This application claims priority to PCT/CA02/00797, filed May 30, 2002, which claims benefit to U.S. provisional application 60/294,475, filed May 30, 2001.

The present invention relates to bioherbicides. More specifically, the present invention relates to fungal bioherbicides and compositions comprising fungal bioherbicides.

BACKGROUND OF THE INVENTION

The use of pesticides to kill insects, weeds and other disease pests is common in agriculture. It has been estimated that Canadian farmers spend more than $750 million on pesticides, and U.S. and European estimates are likely to be several fold higher. On the Canadian prairies, 95% of the land seeded to wheat, barley, canola and flax is treated with one or more pesticides. However, despite extensive pesticide use, weeds continue to cause an estimated one billion dollars in crop losses in Canada alone every year.

Weeds are detrimental to agricultural crops because they are capable of outcompeting crop plants for space, sun and nutrients. Particularly troublesome weeds include Canada thistle (Cirsium arvense) and other members of the Aster family such as perennial sowthistle (Sonchus arvense), and dandelion (Taraxacum officinale).

Canada thistle (Cirsium arvense [L.] Scop.) is an aggressive perennial weed in field crops, pastures and roadsides, and is particularly prevalent in Western Canada where it occurs in about 50% of all fields. Canada thistle causes crop yield losses of about 15 to 60% in cereal, oilseed and pulse crops, depending on weed density. In cereal crops, densities of 6 to 20 Canada thistle plants per square meter result in an 18 to 30% loss in grain yield. In 1937, Canada thistle was designated. as a noxious weed by the Canadian Federal Seeds Act.

Although weeds of the Aster family, for example Canada thistle and dandelion, can reproduce by flowering, they are difficult to eradicate because their extensive root system. The roots are quite brittle and fragment easily during tillage. This results in greater shoot emergence from stimulated buds. Further adding to the difficulties of control, the root fragments carry sufficient food reserves to survive long periods under adverse conditions.

Control of Canada thistle in field crops is currently achieved by pre-seeding, in-crop, and post-harvest chemical control with herbicides, applied at sufficient rates to suppress top growth, or kill the roots. For example, Glyphosate is used as a pre-seeding treatment to kill Canada thistle, or used in-crop on glyphosate tolerant crops. Clopyralid is used for in-crop control to achieve the same effect but has problems with residual activity for some crops in the following year. Other product combinations only provide top growth suppression such as thifensulfuron and tribenuron-methyl or fenoxy-prop and MCPA. Other control options include growing competitive crops and seeding early to get vigorous crop growth before Canada thistle emergence and shallow tilling of soil to reduce root fragmentation and new shoot growth. Also, mowing may be used to control weeds on roadsides, ditches, headlands and fence lines. Controlling patches instead of entire fields is often recommended to reduce costs.

There are a number of drawbacks associated with non-chemical control of Canada thistle in addition to those discussed above. First, there are very few crops which are able to outcompete weeds such as Canada thistle and many crops cannot be seeded early enough to provide the crop with a competitive advantage to Canada thistle. Further, seeding crops earlier than usual may be an inconvenience to farmers. Also, shallow tillage of soil and mowing weeds to kill weeds or prevent weed flowering are only temporary solutions and are at best marginally effective in controlling weeds such as Canada thistle.

There are also several drawbacks associated with the use of chemical herbicides to control weeds such as Canada thistle. Herbicides are expensive and may be too expensive to be used by some farmers. Further, if a farmer uses less than the required dosage of herbicide to kill the weeds, there is an increased risk that some weeds may develop herbicide resistance. There is also an increased risk of herbicide resistance due to overuse of a herbicide. In addition, herbicides are not available for all crops and all situations. For example, there are no effective herbicides available for crops such as peas and lentil whereas some in-crop chemical herbicides only suppress top growth of weeds without controlling root growth, which is a short-term strategy often used for crops such as wheat, barley and canola. Residual herbicidal activity may also limit crop rotation for some crops and some agronomic herbicide practices may increase weed densities. There are also concerns about the short and long term safety of herbicides, both to consumers and the environment.

Environmental issues in the agri-food industry have become a priority with federal and provincial governments, including the development of alternatives for chemical pest control products, with the ultimate goal of reducing chemical pesticide use. Rising economic, environmental and social costs associated with agricultural inputs, spray drift, pesticide residues, government legislation for reduced pesticide use, along with the development of herbicide resistance in weeds make biological control agents attractive strategies for weed control for both agricultural and domestic use.

Broad-leaved weeds in turf situations, such as lawns, parks, and golf courses, disrupt the desired visual uniformity (i.e. are unsightly), create problems in the maintenance of the turf due to clumping and growth habits of the weeds, compete with the turf for light, nutrients, and water. Weeds are also are irritants to humans when allergic reactions to their pollen or the chemicals applied for weed control occur. Important weeds in turfgrass belong to the Compositae (such as dandelion, sowthistle), Caryophyllaceae (such as chickweed), and Rubiaceae, and Convolvulaceae. Typically, control of weeds in turf has been with selective, nonselective, systemic, and contact herbicides applied at various times (pre-plant, pre-emergence, and post-emergence). Public pressure is mounting to prevent the use of chemical herbicides in public places such as parks and homeowners lawns, for example, By-laws have recently passed in Calgary, Alberta, and Halifax, Nova Scotia, both in Canada, against their use. Chemical herbicides used in these areas leads to increased chemical exposure to susceptible groups in the population like children, pets, and the elderly.

A number of bacteria and fungi are natural pathogens of weeds and it has been suggested that bioherbicides, or weed killers made from biological agents rather than chemical agents, may provide an alternative to chemical pesticides. For example, U.S. Pat. No. 6,008,159 discloses controlling annual weeds using the fungus Pyrenophora. U.S. Pat. Nos. 5,993,802 and 5,472,690 teach suppressing the growth of Calmagrostis canadensis using an isolate of a low temperature basidiomycete fungus, or a mycoherbicide (including at least one or both of Fusarium nivalis and Colletotrichum calamagrostidis), respectively. U.S. Pat. Nos. 5,952,264 and 5,635,444 teach controlling crabgrass using the fungus Cochliobolus intermedius, or a fungus selected from the genus Culvularia, respectively. U.S. Pat. No. 5,747,029 teaches controlling sicklepod weeds with the fungus Myrothecium verrucaria. U.S. Pat. No. 5,698,491 and WO 98/08389 discloses controlling nutsedge weeds with the fungus Dactylaria higginsii (WO 98/08389 and U.S. Pat. No. 5,698,491). U.S. Pat. No. 4,606,751 teaches controlling Johnson grass and similar weeds with Bipolaris sorghicola spores. The spores are suspended in a solution of water and surfactant and sprayed onto a field onto which the weed is growing. U.S. Pat. No. 5,795,845 discloses a bioherbicidal composition comprising an invert emulsion carrier and a microorganism which is a weakly or non-pathogenic bacterium or fungus. The composition may be used to control pigweed, plumeless thistle, velvet leaf and ground cherry. U.S. Pat. No. 4,636,386 discloses an isolate of Alternaria for the control of Italian thistle. U.S. Pat. No. 5,994,27 discloses a composition comprising a bioherbicide which is an isolate of Sclerotinia minor which produces foliar wilt and rot in broadleaf weed species so as to inhibit their growth. The bioherbicide may be used to control the growth of broadleaf weeds such as dandelion, broadleaf plantain, ragweed, ivy, knotweed sow thistle and white clover.

Brebaum and Boland (1999, Plant Disease 83:2000) disclose Phoma exigua and Phoma herbarum as pathogens of dandelion (Taraxacum officinale), however, no weed controlling activity was reported using these species.

None of the identified references disclose fungal isolates derived from Phoma macrostoma as biocontrol compositions suitable for use to control Canada thistle, dandelion, or other weed species.

There is a need in the art for novel bioherbicides and biocontrol compositions for controlling weeds. Further there is a need in the art for novel bioherbicides and biocontrol compositions for controlling weed plants for example Canada thistle, perennial sowthistle, dandelion, prairie sunflower, field bindweed, wild buckwheat, and scentless chamomile, cleavers, and chickweed. Further, there is a need in the art for biocontrol compositions comprising a biological control agent and a growth medium for supporting the viability of the biological control agent when the biocontrol composition is employed to control weeds.

It is an object of the present invention to overcome disadvantages of the prior art.

The above object is met by a combination of the features of the main claims. The sub claims disclose further advantageous embodiments of the invention.

SUMMARY OF THE INVENTION

The present invention relates to bioherbicides. More specifically, the present invention relates to fungal bioherbicides and compositions comprising fungal bioherbicides.

