A laboratory evaluation of the pathogenicity of Bacillus popilliae var. rhopaea, the agent of milky disease in Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Two methods of infection, i.e., feeding known numbers of spores and rearing larvae in contaminated peat, were used to bioassay the susceptibility of Rhopaea verreauxi to Bacillus popilliae var. rhopaea at 23°C. The susceptibility of the three larval instars was similar as measured by the ID₅₀ and IC₅₀ values. However, within an instar, newly molted larvae were less susceptible than mature larvae when infected by the contaminated peat method. It is suggested that this was due to reduced food intake. The range of ID₅₀ values for all bioassays with R. verreauxi larvae were 1.1 × 10⁷ to 4.0 × 10⁷ spores per larva, and IC₅₀ values were 3.4 × 10⁶ to 5.0 × 10⁷ spores per g of contaminated peat. The slope of the probit line was always low (0.6 to 1.8) except for young first-instar larvae infected by contaminated peat when the slope was 4.0. Disease per se did not affect food intake, though intake was reduced at high doses of contaminated peat. Young larvae often died without developing symptoms but, with increasing age, infected larvae were more likely to develop symptoms. Bioassays with Othnonius batesi and Rhopaea morbillosa indicated a much lower susceptibility per os than for R. verreauxi. It is concluded that the potential for using B. popilliae var. rhopaea to control R. verreauxi is high, but the bacillus is unlikely to be of value in control of O. batesi or R. morbillosa.     

The development of milky disease under laboratory and field temperature regimes

The effect of temperature on the development of milky disease agent, Bacillus popilliae var. rhopaea, in third-instar Rhopaea verreauxi larvae was studied in two experiments. In the first experiment, only small differences were found between the probit lines of final percentage infection against dose under five temperature regimes (constant 18, 23, and 28°C; alternating 13–23°C, and 18–28°C). The disease developed more slowly at the lower temperatures while alternating temperatures gave shorter ET₅₀ values than the equivalent constant temperatures. In the second experiment, a higher incidence of disease was obtained under field conditions than under any of the laboratory conditions tested. It was concluded that for up to 6 months/year temperature is not likely to limit the development of B. popilliae var. rhopaea in the field, and that, under field conditions, most larvae would achieve a Phase IV infection.     

The in vivo production of Bacillus popilliae var. rhopaea spores

The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23°C, and infecting doses above 10⁶ spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 × 10¹⁰ spores while old third-instar larvae produced about 4 × 10¹⁰ spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per os. Three other host species could be used to mass-produce B. popilliae var. rhopaea spores but all were less efficient than R. verreauxi. Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 × 10¹⁰ spores per larva.     

Bacillus penetrans n. comb. Causing a virulent disease of plant-parasitic nematodes

The prokaryotic and bacterial nature of the incitant of a lethal disease of economically important plant-parasitic nematodes was confirmed by electron microscopy. The organism (formerly Duboscqia penetrans Thorne, Microsporea: Protozoa) was renamed Bacillus penetrans n. comb., Bacillaceae. B. penetrans produces an endospore typical of Bacillus spp., and its life cycle and spore morphology are similar to B. popilliae and B. lentimorbus, the “milky disease” organisms of insects. It differs from the latter pathogens in spore shape, in the dichotomously-branched colonies formed in the host pseudocoelom by nonseparating vegetative cells, and in that the spores are not known to be ingested by nematodes. Substantial reductions in Pratylenchus scribneri populations and in root-knot disease of tomato were obtained in glasshouse tests with the pathogen. Endospore production in vivo presents the first possibility of biological control of pest nematodes with inoculum of a highly specific virulent pathogen.     

Germination and outgrowth of Bacillus popilliae in hemolymph slide mounts

Cabbage looper hemolymph induced rapid germination and outgrowth of spores of Bacillus popilliae. Spores germinated within sporangia but outgrowth occurred from free released spores as well as from spores retained in sporangia. With 37°C, an alkaline pH, and tyrosinase, outgrowth resulted in 1 hr. Of six strains of milky disease bacteria tested, hemolymph mediated germination and outgrowth of only those which are infective perorally for European chafer larvae, indicating a potential use as a screening tool to assess virulence for the chafer.     

