Bacillus Popilliae Research Articles

Growth of Paenibacillus larvae, the causative agent of American foulbrood in honey bees and Bacillus popilliae, the causative agent of milky disease in beetle larvae in lepidopteran cell cultures

AbstractTNM‐FH Lepidopteran insect cell culture medium containing 10% fetal bovine serum (FBS), while allowing limited vegetative growth of Paenibacillus larvae (wild‐type strain), the causative agent of American foulbrood, contained no viable vegetative cells upon subculture, nor were any heat resistant spores produced in this medium alone. However, TNM‐FH medium cotaining embryonic or midgut cells from Trichoplusia ni, hemocytes from Estigmene acrea, ovarian and embryonic cells from Spodoptera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispar, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX‐CELL 405 serum‐free insect cell culture medium alone appeared to contain the appropriate nutrients required for both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occurred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were any heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores (4,000/ml) were detected in the form of cology‐forming units (CFU) on plates following heating to 80°C for 20 minutes. In a parallel study with a wild‐type strain of Bacillus popilliae, vegetative cells grew only in TNM‐FH medium in the presence of mid‐gut BTI‐Tn‐MG and ovarian (Tn‐368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI‐Tn‐MG and Tn‐368 cells were further challenged with four variant cultures of B. popilliae, vegetative growth and limited sporulation were achieved. The BTI‐Tn‐MG cell line in TNM‐FH medium produced as many as 12,000 spores/ml after 21 days in culture.

Mortality Induced by Bacillus popilliae in Cyclocephala parallela (Coleoptera: Scarabaeidae) Held under Simulated Field Temperatures

The bacterium, Bacillus popilliae Dutky, causes milky disease in numerous species of scarabs around the world. Bacillus popilliae induced mortality in naturally infected grubs (third instars) of Cyclocephala parallela Casey was measured when held under simulated field temperatures. Our data show that visual examination in the field underestimates the percentage of grubs actually infected by B. popilliae. 5.6 to 8.2 times as many milky disease infected grubs died during the first 60 days of incubation under simulated field temperatures than did uninfected grubs. These data show that the widely used prevalence value underestimates the total mortality which this bacterium ultimately causes to C. parallela.

Description and Use of a Peroral Injection Technique for Studying Milky Disease

Description and Use of a Peroral Injection Technique for Studying Milky Disease

Lack of Efficacy of In Vivo- and Putatively In Vitro-Produced Bacillus popilliae Against Field Populations of Japanese Beetle (Coleoptera: Scarabaeidae) Grubs in Kentucky

Use of Bacillus popilliae Dutky, causal agent of milky disease in Japanese beetle grubs, has been limited because of its inability to produce infective spores in vitro. Recently, putative milky disease products produced by a patented in vitro process were marketed. We evaluated in vivo- and putatively in vitro-produced commercial spore formulations for efficacy against Japanese beetle grubs in laboratory and field trials. In soil confinement assays, infectivity by feeding (per os) was greater for the traditional spore powder made by maceration of diseased grubs than for the putative in vitro formulation. However, in multi-year field trials on 2 golf courses, neither product was effective in inducing higher levels of milky disease or in reducing grub populations. Application of a full dose (2 g) of spore powder, the amount normally applied every 1.2 m in a grid pattern, within 0.1-m 2 field enclosures failed to induce milky disease in grub populations in soil directly beneath the treated turf. Laboratory-infected 3rd instars in late phases of milky disease continued to feed on grass roots. Putatively in vitro-produced formulations were recalled from the market in 1991 because of questions about the identity of the bacterial spores they contained. We challenge the evidence that commercially available spore powder, applied according to conventional methods, is effective for suppressing localized grub populations in turfgrass, and suggest that standard recommended practices for use of milky disease bacteria should be reevaluated.

