Japanese Society
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of Applied Entomology
Entomology and
and Zoology
Zoology
ofApplied
Appl.Entemol.Zool. 32
625-634(1997)
(4):
Comparative Studiesof Growth Characteristic
and CompetitiveAbility
in Bacillusthuringiensis and Bacilluscereus inSoil
Kaori YARA,iYasuhisaKuNiMi2and HidenoriIwAHANA
Dopartmentofophed
Bibdyat Stibnce,
Fbcudy of4gricttitung
7blvoL]}zlverst'ty
ofAgtimlture
and nchmoig
FLtchag7blvo183,1apan
16 May l997;Accepted2June 1997)
(Received
and
with
We cxamined
the growth characteristics
and
competitive
abilities of Baa'llus
thuri}4gieizsis
BaaiUuscetettsin soi]. The growthand pcrsistenceof the two bacilli,
added
to sterile soil
or without soil bactcria,were
monitored
under
aerobic
conditiens.
When vegetativc
or sporcs
of B. thun'ngi'ensis
andlor
B. cerezar were addecl to steriie soil, both bacilli
increased
their nurnbers.
The finalIovels
ofpopulation
were
greaterfor B. cetetts than forB.
thuritrgz'ensis.
In most of the cases when vegetative cells of B. thuri}agiensis
were
added
at the
same
time as indigenous
soil
bacteria
to steriIe soil, B, thun'ngdensis
sporulated
immediatcly
without any growth. B. thuringt'ensas'
and
indigenous soil
grew only when both B, thun'ngieizsds
bacteriawcre inoculated
at low densities.
On the othcr hand, when vegetativc cclls or speres
of B. ccreus wcre added
at the same
time as indigenous soil bacteria
to sterile soil, B. cereus
even
whcn
indigenous
seil
bacteria
were
inoculatedat high density.These datasuggest
grcw
thatB. thuri}eimsis
islessadaptive to soil habitats
than B. cereus.
IZigr
wonts:
Bacitlus
thapm,
Bact'iitts
ceretts, competition,
soil, interaction,
endotoxin
cells
INTRODUCTION
Bacillus
thun'ngienst's
isa Gram--positive
bacteriumthat upon sporulation
produces a
crystal that istoxic to certain
insects.
StrainsofB. thun'iigz'etzsis
that aflect the
proteinaceous
larvaeofinsects in Lepidoptera,
Diptera,
Coleoptera,
and other orders have been identified
1994).
Accordingly,
B.
thun'i<gz'ensz's
has
been
used as a microbial
(FEDERIci,
pest control agent
throughout the world.
There have also been considerable
eflbrts
made
to isolate
new
B.
thun'iqgi'enst's
strains that have increased
against
target
insect
species
andlor
wider
potency
hostrange. Further,thereisthe potentialto apply DNA technology to create new strains of
B. thun'tqgz'ensis
and to transfer B. theen'iagz'ensis
toxins to create alternative
expression
systems
in
transgcnic plants,
or other
bacteriaand baculoviruses
1993).
In
short,
applica(CARLToN,
tions of B. thzaringz'ensds
seem
likely
to increaseintothe foreseeable
future.
While many studies have investigatedthe molecular
biologyand biochemistryof B.
thure'ngt'ensis,
there has been Qnly limitedresearch
Qn thc ecology
and
behavior ef B.
'
thun'ym' inthe environment.
B. thuringz'ensz's
can be readily isolated
from insects
(DuLgetAGE,
1970;BuRGEs, 1973; VANKovA and PuRRiNl,1979;CHILcoTTand WiGLEy, 1993),sericultural farms(AizAwA
et al., 1961;OHBA et al.,1979, 1981),
soil (DELuccA
et al., 1981;OHBA
and AizAwA, 1986 a, b;TRAvERs et al., 1987;KIKuTA et al., 1989; MARTiN and TRAvERs,
1989;MEADows et al., 199e; HAsTowo et al., }992; CHiLcoTT and WiGLEy, 1993), stored
and HuRsT, 1977; DELuccA
et al.,1982;MEADows et aL, 1990,
productdusts(BuRGEs
i
Prescnt address:
!
To
whom
Mmbnat imtitute
Siciknceq
thnnendea nttkttba
305,Yapan
of4gro-thvironmemlat
should
be addressed.
correspondence
625
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K, YARA
626
1992), deciduousand
ct al.
leavcs (SMITH
and Cou/cHE,1991; OHBA, I996), and aquatic
MARcALIT,
1977;
MARGAi,vi'and DEAN, 1985). It would
ap(GoLDBERG
pear,then, that B. thun'ngiensisisan indigenousbacteriumin each of these environments
1993). However, when viablespores of B. thun'ngienst's
werc
addecl to soil in the
(MEADows,
conifer
environments
field,
thcy
and
decreasedimmediatelyby apremained
constant
fbr a longperiod(PETRAs
proximately1 Iog,after which their number
and CAsiDA,1985).