Stored Bacillus popilliae spores and their infectivity against Popillia japonica larvae

Bacillus popilliae spores were stored for about 7 years under three separate conditions: frozen in sterile distilled water, smeared on glass microscope slides, and stored in loam soil at room temperature. In separate experiments, each of the 7-year-old preparations was fed to Popilla japonica larvae at concentrations of 10³, 10⁵, 10⁷, and 10⁹ spores/g of soil. A significant decrease in the percentage of larvae infected occurred in all of the aged spore tests. B. popilliae spores stored in soil, for the extended period, produced 3% larval infection only at the 10⁹ spores concentration; similar results were obtained from frozen spores. When P. japonica larvae were fed spores stored dried on slides, about 20% of the larvae developed milky disease. When aged frozen spores were artificially injected into larvae, 12% became infected at concentrations of 1 × 10⁶ spores/larvae; dried spores at the same concentration infected about 38% of the insect larvae. We conclude from these data that aged B. popilliae spores are significantly less infective against P. japonica larvae than young spores.     

Media for the detection of Bacillus larvae spores in honey

Bacillus larvae, the causative agent of American foulbrood in honey bees completes its life cycle of germination, outgrowth and sporulation in young honey bee larvae by killing them and often bringing about the destruction of the entire hive. While B. larvae germinates and outgrows on complex organic media in vitro, the literature suggests, for reasons that are not at all clear, that a relatively large number of spores of B. larvae are required to yield each visible colony (colony forming units, CFU) on media. Various researchers have reported that from 16 to 3,000 or more spores of B. larvae are required to yield a single colony on an agar plate. HANSEN in Denmark designed a useful method of spreading approximately 80 mg of honey directly on the surface of a PETRI plate containing “J” agar medium to determine if B. larvae spores are present in the honey. In the present study, selected media were tested for the ability to recover B. larvae spores in honeys in the form of visible colonies (CFU) using HANSEN's strek method. A modification of a medium (TMYGP) developed by DINGMAN and STAHLY, (T-HCL-YGP agar), recovered considerably more viable B. larvae spores in the form of visible colonies (CFU) than HANSEN's “J” medium. When “J” medium was fortified with 0.1% sodium pyruvate, it was comparable to modified T-HCL-YGP medium in its recovery of B. larvae spores. Brain heart infusion agar (BHIA) with the addition of thiamine recovered more spores in the form of viable colonies than did “J” medium but it was not as efficient as T-HCL-YGP medium. Serial dilution from 100 to 10,000 times of weighed samples of honey with deionized water led to higher spore counts (CFU per g honey) than that indicated by undiluted honeys plated at 80 mg levels directly onto the surface of media by the HANSEN procedure.     

The pattern of sporulation of Bacillus popilliae in colonies

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.     

Effect of exposure to soil on potency and spore viability of Bacillus thuringiensis

Ten-gram samples of a clay loam soil were inoculated with Bacillus thuringiensis var. galleriae (H-serotype V) and held at 25°C. Periodically the spores and δ endotoxin protein crystals of B. thuringiensis were extracted from soil samples. Numbers of viable spores were estimated by plate counts and pathogenicity determined by bioassay with larvae of Galleria mellonella. During 135 days, the number of viable spores fell slowly to 24% of the initial numbers, while pathogenicity fell rapidly to <1%, which suggests that the crystals were degraded far more rapidly than spores. Natural soil bacteria increased in numbers during the same period.     

Extrachromosomal DNA in Bacillus thuringiensis var. kurstaki, var. finitimus, var. sotto, and in Bacillus popilliae

Four entomopathogenic bacteria contained extrachromosomal deoxyribonucleic acid (DNA) molecules of various sizes. Bacillus thuringiensis var. kurstaki contained twelve elements banding on agarose gels that ranged from 0.74 to > 50 × 10⁶ daltons, three of which were giant extrachromosomal DNA elements. B. thuringiensis var. sotto contained one giant extrachromosomal DNA element with a molecular size of about 23.5 × 10⁶ daltons and two lesser elements of 0.80 and 0.62 × 10⁶ daltons. B. thuringiensis var. finitimus harbored two giant DNA elements corresponding to >50 × 10⁶ daltons and two lesser bands with relative small size (0.98 and 0.97 × 10⁶ daltons). B. popilliae contained no giant extrachromosomal DNA elements but did contain two smaller elements corresponding to 4.45 and 0.58 × 10⁶ daltons. The possible use of extrachromosomal DNA elements that prove to be autonomous replicons for recombinant DNA studies is discussed.     

Selective Medium for Quantitation of Bacillus popilliae in Soil and in Commercial Spore Powders

A medium consisting of MYPGP agar supplemented with vancomycin was found to be highly selective for Bacillus popilliae, especially for strains originally isolated from Japanese beetle larvae. The medium has proven to be useful for the quantitation of B. popilliae spores in commercial spore powder and in soil.     

