Abstract:
A strain of Colletotrichum coccodes has been discovered which is selectively pathogenic toward eastern black nightshade (Solanum ptycanthum). Formulations comprising propagules of the fungal pathogen are useful for biological control of the eastern black nightshade weed, particularly in agricultural fields.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     Eastern black nightshade (Solanum ptycanthum) is an economically important weed in much of the midwestern United States. It can cause crop yield reductions by competition, but is more notorious for interfering with harvest and lowering the crop quality. The foliage and berries of eastern black nightshade can foul combines and make harvesting nearly impossible. Moreover, the berries contaminate and stain the crop seeds and initiate mold growth during storage. Several species of weedy nightshades, including the eastern black nightshade, have in the past been generically referred to as &#34;black nightshade&#34; (Solanum nigrum L.). In accordance with a recent taxonomic revision, the eastern black nightshade is now designated as a separate species, Solanum ptycanthum. 
     This invention relates to a method of controlling eastern black nightshade by means of infection with a fungal pathogen. 
     2. Description of the Prior Art 
     The merits for using pathogens to control weeds in annual crops have been discussed previously for various fungal species. Daniel et al., U.S. Pat. No. 3,849,104 teaches the use of an endemic anthracnose fungus, Colletotrichum gloeosporioides forma specialis aeschynomene, to control the northern jointvetch weed in rice fields. A related species, C. malvarum, has been reported by Templeton in U.S. Pat. No. 3,999,973 as a control for prickly sida (Sida spinosa L.) or teaweed, particularly in soybean and cotton fields. C. malvarum is also pathogenic toward other noncultivated species, including velvetleaf. C. coccodes has been well documented as a widely distributed pathogen of potato and tomato, as well as 35 other plant species throughout the world. One isolate of C. coccodes has been studied as a potential mycoherbicide for velvetleaf by Gotlief et al. [Proc. Weed Sci. Soc. Am. 37: 68 (1984)]. 
     Walker [Weed Sci. 29: 505-507 (1981)] discloses the use of Alternaria macrospora as a pathogen against spurred anoda. In Walker, U.S. Pat. No. 4,390,360, A. cassiae is disclosed as an effective biological control agent for sicklepod, showy crotalaria, and coffee senna. Finally, Walker teaches in U.S. Pat. No. 4,419,120 that Fusarium lateritium is useful in controlling prickly sida, velvetleaf, and spurred anoda. 
     SUMMARY OF THE INVENTION 
     We have now discovered a strain of Colletotrichum coccodes which is pathogenic toward eastern black nightshade but innocuous toward virtually all crop species for which the nightshade poses agronomic difficulties. This invention is drawn to the use of this fungus as a mycoherbicide for controlling eastern black nightshade and other susceptible species and also to mycoherbicidal compositions comprising C. coccodes propagules. In practice, the target plants are inoculated by treating infested fields with the propagule-containing compositions. 
     In accordance with this discovery, it is an object of this invention to provide a mycoherbicidal alternative to chemical control of eastern black nightshade. 
     It is also an object of the invention to provide a biological herbicide for eastern black nightshade which is nonpathogenic toward crop species. 
     Another object of the invention is to provide a fungal pathogen which can be artificially mass-produced and formulated for both preemergence and postemergence weed control. 
     Other objects and advantages of this invention will become readily apparent from the ensuing description. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The fungal organism for use herein is a strain of C. coccodes isolated from a field of eastern black nightshade experimental plots near Rosemount, Minnesota. In late summer, following an extended period of abundant rainfall and high humidity, this strain was found to be responsible for a disease epihytotic in the plots. Nearly all the eastern black nightshade plants had been killed, and those remaining were in an unhealthy state. 
     The taxonomic characteristics of the isolate are typical of the species. The acervuli growing on stems and roots are rounded or elongated, and attain a diameter of approximately 300μ. The acervular tissue occurs both intra- and subepidermal, disrupting the outer epidermal cell walls of the host. Setose sclerotia are common. The spores are cylindrical with obtuse ends, ranging in length from 16 to 24μ and in diameter from 2.5 to 4.5μ. The spores are hyaline and aseptate and are formed from unicellular hyaline cylindrical phialidic conidiophores. This isolate has been deposited in the Agricultural Research Culture Collection (NRRL) in Peoria, Ill., and has been assigned Accession Number NRRL 15547. For purposes of this invention, any isolate of C. coccodes having the identifying characteristics of NRRL 15547 including subcultures thereof would be effective. 
     The fungal propagules including the spores (conidia) and mycelia can be mass-produced for field inoculations by culturing the C. coccodes on half-strength Emerson&#39;s yeast-starch agar as described below: 
     
