This article was downloaded by: [Eindhoven Technical University]On: 18 November 2014, At: 20:06Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Archives Of Phytopathology And PlantProtectionPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/gapp20

The Efficiency of Viral and BacterialEntomopathogens Formulated withChitinase for Biocontrol of LepidopteranCabbage PestsMargarita Shternshis a , Lubov Ovchinnikova a , Alexander Duzhak b

& Oxana Tomilova aa Department for Biological Control , State Agrarian University ,Novosibirsk, 630039, Russiab Siberian Branch of The Russian Academy of Sciences , Institute ofCytology and Genetics , Novosibirsk, 630090, RussiaPublished online: 09 Sep 2010.

To cite this article: Margarita Shternshis , Lubov Ovchinnikova , Alexander Duzhak & Oxana Tomilova(2002) The Efficiency of Viral and Bacterial Entomopathogens Formulated with Chitinase forBiocontrol of Lepidopteran Cabbage Pests, Archives Of Phytopathology And Plant Protection, 35:2,161-169, DOI: 10.1080/03235400214209

To link to this article: http://dx.doi.org/10.1080/03235400214209

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,

systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14

ISSN 0323-5408 print; ISSN 1477-2906 online © 2002 Taylor & Francis LtdDOI: 10.1080/0323540021000010487

Archiv. Phytopath. Pflanz., Vol. 35, pp. 161–169

* Correspondence to be addressed to: PO Box 793, Krasnoobsk – Novosibirsk, 630500, Russia; e-mail: mars@online.sinor.ru; Tel: (3832) 672847; Fax: (3832) 673922

THE EFFICIENCY OF VIRAL AND BACTERIALENTOMOPATHOGENS FORMULATED WITH

CHITINASE FOR BIOCONTROL OF LEPIDOPTERANCABBAGE PESTS

MARGARITA V. SHTERNSHIS1, LUBOV A. OVCHINNIKOVA1,ALEXANDER B. DUZHAK2 and OXANA G. TOMILOVA1

1Department for Biological Control, State Agrarian University, 630039, Novosibirsk, Russia;2Institute of Cytology and Genetics, Siberian Branch of The Russian Academy of Sciences, 630090,

Novosibirsk, Russia

(Received 5 February 2002)

Preparations of Bacillus thuringiensis subspecies galleriae (Bt), Mamestra brassicae nucleopolyhedrovirus(MbNPV) and chitinase were used in laboratory and field experiments to investigate the efficiency of this triplecomposition against Lepidopteran cabbage pests. Bt subspecies galleriae was shown to be the most relevant amongthree Bt subspecies against the cabbage moth Mamestra brassicae L. (Lepidoptera: Noctuidae), the diamondbackmoth, Plutella xyllostella L. (Lepidoptera: Plutellidae) and the large white butterfly, Pieris brassicae L.(Lepidoptera: Pierilidae). Synergistic toxic effect against pests mentioned above was obtained when the Bt andchitinase were used together. M. brassicae larvae on cabbage were much less susceptible to Bt than other pests. Itwas necessary to include MbNPV in the mixture. Analysis of the different mixtures allowed us to develop theoptimal one containing 2.5×107 spores ml–1 of Bt, 1×107 polyhedra ml–1 of MbNPV and 0.5 mU ml–1 of chitinase.Such triple composition was much more effective against M. brassicae larvae than the mixture of Bt with chitinaseand provided simultaneous biological control of all lepidopteran cabbage pests infesting host plants.

Keywords: cabbage pests; Bacillus thuringiensis; nucleopolyhedrovirus; chitinase

INTRODUCTION

Larvae of cabbage moth, Mamestra brassicae L. (Lepidoptera: Noctuidae), diamondbackmoth, Plutella xyllostella L. (Lepidoptera: Plutellidae) and large white butterfly, Pierisbrassicae L. (Lepidoptera: Pieridae) are common harmful pests for the cabbage crop. M.brassicae larvae are the most harmful. Moreover, very often all species mentioned occursimultaneously.