According to the present invention there is provided a method of controlling one or more broad leaf weeds comprising administering an isolate of Phoma cf. macrostoma, an extract therefrom, an inoculated broth therefrom, or a combination thereof, to the one or more broad leaf weeds, or to soil where said weeds grow.

The present invention is also directed to the method defined above wherein the one or more broad leaf weeds is a species of a family selected from the group consisting of Compositae, Caryophyllaceae, Convolvulaceae, Plantaginaceae and Rubiaceae. Preferably, the one or more broad leaf weeds is selected from the group consisting of Canada thistle, perennial sowthistle, dandelion, scentless chamomile, false cleavers, chickweed, wild buckwheat, plantain, prairie sunflower and field bindweed.

The present invention also provides a biocontrol agent comprising one or more than one Phoma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof, wherein the one or more than one Phoma isolate, or an extract therefrom, or an inoculated broth therefrom, exhibit weed control activity, growth enhancement activity, or both. The present invention also embraces a biocontrol composition, comprising the biocontrol agent just defined, and a medium for supporting viability of said one or more than one Phoma isolate. Preferably, the one or more than one Phoma isolate, is an isolate of Phoma cf. macrostoma. More preferably, the one or more than one Phoma isolate is selected from the group consisting of

• • a) 85-24B (IDAC 230201-1, deposited Feb. 23, 2001),
  • b) 89-25A (IDAC 110401-1, deposited Apr. 11, 2001),
  • c) 94-26(IDAC 230201-2, deposited Feb. 23, 2001),
  • d) 94-44B (IDAC 230201-3, deposited Feb. 23, 2001)
  • e) 94-134(IDAC 230201-4, deposited Feb. 23, 2001),
  • f) 94-359A (IDAC 110401-2, deposited Apr. 11, 2001),
  • g) 95-54A1(IDAC 230201-5, deposited Feb. 23, 2001),
  • h) 95-268B (IDAC 110401-3, deposited Apr. 11, 2001),
  • i) 97-12B (IDAC 230201-6, deposited Feb. 23, 2001),
  • j) 97-15B2(IDAC 110401-4, deposited Apr. 11, 2001), and

    
    
    

    a combination thereof. The Phoma cf. macrostoma isolates are deposited at the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2 Canada.

The present invention also provides a biocontrol agent comprising an extract, or an inoculated broth, from one or more than one Phoma cf. macrostoma isolate. Preferably, the extract or inoculated broth is obtained from the Phoma isolate selected from the group consisting of

• • a) 85-24B (IDAC 230201-1, deposited Feb. 23, 2001),
  • b) 89-25A (IDAC 110401-1, deposited Apr. 11, 2001),
  • c) 94-26(IDAC 230201-2, deposited Feb. 23, 2001),
  • d) 94-44B (IDAC 230201-3, deposited Feb. 23, 2001)
  • e) 94-134(IDAC 230201-4, deposited Feb. 23, 2001),
  • f) 94-359A (IDAC 110401-2, deposited Apr. 11, 2001),
  • g) 95-54A1(IDAC 230201-5, deposited Feb. 23, 2001),
  • h) 95-268B (IDAC 110401-3, deposited Apr. 11, 2001),
  • i) 97-12B (IDAC 230201-6, deposited Feb. 23, 2001),
  • j) 97-15B2(IDAC 110401-4, deposited Apr. 11, 2001), and

    
    
    

    a combination thereof. The Phoma cf. macrostoma isolates are deposited at the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2 Canada.

This invention pertains to the above method wherein the extract is selected from the group consisting of heat killed barley inoculum, a chloroform extract of the Phoma isolate, a methanol extract of the Phoma isolate, and a ethyl-acetate extract of the Phoma isolate, and the inoculated broth is selected from the group consisting of a crude inoculated broth, a filtered inoculated broth, or a centrifuged inoculated broth.

The present invention also pertains to a method of controlling weed development during crop growth comprising:

• • a) adding an effective amount of a biocontrol agent comprising one or more than one Phoma cf macrostoma isolate, an extract therefrom, an inoculated broth therefrom, or combination thereof to soil to produce treated soil, the one or more than one Phoma cf macrostoma isolate, an extract therefrom, or an inoculated broth therefrom, exhibiting weed control activity;
  • b) planting the crops in the treated soil; and
  • c) growing the crop.

According to the present invention there is also provided a method of controlling weed development during crop growth comprising:

• • a) planting the crop,
  • b) adding an effective amount of said biocontrol agent comprising one or more than one Phoma cf macrostoma isolate, an extract therefrom, an inoculated broth therefrom, or a combination therefrom, to soil where the crop is planted, the one or more than one Phoma cf macrostoma isolate, extract therefrom, or inoculated broth therefrom, exhibiting weed control activity;
  • c) growing the crop.

The present invention is also directed to a method of controlling weed development during crop growth comprising:

• • a) adding an effective amount of a biocontrol agent comprising one or more than one Phoma cf macrostoma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof, to a crop seed to produce treated crop seed, the one or more than one Phoma cf macrostoma isolate, extract therefrom, or inoculated broth therefrom, exhibiting weed control activity;
  • b) planting the treated crop seed; and
  • c) growing the crop.

Also included in this invention is the method as just defined wherein the treated crop seed is grass seed, including domestic and specialty turf grass seed, animal pasture or hay seed mixes comprising one or more of timothy, fescue, blue grass, perennial rye grass, bromegrass, canary grass, red top and orchard grass seed.

The methods of the present invention are preferably used to control weed development during growth of a perennial crop. Preferably, the perennial crop is selected from the group consisting of a turf, a perennial grass, and a winter cereal.

The present invention also provides a method of controlling weed development during established crop growth comprising:

• • a) adding an effective amount of a biocontrol agent comprising one or more than one Phoma cf macrostma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof, to the established crop, the one or more than one Phoma cf macrostoma isolate, an extract therefrom, or an inoculated broth therefrom, exhibiting weed control activity, and
  • b) growing the crop.

Also included in this invention is the method as just defined wherein the established crop is grass, including domestic and specialty turf grasses, animal pasture or hay mixes comprising one or more of timothy, fescue, blue grass, perennial rye grass, bromegrass, canarygrass, red top and orchard grass.

The present invention also provides a method of enhancing the growth of a crop, the method comprising:

• • a) adding an effective amount of a biocontrol agent comprising one or more than one Phoma cf macrostma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof to soil to produce a treated soil;
  • b) planting the crop in said treated soil, and
  • c) growing said crop.

The present invention also provides a method of enhancing the growth of a crop, the method comprising:

• • a) planting said crop in soil;
  • b) adding an effective amount of a biocontrol agent comprising one or more than one Phoma cf macrostma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof to the soil where said crop is planted; and
  • c) growing said crop.

The present invention further provides a method of enhancing the growth of an established crop, the method comprising:

• • a) adding an effective amount of a biocontrol agent comprising one or more than one Phoma cf macrostma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof to the established crop; and
  • b) growing the crop.

Also included in this invention is the methods as just defined wherein the crop is grass, including domestic and specialty turf grasses, animal pasture or hay mixes comprising one or more of timothy, fescue, blue grass, perennial rye grass, bromegrass, canarygrass, red top and orchard grass.

The present invention is also directed to a method of enhancing the growth of a crop, the method comprising:

• • a) adding an effective amount of a biocontrol agent comprising one or more than one Phoma cf macrostoma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof, to a crop seed to produce treated crop seed;
  • b) planting the treated crop seed; and
  • c) growing the crop.

Also included in this invention is the method as just defined wherein the treated crop seed is grass seed, including domestic and specialty turf grass seed, animal pasture or hay seed mixes comprising one or more of timothy, fescue, blue grass, perennial rye grass, bromegrass, canary grass, red top and orchard grass seed.

The methods of the present invention also relate to the use of a biocontrol composition comprising one or more than one Phoma cf macrostma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof; and a medium for supporting viability of the one or more than one Phoma cf macrostma isolate.

The present invention also provides for any of the above methods wherein the biocontrol agent or composition is applied to the soil before or after emergence of the weed, preferably before emergence.

The present invention also provides for any of the above methods wherein the biocontrol agent or composition is applied by dusting, rubbing, spreading, drilling, banding, broadcasting, spraying, liquid injection, pouring or soil drenching.

The present invention embraces a coated crop seed comprising one or more Phoma isolates and a binder. The invention also includes a coated crop seed comprising an extract obtained from one or more Phoma isolates and a binder. The crop seed that is coated is preferably grass seed, including domestic and specialty turf grass seed, animal pasture or hay seed mixes comprising one or more of timothy, fescue, blue grass, perennial rye grass, bromegrass, canary grass, red top and orchard grass seed.

This summary does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

FIG. 1 shows the effect of different amounts of inoculum suspension 85-24B on Canada thistle plants. The Rating scale is: 1=healthy, dark green foliage; 2=slightly yellow-green foliage; 3=leaves primarily yellow, some yellow-green; 4=leaves primarily white, a few yellow-green; 5=slants completely white; and 6=plants dead.

