United States Patent Application Publication Pub. No.: Meyer Et Al. Feb .

Feb. 7, 2013 US 2013/0035231 AI 2 eter of 65 mm after 14 days at 20 C. on quarter-strength potato dextrose agar from a single conidial inoculation. [0014] The carrier can be used to form particles for the formulations. The formulation can comprise, for example, the slow-growing strain on the agriculturally acceptable carrier in the form of particles comprising a non-swelling calcined montmorillonite clay material, the particles having a diameter ranging from about 0.200 mm to about 1.000 mm. [0015] It should be appreciated that the invasive grass species can be any invasive grass species prevented, inhibited, or eliminated using the teachings provided herein. In some embodiments, the invasive grass species comprises a Japanese brome (Bromus arvensis ), a ripgut brome (Bromus diandrus or Bromus rigidus), or a medusa head (Taeniatherum caput-medusae). In some embodiments, the compositions and formulations taught herein are designed to kill cheatgrass (Bromus tectorum) and, in some embodiments, the compositions and formulations taught herein are designed to kill red brome (Bromus rubens). [0016] The methods provided herein are the only methods known in the art for killing ungerminated seeds of invasive grass species. As such, the teachings provided herein include methods of treating soil to prevent, inhibit, or eliminate stands of invasive grass species. In some embodiments, the methods comprise administering an effective amount of a P. semeniperda formulation to a soil in need of a prevention, inhibition, or elimination of a stand of an invasive grass species; wherein, the administering is done after a dispersal of seeds of the invasive grass species into the soil and prior to a germinationinducing rain. These embodiments include a co-administering of an effective amount of an emerged seedling control agent. In some embodiments, the emerged seedling control agent can be selected from the group consisting of (i) a burn that is administered either before or after the administration of the P. semeniperda; (ii) a tillage that is administered after the germination-inducing rain, and thus always after the administration of the P. semeniperda; (iii) a pre-emergent herbicide that is administered before, during, or after administration of the P. semeniperda, as long as it's before the germination-inducing rain; (iv) a post-emergent herbicide that is administered after the germination-inducing rain, and thus always after the administration of the?. semeniperda; (v) a second mycoherbicide that is administered before, during, or after administration of the P. semeniperda; (vi) and a bacterial biocontrol that is administered before, during, or after administration of the P. semeniperda. [0017] In some embodiments, the method includes eliminating at least 95% of the seeds of a target invasive species from the soil. And, in some embodiments, the method includes eliminating at least 98% of the seeds of a target invasive species from the soil. [0018] In some embodiments, the soil is an arid, or semiarid, rangeland. In some embodiments, the soil is a cropland soil. And, in some embodiments, the soil supports a desired intact vegetation. The desired intact vegetation can be, for example, a desert, or semi-desert, shrub community. [0019] A variety of methods of sporulating and applying the mycoherbcides were also tested, and valuable formulations and methods of application were identified, In some embodiments, an agricultural, mycoherbicide formulation for killing ungerminated seeds of invasive grass species can comprise a strain of Pyrenophora semeniperda produced using a process comprising sporulation of the strain in a MAM supplement (a modified alpha cell broth of coconut milk and oatmeal); and, an agriculturally acceptable carrier in the form of particles comprising a vermiculite material, the particles having a diameter ranging from about 0.200 mm to about 1.000 mm. And, in some embodiments, an effective amount of such a formulation can be administered to a soil in need of a prevention, inhibition, or elimination of a stand of an invasive grass species. The administration can include a coadministering of an effective amount of an emerged seedling control agent selected from the group consisting of a burn, a tillage, a pre-emergent herbicide, and a post-emergent herbicide, a second mycoherbicide, and a bacterial biocontrol; wherein, the administering is done after a dispersal of seeds of the invasive grass species into the soil and prior to a germination-inducing rain. BRIEF DESCRIPTION OF THE DRAWINGS [0020] FIGS. 1A and 1B illustrate frequency distribution of isolates or strains in different Type 2 virulence categories was quite similar between