The fungus was isolated from diseased coffee senna tissue by surface sterilizing sections of diseased tissue in 0.05% NaOC1 for 1 min, then placing the sections on potato-dextrose agar (PDA) amended with the antibiotics chloramphenicol (0.75 mg∙ml^–1) and streptomycin sulfate (1.25 mg∙ml^–1). The plates were incubated for 48 h at 25˚C. Advancing edges of fungal colonies were transferred to PDA and incubated for 5 days at 25˚C under alternating 12-h light/12-h dark regimens, provided by cool white fluorescent lights. The fungus was sub-cultured on PDA without antibiotics, and preserved under refrigeration in sterilized sandy loam soil (25% water holding capacity), and on sterile silica gel containing skim milk [19]. Conidia of C. gloeosporioides were produced in a 30-L fermenter (B. Braun, Model C-30, Bethlehem, PA, USA) at a 20-L working volume containing 20-L of modified Richards’ medium [20] plus V-8 vegetable juice (15%; v/v) (Campbell’s Soup Co., Camden, NJ, USA), with an aeration rate of 10 L air min^–1 and an agitation of 250 rpm using 2 Rushton-style impellers. A silicon-based antifoam (HODAG FD-62, Lambent Technologies, Gurnee, IL, USA) was used to control foaming, as required. Inoculum for the fermentations consisted of 250 ml of conidia and mycelium grown in identical medium that was produced in 500 ml Erlenmeyer shakeflasks, incubated at 25˚C and 250 rpm for 5 days. Fermenter batch fermentations were maintained at 25˚C for 5 days. Spores were separated from the spent medium by filtering through double-layered cheesecloth. The spore suspension was centrifuged at 2500 x g for 15 min at 15˚C. The medium had an initial pH of 5.5, and pH was not controlled or monitored during culture growth. Conidial concentrations used in the tests were determined with hemacytometers.

Conidial germination was measured by spreading 0.1 ml of a suspension (1.0 × 10⁶ conidia∙ml^–1) on PDA plates, and incubating them at 10˚C, 15˚C, 20˚C, 25˚C, 30˚C, or 35˚C on open-mesh wire shelves of an incubator (Precision Scientific Inc., Chicago, IL, USA). Twelve-hour photoperiods were provided by two-20 W, cool-white fluorescent lamps positioned in the incubator door. Light intensity at plate level was 200 µE∙m^–2∙s^–1 photosynthetically active radiation (PAR) as measured with a light meter (LI-COR Inc., Lincoln, NE, USA). Germinated conidia (500 plate^–1) were counted after 16 h.

For radial growth studies, 5-mm plugs were taken from advancing margins of a 7-day old colony of the fungus and placed in the centers of PDA plates. The plates were incubated at temperatures of 10˚C, 15˚C, 20˚C, 25˚C, 30˚C, or 35˚C as described above for the conidial germination studies.

Colony diameters were measured after 7 days of incubation. In each experiment, five replicate plates for each temperature were utilized. Both experiments were conducted twice, and the results of each experiment were pooled following testing for homogeneity.

In all experiments coffee senna plants were grown from seed in a commercial potting mix contained in peat strips. Each strip contained 12 plants. The potting mix was supplemented with a slow-release fertilizer (14:14:14, NPK). The plants were placed in sub-irrigated trays on benches in the greenhouse. Greenhouse temperatures ranged from 25˚C to 30˚C with 40% to 60% relative humidity (RH). The photoperiod was 12 h with 1650 µE∙m^–2∙s^–1 (PAR) measured at midday.

Five to seven-day old seedlings (cotyledonary to early first-leaf stage) of coffee senna were sprayed until runoff with 1.0 × 10⁷ conidia∙ml^–1 in water. Control plants were sprayed with distilled water only. The plants were placed in darkened dew chambers at 100% RH at temperatures of 15˚C, 20˚C, 25˚C, 30˚C, or 35˚C. The plants were then transferred to environmental growth chambers (Conviron, Model E-7, Pembina, ND) with day/night air temperatures of either 20˚C/10˚C, 25˚C/15˚C, 30˚C/15˚C, 35˚C/ 25˚C, or 40˚C/30˚C. Photoperiods were 14 h with 820 to 840 µE∙m^–2∙s^–1 PAR and a RH of 65%.

