Note: Descriptions are shown in the official language in which they were submitted.
CA 02197882 2006-09-28
52578-14
1
METHYL IODIDE AS A SOIL FUMIGANT
Backc7round of the Invention
The present invention relates generally to the fields
of biology and agriculture. More particularly, the
present invention relates to compositions and methods for
use in fumigation of soils.
The control of plant pathogens, nematodes and weeds
is of central importance to the agriculture industry. In
particular, the substantial reduction or complete
elimination of nematode populations in soils is critical
to initial plant growth, productivity and life-span.
Pathogenic fungi and nematodes develop on the extensive
root systems of both annual and perennial crops, damaging
them severely. Moreover, they persist in the soil after
crop removal and need to be eliminated before replanting
of new crops. Among the fungi and nematodes of particular
significance to agriculture are the following: root rot
pathogens (Phytophthora spp., Pythium spp., Rhizoctonia
spp., Fusarium spp.); vascular wilt pathogens
(Verticillium spp., Fusarium spp.); root knot nematodes
(Meloidogyne spp.); root lesion nematodes (Pratylenchus
vulnus); ring nematodes (Circonemella xenoplax); stubby
root nematodes (Paratrichodorus spp.) ; stem and bulb
nematodes (Ditylenchus dipsaci); cyst nematode (Heterodera
schachtii) ; citrus nematode (Tylenchulus sernipenetrans)
and the burrowing nematode (Radopholus similus).
To date, the only approaches which have been used
successfully to combat plant pathogens and nematodes have
been crop rotation or fallowing for at least four years,
use of pathogen and nematode-resistant crops and soil
fumigation. Rotation has limited value for control in
many cases, because of thewide host range of many species
of fungi and nematodes; moreover, many of the non-host
WO 96/12559 PCTIUS95/1198p
2197882
2
crops provide only a low per acre return. Resistance to
plant pathogens and nematodes is available only in a few
crops, and resistant cultivars may not be developed in the
foreseeable future for many crops of significant
commercial interest. Therefore, soil fumigation remains
the best alternative for control of plant pathogens and
nematodes.
Methyl bromide (CH3Br) is extremely important to
United States agriculture [U.S.D.A. The Biological and
Economic Assessment of Methyl Bromide, U.S.D.A.
Publication (1993)]. It is the most widely used and most
effective universal fumigant in the world. It is used
extensively for soil fumigation, as a commodity quarantine
treatment (export and imports) to control a variety of
pests on numerous crops, and as a structural fumigant for
wood destroying pests.
According to the Montreal protocol of 1991 (as
amended in 1992), methyl bromide (MBr) was categorized as
an ozone depleting chemical with an ozone depleting
potential (ODP) of greater than 0.2 compared to
trichlorofluoromethane (cfc 11), a refrigerant used as a
reference gas having an ODP of 1. Title Five of the Clean
Air Act (Stratospheric Ozone Protection), which was added
in the 1990 amendments thereto, indicates in Section 602
that the U.S. Environmental Protection Agency (EPA) must
list asa Class 1 ozone depleter any substance with an ODP
of 0.2 or greater. Once designated, all production must
be phased out by the year 2000. MBr has an ODP of 0.7;
30-400 of total ozone depletion is said to be as a result
of bromine radicals, which are 30-60 times more efficient
ozone depleters than chlorine [Pyle, J.A. et al.,
In:Scientific Assessment of Ozone Depletion, eds.
Albritton, D.L. et al., World Meteorol. Org., Geneva
(1991), pp. 6.1-6.191.
Evidence on the loss of MEr to the atmosphere after
soil fumigation indicates that of-the total amount applied
~ WO 96/12559 2197882 PCT/US95/11984
3
to the soil for fumigation, approximately 87% is lost to
the atmosphere within seven days [Yagi, K. et al., PNAS
USA 90:8420-8423 (1993)]. On reaching the stratosphere
MBr undergoes photo-oxidation, releasing bromine atoms
which enter the ozone depletion cycle. MBr loss from
fumigated soils is further supported by studies which
indicated a loss of as much as 70% of the applied MBr to
the atmosphere through the tarp and after the tarp is
removed [Rolston and Glauz, Pesticide Science 13:653
(1982)].
In 1990, approximately 64,000,000 pounds of MBr were
used in the U.S., of which 44-49 million pounds were used
for soil fumigation (control of insects, nematodes, weeds,
plant pathogenic microbes and vertebrate and invertebrate
pests), 5 million for post harvest and quarantine
treatments, 4-9 million'pounds for fumigating structures
and 6 million pounds for use as chemical intermediates.
Thus, approximately 80% of the total is used for
agriculturally related purposes.
As currently available alternatives to MBr are less
effective and/or more expensive, the removal of MBr will
be very costly. Annual losses to U.S. producers and
consumers is estimated to be in the region of 1.5 billion
dollars. -This figure does not acdount for the losses due
to post -harvest and quarantine losses as well as
structural fumigation losses. California and Florida are
the largest-users of MBr (approximately 25,000,000 pounds
combined) in the U.S., and hence will be most heavily
affected by its removal. MBr removal would most adversely
affect such commodities as tomatoes, strawberries,
peppers, melons and ornamentals. The loss of MBr would
thus be extremely costly to both agricultural producers
and consumers as well as having a substantial impact on
the U.S. economy. Nonetheless, it is the general
consensus of those working in the field that no approach
is currently available that will achieve the same level
WO 96/12559 PCT1US95/11984.0
4 2197882
of broad-spectrum pest management as methyl bromide;
chemical and non-chemical approaches that are available
can provide some level of agricultural pest management,
but generally with narrower activity and lower crop yields
and quality. Therefore, there is clearly a need for
alternatives to MBr.
It is an object of the present invention to provide
methods and compositions for use in soil fumigation which
ameliorate at least some of the problems attendant to
prior art methods.