The reasons
why
B. thun'ng'ensdscannot multiply in a soil environment
seem
to includethe eflect ofsoil pH (SAi.EH
et al., 1970),moisture
content
(WEsTet al.,
1985; PE'rRAs and CAslDA, 1985),nutrient availability (SALE}I
et al., 1970; AKIBA et al.,
1979),and presence ofindigenous
microorganisms
et
al.,
1985).
CWEsT
In environment,
B. thuringtknsis
seems
to exist most commonly
in a spore-state.
However,
sperulation
are required
germination ofthe spore, vegetative growth,and subsequent
foritto complcte
itslifecycle and persistpermancntly in the ecosystem. There are three
hypotheses concerning
the possible role of B, thzan'ngiensis
in the environment:
that B.
thuringiensis
isan entemopathogen,
a phylloplane
cpiphyte,
or a soil bacterium(MEADows,
1993).
One
could
neither
germinatenor
multiply
but rather
B. thun'ngdensir
in soil was that the clo$est relative ofB.
bacterium.The two bacteriashare similar morpholorequirements,
and
also have a high degree ofDNA
homology and
gies and nutritional
et al., 1988). Despitethese similarities, B. cereus iscommonly
genetic relatedness
(FERGus
isolated,
whereas
B, thuringfezasis
israrely isolatcd
firomsoil (DELuccA
et al., 1981), Furthermore,
B. cereus may be able to grow under conditions that do not permitthe grewth ofB.
thuringiensis
in which B.
CWEsTet al., l985). To date,the only consistent characteristic
thun'ngiensis
and
B, cereus have been fbund to difler
taxonomically
isthe productionby B.
thun'ngiensis,
ofparasporal
crystals endotoxin
et al., 1984; PRiEs'r
et al.,
1988;
(BAuMANN
ZAHNER et al., 1989),
In this study we invcstigated
how the presence of indigenoussoil bacteria
afll]cts the
ability of introduced
bacilli
to germinate and grow in soil, We compared
thc competitive
ability ofB,
thun'ngiensis
against indigenous
soil bacteria
withthat ofB, cereus, and we evaluated the possibility
ofB.
thun'trgz'ensds
as a soil bacterium.
ofthc
fbr studying
reasens
thun'ngz'ensis
isB. cerezts,
a
common
soil
MATERIALS
AND
METHODS
Sbil.The soils used in this experiment
were
collected
from an experimental
farm of
Tokyo University
of Agriculture
and Technology,Tokyo,Japan.Beforeuse, the soil was
air-dried, sieved
<2 mm,
and stored at 5eC. The soil was a silt loam ofpH
5.8,and with a
C:N ratio of 15.0.
Bacten'a.Bacillas
thun'ng'ensis
serovar
aicawai KH was
originally
isolated
from the cadaver of theJapanese silk moth, Antheraea]amamai,
and
continuously
maintained
in our
laboratory.
Bacillvscerezcs K213, which
was
originally
isolated
from soil, was kindlysupplied
by Dr. K. KATo ofthe NationalInstitute
ofAgrobiological
Resources,Japan.
To obtain a mutant
of each
bacillusresistant to two antibiotics, streptomycin
and
rifampicin,
streptomycin-resistant
mutant
was
first
isolated
by spreading a culture of the
l955)platescontaining
parentstrain onto Lennox L agar (LENNox,
O.O19,b
streptomycin.
Isolatedstreptomycin-resistant
mutant
was
cultured
in Lennox L brothcontaining
O.O1gib
NNNG
for 1 h. Then, cultures were
(mutagen,
washed
by sterile distillecl
water
(SDW)by centrifugation fbr20 min at 3,OOO rpm, and
the
JMmethyl-jV'-nitro-IVLnitrosoguanidine)
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CornpetitivcAbilityin B. thun'ngieizsz's'
and
B. cereus
627
rifampicin.
Colonies
agar containing
O.Olg/li
streptomycin
and O.005gtb
Lennox L agar without antibiotics.
For inoculants
bacilli
in Lennox L
ofthe
spores, each ofthe
was cultured
with shaking
brothat 3eOC for20 h. The cultures were platedon Lennox L agar and incubatedat 300C
for7 days,Cellson the plateswere scratched and suspended
in SDW, washed
three times
with SDW by centrifugation
for20 min at 8,OOOrpm, and suspcnded
again in SDW. The
suspension
was heatedat 650C for 15 min
to ki11
any vegetative
cells inthe suspcnsion.
The
plated on Lennox L
were
re-isolated
on
suspension,
designated as spore suspension,
To inoculatevegetative cells of bacilli,
the spore
obtained
was
stored
at
50C
unti1
use,
in Lennox L
in log phase. Cellswere
suspended
in SDW. These
was
suspension
cultured
broth at 30eC fbr 20 h. During this period, the bacilli
were
harvested,
three times with SDW by centrifugation,
and
washed
use.
suspensions
were made
immediatelybefore
Indigenoussoil bacteriawere collected from sampled
soil as fo11ows.
