Isolation of endophytic fungi
Root samples of Malva parviflora and leaf samples of Chenopodium album and Pelargonium graveolens were collected from Abees farm, and leaf samples of Melia azedarach were collected from the garden of the Faculty of Agriculture, Alexandria, Egypt. The samples were washed with tap and distilled water, air dried, surface sterilized by 70% ethanol for 1 min then by 3.0% sodium hypochlorite (NaOCl) for 3 min, and rinsed with sterile distilled water for 1 min three times. The surface-sterilized samples were cut into 5 mm2 surface using a sterile blade and placed in Petri dishes with potato dextrose agar media containing 50 mg/l of ampicillin (Kharwar et al. 2012). Each plate contained four segments of plant pieces with four replicates for each plant. The dishes were incubated at 25 ± 2 °C and observed daily to record the emergence of endophytes and pre-cultured on potato dextrose agar (PDA) media. For testing the effectiveness of surface-sterilized plant samples, 1 ml of the last rinsing water was placed on the PDA media and incubated at 25 ± 2 °C.
Identification of fungal isolates
Morphological observations were carried out, using both macroscopic and microscopic characters, for endophytic fungus culture and spore shapes and types, using the standard manual of Barnett and Hunter (1998). The isolated endophytes were subjected to DNA extraction, using the Qiagen DNA extraction kit (Qiagen, Germany). According to Hafez and Elbestawy (2009), endophytic fungi were identified by amplification of ITS1-5.8SrRNA-ITS4 gene using the universal primers: ITS1 (forward primer: 5-TCC GTA GGT GAA CCT GCG G-3) and ITS4 (reverse primer: 5-TCC TCC GCT TAT TGA TAT G-3). PCR reaction mixture (25 μl) consisted of 12.5 μl PCR Master Mix (2X) Promega Corporation, 1 μl (50 pmol) of each oligonucleotide primer, 1 μl DNA template, and 9.5 μl nuclease-free water. The reaction cycles consisted of initial denaturation (95 °C, 2 min), 35 cycles of denaturation (95 °C, 31 min), annealing (55 °C, 1 min), extension (72 °C, 1 min), and final extension (72 °C, 10 min). The PCR products were analyzed using agarose gel electrophoresis with ethidium bromide (EtBr) staining. The products were purified using the purification kit (Qiagen) and sequenced by DNA sequencer by LGC group (Berlin, Germany). After preprocessing, the resulting DNA sequence was identified using the BLASTN tool of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) and nucleotide collection (nr/nt) database. To create the phylogenetic tree of the endophytic fungi, advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software version 6.0 was used (Tamura et al. 2013).
Pathogenicity of endophytic fungi spore suspension
By dipping technique
The endophytic fungi grown in PDA plates with high spore production levels (Alternaria alternata (MG786545), A. solani (MG786543), Curvularia lunata (MF113056), Nigrospora sphaerica (MF113055), and Stemphylium sp.) were flooded by sterile distilled water containing 0.05% Triton X-100, scrapped with surface-sterilized spatula, and filtered through cheesecloth to remove mycelial debris. Spore suspensions were prepared, and their concentrations were adjusted to 108 spores ml−1 by a hemocytometer then tested against third-instar larvae of S. littorals. Ten larvae were dipped in spore suspensions for 5 s and left to dry on a filter paper. Two control treatments were conducted: with sterilized distilled water and with aqueous solution of 0.05% Triton X-100. The treated larvae were transferred to glass bottles containing fresh castor leaves, with four replicates for each treatment (Baskar et al. 2012). The experiment was checked daily for 2 weeks to keep clean, the castor leaves were changed, the dead larvae were counted, and the mortality percent was calculated. The dead larvae were surface sterilized and placed on the Petri dishes containing PDA to confirm if the mortality was due to the fungi pathogenicity.
By residual film
The same concentration of spore suspensions used for the previous bioassay was used for residual film bioassay. Castor leaves were submerged in spore suspensions for 20 s, left to dry, and transferred to glass bottles with ten starved (4–5 h) third-instar larvae of S. littoralis. Castor leaves treated with sterilized distilled water and with sterilized distilled water containing 0.05% Triton X-100 served as the control treatments with four replicates for all treatments. The results were taken as described above.
Extraction of endophytes secondary metabolites
Based on the results of the previous pathogenicity bioassay, three endophytic isolates from different genera were chosen for fermentation and extraction of their secondary metabolites. Five disks of each endophytic fungus were inoculated in 1 l Erlenmeyer flask containing 300 ml potato dextrose broth (3 l PDB) and incubated at 28 °C for 2 weeks in a shaker incubator at 120 rpm. The culture broth was centrifuged for 10 min at 15000×g, and the supernatant was extracted by the solvent mixture (ethyl acetate to methanol to water, 8:4:1) in an ultrasonic bath at 40 °C for 20 min. The solvent layers were separated by a separatory funnel, and the aqueous layer was extracted twice by ethyl acetate with sonication for 20 min. The ethyl acetate layer was collected and evaporated under vacuum to dry and kept at 4 °C for bioassay.
Pathogenicity of endophyte secondary metabolites
Ethyl acetate extracts were tested against third-instar larvae of S. littoralis by dipping and residual film techniques (Pampapathy et al. 2011). Concentrations of ethyl acetate crude extracts (250, 500, 1000, 2000 mg/l) were prepared in a sterile distilled water containing 0.05% Triton X-100. Ten larvae of S. littoralis were dipped in each extract concentrations for 5 s, left on filter paper for 10 s to dry, and transferred to the Petri dishes containing castor leaves, with four replicates.
The same concentrations of ethyl acetate extracts were used for residual film bioassay. Castor leaves were dipped in each extract concentrations for 20 s, left to dry, and transferred to the Petri dishes containing ten starved (4–5 h) larvae of S. littoralis with four replicates. Control treatments were conducted using castor leaves treated with sterilized distilled water and with sterilized distilled water containing 0.05% Triton X-100. Mortality rate was observed daily for 1 week, and mortality percent was calculated.
Percentage of S. littoralis mortality was subjected to one-way analysis of variance followed by Student-Newman-Keuls test (Cohort Software Inc. 1985) to determine significant differences between mean values at the probability level of 0.05.