5

LABORATORY BIOASSAY AND HOST RANGE TESTS OF THE GRANULOSIS VIRUS OF SPODOPTERA LITTORALIS

By ADLY, M. ABD-ALLA,*' .K. EL-SHEIKH,* S O , ABOL-ELA,* GLLLES/FEDIERE,* JOSE * axxi =IZA, M.F. E L - S m Y * *

* Entomovirology Laborato ept. of Econ. Entomol. & Pesticides, Fac. of Agric., Cairo Univ., Giza, Egypt.

** Dept. of Entomology, National Research Center. Dolrki. Giza, Egypt.

Received 22-2- I99 7)

INTRODUCTION

Few enlomopathogenic viruses, which are specrfic. efficielit and safe to non- target organisms have been exTloited as pest control agents. Some of these agents (e.g., Baculoviruses) have shown potentiality as an excellent alternative to chemical insecticides in the intcgrated pest management programmes (IPM) (Elnagar and El- Sheikh, 1989). A member of the family baculoviridae subfainily Eubaculosirinae. is the granulosis virus (GV), (Francki, et al., 1991). This occluded virus nas strongly proposcd for field application (Tanada. 19G4). S. iittoralis GV was isolated from diseased !amie more than 15 years ago at Bouake in Core d'Ivoire and characterized, in Egypt. through its physical. immunological. and biochemiczl properties as GV-qwdoptera by Aboi-Ela et ai., (1991). Although S. littordis NPV, xhich was isolated in €gyp fourty years a o (Abul-Nasr. 1956). is known by its acute pathogenicity to the Ist instar lanrac, the granulosis virus acts strongly against later instars (Narayanan, 19S5). This fact may be considcred in the management of S. littordis. In Egypt, the granulosis virus :vas neithcr studied sufficiently. nor infectivity tests werc previously conducted. The present work was. thus. designed to evaluate the pathogmicity of the virus and its host range. which are fundaniental facts for better understanding of S. littornlis GV.

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The test insect used in the prcsent study was S. littor-alis (Boisd.) (Noctuidae); the source of its culture was originated from moths collected by a light trap and maintained for severa1 succcssive generations under laboratory controlled conditions of 25 4 2°C and 6570% R.H. LaRae were maintained on asemi- synthetic diet, described by Shorey and Hale (1963), and those for bioassay tcsts, were maintained on the sanie diet ingredients escluding the formaldehydc. All the different dcvelopmental stages of this test insect were maintained as described by Khamiss (1 99 1).

The granulosis Virus used in the present work was available at the Entomovirolog Laboratov3 Dep. of Econ. Entomol., Fac. of A,gic.. Cairo Univ.

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I Bull. ent. Soc. Egypt, Econ. Ser. 24, 1997 (22). Fonds

Cote : 6 x Z53K3 Ex : i

and purified by P. Mons&at and F. Baillon (Personal communication). I GV-infcctcd laniae were homogcnized in b a c r (5OnM Tris, 21nM SDS pH

7.8), the homogenate was filtered through several layers of cotton and muslin (Tonipkins. 199 1). Clarification of the filterate suspension took place by a low centrifugation at 1100 g for 5 min using Beckman J2-2lMIE ccntrifuge, rotor 20 JA. The virus was pelleled from the supernatant by ccntrifiigation at 28000 g for 30 min using the same rotor. The pellet was resuspended in lml Tris buffer (5OnM pH 7.8) deposited on 30-70% (FV/IV) continuous sucrose gradient and centrifiigatcd at 55000 g for 20 min using Beckman L7-65 ultracentrifuge. rotor SW 28. The band containing granules was drawn off with a Pasteur pipette, suspended in Tris buffer. and centrifuged at 43000 g for 30 min using Beckman J2-21MIE ccntrifuge, rotor 20 JA for washing; pelleted granules were then resuspended in Tris buffer. The highly purified viral granules were checked by spectrophotometer DU-70 through 450 nni wave length. and stocked in Tris under -20°C.

Bioassay tests of S, littordis GV :

The highly purified SI GV stock suspension, dilutcd to 4-5 different concentrations, \vas testcd against the 2nd (4-5 day-old), 3rd (6-7 day-old) and 4th (8-9 day-old) instars of S. littoralis larvae pooled out from the virus-free culture. Tested larvae were starved 6-8 hours before exposure to the differcnt virus concentrations.

