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Biological Control


The Microbial World:
Biological Control - Bacillus popilliae

Produced by Jim Deacon
Institute of Cell and Molecular Biology, The University of Edinburgh

This is one of 8 Biocontrol Profiles. It introduces the topic of biocontrol and deals with the commercial use of the milky disease bacterium Bacillus popilliae to control the Japanese beetle, a serious pest of turf, fruit crops and garden ornamentals in the USA.

Other Biocontrol Profiles are:

Biology and Control of Crown gall (Agrobacterium tumefaciens)
Bacillus thuringiensis
Control of Heterobasidion root rot of pine
Biology and control of take-all disease
Catenaria anguillulae, a parasite of nematodes
Pythium oligandrum and other mycoparasites
Fungal tip growth and hyphal tropisms

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Biological Control

Natural environments tend to be balanced environments, where organisms depend on one another and also constrain one another by competition for resources or by parasitism, predation, etc. But human influences can upset these balances, and this is most evident when an exotic organism is introduced on purpose or by accident. Many of the most serious pests, crop diseases or invasive weeds are the result of "introductions" from foreign lands. The newly introduced organisms find a favourable environment, free from their previous constraints, and they proliferate to achieve "pest" status. Entomologists has a useful term for this - they refer to the constraining organisms in the region of origin as "the natural enemy complex".

We can define Biological control (biocontrol) as:

the practice or process by which an undesirable organism is controlled by means of another (beneficial) organism.

In other words, biocontrol is both a naturally occurring process (which we can exploit) and the purposeful use of one organism to control another.

In practice, biocontrol can be achieved by three methods.

  • Inundative release (also termed "classical biocontrol") in which a natural enemy of a target pest, pathogen or weed is introduced to a region from which it is absent, to give long-term control of the problem. An example of this is the use of Bacillus popilliae to control the Japanese beetle in the USA, discussed below.
  • The biopesticide approach in which a biocontrol agent is applied as and when required (often repeatedly), in the same way as a chemical control agent is used. Examples of this include the use of Bacillus thuringiensis, Phlebiopsis gigantea and Agrobacterium radiobacter.
  • Management and manipulation of the environment to favour the activities of naturally occurring control agents. An example of this is seen in take-all control in grass turf.


Control of the Japanese beetle

In this section we discuss the use of a bacterium, Bacillus popilliae, to control a major introduced pest in the USA.

Much of the text below has been copied and updated from a book now out of print [JW Deacon, 1983. Microbial Control of Plant Pests and Diseases. Van Nostrand Reinhold, Wokingham]

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The Japanese beetle, Popillia japonica (Figure A), was accidentally introduced into the USA early this century. Although it is not a problem in its area of origin, the beetle causes serious damage in the USA. It spread rapidly from the initial sightings in New Jersey (1916) and today it is found over roughly half of the country, in almost every state east of the Mississippi. It is a problem as an adult beetle because it feeds on a wide range of plants, eating out the leaf tissues between the leaf veins (Figure B), and it accumulates on ripening fruit causing substantial damage. It is also a problem in the larval stage because the adult beetles lay their eggs in grass turf and the grubs destroy the grass roots, especially on new housing estates where natural enemies are absent.


Figure A. Adult Japanese beetles, about 1-2 cm long. Figure B, feeding damage on foliage. Based on slides provided by Fairfax Biological Laboratory.


By the 1930s the beetle problem had become so serious that a search was begun for a control measure. This led to the discovery of some naturally occurring diseased larvae. The disease was termed milky disease because of the milky white appearance of the grubs, due to a large number of refractile bacterial spores in the haemolymph (insect blood) (
Figures C, D). Two types of bacterium were subsequently isolated from two types of milky disease. Type A disease was characterized by a pure white appearance of the grubs and the bacterium in this case was named B. popilliae. Type B disease differed in that the grubs showed a transition from white to brown over winter and the bacterium causing this disease was named B. lentimorbus. A range of other milky disease bacteria were isolated from beetle hosts throughout the world, but the trend now is to regard all of these as varieties of B. popilliae because they are more closely related to one another than they are to other Bacillus spp.

All these bacteria are specialised pathogens of beetles (Coleoptera), specifically of the scarabaeid beetles (family Scarabaeidae). This family includes the beneficial dung beetles but also some of the most important pasture pests - the chafers. In practice B. popilliae has been used intensively and almost exclusively for control of the Japanese beetle in the USA, and to a lesser degree against the European corn chafer Amphimallon majalis in that country.