Ten grams of
sampled
soil was
shaken
strongly
with
50 ml of SDW ibr 2 min. Serialdilutions
of soil
incubated
48
h.
Approxiextract were spread
onto Lennox L agar platcs,and
at 300C fbr
mately
and
in Lennox L brothat 30eC
ten colonies were selected at random
were cultured
for20 h. Cellswere harvcstedby centrifugation
for20 min at 3,0eO rpm, washed
three
SDW, and resuspended
in SDW.
Experimentatprocedure.Five grams of dried
times
with
soil
placed in 100
were
Erlenmeyer
ml
fiasks
were pluggedwith silicon caps and autoclavcd
20 min), The
(1200C,
soil moisture
was adjusted
to 7egrbby bacteria
inoculants,
B. thuringienst's
or B. cereus and
indigenoussoil bacteria
were inoculated
simultancousiy
intotheautoclaved soil. The inocuIatcdflasks
werc
then incubatedat 250C in darkness.
The concentration
of B. thun'ngiensis,
B.
cereus or indigenous $eil bacteria
105 colony
forTningunit CCFU)lg
of
varied by 103, 104 and
drysoil. Separatecompetitive trialswere run forboth the bacilli
spore and bacilli
vegetativc
cell inoculations.
Ehumeration.
Thc flasks
werc
removed
and the soil was
firmlyshaken with
periodically
ml
min.
of
the
soil
spread
Lennos L agar
50
of SDW
for 2
Serialdilutions
were
Qnto
to estimate
the number
of total viable
cells in soil samples.
The total viable cells of
B. thuringiensis and B, cerezas were estimated
by the drop plate method
(HoBENand
Sosde,LsoGARAN,
1982) using Lennox L agar containing
the appropriate
antibiotics.
To cstimate
the densityofB. thun'agz'ensis
or B. cerezLsspores, serial dilutions
were heatedat 8eOC fbr
flasks,
and
the
5 min
used
three
and
in the drop plate method.
platesfrom the appropriate
The densityofindigenous
B. thuringz'ensi's
or B.
soil
determined by
were
counting
from the drop plate
(obtained
of bacteria
from spread
(obtained
method
using
non-heated
suspen-
Lennox L agar
without
antibiotics).
When the inoculation
densityof indigcnoussoil bacteriawas lower
than that of B. thun'ngiimjts
or B, cerezas,however,subtraction
could
not be used, In such cases
we checked
antibiotics sensitivity ofall colonies
onto the Lennox L agar plateby replating
onto
the plate containing
antibiotics
and
the non-antibiotics-resistant
bacteriawere reas
indigenous
soil
bacteria.
garded
Saturation
densityand growth rate were estimated from a growth curve that contained
all the periodicalenumerations
ofthe
experiments.
Saturationdensitywas estimated as the
mean
densityQf the stationary phaseperiod,and growth rate was estimated as a ratio ofthe
inoculation
saturation
density
to the initial
density.
sion)
from the
cereus
Bacterialnumbers
dilution,
each platecontaining
between 10 to 300 CFU.
bacteriawas determinedby subtracting the number
of total
total number
method
using
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RESULTS
GrowthofB.
thun'nglensis
and B. cereus inautoclaved soil
As a control, each bacilluswas inoculatedinto autoclaved
soil alone.
When B.
thun'ngiknsis
vegetative
cells were
inoculated
IA),
total
B.
thun'ngienst's
began
to
increase
at
CFig.
day 1,achieved 106 CFUIg ofdry soil by day 2,and maintained
this population fbr 10 days
during the cxperimental
B,
thun'ngz'erasz's
spores
began
to
form
on day 2,achievcd
105
period.
CFUIg ofdry soil at day 3, and thereafter increasedgraduallyto achieve nearly total
density.
When B. thun'trgz'ensis
spores were
inoculatedalone(Fig.
IB), total B. thern'ingz'ensi's
began
to increaseat day 2, achieved
106 CFUIg of dry soil by day 3, and maintained
this
fbr
10
days.
B.
thuringzwh's
spores began to increase
at day 3, achieved
10S CFUI
population
this densityfor1O days.
g of dry soil by day 5,and maintained
When B. ceretLs vegetative
ce]ls were
inoculated
alone(Fig.
1C),total B. cerettsbegan to
increaseat day 1,achieved
107CFUfg ofdry soil by day 3,and maintained
this population
fbr10 days, No B. cerettsspore was detectableat day 1, but spore densityreached
the density
of total B. ceretLs at day 2, and
aftcr that fluctuatedalongwith the density
of totalB. ceretLs.
When B. cerezas spores were inoculated alone (Fig.
