Biological Control 63 (2012) 135142

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Biological Control
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Effect of bee-vectored Beauveria bassiana on greenhouse benecials under greenhouse cage conditions
Les Shipp a,, Jean Pierre Kapongo a, Hong-Hyun Park b, Peter Kevan c
a

Agriculture and Agri-Food Canada, Greenhouse and Processing Crops Research Centre, Harrow, ON, Canada N0R 1G0 Crop Protection Division, National Academy of Agricultural Science, RDA, Suwon, 441-707, Republic of Korea c School of Environmental Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1
b

h i g h l i g h t s
" Evaluated bee-vectored Beauveria

g r a p h i c a l a b s t r a c t

bassiana on greenhouse parasitoids and predators. " Mortality levels were similar between treatments for all benecials except Orius. " Parasitism and predation levels were not different between treatments. " Bee vectoring of B. bassiana can be integrated with biological control programs.

a r t i c l e

i n f o

a b s t r a c t
Beauveria bassiana ([Balsamo] Vuillemin [Ascomycota: Hypocreales] Botanigard 22WP formulation]) shows potential as a bee-vectored microbial control agent for control of insect pests on greenhouse tomato and sweet pepper. To integrate this control strategy with existing benecials used in greenhouse vegetable production, it is important to determine the impact of bee vectored Beauveria on commonly used biocontrol agents in greenhouse crops. Therefore, greenhouse trials were conducted to investigate the impact of bee-vectored B. bassiana on the survivorship and parasitism/predation level of Encarsia formosa Gahan (Hymenoptera: Aphelinidae), Eretmocerus eremicus Rose & Zolnerowich (Hymenoptera: Aphelinidae), Aphidius colemani Viereck (Hymenoptera: Braconidae), Orius insidiosus (Say) (Heteroptera: Anthocoridae) and Amblyseius swirskii (Athias-Henriot) (Acari: Phytoseiidae). Two treatments were evaluated: (bee-vectored inoculum [1.37 1010 conidia/g of inoculum] and a control [bees but no inoculum]). The experimental design was a completely randomized block design with three to four replications per treatment over time. The commercial bumblebee pollinator, Bombus impatiens (Cresson) (Hymenoptera: Apidae), was used to vector the microbial control agent to the crop. Over 95% of the ower and leaf samples from the tomato and sweet pepper crops contained detectable levels of Beauveria spores. No signicant differences in mortalities were found between the two treatments for the parasitoid species and A. swirskii. Mortality for O. insidiosus was signicantly greater in the Beauveria treatment compared to the control treatment. Parasitism and predation levels were not signicantly different between to the two treatments. Also, Beauveria did not signicantly impact bee mortality compared to the control treatment. Thus, based on the results presented in the current study, bee vectoring of the entomopathogen, B. bassiana, should be compatible with many of the benecial arthropod control agents that are used in greenhouse integrated pest management. Crown Copyright 2012 Published by Elsevier Inc. All rights reserved.

Article history: Received 31 January 2012 Accepted 13 July 2012 Available online 23 July 2012 Keywords: Amblyseius swirskii Aphidius colemani Encarsia formosa Eretmocerus eremicus Orius insidiosus Beauveria bassiana Bombus impatiens

Corresponding author. Fax: +1 519 738 1235.

E-mail addresses: shippl@agr.gc.ca, Les.Shipp@agr.gc.ca (L. Shipp). 1049-9644/$ - see front matter Crown Copyright 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.07.008

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1. Introduction The use of fungal biological control agents is a rapidly developing eld and is increasingly being adopted and accepted worldwide for management of agricultural pests (Butt et al., 2001; Shah and Pell, 2003; Lacey and Kaya, 2007; Hajek and Delalibera, 2010; Jaronski, 2010). Recently, the phylogenetic classication of fungi, including Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales), is being modied, especially with the use of molecular tools in order to better understand the role of fungi in the environment and how they interact with their hosts (Rehner and Buckley, 2005; Vega et al., 2009). Beauveria bassiana sensu lato is a commonly-used entomopathogenic fungus with a wide host range of >700 (Inglis et al., 2001) including pests such as whiteies, aphids, thrips, corn borer, coffee-berry borer, boll worms, pine caterpillar to Colorado potato beetle (Butt et al., 2001; Shah and Pell, 2003). With a broad host range for B. bassiana, it is important to determine its impact on benecial arthropods (i.e. parasitoids, and predatory bugs and mites). In laboratory trials, there has been numerous reports of B. bassiana infecting benecial insects, including honey bees, parasitic wasps, coccinellids, and predatory bugs and mites (Goettel et al., 1990; James and Lighthart, 1994; Ludwig and Oetting, 2001; Jaronski et al., 2003). Shipp et al. (2003) reported infection levels of 3183% for Encarsia formosa Gahan (Hymenoptera: Aphelinidae), Eretmocerus eremicus Rose & Zolnerowich (Hymenoptera: Aphelinidae), Aphidius colemani Viereck (Hymenoptera: Braconidae), Dacnusa sibirica Telenga (Hymenoptera: Braconidae), Aphidoletes aphidimyza (Rondani) (Diptera: Cecidomyiidae) and Orius insidiosus (Say) (Heteroptera: Anthocoridae) in Petri dish bioassay trials in the laboratory. However, there can be a substantial difference between the physiological host range versus the ecological host rage for a microbial pathogen (Hajek and Butler, 2000). The physiological host range is the list of insect species that can be infected in the laboratory. The ecological host range represents what happens in nature or under eld conditions. With coccinellids, Olla v-nigrum Mulsant (Coleoptera: Coccinellidae) and Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) were not susceptible to the commercial GHA strain of B. bassiana. However, natural B. bassiana isolates from O. v-nigrum were pathogenic to adult O. v-nigrum but not to adult H. axyridis (Cottrell and Shapiro-Ilan, 2003). Traditionally, B. bassiana is thought as a soil fungus and a major mortality factor for Coccinella septempunctata L. (Coleoptera: Coccinellidae) which spends its overwintering months under leaf litter (Roy et al., 2008). Recently, Ormond et al. (2010) found using molecular analyses (ISSR-PCR) that B. bassiana is genetically diverse with both below- ground and above-ground isolates. In laboratory bioassays, it is interesting that adult C. septempunctata demonstrated avoidance behavior to B bassiana on leaf surfaces, in soil and in mycosed C. septempunctata (Ormond et al., 2011). Thus, this defensive behavior may be an adaptation by C. septempunctata to avoid soil and leaf litter in which the density of B. bassiana is high as a survival mechanism. Trials have also shown that the generalist predator, Anthocoris nemorum L. (Heteroptera: Anthocoridae), detects and avoids B. bassiana when it forages on host plants (Meyling and Pell, 2006). In eld trials with honeybee colonies versus single bee trials in the laboratory, exposure of the hives to recommended rates of Beauveria (5 1013 conidia/ha) three times at 5-day interval resulted in an infection rate of 1.2% of the treated bees versus >50% mortality due to Beauveria in laboratory bioassays using lower B. bassiana exposure rates (Jaronski et al., 2003). Similar results were found for A. colemani, E. formosa, O. insidiosus and Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) under greenhouse conditions versus laboratory trials where infection levels decreased by

