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Colletotrichum coccodes is being developed as a bioherbicide to control velvetleaf. An effective and economic process for mass production of C. coccodes sports is required for its possible development as a bioherbicide. A modified Richard's solution containing V-8 juice has been used to produce C. coccodes spores in shake flasks. However, V-8 juice is not available as a bulk commodity and is too expensive for commercial production of a bioherbicide. Soy protein and casamino acids were equal to and can replace V-8 juice for sporulation of C. coccodes . Using the Plackett-Burman experimental design, the effect of the various components in a complex medium on both the sporulation and mycelial biomass of C. coccodes were determined. Carbon concentration and carbon to nitrogen ratio as well as their interaction are critical for C. coccodes growth and sporulation. In medium containing 10 g/l carbon with C:N ratios from 10:1 to 15:1, 8 x 107 C. coccodes spores per ml were produced. Germination of the spores was 90% and they caused extensive shoot dry weight reduction of inoculated velvetleaf. C:N ratios from 10:1 to 15:1 am optimal for C. coccodes sporulation. A cyclone column bioreactor was used to investigate the effects of environmental conditions on spore yield. C. coccodes sporulation was superior with low aeration (∼0.5vvm) and low pH (4.5--5.0) compared with high aeration (2 vvm) and high pH (6.5--7.5). The optimum temperature for sporulation was 24°C. C. coccodes sporulation commences when the dissolved oxygen supply becomes restricted. Spore yields of over 3.5 x 107 spores per ml can be obtained within four days from batch culture. Scale up to a 100 liter cyclone column bioreactor yielded 2.5 x 107 C. coccodes spores per ml within four days.

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Colletotrichum coccodes (Wallr.) Hughes, a foliar pathogen of velvetleaf, is being developed as a bioherbicide. Formulation of living organisms for use as pest control products presents unique problems. This research has achieved the development of an adequate formulation of the pathogen by using kaolin clay or talcum powder (1:2.79 wt/wt) as the fillers to dry conidia. Formulated C. coccodes conidia stored at 4, 30C, or at room temperature in bags permeable to oxygen remained viable and able to infect velvetleaf plants at least six months in storage. Various reported germination stimulants increased germination of formulated conidia, although not significantly, whereas increasing concentrations of cutin resulted in subsequent decreases in germination and appressoria formation of fresh as well as formulated conidia. In controlled environment experiments, 14 day-old velvetleaf seedlings were severely diseased when stearic or oleic acids were added to conidia formulated in kaolin clay or talcum powder, respectively. Combinations of germination stimulants, cutinase and/or pectinase inducers did not significantly increase germination and appressoria formation of C. coccodes conidia. Germination of fresh and formulated conidia increased, although not significantly, with the addition of 1% sucrose.

Resistance responses of Abutilon theophrasti were investigated to determine defense mechanisms of the weed against Colletotrichum coccodes and to verify if some chemical suppression of the resistance mechanism could be exploited to enhance the virulence. Induced resistance in A. theophrasti has been confirmed in treatments with C. coccodes, benzothiadiazole, bentazon, and acifluorfen. Induction of peroxidase and phenylalanine ammonia lyase (PAL) activities in the leaves that did not contact with the inducing agents was observed after the localized stresses to the first leaf or the root of the plant with those agents. alpha-Amino-oxy acetic acid (AOA), 2-deoxy-D-glucose (DDG), mannose, oxalic acid, and analogues of oxalic acid and mannose were tested to enhance C. coccodes virulence. However, the compounds did not enhance C. coccodes virulence or affect A. theophrasti growth. Strong antifungal effects, poor inhibitory effects on plant defense mechanisms, or minor dependence of A. theophrasti on the defense mechanisms that the chemicals affected could be reasons. The efficacy of C. coccodes increased in the presence of 0.25 kg a.i. ha-1 bentazon more than when C. coccodes was applied alone, while the effect of glyphosate was minimal. Peroxidase activity was strongly induced by the treatment of C. coccodes and increased over time. PAL and activation of peroxidase was inhibited in the presence of bentazon, suggesting the synergy effect by bentazon is probably due to the suppression on the two defense-related enzymes. In conclusion, A. theophrasti exploits various biochemical and morphological types of defense mechanisms against C. coccodes infection. However, the activation of the defense responses can be suppressed or by-passed in an integrated weed management system.