ID),total B. ceTezas began to increase
at 1 day, achieved
1O' CFU!g ofdry soil by day 5, and maintained
this population for10
days.B. cerms spores began to increaseat day 2,achieved the same densityas total B. cerezts
at day 3, and
thcreafter fluctuated
along
with
total B ceTeus
B. thun'npm
vs. indigenotLs
soil bacten'a
When vegetative
cells ofB.
thu"'ngiensts
were
added
at the same
timeas indigenoussoil
bacteriato sterile soil, the competitive
outcome
ofB.
thzaringiensis
varied
with both initial
densityofsoil bacteriaand ofvegetative
cells ofB.
thun'ngdensis
2A, Fig.3A).When soil
(Fig.
bacteriawere inoculated at high (I05
CFU/g) and intermediate (10`
CFUIg) density,B.
thorringiensis
hardly increased lessthan 1 ordcr of magnitude
2A,
(Fig. Fig,4A). A single
8
7
87654321o87s54321o
B
6
5
4
-e
F
3
26-
1go)8b7mM.6
D
5
4
3
2
1
oo
246
S
10O2
4G810
lncubation
day6
Fig. 1. Viable count
Inoculant
states
were
ceretts vcgetative
ccll;
oftotal
alone in autoclaved
<e)and spores {O)ofbacilli inoculated
soil,
fo11ows:A, B. thurbrgievists
vegetativc
ccll; B, B. thun'ngz'en.ris
spore;
C, B.
D, B. cereus spore. Arrows denote bclow-detectablelevcl.
as
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Compctitivc Abilityin B. than'tagi'etzsis
and
B. ccreus
4321eo:t
629
4i21o-43E
.1
g,
Nen.s21e
D
I
t･,1･/,11/･1'ttii.,l.lf/111,11t"i/1//'
/･l･1,,/..iiF
11///111,･oi.
Itll/.
.,l/
-1lo
lo4
lnitial
densityof
Fig. 2. Growth
103
les
le"
loS
(CFUIg
soii becteria
soil)
ofsaturation
(ratio
density
to initial
density)
ofbacilli
inoculatedin
bncteria.Inoculant statcs were
as fo11ows:A, B.
thun'ngiensis
vcgctative cell; B, B. thun'ragt'ensiy
spore; C, B. ceretLs vcgctative
cell; D, B. cereus spore.
Ineculationdensities
ofbacilli
were
as fo11ows:
closed colurrms,
103 CFUIg soil; dotted celumns,
10` CFUIg soil; opcn columiis, 10S CFU!g soil.
autoclaved
soil
at
ratcs
the
same
time
as
soil
when
indigenoussoil bacteriawere inoculated at 104 CFUIg and vegthzan'agiensis
were
inoculatedat 103CFU!g. In this case, B. thuringiensis
spores increased
fbr2 daysup to the initialdensityofvegetative
cell ofB. thuringikmsis.
When
soil bacteria
were
inoculated
at low (103
CFUIg) density,
on the other hand, B. thuringdensis
increased
from 1 to 2 ordcrs ef magnitude.
SporedensityofB. thuringz'ensTs'
began to increase
at day 1 and
achieved
total density
of B. thun'ugiensds
by day 2,then maintained
this spore
densitythroughout the experimental
period.
When the spores ofB. thuritigt'ensis
werc inoculated
to autoclaved
soil at the same
time
as indigenoussoil bacteria,
B, thure'agiensis
spores
maintained
almost
their initial
density
throughout the experimcntal
2B, Fig.3B, Fig,4B).There were, however, two
period(Fig.
notable
exceptions,
the first
bcing the simultaneous
inoculation ofB. thuringi'ensis
at 103
CFUIg and indigenoussoil bacteriaat 103CFUIg, and the second beingthe simultaneous
inoculation
ofB. thun'trgzirnsalf
at 103CFUIg and indigcnous
soil bacteria
at 1O` CFUfg, In
both these cases, B. thttn'ngz'ensis
increasedabout 1 order ofmagnitude
at day 10 and spores
of B. thuriJkgzlazst's
maintained
their totaldensitythroughQut the experimental
period.
exception
etative
B,
occurred
cells ofB.
ceretts vs.
indigenoors
soil bacteria
When vegetative cells ofB. ceretLswere inoculatedto autoclaved soil at thesame time as
indigenoussoil bacteria,B. ceraus increased from 1 to'3 orders ofmagnitude
fbr3 days (Fig.
Fig.
2C,
4C).Saturationdensities
ofB. ceretts decreased
as initial
densitiesofindigenous
soil
bacteriaincreased(Fig.
3C). IVhen initialdensities
ofB.
cerezas were
higherthan theSe of
indigenoussoil bacteria,
saturation
densities
and growth rates ofB, cetetts were
not
afllrcted
by the presenceofindigenous
soil bacteria(Fig.
2C, Fig, 3C).The densityofB. ceiezas sporcs
began to increasefor1 day,achieved the total densityofB. ceretLs,and then fluctuated
along
with total density
throughout the experimental
period.
When the B. ceretts spores were added to autoclaved soil at the same time as indigenous
soil bacteria
4D),B. ceretcsincreased 1 order ofmagnitude
on each of days 3,5 or 7,In
CFig.
