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Trichoderma-based biological control agents are compatible with the pollinator Bombus terrestris: A laboratory study
Biological Control 46 (2008) 463–466
Contents lists available at ScienceDirect
Biological Control journal homepage: www.elsevier.com/locate/ybcon
Trichoderma-based biological control agents are compatible with the pollinator Bombus terrestris: A laboratory study Veerle Mommaerts a,*, Gerald Platteau b, Jana Boulet a, Guido Sterk c, Guy Smagghe a,b a
Laboratory of Cellular Genetics, Department of Biology, Faculty of Sciences, Free University of Brussels, Pleinlaan 2, 1050 Brussels, Belgium Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium c Biobest NV, Westerlo, Belgium b
a r t i c l e
i n f o
Article history: Received 7 February 2008 Accepted 16 May 2008 Available online 27 May 2008 Keywords: Trichoderma harzianum Trichoderma polysporum Binab Biological control agents Bumblebee Bombus terrestris Side-effects Survival Sublethal effects
a b s t r a c t The potential value of biofungicides for the control of important plant pathogens, such as the gray mold Botrytis cinerea, requires investigation due to the general requirement for very low pesticide residues in foodstuffs, and concerns over the increasing development of resistance to the different classes of chemical fungicides. Furthermore, the use of bumblebees as pollinators in agriculture and horticulture has become more widespread in recent years, which has generated the need to determine the potential risks of biological control agents against these insects. This study investigated the side-effects of two commercial Trichoderma-based biofungicides against the bumblebee Bombus terrestris. The two commercial compounds tested were Binab-TF-WP and Binab-TF-WP-Konc, both of which contain combinations of Trichoderma harzianum ATCC20476 and Trichoderma polysporum ATCC20475. The bumblebees were exposed to the biocontrol agents under laboratory conditions at their respective maximum concentration in the field (MFRC), and then exposed via three potential routes of exposure: dermal contact, orally via the drinking of treated sugar water, and via treated pollen. The treatments were evaluated through recording worker mortality (lethal effects) and any effects on the production of drones (sublethal effects). The laboratory tests demonstrated that the two Binab products did not cause worker mortality or adversely affect reproduction. In addition, we investigated whether the Trichoderma-based products could survive or grow on the bodies of the adult workers. In no case was survival or growth of the biofungicide observed on the workers. When the effects of these compounds were evaluated against bumblebee larvae (third and fourth instars), no entomopathogenic effects were found. Overall, the results obtained under laboratory conditions indicate that the two Binab products are safe for use in combination with B. terrestris. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Botrytis cinerea Pers, the causal agent of Botrytis bunch rot or gray mold, is one of the most important plant diseases worldwide. This above ground fungus affects a very wide range of economically important crops in both greenhouses and open fields (Mertely et al., 2002; Elad et al., 2004). Although chemical control is still commonly employed for the control of important plant diseases, the application of chemical fungicides has become limited due to the requirement for low pesticide residues within produce and increasing problems associated with resistance to the active principles (Elad et al., 1992; Dianez et al., 2002; Hauke et al., 2004). Therefore, there is significant requirement for the development of alternatives that reduce synthetic fungicide applications and minimize the pressure for resistance development. Promising alternatives to chemical fungicides include biological control * Corresponding author. Fax: +32 2 6292759. E-mail address: [email protected] (V. Mommaerts). 1049-9644/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2008.05.007
agents such as the filamentous fungus Trichoderma spp. Although the natural niche of these organisms is within the soil, it has been documented that antagonistic Trichoderma strains are able to infect above ground plant pathogens such as B. cinerea under laboratory conditions (Elad and Freeman, 2002; Freeman et al., 2004). To date, a number of different Trichoderma-based commercial biofungicides have been developed such as Binab-TF-WP and Binab-TF-WP-Konc, both of which contain combinations of Trichoderma harzianum Rifai ATCC20476 and Trichoderma polysporum Rifai ATCC20475. As reported by Wilson (1997), Dik and Elad (1999), and Shafir et al. (2006), applications of T. harzianum T39 in greenhouse and field experiments resulted in improved levels of suppression of B. cinerea over that achieved using a standard botryticide program when disease levels were low. Honeybees and bumblebees are important pollinators of wild flowers and crop plants worldwide (Corbet et al., 1991). However, due to problems with parasites, Varroa mites and Nosema, honeybee colonies (Apis mellifera L.) are in widespread decline (Watanabe, 1994; Allen-Wardell et al., 1998). These problems have
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contributed to the increasing use of non-Apis species, specifically the bumblebee Bombus terrestris L., to pollinate vegetables such as tomato and pepper, and fruits like strawberry, apple, and pear. Natural pollination results in higher fruit quality and quantity. However, as is the case for chemical pesticides, the potential risks associated with the use of biofungicides towards pollinators need to be determined. The entomopathogenic activities of T. harzianum and T. polysporum were first reported in larvae of Scolytus spp. (Jassim et al., 1990). Although the mechanism remains unclear, Shakeri and Foster (2007) showed that virulence factors involved in the biocontrol of fungal plant pathogens are the same as those for entomopathogenic effects. When Trichoderma-based products are sprayed onto leaves and flowers, bumblebees are exposed to the microorganisms during foraging. Although acute toxicity testing has been undertaken previously, there are limited data on the sublethal effects of Trichoderma spp. on bumblebee colony development and pollination. Therefore, the assessment of these risks is a prerequisite for the development of biorational Botrytis control strategies, and for the production of recommendations for their practical implementation. The aim of the present study was to evaluate the acute toxicity and sublethal effects of two Trichoderma-based biofungicides, Binab-TF-WP and Binab-TF-WP-Konc, against the pollinator B. terrestris. Bumblebee workers were treated under laboratory conditions with the biological control agents at their respective field-use concentration for the control of B. cinerea, and they were exposed to Trichoderma via three potential routes: dermal contact, orally via contaminated sugar water, and orally via contaminated pollen. In addition, entomopathogenic activity was tested against bumblebee larvae (third and fourth instars). Finally, the survival and growth of Trichoderma (derived from both formulations) on adult workers and larvae of B. terrestris were evaluated. 