Title of Invention

A METHOD FOR PRODUCING IMPROVED BIOPESTICIDAL MATERIALS BY FERMENTATION

Abstract The invention discloses a method for producing improved biopesticidal materials by fermentation comprising the steps of: fermenting a bacterium or fungus to obtain a fermentation broth; adjusting the pH of said fermentation broth to a pH of below 2.5 with a biologically acceptable acid, or to a pH of above 8 with a biologically acceptable alkali or base; and heat treating said fermentation broth to a temperature of at least 100°C for at least 15 minutes, then cooling to ambient temperature to obtain a pH-adjusted, heat treated composition having improved biopesticidal activity; with the proviso that said fungus is not Myrothecium verrucaria when the pH of said fermentation broth is adjusted to a pH of below 2.5.
Full Text A METHOD FOR PRODUCING IMPROVED BIOPESTICIDAL MATERIALS
BY FERMENTATION
Field of the Invention
The present invention is directed to a method for producing a new nematocidal
composition particularly useful against plant parasitic nematodes and also a process to
prevent damage resulting from nematode infestation. The method for production of the
composition involves heating a pH-adjusted fermentation broth of microorganisms to a
temperature of at least about 100°C for at least about 15 minutes.
Background of the Invention
Plant parasitic nematodes cause serious economic damage to many agricultural
crops around the world. The nematodes in this group are microscopic worms and are, in
general, obligate parasites of plants. They feed mostly on the roots of host plants;
however, several genera are known to parasitize above-ground parts including stems,
leaves and flowers as well. Almost all the plant species of economic importance are
susceptible to infection by some species of nematodes (notable exceptions are in the
marigolds and asparagus). For example, root knot nematodes (RKN), (Meloidogyne spp.)
are capable of parasitizing more than 3,000 species of crop plants. These plants include
agronomic crops, vegetables, fruits, flowering trees and shrubs. Nematodes reportedly
cause crop loss equivalent to more than six billion dollars in the United States alone and
more than one hundred billion dollars around the world.
The symptoms due to parasitic nematode injury vary widely depending on the
plant host, the nematode species, age of the plant, geographical location and climatic and
external environmental conditions. In general, an overall patchy appearance of plants in a
Considered indicative of nematode infestation. More specifically, nematode injury
; of the roots (abnormal swelling in the tissue due to rapid multiplication
of cells in the cortical region) caused by species of root knot (Meloidogyne spp.)
and cyst (Heterodera spp.) nematodes, lesions (localized, discolored areas) caused by
lesion nematodes (Pratylenchus spp.), suppression of cell division resulting in stubby
roots (Trichodorus spp.), growth abnormalities including crinkling or twisting of above-
ground parts (Aphelenchoides spp.), and even cell necrosis (death) in some cases. Plant
parasitic nematodes may be endoparasitic in nature, as in the case of the root-knot and
lesion nematodes, or ectoparasitic as in the dagger nematode (Xiphinema spp.) and lance
nematode (Hoplolaimus spp.). Nematodes can be vectors of plant viruses and are also i
known to induce disease complexes predisposing plants to infection by other plant *
pathogenic fungi and bacteria.
Chemical nematocides, either soil fumigants or non-fumigants, have been in use
for many years and are among the few feasible options for countering nematodes. At
present, the process involves repeated applications of synthetic chemicals to the ground
prior to planting the crop. These chemicals are extremely toxic to organisms besides
nematodes and many of them may pose serious threats to the environment. With the
renewed emphasis on clean water and air by environmental groups and governmental
agencies, and the detection of many of these active ingredients or the metabolites thereof
in ground water and several non-target organisms, there has been serious concern as to
the manufacture and/or use of these chemicals. One of the most effective, economical,
and widely used nematocides, DBCP (l,2-dibromo-3-chloropropane), found in ground
water has been judged to induce male sterility and possible carcinogenesis. Another
widely used chemical, EDB (ethylene dibromide), has also been found in ground water.
Yet another very common insecticide-nematocide, aldicarb (2-methyl-2-(methylthio)-
propionaldehyde-O-(methylcarbamoyl)oxime), has been found to be acutely toxic.
Aldicarb has been found in ground water in several regions of United States. Carbofuran
(2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) and 1, 3-D (1,3-
dichlorpropane), two very commonly used nematocides, are under special review by EPA
because of their avian toxicity and possible carcinogenic effects. More recently, the
decision by EPA to limit and eventually discontinue the use of the soil fumigant, methyl
bromide, for agricultural purposes presents a threat to the efficiency and quality of
agricultural production in the United States.
Natural isolates such as N-acetyl-D-glucosamine, which may be derived from
microorganisms which are the waste products of industrial fermentation processes, have '
been disclosed as nematocidal in U.S. Patent No. 5,057,141.
