Combined effects of the entomopathogenic fungus ... - Springer Link

BioControl (2007) 52:669–681 DOI 10.1007/s10526-006-9053-1

IOBC 2007

Combined effects of the entomopathogenic fungus, Paecilomyces fumosoroseus Apopka-97, and the generalist predator, Dicyphus hesperus, on whitefly populations Colleen R. ALMA1,*, Mark S. GOETTEL1,2, Bernard D. ROITBERG1 and David R. GILLESPIE1,3 1 Department of Biological Sciences, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada; 2Lethbridge Research Centre, Agriculture and Agri-Food Canada, P.O. Box 3000 Lethbridge, AB, T1J 4B1, Canada; 3Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, P.O. Box 1000 Agassiz, BC, V0M 1A0, Canada *Author for correspondence: e-mail: [email protected]; phone +1-705-876-0298; fax +1-519-638-2606

Received 10 June 2006; accepted in revised form 11 October 2006

Abstract. The effects of intraguild interactions between Dicyphus hesperus Knight (Hemiptera: Miridae) and Paecilomyces fumosoroseus Apopka-97 (PFR-97TM) (Wize) Brown and Smith (Ascomycota: Hypocreales) on Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) populations were investigated in tomato greenhouse microcosms. Conditions were established in which interference or synergy would most likely occur; namely, a high number of available whiteflies were combined with large numbers of both D. hesperus and PFR-97TM. We measured live whitefly density in a factorial repeated measures experiment where plants were provided or withheld releases of D. hesperus and/or applications of PFR-97TM for 6 weeks. Releases of D. hesperus were made at a rate of 10 adults/plant during the first and third week and PFR-97TM suspensions were applied with a backpack sprayer at a rate of 18 107, 1.3 107 and 1.2 107 viable blastospores/ml during the first, third and fourth week, respectively. Results revealed a non-significant interaction effect between D. hesperus and PFR-97TM, indicating that their actions were independent. Individual whitefly reductions of 48% and 35% were achieved by PFR-97TM and D. hesperus, respectively. Collectively, the natural enemies reduced whitefly densities by 62% relative to the controls. The density of D. hesperus adults was unaffected by multiple applications of PFR-97TM. These results suggest that the combination of generalist entomopathogenic fungi and generalist predators has the potential to cause increased pest mortality despite evidence of minimal interference. Key words: biological control, interaction, interference competition, Trialeurodes vaporariorum

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COLLEEN R. ALMA ET AL.

Introduction The greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), is an important pest that causes damage to greenhouse vegetable crops (Malais and Ravensberg, 2003). Crop damage results from phloem feeding and honeydew secretion. Phloem feeding by large populations of T. vaporariorum retards plant vigor and the production of copious amounts of honeydew on leaves and fruits stimulates the growth of sooty mould fungi. Large amounts of fungal growth inhibit the photosynthetic processes of leaves and leads to downgrading of the fruit (Osborne and Landa, 1992; Fransen and van Lenteren, 1993; Poprawski et al., 1998). Many studies have demonstrated the ability of various natural enemies to suppress T. vaporariorum populations, including an aphelinid parasitoid (van Lenteren et al., 1996, 1997), hemipteran predators (Albajes et al., 1996; Sampson and King, 1996; McGregor et al., 1999) and entomopathogenic fungi (Osborne and Landa, 1992; Poprawski et al., 2000; Meekes et al., 2002). Studies have also investigated the interactions between multiple natural enemies of T. vaporariorum (Fransen and van Lenteren, 1994; Sterk et al., 1995; Van de Veire and Degheele, 1996); however, the impact of such intraguild interactions on the ability of the natural enemies to control whitefly populations has not been widely studied. Intraguild interactions occur when two species, which are competing for a shared host/prey, also engage in trophic interactions such as parasitism or predation (Polis and Holt, 1992). Interference among natural enemies used in biological control programs could cause pest mortality to be less or greater than expected. Lack of significant antagonism and synergism indicates behaviours that are autonomous. Dicyphus hesperus Knight (Hemiptera: Miridae) is a generalist predator of small, soft-bodied insects, including whiteflies, that has shown considerable promise as a biological control agent in vegetable greenhouses (McGregor et al., 1999). The generalist entomopathogenic fungus, Paecilomyces fumosoroseus strain Apopka-97 (Wize) Brown and Smith (Ascomycota: Hypocreales) has also exhibited significant potential as a biological control agent of whitefly species. This strain was licensed to Certis USA LLC and developed into a biopesticide, which is currently registered in the United States and Europe under the tradenames PFR-97TM and PreFeRalTM, respectively (Faria and Wraight, 2001). Independently, both D. hesperus and PFR-97TM are effective natural enemies of T. vaporariorum (Bolckmans et al., 1995;

