ASSESSMENT OF THE APPLICATION OF BACULOVIRUSES FOR CONTROL OF LEPIDOPTERA

P1: ARS/ary P2: ARS/spd QC: ARS

October 15, 1998 9:54 Annual Reviews AR074-11

Annu. Rev. Entomol. 1999. 44:257–89Copyright c© 1999 by Annual Reviews. All rights reserved

ASSESSMENT OF THE APPLICATIONOF BACULOVIRUSES FOR CONTROLOF LEPIDOPTERA

Flavio MoscardiEmbrapa—National Soybean Research Center, C. postal 231, Londrina,PR 86001-970, Brazil; e-mail: [email protected]

KEY WORDS: insecta, biological control, nuclear polyhedrosis virus, granulosis virus

ABSTRACT

Baculoviruses, among other insect viruses, are regarded as safe and selectivebioinsecticides, restricted to invertebrates. They have been used worldwide againstmany insect pests, mainly Lepidoptera. Their application as microbial pesticides,however, has not met their potential to control pests in crops, forests, and pastures,with the exception of the nuclear polyhedrosis virus of the soybean caterpillar(Anticarsia gemmatalis), which is used on approximately 1 million ha annu-ally in Brazil. Problems that have limited expansion of baculovirus use includenarrow host range, slow killing speed, technical and economical difficulties forin vitro commercial production, timing of application based on frequent host pop-ulation monitoring, variability of field efficacy due to climatic conditions, andfarmers’ attitudes toward pest control, which have been based on application offast-killing chemical insecticides. Farmer education regarding use of biologicalinsecticides and their characteristics is considered one of the major actions nec-essary for increased use of baculoviruses. Strategies to counteract some of thelimitations of baculoviruses, especially their slow killing activity, have been in-vestigated and are promising. These include the use of chemical or biologicalsubstances added to virus formulations and genetic engineering of the virusesthemselves to express insect toxins or hormones. Such strategies can enhanceviral activity and increase speed of kill as well as reduce larval feeding activ-ity. The use of baculoviruses against Lepidoptera is reviewed, with the utiliza-tion of the nuclear polyhedrosis virus ofA. gemmatalisin Brazil serving as acase-study.

2570066-4170/99/0101-0257$08.00

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258 MOSCARDI

PERSPECTIVES AND OVERVIEW

It has long been known that entomopathogenic viruses can be used as mi-crobial insecticides in lieu of chemical insecticides (6, 6a, 29, 45, 46, 70, 73, 97,126, 178). Those of the family Baculoviridae have been isolated from more than700 invertebrates, including Lepidoptera, Hymenoptera, Diptera, Coleoptera,Neuroptera, Trichoptera, and Thysanura, as well in Crustaceae (120, 132, 160).Some of their characteristics, such as specificity and safeness to nontarget or-ganisms (63, 64), make them desirable agents in integrated pest managementprograms (9, 29, 70, 92, 93, 111a, 118). Until 1995, this family was subdividedinto two subfamilies: Eubaculovirinae, which included the occluded nuclearpolyhedrosis virus (NPV) and granulosis virus (GV); and Nudibaculovirinae,encompassing the nonoccluded baculoviruses (42). Currently, Baculoviridae isdivided into two genera:NucleopolyhedrovirusandGranulovirus(120). Viri-ons of NPV and GV, respectively, are occluded in polyhedral or capsular pro-teinaceous occlusion bodies (OB). NPVs have limited host ranges, usually be-ing restricted to one host species or genus, with the exception of the NPVs ofAutographa californica, Anagrapha falcifera, andMamestra brassicae(29, 63,69). GVs are more specific than NPVs, as they have been reported only fromLepidoptera (15, 61, 106). Larvae normally are infected by ingestion of OBs,although vertical transmission and injection by parasitoids may occur (66, 101).After ingestion, OBs are dissolved in the alkaline midgut contents, releasingthe virions that attach to and enter epithelial midgut cells, where they usuallyreplicate in the nuclei but do not form OBs. Replicated virions reach the basallaminae and apparently use the tracheal system as a major conduit to reachthe hemolymph and cause secondary infections in other tissues (34). OBs areformed in the nuclei of cells (NPVs) or in both nuclei and cytoplasm for someGVs (15, 61). During infection the host larva is debilitated, resulting in reduc-tion of development, feeding, and mobility and increasing exposure to preda-tion (181). Postlarval effects may include lower pupal and adult weights, aswell as reduced reproductive capacity and longevity (139). Infected defoliatinglarvae usually climb to the upper parts of the plants, dying in 5–8 days, althoughcessation of feeding may occur in 2–4 days, depending on biotic and abiotic fac-tors (35, 44, 113). Diseased and dead larvae serve as inoculum for virus trans-mission, which may occur by rain and movement of arthropods on plants, orvia predators and parasitoids (21, 44, 50, 54). Most of the attempts to use bac-uloviruses for insect control have been directed against Lepidoptera, althoughHymenoptera in forests (28, 29) are other examples of successful attempts ofbaculovirus use. Despite their potential, viral insecticides are employed muchless than they could be in crops and forests (29, 46). One exception is theuse of the velvetbean caterpillar,Anticarsia gemmatalisNPV, which has been

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LEPIDOPTERA CONTROL BY BACULOVIRUSES 259

applied extensively in soybean in Brazil (111, 111a). The reasons baculovirususe has limited expansion have been presented in several reviews (e.g. 19, 46,70, 72, 126, 180). The amount of literature on basic and applied aspects of bac-uloviruses is extensive and beyond the scope of this review. The biology andpathogenesis of baculoviruses have been reviewed and discussed in several pub-lications (e.g. 9, 15, 39, 61, 106), as have their diversity and molecular biology(e.g. 18, 106) and the factors that affect their prevalence in the environment(35, 44, 54, 101). Other articles have also focused on practical application ofbaculoviruses in agricultural and forest systems (29, 62, 70, 73, 111a, 118, 126,178). The development of recombinant baculoviruses for insect control hasbeen recently reviewed (20, 67, 105, 106). Also, a comprehensive book hasbeen published on invertebrate cell culture (96), including recent advances on invitro production of baculoviruses. This review emphasizes major examples ofLepidoptera control with baculoviruses, use strategies, problems that restrictuse, and approaches to counteract these limitations. NPV use in Brazil againstthe soybean (velvetbean) caterpillar is detailed as a case-study.

STRATEGIES FOR USE OF BACULOVIRUSIN PEST CONTROL

There are four basic strategies for using baculoviruses for insect control (46, 92,129).

Introduction and EstablishmentThe introduction and establishment of baculovirus in an environment is in-tended to result in permanent suppression of the target pest. However, only afew baculoviruses have been used in this approach (29, 46). There have beenat least 15 reported introductions of baculoviruses, 5 in crops and 10 in forests(45). However, there are controversies regarding the success of these attempts,especially of the methods utilized to evaluate their success (46). In the 1930s,a NPV of the spruce sawfly,Gilpinia hercyniae, was introduced accidentallyinto Canada along with parasitoids, which were imported from Scandinaviaand released for control of the pest (29). This NPV also was grown and ap-plied in selected locations to hasten the collapse of populations of this insect(7). This is the most important example of classical biological control of insectsby baculoviruses: No control measures have been required againstG. hercyniaein Canada for the last 50 years (29). A NPV ofPseudoplusia(Chrysodeixis)includensis possibly the best example of a baculovirus implemented as a clas-sical biocontrol agent in a row crop. This NPV was released on 200–250 haof soybean in Louisiana and provided control 12–15 years later (53; JR Fuxa,personal communication). Despite the value of this approach, it has not been

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260 MOSCARDI

explored to its potential against many insects (29). Its success seems to be re-lated to those viruses with efficient horizontal (insect to insect) and vertical(from generation to generation and year to year) transmission as well as to adiversified complex of natural enemies that interact with introduced viruses tomaintain host populations below damaging levels.

Seasonal ColonizationSeasonal colonization involves the inoculative release of baculoviruses to con-trol hosts for more than one generation, although subsequent releases arerequired when the pathogen population declines (46). It requires efficient repli-cation and transmission of the pathogen in host populations (35, 54). This strat-egy has been successful with the non-occluded virus of the rhinoceros beetle,Oryctes rhinoceros, in coconut palms (183) and with the velvetbean caterpillar,A. gemmatalis, NVP in soybean, among other examples (46).

