Naturally dead R. ferrugineus cadavers were collected from Wenchang, Hainan Island, China (19_32N, 110_47E). The samples were soaked in 70 % alcohol for 1 min and rinsed using sterile distilled water. The cadavers were subsequently surface-sterilized using 0.1 % mercury chloride, followed by three rinses in sterile distilled water. Parts of the tissues were cut and inoculated on Sabouraud Dextrose Agar with Yeast Extract (SDAY) containing 40 g/L dextrose, 10 g/L peptone, 10 g/L yeast, 20 g/L agar and 500 µg/mL streptomycin. These tissues were separately placed on sterile petri dishes sealed with preservative film at 28 ± 1 °C, 75 ± 5 % RH for 6 days. Purification was achieved using a monospore culture, named ZJ-1. Scanning electron microscopy (Hitachi S-3000N) was performed to study morphologic characteristics of ZJ-1 (Driver et al. 2000; Tulloch 1976; Su 2006).
A laboratory population of R. ferrugineus was established by collecting larvae from infected palm trees in the Wenchang suburb (19_32N, 110_47E), Hainan, China (Li et al. 2010). Larvae were reared on sugarcane stem tissues at 28 ± 1 °C, 75 ± 5 % RH. After adults emerged, they were placed in jars and supplied with cotton wicks saturated with 8–10 % honey for feeding. Subsequently, eggs were transferred to a moist sterile filter paper within an unsealed petri dish (12 cm in diameter). Upon hatching, neonate larvae were individually transferred to 50 mL vials containing 10 g weevil’s artificial diet (Martín and Cabello 2006). Approximately 7 days later, laboratory-reared larvae were obtained for further analysis.
DNA extraction and sequencing
Genomic DNA (gDNA) was extracted following the method described by Lee and Taylor (Lee and Taylor 1990). A fragment of the ITS spacer region was amplified using universal primer sets ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′). PCR reactions (50 µL) contained 50 ng of template gDNA, 1 µL of each 10 pM oligonucleotide, 1 µL of 10 mM dNTPs, 1 µL of 2 U/µL Taq DNA polymerase (Sino Geno Max Co., Ltd, Beijing), 5 µL of 10× PCR buffer. The PCR protocol for amplification of ITS regions included initial denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, followed by a final elongation at 72 °C for 10 min. PCR products were kept at 4 °C. The size and quality of PCR products were determined by gel electrophoresis using 1.25 % agarose gel, which was stained with ethidium bromide (0.5 mg/mL) and visualized under UV light. The ITS1-5.8S-ITS2 amplified products were purified using a Fungus Genomic DNA Extraction Kit (Omega) and sequenced in an automated system (Sino Geno Max Co., Ltd, Beijing).
DNA sequences obtained from this work were compared with existing 17 Metarhizium species sequences data in GenBank using the Basic Local Alignment Search Tool (NCBI BLAST) (Destéfano 2004); a summary of these comparisons is shown in Table 1. Phylogenetic relationships among DNA sequences of ZJ-1 were determined using multiple sequence alignment MAGA6.0 via the maximum likelihood method (ML). Confidence levels for generated groups were determined via 1000-repetition bootstrap analysis.
We selected R. ferrugineus larvae of similar size during the feeding period. Conidia of divided purified ZJ-1 were placed in a sterile 10 mL centrifuge tube containing aqueous 0.05 % Tween-80 and the mixture was vortexed to attain homogenization. Conidial concentration was determined using a hemocy to meter. A dilution series of aqueous conidial suspension (1.0 × 108, 1.0 × 107, 1.0 × 106, 2.0 × 105, 1 × 104 conidia/mL) was prepared thorough mixing, then sprayed on larvae. Treated larvae were separately transferred to 50 mL vials containing 10 g weevil’s artificial diet under controlled conditions (28 ± 1 °C, 80 ± 5 % RH). Each trial was performed in triplicate with a total of 30 insects per process. After the treatment, insects were scored as dead at 24 h intervals for 10 days. Meanwhile, dead larvae were selected and tested for potential pathological changes (Anggraeni and Putra 2011).