Abstract:
The present invention is concerned with a novel bioherbicide, and composition thereof for the control of broadleaf weeds in lawns, golf courses, parks, sports fields, other turfed areas, and grass crops such as corn and cereal grains. More specifically, the invention is concerned with a novel isolate Sclerotinia minor IMI 344141, agricultural formulations thereof, and its use as a bioherbicide to control troublesome broadleaf weed species.

Description:
This is a continuation of application Ser. No. 07/648,179, filed on Feb. 1, 1991 now abandoned. 
    
    
     FIELD OF THE INVENTION 
     The present invention is concerned with a novel isolate of Sclerotinia minor, namely Scierotinia minor IMI 344141 particularly effective as a bioherbicide for the control of a broad spectrum of broadleaf weeds without adversely affecting desirable grass species. 
     BACKGROUND OF THE INVENTION 
     Numerous broadleaf weed species, including dandelion (Taraxacum officinale), plantain (Plantago species), knotweed (Polygonum species), ground ivy (Gleocoma hederacea), ragweed (Ambrosia artemisiifolia), white clover (Trifolium repens), black medic (Medicago lupulina) and henbit (Lamium amplexicaule) infest turfgrass areas such as lawns, golf courses, parks and sports fields. Chemical weed control using chemical herbicides is often the easiest, most effective and least expensive way to control broadleaf weeds in turf. Herbicides such as 2,4-D (2,4-dichlorophenoxy acetic acid), mecoprop [2-(4-chloro-2-methylphenoxy) propanoic acid], dichlorprop [2-(2,4-dichlorophenoxy) propanoic acid], dicamba (3,6-dichloro-2-methoxybenzoic acid) and mixtures of these four chemical herbicides are commonly recommended and used to provide broadleaf weed control in turfgrass areas. 
     There is now significant public concern over the safety and use of chemical herbicides, particularly in the urban environment. These public concerns include toxic chemical residues in soils and water, contamination of foodstuffs, adverse effects on non-target organisms and public health. It would therefore be highly desirable to find a herbicidal composition which has a broad spectrum of activity against troublesome broadleaf weed without damaging desirable grass species, and which has a benign effect on the environment and reduces the input of chemical pesticides. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, there is now provided a novel isolate of Sclerotinia minor, namely Sclerotinia minor IMI 344141, which has surprisingly been found to be effective in controlling the growth of undesirable broadleaf weed species without adversely affecting the growth of desirable grass species. 
     In one aspect of the present invention, there is provided an agricultural composition for controlling the growth of dandelion, plantain, ragweed, velvetleaf, sowthistle, ground ivy, knotweed, chickweed, white clover, and the like, and other undesirable broadleaf weeds in lawns, golf courses, parks, sports fields and in agricultural crops of the Poaceae family, such as corn, wheat, oats and barley and other cereal crops. The novel agricultural composition of the present invention comprises an effective amount of the isolate Sclerotinia minor IMI 344141 in association with an agriculturally acceptable carrier. 
     In another aspect of the present invention, there is provided a method for controlling the growth of undesirable broadleaf weeds in turf and other grass crops by applying an effective amount of a composition containing the isolate Sclerotinia minor IMI 344141 preferably in association with an agriculturally acceptable carrier. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 illustrates the effect of temperature on disease level of dandelion when Sclerotinia minor IMI 344141 is applied; 
     FIG. 2 illustrates the effect of age and temperature on disease level of dandelion when Sclerotinia minor IMI 344141 is applied; 
     FIG. 3 illustrates the effect of age and temperature on dry weight of above ground biomass of dandelion when Sclerotinia minor IMI 344141 is applied; 
     FIG. 4 illustrates the effect of age and dew period on disease level of dandelion when Sclerotinia minor IMI 344141 is applied. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Although the merits of using plant pathogens to control weeds in crop production are known for Colletotrichum species (U.S. Pat. No. 3,849,104 and U.S. Pat. No. 3,999,973), Fusarium species (U.S. Pat. No. 4,419,120), Alternaria species (U.S. Pat. No. 4,390,360) and Ascochyta species (U.S. Pat. No. 4,915,724), the use and advantages of using Sclerotinia minor and more particularly the isolate Sclerotinia minor IMI 344141, has not been demonstrated before. No plant pathogens have been used to date as a bioherbicide to control broadleaf weeds in lawns, golf courses, parks and other turfed areas in the urban environment. 
     Sclerotinia minor Jagger is an inoperculate Discomycetes of the Helotiales order which produces sclerotia, which do not incorporate plant tissues, ascospores in asci in stipitate apothecia, and a superficial Myrioconium microconidial state, but no known disseminative conidia. The small size of the sclerotia and their abundant production scattered over the entire colony on agar media are used as taxonomic characteristics to distinguish S. minor from Sclerotinia sclerotiorum and Sclerotinia trifoliorum. The combination of morphological, microanatomical, and cytological characters clearly demonstrate that these three organisms, S. minor, S. sclerotiorum and S. trifoliorum are distinct. 
     Although all of these Sclerotinia species have broad host ranges which overlap to some extent, S. trifoliorum is generally limited to forage legumes, S. minor has been reported as a pathogen of species in Antirrhinum, Apium, Arachis, Brassica, Daucus, Helianthus, Ipomoea, Lactuca, Lycopersicon, Melilotus, Nicotiana, Parthenium, Phaseolus, Solanum, Tragopogon and Tulipa genera, whereas S. sclerotiorum has a worldwide distribution and has been reported as pathogenic to species in at least 148 genera (Farr et al. 1989. Fungi on Plants and Plant Products in the United States. APS Press, St. Paul). 
