© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences, Insect Science, 15, 355-360

L. muscarium and insecticides for B. tabaci control 355

355© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences

Insect Science (2008) 15, 355-360, DOI 10.1111/j.1744-7917.2008.00221.x

Introduction

The sweetpotato whitefly, Bemisia tabaci (Gennadius)(Hemiptera: Aleyrodidae) remains a serious threat to cropsworldwide (Landa et al., 1994; Nomikou et al., 2001). Thedamaging B-biotype is of specific economic concern be-cause it is an effective vector of over 111 viruses fromseveral families, particularly geminiviruses (Alegbejo,2000). Other plant damage includes direct feeding scarsand leaf contamination from honeydew excretion (Naranjoet al., 2002).

The UK has Protected Zone status against B. tabaci anderadication generally involves use of chemical insecticides.

Several active ingredients are currently used within the UKfor treatments of B. tabaci outbreaks (Sharaf, 1986; Buxton& Clarke, 1994; Cheek & Macdonald, 1994; Cannon et al.,2005), but with continuing resistance to chemical insecti-cides being shown by B. tabaci populations (Prabhaker etal., 1985; Osborne & Landa, 1992; Cahill et al., 1994,1996; Ahmad et al., 2002) an integrated strategy using bothbiological and chemical agents is urgently required.

The entomopathogenic fungus Lecanicillium muscariumhas shown significant potential for incorporation into inte-grated pest management (IPM) programs for the control ofB. tabaci, where second instar larvae have proven mostsusceptible to fungal infection (Cuthbertson et al., 2005a).Lecanicillum muscarium has also shown potential forcontrol of B. tabaci larvae in glasshouses on a range ofplant hosts, including poinsettia (Cuthbertson & Walters,2005). However, for complete eradication of this quaran-tine species the control of eggs is required. Previousinvestigations into chemical insecticide compatibility with

Correspondence: Andrew G. S. Cuthbertson, Central Sci-ence Laboratory, Sand Hutton, York Y041 1LZ, UK. Tel:+44 (0)1904 462201; fax: +44 (0)1904 462111; email: a.cuthbertson@csl.gov.uk

Further compatibility tests of the entomopathogenic fungusLecanicillium muscarium with conventional insecticideproducts for control of sweetpotato whitefly, Bemisia tabacion poinsettia plants

Abstract The effect on spore germination of the entomopathogenic fungus Lecanicilliummuscarium following direct exposure for 24 h to the insecticides Majestik, Spray Oil, Agri-50E, Savona and Oberon for the control of both egg and second instar stages of thesweetpotato whitefly, Bemisia tabaci, was determined. Exposure to both Agri-50E andOberon was followed by acceptable spore germination. Infectivity rates of L. muscarium onpoinsettia foliage in the presence of dry residues of the insecticides were also investigated.No significant detrimental effects on the levels of control of B. tabaci were recordedcompared with fungus applied to residue-free foliage. Sequential application of the chemi-cals Savona, Spray Oil and Majestik with the fungus all produced mortalities of second instarB. tabaci above 90%. Incorporation of these chemicals with L. muscarium into integratedcontrol programs for B. tabaci is discussed.

Key words entomopathogenic fungus, integrated pest management, insecticidecompatibility, Lecanicillium muscarium

Andrew G. S. Cuthbertson, Lisa F. Blackburn, Phil Northing, Weiqi Luo, Raymond J. C. Cannon andKeith F. A. WaltersCentral Science Laboratory, Sand Hutton, York Y041 1LZ, UK

© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences, Insect Science, 15, 355-360

356 A. G. S. Cuthbertson et al.

L. muscarium have found varying results. Hall (1981)showed that chemicals such as pirimicarb and white oilcould be ‘tank mixed’ with the fungus for the control ofaphids, and similarly Cuthbertson et al. (2005b) provedthat the fungus could be applied simultaneously withbuprofezin, and also, when used sequentially withimidacloprid gave a high percentage mortality of B. tabacisecond instar larvae. To provide growers and their consult-ants with sufficient information on what to base commer-cially viable control decisions, data on the efficacy of awider range of insecticides for the control of both B. tabacieggs and larvae and their compatibility with L. muscariumis urgently required. This study further investigates thecompatibility of L. muscarium with a range of insecticidalproducts that can be used against both egg and secondinstar larvae of B. tabaci.