FIG. 2 shows the effect of fungal isolates 94-44B, 94-26, 95-54A1 and 97-12B on the root growth ratio of perennial sowthistle.

FIG. 3 shows the effect of fungal isolate 94-44B on foliar biomass of perennial sowthistle.

FIG. 4 shows the effect of fungal isolate 94-44B on shoot emergence of perennial sowthistle.

FIG. 5 shows a graphical representation of weed mortality following application of fungal isolate 94-44B, 89-25A and 97-12B to perennial sowthistle.

DESCRIPTION OF PREFERRED EMBODIMENT

The present invention relates to bioherbicides. More specifically, the present invention relates to fungal bioherbicides and compositions comprising fungal bioherbicides.

The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

By the term “controlling weed growth”, or “weed control activity” it is meant that one or more fungal isolates, an extract therefrom, an inoculated broth therefrom, or a combination thereof, when applied on or near a weed interferes with the normal growth and development of a weed. Examples of weed growth control activity include, but are not limited to, inhibition of root growth, inhibition of shoot growth, inhibition of shoot emergence, reduction of weed biomass inhibition of seed production, or the ability to induce chlorosis, or reduce competitiveness of a weed for water, nutrients, or a combination thereof, that would otherwise be utilized by a crop plant. Alternatively, the fungal isolate may be capable of controlling weeds by killing them. It is preferred that a fungal isolate selectively controls weed growth, and does not have any substantial effect on a plant for which growth is desired, for example a non-target plant such as an agriculturally important plant, or a residential or commercial grass.

Fungal isolates that control weed growth or the exhibit weed control activity may be characterized as having a Weed Control Index (WCI) of between about 20% to 100% (the higher the WCI, the more efficacious the fungal isolate). The WCI includes either an annual WCI (WCIA) or a perennial WCI (WCIP) as defined below. Preferably, fungal isolates are characterized as having a WCI of between about 50% to 100%. More preferably, the fungal isolates have a WCI of between about 70% to 100%. However, it is to be understood that a fungal isolate with a low WCI may still prove effective to help control weed growth and provide a non-target plant a competitive advantage over one or more weeds. Furthermore it may be desirable to use one or more fungal isolates that exhibit a low WCI in order to ensure that a non-target plant is not affected by the bioherbicide.

The weed control Index is determined using either WCIA for evaluation of annual weeds, or WCIP, for evaluation of perennial weeds, as follows:

WCIA={[(100−FFW)+(% M)+(% IOC)]/300}×100%

WCIP={[(100−RW)+(100−FFW)+(% M)+(% IOC)]/400}×100%

where

• • RW—is root weight
  • FFW—is foliar fresh weight;
  • M—is mortality; and
  • IOC—is incidence of chlorosis, as determined by number of plants with a rating of 3-6, where, 1=healthy, dark green foliage; 2=slightly yellow-green foliage; 3=leaves primarily yellow, some yellow-green; 4=leaves primarily white, a few yellow-green; 5=plants completely white; and 6=plants dead.

By “fungal isolate” it is meant a biologically active Phoma species, or a biologically active fragment, component, obtained or isolated from a Phoma spp. By fragment or component of a fungal isolate, it is meant a fragment of the mycelium, or one or more spores, pycnidia, conidia, chlamydospores or a combination thereof, obtained from the fungi. Fungal isolates may be obtained from small chlorotic and necrotic lesions on leaf and stem tissues of a desired weed, for example but not limited to Canada thistle and assayed for weed growth control activity, as described herein or using standard methods as would be known to one of skill in the art. Preferably, the fungal isolates are strains of Phoma cf. macrostoma. Examples of Phoma cf. macrostoma isolates, which may be used according to the present invention, and which are not to be considered as limiting in any manner, include:

• • 85-24B (IDAC 230201-1, deposited Feb. 23, 2001),
  • 89-25A (IDAC 110401-1, deposited Apr. 11, 2001),
  • 94-26(IDAC 230201-2, deposited Feb. 23, 2001),
  • 94-44B (IDAC 230201-3, deposited Feb. 23, 2001)
  • 94-134(IDAC 230201-4, deposited Feb. 23, 2001),
  • 94-359A (IDAC 110401-2, deposited Apr. 11, 2001),
  • 95-54A1(IDAC 230201-5, deposited Feb. 23, 2001),
  • 95-268B (IDAC 110401-3, deposited Apr. 11, 2001),
  • 97-12B (IDAC 230201-6, deposited Feb. 23, 2001),
  • 97-15B2(IDAC 110401-4, deposited Apr. 11, 2001),

    
    
    

    or a combination thereof. The Phoma cf. macrostoma isolates are deposited at the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2 Canada.

By “extract”, it is meant an aqueous or solvent extract, crude or in a more purified state, comprising one or more active compounds obtained from a fungal isolate, that in the proximity of, or when applied onto, a weed is capable of controlling weed growth.

By an “inoculated broth”, it is meant the broth obtained from a culture of one or more than one Phoma isolates (see Example 5) as defined herein, that comprise one or more active compounds capable of controlling weed growth. An inoculated broth may be concentrated using methods known in the art, for example, but not limited to evaporation, roto-evaporation or freeze drying.

By “saturation” it is meant the maximum retention capacity of a soil, and is defined as occuring when the soil pores in the upper part of the soil are filled with water. By “field capacity” or “field moisture capacity”, it is meant the percentage of water remaining in a soil two or three days after having been saturated and after free drainage has essentially ceased.

By “permanent wilting point” it is meant the critical moisture of soil as which plants wilt and fail to recover turgidity when placed in a dark and humid atmosphere.

In a preferred embodiment, the fungal isolate is formulated in a biocontrol composition comprising one or more fungal isolates, one or more extracts obtained from a fungal isolate, an inoculated broth, or a combination thereof, and the biocontrol composition is added to soil, added to compost, added to peat-type pellets, added to or used to coat a planting medium, for example but not limited to wood chips, used to coat or treat plant seed in the presence of a binder, for example but not limited to methylcellulose, starch, clay, sugar or a combination thereof, or applied to a plant, for example but not limited to, spraying or rubbing on a plant, to control weeds. Furthermore, liquid injection may be used to apply one or more isolates, for example, spores or mycelia, extracts obtained from fungal isolates, inoculated broth, or a combination thereof, to soil. Liquid injection may used for perennial applications, for example but not limited to turf grass management.

By the term “biocontrol composition”, it is meant a composition comprising one or more than one biocontrol agent of the present invention within a suitable medium. A biocontrol agent consists of one or more fungal isolates as defined above, an inoculated broth therefrom, an extract therefrom, or a combination thereof. For example, if the biocontrol composition comprises a fungal isolate, then the suitable medium may comprise a growth medium to maintain the viability of the fungal isolate before, and after application of the biocontrol composition to the soil. If an extract of a fungal isolate, one or more fungal isolates, or a combination thereof, is used for administration to a weed or soil, then the suitable medium may comprise stabilizing agents, surfactants and the like as would be known to one of skill in the art. For example, which is not to be considered limiting in any manner, media may include supplemented Agar, pesta, peat prills, vermiculite, clay, starches, potato dextrose broth (PDB), V8® juice broth, whole grain or grain fragments of, for example but not limited to, legume grains including lentil or chickpea, or cereal grain for example, wheat or barley, or corn, or any combination or variant thereof, provided that the medium allows the fungal isolate to remain viable.

Pesta is a term for a granular product made from a cereal grain flour and a biocontrol agent. The process encapsulates biocontrol agents in pasta-like products called pesta (Connick et al., 1991, which is incorporated herein by reference). Bacteria formulated in such media may exhibit extended shelf and field-life (e.g. Connick et al., 1996; Connick et al., 1998). These characteristics are desired in a product which may be stored prior to use or shipped over long-distances prior to being used for weed control in a field. Therefore, the biocontrol compositions comprising fungal isolates of the present invention may be formulated in a suitable medium for example, but not limited to, pesta.