Biopesticides have been developed as an alternative to chemical pesticides. They
are obtained by fermentation and can be used either as crude biomass or purified.
Typically, fermentations are carried out at temperatures in the range of 20 - 40°C. For
example, submerged fermentation at 28-30°C of Paecilomyces funosoroues fungal
isolate ATCC No. 20874 produces fungal biomass for control of nematode infestation as ,
disclosed in U.S. Patent No. 5,360,607; whole fermentation broth from fermentation at
28°C of Streptomyces thermoarchaensis NCIB 12015 is disclosed as nematocidal in U.S.
Patent No. 5,182,207; fermentation broth obtained from fermentation of Streptomyces
cyaneogriseus noncyanogenus NRRL 15773 at 28°C is effective against nematodes as
disclosed in U.S. Patent No. 5,439,934; and fermentation broth obtained by fermentation
of the fungus Myrothecium verrucaria at temperatures of from 25 to 30°C is disclosed as
nematocidal in U.S. Patent No. 5,051,255.
Heating of an infested biomass, as disclosed in U.S. Patent No. 4,229,442, at a
temperature of at least 125°C with water in an amount ranging from 1-5 times the dry
weight of the biomass, may combat nematodes.
However, there is still a need for the development of new and effective
nematocides. It is therefore an object of this invention to provide a method for the
production of nematocidal compositions which are effective and inexpensive.
Brief Summary of the Invention
The invention is directed to a method of producing a nematocide from
fermentation broths of microorganisms by heating a fermentation broth to a temperature
of at least about 100°C after adjusting the pH to about pH 2 or below, or about pH 8 or
above. The nematocide thus produced can be used to prevent plant damage and/or limit
the growth of nematodes.
The present invention is directed to a method for improving biopesticidal activity
of materials produced by fermentation comprising the steps of:
fermenting a bacterium or fungus to obtain a fermentation broth;
adjusting the pH of said fermentation broth to a pH of below about 2.5 with a
biologically acceptable acid, or to a pH of above about 8 with a biologically
acceptable alkali or base; and
heat treating said fermentation broth to a temperature of at least about 100°C for
at least about 15 minutes, then cooling to ambient temperature to obtain a pH-
adjusted, heat treated composition having improved biopesticidal activity;
with the proviso that said fungus is not Myrothecium verrucaria when the pH of said
fermentation broth is adjusted to a pH of below about 2.5.
The invention is also directed to a composition comprising
a heat-treated, pH adjusted fermentation broth of a fungus or bacterium selected from/
the group consisting of Gibberella fujikuroi, Streptomyces erythraeus, Bacillus j
sphaericus. Bacillus thuringiensis and Fusarium moniliforme;
wherein said broth is pH adjusted either to a pH of below about 2.5 with a
biologically acceptable acid, or to a pH of above about 8 with a
biologically acceptable alkali or base;
and wherein said broth is heated to a temperature of at least about 100°C for at
least about 15 minutes, then cooled to ambient temperature.
The invention is also directed to a method for control of nematodes on plants
which comprises the step of administering to the locus, soil or seed of plants in need of
such treatment, a nematocidally effective amount of a composition formed by heating a
pH adjusted fermentation broth from a fungus or bacterium;
wherein said broth is adjusted to a pH of below about 2.5 with a biologically
acceptable acid, or to a pH of above about 8 with a biologically acceptable alkali or base;
and wherein said broth is heated to at least 100°C for at least 15 minutes;
with the proviso that said fungus is not Myrothecium verrucaria when the pH of
said fermentation broth is adjusted to a pH of below about 2.5.
The composition may be administered at a rate of from about 1 to about 200
pounds per acre.
The invention is also directed to a method for producing a nematocidal
composition comprising the steps of:
a) fermenting a bacterium or fungus to obtain a fermentation broth;
b) suspending said broth in an aqueous solution;
c) adjusting the pH of said broth in aqueous solution to a pH below about pH 2.5
with a biologically acceptable acid;
d) heating the pH-treated broth of step c) to a temperature of at least about 100°C
for at least about 15 minutes, then cooling to ambient temperature; and,
e) recovering said composition;
with the proviso that said fungus is not Myrothecium verrucaria.
Preferably, the pH is adjusted below about 2; and most preferably, the pH is
adjusted below about 1.5.
The pH of the composition formed in step d) may be adjusted to a range of about
pH 4 to about pH 8. The fermentation broth of step a) may have water-soluble biomass
and water-insoluble biomass; and the water-soluble biomass may be separated from said
water-insoluble biomass, and the water-insoluble biomass may be suspended in aqueous
solution in step b).
The invention is also directed to a method for producing a nematocidal
composition comprising the steps of:
a) fermenting a bacterium or fungus to obtain a fermentation broth;
b) suspending said broth in an aqueous solution;
c) adjusting the pH of said broth in aqueous solution to a pH above about pH 8
with a biologically acceptable alkali or base;
