Control of Chalkbrood Disease with Natural Products

Home , Ascosphaera, Ascosphaeraceae, Ascosphaera aggregata, Ascosphaera apis, Tea tree oil

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

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.

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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

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

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.

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

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.

Chalkbrood was first reported in south-east Queensland in 1993 and by 1995 it had spread to New South Wales, Victoria and South Australia. More recently, it has been confirmed in Tasmania, Western Australia and the Northern Territory (Hornitzky, 2001). Chalkbrood is common in most beekeeping countries. It occurs widely in the temperate regions of the Northern Hemisphere and has long been known in Europe, Scandinavia and Russia (Betts, 1932) and in New Zealand (Seal, 1957). It was officially recognised in America and Canada in about 1970, and has subsequently been detected in Argentina, Japan, the Philippines, Central America and Mexico (Heath, 1985). Different strains of the fungus may have become established, perhaps in solitary bees which have been cultivated and distributed on a large scale in America for many years, particularly for the pollination of alfalfa. Solitary bees have suffered greatly from their own species of Ascosphaera, probably as a result of the increasingly industrial style of their management. A. apis has been isolated from some species of solitary bee and there may be strains with increased virulence for honey bees among them. Newly emerging healthy adult leafcutter bees have to chew their way through any larvae killed by the fungus, or through contaminated nest material in the tunnel ahead of them. Each young solitary bee can carry many spores on their bodies (Vandenberg et al., 1980), and some of these spores may find their way to honey bees foraging on alfalfa. Although Heath (1985a,b) found no cultural differences of any significance between American and British strains of A. apis, Glinski (1982) reported up to 20-fold differences between the virulence of some of the 40 strains they tested on young honey bee larvae (Bailey and Ball, 1991).

Spores ingested by bee larvae with their food germinate in the lumen of the gut (Heath and Gaze, 1987). The mycelium begins to grow there (Maurizio, 1934), penetrates the gut wall and eventually breaks out of the hind end of the larval body. When they occur, fruiting bodies form on the outside of the dead larvae. Since the optimal temperature for growth and formation of fruiting bodies is about 30°C, A. apis grows best in slightly chilled larvae (Maurizio, 1934). Experiments have shown that brood is most susceptible when chilled immediately after it has been capped (Bailey, 1967). The chilling need be only a slight reduction (from the normal 35°C) for a few hours. This can easily occur, even in warm climates, in colonies that temporarily have insufficient adult bees to incubate their brood adequately. Larvae are most likely to be chilled in early summer when colonies are growing. Drone larvae often suffer most as they are generally on the periphery of brood nests. The smallest colonies are at the greatest risk of becoming chilled because they have relatively large surface areas and the lowest capacity for heat (Hornitzky, 2001). Chalkbrood is aggravated in very small, rapidly expanding colonies (i.e. when the ratio of brood to adult bees is high or when it is increased experimentally), for mating virgin queens, and in observation hives (Heath, 1982a,b). A reduction in the ratio of adult bees to brood has been shown to aggravate chalkbrood (Koenig et al., 1987). Pederson (1976) showed that artificially heating hives in spring diminished the incidence of

the disease. Bailey and Ball (1991) also suggested that other non-lethal factors (e.g. viral or bacterial infection, poisoning, or inadequate food from diseased nurse bees) may slow the rate of development of larvae and increase the risk of the disease.

The migratory nature of many commercial beekeepers within Australia is likely to have contributed to its rapid spread within the continent. While most of the spores formed when a larvae is killed by chalkbrood are ejected from the colony by the house-cleaning bees, some will inevitably become lodged in food stores and find their way to healthy larvae via the nurse bees (Koenig et al., 1986). The spread of chalkbrood within colonies is suppressed by the normal temperature of the brood-nest or the absence of possible predisposing factors. Some further limitation is imposed by the need for at least two different spore strains to germinate within a larvae to form fruiting bodies. Generally, only a few larvae with fruiting bodies are observed in typical outbreaks of the disease. Indeed, combs have been found with all the dead larvae infected with only one fungal strain (Maurizio, 1934).