         ______________________________________Emerson&#39;s Yeast-Starch AgarNutrient            Concentration (g./L.)______________________________________powdered yeast extract (&#34;Difco&#34;)               2soluble starch      7.5K.sub.2 HPO.sub.4   0.5MgSO.sub.4.7H.sub.2 O               0.25distilled water______________________________________ 
    
     The propagules are preferably incorporated into compositions suitable for field application. They can be omitted with any liquid vehicle or solid carrier such as water, emulsions, clay, vermiculite, CaCO 3 , corn cob grits, etc. Both the spores and mycelia lend themselves to formulation as liquid sprays and wettable powders for postemergence treatment. They can also be formulated as controlled-release granules for preemergence weed control. Infection is promoted in the presence of free moisture (dew) for a period of at least about 12 hours. At a temperature of 25° C., the optimal moisture period is 16 to 20 hours. 
     The actual concentration of propagules in the formulated composition is not particularly critical, and is a function of practical considerations such as the properties of the vehicle or carrier, and the method of application. For purposes of formulation and application, an &#34;effective amount&#34; is defined to mean any such quantity of propagules sufficient to infect the target plant and thereby induce the symptoms of the disease described below. 
     It is understood that application of the mycoherbicide does not require immediate direct contact with the target plant. It may be applied in the locus or vicinity of the plant and rely upon natural environmental conditions for infection. In this manner, the herbicide is effective as a preemergent treatment. 
     Though current data indicate that C. coccodes is a relatively selective pathogen of eastern black nightshade, it is within the compass of this invention to treat other undesirable plant species which prove to be susceptible to this disease. For example, as shown by the data in Example 4, below, other species of nightshade are infected and damaged to varying degrees by C. coccodes. 
     When infecting eastern black nightshade, the organism produces typical anthracnose lesions. These lesions are grey, water-soaked-appearing spots, which later become necrotic spots. The lesions tend to spread and eventually kill the plant. Unlike other strains of C. coccodes which are known to be pathogenic toward tomato and potato plants, NRRL 15547 is innocuous toward these species, as well as toward all other major crop species tested, including corn, grain sorghum, rice, soybeans, cotton, eggplant, peppers, and tobacco. 
    
    
     The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims. 
     EXAMPLE 1 
     Pathogen Isolation and Culture 
     Diseased plants of eastern black nightshade were collected from experimental plots near Rosemount, Minn. Infected plant pieces were placed on moist filter paper in plastic petri dishes for several days in a growth chamber to promote sporulation of the pathogen. Then, pieces of plant tissue were placed on the leaves of greenhouse-grown seedlings of eastern black nightshade, accession A37. Pots of seedlings were enclosed in plastic bags containing wet paper towels and placed in shade under a greenhouse bench. After 48 hours, the bags were removed, pots were placed on greenhouse benches, and seedlings were observed for disease development. Diseased seedlings were sent, as fresh shoots pressed between paper towels, to Stoneville, Miss. 
     Colletotrichum coccodes was isolated from a diseased seedling at Stoneville on potato dextrose agar (PDA) (&#34;Difco&#34;) supplemented with streptomycin sulfate (125 mg./L.) and chloramphenicol (75 mg./L.). The fungus was cultured in petri dishes of growth media incubated at 25° C. with 12-hour photoperiods. Light was provided by two 15-watt cool-white fluorescent lamps that were suspended 45 cm. above the cultures. The pathogen produced sclerotia on PDA, but did not sporulate readily on this medium. Abundant conidia were produced on half-strength Emerson&#39;s yeast-starch agar, and these conidia were used as inoculum for greenhouse tests. Conidia were also produced in submerged liquid cultures, using the corn meal-soyflour-sucrose medium and the modified Richard&#39;s medium described below, but sporulation was inconsistent. 
     
