Note: Descriptions are shown in the official language in which they were submitted.
Mo3729CIP
CA-045-CIP
COMBINED USE OF CHEMICALS AND
MICROBIALS IN TERMITE CONTROL
BACKGROUND OF THE INVENTION
The present invention relates to a method for
exterminating termites and to compositions useful in such
extermination.
A number of chemicals which kill termites at specific
concentrations are known. Specific examples of such chemicals
include cyfluthrin (disclosed e.9., in U.S. Patent 4,218,469),
propoxur (disclosed e.g., in U.S. Patent 3,111,539),
fenvalerate (disclosed e.g., in U.S. Patent 4,061,664),
isofenphos (disclosed e.g., in U.S. Patent 3,621,082),
cypermethrin (disclosed in U.S. Patent 4,024,163), and
1-(2-chloro-5-pyridyl methyl)-2-(nitroimino)imidazolidine
(disclosed, e.9. in U.S. Patent 4,742,060).
Certain microbials, specifically some entomophageous fungi
and bacteria are known to be associated with termite colonies
and to cause deleterious effects when certain conditions exist.
See, for example, Ko, W.H., et al, "The Nature of Soil
Pernicious to Coptotermes Formosanus", Journal of Invertebrate
Patholoav, Volume 39, pages 38-40 (1982).
However, each of these known termiticides, bacteria and
fungi has characteristics which makes it commercially
undesirable. For example, many of the known chemicals and
microbials must be used at rates which are too high to be
economical or environmentally desirable. Known termiticides
are also often too slow acting to assure success in their
practical application. The effectiveness of many of the known
termiticides is dependent upon the specific environment in
which they are used and may therefore be detrimentally affected
by uncontrollable factors.
35376LMW1259
2 ~~8~3~~
It has now been found that when specific types of
chemicals and fungi or bacteria selected from specific species
are used in combination to treat a site infested with termites,
unexpected synergism in termite control results. When used in
combination, the application rates can be substantially lower
than those which would be used for an individual chemical
termiticide and individual microorganisms. The effects of the
combined chemical and fungus or bacterium of the present
invention are seen in days rather than weeks. Such
to combinations make it possible to achieve much higher, more
predictable and more economical termite control.
The present invention is particularly advantageous in that
it does not employ chlorinated hydrocarbons, the most widely
used group of termite control chemicals. The present invention
is based upon a new concept in termite control employing
chemical agents with less residual effect than the known
termiticides in combination with biological agents, and
application techniques for retreatment of sites with
diminishing effectiveness.
2o SUMMARY OF THE INVENTION
It is an object of the present invention to provide a
combination of a chemical agent and a biological material which
is an effective termite treatment.
It is also an object of the present invention to provide a
composition which does not present the environmental problems
of chlorinated hydrocarbons and which need not be used in large
quantities to be effective.
It is another object of the present invention to provide a
method for effectively controlling termites.
. These and other objects which will be apparent to those
skilled in the art are accomplished by a composition which
includes (1j an effective amount of a chemical selected from
nitroguanidines, nitromethylenes, pyrazolines and pyrethroids
and (2a) an entomopathogenic fungus, preferably a fungus of the
ConidioboluslEntomophthora) or Metarhizium genus or a
Mo3729CIP
-3-
Paecilomyces or Beauveria or Actinomucor species or (2b) an
entomopathogenic bacterium such as Serratia. The chemical is
generally present in an amount such that it makes up at least
0.01 ppm in the treated medium (e.g., soil) or 1 ppm in the
bait. The fungus or bacterium is generally used in an amount
such that at least I to 100 spores per gram of treated medium
are present upon contact with the termites. The optimum
quantity of fungus or bacterium will depend upon the particular
fungus or bacterium species involved. This treatment may be
aPPlied in the same manner used to apply other known chemical
termiticides. The compositions of the present invention may
also be used in bait formulations and to re-treat previously
treated areas.
DETAILED DESCRIPTION OF THE PREFERRED
EMBODIMENTS OF THE INVENTION
The present invention relates to a termiticide composed of
(1) at least one chemical selected from (a) nitroguanidines
such as 1-(2-chloro-5-pyridylmethyl)-2-(nitroimino)
imidazolidine; (b) nitromethylenes such as 1-(2-chloro-5-
2o PYridylmethyl)-2-(nitromethylene) imidazolidine; (c)
pyrazolines; and (d) pyrethroids such as cyfluthrin and either
(2a) an entomopathogenic fungus such as fungus of the
Conidiobolus(Entomophthora) or Metarhizium or Paecilomyces or
Beauveria or Actinomucor genus or (2b) an entomopathogenic
bacterium such as Serrat_ia.
The termiticide chemicals useful in the practice of the
present invention are known materials and may be made by any of
the known techniques. Specific nitroguanidines and
nitromethylenes and methods for making them are disclosed, for
example, in the following published applications and patents:
EP 464,830; EP 428,541; EP 425,978; DE 36 39 877; DE 37 12 307;
US 5,034,524; EP 386,565; EP 383,091; EP 375,907; EP 364,844;
JP 02.207 083; EP 315,826; EP 259,738; EP 254,859; JP 63
307,857; JP 63 287,764; EP235,725; EP 212,600; EP 192,060; EP
163,855; EP154,178; EP 136,636; US 4,948,798; EP 303,570; EP
Mo3729CIP
20~635~
-4_
302,833; US 4,918,086; EP 306,696; FR 2,611,114; EP 183,972; EP
455,000; JP A3 279,359; JP A3,246,283; W091/17,650; WO
91/104,965; US 5,039,686; EP 135,956; US 5,034,404; EP 471,372;
EP 302,389; JP 3,220,176; Brazil 8,803,621; JP 3,246,283;
s JP A92/9371; and JP 3,255,072.
