52
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 10
9
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
53
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.