The milky disease bacteria are highly pathogenic and also highly persistent in the environment so they can be used for mass release to achieve lasting control. But B. popilliae cannot be produced easily in artificial media, so the inoculum for control programmes is produced in living hosts.


Figure C, larvae of the Japanese beetle in soil; the grubs are about 2-3 cm long. Figure D, a healthy grub (right) and a diseased grub (left). Based on slides provided by Fairfax Biological Laboratory.


The bacterium and its physiology

B. popilliae is a Gram-negative spore-forming rod, 1.3 to 5.2 x 0.5 to 0.8 micrometres. It is a fastidious organism that grows only on rich media containing yeast extract, casein hydrolysate or an equivalent amino acid source, and sugars. Several amino acids are known to be required for growth, as are the vitamins thiamine and barbituric acid. Trehalose, the sugar found in insect haemolymph, is a favoured carbon source though glucose also can be used.

Some varieties of B. popilliae form a crystalline body inside the cell at the time of sporulation and in this respect resemble B. thuringiensis. But the crystal is not thought to play a significant role in infection and certainly it is not as important as in B. thuringiensis. The variety lentimorbus, for example, does not produce a crystal and yet it causes disease. Another difference between B. popilliae and B. thuringiensis is that B. popilliae cannot be induced to sporulate in laboratory media although it does so readily in the diseased host. Actually there are a number of oligosporogenic mutants - ones that produce a few spores - but spores for microbial control programmes are usually produced in living insect larvae - an expensive and time-consuming process.


The host-parasite interaction

B. popilliae causes disease of beetle larvae when they ingest spores in the soil. The spores germinate in the gut within 2 days and the vegetative cells proliferate, attaining maximum numbers within 3 to 5 days. By this time some of the cells have penetrated the gut wall and begun to grow in the haemolymph, where large numbers of cells develop by day 5 to 10. A few spores also are formed at this stage but in the variety popilliae the main phase of sporulation occurs later and is completed by 14 to 21 days when the larva develops the typical milky appearance. In laboratory conditions the larva remains alive until this stage and usually contains about 5 x 109 spores. In field conditions, however, there are reports that larvae sometimes die earlier, before the main phase of sporulation is completed. This is of concern because sporulation stops when the host dies and the larva ultimately releases fewer spores to maintain the level of infestation of a site.


The cause of insect death is not fully known. Physiological starvation caused by the growth of bacterial cells in the haemolymph seems the most likely explanation, and fat reserves of diseased larvae have been shown to be much reduced compared with those of healthy larvae. However, toxins also may be involved because they have been detected in culture filtrates of the bacteria and shown to be lethal on injection. Recently, a crystal protein from sporulating cells of B. popilliae was found to have similarities to one of the Cry toxins of B. thuringiensis (see
B. thuringiensis). Although it does not cause such drastic effects on the insect gut wall as do the B. thuringiensis toxins, it might contribute to pathogenic invasion through the gut wall (Zhang et al., 1997).



Figures E-G.
Injection of healthy larvae of the Japanese beetle, as the first stage in production of commercial spore powders. Based on slides provided by Fairfax Biological Laboratory.


Application for biological control

B. popilliae has been registered for control of the Japanese beetle in the USA since about 1950 - the first registration of any insect pathogen as a microbial control agent. The control strategy is aimed solely against the larvae, so if the beetle itself is causing serious damage a chemical insecticide must be used for short-term control. The bacterial spores are produced commercially in larvae collected from grass turf on golf-courses, airports, etc. The larvae are injected with bacterial cells (Figures E-G), incubated until they develop a milky appearance and then crushed and dried to give a spore powder (Figure H). The spore powders are applied to turf in small heaps at roughly 1-metre spacing (Figures I, J) and the spores are then distributed naturally by wind and rain. They can persist in soil for several years and infect larvae that eat them. Therefore they have the potential to give lasting control of a pest problem, because the spore numbers in soil are boosted periodically when a diseased larva dies.

Commercial "milky spore" powders are marketed under several names, by several companies. For example, Fairfax Biologicals markets its product under the trade name "Doom". Other products include "Milky Spore", "Grub Attack" and "Grub Killer".