For example, US Patent 4,742,060 discloses that
1-(2-chloro-5-pyridylmethyl)-2-(nitroimino) imidazolidine may
be made by reacting a solution of N-(2-chloro-5-pyridylmethyl)
ethylenediamine in toluene with cyanogen bromide at room
1o temperature. The 1-{2-chloro-5-pyridylmethyl)-2-iminoimida-
zolidine hydrobromide thus formed was further reacted with
sulfuric acid and fuming nitric acid. The dichloromethane
solvent was removed and the desired 1-(2-chloro-5-pyridyl-
methyl)-2-(nitroimino) imidazolidine was recovered.
is Chemical termiticides which are suitable for use in the
practice of the present invention are represented by the
formula
2o A\N-C/ { 1 )
R ~~-E
in which
25 R represents hydrogen, an acyl group, a substituted
acyl group, an alkyl group, a substituted alkyl
group, an aryl group, a substituted aryl group, a
heterocyclic group, or a substituted heterocyclic
group;
30 . A represents either (1) a monofunctional group selected
from hydrogen, an acyl group, an alkyl group, an aryl
group, a substituted acyl group, a substituted alkyl
group, a substituted aryl group or (2) a bifunctional
group which is connected to Z;
35 E represents an electron withdrawing group;
Mo3729CIP
2~$~~~~
-5-
X represents N or CH; and
Z represents either (1) a monofunctional group selected
from hydrogen, an acyl group, a substituted acyl
group, an alkyl group, a substituted alkyl group, an
aryl group, a substituted aryl group, a heterocyclic
group, a substituted heterocyclic group, the group
OR, NR2, SR or (2) a bifunctional group which is
connected with either A or X.
Preferred acyl and substituted acyl groups include
to alkyicarbonyl, substituted alkylcarbonyl, arylcarbonyl,
substituted arylcarbonyl, alkylsulfonyl, substituted
alkylsulfonyl, arylsulfonyl, substituted arylsulfonyl,
alkylphosphoryl, substituted alkylphosphoryl, arylphosphoryl,
and substituted arylphosphoryl.
15 Preferred alkyl substituents include: CI-CIO alkyl groups
which are optionally substituted, particularly CI-C4 alkyl
groups which are optionally substituted and most preferably
methyl, ethyl, n-propyl, isopropyl, n-butyl, secondary butyl or
tertiary butyl groups.
20 Preferred aryl groups include phenyl and naphthyl, most
preferably phenyl groups which may optionally be substituted.
Preferred heterocyclic groups include aromatic rings
having up to 10 atoms on the ring with at least one N, 0 or S
atom in the ring (particularly N). Particularly preferred
25 heterocyclic groups are thiophenyl, furyl, thiazolyl,
imidazolyl, pyridyl and benzthiazolyl.
Suitable substituents for the acyl, alkyl, aryl and
heterocyclic groups described above include: alkyl groups
having from 1 to 4 carbon atoms, preferably 1 or 2 carbon atoms
3o such as methyl, ethyl, n-propyl, isopropyl, n-butyl, i-butyl,
and t-butyl; alkoxy groups with from 1 to 6 carbon atoms,
preferably 1 or 2 carbon atoms such as methoxy, ethoxy,
n-propoxy, i-propoxy, n-butoxy, i-butoxy, and t-butoxy;
alkylthio groups having from 1 to 4 carbon atoms, preferably I
3s or 2 carbon atoms, such as methylthio> ethylthio, n-propylthio,
Mo3729CIP
2~~6~a~
-6-
i-propylthio, and n-butylthio, i-butylthio and t-butylthio;
haloalkyl with from 1 to 4 carbon atoms, particularly 1 or 2
carbon atoms, and from 1 to 5, preferably I to 3 halogen atoms
which halogen atoms may be the same or different, particularly
F, C1 or Br (most preferably F), such as trifluoromethyl;
hydroxyl groups; halogen such as fluoride, chloride, bromide
and iodide, particularly fluoride, chloride and bromide; cyano
groups; nitro groups; amino groups; monoalkyl- and
dialkyl-amino groups from 1 to 4, preferably 1 or 2 carbon
io atoms in the alkyl group such as methylamino, methyl-ethyl-
amino, n-propylamino, i-propylamino, and methyl-n-butylamine;
carboxyl groups; alkoxy carbonyl groups with from 2 to 4 carbon
atoms, preferably 2 or 3 carbon atoms, such as carbomethoxy and
carboethoxy; sulfo groups (S03H-); alkylsulfonyl groups with
from 1 to 4, preferably 1 or 2, carbon atoms, such as
methylsulfonyl and ethylsulfonyl; and aryl sulfonyl groups with
from 6 to l0 carbon atoms on the aromatic ring such as
phenylsulfonyl.
In Formula I, A and Z together may form a saturated or an
2o unsaturated heterocyclic ring with from 5 to 7, preferably 5 or
6, atoms in the ring. This ring may contain 1 or 2
heterocyclic atoms such as 0, S, N and N-alkyl. If two atoms
in the ring are not carbon, those two atoms may be the same
(e.g., two N atoms) or different (e.g., one N and one 0).