ï'he lethal concentration (LC) of S. littordis GV was determined for flic 211d and 4/17 lamal instars. A plastic plate measuring 18 s 12 s 1 cm, containing 0.5 cm laycr of dict was divided by a plastic shect into 81 squares 1.5 s 1.5 cm each. Each square was dosed with one of the tested concentrations (e ., 119, 11.9, 1.19, O. 119.

OD4 j o = O. 125 mg capsule/ml, (Chang and Tanada, 1975) in 25 JLI distillicd water dispensed on the diet surface.

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0.0119 OD (1 (Optical density) OD450 = 1.48 s 10 i8 capsule/ml 'and lm1 at 1

The bioassay method described by Elnagar et of., (1983) was used to determine the lethal dose (LD) of Sf GV for the 2nd, 3rd and 4th instars. Each virus dosc was administered on a disc of diet, 0.4 cm diameter and 0.2 cm thick, placed in a well of a microtiter plate. Five p1 of eachvirus dose (119, 23.5, 11.9, 1.19, 0.119, and 0.01 19 OD) was applied on each disc and allowed to air drying before placing the stanied larva. Rate of mortality was calculated according to symptoms of viral infection as nel1 as nucleic probe, and corrected according to Abbott's Fonnula (1 925).

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. Iiost rmgc ofSI GV :

steh z@i&t six insect species from Lepidoptera rcprescnting diEcrext gmera from different families : the lesser cotton ledivorni, S. e-x-igzm (h%.)> the black cutwo-m, Agrotis @don (Hh.) and the maize wom, A&thiti?im Ioreyi (Dup.) all from Nmtuidae, the. greater was moth, Gnlleria itzeloneilrr &.) (Pyralidae), the cabbage butterfly, Artogeia rapm (L.) (Pieridae), and the pink bollwmm, Pectinophora gossypiella (S.) (Gelechiidac).

RESULTS

Obtained results demonstrate that the 2nd and 4th instar larvae of S. littomiis were susceptible to SI GV and mortality rates among testcd larvae, increased with the increase in xirus concentration (Table 1). The highest rates of mortalities 96.76% and 91.21% were achieved €or the 2ndand 4th instar, rcspectiwly, at 119 OD/d . Determination of LC valce was based on the percent mortality obtained from tlic daily recorä of death. Deat!i due to vim infection occurred during the larval stage. The calc'dated LC50 for the 2ndand 4th instar was 0.052 arid 0.813 OD/ml, respectively (Tdde 1). The time rquireci for initial mortality iyas 5 and 8 days. for the 2nd and 4th instar, respective1yz and the last mortalities dcc to virus tïeatment were cor.clnded by the 54th ana 4lst day. The estimated LC25 and LC90 values, for the above mentioried instars were (0.01 and 1.1'7 OD/ml) and (0.039 and 288.4 OD/mi). respxtiveiy flâble 1)- Cbtaïncd d t s pro:cd that the 2i?d instar l a n w were more suc:ept&le io 14rus iníkctiofi than the 4th ones, the LC25 value cf the latter instar ïvas 3.9 timcs mere than that 0f fie former onc. Thc same trend was observcd ivïth the LCSO and LC90, where theirvalues for the 4th instar. n'crc 15.9 and 246.49 tinies more than tha of the 2nd one. Concentration - mortality regression line did not indicate any dífkrence, in the slopc of thc linc, betwen tlic 2nd and 4th instar, which suggests a sidar type of responsc between the two instars to SI GV.

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TABLE ( I ) Mortality percentage and LC25,'LC30 and LC30 values of SI GV versus S. littoralis

i ' - .

~ ~ 3 0 . 9 S-1.1.

4ti7 instar I - -

Rcplicnte Kn. 1

2

3

‘yo i\.lurtnlity st indicated \.¡ru\ cot&ntr;ition.i (OD) Esti:llatc~ti 1.c: v:1lnrs a

119 11.9 1.19 0.119 0.0119 LC25 I.CS0 LC90 24/29* 17/23 29/33 14/40 12 42 (96.55) , (73.91) (78.79) (35) (28.57) 26/30 21/34 (13126) 9 35 8 28

(90.63) (61.76) (50) (25.71’) (28.57) 26/30 (15/28) 8 31 7 31 5 36

(56.67) (53.57) (25.81) (22.58) (13.89)

*NO. of vinis dead larvaeltotal number oftested larvae; perccntages are belwcn brackets.