Biological insecticides based on Bacillus thuringiensis (Bt) are usually applied for thesuppression of lepidopteran insect pests. The efficiency of such preparations depends ingeneral on the composition of Bt subspecies, Cry proteins (Schnepf et al., 1998) and hostnature (Federici, 1996). In Siberia, as in another regions of Russia, the populations of

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14

162 M. V. SHTERNSHIS et al.

cabbage moth are much more resistant to the different Bt subspecies than populations oflarge white butterfly or diamondback moth (Spichenko et al., 1980; Shternshis, 1987a;Kandyibin, 1989). At the same time, the nucleopolyhedrovirus (Baculoviridae) (NPV)appeared to be an effective biological agent against the most harmful cabbage pest M.brassicae, but not for other Lepidoptera. It was also known that the efficacy of NVP wasincreased by the addition of Bt (Krieg, 1971) or enhancers (Shternshis, 1987b; Shapiro &Robertson, 1992).

Development of formulations containing Bt, MbNPV and certain enhancers for biocontrolof all lepidopteran pests of cabbage may increase efficacy. Our strategy is based on thesimilar means of Bt and baculovirus to penetrate the peritrophic matrix (PM) of the midgut.The structural component of PM – chitin partly provides the resistance of insects toentomopathogens (Lehane, 1997; Wang & Granados, 1998). Therefore, the addition of thesubstances, which promote the capacity of entomopathogens to penetrate through PM, couldenhance the insecticidal effect.

The enzyme chitinase destroys the structure of PM (Sampson & Gooday, 1998). whichprobably explains the enhancer effect on the activity of separate NPV (Duzhak et al., 1995;Shapiro et al., 1987), Bt (Smirnoff, 1971) or cloned Cry protein (Regev et al., 1996). Theeffect was expressed in the increased insect mortality without any toxic effect of the chitinasealone. At the same time, the prolonged period of entomopathogen activity was sufficientlyreduced (Duzhak et al., 1995). The addition of the chitinase to the mixture of Bt and NPV wasproposed by us to develop the optimal composition of these three ingredients for theimprovement of microbial control of the most serious pest M. brassicae and two otherLepidopteran cabbage insect pests, P. xyllostella and P. brassicae.

MATERIALS AND METHODS

Insects

Four species of the insects were used in the laboratory experiments and field trials. Thecabbage moth, Mamestra brassicae (L.), the diamondback moth, Plutella xyllostella (L.) andthe large cabbage butterfly, Pieris brassicae (L.) were collected on cabbage fields. The greatwax moth, Galleria mellonella (L.) was reared on the artificial diet as a laboratorypopulation.

Entomopathogens, formulations and other substances

The isolates of Bt subspecies galleriae, Bt subspecies dendrolimus (sotto), Bt subspecieskurstaki, NPV of Mamestra brassicae from the collection of Department for BiologicalControl (Novosibirsk State Agrarian University) and commercial formulation Lepidocide®(based on the Bt concentrated suspension) supplied by “Sibbioprom” (Berdsk, Russia)were used in the laboratory and field experiments. Lepidocide® was registered in Russiaas the commercial standard for lepidopteran pests on cabbage. The strain of MbNPV wasoriginally isolated from M. brassicae larvae field-collected in the Novosibirsk region.MbNPV was produced in vivo and formulated as a liquid concentrate. Crude chitinasepreparation from Streptomyces sp. was supplied by “Biostart” (Novosibirsk, Russia). Foranalysis of the enzyme activity N-acetylglucosamine (Koch-Light Lab., UK), gum-arabik(Sigma, USA) and chitin from crab’s shells (Voikov State Chemical Firm, Russia) wereused.

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14

ENTOMOPATHOGENS WITH CHITINASE 163

Enzyme Assays

The chitinase activity was assayed by the method of Berger et al. (1958) with colloidal chitin(0.5%) as a substrate. The concentration of reducing sugar, expressed asN-acetylglucosamine, was estimated by the reaction with 3.5-dinitrosalicylic acid (Miller,1959). One unit of the chitinase activity is defined as an amount of enzyme that liberates1 �M of N-acetylglucosamine per 1 min at 37°C (pH 5.5 or 9.5).

The lipase activity was assayed by the modified method of Ota & Yamada (1966). Theemulsion of 30% olive oil in 2% gum-arabic solution was used as a substrate.

