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 (Berseem baladi), Trifolium alexandrinum L., and the lucerne (Berseem hagazi) 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 were most cornmon and abundant: The Turnip , segetum Denis & Schiff., the Black Cutworrn, Agrotis ipsilon Hfn., the Brownish Cutworm 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 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 , 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 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). The final concentration ofthe probe is 2/lg ONA/IOO/li i.e., 20ng//l1. -274-

2.7. PCR methods Diseased larvae (0.2 g) were homogenized in 1 III ofH2o and centrifuged for 10 min at 5000 g for clarification. Ten III of the supematant from the undiluted crude extract were used for dilution in 0 1 3 90 III H2 (10- ) and so on to obtain 10-2 and 10- dilutions. Polymerase chain amplification was done on 1 III ofeach dilution, added to 49 III ofMother Mixture (1 III of each primer 100 IlM, 50 III of buffer lOX, 10 III of dntp 10 mM, 20 III ofTaq polymerase 0.5 MIIlI and 408 III oflh0). The following primers were used for the detection ofMlDNV DNA in samples:

2.7.1. From the non-structural protein NS-3 coding sequence DNV 225 Forward (5'- GTG CCA GAG TTT CGT GAT G­ 3') and DNV 264Reverse(5'-TTAGCTTGACCAAGTTGTCC­ 3'). The cycling conditions were: - Predenaturation: 95°C, 5 min, 1 cycle. - Denaturation: 95°C, 50 sec. - Annealing, 49°C, 40 sec. - Polymerisation, Amplification : noc, 40 sec 35 cycles. - Additional Polymerisation: noc, 7 min. PCR products stored ovemight: 4°C

2.7.2. From the viral protein ofthe capsid VP2 and VP3 coding sequence MLDNVVP Forward (5'- GTT ACA TCA ATC AAA CAT TGA TTA TTA ACG- 3') and MLDNVVP Reverse (5'- AGA AGT GTA TGT AAT TCC TAG ACC ATT TTC T-3') The cycling conditions were: - Predenaturation: 94°C, l min, 1 cycle. - Denaturation: 94°C, 45 sec. - Annealing, 55°C, 45 sec. - Polymerisation, Amplification : noc, 1 min 35 cycles. - Additional Polymerisation: noc, 10 min. Following the PCR, the amplified product (amplicon) was analysed after migration in 1.2% agarose gel. -275-

2.8. Cloning ofviral DNA Either complete DNA or restriction fragments were cloned. Complete DNA was blunt ended using Klenow and T4 DNA polymerase (10 U each with 300 ng DNA and 35 :M dNTPs in 50 mM Tris-Hel, pH 8.0, 5 mM MgCI2 and 10 mM DIT) and heated for 15 min at 70 oC and then ethanol-precipitated. Plasmid (pEMBLl9) was digested witb HindI, added to the blunt ended DNA with 1 unit T4 DNA ligase and 0.2 mM ATP and incubated overnight at the room temperature. The presence ofSmaI in the final mix (5 units) reduced considerably the number of religated molecules, and hence background, whereas the proportion ofvector with insert was favored (no SmaI sites regenerated). This method was more effective than dephosphorylation (unpublished observations) but the efficiency was still very low. Transformation ofXI-1 Blue and Sure ceUs (Stratagene) was followed according to the suggestions of the manufacturer. Clones obtained were repetitively recloned to select stable clones. Cloning of restriction fragments which bad only one terminus was considerably easier. Severa] restriction endonucleases (REN's) were used to generate fragments from the viral genome which were subsequently cloned into pUC 19 according to standard methods. BamHI digestion ofMlDNV DNAyielded a large central fragment of about 5.45 kb, lacking only about 300 nts at each end, which was cloned (both orientations). Furthermore, HindlII, EcoRV, and SphI fragments were cloned, into pUC 19 digested with the same restriction enzyme, plus SmaI in order to obtain tenninai fragments.