Figure H. After the larvae have been injected with B. popilliae and incubated to develop milky disease, they are crushed, flash-dried and mixed with a diluent, to produce a commercial spore powder. This powder is applied to the surface of turf (Figures I, J) where it will be washed into the ground. Based on slides provided by Fairfax Biological Laboratory.


The use of B. popilliae has proved remarkably successful. Between 1939 and 1953 over 100 tons of spore powder were applied to turf in over 160,000 sites in the USA as part of a Government programme (Fleming, 1968). Larval numbers in the turf were reduced 10- to 20-fold and the population stabilized at this new low level, with corresponding reductions in the levels of adult beetle damage. However, the treatment is most effective when applied on a region- or state-wide basis (or at least to relatively large areas) to reduce overall the levels of beetle infestation. It is less appropriate for use by small landowners, who may control the larvae in their own turf only to find their trees and shrubs being eaten by beetles from their neighbours' properties. Also, because B. popilliae is obligately dependent on its hosts for sporulation and because some larvae may not ingest spores (or not ingest enough to cause disease) a periodic resurgence and decline of the pest problem can be expected. The success of the control programme must be judged not on this basis but by the fact that over a number of years the mean level of pest damage is lower than it would be in the absence of B. popilliae.

Advantages and disadvantages of B. popilliae

The advantages of B. popilliae include (1) its very narrow host range (which is environmentally desirable) and its consequent lack of effect on beneficial insects; (2) its complete safety for man and other vertebrates (for example, it does not grow at 37oC); (3) its compatibility with other control agents including chemical insecticides and, more recently, insect-pathogenic nematodes (Thurston et al., 1994); (4) its persistence, giving lasting control.

Its disadvantages, however, include (1) the high cost of production in vivo; (2) its slow rate of action; (3) most importantly, its lack of effect on adult beetles which often cause the most obvious and distressing damage, and (4) its relative unattractiveness to the small landowner.

Outstanding problems

There is evidence that the Japanese beetle has re-emerged as a serious pest in some regions where it had been controlled effectively since the initial applications of spore dust in the 1940s (Dunbar & Beard, 1975). Larval densities ranged from 0 to 474 per square metre of turf in 1974 (mean 112), and were sometimes as high as those recorded 25 years earlier, before the control programme was begun. Moreover, in this study only 0.2% of larvae collected from field sites showed symptoms of milky disease compared with 41.5% disease incidence in a survey in 1946 after B. popilliae had been introduced. Spores collected from these few diseased larvae caused only 7 to 17% infection of larvae in laboratory tests, compared with 65 to 67% infection from spores collected from New York State where a decline in the degree of control had not been reported. Even this figure was low in relation to the expected 90% disease incidence at the inoculum level used. Perhaps there has been a reduction in virulence of B. popilliae in field sites over the years, coupled with an increased degree of resistance of the target pest (see Redmond & Potter, 1995). This might be expected by natural selection, because an obligately pathogenic bacterium that kills its host too rapidly would be at a selective disadvantage.


Further reading

Articles:

WE Fleming (1968) Biological Control of the Japanese Beetle. United States Department of Agriculture Technical Bulletin Number 1383. Washington DC.

DM Dunbar & RL Beard (1975) Present status of milky disease of Japanese and other Oriental beetles in Connecticut. Journal of Economic Entomology 68, 453-457.

GS Thurston, HK Kaya & R Gaugler (1994) Characterizing the enhanced susceptibility of milky-disease infected scarabaeid grubs to entomopathogenic nematodes. Biological Control 4, 67-73.

CT Redmond & DA Potter (1995) Lack of efficacy of in-vivo and putatively in-vitro produced Bacillus popilliae against field populations of Japanese bettle (Coleoptera: Scarabaeidae) grubs in Kentucky. Journal of Economic Entomology 88, 846-854.

JB Zhang, TC Hodgman, L Krieger, W Schnetter & HU Schairer (1997) Cloning and analysis of the first cry gene from Bacillus popilliae. Journal of Bacteriology 179, 4336-4341.

Websites

There are many websites on milky disease (type "Japanese+beetle" or "milky+spore" in a search engine). Here are just a few:

Managing the Japanese beetle (not on this server)

Ornamentals and Turf: Insect Pest Management (not on this server)

Milky Spore Japanese Beetle Control -a commercial supplier's site (not on this server)

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This site is no longer maintained and has been left for archival purposes

Text and links may be out of date