Summary of the Invention
In accordance with the present invention, methyl
iodide is employed as a soil fumigant for the effective
control of plant pathogens, nematodes, bacteria and weeds.
Methyl iodide may be employed in substantially the same
manner as is customary for use of methyl bromide, and is
at least as effective as methyl bromide when used in
comparable amounts.
Detailed Description of the Invention
While the present invention is not bound to any
particular theory, methyl iodide appears to be totally
analogous to methyl bromide in its ability to act as a
biocide. The generally accepted mechanism explaining the
activity of a member of the lower alkyl halide series is
that it reacts via bimolecular nucleophilic displacement
(SN2) reaction with functional groups such as NH2 and SH
in various amino acids and peptides in target organisms
[Price, N.R., J. Stored Prod. Res. 21(4):157-164 (1985)].
Methyl iodide reacts at approximately the same rate as
methyl bromide under most SN2 conditions that have been
reported.
There have been several reports in the literature of
the use of methyl iodide as a fumigant for control of
insect populations in stored grain [Lindgren, D.L., J.
Economic Entomol. 31:320 (1938); Lindgren, D.L. et al.,
J. Economic Entomol. 47:923-926 (1954); Lehman, R.S., J.
= WO 96/12559 21 97882 PCTlUS95/11984
Economic Entomol. 35:659-661 (1942); Rajendran, S. &
Muthu, M., Indian J. Ent. 49(3):363-369 (1987); Hassall,
K.A., Ann. Appl.Biol. 43:615-629 (1955)]. Nonetheless,
it would simply not have been possible to predict that an
5 agent having utility in control of insect populations in
stored grain would in fact have any utility whatsoever in
fumigation of soils for elimination of plant pathogens,
nematodes, bacteria and/or weeds.
Soil can modify the chemical activity of fumigants.
Whereas activity of an agent may be high in air, it may
have much less activity in soil [Lehman, R.S., J. Economic
Entomology 35:659-661 (1942)]. Indeed, fumigation of
stored grain and its expectations are relatively simple
when compared to the complexity of fumigating soils and
the expectations from such fumigations. Humidity in
stored grain is uniform throughout the product, whereas
in soil it can vary greatly. In addition, particle size
in stored grain is fairly uniform, as are the airspaces
between particles; this makes fumigation of grain
relatively simple. In soil, the particle sizes and
airspaces vary widely, substantially complicating
fumigation. -
Further, the target organisms in stored grain are
fairly limited in--variety and quite different from the
large variety and number of target organisms in the soil.
Fumigation in stored grain targets insects; fungi,
nematodes and bacteria are not usually a problem when the
humidity is kept low,and weeds would not be affected
by a fumigation that did not also kill the grain. In
soil, fumigation is expected to kill fungi, nematodes,
weed seeds, insects, and vertebrate and invertebrate
pests.
As a consequence, many fumigants used for stored
products are generally not used as soil fumigants. For
example, phosphine is currently registered and used for
stored products but not used in soil where it is
WO 96/12559 PCT/US95/119840
6 2197882
apparently ineffective. Some pests are resistant to
phosphine, and it is not effective under 50 F; moreover,
it requires a fumigation time of 3-5 days and is highly
flammable. Similarly, esters of formate (e.g., methyl
formate) are effective in treating stored products, but
are much less effective in soil. Therefore, it is clear
that compositions useful as stored products fumigants are
not necessarily useful as soil fumigants.
In trials carried out in accordance with the
invention, methyl iodide has proven to be an effective
chemical for the fumigation of five species of soil borne
plant pathogenic fungi, one saprophytic fungus, three
weeds and two nematodes. In the majority of trials in
both the laboratory and the field, MI was effective at
rates that were equivalent to 0.5 to 1.0 lb of methyl
bromide per 100 ft3. In only one trial, on one fungus,
for unknown reasons MI did not eliminate the fungus at any
rate (Table 3); however, this fungus was eliminated in a
different trial (Table 2). In direct comparison field
trials MI was as effective as MBr (Tables 7 and 8) in
eliminating the pathogen. In three laboratory trials, MI
was more effective as a soil fumigant than seven other
alkyl iodides. Therefore, methyl iodide is at least as
effective as methyl bromide in fumigating soil to
eliminate soil borne plant pathogenic fungi.
Methyl iodide absorbs UV radiation most strongly in
the WC range (100 to 280 nm) with a maximum approximating
260 nm, although strong absorption occurs at longer (UVB)
wavelengths (280 to 315 nm). It is these which are
believed to be responsible for tropospheric degradation.
UV absorption_ causes photodegradation, leading to the
formation of methyl radicals and iodine radicals.
The estimated lifetime of methyl iodide in the
troposphere is between about 50 hours and about 8 days,
as compared to methyl bromide with an estimated
atmospheric lifetime of 1.5-years [Lovelock, J.E. et al.,
WO 96/12559 2 1978 p 2 PCT/DS95/11984
7 O
Nature 241.:194-196 (1973); Chameides, W.L. et al., J.
Geophys. Res. 85(12):7383-7398 (1980)]. As a consequence,
MI has not been intimated in stratospheric ozone depletion
[Rassmussen, R.A. et al., J. Geophys. Res. 87(C4):3086-
3090 (1982)]. MI has a vapor pressure of approximately
25 e that of MBr and hence is less volatile, and has a
similar solubility in water. Due to its rapid photolysis
in the troposphere, MI (unlike MBr) is rapidly removed
from the atmosphere. MI occurs at saturated levels in the
ocean and is principally produced by marine algae
[Chameides et al. (1990), supra; Korzh, V.D., Atmospheric
Environ. 18(12):2707-2710 (1984)]; it is postulated that
this is the principal source of MI in the marine boundary
layer. Levels of MI in the atmosphere adjacent to the
marine boundary layer are usually 2.5 times lower [Korzh
(1984), supra].