If the suitable medium is a growth medium, then the growth medium may comprise any liquid, semi-liquid or solid medium which allows the fungal isolates of the present invention to grow or remain viable. Any growth medium known in the art to which is capable of supporting the fungal isolate may be employed. Examples of suitable growth media, which are not to be considered limiting in any manner include potato dextrose agar, potato dextrose broth, V8® juice broth and the like. Preferably, the growth medium is a solid medium, for example but not limited to grain, for example whole grain or fragments thereof, for example but not limited to, legume grains including lentil or chickpea, or cereal grain for example, wheat or barley, or corn (see Example 3). The growth medium should also permit an effective amount of the fungal isolate to remain viable after being applied to the soil of a crop for a suitable period of time, for example but not limited to, up to about 7 days to about 18 months after application. Preferably, the isolate remains viable from about 14 days to about 12 months, and more preferred, from about 14 days to about 90 days. For soil application, spores, mycelia (growing on grain), or spores and mycelia growing on grain may be mixed together and either applied onto, or mixed with, soil. Furthermore, liquid injection may be used to apply one or more isolates, for example, spores or mycelia, extracts obtained from fungal isolates, or a combination thereof to soil. Typical application rates for a fungal isolate that was grown on the preferred growth medium include, but are not limited to, 0.001 kg/m² to 5 kg/m² using a particle size between 49-840 microns and particle viability of 60-100%. The preferred rate of application is 0.1 kg/m² to 1.0 kg/m². However, any application rate that results in weed control activity may be employed.

When one or more fungal isolates are applied using a solid medium, for example hulless barley, the infested barley grain prepared as described in Example 3 may be ground prior to application to soil. Any suitable granule size may be used, for example, from about 50μ to about 1 mm. The preferred viability of the particles used for application is about 60-100%. As shown in Table 20 (Example 3), with smaller granule size, a lower application dose rate (g/m²)will achieve a similar, or better, weed control activity.

It is also contemplated by the present invention that more than one fungal isolate may be used to control weeds. Similarly, a biocontrol composition may comprise more than one fungal isolate. Multiple fungal isolates capable of controlling a specific weed may be used or multiple fungal isolates, each of which is capable of controlling a distinct type of weed may be mixed and used as described herein. It is also preferred that the fungal isolate or biocontrol composition exhibit host selectivity, in that weed control activity is observed in one or more target weeds, while no weed control activity is observed on non-target plants. Examples of non-target plants include agriculturally important plants, and domestic or commercial grasses (Gramineae).

By weed, it is meant any undesired plant. Preferably, a weed is a broad-leaf (dicot) weed, for example but not limited to members of the Compositae, Caryophyllaceae, Polygonaceae, Convolvulaceae, Plantaginaceae and Rubiaceae. More preferably, a weed is selected from the group consisting of:

• Compositae (Composite family): including dandelion [Taraxacum officinale L.], ox-eye daisy [Chrysanthemum leucanthemum], burdocks for example common burdock [Arctium minus], goat's beards [e.g. Tragopogon dubius], cockleburs [e.g. Xanthium strumarium], ragweeds for example common ragweed [Ambrosia artemisiifolia] or giant ragweed [Ambrosia trifolia], scentless chamomile [Matricaria perforata Mérat.], sow-thistles, for example perennial sowthistle [Sonchus arvensis L.], and thistles, for example Canada thistle [Cirsium arvense L.(Scop.)];
• Caryophyllaceae (Pink Family): including chickweed [Stellaria media (L.)Vill.];
• Polygonaceae (Buckwheat Family): including wild buckwheat [Polygonum convolvulus L.];
• Convolvulaceae (Morning Glory Family): including field bindweed [Convolvulus arvensis L.];
• Plantaginaceae (Plantain Family): including plantain [Plantago lanceolata]; or
• Rubiaceae (Rubus family): including false cleavers (Gallium spurium)

Preferably, the fungal isolates, biocontrol compositions, or both, of the present invention are added to the soil where the seed either grows or may grow. The soil may be mixed so that one or more fungal isolates are in close proximity to the root system or root fragments of the weeds. It is also preferable that the fungal isolates be in close proximity to weed seeds when such seeds are present. The fungal isolates and biocontrol compositions of the present invention may be applied to soil or weed by any method known in the art such as, but not limited to dusting, rubbing, spreading, drilling, banding, broadcasting (with or without incorporation), spraying, liquid injection, pouring or soil drenching. The fungal isolates and biocontrol compositions may also be applied at any suitable time, for example but not limited to, during or after soil tillage. Preferably, the biocontrol composition is applied during the spring, or early summer. Solid preparation of the fungal isolate alone, or biocontrol composition for example but not limited to infested barley grain, is added to soil in the amount of about 0.1 kg/m² to about 5 kg/m². Liquid suspensions of about 10³ to about 10⁹ cfu/mL, may be applied at a rate of about 1 L/m² to about 5 L/m². However, any amount that results in weed controlling activity may be applied.

It is also within the scope of the present invention, that extracts obtained from one or more fungal isolates may be formulated and applied to the soil or weed as a liquid, for example as a spray, injection, drench, rubbing, dusting, or as a solid, including autoclaved infested barley granules dusting or rubbing of suitably formulated extracts. As one of skill will be able to determine, appropriate dosages will depend upon the concentration of active components within the extract or solid. Preferably, the extract is derived from either a 4 week old crude broth concentrated about 100× the original volume, or from an extract obtained from a 3:1 ratio of extracted mycelium to methanol. An example, which is not to be considered limiting, of an application rate of such extracts is from about 0.1 to about 2.5 L/m², depending upon the concentration of active ingredients. However, any amount that results in weed controlling activity may be applied.

A biocontrol agent, or a biocontrol composition, of the present invention comprising one or more fungal isolates, one or more extracts obtained from a fungal isolate, an inoculated broth, or a combination thereof may be added to a planting medium, for example compost, or it may be added to or used to coat alternate planting media, for example but not limited to wood chips, landscaping cloth, vermiculite and the like, as would be evident to one of skill in the art. Furthermore, the biocontrol agent or biocontrol composition as described herein may be used to coat or treat plant seed. Coated seed may involve the use of a binder, for example but not limited to methylcellulose, starch, clay, sugar or a combination thereof.

Therefore, according to the present invention, there is provided a method of controlling a range of weeds with a bioherbicide comprising one or more fungal isolates, a biocontrol agent, or a biocontrol composition comprising one or more fungal isolates, an extract obtained from one or more fungal isolates, an inoculated broth, or a combination thereof. Fungal isolates, or a combination thereof, which may be employed to control weed growth include, but are not limited to those listed in Table 1 below.

TABLE 1

Fungal Isolates Information and Target Weeds Affected

Fungal

Deposit

isolate

Target Weed

information

95-54A1

Canada thistle, Scentless chamomile,

IDAC 230201-5*

False cleavers, Chickweed, Field

bindweed, Dandelion, Plantain,

Prairie sunflower,

97-12B

Canada thistle, Dandelion, Scentless

IDAC 230201-6*

Chamomile, False cleavers,

Perennial sowthistle, Chickweed

97-15B2

Canada thistle, Scentless chamomile,

IDAC 110401-4**

False cleavers, Chickweed, Wild

buckwheat, Prairie sunflower

94-359A

Scentless chamomile, Canada thistle,

IDAC 110401-2**

Dandelion

89-25A

Canada thistle, scentless chamomile,

IDAC 110401-1**

dandelion, Prairie sunflower

85-24B

Canada thistle, Dandelion, Scentless

IDAC 230201-1*

chamomile, False cleavers, Prairie

Sunflower, chickweed, Plantain,

wild buckwheat

94-26

Canada thistle, Dandelion, Scentless

IDAC 230201-2*

chamomile, False cleavers,

Perennial sowthistle, Chickweed,

Plantain, wild buckwheat

94-44B

Canada thistle, Dandelion, Scentless

IDAC 230201-3*

chamomile, False cleavers,

Perennial sowthistle, Chickweed,

Wild buckwheat, Plantain, Prairie

sunflower

94-134

Canada thistle, Chickweed, Wild

IDAC 230201-4*

buckwheat,

Scentless chamomile, Plantain, False

cleavers,

95-268B

False cleavers, Chickweed, Wild

IDAC 110401-3**

buckwheat, Scentless chamomile,

Canada thistle, dandelion

*deposited Feb. 23, 2001 at the International Deposit Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2 Canada.

**deposited Apr. 11, 2001 at the International Deposit Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2 Canada.

Referring now to Table 2 (and see Example 2 for associated protocols) there is shown, as an example, weed control activity, as indicated by a reduction of foliar fresh weight, reduction in root weight, chlorosis, or mortality, in Canada thistle by a range of fungal isolates.