EFFECTS OF P. FUMOSOROSEUS+D. HESPERUS ON WHITEFLY

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Van de Veire and Degheele, 1996; McGregor et al., 1999; Sanchez et al., 2003). This study was designed to examine the interactions between D. hesperus and PFR-97TM and how such interactions affected their ability to reduce T. vaporariorum populations on greenhouse tomato plants. Experimental conditions were established in which interference or synergy would be most likely to occur, namely a high humidity, and a high number of available whiteflies were combined with large numbers of both D. hesperus and PFR-97TM. The generalist nature of D. hesperus and P. fumosoroseus are of great importance, as biological control programs usually target multiple pest species; however, in combination, this generalist nature could lead to negative interference. Fungal control agents have the potential to negatively affect insect natural enemies through direct infection, or indirectly, by depleting the prey population (Goettel et al., 1990; Roy and Pell, 2000). In turn, insect natural enemies could negatively affect fungal control agents by consuming prey that are infected with the fungus, thereby removing the fungus from the system (Roy and Pell, 2000).

Materials and methods Insects and fungus A whitefly colony was produced from T. vaporariorum nymphs obtained from Applied Bionomics Ltd (Sidney, British Columbia). Whiteflies were reared on potted tobacco plants, Nicotiana tabacum L. (Solanaceae), and maintained in a rearing room at 26 C, 40% relative humidity (RH), and 24-h light photoperiod. Laboratory colonies of D. hesperus were established using individuals collected from white stem hedgenettle, Stachys albens Gray (Lamiaceae), at an elevation of 300 m near Woody California USA (3543¢ N, 11649¢ W). Colonies for this greenhouse trial were reared in 36 42 30 cm screened Plexiglas cages, and maintained on tobacco plants with Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs as prey. The rearing room was maintained at 22 C, 70% RH, and 16:8 (L:D) photoperiod. Adult females and males used in this greenhouse trial were approximately 7 d post-eclosion. PFR-97TM 20% WDG was obtained from Certis USA, Columbia MD, and consisted of desiccated blastospores of P. fumosoroseus strain Apopka-97 (ATCC 20874) on water dispersible granules. Stock suspensions were prepared in accordance with the product