Materials and Methods

Products and insect cultures

A commercial formulation of L. muscarium (Mycotal,Koppert Biological Systems Ltd, UK; identified as Ver-ticillium lecanii on product label) was used in allexperiments. Following the technique of Cuthbertson et al.(2005a,b), B. tabaci were cultured under strict quarantineconditions in perspex cages (60 cm × 60 cm × 80 cm) onpoinsettia (Euphorbia pulcherrima c.v. Lilo Pink) plants at23 ± 1 oC.

The effect of direct exposure of L. muscarium toconventional insecticides, soap and spray oil products

Following the protocol of Cuthbertson et al. (2005b) theeffect of direct suspension of the fungal spores in insecti-cide solutions was investigated. Lecanicillium muscariumconidia were suspended (107 conidia/mL) in solutions offive conventional insecticides commonly used against B.tabaci in the UK. All insecticides were diluted to recom-mended rates for application to protected ornamentals inthe UK. The suspensions were transferred to beakers,sealed with parafilm and incubated in the dark at 20 oC for24 h after which 10 μL of each mixture was pipetted ontoa sterile Petri dish containing 10% non-bacterial agar. Thedishes were sealed with parafilm and again incubated in thedark for a further 24 h at 20 oC before viability of conidia(germinated spores) from a total of 200 randomly chosenconidia were assessed under the microscope. Each experi-ment (insecticide solution) consisted of two replicates eachfrom three different batches of fresh dilution in order toreplicate the work over time and space (six replicates in

total). The above procedure was repeated using: Majestik(natural plant extract, 2.5 mL/100 mL water, Certis UK);Agri-50E (alginate/polysaccharide, 300 μL/100 mL, FargoLtd, UK); Spray Oil (petroleum oil, 1 mL/100 mL water,Hortichem Ltd, UK); Savona (fatty acids, 2 mL/100 mLwater, Koppert Biological Systems Ltd, UK); Oberon(spiromesifen, 0.05 g/100 mL water, Bayer CropScienceLtd, UK). Separate suspensions of L. muscarium in bothwater and water with a 0.02% solution of the non-ionicwetting agent Agral (Syngenta Crop Protection Ltd,Cambridge, UK; active ingredient: alkyl phenol ethyleneoxide) acted as controls.

The effect of chemical residues on the infectivity of foliarapplications of L. muscarium

Following the protocols of Cuthbertson et al. (2003a,b;2005a,b), 28 poinsettia plants were each infested with twomale and five female B. tabaci adults contained in each offour individual clip cages modelled on those described byMacGillivray and Anderson (1957). The plants were thenincubated for a pre-determined period of time to allow egglaying (Butler et al., 1983; Bethke et al., 1991; Wang &Tsai, 1996). Plants were then divided into two groups,treatment and control, each with 14 plants. Within eachgroup, two plants were assigned for each product treatment.A combination of two sequential treatments as outlined inCuthbertson et al. (2003a; 2005b) were applied to eachplant. The first application consisted of either an insecti-cide applied at the recommended dose rate for applicationor a water control. The plants were sprayed to run-off andthe leaves allowed to dry before returning to the conditionsdefined for infestation of B. tabaci. The second treatmentwas applied 24 h later and consisted of either a suspensionof 107 conidia/mL with 0.02% of the non-ionic wettingagent Agral or a water control, each sprayed until run offusing a Hozelock® Polyspray 2 hand-held sprayer with acone nozzle (Hozelock Ltd., Aylesbury, UK). There wereapproximately 1.5 × 105 conidia per cm2 of leaf surface.Following the second treatment, the plants were returned tothe environmental chamber while still wet and maintainedat 20 ± 1 oC, 85% RH and a 12: 12 L: D period (treatmentswere treated at the start of the dark period). Mortality ofeggs (taken as unhatched) was assessed after 7 days. Theabove procedure was then repeated to test efficacy againstsecond instar larvae, the nymphal stage deemed mostsusceptible to fungal infection (Cuthbertson et al., 2005a).