3488% under greenhouse conditions (Ludwig and Oetting, 2001). Also, Jaronski et al. (1998) reported infection levels of ca. 10% for Orius sp. and chrysopids collected from cotton elds sprayed with B. bassiana (GHA strain). In eld situations, many factors can affect the susceptibility and impact that a Beauveria application can have on benecials including its isolates, insect behaviour, crop type and canopy structure, microclimate and method of application. Since the registration of B. bassiana (GHA strain) in Canada in 2009, B. bassiana is becoming a more common control strategy for greenhouse growers. Both vegetable and ornamental growers use this management option in integrated pest management programs with other biological control agents (parasitoids and predators). Pollinator biocontrol vector technology is a new application system for the delivery of microbial control agents to crops for pest control and disease suppression (Kevan et al., 2008). This delivery system has been demonstrated for bacteria, fungi and viruses (Gross et al., 1994; Butt et al., 1998; Jyoti and Brewer, 1999; Carreck et al., 2007). B. bassiana (GHA strain) shows potential as a bee vectored microbial control agent for control of whiteies, thrips, aphids and Lygus on greenhouse tomatoes and sweet pepper (Al-mazraawi et al., 2006a,b; Kapongo et al., 2008a,b). Also, during the greenhouse cage trials, B. bassiana had a minimal impact on the bumblebee vector, Bombus impatiens (Cresson) (Hymenoptera: Apidae) (Kapongo et al., 2008a,b). To integrate the bee vectoring of microbial control agents with parasitoids and predators, it is important to determine the impact of bee applied Beauveria on commonly used biocontrol agents in greenhouse crops. Therefore, this study investigates the impact of bee-vectored B. bassiana on the survivorship and parasitism/predation level of E. formosa, E. eremicus, A. colemani, O. insidiosus and Amblyseius swirskii (AthiasHenriot) (Acari: Phytoseiidae).

2. Material and methods 2.1. Plant, insect and mite cultures Tomato, Lycopersicon esculentum Mill. (Solanaceae) (cv. Big Dena) and sweet pepper, Capsicum annuum L. (Solanaceae) (cv. Fascinato) plants used in the trials were grown in 20 cm pots containing a mixture of 1:1 sandy loam soil and peat moss. The plants were placed in a separate greenhouse compartment until they produced two sets of owers before being transferred to the trial cages. The plants were maintained at 2123 C and 8085% RH during the trials using an Argus Computer Control System (Argus Control Systems Ltd., White Rock, BC), and were watered and fertilized with Harrow Fertigation Manager (Labbate Climate Control Systems, Leamington, ON) as per standard practices set for commercial greenhouses (Ontario Ministry of Agriculture, Food and Rural Affairs, 2010). Bumblebee (B. impatiens) colonies, provided by Biobest Canada Ltd., Leamington, ON, were used in the cage trials. Each colony consisted of 1 queen and 50 workers. The bees inside the hive were anaesthetized with CO2 and the bees and their nest were transferred from the commercial hive to a wooden hive (32 23 24 cm) tted with an inoculum dispenser (Kapongo et al., 2008a). The bees were kept in the wooden boxes for two days being fed only a sugar solution (50% wt/wt water) to allow the bees to become familiar with the new hive. To assess the parasitism levels of the parasitoids being evaluated, tomato and pepper plants were articially infested with immature whiteies and aphids. The second instar greenhouse whiteies, Trialeurodes vaporariorum Westwood (Homoptera: Aleyrodidae), used in the trials were obtained by collecting adult whiteies from a greenhouse colony at the Greenhouse and

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Processing Crops Research Centre (GPCRC), Harrow, ON and articially infesting tomato plants being grown for the cage trials. To have enough second instars (50100) per plant, the growing point of the plant was enclosed for three days in an organdie sleeve (300 lm mesh) that contained 80 adult whiteies (Fig. 1). After two weeks, the infested plants were transferred into the experimental cages. Sweet pepper plants were infested with green peach aphids, Myzus persicae (Sulzer) (Hemiptera: Aphididae), that were collected from a laboratory colony maintained on pepper plants in a growth chamber (25 C, 60% RH, 16: 8 [L: D]) at GPCRC. Forty third instars aphids, from the laboratory colony, were directly transferred with a ne camel hair brush to the underside of the third leaf from the top of the plant. The aphids were covered with a clip cage (8 cm diameter) to conne them to the leaf until they settled on the new leaf. The clip cage was removed after 24 h. In each trial, infested plants with whiteies or aphids were introduced into the cages to evaluate the parasitism levels. For the predation bioassays, the prey source was rst instar western ower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). To obtain the desired prey stage, adult F. occidentalis were collected from a greenhouse colony that was maintained on yellow potted chrysanthemum (Dendranthema grandiora Tzvelev, var. Chesapeake) at GPCRC and placed on kidney bean (Phaseolus vulgaris L.) leaf disks embedded in 1.5% (w/v) agar Petri dishes (9 cm diameter) with thrips proof screened lids in a controlled environment chamber at 25 1 C. Forty female thrips per leaf disk were held on the leaf disk for two days for egg laying. First instar larvae were obtained four days later, and transferred with a ne brush to the experimental arena for the predation trials. The parasitoid wasps and predators used in the cage trials (A. colemani, E. formosa, E. eremicus, O. insidious, and A. swirskii) were obtained from Biobest Canada Ltd. (Leamington, ON). For A. colemani, newly emerged adults from mummies supplied in the commercial product were released. In the case of E. formosa and E. eremicus, parasitized whitey pupae which were glued on cards were directly introduced into the cages. The O. insidious adults were extracted from the vermiculite mixture in the product bottle and released. The A. swirskii was supplied as loose mites in bulk vermiculite product and were counted using a measuring spoon under the dissecting microscope and released.

