BulL Fac. Agric., Cairo Univ., VoL 54(2003) : 269-282.

EPIDEMIOLOGY AND BIODIVERSITY OF THEDENSOVIRUS 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, Universitédu 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 BahareyaOasis (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 samethat attack the cotton fields.The survey was undertaken during twosuccessive years from April 2000 to May 2002 based on weeklyobservations. Seven species of Noctuidae were most cornmon andabundant: The Turnip Moth, Agrotis segetum Denis & Schiff., theBlack Cutworrn, Agrotis ipsilon Hfn., the Brownish Cutworm Agrotisspinifera Hbn., the Africau Cotton Bollworm, Helicoverpa armigeraHbn., the Alfalfa Semi-looper Autographa gamma L., the LucerneCaterpillar, Spodoptera exigua Hbn. and the Egyptian CottonLeafworm, Spodoptera littoralis Boisd.

Recent studies revealed that the mernbers ofthe Densovirusgenus (DNV) belonging to the specifie subfamily of invertebrateDensovirinae ( Parvoviridae ) showed rernarkable high virulence and

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wide host range for possible use as a viral biopesticide against insectpests.The genomic diversity ofdifferent Egyptian Densovirus isolatesof MlDNV and better understanding of the relations between the hostinsect and the Densovirus pennit us to answer the question: Are thedifferences in impact and virulence against geographical andecological populations ofS. Jittoralis and other species associated withdifferent genetic strains of MlDNV ? We provide evidence thatbiodiversity exists in M1DNV. The use of performant diagnostic toolsat the molecular level (PCR test and nucleic probe) revealed thepresence of Densovirus from A. ipsiJon, A. spinifera, A. gamma, S.exigua, and S. littoralis. The characterization, partial c10ning andsequencing of the genome of these isolates showed two new strainsisolated from S. exigua, and S. littoralis revealed by a sequence of500 nucleotides from the Open Reading Frame ofVP2. This sequencepresents 2 significant nucleotide mutations inducing the change of 2amino acids in the capsid protein. These results suggest that thegenetic biodiversity ofMlDNV is based on new strains ofthis virus.

Key words: Agrotis psi/on, densovirus, molecular diversity,parvoviridae, Spodoptera exigua, S. littoralis, viralepidemioJogy.

1. INTRODUCTION

In Egypt, the most common and abundant noctuid pest speciesof lucerne and clover are similar to those attacking cotton. These twofodder crops (the Egyptian Clover, Trifolium alexandrinum L. duringthe winter and spring seasons, and the luceme alfalfa, Medicago salivaL., as a perennial crop ail year round) are the most important foragesin this country. It was interesting to darify the Noctuid fauna andestablish the fluctuation of population in an isolated locality, i.e., theBahareya Oasis, located about four hundred ki/ometers West from theDelta and Nile Valley. Naturally occurring densoviral diseases in thenoctuid pest populations were recorded and their role in the control ofthe pest was studied. The epidemiological survey was done weekly ailyear round to reveal the presence of any Densovirus in ail thesurveyed species. The Densoviruses are responsible for fatal diseasesof their insect hosts and generally the death appears between four and

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six days after infection (Fédière, 2000).Ail the smaU non-envelopped icosahedral viroses which contain

linear single-stranded DNA genome are classified into the familyParvoviridae (Tijssen and Bergoin, 1995). The Parvovirus ofinvertebrates, isolated from several species of Arthropoda. mainlylepidopterous insects, forms the Densovirinae subfamily (Bergoin andTijssen, 2000). Members of this group are commonly calledDcnsonucleosis virus (DNV) to describe the characteristichistopathologic symptoms, i.e., hypertrophied and densely stainednuclei of sensitive cells in infected larvae (Amargier et al., 1965). Thename was subsequently shortened to Densovirus for aH the group, butthis subfamily consists ofthree genera: Densovirus, Brevidensovirusand Iteravirus (Bergoin and Tijssen, 1998). The reference strain ofthisstudy belongs to the genus Densovirus characterized by a particle of22 nm diamet~r, four capsid proteins of 91, 63, 53 and 47 KDa and agenome consisting of a single-stranded DNA molecule of the size5.95 Kb. The original strain is the Mythimna loreyi DNV designatedas .MlDNV previously isolated in September, 1993, from the maizewonn Mythimna loreyi Dup. collected at the western farm of CairoUniversity in Egypt (Fédière et al., 1995).

