Note: Descriptions are shown in the official language in which they were submitted.
,, ,
1 54~ X52
-1-
DESCRIPTION
CELLULAR ENCAPSULATION
OF BIOLOGICAL PESTICIDES
Background of the Invention
The extraordinary increase in agricultural pro-
ductivity has been a result of many factors, including
significantly better understanding of the methods
involved caitr, agriculture, improved equipment, availa-
bility of fertilizers, and improved pesticides. The
latter factor has not been without detrimental aspects,
hocaever, due to the negative effect on the environment.
There is, therefore, a substantial interest in developing
effective and. environmentally acceptable pesticides.
Among ecologically acceptable pesticides are the
protein toxir,.s produced by various microorganisms, such
as Bacillus thurinli_g ensis. However, the use of B. thurin-
:ZO giensis lysate or spores as a pesticide has significant
drawbacks. The lifetime of the pesticide is relatively
short in the environment, requiring multiple applications
to give adeqL:ate protection. Consequently, these pesti-
cides are not. economical in comparison to more traditional
;)_5 chemical proc;ucts having long residual activities.
Improvements in field longevity would greatly aid in
expanding the: application of biological, or protein toxin-
based pesticides.
As indicated above, there are many requirements for
30 pesticides associated with their particular application.
For example, in many cases it is desirable to have pesti-
cides which have long residual activity in the field
while not accumulating in the environment. In addition,
because of economic, considerations, it is preferable
~34~ 452
-2-
to have pesticides which have a reasonably broad
spectrum of biocidal activity. Also, the pesticide
should degrade to degradation products which are
environmenta:Lly acceptable. Other considerations
include ease of formulation, pesticidal activity,
stability to environmental effects, such as light,
water, organ:~sms, and the like, and effect on beneficial
or innocuous organisms in the environment.
West, Soil Biol. Biochem. (1984) 16:357-360 reports
the results of a study on the persistence of _B._t. toxin
in soil. SeE~ also, West et al., J. of Invertebrate
Pathology (1~~84) 43:150-155. U.S. Patent No. 4,265,880
describes emt>edding live insecticidal pathogens in a
coacervate mi.crobead. Japanese Patent No. 51-5047
describes physical-chemical methods for killing _B.
thuringiensis: spores, while retaining toxicity.
Brief SWrimary of the Invention
Methods and compositions are disclosed for
protecting a~,ricult:ural crops and products from
pests. In one aspect of the invention, whole, i.e.,
unlysed, cells of a toxin (pesticide)-producing or-
ganism are treated with reagents that prolong the
activity of the toxin produced in the cell when the
cell is applied to the environment of target pest(s).
In another aspect of the invention, the vesticides
are produced by introducing a heterologous gene
into a cellular host. Expression of the hetero-
:30 logous gene results, directly or indirectly, in
the intracellular production and maintenance of
the pesticide. These cells are then treated, as dis-
closed above, under conditions that prolong the activity
:35
X34, 052-
- 3-
of the toxin produced in the cell when the cell is
applied to the environment of target pest(s). The
resulting product retains the toxicity of the toxin.
These naturally encapsulated pesticides may then be
formulated in. accordance with conventional techniques
for application to the environment hosting a target
pest, e.g., soil, water, and foliage of plants.
Detailed Disclosure of the Invention
.L 0
In accordance with the subject invention, improved
pesticides are provided, having among their other ad-
vantages an extended residual life, by modifying
naturally-occurring pesticide-producing microorganisms,
.l5 or pesticide-producing microorganisms hosting a hetero-
logous gene capable of expression in the host, where
expression of the gene results, directly or indirectly,
in the production of a pesticide. The subject invention
involves treating t:he organisms with reagents that
~'_0 prolong the activity of the toxin produced in the cell
when the cell is applied to the environment of target
pest(s). The resulting product retains the toxicity
of the toxin.
A wide variety of pesticides can be produced which
~5 will be characterized by being capable of being pro-
duced intracellularly, particularly in a unicellular
microorganism host, such as prokaryotes, e.g., bacteria;
or eukaryotes, e.g., fungi, exemplified by yeast and
filamentous fungi, such as Neurosnora and As~ergillus;
.f0 or protists, such as amoebas, protozoa, algae, and
the like.
.15
1 341 05 2
-4-
The pesticidE~ can be any toxin produced by a microbe.
For example, it can be a polypeptide which has toxic
activity toward a eukaryotic multicellular pest, such as
insects, e.g., coleoptera, lepidoptera, diptera, hemiptera,
dermaptera, and orthoptera; or arachnids; gastropods;
or worms, such as nematodes and platyhelminths. Various
susceptible insects include beetles, moths, flies,
grasshoppers, lice, and earwigs.
The pesticide which is produced in the host cell
may be a polypeptide produced in active form or a pre-
cursor or proform which requires further processing for
toxin activity, e.g., by the pest, as with the crystal
toxin of B. thurin~iensis var. kurstaki. Thus, the gene
may encode an enzyme which modifies a metabolite to
produce a pesticidal composition.