     S. minor is an important pathogen of some crops, including lettuce (Lactuca sativa), peanut (Arachis hypogaea), soybean (Glycine max) and sunflower (Helianthus annus) and can cause losses of up to 70% of the marketable crop. 
     S. minor has a relatively simple disease cycle. Sclerotia in the soil germinate directly to produce hyphae which infect plants, followed by colonization of the infected plants with the production of more sclerotia on the plant tissues which return to the soil. The sclerotia of S. minor can also undergo carpogenic germination to produce apothecia. Apothecia have been reported to occur in nature, but are relatively rare, and therefore are apparently unimportant in the epidemiology of this disease in North America. 
     A sample of Sclerotinia minor Jagger isolate SM-13 has been deposited with the International Mycological Institute in Ferry Lane, Kew, Richmond, Surrey TW9 3AF, United Kingdom, on Jan. 28, 1991, under the Budapest Treaty requirements and has been assigned the accession number IMI 344141. 
     The deposit is available to the public upon the granting of a patent disclosing it. The deposit is also available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a licence to practice the subject invention in derogation of patent rights granted by governmental action. 
     It is most surprising and unexpected that the isolate of S. minor IMI 344141 of the present invention, which has been obtained from lettuce, has such high bioherbicidal broad spectrum activity on broadleaf weeds, since there is no evidence in the scientific literature that such broadleaf weeds are known to be hosts to S. minor. 
     It should be noted that S. minor does not normally grow on leaves but rather in soil where eventually it attacks the foliage which touches the soil and thus it would not be expected that if an isolate was applied to broadleaf weeds, they would be attacked to the point of mortality. 
     It is also surprising that application of the isolate S. minor IMI 344141 of the present invention either alone or in a composition to broadleaf weeds will not damage the surrounding grass species. Accordingly, the isolate S. minor IMI 344141 has the same specificity as known chemical herbicides such as 2,4-D, without the highly undesirable increase in environmental burden. 
     Application of the Bioherbicide. 
     The vehicle used to deliver the infection units of S. minor IMI 344141 of the present invention to the targetted weed species was barley or millet grains invaded by fungal mycelium which are essentially various size granules composed of fungal mycelia and autoclaved plant seeds. The application was broadcast as a granule formulation at an equivalent rate of 100 to 300 g/m 2 . 
     Any other solid media which would facilitate the growth of mycelia of S. minor would be equally effective. Such media are well known in the art. 
     Inoculum Production. 
     Sclerotia of S. minor were dislodged from diseased lettuce tissue and air dried. After surface sterilization, the sclerotia were imbedded in agar (PDA plates) to stimulate germination. The plates were transferred to an incubator set at 21° C. under fluorescent lights. After a four day period the hyphal tips from germinating sclerotia were transferred onto a second set of plates where they grew until the 8.5 cm diameter plate was covered (5 days). Erlenmeyer flasks (250 ml) containing 20 g barley or millet grains and 20 ml water were autoclaved for 20 minutes, cooled, inoculated with mycelium agar plugs (6 mm) from the PDA plates and incubated for 5 days under the same conditions as above. Flasks were shaken everyday to prevent sclerotia formation. Prior to the plant inoculation the inocula was air dried for a period of 2 to 4 hours to obtain individual inoculum units. A constant rate of 2 g/pot of inocula was used throughout the experiments unless otherwise stated. The inoculum was applied in a broadcasting manner over each pot. Pots used for controls received aiutoclaved grains at an equivalent rate of 2 g/pot. 
     Plant Production. 
     Seedlings of test plant species were transplanted (one per pot) in a commercialy prepared potting medium such as peat moss in 10-cm plastic pots and grown in controlled environment chambers. The temperature was set at 21° C./18° C. (day/night) and light intensity at 400 μE m -2  s -1  for 14 hours. Plants were grown for different lengths of time as specified for each experiment. All treatments were replicated from 4 to 16 times in all experiments depending on availability of plant material. 
     Assessment. 
     After one, two and three weeks the effect of the pathogen inoculated on plants was visually rated on a scale of 0 to 5. The proportion of healthy vs diseased tissue served as a means to determine the efficacy of the fungus under set conditions. The statistical analysis performed on the data was a Kruskal-Wallis analysis followed by a multiple comparison test to locate differences among treatments. Plants were kept for further evaluations and in some cases assessment of dry weight of above ground biomass was taken after the third disease rating. 
     The present invention will be further illustrated by the following examples, which are representative, and do not restrict the scope of the invention in any way. 
     EXAMPLE 1 
     Preliminary Experiment. 
     A greenhouse experiment was set up to evaluate the ability of various Sclerotinia species to suppress the growth of broadleaf weeds. Isolates of the genus S. minor, S. sclerotiorum and S. trifoliorum were tested on different weeds including dandelion (Taraxacum officinale), plantain (Plantago major), ground ivy (Glecoma hederacea), and on bluegrass (Poa pratensis) a common desirable grass species in turf. Soil plugs from the campus lawn were removed and transferred into 12,5-cm plastic pots. Each pot contained blue grass and a number of weed species with one being predominant. All pots were placed in a mist frame after receiving 3,5 g/pot of inoculated barley grains. A continuous mist was applied for a 6 hour period during the night. The greenhouse temperature was approximately 23±5° C. The results are summarized in Table 1. 
     
