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Recent isolations of Bacillus pulvifaciens from powdery scales of honey bee, Apis mellifera, larvae
JOURNAL
OF INVERTEBRATE
Recent Isolations
PATHOLOGY
32, 222-223
(1978)
of Bacillus pulvifaciens from Powdery Bee, Apis mellifera, Larvae1
Bacillus pulvifaciens was originally described by H. Katznelson (.I. Bacterial. 59, 153-155, 1950). The organism was isolated from larval scales of honey bees, Apis mellifera, that were dry, powdery, and light brown. Limited attempts to infect bee colonies with material from the powdery scales and culture material did not definitely establish whether the organism was a pathogen or a saprophyte. Later H. Katznelson and C. A. Jamieson (Sci. Agr. 32,219-255, 1952) concluded after more extensive work that the bacillus was not pathogenic to the honey bee, although J. D. Hitchcock (cited by R. E. Gordon, W. C. Haynes, and C. Hor-Nay Pang, U. S. Dept. Agr. Handb. 427, l-283, 1973) isolated additional strains of B. pulvifaciens from powdery scales and has evidence that the organism is pathogenic to honey bees. R. E. Gordon et al. (lot. cit.) examined seven strains of B. pulvifaciens, all from one source (H. Katznelson), and postulated that B. pulvifaciens may form a connection between B. larvae and B. laterosporus, organisms also associated with honey bees. Recently, we isolated B. pulvifaciens from larval remains. Thus, the purpose of this paper is to describe the larvae from which the organisms were isolated and to compare the morphological and physiological properties of our strains with those previously described. A brood comb from a moribund colony in the apiary of an Arizona beekeeper was supplied by Dr. Gordon Waller. Examination of the contents of this comb revealed many powdery scales that were dry and chalky white rather than light brown as de-
scribed by H. Katznelson (lot. cit.). Some scales were positioned on the sides of the cells, and others were on the bottoms extending up one side. All were similar in appearance. Another brood sample was received from Mrs. Diana Menapace of the USDA Bee Laboratory in Laramie, Wyoming. This material was from a bee colony in Iowa and contained a yellow crumbling larva that had been removed from its cell. Scales from both sources were streaked directly onto plates of nutrient agar (D&o), and the plates were incubated at 37°C under aerobic conditions for 10 days. At that time, pure cultures of bacterial colonies that were bright orange were obtained. Twenty strains from the Arizona sample and one from the Iowa sample were maintained on slants of nutrient agar for further study. Gram-stained slides of the isolates revealed ellipsoidal central spores that swelled the sporangia. Many spores had stained remnants of the sporangia adhering to them, which gave the spores a pointed appearance. The isolates were then subjected to the morphological and biochemical tests for the genus Bacillus described by R. E. Gordon et al. (lot. cit.) except that motility was determined in motility test medium (BBL) rather than microscopically. The results of our tests agree with those listed for B. pulvifaciens by R. E. Gordon et al. (lot. cit.). All strains were Grampositive; formed acid from glucose, mannitol, and trehalose; reduced nitrate to nitrite; liquified gelatin; and decomposed casein. Seven of the strains were motile. Also, all but five strains were catalasenegative. These five strains gave a delayed weak-positive reaction by producing a few tiny gas bubbles on a small part of the periphery of the streak after 10% H,O, was added to growth on plates of nutrient
’ Mention of a proprietary product or company name does not constitute an endorsement of this product by the U. S. Department of Agriculture. 0022-201 l/78/0322-0222$01.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
Scales of Honey
222
223
NOTES agar. The bubbles arose from the creamy whitish growth at the periphery that was noted first by H. Katznelson (lot. cit.). Cultures giving the delayed weak-positive reactions were rechecked for purity and found to be pure B. pulvifaciens. R. E. Gordon et al. (lot. cit.) observed this reaction with their strains and stated that it reinforced their idea that B. pulvifaciens may be a connecting link between catalase-positive and catalase-negative species. Thus, powdery scales having different appearances seem to yield B. pulvifaciens strains that have morphological and biochemical reactions consistent with those of
the strains originally isolated by H. Katznelson. This does not, however, rule out the possibility that strain differences might contribute to the variations in color or consistency of powdery scales. KEY WORDS: Bacillus pulvifaciens; honey bee; powdery scale disease. MARTHA GILLIAM DINORAH R. DUNHAM U. S. Department of Agriculture Agricultural Research Service Bee Research Laboratory 2000 E. Allen Road Tucson, Arizona 85719 Received November 16. 1977