Among naturally-occurring toxins are the polypeptide
crystal toxins of B. thuringiensis var. kurstaki, active
against lepidoptera; B.t. var. israelensis, active against
mosquitoes; :B.t. M-7, active against coleoptera; _B.
thurin~iensis var. aizawai, active against spodoptera;
and B. s hae:ricus, active against mosquito larvae. Other
toxins include those of entomopathogenic fungi, such as
beauverin of Beauveria bassiana and destruxins of
I,~etarhizium spp.; or the broad spectrum insecticidal
compounds, such as the avermectins of Streptomyces
avermitilus. Cultures exemplifying the above are as
follows:
Bacillus thurinpiensis var. kurstaki HD-1--
NRRL B-3792; disclosed in U.S. Patent 4,448,885
Bacillus thuringiensis var. israelensis--ATCC 35646
Bacillus thuringiensis M-7--NRRL B-15939
Bacillus thurin~iensis var. tenebrionis--DSM 2803
The following B. thurin~iensis cultures are avail-
able from th~~ United States Department of Agriculture
1 341 05 2
-5-
(USDA) at Texas.
Brownsville, Requests
should
be
made
Co Joe Garcia, otton Insects Research Unit,
USDA, ARS,
C
P.O. Box 1033, Brownsville, Texas 78520 USA.
B. thuringiensis HD2
B. thwringiensis var.finitimus HD3
B. thu~ring_iensisvar.alesti HD4
B. thu:rin~3.ensisvar.kurstaki
H D73
B. thu:ring'~ensisvar.sotto HD770
B. thu:cin iensis var.dendrolimus
H D7
B. thu:ringiensis var.kekenyae
H D5
B. thu:ringiensis var.galleriae
H D29
B. thuringiensis var.canadensis
H D224
B. thuni_ ngiensisvar.entomocidus
H D9
B. thuringiensis var.subtoxicus HD109
B. thuringiensis var.aizawai HD11
B. thuringiensis var.morrisoni
H D12
B. thuringiensis var.ostriniae HDSO1
B. thuringiensis var.tolworthi HD537
B. thuringiensis var.darmstadiensis HD146
B. thunin~ietzsis var.toumanoffi HD201
B. thuringiensis var.kyushuensi:s HD541
B. thuringiensis var.thom~soni HD542
B. thuningiensis var.Pakistani HD395
B. thuz~in~iensis var.israelensis HD567
;05 B. thunin~iensis var.Indiana HD521
B. thurin iensis var.dakota
B. thuringiensis var.tohokuensis HD866
B.. thuringiensis var,kumanotoensis HD867
B. thurin iensis var.tochigiensis HD868
_30 B. thuningiensis var.colmeri HD847
B. thuz~in~iensis var. wuhanensis HD525
:35
~~4; X52_
-6-
Bacillus cereus--ATCC 21281
Bacillus mori.tai--ATCC 21282
Bacillus popi.lliae--ATCC 14706
Bacillus lentimorbus--ATCC 14707
Bacillus sph_aericus--ATCC 33203
Beauveria bassiana--ATCC 9835
Metarhizium anisopliae--ATCC 24398
Metarhizium flavoviride--ATCC 32969
Streptomyces avermitilus--ATCC 31267
The torn need not be the same as a naturally
occurring to}cin. Polypeptide toxins may be fragments
of a naturally-occurring toxin; expression products of
deletion, transver;sion or transition mutations, where
two or fewer number percent of the amino acids may be
changed; or a repetitive sequence capable of processing
by the intended pest host. In addition, fusion products
may be prepared where one, five or more amino acids are
provided at t:he N-terminus to provide, for example,
reduced prote.olytic degradation of the toxin(s). in
some instances, a plurality of the same or different
toxins may be encoded and expressed, where processing
sites may be introduced between each toxin moiety in the
polytoxin.
Illustrative host cells may include either prokary-
otes or eukaryotes" normally being limited to those
cells which do not produce substances toxic to higher
organisms, such as mammals. However, organisms which
produce substances toxic to higher organisms could
be used, where the toxin is unstable or the level of
application sufficiently low as to avoid any possi-
bility of toxicity to a mammalian host. As -
1 341 45 2
_,_
hosts, of particular interest will be the prokaryotes and
lower eukaryotes, such as fungi. Illustrative prokaryotes,
both Gram-negative and -positive, include Enterobacteri-
aceae, such as Escherichia, Erwinia, Shigella, Salmonella,
and Proteus; Bacil.laceae; Rhizobiaceae, such as Rhizo-
bium; Spirillaceae, such as photobacterium, Zymomonas,
Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum;
Lactob acillaceae; Pseudomonadaceae, such as Pseudomonas
and Acetobacter; Azotobacteraceae and Nitrobacteraceae.
Among eukary~~tes are fungi, such as Phycomycetes and.
Ascomycetes, which. includes yeast, such~as Saccharomyc_es and
Schizosaccharomyces; and Basidiomycetes yeast, such as r
Rhodotorula, Aureobasidium, S~orobolomyces, and the like.
Characteristics of particular interest in selecting
a host cell :Eor purposes of production. include ease
of introducing the heterologous gene into the host,
availability of expression systems, efficiency of expres-
sion, stabil:Lty of the pesticide in the host, and
the presence of auxiliary genetic capabilities.
Characteristics of interest for use as a pesticide micro-
capsule include protective qualities for the pesticide,
such as thick cell walls, pigmentation, and intracel-
lular packag~_ng or formation of inclusion bodies; leaf
affinity; la<:k of mammalian toxicity; attractiveness to
pests for ingestion; ease of killing and fixing without
damage to thE~ toxin; and the like. Other considera-
tions includE: ease of formulation and handling,- eco-
nomics, storage stability, and the like.