2. Materials and methods 2.1. Insects A continuous colony of bumblebees, B. terrestris, was maintained under standardized laboratory conditions in complete darkness at 28 ± 2 °C and 60 ± 10% relative humidity. Sugar water (energy source) and commercially available pollen (Apihurdes, Extremura, Spain) were provided as food ad libitum as described in Mommaerts et al. (2006a). 2.2. Biological control agents The Trichoderma-based commercial products were stored and used in accordance with the manufacturers’ guidelines. Table 1 summarizes their commercial name, type of formulation, concentration in colony-forming units (CFU) per gram formulation, and maximum field-use recommended concentration (MFRC) in % formulation and CFU/l. 2.3. Effect on survival and reproduction of bumblebee workers Effects on the survival and reproduction of bumblebee workers were evaluated using artificial nests contained within transparent plastic boxes (15 cm wide, 15 cm deep, 10 cm high). Five young worker bees, taken from the laboratory colony, were placed in each nest as described in Mommaerts et al. (2006a,b). In these nests adult workers were exposed to the biological control agents via three potential routes: (1) contact by topical application, (2) orally via drinking treated sugar water, and (3) orally via eating treated pollen. Subsequently, nests were monitored over a period of 11 weeks. The numbers of workers that died through acute toxicity,
and numbers of drones per nest resulting from reproduction, were determined weekly. Four artificial nests per treatment were used and each treatment was repeated three times. The different commercial Binab products were prepared in water and applied at their MFRC (Table 1). For the contact applications, 50 ll of the aqueous concentration was topically applied on the dorsal thorax of each worker with a micropipette. For the oral treatment, the nests were exposed for 11 weeks ad libitum to either 500 ml of sugar water contaminated with the biological control agent at its MFRC, or to pollen saturated with the prepared MFRC of the biofungicide. In these nests the sugar water and the pollen were replaced weekly with freshly made sugar water and pollen preparations. In addition, a series of negative control treatments were undertaken where workers were treated topically with water alone or fed on untreated sugar water or on pollen treated with water only. In these treatments 0–15% mortality was recorded. In positive control treatments, workers were dosed with imidacloprid (ConfidorÒ) at its MFRC (200 mg/l), which resulted in 100% mortality in all cases. 2.4. Effect on growth and survival of larval instars For these tests young third and fourth instar bumblebee larvae were selected from the laboratory culture. These stages were chosen for their ease of handling. Larval cups were removed from the culture and numbered. The upper side of each cup was opened so that the larvae were completely visible. Subsequently, the larvae were inoculated topically with 50 ll of an aqueous suspension of Binab-TF-WP (2000 spores/ml) or Binab-TF-WP-Konc (330,000 spores/ml). Larval growth and survival were scored twice weekly up to the point of drone formation. In the negative and positive control experiments, larvae were treated by contact with 50 ll of untreated tap water and Beauveria bassiana GHA at 2 1010 spores/ml (Mycotech, Butte, MT), respectively. All experiments were conducted under the standard colony conditions (see above). For each treatment a minimum of 20 larvae were used and each experiment was repeated twice. 2.5. Survival and growth of Trichoderma on the body of bumblebee workers For this test young bumblebee workers were collected from the laboratory colony and dipped for 3 s (achieving complete insect body wetting) in an aqueous suspension of Binab-TF-WP (2000 spores/ml) or Binab-TF-WP-Konc (330,000 spores/ml). In a positive control experiment, workers were topically treated with 1 109 spores (in 50 ll) of B. bassiana GHA. A total of 40 workers were treated for each treatment regime. Following treatment half of the treated workers were killed by decapitation and analyzed individually to determine the quantity of inoculum retained by each bumblebee worker. The remaining insects were maintained at the standard colony conditions of 28 °C and 60% RH for 4 days to allow growth of Trichoderma on the insect body. The experiment was repeated twice. To analyze growth of Trichoderma, bumblebee workers were individually shaken in 15 ml of a sterile physiological solution (0.85% NaCl; pH 7) for 60 min at 200 rpm. The CFUs per bumblebee body were determined by plating out two times 100 ll aliquots of the respective physiological solutions on selective media (Potato Dextrose Agar). 2.6. Statistical analysis Unless otherwise stated, data were analyzed by one-way analysis of variance (ANOVA). Means ± SEM were separated using
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Table 1 Details of the two commercial Trichoderma-based biological control agents, their formulation type and colony forming units (CFU) per gram, and their respective maximum field-use recommended concentration (MFRC) in % formulation and CFU/l Commercial name
Trichoderma strain
Formulation type; concentration (CFU per gram of formulated product)
MFRC (% formulation in water for spraying)
MFRC (CFU per liter of water for spraying)
Binab-TF-WP
T. harzianum ATCC20476 + T. polysporum ATCC20475; 1/1 T. harzianum ATCC20476 + T. polysporum ATCC20475; 1/1
WP; 1 105 CFU/g
0.125
1.25 105
WP; 1 106 CFU/g
0.125
1.25 106
Binab-TF-WPKonc
WP = wettable powder.