The diagnosis of chalkbrood is based on the recognition of disease signs and the identification of A. apis in diseased material (Hornitzky, 2001). Clinical evidence of the disease is characterised by dead larvae in capped cells, infection predominantly found in worker and drone larvae (rather than in queen larvae), small perforations in otherwise normal cell cappings, initially fluffy white, swollen and sponge-like larvae in uncapped cells and later hard and chalk-like "mummies" (either whitish or grey/black). The mummies remain whitish if they are infected with only one strain of the fungus, but will turn grey or black when infected with both strains of the fungus as a result of the production of fruiting bodies. By this stage, the cappings have frequently been removed by the bees. Laboratory diagnosis of chalkbrood disease is based on the identification of the causative agent (A. apis) in diseased material. Diseased material, preferably 'mummies' which have turned grey or black, is mixed with water or a dye.

The microscopic presence of spore cysts is usually sufficient to make a diagnosis. These spore cysts, which are about 60 µm in diameter, contain smaller round bodies known as spore balls (average 12 µm in diameter). The most infective stage of the fungus, the spores (2.9 x 1.4 µm) are found in these spore balls (Gilliam, 1990). In samples where only white “mummies” have been submitted and spore producing bodies cannot be detected microscopically, it may be necessary to grow the fungus in vitro (Hornitzky and Anderson, 2001).

Despite the broad range of experimental work that has been carried out to develop chalkbrood control strategies, there is no specific strategy that has been universally adopted or accepted by beekeepers around the world. Management strategies, chemicals and the use of bees that show resistance to chalkbrood have all been shown to have some benefit although no individual control strategy will ensure a cure for the disease. The effective control of chalkbrood will probably require a combination of control strategies. The fact that A. apis is so widespread makes the possibility of its eradication unlikely. If the disease cannot be eradicated, then any chemical that is considered for use against chalkbrood must be demonstrated to produce minimal residues in honey or other bee products. Long- term use of any chemical for disease control is likely to result in the development of resistance to that chemical. However, the use of a chemical that controls chalkbrood and does not produce residues in honeybee products would be of benefit to beekeepers, even if A. apis did develop resistance. In the long-term, the development of antibiotic resistance by a honeybee pathogen has already occurred in the beekeeping industry in several countries where American Foulbrood has developed resistance to oxytetracycline. The effective control of chalkbrood will probably require a combination of control strategies. This project has attempted to identify and test candidate chemicals for the control of chalkbrood disease.

MATERIALS AND METHODS

Isolates of Ascosphaera apis Two isolates of the Chalkbrood fungus A. apis were obtained from mummies of naturally infected hives in two widely diverse areas of Queensland. One isolate was obtained from a hive located at Fisherman Islands at the mouth of the Brisbane River in Southern Queensland. The second isolate was obtained from the hive of a hobbyist beekeeper in Townsville, North Queensland.

Preparation of test agents Essential oils and other prospective antifungal agents trialled in this work were provided by Craig Davis. The details of these oils are outlined in Appendix 1 of this report. Each of the natural agents trialled in this research were screened for their efficacy against the fungus A. apis by preparing serial dilutions in sterile 0.01% Tween 80. Each dilution of oil was added to 50 mL of molten Sabarouds Dextrose Agar (SDA) which had been held in a water bath at 48oC. Separate dilutions of each product were assayed with the final concentrations of oil in agar being 1000, 500 and 250 ppm.

Preparation of culture plates containing test agents Tri-divided bacteriological petri plates were filled with 15 mL of each dilution of test agent in molten Sabarauds Dextrose Agar (SDA) culture medium, and each dilution was poured in triplicate. The mixtures of molten medium and test dilutions were allowed to set at room temperature.

Preparation of the inoculum Mummified larvae from a naturally infected hive at Fisherman Island, Brisbane were cultured on SDA plates by dabbing the “mummies” onto the agar at various points around the bacteriological plate. The plates were incubated at 30oC in plastic bags for 7-10 days. At the conclusion of the incubation period, plates were examined for fungal growth. Colonies of A. apis grown on SDA plates are 5-7 mm in diameter, and white to pale buff to black colour, and have floccose, matted mycelia (Heath, 1982a,b; Splitour and Olive, 1955). The spores of A. apis were washed off the plates using 0.01% sterile Tween 80 and loosened by shaking the suspension with glass beads for 2-3 hours. The spore suspension was adjusted with sterile Tween 80 so that each inoculum contained approximately 50 x 106 spores/mL, using a Neubauer Counting Chamber.

Inoculation and incubation of plates 100 µL of spore suspension containing approximately 5x106 spores of A. apis was added to plates containing each of three dilutions of test agents, and each dilution was inoculated in triplicate as described above. The spore suspension was spread evenly over the surfaces of the agar using sterile disposable cell scrapers. All plates were sealed in plastic bags and incubated at 30oC for 14 days. At the conclusion of the incubation period, plates were morphologically examined for the presence of A. apis colonies. Fungal colonies grow moderately slowly and are 5 to 7 cm in diameter after 10 days. They produce deeply floccose or matted aerial mycelia, which are usually white to pale buff but may become coral to pale reddish brown with age (Bissett, 1988).