Specific examples of electron withdrawing groups which are
represented by the radical E in Formula I include N02, CN, and
halogenalkyl-carbonyl groups such as the 1,5-halogeno-CI-C6
alkyl-carbonyl groups.
In Formula I, Z and X together may form a heterocyclic
3o ring from 5 to 7, preferably 5 or 6 atoms in the ring. The
ring may contain 1 or 2 heterocyclic atoms such as 0, S, N or
N-alkyl which heterocyclic atoms may be the same or different.
Specific examples of preferred heterocyclic rings include
pyrrolidine, piperidine, piperazine, hexamethyleneimine,
morpholine and N-methylpiperazine.
Mo3729CIP
_7_
Preferred compounds within the scope of Formula I are
those in which
A and Z each represents hydrogen, an alkyl group which may
be substituted, an acyl group which may be
substituted, a phosphonyl group which may be
substituted, or the group NR'R" in which R'and R"
each represents hydrogen or an alkyl group, or
together represent an optionally substituted five or
six membered ring which may have nitrogen or sulfur
to as one of its members,
E represents an electron withdrawing group such as N02,
CN or COCF3,
X represents N or CH,
and
R represents hydrogen, a pyridyl group which may be
substituted, a pyridylalkyl group, a thiazolylalkyl
group, an alkyl thioalkyl group, or a five or six
membered ring having oxygen, nitrogen or sulfur as
one of its members which ring may be substituted with
one or more halogen, alkyl, haloalkyl or N02 groups.
Particularly preferred compounds include those represented by
formula I in which
Z represents NH, CH or NR'R" in which at least one of
R' and R" is an alkyl group,
A represents an alkyl group,
A and Z together represent a nitrogen-containing 5 or 6
membered ring optionally substituted with a thioalkyl
group or a sulfur containing 5 or 6 membered ring
optionally substituted with a thioalkyl group,
30'. E represents CN or N02,
X represents N or CH, and
R represents a 5 or 6 membered ring having 0, N or S as
at least one member of the ring which ring is
optionally substituted with a halogen or alkyl group.
35 and those represented by the formulae
Mo3729CIP
_g_
,A
y ~ ._ (CH2)n N (II)
~C-Z
n
X-E
in which
n represents I or 2,
Y represents any of the substituents described above as
being suitable substituents for the acyl, alkyl, aryl
to and heterocyclic groups in formula I, preferably a
halogen, most preferably chloride and
A, Z, X and E have the same meaning as in Formula I,
and
N
A
Y ~ (CH ) -N / (III)
\ z n y
s c-z
-E
in which A, Z, X, E, Y and n have the same meaning as in
Formulae I and II.
Specific examples of nitroguanidines and nitromethylenes
which may be used in the present invention include:
3-(z-chloro-5-pyridylmethyl)-z-(nitroimino)-thiazolidine;
1-(z-chloro-5-pyridylmethyl)-2-(nitroimino)-imidazolidine;
1-(z-chloro-5-pyridylmethylj-z-(nitromethylene) imidazolidine;
~to3zzgciP
CH3S-C-C-N NH
CH
N02
_ jH3
C1 ~ ~ - CH2-N-C-CH3
N
I
N
RCN
;2H5 NHCH3 ,
Cl ' ~ CH2-N- ~~
N CH
N02
S NH
CH
N02
30
Mo3729CIP
1~
Cl-~ CH2- N-SR ,
N
N
N02
in which
R represents hydrogen, an alkyl substituted alkyl, aryl or
substituted aryl group,
C1 ~ ~ N NH ,
N
N02
Z5
C1 ~ ~ CH2 ~ ,
N -'
2o N
N02
a5 I----1
C ~~ CH2 N NH ,
N
N
N02
Mo3729CIP
-lI- 24~~35~.
C1 ~ ~ CH2------------- N S ,
N
CH
\N02
C1 ~ ~CHZ N ,
~/N
1o N '
CN
C1 ~~H2 N NH ,
~N
N
y
CN
Cl ~ ~ CH2 N S ,
N
CN
C1 ~ \ H2 N NH
3o N
CH-N02 , and
Mo3729CIP
-12- 208fi3~~.
N ---:
--- CH2 N .
C S
C1 N
~ CN
Particularly preferred nitroguanidines and nitromethylenes
are 1-(2-chloro-5-pyridylmethyl)-2-(nitroimino)imidazolidine.
When used in the form of a bait, a nitroguanidine or
nitromethylene is generally used in a quantity such that it
represents at least 0.0001% by weight, preferably from about
0.001 to about 10.0% by weight, and most preferably from about
0.01 to about 1.0% by weight of the total bait components:
When the nitroguanidine or nitromethylene is incorporated
directly into soil or applied directly to a surface being
treated, it is generally used in an amount such that at least
0.01 ppm (parts per million), preferably from about 0.1 ppm to
about 1000 ppm and most preferably from about 1 ppm to about
300 ppm are present in the soil or on the surface being
zo
treated.
