Screening of fungal strains isolated from dead insects for production of plant litter-degrading enzymes

INTRODUCTION

Recently, the enzymatic potential of fungi has gained great attention for their manifold applications in different industries such as paper production (Witayakran & Ragauskas 2009), biofuel generation (Wilson 2009), bioremediation (Strong & Claus 2011), phytopathogen management (Kikot et al. 2009), etc. For instance, fungal strains including Cryphonectria parasitica (Murrill) M.E. Barr, Thermomyces lanuginosus Tsikl., and Rhizopus oryzae Went & Prins. Geerl. are frequently used in food processing (Jensen et al. 2006), detergent production (Johnsen et al. 1997), and pharmaceutical industries (Loureiro et al. 2009), respectively. One of the most predominant fungi used in large-scale production of enzymes is Aspergillus from which 12 different industrial enzymes are currently used in various industrial sectors including the production of rubber, milk and other dairy products, as well as bread (Park et al. 2017).

In addition to their industrial applications, fungi possess an enormous ecological role being parasitic or saprophytic degraders of plant lignocellulosic materials in either aquatic or terrestrial ecosystems (Lundell et al. 2010). Moreover, pectin can be found in lignocellulosic residues to varying degrees based on their original source (Sánchez 2009). The lignocellulolytic enzymes-producing fungi are more prevalent in the phylum Basidiomycota than Ascomycota, in particular, white-rot (e.g., Phanerochaete chrysosporium Burds.) (Leonowicz et al. 1999) and brown-rot fungi (e.g., Fomitopsis palustris (Berk. & M.A. Curtis) Gilb. & Ryvarden) (Yoon & Kim 2005). However, both Dikarya phyla (Ascomycota and Basidiomycota) are differently involved in lignocellulose degradation processes. Ascomycota dominate during the first stages of litter degradation (Koide et al. 2005) by hydrolyzing cellulose (Osono et al. 2003) while Basidiomycota are considered to be prevalent in the later stages of plant litter decomposition (Osono 2007).

Cordycipitaceae belong to the order Hypocreales (Sung et al. 2007) and members of this family are mainly associated with arthropods (Chen et al. 2019), plants (Avery et al. 2011), etc. In particular, some genera such as Akanthomyces Lebert and LecanicilliumW. Gams, H.C. Evans & Zare have been applied as biopesticides because of their well-known entomopathogenecity (Goettel et al. 2008, Helaly et al. 2017) and their inhibitory activity against fungal plant pathogens (Ownley et al. 2010). In this context, the production of cuticle-degrading enzymes has been the subject of many studies (Ali & Moharram 2014; Gandarilla-Pacheco et al. 2015). However, the ability of these fungi to produce plant litter-degrading enzymes has been rarely investigated. The main goal of this study was to evaluate the production of plant litter-degrading enzymes in fungi associated with insects collected from citrus plantations in Guilan province. This study can improve our knowledge about the enzymatic activities of fungi which is important for understanding their role in soil and forest ecosystems.

MATERIALS AND METHODS

Isolation and Identification of Fungi

Plant sucking insects (scales and aphids) infested with fungi were collected from citrus orchards (sweet orange, sour orange, mandarin) and transformed to the laboratory. Insects’ exoskeleton was disinfected using 1 % sodium hypochlorite for three min and then thoroughly rinsed with distilled water. Afterward, the exoskeleton was smashed and small parts were cultivated in potato dextrose agar (PDA). The plates were incubated at 25 °C in a 12:12 light-dark regime.

In samples with fungal structures, the direct culturing method was applied for isolation (Stone et al. 2004). In these cases, a small piece of mycelium or conidial mass on the top of insect bodies was removed using a sterile needle and transferred to or streaked out on agar containing media. After five days of incubation, growing colonies were sub-cultured by transferring a small piece of mycelia to a new PDA medium and incubated at the same conditions for three days. Finally, the hyphal tip technique was used to obtain pure cultures. All cultures were kept at 4 °C for further studies. The fungal strains were identified based on their morphological (Zare & Gams 2004, 2008) as well as molecular features including growth rate, characteristics of the colonies such as shape, color, and pigment production (on PDA medium), the structure of conidia, conidiophore branches, phialides and crystal formation (on PCA medium) that have been already described in Armand (2020). Twelve representative strains were deposited in the Fungal Collection of the Iranian Research Institute of Plant Protection, Iran (refer to Armand 2020 for further information).