d) heating the pH-treated broth of step c) to a temperature of at least about 100°C
for at least about 15 minutes, then cooling to ambient temperature;
e) recovering said composition.
Preferably, the pH is adjusted above about 9 and most preferably the pH is
adjusted above about 10.
The pH of the composition formed in step d) may be adjusted to a range of about
pH 4 to about pH 8. The fermentation broth of step a) may have water-soluble biomass
and water-insoluble biomass; and the water-soluble biomass may be separated from said
water-insoluble biomass, and the water-insoluble biomass may be suspended in aqueous
solution in step b).
For the practice of any aspect of this invention, at least one compound such as
herbicides, antimicrobials, fungicides, insecticides, plant growth regulators or nutrients
may be added to the heat-treated, pH adjusted compositions.
Detailed Description of the Invention
To produce a nematocidal preparation, a substantial amount of biomass is prepared
by submerged fermentation of a bacterium or fungal microorganism. Preferred examples
of microorganisms include Baccillus spp., Myrothecium spp., Gibberella spp.,
Streptomyces spp., and Fusarium spp. More preferred are the Myrothecium spp. and the
Bacillus spp.
More specifically, the composition can be obtained from Gibber ella fujikuroi
(ATCC 12616 or 14164), Streptomyces erythraeus (also known as Saccharopolyspora
erythraea, ATCC 11635 or 31772), Bacillus sphaericus (ATCC 4978), Bacillus
thuringiensis such as Bacillus thuringiensis israelensis (ATCC 35646), Bacillus
thuringiensis kurstaki (ATCC 33679) and Bacillus thuringiensis Berliner (ATCC 19268)
and Fusarium moniliforme (ATCC 10052). The composition can be obtained from any
bacterium or fungal microorganism, since the method breaks down the cell wall of the
microorganism. It is believed that the cell walls of microorganisms (branched
polysaccharides) are partially or completely hydrolyzed under the conditions described
herein, resulting in the formation of nematocidal hydrolysis products. Therefore, the
method is not microorganism-specific.
The bacterium or fungal microorganism is suspended or dissolved in an aqueous
solution, preferably water, and is hydrolyzed by acidification to a pH below about pH 2.0
by means of a biologically acceptable acid such as, for example, sulfuric, hydrochloric or
phosphoric acid, or organic acids such as acetic or formic acid, and heated at a
temperature of at least about 100°C for
a period of at least about fifteen minutes. The material that is treated in this manner is
preferentially the water-insoluble portion of the biomass from the fermentation, and the
insoluble material may be separated from the water-soluble portion of the fermentation
product, but separation is not necessary. The entire fermentation broth produced in the
fermentor may be treated by the process of this invention. After the mixture has been
acidified and heated, it will often be appropriate. to readjust the pH of the mixture to a
more neutral pH to enhance safety of handling and to reduce the risk of damage to plants
which would be treated with the preparation. Said pH adjustment may be carried out by
addition of any biologically-acceptable alkali or base, such as sodium hydroxide,
potassium hydroxide, magnesium oxide, magnesium hydroxide, calcium oxide, calcium
hydroxide, or ammonia solution (ammonium hydroxide).
Alternatively, the treatment can also be achieved by raising the pH to above about
pH 8 by means of alkalization with biologically-acceptable alkali or base such as, for
example, sodium hydroxide, potassium hydroxide, magnesium oxide, magnesium
hydroxide, calcium oxide, calcium hydroxide, or ammonia solution (ammonium
hydroxide), then heating the resulting mixture as above. Again, after heating, it may be
appropriate to adjust the pH of the resulting mixture to a more neutral pH by means of a
biologically acceptable acid such as, for example, sulfuric, hydrochloric or phosphoric
acid, or organic acids such as acetic or formic acid.
The heating step may be performed at a pressure above atmospheric pressure, if
necessary. For example, elevated pressure may be achieved by heating within an
autoclave.
The product thus produced is used to protect plants or control the growth of
nematodes by applying it in solid form or as a suspension in aqueous solution, preferably
water, directly to the surface or the root zone of the soil in which the plants are grown.
An advantage of the method which we have discovered is that the nematocidal
composition is inexpensive and safe. The materials employed in the process include the
fermentation broth, comprising the water-insoluble solids contained in such broth, which
may be waste solids from any industrial fermentation process, such as a fermentation
carried out to prepare pharmaceutical or agricultural products, or foods or beverages, and
ordinary acids and bases. Previously employed industrial processes for production of
nematocidal preparations are chemical syntheses which use dangerous and toxic starting
materials and result in waste streams of high toxicity which must be disposed of.
The method involves a post-treatment of a fermentation material, which can be
industrial fermentation waste, to produce a nematocidal composition.
As used herein the terms "nematocide" or "nematocidal", and the phrases "prevent