Environmental ManipulationEnvironmental manipulation involves changing the host habitat to favor con-servation or augmentation of baculoviruses in a system where they either occurnaturally or have been introduced. Modified cultural practices enhance preva-lence of baculoviruses in insect populations, thereby reducing pest numbers,by aiding viral persistence or assisting its transport from the soil to the insect’sfeeding substrate and leading to viral epizootics (46). These practices includechanges in cultivation, grazing, sowing, and chemical use to aid persistenceand transport of baculoviruses. Examples include the control ofWiseanaspp.in New Zealand pastures (85) and changes in grazing schedule to improve nat-ural control ofSpodoptera frugiperdaby its NPV in Louisiana pastures (48).This approach may be promising, but it has not been used for many insects.This is probably because of the lack of in-depth research on factors relatedto baculovirus epizootics on host populations, including cultural practices andinteractions of the virus with other biological agents.

Microbial InsecticidesApplications of lethal doses of baculoviruses, as many times as needed, havebeen attempted for suppression of the host population, similar to the applicationof chemical insecticides (46, 111a, 129). Baculoviruses differ fundamentallyfrom chemicals, however, because they must be ingested to cause infection,thereby killing the insects more slowly than chemicals. Consequently, theymust be applied against early larval instars to avoid economic damage to plants,which requires frequent monitoring of pest populations. One of the key advan-tages of baculoviruses is that they replicate and persist in the environment andmay be able to maintain host populations below damaging levels with fewerapplications compared with chemicals (29, 46, 116–118). However, most viral

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insecticides are not as cost effective as chemical insecticides (29, 46), except insome developing countries where the cost of labor is low (117, 154).

USE OF BACULOVIRUSES AGAINST LEPIDOPTERA

One of the first attempts to control insects with a virus occurred in 1892, with theintroduction of a NPV into populations ofLymantria monachain pine forestsin Germany (70). In the United States, the first field trials withLymantriadispar(gypsy moth) NPV occurred in 1913 (29). Aerial applications of a NPVagainst the alfalfa caterpillar,Colias eurytheme, were attempted in Californiain the late 1940s (156). In the early 1950s, Californian farmers collected NPV-killed larvae ofC. eurythemeas an inoculum for application of the virus incrude preparations on alfalfa (162). This method was useful for control of thisinsect as well as other lepidopteran species in the United States up to the 1960s(36). In that decade, efforts to develop viruses as microbial insecticides wereintensified. These efforts culminated in the registration in the United States in1975 of the first viral insecticide (Elcar™, by Sandoz Inc.), used against thecotton bollworm,Helicoverpa zea(70, 74). It had a significant influence on thedevelopment and use of other baculoviruses worldwide (118). Several programsof baculovirus use (Table 1) are discussed by type of cropping system and insectgroup. This review is not exhaustive in terms of all programs available and thedetails of each program.

Use in Annual CropsTHE HELICOVERPA/HELIOTHIS COMPLEX The genera Helicoverpa andHeliothisare important on a global basis (29, 74), attacking over 60 crops rang-ing from maize and sorghum to various legumes such as chickpea and pigeonpea to tomatoes, sunflower, and cotton (23). The most important species areHeliothis virescensandHelicoverpa zeain the Americas;Helicoverpa armigerathroughout Australasia, Asia, and Africa;Helicoverpa punctigerain Australasiaand Southeast Asia; andHelicoverpa assultathroughout the Indian subconti-nent, central Asia, and the Middle East (23). Elcar™ (Sandoz Inc.), the NPV ofH. zea, primarily developed for the use in cotton and registered in the USA, in-fects all the majorHelicoverpa/Heliothisspecies and provides efficient controlin soybean, sorghum, maize, and tomato (74), as well as in chickpea and navybeans (161). Elcar failed commercially primarily because of the introductionof the synthetic pyrethroids at the time Elcar was creating a small niche in themarket (70); it also failed because of its slow speed of action and host speci-ficity (46). Sales of Elcar increased from 1975 to 1980, with a total estimatedtreated area of over 1 million ha during this period, but dropped substantiallyin 1981 (74). In 1982, Sandoz Agro Inc. discontinued production, but it can

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262 MOSCARDI

Tabl

e1

Exa

mpl

esof

bacu

lovi

ruse

sde

velo

ped

asm

icro

bial

inse

ctic

ides

toco

ntro

lLep

idop

tera

Hos

tins

ect

Bac

ulov

irus

aC

rops

Com

mer

cial

nam

eC

ount

ryR

efer

ence

s

Ado

xoph

yes

oran

aG

VA

pple

Cap

ex2

Switz

erla

nd29

Ado

xoph

yes

sp.

GV

Tea

—Ja

pan

121

Agr

otis

sege

tum

GV

Chi

na17

9A

nagr

apha

falc

ifer

aN

PVC

otto

n,ve

geta

bles

—U

SA29

,165

Ant

icar

sia

gem

mat

alis

NPV

Soyb

ean

Bac

ulov

iron

,B

razi

l11

6,11

8B

acul

ovir

usN

itral

,C

oope

rvir

us,P

rote

geA

utog

raph

aca

lifo

rnic

abN

PVC

abba

ge,c

otto

n,V

PN80

Gua

tem

ala

29,3

1or

nam

enta

lsB

uzur

asu

ppre

ssar

iaN

PVTe

a,tu

ngoi

ltre

e—

Chi

na17

9C

ydia

pom

onel

laG

VA

pple

,pea

rsC

arpo

viru

sine

Fran

ce79

,71

CY

D-X

USA

Gra

nusa

lG

erm

any

71V

irin

-GyA

pR

ussi

a41

Eri

nnyi

sel

loG

VC

assa

va—

Bra

zil

140

Ven

ezue

la15

2H

elic

over

paze

aN

PVC

otto

nE

lcar

USA

74G

emSt

arU

SAH

elio

this

vire

scen

sN

PVC

otto

nE

lcar

USA

74G

emSt

arU

SAH

elic

over

paar

mig

era

NPV

Cot

ton,

tom

ato

—C

hina

179,

184

Vir

in-H

SR

ussi

a41

Hom

ona

mag

nani

ma

GV

Tea

—Ja

pan

121

Hyp

antr

eacu

nea

NPV

Fore

st,m

ulbe

rry

Vir

in-A

BB

Rus

sia

41

b

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Lym

antr

iadi

spar

NPV

Fore

sts

Gyp

chek

USA

29,1

35D

ispa

rvir

usC

anad

a29

Vir

in-E

NSH

Rus

sia

41C

hina

179

Mam

estr

abr

assi

cae

NPV

Cab

bage

Mam

estr

inFr

ance

29V

irin

-EK

SR

ussi

a41

Org

yia

pseu

dots

ugat

aN

PVFo

rest

sT

MB

ioco

ntro

l1U

SA70

Vir

tuss

Can

ada

29P

htho

rim

aea

oper

cule

lla

GV

Fiel

dan

dst

ored

PTM

bacu

lovi

rus

Peru

3po

tato

esE

gypt

152

Tun

isia

152

Pie

ris

rapa

eG

VC

abba

geC

hina

179

Plo

dia

inte

rpun

ctel

laG

VSt

ored

alm

onds

—U

SA29

and

rais

ins

Plu

tell

axy

lost

ella

GV

Cab

bage

Chi

na17

9Sp

odop

tera

exig

uaN

PVO

rnam

enta

lsSP

OD

-XU

SA17

2an

dve

geta

bles

Shal

lot,

gard

enpe

a,T

haila

nd17

2gr

ape,

Chi

nese

kale

Flow

ers,

orna

men

tals

The

Net

herl

ands

172

Spod

opte

rafr

ugip

erda

NPV

Mai

ze—

Bra

zil

170

Spod

opte

rali

ttor

alli

sN

PVC

otto

nSp

odop

teri

nA

fric

a29

Spod

opte

rali

tura

NPV

Veg

etab

les,

cotto

n,C

hina

179

rice

,pea

nuts

Spod

opte

rasu

nia

NPV

Veg

etab

les

VPN

82G

uate

mal

a29

a GV

,Gra

nulo

viru

s;N

PV,n

ucle

arpo

lyhe

dros

isvi

rus.

bT

hese

NPV

sha

vebe

ende

velo

ped

prim

arily

for

use

agai

nst

lepi

dopt

eran

spec

ies

othe

rth

anor

igin

alho

sts

beca

use

ofth

eir

wid

eho

stra

nge

(see

text

).

