Chalkbrood Research at Madison, Wisconsin*
by FLOYD E. MOELLER and PAUL H. WILLIAMS
Agricultural Research Service, U.S. Department of Agriculture, and the Department of Plant Pathology, Colleges of Agricultural and Life Sciences, University of Wisconsin, Madison 53706
CHALKBROOD disease of honey bees, Apis mellifera L., is caused by a fungus, Ascosphaera apis (Maassen ex Claussen) Olive & Spiltoir, originally known as Pericystis apis, that infects many insect species in the larval stage. Such species as alfalfa leafcutter bees, Megachile rotundata (F.) (M. pacifica), and alkali bees, Nomia melanderi Cockerell, may be affected by chalkbrood. Presently some uncertainty exists as to whether this is the same organism or whether specific strains exist.
The disease was noted in the early 20th century in Europe where beekeepers called it stonebrood or chalkbrood. The dead bee larvae were mummified and largely replaced by fungal mycelium, which gave them a grayish or chalky color, hence the name chalkbrood. Most of the European reports indicate that chalkbrood is seldom serious and that the bees will usually clean up the disease without special treatment (Claussen 1921). Weak colonies or colonies weakened by other diseases are most susceptible according to Deans (1940). Incidence of chalkbrood in parts of Germany was associated with the damp, cool oceanic climate (Dreher 1938).
Bailey (1963) reported that chalkbrood occurred only in Europe. However, Seal (1957) reported it in New Zealand. First official records of the occurrence of chalkbrood in the United States were from Utah on wild bees in 1965 (Baker and Torchio 1968) and from California on honey bees in the spring of 1968 in Tehama County. This first report was by Homer L. Foote, state apiary inspector of California, and was in a single apiary. Gerald Thomas of the University of California at Berkeley, Division of Plant Pathology, confirmed it (unpublished report). Since then, we have reports of the disease in Minnesota and North Dakota in 1969; Montana, Wyoming, and British Columbia in 1971; Florida in 1972; Wisconsin in 1973; and Illinois in 1975. Apparently the disease is now widespread in Canada and the United States.
Cool, humid weather conditions do not appear to influence the occurrence of chalkbrood disease. Curiously, many western states have been drier and warmer recently but have plenty of chalkbrood. Is this a more virulent strain of the fungus? (The inside of the active honey bee colony has a relatively constant humidity level regardless of climate.)
The occurrence of chalkbrood was thoroughly reviewed by Hitchcock and Christensen (1972).
The USDA Agricultural Research Service is currently investigating etiology and controls for chalkbrood in honey bees at Laramie, Wyoming; Madison, Wisconsin; and Tucson, Arizona. At Beltsville, Maryland, Dr. L. Batra of the Mycology Laboratory identified Ascospaera apis affecting leafcutter bees. His data have been turned over to Dr. Robbin Thorp of the University of California at Davis.
Life Cycle of Ascosphaera apis
The life cycle of the chalkbrood organism is not clearly defined in nature. This is one area that will be studied at Madison during the research on control procedures.
In culture, A. apis exists as a dense grayish-white mycelium containing aerial, surface, and subsurface hyphae. Surface hyphae are 4 to 8 microns wide, and vegetative nuclei are very small. It is morphologically heterothallic, that is, it has + and – strains (or male and female strains) that, when inoculated onto the same agar plate, will show a black line of spore cysts where the mycelia of the two strains make sexual union. No apparent morphological differences exist between the two types when seen in culture.
When the hyphac of compatible types come close together, some of the female hyphae produce large protuberances (ascogonial primordia) that elongate and grow toward the male hyphae. The formation of the ascogonium and its contact with the male hypha take only 15 to 20 minutes. The nutriocyte reaches maximum size in about one day and then begins a complex series of cell divisions and delineations that terminate in the formation of asco-spores contained in numerous spore balls enclosed in a spore cyst that appears black because of the thick, dark colored wall.
The spores of A. apis require a nearly anaerobic environment for germination, but the mycelium requires an aerobic environment for growth (Bailey 1967). Thus, optimum temperature for growth is about 30ºC (86ºF), and spores germinate best at 35ºC (95ºF). However, when the temperature is lowered to 25ºC (77ºF), oxygen can move more readily into the agar, which allows the mycelium to grow.