The present work was planned to confirm the naturalpolyspecificity of MlDNV and its high virulence in the field tocomplete its biological characterization and to provide moreknowledge 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 ofiosect specimensThe study was conducted at two different localities in the El­

Bahareya Oasis, Giza Governorate, Egypt. After seeding berseem, 4plots of normal growth were chosen in each village. Weekly samplingwas conducted throughout the year. The vegetative method wasfoJlowed for sampling larvae. In this mehtod, the berseem plants ofone of the 4 chosen plots in both areas were cut above ground andshaked over a white plastic sheet as weU as inspected carefully for thepresence of any insect material (larvae and/or pupae) in a similar way

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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. Larvaewere transferred to the laboratory, where they were separatedindividually in c1ean sterilized polypots containing artificial diet(Shorey and Hale, 1965). Oaily follow up was routinely made onfield-collected larvae, and those dying or showing any diseasesymptoms, were immediately picked up, kept in c1ean sterilizedplastic tubes, labelled and then kept in the deep freezer until theyrequired for virus screening.

Larvae which pupate (i.e., healthy) were maintained underlaboratory conditions till emergence to adults and identified. Stockcultures of the different species were established to be used as testinsects of virus bioassay. Sometimes, speciemens were compared withthose of the reference collection with the help of "lnsect ClassificationResearch Department" team, attached to the Plant Protection Researchlnstitute (PPRl), of the Agriculture Research Centre (ARC), Ministryof Agriculture and Land Reclamation, Egypt.

2.2. Virus isolatesAli the new isolates or strains of Densovirus were sampled

from larvae collected during pest infestations in the fields as weil asfrom naturally dead larvae during mass-rearing. For biochemicalcomparison, the strain of Mythimna loreyi DNV designated asMlDNV was the original strain maintained in our laboratory for sevenyears (Fédière et al., 1995).

2.3. Virus purificationThe diseased larvae were homogenized in Tris (0.05M)-SOS

(0.06%)Buffer, pH 7.8. After filtration through cheese cloth andclarification (9000 g for 5 min), the virus was concentrated by a highspeed centrifugation (Ti 55 Beckman rotor, 35.000 rpm for 1.5 hr).The viral pellets, resuspended in Tris Buffer were dispersed byultrasonication and then clarified (9000 g for 5 min). The resultingsupematant containing virus particles was layered onto a 15 - 45 %sucrose gradient and ultracentrifuged (SW 28 Beckman rotor, 27000rpm for 2.5 hr). The virus band was collected and the purified viruswas concentrated as above in Tris-Buffer, and the concentration of the

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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 extractionThe 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 of 2%, then Iysed with Sarcosyl]0% from the final volume. This mixture was incubated in water bathat 50 oC for ].5 br. The DNA solution was deproteinized by mixingwith a suspension of a phenol 1chloroform and gently shaked at theroom temperature. The mixture was centrifuged for 5 min at 5 000rpm .The nucleic acid was precipitated by the addition of2 volumes oficed absolute ethanol in the presence of sodium acetate (O.3M final)for 14 hours at -20 oc. After centrifugation (28 000 rpm, ]0 min), thepellet was washed in 70% ethanol. The pellet was dried bycentrifugation under vacuum, and incubated in TE buffer (]5 mMTris·HCL, ] mM EDTA, pH 7.5). Concentration ofONA was finallymeasured according to its optical density (0.0) through 260 nmwavelength and kept at -20 oC.

2.5. Restriction enzyme digestion and electrophoresis of the viralDNA

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 Tris­BOTA-Phosphate buffer (TEP) (90mM Tris-phosphate, 20mM EOTA,pH 8.0). Electrophoresis was conducted at 50 V for 2 hours. The gelwas visualized and photographed under a short wave UVtransilluminator. The size of the ONA fragments was estimated bycomparison with standard marker ONA (Roche) : Marker III andMarker VII.

2.6. DNA probe testsThe preparation of MlDNV ONA probe, original template

ONA extracted from MlDNV was used at a concentration of 40.D.260.The digoxygenin-Iabelled ONA probe was applied according

to the protocol recommended by the supplier (Roche). The finalconcentration of the probe is 2/lg ONA/IOO/li i.e., 20ng//l1.