The activity of protease was assayed by the modified method of Kunitz et al. (cited inKaversneva, 1971). One unit of protease activity is defined as an amount of enzyme thatliberates 1 �M of L-thyrosine per 1 min at 37°C (pH 5.5 or 9.5).

The universal buffer: 5 �M Na2HPO4; 5 �M H3BO3; 3.6 �M citric acid; 25 �M NaOH; pH5.5 or 9.5 (Skujins et al., 1973) was used in all assays.

Petri dish bioassays

The effect of different concentrations of Bt and its mixture with chitinase was determinedfirst in a Petri dish bioassay. For bioassay involving G. mellonella, the artificial diet (3 g)mixed with 1 ml of test Bt, and its combination with chitinase was introduced in Petri dishes(diameter = 8,5 cm). Fifteen second instar G. mellonella larvae, starved for 24 h, wereintroduced in to each Petri dish. In the control experiments, the bioassay was performed inthe same way but without the Bt or chitinase. The experiments were carried out at 28°C andmortality was scored after 1, 3, 5 and 7 days.

For bioassay involving cabbage pests, lepidopteran larvae were fed on cabbage leaves,wetted with suspensions of entomopathogens with or without chitinase. Ten or fifteensecond-instar or third instar larvae were introduced into each Petri dish with pieces ofcabbage leaves treated with tested suspensions. The control experiments were set in the sameway but leaves were treated with water or chitinase. The experiments were carried out at24 ± 2°C and the mortality of larvae was scored at 3, 5, 7 and 10 days. Control insectmortality was not more than 3–7%.

Field trials

The field trials were conducted on commercial cabbage plantations in July 1999.Experimental plots were 25 m2 and separated from other plots by 1 m. A randomizedcomplete block design was used to assign treatments to four replicates. Immediately beforeapplication and at 3, 5, 7 and 10 days after treatment, lepidopteran larvae were counted on100 plants per plot.

The test composition containing MbNPV (1 × 107 polyhedra ml–1), Bt galleriae (2.5 × 107

spores ml–1) plus chitinase (0.5 mU ml–1) and the standard formulation Lepidocide® (1.25 ×108 spores ml–1) were applied at an application rate of 300 L ha–1 using the hand sprayer Orion-6 (Quazar Corp, Warsaw, Poland). The treatments of cabbage plots were done twice in 10 dayintervals. The control plots were left untreated. Chitinase control was not included in field trialsbecause all laboratory experiments showed no chitinase influence on insect mortality.

Data analysis

Percent mortality was corrected for control mortality using Abbott’s (1925) formula. Theaverage lethal concentration (LC50) was estimated by Probit analysis. Data were subjected to

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14

164 M. V. SHTERNSHIS et al.

an analysis of variance (ANOVA). The significance between treatments was determinedusing F-test at P < 0.05. Standard errors of the mean (SE) are presented in parenthesis.

RESULTS

Enzyme assay

The chitinase, protease and lipase activities were analyzed in a crude enzyme preparationfrom Streptomyces sp at pH 5.5 or pH 9.5 (Table 1). High chitinase activity was observedat pH 5.5 and at pH 9.5. However, only traces of protease activity were observed at pH5.5 and none at pH 9.5. No lipase activity was observed at both pH values. Therefore, weused the designation “chitinase” in this paper for crude enzyme preparation obtained fromStreptomyces sp.

Selection of relevant Bt

The effect of Bt subspecies galleriae, Bt subspecies dendrolimus and Bt subspecies kurstakiagainst M. brassicae larvae is presented in Table 2. Bt subspecies galleriae (LC50 = 5.9×107 at3d day) was shown to be the most active in causing the mortality of M. brassicae larvae.

Analysis of the chitinase potency combined with the MbNPV and Bt

The addition of chitinase (0.5 mU ml–1) to the MbNPV suspension (1×106 polyhedra ml–1)causes the same level of M. brassicae larval mortality as the MbNPV concentratedsuspension (1×107 polyhedra ml–1) alone (F = 17.2; df = 3, 8; P < 0,05) (Figure 1). Theaction of NPV + chitinase formulation was significantly accelerated compared to the NPValone. In the latter case, larval mortality was observed 2 days later.