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264 MOSCARDI

reintroduce Elcar into the market as cases of host resistance to pyrethroids in-crease worldwide (23, 29, 59, 70). In 1996, Biosys introduced GemStar™ LC,a liquid concentrated formulation ofH. zeaNPV, for control ofH. zeaandH. virescensin US cotton. In 1997, biopesticide assets of Biosys were soldto Thermo Trilogy Corporation, which is continuing sales and is investigatingthe introduction of GemStar in Australia for use in cotton (MB Dimock, per-sonal communication). In Australia, Elcar has been registered for the controlof H. armigeraand has been tested in certain crops, but it currently is un-available commercially (161). The first virus commercially developed in thePeople’s Republic of China (PR China) was aH. zeaNPV isolate from theUnited States provided to Chinese institutions in the late 1970s (CM Ignoffo,personal communication). Currently, either this isolate or an indigenous isolateof H. armigeraNPV has been produced onH. armigeralarvae reared on arti-ficial diet and formulated as a wettable powder or as an emulsion. This virus hasbeen applied to aproximately 100,000 ha annually in PR China (172), mainly tocontrolH. armigeraandH. assultaon cotton, but also on tobacco, cayenne pep-per, and tomato (184). Isolates ofH. armigeraNPV have also been producedand used in Thailand and Vietnam (31).

THE SPODOPTERA COMPLEX Several species in the genusSpodoptera—includingS. frugiperda, S. exigua, S. littoralis, andS. litura—are also importanton many crops. On several plants, such as maize, rice, wheat, vegetable crops,and pastures, the most important one in the Americas isS. frugiperda. In Brazil,an indigenous isolate ofS. frugiperdaNPV (SfNPV) has been used to controlthe insect on maize (170). It is produced inS. frugiperdalarvae reared on anartificial diet and is processed as a wettable powder to be applied at 2.5× 1011

OB/ha against first to second instar larvae. The SfNPV has been used in ap-proximately 5,000–10,000 ha annually up to 1995 and in approximately 20,000ha annually thereafter. The most important limitation of this program is the costand difficulty of producing the virus, as the insect is cannibalistic and mustbe reared individually on an artificial diet (F Valicente, personal communica-tion). NPVs also have been developed and used againstS. litura (China, India,Taiwan),S. littoralis (Egypt),S. exigua(United States, Guatemala, Thailand),andSpodoptera sunia(Guatemala). In Thailand, aS. exiguaNPV is produced inthe laboratory to be distributed to farmers for field production of the virus (31).A S. exiguaNPV was developed in the United States through collaborationbetween Crop Genetics International (CGI) and DuPont from 1992 to 1994. In1994, CGI began selling SPOD-X™ for control of the insect on greenhouse-grown cut flowers and ornamental plants in The Netherlands. In 1995, CGImerged with Biosys, and SPOD-X was introduced in the United States for con-trol of S. exiguaon cotton and vegetable crops. As Biosys assets were bought

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by Thermo Trilogy Corporation in 1997, this company is currently marketingSPOD-X (MB Dimock, personal communication). In Guatemala, Agricola ElSol commercializes aS. suniaNPV (VPN 82) for control of bothS. suniaandS. exiguaon vegetables, which is used on approximately 3000 ha annually (REstrada, personal communication). This company also produces another for-mulation (VPN 80), based on the NPV of the alfalfa looper,A. californica, andis intended for control ofS. exiguaandTrichoplusia ni(29). Formulations of theNPV of the cotton leafworm,S. littoralis, have been developed (29, 104). Cur-rently, Spodopterin® is produced by Natural Plant Protection at approximately100 ha equivalents/year for field trials (M Guillon, personal communication).In PR China, a NPV ofS. liturawas successfully applied to control this insectin vegetables, cotton, rice, and peanuts (179).

OTHER INSECTS IN ANNUAL CROPS Another important group of Lepidopterain several crops is the semi loopers (Noctuidae: Plusiinae), includingT. ni,Rachiplusia nu, A. californica, C. includens, Chrysodeixis chalcites, andThy-sanoplusia orichalcea. A NPV of T. orichalceaehas been produced and used inZimbawe (F Hunter, personal communication). Partially purified suspensionsare formulated by spray drier with 6% lactose. The distribution of the formula-tion is made at 1 g per farmer, who dilutes it in 100 ml of water. Soybean leafletsare immersed in this suspension and used to feed third instar larvae. Contami-nated dead larvae are distributed in the field at the first sight of larval infestationto initiate epizootics in host populations. Approximately 10,000–15,000 ha havebeen treated annually with this NPV (29). In the 1950s, NPV-killed caterpillarsof the cabbage looper,T. ni, were collected by farmers in California as an in-oculum to spray the virus on cotton, potato, Brassicae, and other crops (36). Ifa virus is considered for the control of cabbage looper, it will probably be theA. californicaNPV or theA. falciferaNPV (AfNPV), because both have fieldefficacy against other insect pests (29, 69). The AfNPV was patented by the USDepartment of Agriculture (USDA), and a license for the patent was awardedto Sandoz Ag and then developed by Biosys (29; PV Vail, personal commu-nication). BesidesH. zeaandH. virescens, AfNPV showed cross infectivityto other cotton and vegetable pests, such as the pink bollworm,Pectinophoragossypiella, S. exigua, andT. ni, providing a stimulus to develop this virus asa microbial insecticide (165). Later studies (166) demonstrated that the raisinmoth,Cadra figurella, navel orangeworm,Amyelois transitella, codling moth,Cydia pomonella, and Indian meal moth,Plodia interpunctella, were suscep-tible to this virus. AfNPV also may be more efficient thanB. thuringiensisagainst the corn earworm,H. zea(128). A GV of the cabbage worm,Pierisrapae, has been mass produced in PR China as PrGV since 1978 and applied inmany regions, totaling 100,000 ha (179). GVs ofPlutella xylostella(cabbage)

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andAgrotis segetumhave been used in large areas of central and northwestPR China, respectively (179). A GV of the cassava hornworm,Erinnyis ello,has been used in Brazil, and its use was first implemented in Santa Catarina(a state of Brazil), based on replication of the GV in the laboratory or in cas-sava fields (140). This GV also is produced in the field in the state of Parana(S Torrencillas, personal communication). It has been used in crude prepara-tions (10 larval equivalents/ha), providing good control of the pest with onlyone application during the season. Its use reached around 20,000 ha in 1987,and currently it is applied on 5000–8000 ha in the south, but it is also used incommercial cassava plantations in the northeast. This GV also has been testedin Colombia (11) and is being used on large cassava plantations in Venezuela,with nearly 100% reduction in insecticide use (152). A NPV ofM. brassicae,a pest of cabbage, has been registered in France by National Plant Protection,under the name Mamestrin®. This NPV is also effective against other impor-tant hosts such asH. zea, H. virescens, H. armigera, S. exigua, and the pinebeauty moth,Panolis flammea(29, 63). In 1997, Mamestrin was produced atapproximately 50 ha equivalents/day, and a quantity sufficient to treat approx-imately 2000 ha was sold (M Guillon, personal communication). A NPV ofM. brassicae(Virin EKS) has also been used in Russia (41).

The potato tuber moth (PTM),Phthorimaea operculella, is an importantpest of potatoes worldwide. Its larvae damage plant shoots, bore into stems,mine leaves, and attack tubers in the field and in storehouses (134). A GVisolated from PTM has been developed as a microbial insecticide in Peru bythe International Potato Center (CIP). Studies with this GV have shown highefficacy in protecting the potato crop in the field or potato tubers under storage(3, 134). This virus is currently produced at CIP by infecting PTM larvae rearedon potato tubers. The formulated product is widely used in Colombia, Ecuador,Peru, and Bolivia. It is also being produced in the Middle East and used incountries such as Tunisia and Egypt (152).