plant damage" and "control of growth", with respect to nematodes, include not only the
rapid, direct killing of nematodes, but also the concept of repelling nematodes, the
prevention or effective control of their multiplication or reproduction, the prevention of
nematode egg hatching, and confusing or immobilizing the nematodes so that they are
prevented from finding a mate or a plant to parasitize.
The methods of using the compositions of this invention for nematode control are by
application to any field, fruit, vegetable, floral or ornamental crop or nursery crop that is
sensitive to attack by plant parasitic nematodes, particularly the Meloidogyne species.
Methods of application are well-known in the art and include direct application to the
soil, either as a liquid or a dried solid, controlled release of the bioactive components
from solid formulations into the surrounding soil, application to the plant roots directly
before planting in the soil, foliar application and the like.
The term "soil", used herein is intended to include all media capable of supporting
the growth of plants and may include humus, sand, silt, loam, manure, compost and
commercial potting mixtures among others.
The term "fermentor", as used herein refers to apparatus used for various types of
fermentation methods including, but not limited to, shaken culture, solid-state, continuous
and batch fed methods that are contemplated for production of the fermentation broths of
this invention in both laboratory and large scale fermentation processes.
The term "biologically acceptable acid", as used herein refers to acids such as
sulfuric acid, phosphoric acid, hydrochloric acid, acetic acid or formic acid.
The term "biologically acceptable alkali or base", as used herein refers to bases such
as sodium hydroxide, potassium hydroxide, magnesium oxide, magnesium hydroxide,
calcium oxide, calcium hydroxide or ammonium hydroxide.
The process of this invention may utilize various media for the initial culture growth
and can consist of potato-dextrose agar, hay infusion agar, corn meal agar, leaf litter agar,
PCNB agar, soil infusion (modified), or Yeast Malt Agar as are defined in the Manual of
Industrial Microbiology and Biotechnology. Demain and Solomon, American Society of
Microbiology, Washington, D.C., 1986.
According to one embodiment of this invention, fermentation is carried out in shake-
flasks or in stationary-vat fermentors. In shake-flasks, aeration is provided by agitation
of the flask which causes mixing of the medium with air. In the stationary fermentors,
agitation is provided by impeller means such as a disc turbine, vaned disc, open turbine,
or marine propeller; and aeration is accomplished by injecting air or oxygen into the
fermentation mixture.
The fermentation medium consists of suitable sources of carbon, nitrogen, inorganic
salts, and growth factors assimilable by the microorganism. Suitable examples of carbon
sources are various sugars such as dextrose, glucose, lactose, and maltose, starch, dextrin,
com meal and glycerol.
The sources of nitrogen can be of organic, inorganic or mixed organic/inorganic
origin. Examples of nitrogen sources that can be used in the culture medium are soybean
meal, com steep liquor, peanut meal, cottonseed meal, com germ meal, fish meal, lard
water, and various ammonium salts.
The inclusion of certain amounts of minerals and growth factors in the fermentation
medium is also helpful. Crude medium ingredients such as distillers' solubles, com steep
liquor, fish meal, yeast products, peptonized milk and whey contain not only minerals but
growth factors. However, inorganic salts such as potassium phosphate, sodium chloride,
ferric sulfate, calcium carbonate, cobalt chloride, magnesium sulfate, and zinc sulfate can
be added to the fermentation medium.
Solid materials, such as calcium carbonate may be added in this process, to help with
pH control, which sometimes favors particular types of pellet formation for best results.
The process of producing the fermentation materials for use in this invention, while
utilizing a shaken culture fermentation technique may also use such a technique for the
initial stages or inoculum production as well. Production cultures are started from
specially grown inocula. Growth is generally rapid at first. It then slows down and a
stationary phase is usually reached. The production yield depends on the quantity of cells
present, their specific activity, and the span of their product-forming capacity.
The inoculum is placed in a liquid medium which is selected empirically for its
ability to allow the recovery of the majority of the cells in the population. The spores
produced on an initial growth medium, such as potato-dextrose agar, are transferred into
growth medium contained in a flask (termed seed flask), that would allow for the
germination and initial growth of culture. The germinated spores in an active growth
state are then transferred to Erlenmeyer flasks (shake-flasks) or stationary-vat fermentors
containing the specific fermentation medium. A 1-2% inoculum is typically produced for
the fermentation stage of development.
The inoculum medium is within the purview of those skilled in the art, and
additional information may be found in the Manual of Industrial Microbiology and
Biotechnology, pages 31-40, supra.
A wide range of shaker-culture apparatus may be used in the practice of this