TABLE I Enzyme assay in preparation obtained fromStreptomyces sp.

pH Chitinase,mU mg–1

Protease,mU mg–1

Lipase,mU mg–1

5.5 12.3 0.2 0.09.5 10.0 0.0 0.0

TABLE II LC50 of Bt for 2-instar M. brassicae larvae

Bacillusthuringiensissubspecies

Observation duration (days)

3

LC50 Ig LC50 ± SE(mean ± SE)

5

LC50 Ig LC50 ± SE(mean ± SE)

Galleriae 5.9 × 107 7.77 ± 0.2882 2.9 × 107 7.47 ± 0.2027Dendrolimus 7.5 × 108 8.40 ± 0.3054 4.6 × 108 8.67 ± 0.3032Kurstaki 1.7 × 109 9.23 ± 0.1831 1.2 × 109 9.08 ± 0.2179

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14

ENTOMOPATHOGENS WITH CHITINASE 165

The most effective concentration of the chitinase was shown to be 0.5 mU ml–1 and wassimilar to that of the MbNPV. When chitinase at a concentration of 0.5 mU ml–1 was addedto the Bt preparation (1×108 spores ml–1), the larval mortality was enhanced greatly at thethird day (F = 143.7; df = 4, 10; P < 0,05) (Table 3).

The potency of the chitinase (0.5 mU/ml) in combination with each of three Bt subspecieswas tested against second instar M. brassicae larvae (Table 4). The chitinase without

FIGURE 1 The dynamics of M.brassicae larval mortality. 1 – MbNPV (106 polyhedra ml–1) 2 – MbNPV (107

polyhedra ml–1), 3 – the mixture of MbNPV (106 polyhedra ml–1) with chitinase (0.5 mU ml–1).

TABLE III The influence of Bta with chitinaseb on larval mortalityc ofG. mellonella

Treatment Mortality, %Observation duration (days)

3 5 7

Bt subspecies galleriae 64.4 73.3 86.4Bt + chitinase (0.5 mU ml–1) 95.6 100.0 100.0Bt + chitinase (2.5 mU ml–1) 88.9 93.3 93.2Bt + chitinase (25 mU ml–1) 82.2 95.6 95.5

a The concentration of Bt subspecies galleriae suspension was 1 × 108 spores ml–1.b Chitinase alone had no effect on insect mortality.c Mortality rates were corrected for control mortality.

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14

166 M. V. SHTERNSHIS et al.

entomopathogens had no effect on the insect larvae in the investigated doses. The mosteffective combination was Bt subspecies galleriae plus enzyme. It was shown that thiscombination led to the highest level of M. brassicae mortality at 3d, 5th, 7th and 10th dayof the treatment (F3 = 30.8; F5 = 33.7; F7 = 59.2; F10 = 53; df = 7, 16; P < 0,05), whereas theefficiency of Bt subspecies kurstaki with the chitinase was the lowest (Table 4).

Mixture formulation and bioassay

The insecticidal activity of our mixture was examined against three species of Lepidopteranlarvae: M. brassicae, P. brassicae and P. xyllostella (Table 5). The most significant increaseof M. brassicae mortality was observed on the 7th day after treatment.

When mortality readings were made 3d day after treatment (Table 5) the compositionshowed the synergistic effect for M. brassicae compared to the components alone, whereas

TABLE IV Larval mortality of M. brassicae after exposure to Bt with chitinase

Treatment Mortality, %Observation duration (days)

3 5 7 10

Bt subspecies: dendrolimusa 26.7 36.6 46.7 53.3dendrolimusb + chitinase (0.5 mU ml–1) 33.3 50.0 60.0 70.0kurstakia 10.0 13.3 23.3 46.7kurstakib + chitinase (0.5 mU ml–1) 13.3 16.7 26.7 50.0galleriaea 36.7 53.3 60.0 66.7galleriaeb + chitinase (0.5 mU ml–1) 56.7 63.5 76.7 83.3chitinase (0.5 mU ml–1) 0.0 0.0 0.0 0.0

a The concentration of Bt in suspension was 1 × 108 spores ml–1.b The concentrations of Bt in suspension was 1 × 107 spores ml–1.