Comparison of Pulsed-Field Gel Electrophoresis DNA Fingerprints of Field Isolates of the Entomopathogen Bacillus popilliae

Eight strains of the entomopathogen Bacillus popilliae were analyzed by pulsed-field gel electrophoresis, and genomic size estimates of ;sim2,600 to 3,500 kb were obtained. The type strain, ATCC 14706, had a genomic size of 3,395 kb. For the six New Zealand isolates, the degree of similarity in the pulsed-field gel electrophoresis fingerprints may correlate with the geographical closeness of the sites of isolation. The plasmid profiles of the New Zealand isolates were also compared; four of the six strains carry plasmids in the 3.6- to 9.7-kb size range.

Bacillus and Serratia species for Scarab control

Few microorganisms are commercially available for use against white grubs (larvae of Scarabaeidae). Entomopathogenic bacteria, particularly Bacillus popilliae, have been used the longest for white grub suppression. Other bacteria, namely B. thuringiensis and Serratia spp. offer promise for future control. This papes examines two genera of bacteria (Bacillus and Serratia) from the historical and current perspective. Bacillus popilliae, the firs microbial control agent registered in the United States, has a long history of use in suppressing populations of the Japanese beetle, Popillia japonica. However, lack of in vitro production and the slow and sporadic nature of its activity, severely limits its utilization. B. thuringiensis, the most widely used microbial pesticide, has not been used for scarab, control. However, strains with scarab activity have recently been discovered. Scarab larvae have been collected in the United States with signs and symptoms similar to those characteristic of amber disease (caused by Serratia entomophila) in the New Zealand grass grub, Costelytra zealandica. A total of 147 bacteria have been obtained from the digestive tracts of larvae of the Japanese beetle and masked chafers, Cyclocephala spp., as well as from larvae and soil collected in Japan and China. Seventy five of these have been identified as Serratia spp. Most (40) of the remaining bacteria are in the genus Enterobacter. A majority of the bacteria (73) and of the Serratia (38) came from P. japonica.

Inhibitory Effects of Turf Pesticides on Bacillus popilliae and the Prevalence of Milky Disease

Fourteen pesticides (fungicides, herbicides, and insecticides) were tested to determine whether they had deleterious effects on the bioinsecticide Bacillus popilliae, the causal agent of milky disease. All of these pesticides reduced levels of spore viability, spore germination, and/or vegetative cell growth when they were tested over a range of concentrations from 0 to 1,000 ppm of active ingredient, and the fungicides had the greatest detrimental effects. As determined by tests in water, the level of spore viability was significantly reduced by chlorothalonil, iprodione, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and 2-[(4-chloro-o-tolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid. In tests performed with iprodione, loss of viability was evident at concentrations less than the concentration calculated to result from recommended use. Tests performed in soil demonstrated that triadimefon, chlorothalonil, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and pendimethalin at concentrations resulting from recommended rates of application reduced spore titers. Spore germination did not occur in the continued presence of 2-[(4-chloro-otolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid, isofenphos, and chlordane, whereas exposure of spores to triadimefon or pendimethalin for 2 days stimulated germination. The tests to determine effects on spore germination were inconclusive for all other pesticides. Triadimefon, chlorothalonil, iprodione, pendimethalin, and chlorpyrifos at concentrations less than the concentrations recommended for use inhibited vegetative cell growth of B. popilliae, and chlordane at a concentration that was twice the concentration expected to result from the recommended rate of application repressed cell growth. My data support the hypothesis that use of synthetic pesticides can contribute to a low incidence of milky disease in white grubs.

Biology of Pseudoheteronyx sp. (Coleoptera: Scarabaeidae) on the Central Highlands of Queensland

ABSTRACTThe life cycle, development rates, fecundity and survivorship of Pseudoheteronyx sp. were studied in the laboratory and field. Adults fed on leaves of Asteraceae (sunflower and Parthenium hysterophorus L., an introduced weed). Larvae in the soil fed on cereal roots and on crop residues but developed faster on living wheat roots compared to wheat stubble. the shortest development time for larvae was 8 months under laboratory conditions. Teneral adults appeared in the field at the start of the summer wet season (usually December). Sex ratio was 1:1. Size could not be used to separate the sexes, which were morphologically similar. Adults began laying eggs about 8 weeks after adult emergence. Under moist soil conditions in the laboratory, adults laid eggs for 1 year except during 4–6 weeks in winter when mean monthly temperature dropped below 20°C. When confined in groups of up to 20 adults, Pseudoheteronyx sp. laid an average 18.5 eggs per female when fed sunflower leaves, and 3.0 eggs per female when fed leaves of Parthenium. Individually confined females fed sunflower leaves laid 10–51 eggs during their life. Natural enemies recorded in the field included fungal pathogens (Metarhizium, Beauveria, Hirsutella and Cordyceps spp.) and milky disease (Bacillus popilliae).