         ______________________________________Corn Meal-Soyflour-Sucrose Medium.sup.1Nutrient     Concentration (g./L.)______________________________________corn meal    15soybean flour        15sucrose      30CaCO.sub.3    3______________________________________ .sup.1 Nutrients were dissolved or dispersed in distilled water and the medium pH was approximately 7.0. 
    
     
         Modified Richard&#39;s Medium.sup.1Nutrient          Concentration (g./L.)______________________________________sucrose           50KNO.sub.3         10KH.sub.2 PO.sub.4 5MgSO.sub.4.7H.sub.2 O             2.5FeCl.sub.3        0.02&#34;V-8&#34; juice       150      ml./L.______________________________________ .sup.1 Nutrients were dissolved in distilled water and the solution was adjusted to pH 6 with 50% (w/v) NaOH. 
    
     EXAMPLE 2 
     Seedlings for host range tests at Stoneville, Miss., were greenhouse-grown in 5.5-cm. square peat pots containing a commercial blend of peat, pine bark, and perlite with one seedling per pot. Aqueous suspensions were prepared comprising conidia of the eastern black nightshade isolate of C. coccodes produced by culture on the half-strength Emerson&#39;s yeast-starch agar in 0.05% (w/v) oxysorbic (20 POE) polyoxyethylene sorbitan monooleate surfactant (&#34;Tween 80&#34;) at a concentration of 1×10 6  conidia/ml. The seedlings were inoculated by wetting the foliage with the suspension in the third- to fourth-leaf stage. Immediately following inoculation, the plants were placed in dark dew-chambers (25° C.) for 24 hours, then moved to greenhouse benches and evaluated for disease symptoms 14 days after inoculation. Susceptible plants displayed anthracnose symptoms (necrotic lesions) usually within 3 days following inoculation. 
     A randomized complete block design was used with three replicates consisting of 12 plants of each species per replicate. The experiment was conducted twice. The results are reported in Table I, below. 
     EXAMPLE 3 
     The procedure of Example 2 was repeated except that the seedlings were inoculated by injecting approximately 20 μL. of the conidial suspension into the stems. 
     A randomized complete block design was used with three replicates consisting of 12 plants of each species per replicate. The experiment was conducted twice. The results are reported in Table II, below. 
     EXAMPLE 4 
     Tests were conducted at St. Paul, Minn., to determine the reaction of weeds of the Solanaceae to the pathogen. Seedlings were started in peat pellets in the greenhouse, then transplanted to 250-ml. plastic cups of potting soil with one seedling per cup. The plants were inoculated by wetting the foliage with a conidial suspension containing 0.05% (w/v) of &#34;Tween 80&#34; and having approximately 1×10 6  conidia/ml. Immediately after inoculation, plants were placed in a dark dew-chamber on a greenhouse bench for 24 hours before being returned to the greenhouse bench. Temperatures in the greenhouse were 22±4° C. and humidity was 30 to 50%. Natural light was supplemented with light from fluorescent tubes to give a 16-hour photoperiod. 
     