In vitro assay of the inhibitory levels of test agents against A. apis Test agents which failed to inhibit the growth of A. apis after 14 days incubation at a screening concentration of 1000 ppm were considered to be ineffective in preventing growth of this fungus. Plates containing diluted test agents which showed no growth of A. apis at concentrations of 500 ppm and 250 ppm were subjected to further in vitro assay techniques to determine the Minimal Fungicidal Concentration (MFC) which would prevent growth of A. apis. These products were further serially diluted in sterile 0.01% Tween 80 as described previously in this report so that the final concentrations in molten agar were 125, 62.5 and 31.25 ppm. The culture plates containing the diluted test agents, the inoculum preparation and the inoculation were prepared and treated as described previously in this report.

RESULTS

The Minimal Fungicidal Concentration of the various test agents The Minimal Fungicidal Concentrations (MFC’s) of the forty-nine natural essential oils, a banana extract and a sample of monolaurin, as determined in the in vitro assay techniques against A. apis (the isolate obtained from Fisherman Islands) are outlined in Appendix 1.

The most active antifungal test agents in this study were Nepalese Lemon Grass Oil, Lemon Scented Eucalyptus (Eucalyptus citrodora), Lemon Scented Tea Tree (Leptospermum petersonii) 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, the banana extract and the monolaurin preparation were shown to be ineffectual against Ascosphaera apis in this in vitro test system. The pictures below are typical growth presentations of Ascosphaera apis in the in vitro system employed in this investigation.

DISCUSSION

The chalkbrood disease of honeybee (Apis mellifera L.) brood is caused by the fungus Ascosphaera apis (Maassen ex Claussen) Olive et Spiltoir, and can seriously hinder colony build-up. Thus, it reduces both pollination activity and the honey crop (Mehr et al., 1978). While a range of chemicals (both artificial and natural) are reported to have activity against A. apis, no therapeutic agents are registered in Australia for use against chalkbrood. There are a number of techniques that can be used to minimise the effects of chalkbrood. Strengthening badly diseased colonies can be achieved by adding young adult bees or hatching brood, by feeding sugar syrup and by not allowing bees to winter in too large a brood chamber (Seal, 1957). Enlargement of the colony entrance to aid ventilation (Gochnauer et al., 1975) and, in severe cases, destruction of affected combs has been recommended (Betts, 1951). New comb can reduce the incidence of chalkbrood (Nelson and Gochnauer, 1982; Koenig et al., 1986). Honey can be rendered free of viable A. apis spores by holding it in water baths at 70°C for 2 hours (Anderson et al., 1997). Hive materials and bee products can be rendered free of viable chalkbrood spores using γ-irradiation with Cobalt 60 (Katznelson and Robb, 1962), high-velocity electron beams (Shimanuki et al., 1984) and ethylene oxide fumigation (Gochnauer and Margetts, 1980). Methyl bromide has been used to fumigate hive equipment but chemical residues were detected in wood and wax (Faucon et al., 1982).

Bailey and Ball (1991) have reported that bees can be selected for resistance towards disease, as this has been demonstrated by Rothenbuhler and his colleagues (1964) with American foulbrood. Unfortunately, resistance towards chalkbrood disease, which is the most likely of all microbial diseases to kill a bee colony, is at least partly determined by recessive factors (Hornitzky, 2001). This suggests that death of infected colonies has enabled the species to survive better than natural selection for resistance. Whatever the reason, it makes the maintenance of resistant strains more difficult than if resistance were due to dominant genes. There has been considerable interest in the development of disease-resistant bees, both overseas and in Australia. Rothenbuhler (1964) reported that the efficiency of the hygienic behaviour of adults in removing diseased larvae was further separable into a factor for uncapping the cells and a factor for removing the larvae. This behavioural characteristic has been further examined by Spivak and Gilliam (1993) who determined the effect of changing colony strength on hygienic behaviour, responses to freeze-killed and live brood of hygienic and non-hygienic bees, the effect of adding hygienic bees to non-hygienic colonies and the response of feeding Ascosphaera apis (the cause of chalkbrood) to hygienic and non-hygienic bees. Spivak and Downey (1998) promoted the importance of propagating colonies that remove pupae infested by Varroa mites and show resistance to chalkbrood and American foulbrood. They evaluated two commonly used field assays (the freeze-killed brood assay and the pierced brood assay) to screen colonies for hygienic behaviour. Both involve determining the time required for worker bees to remove dead capped brood from a section of comb. Their results indicated that bees from non-hygienic lines can be induced to express hygienic behaviour only if a sufficiently strong stimulus is present. The hygienic colonies removed significantly more freeze-killed brood than the commercial colonies, had significantly less chalkbrood, had no American foulbrood, and produced significantly more honey than the commercial colonies. Studies of the natural hygienic behaviour of Australian honeybees were carried out by Oldroyd (1996). Ten strains of Australian commercial honeybees were evaluated for hygienic behaviour. Dead pupae were inserted into the colonies. Most colonies (80%) were non-hygienic, but two strains gave a good overall performance in the test and comprised colonies that were highly hygienic. The results suggest that hygienic behavioural morphs exist in Australia's commercial bee strains, and that selective breeding should be able to produce suitable genotypes.