invention. The main types of apparatus are based on either rotary or reciprocating
shaking machines. The process herein preferably uses rotary shakers in which the flasks
move in orbits of about 50 mm at about 200 to about 250 rpm, (but may vary between
100 and 500 rpm). The culture moves smoothly around the inside of the flask (wnich is
usually an Erlenmeyer flask). The scale-up of the fermentation process is well known to
those skilled in the art.
The purpose of shaking in submerged culture is to supply oxygen and nutrients to the
growing cells. In shaken cultures, the medium in the fermentation flasks is inoculated
with cells or spores, as is the case herein. The strain used as an inoculum is held as a
master culture, in the freeze-dried state or at reduced temperatures, such as -70°C. The
optimal spore concentration to be used for the inoculum is easily determined by those
skilled in the art by routine experimentation.
The biopesticidal compositions of the present invention can be used against plant
parasitic nematodes, including, for example, Meloidogyne spp., Pratylenchus spp.,
Radopholus similis, Ditylenchus dipsaci, Heterodera spp., Xiphinema spp., Globodera
spp. and Hoplolaemus spp.
The processed fermentation materials prepared according to the process of this
invention can be used to control nematodes for a variety of agricultural applications on
many different plants and fruits including, but not limited to, artichokes, aubergines,
banana, barley, beet roots, cacao, carrots, cassava, celery, chickpea, citrus, coconut,
coffee, cole crops, com, cotton, cowpea, eggplant, field bean, forages, ginseng, grape,
guava, various lettuces, melons, millet, oat, okra, ornamentals, papaya, peanut, pepper,
pigeon pea, pineapple, potatoes, rice, rye, sorghum, soybean, sugar beet, sugar cane,
sweet peppers, sweet potato, tea, tobacco, tomatoes, turf, wheat and yam. Cultivated
flowers can be protected according to the present invention, such as carnations, rose
bushes, gerberas, chrysanthemums, pot plants, philodendrons, ferns, figs, pothos,
sanseverias, and cacti; examples of nursery plants would include all the ornamental and
flowering shrubs.
The bioactive materials can be incorporated into the soil of flower pots or containers,
by direct application to the area to be treated at the time of planting, or up to several days
earlier, or by application in a controlled release form. Application to field or orchard
crops can be by granule dispersement on the surface with turnover of the soil by a claw
cultivar or a light plow, generally to about 10 cm up to about 20 cm depth of soil. As the
nematocide is water soluble, a drip irrigation method for application is also possible.
The compositions of the present invention can be in a suitable form for direct
application or as a concentrate or primary composition which requires dilution with a
suitable quantity of water or other diluent before application. The pesticidal
concentration will vary depending upon the nature of the particular formation,
specifically whether it is a concentrate or to be used directly.
The nematocidally effective amount of the active materials will depend upon the
population of the nematode expected to be encountered, the nematode type, soil, crop,
and moisture. In general, the composition may be applied at a field rate of from about 1
to about 200/lbs per acre; preferably at a rate of from about 5 to about 100/lbs per acre
and most preferably at a rate of from about 10 to about 60 lbs/acre.
The nematocidal compositions may be in the form of a suspension, a solution, an
emulsion, a dusting powder, a dispersible granule, a wettable powder, an emulsifiable
concentrate, an aerosol or impregnated granule, formulated by techniques well known to
those skilled in the art.
Additives to these compositions may include surface active agents, inert carriers,
preservatives, humectants, feeding stimulants, attractants, encapsulating agents, binders,
emulsifiers, dyes, U. V. protectants, buffers, flow agents, or other components which
facilitate product handling and application for protection against nematodes.
Examples of inert carriers include inorganic minerals such as kaolin, mica, gypsum,
fertilizer, phyllosilicates, carbonates, sulfates, or phosphates; organic materials such as
sugar, starches or cyclodextrins; or botanical materials such as wood products, cork,
powdered corn cobs, rice hulls, peanut hulls and walnut shells.
Suitable surface active agents include anionic compounds such as carboxylates, for
example an alkali metal carboxylate of a long chain fatty acid; an N-acylsarcosinate;
mono- or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such
esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or
sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkylphenol sulfates;
lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as alkyl benzene
sulfonates or lower alkyl naphthalene sulfonates such as butyl naphthalene sulfonate;
salts or sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-
formaldehyde condensates or more complex sulfonates such as the amide sulfonates.
Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty
acid amides or fatty alkyl- or alkenyl- substituted phenols with ethylene oxide, fatty
esters of polyhydric alcohol ethers, such as sorbitan fatty acid esters, condensation
products of such esters with ethylene oxide, such as polyoxyethylene sorbitan fatty acid
