WO1995000020A2

WO1995000020A2 - Method for control of conidial dispersal and improved maintenance of entomopathogenic fungal products

Info

Publication number: WO1995000020A2
Application number: PCT/US1994/006886
Authority: WO
Inventors: Sarjit Johal; Lorraine M. Marold
Original assignee: Ecoscience Corporation
Priority date: 1993-06-17
Filing date: 1994-06-17
Publication date: 1995-01-05
Also published as: AU7112294A; WO1995000020A3

Abstract

A class of economical, non-toxic materials which control conidial dusting without creating physical barriers or altering the geometry of devices such as contamination chambers or mycelia pellets is disclosed. These materials can be applied to previously unmodified, conidial layers (such as conidial 'lawns'), active solid substrate fungal cultures, and sporulated mycelial pellets of various sizes and dimensions, as well as such products as contamination chambers and sheets of natural or synthetic matrixes such as sponges, nonwoven cloths, and screens.

Description

METHOD FOR CONTROL OF CONIDIAL DISPERSAL AND

IMPROVED MAINTENANCE OF ENTOMOPATHOGENIC FUNGAL

PRODUCTS

Background of the invention

The present invention is generally in the area of methods for the control and maintenance of conidia on fungal impregnated surfaces.

The employment of entomopathogenic fungi for biological control of pests has been known and successfully used in low technology, labor intensive operations throughout the world for some time. Spurred by a new set of directives and marketplace incentives such as safety and environmental compatibility, as well as consumer preferences, there is now a strong interest in the development and commercialization of sophisticated biological pest control agents. Some applications where attributes such as safety and specificity are favored are in the residential, commercial, and institutional industrial food-related markets, such as food processing and storage, and medical markets. Successful entry of biocontrol agents into these markets requires that the products have, and retain, high insecticidal activity, acceptable appearance, and good shelf-life, as well as easy and safe handling.

The entomopathogenic fungi classified as Hyphomycetes, such as Metarhizium anisopliae, display good, broad spectrum insecticidal activity. These fungi have a biphasic biological cycle consisting of a mycelial vegetative phase and an asexual conidiospore reproductive phase. Conidial production and subsequent shedding are the terminal events in the normal growth cycle of these fungi. The reproductive propagules, the conidia, are essential to the infective process. Specifically, when insects such as cockroaches, ants and termites are out foraging, they come into contact with and rub against the fungus. In so doing the insects contact the fungal conidia which then attach to the surface of the insect. The spores then infect the susceptible insect host through the insect cuticle. Several steps are recognized in the development of the infection of an insect, including adhesion of the spore to the cuticle; formation and penetration of the cuticle by the germ tube; and growth of the fungus within the body. The initial fungus-host interaction is random. Upon contact, however, the conidiospore(s) of Metarhizium anisopliae and Beauveria bassiana , among others, have evolved spore surface (or coat) physicochemical attributes which enhance the attachment of the spore to the insect cuticle. In the absence of attachment and germination, pesticidal activity is impossible.

In nature, conidial dispersion ("dusting") is a highly desirable attribute to assure survival of the species. Broad dissemination of the conidia insures propagation and survival of the fungi. Dispersion also favors and enhances the probability of infecting a susceptible insect host and promoting further propagation. These factors have been used to advantage commercially to enhance the utilization of conidia in products for the biological control of insects. For example, the contamination chambers described in U.S. Patent Nos. 5,057,316 and 5,057,315 are based on a method for control and extermination of insects by infection of the insects with a fungus that can be pathogenic when administered to the insects in a sufficiently high concentration by means of a contamination chamber.

However, uncontrolled conidial dispersal is problematic and poses difficulties for many commercial applications and product forms. For example, it is unacceptable to have leakage and dusting of fungal conidia from defined use containers, such as the contamination chambers and systems which are described in U.S. Patent Nos. 5,057,316 and 5,057,315, or sporulated mycelial pellets, during use, transportation, and package handling. Moreover, the uncontrolled release of conidia, the preferred active biological control agents in the chambers, will ultimately reduce the efficacy and duration of activity in use of this and similar products.