2.9. Sequencing and analysis of À'DDNV DNA Both directions were sequenced using the primer-waiker method. Areas which yieided compressions, particuiarly in the extremities, required special approaches. Among the aitematives attempted were Taq and Bsm polymerases (at 75EC) and nucleotide analogues, such as deaza dGTP, deaza dATP and dITP, during the reaction steps, according to the recommendations of the supplier. Moreover, conditions of increased denaturation during electrophoresis were attempted, such as the inclusion offormamide up to 50% in the gel. Autoradiograms were read with an lBi Gel Reader and the sequences were anaIyzed with Sequence Analysis programs (lBI) and DNASIS. A more advanced and comprehensive computer analysis of -276-

the sequences was obtained with the UWCGG programs (University of Wisconsin).

3. RESULTS AND DISCUSSION

Population fluctuation of Noctuid fauna oflucerne and clover was studied at two different localities namely EI-Jaffara and El-Kasaa villages belonging to the El-Bahareya Oasis, Giza Governorate, Egypt. The obtained results indicated remarkable infestations with eight different noctuid species. The , Agrotis segetum Denis & Schiff., the Black Cutworm, Agrotis ipsilon Hfn., the Brownish Cutworm Agrotis spinifera Hubner, the Large Cutworm Agrotis pronuba L., the African 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. were the most common and abundant species that occurred. These species attack also cotton fields in the Delta and Nile Valley. The observed fluctuation of the population (Fig. ]) indicates one infestation peak of S. littoralis around mid June, two infestation peaks around mid May and mid September for S. exigua, two infestation peaks around the beginning of July and the end of September for JI armigera, and aIl the Agrotis species appeared during the end of auturnn and al! the winter. The other species appeared at the end of the spring and the beginning ofthe summer. The epidemiological studies were done by PCR tests and nucleic probe to reveal the presence of any viral strains or isolates of Densovirus tested in the noctuid fauna species. Screenings revealed the presence of only one species of Densovirus, i.e., MlDNV hopefully not introduced, from A. ipsilon, A. spinifera, S. exigua, S. littoralis and A. gamma. Screening attempts for any other Densoviruses from naturally dead and dying speciemens of the mass-rearing culture of our laboratory have revealed the presence of isolates from the Greater -277- 800 l1------,

700 ..:.: Il> Il> ~ :: 600 c Q.. Ne N 500 ..,. s.. IiJS.I. Il> Q. .S.e Il> ~ 400 OH.a.

~t DA.i. .... c .A.s ~ 300 .ce :l= ~ 200 ~ s.. ,Il> -

01 1. IL.. .1 .. __ U Jan. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct Nov. Dec Time of the year Fig. (1): Seasonal fluctuation in the population of the most abundant five noctuid species survyed on lucern and c10ver fields in the EI-Bahareya Oasis throughout the growing seasons 2000 - 2002: Spodoptera littoralis (S.I.), S. exigua (S.e.), Helicoverpa armigera (H.a.) . A. ipsilon (A.i.), Agrotis segetum (A.s.). -278-

Wax Moth Galleria mellonella L., the Silk Worm Bombyx mari L., the Pink Bollworm Pectinophora gascsypiella Saunders, the three Egyptian Corn Borers, Sesamia cretica Led., Chilo agamemnon Bles., Ostrinia nubilalis Hbn. and the Potato Tuber Moth Phthorimaea aperculella Zeller. Purified viral suspensions were obtained from ail these different insect species. Polyacrylamide gel electrophoresis of the capsid proteins does not bring any difference with those ofMlDNV. The DNA extracted from the virions was characterized using 8 restriction endonucleases. AIl the restriction profiles were identical with those of MlDNV. After partial cloning, genome sequencing was done for some isolates. At the nucleotidic level of the two strains isolated from S. littaralis and S. exigua naturally found in the same region of the Bahareya Oasis, a sequence of 500 nucleotides from the Open Reading Frame of VP2 presents 2 significant nucleotide mutations which induce the change of 2 amino acids in the capsid protein. The complete sequencing is now underway for these two strains of MlDNV.