Host organisms of particular interest include
yeast, such as Rhodotorula sp., Aureobasidium sp.,
Saccharornyce~~ sp., and Sporobolomyces sp.; phylloplane
organisms such Pseudomonas sp., Erwinia sp. and Flavo-
bacterium sp.; or such other organisms as Escherichia,
X341452
_$_
Lactobacillus sp., Bacillus sp., and the like. Speci-
fic organisms include Pseudomonas aeru~inosa, Pseudomonas
fluorescens, Saccharomyces cerevisiae, Bacillus thurin-
~iensis, Escherichia coli, Bacillus subtilis, and the
like.
The cell will usually be intact and be substantially
in the proliferative form when killed, rather than in a
spore form, ,although in some instances spores may be
employed.
The cel:Ls may be inhibited from proliferation
in a variety of ways, so long as the technique does not
deleteriousl~~ affect the properties of the pesticide,
riot diminish the cellular capability in protecting the
pesticide. '.:he techniques may involve physical treat-
ment., chemic<~1 treatment, changing the physical charac-
ter of the cE:ll or leaving the physical character of
the cell substantially intact, or the like.
' Various techniques for inactivating the host
cells includ~s heat, usually 50°C to 70°C; freezing; UV
irradiation; lyophilization; toxins, e.g., antibiotics;
phenols; ani:Lides, e.g., carbanilide and salicylanilide;
hydroxyurea; quaternaries; alcohols; antibacterial dyes;
EDTA and ami<iines; non-specific organic and inorganic
2S chemicals, such as halogenating agents, e.g., chlorinating,
brominating c>r iod:inating agents; aldehydes, e.g.,
glutaraldehyde or :formaldehyde; toxic gases, such as
ozone and ethylene oxide; peroxide; psoralens; desiccating
agents; or the like,which may be used individually or
in combination. The choice of agent will depend upon
the particular pesi:icide, the nature of the host cell,
the nature of the modification of the cellular structure,
:35
1 341 45 2
- 9-
such as fixing and preserving the cell wall with cross-
linking agents, or the like.
The cells generally will have enhanced structural
stability which will enhance resistance to environmental
degradation in the field. Where the pesticide is in a
proform, the method of inactivation should be selected so
as not to inhibit processing of the proform to the mature
form of the pesticide by the target pest pathogen. For
example, formaldehyde will crosslink proteins and could
inhibit processing of the proform of a polypeptide
pesticide. The method of inactivation or killing retains
at least a substantial portion of the bioavailability or
bioactivity of the toxin.
The hetE~rologous genes) may be introduced into the
host in any ~~onvenient manner, either providing for
extrachromosomal maintenance or integration into the
host genome. (By heterologous is intended that the gene
is not present in the host into which it is introduced,
nor would the gene normally be found in such host. That
is, even if t:he host organism and the source of the
heterologous gene exchange information, the heterologous
gene would nc>rmall:y not be found in the wild-type host
cells in nature. 'Usually, the term heterologous will
involve species of different genera as host and gene
source.)
Various constructs may be used, which include
replication systems from plasmids, viruses, or eentro-
meres in comf~ination with an autonomous replicating
segment (ors) for stable maintenance. Where only
integration i.s des:ired, constructs can be used which
may provide for replication, and are either transposons
or have transposon~-like insertion activity or provide
for homology with the genome of the host. Frequently,
DNA sequences are employed having the heterologous
1 3~1 X52
-lo-
gene between sequences which are homologous with se-
quences in the genome of the host, either chromosomal
or plasmid. Desirably, the heterologous genes) will
be present in multiple conies. See for example, U.S.
Patent No. 4;,399,216. Thus, conjugation, transduction,
transfection and transformation may be employed for
introduction of the heterolojous gene.
A large number of vectors are presently available
which depend upon eukaryotic and prokaryotic replication
systems, such as ColEl, P-1 incompatibility plasmids,
e.g., pRK290, yeast 2m a plasmid, lambda, and the like.
Where are extrachromosomal element is employed, the
DNA construct will desirably include a marker which
allows for a selection of those host cells containing
:15 the construct. The marker is commonly one which provides
for biocide resistance, e.g., antibiotic resistance or
heavy metal resistance, complementation providing
prototrophy to an auxotrophic host, or the like. The
replication systems can provide special properties, such
as runaway replication, can involve cos cells, or other
special feature.
Where the heterologous genes) has transcriptional
and translational initiation and termination regulatory
signals recognized by the host cell, it will frequently
be satisfactory to employ those regulatory features in
conjunction with the heterologous gene. However, in
those situations where the heterologous gene is modified,
as for example, removing a leader sequence or providing
a sequence which codes for the mature form of the
pesticide, where the entire gene encodes far a precursor
~.t will freqmantly be necessary to manipulate the DNA
sequence, so that a transcriptional initiation regulatory
sequence may he provided which is different from the
?~5
~ 34 ~ ~5 2
-11-
natural one.
