Responses of Production and Storage Walnut Pests

to Bacillus thuringiensis Insecticidal

Crystal Protein Fragments

P. V. Vaill, J. S. Tebbetsl, D. F. Hoffmannl,

and A. M. DandeIc.ar,

lHorticulturalCrops ResearchLaboratory, USDA-ARS,2021 SouthPeach Avenue, Fresno,

California 93727

2Department of Pomology, University of California, Davis, California 95616

206-- ---- ---

ABSTRACT

Recent advances in genetic engineering have provided the opportunity to induce walnut

plants to produce Bacillus thuringiensis Insecticidal Crystal Protein Fragments (ICPFs) for

insect' control. We studied the effects of two ICPFs CryIA(b) and CryIA(c) previously

shown to be encoded by the CryIA(b) and CryIA(c) genes in the Bacillus thuringiensis. strains

HD-l and HD-73respectively. The lethal effects on larvae of codling moth, Cydia

pomonella (L.), navel orangeworm, Amyelois transitella (Walker), and the major postharvest

pest Indianmea1 moth, Plodia interpunctella (Hubner) were investigated. Both proteins were

toxic to the three species tested. Indianmea1 moth larvae were most susceptible and navel

orangewormthe least; CryIA(b)was consistentlymore toxic to navel orangeworm. Similar.

relationshipsresulted whenICPFs were incorporatedinto the diet. Both ICPFs caused

decreased rate of developmentof navelorangewonn. Effectson pupal weightoccurred only

at the highest dose (100 ng/cm2). Neither ICPF affected frequencyof matingor fecundity.

In addition to the lethal effects, the extended development times observed could have

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considerable effects on the population dynamics of the navel orangeworm and possibly other

species.

Key words: Bacillus rhuringiensis; endotoxin; insecticidal crystal protein fragment; Plodia

inrerpuncrel1a;Amyelois transitel1a; Cydia pomonel1a; biological control; walnut; transgenic

plants

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1ntroouction

The insecticidal activity of the bacterium Bacillus thuringlensis (Bt) resides in the

crystaline inclusion body (Angus, 1954; Dulmage, 1981) containing a protein known as the

<>-endotoxinand more recently referred to as insecticidal crystal protein fractions (lCPp). Bt

encoded JCPFs are considered safe biocontrol agents because they have no known effect on

nontarget organisms. Productscontainingcrystalsand sporesof Bt are have have been used

commercially for many years in products such as Dipell8and Thuricidel8 and are exempt

from residue tolerance. Different strains of Bt make insecticidal proteins that are effective

against a particular group of insect pests (Aronson et al., 1986); var israelensis is most

active againstDiptera(Yamamotoand McLaughlin, 1981;Goldbergand Margallt, 1977); var

kurstaJd and var berliner are ~ost active against Lepidoptera (Vaeck et al., 1987; Fischhoff

et al., 1987); and var tenebrionis and var san diego against Coleoptera (Krieg et al., 1983;

Herrnstadt et al., 1986).

The Bt encoded JCPF is first synthesized by the bacteria as the o-protoxin thus it is not

active in its nativeform. Onceit is ingestedby the target insect it is first solubilizedin the

gut and then acted uponby midgutprotease(s) to release the active componentof the toxin

(Lilley et al., 1980). Several genes encoding individual JCPFs effective against lepidopteran

insects have been isolated from different strains of Bt and their DNA sequence determined

(Schnepfet al., 1985;Shibanoet al., 1985;Adang et al., 1985;Thorne et al., 1986;

Wabiko et al., 1986;Hofteet al., 1985; Geiser et al., 1986). Very recently transgenic

plants expressingthe JCPFsfrom Bt were obtainedin tobacco(Vaecket al., 1987), tomato

209

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(Fischhoffet al., 1987),and cotton (Fedak et al., 1990;T. J. Henneberry, personal

communication). These transgenic plants expressed sufficient quantities of ICPF protect the

plants from damagecausedby the feedinglarvae of severallepidopterous pests.

Recently transgenic walnut plants have been regenerated carrying genes encoding

resistance to the antibiotic kanamycin1K(McGranahan et al., 1988, 1990) thus providing

further impetus to the possibility of engineering walnuts to contain and produce insecticidal

crystal protein fragments(JCPF) for control of walnutpests. In addition, McGranahan

demonstrated that such transgenicplants could be propagatedusing in vitro techniques.

Dandekar (1989) also demonstrated that the genes responsible for the production of ICPF$

could be incorporated into the walnut genome.