CONCLUSION The present study provided an evidence that biodiversity exists in MlDNV. The genetic biodiversity of the MlDNV viral strains is probably associated with the geographical distribution ofthis pest in Egypt and the host-crops, which provide the wide host range of this virus. These results suggested that some new isolates of the MIDNV are, in fact, new strains, when significant mutations are observed. The confirmation of the natural polyspecificity of MlDNV in the field completes its biologica! characterization and would provide a better known pathogen for the use as a biocontrol agent against S. littoralis larvae and against other lepidopterous pests. Regarding its high virulence, it is possible that MlDNV may represent an important complementary agent alongside with NPV and GV(Nucleopolyedro Virus and Granulo Virus) in the 1PM programme of S. littoralis. -279-

4. REFERENCES

Abul-Nasr S.E. and Naguib M.A. (1968). The population density of larvae and pupae of Spodoptera littoralis (Boisd.) in c10ver fields in Egypt. Bull. Soc. Ent. Egypte, LB, 297 - 312. Amargier A., Vago C. and Meynadier G. (1965). Etude histopathologique d'un nouveau type de virose mis en évidence chez le lépidoptère Galleria mellone/la. Arch. Gesamte. Virusforsch., 15: 659-667. Bergoin M. and Tijssen P. (1998). Biological and molecular properties of Densoviruses and their use in protein expression and biological control. In Miller LK, Bail LA (eds): The insect viruses. New York, Plenum Press, pp 141-169. Bergoin M. and Tijssen P. (2000). Molecular Biology ofDensovirinae. In S. Faisst, J. Rommelaere (eds): Parvoviruses. From Molecular Biology to Pathology and Therapeutic Uses. Contribution to Microbiology. Karger, Basel, SwitzerJand. pp 11-32. Fédière G. (2000). Epidemiology and PathoJogy ofDensovirinae. In S. Faisst, J. Rommelaere (eds): Parvoviruses. From MolecuJar Biology to Pathology and Therapeutic Uses. Contribution to Microbiology. Karger, Basel, Switzerland. pp 1-11. Fédière G., EI-Sheikh M., Aboi-Ela S., Salah M.,Masri M. and Veyrunes J. C. (1995). Isolation ofa new Densonucleosis Virus from Mythimna loreyi Dup. (Lep. Noctuidae) in Egypt. Bull. fac. Agric., Cairo Univ., 46(4): 693-702. Shorey RH. and Hale R. L. (1965). Mass rearing ofnine noctuid species on a simple artificial medium. J. Econ. Entomol., 58: 522 - 524. Tijssen P. and Bergoin M. (1995). Densonucleosis viruses constitute an increasing diversified subfamily among the parvoviruses. Sernin. Viral., 6: 347-355. -280-

