BulL Fac. Agric., Cairo Univ., VoL 54 (2003) : 269-282. EPIDEMIOLOGY AND BIODIVERSITY OF THE DENSOVIRUS MlDNV IN THE FIELD POPULATIONS OF Spodoptera littoralis AND OTHER NOCTUID PESTS (Received: 29.6.2002) By R. EI-Mergawy, Yi Li*, M. EI-Sheikh, M. EI-Sayed, S. Aboi-Ela, Max Bergoin**, P. Tijssen* and G. Fédière Center ofVirology, Institut de Recherche pour le Développement (IRD)­ Faculty ofAgriculture, Cairo University, Giza, Egypt. * Centre de Microbiologie, INRS - Institut Armand-Frappier, Université du Québec, Laval, Qébec, H7VIB7, Canada. ** Laboratoire de Pathologie Comparée, Université de Montpellier II. lldontpellier, Cadexs France. ABSTRACT The viral epidemiology ofMlDNV was studied in the Bahareya Oasis (Western desert) on two host plants: the Egyptian clover (Berseem baladi), Trifolium alexandrinum L., and the lucerne alfalfa (Berseem hagazi) Medicago sativa L. on which the pests are the same that attack the cotton fields.The survey was undertaken during two successive years from April 2000 to May 2002 based on weekly observations. Seven species of Noctuidae were most cornmon and abundant: The Turnip Moth, Agrotis segetum Denis & Schiff., the Black Cutworrn, Agrotis ipsilon Hfn., the Brownish Cutworm Agrotis spinifera Hbn., the Africau Cotton Bollworm, Helicoverpa armigera Hbn., the Alfalfa Semi-looper Autographa gamma L., the Lucerne Caterpillar, Spodoptera exigua Hbn. and the Egyptian Cotton Leafworm, Spodoptera littoralis Boisd. Recent studies revealed that the mernbers ofthe Densovirus genus (DNV) belonging to the specifie subfamily of invertebrate Densovirinae ( Parvoviridae ) showed rernarkable high virulence and -269- -270- wide host range for possible use as a viral biopesticide against insect pests.The genomic diversity ofdifferent Egyptian Densovirus isolates of MlDNV and better understanding ofthe relations between the host insect and the Densovirus pennit us to answer the question: Are the differences in impact and virulence against geographical and ecological populations ofS. Jittoralis and other species associated with different genetic strains of MlDNV ? We provide evidence that biodiversity exists in M1DNV. The use ofperformant diagnostic tools at the molecular level (PCR test and nucleic probe) revealed the presence of Densovirus from A. ipsiJon, A. spinifera, A. gamma, S. exigua, and S. littoralis.The characterization, partial c10ning and sequencing of the genome of these isolates showed two new strains isolated from S. exigua, and S. littoralis revealed by a sequence of 500 nucleotides from the Open Reading Frame ofVP2. This sequence presents 2 significant nucleotide mutations inducing the change of2 amino acids in the capsid protein. These results suggest that the genetic biodiversity ofMlDNV is based on new strains ofthis virus. Key words: Agrotis psi/on, densovirus, molecular diversity, parvoviridae, Spodoptera exigua, S. littoralis, viral epidemioJogy. 1. INTRODUCTION In Egypt, the most common and abundant noctuid pest species of lucerne and clover are similar to those attacking cotton. These two fodder crops (the Egyptian Clover, Trifolium alexandrinum L. during the winter and spring seasons, and the luceme alfalfa, Medicago saliva L., as a perennial crop ail year round) are the most important forages in this country. It was interesting to darify the Noctuid fauna and establish the fluctuation ofpopulation in an isolated locality, i.e., the Bahareya Oasis, located about four hundred ki/ometers West from the Delta and Nile Valley. Naturally occurring densoviral diseases in the noctuid pest populations were recorded and their role in the control of the pest was studied. The epidemiological survey was done weekly ail year round to reveal the presence of any Densovirus in ail the surveyed species. The Densoviruses are responsible for fatal diseases of their insect hosts and generally the death appears between four and -271- six days after infection (Fédière, 2000). Ail the smaU non-envelopped icosahedral viroses which contain linear single-stranded DNA genome are classified into the family Parvoviridae (Tijssen and Bergoin, 1995). The Parvovirus of invertebrates, isolated from several species of Arthropoda. mainly lepidopterous insects, forms the Densovirinae subfamily (Bergoin and Tijssen, 2000). Members of this group are commonly called Dcnsonucleosis virus (DNV) to describe the characteristic histopathologic symptoms, i.e., hypertrophied and densely stained nuclei ofsensitive cells in infected larvae (Amargier et al., 1965). The name was subsequently shortened to Densovirus for aH the group, but this subfamily consists ofthree genera: Densovirus, Brevidensovirus and Iteravirus (Bergoin and Tijssen, 1998). The reference strain ofthis study belongs to the genus Densovirus characterized by a particle of 22 nm diamet~r, four capsid proteins of91, 63, 53 and 47 KDa and a genome consisting of a single-stranded DNA molecule of the size 5.95 Kb. The original strain is the Mythimna loreyi DNV designated as .MlDNV previously isolated in September, 1993, from the maize wonn Mythimna loreyi Dup. collected at the western farm ofCairo University in Egypt (Fédière et al., 1995). The present work was planned to confirm the natural polyspecificity of MlDNV and its high virulence in the field to complete its biological characterization and to provide more knowledge on the pathogen candidate as a biocontrol agent against S. littoralis larvae on cotton as weil as other noctuid pests. 