A caide variety of transcriptional initiation
sequences exist for a wide variety of hosts. The
sequence can provide for constitutive expression of the
pesticide or regulated expression, where the regulation
may be induc:ible by a chemical, e.g., a metabolite, by
temperature, or by a regulatable repressor. See for .
example, U.S" Patent No. 4,374,927. The particular
choice of thE~ promoter will depend on a number of
factors, the strength of the promoter, the interfer-
ence of the F>romater with the viability of the cells,
the effect of regulatory mechanisms endogenous to
the cell on t:he promoter, and the like. A large number
of promoters are available from a variety of sources,
:15 including con~inercial sources .
The cellular host containing the heterologous
pesticidal gene may be grown in any convenient nutri-
ent medium, where t:he DNA construct provides a selective
advantage, providing for a selective medium so that
substantially all or all of the cells retain the hetero-
logous gene. These cells may then be harvested in
accordance with conventional ways and modified in the
various manners described above. Alternatively, the
cells can be fixed prior to harvesting.
The meth~~d of treating the host organism containing
the toxin can fulfill a number of functions. First, it
may enhance snructural integrity. Second, it may provide
for enhanced i~rotE~olytic stability of the toxin, by modi-
fying the to:cin so as to reduce its susceptibility to pro-
teolytic degradation and/or by reducing the proteolytic
activity of proteases naturally present in the cell. The
cells are pref=erably modified at an intact stage and when
there has been a substantial build-up of the toxin
protein. The:;e modifications can be achieved in a
..
1341 X52
-12-
variety of ways, such as by using chemical reagents
having a broad spectrum of chemical reactivity. The
intact cells can be combined with a liquid reagent
medium containing the chemical reagents, with or without
agitation at temperatures in the range of about -10 to
60°C. The reaction time may be determined empirically
and will vary widely with the reagents and reaction
conditions. Cell concentrations will vary from about
10E2 to 10E1~ per ml.
Of particular interest as chemical reagents are
halogenating agents, particularly halogens of atomic
no. 17-80, lore particularly, iodine can be used under
mild conditions and for sufficient time to achieve the
desired results. Other suitable techniques include
treatment wi~h aldehydes, such as formaldehyde and
glutaraldehy~le; anti-infectives, such as zephiran
chloride and cetylpyridinium chloride; alcohols, such
as isopropyl and ethanol; various histologic fixatives,
such as Bouin's fixative and Helly's fixative (See:
Humason, Gre~_chen L., Animal Tissue Techniques, W.H.
Freeman and e~ornpany, '1967) ; or a combination of
physical (heat) and chemical agents that prolong:the
activity of v~he toxin produced in the cell when the cell
is applied to the environment of the target pest(s).
For hal~~genation with iodine, temperatures will
generally range from about 0 to 50°C, but the reaction
can be conveniently carried out at room temperature.
Conveniently, the iodination may be performed using
triiodide or iodine at 0.5 to S~ in an acidic aqueous
medium, part:LCUlarly an aqueous carboxylic acid solution
that may var;,~ from about 0.5-5M. Conveniently, acetic
acid may be used, although other carboxylic acids,
:35
~34~ X52
-13-
generally of from about 1 to 4 carbon atoms, may also be
employed. The time for the reaction will generally
range from less than a minute to about 24 hrs, usually
from about 1. to 6 hrs. Any residual iodine may
be removed try reaction with a reducing agent, such
as dithionite, sodium thiosulfate, or other reducing
agent compatible with ultimate usage in the field.
In addition, the modified cells may be subjected to
further treatment" such as washing to remove all of the
reaction medium, isolation in dry form, and formulation
with typical stickers, spreaders, and adjuvants generally
utilized in agricultural applications, as is well kno~,rn to
those skilled in t:he art.
Of particular interest are reagents capable of cross-
linking the cell wall. A number of reagents are known in
the art for this purpose. The treatment should result in
enhanced stability of the pesticide. That is, there
should be enhanced persistence or residual activity of
the pesticide undE:r field conditions. Thus, under condi-
'0 tions where the pE:sticidal activity of untreated cells
diminishes, the activity of treated cells remains for
periods of from 1 to 3 times longer.
The cel:Ls may be formulated in a variety of ways.
They may be employed as wettable powders, granules or
dusts, by mi:{ing with various inert materials, such as
inorganic minerals (phyllosilicates, carbonates, sulfates,
phosphates, and the like) or botanical materials (pow-
dered corncobs, nice hulls, walnut shells, and the like).
The formulat:Lons may include spreader-sticker adjuvants,
stabilizing agents, other pesticidal additives, or~sur-
factants. L~:quid formulations may be aqueous-based or
non-aqueous and employed as foams, gels, suspensions,
emulsifiable concentrates, or the like. The ingredients
'34~a~2
-14-
may include rheological agents, surfactants, emulsifiers,
dispersants, or polymers.
The pesticidal concentration will vary widely
depending upon they nature of the particular formulation,
particularly whether it is a concentrate or to be used
directly. The pesticide will be present in at least 1%
by weight and may be 100% by weight. The dry formulations
will have fr~~m about 1-95% by weight of the pesticide
while the li~~uid formulations will generally be from
about 1-60% by weight of the solids in the liquid phase.
The formulat:ions will generally have from about lE2 to
about lE4 ceLls/mg. These formulations will be admini-
stered at about :? cz (liquid or dry) to 2 or more lb/ha.
The foryulations can be applied to the environment
of the pest(;s), e.g., plants, soil or water, by spraying,
dusting, spr=inkling or the like.