Tukey–Kramer post hoc tests (p = 0.05), using SPSS 15.0 software (SPSS Inc., Chicago, IL).
Trichoderma to survive and grow on bumblebee bodies. In the positive control experiment using B. bassiana GHA, 40 ± 15% of the original inoculum was recovered 4 days after treatment.
3. Results 4. Discussion 3.1. Lethal effects on bumblebee workers No toxicity was scored for bumblebee workers when treated with the two Binab products at their MFRC by topical application or orally via treated sugar water or pollen. At the end of 11 weeks, the numbers of dead workers never exceeded 15%, a value similar to the respective control nests treated with water only. 3.2. Sublethal effects on reproduction Topical treatment with the two Binab products, or oral treatment via treated sugar water and pollen, had no negative effect on bumblebee reproduction. After 11 weeks the production of drones in the nests treated with Binab-TF-WP and Binab-TF-WPKonc was not significantly different (p > 0.05) from the control nests (Table 2). However, it should be noted that, in all cases, treatment with Binab tended to have a stimulating effect on reproduction when compared to control nests although this trend was not statistically significant. 3.3. Effect on growth and survival of larval instars The two Binab products did not exert entomopathogenic activity against third and fourth instar bumblebee larvae and no mortality was recorded. As a result, all larvae successfully developed into drones regardless of treatment. In contrast, all larvae treated with the entomopathogenic fungi B. bassiana GHA died after 1 week. 3.4. Survival and growth of Trichoderma on the body of bumblebee workers The quantity of inoculum recovered from the bodies of bumblebee workers was calculated to be 533 ± 105 spores for Binab-TF-WP and 4933 ± 554 spores for Binab-TF-WP-Konc. Following incubation of the bumblebee workers for 4 days, zero CFUs were counted when plating out on a selective medium, indicating the failure of Table 2 Effect by topical contact and orally via sugar water and via pollen of the two Trichoderma-based products after 11 weeks on the production of drones per nest when treated at their respective MFRC Products
Contact
Sugar water
Pollen
Binab-TF-WP Binab-TF-WP-Konc Control
38.5 ± 0.7a 37.1 ± 2.4a 33.5 ± 3.0a
35.8 ± 2.5a 37.2 ± 2.7a 32.9 ± 1.6a
35.4 ± 1.9a 35.6 ± 1.1a 30.1 ± 0.4a
Data are expressed as means ± SEM based on three independently repeated experiments using 4 nests each with each nest consisting of five workers. Across products and routes of exposure, no statistically significant differences were recorded (values followed with a similar letter (a)) after ANOVA (F = 2.042, df = 26, p = 0.099) and a post hoc Tukey test at p = 0.05.
In order to use a new biocontrol strategy where the biological agents are sprayed onto plants for the control of phytopathogens, it is necessary to assess any potential harmful effects on beneficial pollinators that may occur. Although such tests are of high importance, it is only in recent years that some information has became available with respect to the impact of Trichoderma against pollinators such as honeybees and bumblebees. For honeybees, Van der Steen et al. (2003) reported that T. harzianum T22 had no lethal or sublethal effects on adult workers or on colony reproduction. Similarly, Dedej et al. (2004) and Ngugi et al. (2005) found that honeybees were not affected by Bacillus subtilis QST713. However, Hokkanen et al. (2004) observed high worker mortality in B. terrestris following treatment with the entomopathogenic fungi B. bassiana and M. anisopliae. Such negative effects would clearly impair pollination and crop production. On the other hand, the current study clearly demonstrates that the Binab products, containing T. harzianum ATCC20476 and T. polysporum ATCC20475, when used at their recommended concentrations for the control of B. cinerea, are not harmful to bumblebees via the three possible routes of exposure. Moreover, several Trichoderma isolates, such as Trichodex that contains T. harzianum T39 (used at 109 CFU/g), have been examined by different workers in conjunction with honeybees with no adverse effects recorded (Hjeljord et al., 2000; Shafir et al., 2006). In addition, it is of interest that the current research showed that Trichoderma is not able to survive and grow on the bumblebee bodies. This can be explained by the fact that the spores contained in the Binab products do not develop at temperatures above 30 °C; at the normal colony conditions the respective temperatures of legs, thorax and head were 30–32 °C, 32–34 °C and 38–40 °C by use of an infrared camera (Mommaerts V. and Smagghe G., unpublished). In the positive control treatments that used B. bassiana, only 40% of the spores were recovered 4 days after exposure, which is probably a consequence of the cleaning behavior of the bumblebee workers. There were no negative effects on reproduction when B. terrestris was exposed to the MFRC of the two Trichoderma-based biofungicides investigated here. Whilst it would be beneficial to include additional studies to ensure that there are no sublethal effects following long-term exposure to these formulations, we believe that these maximum challenge trials are a strong indication of compatibility. However, before drawing final conclusion, field tests are required as exposure in the laboratory is markedly different from exposure under field conditions. Since Trichoderma-based biofungicides are safe for B. terrestris, opportunities exist to use bumblebees as vectors for their dissemination. This has been successfully tested in field strawberries where honeybees have been used to deliver Trichoderma for the control of Botrytis (Kovach et al., 2000), and a similar system may be envisioned with bumblebees in protected horticultural crops.