The management of honeybee colonies is becoming increasingly dependent on the use of chemotherapeutic agents. For example, menthol is sometimes used for the control of the tracheal mite (Acarapis woodi) and fluvalinate has been used for the control of Varroa jacobsoni. This increasing dependence on the use of chemotherapeutic agents has created several problems: profit margins of beekeepers have been reduced as expenditures for chemicals have risen;

labour requirements have increased as the number of treatments increases; beekeepers have become vulnerable to the evolution of resistance in the parasite and pathogen populations due to the limited number of available chemotherapeutics; toxicological hazards to beekeepers and bees have increased; and chemical residues have contaminated some honey crops (Gamber, 1990).

Research has shown that many pathogens and parasites can be controlled by selection for resistance or tolerance in the honeybee population. However, this work has not translated into commercial application. Elimination of pathogens from equipment (without bees) has been accomplished using ethylene oxide and γ-irradiation. These methods have achieved only a limited degree of commercial success (Calderone et al., 1994).

The identification of effective natural compounds will increase the number of available chemotherapeutic agents, and reduce the threat of resistance in parasite and pathogen populations by providing alternatives with greater public acceptance. Identification of compounds with activity against more than one organism may reducte in the total amount of chemicals required for parasite and pathogen management (Calderone et al., 1994). Natural plant extracts may play a significant role in the management of honeybee parasites and pathogens. The translation of the results of in vitro studies into a successful management strategy requires the resolution of several complex problems. These include the demonstration of in vitro activity at levels that are not toxic to honeybee larvae, pupae and adults, and that do not affect the flavour of the honey. The development of an adequate delivery system to ensure the consumption of extracts in food supplements by adult bees is also important. Typically, the consumption of supplements containing extracts at concentrations above 0.5% is greatly reduced. Additionally, formulations must be competitive with naturally-occurring nectar and pollen flows.

In America, the management of honeybee colonies is becoming increasingly dependent on the use of chemotherapeutic agents. In addition to the use of oxytetracycline for the control of brood diseases, the recent introduction of two parasitic mites has resulted in the use of additional agents. This increasing dependence on the use chemotherapeutic agents has created several problems. The profit margins of beekeepers have been reduced as expenditures for chemicals have risen. The labour requirements have increased as the number of treatments have increased. Beekeepers have become vulnerable to the evolution of resistance in the parasite and pathogen populations due to the limited number of chemotherapeutics available for control of each individual organism. In addition, toxicological hazards to beekeepers and bees have increased and chemical residues have contaminated some honey crops. Chalkbrood, a fungal disease caused by the pathogen Ascosphaera apis, attacks larval honeybees. Symptoms tend to be seasonal, and significant decreases in honey yield and pollination capacity may occur. American Foulbrood and European Foulbrood have been prevented by the prophylactic use of oxytetracycline. Treatment of the active disease is problematic as oxytetracycline eliminates the vegetative stage of American Foulbrood, but does not affect the spore stage, and suspension of the antibiotic treatment generally results in recurrence. Management of European Foulbrood with antibiotics is generally more successful, with symptoms usually disappearing either entirely or for long periods. Unfortunately, the development of antibiotic-resistant strains of these foulbrood pathogens has recently become a serious threat. There is no chemotherapeutic agent currently registered for the control of chalkbrood.