The following examples are offered by way of illustra-
tion and not by way of limitation.
Example 1
After treatment of intact spore-containing cells
(prior to auoolysis) of B. thuringiensis with lugol's
iodine, the c=ells are killed; however, they retain
toxicity to =Crichoplusia _ni larvae.
The intact cells of Bacillus thuringiensis (HD-1)
were harvested just prior to autolysis of the sporulating
cells by cent=rifugation and the cell pellet suspended in
deionized wat=er to give a concentration of 6.O x 10E9
cells/ml. An aliquot of the cell suspension was diluted
to 1.5 x lOEFt cells/ml and exposed to 1% lugol's iodine
for 4 hr at room temperature (a 1% lugol's solution
contains 1.0 g potassium iodide, 0.5 g iodine and 1.0
ml glacial acetic acid per liter.) The treated cells
~ ~4 ~ °5 z
-15-
were washed ,and resuspended in sterile deionized water to
give a cell concentration of 6.0 x 10E9. No viable
cells were detected by plate counts on nutrient agar
after the 4 hr iodine treatment. Lugol's treated and
untreated control cells were then bioassayed for toxicity
to T, ni larvae.
Since tile cells of the subject invention are
naturally-occurring cells, it would not be necessary
to treat there under killing conditions in order to
realize the benefits of the subject invention. Thus,
treatment of the cells, as described herein, can be
optimized by a person skilled in the art to achieve the
highest leve~_ of prolongation of toxin (pesticidal)
activity in t:he environment of the target pest(s).
B. Bioassay procedure
Dilutions of lugol's killed cells or untreated
live HD-1 cells were mixed with a constant volume of
larval diet c:up. A single S day ald _T. _ni Iarva was then
added to each cup. Eighteen larvae were tested per
dilution. The larvae were examined after six days and
the total number o:E larvae killed was recorded. The
results are shown :in Table 1. They are given in percent
larvae killed.
30
~ 34 ~ ~5 2
-1G-
Table 1
Bioassay of Lugol's-Treated Intact Spore-Containing
Bacillus .thuringiensis (HD-1) Cells
Cell Dilution 1.0E9 10E8 10E7 10E6 10E5
HD-1
Untreated 1.00 100 68.8 0 0
Lugo l ' s
Treated 94.4 61.0 0 0 0
Example 2--Stability Testing
Intact, spore-containing cells of _B._t. HD-1 were treated
caith 1% lugol's solution for 4 hr at room temperature,
washed in dei,onized water, and stored in the refrigerator
for 52 days. After this period the cells remained whole,
and there was no evidence of lysis (release of spores
and crystal).
Intact, spore-containing cells of _B._t. HD-1 were
harvested by centrifigation and the resulting pellet
suspended in sterile deionized water (10E10 cells/ml),
heated to 70'C for 30 min, and stored in the refrigera-
tor for 9 da:,~s. After this period, virtually alI of the
cells have l:,~sed, releasing spores and crystals.
Example 3
Soil Experiment Procedure
B.t. HD-1 preparation:
An intact spore-containing culture of _B._t. HD-1
was harvested by centrifugation and the cell pellet resus-
pended in 400 ml of 1% lugol's iodine (4 x 10E8 cells/ml).
The iodine-cell suspension was stirred for 3 hr at room
temperature, washed 3 times and resuspended in 400 ml
~ 34 1 05 2
-17-
sterile 0.1. M sodium phosphate buffer, pH 6.9. No
viable B.t. fID-1 cells were detected on nutrient agar
(l0E-1) after treatment with iodine, and microscopic
examination revealed that all cells were intact (unlysed).
Dipe l* preparation
Dipel*(0.1 ~;, Abbott Laboratories, 16,000 Interna-
tional Units of fotency/mg. List x/5188), which contains
B.t. HD-1 ce_Lls, wa,s measured into 400 ml of sterile
0.1 M sodium pho~;phate buffer.
Experimental Design:
1) Non--sterile soil preparation: 40 g of
soil was plac=ed in a sterile 500 ml flask and 200 ml of
experimental sol_ati_on added .
2) Sterile soil preparation: 40g of soil
was placed in a st=erile 500m1 flask and autoclaved for
1 hr prior to adding 200m1 of experimental solution.
Flas)cs containing soil suspensions were incubated on a
gyratory shaker (200RPM) at room temperature. Samples
(30-40m1) of each soil suspension were filtered through
4x cheeseclcth and sprayed onto the leaves of lettuce
plants for subsequent measurement of toxicity against
larvae of T. ni .
Measurement _of Bst- Toxin by Feeding Inhibition on_a
Leaf Inhibition Assay
Leaves of Romaine lettuce seedlings are sprayed
with freshly prepared standard concentrations of Dipel*
(1x 0.19g/100m1, 1/10x, 1/20x, and 1/100x), and experi-
mental solutions 24 hr before use.
Le,3ves treated with standards or experimental
solutions ar~~ removed from the plant, weighed individually,
and placed in individual petri dishes. Ten starving,
7 day old T. ni Larvae are applied to each leaf and
allowed to feed on the leaf for approximately 24 hrs at
25°C. At the end of the feeding period the leaves are
re-weighed and tine average weight loss determined for
each trear_ment.