OF INVERTEBRATE
Recent Isolations
PATHOLOGY
32, 222-223
(1978)
of Bacillus pulvifaciens from Powdery Bee, Apis mellifera, Larvae1
Bacillus pulvifaciens was originally described by H. Katznelson (.I. Bacterial. 59, 153-155, 1950). The organism was isolated from larval scales of honey bees, Apis mellifera, that were dry, powdery, and light brown. Limited attempts to infect bee colonies with material from the powdery scales and culture material did not definitely establish whether the organism was a pathogen or a saprophyte. Later H. Katznelson and C. A. Jamieson (Sci. Agr. 32,219-255, 1952) concluded after more extensive work that the bacillus was not pathogenic to the honey bee, although J. D. Hitchcock (cited by R. E. Gordon, W. C. Haynes, and C. Hor-Nay Pang, U. S. Dept. Agr. Handb. 427, l-283, 1973) isolated additional strains of B. pulvifaciens from powdery scales and has evidence that the organism is pathogenic to honey bees. R. E. Gordon et al. (lot. cit.) examined seven strains of B. pulvifaciens, all from one source (H. Katznelson), and postulated that B. pulvifaciens may form a connection between B. larvae and B. laterosporus, organisms also associated with honey bees. Recently, we isolated B. pulvifaciens from larval remains. Thus, the purpose of this paper is to describe the larvae from which the organisms were isolated and to compare the morphological and physiological properties of our strains with those previously described. A brood comb from a moribund colony in the apiary of an Arizona beekeeper was supplied by Dr. Gordon Waller. Examination of the contents of this comb revealed many powdery scales that were dry and chalky white rather than light brown as de-
scribed by H. Katznelson (lot. cit.). Some scales were positioned on the sides of the cells, and others were on the bottoms extending up one side. All were similar in appearance. Another brood sample was received from Mrs. Diana Menapace of the USDA Bee Laboratory in Laramie, Wyoming. This material was from a bee colony in Iowa and contained a yellow crumbling larva that had been removed from its cell. Scales from both sources were streaked directly onto plates of nutrient agar (D&o), and the plates were incubated at 37°C under aerobic conditions for 10 days. At that time, pure cultures of bacterial colonies that were bright orange were obtained. Twenty strains from the Arizona sample and one from the Iowa sample were maintained on slants of nutrient agar for further study. Gram-stained slides of the isolates revealed ellipsoidal central spores that swelled the sporangia. Many spores had stained remnants of the sporangia adhering to them, which gave the spores a pointed appearance. The isolates were then subjected to the morphological and biochemical tests for the genus Bacillus described by R. E. Gordon et al. (lot. cit.) except that motility was determined in motility test medium (BBL) rather than microscopically. The results of our tests agree with those listed for B. pulvifaciens by R. E. Gordon et al. (lot. cit.). All strains were Grampositive; formed acid from glucose, mannitol, and trehalose; reduced nitrate to nitrite; liquified gelatin; and decomposed casein. Seven of the strains were motile. Also, all but five strains were catalasenegative. These five strains gave a delayed weak-positive reaction by producing a few tiny gas bubbles on a small part of the periphery of the streak after 10% H,O, was added to growth on plates of nutrient
’ Mention of a proprietary product or company name does not constitute an endorsement of this product by the U. S. Department of Agriculture. 0022-201 l/78/0322-0222$01.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
Scales of Honey
222
223
NOTES agar. The bubbles arose from the creamy whitish growth at the periphery that was noted first by H. Katznelson (lot. cit.). Cultures giving the delayed weak-positive reactions were rechecked for purity and found to be pure B. pulvifaciens. R. E. Gordon et al. (lot. cit.) observed this reaction with their strains and stated that it reinforced their idea that B. pulvifaciens may be a connecting link between catalase-positive and catalase-negative species. Thus, powdery scales having different appearances seem to yield B. pulvifaciens strains that have morphological and biochemical reactions consistent with those of
the strains originally isolated by H. Katznelson. This does not, however, rule out the possibility that strain differences might contribute to the variations in color or consistency of powdery scales. KEY WORDS: Bacillus pulvifaciens; honey bee; powdery scale disease. MARTHA GILLIAM DINORAH R. DUNHAM U. S. Department of Agriculture Agricultural Research Service Bee Research Laboratory 2000 E. Allen Road Tucson, Arizona 85719 Received November 16. 1977