TABLE V Larval mortality of insects after exposure to different formulations

Treatment Species ofinsects

Mortality, %Observation duration (days)

3 5 7

Bt subspecies galleriae M. brassicaea 3,3 10.0 33,3(2,5×107 spores ml–1) P. xyllostella 30.0 43,3 63,3

P. brassicae 33,3 46,7 66,6

Bt (2,5×107 spores ml–1) + M. brassicaea 10.0 23,3 46,7chitinase (0,5 mU ml–1) P. xyllostella 50.0 66,7 86,7

P. brassicae 53,3 76,7 83,3

Composition: Bt (2.5×107 spores ml–1) + M. brassicaea 20.0 43,3 83,3chitinase (0,5 mU ml–1) + P. xyllostella 63,3 86,7 100.0MbNPV (1×107 polyhedra ml–1) P. brassicae 66,7 100.0 100.0

Chitinase (0,5 mU ml–1) M. brassicaea 0.0 0.0 0.0P. xyllostella 0.0 0.0 0.0P. brassicae 0.0 0.0 0.0

a third instar larvae were used

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14

ENTOMOPATHOGENS WITH CHITINASE 167

at days 5 and 7, the additive effect was observed (F5 = 3.3; F7 = 23.8; df = 3, 8; P < 0,05). Thesignificant increase of P. xyllostella and P. brassicae mortality was also observed at 5th and7th days after treatment (for P. brassicae: F5 = 83.8; F7 = 345.8; for P. xyllostella: F5 = 166.6;F7 = 88.5; df = 3, 8; P < 0,05).

Field trials

The action of the mixture against three species of cabbage larvae in a field was compared tothe action of commercial Bt-formulation Lepidocide® as a standard. The treatment of fieldplants with the mixture resulted in a significant increase of M. brassicae larval mortality(Table 6) at 3d and 7th day after treatment (F3 = 4.4; F7 = 40; df = 4, 19; P < 0.05). The effectof the mixture against other species of insects tested was equal or to some extent lower incomparison with the commercial formulation.

DISCUSSION

The combined mixtures of Bt and NPV were investigated earlier as a basis for the improvementof microbial insect control (Su, 1982; Tanada, 1984; Geervliet et al., 1991; Kolodny-Hirsch etal., 1997). The results obtained by different groups had some contradictions. Bt and NPVmixtures at different combinations showed not only synergistic or additive effects but alsoantagonistic effects (Geervliet et al., 1991). It was also revealed that the activity of thebiological agents was greatly enhanced by the addition of chitinase to Bt (Smirnoff, 1971), Cry-toxins alone (Regev et al., 1996) or baculoviruses (Shapiro, 1987). In the study reported here,we evaluated the combined action of Bt, MbNPV, and chitinase.

Previous evidence indicated that the insecticidal effect of Bt and some baculoviruses wasenhanced by the addition of chitinase to the mixtures with the optimal concentration0.5 mU ml–1 (Duzhak et al., 1995). Some data showed that the pathological process in theinsects could be under the influence not only of the chitinase but also on the proteolytic(Kumar & Venkateswerlu, 1998; Wang & Granados, 1998) and lipolytic (Ivanov, Kuzmanova& Kamberov, 1990) enzyme activity in the preparation. We examined the possible existanceof such enzymes in the chitinase preparation. All activities were tested at pH 5.5 and 9.5. Theexperimental data revealed that the crude enzyme preparation was characterized only by the

TABLE VI Evaluation of the pathogen-chitinase against cabbage insect pests in the field

Treatment Species ofinsects

Mortality, %Observation duration (days)

3 5 7 10

Composition M. brassicae 16.8 39.1 65.9 75.2P. xyllostella 29.7 45.6 55.6 74.1P. brassicae 32 47.9 66.3 80.4

Lepidocide® M. brassicae 8.4 29.2 41.7 66.9P. xyllostella 44.7 55.6 64.4 74.7P. brassicae 45.3 76.7 86.5 96.2

a Mortality rates were corrected for control mortality.

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14

168 M. V. SHTERNSHIS et al.

chitinase activity (Table 1). Moreover, the neutral effects of the chitinase preparation againstthe larvae and entomopathogens allowed us to exclude the possible contamination of themixture with antibiotics, toxins and biologically active metabolites.