TABLE 2

Effect of fungal isolates on foliar fresh weight (FFW), root weight

(RW), mortality (M) and incidence of Chlorosis (IOC) on Canada

Thistle plants, and associated Weed Control Index (WCIP)

Isolates

% FFW*

% RW*

% M

% IOC

WCIP**

Control

100 ± 4 

100 ± 5 

 1 ± 1

0

0

85-24B

22 ± 6

25 ± 4

57 ± 7

86 ± 6

74

94-26

23 ± 7

26 ± 6

72 ± 7

83 ± 6

76

94-44B

 8 ± 3

15 ± 2

80 ± 5

96 ± 2

88

94-134

20 ± 8

26 ± 5

 59 ± 11

74 ± 9

72

95-54A1

 20 ± 10

23 ± 8

79 ± 8

82 ± 8

79

97-12B

 59 ± 13

 53 ± 11

 39 ± 11

 61 ± 11

47

89-25A

 76 ± 13

 63 ± 11

23 ± 8

 36 ± 10

30

94-359A

 69 ± 13

 63 ± 10

18 ± 9

 43 ± 13

32

95-268B

31 ± 7

32 ± 6

58 ± 8

75 ± 7

68

97-15B2

17 ± 4

17 ± 4

81 ± 8

89 ± 6

85

*% of control

**WCIP = {[(100 − RW) + (100 − FFW) + (% M) + (% IOC]/400} × 100%.

From Table 2, it can be noted that several fungal strains, for example but not limited to, 85-24B, 94-26, 94-44B, 94-134, 95-54A1, 97-12B, 95-268B and 97-15B2 are capable reducing foliar fresh weight in Canada thistle from about 40% to about 92%, and of suppressing root weight by about 47% to about 85% compared to the uninoculated control. These fungal isolates are characterized as having a WCIP from about 47 to 88% and they are effective in controlling weed growth which is supported by the observation that up to about 80% of the plants are killed by the treatment. Fungal isolates 89-25A and 94-359A are also effective at suppressing foliar fresh weight, root weight, and exhibit a WCIP of about 30-32%.

Therefore, the present invention is directed to a bioherbicide comprising fungal isolates 85-24B, 94-26, 94-44B, 94-134, 95-54A1, 97-12B, 89-25A, 94-359A, 95-268B, 97-15B2, or a combination thereof for the control of Canada thistle.

Referring now to FIG. 1, there is shown the effect of an inoculum suspension comprising fungal isolate 85-24B on Canada thistle. FIG. 1 demonstrates that damage to Canada thistle was greater at higher inoculum levels. A dose in the range of about 5 g/0.01 m² to about 50 g/0.01 m² or higher is capable of controlling Canada thistle. Similar result were also observed by applying granules of infested barley grain to the soil.

Without wishing to be bound by theory, the fungal isolates as described herein may have the ability to weaken Canada thistle, or a range of other perennial or annual weeds, as described below, by affecting processes involved in plant growth and development, for example photosynthesis, the accumulation of storage products in the roots, reducing shoot emergence, reducing root growth, inducing symptoms of chlorosis (yellowing of plant leaves).

Characterization of the weed control activity of several fungal isolates of the present invention indicates that weed control activity of a fungal isolate may last about one growing season, depending upon the time of application of the fungal isolate to the soil or plant. With reference to Table 18 (Example 3), it is shown that spring or summer application of a fungal isolate exhibits weed control activity over one or more growth seasons. Fall application results in no observed weed control activity. Furthermore, as shown in Table 19 (Example 3), weed control activity increases with higher soil moisture content.

A number of fungal isolates were also tested to determine their efficacy at controlling weeds other than Canada thistle, for example, members of the Aster family including Sonchus arvense (perennial sowthistle), Helianthus (prairie sunflower), Taraxacum officinale (dandelion), Matricaria perforata (scentless chamomile), and other plants, including chickweed (Stellaria media), wild oats, green foxtail, and false cleavers (Gallium spurium).

Fungal isolates 85-24B, 89-25A, 94-26, 94-359A, 94-44B, 94-134, 95-54A1, 95-268B, 97-15B2 and 97-12B were applied to perennial sowthistle using the inoculum mat bioassay described in Example 2. As shown in FIG. 2, fungal isolates 94-44B, 94-26, 95-54A1 and 97-12B reduced the weight of roots compared to the uninoculated control in greenhouse trials. A similar reduction in root weight was also observed with most of the other fungal isolates as indicated in Table 8 of Example 2. Further, fungal isolate 94-44B significantly reduced foliar biomass (FIG. 3) and reduced shoot emergence (FIG. 4) relative to the control. Three isolates (94-44B, 89-25A, and 97-12B) increased the mortality of the weed as shown in FIG. 5 and Table 8.

Therefore, fungal isolates 94-44B, 89-25A, 94-26, 95-54A1 and 97-12B may be used to control perennial sowthistle (Sonchus arvensis). In a preferred embodiment, fungal isolate, or a biocontrol composition comprising 94-44B is used to control perennial sowthistle.

Similar results have been observed of the effect of these fungal isolates on other weeds, both perennial and annual weeds, as demonstrated by determining the WCI's for a range of weed species, for example as shown in Table 3 (also see Example 2).

TABLE 3

Weed control index (WCI) of fungal isolates on scentless chamomile

(SC), false cleavers (FC), prairie sunflower (SF), chickweed (CH),

wild buckwheat (WB), field bindweed (FB), perennial sow thistle

(PST), dandelion (DA), and Canada thistle (CT).

Isolate

SC*

FC*

SF*

CH*

WB*

FB*

PST**

DA**

CT**

No fungus

7

4

0

0

0

 3

0.1

0

0

85-24B

79

30

84

76

92

nd

7

62

74

94-26

27

48

nd

91

39

nd

24

47

76

94-44B

82

64

69

99

96

nd

58

59

88

94-134

82

54

6

59

85

nd

8

15

72

95-54A1

98

54

72

59

85

nd

8

15

72

97-12B

93

43

9

57

15

nd

34

61

47

89-25A

71

13

70

nd

13

nd

15

43

30

94-359A

50

18

3

nd

17

13

6

25

32

95-268B

72

92

0

97

81

nd

0

45

68

97-15B2

91

72

53

65

55

nd

7

9

85

nd = not determined

*WCIA(%) = {[(100 − FFW) + (% M) + (% IOC)] ÷ 300} × 100%.

**WCIP (%) = {[(100 − RW) + (100 − FFW) + (% M) + (% IOC)] ÷ 400} × 100%.

Therefore, the present invention is also directed to a bioherbicide comprising fungal isolates 85-24B, 94-26, 94-44B, 94-134, 95-54A1, 97-12B, 89-25A, 94-359A, 95-268B, 97-15B2, or a combination thereof, for the control of any susceptible weed, both annual and perennial. Preferably the weed is a broad leaf weed. More preferably, the broad leaf weed is from Compositae, Caryophyllaceae, Polygonaceae, Plantaginaceae, Rubiaceae, or Convolvulaceae, for example but not limited to, scentless chamomile, false cleavers, chickweed, wild buckwheat, field bindweed, perennial sow thistle, dandelion, and Canada thistle.

Using the inoculum mat bioassay (method outlined in Example 2), a number of fungal isolates were tested for their ability to control sunflower (Helianthus) weeds. Germination of seed was affected by fungal isolate 85-24B, which reduced sunflower seed germination by about 10%. Five fungal isolates (85-24B, 94-44B, 89-25A, 95-54A1, 97-15B2) reduced foliar biomass in prairie sunflower (also see Table 10(B), Example 2). Thus, fungal isolates 85-24B, 94-44B, 89-25A, 95-54A1, 97-15B2 may be used to control prairie sunflower.

It has also been observed that these fungal isolates and biocontrol compositions comprising the fungal isolates of the present invention are specific for a target group of weed plants, for example, those of the Aster (Compositae) family. Generally, the fungal isolates of the present invention were not effective in controlling growth of grasses, for example, wild oats, and green foxtail (see Table 12 and 13, respectively). However, 94-44B exhibits weed control activity in wild oats, and 85-24B and 95-359A exhibits weed control activity in green foxtail (see Table 17, Example 2 for summary of WCI's).

The fungal isolates of the present invention also exhibit selectivity in that, even under high inoculum loads (significantly higher than that used under field conditions), isolates can be identified that induce negligible, or no, disease symptoms in crop plants (see Tables 22 and 23, Example 4). Example of crop plants tested include:

• • 1) Cereal and other monocots

    • Wheat—cvs. Katepwa, AC Domain, AC Karma, Biggar, Kyle
    • Barley—cvs. Harrington, Silky
    • Oat—cvs. Derby or Walden
    • Millet—cvs. Minco or Prairie Gold
    • Canary seed—cv. Keet

  • 2) Oilseed crops

    • Canola—cvs. AC Excel, AC Parkland
    • Mustard—cvs. Cutlas, Ochre
    • Flax—cv. Vimy
    • Sunflower—cvs. Cargill SF270 or IS7111
    • Safflower—cv. Lethbridge

  • 3) Pulse crops

    • Lentil—cvs. Laird, Eston
    • Field pea—cv. Express
    • Chickpea—cv. Sanford
    • Faba bean—cv. CDC Fatima

  • 4) Forage crops

    • Clovers—yellow clover cv. Norgold, white clover cvs. Polara and Sonja, common clover,
    • red clover cvs. Altaswede or Florex
    • Birdsfoot trefoil—cv. Cree
    • Alfalfa—cv. Beaver

For example, which is not to be considered limiting in any manner, 94-44B is suitable for use with cereal crops, as even under high inoculum loads no disease symptoms are observed. However, the use of 94-44B under high inoculum loads may not be desired for use on pulse crops, as pulse crops exhibit some disease symptoms under these conditions. If lower inoculum loads of 94-44B are used, then the disease symptoms in pulse are minimized and this fungal isolate may be used with pulse crops. At reduced inoculum loads, these isolates still exhibit weed control activity. One of skill in the art may manipulate the dosage to optimize the balance between obtaining weed control activity and avoiding disease symptoms in a non-target crop, agriculturally or commercially important plant.