The control of Monomorium pharaonis (Hymenoptera: Formicidae) with Bacillus thuringiensis

In this study, the effect of different preparations made from Bacillus thuringiensis var. thuringiensis (strains: CCEB 555 and CCEB 058) on ants, Monomorium pharaonis, under laboratory conditions is reported. The different preparations tested consisted of (1) a liquid culture of the strain B. thuringiensis CCEB 555 (containing spores and exotoxin), (2) the supernatant of the culture broth of strain CCEB 555 (containing exotoxin), and (3) the biological preparation “Bathurin” prepared from the strain B. thuringiensis CCEB 058 (containing spores and inclusions, without exotoxin). The preparations were used either pure or in alternation with borax, i.e., 1 wk borax, 3 wk the respective preparation for several months. All preparations were found to be toxic to M. pharaonis and their effect was characterized by a slow extinction of the ant colony. Administration of “Bathurin” (1.3%) yielded a 100% mortality after 20 wk. Using a liquid culture of B. thuringiensis var. thuringiensis, 100% mortality was recorded after 21 wk, a period of time which did not differ from that obtained with the supernatant of the culture containing exotoxin. The alternation with borax was found to accelerate ant mortality by 9–10 wk after administration. In all experiments, the worker ants died first, the queen ants surviving them by 1–3 wk.In experiments employing worker ants only, a 100 and 98% mortality, respectively, occurred within 3 wk after administration of a liquid culture of B. thuringiensis and “Bathurin” supplemented with borax.     

Nonoccluded virus-like particles in the mollusc Agriolimax reticulatus (Stylommatophora: Limacinae)

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.     

Occurrence of resistance to neomycin and kanamycin in Bacillus popilliae and certain serotypes of Bacillus thuringiensis: Mutation potential in sensitive strains

Serotypes of Bacillus thuringiensis and the commercial strain of Bacillus popilliae were examined for their inherent resistance to antibiotics and their mutation potential in respect to neomycin and kanamycin, the presence of which would preclude the use of plasmids marked by genes for resistance to the antibiotics. Clones on initial plates were detected by the recurrence of resistance colonies at superimposable sites on serial replicaplates containing the antibiotic. Susceptible strains were selected for the determination of their antibiotic-resistance mutation potential. Three varieties of B. thuringiensis were found to be doubly resistant, seven varieties were singly resistant (Neo^r), and three other varieties, including B. popilliae, were susceptible to both antibiotics. Estimates of mutation ratios revealed that three serotypes developed no resistant mutants to either antibiotics in populations as high as 3.0 × 10¹⁰; seven other serotypes developed no resistance to kanamycin in populations as high as 4.6 × 10⁹ cells. Three other serotypes exhibited mutation ratios as high as 1.6 × 10^?2. We were unable to determine the mutation ratio for B. popilliae.     

Production,long term preservation and synchronous germination of aerial spores of Streptomyces

Vigorous vegetative growth of various Streptomyces species (S. auroefaciens, S. collinus and S. granaticolor) was achieved in a new semisynthetic liquid medium. Unlike the media commonly used for the cultivation of the submerged mycelia of different streptomycetes, this one does not contain insoluble material which enables direct and reliable measurement of net production of biomass. The medium was formulated to meet the nutritional requirements of all the three species. Is also supported production of antibiotic in each of the strains. A method for bulk preparation of Streptomyces aerial spores, involving cultivation on agar plates covered with cellophane, was developed. Advantage of this method lies in higher yields of spores, their higher purity and easier harvesting. The spores were activated by amild treatment with an Ultra-Turrax homogenizer resulting in the breakage of fibrous sheath, suspended in 20% glycerol, and stored at ?60°C. Thus, treated spores germinated synchronously even after several months of the storage. Hence, such spore material may be used for precise inoculation in a large series of experiments implying synchronous germination, and the inoculations can be carried out from the same batch over a long period.     