Assessment of efficacy

The numbers of live and dead (defined by the presence ofmycelial mass where L. muscarium was applied) larvae

© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences, Insect Science, 15, 355-360

L. muscarium and insecticides for B. tabaci control 357

were recorded 7 days after the second treatment for eachreplicate in each treatment group. Treated eggs were alsoassessed 7 days after treatment and noted as live (hatched)or dead (unhatched). Data underwent the non-parametricKruskal-Wallis rank sum test to compare the effect of thedifferent insecticides.

Results

Direct suspension of L. muscarium in chemical products

There was a significant difference in the percentagespore germination of L. muscarium following directexposure to the different active ingredients (Fig. 1).Following exposure to Agri-50E spore germination wasgood (79%); however, it was still significantly lowerthan that recorded for the water control (Kruskal-Wallischi-squared = 6.2, df =1, P = 0.013). Exposure toMajestik, Savona and Spray Oil had spore germinationpercentages (6.8%, 23% and 47.5% respectively)that were all significantly lower than the watercontrol (Kruskal-Wallis chi-squared = 13.0, df =1,P < 0.001). Spore germination following exposureto Oberon was higher (64%), but still significantlylower than in the water control (Kruskal-Wallis chi-squared = 8.3, df = 1, P = 0.004).

Responses of L. muscarium to dry insecticide residues

Mortality of Bemisia tabaci eggs Sequential applica-tion of a chemical product followed 24 h later with anapplication of L. muscarium resulted in increased mortality

of B. tabaci eggs in all cases (Fig. 2A), for example,significantly with Oberon, (Kruskal-Wallis chi-squared =5.3, df = 1, P = 0.02) compared to the application ofchemical alone. Only two treatments (Spray Oil on its ownand Spray Oil and fungus) resulted in significantly highermortality than the fungus control (Kruskal-Wallis chi-squared = 5.7, df = 1, P = 0.02). In two cases (Savona andSpray Oil) the chemical on its own and the sequentialtreatment resulted in almost the same level of mortality.Sequential application of Agri-50E and Majestik with thefungus resulted in increased mortality of B. tabaci eggs;however, only Agri-50E produced a significant increase(Kruskal-Wallis chi-squared = 5.3, df = 1, P = 0.02 andKruskal-Wallis chi-squared = 3, df = 1, P = 0.08,respectively). Agri-50E was noted to cause some foliagedamage (yellowing and curling of leaves) to plant material(Cuthbertson, A.G.S., pers. observ.).

Mortality of second instar Bemisia tabaci Highermortalities were recorded for all chemical products testedagainst second instar B. tabaci than eggs (Fig. 2B). Allchemical products produced over 80% mortality of lar-vae (Fig. 2B) except Oberon and Agri-50E (68% and61% respectively). The addition of the fungus in sequen-tial treatments in some cases (Spray Oil and Majestik)resulted in further increases in mortality. Sequentialapplications of fungus with Savona, Spray Oil andMajestik all resulted in mortalities of greater than 90%.However, only Spray Oil with sequential application ofthe fungus caused significantly higher B. tabaci mortal-ity than application of the chemical product alone(Kruskal-Wallis chi-squared = 4.1, df = 1, P = 0.04).