Also contemplated by the present invention is the use of an inoculated broth, or an extract from one or more of the fungal isolates of the present invention as a weed control agent. As described in Example 5, an inoculated broth (including a concentrated inoculated broth), aqueous or solvent extracts, obtained from one or more fungal isolates as described herein, and reconstituted in an appropriate medium, for example, but not limited to water or methanol, may be applied to the soil or leaf of a plant and exhibit weed control activity. It is also contemplated that an inoculated broth, or an extract of the present invention may be combined with a chemical herbicide, or a fungal isolate, including non-Phoma isolates as a weed control agent.

Also contemplated by the present invention is a method using one or more than one Phoma cf macrostma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof for controlling weed development during growth of an establishing or established crop, for example, as listed above, and including, but not limited to a grass, such as, but not limited to domestic and specialty turf grasses, animal pasture or hay mixes comprising one or more of timothy, fescue, blue grass, perennial rye grass, bromegrass, canary grass, red top and orchard grass, as illustrated, for example, in Table 18A (Example 3).

The present invention further contemplates a method using one or more than one Phoma cf macrostma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof for enhancing the growth (e.g. increasing biomass) of an establishing or established crop, for example, as listed above, and including, but not limited to a grass, such as, but not limited to domestic and specialty turf grasses, animal pasture or hay mixes comprising one or more of timothy, fescue, blue grass, perennial rye grass, bromegrass, canary grass, red top and orchard grass, as illustrated, for example, in Tables 18A (Example 3), 21(C) and 21(D) (Example 3). Without wishing to be bound by theory, enhancement of growth may be a result of an increase in the rate of germination.

Also contemplated by the present invention is a method using one or more than one Phoma cf macrostma isolate, an extract therefrom, an inoculated broth therefrom, or a combination thereof for controlling weed development during growth of an establishing or established crop by foliar spray application before or after emergence of the weed. The crop can be, for example, one of the crops listed above, and including, but not limited to a grass, such as, but not limited to domestic and specialty turf grasses, animal pasture or hay mixes comprising one or more of timothy, fescue, blue grass, perennial rye grass, bromegrass, canary grass, red top and orchard grass, as illustrated, for example in Table 21(D) (Example 3).

The application of an inoculated broth, or an extract of the present invention, when combined with an fungal isolate, exerts weed controlling activity in a rapid manner, when compared to applying an isolate on its own. Without wishing to be bound by theory, the inoculated broth, or extract is able to function in a more rapid manner, since there is no requirement for growth of the fungal isolate, and associated production of the one or more compounds from the growing fungal isolate prior to noting weed controlling activity. Such a combined application provides a rapid and more consistent weed controlling activity during the period of exposure of a plant to the extract-isolate, or inoculated broth-isolate mixture.

The present invention also contemplates the use of a fungal isolate or any combination of fungal isolates with one or more chemical herbicides. Further, the present invention contemplates biocontrol compositions comprising a fungal isolate of the present invention, or a plurality of fungal isolates with one or more chemical herbicides and a growth medium for supporting the viability of the fungal isolates. The present invention also encompasses the use of a fungal isolate or any combination of fungal isolates with one or more non-Phoma fungal strains. Further, the present invention contemplates biocontrol compositions comprising a fungal isolate of the present invention, or a plurality of non-Phoma fungal isolates with one or more herbicides and a growth medium for supporting the viability of the fungal isolates.

The above description is not intended to limit the claimed invention in any manner, Furthermore, the discussed combination of features might not be absolutely necessary for the inventive solution.

The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.

EXAMPLES

Example 1

Isolation, Storage and Growth of Fungal Isolates

Fungal Isolates

Fungal strains were isolated from small chlorotic and necrotic lesions on leaf and stem tissues of Canada thistle plants collected from fields, pastures, and roadsides. Purified fungi isolated from the plant tissues were verified to cause the disease symptoms using Koch's postulates.

TABLE 4

Information on fungi isolated from Canada thistle.

Host Growth

Original

Name

Location

Habitat

Stage

symptoms

85-24B

Erwood, SK

not recorded

flowering

leaf spots,

chlorosis

94-26

Chatham, ON

roadside

vegetative

chlorosis

94-44B

Melfort, SK

roadside

bolting

chlorosis

94-134

St. Quentin, NB

waste field

bolting

chlorosis

95-54A1

Coldbrook, NS

pasture

bolting,

foliar

flowering

necrosis

97-12B

Valleyview, AB

fallow field

flowering

stem lesion

89-25A

Ituna, SK

field crop

vegetative

leaf spots

94-359A

RM 157, SK

barley field

seed setting

leaf spot,

stem lesion

95-268B

Rosthern, SK

filed crop

flowering

leaf spot

97-15B2

Westlock, AB

pasture

flowering

stem lesion,

top dieback

Isolation of Fungal Isolates

Fungal isolates, obtained as outlined above are surface sterilized for 2 minutes in 0.5% sodium hypochlorite, rinsed in sterile distilled water, and placed on Difco PDA (Potato Dextrose Agar) plates at 24° C. with 12 hours light, for 3-7 days. Fungi growing from the tissues are individually transferred to fresh media and allowed to grow to maturity. Isolates are screened for pathogenicity using Koch's postulates with a detached leaf bioassay. Freshly cut leaves from Canada thistle are surface sterilized and placed on a moist Whatman #3 filter paper in a glass petrie dish. An agar plug from the purified fungal culture is placed on the centre of each leaf, the dish is sealed to prevent moisture loss, and incubated as described above. The development of disease symptoms is observed. Multiple copies of the purified pathogenic cultures are placed in cyropreservation storage as outlined below.

Purified fungal cultures are stored by cryopreservation of spores and mycelia at −78° C. using a 1:1 mixture of 10% skim milk (w/v) to 40% glycerol (v/v) solutions.

The following 10 fungal cultures were identified by CBS, The Netherlands as Phoma cf. macrostoma Montagne, and were deposited within the International Deposit Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2 Canada as follows:

• • 85-24B (IDAC 230201-1, deposited Feb. 23, 2001);
  • 89-25A (MAC 110401-1, deposited Apr. 11, 2001);
  • 94-26(IDAC 230201-2, deposited Feb. 23, 2001);
  • 94-44B (IDAC 230201-3, deposited Feb. 23, 2001);
  • 94-134(IDAC 230201-4, deposited Feb. 23, 2001);
  • 94-359A (IDAC 110401-2, deposited Apr. 11, 2001);
  • 95-54A1(IDAC 230201-5, deposited Feb. 23, 2001);
  • 95-268B (IDAC 110401-3, deposited Apr. 11, 2001);
  • 97-12B (IDAC 230201-6, deposited Feb. 23, 2001); and
  • 97-15B2(IDAC 110401-4, deposited Apr. 11, 2001).

    
    
    

    Culturing and Storing Fungal Isolates

Flasks containing 125 mL of Potato Dextrose Broth (PDB) or V8® juice broth (Dhingra, O.D., and Sinclair, J.B. 1995. Basic Plant Pathology Methods, Second Edition, CRC Press Inc., Boca Raton, Fla. ) were inoculated with 1×10⁵ conida/L and incubated on a shaker (150 rpm) under ambient room conditions (20° C.) for a minimum of 7-14 days, but more preferably for 14-28 days. The contents of each flask are then vacuum filtered to separate spores and mycelium. Conidial concentration and fresh weight of mycelium are measured.

Fungi may be grown on solid or liquid media. For solid media culture, all fungal isolates are grown on Difco Potato Dextrose Agar (PDA; prepared as per manufacturer's directions) augmented with 3 ml 85% lactic acid per liter of media. A vial of fungal culture taken from cyropreservation is thawed to room temperature and the contents are aseptically distributed by pouring or pipetting the contents onto three or more prepared plates. The inoculum is spread over the surface of each plate using a sterile glass hockey stick. Agar plates are incubated either on a lab bench at ambient room temperature or in an incubator at 23/18° C. with 12 hours light (20 W cool white fluorescent bulbs) for one to two weeks.