A broad range of chemicals have been tested either in honeybee colonies and/or on A. apis in culture for the control of chalkbrood. Many approaches have been tried in the effort to find a means of preventing and/or controlling chalkbrood. For example, development of chalkbrood-resistant stocks (Herbert et al., 1977), control of broodnest temperatures and disinfection (Pedersen, 1976), hive ventilation (Morris, 1977), and chemical therapy (e.g. sodium propionate and sorbic acid (Taber et al., 1975), citral and geraniol (Gochnauer et al., 1979), and sorbic acid and griseofulvin). Heath (1982a) has produced a comprehensive list of chemicals in his review of chalkbrood (Appendix 2). Since this review, a number of other studies have demonstrated the activity of chemicals against chalkbrood. Trichloroisocyanuric acid (which releases chlorine gas when exposed to air) was able to control the progression of chalkbrood in the hive (Tanaka et al., 1984; Serrano et al., 1995). The effect, however, was dependent on the number of spores and the degree of aggregation of spores in spore balls. Vapours of propionic acid were also shown to be efficacious (Kajikawa and Nakane, 1986). Herbert et al. (1985, 1986) have shown that certain alkyl amines not only stimulate the removal of chalkbrood cadavers but also inhibit the growth of the fungus in vitro. A World Patent granted to the Alcide Corporation (Kemp and Kross, 1997), entitled “Compositions and methods for prevention of diseases associated with honeybees”, proposes to treat chalkbrood with a protic acid and chlorite ions. Researchers in Spain (Jimenez et al., 1994) demonstrated that that imidazole derivatives possessed appreciable antifungal activity against A. apis. Glinski and Clunielewski (1996) demonstrated that an imidazole derivative (Klotrimazol) stimulates defence reactions in the honeybee. Phagocytosis and the level of innate and inducible hemolymph immune proteins increased in worker bees exposed to Klotrimazole. Jendrejak and Kopernicky (1998) evaluated the efficacy of 85% formic acid (Apiform) against A. apis. The medication was applied thorough a wick in the centre of the honey chamber. They reported 100% effectiveness in 1994 and 87.9% in 1997 where affected combs were removed and bottom boards of hives were cleaned before treatment. Thurber (1979) reported good chalkbrood control from feeding sodium propionate and potassium sorbate and benomyl accompanied by some rather elaborate management techniques including extensive colony sanitation measures. While Taber et al. (1975) have also reported success in chalkbrood control using sodium propionate and potassium sorbate, Menapace and Hale (1981) have reported that sorbic acid is definitely not a reliable control for chalkbrood in all cases and in all geographical areas. None of these treatments have proved efficacious to the point where it has been universally accepted. A chemical which is effective against chalkbrood, does not produce residues in bee products and is not harmful to bees is yet to be found.

The lack of any effective control agent for chalkbrood has resulted in an increased interest in the investigation of alternative control strategies. Essential oils and oleoresins of many plants are known to exhibit significant antimicrobial activity against a wide spectrum of microorganisms. Clove oil has been found to effectively inhibit the growth of many Gram-negative bacteria (Forag et al., 1989), several species of Penicillium (Azzouz and Bullerman, 1982), as well as the spore-forming Clostridium botulinum (Ismaiel and Pierson, 1990). Cinnamon oil exhibits activity against mycotoxigenic moulds, Penicillium spp. and C. botulinum. Thymol, a major component of thyme oil, is highly active against Aspergillus parasiticus (Buchanan and Shepherd, 1981), Staphylococcus aureus (Karapinar and Aktug, 1987) and C. botulinum. The literature cites references which have investigated the susceptibility of a broad range of fungi to natural products, including citral in lemon peel (Rodov et al., 1995), sorbic acid and essential oils (Kubo and Lee, 1998).

Evaluation of plant extracts for the control of honeybee pathogens and parasites is limited. Several studies have shown essential oils to be effective in controlling bee diseases such as American Foulbrood disease (Carpana et al., 1996; Floris et al., 1996, Muszynska, 1999), tracheal mite (Acarapis woodi), Varroa jacobsoni and Chalkbrood (Higes et al., 1998). Colin et al. (1989), using in vitro tests, demonstrated fungicidal activity of essential oils of Thymus vulgaris, Satureja montana and Origanum vulgare against chalkbrood. Field trials demonstrated that application of Satureja montana resulted in a reduction in the number of chalkbrood-infected larvae observed at entrances of infected colonies. Calderone et al. (1994) tested 8 plant extracts for their activity against A. apis and found that cinnamon oil completely inhibited the growth of A. apis at 100 ppm for 168 h. Bay oil, citronellal, clove oil, origanum oil and thymol inhibited all growth at 1,000 ppm for 168 h.