                       TABLE 1______________________________________Effect of different isolates of Sclerotinia on various weeds and on blue grass.        Mean disease level.sup.a            Trial # 1     Trial # 2  Isolates.sup.b Isolates.sup.bHost Name    1      11     13    1    11   13______________________________________Taraxacum officinale        2      3      4     3    4    5  Plantago major            4   2      5     2      2      5  Glecoma hederacea         0   0      2      1      1      3  Poa pratensis          0   0      0      0      0      0______________________________________ .sup.a Disease rating scale: 0: no disease, 1: 125% of necrotic tissue, 2 2650%, 3: 5175%, 4: 7699%, 5: dead plants. .sup.b Isolate #1: S. trifoliorum Isolate #11: S. sclerotiorum Isolate #13: S. minor IMI 344141 of the present invention 
    
     In this experiment, isolate of S. minor IMI 344141 consistently caused the greatest amount of disease on the three weed species tested as illustrated in Table 1. The desirable grass species Poa pratensis was not affected by S. minor. 
     Therefore, S. minor IMI 344141, was the isolate selected for further evaluation. It severely damaged or killed the broadleaf weed species without any harm to the grass crops. 
     EXAMPLE 2 
     Experiments were conducted to evaluate the effect of the age of the host plant and environmental parameters of dew period duration and dew period temperatures on disease development caused by S. minor IMI 344141 when inoculated onto dandelion. The experimental variables are given in Table 2. 
     
                       TABLE 2______________________________________Post Inoculation Variables For Dandelion Experiments.                Dew period    Inoculum  Host age  duration                                temperature  Experiment #  carrier (days)  (hrs)  (° C.)______________________________________1         Barley   17        18      18, 21, 24  2               Barley         15, 21        18         18, 21, 24                                 3               Barley         14,                                21, 45  6, 12, 18      21______________________________________ 
    