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630
10s
et al,
10
r1.
lal
i:l
A
6F.g4.a,2VogVIVPpt.th
Bl
//i
1
1"''t
/tt･'I.-t---
1.･s,''1･.･1･1
,l･･
t
2i,l10
/ti-/['
D
c
B6gzo-
-8V6E'W'4gB,8
o
1o3
le4
10]
loS
lnitial
density of
loS
soi])1o4
(CFU/g
soil bacteria
Fig.3. Saturation
densitics
CFUIg drysoil) of bacilliinoculatcdin autoclavcd
(log
Inoculant statcs wcrc
as fbllows:
A, B. thurng'ensis
veg'etativc
samc
time as soil bacteria.
the
soil at
ce]1;
spore.
Inoculation densitiesof
B, B. thun'ugt'ensis
spore;
C, B. cerezas vcgetative
celll D, B. cereus
1CrVCFU!g soil; opcn
bacilliwere as fbllows:
closed
coLumns,
10/'
CFUIg soi]; do"cd columns,
columns,
10S CFUIg soil.
1086
A-io
..-
B
---y.+-H
t.
8
6
"'--t'N
ts=e
4
42
=tbr}-M
"
s-2&o)106-ut)B
c
-ri'--le108
-He
6
6
4
4z
D
-H
"
--,
-ete'f
2
o
o
02days
810tncubation
o246
46810
Fig. 4. Viable count
of total
sDil at thc
(e)aiid spores (O)ofbacilli inoculatedin autoclaved
time as soil bactcria {i).
Inoculant concentration
of' both bacilli
and
indigcnous soil
bacteriawcrc
1OrlCFUIg dry soil. Statesofi]]oculant
wcrc
as fo11ows:A, B. thun'tlg.
ien,siy
vegetaB.
cereus
tive cell; B,
thutitrgiknsab'
spore;
C, B
vegetative
cell; D, B. cereus sporc,
same
the cases
when
B, cereus
was
inoculatedat high (105
CFUIgl
or
intermediate
(104CFU/g)
density and indigenous soil bacteria were
inoculated at 10:`CFU!g,
their initial
densities(Fig.
2D, Fig. 3D). In some
of the experimental
creased
at
expcrimental
In all
day 1O.Spore clensity
ofB,
period.
the
competitive
trials,the
B.
cereas
cases,
fluctuatccl,
as did total density,
throughout
cerents
outcome
maintained
B. ceTeus de-
ofindigenous
soil
bacteriawas
as
the
follows.
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Competitive Ability
in B. thun'tag2'ensds
and
B. ce,eus
densityofipdigenous
a few daysmcreased
IO days,
soil
bacteriaincreasedto 10e CFUIg
grad ually
to 109 CFU/g
ofdry
soil and
ofdry
631
soil
maintained
fbr 1 day,then
after
this population fbr
DISCUSSION
In most
were
of the cases when
vegetative
cells
ofB.
thun'ngileizsis
and indigenous soil bacteria
to sterile soil at the same time, B. thun'ngdensis
sporulated
immediatelywithout
added
multiplied
only when
growth.B. thuringdensis
bothB. thuringiensis
and indigenous soil
ba,cteria
were inoculated
at low densities,
Such a situation seems to be very rare in natural
soil habitats
because 106-le9bacteriaper gram are ft)undin soil habitats(A'rLAs
and
BARTHA, I987). Furthermore,B, thun'agilrrzsis
vegetative
cells have been shown
to disappear
rapidly, losm 91g'b
oftheir
24 h ofincubation
g
population in thc first
in natural soil (WEsT
et al., 1984). Therefbre,
itappears that B. thunngzenst's
vegctative
cells cunnot
persistfora
Iong pe-od and grow in a natural
soir environment.
In .o.rdert.oesamine the possibility
of germination, spores
ofB.
thun'npm's were set in
corppet.rtron
with mdigenous
soil bacteria.
ds a result, B, thun'ngilansis
did not gcrminatebut
maintamed
their initial
density and remained
in the spore state. B. thuringiensis
increased
slightly
only when
indigenous soil bacteria
were inoculated
at low densities,
so itseems
that
B. thun'ngilansis
spere
can neither
germinate nor grow in natural soil environments.
In contrast to the above, the competitive trialsofB. ceraus, which was thought to be a
soil bacterium,
against indigenous
soil bacteriasuggested
that B, cereus ismore characteristic
of soil bacteria
than isB. thun'ngilensts.
Vegetative cells ofB. cereus grew even ifindigenous soil
bacteria
were
inoculatedat high density,
under
which
condition
B. thun'ngibnsas
was unable
to
When
spores of B. ceieus were set incompetition
with
indigenous
soil
bacteria,
B ceretcs
grow.
mcreased
their number
even
ifindigcnoussoil bacteriawcre inoculated at high density,
under
which
condition
B. thun'izgi'ensab
never
we could
not
germinated.In these experiments,
any
3z",E･:m..g.e,:'/alP,ag'h?",,b
m
a spore
state,
AFclB" (l99.2)
reported
.
thunngzensis
was inoculated
that B. thuringibnsis
sotto suppressed
their multiplication
when
B.
in sterilized soil in addition to a little
nonsterilized
soil. WEsT et
al, (1985)
showed
that B. thun'ngdensis
could
not
B. cereus
grow in non-sterilized soil, whereas
Similar results were obtained
in
our
experiments,
suggesting
that
the
pould. soil bacteriaisa major factorinhibitingthe
presence of
ofB.
thun'ngqiensis
in natural
growth
;::igenous
In
these
it appears
gxperiments,
thatB. thuringtensalf
has lesscompetitive
ability
against
mai',,',,P8,:SS.OSBb.a:,e,,,rla,g2
gfi,2bg//?i,g,%
toxin. The
'
ofcrystal
toxin synthcsis isabout 33 to 43% ofthe
overall
rate of
et al.,1986).