Many factors affect the release of conidia. An important factor is the conditions under which the fungus is stored. Overall product consistency and stability cannot be controlled and regulated over time under variable environmental conditions. For example, the moisture content in a contamination chamber significantly influences the amount of dusting; the drier the conditions in the chamber, the greater the dusting (i.e., the release of conidia). Hence, two identically manufactured biological pest control chambers may display significantly different performance characteristics when stored and used in different environments.

While a variety of approaches can be envisioned to reduce or alleviate dusting, such as physical containment or treatment with chemical agents, very few, if any, can accomplish this task without negatively impacting the biological characteristics of agents such as fungal conidia or making the biological agent inaccessible to the pest. For example, increasing containment of the conidia by physical barriers such as by decreasing the size of the openings into a contamination chamber might reduce access to insects, thereby limiting frequency of exposure and insecticidal activity overall. Alternatively, most chemicals, such as some surface active agents, which reduce dusting negatively impact conidial adhesion, penetration of the cuticle, and/or growth, all of which are features necessary for insecticidal activity. It is therefore an object of the present invention to provide an effective, economical method and means to control conidial dispersion from fungal impregnated surfaces, mycelial pellets or any other surface where a layer of conidia is used to serve as a contact infection site for insect pests.

It is a further object of the present invention to provide a method and means to control conidial dispersion without reducing biological activity, particularly for the control of insects.

It is another object of the present invention to provide a means and method to control conidial dispersion which enhances stability and shelf life in select product forms such as contamination chambers.

It is still another object of the present invention to provide a means and method to control conidial dispersion which can be applied to either fully sporulated fungal surfaces or unsporulated fungal surfaces which ultimately progress to produce conidia.

Summary of the Invention

A class of economical, non-toxic materials which control conidial dusting without creating obstructive physical barriers or altering the geometry of devices such as contamination chambers or mycelial pellets is disclosed. These materials can be applied to previously unmodified, conidial layers (such as conidial 'lawns-'), active solid substrate fungal cultures, and sporulated mycelial pellets of various sizes and dimensions, as well as such products as contamination chambers and sheets of natural or synthetic matrixes such as sponges, nonwoven cloths, and screens. Brief Description of the Drawings

Figure 1 is a graph of the percent water retention over time (days) for fungal cultures in contamination chambers treated with 0.6 g light mineral oil/11 cm² (dark squares), 1.2 g light mineral oil/11 cm² (light squares) , or untreated controls (dark triangles) .

Detailed Description of the Invention

The present invention is the discovery of materials which can be used to control and/or inhibit release of entomopathogenic fungal conidia from contamination chambers, materials, or surfaces, in a cost effective manner, which does not significantly decrease the biological activity of the conidia.

Sources of conidia to be treated.

The starting materials for the production of a controlled release system or station such as a contamination chamber as disclosed in U.S. Patent No. 5,057,315 are actively growing solid substrate cultures or active sporulated solid state cultures which have a layer of conidia on the solid substrate surface or sporulated mycelial cultures of entomopathogenic fungi. This includes, but is not limited to, fungi such as Metarhizium anisopliae and Beauveria baεεiana and related Hyphomycetes or other taxonomic classification of fungi which sporulates.

The fungal cultures are produced by procedures known to those skilled in the art. For example, on standard agar based nutritive media formulations, solid state (substrate) fermentations on a variety of nutrient sources such as straw or cereals, and the submerged fermentation for the production of mycelium which is then incubated under appropriate conditions to effect sporulation.

When a solid state culture medium is inoculated with spores, the fungus will produce a layer of mycelia after several days of growth under the appropriate conditions. Conidia form on the mycelial layer. A uniform layer several millimeters thick will be produced in the proper environment.

These mixed conidia-mycelia and/or substrate combinations can be used immediately or stored under the proper conditions, as may be required, appropriate or preferred for maintenance of viability. The preferred physiological state is that which yields the highest percent viability and rapid germination rate.