     In experiment #1, the dandelion plants were rapidly and severely affected by S. minor IMI 344141 (FIG. 1) with most plants killed or severely damaged after 4 days. The level of disease was greater when the dew period temperature was 21° C., but high levels of disease occurred at cooler (18° C.) and warmer (24° C.) temperatures. 
     In a similar experiment #2, disease level was very high over all dew period temperatures tested and there was no significant differences between two-week-old and three-week-old dandelion plants, both ages were equally susceptible to S. minor IMI 344141 (FIG. 2). The effect of the bioherbicide on dandelion control is clearly illustrated in FIG. 3 where the biomass of control (C) and treated (T) are compared. Two-week-old dandelion plants are killed by the treatment under all three dew-period temperatures tested and essentially all of the above-ground biomass is also destroyed when treated at the three-week-old stage. 
     In experiment #3, even when the dew period is relatively short (6 hr), extensive disease develops on two-week-old seedlings (FIG. 4). Dew-period duration appears to have a greater effect when the plant is three weeks old, although the results are not statistically different according to the analysis used. However, as the dandelion plants mature (45 days old), the longer dew periods provide higher disease levels. 
     EXAMPLE 3 
     Similar experiments as to those described in Example 1 and 2 on dandelion plants were conducted on the following additional broadleaf species: Plantago major, Ambrosia artemisiifolia, Plantago lanceolata, Sonchus arvensis, Polygonum aviculare, Trifolium repens, Glecoma hederacea, Oxalis sp and Cerastium vulgatum. The plants tested, their age, the inoculum type, the dew-period duration and dew-period temperature, and the results are summarized in Table 3. 
     
                       TABLE 3______________________________________Experiments using Sclerotinia minor IMI 344141 (isolate #13)  on weed species other than dandelion.                                       MeanAge         Disease      (host)                 Dew period    Mortality RatingHost    (days)  Inoculum (Time)                          (° C.)                               (%)    (0-5).sup.a______________________________________Plantago   27      whole    12 hrs                          21   100     5  major     barley  cracked      12 hrs 21      100           5  barley32       whole     6 hrs  21       33          3.0  barley    12 hrs 21       33          2.7    18 hrs 21      100           534       whole        12 hrs 18       40          3.5  barley     21       25          3.7     24       30          3.634       whole        18 hrs 18       70          4.4  barley     21       90          4.8     24       50          4.238       whole      --.sup.b -- 17          3.2  barley     6 hrs  21       20          3.5  cracked        -- --  17          2.8  barley     6 hrs  21       80          4.345       whole        24 hrs 21      100           5  barley  Ambrosla             23       whole        12 hrs 21      100                                      5  artemisiifolia                  barley  cracked      12 hrs 21       89          4.9  barley37       whole        18 hrs 21       46          3.1  barley40       whole          6 hrs  21         0          2.0  barley    12 hrs 21         0          1.5  Plantago  30       cracked        -- --           50          3.5                                       lanceolata     barley                                               6 hrs  21       25                                            2.0    12 hrs 21       20          2.2    18 hrs 21       40          2.8  Sonchus     22       cracked      12 hrs 21      100           5                                       arvensis     barley    18 hrs 21      100           5  Polygonum 13        cracked      -- --          0          0  aviculare     barley     6 hrs  21      0          0    12 hrs 21           33          1.7    18 hrs 21             0          028       cracked      --   --   0          0  barley    18 hrs 21           25          1.3  whole        --  --    0          0  barley    18 hrs 21           75          4.043       whole        18 hrs 21           50          3.3  barley  Trifolium     27       cracked      --  --   0          3.0  repens     barley     6 hrs  21           33          4.0    12 hrs 21             0          2.0    18 hrs 21           33          3.0  Glecoma    10       cracked        6 hrs  21             0          0                                       hederacea     barley    12 hrs 21             0          0.8    18 hrs 21             0          0.523       cracked      --   --            0          0.3  barley    18 hrs 21             0          0.3  whole        --   --          0          0  barley    18 hrs 21            0           045       whole        24 hrs 21           50          4.0  barley  Oxalis sp             28       cracked      --   --        0                                      0.3  barley    18 hrs 24         0          0.3  Cerastium    45      whole        24 hrs 21          0          3.0                                       vulgatum     barley______________________________________ .sup.a Disease rating scale: 0  no disease, 1  1 to 25% necrotic tissue1  26 to 50% necrotic tissue, 3  51 to 75% necrotic tissue, 4  76 to 99% necrotic tissue, and 5  dead plants. .sup.b --did not receive a dew period. 
    
     All weed species tested, with the exception of the Oxalis sp. were susceptible to S. minor IMI 344141 and were severely damaged or destroyed. Mortality and disease rating were higher when plants received a relatively long dew period (≧18 hr) at a relatively warm temperature (≧21° C.). Older plants tended to be more tolerant to the disease.