertilizers for enhancing root development (Arnold et al., strategies is a critical factor because of the rising prices 2003; Benítez et al., 2004; Harman et al., 2004; Barari, of crude oil as well as for reducing environmental pollu- 2016). Trichoderma can also grow on various substrates tion. Therefore, bioconversion of lignocellulosic residues * Corresponding author: Department of Genetics, Faculty of Agriculture, Menoufia University, Shibin, El-Kom, Egypt; e-mail: [email protected] 118 O.A. Hewedy, A.M. El-Zanaty, A.I. Fahmi is initiated primarily by microorganisms such as Tricho- cellulosic polymers into bioethanol via glucose or plat- derma reesei and Aspergillus niger. T. reesei is a Tri- form chemicals is considered as an important alternative choderma species that is commercially used for the pro- method to reduce carbon dioxide emission and reduce duction of cellulolytic enzymes required for making bio- the overdependence on fossil fuels (Schuster et al., fuels and is also used in other industries (Dashtban 2012). The traditional methods used for eliminating cel- et al., 2009; Druzhinina et al., 2018c). lulolytic materials and biological wastes have a negative Cellulose and hemicellulose polysaccharides are influence on the environment and climate. Moreover, β-(1,4)-linked glucose polymers with diverse composi- global energy demand is increasing with the rapidly gro- tions. Evidently, they constitute 60–80% of the plant cell wing human population, and recently, the rapid con- wall. This lignocellulosic biomass, which includes forest sumption of fossil fuels, for instance, has resulted in its wastes and residues of crops, is considered the world’s depletion, which has raised questions concerning en- most abundant natural biopolymer and renewable re- vironment safety. Lignocellulosic biomass has emerged source on the Earth (Singhania et al., 2013). It is a pro- as an alternative solution for these issues because of the mising sustainable source of substrate in the production high cost of cellulase preparation and difficult fermenta- of bioethanol (Gusakov, 2013). Nevertheless, the high tion process and to reduce the destructive effect on en- price of cellulase extraction and processing increases the vironment due to the burning of rice straw for instance. cost of biofuel production compared to that of fossil fuels, It is therefore necessary to enhance the hydrolytic effi- which limits the commercial production of biofuel on ciency of promising strains such as Trichoderma for a large scale (Wu et al., 2011). A promising strategy for ef- lignocellulosic biomass. ficient utilization of renewable resources is the hydrolysis The present study evaluated the diversity of Tricho- of lignocellulosic biomass by filamentous fungi such as derma isolates in several Egyptian locations. Thus, the Trichoderma. Cellulase enzymes comprise a mixture of present work focused on the identification of representa- three major enzyme classes: endoglucanase (EC 3.2.1.4) tive Egyptian Trichoderma isolates, determination of that attack multiple internal sites in the regions of the their favorable growth conditions, and screening of the cellulose fiber, cellobiohydrolase (EC 3.2.1.91), and gluco- cellulase activity of fungi from native environmental sidase (EC 3.2.1.21) (El-Zawahry et al., 2010; Adsul et al., sources at different geographical locations to select the 2019). These enzymes are used not only in the biodegra- promising isolates of cellulase production. dation of wastes, but they also have extensive applications in animal feed; textiles; grain alcohol fermentation; Materials and methods pulp and paper processing; starch processing; and pharmaceutical, malting, and food industries (Gordillo- Fungal isolates and collection sources Fuenzalida et al., 2019; Zhanga et al., 2019). Tricho- A library of 40 Trichoderma isolates was randomly derma can produce high yields of endo/ exoglucanases, collected from the rhizosphere soil samples associated but its low yield of β-glucosidase has diminished its in- with various crop residues located at 38 different geo- dustrial value (Dashtban and Qin, 2012; Tiwari et al., graphic habitats in Egypt (Table 1, Fig. 1). Each sample 2013; de Souza et al., 2018). By using a series of chro- contained 200 g of soil collected from a depth of approxi- matographic procedures, four endoglucanases (Endo I, mately 20 cm. The samples were placed in sterile poly- II, III, and IV) and one exoglucanase (Exo II) were iso- ethylene bags, transported to the laboratory, and stored lated from a commercial cellulase preparation derived at 4EC until isolation according to the method of Elad from T. viride (Kim et al., 1994). Additionally, T. reesei et al. (1981). Briefly, approximately 10 g of each homo- is considered as one of the most industrially useful geneous soil sample was weighed and transferred to Trichoderma species for cellulase processing (Mandels a 250 ml Erlenmeyer flask containing 100 ml of sterile and Reese, 1957; Gomes et al., 1992; Keshavarz and water and shaken at 150 rpm for 2 h to make the stock Khalesi, 2016; Druzhinina and Kubicek, 2017b). solution. Subsequently, a series of 10-fold dilutions of Glucose is the most abundant monosaccharide pro- the suspension were prepared for each sample, and duced in plant tissues and most algae during photosyn- 100 μl of each dilution was plated on Potato Dextrose thesis. Therefore, the conversion of cellulosic and hemi- Agar (PDA). The isolates of Trichoderma spp. were sub- Discovering new taxa of Trichoderma strains 119 Table 1. Different crop residues for Trichoderma isolation collected from different geographical locations No. of location(s) No. of Trichoderma Isolate code Habitats Crop residues Soil type surveyed isolates EGY-T1 Quwesna corn Peat 3 3 EGY-T2 Banha rice straw Clay Loam 4 4 EGY-T3 Berket-Elsaba berssem Loamy 2 1 EGY-T4 Tanta rice straw Clay Loam 3 1 EGY-T5 Kafer-Elshikh rice straw Clay Loam 3 1 EGY-T6 Behra tomato Loamy 5 1 EGY-T7 Alexandria rice straw Clay 1 1 EGY-T8 Menia tomato Loamy 3 1 EGY-T9 Cairo grape Sandy

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