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Obviously, this would require the development of an efficient dissemination device and appropriate formulations to provide efficient applications. Finally, if biological control agents are to be applied in greenhouses for the control of plant pathogens, it is important for the grower that pollination remains unimpaired. Therefore, adequate testing for potential harmful effects of these agents towards bees are necessary but, in addition, we believe that product quality control is also very important as contaminants present with the formulation can be detrimental for pollinators. Overall, the results obtained under laboratory conditions presented here indicate that the two Binab products are safe for use in combination with B. terrestris. Acknowledgments The authors appreciate the gifts of Binab-TF-WP and Binab-TFWP-Konc by Bio-innovation (Helsingborg, Sweden). We are also indebted to Kurt Put and Kris Jans (both Biobest) for their help during the experiments and to Dr. Howard Bell (CSL, York) for his careful editing. Comments from three anonymous referees were also helpful. This research was supported in part by the Research Council of VUB (Belgium) and the Centre of Public Research Gabriel Lippmann (Belvaux, Luxembourg). References Allen-Wardell, G., Berhardt, P., Bitner, F., Burquez, A., Buchmann, S., Cane, J., Cox, P.A., Dalton, V., Feinsinger, P., Ingram, M., Inouye, D., Jones, E.C., Kennedy, K., Kevan, P., Koopwitz, H., Medellin, R., Medellin, S.M., Nabhan, G.P., 1998. The potential consequences of pollinator declines on the conservation of biodiversity and stability of food crop yields. Conservation Biology 12, 8–17. Corbet, S.A., Williamson, I.H., Osborne, J.L., 1991. Bees and the pollination of crops and wild flowers in the European community. Bee World 72, 47–59. Dedej, S., Delaplane, K.S., Sscherm, H., 2004. Effectiveness of honey bees in delivering the biocontrol agent Bacillus subtilis to blueberry flowers to suppress mummy berry disease. Biological Control 31, 422–427. Dianez, F., Santos, M., Blanco, P., Tello, J.C., 2002. Fungicide resistance in Botrytis cinerea isolates from strawberry crops in Huelva (southwestern Spain). Phytoparasitica 30, 529–534. Dik, A.J., Elad, Y., 1999. Comparison of antagonists of Botrytis cinerea in greenhouse grown cucumber and tomato under different climatic conditions. European Journal of Plant Pathology 105, 123–137. Elad, Y., Freeman, S., 2002. Biological control of fungal plant pathogens. In: Kempken, F. (Ed.), The Mycota, a Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research. XI. Agricultural Applications. Springer, Heidelberg, Germany. Elad, Y., Yunis, H., Katan, T., 1992. Multiple fungicide resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of Botrytis cinerea in Israel. Plant Pathology 41, 324–332.
Elad, Y., Williamson, B., Tudzynski, P., Delen, N., 2004. Botrytis spp. and diseases they cause in agricultural systems. An introduction. In: Elad, Y., Williamson, B., Tudzynski, P., Delen, N. (Eds.), Botrytis: Biology, Pathology and Control. Kluwer Academic Publishers, Dordrecht, The Netherlands. Freeman, S., Minz, D., Kolesnik, I., Barbul, O., Zveibil, A., Mayon, M., Nitzani, Y., Kirshner, B., Rav-David, D., Bilu, A., Dag, A., Shafir, S., Elad, Y., 2004. Trichoderma biocontrol of Colletotrichum acutatum and Botrytis cinerea and survival in strawberry. European Journal of Plant Pathology 110, 361–370. Hauke, K., Creemers, P., Brugmans, W., Van Laer, S., 2004. Signum, a new fungicide with interesting properties in resistance management of fungal diseases in strawberries. Communications in Agricultural and Applied Biological Sciences, Ghent University 69, 743–755. Hjeljord, L.G., Stensvand, A., Tronsmo, A., 2000. Effect of temperature and nutrient stress on the capacity of commercial Trichoderma products to control Botrytis cinerea and Mucor piriformis in greenhouse strawberries. Biological Control 19, 149–160. Hokkanen, H., Zeng, Q.Q., Menzler-Hokkanen, I., 2004. Assessing the impact of Metarhizium and Beauveria on bumblebees. In: Hokkanen, H., Hajek, E.A. (Eds.), Environmental Impacts of Microbial Insecticides, Needs and Methods for Risk Assessment, Progress in Biological Control, vol. 1. Kluwer Academic Publishers, Dordrecht, The Netherlands. Jassim, H.K., Foster, H.A., Fairhurst, C.P., 1990. Biological control of Dutch elm disease: larvicidal activity of Trichoderma harzianum, Trichoderma polysporum and Scytalidium lignicola in Scolytus scolytus and Scotylus multistriatus reared in artificial culture. Annals of Applied Biology 117, 187–196. Kovach, J., Petzoldt, R., Harman, G.E., 2000. Use of honey bees and bumble bees to disseminate Trichoderma harzianum 1295-22 to strawberries for Botrytis control. Biological Control 18, 235–242. Mertely, J.C., MacKenzie, S.J., Legrand, D.E., 2002. Timing of fungicide applications for Botrytis cinerea based on development stage of strawberry flowers and fruit. Plant Disease 86, 1019–1024. Mommaerts, V., Sterk, G., Smagghe, G., 2006a. Hazards and uptake of chitin synthesis inhibitors in bumblebees Bombus terrestris. Pest Management Science 62, 752–758. Mommaerts, V., Sterk, G., Smagghe, G., 2006b. Bumblebees can be used in combination with juvenile hormone analogues and ecdysone agonists. Ecotoxicology 15, 513–521. Ngugi, H.K., Dedej, S., Delaplane, K.S., Savelle, A.T., Scherm, H., 2005. Effect of flowerapplied Serenade biofungicide (Bacillus subtilis) on pollination-related variables in rabbiteye blueberry. Biological