Discussion

For the successful introduction of any IPM program, com-prising either invertebrate or pathogenic control agents,information is not only needed on the biology of thebiological agent in question but also on its potential to becombined with other control agents, including chemicals(Cuthbertson et al., 2003c; Cuthbertson & Murchie, 2006a,b). Clarification of the effects of chemical insecticides onthe wide variety of entomopathogenic fungi is necessary,as few in-vitro tests have been performed (Olmert &Kenneth, 1974; Hall, 1981; Anderson & Roberts, 1983; Liet al., 2004; Cuthbertson et al., 2005b). Variousbiopesticides now based on L. muscarium (formerly Ver-ticillium lecanii) are often utilised in greenhouses to man-age a range of invertebrate pest species (Osborne & Landa,1992). Entomopathogenic fungi have the capacity to sup-press and, and in some cases, to provide good control ofwhiteflies in both greenhouse and field crops (Cuthbertson

Fig. 1 Lecanicillium muscarium spore germination following24 h exposure of the entomopathogenic fungus to chemicalinsecticides: Oberon (0.05 g/100 mL water); Savona (2 mL/100 mLwater); Spray Oil (1 mL/100 mL water); Agri-50E (300 μL/100mL water); Majestik (2.5 mL/100 mL water); Agral control(200 μL/1L water); or water control. Bars are standard errors (±)of the mean.

© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences, Insect Science, 15, 355-360

358 A. G. S. Cuthbertson et al.

Fig. 2 The effect of applications of Lecanicillium muscarium (ca. 1.5 × 105 spores per cm2 of leaf area) to poinsettia plants with chemicalresidues on the mortality of (A) Bemisia tabaci eggs and (B) second instar B. tabaci larvae. The second treatment application was applied24 h following the first treatment. Bars are standard error (±) of the mean.

& Walters, 2005). Therefore, there is a need to determinethe potential for combining entomopathogenic biocontrolagents with chemical insecticides to form components ofIPM strategies.

This study investigated the compatibility of L. muscariumwith several insecticide products commonly used withinthe UK for B. tabaci control. All of the insecticide products

tested were applied at the highest legal UK applicationrates to establish a worse case test: lower rates of insecti-cide use may have lower impacts on the activity of L.muscarium. Direct exposure of L. muscarium for 24 h toMajestik, Spray Oil and Savona resulted in very low sporegermination, unsuitable for commercial use (Cuthbertsonet al., 2005b). However, the chemicals Oberon and Agri-

© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences, Insect Science, 15, 355-360

L. muscarium and insecticides for B. tabaci control 359

50E produced acceptable spore germination. This studyhas shown that direct‘tank-mixing’of L. muscarium withthese products is a viable IPM option.

The implementation of an IPM scheme may requiresequential rather than simultaneous applications of insec-ticides and entomopathogenic fungi (Hall, 1981). Apartfrom the study of Cuthbertson et al. (2005b) few previousstudies have tested the effect of dry insecticide residue onfungal activity. In the current study, when L. muscariumwas applied to plants sprayed 24 h earlier with a standardapplication of one of five contact insecticides, no signifi-cant reduction in infectivity (mycelial growth) was detected.Therefore, L. muscarium could be applied sequentiallywith Oberon, Savona, Spray Oil, Agri-50E and Majestikfor the control of B. tabaci, with second instars againproving highly susceptible to fungal attack, as found byCuthbertson et al. (2005a). However, after application ofAgri-50E commercially unacceptable foliage damage wasrecorded (Cuthbertson, A.G.S., pers. observ.). It is there-fore unlikely that this product would be a candidate forfurther research into development of IPM strategies forpoinsettia plants. However, other plant species may proveto be more tolerant of this product and therefore investiga-tion of its use on different host plants may prove viable.

In summary, this study has added significantly to theknowledge base (Cuthbertson et al., 2005b), concerningthe compatibility of L. muscarium with chemicalinsecticides. Two approaches have been identified. First,Oberon can be applied simultaneously with L. muscariumfor control of B. tabaci eggs on poinsettia plants. Second,significantly higher levels of control of second instarB. tabaci can be achieved by the application of L. muscariumto poinsettia foliage previously treated with Spray Oil thanafter application of either treatment alone.