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label. Blastospores were quantified and the viability of the suspensions was determined using the guidelines outlined by Goettel and Inglis (1997). In brief, blastospore counts were performed from three independent dilutions of the stock suspension using an Improved Neubauer haemocytometer. Blastospore viability was determined by plating 0.1 ml aliquots of diluted suspension onto three potato dextrose agar plates amended with 0.005% benomyl. After incubation at 27 C for 24 h, 500 spores were examined per plate and scored as either viable or non-viable. A blastospore was deemed viable if a germ tube was present. Experimental procedure The experiment was replicated in two greenhouse compartments (each 3.2 12 m) at the Pacific Agri-Food Research Centre (Agassiz, British Columbia). Each compartment contained 24 tomato plants, Lycopersicon esculentum Mill. (Solanaceae) cultivar Rhapsodie (Enza Seeds), planted in two rows and grown in hydroponics culture in rockwool substrate. The distance between rows was 2.2 m and the distance between plants within a row was 0.9 m. Plants were secured to vertical nylon strings hung from horizontal ceiling wires 3 m above the greenhouse floor. Prior to the experiment, plants were lowered to the approximate height of 1.5 m by lowering the vertical strings and moving the plants sideways. Each plant was then enclosed with Agryl P17 spunbond row-cover fabric (BBA Fibreweb Inc. London, UK; average opening 0.1±0.06 mm) suspended from the horizontal ceiling wires. The vertical fabric edges were secured with clothespins to form a cage approximately 1 1 3 m tall. Each plant was completely enclosed but could be closely examined by releasing the necessary clothespins, and re-securing the flap edges once inside the enclosure. The plants were individually caged so that each plant could serve as a replicate in the statistical analyses as the Agryl cloth prevented insects from entering or leaving the plant cages. Each plant was infested with 60 newly-emerged, unsexed, adult whiteflies on 29 August 2003. Following infestation, all plants were randomly assigned to one of the following factorial treatment combinations: (1) an application of PFR-97TM suspension and a release of D. hesperus adults; (2) an application of PFR-97TM suspension and no release of D. hesperus adults; (3) no application of PFR-97TM suspension and a release of D. hesperus adults; and (4) no application of PFR-97TM suspension and no release of D. hesperus adults. Each factorial treatment combination was replicated six times in each


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compartment. Adults of D. hesperus were collected from laboratory colonies and placed into collecting vials. Each collection vial contained five males and five females. One vial was then placed in the middle of each plant, the lid was removed allowing the adults to escape, and the empty vial was collected after 30 min. Releases of D. hesperus adults were made on 23 September and 10 October. Two early releases were sufficient to allow D. hesperus to establish on tomato plants and both sexes of D. hesperus were released to provide a representative adult population. PFR-97TM suspensions were applied to the plants on 23 September, 10 October, and 17 October as recommended on the PFR-97TM label, which suggests that applications be made once a week for 2–3 weeks consecutively. The first application was made at 15 times the recommended label rate (31.5 g/ l) with the intent of exposing D. hesperus to maximum challenge conditions, while the second and third applications were made at the recommended label rate (2.1 g/l). Spray applications were made at 1800 h, by spraying the underside of the leaves until run-off, using a single nozzle seven litre backpack sprayer with a 1 mm spray orifice and an application pressure of between 68.9 and 137 KPa (Hudson Industrial Sprayer # 65010). Plants that were not sprayed with PFR97TM were sprayed with water using a separate identical sprayer in the same manner and time frame as the PFR-97TM applications. Greenhouse vents were closed and three containers of water placed in the central aisle during spraying and 12 h after spraying to increase relative humidity and promote fungal growth and infection. Actual blastospore deposition for each PFR-97TM spray application was determined by randomly selecting four plants per compartment and pinning three discs of 2.6% water agar (10 mm diam. 6 mm ht.), per plant, to the underside of three randomly chosen leaves prior to spraying. The discs were then collected after spraying and examined immediately. Direct blastospore counts were made from each agar disc; counts were first averaged within the plants, then averaged among the plants, and expressed as viable blastospores/mm2. To ensure that cross-contamination during spraying did not occur and to serve as positive controls, one leaf lobe per plant was collected immediately after each spray application. Each leaf lobe was placed in a standard Petri dish containing two moist Whatman filter papers, and incubated at 27±0.54 C, 42 ± 1.8% RH, and 16:8 (L:D) photoperiod for five days. Leaf lobes were then examined under a binocular dissecting microscope for the presence of fungal infection and the