The successof the above investigatorsin obtainingtransgenicplants and developing in

vitro propagation techniques led us to consider the possibility of developing transgenic plants

containingICPFs. Productionand storage pests of walnut (codlingmoth, navelorangeworm,

and Indianmealmoth)were consideredas candidatesfor control. Potential benefits of this

research would be through (1) increased production efficiency associated with reduction of

costs associated with insecticide applications; (2) less impact on natural enemies such as

Trioxys pallidus (Haliday) (Hymenoptera: Aphidiidae) a parasitoid of the walnut aphid

(Chramaphis juglandicola) (Kaltenbach» (Homoptera: Aphidiidae) (van den Bosch et al.,

1979); and (3) postharvest insect control impacting on quality and exportation issues.

We report the insecticidal effects of two purified ICPFs that could be engineered into

walnut germplasm. These studies documentlethal and sublethaleffects of ICPF necessary to

210

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evaluate efficacy of the transgenic walnut stocks in sitU or on extracts of the plant material.

They also provide further insight into the impact ICPFs might have on insect populations.

Materials and Methods

walnutpests. The ICPFs producedby strainsHD-l (CryIA(b» and HD-73 (CryIA(c» of Bt

were chosen for this study. The active component of the toxin protein is the N-termina1113

of the protein. Purified active toxin fragmentsof the Bt toxins of HD-l an~ HD-73 were

obtainedfrom D. A. Fischhoff, MonsantoAgriculturalCo., St. Louis, MO 63198. The

active toxin fragments were obtained after tryptic digestion (with the protease Trypsin) of the

purified toxin followedby separationby columnchromatography. The purified toxin

fragmentswere stored in l00.mM sodiumcarbonate(pH 10.0), 50% glycerol, and 10 mM

dithiothreitolfrozen at -80°C. Dilutionswere made in 100 mM sodiumcarbonate buffer (pH

10.0) containing10 mM dithiothreitol. Two controlswere used, buffer alone and buffer

with dithiothreitol, to ensure that the buffer or dithiothreitol were not toxic to the insects.

The test insects, codling moth, navel orangeworm,and Indianmealmoth were reared

and the bioassaysconductedat the USDA-~RS HorticulturalCrops Research Laboratoryat

Fresno, CA. The susceptibilityof each of these insect species to the Bt toxins HD-1 and

HD-73 was determinedper os. Serial dilutionsof each protein were layered onto the surface

of an agar-baseddiet (Bioserv#9370). One neonatallarva was placed in each of 20 vials

(containingca. 1 ml of surface-contaminated(diet) for each insect speciesand dilution. The

larvae were then incubatedat 26.7°C and mortalityrecorded daily. The tests were replicated

211

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twice to determine the LCsoor L~5 at specific time intervals; dose is expressed as ng/cnr of

diet surface.

Similarily, diet incorporation tests were conducted except that dilutions of the ICPF

were incorporated into the diet prior to solidification. Observations, replicates, data

acquisition, and analysis were also similar; however, the doses are expressed as nglcm'.

Sublethal effects. The effects of sublethal exposures of the navel orangeworm to ICPFs

was determined. Neonatallarvae were placedon agar diet surfacecontaminatedwith ICPFs

at 10, 50, and 100ng/cmZand allowedto pupate and emerge as adults. A control group of

larvae was also includedon the agar diet withoutICPF. Growthand developmentof the

survivors in each group was noted and recorded (Le., developmentalstage, 50% adult

emergence, pupal weight,and.adult fecundity(in crosses only». All dose-mortalitydata

were analyzedusing the probit analysismodel (pOLO-PC1987). Other data were subjected

to analysis of variance and Tukey's studentized range test (SAS Institute 1987).

Results and Discussion

Susceptibilityto insecticidalcrystal protein fragments. The data that we have obtained. .

on feeding studieswith ICPFs from CryIA(b)and CryIA(c)isolated from HD-1 and HD-73

strains of Bt clearly show that both proteins are toxic to the target insects at relatively low

concentrations(fable 1). Basedon overlap of the 95% CL, we found no consistent

statistical differences in toxicity between CryIA(b) and CryIA(c) to any of the three insect

species tested, with the exception of navel orangeworm where Cryla(b) was consistently

more toxic than CryIA(c). Indianmealmoth was the most susceptiblespecies and navel

212

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orangeworm the least susceptible to either of the ICPFs. Furthermore surface-contaminated

diet stored at 26.7°C for eight days showed no loss of activity when infested with neonatal

larvae, indicatingthat the protein remainedactive during the duration of the study. Results

from the diet-incorporation studies were similar to those from the surfa.ce-contaminated tests

(Table 2). A comparison of the LDsoSshowed no significant difference between the ICPFs to

the insect species tested.

Sublethal effects. Sublethal concentrations of ICPFs reduced the rate of development

of the navel orangeworm(Table3). Pupae and sixth instars (L6) were present in the control

group after 21 days comparedto the presenceof more immaturestages in those groups

feeding on ICPFs. The rate of developmentdecreasedwith increased dose of ICPF.