Treatment of conidia to reduce dusting.

Any of these forms of active viable sporulated or unsporulated (freshly inoculated) cultures can be processed by treatment with non-polar, hydrophobic organic coating such as oils and related materials, for example, paraffinic oils such as mineral oils, petrolatums, mineral waxes, vegetable and animal oils, waxes, and fats, and derivatives thereof having the same physical and chemical properties that yield the desired effects, i.e., reducing dusting while not reducing conidial viability. The most preferred compound is light white mineral oil N.F., available as mineral oil having a density of 0.84 g/ml. For ease of reference herein, these materials are referred to as "oils".

In the presence of a thin coating of any one or more of these materials which encapsulates but does not suspend the conidia, no significant conidial dusting is observed for prolonged periods. It is preferred that there be less than fifty percent of the dusting of the untreated conidia following treatment with the oil, more preferably less than 10%, and most preferably less than 5% for a commercially useful product. In the preferred treatments, conidial viability is maintained and is totally unaffected as determined by germination and insect bioassay test results. Too heavy a coating leads to a delay in the insecticidal activity of the conidia. A key product characteristic, stability under standard storage and use conditions, is enhanced with the application of these materials. One reason for this extended use is that random dispersal is substantially reduced, thereby maintaining optimal conidial concentration available for contact and adhesion to the insect. Another reason is that the conidia are protected from the vagaries of the environment, including humidity detrimentally affecting viability of the conidia, usually relative humidity greater than 10% and less than 60%, and unintended desiccation by this treatment of the conidia.

The quantity of protective material which is applied to a conidial layer grown on a solid substrate or a mycelial pellet is in the range of between 0.00009 and 0.45 grams/cm², more preferably in the range of between 0.00045 and 0.045 grams/cm², and most preferably in the range of between 0.003 and 0.022 grams/cm². This quantity of oil is sprayed or applied to the conidial surface using any commercially available industrial application system or methodology which applies a uniform coating. Care must be taken to avoid application systems such as aerosol spray cans which could disrupt the conidial surface. The treated material is then packaged, stored, and can be used in a less stringent manner than the untreated product.

Substrates to which conidia are applied.

As described herein, the fungal culture itself may be treated, or the conidia may be applied to another substrate and treated with oils to reduce dusting. The oils can be applied to previously unmodified, conidial layers (such as conidial 'lawns'), active solid substrate fungal cultures, and sporulated mycelial pellets of various sizes and dimensions, as well as such products as contamination chambers and sheets of natural or synthetic matrixes such as sponges, nonwoven cloths, and screens. As discussed above, the conidial lawn may be within a contamination chamber, for example, of the type described in U.S. Patent Nos. 5,057,316 and 5,057,315, the teachings of which are incorporated herein. Conidia are applied to form a composite, such as by applying an aqueous suspension of conidia to a sponge, then covering and drying the sponge to yield conidia in a concentration of between 10⁴ and 10⁹ conidia/10 cm² for most applications. Examples of polymeric matrices having conidia on and/or within the matrix which can be treated include the alginate pellets described in U.S. Patent No. 4,718,935 to Walker, et al., Patel, et al., J. Amer. Mosquito Control Ass. 6,101 (1990), and EPA 92100010.5 by Eyal, et al., the teachings of which are incorporated herein. A polymer substrate with conidia thereon which is suitable for treatment is described by U.S. Patent No. 4,921,703 to Higuchi, et al., the teachings of which are incorporated herein. Pure mycelial cultures, described by Australian patent application AU-8-81386/87, the teachings of which are incorporated herein, can also be treated.

The present invention will be more clearly understood by reference to the following non-limiting examples, which demonstrate the best mode of the methods and materials for treatment of conidia to inhibit dusting described herein. Example 1: Treatment of conidia with light white mineral oil.