2.2. Microbial inoculum A commercial formulation of Beauveria bassiana, GHA strain (BotaniGard 22WP, Laverlam International Corporation, Butte, MT) containing 4.4 1010 conidia of B. bassiana/g of product was used as the microbial control agent in the trials. Viability of the B. bassiana was determined before making the inoculum for each trial (Kapongo et al., 2008a). This allowed re-adjustment of the amount of the product used to make the appropriate concentration for each trial. Spore germination from the viability tests ranged from 95% to 98% for each of the trials. The active inoculum of B. bassiana was made by mixing BotaniGard 22WP and autoclaved corn our (<100 lm particle size) for a nal concentration of 1.37 1010 conidia of B. bassiana/g of inoculum. 2.3. Greenhouse cage trials The cage trials were conducted in two greenhouse compartments (13 8 m) containing two to three ne-mesh screened cages (520 240 220 cm high) per compartment at GPCRC from spring through autumn of 2008. Each cage (treatment) contained 32 potted sweet pepper or tomato plants that were arranged in two double rows, each with eight plants per row. Each double row contained three plants that were articially infested with second instar whiteies (for trials with tomato) or third instar of green peach aphids (for trials with sweet pepper). Each natural enemy species was evaluated with two treatments (bee-vectored inoculum [1.37 1010 conidia/g of inoculum] and a control [bees but no inoculum]). The experimental design was a completely randomized block design with three to four replications per treatment over time. Each colony of bumblebees was introduced on day 1 after the plants, including infested plants, were placed in the cages. On day 2, 1,000 E. formosa, E. eremicus, A. colemani, and A. swirskii or 750 O. insidious were released per cage. The wooden inoculum dispensers (21 12 8.5 cm height) lled with 25 g of the inoculum were attached on day 3 to the exit/entry opening of the wooden hives after which the bees were allowed to forage and disseminate the inoculum. On day 6, the hives were closed and the dispensers were removed. The bees were fed pollen patties (pollen grains mixed with 50% wt/wt sugar solution) until the dispensers were replaced. After that, the rst set of samples was collected. At each sampling date, 60 parasitoids or 70 predators per cage were collected to determine percentage mortality, predation level, internal inection level of the benecials, and the number of B. bassiana propagules per group of ve individuals. It was not always possible to collect 70 predators, especially A. swirskii, at each sampling period. Also, ve bees were collected individually per treatment as they exited the dispenser to determine the number of B. bassiana propagules per each bee. Six individual owers and leaves were collected from six plants (three in each double row) per cage which were selected randomly at each sampling date. One ower and one leaf were collected from the same selected plant. In addition, six whitey or aphid infested plants were removed and replaced with six new infested plants. On day 10, the dispensers were relled with the B. bassiana inoculum and the hives were open for the bees to forage and disseminate the inoculum. On day 13, the hives were closed and the dispensers were removed. After that, the second set of samples was collected. 2.4. Sample processing and assessment of mortality

Fig. 1. An organdy sleeve, leaf cage (300 lm mesh) afxed to a young leaf of tomato plant containing 80 adult whiteies. The leaf cages were used to obtain second instar nymphs for parasitism trials after exposure to Encarsia and Eretmocerus in the cage trials.

For each sampling date, 30 individuals of each parasitoid species (E. formosa, E. eremicus, and A. colemani) were individually placed in vented plastic vials to examine daily mortality. The parasitoids were fed honey that was provided in the form of a streak

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on the underside of the vial lid. The honey was renewed every second day and vials were kept in a controlled environment chamber at 25 1 C, 80 10 RH for 7 days, the time necessary for spores of B. bassiana to induce death of the host. Thirty individuals of O. insidious were individually maintained in vented plastic vial in which a young bean stem was placed and a honey streak was placed on the lid of plastic vial. The bean stem and honey streak were renewed every second day. Mortality was checked daily for 7 days. Thirty individuals of A. swirskii were individually conned on a bean leaf disk (1.5 1.5 cm) that was oated upside down on a water saturated sponge in a 60 ml cup (Solo, Highland Park, IL) and checked daily for mortality. The bioassay lasted 7 days. Both predatory species were kept in a controlled environment chamber at 25 1 C (day) and 20 1 C (night), 60 5 RH, 16: 8 (L: D) during the bioassay. Dead benecials were placed on solid selective media for B. bassiana culture (Beilharz et al., 1982) and incubated at 25 C for 7 days. The appearance of white mycelium on the dead insects or mites indicated B. bassiana infection. Twenty individuals of each natural enemy species (by groups of ve), and the six ower, six leaf, and ve bee samples were washed separately, serially diluted, and plated on solid selective media to determine the number of externally carried spores (Kapongo et al., 2008a). The remaining 10 individuals of each natural enemy species per cage were surface-disinfected to determine internal infection rate. After disinfection, they were plated on selective media to assess for the presence of mycelia (Shipp et al., 2003). Mortality of the bumblebees used in the trials was determined after the second sampling period. At this time, each bumblebee colony was transferred back into the original commercial hive boxes and fed pollen patties for three weeks to simulate a typical time period in which the hives would be kept in commercial greenhouses. The hives were maintained in controlled environment chambers at 23 1 C. Incidence of bee mortality was recorded weekly. 2.5. Assessment of parasitism and predation levels To assess the parasitism level of E. formosa, E. eremicus, and A. colemani, six infested plants per sampling period for each cage were transferred in another greenhouse for 23 weeks to allow any parasitized whitey scales to turn black or yellowish brown and for aphid mummies (indicative of parasitism). Leaf samples were randomly selected and the numbers of parasitized scales or aphid mummies were counted under a dissecting microscope (10). Approximately 200500 whitey pupae or 6075 aphids per plant for each of the six infested plants per sampling period were assessed for parasitism. The additional 10 individuals of the predatory species (A. swirskii and O. insidious) were evaluated for their predation rate on F. occidentalis. In the A. swirskii predation bioassay, 15 rst instar larvae of F. occidentalis and one female predatory mite were conned on a bean leaf disk (1.5 1.5 cm) that was placed upside down on a water saturated sponge in a

60 ml cup (Solo). For Orius, 30 rst instar thrips were released on bean leaf disk (2 2 cm) that was placed upside down on solidied water agar in a Petri dish (9 cm diameter). Both containers were enclosed with vented lids. The numbers of consumed prey were recorded after 24 h.

