Control of chalkbrood disease with natural products A report for the Rural Industries Research and Development Corporation by Dr Craig Davis and Wendy Ward December 2003 RIRDC Publication No 03/107 RIRDC Project No DAQ-269A © 2003 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 58672 X ISSN 1440-6845 The control of chalkbrood disease with natural products Publication No. 03/107 Project No. DAQ-269A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone (02) 6272 3186. Researcher Contact Details: Dr Craig Davis Wendy Ward Centre for Food Technology Animal Research Institute 19 Hercules Street, Hamilton 4007 665 Fairfield Road, Yeerongpilly 4105 Phone: (07) 3406 8611 Phone: (07) 3362 9446 Fax: (07) 3406 8677 Fax: (07) 3362 9440 Email: [email protected] Email: [email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4819 Fax: 02 6272 5877 Email: [email protected] Website: http://www.rirdc.gov.au Published in December 2003 Printed on environmentally friendly paper by Canprint ii Foreword Chalkbrood of honeybees (Apis mellifera) is caused by the fungus Ascosphaera apis. It was first diagnosed in Australia in 1993 and is now endemic in most areas of Australia. Although chalkbrood is not usually fatal to honeybee colonies, it can cause substantial production losses. The aims of the present work were to conduct a laboratory study on the efficacy of natural products against the chalkbrood fungus Ascosphaera apis, and to develop an appropriate field treatment plan to evaluate any products identified in the first section of the investigation. This project was funded from RIRDC Core Funds which are provided by the Australian Government. This report is an addition to RIRDC’s diverse range of over 1000 research publications and, forms part of our Essential Oils and Plant Extracts R&D program, which aims to support the growth of a profitable and sustainable essential oils and natural plant extracts industry in Australia. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at www.rirdc.gov.au/reports/Index.htm purchases at www.rirdc.gov.au/eshop Simon Hearn Managing Director Rural Industries Research and Development Corporation iii Acknowledgements There are a number of people whose skills and expertise in specialised areas have contributed to this project. Denis Rogers prepared all of the media used in this reported work. The expert technical assistance of Gary Everingham has been an invaluable contribution to this work. The financial support provided by RIRDC is gratefully acknowledged. Abbreviations A. mellifera Apis mellifera A. apis Ascosphaera apis oC degrees Celcius cm centimetre CO2 carbon dioxide g gram h hour MFC Minimal Fungicidal Concentration mL millilitre ppm parts per million SDA Sabarauds Dextrose Agar µL microlitre µm micrometre iv Executive Summary Chalkbrood is a highly contagious disease of the honeybee Apis mellifera caused by the heterothallic fungus Ascosphaera apis. It was first identified in Queensland in 1993, and, since that time, the disease has spread throughout Australia. Although not usually fatal, the disease causes reduced honey production. The effects of chalkbrood can be controlled by improved management techniques such as strengthening colonies with bees or hatching brood and enlarging colony entrances to aid ventilation. While chalkbrood infections have been related to stress factors, sensible management practices can reduce the numbers of spores of Ascosphaera apis in infected hives and hive equipment. Some hives appear to be more resistant to chalkbrood disease than others due to the ability of their adult bees to uncap and remove affected brood. The disease appears to be most prevalent in the spring when the brood area is increasing. The presence of chalkbrood in a colony can prevent normal colony growth and can seriously affect the honey production of the hive. While a broad range of chemicals have been used either in hives or in the laboratory to control chalkbrood, no chemicals for the treatment of chalkbrood have been registered for use in Australia and no specific strategy has been universally adopted or accepted by beekeepers around the world. Chalkbrood-resistant bees have been shown to exist naturally in Australia, but the large-scale production of such bees either overseas or in Australia has been slow. The thermal destruction (time/temperature) parameters have been determined for Ascosphaera apis in honey and the sensitivity of Ascosphaera apis to γ-irradiation using Cobalt 60 has been estimated. Such treatments of honey or other bee products can reduce the spread of the disease. There has been an increased interest in the investigation of alternative