Bailey (1967) indicated that infection of the honey bee larva begins when chalkbrood spores are ingested with the larval food. However, the germinated spores are generally voided harmlessly with the feces because the mycelium cannot grow in the anaerobic environment of the gut. Spores apparently germinate in the lumen of the gut, especially at the hind end that does not communicate with the rectum until after the larva is sealed in the cell to pupate. Thus, Bailey (1967) contends that A. apis is rarely lethal unless larvae are chilled for a brief period shortly after sealing.
Transmission of spores may be by wind from mummies carried to the exterior of the hives. Spores could he picked up by foraging bees at nectar, pollen, or water sources and passed on to larvae in their food; or infection could be spread by adult bees with contaminated mouth and body parts. At Madison, colonies in outyards not showing chalkbrood in 1974 were requeened with queens reared in colonies at the homeyard that contained chalkbrood. In 1975, all these outyards showed some colonies with chalkbrood, an indication that the infection had spread to them via queens. In the spring of 1975, colonies clean in 1974 were requeened with queens purchased from a breeder who had chalkbrood in his outfit. During the summer of 1975, these colonies showed some chalkhrood, further evidence that queens may disseminate the disease. Spread via queens could account for the rapid spread throughout the country.
Toumanoff (1951) states that spores remain viable for 15 years.
Effect on Colonies
The effect of chalkbrood on colony populations is difficult to measure, though any disease that kills brood will directly affect colony strength. Chalkbrood in some areas and at some seasons may produce severe mortality of brood. For example, reports from beekeepers in the prairie provinces of Canada and certain localities in the United States have indicated severe brood kill from chalkbrood.
At localities where genetically inbred honey bee stocks are maintained (Madison, Wis., and formerly Davis, Calif.), chalkbrood may cause alarming losses of brood. The inbred lines are often physiologically weaker and seem to be excellent targets for chalkbrood. It is likely that strains of bees vary in their susceptibility to chalkbrood. At Madison and elsewhere, mortality has been high among drone larvae.
Other stress factors seem to influence the development of chalkbrood. In 1975, sacbrood disease common over the years in some inbred lines at Madison, was not noted; instead chalkbrood was present. Does this mean that larvae weakened by sacbrood virus succumb to chalkbrood fungus before the sacbrood can be noted?
Peculiarities of Chalkbrood Affecting Research
Chalkbrood disease is particularly difficult for the researcher because the active disease can only be identified by mummies in the brood cells (white, gray, or black). Also, evaluation of inoculation or treatment is difficult because of the tendency of colonies to remove all traces of diseased material following hive manipulation or requeening, or during heavy sirup feeding or honey flows. Some strains of bees that are heavy propolizers sometimes propolize chalkbrood mummies into the cells. We do not know the final disposition of such propolized mummies. Propolized mummies may serve as a reservoir for infection at a later time.
Chemotherapeutic Tests to Date
A laboratory study with caged bees was made at Madison in February 1974 to determine the effect of the fungicide benomyl on Nosema apis Zander. A report of this research follows but is not intended as a recommendation to beekeepers. Benomyl (methyl-l-(butylcarbamoyl) -2- benzimidazolecarbamate) fed in sugar sirup had no effect on Nosema apis at any level from 250 to 2000 ppm. However, the results furnished information concerning the toxicity of benomyl to adult honey bees that was useful in testing the material for control of chalkbrood fungus. At 250 ppm, benomyl was not toxic to adult bees, but 500, 1000, and 2000 ppm did increase bee mortality. Earlier Giauffret and Taliercio (1967) had described the effect of thiabendazole (2-(4-thiazolyl) benzimidazole) on Ascosphaera apis and suggested that 500 ppm was nontoxic to brood. We therefore tested both benomyl and thiabendazole in August 1974 for possible use in controlling chalkbrood of honey bees.
The treatments were administered to chalkbrood infected nucs and small colonies as follows:
0.5 g/liter of thiabendazole
= 500 ppm thiabendazole in water.
0.5 g/liter of Benlate® = 250 ppm benomyl in water.
1. Spray bees and interior of hive at weekly intervals and repeat 3 times; use materials suspended in water.
2. Feed material in dust: 0.5 g/2 lb. soybean flour.
4 replicates of each of treatments 1 and 2 = 8 units (nucs) for each material or a total of 16 units.
4 check units (total 20 test units).
In addition, the soil around the area where colonies are kept for queen rearing was sprayed weekly with Benlate, 1 g/liter of water. Treatments were made on August 9, 19, and 23. No brood toxicity resulted from the use of the materials. Thiabendazole slightly improved and Benlate dust cleared the infection in the colonies. The same was not true with the nucs.