2.6. Statistical analysis The number of colony forming units (CFU) on the bee, ower, leaf, and natural enemy samples, and the predation rates of O. insidious and A. swirskii were rst subjected to square-root transformation before analysis. Percent mortality of B. impatiens, E. formosa, E. eremicus, A. colemani, A. swirskii, and O. insidious and parasitism data were both subjected to arcsine square-root transformation before analysis. The transformed data were analyzed using a two-way ANOVA (PROC GLM) to examine the effects of treatment and trial differences over time, and one-way ANOVA (PROC GLM) was performed when all data were obtained from the same trial. Mean values were compared using the least significant difference (LSD) test (SAS Institute, 2008). The type 1 error rate was set at probability level of 0.05.

3. Results 3.1. Colony-forming units of B. bassiana deposited on owers and leaves by B. impatiens B. bassiana were detected on 95% of the tomato owers and 98% of the pepper owers. Spore counts of B. bassiana were 7.6 103 to 1.2 104 CFU per ower for tomato, and 5.6 103 to 1.7 104 CFU per ower for pepper (Table 1). The average counts of active B. bassiana spores delivered by bumble bees were 397.7 to 508.4 and 105.6 to 449.1 CFU cm2 to leaves in the tomato and pepper cages, respectively (Table 1). Spores were found on 95% and 100% of the sampled tomato and pepper leaves, respectively. No spores were detected on any of the samples collected from the control treatments indicating that there was no cross contamination among samples during collection and processing.

3.2. Colony-forming units of B. bassiana recovered from bees and benecials Bumblebees emerging from the dispenser at the exit/entry of the hive carried 9.7 105 to 1.8 106 and 1.3 to 3.4 106 CFU of B. bassiana in the tomato and pepper cages, respectively. Essentially, all of the sampled bumblebees carried viable spores in both crops. The mean numbers of B. bassiana spores per group of ve adult biocontrol agents for each of the benecial species ranged from 98.3 to 206.6 CFU (Table 1).

Table 1 Mean (SE) number of colony forming units (CFU) of Beauveria bassiana from washings of insect or plant samples collected from the trial cages exposed to bumblebee-vectored B. bassiana. Insect/plant samples Tomato Encarsia formosa (4 reps.) 9.7 105 (1.4 105) n = 40 7.6 103 (1.0 103) n = 48 508.4 (106.2) n = 48 163.9 (10.9) n = 32 Eretmocerus eremicus (4 reps.) 1.8 106 (1.8 105) n = 40 1.2 104 (2.2 103) n = 48 397.7 (67.0) n = 48 173.8 (5.9) n = 32 Pepper Aphidius colemani (3 reps.) 3.4 106 (7.0 105) n = 30 1. 7 104 (4.0 103) n = 36 105.6 (14.3) n = 36 189.8 (11.5) n = 24 Orius insidiosus (3 reps.) 1.3 106 (3.2 105) n = 30 8.9 103 (1.6 103) n = 36 383.4 (72.4) n = 36 206.6 (12.8) n = 24 Amblyseius swirskii (3 reps.) 1.3 106 (2.6 105) n = 30 5.6 103 (9.3 102) n = 36 449.1 (88.4) n = 36 98.3 (3.8) n = 24

Bees Flowers Leaves (per cm2) Benecials (per 5 adults)

n = number of samples of groups of 5 individual benecials.

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Table 2 Mean (SE) percent mortality, mycosis and internal infection of parasitoid and predator samples collected from greenhouse cages that were exposed to bumblebee-vectored B. bassiana. Treatment Encarsia formosa (4 reps.) Control Mortality 2.7(1.4)a n = 240 (8 sets) 0 0 n = 80 Beauveria 2.9(0.7)a n = 240 (8 sets) 36.9 (11.0) 10.6 (3.3) n = 80 Eretmocerus eremicus (4 reps.) Control 13.3(6.5)a n = 240 (8 sets) 0 0 n = 80 Beauveria 16.4(6.8)a n = 240 (8 sets) 51.7 (17.5) 13.8 (2.6) n = 80 Aphidius colemani (3 reps.) Control 72.8(3.4)a n = 180 (6 sets) 0 0 n = 60 Beauveria 76.1(1.3)a n = 180 (6 sets) 53.0 (3.4) 13.3 (3.3) n = 60 Orius insidiosus (3 reps.) Control 16.9(2.3)a n = 152 (6 sets) 0 0 n = 60 Beauveria 41.2(3.9)b n = 161 (6 sets) 58.1 (2.9) 15.0 (4.3) n = 60 Amblyseius swirskii (3 reps.) Control 12.0(4.8)a n = 94 (6 sets) 0 0 n = 60 Beauveria 13.7(4.4)a n = 92 (6 sets) 36.1 (8.0) 6.7 (3.3) n = 60

Mycosis Internal infection

n = number of individuals evaluated. Two sets of samples were collected per replication. Within a species row, means followed by different letters are signicantly different at P < 0.05.