As with other halogens, the postulated chemistry of
iodine if it reached the stratosphere suggests that it
would be very effective in ozone destruction [Rolston &
Glauz (1982), supra]. However, the above reasons and the
very short life of MI in the atmosphere negate the
likelihood of any substantial migration of MI to the
stratosphere. As the atmospheric life of MBr is
approximately 1.5 years, it clearly has an ozone-depleting
potential several orders of magnitude higher than MI.
Studies on trifluoromethyl iodide have not shown any
involvement of this substance with ozone depletion. This
substance is similarly broken down by solar radiation to
reactive radicals; as with CH3I, it does not reach the
stratosphere due in part to itsshort tropospheric half
life.
Application of MI in accordance with the present
invention may be effected by a number of different
procedures as are currently routinely employed for soil
treatments with MBr. Thus, for example, MI may be applied
to the soil by tractor mounted injectors on tynes,
WO 96/12559 PCTIUS95/11984
2197882
8
manually in canisters and via an existing irrigation
system or as a gas through lay flat tubing. MI may
advantageously be pre-heated by passage through a heat
exchanger prior to delivery; pre-heating vaporizes MI for
more rapid and even distribution and increases its
activity. In addition, MI may be dissolved in suitable
solvents (e.g., lower alcohols, acetone, mixtures of water
with acetone or alcohol, etc.) to assist in dispersion of
the material in the soil. Further, it is contemplated as
within the scope of the invention to apply mixtures of MI
with other fumigants (e.g., carbon disulfide or
chloropicrin) in ratios comparable to those currently
employed with MBr. For example, a mixture of 67;; MI and
33%- chloropicrin would beeffective, as would a mixture
of about 98% MI with 2%- chloropicrin as a warning agent.
in general, it is preferred that tarping be undertaken
immediately following fumigation. The duration of the
fumigation treatment and the application and removal of
tarps should be consistent with contemporary practice in
connection with MBr treatments.
A wide range of application rates of MI have been
found suitable in accordance with the present invention.
Those working in the field would of course be readily able
to determine in an empirical manner the optimum rates of
application for any given combination of crops, soils and
plant pathogens. in general, application of MI is
preferably effected at a rate of about 2 lb/acre to about
2000 lb/acre (2.23 kg/hectare to about 2250 kg/hectare),
more preferably about 500 lb/acre to about 1500 lb/acre
(560 kg/hectare to about 1680 kg/hectare), and most
preferably about 600 lb/acre to about 1200 lb/acre (670
kg/hectare to about 1340 kg/hectare). Applications of MI
at rates substantially in excess of about 2000 lb/acre
(2250 kg/hectare) would not be expected to provide any
significant advantage over applications within the
preferred ranges specified herein, but are nonetheless
WO 96n2559 2197882 PCT/US95/11984
9
regarded as well within the scope of the present
invention.
Soil fumigation with MI in accordance with the
present invention has been found to be extremely effective
in the substantial or complete elimination of a wide
variety of plant pathogens. For purposes of the present
invention, substantial elimination of a plant pathogen is
intended to mean reduction in the population of the
pathogen by about 90%, more preferably about 95%, and most
preferably about 100%-. In general, treatment in
accordance with the present invention by application of
ari amount of MI within the preferred ranges specified
herein results in almost complete elimination of plant
pathogen populations within the present limits of
customary means employed for the detection thereof.
Plant pathogenic organisms successfully controlled
or eliminated by treatments in accordance with the present
invention- include, but are not limited to, nematodes,
fungi and weeds. Particular plant pathogens and nematodes
controlled or eliminated by application of MI include, but
are not limited to, the following: root rot pathogens
(Phytophthora spp., Pythium spp., Rhizoctonia spp.,
Fusarium spp.) ; vascular wilt pathogens (Verticillium
spp., Fusarium spp.); root knot nematodes (Meloidogyne
spp.); root lesion nematodes (Pratylenchus vulnus); ring
nematodes (Circonemella xenoplax); stubby root nematodes
(Paratr.ichodorus spp.); stem and bulb nematodes
(Ditylenchus dipsaci); cyst nematode (Heterodera
schachtii); citrus nematode (Tylenchulus semipenetrans)
and the burrowing nematode (Radopholus similus). While
the definition of "weed" in agriculture is of course
purely contextual, among the types of plants generally
sought to be controlled or eliminated the following should
be mentioned: cheeseweed (Malva spp.), field bindweed
(Convo2vulus arvensis), annual bluegrass (Poa annua), etc.
MI treatment is also useful in the control of other
WO 96/12559 2197882 PCTIUS95/11984*
pathogens, such as crown gall (Agrobacterium turnefaciens)
and other plant pathogenic bacteria. Finally, as
previously reported in the literature treatment with MI
may also reduce or eliminate the populations of a variety
5 of insects. Insects of particular interest in agriculture
which are controlled or eliminated during a treatment in
accordance with the present invention include, but are not
limited to, the following: fungal gnat larvae, soil mealy
bugs, phylloxera, ants, termites and animal parasites,
10 etc.
The invention may be better understood with reference
to the accompanying examples, which are intended for
purposes of illustration only and should not be construed
as in any sense limiting the scope of the invention as
defined in the claims appended hereto.
Examples
Fungi used were maintained as stock cultures and
transferred to 15 cm potato dextrose agar (PDA) petri
plates as needed. Plate colonies were allowed to grow at
ambient laboratory temperature (ca. 25 C). When 3/4 of
the agar surface was covered the cultures were considered
ready for use. Circular plugs, 18 mm in diameter, were
cut from the leading edge of colony growth with a sterile
cork borer and used to inoculate sterile millet seed.
Three hundred ml of white millet seed was placed in
950 ml (1 qt) Mason canning jars, rinsed with distilled
water and drained. The jars were sealed with canning lids
and rings. The lids had 12mm holes plugged by non-
absorbent cotton. The jar tops were then covered-with a
double layer of heavy brown paper secured by masking tape.