M. anisopliae was grown on standard (100 millimeter) nutritive agar plates for the production of conidia. The plates were used without further treatment after conidiation (as defined by the development of a full dark green conidial lawn) and were stored at room temperature (approximately 25°C) . About twenty drops of light white mineral oil were applied across the conidial surface using a pipette. The plate was then tilted to assure complete coverage. An oil treated plate was then placed in a biocontainment hood. No conidial dispersion was visually observed after the sample was treated with oil. Without treatment conidia would have immediately dispersed, causing a dusting effect.

Evaporative water losses were also significantly reduced relative to untreated plates. Conidial germination as a measure of viability was determined qualitatively by sampling the treated material with a sterile cotton swab which was then used to inoculate standard nutritive agar plates for the cultivation of fungi. Percent germination of the treated sample was monitored for thirty-five days. Rapid germination was observed throughout this period. Example 2: Treatment of conidia.

Freshly harvested conidia of M . anisopliae were added to yield a spore density of about 10⁴ colony forming units (CFU) per milliliter to molten nutritive agar-based medium at a temperature of 40°C. The inoculated media was then immediately dispensed into sterile disposable petri dishes (60 millimeter diameter) and allowed to solidify. The plates were then used in multiples of six for each of the treatments outlined below. The surface area in each plate being treated is 30 cm². Treatment 1; Light white mineral oil.

One drop of light white mineral oil (between 0.02 and 0.025 g) was applied to the agar surfaces. The droplets were then evenly distributed over the agar surfaces with sterile glass spreaders. Treatment 2: Petrolatum.

Molten Protopet (White IS) , a petrolatum (homogeneous semi-solid mixture of oily and waxy hydrocarbons (Sonneborn Division, Witco Corp., New York, N.Y.), was applied to the surfaces. The material was applied to the conidia with a prewarmed glass pipette and distributed with a prewarmed glass spreader. The amount of material applied could not be controlled with accuracy due to the variable viscosity of the material and therefore ranged from heavy to light applications. The material became increasingly more viscous upon cooling. Treatment 3: Polyethylene qlycol.

Polyethylene Glycol (average molecular weight 8,000), at 10 percent (weight per volume) in water was added to the surfaces of the plates. The plates were tilted slightly after application of the material to insure even distribution. Controls

Six plates with no further treatment served as controls.

All plates were incubated at 25°C, 75 percent relative humidity (RH) for 35 days, followed by 4 days at room temperature (RT) before the plates were examined for growth and signs of conidial dispersion (dusting) .

Results

The conidial inoculum germinated and led to fungal growth in the presence of all of the treatments. The pattern of growth appeared normal and included the development of mycelia which subsequently sporulated to produce a uniform conidial overlay ent.

The following observations and modifications to the method were then made. One drop of mineral oil was insufficient to prevent spore dusting and the application of larger amounts of material is required. Plates receiving heavy applications of the Protopet did not show fungal growth whereas plates with a light application resulted in the growth of dark green conidia with no dusting observed. Polyethylene glycol did not work and the medium was completely dehydrated and shriveled. Conidia were widely dispersed producing a dust-like appearance. Example 3: Application of oils to conidia in contamination chambers.

Nutritive agar-based media inoculated with M . anisopliae conidia prepared as described in Example 2 was dispensed into sterile plastic trays containing 6 wells, each 11 cm² in surface area. The filled trays were incubated for 13 days at 26°C, 75 percent relative humidity for the growth of the fungus and the production of spores.

The heavily sporulated colonies from 12 wells (2 trays) were each overlaid with approximately 0.6 grams of light mineral oil/11 cm², Treatment A. Similarly, 12 wells were overlaid with approximately 1.2 grams of light mineral oil/11 cm², Treatment B. Growth in wells of two other trays were left untreated and served as controls. After the treatment all of the trays were weighed and placed without covers in a biocontainment hood which was in full operation except for the absence of hood illumination.

The trays were weighed again after 5 and 7 days in the hood. After seven days in the hood the trays were removed and stored in a large covered box. The trays were removed from the box and reweighed after an additional 7 days (a total of 14 days) and 14 days (a total of 21 days) . The same schedule described above for the weight determinations was used for the timing of viability tests using material from a well selected at random.