Camphor inhibited all growth at 10,000 ppm for 168 h, and α-terpinene inhibited all growth for 72 h at 10,000 ppm. Several compounds reduced growth in a dose-dependent manner at levels below their threshold values. By comparison, an in vitro examination of the efficacy of a number of essential oils in Argentina (Larran et al., 2001) found that coriander oil was the most effective at controlling fungal growth. The oils tested were found to be effective only at concentrations greater than 700 ppm. Larran et al. (2001) found that there was no discernible difference between the susceptibility of different Ascosphaera apis strains to antifungal agents.

In our investigation, two strains of Chalkbrood fungus were obtained from mummies of naturally infected hives in two widely diverse areas of Queensland (Fisherman Islands at the mouth of the Brisbane River in Southern Queensland and Townsville in North Queensland). Both strains had similar susceptibility to the various prospective antifungal agents examined. 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. Previous studies have suggested that Thyme oil (Colin et al., 1989) and origanum and cinnamon oils (Calderone et al., 1994) are efficacious against the chalkbrood fungus, but these observations have not been confirmed in our current investigation.

Some previous studies have investigated the presence of citral (and related compounds in the honeybee). The Nassanoff scent gland secretions of adult worker honeybees contain geraniol, citral (geranial and neral in proportions of about 2:l), nerolic and geranic acids (Boch and Shearer, 1962, 1964; Shearer and Boch, 1966). Iso-pentyl acetate and 2-heptanone are present in the sting and mandibular glands, respectively (Boch et al., 1962; Shearer and Boch, 1965). The vapours from citral and geraniol have been shown to inhibit the growth of certain wood-rotting fungi (Stranks, 1977). In vitro tests showed that citral and geraniol inhibited the fungus Ascosphaera apis which causes chalkbrood disease in the honeybee Apis mellifera. Gochnauer et al. (1979) tested natural oils (oil of citronella, Melissa oil) which contain citral, sorbic acid and sodium propionate. The vapours of 5 µL of citral or 10 µL of geraniol per culture dish prevented vegetative growth. Daily applications of 30 µL of citral per dish killed sporulated cultures within 48 hr. However, A. apis spores in dried larval remains (mummies) survived 96-hr exposure to the vapours of 30 µL of citral per dish per day. Vapours of a geranic and nerolic acid mixture, 2-heptanone, iso-pentyl acetate, octanoic acid, and citronella and melissa oils were less inhibitory than citral or geraniol. These compounds have been previously reported to be effective in the control of chalkbrood disease (Taber et al., 1975). Iso-pentyl acetate was ineffective at all levels tested. It is tempting to speculate that the Nassanoff gland scent secreted by honeybees might repress A. apis in the honeybee colony. However, observations by Renner (1960) indicate that the scent gland secretion plays a role in mutual attraction of bees outside their hive, but bees inside the hive keep their scent glands closed (Gochnauer et al., 1979). Viable fungus persists in the fumigated mummies probably because of the solid mycelial growth and heavy spore content of the infected larvae (Thorstensen, 1976). However, since hive bees remove infected chalkbrood mummies intact and without chewing them, it seems that surface sterilisation is sufficient to retard transmission of spores by bees within the hive. Geraniol or citral may be able to be administered within the hive without severely disrupting the social organisation of the honeybee colony, while the alarm substances 2-heptanone and iso-pentyl acetate could probably not be used even if they were effective fungistats. It appears that citral, added to colony food supplies, might effectively suppress the growth or kill A. apis in susceptible honeybee larvae, in which case a

preventive therapy of the disease would be feasible. The efficacy of citral and geranial (an isomer of citral) against Ascosphaera apis has been previously been reported (Gochnauer et al., 1979; Menapace and Hale, 1981). These studies showed that citral was efficacious in controlling Ascosphaera apis infection in vitro, but that citral in combination with sodium propionate and potassium sorbate did not prevent or control chalkbrood under field conditions.