The jars were placed in a deep, autoclavable plastic pan
to which water was added until it passed the level of seed
ir. the jars. The seed was sterilized for 30 min at 250
C and 1 atm of pressure. After sterilizing, the seed was
cooled to room temperature and 100 ml of a 1:9 sterile V-8
juice-water mixture was added to each jar. The millet was
WO 96/12559 2197882 PCT/US95/11984
11
then inoculated with 10 circular agar plugs of the
appropriate fungus and incubated at laboratory
temperatures until used or discarded. Jars were shaken
periodically to distribute the fungal growth. Seed not
used within 30 days was discarded. For smaller amounts
of inoculum, 100 ml of seed was used and incubated in 500
ml Erlenmeyer flasks. These flasks were sealed with a
cotton plug and covered with aluminum squares.
When used, millet seed cultures were removed from the
jars, broken up by hand into individual seeds and added
to the appropriate soil for the experiment. The seed
culture was thoroughly mixed into the soil at a ratio of
300 ml to 3.5 1 of soil.
Soils used for inoculum were a 1:1 potting mix of
topsoil and sawdust or wood shavings for the laboratory
experiments and field soil sieved through a No. 10 screen
for field trials. Moisture in the inoculum soil ranged
from 8.4% to 32%, depending on the trial. Soils were
sterilized by autoclave before adding inoculum.
Inoculum containers were made from 45 ml clear
plastic vials (No. 55-12, Thornton Plastic Co., Salt Lake
City, Utah). Each vial was perforated by sixteen 1 cm
holes using an Unger electric soldering iron with 1/2 cm
tip. The holes were distributed in two rows of 4 and two
of three (on opposite sides) with one hole in the bottom
and one in the white plastic snap cap.
After the vials were filled with inoculum, those used
in laboratory trials were placed on a 1 cm layer of
potting mix in 1893 ml (2 qt) Mason canning jars and
covered with the same soil to a depth of 1 to 1 1/2 cm.
The jars were placed under a fume hood and a measured
amount of the fumigant was injected into each jar using
a micropipette with the appropriate tip. The fumigant was
placed on the soil just inside the mouth of the jar. The
jars were sealed immediately with a solid canning lid and
ring and placed horizontally on the laboratory bench to
_. ..._ ....... .._.. ....-_ :_.< .-._...
2197882
WO 96/12559 PCTIUS95/11984
12
incubate. Incubation was for 1, 2 or 3 days depending on
the trial. Each experiment contained 4 replications of
25 seeds each per treatment.
After fumigation the vials were removed from the soil
and ventilated under the hood for one hour. After
ventilating the seed were separated from the soil by
sieving through a No. 10 soil sieve. Twenty five seeds
from each replicate were chosen and placed on agar in 15
cm petri plates. For Pythium spp. PARP medium was used,
and for Phytophthora PARPH medium [Jeffers & Martin, Plant
Disease 70:1038-1043 (1986)1; for Rhizoctonia, a medium
was used as reported in the literature [Ko & Hora,
Phytopathology 61:707-710 (1971)]. Other fungi were
plated on 1/4 strength PDA medium [Plant Pathologists
Pocketbook (1968) Commonwealth Mycological Institute, p.
2397. After plating seeds were incubated at laboratory
temperatures and surveyed for growth after 2 days. Seeds
showing growth were counted and the plates checked until
no more growth appeared, usually 3 - 4 days. After the
results were recorded the plates were disposed of by
sterilization.
In field trials the inoculum was prepared as
described aboveand placed at depths of 2.5, 15 and 30 cm
half way between the center and one corner of each plot.
The plots were 3 x 3 m and the corner for placement of the
inoculum was chosen randomly. Field trials were block
randomized with 4 replications per treatment. After the
fumigant was applied the plots were covered with 4 mil
clear polyethylene plastic sheeting with the edges buried
7 cm.
Methyl bromide was prepared by storing 454 g
containers and laboratory glass beakers 14 h in a portable
ice chest with frozen COZ_ When used, the treatment
amount was measured, poured into a chilled beaker, placed
on the soil surface in the center of the plot, and covered
with an inverted 15 cm black plastic plant pot. Methyl
2197882
WO 96/12559 PCT/US95/11984
13
iodide was treated the same way but was not prechilled.
The plot was then covered with plastic sheet. The control
was no treatment covered with plastic. After 4 days the
plastic was removed and the plots were allowed to aerate
for 2 days. The inoculum vials were then removed and
evaluated as described.
All fumigation concentrations were based on a methyl
bromide application rate of 0.454 kg/2.8 m3 (1 lb/l00
ft'), equal to 4.78 moles/2.8 m3 for field trials and 1.69
.M/ml for laboratory trials.
WO 96/12559 219/-7 pDpU2 PCT/US95/11984S
I 14
Example 1
This series of trials utilized Phytophthora cinnamomi
and Rhizoctonia solani as the test organisms. MI
concentrations used were 1.69, 1.27, 0.84 and 0.42 EcM/ml.
Fumigation time periods were 24, 48 and 72 hours.
In this series all non-treated controls for both
Phytophthora and Rhizoctonia had a 1002; recovery rate
based on an average of 4 replications of 25 seeds each.
Cultures of Phytophthora and Rhizoctonia fumigated 1 day
at 0.42 M/ml had recovery rates of 191 and 72%,
respectively. After 2 days both had no recovery, while
after 3 days at this concentration Rhizoctonia had a 1%
recovery rate. All other concentrations were completely
effective with no recovery of either fungus.
Example 2
This series of trials utilized P. cinnamomi, R.
solani and P. citrophthora as the test organisms. MI
concentrations were 1.69, 1.27, 0.84, 0.42 and 0.21 M/ml.
Fumigation time periods were 24, 48 and 72 hours.
