First Isolation, In Egypt, of a granulovirus (AgseGV) from Agrotis segetum Schiffermuller (Lepidoptera: Noctuidae)

A new Granulovirus was isolated from cutworm Agrotis segetum larvae collected from the field in Qalubaya governorate. Transverse section of larvae showed infected fat body, epidermis and mid-gut cells. Transmition electromicroscope evealed ovoid occlusion bodies (granules), each granule contains a single rod-shaped virion with one nucleocapsid, typical of granuolosis virus. Also, the scanning electromicroscopy of apurified viral suspension confirmed a baculovirus morphology. The size of virus particles in the present isolation ranged between 1.9 m to 2.5m. Preliminary bioassay indicated that A. ipsilon neonate larvae are susceptible to AgseGV and the calculated LC50 value was 3.79x10capsules/ml.


INTRODUCTION
Both Agrotis segetum and A. ipsilon are important pest of many vegetable and field crops.In Egypt, A. ipsilon commonly called black cutworm is the most abundant species followed by A. spinifera and A. segetum; infesting about 50 plant species (El-Malki et al., 1998).
Several species of entomopathogenic viruses have been isolated from larvae of species Agrotis (Ignoffo and Garcia, 1979) and some have been evaluated as possible biological control agents of cutworms (Thomsen et al., 1998).Viruses with biocontrol potential in the field, belonging to the family Baculoviridae, (polyhedrosis and granulosis) have been isolated from various Agrotis species (Caballero et al., 1991;Bourneret al., 1992;Boughton et al., 1999).
In Egypt, serious attempts did not lead to baculovirus isolation, but did not rule out the possibility of virus presence at very low level (Khattab, 1988) but reported non-occluded virus like particles about 30 nm. in A. segetum.Sherlock (1983) also reported non-occluded virus-like particles 30-40nm or 40-50 nm in Agrotis spp.
The reported AgseGV is also registered as "Virin-OS", a wettable powder, in the former Soviet Union for use against A. segetum in cotton (Lipa, 1991).
In Egypt, however baculoviruses have never been reported or isolated on Agrotis spp.
The presen treport describes a new granulovirus, (AgseGV EG ) in Egypt which is isolated from A. segetum and readly infects A. ipsilon neonate larvae.

Source of virus isolation:
Larvae cutworm Agrotis spp. was collected from the field in Qalubaya governorate, reared individually on semi-artificial diet described by Shorey and Hale (1965).Virus-dead larvae identified as A. segetum were triturated and occlusion bodies (OB's) were purified as described by Khattab (1988).The haemolymph smears of dead larvae were stained with Giemsa's stain and examined under light microscope for presence of any virus inclusion bodies (VIB).Newly emerged adult moths from survived larvae were kept in a mating cage for oviposition and deposited eggs were collected daily and hatched larvae were transferred to the semi-artificial diet.

Virus clarification and purification
Dead larvae were blended in distilled water at 4°C, filtered through several layers of muslin cloth then centrifuged at 1000 rpm for 5 min and the pellet was discarded.The VIB was pelleted from the supernatant at 4000 rpm for 30 min.and the pellet was resusp ended in Tris buffer ( 50 et al., 1980).

Bioassays:
A1.0 ml volume of each dose of virus suspension was spread over the diet surfacein the cells of a microtitre plate.One neonate A. ipsilon larva was placed on each cell then the plates were covered with tissue paper and glass plate fixed with rubber bands.All treatments were incubated at 25 2°C.and mortality due to virus was recorded against untreated diet-control treatment.All treatments included 2.5% wetter-sticker (Triton X -100) to reduce clumping.

RESULTS AND DISCUSSION
In the present study, all of A. segetum larvae died due to virus disease were fifth and six instars (Fig. 1).Transverse section of A. ipsilon larvae infected with virus examination by phase contrast microscope showing infected Fat body (F), Epidermis (EP) and the cells of the mid-gut (MG) (Fig. 3).
Transmition electron microscopic showed the major morphological properties of typical GV granules isolated from A. segetum larvae.Section through separated virus showed virions and granules in longitudinal and transverse section.Each virion contains one nucleocopsid (NC) (Fig. 4, A).
The occlusion bodies (OB,s) were avoidale, each granule contained single rod-shaped virion with one nucleocapsid indicating a granulovirus (Fig. 4 and B, C).Scanning electron microscopic examination showed the occlusion bodies (OB,s) and the size of the granule ranged 1.9 m to 2.5 m (Fig. 5).The size of the granule seemed significantly larger than the average granulosis inclusion bodies (Akermann and Smirnoff, 1983) which resembling a distinguished granule size 0.2-0.4μM.Table (1) shows the mortality response of A. ipsilon neonate larvae to the isolated GVat different tested concentrations ( capsule's/ml).The calculated LC 50 value is 3.79 x 10 6 capsule's/ml diet and the slope value of the regression line is 0.9877 (Fig. 6).In conclusion, the isolated GV is assumed AgseGV as reported here first time in Egypt from A. segetum.The reasonable susceptibility of A. ipsilon to AgseGV EG suggests a promising potential of this virus in pest management of cutworms.The author suggests the name (AgseGV EG ) for the reported isolate in Egypt.

First
Fig.1: Agrotis segetum, Adult, larva and pupa.The naturally infected larvae of A. segetum showed the typical symptom of virus infection.Some were extremely fragile rupturing to release containing VIB when examined in smears.At this time larvae are long swollen and whiten creamy, death occurred within a few days (Fig.2).

Fig. 2 :
Fig. 2: Agrotis ipsilon, Healthy larvae (far right), and different signs due to the presence of granules.

Fig. 4 :
Fig. 4: Electron micrographs illustrating the major morphological properties of Agrotis segetum (AgseGV EG ) A: Section through AgseGV EG virus showing virions and granules (1000X).B: Longitudinal section through an occlusion body with a single embedded virion.Note the ''nipple' and the 'claw' ends of the nucleocapsid (40000X).C: Longitudinal and transverse section through an occlusion body (25000X).