For liquid media culture, 125 ml of Difco PDB is placed in a in a 500 ml Erlenmeyer. The flask is inoculated with either a spore suspension or agar plugs. For the spore suspension, an mature Difco PDA plate is flooded with sterile distilled water and the spores are gently dislodged with a sterile glass hockey stick. The spore suspension is diluted to a concentration of 1×10⁶ spores/ml and then. 1 ml of the diluted spore solution is added to each flask of liquid medium. To inoculate the liquid medium with agar plugs, 5-8 mm diameter agar plugs are taken from a mature culture on a Difco PDA plate. The inoculated flasks are incubated on a bench top shaker at 150 rpm for 2 weeks at ambient light and temperature conditions.

Mycelia (10 g) from the liquid cultures is placed in 20 mL of 5% skim milk: 20% glycerol cryo-preservation solution and homogenized. The samples are frozen and stored at −18° C. and −73° C. for 30 days and compared to a control prepared immediately after homogenization. Following storage for 30 days, viability is determined by spreading 500 μL of the suspension on a plate of ½ strength PDA, incubating for 4 days, and assessing mycelial growth and conida production. As an example, there was no loss in viability of fungal isolate 85-24B grown in PDB or V8 juice broth after storage at −18° or −73° C. for a period of 1 month.

Example 2

Control of Weed Growth using Fungal Isolates

Effect of Dose on Weed Control

Fungal isolates are grown on PDA and lactic acid for 10 to 14 days. The agar plates cultured with fungus are weighed into doses of 50 g (equivalent to an entire agar plate), 10 g, 5 g, 2.5 g, and 0 g (control), then macerated with sterile distilled water and each dose of the inoculum suspension is brought to a final volume of 50 mL. As an example, 85-24B is tested.

Roots are cut into appropriate lengths, for example roots of Canada thistle are 10 cm long, weighed and placed in 10 cm square pots filled with soil. A dose of the inoculum suspension is poured over the surface of the roots, covered with 1 cm soil, watered to saturation and placed in a greenhouse (20° C. day, 15° C. night; 16 hr daylight) with 6 replicates. Plant are rated for shoot emergence, chlorosis and death at 2, 4, and 6 weeks. At 6 weeks, roots are harvested and weighed. Results from this study using isolate 85-24B are presented in FIG. 1 ( the Rating scale is 1=healthy, dark green foliage; 2=slightly yellow-green foliage; 3=leaves primarily yellow, some yellow-green; 4=leaves primarily white, a few yellow-green; 5=plants completely white; and 6=plants dead).

These results demonstrate that fungal isolates of the present invention can control weed growth, and that this effect is more prominent with increased amounts of inoculum administered to the roots.

Inoculum Mat Bioassay

Roots are washed for 1 hour under running tap water to remove excess soil, and cut into 10 cm lengths each length with at least one bud. The weight of 2-10 cm root lengths, keeping similar root weights for all pots used in a replicate, is recorded A two week old inoculated agar plate is inverted over the root pieces. The control is an agar plate that was not inoculated with a fungus. The agar plate and roots are covered with 2-3 cm of soil mix, and the pots placed in a greenhouse at 20/15° C. and natural light. The total number of shoots or plants, number of shoots or plants that died, total number of shoots or plants with symptoms (i.e. chlorosis, necrosis, lesions) at 2, 4 and 6 weeks after root inoculation is recorded. After six weeks, foliar biomass and root weight were taken. Data analyzed for several parameters:

• • i) % root growth: [final root weight of treatment/start root weight of treatment]÷[final root weight of control/start root weight of control]×100);
  • ii) foliar biomass;
  • iii) shoot emergence as % of control; and
  • iv) % shoots with symptoms.

The effects of several fungal isolates of the present invention on Canada thistle growth and development are shown in Table 5.

TABLE 5

Effect of root inoculation of Phoma isolates on disease development

of Canada thistle, assayed using inoculum mat bioassay.

Root Zone Application

Chlorosis

Foliar fresh wt

RW (% of

Isolate

(scale 1-6)^z

(g)

control)*

Experiment A

Control

1a^y

0.67

a

100

a

95-54A1

5 b

0.24

b

66

a

Experiment B

Control

1 a

2.6

a

100

a

97-12B

6 b

0.0

b

14

b

Experiment C

Control

1a

49.1

a

100

a

89-25A

4 c

17.0

c

29

c

94-359A

1a

44.8

ab

70

b

97-15B2

3 b

35.8

ab

47

bc

*RW—root weight;

^zRating scale of increasing chlorosis starting from 1 = green, healthy to 6 = white, dead.

^yDifferent letters within a column for each experiment indicate significant differences at P < 0.05 using Duncan's Multiple Range Test.

The results in Table 5 demonstrate that the isolates were effective in weed control activity, for example controlling Canada thistle growth and development, when applied to soil.

A) Comparison of Fungal Strains for Canada Thistle Control

Using the inoculum mat bioassay, a range of fungal isolates were tested for weed control activity using Canada thistle as a weed.

The results using the above bioassay, on the effect of several fungal isolates on Canada thistle growth and development are shown in Table 6.

TABLE 6

Comparison of fungal isolates and untreated control for reduction in root weight, foliar biomass, mortality,

and expression of disease symptoms in Canada thistle conducted in six greenhouse experiments.

Control

85-24B

94-26

94-44B

94-134

95-54A1

97-12B

89-25A

95-359A

95-268B

97-15B2

Root weight (% of control)

Expt 1

100

a*

41

b

84

a

18

b

 nt**

nt

68

ab

73

a

85

a

51

ab

nt

Expt 2

100

a

33

cd

8

d

11

d

nt

nt

53

bc

34

cd

28

cd

46

c

nt

Expt 3

100

a

23

b

19

b

16

b

nt

nt

37

b

83

a

77

a

23

b

nt

Expt 4

100

a

6

b

10

b

12

b

28

b

8

b

nt

nt

nt

17

b

14

b

Expt 5

100

a

30

b

24

b

26

b

32

b

16

b

nt

nt

nt

42

b

20

b

Expt 6

100

a

13

c

12

c

9

c

17

c

46

b

nt

nt

nt

14

c

17

c

Mean

100 ± 5 

25 ± 4

26 ± 6

15 ± 2

26 ± 5

23 ± 8

53 ± 11

63 ± 11

63 ± 10

32 ± 6

17 ± 4

Foliar fresh wt (g)

Expt 1

5.2

ab

2.2

c

5.3

ab

0.5

d

nt

nt

4.7

ab

4.1

b

4.8

ab

2.4

c

nt

Expt 2

4.1

a

1.4

cde

0.2

e

0.1

e

nt

nt

2.4

bc

1.7

cde

0.8

de

1.8

cd

nt

Expt 3

3.0

a

1.1

b

0.4

b

0.1

b

nt

nt

0.9

b

3.2

a

2.8

a

0.6

b

nt

Expt 4

11.8

a

0

c

0

c

0

c

4.4

b

0.2

c

nt

nt

nt

2.3

bc

1.4

bc

Expt 5

8.6

a

0.7

b

0.8

b

2.4

b

1.6

b

0.3

b

nt

nt

nt

3.5

b

1.1

b

Expt 6

11.1

a

0.5

c

0.8

c

0.7

c

0.4

c

5.9

b

nt

nt

nt

1.8

c

1.7

c

Mean

7.2±

1.0±

1.3±

0.6±

2.1±

2.2±

2.7±

3.0±

2.8±

2.0±

1.4±

Mortality (%)

Expt 1

0

20

bc

0

38

c

nt

nt

7

ab

8

ab

10

ab

27

bc

nt

Expt 2

5

a

40

b

95

c

100

c

nt

nt

39

b

50

b

45

b

27

ab

nt

Expt 3

0

40

b

78

c

85

c

nt

nt

39

b

50

b

45

b

27

ab

nt

Expt 4

0

92

bc

100

c

100

c

60

b

92

bc

nt

nt

nt

88

c

87

bc

Expt 5

0

72

d

78

d

78

d

40

bc

90

d

nt

nt

nt

64

cd

73

d

Expt 6

2

a

84

c

82

c

90

c

77

c

55

bc

nt

nt

nt

80

c

83

c

Mean

1 ± 1

57 ± 7

72 ± 7

80 ± 5

 59 ± 11

79 ± 8

39 ± 11

23 ± 8 

18 ± 9 

58 ± 8

81 ± 8

Disease Symptoms (% shoots with chlorosis)

Expt 1

0

87

b

23

a

100

b

nt

nt

33

a

32

a

20

a

70

b

nt

Expt 2

0

80

bc

95

c

100

c

nt

nt

70

bc

50

b

100

c

63

b

nt

Expt 3

0

67

b

90

bc

100

c

nt

nt

80

bc

25

a

10

a

85

bc

nt

Expt 4

0

100

c

100

c

100

c

60

b

96

c

nt

nt

nt

88

c

87

c

Expt 5

0

100

c

88

bc

78

bc

73

bc

100

c

nt

nt

nt

68

b

90

bc

Expt 6

0

88

c

100

c

100

c

90

c

49

b

nt

nt

nt

80

c

90

c

Mean

0 ± 0

86 ± 6

83 ± 6

96 ± 2

74 ± 9

82 ± 8

61 ± 11

36 ± 10

43 ± 13

75 ± 7

89 ± 6

*For each experiment, different letters in a row indicate significant differences among the isolates and the control by Duncan's multiple range test at P < 0.1.