Acknowledgments

We thank Koppert Biological Systems Ltd. (UK) for sup-plying the formulation of Lecanicillium muscarium(marketed as Verticillium lecanii on product label) used inthis study and Mr. Richard Natt (CSL HorticulturalManager) and his team for the provision of plant material.The work was funded by Plant Health Division, Defra.Bemisia tabaci were held under quarantine license number:PHL 251B/5328(02/2006) Amended (04/2006).

References

Ahmad, M., Arif, M.I., Ahmad, Z. and Denholm, I. (2002) Cottonwhitefly (Bemisia tabaci) resistance to organophosphate and

pyrethroid insecticides in Pakistan. Pest Management Science,58, 203-208.

Alegbejo, M.D. (2000) Whitefly transmitted plant viruses inNigeria. Journal of Sustainable Agriculture, 17, 99-109.

Anderson, T.E. and Roberts, D.W. (1983) Compatibility ofBeauveria bassiana isolates with insecticide formulationsused in Colorado potato beetle (Coleoptera: Chrysomelidae)control. Journal of Economic Entomology, 76, 1437-1441.

Bethke, J.A., Paine, T.D. and Nuessly, G.S. (1991) Comparativebiology, morphometrics and development of two populationsof Bemisia tabaci on cotton and poinsettia. Annals of theEntomological Society of America, 84, 407-411.

Butler, G.D., Henneberry, T.J. and Clayton, T.E. (1983) Bemisiatabaci: development, oviposition, and longevity in relation totemperature. Annals of the Entomological Society of America,76, 310-313.

Buxton, J. and Clarke, A. (1994) Evaluation of insecticide dips tocontrol Bemisia tabaci on poinsettia cuttings. Pesticide Science,42, 141-142.

Cahill, M., Byrne, F.J., Denholm, I., Devonshire, A.L. andGorman, K.J. (1994) Insecticide resistance in Bemisia tabaci.Pesticide Science, 42, 137-139.

Cahill, M., Gorman, K., Day, S., Denholm, I., Elbert, A. andNauen, R. (1996) Baseline determination and detection ofresistance to imidacloprid in Bemisia tabaci (Homoptera:Aleyrodidae). Bulletin of Entomological Research, 86, 343-349.

Cannon, R.J.C., Eyre, D., MacLeod, A., Matthews, L., Malumphy,C., Cheek, S. and Bartlett, P.W. (2005) Interceptions andoutbreaks of Bemisia tabaci in the UK. The BCPC Interna-tional Congress - Crop Science and Technology 2005. Iasgow,UK, October 31-November 2. Congress Proceedings Volume2, 1007-1012. ISBN 1 901396 65 7.

Cheek, S. and Macdonald, O. (1994) Management of Bemisiatabaci. Pesticide Science, 42, 135-137.

Cuthbertson, A.G.S. and Murchie, A.K. (2006a) A preliminarystudy into the direct effect of chemical pesticides on thepredatory mite Anystis baccarum. International Journal ofUltra Scientists of Physical Sciences, 18, 177-180.

Cuthbertson, A.G.S. and Murchie, A.K. (2006b) Environmentalimpact of an orchard winter wash and early season pesticideapplications on both a beneficial and a pest mite species inBramley apple orchards. International Journal of Environ-mental Science and Technology, 3, 333-339.

Cuthbertson, A.G.S. and Walters, K.F.A. (2005) Pathogenicity ofthe entomopathogenic fungus, Lecanicillium muscarium, againstthe sweetpotato whitefly Bemisia tabaci under laboratory andglasshouse conditions. Mycopathologia, 160, 315-319.

