Screening of tropical isolates of Metarhizium anisopliae for virulence to the red palm weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae)

Fungal morphology of fungi

Sporulation color in Metarhizium can be light to very dark green (Bischoff et al. 2006), and while green sporulation is characteristic of Metarhizium spp., it cannot be considered as diagnostic, because other fungi also produce green spores. The morphology of strain ZJ-1 was typical of the genus M. anisopliae. It grew fast on SDAY and PDA plates. Round colonies on agar plates were white or cream, with villous and gossypine aerial hyphae on the surface. Punctiform olive green formed in the middle of the colony, which turned darker during growth and resulted in a tawny color at the back of the plates.

The morphology of hyphae and conidial of M. anisopliae ZJ-1 from R. ferrugineus cadavers were showed. The mycelium was smooth and hyaline, well-branched and separated, with major hyphae up to 2.0–3.2 μm wide. Conidiophores were podgy, simple, or highly branched, with 2–5 sterigma at the branch top. Conidia were colorless, elliptical, and cylindrical, with obtuse ends. Most conidia were 4.8–5.5 μm × 2.0–2.5 μmin length. Growth temperatures were between 10 and 38 °C, with an optimum of 28 °C.

Molecular identification

An approximately 685 bp fragment was amplified by ITS (ITS1-5.8S-ITS2) PCR from strain ZJ-1. The ITS sequence was submitted to GenBank (accession No. JN377427). The ZJ-1 sequence bore 93 % similarity to that of M. anisopliae (FJ545294) according to an NCBI Blast search. This result indicated that strain ZJ-1 could represented a new species and possibly a member of a new genus.

The dendrogram bifurcated into two distinct clades (1 and 2) with a dissimilarity of 0.02 (Fig. 1). Clade 1 consists of four additional clusters, in which isolates ZJ-1, M. anisopliae, M. pinghaense, M. album, and M. robertsii are identical (cluster I); M. guizhouense, M. majus, M. acridum, and M. minus show a high similarity (cluster II, cluster IV); and cluster IV separates from the other clusters. Clade 2 is represented by two large clusters (V and VI); these sequences show the lowest similarity to the ITS sequences of the isolate ZJ-1 used in this investigation. The origin of 17 kinds of Metarhizium ITS sequences are shown in Table 1.

Name	Location	Bankit	Name	Location	Bankit
Metarhizium pinghaense	Italy	KJ588068	Metarhizium anisopliae	Algeria	LK995311
Metarhizium album	Brazil	AY781687	Metarhizium robertsii	Poland	JX438656
Metarhizium guizhouense	China	JN180461	Metarhizium majus	Brasil	EF051727
Metarhizium brunneum	Poland	JX438652	Metarhizium pemphigi	Brazil	AY781676
Metarhizium lepidiotae	Japan	AB524442	Metarhizium acridum	USA	FJ617343
Metarhizium frigidum	USA	FJ617345	Metarhizium minus	China	HM055454
Metarhizium cylindrosporae	Thailand	HQ165694	Metarhizium rileyi	USA	NR119513
Metarhizium carneum	Brazil	KR093921	Metarhizium marquandii	China	KT719207
Metarhizium flavoviride	Greece	AF516291

Laboratory bioassays

Results showed that conidia of M. anisopliae ZJ-1 killed 60 % of test specimens at the lowest concentration (1 × 10⁶ conidia/mL) 10 days post-inoculation. The mortality rate increased with conidial concentration (P < 0.05). Conidia were found to be highly virulent to R. ferrugineus and caused approximately 100 % mortality 8 days post-inoculation of 1 × 10⁸ conidia/mL conidia. In contrast, the control group showed significantly lower mortality rates (3.33 ± 2.87 %) (Fig. 2). LT50 values for M. anisopliae ZJ-1 were 1.66 day (1 × 10⁸ conidia/mL) (Table 2). Probit analysis showed that LD50 values were 2175.29 conidia/larvae 3-day post-treatment.

Spores concentration	LT50 (day)	Correlation coefficient R	95 % CL
			Lower	Upper
1 × 108	1.66	0.9967	1.46	1.87
1 × 107	2.44	0.9969	2.20	2.70
1 × 106	3.78	0.9764	3.46	4.12
1 × 105	4.73	0.9923	4.23	5.31
1 × 104	7.14	0.9810	6.37	8.01

LT50, lethal time for 50 % mortality

Entomopathogenic fungus

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).

Experimental insects

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.

Laboratory bioassays

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).