Pyrethroids which may be used in the compositions of the
present invention are known. Both naturally occurring and
synthetic pyrethroids are suitable. Examples of suitable
zs pyrethroids include: pyrethrins, cinerins, jasmolins, allethrin
[2-methyl-4-oxo-3-(2-propenyl)-2-cyclo-penten-1-yl-2,2-
dimethyi-3-(2-methyl-1-propenyl)cyclopropanecarboxylate],
bioallethrin [D-trans-allethrin], barthrin [6-chloro-1,3-
benzodioxol-5-yl)methyl 2,2-dimethyl-3-(2-methyl-1-propenyl)-
3o cyclopropanecarboxylate], tetramethrin [(1,3,4,5,6,7-hexahydro-
1,3-dioxo-2H-isoindol-2-yl)methyl 2,2-dimethyl-3-(2-methyl-1-
propenyl)cyclopropanecarboxylate], furamethrin [[5-(2-
propyenyl)-2-furanylJmethyl 2,2-dimethyl-3-(2-methyl-1-
Mo3729CIP
20~~35~
-13-
propenyl)cyclopropanecarboxylate], resmethrin [[5-(phenyl-
methyl)-3-furanyl]methyl 2,2-dimethyl-3-(2-methyl-1-
propenyl)cyclopropanecarboxylateJ, bioethanomethrin
[[5-(phenylmethyl)-3-furanyl]methyl 3-(cyclopentylidiene-
methyl)-2,2-dimethyl-cyclopropanecarboxylate], phenothrin
[(3-phenoxyphenyl)methyl 2,2-dimethyl-3-(2-methyl-1-propenyl)-
cyclopropanecarboxylateJ, fenpropanate (cyano(3-phenoxyphenyl)-
methyl 2,2,3,3-tetramethylcyclopropanecarboxylateJ, permethrin
[(3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethyl-
1o cyclopropanecarboxylate], cyfluthrin [cyano(4-fluoro-3-phenoxy-
phenyl)methyl-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane-
carboxylate], cypermethrin [cyano(3-phenoxyphenyl)-methyl
3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropane-carboxylate],
decamethrin [cyano(3-phenoxyphenyl)methyl 3-(2,2-dibromo-
ethenyl)-2,2-dimethylcyclopropanecarboxylate], fenvalerate
[cyano(3-phenoxyphenyl)methyl 4-chloro-a-(1-methylethyl)-
benzeneacetate], and cyhalothrin [cyano(3-phenoxy-phenyl)-
methyl 3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl-
cyclopropanecarboxylate]. Particularly preferred pyrethroids
2o are cyfluthrin and fenvalerate.
The pyrethroid is generally used in bait compositions in a
quantity such that at least 0.0001% by weight, preferably from
about 0.01 to about 10.0% by weight, and most preferably from
about 0.01 to about 1.0% by weight of the bait composition is
25 PYrethroid. Where the pyrethroid is added directly to soil or
applied directly to a surface to be treated, the pyrethroid is
generally used in a quantity such that at least 0.01 ppm,
preferably from about 0.1 to about 1000 ppm, and most
preferably from about 1.0 to about 300 ppm are present.
30 . Any of the known pyrazolines may also be employed in the
termite treatment of the present invention. Such pyrazolines
are, for example, disclosed in published European Patent
Application 0,438,690. Examples of suitable pyrazolines
include those represented by the formulae:
Mo3729CIP
~08~3~~
-14-
a
N
N
N-CO-NH ~ CF3
~N~
a
~o
a / F
N
~N ~ N-CO-N OCF O N-C0-NH O 0CF3
\ ~ 3
~ Ni N
F2CI~0 F
F~ N
-CO-NN ~ CF3
F O
30
Mo3729CIP
~~~6~~1
-15-
C1
N
N~
N-CO-NH~O~C1
N
to
F2CH0
C1
N N
15w,.N/
N-CO-N~ CF3 \ ~ -CO-HN~ C1
N ~ ~ ~/N
F2CH0
Br
N
N
N
J-CO-N~1 N-CO-N~CF3
~N ~~
C1
Mo3729CIP
16
C1
i
N
~ N ~ N-CO-NH ~ F3
~ N~
C
to in which a represents a halogen atom, an alkyl group, a halogen
group or a nitro group.
The pyrazoline is generally used in bait compositions in a
quantity such that at least 0.0001% by weight, preferably from
about 0.001 to about 10.0% by weight, and most preferably from
~s about 0.01 to about 1.0% by weight of the bait composition is
pyrazoline. Where the pyrazoline is added directly to the soil
or applied directly to the surface to be treated, the
pyrazoline is used in a quantity such that at least about 0.01
ppm,. preferably from about 0.1 to about 1000 ppm, and most
20 Preferably from about 1.0 to about 300 ppm are present.
The fungi employed in the termite treatment of the present
invention occur naturally in soil and may be readily isolated
therefrom. Species of the genus Conidiobolus (Entomophthora)
useful in the termiticides of the present invention include:
2s Conidiobolus coronatus, Conidiobolus virulenta, and
Conidiobolus obscura. Conidiobolus coronatus is particularly
preferred.
Species of the genus Metarhizium useful in the present
invention occur naturally in soil and may be readily isolated
30 therefrom. Various strains of Metarhizium anisopliae are
useful in the present invention. The strains F 52 (BIO 1020,
DSM Number 3884) and the MADA strain (received from the
University of Florida)(CBS Number 326, Baarn, Netherlands) of
Metarhizium anisopliae are most preferred.
Mo3729CIP
-17-
Species of the genus Paecilomvces useful in the
termiticides of the present invention include Paecilomvces
farinosus which naturally occurs in soil and may be readily
isolated from soil or from diseased and sporulating termites by
methods known to those in the art.