**nt—not tested.

These results demonstrate that a range of Phoma isolates have a negative impact on root weight, foliar fresh weight, chlorosis, and mortality in Canada thistle, and may be used to control the growth and development of Canada thistle.

The above results were averaged (Table 7). These results indicate that a range of fungal isolates exhibit weed control activity, in that the WCIP is greater than 20%.

TABLE 7

Comparison of 10 fungal strains for control of Canada thistle using

the inoculum mat bioassay. Means and standard error calculated from

data collected in 3-6 trials, each trial with 5 replicates.

RW*

FFW

% of

% of

Mortality

IOC

WCIP

Treatment

control

control

%

%

%

Control

100 ± 5 

100 ± 4 

 1 ± 1

 0 ± 0

0

85-24B

25 ± 4

22 ± 6

57 ± 7

86 ± 6

74

94-26

26 ± 6

23 ± 7

72 ± 7

83 ± 6

76

94-44B

15 ± 2

 8 ± 3

80 ± 5

96 ± 2

88

94-134

26 ± 5

20 ± 8

 59 ± 11

74 ± 9

72

95-54A1

23 ± 8

 20 ± 10

79 ± 8

82 ± 8

79

97-12B

 53 ± 11

 59 ± 13

 39 ± 11

 61 ± 11

47

89-25A

 63 ± 11

 76 ± 13

23 ± 8

 36 ± 10

30

94-359A

 63 ± 10

 69 ± 13

18 ± 9

 43 ± 13

32

95-268B

32 ± 6

31 ± 7

58 ± 8

75 ± 7

68

97-15B2

17 ± 4

13 ± 6

81 ± 8

89 ± 6

85

*RW—root weight;

FFW—foliar fresh weight;

IOC—incidence of chlorosis;

WCIP—weed control index perennial (WCIP % = {[(100 − root weight) + (100 − foliar fresh weight) + (% mortality) + (% incidence of chlorosis)] ÷ 400} × 100%.)

B) Comparison of Weed Control Activity of Fungal Strains in a Range of Plants

The weed control activity of a range of fungal isolates on several annual and perennial weeds and other plants is examined using the mat bioassay. The plants tested are:

Perennial sow thistle

Table 8

Dandelion

Table 9

Scentless chamomile

Table 10(A)

Prairie Sunflower

Table 10(B)

False Cleavers

Table 11

Wild Oats

Table 12

Green Foxtail

Table 13

Chickweed

Table 14

Wild Buckwheat

Table 15

Field Bindweed

Table 16

Plantain

Table 16A

Summary of WCI's

Table 17

In Tables 8-17, the following acronyms are used:

• • RW—root weight;
  • FFW—foliar fresh weight;
  • IOC—incidence of chlorosis;
  • WCIP—weed control index perennial (WCIP %={[(100−root weight)+(100−foliar fresh weight)+(% mortality)+(% incidence of chlorosis)]÷400}×100%.)
  • WCLA—weed control index annual (WCIA %={[(100−foliar fresh weight)+(% mortality)+(% incidence of chlorosis)]+300}×100%.)
  • Pooled S.E.=Mean pooled standard error among isolates and control

TABLE 8

Comparison of fungal strains for control of perennial sow thistle.

RW

FFW

Isolate

(% of C)

(% of C)

Mortality %

IOC %

WCIP %

Control (C)

100

100

3

0

0.1

85-24B

78

100

7

0

7

94-26

67

72

0

33

24

94-44B

22

20

33

40

58

94-134

68

100

0

0

8

95-54A1

67

100

10

10

13

97-12B

57

76

27

40

34

89-25A

92

87

20

17

15

94-359A

100

98

13

13

6

95-268B

100

100

0

0

0

97-15B2

72

100

0

0

7

Pooled S.E.

12

14

4

6

TABLE 9

Comparison of fungal strains for control of dandelion.

RW

FFW

Isolate

(% of C)

(% of C)

Mortality %

IOC %

WCIP %

Control (C)

100

100

0

0

0

85-24B

55

17

19

100

62

94-26

63

50

12

90

47

94-44B

63

24

24

97

59

94-134

88

80

7

21

15

95-54A1

47

35

45

80

61

97-12B

48

21

35

78

61

89-25A

70

31

13

74

43

94-359A

73

66

15

38

25

95-268B

57

37

10

75

45

97-15B2

79

96

4

12

9

Pooled S.E.

8

13

6

8

TABLE 10(A)

Comparison of fungal strains for control of scentless chamomile.

FFW

Isolate

(% of C)

Mortality %

IOC %

WCIA %

Control (C)

100

12

8

7

85-24B

28

83

83

79

94-26

66

27

19

27

94-44B

15

86

75

82

94-134

16

83

80

82

95-54A1

0

100

94

98

97-12B

5

93

91

93

89-25A

26

81

60

71

94-359A

45

49

46

50

95-268B

18

67

67

72

97-15B2

14

91

95

91

Pooled S.E.

15

15

14

TABLE 10(B)

Comparison of fungal strains for control of Prairie Sunflower.

FFW

Isolate

(% of C)

Mortality %

IOC %

WCIA %

Control (C)

100

0

0

0

85-24B

19

85

88

84

94-26

nd

2

0

nd

94-44B

32

65

74

69

94-134

85

0

4

6

95-54A1

21

51

86

72

97-12B

100

11

15

9

89-25A

27

62

75

70

94-359A

100

0

8

3

95-268B

100

0

0

0

97-15B2

44

60

44

53

Pooled S.E.

15

10

10

TABLE 11

Comparison of fungal strains for control of false cleavers.

FFW

Isolate

(% of C)

Mortality %

IOC %

WCIA %

Control (C)

100

12

0

4

85-24B

66

33

24

30

94-26

43

45

42

48

94-44B

35

65

63

64

94-134

42

63

40

54

95-54A1

41

78

76

71

97-12B

48

45

31

43

89-25A

97

23

13

13

94-359A

90

29

14

18

95-268B

6

93

89

92

97-15B2

29

72

74

72

Pooled S.E.

16

10

11

TABLE 12

Comparison of fungal strains for control of wild oats.

FFW

Isolate

(% of C)

Mortality %

IOC %

WCIA %

Control (C)

100

0

0

0

85-24B

100

0

0

0

94-26

na

4

0

nd

94-44B

100

3

61

21

94-134

94

0

0

2

95-54A1

100

0

0

0

97-12B

100

0

0

0

89-25A

100

0

0

0

94-359A

100

0

0

0

95-268B

96

13

0

17

97-15B2

96

0

0

4

Pooled S.E.

18

1

1

TABLE 13

Comparison of fungal strains for control of green foxtail.

FFW

Isolate

(% of C)

Mortality %

IOC %

WCIA %

Control (C)

100

3

0

1

85-24B

48

37

39

43

94-26

na

52

0

nd

94-44B

72

0

3

10

94-134

100

0

0

0

95-54A1

100

6

0

2

97-12B

95

18

0

8

89-25A

100

3

0

3

94-359A

50

36

43

43

95-268B

100

0

0

0

97-15B2

100

0

0

0

Pooled S.E.

20

4

2

TABLE 14

Effect of fungal isolates on control of chickweed.

Emergence

Chlorosis

FFW

Mortality

WCIA

%

%

%

%

%

Control (C)

57

0

100

3

0

85-24B

55

73

15

70

76

94-134^z

65

34

29

70

91

94-26

45

87

1

88

99

94-44B

35

100

0

96

59

95-54A1

60

73

3

82

84

97-12B

36

50

18

40

57

95-268B

69

97

2

97

97

97-15B2

64

60

22

56

65

^zMean of two trials

TABLE 15

Effect of fungal isolates on control of wild buckwheat.

Emergence

Chlorosis

FFW

Mortality

WCIA

%

%

%

%

%

Control (C)

54

0

100

0

0

85-24B

41

88

1

91

92

94-134

32

87

5

74

39

94-26

40

22

32

28

96

94-44B

49

95

1

95

85

95-54A1

55

5

78

5

11

97-12B

40

4

61

0

15

89-25A

39