esters, block copolymers of ethylene oxide and propylene oxide, and acetylenic glycols.
Examples of cationic surface active agents include an aliphatic mono-, di-, or polyamine
as an acetate, naphthenate or oleate; an oxygen containing amine such as an amine oxide
of polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a
carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.
It is also contemplated that the materials of this invention may be used in
combination with other essential biologicals or beneficial microorganisms or active
ingredients, such as herbicides, anti-microbials, fungicides, insecticides, plant growth
regulators or nutrients.
The compositions of this invention may also be formulated as active mixtures which
may include finely divided dry or liquid diluents, extenders, fillers, conditioners, and
excipients, including various clays, diatomaceous earth, talc and the like, or water and
various organic liquids and mixtures thereof.
Of course, the present invention is not limited to the particular embodiments and
modes of operation described herein and it is possible to imagine a number of variations
in the details without departing from the scope of this invention.
The examples below are presented to describe preferred embodiments and utilities of
the invention and are not meant to limit the invention unless otherwise stated in the
claims appended hereto.
Example 1
Filter cakes (washed, damp fermentation solids) from two fungal fermentations were
re-suspended in three times their weight of water, adjusted to pH 2.0 with sulfuric acid
and heated in an autoclave at 121°C for three hours. The samples were cooled, re-
adjusted to pH 4.05 - 4.10 with sodium hydroxide solution and made up with distilled
water to a final weight eight times that of the cake used (12.5% concentration). Groups
of approximately 50 root-knot nematodes for each replicate were incubated for 24 hours
in these suspensions as well as a distilled water control and counted to determine
mortality. The heat-treated fungal fermentation solids showed strong nematocidal
activity, as indicated in Table 2.
Example 2
Dry material was recovered by lyophilization from a suspension of fermentation
solids from an unclassified Streptomyces species. The solid material (4.56 g) was re-
suspended in 40 mL of water, adjusted to pH 1.9 with sulfuric acid and heated at 121-
122°C for three hours. After cooling, the pH was re-adjusted to 4.0 with sodium
hydroxide solution, giving a final suspension containing 9% by weight of the
fermentation solids. Groups of approximately 50 root-knot nematodes for each replicate
were incubated for 24 hours with this suspension or with an antibiotic control solution
(100 units of penicillin plus 0.1 mg of streptomycin per mL) and then counted to
determine mortality. The heat-treated fermentation solid suspension was effective in
killing the plant-parasitic nematodes, as shown in Table 3.
Example 3
Nematocidal Activity of Alkali Digested Fermentation Samples (Contact Assay)
Myrothecium fermentation broth was heated in basic (alkaline) conditions, and the
efficacy of the resulting materials was compared in a nematocidal assay (% mortality of
root knot nematodes). The control broth sample was adjusted to neutral pH (pH 7), and
other samples were adjusted to basic (pH 12) conditions. Half of each sample was heated
at 121°C for two hours. Once cooled, all the samples were readjusted to neutral pH and
lyophilized (dried to a powder). The powders were reconstituted in water to equal
concentrations by weight and tested in a contact assay. The contact assay consisted of
RKN incubated for 24 hours in five concentrations of each material, with multiple
replications. The nematodes were counted, and the number of dead nematodes
determined for each concentration for every sample. Data from this test was analyzed by
PROBIT analysis to determine the LC50 (lethal concentration or dose to obtain an
estimated 50% mortality of the nematodes within 95% confidence range). Fermentation
broth when heated in basic condition showed increased toxicity to nematodes, indicated
by a lower LC50 value for these materials, as shown in Table 7. Materials that were pH
adjusted but not heated did not show a significant increase of toxicity to nematodes.
Table 3
When plant parasitic nematodes are placed in direct contact with the compositions so
produced, the nematodes are paralyzed or killed, depending on the concentration of the
composition applied. When the soil in which potted plants are growing is treated with a
suspension of the product produced according to the method of this invention, and then
inoculated with plant parasitic nematodes, after a growing period of several days, the
plants are healthier, with larger and heavier aerial parts than untreated plants similarly
inoculated with nematodes. The roots of the treated plants also exhibited fewer root galls
(symptoms of nematode attack and invasion) than the untreated plants. In the field, the
composition produced may be applied as a suspension in water or as a dry granular
material to the plants to be protected from parasitic nematodes.
Example 4
Portions of whole fermentation beer from the submerged fermentation of
Myrothecium sp. were adjusted to pH=s of 4, 8, 9,10 or 11 (V0.1) with sulfuric acid or
sodium hydroxide and heated in an autoclave for two hours at 121°C. Control samples