trials, according to some embodiments. [0021] FIG. 2 illustrates the negative relationship between Type 2 virulence and mycelial growth index, according to some embodiments. [0022] FIG. 3 illustrates the proportion of stromata produced by a 50/50 mix of a strain with high Type 2 virulence and a strain with low Type 2 virulence after co-inoculation onto dormant vs. non-dormant cheatgrass seeds, according to some embodiments. [0023] FIGS. 4A and 4B illustrate the density of killed and viable carryover cheatgrass seeds in Utah seed banks as a function of the administered dose of a P. semeniperda inoculum, according to some embodiments. [0024] FIGS. SA and SB illustrate the proportion of carryover cheatgrass seeds in Utah seed banks killed as a function of the administered dose of a P. semeniperda inoculum, according to some embodiments. [0025] FIG. 6 illustrates proportion of carryover red brome seeds killed as a function of the administered dose of a P. semeniperda inoculum, according to some embodiments. [0026] FIGS. 7A-7C illustrate the density of killed and viable carryover cheatgrass seeds in burned and unburned treatments for a Washington seed bank as a function of the administered dose of a P. semeniperda inoculum, according to some embodiments. [0027] FIG. 8 shows the results of the formulation field test comparisons, according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION [0028] This application is generally directed to a selective, mycoherbicide formulation for killing ungerminated seeds of invasive grass species. In some embodiments, the teachings are directed to an agricultural, mycoherbicide formulation for killing ungerminated seeds of invasive grass species, the formulation comprising a slow-growing strain of Pyrenophora semeniperda, a fast-growing strain of Pyrenophora semeniperda, and an agriculturally acceptable carrier. As such, in some embodiments, the teachings provided herein are directed to a composition comprising a mixture of a slowgrowing strain of Pyrenophora semeniperda and a fast-growing strain of Pyrenophora semeniperda. In some embodiments, the mixture of strains can be formed into any ratio of fast strain to slow strain that provides a desired result with regard to the teachings provided herein. In some embodi-

US 2013/0035231 AI Feb. 7, 2013 3 ments, the fast strain: slow strain ratio can range from 6:1 to 1:6,from5:1 to 1:5, from4:1 to 1:4,from3:1 to 1:3, from2:1 to 1:2, to 1:1. [0029] The mixture of strains provides a mixture of virulence, with regard to both type and level of virulence. The term "low Type 1 virulence" can be used to refer to less than 10% dormant seed mortality when applied at 1:6400 inoculum dilution after incubation at 20 C for 2 weeks. The term "high Type 1 virulence" can be used to refer to greater than 30% dormant seed mortality at 1:6400 inoculum dilution after incubation at 20 C for 2 weeks. The term "intermediate Type 1 virulence" can be used to refer to a range from about 10% to about 30% dormant seed mortality when applied at 1:6400 inoculum dilution after incubation at 20 C for 2 weeks. The term "low Type 2 virulence strains" can be used to refer to strains that can only kill 10% of non-dormant cheatgrass seeds after inoculation at saturating loads and incubation for 4 weeks at about 20 C. The term "high Type 2 virulence strains" can be used to refer to strains that can kill 30% of non-dormant cheatgrass seeds after inoculation at saturating loads and incubation for 4 weeks at about 20 C. And, the term "intermediate Type 2 virulence strains" can be used to refer to strains that can kill 10-30% of non-dormant cheatgrass seeds after inoculation at saturating loads and incubation for 4 weeks at about 20 C. One of skill will appreciate that the time and temperature can be varied, for example, from about 15 C. to about 25 C.; from about 15 C. to about 20 C., from about 20 C. to about 25 C., or any range therein; and, from about 7 days to about 14 days, from about 10 days to about 30 days, from about 14 days to about 28 days, or any range therein, in some embodiments, where the relative measure should be taken at the same, or substantially the same, time and temperature between samples. [0030] Type 1 virulence measured using this method is weakly positively correlated with ability to kill seeds at low loads using bulk inoculum in laboratory infection trials in seed zone microcosms (steel rings containing autoclaved natural seed zone cores from the field using the ring bioassay procedure of Beckstead eta!., Journal of Ecology (1) 98:168177 (201 0). Type 1 virulence and Type 2 virulence are not positively correlated among strains. However, Type 1 virulence is weakly positively correlated with mycelial growth rate. [0031] Ungerminated