The influence of the separate components (Bt, MbNPV, and the chitinase) on the totalefficiency of the combined preparation was tested against M. brassicae larvae in thelaboratory experiments. The insecticidal activity of the combined preparation in comparisonwith its separate components against larvae showed the synergistic effect at the third and fifthday after feeding. Further laboratory experiments of the proposed formulation for bioassay ofP. brassicae and P. xyllostella larvae revealed a considerable activity of the combinedpreparation against these cabbage pests (Table 5). However, no synergistic effect wasobserved in the bioassay mentioned above. The results presented here were in a goodagreement with our previous data which revealed the enhancement of the MbNPV activity bychitinase (0.5 mU ml–1) on the laboratory insect population (Duzhak et al., 1995) and supportthe hypothesis that the enzyme raised PM permeability. Chitinase alone has no effect oninsect mortality and this is in agreement with other reports (Shapiro et al., 1987; Duzhak etal., 1995; Sampson & Gooday, 1998).

The field trials of the mixture were conducted on cabbage plots infested with three speciesof lepidopteran pests. Our results clearly indicate the efficacy of the mixture against thesepests. The mortality of the larvae varied from 74.1 to 80.4 percent. Previously, Spichenko et.al. (1980) demonstrated that in Western Siberia, a mixture of Bt (1×108 spore ml–1) andMbNPV (1×107 polyhedra ml–1) appeared to be more effective than individual pathogensagainst M. brassicae and P. xyllostella larvae. However, no additive or synergistic effect wasobserved for this mixture. Our results are in good agreement with the findings of Spichenkoet. al. (1980), concerning high efficacy of Bt subspecies galleriae and MbNPV mixtureagainst cabbage pests. However, we demonstrated the synergistic action of the Bt andchitinase against M. brassicae, in addition to the synergistic action of the chitinase withMbNPV and the complementary action of Bt with MbNPV against M. brassicae.Furthermore, the proposed addition of chitinase to Bt and NPV mixtures provides asignificant reduction of entomopathogen concentration. The original combination contains5-fold lower amounts of Bt-spores and 10-fold lower amounts of polyhedra per ml comparedto the values recommended for the standard commercial formulations. Thus the data obtainedin this study clearly indicate the advantage of the mixture, containing Bt, MbNPV and thechitinase in the microbial insect control of Lepidoptera on cabbage.

Acknowledgment

This work was partly supported by The Federal Russian Programme “Integration ofFundamental Research and Education”, (FCP “Integracia”).

References

Abbott, W. S. (1925) A method of computing the effectiveness of an insecticide. Journal of Economic Entomology,18, 265–267.

Berger, L. R. & Reynolds, D. M. (1958) The chitinase system of a strain of Streptomyces griseus. Biochimica etBiophysica Acta, 29, 522–534.

Duzhak, A. B., Salganik, R. I., Shternshis, M. V., Ermakova, N. I., Tsvetkova V. P., Andreeva, I. V. & Kushnikova,T. A. (1995) The influence of enzyme preparation of chitinase on biological insect control. Siberian Journal ofEcology, 2, 457–461.

Federici, B. (1996) Host specificity in microbe-insect interaction. Bioscience, 46, 410–421.

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14

ENTOMOPATHOGENS WITH CHITINASE 169

Geervliet, J. B. F., Vlak, J. M. & Smith, P. H. (1991) Effects of Spodoptera exigua nuclear polyhedrosis virus andBacillus thuringiensis subsp. aizawai mixtures on mortality of beet armyworm. Mededelingen FaculteitLandbouwwetenschappen Rijks Universiteit Gent, 56, 305–311.

Ivanov, A., Kuzmanova, I. & Kamberov, E. (1990) Bacillus thuringiensis phospolipase C: the substrate specificityand entomopathogenicity. Biotechnology Moscow, 5, 69–72.

Kandyibin, N. V. (1989) Bacterial Insect and Rodent Pest Control. Agropromisdat, Moscow.Kaversneva, E. D. (1971) Standard method for determination of proteolytic activities of complex protease

preparation. Pricladnaya biokhimia i microbiologia Moscow, 7, 225–228.Kolodny – Hirsch, D. M., Sitchawat, T., Jansiri, T., Chenrchaivachirakul, A. & Ketunuti, U. (1997) Field evaluation

of a commercial formulation of the Spodoptera exigua (Lepidoptera: Noctuidae) nuclear polyhedrosis virus forcontrol of beet armyworm on vegetable crops in Thailand. Biocontrol Science and Technology, 7, 475–488.