Coffee senna seedlings in the cotyledonary to early first leaf stage of growth were sprayed until runoff occurred with a spray mix containing 1.0 × 10⁷ conidia∙ml^–1. Control plants were sprayed with distilled water. The inoculated plants were then placed in darkened dew chambers at 25˚C and 100% RH for periods of 4, 8, 12, 16, 20, or 24 h. Following the dew period inoculation, the plants were placed on sub-irrigated trays in the greenhouse and monitored for 14 days for disease development and mortality. Greenhouse temperatures were 28˚C to 32˚C, with 40% to 60% RH at day lengths of 12 h with an average of 1650 mol∙m^–2∙s^–1 photosynthetically active radiation at mid-day. Mortality and dry weight reductions were recorded after 14 days after treatment. The experiment was conducted twice with 3 sets of 12 plants for each experiment.

Coffee senna plants in the cotyledonary, 1 to 2 true-leaf, 3 to 4 true-leaf and 5 to 7 true-leaf stages of growth were sprayed with conidial suspensions of 1.0 × 10³ to 1.0 × 10⁷ conidia∙ml^–1 and held in a dew chamber for 16 h at 25˚C. Control plants were sprayed with distilled water only. Plants were moved to the greenhouse, and mortality and dry weight reductions were recorded after 14 days after treatment. Experiments were conducted twice with 3 sets of 12 plants for each experiment.

Two field experiments were conducted on a Dundee very fine sandy loam (Aeric Ochraqualf) soil at the USDAARS, Southern Weed Science Experimental Farm at Stoneville, MS. Plots consisted of four rows of soybeans (cv. “Centennial”), 12.2 m long and 1 m apart, with the two center rows receiving treatment. All rows were planted with scarified coffee senna seed at a density of about 100 seeds∙m^–1 of row. Treatments consisted of: 1) 1.0 × 10⁵ conidia∙ml^–1 in distilled water and 0.2% Silwet L-77 surfactant; 2) 1.0 × 10⁶ conidia∙ml^–1 in distilled water and 0.2% Silwet L-77 surfactant; 3) 1.0 × 10⁷ conidia∙ ml^–1 in distilled water and 0.2% Silwet L-77 surfactant; 4) 0.2% Silwet L-77 only; and 5) distilled water only. The seedlings were in the cotyledonary to first-leaf growth stage, and were sprayed until fully wetted (approximately 450 l∙ha^–1), resulting in inoculum densities of 9.0 × 10¹² conidia∙ha^–1 in those plots that received fungal treatments. Applications were made at midday with a hand-held pressurized sprayer. For the first experiment, environmental conditions at the time of inoculation and for 24 h following treatment were: temperature at inoculation, 29˚C with a RH of 69%. The high temperature for the 24 h period was 31˚C and the low temperature was 25˚C. Maximum RH was 94%, with a short dew period of 3 h. For the second experiment, environmental conditions at the time of inoculation and for 24 h following treatment were: temperature at inoculation, 34˚C with a RH of 58%. The high temperature for the 24 h period was 34˚C, and the low temperature was 24˚C. Maximum RH was 92%, with a short dew period of 4 h. The plants were monitored for disease development at 7 day intervals for 21 days. A randomized complete block design was utilized, and the treatments were replicated four times. Data over the 2 years from the two experiments were pooled following subjection to Bartlett’s test for homogeneity, and analyzed using analysis of variance. Percentage data of coffee senna control were determined in two randomly selected 1-m² subplots from each treated plot 1, 2, and 3 weeks after treatment. The experimental plots were furrow-irrigated as required to maintain healthy coffee senna and soybean plants throughout the growing season. Significant differences were determined using Fisher’s Protected Least Significant Difference (FLSD) at P = 0.05.