Use in Fruit CropsThe codling moth,C. pomonella, is a worldwide key pest of apples, pears, andwalnuts. Control is achieved by frequent applications of chemical insecticidesbecause penetration of fruit by a single larva usually makes it unacceptable formarket (70, 71, 138). A GV isolated fromC. pomonella(CpGV) was highly vir-ulent to the insect and killed it rapidly, protecting fruit from economic damage,in numerous field trials in several countries (37, 38, 70, 81). The developmentof this GV has been reviewed previously (29, 37, 70, 71). Currently, commer-cial formulations of CpGV are available in France (Carpovirusine®, by NaturalPlant Protection), in Switzerland (Madex®, by Andermatt Biocontrol AG), inGermany (Granusal®, by Behringwerke AG) (71), and in Russia (Virin-CyAP)

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(41). Registration of Carpovirusine® has also been granted in Belgium, TheNetherlands, Switzerland, Spain, Poland, Hungary, and Argentina. NaturalPlant Protection produced approximately 400 ha equivalents/day, and it hasbeen sold to treat 10,000 ha in 1996, 30,000 ha in 1997, and possibly as manyas 60,000 ha in 1998 (M Guillon, personal communication). Apparently Natu-ral Plant Protection succeeded in producing a cost-competitive product thatprovides good protection against the pest (14). Potential for use of this GV ishigh because of the importance ofC. pomonellaworldwide, but it has beenused on a small scale. The fact that various companies have registered thisvirus in many countries is important because as demand for the GV increases,these companies will be able to respond and make it available on a larger scale.A GV for the control of summer fruit tortrix,Adoxophyes orana, is sold byAndermatt Biocontrol in Switzerland under the name Capex® 2 (29). A GVof the Indian meal moth,P. interpunctella, has potential to control this insectin stored in-shell nuts and in raisins (167, 169). A formulation of this GV waspatented by the USDA and is being registered by the company John Evans (29).

Use in Tea CropsParaguay tea (Ilex paraguariensis) is an important crop in northeasternArgentina, and the hornwormPerigonia luscais one of its key pests. A NPV ofthis insect was found in 1988, and in 1992 approximately 900 ha were treatedin preliminary trials on farms in the Province of Corrientes (153). The viruswas applied aerially using the hemolymph of 15 infected last instar larvae perhectare. A large amount of the virus was obtained by collecting larvae in thesetreated fields and storing them frozen until the subsequent season, when thetreated area reached 2362 ha. In Japan, two pests of tea, the smaller tea tortrix(Adoxophyessp.) and the oriental tea tortrix (Homona magnanima), are beingcontrolled by their GVs in Kagoshima county (121). From 1990 to 1993, five GVproduction facilities were established in Japan and were operated at tea grow-ers’ expense. The treated area increased from 460 ha (6.1% of tea area) in 1991to 3806 ha (50.2%) in 1993, 4880 ha (64.4%) in 1994, and 5850 ha (77.2%) in1995. Over 80% control has been achieved at 1000 larval equivalents of the GVsper ha in 2000 liters of water/ha. A NPV ofBuzura suppressariahas been usedin China to control this insect on tea and tung oil trees in over 20,000 ha (179).

Use in ForestsForest Lepidoptera subjected to applications of baculoviruses include the gypsymoth, L. dispar; the Douglas-fir tussock moth,Orgyia pseudotsugata; thespruce budworm,Choristoneura fumiferana; the western spruce budworm,Choristoneura occidentalis; the jackpine budworm,Choristoneura pinus; thepine beauty moth,P. flammea(29, 70); and the fall webworm,Hyphantrea

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cunea(41). A NPV ofO. pseudotsugatawas registered in the United States in1976 (TM BioControl-1) and was produced by private companies under a con-tract with the USDA (70). TM BioControl-1 and Virtuss, developed in Canada,were registered in 1983 by the Canadian Forest Service (29). Between 1971and 1982, 1508 ha were treated with TM Biocontrol-1 in the United Statesand Canada, and between 1974 and 1982, 165 ha were treated with Virtussin Canada. This virus was used on approximately 200, 900, and 650 ha in1991, 1992, and 1993, respectively, in Canada. It is effective againstO. pseu-dotsugata(123), but because of the cyclic nature of the host, with long spellsbetween outbreaks, private industries have not shown interest in production andcommercialization of this NPV (29). The NPV of the gypsy moth,L. dispar,has been developed in the United States, Canada, certain European countries(29, 135), China (179), and Russia (31, 41). Efforts by the USDA to explore itsfeasibility as a microbial insecticide began in the late 1950s (135). Registrationof Gypchek was granted in 1978 in the United States to the USDA Forest Ser-vice, based on spray trials in Pennsylvania between 1973 and 1978. Up to 1987,recommendation for Gypchek use was based on two applications of 2.5× 1011

POB/ha to two applications of 1.25× 1012 PIB/ha, 7 to 10 days apart, againstsecond instar larvae, in a volume of 18.8 liters/ha and a tank mix containingmolasses, a sunscreening agent, and a sticker (29, 135). This product is efficientagainst the insect (130) and is continuously being improved (e.g. processing ofNPV-killed larvae and formulation), and recommendation of two applications3 days apart has delivered more active NPV to the insect in a 5- to 6-day period(see details in 135). From its registration in 1978 through July 1996, Gypchekwas used on approximately 19,200 ha in the United States and approximately460 ha in Canada (RC Reardon, J Podgwaite, personal communication). Themain impeding factor for expansion of its use is the lack of a commercial source(29). In 1990, a product developed in Canada, Disparvirus, was submitted forregistration, which was granted in 1997 (JC Cunningham, personal commu-nication). Between 1982 and 1983, 1240 ha were treated experimentally witheither Gypchek or Disparvirus in Ontario (29). No viruses were applied onCanadian forests against Lepidoptera in 1995 and 1996 (JC Cunningham, per-sonal communication). The development of the NPV of the spruce budworm,C.fumiferana(CfNPV), is of interest in eastern Canada because of the importanceof the pest. Several trials were conducted over an area of 2140 ha at dosagesranging from 2.5× 1010 OB/ha to a double application of 3.4× 1012 OB/hafollowed by 2.3× 1012 OB/ha. At an average dosage of 7.5× 1011 OB/ha,there was adequate population reduction in some tests but negligible foliageprotection (29). The most important problem with CfNPV is that applicationshave to be made against fourth instar larvae at budflush, when they are exposedto the virus. Sprays against needle-mining second instars are not effective, as

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there is no time for horizontal transmission of the virus, and its prevalence de-creases annually because there is limited vertical transmission (29). This virusalso infectsC. occidentalisandC. pinus. Trials between 1976 and 1982 withCfNPV for C. occidentaliscontrol, and in 1985 forC. pinus, were disappoint-ing because virus epizootics did not terminate budworm outbreaks and attemptsto initiate epizootics were not successful (124; JC Cunningham, BL Cadogan,unpublished data). In Scotland, a NPV of the pine beauty moth,P. flammea,was produced inM. brassicaebecause of problems in rearingP. flammeaonan artificial diet. Around 905 ha were sprayed against the insect between 1986and 1988 in Scotland, at 2.2× 1011 OB/ha (29). As this species is also suscep-tible to M. brassicaeNPV, and Mamestrin® is currently produced in France,this NPV can be used to controlP. flammeaon lodgepole pine. Virin EKS, aM. brassicaeNPV produced in Russia, was used on the Isle of Lewis in 1992(29).

USE OFA. GEMMATALISNPV IN SOYBEANIN BRAZIL: A CASE-STUDY

In Brazil,A. gemmatalisand stink bug species (Heteroptera: Pentatomidae) arefrequent and abundant in soybean, demanding frequent insecticide applications(111). With the establishment of an integrated pest management (IPM) programin Brazil in the mid-1970s (58, 88, 111), the number of insecticide applicationson the crop fell from an average of approximately six to approximately two inIPM-assisted areas in a 4-year period. Use of insecticides againstA. gemmatalishas progressively changed to more selective chemicals, such as insect growthregulators (IGRs) and biologicals, like the NPV ofA. gemmatalis(AgNPV), andto a lesser extentB. thuringiensis. The AgNPV occurs naturally in Brazil inA. gemmatalispopulations with pathogenesis similar to other NPVs (4, 22).Initial experiments with the AgNPV demonstrated its potential to control theinsect (22, 112). Its development and implementation as a microbial insecticidewere carried out by Embrapa (Brazilian Organization for Agricultural Research)(see below).