Table 3 Mean (SE) percentages of parasitized pest insects (whiteies and green peach aphids) by Encarsia formosa, Eretmocerus eremicus, and Aphidius colemani in greenhouse cages exposed to bumblebee-vectored Beauveria bassiana, and numbers of consumed larvae of western ower thrips by Orius insidiosus and Amblyseius swirskii collected from the greenhouse cages. Treatment Control Beauveria Encarsia formosa (4 reps.) 50.7 (1.9)a n = 17,734 (48 plants) 55.6 (1.6)a n = 23,347 (48 plants) Eretmocerus eremicus (eps.) 45.1 (2.2)a n = 10,442 (48 plants) 46.6 (2.2)a n = 12,526 (48 plants) Aphidius colemani (3 reps.) 91.4 (0.7)a n = 2,097 (36 plants) 91.4 (1.5)a n = 2,686 (36 plants) Orius insidiosus (3 reps.) 19.5 (0.6)a n = 60 21.1 (0.7)a n = 60 Amblyseius swirskii (3 reps.) 5.1 (0.4)a n = 37 6.2 (0.4)a n = 58

n = number of individual whitey scales/aphids or predators evaluated in the parasitism or predation trials. Within a column, means followed by different letters are signicantly different at P < 0.05.

Table 4 Mean (SE) percent mortalities of Bombus impatiens exposed to the Beauveria bassiana (1.37 1010 conidia B. bassiana/g of inoculum) during the greenhouse cage trials. Treatment Control Beauveria Encarisa formosa (4 reps.) 18.2 (1.3)a n = 610 (4 hives) 20.7 (0.9)a n = 524 (4 hives) Eretmocerus eremicus (4 reps.) 16.8 (0.7)a n = 626 (4 hives) 18.5 (0.8)a n = 573 (4 hives) Aphidius colemani (3 reps.) 17.9 (0.6)a n = 480 (3 hives) 20.0 (1.1)a n = 360 (3 hives) Orius insidiosus (3 reps.) 19.2 (1.5)a n = 474 (3 hives) 19.6 (1.3)a n = 442 (3 hives) Amblyseius swirskii (3 reps.) 18.4 (1.1)a n = 442 (3 hives) 18.6 (2.0)a n = 451 (3 hives)

n = number of bee hives used, and number of bees that were found in the hive 3 weeks after the second sampling period for each trial. Within a column, means followed by different letters are signicantly different at P < 0.05.

3.3. Effect of bee vectored B. bassiana on benecials Percent mortalities of benecials collected from the cages with or without bee-vectored B. bassiana are presented in Table 2. No signicant differences between treatments were found for the following four species (two-way ANOVA, E. formosa: F1,12 = 0.29; P = 0.5981, E. eremicus: F1,10 = 0.00; P = 0.9454, A. swirskii: F1,6 = 0.62, P = 0.46; one-way ANOVA, A. colemani: F1,10 = 0.76, P = 0.4042). However, mortality of the predatory bug, O. insidiosus in the Beauveria treatment was signicantly higher (two-way ANOVA, F1,6 = 43.39; P = 0.0006) compared to the control treatment. The mean percentage of dead benecials exhibiting mycosis in the Beauveria treatments ranged from 36.9% to 58.1% while none of the dead benecials in the control treatments showed mycosis. With respect to surface sterilized benecials, internal infections were found in 6.715.0% of the adults from the Beauveria treatments and none in the control treatments. 3.4. Effect on parasitism and predation level of benecials Mean percentages of parasitized whitey nymphs by Encarsia and Eretmocerus and green peach aphid by Aphidius, and the numbers of rst instar thrips predated by Orius and A. swirskii are presented in Table 3. The release of Encarsia jointly with bee vectored Beauveria in a greenhouse cage did not affect the parasitism level of

second instar nymphs of whiteies. No signicant difference was found between treatments (50.7 1.9% vs 55.6 1.6%) (two-way ANOVA, F1,89 = 0.09; P = 0.7707). The same trend was observed with Eretmocerus (45.1 2.2% vs 46.6 2.2%) (two-way ANOVA, F1,89 = 0.12; P = 0.7351). With A. colemani, parasitism levels between the two treatments for greenhouse peach aphid were also similar and not signicantly different (91.4 0.7% parasitized nymphs in the control vs 91.4 1.5% in the Beauveria treatment) (one-way ANOVA, F1,70 = 0.32; P = 0.5705). The predation levels of O. insidious and A. swirskii were not affected after they were exposed in greenhouse cages to bumblebees vectoring Beauveria for insect pest control. Predation level (mean numbers) of rst instar western ower thrips did not statistically differ between the cages where bees vectored Beauveria and the control (two-way ANOVA, F1,114 = 2.10; P = 0.1499) with a mean of 19.5 0.6 vs 21.1 0.7 consumed thrips per day by a single adult O. insidiosus in the control and Beauveria treatments. Predation trials with A. swirskii showed similar tend as with O. insidiosus with means of 5.1 0.4 for the control vs 6.2 0.4 for the Beauveria treatment (two-way ANOVA, F1,91 = 3.18; P = 0.0781). 3.5. Effect of bee vectored B. bassiana on the bumblebee carriers The mean percentages of dead bees when exposed to the two treatments (control without Beauveria and a treatment with bees

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vectoring Beauveria) in greenhouse cage trials are listed in Table 4. None of the dead bees from the control treatments exhibited mycosis and thus, were not infected with Beauveria. Some of the dead bees in the Beauveria treatment exhibited mycosis. However, this did not negatively impact the total number of dead bees in the Beauveria treatment cages. In the parasitoid treatments cages with and without Beauveria, no signicant difference in bee mortality was found between the two treatments (two-way ANOVA, E. formosa: F1,2 = 4.43; P = 0.1699, E. eremicus: F1,2 = 1.10; P = 0.4047 and one-way ANOVA, A. colemani: F1,4 = 2.57; P = 0.1845). For the predator treatment cages, the same results were found (two-way ANOVA, O. insidiosus: F1,2 = 0.07; P = 0.8173 and A. swirskii: F1,2 = 0.01; P = 0.9277).