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number of dead whitefly nymphs. Herein, leaf lobes refer to the sub parts of the tomato leaf conventionally known as a leaflet. Sampled lobes were approximately 40 cm2. The temperature and RH within the plant canopy were measured using two dataloggers (HOBOTM, 2 channel; Onset Incorporated) in each compartment, placed unprotected in the mid-canopy of different plants. Readings were taken every eight h, except during the 12 h period following each spray application when readings were taken hourly. Evaluation of treatments Plants were monitored weekly for 6 weeks commencing on 26 September, three days after the first application of the treatment combinations. Twelve leaf lobes per plant were sampled in situ and nondestructively by counting the number of live whitefly nymphs (1st–4th instar nymphs) per leaf lobe using a 10 magnifier. Leaf lobes were selected by dividing the plant equally into four horizontal strata and from each stratum randomly sampling three leaf lobes from different leaves. This ensured uniform sampling between plants and an accurate representation of the whitefly density within each plant. A twofactor repeated measures analysis of covariance was used to determine the influence of the PFR-97TM and D. hesperus factorial treatment combinations on whitefly density over the 6 week trial period. To control for preliminary differences in the whitefly density between the factorial treatment combinations, the initial whitefly density was used as a covariate factor (F1,263=5.3, P=0.0001). The initial whitefly density was determined four days prior to the first application of the treatment combinations. A two-factor analysis of variance was used to determine the effects of the PFR-97TM and D. hesperus factorial treatment combinations on whitefly density in the final week of the trial period. The number of live D. hesperus adults per plant, for every plant, were counted in situ and non-destructively by inspecting the whole plant. A single-factor repeated measures analysis of variance was performed to determine the effects of PFR-97TM on numbers of D. hesperus adults. Recovered, dead D. hesperus adults were surface sterilized for two min in 0.2% sodium hypochlorite, rinsed twice in sterile water, and plated on 2% water agar at 25 C, and 100% RH for seven days to encourage sporulation of P. fumosoroseus on infected cadavers. Plants were also examined for the presence of nymphs to determine if eggs laid by D. hesperus females on


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PFR-97TM-treated plants developed into nymphs. Statistical analyses were not performed on the nymph population as the nymphs could not be accurately quantified. Temperature and RH were each analyzed using a one-factor analysis of variance to ensure that the environmental conditions were similar between greenhouse compartments. For each spray application, a one factor analysis of variance was performed to determine if actual application rates of PFR-97TM (spore deposition) differed between greenhouse compartments. All statistical analyses were performed using JMP Version 5: SAS Institute, 2002, Toronto Canada. All tests were judged at P < 0.05, with greenhouse compartments as the blocking factor. Inspection of the data sets, particularly the normality of the variance and distribution of the residuals, indicated that data transformations were unnecessary.

Results The temperature and RH, measured within the plant canopy, was comparable between greenhouse compartments (temperature: F1,522=2.3, P=0.13; RH: F1,522=1.2, P=0.28). The mean temperature was 21 C with a minimum and maximum value of 16 C and 31 C, respectively. The mean RH was 57% with a minimum and maximum value of 28% and 85%, respectively. During the 12 h period following each spray application, the mean temperature was 21 C (20–22 C range), with a mean RH of 72% (68–74% range). Actual application rates, as measured by spore deposition, did not vary significantly between greenhouse compartments (application 1: F1,12=1.9, P=0.11; application 2: F1,12=0.67, P=0.53; application 3: F1,12=0.77, P=0.49), and therefore, were averaged across compartments (Table 1). Mean blastospore viability averaged 81%. Leaf lobes removed from plants after each PFR-97TM application and incubated under laboratory conditions revealed no cross-contamination between PFR-97TM-treated plants and water-treated control plants, as T. vaporariorum nymphs on control lobes did not show signs of fungus infection. Conversely, dead T. vaporariorum nymphs on PFR97TM-treated leaves exhibited signs of fungal infection, such as the presence of mycelia, which was verified to be P. fumosoroseus. Nymph mortality was noted on PFR-97TM-treated lobes (55–66%), but was low on control lobes (less than 15%).

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Table 1. Blastospore viability, concentration in suspension, and blastospores deposited per mm2 (mean ± SEM) for three sequential PFR-97TM foliar applications Application Date

Blastospore Viability (%) (n = 3)

Concentration No. viable blastospores (107)/ml (n = 3)

Spore deposition No. viable blastospores (103)/mm2 (n = 8)

23 September 10 October 17 October

79±1.9 83±1.4 82±0.69

19±0.80a 1.3±0.14b 1.2±0.16b

7.7±0.57 1.6±0.16 1.5±0.14

a

The first foliar application of PFR-97TM was 15 times the recommended label rate in order to expose the non-target arthropod, Dicyphus hesperus, to a maximum challenge concentration. b The concentrations of the second and third foliar application of PFR-97TM were made at the recommended label rate of 2.1 g/l water.

