Isolation and identification of L-asparaginase-producing endophytic fungi from the Asteraceae family plant species of Iran

Isolation and identification of fungal endophytes

From May until September 2015, the plant specimen (stem, root, leaf and flower) were obtained from seven healthy medicinal plants: Matricaria chamomilla, Matricaria parthenium, Anthemis triumfetii,, Anthemis altissima, Achillea millefolium, Achillea filipendulina and Cichorium intybus growing in the natural ecosystem in Northeastern of Iran. The samples were stored in polyethylene bags at 4 °C (Waksman, 1916). Samples were washed thoroughly in distilled water. The Surface sterilization was performed by sequential immersion of samples in sodium hypochlorite (3 times in 5% NaOCl for 3–8 min each, depending on the type of samples) followed by 75% ethanol for 1 min and rinsed five times in sterile distilled water. The samples were dried on sterile blotters under the laminar air flow. The surface-sterilized samples were cut into about 0.5 × 1 cm² using a sterile scalpel. 200 segments from stem, leaf, root and flower (four segments per Petri plate) for each plant species were placed equidistantly on Potato Dextrose Agar medium (PDA, Merck, Darmstadt, Germany) supplemented with tetracycline (50 mg/L) to inhibit bacterial growth. Three replicates of Petri dishes were used per plant sample. The petri plates were incubated at 28 ± 2 °C with 12 h light and dark cycles for up to 6 to 8 weeks. To verify sterility controls were prepared by spreading 100 µL aliquots of the water from final rinse solutions onto PDA medium plates and incubated for 2 weeks at 28 ± 2 °C with 12 h light and dark cycles. The absence of fungal growth in controls indicated effective sterilization, while mycelial growth from plant samples was indicative of endophyte isolation. Colonies that emerged from tissue segments were picked up and transferred to antibiotic-free PDA to enable identification. Colonization Frequency (CF) of endophytes was calculated as described by Khan et al. (2010).

${\displaystyle \text{Colonization Frequency}\mathrm{of}\text{Fungi}\left(\text{\%}\right)=\frac{\text{Number of}\text{Isolates}\mathrm{of}\text{Taxon from}\text{Each}\text{Segment}}{\text{Total}\text{Number of}\text{Segments}}\times 100}$

Identification was achieved using morphological and molecular methods. Morphological identification of isolates was performed based on the fungal colony morphology, characteristics of the spores and reproductive structures using standard identification manuals (Barnett & Hunter, 1999; Bensch et al., 2012; Boerema et al., 2004; Simmons, 2007; Booth, 1971).

Molecular identification of endophytic fungi

Endophytic fungal isolates were grown in 200 mL of potato dextrose broth (PDB) for 7 days at 28 °C. The mycelia were washed with distilled water and ground with liquid nitrogen. The nucleic acid was extracted using the cetyl trimethyl ammonium bromide (CTAB) method (Dayle et al., 2001). Strains were sequenced with four: molecular markers, including ITS (Internal transcribed spacer), LSU (partial large subunit nrDNA), TEF1 (Translation elongation factor) and TUB (β-tubulin) using primer sets listed in Table 1. PCR amplifications were performed on a GeneAmp PCR System 9600 (Perkin Elmer, USA) in a total volume of 12.5 µL solution containing 10–20 ng of template DNA, 1 × PCR buffer, 0.7 µL DMSO (99.9%), 2 mM MgCl2, 0.5 µM of each primer, 25 µM of each dNTP and 1.0 U Taq DNA polymerase (NEB). The amplification process was initiated by pre-heating at 95 °C for1 min, followed by 40 cycles of denaturation at 95 °C for 30s, with primer annealing at the temperature stipulated in Table 2, extension at 72 °C for 10s, and a final extension at 72 °C for 5 min. The products of the PCR reaction were then examined by electrophoresis using 1% (w/v) agarose gel, stained with gel red (Biotium®) and visualized with a UV transilluminator (UVP MultiDoc-It™, Analytik Jena, Germany). BLAST analysis was carried out in the NCBI database. All sequences were deposited in NCBI’s GenBank Database.

Screening of L-asparaginase-producing endophytes

The isolated endophytic fungi were screened for their ability to produce asparaginase. Mycelial plug was inoculated onto Modified Czapex Dox (McDox) agar [agar powder (20.0 g/ L⁻¹), glucose (2.0 g/L⁻¹), L-asparagine (10.0 g/L⁻¹), KH₂PO₄ (1.52 g/L⁻¹), KCl (0.52 g/L⁻¹), MgSO₄⋅7H₂O (0.52 g/L⁻¹), CuNO₃⋅3H₂O (0.001g/L⁻¹), ZnSO₄⋅7H₂O (0.001 g/L⁻¹), FeSO4⋅7H2O (0.001 g/L⁻¹)], l-asparagine (10.0 g/L⁻¹) and 0.3 mL of 2.5% phenol red dye (indicator). Controls were prepared by inoculating mycelial plugs on Czapex Dox agar without asparagine. Triplicates for each isolate were prepared. All Petri plates were incubated at 26 ± 2 °C. After 5 days of incubation, the diameter of the pink zone was evaluated (Gulati, Saxena & Gupta, 1997).