In June 1975, two more tests were made with benomyl feeding.
Test No. 1 was conducted with 3 groups of ten 4-frame nuclei set up June 10 on ethylene oxide-fumigated comb as follows:
Treatment 1 (check group). Bees shaken from chalkbrood-infected colonies directly into clean ETO-fumigated equipment and fed plain sugar sirup (0.9 gal) containing fumagillin (Fumidil B®) (ten 4-frame nuclei).
Treatment 2. Bees shaken from chalkhrood-infected colonies directly into clean ETO-fumigated equipment and fed 0.9 gal of sugar sirup containing 250 ppm benomyl (0.5 g Benlate/liter) plus Fumidil B (ten 4-frame nuclei).
Treatment 3. Bees shaken from chalkbrood-infected colonies into package cages. Bees were held off combs for 24 hours while feeding sugar sirup containing 250 ppm benomyl plus Fumidil B. After 24 hours, bees were hived in nuclei on clean equipment and fed plain sugar sirup with Fumidil B (0.9 gal) (ten 4 – frame nuclei). All package units had one pound of bees and a laying queen.
On June 23, all the check colonies except 3 showed 2 to 10 cells of chalkbrood. All but 4 colonies bulk-fed Benlate sirup continuously after being established showed no chalkbrood. The 4 infected units showed 1 to 8 cells of chalkbrood. All colonies held for 24 hours off the combs on Benlate sirup and subsequently hived and fed plain sirup showed no chalkbrood.
Test No. 2 was conducted with 4 package colonies set up June 10. All were heavily infested with chalkbrood. Two of the colonies were bulk-fed sugar sirup containing 250 ppm benomyl (1.8 gal). The other two were not so treated. On June 17, the two treated colonies showed no chalkbrood, and brood quality was excellent. The other two had numerous chalkbrood mummies scattered through the comb. On June 30, one treated colony was still clean; the other showed 3 cells of chalkbrood.
The treatment with 250 ppm benomyl appeared to have a positive effect in reducing chalkbrood.
At Madison, cooperative work with the University Department of Plant Pathology on chalkbrood disease is progressing. Currently we are testing both plant and mammalian systemic fungicides for their effect on the organism. Promising candidate materials will be tested in the field this summer. We plan to define more closely the life cycle of A. apis and the chalkbrood disease cycle.
Bailey, L. 1963. Infectious Diseases of the Honeybee. Land Books Ltd. London. 176 p.
Bailey, L. 1967. The effect of temperature on the pathogenicity of the fungus, Ascosphaera apis, for larvae of the honey bee. Apis mellifera. Proc. mt. Colloq. Insect Pathol. Microb. Control (Wageningen, 1966): 162-167.
Baker, G. M. and P. F. Torchio. 1968. New records of Ascosphaera apis from North America. Mycologia 60: 189-190.
Claussen, P. 1921. Entwicklungsgeschictliche Untersuchungen uber den Erreger der ais “Kalkbrut” bezeichneten Krankheit der Bienen. Arb. Biol. Reichsanst. Land Forstwirtsch. 10(6): 467-521.
Deans, A. S. C. 1940. Chalkbrood. Bee World 21(4): 46.
Dreher, K. 1938. Auftreten von Bienenkrankheiten in Niedersachsen und Braunschweig im Jahre 1937. Niedersachsische Imker 73(12): 282-284.
Giauffret, A. and Taliercio, Y. P. 1967. Les mycoses de l’abeille (Apis mellifera L.): etude de quelques antimycosiques [Fungal diseases of the honey bee (Apis mellifera L.): study of some antimycotics]. Bull. Apic. 10(2): 163-174.
Hitchcock, J. D. and M. Christensen. 1972. Occurrence of chalk brood (Ascosphaera apis) in honey bees in the United States. Mycologia 64(5): 1193-1198.
Seal, D. W. A. 1957. Chalk brood disease of bees. N.Z. J. Agric. 95(6): 562.
Toumanoff, C. 1951. Lea Maladies des Abeilles. Revue Francaise Apic. Num. Spec. 68: 325 pp.
*Mention of a proprietary product in this paper does not constitute a recommendation of this product by the USDA.
Reprinted from October, 1976, American Bee Journal
Vol. 116 (10): 484, 486 and 495