*Trade-mark
D
~ 3~ ~ ~5 2
-18-
Under the conditions described above, leaf weight
loss is a lob linear function of toxin concentration
over concentrations of toxin equivalent to 0.19 mg/ml-
0.019 mg/ml ;~~ipe:L (16,000 international units of potency
per mg). This, the freshly prepared Dipel concentrations
sprayed onto lettuce plants and run with each assay
serve as standards by which the concentration of _B._t.
HD-1 toxin on leaves sprayed with experimental solu-
tions can be equated. The data in Table 2 show the
percentage of the original toxicity (day I) remaining
at various times after incubation in soil and subse-
quently assa,~ed with the leaf inhibition assay.
Leaves other than lettuce plant leaves can be used
in assaying for B.t. toxin efficacy.
20
'0
-19-
~~4~ X52
r
A
x
~I
~I
v cr o o ,ar~
a~ .~ a o 0
L C N r-1r~ H
N O
O
~ z
N ~n
v
G t~.
rl al
~b a
O p.
i--~
..
~ U
O
OD Dl
r1 G C
~-a O v
O G
'U ..-1
U ~ Sa O M O 1~
cn v O M o000
'"'~ri
CY ~
y
T v
N
U .r-i h.
.
ri U
O >C
H O n
N C aJ
W O r-1
H O r-a .ry
~' ~'
v G rr1M I
U '~ Sa a p
C o0 c0 u7 .-iO V1
U '~ t~.G
a S-~ r~ d O
O w ~ z
ly p"
tn W '~-1
Sr O ~7 m-I
v
a. se ,J
sJ
U
N
U .-1
~r r~
O 1r O r-tY ~
C1 ~ O a0 w
cn .u r-i
C/1
C
iJ O
la ri
C7 J..r
iJ fQ
V7 ,D
O
1r U
I
U G
I
17 H
~~~1 00U1
<, ri .-.r
G7 O
'~
i~. f17
I
CS
1341 052 _
-20-
Inactivation of t:he protein-crystal of _B._t. HD-1 in
soil has been shown to be due to the activity of soil
microorganisms (~~est, 1984). The results of Table II
demonstrate that when Dipel and a lugol's iodine-
treated pre-~.ysed B.t. HD-1 preparation were incubated
under similar conditions, the toxicity of the Dipel
(spore-crystal) preparation degenerated rapidly, cahile
the toxicity of thE~ lugol's iodine-treated cells
remained essential:Ly unchanged
It is evident from the above results that chemical
treatment of whole microbial cells can be performed
in such a way as to retain polypeptide toxin activity,
while rendering thE: cell stable to storage conditions.
This provides increased residual activity of toxin
:LS activity under field conditions.
Example 4--Construction of a Heterolo~ous Gene and
Transformation into A Suitable Host.
A construction began with a clone of Pseudomonas
:?0 aeruginosa, available from Northern Regional Research
Laboratories (NRRL B-12127), containing a broad host range
shuttle plasmid pRa1614 (J. Bact. [1982) 150:60; U.S.
Patent No. 4,374,200) . The plasmid has unique HindIII,
BamHI, and Sall and PvuII restriction sites, a PstI inser-
~'.S tion, which ivncludes the carbenicillin resistance gene and
a P. aeruginosa replication system, where the HindIII,
BamHi and Sal=I restriction sites are in a tetracycline
resistance gene. 'The remainder of the plasmid is
derived from pBR322. A second plasmid, pSMl-17, has
SO been deposited as a clone of E. coli (NRRL B-15976).
This deposit was made with the permanent collection of
the Northern Regional Research Laboratory, U.S. Depart-
ment of Agric:ultur~e, Peoria, Illinois 61604, USA.
~' S
f
~'~'~~ X52
-21-
Plasmid pSMl-17 confers ampicillin resistance to E. coli
and contains a 6..8 Kbp HindIII DNA fragment that includes
the d-endotoain J;ene from the 50 and plasmid of Bacillus
thuringiensis HD73. Sufficient toxin is expressed from
this gene in E. coli to make the intact cells toxic to
cabbage loopers. A further modification of the DNA
fragment was done to enhance toxin expression and to
accomplish a}cpression of toxin in an alternate host,
Pseudomonas f-luorescens . In order to eliminate unwanted
DNA from the 6.8 Kbp fragment, pSMl-17 was digested with
BamHl to open the circular plasmid at a site nearest to
the 5' end of.-' the toxin gene, and was treated with the
exonuclease Fia131 to reduce the 6.8 Kbp HindII insert to
about 2.9 Kbp. Tha_ free ends of the DNA were filled in
a5 with Klenow polymerase, BamHl linkers were added, and
the linear DL'JA caas ligated to reform the circular
plasmid. ThE: resultant vector, pFG270, was digested with
Xhol to open the circular plasmid at a site nearest to
the 3' end of the toxin gene. Sall linkers were added,
?0 the plasmid eras ligated to its circular form, and the
resultant vector, pFG10.6 was amplified in _E. coli.
pFG10.6 was digested completely with Sall and BamHI and
the resulting 2.1.1Zbp fragment containing the toxin gene
was purified by ge~1 electrophoresis and ligated into the
>5 BamHI and Sal_I sites of plasmid pR01614. The resultant
plasmid, pCHa!.1, w;as amplified in E. coli and used
to transform Pseudomonas fluorescens to carbenicillin
resistance and the ability to synthesize d-endotoxin.