Morphological variants of Moniliophthora roreri on artificial media and the biotroph/necrotroph shift

Moniliophthora roreri (Mr) causes frosty pod rot of Theobroma cacao in a hemibiotrophic association. The Mr biotroph-like phase has not been studied in culture. Mr spores (isolates Co12, Co52, and B3) were germinated on high (V8) and low (BPMM) nutrients with different media hardness (0.5% to 3% agarose). Germination was high on V8 media. Hardness affected germination on BPMM. Most colonies on V8 were slow-growing, failing to sporulate. Colony morphology depended on the isolate. On BPMM, exaggerated mycelia formed of limited length with enlarged cells. On agarose, rapidly expanding sporulating necrotrophic colonies formed rarely. Co12 and B3 spores were germinated on V8 and BPMM with low melting point (LMP) agarose. Slow-growing colonies of B3 on BPMM were unstable on LMP agarose, often forming slow-growing/rapidly expanding hybrids. Slow-growing colonies are hypothesized to represent the biotrophic phase. One nucleus was common in Mr cells, other than spores. Binucleate cells were occasionally observed in aged cells of slow-growing mycelia. Co52 cells often had more than two nuclei per cell after germination. Mr mycelia cells typically carry a single nucleus, being considered haploid. Biotroph- and necrotroph-like mycelia displayed differential gene expression but results were inconsistent with published in vivo results and require further study.     

Milky Disease Bacteria

A comparative study was made of all available milky-disease species and strains that have been isolated around the world from beetle larvae (family Scarabaeidae). Included in the study were Bacillus popilliae Dutky, B. lentimorbus Dutky, and B. lentimorbus var. maryland from the United States; B. euloomarahae Beard and B. lentimorbus var. australis Beard from Australia; B. fribourgensis Wille from Switzerland; and New Zealand milky disease (Dumbleton). The organisms were classified into three groups: (i) those containing parasporal bodies, including B. popilliae Dutky, B. fribourgensis Wille, and New Zealand milky disease (Dumbleton); (ii) those without a visible parasporal body and with spore morphology similar to B. lentimorbus Dutky, including B. lentimorbus var. australis Beard; and (iii) those with very tiny spores and no parasporal body, including B. euloomarahae Beard and B. lentimorbus var. maryland. All available milky-disease species and strains were cultivated in vitro on Brain Heart Infusion Agar plates. However, the most fastidious organisms—B. euloomarahae and B. lentimorbus var. maryland—could not be grown until they were passed through a life cycle in larvae of a large scarabaeid beetle infesting rotting wood. Then they remained stable for only one or two subcultures. All the milky-disease organisms produced larger cells in vitro than they did in vivo. The pattern of sugar fermentations was similar for all milky-disease species. It appears that there is a very low percentage of strains of B. popilliae, B. lentimorbus, and the other milky-disease organisms that have the inherent genetic makeup to permit them to sporulate on artificial media, if conditions are favorable. Among these conditions are a sufficiently high cell population and a reduced oxygen tension. Spores produced in vitro may have a low virulence via the normal ingestion pathway, even though they show apparent virulence when injected directly into the hemocoel.     

Detection of Bacillus thuringiensis in soil by immunofluorescence

Persistence of viable and heat-killed vegetative cells, parasporal crystals, and spores of Bacillus thuringiensis in soil was monitored by immunofluorescence. The rates of disappearance of the different bacterial components decreased in the following order: viable cells, heat-killed cells, parasporal crystals, and spores. Vegetative cells disappeared at rapid, exponential rates; viable cells autolysed, whereas heat-killed cells were digested by an actinomycete-like, soil microorganism. Parasporal crystals disappeared at a slower, nonexponential rate. Numbers of spores remained unaltered throughout 91 days incubation at 25°C and no germination was detected in this period.     

The preparation,germination properties and stability of superdormant spores of Bacillus cereus

Aims: To determine yields, germination and stability of superdormant Bacillus cereus spores. Methods and Results: Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or l ‐alanine in 2–5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca‐DPA, dodecylamine, l ‐alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat‐activated spores were germinated. Superdormant spores stored at 4°C slowly recovered some germination capacity, but recovery was slowed significantly at ?20°C and ?80°C. Conclusions: Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Significance: Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.     

Virulence of entomopathogenic Fungi (Fungi imperfecti) for the adults of Acanthoscelides obtectus (Coleoptera: Bruchidae)

Tests with the bean weevil, Acanthoscelides obtectus, in which the hosts were exposed indirectly to various dilutions of conidia of four entomopathogenic fungi showed that mortality was a function of the concentration of the inoculum. In these tests a given spore suspension was sprayed on the internal surfaces of a Petri dish. Adult weevils of a known age were placed in the dish, held there for 24 hr, then removed and kept at 20°C. After 20 days, the host mortality was determined. From the data obtained, it was possible to trace a probit regression line of the mortality in relation to the increasing spore concentration. Infection was observed in hosts exposed to a concentration of approximately 5 × 10⁶ spores/ml up to a maximum of about 1 × 10⁹ spores/ml. The A. obtectus was susceptible to infection by spores of Beauveria bassiana, B. tenella, Metarrhizium anisopliae, and Paecilomyces fumoso-roseus.