Characterization of a Granulovirus Isolated from Epinotia aporema Wals. (Lepidoptera: Tortricidae) Larvae

Abstract

A granulovirus (GV) isolated from Epinotia aporema (Lepidoptera: Tortricidae)-a major soybean pest-was studied in terms of its main morphological, biochemical, and biological properties. The ovoidal occlusion bodies were 466 by 296 nm in size, and their most prominent protein had an apparent molecular mass of 29 kDa. Its amino-terminal sequence was remarkably homologous to that of the granulins of other GVs. The DNA genome size was estimated to be 120 kbp. The high specificity and pathogenicity of this newly described granulovirus (EpapGV) indicate that it is indeed a good candidate for the biological control of this pest.The bean shoot borer, Epinotia aporema (Wals.), is a serious pest of soybean and other legume crops in South America. In Argentina, damages caused by this tortricid decrease soybean yields up to 40%, depending on population level and environmental conditions (18). Currently, broad-spectrum chemical insecticides are used to control this caterpillar, delaying or suppressing field colonization by natural enemies. This scenario compromises the implementation of a proper integrated pest management scheme for soybean crops, in areas where the incidence of E. aporema reaches high population levels (25).Among pathogens, a granulovirus (GV) was detected as the main mortality factor (6, 26). In general, the potential of baculoviruses for pest control has been well documented and they have proven to be effective against many pests (9,15,24). In this regard, preliminary experiments showed that E. aporema GV (EpapGV) could be a safe alternative for inclusion in soybean pest management (25). However, former studies were directed towards evaluating its virulence, and none of the previously cited reports addressed the characterization of the virus. As an initial point in our research, we identified and characterized a GV isolate from E. aporema, indigenous to the main soybean area of Argentina.Virus isolation and multiplication. A colony of E. aporema caterpillars reared on artificial medium (12) was established in our laboratory. EpapGV was isolated from a single E. aporema larva collected in Oliveros (Santa Fe, Argentina). Viral amplification was carried out, allowing fourth-instar larvae to feed on formalin-free artificial diet, superficially contaminated with 4,000 granules per mm 2 . Granules were purified from homogenized larvae by two cycles of centrifugation on continuous 40 to 65% (wt/wt) sucrose gradients at 100,000 ϫ g for 1 h. Virions were released by hydrolyzing granules in 0.1 M Na 2 CO 3 -0.17 M NaCl-0.01 M EDTA (pH 10.5) at 37°C for 30 min. Following the dissolution of the granules, the suspension was neutralized by addition of 1 M HCl, and undissolved material was removed by low-speed centrifugation.Electron microscopy. Tissues were dissected from infected and control larvae, fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3)-0.25 M sucrose for 3 h, postfixed in 1% osmium tetroxide for 1 h, dehydrated through an ethanolpropylene oxide series, and embedded in Epon-Araldite resin. Ul...

Histopathology of Anticarsia gemmatalis Hübner (Lepidoptera; Noctuidae) treated with Nucleopolyhedrovirus and Bacillus thuringiensis serovar kurstaki

“…These cells were rounded and developed nuclear hypertrophy. Same data was observed by Sciocco-Cap et al (23), showing in the histological analysis of the 4th instar of Epinotia aporema larva (Lepidoptera, Tortricidae) infected with Baculovirus that were presented the same phases of cellular disruption.…”

Detection and kinetic analysis of Epinotia aporema granulovirus in its lepidopteran host by real-time PCR

“…The virus used in this study was propagated in and purified from initially healthy larvae infected by diet surface contamination with the characterized EpapGV isolate as reported previously [6,19]. The virus titer was estimated using a dark-field microscope by counting the number of OBs in a Petroff-Hausser chamber.…”

“…Crocidosema) aporema Wals. (Lepidoptera: Tortricidae) [6,12,16,19]. Initial studies on virus-host interactions were performed using optical and electron microscopy [6,19], and an attempt to measure the accumulation of the major viral protein (granulin) after per os ingestion involved the production of polyclonal antibodies [17].…”

Biology and morphometrics of the immature stages of Epinotia aporema on artificial diet

“…A recently discovered granulovirus of E. aporema (EpapGV) (Sciocco-Cap et al 2001;Goldberg et al 2002) has been shown as a promising biological control agent when ingested by early larval instars. This led to the establishment of a laboratory-reared insect colony supplied with artificial diet, both to perform pathogen-host interaction studies and for virus multiplication.…”

“…The available information on E. aporema life cycle is limited and mostly restricted to its occurrence and development on soybean crops under variable conditions (Morey, 1972;Iede & Foerster, 1982). Nevertheless, some basic aspects of the insect biology and morphometrics are still poorly documented.Traditionally, various chemical insecticides have been used against this moth, but the interest in more environmentally friendly biopesticides such as Bacillus thuringiensis and baculoviruses has increased in the past few years (Ibarra et al 1992;Sciocco-Cap et al 2001;Sauka et al 2007). A recently discovered granulovirus of E. aporema (EpapGV) (Sciocco-Cap et al 2001;Goldberg et al 2002) has been shown as a promising biological control agent when ingested by early larval instars.…”