Pseudomonas fluorescens (pCH2.1) was deposited with
NRRL and was given the accession number NRRL B-15977.
plasmids pSM:I-17 and pCH2.1 were deposited in the
repository on July 2, 1985.
_! 5
X341052
-22-
In the following illustrative experiments P. fluorescens
(pCH2.1) was used in conjunction with controls of untrans-
formed cells.
The culture dleposits E. coli(pSMl-17)--NRRL B-15976
and P. fluorescens_(pCH2.1)--NRRL B-15977 were made in the
permanent collection of the NRRL repository, to be main-
tained for at least thirty years. These deposits are
available to the public upon the grant of a patent dis-
closing them. The: deposits are also available as
required by foreign patent laws in countries wherein
counterparts of tt~~e subject application, or its progeny,
are filed. However, it should be understood that the
availability of a deposit does not constitute a license
to practice the subject invention in derogation of patent
rights granted by governmental action.
Example 5--Treatment and Testing of Microbes Hosting a
Heterologous Gene.
The Pseudomonads were either killed by 1% lugol's
iodine (1008, KI, _'i0 g I2, 100 ml glacial acetic acid in
1 L sterile distilled water) or by treatment with a 2%
formalin solution, each at ambient temperatures.
Cell pE:llets from two liter broth cultures of
P. fluorescEm_s with and without _B._t. toxin are divided
in half. Half the cells are treated with 1% lugol's
iodine for ~+ hr, while the other half are held on ice.
Washed lugo:L's treated cells and untreated cells are
repelleted :Ln 10 ml of sterile deionized water.. Nine
ml of this suspension (10E8-10E12 cells/ml) are sprayed
on three young lettuce plants. The three sprayed plants
are placed :gin a single enclosed chamber and 50-75
Trichoplusia _ni larvae are applied to the plants. Plants
are considered protected if there is no visible loss of
foliage over the observation period.
~3~'~ ~~2
-23-
TABLE 3: Plant Assay 1 - P. fluorescens.
4t Viable Total
11
Microorganism Treatment Cells/ml Larvae Protection
__ _____________________._______________________________________
____
P. fluorescens Live 2.5 x 10E1175 Not protected
_P, fluorescens Live 3.1 x 10E1175 Protected
+ Iit Toxin
P fluorescens 1% Lugol's 0 75 Not protected
Iod ine
4 hrs
P. fluorescens I% Lugol's 0 75 Protected
+ Bt Toxin Iodine
4 hers
* Set up day 25 2-day larvaeapplied plants,
1, to
day 3, 50 5-daffy applied plants,
larvae to
day 10, observation
reported.
In the next study, newly hatched cabbage loopers
are placed in a Petri dish containing droplets of the
material to 'be bio~assayed. The larvae imbibe the liquid
and are then removed to small cups containing larval
diet. The larvae are examined after seven days and the
total number of animals killed is recorded. The follow-
ing Table 4 indica~.tes the results.
35
1341 052
TABLE 4': Bioassay 1.~ fluorescens
- P.
~/ Larvae
Pficroorganism Treatment Killed/ % Larvae Viable Cell
-_---______ Total Killed Count/ml
_____.______ ___________-_____--_____r.__________
S P. fluorescens Live 0/15 0 8 x
1 0E11
P. fluorescens Live 11/18 62 2
5 x 10E12
+ Bt Toxin .
P. fluorescens 1% Lugol's 0/15 0 0
4 hrs
P, fluorescens1% Lugol's 8/13 62 0
+ Bt Toxin 4 hrs
Bioassay 2 - P. fluorescens
!l Larvae
Microorganism Treatment Killed/ % Larvae Viable Cell
Total Killed Count/ml
P. fluorescens Live 0/15 0 3
1 7 x
0E11
.
_P. fluorescens Live 3/20 1.5 4.5 x 10E11
+ Bt Toxin
P. fluorescens 1% LuF;ol's 0/15 0 0
4 hrs
P. fluorescens 1% LuF;ol's 8/15 53 0
+ Bt Toxin 4 hrs
' -25- 1 3 4 1 4 5 2
Bioassay 3 - P. fluorescens
ll Larvae
Microorganism Treatment Killed/ % Larvae Viable Cell
Total Killed Count/ml
____________ _._____________________________________~__________
S P. fluorescens Live-frozen 9/10 90 2
5 x 10E12
+ Bt Toxin, 14 days .
P. fluorescens 1% Lugol's 1/15 0
7
4 hrs
P. fluorescens 1% Lugol's 11/15 73 0
+ Bt Toxin
P. fluorescens 2% formalin 0/15 0 0
4 hrs
P. fluorescens 2% formalin 10/15 67 0
+ Bt Toxin 4 hrs
Bioassay 4 - P, fluorescens
II Larvae
Microorganism Treatment Killed/ % Larvae Viable Cell
Total Killed Count/ml
P'. fluorescens Live 6/14 30 3 x
1 0E11
P. fluorescens Live 20/20 100 3 x 10E10
+ Bt Toxin
P. fluorescens 1% Lugol's 0/20 0 ~ 0
4 hrs
P. fluorescens 1% Lug;ol's 18/20 90 0
2.'p + Bt Toxin 4 hrs
In the next study, different methods of
killing the cells were employed to determine the effect
on cell stability to s;onication. _P. fluorescens strain
33 is an untransfoxmecl cell not containing the Bt
-26-
toxin, while strain pCH contains the 2.1 kb toxin
gene. In the first study, stationary phase cultures of
strain 33 and strain pCH were harvested by centrifugation
and cell pellets suspended in sterile deionized water
S at concentrations of 10E10 and 10E9, respectively.