controls strategies. A compound for control of chalkbrood should have the following three characteristics. First, it must completely control the disease, or more realistically, keep it below the natural infection rate. Second, the control must be convenient to use, since practices such as applying chemicals and cleaning the bottom boards of colonies every week are not practical for commercial beekeepers with large numbers of colonies. Third, the control must not be more expensive than the natural loss due to the disease. This project has investigated the antifungal efficacy of over 50 natural products and found that a number of essential oils were particularly efficacious at controlling the in vitro growth of Ascosphaera apis. Of these, citral-containing oils were the most active, with growth inhibition at 250 ppm. These findings need to be progressed to field studies to evaluate product efficacy in the hive and to determine whether residues are a problem with this form of disease control. The most active antifungal test agents in this study were Nepalese Lemon Grass oil, Lemon Scented Eucalyptus (Eucalyptus citrodora) oil, Lemon Scented Tea Tree (Leptospermum petersonii) oil and a particular fraction of a New Zealand Manuka (Leptospermum scoparium) oil. All of these agents presented with a Minimal Fungicidal Concentration against Ascosphaera apis of 250 ppm. Citral is the major component of the former three oils while the active chemical in the New Zealand Manuka (Leptospermum) oil is a unique terpenoid agent (leptospermone). Interestingly, a number of other oils which should have contained significant levels of citral (East Indian Lemon Grass oil, cold-pressed Lemon oil, Natural Citral and lemon essential oil) returned negative MFC scores at 1000 ppm or greater. Several other oils exhibited moderate antifungal activity (active at 500 ppm) against Ascosphaera apis (Citricidal, several Tea Tree oils, a number of other New Zealand Leptospermum oils, a ginger oil and a lavender oil). The remaining essential oils were shown to be ineffectual against Ascosphaera apis in this in vitro test system. This project has also proposed a field test system to assess the efficacy of the most active antifungal agents identified in the in vitro assay system presented in this report. There are few reports in the literature of field trials of natural products against bee diseases, and even fewer investigating the efficacy of natural products against the causative agent of chalkbrood in an apiary system. v Contents FOREWORD III ACKNOWLEDGEMENTS IV ABBREVIATIONS IV EXECUTIVE SUMMARY V INTRODUCTION 1 MATERIALS AND METHODS 3 Isolates of Ascosphaera apis 3 Preparation of test agents 3 Preparation of culture plates containing test agents 3 Preparation of the inoculum 3 Inoculation and incubation of plates 3 In vitro assay of the inhibitory levels of test agents against A. apis 3 RESULTS 4 The Minimal Fungicidal Concentration of the various test agents 4 DISCUSSION 5 RECOMMENDATIONS 10 APPENDIX 1. 11 Minimal Fungicidal Concentrations of various natural agents against A. apis. 11 APPENDIX 2. 12 Chemical treatments tested on A. apis in culture or in bee colonies (Heath, 1982a). 12 APPENDIX 3. 13 A proposal for the field assessment of natural antifungal agents. 13 REFERENCES 14 vi INTRODUCTION The chalkbrood disease of honeybees (Apis mellifera) has been recognised since the early 1900s and has been extensively studied over the years. The current knowledge of chalkbrood disease, the control strategies and the epidemiology of the disease have recently been reviewed for the RIRDC by the NSW Department of Agriculture (Hornitzky et al., 2001). Chalkbrood of honeybees (Apis mellifera) is a fungal disease, which affects their larvae. It is caused by Ascosphaera apis (Spiltoir, 1955), a heterothallic organism that sporulates only when mycelia of opposite sexes (designated + and -) come together. Individual spores (2-4 µm in diameter) form within dark brownish green fruiting bodies known as spore cysts or ascocarps (Bailey and Ball, 1991). Young honeybee larvae (3-4 days old) are most susceptible to chalkbrood infection. Spores of A. apis are ingested with food and germinate in the gut of the larvae. These spores only form when two different strains of mycelia of A. apis (+ and -) touch each other. The spores are approximately 60 µm in diameter and can remain infective for 15 years (Bailey and Ball, 1991). The infected larvae are transformed into white chalk-like mummies when the mycelium of only one strain has infected the larvae. The mummies became grey/black when infected with both the + and the – strains of A. apis.