Even though most crystal toxin genesare on
proteinsynthesis (ARoNsoN
which
are easily lostor altered, as a rule, B. thuringz'ensts
plasmids,
tenaciouslyretains crystal
1994),
CMARTiN, The crystal toxin must contribute to the survival ofB. thun'ugiensds
in the
environment
despitethe obvious
cost to the cell in tcrms of increased
genome size and
synthesis
large
body
during
nutrient
stress (sporulation).
protcin
Based
on the principle
pfa
ofallocation
(CoDyand DiAMoND, 1975),there seems to be a trade-off between the ability
to compete
and the a bility
to p roduce proteinaceous crystal toxin.B, thun'iagz'ensis
appears
to
crystal
rate
total
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K. YARA ct
632
al.
crystal toxin rather than in competiitsenergy in theproduction ofproteinaceous
thus is]esscornpctitive
than B. cereus, MAR'i'IN
indigenoussoil bacteria,
and
and
that
suggcsted
that B. thun'trgz'ensis
isa soil bacterium,
proteinaceous crystal toxin
(1994)
may
be stored as a food foruse ingermination, as a directantagonist against ether microbes
suggest ¢ d that
or as baitto divert
predatorsinthe soil. On the other hand,FEDERJci(1994)
is
to
ki11
host
insects,
the natural role ofproteinaceous
crystal toxin
providingB. thun'tqgzknsab
is taken to be an entoresources
for growth and reproduction.
IfB, thun'ngiensis
with
over non-toxic
spore-formers
in the
mopathogenic
bactcrium,then itgains an advantage
of
the
of
crystal
toxin.
FEITELsoN
et al.
environmcnt
of an insectgut because
presence
and
Emimalhave discoveredB. thuriugiensds
strains that show activity against plant(1992)
mites.
Whether
B.
thun'iLgz'ensis
is an
Protozoa,
flatworms,
and
ncmatodes,
parasitic
crystal
toxin
must
contribute
to
its
survival
in
the
entomopathogenic
or soil bacterium,
expend
tion against
cnmronmcnt.
B. thun'tngi'enszs'
seems
to maintain
to complete
a full
lifecycleof
a spore
state
in the
soil
environment,
where
as
B, eereus
and
germination,
in order
to maintain
itsenvironmental
longevity.
IfB. thun'ugiensisisa soil
sporulation
from this classification than B. cereus.
bacterium,itwould seem to gain fewer advantages
used
in these cxperiments
was
isolatedfrom insect
However, because the B. thun'ugiensis
be
needed
using
various
serovarieties
ofB.
thun'ngiensds,
cadaver,
furtherexperiments
will
especially
B. thzaringibnsis
isolated
from soil environments.
seems
spore
vegetative
cell
multiplicatioll,
REFERENCES
K, KuRATA (1961)
IsolationofBacitlus thun'tzgdensifrom the dustofsilkworrn
and
farrners, f Sbtibutt,
Sti.ZPn.
30: 45IL455 (inJapanese
with English summary).
AKiim, Y., Y. SEKulMA,K. AIzAwA and N. FLulyosHl(1979)Microbial ecological studies en BactVtzas
thuti)4siibttsis.
thuringdezasiy
in
soil
cxtracts.
AmpL
MitomoL
Zbol.
23: 220III.Efflrct
ofpH
on thc growth ofBacillus
.lpn.J
with
English
summary).
223 (inJapanese
AKIBA, Y. (1992)
Studieson thc microbial
ccology
of Bacillilts
thun'ngiensas'.Butt.
SZiiinnza
SeTicztde.
Etp,Sn. 40:
English
summary).
79-143 (inJapancse
with
ARoNsoN, A.I.,W. BEcK/NcANand P. DuNN (1986)
Bacilins
thupmis and related insectpathogens.Miicrobthl.
Rev, 50: l-24.
Mi'crebial
EbotcgT:
fibundomentats
and Apatications,
2nd ed. The Berijarriinl
ATL/ts,
R.M. and R. BAR'i'HA(1987)
Cummlngs Publishing,Mcnlo Park, California,533 pp.
BAuMANN,
L, K. OKAMoTo, B.M. UNTERMAN, M. LyNcH and P. BAupttANN (1984}Phcnotypic characterizaR!thol.44/ S29-341.
tienof Baalllus
thutipgfensis
and
Baciltus
ceteus.
f imertebr.