Cuthbertson, A.G.S., Head, J., Walters, K.F.A. and Murray, A.W.A. (2003a) The integrated use of chemical insecticides and theentomopathogenic nematode, Steinernema feltiae, for the con-trol of sweetpotato whitefly, Bemsia tabaci. Nematology, 5,

© 2008 The AuthorsJournal compilation © Institute of Zoology, Chinese Academy of Sciences, Insect Science, 15, 355-360

360 A. G. S. Cuthbertson et al.

713-720.Cuthbertson, A.G.S., Head, J., Walters, K.F.A. and Gregory, S.A.

(2003b) The efficacy of the entomopathogenic nematode,Steinernema feltiae, against the immature stages of Bemisiatabaci. Journal of Invertebrate Pathology, 83, 267-269.

Cuthbertson, A.G.S., Fleming, C.C. and Murchie, A.K. (2003c)Detection of Rhopalosiphum insertum (apple-grass aphid)predation by the predatory mite Anystis baccarum using mo-lecular gut analysis. Agricultural and Forest Entomology, 5,219-225.

Cuthbertson, A.G.S., Walters, K.F.A. and Northing, P. (2005a)The susceptibility of immature stages of Bemisia tabaci to theentomopathogenic fungus Lecanicillium muscarium on to-mato and verbena foliage. Mycopathologia, 159, 23-29.

Cuthbertson, A.G.S., Walters, K.F.A. and Deppe, C. (2005b)Compatibility of the entomopathogenic fungus Lecanicilliummuscarium and insecticides for eradication of sweetpotatowhitefly, Bemisia tabaci. Mycopathologia, 160, 35-41.

Hall, R.A. (1981) Laboratory studies on the effects of fungicides,acaricides and insecticides on the entomopathogenic fungus,Verticillium lecanii. Entomologia Experimentalis andApplicata, 29, 39-48.

Landa, Z., Osborne, L., Lopez, F. and Eyal, J. (1994) A bioassayfor determining pathogenicity of entomogenous fungi onwhiteflies. Biological Control, 4, 341-350.

MacGillivray, M.F. and Anderson, G.B. (1957) Three usefulinsect cages. Canadian Entomologist, 89, 43-46.

Naranjo, S.E., Ellsworth, P.C., Chu, C.C. and Henneberry, T.J.(2002) Conservation of predatory arthropods in cotton: role ofaction thresholds for Bemisia tabaci (Homoptera: Aleyrodidae).

Journal of Economic Entomology, 95, 682-691.Nomikou, M., Janssen, A., Schraag, R. and Sabelis, M.W. (2001)

Phytoseiid predators as potential biological control agents forBemisia tabaci. Experimental and Applied Acarology, 25,271-291.

Olmert, I. and Kenneth, R.G. (1974) Sensitivity of theentomopathogenic fungi, Beauveria bassiana, Verticilliumlecanii, and Verticillium sp. to fungicides and insecticides.Environmental Entomology, 3, 33-38.

Osborne, L.S. and Landa, Z. (1992) Biological control of white-flies with entomopathogenic fungi. Florida Entomologist, 75,456-471.

Prabhaker, N., Coudriet, D.L. and Meyerdirk, D.E. (1985) Insec-ticide resistance in the sweetpotato whitefly, Bemisia tabaci(Homoptera: Aleyrodidae). Journal of Economic Entomology,78, 748-752.

Sharaf, N. (1986) Chemical control of Bemisia tabaci. Agricul-ture Ecosystems and Environment, 17, 111-127.

Wang, K.H. and Tsai, J.H. (1996) Temperature effect on devel-opment and reproduction of silverleaf whitefly (Homoptera:Aleyrodidae). Annals of the Entomological Society of America,89, 375-384.

Li, W., Wang, X.F. and Sheng, C.F. (2004) Impact of sixteenchemical pesticides on conidial germination of twoentomophthoralean fungi: Conidiobolus thromboides andPandora nouryi. Biocontrol Science and Technology, 14,737-741.

Accepted February 18, 2008