Development and Implementation of the ProgramA pilot program for AgNPV use was conducted during the 1980/1981 and1981/1982 seasons, on 21 farms in the Brazilian states of Parana and RioGrande do Sul, comparing AgNPV, insecticide, and untreated 1-ha paired plots(110, 114, 116). In all AgNPV plots, reductions of over 80% ofA. gemmatalislarval populations were attained and yields were not significantly different frominsecticide-treated plots, whereas yields in untreated plots were greatly re-duced. AgNPV field tests for more than 20 years (22, 43, 110, 112, 116, 148)

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270 MOSCARDI

have demonstrated its value as a biological insecticide. Implementation of theAgNPV began in the 1982/1983 season, when approximately 2000 ha of soy-bean were treated. Initially, small amounts of AgNPV were produced in hostlarvae reared on artificial diet (110). Frozen killed larvae were distributed toextension officers for treatment of demonstration plots and virus productionin the field, which in turn provided inoculum to treat other areas in the sameseason or to collect and store as dead larvae frozen for use in the subsequentseason. Applications were made at 50 larval equivalents/ha (approximately 1.5× 1011 OB/ha), when most larvae were in the first three instars and there werefewer than 20 larvae/m of row (110). Approximately 20,000 ha were treatedin the 1983/1984 season. Use increased substantially in the 1984/1985 season(approximately 200,000 ha) after the implementation of regional NPV produc-tion units (110). The treated area increased to 500,000 ha in the 1986/1987season, to 700,000 ha in 1988/1989, and to 1 million ha in 1989/1990, re-maining approximately the same through the 1996/1997 season (116–118).In the 1997/1998 season, AgNPV use reached approximately 1.2 million ha(F Moscardi, unpublished data). Until 1985, the AgNPV was used as crudepreparations. Afterwards, a kaolin-based wettable powder formulation has beenused predominantly (110). In 1991, after a contractual agreement with Embrapa,five private companies began commercialization of the AgNPV (116–118).The AgNPV also is produced in the field in Paraguay by the farmer coopera-tive Colonias Unidas, at Obligado, with annual application to approximately50,000 ha (154). Additionally, virus produced in Brazil by Nitral and Geratecalso has been used annually on approximately 100,000 ha in Paraguay. It wasused on approximately 3500 ha in Northern Argentina (118), but currentlyAgNPV use in this country is negligible. Introduction-establishment of AgNPVwas attempted in the United States, where AgNPV does not occur naturally(49) and the agricultural situation is not favorable for a microbial insecticideapproach (market size and costs). AgNPV spread 1 m/day after application (50)and survived soil manipulations and overwintering (51) to initiate new infec-tions up to 3 years after release. However, lack of host insects or rainfall causedit to die out.

Laboratory Versus Field ProductionDifferent methods of AgNPV production, in laboratory, in field screen cages,and in farmer’s fields, have been developed (110). Laboratory production wasadopted by one of the companies (Geratec) that commercialize the NPV, but itwas discontinued because of high costs (e.g. labor, equipment, and diet) (115).Production in screened soybeans has not been adopted by private industriesbecause it involved the establishment of an insect rearing facility and NPVyields were not sufficient to satisfy a large and increasing demand. Currently,

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production in farmer’s fields is the only method utilized by private companiescommercializing the AgNPV. This method has been improved to allow highvirus yields at a low cost. It involves virus application on soybean infested withA. gemmatalislarvae, collection of the dead larvae, and storage in large roomsat−4 to−8◦C until processed as a formulation. Formulated batches undergoquality control at Embrapa, based on OB per gram and biological activity onA. gemmatalislarvae compared with an AgNPV standard. For registration,the product underwent a series of tests required by the Brazilian legislature(116). In the 1996/1997 season, conditions were favorable for field produc-tion, and approximately 35 metric tons of AgNPV-dead larvae were collected,which correspond to approximately 1,750,000 ha equivalents of AgNPV. Pro-duction at the field level is currently at an average of 1.8 kg (approximately100 ha equivalents) of AgNPV-killed larvae/person/day, at a mean cost of US$15.00/person/day. Cost of the formulated product is about US $0.70/ha, andit reaches the farmer at a mean cost of US $1.20–$1.50/ha, which is lowerthan the cost of chemical insecticides. Despite its advantages, AgNPV yieldsin each soybean season are dependent on abiotic and biotic factors that affecthost abundance (115), resulting in production levels ranging from 650,000 to1,750,000 ha equivalents in the last 7 years. Therefore, laboratory productionwould be important in complementing AgNPV field production in seasons withlower insect numbers. Recent research in laboratory production indicates thatAgNPV can be produced with higher efficacy and lower costs (115).

The Reasons for the Success of AgNPV Use in BrazilAgNPV is highly virulent to host larvae at a low dose (1.5× 1011 OB/ha),and it is efficiently transmitted in the host population by natural enemies andabiotic factors (21, 50, 110). As a result, usually one AgNPV application is suf-ficient to control the insect, compared with a mean of 1.8 chemical applications(L Morales, unpublished data). As a leaf feeder,A. gemmatalisis exposed toapplied AgNPV, and usually no other key insects occur simultaneously on thecrop (111, 111a). Also, soybean tolerates considerable defoliation with no re-duction in yield (88). The implementation of a soybean IPM program in Brazilin the mid-1970s (58, 88, 111) facilitated farmer acceptance of the AgNPV. Theofficial extension services and the strategies developed for farmer education re-garding AgNPV use were also important in the implementation of the program,which has been most successful where the extension services are proactive.The amount of AgNPV used in Parana corresponds to 50% (approximately500,000 ha) of the total application in Brazil and to approximately 21.0% ofthe total insecticide applications used to controlA. gemmatalisin this state(L Morales, unpublished data).

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272 MOSCARDI

Problems That Limit the Expansion of AgNPV UseAlthough the current annual AgNPV-treated area is large, compared with viralinsecticide programs worldwide, the estimated demand for its use in Brazil is forapproximately 4.0 million ha (approximately 35% of soybean area). Therefore,current field and laboratory production need to be improved without significantincrease in cost of the final product. AgNPV speed of kill is also an importantlimitation. Usually, farmers initially react negatively to this characteristic, andfrequently they are not prepared for a more extensive scouting of larval popu-lations to time viral application against early instars and at specified populationintensities (117, 118). The cases of unsuccessful use of the AgNPV are mostlyrelated to inappropriate timing of application, to use in regions with low meantemperatures (around 20◦C) where it takes longer to kill the insects (84), and toyears of extended drought periods (F Moscardi, unpublished data). The latter as-pect seems to affect viral efficacy by its rapid inactivation due to solar radiation(80), reduced dissemination of the pathogen, slower soybean development, andquicker insect outbreaks. These factors demand considerable efforts for farmereducation regarding AgNPV use. Since implementation of the program, farmereducation has been the major factor related to the increase in AgNPV use,through a coordinated effort between research and extension organizations. Inregions where this effort is weak, the use of the AgNPV has been negligible.

Possibility ofA. gemmatalisPopulations DevelopingResistance to AgNPVNo cases of resistance to baculoviruses used as microbial insecticides have beenreported in the field, but these agents have not been used extensively or fre-quently enough applied to allow an adequate evaluation of this phenomenon(47). On the other hand, examples of development of resistance to baculovirusesby host populations in the laboratory, through selection pressure trials, have be-come frequent (47). Initial studies with the AgNPV comprised bioassays withA. gemmatalispopulations collected from different regions of Brazil and theUnited States, with different histories of exposure (from 0 to 8 years) to AgNPVas a microbial insecticide. These Brazilian populations were equally suscepti-ble to the virus (1), and two US populations (Texas and Louisiana) also did notdiffer in their susceptibility to the AgNPV. In another study, two populationsfrom Brazil and one from the United States were submitted to selection pressureby the same AgNPV isolate in the laboratory and compared with respective non-selected populations (2). In the Brazilian colonies ofA. gemmatalis, resistancecould be detected in the F4 generation and increased to a resistance ratio (RR) ofover 2000 in F15, the highest reported in studies with baculoviruses (47). TheRR for a population in Louisiana (United States) reached a plateau at a much

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lower level (fivefold), probably because of little exposure ofA. gemmatalistoAgNPV in the United States, where it does not occur naturally (49). After 16generations of selection pressure in one of the Brazilian populations (Serta-nopolis), the RR were 3000 times higher. When this population was releasedfrom AgNPV pressure, resistance decreased substantially in 11 generations(F Moscardi, AR Abot, DR Sosa-Gómez, unpublished data). When exposureto AgNPV was discontinued in the US population, the resistant insects returnedto their original level of susceptibility in three generations (52). Therefore, re-sistant populations ofA. gemmatalisfrom Brazil and the United States differsubstantially in their response to the discontinuation of exposure to the AgNPV.Through backcrossing highly resistant Brazilian populations ofA. gemmataliswith the respective nonselected populations, resistance was lost in four gener-ations (F Moscardi, AR Abot, DR Sosa-Gómez, unpublished data). Thus, oneof the major factors retarding the development of resistance in the field mightbe the migration of adults from soybean areas not exposed to applications ofAgNPV to areas where the pathogen has been extensively applied.