were pH adjusted but not heated. After heating, the samples were cooled to room
temperature, and all samples were adjusted to pH 4.1 (VO. 1) using sulfuric acid. Then
samples were diluted to a final concentration of 2% solids. Groups of Meloidogyne
incognita (root-knot nematodes, RKN) were incubated in the suspensions as well as a
dilute antibiotic control solution (1 unit/mL of penicillin and 0.1 mg/mL of streptomycin),
and after 24 hours live and dead individuals were counted. Beer samples heated at
elevated pH demonstrated increased nematocidal activity over non-heated samples, as
shown in Table 8.
Table 4
Whole-Beer Alkaline Heat Treatment
The present invention is illustrated by way ot the foregoing description and
examples. The foregoing description is intended as a non-limiting illustration, since
many variations will become apparent to those skilled in the art in view thereof. It is
intended that all such variations within the scope and spirit of the appended claims be
embraced thereby.
Changes can be made in the composition, operation and arrangement of the
method of the present invention described herein without departing from the concept and
scope of the invention as defined in the following claims:
We claim:
1. A method for producing improved biopesticidal materials by fermentation
comprising the steps of:
fermenting a bacterium or fungus to obtain a fermentation broth;
adjusting the pH of said fermentation broth to a pH of below 2.5 with a
biologically acceptable acid, or to a pK of above 8 with a biologically acceptable alkali or
base; and
heat treating said fermentation broth to a temperature of at least 100°C for at
least 15 minutes, then cooling to ambient temperature to obtain a pH-adjusted, heat
treated composition having improved biopesticidal activity;
with the proviso that said fungus is not Myrothecium verrucaria when the pH of
said fermentation broth is adjusted to a pH of below 2.5.
2. The method as claimed in claim 1 wherein said fungus or bacterium is selected
from the group consisting of Gibberella fujikuroi, Streptomyces erythraeus, Bacillus
sphaericus. Bacillus thuringiensis and Fusarium moniliforme.
3.. A method for producing a nematocidal composition comprising the steps of:
a) fermenting a bacterium or fungus to obtain a fermentation broth;
b) suspending said broth in an aqueous solution;
c) adjusting the pH of said broth in aqueous solution to a pH below pH 2.5 with
a biologically acceptable acid;
d) heating the pH-treated broth of step (c) to a temperature of at least 100°C for
at least 15 minutes, then cooling to ambient temperature; and,
e) recovering said composition in a conventional manner;
with the proviso that said fungus is not Myrothecium verrucaria.
4. The method as claimed in claim 3 wherein said pH is adjusted below 2.
5. The method as claimed in claim 3 wherein said pH is adjusted below 1.5.
6. The method as claimed in claim 3 optionally comprising adjusting pH of said
composition formed in step (d) to a range of pH 4 to pH 8.
7. The method as claimed in claim 3 optionally comprising adding at least one
compound to said composition of step (e), said compound selected from the group
consisting of herbicides, antimicrobials, fungicides, insecticides, plant growth regulators
and nutrients.
8. The method as claimed in claim 3 wherein said fermentation broth of step (a)
has water-soluble biomass and water-insoluble biomass.
9. The method as claimed in claim 8 wherein said water-soluble biomass is
separated from said water-insoluble biomass, and said water-insoluble biomass is
suspended in aqueous solution in step (b).
10. A method for producing a nematocidal composition comprising the steps
of:
a) fermenting a bacterium or fungus to obtain a fermentation broth;
b) suspending said broth in an aqueous solution;
c) adjusting the pH of said broth in aqueous solution to a pH above pH 8 with a
biologically acceptable alkali or base;
d) heating the pH-treated broth of step (c) to a temperature of at least 100°C for
at least 15 minutes, then cooling to ambient temperature;
e) recovering said composition in a conventional manner.
11. The method as claimed in claim 10 wherein said pH is adjusted above 9.
12. The method as claimed in claim 10 wherein said pH is adjusted above 10.
13. The method as claimed in claim 10 optionally comprising adjusting pH of
said composition formed in step (d) to a range of pH 4 to pH 8.
14. The method as claimed in claim 10 optionally comprising adding at least one
compound to said composition of step (e), said compound selected from the group
consisting of herbicides, antimicrobials, fungicides, insecticides, plant growth regulators
and nutrients.
15. The method as claimed in claim 10 wherein said fermentation broth of step (a)
has water-soluble biomass and water-insoluble biomass.
16. The method as claimed in claim 15 wherein said water-soluble biomass is
separated from said water-insoluble biomass, and said water-insoluble biomass is
suspended in aqueous solution in step (b).
The invention discloses a method for producing improved biopesticidal materials by
fermentation comprising the steps of:
fermenting a bacterium or fungus to obtain a fermentation broth;
adjusting the pH of said fermentation broth to a pH of below 2.5 with a biologically
acceptable acid, or to a pH of above 8 with a biologically acceptable alkali or base; and
heat treating said fermentation broth to a temperature of at least 100°C for at least 15
minutes, then cooling to ambient temperature to obtain a pH-adjusted, heat treated composition
having improved biopesticidal activity;
with the proviso that said fungus is not Myrothecium verrucaria when the pH of said
fermentation broth is adjusted to a pH of below 2.5.