Control 33, 32–38. Shafir, S., Dag, A., Bilu, A., Abu-Toamy, M., Elad, Y., 2006. Honey bee dispersal of the biocontrol agent Trichoderma harzianum T39: effectiveness in suppressing Botrytis cinerea on strawberry under field conditions. European Journal of Plant Pathology 116, 119–128. Shakeri, J., Foster, H.A., 2007. Proteolytic activity and antibiotic production by Trichoderma harzianum in relation to pathogenicity to insects. Enzyme and Microbial Technology 40, 961–968. Van der Steen, J.J.M., Langerak, C.J., Van Tongeren, C.A.M., Dik, A.J., 2003. Aspects of the use of honeybees and bumblebees as vector of antagonistic microorganisms in plant disease control. In: Bruin, J. (Ed.), Proceedings of the Netherlands Entomological Society Meeting. Uitgeverij Nederlandse Entomologische Vereniging, Amsterdam, The Netherlands. Watanabe, M.E., 1994. Pollination worries rise as honey bees decline. Science (Washington, DC) 265, 1170–1171. Wilson, M., 1997. Biocontrol of aerial plant diseases in agriculture and horticulture: current approaches and future prospects. Journal of Industrial Microbiology and Biotechnology 19, 188–191.
Contents lists available at ScienceDirect
Biological Control journal homepage: www.elsevier.com/locate/ybcon
Trichoderma-based biological control agents are compatible with the pollinator Bombus terrestris: A laboratory study Veerle Mommaerts a,*, Gerald Platteau b, Jana Boulet a, Guido Sterk c, Guy Smagghe a,b a
Laboratory of Cellular Genetics, Department of Biology, Faculty of Sciences, Free University of Brussels, Pleinlaan 2, 1050 Brussels, Belgium Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium c Biobest NV, Westerlo, Belgium b
a r t i c l e
i n f o
Article history: Received 7 February 2008 Accepted 16 May 2008 Available online 27 May 2008 Keywords: Trichoderma harzianum Trichoderma polysporum Binab Biological control agents Bumblebee Bombus terrestris Side-effects Survival Sublethal effects
a b s t r a c t The potential value of biofungicides for the control of important plant pathogens, such as the gray mold Botrytis cinerea, requires investigation due to the general requirement for very low pesticide residues in foodstuffs, and concerns over the increasing development of resistance to the different classes of chemical fungicides. Furthermore, the use of bumblebees as pollinators in agriculture and horticulture has become more widespread in recent years, which has generated the need to determine the potential risks of biological control agents against these insects. This study investigated the side-effects of two commercial Trichoderma-based biofungicides against the bumblebee Bombus terrestris. The two commercial compounds tested were Binab-TF-WP and Binab-TF-WP-Konc, both of which contain combinations of Trichoderma harzianum ATCC20476 and Trichoderma polysporum ATCC20475. The bumblebees were exposed to the biocontrol agents under laboratory conditions at their respective maximum concentration in the field (MFRC), and then exposed via three potential routes of exposure: dermal contact, orally via the drinking of treated sugar water, and via treated pollen. The treatments were evaluated through recording worker mortality (lethal effects) and any effects on the production of drones (sublethal effects). The laboratory tests demonstrated that the two Binab products did not cause worker mortality or adversely affect reproduction. In addition, we investigated whether the Trichoderma-based products could survive or grow on the bodies of the adult workers. In no case was survival or growth of the biofungicide observed on the workers. When the effects of these compounds were evaluated against bumblebee larvae (third and fourth instars), no entomopathogenic effects were found. Overall, the results obtained under laboratory conditions indicate that the two Binab products are safe for use in combination with B. terrestris. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Botrytis cinerea Pers, the causal agent of Botrytis bunch rot or gray mold, is one of the most important plant diseases worldwide. This above ground fungus affects a very wide range of economically important crops in both greenhouses and open fields (Mertely et al., 2002; Elad et al., 2004). Although chemical control is still commonly employed for the control of important plant diseases, the application of chemical fungicides has become limited due to the requirement for low pesticide residues within produce and increasing problems associated with resistance to the active principles (Elad et al., 1992; Dianez et al., 2002; Hauke et al., 2004). Therefore, there is significant requirement for the development of alternatives that reduce synthetic fungicide applications and minimize the pressure for resistance development. Promising alternatives to chemical fungicides include biological control * Corresponding author. Fax: +32 2 6292759. E-mail address: [email protected] (V. Mommaerts). 1049-9644/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2008.05.007