Possibilities of Changes in AgNPV Genome and VirulenceWhen a program of baculovirus use at the farmer level is begun, it has been rec-ommended (141) that a large amount of an initial virus inoculum be producedto allow storage of aliquots of the original isolate for subsequent large-scaleproductions. This procedure would avoid loss of virulence of the pathogen dueto its sequential passages through the host by selection of less-virulent variants.This was possible in the beginning of the AgNPV program, as the amount ofvirus produced was not high. However, as the program progressed, this wasnot possible because of the large amounts of virus required for AgNPV fieldproduction. Currently, about 30,000 ha of soybean are treated each year inBrazil for AgNPV production. Therefore, AgNPV has been produced in thefield from inoculum multiplied in the previous season. Analysis of AgNPVisolates showed that the virus has varied in certain locations of the genome, in-dicating that the AgNPV has changed genetically in relation to the original wildisolate (56, 99, 100). Recombination among isolates of AgNPV may explainthe high variability of wild isolates of this virus (27). Genomic analysis of Ag-NPV variants obtained from field multiplication during many years indicatedthat this virus has maintained considerable stability (S Araujo, MEB Castro,ML Souza, unpublished data). Bioassays or field tests with batches of AgNPV,obtained each year from field-collected larvae from 1979 to 1995, showed thatits virulence to the host was not altered significantly (8, 13). Although changesin virulence have not been detected, monitoring virus genomic changes andwhat they represent in practice will be important for the continuing success ofthe AgNPV program.

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274 MOSCARDI

FACTORS THAT INFLUENCE FIELD EFFICACYOF BACULOVIRUSES

Inoculum Potency, Application Timing, and Plant SubstrateThe genetic variability among baculoviruses isolates may result in great differ-ences in virulence to a given host. Up to 2900-fold differences in activity wereobserved among several geographical isolates ofL. disparNPV (142). Selectionof the most potent isolates has been an important initial step in implementa-tion of programs of baculovirus use, followed by determination of the adequatedosage for host control. Dosage determination involves years of field researchin different ecological regions and under various conditions of insect infestationin different plant stages (110, 111a, 114, 118). If adequate steps are not takenfor each virus-insect-plant system, including proper protocols for quality con-trol of produced virus batches, the dosage may be unnecessarily high, resultingin a high cost for the final product, or the dosage may be lower than necessary,leading to variable host control. One of the AgNPV products (Multigen®) wasregistered by Agrogen in 1989 to controlA. gemmatalisin Brazil. However, thisproduct failed in the field, probably because of inadequate dosage determina-tion and quality control of the liquid NPV formulation, as it was produced in anunnatural host, the sugarcane borer,Diatraea saccharalis(116, 154). Timingof applications at appropriate host age structure and density is also a key featurefor successful use of baculoviruses, because susceptibility to baculoviruses isgreatly reduced as larvae mature (54, 74, 116), and insects infected in late instarscause economic damage to host plants. Insects of cryptic habits (borers, miners,etc) usually are difficult to control in the field because of the low probabilityof larvae ingesting a lethal dose of the pathogen before boring or mining intoplant parts. For these insects, applications must be directed against the first twoinstars, when they are still feeding externally on plant parts, as is the case ofHelicoverpa/Heliothisin cotton and maize (74),C. pomonellain apple and pears(138),D. saccharalisin sugarcane (5), and the spruce budworm (29, 30). Thus,monitoring adult populations and egg counts are usually needed to direct appli-cations early enough to prevent economic damage (37, 81, 138). Plants that, byleaf distribution and branch architecture, allow less solar radiation penetrationusually provide longer persistence of baculoviruses, while virus deposits on pe-ripheral leaves are less protected against solar radiation (80). For instance, theH. zeaNPV deposited in the calix, bracts, flowers, or lower leaf surface ofcotton resulted in higher NPV persistence (up to 10-fold) than NPV depositedon the upper surface of terminal leaves (74). Substances present in plants mayalso inactivate baculoviruses. The effects of plant substrates on baculoviruseshave been attributed to insect stress on certain host plants or to antimicrobial

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substances (e.g. tanins, rutin, chlorogenic acid) (40, 86, 87, 127, 133). Theseeffects have been studied mainly in the laboratory, but there is a need for morestudies in the field to evaluate their extent on baculoviruses, especially thosewith wider host ranges or those that can be used on different host plants of agiven insect species or complex.

Solar Radiation and TemperatureSolar radiation is the major factor affecting field persistence of baculoviruses.Viral activity can be completely lost in less than 24 h, but mean half-life gener-ally has varied from 2 to 5 days (80). Ultraviolet radiation in region B (UV-B)(280–310 nm) inactivates baculoviruses; however, the UV-A (320–400 nm)may also be critical in baculovirus deactivation (142). Among other factors(75), sunlight generation of highly active radicals (e.g. peroxides, singlet oxy-gen, hydroxyls) may be involved in degradation of baculoviruses (76). Manysubstances have been tested as sunscreening agents to extend viral activityin the field (80, 142). The ones that provided the best protection against UVwere uric acid, p-aminobenzoic acid, 2-hydroxy-4-methoxy-benzophenone, 2-phenylbenzimidazole-5-sulfonic acid, folic acid, and Tinopal DCS. Among79 dyes, lissamine green, acridine yellow, brilliant yellow, alkali blue, andmercuriocrome provided effective protection against UV, whereas Congo Redprovided total protection to the NPV ofL. dispar. Those considered effectiveshowed higher capacity to absorb UV-A than the less-effective dyes. Recently,research with fluorescent brighteners of the stilbene group showed that theyare efficient protectants of baculoviruses against solar radiation because theyabsorb UV-A and UV-B (142). Extended activity in the field is desirable inbaculovirus formulations. However, increased protection may not result in sub-stantial returns, especially if it implies adding significant costs to the finalproduct (46). In some systems, despite the rapid deactivation of applied bac-ulovirus, high larval mortality results in a subsequent great load of virus in thepest environment. When this effect is added to efficient horizontal transmis-sion, season-long control of the insect can result, especially when the host planttolerates high damage levels (117, 118). Temperature effects on baculovirusesare not as important as solar radiation (80), but they can affect the success ofapplied viruses by increasing the lethal time of the virus in regions with lowmean temperatures, and by inhibiting the infection at low or high temperatures(12). Inhibition of viral replication has been demonstrated in some insects, suchas the GV ofP. rapae(36◦C), the NPV ofT. ni (39◦C) (158), the NPV ofH. zea(40◦C) (74), and the NPV ofA. gemmatalis(10◦ and 40◦C) (84). In regionswith low mean temperatures (approximately 20◦C), peak mortality by AgNPVmay be delayed by as much as 4 days when compared with warmer regions

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(approximately 27◦C), causing difficulties for implementation of AgNPV use incooler areas.

Application TechnologyMany factors can influence the outcome of a virus application, including type ofequipment, crop stage, virus formulation, and climate (151, 182). Aerial appli-cation of NPVs in forests has been improved because of better formulation andreduction in application volume (29, 135). Trials in Brazil showed that aerialapplication of the AgNPV could be efficient in water at a minimum volume of15 liters/ha (147) or soybean oil at 5 liters/ha (60). Unconventional deliverymethods have been attempted with viruses, profiting from the ability of insectsto carry virus particles and spread the pathogen in host populations (33, 79).This approach (autodissemination) has been successful with the scarabaeidO. rhinocerosin Southeast Asia in coconut (183). Autodissemination by adultshas also been tried withH. zeaNPV on cotton (57) and withA. gemmatalisNPV on soybean (F Moscardi, unpublished data), involving light traps andcontamination devices containing dried or liquid virus preparations. In bothcases, observed larval infection by baculoviruses was>50% within 20 m ofthe contamination device, decreasing to<10% at distances over 100 m fromthe virus source. This approach was also attempted with the AcNPV to con-trol the tobacco budworm in tobacco, where it was effective in causing somelarval mortality by AcNPV but not at a level high enough to be economicallyeffective (79). Autodissemination of the Indian meal moth,P. interpunctella,GV was proposed in which male moths are attracted to virus-baited pheromonelures contaminated with the GV, which is transferred to females during copu-lation, causing larval food to be contaminated during oviposition (167). Area-wide management ofHelicoverpa/Heliothisis being tried by virus applicationson spring hosts of the pest to reduce their populations before they attack cottonand other crops (10). Use of the NPVs ofS. frugiperdaandH. zeain irriga-tion water on maize was relatively successful (65). In Brazil, this method hasbeen adapted to controlS. frugiperdaby its NPV in maize fields, with promisingresults (171). Although unconventional delivery of baculoviruses is an approachto be considered for inoculative releases of baculoviruses in insect populations,its practical usefulness still depends on further research.

