STATE PLANT BOARD
E-703 September 1946
United States Department of Agriculture
Agricultural Research Administration
Bureau of Entomology and Plant Quarantine
EFFECT OF THE SUN'S RAYS UPON THE VIABILITY OF SPORES OF
BACILLUS POPILLIAE DUTKY, THE ORGANISM CAUSING
MILKY DISEASE OF JAPANESE BEETLE LARVAE
By Ralph T. White, Division of Fruit Insect Investigations
The method of distribution of milky disease spore dust used
lby the Bureau of Entomology and Plant Quarantine, and by certain
agencies cooperating with the Bureau, for the control of the
Japanese beetle (Popillia japonica Newm.) usually consists in
placing spore dust in spots at intervals on the surface of turf or
Boil and depending upon heavy dews or rains to wash the material
into the soil. Such spots have remained visible during drought
periods for as long as 10 days. Although the dust layer has an
appreciable thickness, and a portion sifts through the grass, a
large portion remains exposed for a time to the direct rays of the
sun. Another method of distribution given consideration consists
in applying the material over an area as a fine dust. By this
method the material would be spread out as a very thin film, and
practically all of it wculd be exposed to the rays of the sun until
washed in by rain or covered in some other manner.
In August 1943 tests were started to determine the effect of
direct sunlight on the spores of Bacillus popilliae Dutky in the
spore-dust mixture. preliminary tests were carried on in that year
and more extensive ones in 1944. Since the results were in general
agreement, only the more extensive tests will be reported.
The spore mixture was dusted very thinly over shallow trays
and exposed out of doors to full sunlight for various periods from
1 to 80 hours. These trays had removable covers of perforated
cellophane to prevent the blowing of the fine spore dust. Dust to
be exposed to the sun for more than 6 hours was brought indoors at
the end of each 6-hour period, remixed, redusted over the trays,
and returned to the sunlight until the required number of hours' ex-
posure had accumulated. Periods during which the sun was even
partially hidden by clouds were deducted from the time of exposure.
A similar series of spore-dust samples, not exposed to sunlight,
was used to test the viability of the spores in the original material.
A parallel series, in which equal samples of the original spore
dust were placed in an oven at 140O F. for the same periods as those
exposed in the sunlight, was also run to test the effect of heat on
the viability of the spores.
At the close of the various exposure periods during July and
st 1944, the samples were placed in new 2-ounce tins, labeled,
and put aside until November 27, 1944, when enough healthy third-
iL:S-tar larvae from one source were available for running biological
t on all series simultaneously.
T each series 40 grams of the dust was incorporated into 2
kil.-...,-- of soil together with 20 grams of equal parts of white
el ". s.. red top grass seed. This soil was then divided into 50
,.;.al ,rts and placed in 2-ounce tins. One healthy larva was placed
in eac tin and allowed to feed for 37 days, or until it became
i or died. There were 14 exposure periods requiring 50 larvae
each or a total of 700 larvae for each of the 3 treatments. The
saie ufoui.it of pure chalk and talc without spores was used with a
series of 700 larvae to determine the normal disease incidence that
,^L .t be expected from the larvae and soil used in the tests.
The results of the tests are summarized in table 1.
Tab'e l --Incidence of milky disease among Japanese beetle larvae
In soil containing milky disease spores that had been given
various exposures to sunlight and heat; 50 larvae in each lot.
SNumber of infected larvae in soil containing spore dust
Hours of : (chalk plus : Exposed : Not exposed : Heated at
exposure : talc, no : to sunlight : to sunlight : 140 F.
. ....spo r e s ) : ..:. ........
1 4 38 37 41
2 2 43 37 42
4 2 36 38 36
6 2 37 42 38
8 3 39 36 42
16 3 28 37 41
24 4 28 39 40
5 31 42 43
40 2 29 39 39
0 2 22 44 40
56 3 24 41 38
4 22 44 38
S3 21 44 38
;G 20. 3 32
Tot a]s 42 418 556 553
E'r i:t ages 6 79.4
The data indicate that there is no reduction in viability
when milky disease spores are exposed to the sun for 8 hours or
less, that there is a significant reduction in viability when they
are exposed from 16 to 40 hours, and that there is a further marked
reduction in viability when they are exposed for 48 hours or more.
The infection rate in the series exposed to 140 F. for as
much as 80 hours was similar to that obtained from impregnated spore
dust not exposed to the sun. This indicates that the effect of
heat alone on the potency of Bacillus popilliae is of minor impor-
tance under field conditions. However, the effect of temperature
in combination with exposure to direct sunlight on milky disease
spore dust was not included in this study. Neither was any attempt
made to detennrmine the particular factor or factors responsible for
the effect of the sun on viability of the spores. The disease inci-
dence of 6 percent occurring in the check series compares very
favorably with that in numerous other check series run during the
same period in other work.
Another series of tests was run to determine the effect of the
sun on the viability of the spores in deposits of dust applied by
the accepted method of spore-dust distribution used by the Bureau
and cooperating agencies. Deposits of spore-dust material 1/32,
1/16, and 1/8 inches in thickness were exposed to direct sunlight
as previously described, for 56, 64, and 72 hours, respectively.
This material was left undisturbed for the entire exposure period.
The exposed spore dust was then mixed with soil as in the other
tests, 50 larvae being used for each series. These results are
given in table 2, and may be compared with those of the checks used
in similar exposure periods with a thin film of dust (56, 64, and
72 hours, table 1).
Table 2.--Incidence of milky disease among Tapanese beetle larvae
in soil containing nilky disease spores that had been exposed
to the suns rays in layers varying in thickness;
50 larvae in each lot
Thickness of : Hours of of infected
spore-dust : exposure larvae
layer (inch) : to sun recovered
1/32 56 35
1/16 56 37
1/8 56 36
UNIVERSITY OF FLORIDA
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3 1262 09238 7231
These results indicate that sunlight does not materially
reduce the viability of the spores of Bacillus popilliae in the
standard milky disease spore-dust mixture applied for the control
of grubs of the Japanese beetle in accordance with the spot method
followed by the Bureau and cooperating agencies. Most of the dust
is deposited on the turf or soil in layers of sufficient thickness
to prevent damage to any except the thin top layer of spores. On
the other hand, a considerable reduction in the viability of the
spores may result when the dust mixture is broadcast in a very thin
film, if several sunny days pass before the material can be washed
in by rains or otherwise worked into the soil or turf.