agents such as the filamentous fungus Trichoderma spp. Although the natural niche of these organisms is within the soil, it has been documented that antagonistic Trichoderma strains are able to infect above ground plant pathogens such as B. cinerea under laboratory conditions (Elad and Freeman, 2002; Freeman et al., 2004). To date, a number of different Trichoderma-based commercial biofungicides have been developed such as Binab-TF-WP and Binab-TF-WP-Konc, both of which contain combinations of Trichoderma harzianum Rifai ATCC20476 and Trichoderma polysporum Rifai ATCC20475. As reported by Wilson (1997), Dik and Elad (1999), and Shafir et al. (2006), applications of T. harzianum T39 in greenhouse and field experiments resulted in improved levels of suppression of B. cinerea over that achieved using a standard botryticide program when disease levels were low. Honeybees and bumblebees are important pollinators of wild flowers and crop plants worldwide (Corbet et al., 1991). However, due to problems with parasites, Varroa mites and Nosema, honeybee colonies (Apis mellifera L.) are in widespread decline (Watanabe, 1994; Allen-Wardell et al., 1998). These problems have
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contributed to the increasing use of non-Apis species, specifically the bumblebee Bombus terrestris L., to pollinate vegetables such as tomato and pepper, and fruits like strawberry, apple, and pear. Natural pollination results in higher fruit quality and quantity. However, as is the case for chemical pesticides, the potential risks associated with the use of biofungicides towards pollinators need to be determined. The entomopathogenic activities of T. harzianum and T. polysporum were first reported in larvae of Scolytus spp. (Jassim et al., 1990). Although the mechanism remains unclear, Shakeri and Foster (2007) showed that virulence factors involved in the biocontrol of fungal plant pathogens are the same as those for entomopathogenic effects. When Trichoderma-based products are sprayed onto leaves and flowers, bumblebees are exposed to the microorganisms during foraging. Although acute toxicity testing has been undertaken previously, there are limited data on the sublethal effects of Trichoderma spp. on bumblebee colony development and pollination. Therefore, the assessment of these risks is a prerequisite for the development of biorational Botrytis control strategies, and for the production of recommendations for their practical implementation. The aim of the present study was to evaluate the acute toxicity and sublethal effects of two Trichoderma-based biofungicides, Binab-TF-WP and Binab-TF-WP-Konc, against the pollinator B. terrestris. Bumblebee workers were treated under laboratory conditions with the biological control agents at their respective field-use concentration for the control of B. cinerea, and they were exposed to Trichoderma via three potential routes: dermal contact, orally via contaminated sugar water, and orally via contaminated pollen. In addition, entomopathogenic activity was tested against bumblebee larvae (third and fourth instars). Finally, the survival and growth of Trichoderma (derived from both formulations) on adult workers and larvae of B. terrestris were evaluated. 2. Materials and methods 2.1. Insects A continuous colony of bumblebees, B. terrestris, was maintained under standardized laboratory conditions in complete darkness at 28 ± 2 °C and 60 ± 10% relative humidity. Sugar water (energy source) and commercially available pollen (Apihurdes, Extremura, Spain) were provided as food ad libitum as described in Mommaerts et al. (2006a). 2.2. Biological control agents The Trichoderma-based commercial products were stored and used in accordance with the manufacturers’ guidelines. Table 1 summarizes their commercial name, type of formulation, concentration in colony-forming units (CFU) per gram formulation, and maximum field-use recommended concentration (MFRC) in % formulation and CFU/l. 2.3. Effect on survival and reproduction of bumblebee workers Effects on the survival and reproduction of bumblebee workers were evaluated using artificial nests contained within transparent plastic boxes (15 cm wide, 15 cm deep, 10 cm high). Five young worker bees, taken from the laboratory colony, were placed in each nest as described in Mommaerts et al. (2006a,b). In these nests adult workers were exposed to the biological control agents via three potential routes: (1) contact by topical application, (2) orally via drinking treated sugar water, and (3) orally via eating treated pollen. Subsequently, nests were monitored over a period of 11 weeks. The numbers of workers that died through acute toxicity,
and numbers of drones per nest resulting from reproduction, were determined weekly. Four artificial nests per treatment were used and each treatment was repeated three times. The different commercial Binab products were prepared in water and applied at their MFRC (Table 1). For the contact applications, 50 ll of the aqueous concentration was topically applied on the dorsal thorax of each worker with a micropipette. For the oral treatment, the nests were exposed for 11 weeks ad libitum to either 500 ml of sugar water contaminated with the biological control agent at its MFRC, or to pollen saturated with the prepared MFRC of the biofungicide. In these nests the sugar water and the pollen were replaced weekly with freshly made sugar water and pollen preparations. In addition, a series of negative control treatments were undertaken where workers were treated topically with water alone or fed on untreated sugar water or on pollen treated with water only. In these treatments 0–15% mortality was recorded. In positive control treatments, workers were dosed with imidacloprid (ConfidorÒ) at its MFRC (200 mg/l), which resulted in 100% mortality in all cases. 