A preferred species of the Beauveria genus is B_eauveria
bassiana which may also occur naturally in soil and may be
readily isolated from soil or from diseased and sporulating
termites by methods known to those skilled in the art.
1o The Actinomucor species is also useful in the practice of
the present invention.
Among the bacteria useful in the present invention are
those of the Serratia species which occur naturally in soil and
may be readily isolated from soil or from diseased and
sporulating termites by methods known to those skilled in the
art.
The fungus or bacterium should generally be present in an
amount and form such that at least 101-102 spores, preferably
103-105 spores per grams of media are present. The optimum
2o amount will, of course, depend on the species used.
The termite treatment of the present invention may be
applied in the form of a powder, solution, suspension,
emulsion, foam, paste, granules, aerosols, natural and
synthetic materials impregnated with active compound and fungus
and very fine capsules in polymeric substances. When in powder
or granular form, the fungus may be added to the solid
nitroguanidine, nitromethylene, pyrazoline or pyrethroid
formulation in an appropriate amount. Other known additives
for termiticides such as extenders, attractants, feeding
so'. stimulants, pheromones, may optionally be included in the final
composition. Examples of suitable powder vehicles include
clay, talc, lime and pyrophyllite.
The termiticide of the present invention may also be used
in a variety of liquid forms. Suitable liquid vehicles for the
termiticide include water and inert solvents. Other additives
Mo3729CIP
~o~~~~~
_Ig_
which are commonly used in liquid insecticide formulations,
e.g., emulsifiers may also be included in liquid termiticide
formulations of the present invention. Liquid formulations of
the chemical and/or fungal agent may also include lignins,
hydrocelluloses, bentonites, pectins, or any other material
which causes the formulation to solidify after application.
The chemical compound and the fungus or bacterium could be
sequentially applied to the medium. When this technique is
used, either the chemical compound or the fungus can be applied
1o first. The interval between application of the chemical and
fungus may be as short as a few minutes or as long as a few
days or even a few weeks. If an appropriate strain of the
fungus or bacterium is already present as a naturally occurring
material with the necessary spore titer in the medium to be
treated, addition of fungus or bacterium is unnecessary and
only the chemical compound need be applied. These treatments
may also be made repeatedly in regular or irregular intervals
to assure long-lasting effect.
The termiticides of the present invention are effective
2o against all types of termites but have been found to be
particularly effective against the subterranean termite
Reticulitermes flavipes and the formosan termite Coetotermes
formosanus.
The data presented below on the effects of the combined
applications of chemical and biological agents reveal an
impressive degree of synergism. It would take application
rates higher by several powers of ten if either of these agents
were used alone for termite control to achieve termite control
comparable to that obtained by the combined application of the
so present invention.
Having thus described our invention, the following
examples are given as being illustrative thereof. All parts
and percentages given in these examples are parts by weight and
percentages by weight, unless otherwise indicated.
Mo3729CIP
2086~~~.
-19-
EXAMPLES
Examples 1 through 5 demonstrate the high degree of .
termite extermination in short periods of time achieved by the
use of a chemical termiticide in combination with a fungus as
s compared to extermination with chemical termiticide alone or
fungus alone. Examples 6 and 7 illustrate the interaction of
Conidiobolus coronatus and various chemical termiticides in
termite extermination. Examples B, 9 and 10 illustrate the
interaction of 1-(2-chloro-5-pyridylmethyl)-2-(nitroimino)-
lo imidazolidine and two fungal pathogens of the Metarhizium genus
and the Conidiobolus genus in quantitative terms. Example 11
illustrates the synergy of 1-(2-chloro-5-pyridylmethyl}-Z-
(nitroimino)imidazolidine with three additional fungal species
and one species of bacterium. Example 12 illustrates the
15 synergy between microbes in non-sterile soil and various ,
nitromethylenes or nitroguanidines.
Exams 1p a 1
100 g of sterile soil were used in each of the samples
described in Table 1. The control sample A was not treated
2o with nitroguanidine, nitromethylene, pyrethroid, pyrazoline or
fungus. 1-(2-chloro-5-pyridylmethyl)-2-(nitroimino)-
imidazolidine was added in various concentrations (indicated in
Table 1) directly to the soil in samples B, C, a, E and F. No
fungus was added to the soil in these samples. Filter paper
25 discs which had been soaked in a solution of 1-(2-chloro-
5-pyridylmethyl)-2- (nitroimino)-imidazolidine having the
concentration indicated in Table 1 were placed on top of the
soil in samples G, H and I. No fungus was present in soil
samples G, H or I. No vitro- guanidine was added to soil
30' sample J but soil from a container used in a previous test in
which the fungus Conidiobolus coronatus had shown up naturally
was included in sample J. In soil sample K, soil from a
previous test in which the fungus Conidiobolus coronatus had
shown up naturally was used but in addition, the termites were
Mo3729CIP
-2~- 2U~fi3~1
fed with filter paper that was soaked with 0.01% of the
nitroguanidine.
1 gram of subterranean termites Reticulitermes flavipes
was then added to each of the soil samples A through K. The
observations made over the next 18 days are summarized in Table
1.
It is evident from the data in Table 1 that 1 ppm nitro-
guanidine must be present in soil in order to achieve
significant effect upon the termites and an eventual (after 18
to days exposure) 100% mortality. Use of higher rates (e.g. 10 to
100 ppm) of nitroguanidine in the soil did not significantly
change this result.