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2.7. PCR methodsDiseased larvae (0.2 g) were homogenized in 1 III ofH2o and

centrifuged for 10 min at 5000 g for clarification. Ten III of thesupematant from the undiluted crude extract were used for dilution in90 III H2

0 (10-1) and so on to obtain 10-2 and 10-3 dilutions.

Polymerase chain amplification was done on 1 III of each dilution,added to 49 III of Mother Mixture (1 III of each primer 100 IlM, 50 IIIof buffer lOX, 10 III of dntp 10 mM, 20 III ofTaq polymerase 0.5MIIlI and 408 III oflh0).

The following primers were used for the detection of MlDNVDNA in samples:

2.7.1. From the non-structural protein NS-3 coding sequenceDNV 225 Forward (5'- GTG CCA GAG TTT CGT GAT G­

3') and DNV 264Reverse(5'-TTAGCTTGACCAAGTTGTCC­3').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 codingsequenceMLDNVVP Forward (5'- GTT ACA TCA ATC AAA CAT

TGA TTA TTA ACG- 3') and MLDNVVP Reverse (5'- AGA AGTGTA 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) wasanalysed after migration in 1.2% agarose gel.

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2.8. Cloning of viral DNAEither complete DNA or restriction fragments were cloned.

Complete DNA was blunt ended using Klenow and T4 DNApolymerase (10 U each with 300 ng DNA and 35 :M dNTPs in 50 mMTris-Hel, pH 8.0, 5 mM MgCI2 and 10 mM DIT) and heated for 15min at 70 oC and then ethanol-precipitated. Plasmid (pEMBLl9) wasdigested witb HindI, added to the blunt ended DNA with 1 unit T4DNA ligase and 0.2 mM ATP and incubated overnight at the roomtemperature. The presence of SmaI in the final mix (5 units) reducedconsiderably the number of religated molecules, and hencebackground, whereas the proportion ofvector with insert was favored(no SmaI sites regenerated). This method was more effective thandephosphorylation (unpublished observations) but the efficiency wasstill 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 terminuswas considerably easier. Severa] restriction endonucleases (REN's)were used to generate fragments from the viral genome which weresubsequently cloned into pUC 19 according to standard methods.BamHI digestion of MlDNV DNAyielded a large central fragment ofabout 5.45 kb, lacking only about 300 nts at each end, which wascloned (both orientations). Furthermore, HindlII, EcoRV, and SphIfragments were cloned, into pUC 19 digested with the same restrictionenzyme, plus SmaI in order to obtain tenninai fragments.

2.9. Sequencing and analysis of À'DDNV DNABoth directions were sequenced using the primer-waiker

method. Areas which yieided compressions, particuiarly in theextremities, required special approaches. Among the aitemativesattempted were Taq and Bsm polymerases (at 75EC) and nucleotideanalogues, such as deaza dGTP, deaza dATP and dITP, during thereaction steps, according to the recommendations of the supplier.Moreover, conditions of increased denaturation during electrophoresiswere attempted, such as the inclusion of formamide up to 50% in thegel. Autoradiograms were read with an lBi Gel Reader and thesequences were anaIyzed with Sequence Analysis programs (lBI) andDNASIS. A more advanced and comprehensive computer analysis of

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the sequences was obtained with the UWCGG programs (Universityof Wisconsin).

3. RESULTS AND DISCUSSION

Population fluctuation of Noctuid fauna of lucerne and cloverwas studied at two different localities namely EI-Jaffara and El-Kasaavillages belonging to the El-Bahareya Oasis, Giza Governorate,Egypt.

The obtained results indicated remarkable infestations witheight different noctuid species. The Turnip Moth, Agrotis segetumDenis & Schiff., the Black Cutworm, Agrotis ipsilon Hfn., theBrownish Cutworm Agrotis spinifera Hubner, the Large CutwormAgrotis pronuba L., the African Cotton Bollworm, Helicoverpaarmigera Hbn., the Alfalfa Semi-looper Autographa gamma L., theLucerne Caterpillar, Spodoptera exigua Hbn. and the Egyptian CottonLeafworm, Spodoptera littoralis Boisd. were the most common andabundant species that occurred. These species attack also cotton fieldsin the Delta and Nile Valley.