Documents:

in-pct-2002-1240-kol-granted-abstract.pdf

in-pct-2002-1240-kol-granted-assignment.pdf

in-pct-2002-1240-kol-granted-claims.pdf

in-pct-2002-1240-kol-granted-correspondence.pdf

in-pct-2002-1240-kol-granted-description (complete).pdf

in-pct-2002-1240-kol-granted-examination report.pdf

in-pct-2002-1240-kol-granted-form 1.pdf

in-pct-2002-1240-kol-granted-form 18.pdf

in-pct-2002-1240-kol-granted-form 3.pdf

in-pct-2002-1240-kol-granted-form 5.pdf

in-pct-2002-1240-kol-granted-gpa.pdf

in-pct-2002-1240-kol-granted-reply to examination report.pdf

in-pct-2002-1240-kol-granted-specification.pdf

in-pct-2002-1240-kol-granted-translated copy of priority document.pdf


Patent Number 223418
Indian Patent Application Number IN/PCT/2002/1240/KOL
PG Journal Number 37/2008
Publication Date 12-Sep-2008
Grant Date 10-Sep-2008
Date of Filing 01-Oct-2002
Name of Patentee VALENT BIOSCIENCES, CORP.
Applicant Address 870 TECHNOLOGY WAY, LIBERTYVILLE, ILLINOIS 60048
Inventors:
# Inventor's Name Inventor's Address
1 WARRIOR PREM 14584 NORTH SOMERSET CIRCLE GREEN OAKS, ILLINOIS 60048
2 HEIMAN DANIEL F 407 DRAKE LIBERTY VILLE, ILLINOIS 60048
3 REHBERGER LINDA A 4001 MILLER DRIVE GLENVIEW, ILLINOIS 60025
4 HANSEN JAMES R 248 MINER STREET BENSENVILLE ILLINOIS, 60106
5 MCVICKER KEVIN A 43 RAMSGATE PALOS PARK, ILLINOIS, 60464
6 JOHNSON RONALD E 26 HARVEY STREET, GRAYSLAKE ILLINOIS 60030
PCT International Classification Number A61K 35/74,35/84
PCT International Application Number PCT/US01/11699
PCT International Filing date 2001-04-10
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/196,257 2000-04-11 U.S.A.