When the nitroguanidine was used in the form of a bait
(i.e. on treated filter paper), smaller amounts of nitro-
guanidine (i.e. 0.001%; 0.003%; and 0.010°!°) were as effective
as the larger amounts used to treat the soil.
The data in Table 1 also show that when only fungus was
applied to the soil (i.e., no nitroguanidine was used), the
results were the same as those observed in the untreated
2o control. That is, intense tunneling and location at the bottom
of the sample container were observed. However, when a filter
paper disc having 0.01% nitroguanidine present thereon was
placed on soil in which the fungus was present, 100% of the
termites in the sample were dead within two days.
30
Mo3729CIP
~~8~33~.
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Mo3729CIP
~~~s~~~
-22-
EXAMPLE 2
The procedure of Example l was repeated using both sterile
soil samples and soil samples in which Coni.diobolus coronatus
was added in the form of 1 drop of crude spore suspension.
s This crude spore suspension was obtained by rinsing spores from
one petri dish culture with 50 cc sterile water. 1-(2-chloro-
5-pyridylmethyl)-2-(nitroimino) imidazolidine in various
concentrations (indicated in Table 2) was added directly to the
soil. The results of these tests are reported in Table 2.
1o The data presented in Table 2 show that change in termite
behavior in the sterile soil samples was first observed at the
1 ppm nitroguanidine level. However, 100 ppm nitroguanidine
were necessary to cause substantial mortality after 6 days. In
the soil inoculated with the fungus, substantial mortality
15 resulted at the 1 ppm level, with total mortality of the
termites achieved at 3 ppm. Without a nitroguanidine
treatment, termites in the soil inoculated with fungus behaved
like those in the untreated check.
25
Mo3729CIP
'23'
0
o ~
W
+ +
~o o ~n ~ 0 0 0
.
o
N U CG i N 1~ O O O
7-O O ~ '_'~'"~'-,
a Z ~"~
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a
i
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Mo3729CTP
-24-
EXAMPLE 3
100 grams of soil were used in each of the samples
described in Table 3. Half of the soil samples were made with
sterile soil. The other half were made up of sterile soil
which had been inoculated with one drop per sample of crude
spore suspension of Conidiobolus coronatus. The crude spore
suspension was obtained by rinsing spores from a petri dish
culture with 50 cc of sterile water. Filter paper discs were
saturated with solutions of 1-(2-chloro-5-pyridylmethyl)-2-
io (nitroimino) imidazolidine having the concentration indicated
in Table 3. 1 gram of subterranean termites Reticulitermes
flaviQes was added directly to each sample. The termites in
each sample were then observed for a period of 6 days. The
termite mortality for each sample is given in Table 3.
Results: Without any nitroguanidine, the termites did not
show any response to the soil inoculation with Conidiobolus
coronatus during the 6 day observation period. When treated
filter paper baits were added to the sterile soil without the
fungus, first symptoms of behavioral change showed up in the
0.001% treatment, but mortality remained low (8%) even at rates
100 times higher (0.1%). However, in the fungus inoculated
soil, mortality was 95% at the 0.001% nitroguanidine level
after 6 days.
30~ .
Mo3729CIP
~:p~~~5~.
-25- '
N 3~ I
r O I
Q oV
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a
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Mo3729CIP
2~~63~1
-26-
EXAMPLE 4
The effect of exposing termites directly to actively
growing fungus in the presence and absence of
1-(2-chloro-5-pyridylmethyl)-2-(nitroimino) imidazolidine was
s studied. In these experiments, an agar plate with no fungus
and no nitroguanidine was used as the control. One sample was
an agar plate with a filter paper disk treated with
1-(2-chloro-5-pyridylmethyl)-2-(nitroimino) imidazolidine
(0.01% active ingredient)-. A third agar plate was inoculated
io with Conidiobolus coronatus and a fourth agar plate was
inoculated with the Conidiobolus coronatus fungus and treated
with a filter paper disk containing 0.01% of the
nitroguanidine. Termites (Reticulitermes flavipes) were added
to each agar plate and observed for 4 days. The results of
15 these tests are reported in Table 4.
In the non-inoculated Control sample, normal behavior of
the termites with immediate extensive tunneling took place. No
mortality was observed after 4 days. In the plate treated only
with fungus, 25% mortality was observed after 4 days. In the
20 plate treated with with nitroguanidine bait only, the termites
exhibited severe intoxication and tunneling was not present.
100% mortality occurred within 1 day on the fungus plus
nitroguanidine treated agar and massive sporulation occurred
within 4 days on the dead termites.
2s
30'
Mo3729CIP
-a7_
r
N N
0
ct
N
r
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Mo3729CIP
20~63~~
_28_
EXAMPLE 5
4 kilograms of sandy soil were sterilized by means of an
autoclave. The sterilized soil was then divided into two equal
portions. In one portion of the soil, the moisture content was
adjusted to 10% by adding 10 ml of distilled water to each 100
grams of soil. The second portion of sterilized soil was
inoculated with a crude slurry of Conidiobolus coronatus spores
and mycelium. This slurry was made by scraping fungal growth
from agar cultures on petri dishes into 50 ml of distilled
1o water. The slurry was then added to the sterilized soil at a
rate of 10 ml per 100 gram of soil. Each of the 2 kg batches
of soil was then distributed in 20 plastic cups so that 100 g
of soil were present in each cup. Ten cups of each batch
received filter paper disks which had been treated with 0.01%
1-(2-chloro-5-pyridylmethyi)-2-(nitroimino) imidazolidine. The
remaining samples received filter paper disks which had been
soaked in distilled water. Approximately 1 gram of live
Reticulitermes flavipes was added to each sample. Observations
of mortality, activity and fungal growth were made every few
2o days for a week. The results of these tests are reported in
Table 5.