Developmental times to 50% adult emergence are shown in Table 4 and further illustrate the

effect of exposure to ICPFs ori the life cycle of the navel orangeworm. Compared to

controls, developmentaltime was 5.5 to 10.5 days longer with CryIA(b) and 10.5 to 14.5

days longer with CryIA(c). Thus, CryIA(c)further delayeddevelopmentca..five days

compared to CryIA(b). .

Although ICPFs slowed navel orangeworm development, there was little or no effect

on adult size, mating,or fecundityin survivors. Significantdifferences occurred in pupal

weights at the highestdose (100 ng/cm2)(Table5). Frequencyof mating was unaffected,

ranging from 75 to 100%in the control and all treated groups. No difference in fecundity

was observedbetweenany group. The number of eggs per female and the number of viable

eggs per mated female were not significantly different (Table 6).

213

The recent advances in developing transgenic plants expressing the Bt toxin at levels

required for protection from economic damage have primarily been on crops such as cotton

(Henneberry, personal communication), tobacco (Vaeck, 1987), and tomato (Fischhoff et ai.,

1987). Dandekar (1989) has shown that a gene responsible for the production of an ICPF

can be incorporated into the walnut genome. Thus the potential exists for developing

transgenicwalnutplantscapableof controllingboth productionand postharvestpests. We do

not know if the ICPFs will be differentially expressed (Le., vegetative versus reproductive

tissues) in walnut cultivars,nor do we know if ICPFs will persist or accumulatein the

targeted plant organs.

Our data show the relative susceptibility of the Indianmeal moth, codling moth, and

navel orangeworm. The Indianmeal moth, a major postharvest pest of walnuts, is highly

susceptible to the ICPFs and could be controlled if sufficient quantities of ICPF are available

in the nut meats. The navel orangeworm is the least susceptible production pest and is

considered the key insect in the screening of engineered walnuts. The sublethal effects of the

ICPFs on navel orangeworm were also identified and provide another alternative for

screening of desirable biological effects of engineered plants. The increased developmental

times could significantly reduce the number of generations per year as well as provide

increased time for predators or parasitoids and pathogens to have an effect on populations.

An increase in generation time alone would be beneficial, particularly if the other two species

responded similarly.

214

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If sufficient quantities of the ICPF were produced in walnut fruit it is quite possible

that significant reductions in the use of pesticides could result in both the production and

postharvest sectors. Reduction in production pesticide use would provide additional benefits

by not upsetting biological control of other species such as the walnut aphid (van den Bosch

et aI., 1978).

There are several potential problems associated with the use of ICPF containing

walnuts. We do not know if ICPF(s) produced in walnutsis identical to those tested in these

studies. Resistanceof the Indianmealmoth to Bt has been reported (McGaughey, 1985)and

several production pests can be selected for resistance based on laboratory studies (Sims and

Stone, 1991). Recently Tabashnik et aI. (1990) reported on the field resistance of PIUleIla

xylostella (L.) to commercial formulations of the insecticidal spore-crystal protein complex of

Bt subsp. kurstaki. Since populations could be continually exposed to ICPFs the potential for

development of resistance exists. Possibly the combined use of another microbial control

agent such as the granulosisvirus of codling moth (Vailet aI., 1991)could reduce the

potentialrisk of resistantpopulationsdeveloping. In addition, we do not know if the quality

of nuts containingICPF might be altered.

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.

AckDowiedgments"

The authors wish to express their gratitude to D. A. Fischhoff, Monsanto Agricultural

Company, St. Louis, Missouri for graciously supplying the purified ICPFs used in this study.

216

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Table 1. Estimated LCsoand LC9Swith 95 % CL for three lepidopteran insects when fed purified insecticidal crystal protein

fragments from Bacillus thuringiensis var. kurstaki, surface-layered on agar dier

Insect

Indianmeal moth

-----------------------------------------------------------------------------------------------------------------------------------

.40 to 160 neonate larvae exposed per dose; four to six doses.

ExposureICPF LDso(ng/cm2) Slope (:I: SE)

(days)-3 CryIA(b) 7.39 (5.71-9.57) 149 (90.9-244) 1.261 (0.102)

CryIA(c) 3.27 (2.23-4.79) 199 (102-388) 0.922 (0.089)

Codling moth 7 CryIA(b) 34.7 (24.9-48.4) 1,675 (879-3,192) 0.977 (0.081)

I'VCryIA(c) 59.0 (41.6-83.6) 7,665 (3,392-17,321) 0.778 (0.064)I'V->

14 CryIA(b) 13.8 (11.0-17.4) 106 (70.1-160) 1.862 (0.181)

CryIA(c) 9.88 (7.82-12.47) 89.7 (60.3-133) 1.717 (0.161)-----------------------------------------------------------------------------------------------------------------------------------

Navel orangeworm 7 CryIA(b) 307 (166-561) 2,731 (1,221-16,813) 1.733 (0.237)

CryIA(c) 1,412 (1,093-1,824) 29,186 (16,336-52,144) 1.250 (0.108)

14 CryIA(b) 128 (110-150) 428 (310-591) 3.140 (0.017)

CryIA(c) 287 (142-572) 1,786 (809-14,080) 2.072 (0.332)

"55 neonate larvae exposed per dose; three to four doses.