Upon collecting data it was found that the
Rhizoctonia culture was contaminated with an Aspergillus
sp. so data was collected on that species. All non-
treated controls for all three organisms for all three
time periods were 100; viable. The lowest concentration
of 0.21 M/ml MI (= 0.125 lb MBr/100 ft2) was ineffective
for all three time periods for P. citricola and the 1 day
and 2 day periods for P. cinnamomi and Aspergillus sp.
with 100% recovery. At 3 days at this concentration both
P. cinnamomi and Aspergillus had a recovery rate of 55%.
At 0.42 M/ml MI (=0.250 lb MBr/100 ft2) P. citricola had
a 54% recovery after 1 day and 0 after 2 and 3 days, while
P. cinnamomi had 65% at 1 day and 0 at 2 and 3 days;
Aspergillus had 25% at 1 day and 0 after 2 and 3 days.
At 0.84 M/ml MI (=0.5 lb MBr/100 ft2) there was no
recovery of P. citricola, while P. cinnamomi had a 25%
recovery after 2 days but 0 for day 1 and 3; Aspergillus
2197882
wo 96/12559 PCT/US95/11984
had a 20% recovery after 1 day but 0 for days 2 and 3.
Concentrations of MI at 1.27 M/ml (=0.75 lb MBr/100 ft2)
and 1.69 M/ml (= 1.0 lb MBr/100 ft2) for all time periods
had 0 recovery (Table 1).
5 In all of the tables, numbers followed by different
letters are significantly different at p=.05 using the
Duncan-Waller T test.
TABLE 1
10 P. citricola
M/ml MI Davs Recovery % Note
0 1 100 a
15 0 2 100 a
0 3 100 a
0.21 1 100 a
0.21 2 100 a
0.21 3 100 a
0.42 1 54 bc
0.42 2 0 d
0.42 3 0 d
0.84. 1 0 d
0.84 2 0 d
0.84 3 0 d
1.27 1 0 d
1.27 2 0 d
1.27 3 0 d
1.69 1 0 d
1.69 2 0 d
1.69 3 0 d
WO 96/12559 21 9788 2 PCT/US95/11984*
16
TABLE 1 (Contd.)
P. cinnamomi
ml MI Davs Recovery o Note
0 1 100 a
0 2 100 a
0 3 100 a
0.21 1 100 a
0.21 2 100 a
0.42 1 65 b
0.21 3 55 bc
0.84 2 25 cd
0.42 2 0 d
0.42 3 0 d
0.84 1 0 d
0.84 3 0 d
1.27 1 0 d
1.27 2 0 d
1.27 3 0 d
1.69 1 0 d
1.69 2 0 d
1.69 3 0 d
- - - - -
W 96'12559 2 1 9 7 8 8 2 PCTIUS95111984
17
TABLE 1 (Contd.)
AsnercTillus
ml MI Davs Recovery s Note
0 1 100 a
0 2 100 a
0 3 100 a
0.21 1 100 a
0.21 2 100 a
0.21 3 55 bc
0.42 1 25 cd
0.84 1 20 d
0.42 2 0 d
0.42 3 0 d
0.84 2 0 d
0.84 3 0 d
1.27 1 0 d
1.27 2 0 d
1.27 3 0 d
1.69 1 0 d
1.69 2 0 d
1.69 3 0 d
:.:_f.'. . . _. .__... . _.
WO 96/12559 2 1 9 7 8 8 2 PCTIUS95/11984fl
18
Example 3
This series of trials utilized P. cinnamomi, P.
citricola, P. parasitica and R. solani. MI concentrations
were 1.69, 1.27, 0.84, 0.42, and 0.21 M/ml. Fumigation
time periods were 24, 48 and 72 h.
Control recovery for P. cinnamomi was 100 % at 1 day,
99% at 2 days and 100% at 3 days. The P. cinnamomi
recovery rate at 0.21 AM/ml MI (= 0.125 lb MBr/100 ftz)
was 62% after 1 day, 64% after 2 days and 62% after 3
days. At 0.42 AM/ml MI (=0.25 lb MBr/100 ft2) the rate
after i'day was 39%, after 2 days 23% and after 3 days 5%.
There was no recovery at the higher concentrations of MI
at 0.84 AM/ml (=0.5 lb MBr/100 ft2), 1.27 M/ml (=0.75 lb
MBr/100 ft2) and 1.69 M/ml (= 1.0 lb MBr/100 f t2) for any
time period (Table 2).
For P. citricola recovery rates after 1 day were
control 100%, 0.21 M/ml MI 100%, 0.42 AM/ml MI 100%, 0.84
AM/ml MI and higher 0%. After day 2 the control was 100%,
0.21 /CM/ml MI was 85%, 0.42 M/ml MI was 4% with all
higher concentrations 0%. After 3 days the control was
99%, the 0.21 M/ml MI was 61% and all other
concentrations were 0 (Table 2).
For P. parasitica recovery for the control, 0.21 and
0.42 AM/ml MI after 1 day were all 100% and all higher
concentrations were 0. After 2 days the control and 0.21
M/ml MI recovery was 100%, and at 0.42 AM/ml MI it was
54%; all other concentrations were 0. After 3 days
exposure recovery of the control was 98%, 0.21 M/ml MI
was 100% and 0.42 AM/ml MI was 76%; all other
concentrations were 0 (Table 2).
For Rhizoctonia after 1 day recovery was 100% for the
control, 0.21 and 0.42 M/ml MI and 29% for 0.84 AM/ml MI.
All other concentrations were 0. After 2 days the control
and 0.21 AM/ml MI were 100 %, 0.42 was 93% and all other
= WO 96/12559 . 2 19 7 8 Q2 PCT/US95/11984
19 v
concentrations were 0. After 3 days the control and 0.21
M/ml MI were recovered at 100o and 0.42 at 48%; all other
concentrations were 0 (Table 2).