Results: As expected, in the controls the termites
behaved normally over the 7 day observation period. The same
was true in the "fungus only" treatment with the exception that
2s 209'° final mortality was observed. The same degree of mortality
resulted from the "chemical only" treatment, although
intoxicative effects were observed from the early stages of the
study. The "fungus plus chemical" treatment resulted in 100f°
mortality within 2 days.
Mo3729CIP
-29-
N I o000
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a .-
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=
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208351
Mo3729CIP
20863~~
-30-
EXAMPLE 6
The interaction between the fungus Conidiobolus coronatus
and various known termiticides (listed in Table 6) was studied.
In these tests, 100 gram soil samples were used. Conidiobolus
coronatus was added to. half of the soil samples according to
the procedure explained in Example 2. No fungus was added to
the other half of the samples. The chemical termiticide was
then added to each of the samples (i.e., both those which had
been inoculated with fungus and those which had not been
1o inoculated with fungus) in an amount sufficient to reach the
rate reported in Table 6. 1 g of the subterranean termite
Reticulitermes flavioes was then added to each sample. The
samples were then observed over a period of 7 days. The
observations made are reported in Table 6.
The carbamates and the organophosphates used in this test
had little effect on the termites or they showed no difference
in response between fungus-free and fungus-added soil. In
contrast, the pyrethroid used (cyfluthrin) resulted in no kill
without the fungus, but 100% mortality when the fungus was
added to the soil.
25
Mo3729CIP
2~863j1
N
0 00 00 000
00 0
U
r
h N f'
O
O t
C * fl.
N
7
4- O O O ~ O O
- O O O
L
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C O
r
r
i
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tO O~ ~' C
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+~ 4- r, r
r
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ct N
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p
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N
y 4- O OO OO NOO
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N ~ i
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r0 A
n ~ d U
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U U N O d * * .H CV
M ~' 117
Njo3729CIP
208fi35~.
-32-
EXAMPLE 7
The procedure of Example 6 was repeated using only
pyrethroid termiticides. The results are reported in Table 7.
Results: Each of the three pyrethroids used (cyfluthrin,
fenvalerate and cypermethrin) showed significant differences in
activity between fungus-free and fungus-inoculated soil.
Cyfluthrin was the most active compound and provided at least
times better termite control in the fungus-added soil than
the fungus-free soil at all three observation dates.
to
20
30
Mo3729CIP
-33-
N
O ~~~ ~~~ ~
r-1 N '-1 r1
H r-1 r1
C O O O O O O
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Mo3729CIP
~0~535~
-34-
EXAMPLE 8
The following scale was used to evaluate the results of
the tests reported in this Example and in Example 10.
RATING % MORTALITY SUBJECTIVE EVALUATION
1 98-100 Excellent
2 90-97 Very Good
3 80-89 Good
4 65-79 Satisfactory
5 45-64 Unsatisfactory
6 30-44 Unsatisfactory
7 20-29 Poor
8 3-19 Poor
9 0-2 No Effect
100 gram soil samples were used in this test. Some of the
samples were injected with spores of Conidiobolus coronatus and
some of the samples were injected with spores of Metarhizium
a_nisopliae (MADA strain, CBS Number 326). The number of spores
2o injected for specific samples are recorded in Tables 8A arsd 8B.
These samples were then treated with filter paper disks which
had been soaked in solutions of 1-(2-chloro-5-pyridylmethyl)-
2-(nitroimino) imidazolidine having varying concentrations.
The concentration of the solution used to prepare the disk for
z5 specific samples is reported in Tables 8A and 8B. 1 gram of
the termites Reticulitermes flavi~es was then added to each of
the samples. The samples were then rated using the above-given
scale after 7 days. The results are reported in Tables 8A and
8B. They show that for Conidiobolus by itself {Table 8A),
3o spore densities of 105 per gram of soil have little effect on
the termites. However, in soil treated with only a 1-
(2-chloro-5-pyridylmethyl)-2-(nitroimino)-imidazolidine bait at
concentrations which are normally ineffective, 100% mortality
resulted at spore concentrations in the sail as low as 101.
~5 That means spore densities at least 4 powers of ten less were
Mo3729CIP
required when nitroguanidine treated baits were also added to
the soil sample. When Metarhizium was added to the soil
samples (Table 8B), spore densities could be several powers of
ten lower where nitroguanidine treated baits were also added to
the soil sample.
to
20
30
Mo3729CIP
-36-
20~6351
°I~~OOCO
I °°. 0001
N ~ '
C +~ '
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2~~~~5~
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M03729GIP
~oso~~~
-38-
EXAMPLE 9
The procedure of Example 8 was repeated using two
different strains of Metarhizium anisopliae-- the MADA strain
(CBS Number 326) and the BIO 1020 strain (DSM Number 3884) and
Conidiobolus coronatus. However, instead of using various
concentrations of the nitroguanidine, the filter paper discs
were soaked in either distilled water or a 0.001% solution of
1-(2-chloro-5-pyridylmethyl)-2-(nitroimino) imidazolidine. The
results observed over a 10 day period are reported in Table 9.
to
20
30
Mo3729CIP
-39-
aooo ~ o..~ ,~.o~ O ~ ~
O O1 W f1O ~D01 O tJtDM VI
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Mo3729CIP
2p~635~.