4. Discussion The broad pest host range for B. bassiana makes this entomopathogen an excellent choice as a management tool for greenhouse crops (Inglis et al., 2001). In greenhouse crops, pest management programs are an integrated approach with biological control as the main control strategy. In an earlier study in commercial greenhouse tomato operations, it was demonstrated that bee vectoring of B. bassiana did not reduce pollination efciency, fruit quality or yield (Kapongo, 2007). Thus, it is important to demonstrate if bee vectoring of B. bassiana can be integrated with the current biological agents that are being used in greenhouse crops. In the present study, bee vectoring of B. bassiana did not have any statistically negative impact on the survival and parasitism rates of the parasitoids (E. formosa, E. eremicus and A. colemani) and the predatory mite (A. swirskii) compared to the control treatment (bees, but no Beauveria). Mortality of the Aphidius was high in both treatments, but this parasitoid was sensitive to handling and its longevity is only approximately 10 days at 1822 C (Malais and Ravensberg, 2003). Only the benecials that died in the Beauveria treatment exhibited any fungal mycosis. From laboratory and greenhouse bioassays, we know that B. bassiana can infect E. formosa, E. eremicus and A. colemani; although infection levels are substantially lower in a greenhouse crop setting (Ludwig and Oetting, 2001; Shipp et al., 2003). The critical question is Does B. bassiana negatively impact the performance of these biocontrol agents?. In a eld study on melon, B. bassiana (GHA strain) was evaluated for control of Bemisia argentifolii Bellow and Perring (Hemiptera: Aleyrodidae) and its impact on introduced Eretmocerus mundus Howard (Hymenoptera: Aphelinidae). The study was conducted at two sites in the Imperial Valley of California. At both sites, parasitism rates were not signicantly different from the untreated or carrier controls even though Eretmocerus and their whitey hosts were less abundant in the Beauveria treated plots (Jaronski et al., 1998; Jaronski et al., 2003). Labb et al. (2009) also found that spray application of GHA strain did not negatively affect the parasitism rate by E. formosa on T. vaporariorum in greenhouse tomato. In fact, parasitism levels were consistently greater by ca. 25% in the Beauveria treated compartment compared to the control. Similar results have been reported for A. colemani against Aphis gossypii Glover on Asiatic lilies. Fogg et al. (1998), Murphy et al. (1999) both found that spray application of B. bassiana (GHA) did not signicantly affect the parasitism rates of A. colemani on A. gossypii. With predatory mites, laboratory leaf bioassays and greenhouse cucumber crop trials found that B. bassiana (Naturalis-L) did not kill Amblyseius cucumeris Oudemans (Acari: Phytoseiidae) or affect their population levels on cucumber leaves (Jacobson et al., 2001). Shipp et al. (2003) report only 5% infection level of A. cucumeris in laboratory bioassay trials under 97.5% RH. In the current study, there was no signicant difference in mortality rates between the

control and the bee vectored Beauveria. Some of the dead A. swirskii in the bee vectored treatment exhibited mycosis (36%), but none in the control treatment. Also, predation rates on rst instar F. occidentalis between the two treatments were not statistically different and were representative of predation rates reported in other studies. Some studies have reported more substantial impacts of B. bassiana on P. persimilis. However, these studies all used different strains of Beauveria. Shipp et al. (2003) found that B. bassiana (GHA) has a minimal impact on adult and immature P. persimilis (89%). Forty-three percent mortality was reported for the Naturalis strain (Duso et al., 2008) and strain DSM 15126 from South America (Numa Vergel et al., 2011). In addition, Numa Vergel et al. (2011) report 14% infection for Neoseiulus californicus (McGregor) (Acari: Phytoseiidae). Thus, strain origin and species of predatory mite can inuence the infection rate by B. bassiana. In the current study, the only predator or parasitoid species that was signicantly impacted by Beauveria was O. insidiosus (41% mortality) compared to the control (17% mortality). The reason for this is not known. Perhaps, due to the larger body mass, Orius can pick up more spores of Beauveria and thus, increase the chance for successful germination. In this study, Orius had the highest CFU counts from the sample washings (Table 1.). Also, Jaronski (2010) reported that insect cuticle surface can affect the germination success of spores and there may be possible differences in the cuticular surfaces among the different benecial species. In greenhouse trials on chrysanthemum plants, Ludwig and Oetting (2001) found only 3.516.5% infection level with Beauveria (Naturalis-O strain). In laboratory trials with the GHA strain, Shipp et al. (2003) reported infection levels of 6177%. However, exposure of adult Orius to bee-vectored B. bassiana did not signicantly affect its predation level as measured in the laboratory bioassays. With respect to the impact of B. bassiana on the bee carriers, no signicant difference in bee mortality was found between the two treatments. At the recommended rate of 1.37 1010 conidia of B. bassiana/g of inoculum, previous studies have shown that any negative effect on bumblebee survival is non-existent or minimal (Kevan et al., 2008; Kapongo et al., 2008a,b). It is also important to understand that the number of bumblebees used in greenhouses often exceeds the actual number required for effective pollination. The current study only examined the direct effects of bee vectored Beauveria on the benecials and its impact on their parasitism or predation level. We did not investigate the sublethal effects of Beauveria on the benecials. Numa Vergel et al. (2011) reported that B. bassiana (strain DMS 15126) from Columbia decreased the egg laying capacity of the predatory mite, N. californicus. This area of research needs to be investigated more to determine the actual signicance of sublethal effects. It is important to remember in the greenhouse setting, benecials are usually introduced on a regular schedule on a weekly or biweekly basis. Also, the longevity of the adult stage for the parasitoids is can be quite short (714 days) or even lower at temperatures above 25 C (Malais and Ravensberg, 2003). Several studies have reported that benecials can detect potentially harmful pathogens, such as fungi, and exhibit avoidance behavior. Non-choice laboratory trials showed that the predatory mirid bug, Dicyphus hesperus Knight (Hemiptera: Miridae), generally avoided feeding on whitey scales which were infected with B. bassiana (GHA strain) (Labb et al., 2006). Similarly, female Eretmocerus sp. can detect B. bassiana infected whitey nymphs and will not oviposit in them (Jaronski et al., 2003). The adult coccinellid, C. septempunctata, was found to demonstrate avoidance behavior of leaf surfaces and soil containing B. bassiana and mycosed C. septempunctata (Ormond et al., 2011). Meyling and Pell (2006) also showed that the predatory anthocorid bug, A. nemorum, exhibited similar avoidance behavior to leaf surfaces containing B. bassiana.