Aliquots of cells were exposed to a 70°C water bath for
varying amowntsof time and cell viability measured by
plate counts on King's B agar. (See King, E~:O. et al.(1954) J.
Lab. ~ Clin. Med. 44:304. The medium has the following
ingredients: Proteose peptone No. 3, 2.0 percent; Bacto
agar, 1.5 percent; glycerol, C.P., 1.0 percent; K2HP04
[anhydrous], 0.15 percent; MgS04~7H20, 0.15 per cent;
adjusted to ~pH 7.2.) Strain 33 was substantially
completely killed within 5 min, while strain pCH showed
substantially_no viable cells within 10 min.
Cells of strain 33 (untreated, heat treated at
70°C, or lugol's treated [1% lugol's iodine, 2 hr,
room temperature]) were suspended in 10 ml deionized
water to give a cell concentration of approximately
10E9/ml. The cell suspension was then subjected to
sonication for 5 min on a Bronson 200 Sonifier (using
a microprobe output 10; 50% duty cycle, pulse mode).
The optical density (575 nm) of the cell suspension
was measured before and after the sonication treat-
ment. A decrease in optical density indicates cell
disruption. The following Tables 5 and 6 give the
results.
35
. 1349 052
-27-
TADL~ 5: Sonication - f. fluorescens 33
Optical Density (575nm)
Treatment Pre-sonication Post-sonication
_ _ _ _____________________________ __________
Live 0.24 0.05
Lugol's 0.20 0.19
(1%, 2hr, room temp)
Heat 70C
1 0.22 0.06
3 0.21 0.07
5 0.22 0.09
0.20 0.10
30 0.21 0.11 '
60 0.19 0.13
90 0.22 0.13
15 Cells of strain pCH (5x10E10/ml) were heat
treated in a 70°C water bath and assayed for toxicity
against T. ni larvae:. The bioassay procedure involved
newly hatched cabbage loopers which were placed in a
petri dish containing droplets of the materials to be
bioassayed. The larvae were allowed to imbibe the
liquid and were them removed to small cups containing
larval diet. The larvae were examined after six days
and the total number of animals killed recorded.
' ~'~4 ~ X52
-28-
TABLE 6: Bioassay Heat Treated P. fluorescens
Treatment (70°C) ~~ Larvae Killed/
Time (min) ~~ Viable Cells/Total Cells Total Larvae
lx % Killed
3 2 x 10E5 / 5.5 c 10E10 8/8 100
5 2 x 10E6 / 5.5 x 10E10 8/8 100
0 / 5.5 x 10E10 8/8 100
l0
Analysis of the persistence of the lugol's treated
cells of P. fluorescens + B.t. toxin (strain pCH) was
done in the greenhouse by (1) spraying leaves of Romaine
lettuce with the pesticidal cells, (2) exposing the
treated plants to the greenhouse environment for up to
19 days (the greenhouse was covered with a UV transpar-
ent roof), anal (3) assaying the cabbage looper pesti-
cidal activity remaining on the leaves with the leaf in-
hibition assay. Measurement of B.t. toxin by feeding
:20 inhibition is~ as described previously, and the method
for spraying the plants is described in Example 5. In
this experiment a toxin activity level of 1.0 is equi-
valent to thE: pesticidal activity achieved with 0.19 g
of Dipel/100 ml of spray applied at a rate of a~proxi-
mately 10 mlfplant (i.e., just prior to run-off).
As shown in Table 7, after 11 days the Pseudomonas
preparation is st:i:l1 fully active whereas the Dipel
example has lost over 2/3 of its initial activity.
We interpret these data to mean that the fixed Pseudo
moms cell i~~ in some manner protecting the pesticidal
toxin from inactivation on the leaf surface.
~ "~4 ~ ~5 2
-29-
Table 7: Greenhouse Persistence data
Activity
Day pCH Dipel
1 0.75 1.0
5 1.15 0.85
8 1.1 1.2
11 1.0 0.3
19 0.13 <0.01
l0
It is evident from the above results that the
killed bacter is containing toxin provide many advan-
tages, while still retaining all or a substantial
proportion of the mesticidal activity of the live
bacteria. M<<intenance of dead bacteria for storage,
transportation and application is more convenient and
economical than employing the live organisms. At
the same time, the organisms provide encapsulated
protection of the toxins, so as to maintain the toxins'
bioactivity for long periods of time. In addition,
denaturation of the proteases prevents proteolytic
degradation ~of the polypeptide pesticides. Various
means for killing the organisms without disrupting
their structure oz' denaturing the protein pesticide can
be employed to provide the desired,pesticidal activity,
while providing for a convenient pesticidal composition
for use in formulation and application to plants and
plant products.
Although the foregoing invention has been
described in some detail by way of illustration and
example for purposes of clarity of understanding, it
will be obvious that certain changes and modifications
may be practiced within the scope of the appended
claims.