W0 96/12559 21// U J L PCl'/US95/119841*
TABLE 2
P. citricola
5
am/m1 MI Davs Recoverv % No e
0 1 100 a
0 2 100 a
0 3 100 a
10 0.21 1 100 a
0.42 1 98 a
0.21 2 85 ab
0.21 3 61 c
0.42 2 4 d
15 0.42 3 0 d
0.84 1 0 d
0.84 2 0 d
0.84 3 0 d
1.27 1 0 d
20 1.27 2 0 d
1.27 3 0 d
1.69 1 0 d
1.69 2 0 d
1.69 3 0 d
- - - - -
WO 96/12559 2197 8 8 2 PCT/US95/11984
~ 21
TABLE 2 (Contd,)
P. cinnamomi
M/ml MI Dave Recovery 's Note
0 1 100 a
0 2 100 a
0 3 100 a
0.21 2 72 bc
0.21 1 62 c
0.21 3 62 c
0.42 1 39 c
0.42 2 23 d
0.42 3 5 d
0.84 1 0 d
0.84 2 0 d
0.84 3 0 d
1.27 1 0 d
1.27 2 0 d
1.27 3 0 d
1.69 1 0 d
1.69 2 0 d
1.69 3 0 d
2197882
WO 96/12559 PCT/U895/11984
1*
22
TABLE 2 (Contd.)
P. Parasitica
uM/ml MI Days Recovery % Note
0 1 100 a
0 2 100 a
0 3 100 a
0.21 1 100 a
0.21 2 100 a
0.21 3 100 a
0.42 1 100 a
0.42 3 76 ab
0.42 2 54 bc
0.84 1 0 d
0.84 2 0 d
0.84 3 0 d
1.27 1 0 d
1.27 2 0 d
1.27 3 0 d
1.69 1 0 d
1.69 2 0 d
1.69 3 0 d
~ WO 96/12559 2 1 9 7 8 8 2 PC1'/US95/11984
23
TABLE 2 (Contd.)
R. Solani
M/ml MI Da s Recoverv Note
0 1 100 a
0 2 100 a
0 3 100 a
0.21 1 100 a
0.21 2 100 a
0.21 3 100 a
0.42 1 100 a
0.42 2 100 a
0.42 3 48 bc
0.84 1 29 c
0.84 2 0 d
0.84 3 0 d
1.27 1 0 d
1.27 2 0 d
1.27 3 0 d
1.69 1 0 d
1.69 2 0 d
1.69 3 0 d
WO 96/12559 2197882 PCT/US95/11984*
24
Example 4
These trials utilized P. citrophthora, P. citricola,
P. parasitica and R. solani. MI concentrations were 1.69,
1.27, 0.84, 0.42, and 0.21 M/ml. Fumigation time periods
were 24, 48 and 72 hours.
After 1 day recovery of P. citrophthora was 100% for
the control and 0.21 AM/ml MI. After 2 days control
recovery was 100% and at 0.21 AM/ml MI it was 32%. After
3 days recovery was 100% for the control and 10% for 0.21
AM/ml MI. All other exposures were 0.
For P. citricola the control at 1 day exposure was
recovered 100% and 0.21 AM/ml MI recovery was 33%. After
2 days recovery was 100% for the control, 1% for 0.21
AM/ml MI and 2% for both 0.84 and 1.69 AM/ml MI. After
3 days recovery for the control was 100%; recovery at all
other exposures was 0.
For P. parasitica recovery in the control was 100%
for all three time periods while at 0.21 AM/ml MI recovery
at 1 day was 98% and at 2 days was 19%. All other
exposures were 0.
In this trial recovery of Rhizoctonia was 100% for
all three time periods for the control and 0.21 AM/ml MI
and for 1 day of 0.42 AM/ml MI. Recovery at 2 days of
0.42 AM/ml MI was 32% and at 3 days was 91%. At 0.84
AM/ml MI recovery was 30%, 44% and 45% for 1, 2 and 3
days, respectively. At 1.27 AM/ml MI recovery was 17%,
43% and 68% for the same three time periods. At 1.69
AM/ml MI recovery was 20% at one day, 53% at 2 days and
78% at 3 days.
WO 96/12559 2 1 9 7 8 8 2 PCP/US95/11984
TABLE 3
P. citricola P. citrophthora
M/ml day recove note M/ml day recove note
5 MI a ry% MI s ry %
0 1 100 a 0 1 100 a
0 2 100 a 0 2 100 a
0 3 100 a 0 3 100 a
0.21 1 33 b 0.21 1 100 a
10 1.69 2 2 c 0.21 2 10 b
0.21 2 1 c 0.21 3 10 b
0.21 3 0 c 0.42 1 0 c
0.42 1 0 c 0.42 2 0 c
0.42 2 0 c 0.42 3 0 c
15 0.42 3 0 c 0.84 1 0 c
0.84 1 0 c 0.84 2 0 c
0.84 2 0 c 0.84 3 0 c
0.84 3 0 c 1.27 1 0 c
1.27 1 0 c 1.27 2 0 c
20 1.27 2 0 c 1.27 3 0 c
1.27 3 0 c 1.69 1 0 c
1.69 1 0 c 1.69 2 0 c
1.69 3 0 c 1.69 3 0 c
,. .