PROBLEMS THAT LIMIT EXPANSIONOF BACULOVIRUS USE

Some characteristics of baculoviruses keep industry interest low in their com-mercialization as microbial insecticides. The host-specificity of baculoviruses

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(63) is desirable in IPM programs, but their potential market is restricted,as are economic returns, a factor that might have influenced the decision bysome private companies to discontinue development or sales of some viralinsecticides (29, 46, 70, 180). However, this factor alone does not seem to applyfor pests such as theHelicoverpa/Heliothis complex and the codling moth,C. pomonella, as potential market areas for baculoviruses of these pests are large(23, 70, 74). Currently, industry interest in baculoviruses is increasing becauseof the buildup of insect resistance to insecticides, banning of various insecti-cides in many countries, and overall society demand for safer pesticides andless-toxic chemical residues in food and water. The availability of baculoviruseswith fairly wide host ranges, such as the NPVs ofA. californica, A. falcifera,andM. brassicae, also has attracted increased industry interest (29). Anotherlimiting factor for industry is the large-scale production of baculoviruses. Cur-rently, this can be accomplished primarily in vivo, mostly on insects rearedon artificial diets (17) and, in some cases, under field conditions (116–118,154). Production in laboratory-reared insects has progressed technically andcostwise, but it is still expensive to allow baculoviruses to be cost-competitivewith chemicals (17, 115, 141, 174). Industry is interested in producing bacu-loviruses in vitro, in insect cells in bioreactors, because such systems wouldallow selection of cell clones with high baculovirus yield potential and free fromundesirable contaminants, thereby reducing the number of personnel neededto handle various tasks of in vivo production (174). Despite recent progress inin vitro commercial production of baculoviruses (17, 83, 136, 174), it is still notfeasible technically and economically. The main problems are lack of low-cost,serum-free media and suitable cell lines and virus strains and the need to scaleup production of highly active virus with high titers per milliliter of medium(17, 83, 174). Insect cells have been grown andA. falciferaNPV produced inup to 100-liter bioreactors, and the resulting OBs had similar activity to OBsproduced in vivo againstT. ni larvae (174). Currently,A. falcifera NPV hasbeen produced in 150-liter tanks, and work is in progress to scale up its produc-tion in 2,000- to 50,000-liter tanks (83). Also, ABC (Australian BiopesticideCompany) has successfully scaled upH. armigeraNPV to 150 liters in biore-actors, and ABC intends to scale up its production to a commercial level in1999 (F East, personal communication). When available at competitive costs,in vitro–produced baculoviruses will face some of the problems that limit expan-sion of in vivo–produced wild-type viruses, as discussed herein. An importantfactor that limits industry interest and farmer acceptance of baculovirus pesti-cides is their slow speed in stopping pest damage and killing the hosts. Evenin crops with high tolerance to damage, farmers are initially reluctant to use abaculovirus insecticide.

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278 MOSCARDI

STRATEGIES TO COUNTERACT LIMITATIONSOF BACULOVIRUSES

Mixture of Baculoviruses with Reduced Dosagesof InsecticidesMost insecticides mixed with baculoviruses usually do not affect viral activityon hosts (66, 82, 116). Trials with the NPV ofA. gemmatalisin Brazil showedthat several insecticides at one fourth to one sixth the recommended dosages,when mixed with the AgNPV, controlled larval populations and maintainedyield potential of soybean when the insect populations were above thresholdsrecommended for virus application (116, 149). This approach has been tested inother countries (82) but is apparently employed mainly in Brazil (approximately20% of the AgNPV treated area in Parana state) (L Morales, unpublished data).This strategy may be important because baculoviruses can be used in situationsin which the recommendation is to apply chemical insecticides, and the sublethaldosages of insecticides will be less hazardous to humans and the environment.

Substances That Enhance Baculovirus Activityin Host InsectsCertain substances, such as boric acid (109, 143), chitinase (144), extracts ofneem tree (Azidarachta indica) (24, 146), and optical brighteners of the stil-bene group (142, 145), have enhanced baculovirus activity. Studies with a pro-tein associated with a Hawaian isolate of the GV ofPseudaletia unipunctademonstrated that this protein increased the virulence of a NPV in this insectand was denominated “viral enhancing factor” (159). Later, this substance wasnamed enhancin and was shown to cause ruptures in the peritrophic membrane,thereby enhancing the virulence ofA. californicaNPV and the NPV and GV ofT. ni (55). Enhancin has been characterized as a metalloprotease (94) and hasgood potential for use in engineered constructs to improve virulence and speedof kill of baculoviruses. Mixtures of baculoviruses with optical brighteners ofthe stilbene group seem to have excellent potential for use in formulations ofbaculoviruses because they can enhance viral activity at concentrations as lowas 0.01% (145), reduce time to kill the host, and provide protection against UVsolar radiation (142). Field trials have confirmed the enhancement ofL. disparNPV by optical brighteners (173). These substances have enhanced other bac-uloviruses, including the NPVs ofH. zea, H. virescens, A. californica, S. exigua,T. ni, C. includens, A. falcifera, andA. gemmatalis(52, 142, 168, 185). Fur-thermore, these substances may expand the host range of some baculoviruses(142). Another important aspect of optical brighteners is that they may brake re-sistance developed by insects to baculoviruses. A population ofA. gemmatalis

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selected in the laboratory for high resistance to AgNPV (2) regained suscepti-bility when the virus was mixed with certain optical brighteners (F Moscardi,L Morales, DR Sosa-Gómez, unpublished data). In another study, an opticalbrightener lowered the 50% lethal concentration in virus-resistantA. gemmatalisto a level below that in the susceptible population (52). The characteristics ofoptical brighteners, as well as other chemicals and organic substances, opennew arenas for improvement of baculovirus formulations.

In Vivo Sequential Passage of Baculoviruses on Hostand Non-Host InsectsIn vivo sequential passages of wild isolates can increase virulence and reducethe time to kill natural hosts as well as other species that normally have lowsusceptibility to a given baculovirus (89, 100, 108, 125, 142). Apparently, se-quential passages select more active isolates present in heterogeneous wildvirus populations, resulting in more homogeneous virus populations againstthe host or non-host species (142). TheA. gemmatalisNPV increased activityagainst the sugarcane borer (D. sacharalis) after sequential passage in this host,reaching activity levels comparable to a GV that naturally infectsD. saccharalis(125). Attempts to develop an isolate ofA. californicaNPV (AcNPV) that wouldbe effective against a lepidopteran complex (A. gemmatalis, C. includens, andR. nu) in soybean were not successful. The virulence of AcNPV passed seriallythrough each species increased dramatically after five passages, with activi-ties close to or above those of natural NPVs associated with each host, butvariants obtained from each species were not effective against the other twospecies (108). Although baculovirus activity and host range may be improvedthrough serial passages of wild-type isolates either on host or non-host insects,this method has not been adequately explored for many viruses being used asmicrobial insecticides. On the other hand, research on genetically engineeredbaculoviruses gained momentum in the 1990s, as a means to overcome someof the shortcomings of baculoviruses as microbial insecticides.