T. vaporariorum nymphs

No. of Trialeurodes vaporariorum n ymp hs pe r l eaf l o be

Initial live T. vaporariorum density was approximately 10 nymphs/leaf lobe, which increased to a final density of 95 nymphs/leaf lobe on control plants (Figure 1). Results over the 6 week trial period indicated that there was a non-significant interaction effect between D. hesperus and PFR-97TM (Figure 1: F1,35=0.28, P=0.60). When the effects of 120 105 90 75

D P D+P C

60 45 30 15 0 23 Sept.

30 Sept.

10 Oct.

17 Oct.

24 Oct.

31 Oct.

Sampling Date

Figure 1. Mean (±SEM) density of live Trialeurodes vaporariorum nymphs which received one of the following treatment combinations: releases of D. hesperus adults and no application of PFR-97TM suspension (D); applications of PFR-97TM suspension and no release of D. hesperus adults (P); applications of PFR-97TM suspension and releases of D. hesperus adults (D + P); and no application of PFR-97TM suspension and no release of D. hesperus adults (C). Black arrows signify releases of D. hesperus adults and grey arrows signify foliar applications of PFR-97TM blastospore suspensions made during the 6 week trial period. Sample size for each bar is 12 plants.


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D. hesperus and PFR-97TM were analyzed over time, PFR-97TM resulted in a 28% reduction in live T. vaporariorum densities relative to the controls (Figure 1: F1,35=9.9, P=0.0033), whereas, D. hesperus did not cause a significant reduction in live T. vaporariorum densities (Figure 1: F1,35=1.8, P=0.19). The effect of D. hesperus in the final week of the 6 week trial period was significant; D. hesperus reduced live T. vaporariorum densities by 35% relative to the controls. Similarly, PFR-97TM independently caused a 48% reduction in live T. vaporariorum densities (Figure 1: F1,35=10, P=0.0025). Together, the natural enemies reduced whiteflies by 62% and exhibited a non-significant interaction effect (Figure 1: F1,35=0.69, P=0.41). D. hesperus adults In total, 15 D. hesperus cadavers were recovered. Of those, seven were from plants that received spray applications of PFR-97TM. After an incubation period, none of the cadavers exhibited symptoms of infection by P. fumosoroseus. Overall, PFR-97TM applications did not affect the number of live D. hesperus adults per plant (Table 2: F1, 20=0.73, P=0.40). A single D. hesperus was detected on no-predator plants on one occasion and was removed. Otherwise, predators did not invade Table 2. Mean density (±SEM) of live Dicyphus hesperus adults under the presence or absence of foliar PFR-97TM applications on caged tomato plants in the greenhouse Sampling Date

23 30 10 17 24 31

September September October October October October

Mean Number of Dicyphus hesperus adults per planta PFR-97TM absent

PFR-97TM present

2.1±0.38a 2.6±0.42a 3.6±0.49a 7.0±0.54a 5.7±0.37a 5.1±0.51a

2.2±1.3a 3.0±0.36a 3.1±0.34a 6.5±0.53a 5.2±0.44a 4.8±0.45a

Sample size for each sampling date was 12 plants. Spray applications were made on 23 September, 10 October, and 17 October Adults of D. hesperus (10/plant) were released onto the plants on 23 September and 10 October a Means within a row followed by the same letter are not significantly different (repeated measures 1-ANOVA in JMP Version 5: SAS Institute, 2002; P < 0.05 was used for comparisons).

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no-predator plants, as demonstrated by the lack of characteristic symptoms of predation. Nymphs were noted on both PFR-97TM-treated plants and control plants within 5 weeks.