WO 96/12559 2 i 9 788~ p~~S95/11984~
26
P. parasitica R. solani
M/ml day recove note M/ml day recove note
MI s ry ~ MI s ry $
0 1 100 a 0 1 100 a
0 2 100 a 0 2 100 a
0 3 100 a 0 3 100 a
0.21 1 98 a 0.21 1 100 a
0.21 2 19 b 0.21 2 10 a
0.21 3 0 b 0.21 3 100 a
0.42 1 0 b 0.42 1 100 a
0.42 2 0 b 0.42 3 91 ab
0.42 3 0 b 1.69 3 78 abc
0.84 1 0 b 1.27 3 68 bcd
0.84 2 0 b 1.69 2 53 cde
0.84 3 0 b 0.84 3 45 def
1.27 1 0 b 0.84 2 44 def
1.27 2 0 b 1.27 2 43 def
1.27 3 0 b 0.42 2 32 ef
1.69 1 0 b 0.84 1 30 efg
1.69 2 0 b 1.69 1 20 fg
1.69 3 0 b 1.27 1 17 fg
W096/12559 - 2197882 PCT/US95111984
27
Example 5
Alkyl iodides tested were methyl iodide, 1-
iodoethane, 1-iodopropane, 2-iodopropane, 1-iodobutane,
1-iodopentane, diiodomethane, and 1-iodo-2-methylpropane.
Inoculum was preparecl and trials were performed as in
Example 1. The chemicals were compared on a molar basis
with rates of 1.27 and 0.42 M/ml (equal to 3/4 lb and 1/4
lb methyl bromide/100 ft3, respectively) The test
organism was Phytophthora parasitica. Soil moisture was
24%. Fumigation exposure was 48 hours with 4 replications
of 25 seeds each per treatment.
In this trial methyl iodide was the most effective
compound with 0 recovery at both concentrations (1.27 and
0.42 M/ml = to 3/4 lb MBr and 1/4 lb MBr/100 ft3) . This
was followed by diiodomethane with a 62"s recovery at the
high concentration. All other concentrations were not
significantly different from the control (Table 4).
er: . . __._ _. . . .
WO96/12559 219 7 8 8 2 PCT/US95/11984*
28
TABLE 4
Chemical M/ml MI % survival Note
None 0 97 a
1-iodo-2- 1.27 93 a
methyl-propane
1-iodo-2- 0.42 98 a
methyl-propane
1-iodo-pentane 1.27 92 a
1-iodo-pentane 0.42 96 a
1-iodo-butane 1.27 90 a
1-iodo.-butane 0.42 93 a
2-iodo-propane 1.27 94 a
2-iodo-propane 0.42 92 a
1-iodo-propane 1.27 94 a
1-iodo-propane 0.42 91 a
1-iodo-ethane 1.27 81 a
1-iodo-ethane 0.42 82 a
Di-iodo- 0.42 84 a
methane
Di-iodo- 1.27 62 b
methane
Methyl iodide 0.42 0 c
Methyl iodide 1.27 0 c
-
Example 6
The soil was prepared for this trial as in Example
5; soil moisture was 32%. Rates used were 1.27 and 0.42
M/ml for methyl iodide and 2.54 and 1.27 M/ml for all
other chemicals. Fumigation exposure was 48 hours with
4 replications of 25 seeds each per treatment. Methyl
iodide was again the most effective compound with 0
recovery at both rates (1.27 and 0.42 M/ml = to--3/4 lb
and 1/4 lb/100 ft3). This was followed by 1-iodoethane at
2.54 M/ml (= to 1.5 lb MBr/100 ft3). All other
2197882
WO 96/12559 PGT/US95/11984
29
concentrations were not significantly different from the
control.
TABL$ 5
Chemical M/ml MI ~ survival Note
None 0 98 a
1-iodo-2- 2.54 100 a
methyl-propane
1-iodo-2- 1.27 100 a
methyl-propane
1-iodo-pentane 2.54 100 a
1-iodo-pentane 1.27 100 a
1-iodo-butane 2.54 100 a
1-iodo-butane 1.27 100 a
2-iodo-propane 2.54 99 a
2-iodo-propane 1.27 100 a
i-iodo-propane 2.54 100 a
1-iodo-propane 1.27 100 a
Di-iodo- 2.54 100 a
methane
Di-iodo- 1.27 100 a
methane
1-iodo-ethane 1.27 100 a
1-iodo-ethane 2.54 0 b
Methyl iodide 0.42 0 b
Methyl iodide 1.27 0 b
WO 96/12559 PCT/US95/1198.0
2197882
Example 7
This trial was a comparison of inethyl iodide,
diiodomethane and 1-iodoethane at 0.42, 0.84, 1.27, 1.69,
and 2.11 M/ml (equal to 1/4, 1/2, 3/4, 1 and 1 1/4 lb
5 methyl bromide/100 ft'). Phytophthora parasitica was used
as the test organism. Soil moisture was 32~; with a
fumigation time period of 48 hours. There were 4
replications per treatment. Methyl iodide applications
at all concentrations were the best treatments and were
10 significantly different from all other treatments. This
was followed by diiodomethane and 1-iodoethane at 2.11 and
diiodomethane at 1.69 and 1.27 M/ml. All other
treatments were not significantly different from the
control.
15 TABLE 6
Chemical M/ml MI % survival Note
None 0 100 a
Di-iodo-methane 0.42 100 a
20 Di-iodo-methane 0.84 100 a
1-iodo-ethane 0.42 100 a
1-iodo-ethane 0.84 100 a
1-iodo-ethane 1.27 100 a
1-iodo-ethane 1.69 100 a
25 Di-iodo-methane 1.27 87 b
Di-iodo-methane 1.69 78 bc
1-iodo-ethane 2.11 70 c
Di-iodo-methane 2.11 66 c
Methyl iodide 0.42 0 d
30 Methyl iodide 0.84 0 d
Methyl iodide 1.27 0 d
Methyl iodide 1.69 0 d
Methyl iodide 2.11 0 d
WO 96/12559 2 1 978Vp2 PCT/US95/11984
31
Examole 8
The test soil'for this field trial was a sandy loam
averaging 5.85% moisture at 15 cm. The trial was a
randomized block with 7 treatments of 4 replications each.
The test organism was Phytophthora parasitica prepared as
described above and incubated on the laboratory bench
overnight before placement in the field. Fumigants used
were methyl bromide at 454, 227 and 113.5 g/9 m2 (1, 1 /2
and 1/4 lb/100 ft2 ) and methyl iodide at 684, 342 and 171
g/9 mZ (1.5, 0.75 and 0.325 lb/10o ft2). These rates are
4.8, 2.4 and 1.2 moles.
