Eugene S. Sharpe

Biology of Bacillus popilliae

Publisher Summary This chapter discusses the biology of bacillus popilliae. Bacillus popilliae is a pathogen of various scarabaeid beetles. Bacillus popilliae represents a unique category among bacteria. It is gram-variable, facultatively anaerobic, catalaseless, insecticidal, and it forms endospores along with parasporal crystals. Whether parasporal crystals are toxic to larvae is not yet fully determined however, some data indicate that there is pathogenicity associated with the crystals. Also, characterization of the inclusion bodies is incomplete, regardless, it is known that they are predominantly protein and can be separated in three cathodic components when solubilized crystals are subjected to high-voltage electrophoresis. They contain no lipid or nucleic acids however, they contain glycoprotein components. Of the 17 amino acids present, glutamic acid and aspartic acid predominate, followed in order of quantity by leucine, valine, glycine, threonine, isoleucine, alanine, and arginine; no unusual amino acids are present.

Growth pattern of Bacillus popilliae in Japanese beetle larvae.

Abstract Induction of milky disease in 50% of Japanese beetle (Popillia japonica) larvae by feeding requires about 109 spores of Bacillus popilliae per gram of soil. The infectious process occurs in four phases: (1) An initial incubation phase of about 2 days during which there is no evidence of infection in the hemolymph. (2) A vegetative phase of poliferation in the hemolymph which lasts until day 5 when prespores occur and a few spores first are observed. (3) An intermediate phase between day 5 and day 10 characterized by concomitant vegetative growth, prespore formation, and sporulation; maximum vegetative populations of about 109 cells per ml hemolymph occur during this phase but the number of spores exceed that of vegetative cells by the end of the phase. (4) Thereafter, a sporulation phase which terminates by day 14 to day 21 with typical milkness and death of larvae; vegetative populations steadily decline and large numbers of spores accumulate during this phase. Milky larvae contain an average of 5 × 1010 spores per ml hemolymph. Throughout the process microscopic evidence indicates many vegetative cells die without forming spores; dead cells disappear from the hemolymph by some unknown lytic or phagocytic process. Thus the massive spore populations which characterize milky disease result from accumulation of spores during a prolonged period of simultaneous vegetative growth and sporulation rather than from an extended period of exclusively vegetative growth followed by sporulation of most cells.

BACTERIA, SPIROCHETES, AND RICKETTSIA AS INSECTICIDES

Most bacteria pathogenic to insects are classified in the families Pseudomonadaceae, Enterobacteriaceae, Lactobacillaceae, Micrococcaceae, and Bacilliaceae; spirochetes and rickettsia are in the families Spirochaetaceae and Rickettsiaceae, respectively. Except for Bacilliaceae, these families contain nonsporulating microorganisms. Most spore-forming bacteria pathogenic to insects belong to the family Bacilliaceae. The identification of microorganisms associated with insects is inadequate because of inaccurate descriptions. A comprehensive evaluation of bacterial classification and identification can be found on the sixth and seventh editions of Bergey’s Manual of Determinative Bacter i~ logy .~ ,~ However illogical the existing scheme may be, we feel strongly that it should be adhered to by the insect pathologist, and the use of generalized names without reference to the specific causative disease agent should not continue. The incorrect hypothesis among insect pathologists that insects harbor special microorganisms is probably the main cause of misnamed bacterial species. Often, microorganisms from diseased insects are named for the insect from which they are isolated and are incriminated as pathogens. This practice should be discouraged. The procedures outlined in Koch’s canons for demonstrating that a disease is caused by a microorganism are as follows: finding the specific microorganism in all cases of the disease; isolating it in “pure culture” (description of the pure culture isolate should be by established taxonomic procedures) ; inoculating (or feeding) the isolate into the host insect and experimentally producing the original disease; and finally, reisolating the microorganism from the experimentally diseased insect and demonstrating it to be the same as the pure culture isolate previously inoculated. In this communication, we shall, when possible, ignore unauthenticated bacterial species claimed to be insecticidal. Bucher7z9 classified bacterial insect pathogens as either obligate, facultative, or potential. We shall discuss, according to Bucher’s categorization, nonsporulating bacterial pathogens, including true bacteria, spirochetes, and rickettsiae and sporulating bacterial pathogens, including “crystalliferous” types.

Infectivity of spores of Bacillus popilliae produced on a laboratory medium

Abstract No occurrence of milky disease resulted when third-instar larvae of the Japanese beetle, Popillia japonica, were exposed to soil containing 2 billion spores/kg of strain NRRLB-2309M of the milky disease bacterium, Bacillus popilliae, produced on a laboratory medium, or when the larvae imbibed 200,000 of the spores of strain NRRL B-2309M in aqueous suspension; however, 24% of the larvae were infected when they were injected with 10,000 of the spores. In contrast, spores produced commercially 2

The pattern of sporulation of Bacillus popilliae in colonies

Abstract Spores accumulate periodically in colonies of Bacillus popilliae after 3 days of vegetative growth on solid medium. Sporulation occurs on the surface and primarily in a ring near the periphery, causing slight changes in colony contour. The formation of mature spores and their acquisition of resistance to drying and to heat occur in a stepwise manner. A high level of prespore forms persists in mature colonies. Sporulation in colonies is as efficient as early stages of sporulation in larvae, but efficiency in vivo must increase as milky disease progresses.

Germination and outgrowth of Bacillus popilliae spores in microscope slide culture

A novel slide-culture technique was used to study germination and outgrowth of Bacillus popilliae spores without disturbing the microenvironment. Infective spores formed in larvae required 24 hr to begin outgrowth, whereas noninfective spores from colonies initiated outgrowth in 12 hr. Dissolution of the paraspore coincided with outgrowth and not with germination of the attached spore. Germination and outgrowth were asynchronous events and required several days in all cell populations, except for free spores.