BuRGEs, H,D, (1973)
Enzootic discasesof insects.
In Ragalatihn
Ilat)ulations
BuwA,
of'insect
byMthrooqgtinisnas
(L.A.
New
York,
31-49.
ed.). The New York Academy of Sciences,
Jr,,
pp.
BuRGEs, H.D. andJ,A.
HuRs'r(1977)EcologyofBacitlvs thurklgiensir
in storage moths.
IlathoL
30:
f imertebr.
130-139.
aARLToN, B.C. (1993)Devcloprncnt of improved bioinsecticides
based on BaciUus thun'ngietzsis.
In 1)last
Cbntiol
ievith thhanced linvironmental
Snj?ty
(S.O.DuKii,JJ. MENN andJ.R. PuMMER, eds,), American Chemical
Society,Washington, D.C., pp,258-266,
CHiLcoTT, C.N, and PJ. WIGmy (1993)
Isolationand toxicity ofBacittus thuriugiensis
from soil and insect
habitatsin New Zcalmid, f imcrtebr.
Ilathet.
61: 244-247.
DmMoNi) (l975)
Ecotqg7and Evolumbnofthmmunities,
Cor)y,M.L. andJ.M.
Thc BclknapPrcssofHarvard
UniversityPress,Lendon, 545 pp.
DELuccA, AJ,,J.G. Sm{oNsoN ancl A.D, LARsoN {l981)Bacillus
thun'ngiensds
distributien
in soils ofthe
United
States.thn.1.MinvebioL27:865-870.
DELuccA, AJ,,M,S, PALMGREN and A, CiEGLERC1982)Ba"'aus
thun'tlgt'ensis
in grain elcvator dusts, thn.1.
AIzAwA,K.,T. TAKAsu
rearing
houscs
of
NII-Electronic Library Service
Japanese Society
JapaneseSociety
of Applied Entomology
Entomology and
and Zoology
Zoology
ofApplied
Abilityin B. thun'ngz'ensds
and
Competitive
B. cereus
imobibl. 28:452"456,
Dui.MAGE, H.T. (1970)
Insccticidal
activity
fiivertebr.
Ilathol.
15:232-239.
of
HD-1,
a
ncw
633
isolateof Baat'll"s
thurir<gt'ensis
variety
alesti.
Y/
FEDERIcl,
B.A. C1994)
Bacillus
thuringiensis/Biology,
application,
and
devclopment,
In
prospccts forfurther
lin)ceedi4gs
2nd thnber,zz
Adeetiqg
on Bacz'lkcs'
thurbagiensiy
ofthe
(RJ.AKHuRs'r,ed,). CSIRO, CanberTa, pp. I15.
FEnELsoN,J.S,,J.?AyNE and
L.KIM (1992)
BaciUus
thorn',{gte'
nsis: Insects
and
beyond. Bto17lecimoig
10:271275.
FERGus,G.P.,M. GooDFEu.ow and C. ToDD (1988)
A numcrical classMcation
ofthe
genus Bacitins.
f Gen.
Microbint.
134: 1847-1882.
andJ.
MARGALrr {1977)A bacterialspore demonstrating rapid larvicidal
activity against
AnqPhetessetgenti4 Uinnotaenin
utrgttabulata, Clattax
unin.ttatus, Aedesaag)pti and
thtexpipim. Mbsgecito
ims 637:
355-358.
HAsTowo, S.,B.W. LAy and M. OHBA (1992)
Naturally
occurring
Batitlus
thun'ngtettsis
in Indonesia.YlAAPL
GoinBERG,L.H.
Bacteriot.
73: 108-113.
and
P. SoMAsoGARAN
HoBEN, HJ.
enumeration
ofRhdaobium
spp.
(1982)Comparisen of
in ineculants
made
the
pour,
sprcad,
and
from prestcrilized
peat.
drep platc methods
for
4kPLEmbiron.Mi'crobioL44:
1246-1247,
KmuTA, H., S, AsANo and T, IIzuKA <1989)
IsolatesofBacitlas' thun',-gz'ens'is
from soil at Hokkaido. .1/
Ric.
Agn'c.HbkhaicloUhin.16: 383-389 <inJapanese
with English summary),
LENNox, E.S. (1955)Transduction of Iinkcdgenetic characters of the hest by bactcriophagc Pl. n'retlgy
1:
l90-206.
MARGAllT,J. and D. DEAN (1985)
The story ofBacilkts thun'agiensisvar, dsruetensis
(B.t.i.),
YLAm, Mbsg. Chnt.
Assoc.1: 1-7.
MARI'IN,P.A.SV. (1994)
An iconoclastic
view of Bat'itlas
thuriugiensab
ecology.
Am. E}ztomot.
40: 85-90.
MARTiN, P.A.W, and R.S.TRAvERs (1969)
Worldwide abundancc
and
distribution
ofBact'Uus
thuringiensis
isolates.