The observed fluctuation of the population (Fig. ]) indicatesone infestation peak of S. littoralis around mid June, two infestationpeaks around mid May and mid September for S. exigua, twoinfestation peaks around the beginning of July and the end ofSeptember for JI armigera, and aIl the Agrotis species appearedduring the end of auturnn and al! the winter. The other speciesappeared at the end of the spring and the beginning of the summer.The epidemiological studies were done by PCR tests and nucleicprobe to reveal the presence of any viral strains or isolates ofDensovirus tested in the noctuid fauna species. Screenings revealedthe presence of only one species of Densovirus, i.e., MlDNVhopefully not introduced, from A. ipsilon, A. spinifera, S. exigua, S.littoralis and A. gamma.

Screening attempts for any other Densoviruses from naturallydead and dying speciemens of the mass-rearing culture of ourlaboratory have revealed the presence of isolates from the Greater

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:: 600c

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Il>~ 400t~

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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 c10verfields 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.).

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Wax Moth Galleria mellonella L., the Silk Worm Bombyx mari L.,the Pink Bollworm Pectinophora gascsypiella Saunders, the threeEgyptian Corn Borers, Sesamia cretica Led., Chilo agamemnon Bles.,Ostrinia nubilalis Hbn. and the Potato Tuber Moth Phthorimaeaaperculella Zeller.

Purified viral suspensions were obtained from ail these differentinsect species. Polyacrylamide gel electrophoresis of the caps idproteins does not bring any difference with those of MlDNV. TheDNA extracted from the virions was characterized using 8 restrictionendonucleases. AIl the restriction profiles were identical with those ofMlDNV.

After partial cloning, genome sequencing was done for someisolates. At the nucleotidic level of the two strains isolated from S.littaralis and S. exigua naturally found in the same region of theBahareya Oasis, a sequence of 500 nucleotides from the OpenReading Frame of VP2 presents 2 significant nucleotide mutationswhich induce the change of 2 amino acids in the capsid protein. Thecomplete sequencing is now underway for these two strains ofMlDNV.

CONCLUSIONThe present study provided an evidence that biodiversity

exists in MlDNV. The genetic biodiversity of the MlDNV viral strainsis probably associated with the geographical distribution ofthis pest inEgypt and the host-crops, which provide the wide host range of thisvirus.

These results suggested that some new isolates of the MIDNVare, in fact, new strains, when significant mutations are observed.

The confirmation of the natural polyspecificity of MlDNV inthe field completes its biologica! characterization and would providea 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 mayrepresent an important complementary agent alongside with NPV andGV(Nucleopolyedro Virus and Granulo Virus) in the 1PM programmeof S. littoralis.

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4. REFERENCES

Abul-Nasr S.E. and Naguib M.A. (1968). The population density oflarvae and pupae of Spodoptera littoralis (Boisd.) in c10verfields in Egypt. Bull. Soc. Ent. Egypte, LB, 297 - 312.

Amargier A., Vago C. and Meynadier G. (1965). Etudehistopathologique d'un nouveau type de virose mis en évidencechez le lépidoptère Galleria mellone/la. Arch. Gesamte.Virusforsch., 15: 659-667.

Bergoin M. and Tijssen P. (1998). Biological and molecular propertiesof Densoviruses and their use in protein expression andbiological control. In Miller LK, Bail LA (eds): The insectviruses. New York, Plenum Press, pp 141-169.

Bergoin M. and Tijssen P. (2000). Molecular Biology ofDensovirinae.In S. Faisst, J. Rommelaere (eds): Parvoviruses. FromMolecular Biology to Pathology and Therapeutic Uses.Contribution to Microbiology. Karger, Basel, SwitzerJand. pp11-32.

Fédière G. (2000). Epidemiology and PathoJogy ofDensovirinae. In S.Faisst, J. Rommelaere (eds): Parvoviruses. From MolecuJarBiology to Pathology and Therapeutic Uses. Contribution toMicrobiology. Karger, Basel, Switzerland. pp 1-11.

Fédière G., EI-Sheikh M., Aboi-Ela S., Salah M.,Masri M. andVeyrunes J. C. (1995). Isolation of a new Densonucleosis Virusfrom 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 noctuidspecies on a simple artificial medium. J. Econ. Entomol., 58:522 - 524.

Tijssen P. and Bergoin M. (1995). Densonucleosis viruses constitutean increasing diversified subfamily among the parvoviruses.Sernin. Viral., 6: 347-355.

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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