seeds can be dormant seeds, and the determination of whether a seed is ungerminated or dormant can be made using any method known to one of skill. The term "dormant seeds" can be used to refer to seeds that require at least 6-8 days to germinate under optimum temperature conditions of about 20 C., and the term "non-dormant seeds" can be used to refer to seeds that can germinate in 1-6 days under optimum temperature conditions of about 20 C. The terms "composition," "compound," "inoculant," and "pathogen" can be used interchangeably in some embodiments and, it should be appreciated that a "formulation" can comprise an inoculant or pathogen presented herein. In some embodiments, the formulation is administered in combination with burning, tillage, or an herbicide, and the combination administration can be referred to as a "system." Likewise, in some embodiments, the active components can also be referred to as an "agent," a "bioactive agent," or an "herbicide" whether alone, or in an agriculturally acceptable composition or formulation. An inoculant or formulation as taught herein should be stable. [0032] [0033] An inoculant or formulation can be considered as "stable" if it loses less than 10% of its original activity. For example, the stability of a formulation can be measured by comparing its activity immediately after making the formulation to its activity at the time of administration, and this can include a reasonable shelf life, in some embodiments. In some embodiments, the formulation can be considered as stable if the formulation loses less than 5%, 3%, 2%, or 1% of its original activity when comparing its activity immediately after making the formulation to its activity at the time of administration, and this can include a reasonable shelflife, in some embodiments. [0034] An inoculant or formulation can be considered as "substantially stable" if its formulation loses less than about 10% of its original activity, as long as it can perform its intended use to a reasonable degree of efficacy, as determined as useful by one of skill. In some embodiments, a "reasonable degree of efficacy" can be 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage therein, in 1% increments, where the term "efficacy" can be used to refer to the percentage of seeds killed using the ring bioassay procedure of Beckstead eta!., Journal of Ecology (1) 98:168-177 (2010). The loss can be measured, as above, by comparing its activity after making the formulation to the time of administration, and this can include a reasonable shelf life, in some embodiments. In some embodiments, a formulation or inoculant can be considered as substantially stable if it loses greater than about 2%, about 5%, about7%, about 9%, about 10%, of its original activity. The loss may be measured by comparing its activity after making the inoculants or formulation to the time of administration, and this can include a reasonable shelflife, in some embodiments. In some embodiments, an inoculant or formulation is stable or substantially stable, if useful for a period ranging from about 1 month to about 3 months, from about 1 month to a year, from 3 months to a year, from 3 months to 2 years, from 3 months to 3 years. [0035] Making the Inoculant One of skill will appreciate that, at least from the teachings provided herein, there are a wide variety of possible formulations that can be selected and designed for administration at a given target site, the selection of which is, at least in part, dependent on the site to be treated. The design of the formulation can include for example, (i) identifying the target site; (ii) identifying the present condition of the target site, such as whether it has intact vegetation or is post-burn; (iii) selecting an inoculant, for example, slow strain, fast strain, or mix of slow and fast strains; (iv) selecting a carrier; and (v) selecting a co-treatment for elimination of seedlings, such as burning, tillage, or herbicide, for co-administration at the target site. [0036] [0037] The term "target site" can be used to refer to a select location to be treated, in which the target site comprises a seed bank having annual grass weed seeds, referred to herein as "a target invasive species", that could be killed, infected, or otherwise reduced in carryover using the methods provided herein. One of skill will appreciate that the target site can include any location arid, semi -arid, or otherwise, that would benefit from any of the compositions or formulations provided herein. The terms "treat," "treated," "treating," and "treatment" can be used interchangeably and refer to the administering or application of the formulations taught herein directed to the prevention, inhibition, or elimination of stands of invasive grass species.