Characterizing the Enhanced Susceptibility of Milky Disease-Infected Scarabaeid Grubs to Entomopathogenic Nematodes

Grubs of the scarabaeid Cyclocephala hirta infected with the milky disease bacterium, Bacillus popilliae (Bp), were more susceptible to the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema glaseri than healthy grubs. Increased susceptibility was due to greater ease of nematode penetration through the midguts of Bp-infected insects and occurred after > 10 days exposure to B. popilliae spores. By that time, the bacterium was proliferating rapidly in the insect hemolymph and possibly stressed the insect by physiological starvation. Attraction of the nematodes to the insects and cuticular penetration by the nematodes were ruled out as possible reasons for the enhanced susceptibility of Bp-infected grubs. In greenhouse pot trials, S. glaseri killed more Bp-infected grubs than healthy grubs, whereas no differences were noted with H. bacteriophora. Thus, to achieve the best grub control in the presence of B. popilliae, application of S. glaseri is recommended. The compatibility of B. popilliae and entomopathogenic nematodes in the field is ensured by the existence of a diseased insect class with a low level of susceptibility to the nematode, the presence of B. popilliae in grubs not interfering with nematode progeny production, and the ability of B. popilliae spores to survive invading nematodes and their associated bacteria.

Milky Disease Bacterium as a Stressor to Increase Susceptibility of Scarabaeid Larvae to an Entomopathogenic Nematode

Mortality of Cyclocephala hirta by the entomopathogenic nematode Heterorhabditis bacteriophora was enhanced in grubs infected with the milky disease bacterium, Bacillus popilliae (Bp). In concentration-response assays with the nematode, LC50 values and 95% fiducial limits were 4.0 (<0.1, 14.6) infective juveniles per grub for Bp-infected insects and 47.7 (32.0, 64.7) for non-Bp-infected insects. The increased mortality of Bp-infected insects was partially attributed to rapid penetration of H. bacteriophora through the midgut wall. In in vitro assays with ligated midguts, the time required for H. bacteriophora to penetrate the midgut wall of Bp-infected grubs was 0.6 ± 0.1 hr, whereas the time required in non-Bp-infected grubs was 1.3 ± 0.1 hr. Total nematode progeny production per milligram grub tissue did not differ significantly between Bp-infected and non-Bp-infected grubs. However, the proportion of progeny remaining trapped in the non-Bp-infected cadavers at the end of the assay was greater than the proportion remaining in Bp-infected cadavers (0.52 ± 0.04 vs 0.13 ± 0.05, respectively). These data indicate that coexistence of these two pathogens within a host can occur and that B. popilliae could be used as a stressor on C. hirta to increase its susceptibility to H. bacteriophora.

Impact ofBacillus popilliae, Rickettsiella popilliaeand entomopathogenic nematodes on a population of the scarabaeid,Cyclocephala hirta

Larvae of the scarabaeid, Cyclocephala hirta, are major pests of turfgrass in California. A field test was conducted against third instars that included the following treatments: untreated control; chemical insecticide (bendiocarb); milky disease bacterium (Bacillus popilliae); and entomopathogenic nematodes (Steinernema feltiae and Heterorhabditis bacteriophora). There were no significant differences in population reduction among the treatments, but the larval population in all plots showed a dramatic decline. The C. hirta population had a natural occurrence of milky disease and blue disease caused by Rickettsiella popilliae. The prevalence of blue disease during the course of the study averaged < 10% but that of milky disease averaged about 20%. More significantly, the soil from all treatment plots when bioassayed for B. popilliae showed that 67–90% of the larvae became infected with this bacterium. None of the larvae became infected with the blue disease organism. We conclude that B. popilliae was occurring in epizootic proportions in our field tests and was a significant mortality factor in causing the decline of the C. hirta population.