Results show that the rate of mortality: among tested larvae increased directly with the increase in virus dose. The time required for initial mortality was 12, 6 and 9 days and all mortalities due to virus treatmcnt occurrcd during the larval stage and ,

concluded by the 34th, 25th and 41111 days for the 2 n d 3rd and 4th instar, respectively, and according to the dose Icvcl. Data in Tablcs (2,3 and 1) show the estimated LD 25, LD50 aEd LD90 V ~ I L I C S of thc 2nd: 3rd and &li instsrs, respectivcly. LD25 value for the Itli instar was 183 and 160 times 1;iore than that of ihc 2176 and 3rd instars. respectively. Iiowevcr. the LD25 valuc of the 3rd instar was only 1.15 timcs that of the 2ndonc. Also, the Ln50 for the 4lk instar was 38.65 and 24.95 times that of the 2nd and 3rd instars, respectively? while the LD50 vahie of the 3rd insur was only 1.55 times that of the 2nd one. The LC90 of the 4th instar ? v a 1.7 and 1.57 times that ofthe 2ndand 3rd instars, respectiveiy. Thc LD90 for ille 3rd instar was 1.09 times that the 2nd one. Analysis of dose - mortalit?- data indicate no difference in the slope of the regression lincs of the 2izcland 3rd instars, wliile a clear difference in that slope occurred betivxn the 2/?d and 4tl7 instars.

Thcre was an inverse relationship bctween the vins dose (or concentration) and the tinic rcquired for 25% mortality (LT25) and 50% mortality (LTjO). Results in Tables (5 & 6) indicate that the prolonged duratioil of larval instars due to GV infection, was more comnion with the lower infcctious doses.

Host range of S. littoidis GV :

Results of the host range study arc prcscnted in Table (7). A test insect was coiisidcrcd to be a host of SI GV, when the characteristic symptoms of GV disease were obscrved on the test larva, in addilion to the detection of virus in dead larvae. Rcsults in Table (7) show that, SI GV could infect only the 2 insect species bclonging to family Noctuidae, (S. litturdis and S. exiguo) using the high conccntra,ion 119 OD.

Total 83/91 I (31.21)

53/85 47/90 30/106 251102 0.039 1 0.8265 288.4 (92.35) (52.22) (28.58) I (24.51) I

Replicntc Yo Mortali@ at incIic;ted virus doses (OD) xo. 113 I 11.9 1.19 I 0.119 I 0.0119 1 28/34* 14/23 15/33 5 14 6 22

(45.45) 1 (32.56) 1 (27.27) 2 10 10 4 G 3 9 4 14 2 8

(100) 1 (66.67) (33.33) I (28.57) (25) 3 31/32 19/25 19Gï 14140 9 38 I (96.57) (70.37) (51.53) (35) (24) 4 28/26 13/30 10 29 7 22 (6120)

(96.15) (43.33) (34-48) (31.81) 30% Total 9W102 50/56 4ïilOS 31/90 23/58

(91.36) (53.94) I (37.87) (27.78) I (18.75)

(52.35) I (60.86)

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Estiniated LD values LD25 LDJO LD90

0.09 1.62 135.98

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Insect instar Tested dose (OD) . LT25 (in days) LT50 (in days)