All experiments utilized a randomized complete block experimental design, with a minimum of three replications of 12 plants for each experiment. All experiments were repeated. Data were subjected to the analysis of variance and values are presented as the means of replicated experiments. Duncan’s multiple range test at the 0.05 level of probability was used to separate means in the dew and air temperature experiment. Confidence limits (95% level) were used to analyze the data in remaining experiments.

The fungus was readily isolated from diseased tissue and it sporulated abundantly on PDA. When reinoculated onto healthy seedlings, the fungus was highly virulent and killed all inoculated plants within 5 days, while the controls remained healthy (data not shown). The organism produced typical anthracnose lesions on leaves and stems, with acervuli scattered throughout the lesions. The acervuli were glabrous (non-setose) and were slightly sunken in the host tissue. Conidia were elliptical, with rounded ends, and range in size from 15 to 19 µm by 4 to 6 µm and averaged 16 by 6 µm.

Maximal germination of conidia on PDA occurred at 25˚C (86%) and 30˚C (79%), with significant decreases in germination at the other temperatures (Figure 1(A)). The fungus grew well at 20˚C to 30˚C, but growth was significantly reduced when incubated at 10˚C or 15˚C and slow growth occurred at incubation temperatures of 35˚C or higher (Figure 1(B)). The fungus also sporulated prolifically under submerged culture in modified Richard’s

medium. Conidial yields averaged 3.0 × 10⁸ conidia∙ml^–1 after 7 days at 25˚C and 250 rpm (data not shown). This growth is comparable to conidial yields produced by other C. gloeosporioides formae speciales that have been evaluated as bioherbicides against other weeds [22,23].

Optimal day, night, and dew temperatures for maximum weed control (100%) were 30˚C/25˚C/20˚C (Figure 2). Both weed mortality and biomass reduction were significantly reduced when the day/dew/night temperatures were lowered to 20˚C/15˚C/10˚C. No pathogenesis or mortality occurred on coffee senna seedlings under a day/dew/night temperature regime of 40˚C/35˚C/30˚C (Figure 2).

The fungus killed coffee senna over a broad range of dew period durations conducted at 25˚C (Figure 3). Although some infection occurred at all dew periods tested, a dew period of at least 12 h was required to kill about 80% of fungus-inoculated plants. Complete mortality was not achieved at any dew period, but many plants were severely stunted, which resulted in greatly reduced biomass.

In greenhouse tests the fungus had no effect on other weed and crop plants tested except for sicklepod, which was slightly affected by the pathogen, and wild senna, which was nearly as susceptible as coffee senna (data not

shown). Plants were grown to the cotyledonary to thirdleaf growth stage and tested as described in the material and methods. Severe disease developed on coffee senna and wild senna within 2 to 3 days of inoculation, and seedlings were killed within 4 to 6 days.

Weed control (mortality) under greenhouse conditions was significantly increased at all growth stages by increasing the fungal inoculum concentration (Figure 4). Coffee senna seedlings in the 5 to 7 leaf stage were more resistant to infection than younger plants. Weed control was significantly less than that achieved with plants at

earlier growth stages at the inoculum concentrations tested. Similar results were found on the dry weight reductions of plants at these growth stages and conidia concentrations (data not shown).

In field experiments, coffee senna plants treated with the highest inoculum concentration (1.0 × 10⁷ conidia∙ml^–1) were controlled over 80% at 21 days after treatment, while only 40% and 20% weed control was achieved with 1.0 × 10⁶ conidia∙ml^–1 and 1.0 × 10⁵ conidia∙ml^–1, respectively, following this time period (Figure 5). The fungus sporulated profusely on infected tissue, and killed greenhouse-grown coffee senna seedlings when re-inoculated onto them, thus fulfilling Koch’s Postulates. No disease or mortality occurred to untreated control plants, or to plants treated with surfactant only (data not shown).