                       TABLE I______________________________________Reaction of Various Plant Species to the Foliar Spray ofColletotrichum coccodes Pathogen                       DiseaseSpecies                     Rating.sup.a______________________________________GramineaeCorn ( Zea mays L. `XL 394`)                       NSGrain sorghum [Sorghum bicolor (L.) Moench `Texas                       NSC 424`]Rice (Oryza sativa L. `Starbonnet`)                       NSLeguminosaeSoybean [Glycine max (L.) Merr. `Centennial`]                       NSMalvaceaeVelvetleaf (Abutilon theophrasti Medic.)                       NSCotton (Gossypium hirsutum L. `DPL 61`)                       NSSolanaceaeEastern black nightshade (Solanum ptycanthum Dun.)                       SEggplant (Solanum melongena L. `Black Beauty`)                       NSPepper (Capsicum annuum L.) NSPetunia (Petunia hybrida Vilm. `Confetti                       NSmultiflora`)Potato (Solanum tuberosum L. `Red LaSoda`)                       NSTobacco (Nicotiana tabacum L.)`NC 95`                     NS`NC 2326`                   NSTomato (Lycopersicon esculentum Mill.)`Rutgers`                   NS`Marglobe`                  NS`Better Boy`                NS`Patio`                     NS______________________________________ .sup.a Rating scale: NS = No disease symptoms, S = susceptible. 
    
     
                       TABLE II______________________________________Reaction of Various Plant Species to InjectedColletotrichum coccodes Pathogen                       DiseaseSpecies                     Rating.sup.a______________________________________MalvaceaeVelvetleaf ( Abutilon theophrasti Medic.)                       NSSolanaceaeEastern black nightshade (Solanum ptycanthum Dun.)                       SPotato (Solanum tuberosum L. `Red LaSoda`)                       NSTomato (Lycopersicon esculentum Mill.)`Rutgers`                   NS`Marglobe`                  NS`Better Boy`                NS`Patio`                     NS______________________________________ .sup.a Rating scale: NS = No disease symptoms, S = susceptible. 
    
     Two experiments were conducted. Eastern black nightshade, accession A37, was included in both experiments. For each species or accession of weeds, 15 plants were inoculated and five plants, wetted only with water, served as controls. The experiments were in completely randomized designs with 15 single-plant replicates of each species or accession. Visual ratings of disease injury (0=no effect, 5=dead) were made 9 days after inoculation. The results are reported in Table III, below. 
     EXAMPLE 5 
     To study the effect of dew period duration on disease development, seedlings of eastern black nightshade in the third- to fourth-leaf stage were inoculated by wetting the foliage with conidial suspensions of the pathogen (1×10 6  conidia/ml.) and placed in dark, dew-chambers at 25° C. After 0, 4, 8, 12, 16, and 24 hours, groups of 12 inoculated and 12 control plants were removed from the dew-chambers, placed on greenhouse benches, and evaluated for disease development 14 days after inoculation. In general, maximum disease development (95-100% of plants killed) occurred for the dew periods of 16 hours or longer, with 40% of the plants being killed after a dew period of 12 hours. 
     It is understood that the foregoing detailed description is given merely by way of illustration and that modification and variations may be made therein without departing from the spirit and scope of the invention. 
     
                       TABLE III______________________________________Reaction of Weeds of the Solanaceae to Foliar Sprayof the Colletotrichum coccodes                     True leaves                                Injury                     when treated                                rating.sup.bSpecies         Accession.sup.a                     (no.)      (0-5)______________________________________First experimentEastern black nightshade           A37       4          4.8( S. ptycanthum Dun.)Eastern black nightshade           A23       4          4.4(S. ptycanthum Dun.)No common name  A14       4          3.5(S. interius Rydb.)Hairy nightshade           A32       5          2.5(S. sarrachoides Sendtner# SOLSA)Hairy nightshade           A29       5          2.2(S. sarrachoides Sendtner)American nightshade           A59       4          2.3(S. americanum Mill.# SOLAM)Black nightshade           A3        4          1.7(S. nigrum L. subsp. nigrum)Black nightshade           ND3       3          2.4[S. nigrum L. subsp.schultessi (Opiz) Wessely]Second ExperimentEastern black nightshade           A37       4          4.9(S. ptycanthum Dun.)Cutleaf nightshade           --        4          1.9(S. triflorum Nutt.# SOLTR)Jimsonweed      --        3          1.1(Datura stramonium L.# DATST)Buffalobur      --        3          0.6(S. rostratum Dun.# SOLCU)______________________________________ .sup.a Accession numbers identify strains of weeds maintained by Robert N Andersen in Minnesota. .sup.b 0 = No effect, 5 = Dead; LSD (0.05) = 0.1.