2.4. Effect on growth and survival of larval instars For these tests young third and fourth instar bumblebee larvae were selected from the laboratory culture. These stages were chosen for their ease of handling. Larval cups were removed from the culture and numbered. The upper side of each cup was opened so that the larvae were completely visible. Subsequently, the larvae were inoculated topically with 50 ll of an aqueous suspension of Binab-TF-WP (2000 spores/ml) or Binab-TF-WP-Konc (330,000 spores/ml). Larval growth and survival were scored twice weekly up to the point of drone formation. In the negative and positive control experiments, larvae were treated by contact with 50 ll of untreated tap water and Beauveria bassiana GHA at 2 1010 spores/ml (Mycotech, Butte, MT), respectively. All experiments were conducted under the standard colony conditions (see above). For each treatment a minimum of 20 larvae were used and each experiment was repeated twice. 2.5. Survival and growth of Trichoderma on the body of bumblebee workers For this test young bumblebee workers were collected from the laboratory colony and dipped for 3 s (achieving complete insect body wetting) in an aqueous suspension of Binab-TF-WP (2000 spores/ml) or Binab-TF-WP-Konc (330,000 spores/ml). In a positive control experiment, workers were topically treated with 1 109 spores (in 50 ll) of B. bassiana GHA. A total of 40 workers were treated for each treatment regime. Following treatment half of the treated workers were killed by decapitation and analyzed individually to determine the quantity of inoculum retained by each bumblebee worker. The remaining insects were maintained at the standard colony conditions of 28 °C and 60% RH for 4 days to allow growth of Trichoderma on the insect body. The experiment was repeated twice. To analyze growth of Trichoderma, bumblebee workers were individually shaken in 15 ml of a sterile physiological solution (0.85% NaCl; pH 7) for 60 min at 200 rpm. The CFUs per bumblebee body were determined by plating out two times 100 ll aliquots of the respective physiological solutions on selective media (Potato Dextrose Agar). 2.6. Statistical analysis Unless otherwise stated, data were analyzed by one-way analysis of variance (ANOVA). Means ± SEM were separated using
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Table 1 Details of the two commercial Trichoderma-based biological control agents, their formulation type and colony forming units (CFU) per gram, and their respective maximum field-use recommended concentration (MFRC) in % formulation and CFU/l Commercial name
Trichoderma strain
Formulation type; concentration (CFU per gram of formulated product)
MFRC (% formulation in water for spraying)
MFRC (CFU per liter of water for spraying)
Binab-TF-WP
T. harzianum ATCC20476 + T. polysporum ATCC20475; 1/1 T. harzianum ATCC20476 + T. polysporum ATCC20475; 1/1
WP; 1 105 CFU/g
0.125
1.25 105
WP; 1 106 CFU/g
0.125
1.25 106
Binab-TF-WPKonc
WP = wettable powder.
Tukey–Kramer post hoc tests (p = 0.05), using SPSS 15.0 software (SPSS Inc., Chicago, IL).
Trichoderma to survive and grow on bumblebee bodies. In the positive control experiment using B. bassiana GHA, 40 ± 15% of the original inoculum was recovered 4 days after treatment.
3. Results 4. Discussion 3.1. Lethal effects on bumblebee workers No toxicity was scored for bumblebee workers when treated with the two Binab products at their MFRC by topical application or orally via treated sugar water or pollen. At the end of 11 weeks, the numbers of dead workers never exceeded 15%, a value similar to the respective control nests treated with water only. 3.2. Sublethal effects on reproduction Topical treatment with the two Binab products, or oral treatment via treated sugar water and pollen, had no negative effect on bumblebee reproduction. After 11 weeks the production of drones in the nests treated with Binab-TF-WP and Binab-TF-WPKonc was not significantly different (p > 0.05) from the control nests (Table 2). However, it should be noted that, in all cases, treatment with Binab tended to have a stimulating effect on reproduction when compared to control nests although this trend was not statistically significant. 3.3. Effect on growth and survival of larval instars The two Binab products did not exert entomopathogenic activity against third and fourth instar bumblebee larvae and no mortality was recorded. As a result, all larvae successfully developed into drones regardless of treatment. In contrast, all larvae treated with the entomopathogenic fungi B. bassiana GHA died after 1 week. 3.4. Survival and growth of Trichoderma on the body of bumblebee workers The quantity of inoculum recovered from the bodies of bumblebee workers was calculated to be 533 ± 105 spores for Binab-TF-WP and 4933 ± 554 spores for Binab-TF-WP-Konc. Following incubation of the bumblebee workers for 4 days, zero CFUs were counted when plating out on a selective medium, indicating the failure of Table 2 Effect by topical contact and orally via sugar water and via pollen of the two Trichoderma-based products after 11 weeks on the production of drones per nest when treated at their respective MFRC Products
Contact
Sugar water
Pollen
Binab-TF-WP Binab-TF-WP-Konc Control
38.5 ± 0.7a 37.1 ± 2.4a 33.5 ± 3.0a
35.8 ± 2.5a 37.2 ± 2.7a 32.9 ± 1.6a
35.4 ± 1.9a 35.6 ± 1.1a 30.1 ± 0.4a
Data are expressed as means ± SEM based on three independently repeated experiments using 4 nests each with each nest consisting of five workers. Across products and routes of exposure, no statistically significant differences were recorded (values followed with a similar letter (a)) after ANOVA (F = 2.042, df = 26, p = 0.099) and a post hoc Tukey test at p = 0.05.