Problems with in vitro production of spores of Bacillus popilliae for use in biological control of the Japanese beetle

Bacillus popilliae spores have been used in the United States for the biological control of the Japanese beetle for over 50 years. Lack of efficient sporulation in vitro has been a major limiting factor in expanding the use of this bacterium. Previously, commercial spore powders were produced by maceration of milky disease infected larvae containing large numbers of spores. Recently, (since 1985) a commercially available spore powder produced by a patented in vitro procedure has been marketed. However, most batches of the in vitro spore powder and primary fermentation products that we examined contained a bacterium identified as Bacillus polymyxa, but no B. popilliae. We also examined four cultures submitted to the American Type Culture Collection (Rockville, MD) at the time of patent application as representative sentative of the strains of B. popilliae produced in vitro. One of these, referred to as the preferred strain, was identified as B. polymyxa. The other ATCC strains were identical in their characteristics to Bacillus amylolyticus. Both B. polymyxa and B. amylolyticus spores, and in vitro-produced milky disease spore powder containing these bacteria rather than B. popilliae, caused no milky disease in Japanese beetle larvae. The patented procedure should be reexamined since there is no evidence that it is effective in producing infective B. popilliae spores.

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.

Distribution of Milky Disease and Green Muscardine Disease of White Grub in Central Gujarat

Bacillus popilliae var holotrichiae and Metarhizium anisopliae var anisopliae were found well distributed in three districts of Central Gujarat (23°N, 73°S)causing milky disease and green muscardine disease respectively, in Holotrichia consanguinea populations. The incidence of milky disease varied, from 2.0 to 36.0 per cent and green muscardine disease 1.9 to 15.3 per cent in different villages durlna 1985-87. A few arubs were also found simultaneously infected by both the pathogens.

Epizootiological Investigations of the Microsporidium Ovavesicula popilliae and Bacterium Bacillus popilliae in Field Populations of the Japanese Beetle (Coleoptera: Scarabaeidae)

Japanese beetle, Popillia japonica Newman, population densities and prevalence of the pathogens Ovavesicula popilliae Andreadis & Hanula and Bacillus popilliae Dutky are reported from three locations in Connecticut over a 3-yr period. Populations decreased to very low levels ( 50%). Conversely, prevalence of B. popilliae was <30% throughout most of the study. Ovavesicula popilliae reduced fecundity 50% in heavily infected females in the laboratory. In addition, maximum prevalence of O. popilliae in adults, collected in attractant-baited traps, peaked several weeks after peak adult emergence in the field, suggesting that O. popilliae slows development. However, no differences were detected in the weight or development of field-infected third instars reared in the laboratory. It is suggested that the effect of O. popilliae on its host is modified by environmental stress.

Biological control of grass grub in Canterbury

The grass grub (Costelytra zealandica) is a major pest of Canterbury pastures. Grass grub numbers are low in young pastures and then commonly rise to a peak 4-6 years from sowing, before declining. Grass grub numbers in older pastures fluctuate but rarely reach the same levels as the early peak. Biological control agents such as bird and invertebrate predators, parasites and diseases cause mortality in grass grub populations; the effect of predators and parasites is limited. Pathogens are common in grass grub populations. Amber disease, caused by the bacteria Serratia spp., was the disease most frequently found in population surveys in Canterbury, while milky disease (Bacillus popilliae) and protozoan diseases were less common. The level of amber disease was reduced in pastures where insecticide had been applied. Inundative applications of entomophagous nematodes and the bacterium Serratia entomophila have been tested against grass grub. A bacterial productbased on S. entomophila is now being marketed for grass grub control. Keywords grass grub, biological control, pathogens

INCIDENCE OF BACILLUS POPILLIAE IN DIFFERENT LIFE STAGES OF FLORIDA SUGARCANE GRUBS (COLEOPTERA: SCARABAEIDAE)