US 2013/0035231 AI Feb. 7, 2013 4 [0038] In some embodiments, the formulation comprises a slow-growing strain of Pyrenophora semeniperda, a fastgrowing strain of Pyrenophora semeniperda, and an agriculturally acceptable carrier. In these embodiments, the formulation functions to kill ungerminated seeds of an invasive grass species. As such, in some embodiments, the formulation comprises a composition having a mixture of a slow-growing strain of Pyrenophora semeniperda and a fast-growing strain of Pyrenophora semeniperda. [0039] The relative growth rates of strains can be determined using any method known to one of skill. In some embodiments, for example, the slow-growing strain is limited to reaching a mycelial colony diameter of 50 mm after 14 days at 20 C. on quarter-strength potato dextrose agar from a single conidial inoculation. And, in some embodiments, for example, the fast-growing strain is limited to reaching a mycelial colony diameter of 65 mm after 14 days at 20 C. on quarter-strength potato dextrose agar from a single conidial inoculation. [0040] The term "fast-growing strains" can be used to refer to strains that reach a mycelial colony diameter 65 mm after 14 days at about 20 C. on quarter-strength potato dextrose agar from a single conidial inoculation. The term "slowgrowing strains" can be used to refer to strains that reach a mycelial colony diameter of 50 mm after 14 days at about 20 C. on quarter-strength potato dextrose agar from a single conidial inoculation. The term "intermediate-growing strains" can be used to refer to strains that reach a mycelial colony diameter of 50 to 65 mm after 14 days at about 20 C. on quarter-strength potato dextrose agar from a single conidial inoculation. [0041] The compositions of the invention may be formulated as granules, wettable powders, emulsifiable concentrates, powders or dusts, flowables, solutions, suspensions or emulsions, or as controlled release forms such as microcapsules. In some embodiments, these formulations can contain from about 0.5% to about 95%, from about 1.0% to about 98%, from about 2.0% to about 90%, from about 5.0% to about 85%, from about 10.0%to about 80%, from about 15% to about 7 5%, from about 20% to about 65%, from about 25% to about 50%, from about 30% to about 40%, or any range therein, by weight of active ingredients. The optimum amount for any given compound will depend on formulation, application equipment and nature of the invasive species to be controlled. [0042] The term "about" can be used to refer to a possible variation in an amount or condition that would not be expected by one of skill in the art to create a significant difference in performance of a recited claim or claim limit such as, for example, in a function, a measure, or an effect. Likewise, the terms "substantial" or "substantially" can be used to (i) refer to a difference in an amount or condition that would be expected by one of skill in the art to create a significant difference in performance of a recited claim or claim limit such as, for example, in a function, a measure, or an effect; or (ii) an approximate equivalence in an amount or condition that would not be expected by one of skill in the art to create a significant difference in performance of a recited claim or claim limit such as, for example, in a function, a measure, or an effect. [0043] An "agriculturally acceptable carrier" is a diluent, adjuvant, excipient, or vehicle with which the inoculant is administered. A carrier is agriculturally acceptable when it has been determined to be environmentally safe including, but not limited to, safe to plants, animals, and fungi. Such carriers can be readily identified by those of skill. An example of an agriculturally acceptable carrier is AGSORB (available from Monsanto Agricultural Co.), available, for example, in compositions that comprise montmorillonite, attapulgite, and hydrous aluminosilicate. In some embodiments, an agriculturally acceptable carrier can include agricultural waste products such as, for example, corncob grits. In some embodiments, an agriculturally acceptable carrier is a solid having a bulk density that is less than 1.0 g/ml, 0.8 g/ml, 0.6 g/ml, or 0.4 g/ml. In some embodiments, the carrier can be used to form particles for the formulations. The formulation can comprise, for example, the slow-growing strain on the agriculturally acceptable carrier in the form of particles comprising a calcined montmorillonite material, the particles having a diameter ranging from about 0.200 mm to about 1.000 mm. [0044] Examples of solid carriers include fertilizer, sand, Fuller's earth, attapulgite clay, bentonite clays, montmorillonite clay, vermiculite, perlite, calcium carbonate, brick, pumice, pyrophyllite, kaolin, dolomite, plaster, wood flour, wheat flour, ground corn cobs, ground peanut hulls, ground walnut shells, cotton seed hulls, lignin, sodium silicate, magnesia, mica, iro

c12) Patent Application Publication Meyer et al. (10) Pub. No.: US 2013/0035231 A1 (43) Pub. Date: Feb. 7, 2013 (54) ANNUAL BROME CONTROL USING A NATIVE FUNGAL SEED PATHOGEN (75) Inventors: Susan Elizabeth Meyer, Elk Ridge, UT (US); Suzette Clement, Mapleton, UT