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Bumblebees (Bombus Impatiens Cresson) As Vectors Of Commercial Bumble Bees as Vectors of the Microbial Antagonist Clonostachys rosea for Management of Botrytis Blight in Wild Blueberry (Vaccinium angustifolium) by Kevin William Reeh Submitted in partial fulfilment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia in co-operation with Nova Scotia Agricultural College Truro, Nova Scotia June 2012 © Copyright by Kevin William Reeh, 2012 DALHOUSIE UNIVERSITY NOVA SCOTIA AGRICULTURAL COLLEGE The undersigned hereby certify that they have read and recommend to the Faculty of Graduate Studies for acceptance a thesis entitled “Commercial Bumble Bees as Vectors of the Microbial Antagonist Clonostachys rosea for Management of Botrytis Blight in Wild Blueberry (Vaccinium angustifolium)” by Kevin William Reeh in partial fulfilment of the requirements for the degree of Master of Science. Dated: May 10, 2012 Supervisor: _________________________________ Readers: _________________________________ _________________________________ _________________________________ ii DALHOUSIE UNIVERSITY AND NOVA SCOTIA AGRICULTURAL COLLEGE DATE: May 10, 2012 AUTHOR: Kevin William Reeh TITLE: Commercial Bumble Bees as Vectors of the Microbial Antagonist Clonostachys rosea for Management of Botrytis Blight in Wild Blueberry (Vaccinium angustifolium) DEPARTMENT OR SCHOOL: Department of Environmental Sciences DEGREE: M.Sc. CONVOCATION: October YEAR: 2012 Permission is herewith granted to Dalhousie University to circulate and to have copied for non-commercial purposes, at its discretion, the above title upon the request of individuals or institutions. I understand that my thesis will be electronically available to the public. The author reserves other publication rights, and neither the thesis nor extensive extracts from it may be printed or otherwise reproduced without the author’s written permission. The author attests that permission has been obtained for the use of any copyrighted material appearing in the thesis (other than the brief excerpts requiring only proper acknowledgement in scholarly writing), and that all such use is clearly acknowledged. _______________________________ Signature of Author iii Table of Contents LIST OF FIGURES ............................................................................................................ vi ABSTRACT ....................................................................................................................... ix ACKNOWLEDGEMENTS ................................................................................................. x Chapter 1. Introduction .................................................................................................... 1 1.1 Bees as Vectors for Biopesticides ......................................................................... 1 1.1.1 Dispenser Designs .......................................................................................... 5 1.1.2 Impact on Vectoring Pollinators .................................................................. 10 1.2 Wild Blueberry .................................................................................................... 10 1.3 Grey Mould / Botrytis Blight .............................................................................. 13 1.4 Clonostachys rosea ............................................................................................. 18 1.5 Research Scope and General Objectives ............................................................. 19 Chapter 2. Efficacy and Compatibility of Clonostachys rosea for Integrated Management of Botrytis Blight in Wild Blueberry (Vaccinium angustifolium) ............... 21 2.1 Introduction ......................................................................................................... 21 2.2 Material and Methods.......................................................................................... 23 2.3 Results ................................................................................................................. 28 2.4 Discussion ........................................................................................................... 31 Chapter 3. Bumble bee Biovectoring for Disease Management in Wild Blueberry ..... 35 3.1 Introduction ......................................................................................................... 35 3.2 Materials and Methods ........................................................................................ 38 iv 3.2.1 2010 Field Experiment ................................................................................. 38 3.2.2 Semi-Field Experiment ................................................................................ 42 3.3 Results ................................................................................................................. 55 3.4 Discussion ........................................................................................................... 65 Chapter 4. General Discussion ...................................................................................... 72 4.1 Pesticides in Agriculture ..................................................................................... 72 4.2 Botrytis Management .......................................................................................... 74 4.3 Rationale and summary of current research ........................................................ 74 4.4 Recommendations ............................................................................................... 76 References ...................................................................................................................... 82 v LIST OF FIGURES Figure 1.1. The microbial dispenser for Apis mellifera hives, used by Peng et al. (1992) to disseminate Clonostachys rosea into strawberries for control of Botrytis cinerea. Inoculum dispenser shown A) separately; B) attached to a half-size hive. (from Peng et al. 1992) .................................................................................................................................... 7 Figure 1.2. The side-by-side passageways (SSP) dispenser design tested for Bombus terrestris using the microbial antagonist Trichoderma harzianum (MacCagnani et al. 2005). A: One-way exit from hive; B: Entrance to colony box; C: Opening used by outgoing and incoming bees. (from MacCagnani et al. 2005) ............................................. 8 Figure 1.3. The overlapping-passageways (OP) dispenser design tested for Bombus terrestris using the microbial antagonist Trichoderma harzianum (MacCagnani et al. 2005). A: Entrance to colony box; B: One-way exit from hive. (from MacCagnani et al. 2005) .................................................................................................................................... 9 Figure 1.4. The life cycle of Botrytis cinerea (Agrios 1997). ........................................... 17 Figure 2.1. Percentage of blossoms infected with Botrytis cinerea (±SD) 3 days after incubation (22° C; dark), according to treatment with Clonostachys rosea. Bars with different letters are significantly different (α = 0.05; Tukey Kramer HSD). ..................... 29 Figure 2.2. The number of colony-forming units of Clonostachys rosea germinated (±SD) according to the fungicide present in Kings B-glucose media. Bars with different letter groupings are significantly different (α = 0.05; Tukey Kramer HSD). .................... 30 Figure 3.1. Wooden dispenser for mounting on a bumble bee hive for bee vectoring of microbial biological controls. (A) Lower-level maze (filled with powdered biopesticide) – closed except during re-fill; (B) Upper-level return to hive; (C) Gap in the partition allowing exit from lower-level maze to the upper level (other side of Plexiglas). ............ 50 Figure 3.2. Koppert Biological Inc. top-loading mechanical biopesticide dispenser attached to a bumble bee hive for bee vectoring of biopesticides. (A) Dispenser exit tunnel wired with electronic sensor, and filled with biopesticide; (B) Return entrance to hive; (C) Top-loading reservoir for biopesticide, attached to gate mechanism and battery pack with electronics.......................................................................................................... 51 Figure 3.3. PK biopesticide dispenser attached to bumble bee hive for bee vectoring of biopesticides. (A) Exit from lower-level biopesticide cartridge; (B) Return entrance; (C) Access panel to biopesticide cartridge.*Note: Bungee cord was added mid-bloom to maintain a tight fit between the dispenser and hive, due to cardboard sagging. ................ 52 vi Figure 3.4. Pollination tunnels (Multi Shelter Solutions, Palmerston, ON) used for 2011 semi-field experiment. ....................................................................................................... 53 Figure 3.5.Vented Plexiglas cage used to protect bumble hives in semi-field experiment. (A) Vented front panel that slides down over front of hive to prevent bees from accessing the tunnel between experimental pollination periods; (B) Dish of supplemental pollen; (C) Vial of 50% honey solution. .............................................................................................. 54 Figure 3.6. Pollen weights (± SD) recovered from B. impatiens returning to hives fitted with different dispensers (used to load bees with microbial biopesticides) containing powdered Clonostachys rosea. No significant differences (α = 0.05). .............................
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