Sampling sites and collection of soil samples
Ten soil samples were collected from 2 different cultivated orchards with tomato, cabbage in Ismailia Governorate (Lat.: 30° 31′ 57.28′′ N, long.: 31° 49′ 12.55′′ E) during 2019. Within the given site, approximately 3 soil samples of 250 g each were taken and placed in polyethylene bags, sealed with an adhesive tape and transported to the laboratory.
Soil samples were processed using “Galleria baits method” (Zimmermann 1986). Approximately 240 g soil, dampened with distilled water was placed in 250 g plastic container and 5 last instar larvae of Galleria mellonella (L.) were placed on the soil. Soil samples were checked every 3 days and dead larvae were removed. Then, they were placed on damp filter paper within a sterile petri dish and incubated at 26 ± 2 °C for 7–14 days (De La Rosa et al. 2000). After the incubation period, fungi were isolated from larvae showing external mycelial growth on Sabouraud dextrose agar medium with 1% yeast extract (SDAY) in addition to 50 μg/ml chloramphenicol and 50 μg/ml streptomycin (Meyling and Eilenberg 2007).
Molecular identification of Beauveria bassiana
Ribosomal regions were amplified by PCR from the extracted genomic DNA with some modification (Liu et al. 2000). Polymerase chain reactions (PCR) for the ITS rDNA region using forward primer TW81 (5-GTTTCC GTA GGT GAA CCT GC-3) and reverse primer AB28 (5-ATA TGC TTA AGT TCA GCG GGT-3) (Joyce et al. 1994) were used.
PCR reaction mixture consisted of 1 μl of DNA (50 ng/μl), 10 μl of Master mix BioMix™ (Bioline), and 1 μl (25 pM) each of the forward and reverse primers and made up to a final volume of 20 μl with sterile double distilled water. PCR conditions included initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 45 s., 55 °C for 1 min, and72 °C for 1 min, followed by a final extension at 72 °C for 5 min. The products were analyzed on 1.2% agarose gels with TAE buffer.
The PCR products were purified using Wizard® SV Gel and PCR clean-Up system Kit (Promega) following the manufacturer’s instructions. PCR products were sequenced in both directions by the Macrogen Inc. service, South Korea. The data obtained were evaluated with Finch TV (Blast) programs. The identity of approximately 532 bp sequence was confirmed by a BLAST (Basic Local Alignment Search Tool) search at NCBI and the obtained sequences were submitted and located at the NCBI database with the accession number.
The endophytism of B. bassiana in rice plants by seed inoculation
Two hundred rice seeds were surface sterilized by immersion in 70% ethanol for 1 min with constant shaking, then 15 min in 2% sodium hypochlorite (NaOCl), followed by 3 washes in sterile water; seeds were then soaked for 24 h in sterile distilled water. Spore concentrations of fungus were zero (control), and 5 × 107 spores/ml with 0.01% sterile aqueous Triton X-100 (treatment) based on inoculum concentrations used in previous studies of endophytic entomopathogens (Gurulingappa et al. 2010). The beakers containing the soaked seeds and the fungal concentration were placed in a dark environment chamber at 28 °C until the next day for planting. For the control, rice seeds were immersed with sterile water. Soaked seeds were planted in individual pots (15 cm diameter) containing sterilized soil consisting of peat moss, vermiculite, and clay. All plants were grown in a greenhouse at 25 °C with natural photoperiod for the duration of the experiment. Pots were placed in a completely randomized design, watered as needed, and no fertilizer was applied throughout the experiments.
Evaluation for endophytic presence of B. bassiana in rice
Diagnostic PCR analysis was applied to confirm the presence of the target endophytes in 50 experimental plants in the greenhouse experiments after 30 days; DNA was extracted from B. bassiana, B. bassiana-treated rice plants and untreated rice plants (negative control) utilizing the CTAB protocol (Rogers and Bendich 1985). Primers for ITS rDNA region were designed as follows: forward primer TW81 (5-GTTTCC GTA GGT GAA CCT GC-3) and reverse primer AB28 (5-ATA TGC TTA AGT TCAGCG GGT-3) (Joyce et al. 1994) and conserved region of fungal β-tubulin gene (360bp) (Bt2a-fwd: 5′-GTAACCAAATCGGTGCTGCTTTC-3′and Bt2b-rev: 5′-ACCCTCAGTGTAGTGACCCTTGGC-3′) (Glass and Donaldson 1995) were used.
PCR reaction was set up in a total volume of 20 μl. The reaction mixture consisted of 1 μl of DNA (50 ng/μl), 10 μl of Master mix BioMix™ (Bioline), and 1 μl (20 pM) each of the forward and reverse primers and filled to a final volume of 20 μl with sterile double distilled water.
The PCR program for ITs primers was set as described above and for β-tubulin gene as follows: an initial cycle of 94 °C for 4 min, followed by 30 cycles at 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 30 s, followed by a final extension at 72 °C for 2 min. PCR products were visualized on a 2% agarose gel to determine the presence of the inoculated fungal endophytes based on amplification of a DNA fragment of the expected size (positive control). PCR products were then sequenced in both directions using the same primers as used for the initial PCR reactions. The sequences were compared to the entries in GenBank database using BLASTn program.
Detection of B. bassiana by light micrograph in treated rice leaves
It was intended to carry out a comparative microscopically analysis on plant materials which showed the most prominent response of the investigated treatment.
Specimens were killed and fixed at least for 48 h in FAA (10 ml formalin, 5 ml glacial acetic acid, 50 ml ethyl alcohol 95%, and 35 ml distilled water). The specimen was washed in 50% ethyl alcohol, dehydrate in normal butyl alcohol series, embedded in paraffin wax of melting point 56 °C, sectioned to a thickness of 20 μm, double stained with crystal violet/erythrosine combination, cleared in xylem, and mounted in Canada balsam (Nassar and El-Sahhar 1998). Sections were microscopically analyzed and photomicrographed.