Table 2. Estimated LCj()and LC95with 95% CL for three lepidopteran insects when fed purified insecticidal erystal protein

fragments from Bacillus thuringiensis var. leurstaki, incorporated into agar dier

InsectExposure

ICPF LD50(ng/em3) Slope (:f: SE)(days)

Indianmeal moth 3 CryIA(b) 1.53 (1.00-2.24) 13.14 (7.62-32.72) 1.764 (0.270)

CryIA(e) 2.42 (1.43-3.92) 75.34 (35.63-236.99) 1.101 (0.138)

Codling moth 11 CryIA(b) 0.83 (0.51-1.29) 23.97 (11.94-67.21) 1.125 (0.131)

NN

CryIA(e) 0.96 (0.58-1.55) 41.47 (19.37-128.34) 1.007 (0.117)N

Navel orangeworm 11 CryIA(b) 6.54 (4.64-9.19) 48.47 (29.42-104.39) 1.891 (0.242)

CryIA(e) 8.65 (6.03-12.39) 80.28 (46.99-180.58) 1.700 (0.210)

~1 -L6 denotes larval instar; P =pupa.

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Table 3. Effectsof purified insecticidalcrystalprotein fragmentson larval developmentof

navel orangewonnafter 21 days incubationat 26.7°C

Dose % of each instar or stage at 21 dayICPF

(ng/cm L1 L2 L3 U 1.5 L6 P

Control 0 0 0 0 0 0 85 15

CryIA(b) 10 0 .0 0 1 1 98 0

50 0 1 7 21 39 32 0

100 0 13 37 40 9 1 0

CryIA(c) 10 2 2 3 2 10 81 0

50 5 13 26 35 18 3 0 r

100 3 33 44 15 5 0 0

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Table 4. Time required for 50% adult emergencewhen navel orangewormlarvae were fed

purified insecticidalcrystal protein fragments

Dose Time Difference in developmentaltime (days)ICPF

(ng/cm (days) ICPF vs control BetweenICPFs

Control 0 28.5

CryIA(b) 10 34.0 +5.5

50 37.5 +9.0

100 39.0 + 10.5

CryIA(c) 10 39.0 +10.5 +5.0

50 42.0 +13.5 +4.5

100 43.0 + 14.5 +4.5

Table 5. Effects of two purified insecticidal crystal protein fragments on pupal weights of

female or male navel orangeworms

Dose (ng/cm2) Mean (:t: SEM) pupal weight (mgf

50

Protein Dose

Control 0

Cry IA (b) 10

100

Cry IA (c) 10

50

100

~eans (withincolumns)followedby the same letter are not significantlydifferent (p >

0.05) based on Tukey's studentized range (HSD) test (SAS Institute, 1987).

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Female Male

63.6 (2.0) a 50.3 (1.4) a

62.2 (1.7) ab 47.1 (1.3) ab

59.3 (2.3) ab 50.4 (1.8) a

58.1 (2.2) be 46.3 (1.7) ab

60.4 (1.9) ab 49.5 (1.8) ab

62.3 (2.2) ab 48.0 (1.8) ab

53.0 (2.6) c 45.8 (1.9) b

"Means followedby the same letter in each columnare not significantlydifferent (p > 0.05)

based on Tukey's studentized range (HSD) test (SAS Institute 1987).

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Table 6. Effectsof purified insecticidalcrystal protein fragmentsfed to larvae on fecunidty

of navel orangewonnmoths

x matings Mean (+ SEM) fecundityDoseICPF n (+ SEM)

(nglcmEggs per 9 Per mated 9

per 9

Control 0 21 0.9 (0.1) 263 (31) a" 257 (26) a

CryIA(b) 10 39 1.0 (0.0) 311 (14) a 245 (23) a

50 30 1.1 (0.1) 302 (19) a 254 (23) a

100 39 0.9 (0.1) 291 (11) a 233 (10) a

CryIA(c) 10 27 0.9 (0.1) 318 (20) a 249 (36) a

50 27 0.9 (0.1) 301 (25) a 248 (28) a

100 9 0.8 (0.4) 258 (50) a 165 (31) a