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For bumblebees, the sublethal effect of B. bassiana (GHA strain, suspo-emulsion formulation) was evaluated with Bombus terrestris L. (Hymenoptera: Apidae) (Mommaerts et al., 2009). Laboratory bioassay trials found that sugar water and pollen treated with Botanigard at the Maximum Field Recommended Concentration (2.5 1010 CFU L1) have minimal impact on the reproductive capacity of B. terrestris (mean number of drones produced) according to the IOBC classication rating (<25%) (Mommaerts et al., 2009). It would be interesting to conduct similar experiments with the WP formulation of Botanigard and B. impatiens as results many vary with the bee species and Beauveria product formulation. In summary, based on the results presented in the current study, bee vectoring of the entomopathogen, B. bassiana, is compatible with many of the benecial arthropod control agents that are used in greenhouse integrated pest management (IPM). When Orius or other predatory bugs are used, bee-vectored B. bassiana may not be compatible. Bee vectoring of microbial control agents should be viewed as another tool for the IPM toolbox that can be potentially integrated with the current biological control programs being implemented. Acknowledgments We thank Biobest Canada Ltd. for providing the bee colonies and Laverlam International Corp. USA for supplying the Botanigard 22WP. Also, thank Y. Zhang, K, Shires, W. Romero and C. Meharg for technical assistance. The project was funded in part by Agriculture and Agri-Food Canada Matching Investment Initiative and a collaborative project between the Rural Development Administration of the Republic of Korea and Agriculture and Agri-Food Canada (PJ006848032011). This is contribution #55 of NSERC-CANPOLIN. References
Al-mazraawi, M., Shipp, L., Broadbent, B., Kevan, P., 2006a. Biological control of Lygus lineolaris (Hemiptera: Miridae) and Frankliniella occidentalis (Thysanoptera: Thripidae) by Bombus impatiens (Hymenoptera: Apidae) vectored Beauveria bassiana in greenhouse sweet pepper. Biological Control 37, 8997. Al-mazraawi, M., Shipp, L., Broadbent, B., Kevan, P., 2006b. Dissemination of Beauveria bassiana by honey bees (Hymenoptera: Apidae) for control of tarnished plant bug (Hemiptera: Miridae) on canola. Environmental Entomology 35, 15691577. Beilharz, V.C., Parbery, D.G., Swart, H.J., 1982. Dodine: a selective agent for certain soil fungi. Transactions of British Mycology Society 79, 507511. Butt, T.M., Carreck, N.L., Ibrahim, L., Williams, I.H., 1998. Honey-bee-mediated infection of pollen beetle (Meligethes aeneus Fab.) by the insect-pathogenic fungus Metarhizium anisopliae. Biocontrol Science and Technology 8, 533538. Butt, T.M., Jackson, C.W., Magan, N. (Eds.), 2001. Fungi as Biocontrol Agents: Progress, Problems and Potential. CABI Publishing, Wallingford, UK, p. 390. Carreck, N.L., Butt, T.M., Clark, S.J., Ibrahim, L., Isger, E.A., Pell, J.K., Williams, I.H., 2007. Honey bees can disseminate a microbial control agent to more than one inorescence pest of oilseed rape. Biocontrol Science and Technology 17, 179 191. Cottrell, T.E., Shapiro-Ilan, D.I., 2003. Susceptibility of native and an exotic lady beetle (Coleoptera: Coccinellidae) to Beauveria bassiana. Journal of Invertebrate Pathology 84, 137144. Duso, C., Malagnini, V., Pozzebon, A., Castagnoli, M., 2008. Comparative toxicity of botanical and reduced-risk insecticides to Mediterranean populations of Tetranychus urticae and Phytoseiulus persimilis (Acari: Tetranychidae, Phytoseiidae). Biological Control 47, 1621. Fogg, E.K., Murphy, B.C., Alexander, C.L., Parrella, M.P., Giraud, D., 1998. The integration of parasitoids and fungi for control of Aphis gossypii (Homoptera: Aphididae). Innovation in Biological Control Research, California Conference on Biological Control, Berkeley, CA, pp. 170173. Goettel, M.S., Poprawski, T.J., Vandenberg, J.D., Li, Z., Roberts, D.W., 1990. Safety to nontarget invertebrates of fungal biocontrol agents. In: Laird, M., Lacey, L.A., Davidson, E.W. (Eds.), Safety of Microbial Insecticides. CRC Press, pp. 209232. Gross, H.R., Hamm, J.J., Carpenter, J.E., 1994. Design and application of a hivemounted device that uses honeybees (Hymenoptera: Apidae) to disseminate Heliothis nuclear polyhedrosis virus. Environmental Entomology 23, 492501. Hajek, A., Butler, L., 2000. Predicting the host range of entomopathogenic fungi. In: Follett, P.A., Duan, J.J. (Eds.), Nontarget Effects Of Biological Control. Kluwer Academic Press, pp. 263276. Hajek, A.E., Delalibera Jr, I., 2010. Fungal pathogens as classical biological control agents against arthropods. BioControl 55, 147158.