In order to use a new biocontrol strategy where the biological agents are sprayed onto plants for the control of phytopathogens, it is necessary to assess any potential harmful effects on beneficial pollinators that may occur. Although such tests are of high importance, it is only in recent years that some information has became available with respect to the impact of Trichoderma against pollinators such as honeybees and bumblebees. For honeybees, Van der Steen et al. (2003) reported that T. harzianum T22 had no lethal or sublethal effects on adult workers or on colony reproduction. Similarly, Dedej et al. (2004) and Ngugi et al. (2005) found that honeybees were not affected by Bacillus subtilis QST713. However, Hokkanen et al. (2004) observed high worker mortality in B. terrestris following treatment with the entomopathogenic fungi B. bassiana and M. anisopliae. Such negative effects would clearly impair pollination and crop production. On the other hand, the current study clearly demonstrates that the Binab products, containing T. harzianum ATCC20476 and T. polysporum ATCC20475, when used at their recommended concentrations for the control of B. cinerea, are not harmful to bumblebees via the three possible routes of exposure. Moreover, several Trichoderma isolates, such as Trichodex that contains T. harzianum T39 (used at 109 CFU/g), have been examined by different workers in conjunction with honeybees with no adverse effects recorded (Hjeljord et al., 2000; Shafir et al., 2006). In addition, it is of interest that the current research showed that Trichoderma is not able to survive and grow on the bumblebee bodies. This can be explained by the fact that the spores contained in the Binab products do not develop at temperatures above 30 °C; at the normal colony conditions the respective temperatures of legs, thorax and head were 30–32 °C, 32–34 °C and 38–40 °C by use of an infrared camera (Mommaerts V. and Smagghe G., unpublished). In the positive control treatments that used B. bassiana, only 40% of the spores were recovered 4 days after exposure, which is probably a consequence of the cleaning behavior of the bumblebee workers. There were no negative effects on reproduction when B. terrestris was exposed to the MFRC of the two Trichoderma-based biofungicides investigated here. Whilst it would be beneficial to include additional studies to ensure that there are no sublethal effects following long-term exposure to these formulations, we believe that these maximum challenge trials are a strong indication of compatibility. However, before drawing final conclusion, field tests are required as exposure in the laboratory is markedly different from exposure under field conditions. Since Trichoderma-based biofungicides are safe for B. terrestris, opportunities exist to use bumblebees as vectors for their dissemination. This has been successfully tested in field strawberries where honeybees have been used to deliver Trichoderma for the control of Botrytis (Kovach et al., 2000), and a similar system may be envisioned with bumblebees in protected horticultural crops.
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Obviously, this would require the development of an efficient dissemination device and appropriate formulations to provide efficient applications. Finally, if biological control agents are to be applied in greenhouses for the control of plant pathogens, it is important for the grower that pollination remains unimpaired. Therefore, adequate testing for potential harmful effects of these agents towards bees are necessary but, in addition, we believe that product quality control is also very important as contaminants present with the formulation can be detrimental for pollinators. Overall, the results obtained under laboratory conditions presented here indicate that the two Binab products are safe for use in combination with B. terrestris. Acknowledgments The authors appreciate the gifts of Binab-TF-WP and Binab-TFWP-Konc by Bio-innovation (Helsingborg, Sweden). We are also indebted to Kurt Put and Kris Jans (both Biobest) for their help during the experiments and to Dr. Howard Bell (CSL, York) for his careful editing. Comments from three anonymous referees were also helpful. This research was supported in part by the Research Council of VUB (Belgium) and the Centre of Public Research Gabriel Lippmann (Belvaux, Luxembourg). References Allen-Wardell, G., Berhardt, P., Bitner, F., Burquez, A., Buchmann, S., Cane, J., Cox, P.A., Dalton, V., Feinsinger, P., Ingram, M., Inouye, D., Jones, E.C., Kennedy, K., Kevan, P., Koopwitz, H., Medellin, R., Medellin, S.M., Nabhan, G.P., 1998. The potential consequences of pollinator declines on the conservation of biodiversity and stability of food crop yields. Conservation Biology 12, 8–17. Corbet, S.A., Williamson, I.H., Osborne, J.L., 1991. Bees and the pollination of crops and wild flowers in the European community. Bee World 72, 47–59. Dedej, S., Delaplane, K.S., Sscherm, H., 2004. Effectiveness of honey bees in delivering the biocontrol agent Bacillus subtilis to blueberry flowers to suppress mummy berry disease. Biological Control 31, 422–427. Dianez, F., Santos, M., Blanco, P., Tello, J.C., 2002. Fungicide resistance in Botrytis cinerea isolates from strawberry crops in Huelva (southwestern Spain). Phytoparasitica 30, 529–534. Dik, A.J., Elad, Y., 1999. Comparison of antagonists of Botrytis cinerea in greenhouse grown cucumber and tomato under different climatic conditions. European Journal of Plant Pathology 105, 123–137. Elad, Y., Freeman, S., 2002. Biological control of fungal plant pathogens. In: Kempken, F. (Ed.), The Mycota, a Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research. XI. Agricultural Applications. Springer, Heidelberg, Germany. Elad, Y., Yunis, H., Katan, T., 1992. Multiple fungicide resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of Botrytis cinerea in Israel. Plant Pathology 41, 324–332.
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