2. MATERIAL AND METHOOS 2.1. Experimental trials, sampling methods and identification of iosect specimens The study was conducted at two different localities in the El­ Bahareya Oasis, Giza Governorate, Egypt. After seeding berseem, 4 plots of normal growth were chosen in each village. Weekly sampling was conducted throughout the year. The vegetative method was foJlowed for sampling larvae. In this mehtod, the berseem plants of one of the 4 chosen plots in both areas were cut above ground and shaked over a white plastic sheet as weU as inspected carefully for the presence ofany insect material (larvae and/or pupae) in a similar way -272- of sampling procedure previously described by Abul-Nasr and Naguib (1968). Collected insects were placed in plastic boxes 18 cm x 8 cm, provided with berseem and covered with its perforated lids. Larvae were transferred to the laboratory, where they were separated individually in c1ean sterilized polypots containing artificial diet (Shorey and Hale, 1965). Oaily follow up was routinely made on field-collected larvae, and those dying or showing any disease symptoms, were immediately picked up, kept in c1ean sterilized plastic tubes, labelled and then kept in the deep freezer until they required for virus screening. Larvae which pupate (i.e., healthy) were maintained under laboratory conditions till emergence to adults and identified. Stock cultures of the different species were established to be used as test insects of virus bioassay. Sometimes, speciemens were compared with those ofthe reference collection with the help of"lnsect Classification Research Department" team, attached to the Plant Protection Research lnstitute (PPRl), ofthe Agriculture Research Centre (ARC), Ministry ofAgriculture and Land Reclamation, Egypt. 2.2. Virus isolates Ali the new isolates or strains of Densovirus were sampled from larvae collected during pest infestations in the fields as weil as from naturally dead larvae during mass-rearing. For biochemical comparison, the strain of Mythimna loreyi DNV designated as MlDNV was the original strain maintained in our laboratory for seven years (Fédière et al., 1995). 2.3. Virus purification The diseased larvae were homogenized in Tris (0.05M)-SOS (0.06%)Buffer, pH 7.8. After filtration through cheese cloth and clarification (9000 g for 5 min), the virus was concentrated by a high speed centrifugation (Ti 55 Beckman rotor, 35.000 rpm for 1.5 hr). The viral pellets, resuspended in Tris Buffer were dispersed by ultrasonication and then clarified (9000 g for 5 min). The resulting supematant containing virus particles was layered onto a 15 - 45 % sucrose gradient and ultracentrifuged (SW 28 Beckman rotor, 27000 rpm for 2.5 hr). The virus band was collected and the purified virus was concentrated as above in Tris-Buffer, and the concentration ofthe -273- final suspension was measured on the spectrophotometer at 260 nm. The purified viral suspensions were stored at - 20 C in Tris Buffer. 2.4. DNA extraction The extraction of the nucleic acid from the purified virus was carrled out using the suspension of virus, mixed with proteinase K (2mg Iml ) for a final concentration of2%, then Iysed with Sarcosyl ]0% from the final volume. This mixture was incubated in water bath at 50 oC for ].5 br. The DNA solution was deproteinized by mixing with a suspension of a phenol 1chloroform and gently shaked at the room temperature. The mixture was centrifuged for 5 min at 5 000 rpm .The nucleic acid was precipitated by the addition of2 volumes of iced absolute ethanol in the presence ofsodium acetate (O.3M final) for 14 hours at -20 oc. After centrifugation (28 000 rpm, ]0 min), the pellet was washed in 70% ethanol. The pellet was dried by centrifugation under vacuum, and incubated in TE buffer (]5 mM Tris·HCL, ] mM EDTA, pH 7.5). Concentration ofONA was finally measured according to its optical density (0.0) through 260 nm wavelength and kept at -20 oC. 2.5. Restriction enzyme digestion and electrophoresis ofthe viral DNA 1 ]lg of the viral ONA (l 0.0.260 of ONA = 50 /lg/ml) was digested in a volume of 20 /lI as recommended by the supplier (Roche). Electrophoresis was carried out using 1% agarose gel in Tris­ BOTA-Phosphate buffer (TEP) (90mM Tris-phosphate, 20mM EOTA, pH 8.0). Electrophoresis was conducted at 50 V for 2 hours. The gel was visualized and photographed under a short wave UV transilluminator. The size of the ONA fragments was estimated by comparison with standard marker ONA (Roche) : Marker III and Marker VII. 2.6. DNA probe tests The preparation of MlDNV ONA probe, original template ONA extracted from MlDNV was used at a concentration of 4 0.D.260.The digoxygenin-Iabelled ONA probe was applied according to the protocol recommended by the supplier (Roche).