Inglis, G.D., Goettel, M.S., Butt, T.M., Strasser, H., 2001. Use of hyphomycetous fungi for managing insect pests. In: Butt, T.M., Jackson, C., Magan, N. (Eds.), Fungi as Biocontrol Agents: Progress, Problems and Potential. CABI Publishing, Wallingford, UK, pp. 2369. Jacobson, R.J., Chandler, D., Fenlon, J., Russell, K.M., 2001. Compatibility of Beauveria bassiana (Balsamo) Vuillemin with Amblyseius cucumeris Oudemans (Acarina: Phytoseiidae) to control Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) on cucumber plants. Biocontrol Science and Technology 11, 391 400. James, R.R., Lighthart, B., 1994. Susceptibility of the convergent lady beetle (Coleoptera: Coccinellidae) to four entomopathogenic fungi. Environmental Entomology 23, 190192. Jaronski, S.T., 2010. Ecological factors in the inundative use of fungal entomopathogens. BioControl 55, 159185. Jaronski, S.T., Goettel, M.S., Lomer, C.J., 2003. Regulatory requirements for ecotoxicological assessments of microbial insecticides how relevant are they? In: Hokkanen, H.M.T., Hajek, A.E. (Eds.), Environmental Impacts of Microbial Insecticides: Need and Methods for Risk Assessment. Kluwer Academic Publishers, The Netherlands, pp. 237260. Jaronski, S.T., Lord, J., Rosinska, J., Bradley, C., Hoelmer, K., Simmons, G., Osterlind, R., Brown, C., Staten, R., 1998. Effect of Beauveria bassiana-based mycoinsecticide on benecial insects under eld conditions. Proceedings of the 1998 Brighton Conference Pests and Diseases 2, pp. 651657. Jyoti, J.L., Brewer, G.J., 1999. Honey bees (Hymenoptera: Apidae) as vectors of Bacillus thuringiensis for control of banded sunower moth (Lepidoptera: Tortricidae). Environmental Entomology 28, 11271176. Kapongo, T.J-P., 2007. Vectoring of fungal agents by bumble bees for pest control and disease suppression in greenhouse tomato and sweet pepper. Ph.D. dissertation, University of Guelph, Ontario. Kapongo, J.-P., Shipp, L., Kevan, P., Broadbent, B., 2008a. Optimal concentration of Beauveria bassiana vectored bumble bees in relation to pest and bee mortality in greenhouse tomato and sweet pepper. BioControl 53, 797812. Kapongo, J.-P., Shipp, L., Kevan, P., Sutton, J.C., 2008b. Co-vectoring of Beauveria bassiana and Clonostachys rosea by bumble bees (Bombus impatiens) for control of insect pests and suppression of grey mould in greenhouse tomato and sweet pepper. Biological Control 46, 508514. Kevan, P.G., Kapongo, J.-P., Al-mazraawi, M., Shipp, L., 2008. Honey bees, bumble bees and biocontrol. In: James, R.R., Pitts-Singer, T.L. (Eds.), Bee Pollination in Agricultural Ecosystems. Oxford University Press, New York, pp. 6579. Labb, R.M., Cloutier, C., Brodeur, J., 2006. Prey selection by Dicyphus hesperus of infected or parasitized greenhouse whitey. Biocontrol Science and Technology 16, 485494. Labb, R.M., Gillespie, D.R., Cloutier, C., Brodeur, J., 2009. Compatibility of an entomopathogenic fungus with a predator and a parasitoid in the biological control of greenhouse whitey. Biocontrol Science and Technology 19, 429 446. Lacey, L.A., Kaya, H.K. (Eds.), 2007. Field manual of techniques in invertebrate pathology: application and evaluation of pathogens for control of insects and other invertebrate pests (Second Edition). Springer, Dordtecht, The Netherlands, p. 868. Ludwig, S.W., Oetting, R.D., 2001. Susceptibility of natural enemies to infection by Beauveria bassiana and impact of insecticides on Iphesius degenerans (Acari: Phytoseiidae). Journal of Agriculture and Urban Entomology 18, 169 178. Malais, M.H., Ravensberg, W.J., 2003. Knowing and recognizing: The biology off glasshouse pests and their natural enemies. Koppert B. V., The Netherlands, p. 288. Meyling, N.V., Pell, J.K., 2006. Detection and avoidance of an entomopathogenic fungus by a generalist insect predator. Ecological Entomology 31, 162171. Mommaerts, V., Sterk, G., Hoffmann, L., Smagghe, G., 2009. A laboratory evaluation to determine the compatibility of microbiological control agents with the pollinator Bombus terrestris. Pest Management Science 65, 949955. Murphy, B., Von Damm-Kattari, D., Parrella, M., 1999. Interaction between fungal pathogens and natural enemies: Implication for combined biocontrol of greenhouse pests. IOBC/WPRS Bulletin 22 (1), 181184. Numa Vergel, S.J., Bustos, R.A., Rodriguez, C.D., Cantor, R.F., 2011. Laboratory and greenhouse evaluation of the entomopathogenic fungi and garlic-pepper extract on the predatory mites, Phytoseiulus persimilis and Neoseiulus californicus and their effect on the spider mite, Tetranychus urticae. Biological Control 57, 143149. Ontario Ministry of Agriculture, Food and Rural Affairs, 2010. Growing greenhouse vegetables. Publication 836. Queens Printer, Toronto, Canada, p. 146. Ormond, E.L., Thomas, A.P.M., Pell, J.K., Freeman, S.N., Roy, H.E., 2011. Avoidance of a generalist entomopathogenic fungus by the ladybird, Coccinella septempunctata. FEMS Microbiology Ecology 77, 229237. Ormond, E.L., Thomas, A.P.M., Pugh, P.J.A., Pell, J.K., Roy, H.E., 2010. A fungal pathogen in time and space. the population dynamics of Beauveria bassiana in a conifer forest. FEMS Microbiology Ecology 74, 146154. Rehner, S.A., Buckley, E., 2005. A Beauveria phylogeny inferred from nuclear ITS and EFI-a sequences: evidence for cryptic diversication and links to Cordyceps teleomorphs. Mycologia 97, 8498. Roy, H.E., Brown, P.M.J., Rothery, P., Ware, R.L., Majerus, M.E.N., 2008. Interactions between the fungal pathogen Beauveria bassiana and three species of coccinellid: Harmonia axyridis, Coccinella septempunctata and Adalia bipunctata. BioControl 53, 265276. SAS Institute, 2008. SAS Users Manual, Version 9.2. SAS Institute, Cary NC.

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L. Shipp et al. / Biological Control 63 (2012) 135142 Vega, F.E., Goettel, M.S., Blackwell, M., Chandler, D., Jackson, M.A., Keller, S., Koike, M., Maniania, N.K., Monzn, A., Ownley, B.H., Pell, J.K., Rangel, D.E.N., Roy, H.E., 2009. Fungal entomopathogens: new insights on the ecology. Fungal Ecology 2, 149159.

Shah, P.A., Pell, J.K., 2003. Entomopathogenic fungi as biological control agents. Applied Microbiology and Biotechnology 61, 413423. Shipp, J.L., Zhang, Y., Hunt, D.W.A., Ferguson, G., 2003. Inuence of humidity and greenhouse microclimate on the efcacy of Beauveria bassiana (Balsamo) for control of greenhouse arthropod pests. Environmental Entomology 32, 1151 1163.