All life stages of Cyclocephala parallela Casey and Ligyrus subtropicus Blatchley were collected from commercial sugarcane fields in southern Florida from May, 1985 to May, 1988. These specimens were examined using phase contrast microscopy for the presence of the milky disease bacteria, Bacillus popilliae Dutky. In addition, field soil temperatures were measured, and the survival of milky grubs under simulated field temperatures was determined in a laboratoiy. Bacillus popilliae was not detected in the eggs or pupae of either grub species. The bacterium was found in 0.2% and 0.3% of adults in C. parallela and L. subtropicus respectively. The highest incidence of B. popilliae in both species was observed in the larval stage being 6.1% and 1.9% in C. parallela and L. subtropicus respectively. Survival data showed that B. popilliae was not limited in its development in infected third instar grubs by soil temperatures at any time during the year in Florida sugarcane fields.

Pest management of soil-inhabiting insects with microorganisms

Although more than 90% of insects have a stage in the soil, there are very few microorganisms now being used to manage soil insects. The first successful use of a microorganism for suppression of a pest was the utilization of milky-disease bacteria, Bacillus popilliae Dutky, for control of Japanese beetle larvae in turf. More extensive use of this bacterium has been hampered by its narrow host range, slow build-up in the soil, and an inability to produce it in vitro. Recent developments may have solved the production problems and will open up increased use of B. popilliae products. Although various strains of Bacillus thuringiensis Berliner are available for control of foliage-feeding Lepidoptera, aquatic Diptera, and the Colorado potato beetle, none have found a use in the soil environment. Other bacteria, such as Serratia spp. are being examined for control of the grass grub in New Zealand. Fungi are often associated with dead and dying insects in the laboratory and field. Both Beauveria spp. and Metarhizium anisopliae (Metschnikoff) Sorokin are used in pest-management programs in South America, Europe and Asia, but not primarily for control of soil-inhabiting insects. Beauveria has the potential for future development against a number of pests in the U.S.A. Viruses are available for control of several pine-feeding sawflies and certain lepidopterous pests. They are also important in the management of Oryctes rhinoceros (L.), a pest of coconut palms. Although protozoa have been detected in numerous soil insects, they do not appear to have much potential for direct use in pest management. One exception is the use of Nosema locustae Canning for control of grasshoppers in the Western U.S.A. and in Africa. If microorganisms are to be utilized to their full potential, we need to fill in the gaps in our knowledge of the soil ecology.

Incidence of Bacillus popilliae in Ligyrus subtropicus and Cyclocephala parallela (Coleoptera: Scarabaeidae) in Florida Sugarcane Fields

Bacillus popilliae Dutky was the major pathogen found in grubs of Cyclocephala parallela Casey and Ligyrus subtropicus Blatchley in Florida sugarcane fields. The milky disease was widespread among fields in the sugarcane-producing region, and two different B. popilliae varieties infected the grubs. The type A milky disease, containing a parasporal crystal and spore in the sporangium, was the prevalent variety infecting C. parallela. The type B variety, possessing only a centrally located spore within the sporangium, infected mainly L. subtropicus grubs. The overall infection rate of B. popilliae was significantly greater in C. parallela than in L. subtropicus.

Studies on transfection and transformation of protoplasts of Bacillus larvae, Bacillus subtilis, and Bacillus popilliae.

Protoplasts of Bacillus larvae NRRL b-3555 and Bacillus subtilis RM125 (restrictionless, modificationless mutant) were transfected with DNA from the B. larvae bacteriophage PBL1c in the presence of polyethylene glycol. B. subtilis 168 and Bacillus popilliae NRRL B-2309M protoplasts could not be transfected with PBL1c DNA. Protoplasts of B larvae NRRL B-3555 were transformed with plasmids pC194 and pHV33 in the presence of polyethylene glycol. The frequency of transformation was much higher when the plasmids were isolated from B. larvae NRRL B-3555 transformants than when they were isolated from B. subtilis 168. These results indicate that the restriction-modification systems found in B. larvae NRRL B-3555 and B. subtilis 168 may be different. Conditions for protoplast formation and cell wall regeneration were developed for B. popilliae NRRL B-2309S. However, no transformation occurred with plasmids pC194 and pHV33 (isolated from B. subtilis 168).