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o~ w--- JJy..JI (Densovirus) ù"J..>#~~\ ~4J :i...... \,.)~ ~ ~~J (~.Ji.l\ "I~I) ~~I ul..I)\ A.ik., ~ (MlDNV) /..):.11 o.lJ.l ~ 4 1jiJ ',?:.11 J ',?j~\ f':!""~\J ',?~I f':!""~1 L.A ~4i ~~ ~ ',?~i .l....i1J .~1 J~ ~~ ~I ..ill:jPJj~ ~yh.ll wli';\ Ü" tl.,iÎ û...... p.. , ~La:i.a ~ J)b - ~-*",'ll w1~L.;..J1 ~ ~ ',?i\l - ~I ."• ." ("\.:. .J.lL.. ~ J"• •• ("\.:. J.1y Î 0"" 0jiill ~ .ll.oÎ y.....s.'ll ~ ~~ t \.,il ~ 4 1jJ ùC y.o=Jl ~ w~i .l9J ~~ ul~\ .d...p) ~üll ( Noctuidae) ~\ ~~ Ü" 0.)JJ \.:.~ ,,,I.l~\ ~.)W\ ;;.lJ.l.-.11 ,J...... <..)Wl <.:JilI1 o.l~ :~ tl.,.ï'll o~J .~'l' o~J~ ,~Yi1\ ~ (":!""'~\ ;;~J.l '~Y''l\ j)ll ;;~J.l ,~\ ~.)W\ "~J~\J .""pl ~I -:;')J "~J~ ~ ',?~I ~I -:;')J ,\~ ~\ ~I\ ~~\ ~\ ~ I..>"J~~. ~... ,- 1 L..,u\:i.ll. uL...J~.. U_'i uL..I.) U-....:..:i ~ ~L;,. ~~ u.:.,:j JI ~ ~I_, Densovirus genus (DNV) invertebrate Densovirinae d":1')W)IJ\ ~ .;ïlIJ ~~I wL..J..># ~~ 4.).l! W\.:. ~Y' o~ u~.l! ( Parv;viridae ,-:?~..>#"!,.)4 ~\.:.) üU')1 ~ ~J..>#;.jJF'- ~ ("\~ è""'l J JJI"c ",,~J JJ:.".:J.o '-'~ ~ ~ _,~ll .~ Ml·ù"J~~. 4.....-.-~.JCü'1' r..?"~-',-'I t .J-U' -LIU"-' _~,'.- li 0\ L..i.l ~ ~~I I..>"J..>#J JJWI ~ uU)Wl J..;:.9'l1 ~\ I~J DNV "W ~yJl "~J ..»ï~\ ~ u~'1\ J40 :J\:i.l\ J\y..J1 ~ ~ ü'1)l..J4 ~ji ~fi\ ~Î I.3')J ;;~J~ ~)fi:!'1IJ ~\~I übl.lJÜ.l\ -281-

Ml ....JI t."l 0-" (,,)J'..l.J:!S~.ill (Different genetic strains) ~\ ~\.J.,ll .DNV , '.- Il ..~ .. II \... J-,:i ~Il . -,.:.·.~II ul ~1 I~I ù..1S. ~ ~..fiW Ij."...... -.... '-r" ~ ~ J f'. J ~ ~.ll\ (,,)J'J.J:!S ~lji (J&- (IjJ~l ~I .J~\J PCR _II .J~I ~) ,~I ~).ill o~J.ll1 'l'by.J\ ~).ill b~J.ll1 :~tJl ~~I t'."lY\ Ü'" JS ü--bil1 j.JJ o~J~ ~ -.?~I ~I j.JJ b~~ '~Yill -.....i...... : ~~I b~J~ -II oL...t. ' ~ ~ '.- 1\ ~ .. 1<11 ,._..:.=-.1\ ù..1S., ,,<1\ ()--L. u'J',...r--- f'~ ~ J _..fiW ~ J ~ Ij~ -.?~I ~I j.JJ o~J~J -.?~I ~\ j.JJ b~J~ Ü'" JS Ü'" tJjc. ~ Open Reading Frame c~\ 01'1.;11 ).l::.J "Ü'" ,,~~~ o. • ....J ~~ .', ,~,.~ -01< "- .~L l ~\\ (VP2) ~t:i.I, A...w .. ~ll o~ .. ~~~ ~J""" f' i, r.r J -- J~ -" ( inducing) .,.Je \hj ùJllll ( 2 significant nucleotide mutations) ~lA t~1 J ~tûl, /)~ CytJJ .~lSll 0iJJ-Y. ~ ~ï ~.J:!Li ~J~ ~ J 111'!J~1 (,,)J'J.J--:!À1 ( genetic biodiversity ) ~\.J.,ll -.?~1 .(,,)J'J.J:!il1 I~ o~~ u'J"J- ~ ~ ùfi:l ( Ml DNV )t."l .;t:i.I\ ~.Ali (0 f) ~l - l..J-Alill ~~ -l...... c1.J)1 ~ ~\ ~I ." A\' -n '\ :(" ··'i J.!-Y.J ) El-Mergawy R., Li Y., El-Sheikh M., El-Sayed M., Abol-Ela S., Bergoin M., Tjissen P., Fédière Gilles (2003) Epidemiology and biodiversity of the densovirus MlDNV in the field populations of Spodoptera littoralis and other noctuid pests Bulletin of Faculty of Agriculture - University of Cairo, 54 (2), 269-281 ISSN 0526-8613