Weight losses were determined, average weight losses were computed and percent weight losses calculated. Average weight retention (in percent) per tray was derived from the weight loss data.

The results for moisture retention are shown in Table 1. It is evident that the application of the oil serves as a barrier which significantly reduces water loss. Moreover, this barrier effect is dose dependent. The oil treatments did not appear to negatively impact conidial viability as measured by germination. Conidia from all of the treatments at day twenty-one retained high germination activity. A qualitative method which consisted of inoculating a potato-dextrose-agar (PDA) plate and observing rate and density of growth was employed for monitoring germination and indicating viability.

Table 1: Effect of Treatments on Percent Water Retention of conidia

STORAGE TIME IN DAYS

5 7 14 21

Treatment A 81 76 61 46

Treatment B 87 85 73 61

Control 32 32 32 32

The results are also shown in Figure 1. Example 4: Evaluation of different materials for control of dispersal, viability and efficacy of treated conidia.

Studies were conducted using wells and general conditions described in Example 3. Dusting Assays;

The amount of conidia lost by shedding (or dusting) was assessed by opening the contamination chamber and lightly tapping each chamber while holding it vertically and then dropping each chamber onto a tray from approximately four inches four times and again tapping each chamber while held vertically. Germination Assays;

Conidia were brushed into potato-dextrose-agar (PDA) plates which had been divided into quadrants. The plates, in duplicate, were then incubated for a total of 13 hours at 28°C, 75 percent Relative Humidity, whereupon conidia were observed microscopically (200X) for the presence of germ tubes, indicative of viable conidia. A minimum of two hundred conidia were examined from each of two sectors from each of two plates. Conidia were then scored for the presence or absence of germ tubes from which average percent germination was determined. Conidia were examined for germination prior to incubation to assure the absence of premature germination which would have invalidated the test results. Insect Bioassav;

Standard laboratory shoe box assay using twenty adult cockroaches.

Materials:

Polystyrene shoe storage boxes (12.5 x 6.75 x 3.6 in) having air holes in the top covered with Polyester mosquito mesh netting.

Adult German cockroaches (Blatella germanica ; JK-Consulting, Amherst, MA) , fed Purina Lab Chow (Purina #5001; Purina Mills, Inc., St. Louis, MO) with free access to distilled water in a test tube stoppered with tissue.

Environmental chamber with controlled temperature and humidity and continuous data recorder (28°C and 75% RH) into which the shoe boxes were placed. METHODS:

Vertical sides of shoe boxes were coated with a thin layer of petroleum jelly to keep roaches off. A pellet of autoclaved Purina Lab Chow and a water tube were added to each shoe box. Twenty cockroaches were added to each box. The shoe boxes of cockroaches were placed in the environmental chamber. One infection chamber as described in U.S. Patent Nos. 5,057,315 and 5,057,316 was added to each of four shoe boxes of cockroaches. Four additional shoe boxes were used as controls.

Shoe boxes of cockroaches were incubated at 28 ± 3°C and 75 ± 15% RH under a 10 hour photoperiod. Cockroach mortality was recorded weekly for 6 weeks. The criteria for 'dead' was if no movement was observed when the insect was prodded with a blunt instrument.

Results.

The results of these studies are presented in Table 2. The results show that the oils (wheat germ and mineral) outperformed all other materials with regards to control of dispersion. These materials did not negatively impact percent germination and viability, however.

Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the following claims.