-ao-
EXAMPLE 10
The procedure of Example 8 was repeated using Metarhizium
anisopliae (MADA strain, CBS Number 326) and
1-(2-chloro-5-pyridylmethyl)-2-(nitroimino) imidazolidine in
the quantities indicated in Table 10. The results observed
after 5 days are reported in Table 10 using the same scale as
was recited in Example 8.
The results shown in Examples 9 and 10 confirm that by the
addition of low strength 1-(2-chloro-5-pyridylmethyl)-2-
to nitroimino imidazolidine baits, spore concentrations
irrespective of fungus species or strain, can be lowered by up
to 4 powers of ten and more eTfective termite control will
still be obtained than is obtained with soil treated with only
water soaked baits.
20
30
Mo3729CIP
-41-
N
M
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m
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v
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Mo3729CIP
20~6~5~.
EXAMPLE 11
The procedure of Example 5 was repeated using the fungi
Actinomucor sp., Paecilo_myces farinosus, and Beauveria bassiana
and the bacterium Serratia so. The filter paper discs were
soaked in either distilled water or a 0.001% solution of
1-(2-chloro-5-pyridylmethyl)-2-(nitroimino) imidazolidine. The
results observed over a 7 day period are reported in Table 11.
In Table 11, the rating system used and reported in the
left column of each box was as follows:
to - Normal: no change as compared to untreated
control.
+ Slight: some termites on surface or feeding
and tunneling somewhat affected.
++ Moderate: many termites on the surface with
substantial reduction in feeding and
tunneling; intoxication symptoms
(sluggish, ataxial).
+++ Severe: all termites on the surface with no
feeding and tunneling; increasing
2o intoxication symptoms.
++++ Lethal: 95-100% of termites dead or moribund.
The numbers reported in the right column of each box in Table
11 are the % mortality.
30
Mo3729CIP
-43-
Z ~o
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t
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Mo3729CIP
-45-
Z~~~~~1
EXAMPLE 12
The procedure of Example 5 was repeated using filter paper
disks dipped into various concentrations of several different
nitromethylene compounds and two soil types. One type of soil
was unsterile as taken from the filed. The second type of soil
was autoclaved. The concentrations of each nitromethylene and
the results observed after 4 days are reported in Table 12.
The nitromethylenes used in this Example were as follows:
io
Compound Formula
n
A Cl ~~ CH2 N NH
N
N
CN
g C1 ~~-CH~--N NH
N
CH
\N02
HN N --~~ ~ Cl
so' C ~ N -
N02
Mo3729CIP
-4s- ~p$~351
r-
CH3-S-CH2-CH2-N NH
C
~N02
- CH3
. E C1 ~ ~CH2-N-C-CH3
N
CN
NHCH3
1s - ~ =CN02
F C1 ~ ~CH2-N
N ~!~ C
~CH3
G C1 ~~CH2 N S
~N
N
as \CN
Mo3729CIP
2~~6~~~
H C1 ~ ~H2 N N-CH3
~N
C\
N02
to
I Cl ~ ~CH2 N NH
N
N
\N02
The degree of Intoxication is reported in Table 12 using
the same rating system as was used in Table 11.
25
Mo3729CIP
_48_
Table 12
20~~3~~.
Mortality
after days
4
Nitromethylene % NitromethyleneDegree SterijeNon-St erile
Early
Soil Soil
A 0.001 + 10 FF 100 FF
B 0.1 ++++ 99 FF 100 FF
0.01 ++++ 25 FF 100 FF
C 0.1 +++ 62 FF 100 FF
0.01 +++ 59 FF 100 FF
0.001 ++ 35 FF 100 FF
D 0.1 ++++ 96 FF 100 FF
0.01 +++ 74 FF 100 FF
E 0.1 ++++ 74 FF 100 FF
0.01 +++ 42 FF 100 FF
0.001 +++ 24 FF 100 FF
is
F 0.1 +++ 22 FF 100 FF
0.01 ++ 28 FF 100 FF
G 0.1 ++++ 41 FF 100 FF
0.01 +++ 21 FF 100 FF
H 0.1 ++ 12 FF 100 FF
2o 0.01 + 6 FF 100 FF
0.001 - 0 7 FF
I 0.1 +++ 18 FF 100 FF
0,01 +++ 13 FF 100 FF
0.001 +++ 20 FF 100 FF
25 Control 0 4
0.1~° Sol vent - -
Water - - 0 0
1) Soil sterilization process was obviously incomplete.
30 '~F" indicates spontaneous collapse of termite population due
to occurrence of entomophagus fungi, mostly of the
Metarhizium and Conidiobolus type. Each F represents one
of 2 replicates.
Mo3729CIP
20863~~
-49-
Although the invention has been described in detail in the
foregoing for the purpose of illustration, it is to be
understood that such detail is solely for that purpose and that
variations can be made therein by those skilled in the art
without departing from the spirit and scope of the invention
except as it may be limited by the claims.
io
20
30
Mo3729CIP