Krieg, A. (1971) The interaction of pathogens, in Microbial Control of Insects and Mites (Burges, H. D. and Hussly,N. W., Eds.) Academic Press, New York, pp. 365–372.

Kumar, N. S. & Venkateswerlu, G. (1998) Endogenous protease-activated 66-kDa toxin from Bacillus thuringiensissubsp. kurstaki active against Spodoptera littoralis. FEMS Microbiology Letters 159, 113–120.

Lehane, M. I. (1997) Peritrophic matrix structure and function. Annual Review of Entomology 42, 525–550.Miller, G. L. (1959) Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Analytical

Chemistry 31, 426–428.Ota, V. & Yamada, K. (1966) Lipase from Candida paralipolytica. Part I. Anionic surfactants as the essential

activator in the systems emulsified by polyvinyl alcohol. Agricultural & Biological Chemistry 30, 351–358.Regev, A., Keller, M., Strizhov, N., Sneh, B., Prudovsky, E., Chet, I., Ginzberg, I., Koncz-kalman, Z., Koncz, C.,

Schell, I. & Zilberstein, A. (1996) Synergistic activity of a Bacillus thuringiensis �-endotoxin and a bacterialendochitinase against Spodoptera littoralis larvae. Applied and Environmental Microbiology 62, 3581–3586.

Sampson, M. N. & Gooday, G. W. (1998) Involvement of chitinases of Bacillus thuringiensis during pathogenesisin insects. Microbiology 144, 2189–2194.

Shapiro, M., Preisler, H. K. & Robertson, I. L. (1987) Enhancement of baculovirus activity on gypsy moth(Lepidoptera: Lymantriidae) by chitinase. Journal of Economic Entomology 80, 1113–1116.

Shapiro, M. & Robertson, I. L. (1992) Enhancement of gypsy moth (Lepidoptera: Lymantriidae) baculovirus activityby optical brighteners. Journal of Economic Entomology 85, 1120–1124.

Schnepf, E., Crickmore, N., Van Rie, I., Lereclus, D., Baum, I., Fietesson, I., Zeigler, D. R. & Dean, D. H. (1998)Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews, 62,775–806.

Shternshis, M. V. (1987a) The pest microbiological control with the intensive technologies of crop cultivation.Vestnic selskokhozyaistvennoi nauki, Moscow, 9, 54–59.

Shternshis, M. V. (1987b) The principles of intensification of microbiological insect control. Mededelingen FaculteitLandbouwwetenschappen Rijks Universiteit Gent 52, 395–401.

Skujins, J., Pukite, A. & Mclaren, A. D. (1973) Adsorbtion and reactions of chitinase and lysozyme on chitin.Molecular and Cellular Biochemistry 2, 221–228.

Smirnoff, W. A. (1971) Effect of chitinase on the action of Bacillus thuringiensis. Canadian Entomologist 103,1829–1831.

Su, T.-M. (1982) Use of bacteria and other pathogens to control insect pests in China, in Microbial and ViralPesticides (Kurstak, E. Ed.) Academic Press, New York, pp. 317–333.

Spichenko, L. N., Gouli, V. V. & Krupko, L. A. (1980) Application of bacterial formulations with VIRIN-EKSagainst lepidopteran cabbage insect pests, in Biological Control of Pests Organisms (Gouli, V. V. Ed.),Novosibirsk, pp. 16–19.

Tanada, Y. (1984) Bacillus thuringiensis: integrated control – past, present and future, in Comparative pathobiology(Cheng, T.C., Ed.) Plenum Publishing Corporation, 7, pp. 59–90.

Wang, P. & Granados, R. R. (1998) Observation on the presence of the peritrophic membrane in larval Trichoplusiani and its role in limiting baculovirus infection. Journal of Invertebrate Pathology 72, 57–62.

Dow

nloa

ded

by [

Ein

dhov

en T

echn

ical

Uni

vers

ity]

at 2

0:06

18

Nov

embe

r 20

14