Table 2: Dust Retention, Viability and Efficacy After Application of Chemical Additives Following Storage at Room Temperature

Material Chemical Conidial Dust % Germination Efficacy^** Additive Released (g ) per (mg/chamber) Chamber

LT₅₀ LT₉₀ days days

5 days 3 weeks 5 days 8 weeks

Wheat Germ Oil 0.07 + 0.8 95.1 97.8 15 26 Mineral Oil 0.02 + 1.3 94.9 94.1 24 28 Canola Oil⁴ 0.14 # # # # # (aerosol spray) 0.1% Triton™ 0.17 - # # # # # X100 Surfactant Tween™ 0.33 + 7.9 95. ,2 93. ,8 14 25 Polyethylene 0.27 # # # # # Glycol (PEG) Xanthan Gum 0.30 # # # # # Control + 22.5 83.8 96.7 15 25 Control, + 141.3 88.8 92.9 14 26 unsealed bags

Bioassay was standard lab shoe box assay using 20 adult cockroaches at 28°C and 75% RH. + indicates insignificant conidial release. indicates unacceptably high conidial release. # Due to high initial release of conidia, this material was not tested further. & aerosol spray obtained at retail outlet; did not provide uniform coverage; results therefore not representative of effect of canola oil.

Claims

We claim:

1. A method for the treatment of conidia to reduce dusting comprising applying to viable conidia of a fungus having a biphasic biological cycle consisting of a mycelial vegetative phase and an asexual conidiospore reproductive phase, isolated from the fungal mycelia, an effective amount to reduce random dispersion not resulting from direct contact with the conidia, of the conidia by at least 50% but not to suspend or inhibit the biological activity of the conidia of an oil selected from the group consisting of paraffinic oils and waxes, petrolatums, vegetable oils, vegetable waxes and vegetable fats, animal oils, animal waxes, and animal fats, and synthetic derivatives thereof of the oils, waxes and fats.

2. The method of claim 1 wherein the compound is applied in a concentration range of between 0.00009 and 0.45 grams/cm^2*

3. The method of claim 2 wherein the compound is applied in a concentration range of between 0.00045 and 0.045 grams/cm².

4. The method of claim 3 wherein the compound is applied in a concentration range of between 0.003 and 0.022 grams/cm².

5. The method of claim 1 wherein the compound is mineral oil.

6. The method of claim 1 wherein the conidia is present on a fungal culture.

7. The method of claim 6 wherein the fungal culture is contained within a chamber having openings allowing insects to enter the chamber and contact the conidia and including a nutrient medium supporting the fungal culture.

8. The method of claim 1 wherein the conidia is present on the surface of a non-nutritive substrate.

9. The method of claim 8 wherein the substrate is selected from the group consisting of sheets of natural or synthetic matrixes, nonwoven cloths, and screens.

10. The method of claim l wherein the conidia is dispersed on or within a polymeric matrix.

11. A composition comprising viable isolated fungal conidia of a fungus having a biphasic biological cycle consisting of a mycelial vegetative phase and an asexual conidiospore reproductive phase, isolated from the fungal conidia, in combination with an effective amount to reduce random dispersion not resulting from direct contact with the conidia, of the conidia by at least 50% but not to suspend or inhibit the biological activity of the conidia of an oil selected from the group consisting of paraffinic oils and waxes, petrolatums, vegetable oils, vegetable waxes and vegetable fats, animal oils, animal waxes, and animal fats, and synthetic derivatives thereof of the oils, waxes and fats.

12. The composition of claim 11 wherein the compound is present in a concentration range of between 0.00009 and 0.45 grams/cm²

13. The composition of claim 12 wherein the compound is present in a concentration range of between 0.00045 and 0.045 grams/cm².

14. The composition of claim 13 wherein the compound is applied in a concentration range of between 0.003 and 0.022 grams/cm².

15. The composition of claim 11 wherein the compound is mineral oil.

16. The composition of claim 11 wherein the conidia is present on a fungal culture.

17. The composition of claim 16 wherein the fungal culture is contained within a chamber having openings allowing insects to enter the chamber and contact the conidia and including a nutrient medium supporting the fungal culture.

18. The composition of claim 11 wherein the conidia is present on the surface of a non-nutritive substrate.

19. The composition of claim 18 wherein the substrate is selected from the group consisting of sheets of natural or synthetic matrixes, nonwoven cloths, and screens.

20. The composition of claim 11 wherein the oil is in a concentration which encapsulates but does not suspend the conidia.

21. The composition of claim 11 wherein the conidia is dispersed on or within a polymeric matrix.