There is some anecdotal evidence in Australia that banana fruit placed in hives is an effective control for chalkbrood. Our research has failed to find any inhibitory action of banana extract when it is incorporated into an in vitro culture system. Any beneficial effect that banana fruit placed within the hive might have in controlling or reducing the severity of chalkbrood would most probably arise from volatiles evolved by the fruit. It is not inconceivable that the fruit might evolve one or more volatile compounds that are inhibitory to the spore germination or mycelial growth of the causal fungus A. apis. Such fungistatic or fungitoxic/fungicidal volatiles might be the normal products of fruit metabolism or the products of microbial degradation of the fruit as it senesces. The banana fruit contains at least 200 individual volatile components (Palmer, 1971). Many more volatile compounds, including fermentation products, could arise from microbial action (Sureh and Ethiraj, 1991). Fermentation products (ethanol and acetaldehyde) have been shown to inhibit mould growth on oranges (Yuen et al., 1995).

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 (see Appendix 3). 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.

RECOMMENDATIONS

Further work needs to be carried out to identify and test candidate chemicals for their ability to control chalkbrood and whether they leave residues in honeybee products in field conditions. The propagation of chalkbrood-resistant bees in the beekeeping industry needs to be further investigated. Beekeepers should also be trained in assessing whether their colonies have chalkbrood resistance traits. These chalkbrood-resistant bees could then be propagated, provided they also have the necessary production capabilities.

These results suggest that plant extracts might play a significant role in the management of honeybee diseases. The oils used in this study should be considered an alternative option for the control of this important disease.

Appendix 3 provides a draft outline for a field trial to assess the efficacy of the most promising products from the in vitro investigations presented in this study. Support for a field trial of the most promising natural products to have been identified in this laboratory screening may afford us an additional defence against this fungal pathogen of the honeybee.

APPENDIX 1. Minimal Fungicidal Concentrations of various natural agents against A. apis.

OIL IDENTIFICATION MFC (µL/L) 1 Lemon Grass Oil (East Indian) 1000 2 Lemon Grass Oil (Nepal) 250 3 Lemon Cold Pressed >1000 4 Natural Citral 1000 5 Citricidal 500 6 Lemon Scented Eucalyptus (Eucalyptus citrodora) 250 7 Lemon Scented Tea Tree (Leptospermum petersonii) 250 8 Tea Tree (Melaleuca) Oil >1000 9 Tea Tree Oil (TT92079) 500 10 Tea Tree Oil (Fraction Y) 500 11 Tea Tree Oil (38% T) 500 12 Tea Tree Oil (Pacific Oils) >1000 13 Tea Tree Oil (ANDO) >1000 14 Tea Tree Oil (Manuka & Kanuka) >1000 15 Coast Manuka Oil (Leptospermum scoparium) 500 16 Manuka Oil (Fraction 8) 250 17 Coast Manuka Oil (100% Type A) 500 18 Manuka Oil 1000 19 Manuka Oil (Fraction 1) 500 20 Kanuka Leaf Oil (Kunzea ricoides) 500 21 LEMA Oil >1000 22 Leptospermum polygalifolium 1000 23 Melaleuca alternifolia >1000 24 Celery Seed Oil (I) >1000 25 Celery Seed Oil (II) >1000 26 Thyme (Thymus vulgaris) >1000 27 Ginger Oil (P17077) >1000 28 Ginger Oil (CO2 extract) >1000 29 Ginger Oil (I) 500 30 Ginger Oil (II) >1000 31 Sweet Fennel Oil (P14137) >1000 32 Lemon Essential Oil >1000 33 Mandarin Cold Pressed (Citrus reticulator) >1000 34 Lavender (Lavender augustifolia) 500 35 Orange Navel Cold Pressed (Citrus sinensis) >1000 36 Grapefruit Cold Pressed (Citrus x Paradisa) >1000 37 Leptospermum livistigii >1000 38 Bakea virgata >1000 39 Leptospermum Spp. >1000 40 Leptospermum petersonii >1000 41 Eucalyptus globulus >1000 42 White Thyme (Thymus vulgaris) >1000 43 Sage (Salvia officinalis) >1000 44 Chamomile (Matricaria chamomilia) >1000 45 Rosemary (Rosmarinus officinalis) >1000 46 Oregano (Origanum vulgara) >1000 47 Peppermint (Mentha piperita) >1000 48 Pine (Pinus pinasta) >1000 49 Cinnamon (C. zeylanicun) >1000 50 Banana extract >1000 51 Monolaurin >1000

APPENDIX 2. Chemical treatments tested on A. apis in culture or in bee colonies (Heath, 1982a).