Estimation of L-asparaginase activity

The L-asparaginase positive fungal isolates were inoculated using 5 mm fungal mycelial plugs into 200 mL of McDox broth and incubated for 5 days at 36 ± 2 °C and 120 rpm. L-asparaginase was estimated by Nesslerization as described by Imada et al. (1973). After incubation, 100 µl of broth (crude enzyme) was pipetted into 2 ml tubes. After that, 100 µl of Tris HCl (pH 7), 200 µl of 0.04 M asparagine and 100 µl of sterile distilled water (SDW) were added. The mixture was incubated at 37 ± 2 °C for 1 h. After incubation, 100 µl of 1.5 M Trichloroacetic Acid (TCA) was then added to stop the enzymatic reaction. Finally, 100 µl of the mixture was pipetted into fresh tubes containing 750 µl SDW and 300 µl of Nessler’s reagent and incubated at 28 ± 2 °C for 20 min and the amount of enzyme activity was measured by determining the absorbance of samples at 450 nm using UV-Visible spectrophotometer (Jenway model 6315). One unit of asparaginase is expressed as the amount of enzyme that catalyzes the formation of 1 µmol of ammonia per minute at 37 ± 2 °C (Theantana, Hyde & Lumyong, 2007). ${\displaystyle \text{Units}∕\text{ml enzyme}=\frac{\left(\mathrm{\mu }\text{mol of NH}3\text{liberated}\right)\left(0.6\right)}{\left(0.1\right)\left(60\right)\left(0.2\right)}}$

0.6 = Initial volume of enzyme mixture in mL

0.1 = Volume of enzyme mixture used in final reaction in mL

60 = Incubation time in minutes

0.2 = Volume of enzyme used in mL

Statistical analysis

The final experiment was conducted using a completely randomized design with triplicates for each parameter assessed. The data were statistically analyzed using the software Statistical Package for the Social Sciences (SPSS) version 17.0. One-way ANOVA with least significant difference (LSD_(0.01)) were applied to analyze all the data collected.

Identification of fungal endophytes

Endophytes were obtained from all seven medicinal plant species with a total of 837 isolates from 200 each of leaf, stem and root segments. Endophytes were mostly recovered from A. altissima (241 isolates), followed by A. millefolium (163 isolates), A. triumfetii (121 isolates), C. intybus (132 isolates), A. filipendulina (90 isolates), M. chamomilla (59 isolates) and M. parthenium (31 isolates) (Table 2). Due to the large number of fungal endophytes, the isolates were further classified into 84 morphotypes based on the different morphological and cultural characteristics. Eighty-four endophytic fungal species belonging to Ascomycota and Basidiomycota were identified using morphological and molecular methods. Few endophytic fungi such as Acremonium sclerotigenum, Alternaria burnsii, Bjerkandera adusta, Colletotrichum tanaceti, Epicoccum nigrum, Fusarium acuminatum, Paraphoma chrysanthemicola, Plectosphaerella cucumerina and Stemphylium amaranthi showed wide distributions in the host plants and were isolated from most plants studied. Also, a higher number of endophytes were recovered from stem tissues of all seven plant species (Table 2). The percent colonization frequency of endophytes varied in the plant parts with stem fragments harboring 55.6% of endophytic isolates followed by leaves with 31.1% and least for the isolates from flower samples. Many isolates belonged to the genera Alternaria, Fusarium, Phoma, Chaetosphaeronema and Plectosphaerella which colonized more than one plant part. The isolates of Fusarium were recovered from stem, leaf, flower and root while Phoma spp. was obtained from stem and leaf sample. Tissue specificity was also observed for some endophytes. This was most evident in the Septoria species that were found only in the stem tissues. Basidiomycetous endophytes such as Trametes versicolor, Bjerkandera adusta, Trichaptum biforme and Schizophyllum commune was isolated from stem tissues. Fusarium spp. was found as the dominant endophytes with 140 isolates, followed by Alternaria spp. (105 isolates). The results indicated that the species composition and frequency of endophyte species was found to be dependent on the tissue and host plant.

Screening of L-asparaginase-producing endophytes by qualitative plate assay

All endophytic fungal isolates were screened for their ability to produce L-asparaginase by qualitative rapid plate assay. Of the eighty-four fungal endophyte isolates tested for L-asparaginase activity (Table 3), thirty-eight isolates were positive for extracellular L-asparaginase and formation of pink zones was evident on Modified Czapex Dox (McDox) medium. The pink zone diameter varied from 15.3 to 58.2 mm (Table 3). Fusarium proliferatum showed maximum enzyme activity (Fig. 1), followed by Plenodomus tracheiphilus. All six Fusarium species in this study could produce L-asparaginase and the pink zones were measured above 27.3 mm. Forty-six isolates did not produce L-asparaginase, including all endophytic fungi isolated from M. parthenium. Also, our results demonstrated that the basidiomycetous endophytes did not produce L-asparaginase. Thirty-eight fungal strains exhibiting positive enzyme activities were selected for quantitative assay of L-asparaginase.

Estimation of L-asparaginase production by Nesslerization

L-asparaginase activities of thirty-eight fungal endophytes were recorded to range of 0.019–0.492 unit/mL⁻¹ (Table 3). The isolates of F. proliferatum obtained from A. altissima, exhibited a maximum enzyme activity with 0.492 unit/mL⁻¹, followed by P. tracheiphilus isolated from A. altissima (0.481 unit/mL⁻¹). F. oxysporum and Cladosporium limoniforme exhibited moderate enzyme activity, while Septoria tormentillae showed the least activity with 0.019 unit/mL⁻¹ of enzyme (Fig. 2). Results showed that there were significant differences among the isolates at 1% (Table 3). The percentage of L-asparaginase-producing fungal endophytes was 45.2% of the total isolated endophytes (84 isolates) with 2.5%, 3.5%, 15%, 14.2%, 3.5% and 6.5% respectively for M. chamomilla, A. triumfetii, A. altissima, A. millefolium, A. filipendulina and C. intybus.