Enzymatic screening

Cellulolytic and pectinolytic activities were evaluated using the MEA medium (30 g of malt extract with 20 g of agar in 1 L H₂O) amended separately with either 7.5 g carboxymethyl cellulose (CMC), 7.5 g cellobiose (CEL), 5 g Avicel (AVL) and 5 g pectic acid (PGA) per liter. CMC, cellobiose, AVL and PGA were considered as indicators of Endo-1,4-β-glucanases (CM-cellulase, endoglucanae and endocellulase), β-Glucosidases and Cellobiodydrolase (exocellobiohydrolase, exocellulase and Avicelase) and pectinases, respectively. Seven-day-old mycelia were transferred to the medium and kept at room temperature for three weeks. To estimate the enzyme activity, 15 ml of Congo Red (1 mg ml^-1) was poured into the medium as an indicator. Subsequently, petri dishes were gently shaken for 15 min and finally rinsed with distilled water. Thereafter, 30 ml of 1 M NaCl were added (Pointing 1990).

The ligninolytic activity was also measured using the mentioned medium amended with 0.1 % wt/vol 2,2′--Azino-bis 3-ethylbenzothiazoline-6-sulfonic acid di-ammonium salt (ABTS). Seven-day-old mycelia were transferred to the medium, kept at room temperature for three weeks, and checked daily (Masigol et al. 2019). The results were considered positive (production of laccases) when the area around the colonies turned blue.

Removal of color from the medium by 0-33, 33-66 and 66-100% in all tested plates were detected as weak, medium and strong reactions, respectively (Masigol et al. 2019). This considers the different growth rates of the tested strains for the five different substrates to cluster the strains by using hierarchical cluster analysis in SPSS 16.0 according to the Ward method (Ward 1963).

RESULTS

Considering all five substrates testing for Endo-1,4-β-glucanase, β-Glucosidase, Cellobiodydrolase, pectinase and laccase activities of the fungal strains, nearly 35.52 % of all strains did not show any positive reactions. In addition, 35.52 %, 1.52 %, 6.57 %, 9.21 % and 2.63 % of the strains revealed one, two, three, four and five positive reactions, respectively. Based on the hierarchical clustering analysis of five enzymatic activities, the strains were placed in two clades. One clade, mainly consists of A. lecanii and Akanthomyces sp. strains, while the other contains A. muscarius with some minor exceptions (Fig.1a). 

About 71 %, 76 % and 86 % of all strains did not reveal any cellulo-, pectino- and lignolytic activities, respectively. On average, 13 %, 5 %, and 11 % of the strains showed high, medium and low cellulolytic activity, respectively. In addition, high, medium and low pectinase activity was observed in 9 %, 3 %, and 12 % of the tested strains, respectively. Only 7.89 %, 2.63 %, and 3.94 % of the strains showed a low, medium and high ligninolytic activity, respectively (Fig. 1b).

A Venn diagram (Fig. 1c) shows all possible logical combinations between different enzymes. Regarding cellulolytic activity, 12 strains (nearly 16 %) produced Endo-1,4-β-glucanase, β-Glucosidase as well as Cellobiodydrolase, including O1103-1, R128-1, R128-2 and Sh901-3 strains with strong activities for all mentioned enzymes. In addition, two strains including F901-2 and 243 were able to produce cellulolytic enzymes, pectinase as well as laccase (Fig. 1c). Interestingly, A. muscarius and A. lecanii showed different enzymatic activities (Fig. 1d). In general, the enzymatic diversity of A. muscarius strains were higher than A. lecanii strains.