Discussion In the whitefly-tomato system, the generalist predator, along with the generalist entomopathogenic fungus, independently reduced the herbivore population as evidenced by their non-significant interaction effect and combined whitefly mortality. Both PFR-97TM and D. hesperus caused moderate reductions in whitefly populations, and the combination of the two caused a further reduction. Multiple spray applications of PFR-97TM did not reduce the number of D. hesperus adults despite the initial high application rate. These results suggest that the use of generalist entomopathogenic fungi and generalist predators have the potential to cause greater pest mortality when used in conjunction, even though some level of interference may occur. PFR-97TM reduced whitefly densities quickly and effectively. Previous studies have showed similar results with single or multiple applications of PFR-97TM or PreFeRalTM. An application of PreFeRalTM at a concentration of 2 106 colony forming units/ml reduced T. vaporariorum densities by 38% in 3 weeks (Bolckmans et al., 1995). Van de Veire and Degheele (1996) reported a 63% reduction of T. vaporariorum densities 2 weeks after the second application when sprayed at 1 g/l water. Conversely, the effect of D. hesperus was relatively slow and of a smaller magnitude, which was attributed to prey saturation and/or the time it takes D. hesperus populations to establish themselves on host plants. McGregor et al. (1999) reported that it took 5–6 weeks for D. hesperus adults to establish populations on greenhouse tomato plants containing T. vaporariorum. Adults of D. hesperus were difficult to quantify due to their rapid movement across plant surfaces, which caused our underestimation of the population density (Table 2). Eggs of D. hesperus would be tolerant to PFR-97TM foliar applications because females inserted eggs in the tissue of treated plants. Nymphs were noted on both PFR-97TM-treated plants and control plants within 5 weeks. First and second instar nymphs of Macrolophus caliginosus Wagner (Hemiptera: Miridae) experienced a 26% control-corrected mortality after being placed on greenhouse vegetable plants that were sprayed with PreFeRalTM at a concentration of 5 106 colony forming units/ml (Sterk et al., 1995). Under the same experimental


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conditions, PreFeRalTM did not cause significant nymph mortality of Orius insidiosus (Say) and Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) (Sterk et al., 1995). These results indicate that D. hesperus could likely establish populations on greenhouse tomato crops that are treated with PFR-97TM, and that treatments would likely have little effect on established populations. A lack of a negative interaction suggests that either PFR-97TM does not readily infect D. hesperus or that D. hesperus discriminates between nymphs that are and nymphs that are not infected with P. fumosoroseus. Studies have shown that other hemipterans are tolerant to a one-time application of P. fumosoroseus. Prey discrimination is more commonly noted in parasitoids than predators; however, Roy et al. (1998) reported that fourth instar larvae of the seven-spot ladybird, Coccinella septempunctata L. (Coleoptera: Coccinellidae) were able to discriminate between healthy and sporulating aphids. Nonetheless, the combined mortality is less that fully additive, sensu Ferguson and Stiling (1996), suggesting some form of interference between the two natural enemies. Further investigation of D. hesperus’ discriminatory abilities and defense mechanisms to PFR-97TM is therefore warranted.

Acknowledgements This work was supported by the NSERC Biocontrol Network, Agriculture and Agri-Food Canada (AAFC), and the British Columbia Greenhouse Research Council. We would like to thank Ian Bercovitz for his assistance with the statistical analyses. We are very grateful to Jasmin Gee for her assistance with the collection of experimental data and to Randy Thompson for his periodic assistance. The commercial formulation PFR-97TM was provided by Certis USA, Columbia MD. References Albajes, R., O. Alomar, J. Riudavets, C. Castan˜e´, J. Arno´ and R. Gabarra, 1996. The mirid bug Dicyphus tamaninii: an effective predator for vegetable crops. IOBC/ WPRS Bull. 19(1), 1–4. Bolckmans, K., G. Sterk, J. Eyal, B. Sels and W. Stepman, 1995. PreFeRal, (Paecilomyces fumosoroseus (Wize) Brown and Smith, strain Apopka 97), a new microbial insecticide for the biological control of whiteflies in greenhouses. Med. Fac. Landbouww. Univ. Gent. 60: 707–711.

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