Substances(s) Concn Procedure sorbic acid 0.05% in culture sorbic acid 0.1% fed to colonies in syrup sorbic acid 5.0% aqueous solution on culture sorbic acid and sodium propionate 0.1% fed to colonies in pollen cakes sorbic acid and sodium propionate 0.2% fed in 225 g pollen supplement sorbic acid and sodium propionate 0.1% fed to colonies in syrup methyl parahydroxybenzoate 0.05% in culture methyl parahydroxybenzoate 0.5% in aqueous solution on culture for 5min Ascorbic acid in culture thiabendazole 1.0% in sucrose dusted over colonies thiabendazole 0.2% in 225 g pollen supplement thiabendazole benomyl 0.25% fed to colonies in syrup benomyl 0.25% as above and also sprayed over frames dinocap 0.25% fed to colonies in syrup Acti-dione griseofulvin in culture Nipagin 2.0% fed to colonies in syrup Nipagin & potassium sorbate fed to colonies in syrup cycloheximide 0.0025% in culture cycloheximide 0.01% fed to colonies in syrup Amphotericin B in culture Nystatin in culture Mycostatin (=Nystatin) 1 million IU/L fed to colonies in syrup Mycostatin 0.05% aqueous solution on culture for 24 h Mycocidin 100-150 g/col sifted over colony citral 5 uL/dish vapour on culture geraniol 10 uL/dish vapour on culture 3P (polyfungine cholate) 40% sprayed on brood in syrup Amophotericin B in culture Lastanox (bis-tributyl-stannic oxide) 2.0% aqueous solution for 5 min on cultures Nitrofungin 2.0% aqueous solution for 24 h on cultures Chinosol 0.05% sprayed over bees Soloxin fed to colonies in syrup formalin 0.2% aqueous solution for 1 h on cultures Fesia-form 4.0% 250 mL sprayed over combs of colony allylisothio-cyanate 1.0% aqueous solution for 1 h on cultures boric acid 0.5% aqueous solution for 24 h on cultures salicyclic acid 1.0% aqueous solution for 24 h on cultures thymol 0.7% in culture thymol 0.7% sprayed over combs thymol 0.8% sprayed on colonies propolis 15% alcoholic solution on culture for 24h copper sulphate 0.1% fed to colonies in syrup acetic acid (glacial) vapour on culture for 1 h

Appendix 3. A proposal for the field assessment of natural antifungal agents.

Beehives and colonies of bees. Beehives will be placed on benches to isolate them of the ground, and located in the Animal Research Institute, Yeerongpilly. All beehives will be populated by homogenous colonies of bees (Apis mellifera L.) and with queens of the same age. Field trials will be undertaken in spring when the maximum incidence of the disease is observed and the greatest growth of the colonies occurs.

Essential oils. The essential oils with antifungal potential will be incorporated into honeybee food as vehicles and applied to the beehives in plastic containers. In a similar study by Higes et al. (1998), three types of foods were provided – a syrup, a semi-solid food and a pollen supplement. The syrup (50% water, 50% honey) containing 0.1% of essential oil (500 mL per beehive) was the only food which was totally consumed by the bees in 24 h. The syrup with higher concentrations of essential oil was rejected by the bees. The control beehives consumed all of the syrup in 24 h, while the semi-solid food and the pollen supplement were partially consumed (30% and 10%, respectively). In the study proposed here, a syrup formulation will be used and the amount of food consumed by the bees will be recorded daily.

Infection with Ascospherosis apis. Beehives will be inoculated three times per week over a four- week period, with 8 mL of a suspension of A. apis spores (five black mummies will be macerated in 5 mL of distilled water to obtain a concentration of about 106 spores per mL). The beehives will then be divided randomly in two groups and each will receive the spore suspension for a further four weeks. Homogenous portions of honeycombs with 30-100 larvae of the same age will be extracted from the beehives and placed subjected to a thermal shock (22 ± 2°C for 24 h). The effectiveness of the essential oils in controlling ascospherosis will be determined by comparing the percentage of healthy larvae in the infected hives with those in the control hives.

Development of the ascospherosis and tests of effectiveness in field. The main problem with producing ascospherosis in the field is the method of administration of the fungicidal molecules to the digestive tract of the larvae in the 24 h prior to hatching (when they are most susceptible to develop of the disease). The honeybee colony must incorporate these substances into its food chain and interchange them with the larvae as they hatch. This method of administration assists the beekeeper by reducing handling, minimising the cooling of the young and avoids the problem of looting. Ascospherosis is clearly a multifactorial disease, the correct interpretation of the clinical tests in the field needs to use a technique that assures the controlled development of the disease and the presence of typical symptoms in a known number of larvae.

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