Environ,
throbioL55:2437-2442,
4tipl.
MEADows,
M.P.
thuringiensds'
in thc
(1993}Bacittus'
environmcnt:
ecology
and
riskassessment.
In Bacitlus
thurkagdensis}
An Ehvironmental
Bmpesticide:
7heozy
and kactabe
EN]visTLE,J.S.
CoRy, MJ. BAiLEyand A.
(P.
HIGGs,eds.).John Wiley and Sons,London,pp. 19a-220.
MEADows, M.P,,DJ. E-s,J, BuirF,
P.JARRETrand H.D. BuRGEs (1992)Disuibution,frequencyand diversity of Bacrllus
tharab{gie,isis
in an animal feedmill. opL E}iviron.
MicrobioL58: 1344-1350.
MEADows, M.P., DJ. ELus, NJ. PETHyBRIDc}4 K. BERNHARD, H.D. BuRGEs and P.JARREITr
{1990)Activityof
ncw
isolates
of BaciUzas
thuritagi'ettsds
against
fivcinscctpests.
In Pinc.5th intematibnat
Cotloeaiumon imertebr.
fathol.and imobial Cbntrot,
p. 497.
OHBA, M. (1996)Bacdiins
thun'ikgz'ensis
leaves:a possible
source
of
populationsnaturally occurring on mulberry
the
withsilkworm-rearing
insectaries.
80:56-64,
1/AmpL Bactetiol.
K. AIzAwA (1986a) Insecttoxicity of Baciltus
thuringdensab'
isolated
from soils ofJapan.
populationsassociatcd
OHBA, M.
and
.1/
invcrtebr.
Ilathol.
I7: 12-20.
OHBA, M. and K. AIzAwA (1986
b) Distribution
of BaciZtiLs
thupmis in soils ofJapan,
Rithot,
47:
YZinvertebr.
277-282.
OHJjA,M,, K. AizAwA and T, FuRusAwA C1979)
Distributionef Bacitlus
tharkrgibnsis
serotyT)es in Ehime Prefecture,Japam.
Ehtornol.
thoL 14:340-345.
4tipl,
OHBA, M.,K. AIzAwA and S.SHIMIzu(1981)
A new subspccics ofBact'llas thu,i}agiensis
isolatcd
inJapan:Bact'lltts
thuri4gt'ensig
subsp.
tohohae,tsis
l7). f invertebr.
IlatheL38: 307-309,
(serotype
Pz'rRAs,S.F.andL.E.
CAsiDA,Jr.C1985)
SurvivalofBaczllzas'
thuriTrgz'ensts'
in seil.
spores
50: 1496-1501.
PRiEs'i}
F,G., M. GooDFELLow and C. Ton]]
Environ.
imobtot.
42t)l.
classification
of the gcnus Baa'tltts.
{1988)A numcrical
f Gen.
Microbibl.
134: 1847-1882.
Fate of Baaitlus
SAi.EH,
S.M,,R.F, HARiss and O,N, AI,LEN (1970)
thun'izgt'ensis
in soil: cflbct of soil pH and
organic
amendment.
thn.f imobioL 16:667-680.
NII-Electronic Library Service
Japanese Society
JapaneseSociety
of Applied Entomology and
and Zoology
Zoology
ofAppliedEntomology
634
K. YARA
et aL
SMITH,R.A. and G.A. CoucHE (1991)
Thc phylloplanttas a source of Bacittn.s
thun'ngiensdsvariants.
4tipL
E}zviron,
Mibrobdol.
57: 31 IL331.
TRAvERs, R.S., P.A.W. A(AR'i'iN
ancl C.F.REIcHiti,DERFER
Selcctivc
(1987)
processfbrthe cMcicnt iselationof
soil Bactltas
specics.
Ampl. E}!viron.
Ml'crobiot.
53: 1263-I266.
VANKovA,J. and K. PuRRiNi{1979)
Naturalepizootics causcd by bacilli
of the species
Baaaftus
thun'rlgi'ensis
and
Baa'ilas
cereus.
Zl
ang.
E}it.88: 2I6-221,
WEsT, A.W., H.D. BuRGEs, RJ, WI{TE and C.H. Wy]oRx C1985)Sur'vival
of BaciUusthun'trgve'
n,sds and
Bacillvs
cereus sporc
inoculain soil: efflect ofpH, moisture, nutricnt availability and indigenous
microorganism.
SbilBinl.Biothem.17: 657-665.
WEs'r,A.W., H,D. BuRc;Es,RJ. IVHTTEand C.II.WyBoRN (1984)PersistenccofBact'zavfts thun',agz'ezayir
parasporal
crystal
insecticidaL
activity in soil.
Ilathol.
44:
1/1}tvertebr.
l28-133.
ZAHNER,V., H. MoMEN, C.A. SALLEsand L. RtxBINovr]'cH
(1989}A comparativc
Batillus
ceretts and
BaciUusthun'ngievzsis,f 4pt,l.
Bacten'ot.
67, 275-282.
study
of enzymc
variation
in
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