2nd 5.9 x 10-2 20.53 32.27

I

5.9 x IO-' 18.78 2s. 1

5.9 x 10-3" 35.97 5.9 >I 10-1 10 15

5.9 10-3* 16.98

1.19 x lo-'* 17.12

3rd 5.9 x 10-2 14.71 23.1

4th 5.9 x 10-1 16 26.22

Insect instar Tested Cone. (OD) LT25 (in dags) LT50 (in days) 119 23.15 37.37

%Id 1.19 24.2 36.32

119 11.46 18.12 4th 1 11.9 13.02 21.46

0.0119* 18.85 -

0.0119" 19 *

parallcl to those of Crook and Brown (1982), and Siicppard and Stairs (1977). Based. on thc LC50 values, the 2nd .instar lnnzc wcre m r c susceptible to SI GV than tlic 4th oncs, but the LDSO values for thc 2nd and 3rd instars wcre similar. Thc LD5O for 3rd instar was 1.35 times. that ofthe 3ndoi1c, whilc tlic LD50 for 4th instar \vas 33.65 and 24.9 than &zit of thc 2nd and 3i-d inslars. rcspcctivcly. The LDjO €or the 217d and 3rd instars werc approsimaicly siii?iIar because in the bioassay tcsts, both the late 2nd and early 3rd insrar larvac were used. Howcvcr, a noliccnblc dilTercncc in the LDSO vaiues between tlic 3rd and 4/17 larsal instars were obsenrcd. These results agree with ~~IGSC of Easwarnmocrti~y and Jayaraj (1993), who reported îhat the LD50 values of the 3rd and 4fh instar larvae of Chilo scrcc~~nripì~n,~ts irdicm G'? were 533.3 and 2666.9 IBs/lana. rcspecti\.dy. In this rzspect, EInagar et al., (2953) wggested that youn'gcr lanrae, are likely to be the suitable target for virus treatmezi where less virus conccntrrtion is necdcd and faster mortality could be attained M o & serious darnagc occurs to tlic crop by older larvae.

In corr.i;arisoii with the hiVs. GVs arc gczcra!ly nicrc sclcctive viruscs, (Ignoffo. 1365, Payne et al., 1951 and Groner. 19S6). Obtaincd rcsiilts also dci:ionstratcd t!iat Si GV infected the larvae of the aitcrnativc liost S exigiiltn. this resili agrees x i th ihc findinss of SmitIi 2nd Rivers (!956): Rip et n/., 1973). Crook (1366). Zethner and Cgaard (1982) and Easnaramoorlh:; and Jtìyaraj (19S7j ivitk other GVs. Thc SI GV failed to cause' infection il: iixcct-spccics from ciifferent gcmn belonging to the sm~c family c - s ~ : A . ipsi'lm o r M Ioreji (Noctuidnc). In contrast, L. FU)>IOSWIIQ GV iirfects i n sxu from difCcient gcncrii ii? tlic sanic fsmiiy (Tortricidnc) (Eubcr i97S). €'!odia in~eipiictellrr aiid C n c h cnittelkc GV's h ~ c B cross transmission among their hosts (Hunter and EolFii1nll 1972). In most C ~ S C S in contrast to NTV-:lncíuced momiity? mostly in the Is1 2nd 2nd instars. GV-treatccd lanac did not die before tke last instar, so a remarkable cspnding in thc duration of infectcd lamal instars \tas clearly observcd. rvhilc thosc oc control had bce!i cincrgcd to xlults. Mortali& in lie tg GV never occurrcd bcforc the 4111 instar. but mostly just before p mm, 1932). Prcscnt rcsults on time mortality arc in harmony with' th . by Ea" (19SZ) ancl Bem (1963). The GV-infectcd larvae showed a chanse into a whitish or brownish coIour, particularly 011 thc ventral side, and sone of tlicsc 1;iwac wcrc Iargcr tllnn the 11GrilMl oncs. It is possible that, SI GV may represent an iniportant coinplemcntary agent alongside with S. littomfis NFW ín the IPM prograinnlc oftliis pest.

SUMiMFiRY

Standard bioassay studies on S. iittoralis ,".dosis virus (SI GV) wcre carried out in the laboratory to determine values of lhe lethal concentration (LC) and Ictkal dose. (LD) of virus for th rent instars of S Iitiom1i.s lame. The LC25, LC50 and LC90 values \ve&. ined for both tlic 2nd and 4th larval iiistar, tliesc values were 0.01.0.052 nd 0.036,0.831 tk 288.4 ODA.5 cm2

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29 -.

' of diet, rcspectively. The LDjO values wcrc determined for the 2nd, 3rd and 4th larval instar; as 5.2 s 8.1 s 10-3 and 2 s 1O-I OD/lanla. rcspectively. Mcdian lcthal time. incrcascci with decreased virus dose. and was longer for younger instars than older o m . The host range of 5'1 GV nas studied €or Spodoptero migita, Pectimpliorn gossypiclla, Gnllerin iiielonc!ln, ..lgsotis ipsiloit, Artogein rnpne, and A{vll;imi?n loreyi. Tnc S! GV infcctcd only S exigrm wvliich belongs to the sanie genus o€ thc honiolegous host.

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