Methyl iodide and methyl bromide were similar in
performance. There were low percentages of recovery in
six fumigated plots. At 2.4 M both MI and MEr had two
plots with recovered organisms. MI had a 1 percent
recovery at 4.8 M and MBr had a 1 percent at 1.2 M. The
highest rate of recovery for MBr was 31. at 2.4 M and a 12
inch depth, while for MI it was 4% at 2.4 M at 6 inches.
All controls were recovered at 100% (Table 7).
WO 96/12559 219 7882 PCTIUS95/11984
32
TABLE 7 (field trial 1)
Chemical M/100 ft3 depth (in) % recovery Note
Control 0 1 100 a
Control 0 6 100 a
Control 0 12 100 a
MI 2.4 6 4 b
MBr 2.4 12 3 bc
MI 2.4 1 2 bc
MBr 2.4 6 1 bc
MI 4.8 6 1 bc
MI 1.2 12 1 bc
MBr - 1.2 1 0 c
MBr 1.2 6 0 c
MBr 1.2 12 0 c
MBr 2.4 1 0 c
MBr 4.8 12 0 c
MI 1.2 1 0 c
MI 1.2 6 0 c
MI 2.4 12 0 c
MI 4.8 1 0 c
MI 4.8 12 0 c
MBr 4.8 1 0 c
MBr 4.8 6 0 c
PCT/IIS95/11984
WO 96/12559 2197882
33 Example 9
in this field trial, soil moisture averaged 9.5%
between 15 and 30 cm. Methyl bromide was applied as in
Example 8. Methyl iodide was mixed with 95% ethanol and
poured in a cross pattern across the plot for better
distribution. Fumigant rates were as in Example B. The
ethanol was mixed at 160, 80 and 40 ml for the high,
medium and low rates, respectively. Controls were non-
treated and ethanol at 160 ml/plot. Plots were fumigated
for 4 days and aerated 1 day before plating.
MI and MEr were again similar in performance,
although the percent recovery in fumigated plots ranged
from 24 to 45% at rates of_1.2 M for 4 plots (2 MI and 2
MBr) and 2.4 M for one plot (MBr). Controls at 6 and 12
inch depths were recovered at 99 to 100%. All treatments
at 1 inch depth had 0% recovery due to the effects of
solarization (Table 8).
wo 96/12559 2 19 7 8 8 2 PCT/US95/11984*
34
TABLE 8 (field trial 2)
Chemical M/100 ft' depth (in) % Note
recovery
Control 0 6 100 a
Control 0 12 99 a
Ethanol 0 6 99 a
Ethanol 0 12 99 a
MI 1.2 12 45 b
MEr 2.4. 12 25 bc
MEr 1.2 6 25 bc
MBr 1.2 12 25 bc
MI 1.2 6 24 bc
MEr 1.2 1 0 c
MBr 2.4 1 0 c
MBr 2.4 6 0 c
MBr 4.8 1 0 c
MBr 4.8 6 0 c
MBr 4.8 12 0 c
MI 1.2 1 0 c
MI 2.4 1 0 c
MI 2.4 6 0 c
MI 2.4 12 0 c
MI 4.8 1 0 c
MI 4.8 6 0 c
MI 4.8 12 0 c
Control 0 1 0 c
Ethanol 0 1 0 c
WO 96/12559 PCT/US95/11984
35 2197882
Example 10
The effects of MI fumigation on three weed seeds were
determined. The percent survival of these seed after
fumigation with different concentrations of MI is reported
M1
in Table 9. The percent survival is calculated by
dividing the number of treated germinated seeds by the
number of untreated germinated seeds.
Treatmen Weed Species
t
M/ml MI Annual Cheeseweed Field
Bluegrass Bindweed
1.69 0 0 3.6
1.27 0 0 1.8
0.84 0 0 0
0.42 0 0 1.8
0.21 0 0 5.4
Bxamnle 11
The effects of MI treatment on the nematode
Meloidogyne incognita were determined. The percent
survival after fumigation at different concentrations of
MI are reported in Table 10. The percent survival was
calculated by dividing the number of treated surviving
nematodes by the number of untreated surviving nematodes.
M/ml MI percent survival
0.052 0
0.026 0
0.013 0
0.006 55
0.003 65
WO 96/12559 2197882 - PCT/US95/11954*
36
Example 12
The effects of MI on the citrus nematode Tylenchulus
semipenetrans were determined. The numbers surviving
after fumigation at different concentrations of MI are
reported in Table 11.
Rate (lb/ac - Mean Fisher's
M/container) protected LSD
ip=.05
25 lb/ac (0.95 0.000 a
M)
15 lb/ac (0.57 0.250 a
M)
5 lb/ac (0.19 4.000 a
M)
2 lb/ac (.072 64.750 b
M)
0 lb/ac (0 M) 223.000 c
Example 13
The effects of methyl iodide, methyl bromide, clear
and black plastic covers on survival of weeds in the soil
were examined.
Plastic3 No Treatment Methyl Methyl
Bromide' Iodide
None 23 Not used Not used
Clear 1.5 4.75 5
Black 2.25 4.75 4.5
14 mil thick. ZMethyl bromide and methyl iodide were used
at 4.8 M/100 ft2. 'Rating 1-5: 1 = dense weed population;
5 = no weeds.
While the present invention has been described with
reference to preferred embodiments and illustrative
examples, it should be understood that one of ordinary
skill in the art after reading the foregoing specification
would be able to effect various changes, substitutions of
equivalents and modifications to the methods as described
WO 96/12559 PCT/US95/11984
37 2197882
herein. Therefore, it is intended that the scope of the
invention not be limited by reference to the illustrative
examples, but rather with reference to the accompanying
claims.