Genetic Engineering of BaculovirusesReducing time to kill host insects and their feeding capacity apparently is themain target involving engineered baculoviruses (20, 105, 106, 175). Researchin this area has involved the expression of genes coding for foreign proteinsin baculovirus genomes, such as insect-specific toxins (arthropod venoms,B. thuringiensistoxins), insect hormones (diuretic, eclosion, and prothoracichormones), juvenile hormone esterase (JHE), as well as deletion of ecdystroidUDP-glucosyltransferase (egt) (20). So far, the most promising results havebeen obtained with the toxin AaIT derived from the scorpionAndroctonusaustralis (95, 102, 157), with the toxin TxP1 from the mitePyemotes tritici

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280 MOSCARDI

(131, 163), and withegt deletion that prevents infected insects from molting(122, 164). Private industries are particularly interested in these developmentsbecause engineered viruses would be more effective in short-term effect on hostinsects, and they would be applied more frequently than current wild-type bac-uloviruses (17) because of their lower production of polyhedral inclusion bodiescompared with wild viruses (26, 78, 122). Recent studies with recombinant bac-uloviruses have shown that they killed the host faster, but the mean number ofpolyhedra produced in larvae infected with recombinant viruses was approxi-mately 20%–60% less than those obtained in larvae inoculated with the wild-type virus (25, 90). Therefore, one of the most important characteristics of wild-type baculoviruses, their ability to recycle and persist in the host environment,would be impaired in recombinant baculoviruses. Time to kill the host has beenreduced by 25%–40% in some insect species by recombinant baculovirusesexpressing JHE, AaIT, and TxP1 (16, 77, 91, 131, 157, 164). However, someof these viral constructs have failed to reduce speed of kill and host feeding(32, 137). Although significant reductions in speed of kill were observed withA. californicaNPV expressing AaIT onPseudoplusia includenslarval instars2–5, more than 80% of the insects were still alive after 6 days, which wasinsufficient to allow competition with most commercial insecticides (91). TheAaIT gene expressed inA. californicaNPV increased speed of kill by 25% insecond instarT. ni larvae but did not increase infectivity or host range (16, 157).A strain ofH. virescensresistant to pyrethroids was more sensitive to a recom-binant virus (AcAaIT) than a suscetible strain was (103). Feeding damage tolettuce and cotton byH. virescensinfected withA. californicaNPV expressingJHE was reduced by 50% and 36%, respectively, compared with the wild-typeAcNPV (20). However, laboratory studies with the AcAaITNPV inT. ni larvaeresulted in only 5% larval feeding reduction when compared with the untreatedlarvae (77). Therefore, it is evident that the effect of engineered baculoviruseson speed of kill and host feeding will vary with the target insect, and initialselection from the natural pool of isolates may be important for their success.

There have been a few field experiments with genetically modified bac-uloviruses (25, 137a). Trial with anA. californicaexpressing AaIT resulted in aquicker speed of kill and a significant reduction of crop damage byT. ni larvae(26). Tests with a polyhedrin-minus AcNPV indicate that unprotected virions ofgenetically engineered baculoviruses may be an approach to reducing the timethese recombinant viruses remain active, thus reducing the likelihood of neg-ative environmental effects (176). However, nonoccluded virions are rapidlyinactivated by sunlight and may be of no interest to industry (25). Lowerpersistence and dispersal of a modifiedA. californica NPV, lacking the p10gene, have been demonstrated in the field (119). In recent years, recombinantbaculovirusess have been field tested in the United States and in the United

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Kingdom, but they have not been applied experimentally over large areas orregistered for commerical use (137a).

CONCLUSIONS AND FUTURE PROSPECTS

Baculoviruses have been developed as microbial insecticides in different coun-tries against lepidopteran pests of forests and crops. Despite their potential,baculovirus use generally has been disappointing. The most significant devel-opments were made with the NPV ofA. gemmatalisin soybean (area wide), theNPV of Helicoverpa/Heliothiscomplex, the NPV ofL. dispar, and the GV ofC. pomonella. These programs show that the use of baculoviruses as microbialinsecticides is a viable alternative to chemical insecticides. However, expansionof baculovirus use will depend on further research or actions on key limiting is-sues. Most important developments in the next 5 years will probably occur in theareas of recombinant baculoviruses and in the in vitro commercial production ofthese agents. On the other hand, methods to increase field efficacy of wild-typebaculoviruses, such as adding enhancing substances to formulations, deservefurther exploration. Considering that the use of baculoviruses may increase sub-stantially in the next 10 years, research should also be directed to determinethe possibilities of development of resistance by field populations of insects toviral insecticides and to the mechanisms involved in this phenomenon, so thatproper management of insect resistance to viral insecticides can be implementedbefore resistance becomes a problem. Also, the possible development of resis-tance by insects to toxins and hormones expressed by engineered baculoviruseswarrants investigation (172). Risk assessment protocols are needed for genet-ically engineered baculoviruses (25, 107, 137a, 172, 177). Environmental risksof these modified baculoviruses are low (17, 83, 172), but the possibilities ofimproving wild-type baculovirus formulations may provide better alternativesto engineered viruses. Certainly, it will depend on the virus-insect-crop systeminvolved, but engineered and in vitro–produced baculoviruses will face manyof the same problems involved in the previous use of baculoviruses as micro-bial insecticides. Because of their characteristics, engineered baculovirusesare more likely to be produced in vitro (17). Therefore, current research ef-forts toward producing these agents in vitro will have to be intensified to allowcommercial production and large-scale use of engineered baculoviruses at com-petitive costs with chemical insecticides. Global farmer education toward useof biological agents for pest control will be a key feature for expansion ofbaculovirus use worldwide, whether it is a wild-type or a genetically modifiedproduct. Only past experience and further field data, proper risk assessment(137a), and society response to these modified baculoviruses will provide anappropriate perspective of engineered viruses as pest control agents.

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282 MOSCARDI

ACKNOWLEDGMENTS

I thank Drs. James Fuxa, Lawrence Lacey, Carlo Ignoffo, John Cunningham,and Daniel Sosa-Gómez for reviewing and making important comments to im-prove the manuscript. Also, thanks are due to R Lecuona, G Biache, M Guillon,J Podgwaite, C Nielsen-LeRoux, W Whitlock, P Vail, S Young, K Maramorosch,P Yi, J Maruniak, F Valicente, M Dimock, S Weiss, L Morales, E Vargas Osuna,R Cisneros, S Torrencillas, F Hunter, and F East, who provided useful informa-tion on baculovirus research and use. This work was supported by Embrapa andthe Brazilian National Council for Scientific and Technological Development(CNPq) and was approved for publication by the Technical Director of EmbrapaSoja as manuscript 003/98.

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Annual Review of Entomology Volume 44, 1999

CONTENTSMites in Forest Canopies: Filling the Size Distribution Shortfall? David Evans Walter, Valerie Behan-Pelletier 1

Insects as Food: Why the Western Attitude is Important, Gene R. DeFoliart 21

Emerging and Resurging Vector-Borne Diseases, Norman G. Gratz 51

Insect Pests of Pigeonpea and Their Management, T. G. Shanower, J. Romeis, E. M. Minja 77

The Evolution and Development of Dipteran Wing Veins: A Systematic Approach, Julian Stark, James Bonacum, James Remsen, Rob DeSalle 97

Odor-Mediated Behavior of Afrotropical Malaria Mosquitoes, Willem Takken, Bart G. J. Knols 131

Pathogens and Predators of Ticks and Their Potential in Biological Control, M. Samish, J. Rehacek 159

The Role of Stingless Bees in Crop Pollination, Tim A. Heard 183

Bionomics of the Anthocoridae, John D. Lattin 207

Adaptative Strategies of Edaphic Arthropods, M. G. Villani, L. L. Allee, A. Díaz, P. S. Robbins 233

Assessment of the Application of Baculoviruses for Control of Lepidoptera, Flávio Moscardi 257

Hyperparasitism: Multitrophic Ecology and Behavior, Daniel J. Sullivan, Wolfgang Völkl 291

Density-Dependent Physiological Phase in Insects, S. W. Applebaum, Y. Heifetz 317

Recent Advances in Cassava Pest Management, Anthony C. Bellotti, Lincoln Smith, Stephen L. Lapointe 343

Mate Choice in Tree Crickets and Their Kin, W. D. Brown 371

CONGRUENCE AND CONTROVERSY: Toward a Higher-Level Phylogeny of Diptera, D. K. Yeates, B. M. Wiegmann 397

The Insect Voltage-Gated Sodium Channel As Target of Insecticides, Eliahu Zlotkin 429

Management of Plant Viral Diseases Through Chemical Control of Insect Vectors, Thomas M. Perring, Ned M. Gruenhagen, Charles A. Farrar 457

Influence of the Larval Host Plant on Reproductive Strategies in Cerambycid Beetles, L. M. Hanks 483

Insect P450 Enzymes, René Feyereisen 507

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Risk-Spreading and Bet-Hedging in Insect Population Biology, Keith R. Hopper 535

Nutrition and Culture of Entomophagous Insects, S. N. Thompson 561

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ASSESSMENT OF THE APPLICATION OF BACULOVIRUSES FOR CONTROL OF LEPIDOPTERA

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