Field and growth bench experiments were performed to assess the effect of a selective fungal pathogen of Abutilon theophrasti (velvetleaf) on various aspects of intra- and interspecific competition between this vigorous agricultural weed and soybean (Glycine max). In the absence of the foliar pathogen, Colletotrichum coccodes, A. theophrasti and soybean responded differently to the presence of conspecies or to individuals of the other species. In pure stand, the deleterious effects of intraspecific competition on reproductive output were substantially greater for A. theophrasti than for soybean, especially at lower monoculture densities. In mixtures, however, A. theophrasti reproductive performance was markedly higher than at equivalent monoculture densities, particularly at the lower mixture densities. Soybean reproduction at these lower mixture densities (10 to 20 plants m$ sp{-2}$) was severely curtailed compared with reproductive output at equivalent pure stand densities. A. theophrasti reproductive output was limited more by the presence of conspecies than by the presence of soybean, whereas the opposite trend was observed for soybean. In pure stand, application of C. coccodes had limited impact on either A. theophrasti or soybean yield. However, application of the fungal pathogen in A. theophrasti monocultures caused significant (30-44%) aboveground biomass reductions within five weeks of inoculation, in two of the three years in one field study. Eight weeks following C. coccodes inoculation, A. theophrasti biomass within inoculated monoculture plots did not differ significantly from biomass within uninoculated control plots, although height hierarchies were significantly more developed. In mixtures, C. coccodes applications caused reductions in A. theophrasti growth and reproduction when provided with an adequate dew period. Alternatively, soybean yield losses within inoculated mixture plots were generally lower than for uninoculated control plots, althoug

Colletotrichum coccodes strain DAOM 183088 is considered a potential bioherbicide for velvetleaf (Abutilon theophrasti), a devastating weed in North American corn and soybeans. Risk assessment studies have created a demand for an accurate and robust method to monitor this strain, and to distinguish it from indigenous background population of microorganisms present in the field. Safe biological control management of velvetleaf also requires comprehensive understanding of the pathogenicity determinants employed by this host-specific fungus to establish infection on velvetleaf, an aspect central to a safe biocontrol strategy task. In this study, molecular markers were designed that allow strain specific identification of the bioherbicide strain of C. coccodes and its identification within complex plant and soil matrices. An assay was developed to quantify C. coccodes from deliberate release field soil samples, in which biases caused by soil-originating PCR inhibitors were monitored on a sample per sample basis. The developed external control assay allowed for the estimation of target C. coccodes DNA quantities with normalization for the presence of PCR inhibitory compounds. Kinetic growth curves of disease development were performed for C. coccodes wild-type and T20-a (genetically engineered for hypervirulence with the NEP1 (necrosis and ethylene inducing peptide) gene) strains on velvetleaf leaves over a period of 14 days after C. coccodes infection. The wild-type strain was more efficient at infecting velvetleaf than the transgenic T-20a strain, while expression of NEP1 could not be detected suggesting that the introduced gene may not be transcriptionally active in the transformed strain, a result in conflict with previous observations. Velvetleaf and C. coccodes genes specifically upregulated at 12 and 24 h after fungal infection were cloned and differentially screened by microarrays. The resulting EST collection was sequenced and assigned to putative functions. Early gene up-regulation was confirmed by QRT-PCR analysis for type 3 metal lothionein, EREB, WRKY, and bZIP transcription factors, reticuline oxidase, ascorbate peroxidase, and ACC oxidase gene candidates. In addition, type 2, type 3 metallothionein, and bZIP gene expression profiles were investigated over a period of 14 days after C. coccodes infection, and the results indicated that C. coccodes altered the expression of all three gene analyzed.

Experiments were conducted in controlled and field conditions to determine the effect of seed size, a fungal pathogen (Colletotrichum coccodes), and soybean interspecific competition on the population dynamics of Abutilon theophrasti (velvetleaf). Seed size differences among ten individual A. theophrasti plants significantly (P < 0.001) affected seed germination and dormancy. Higher seed viability (98%) was observed among seeds having a weight above 6.0 mg. The response of A. theophrasti plants that originated from two extreme seed size groups (small <7mg and large >12mg) to the pathogen, C. coccodes, did not change over generations, and the most vigorous plants produced heavier seeds regardless of the initial seed size or infection with C. coccodes. Under field conditions, the application of C. coccodes and the herbicide, bentazon, did not affect the vegetative and reproductive biomass of A. theophrasti plants when grown in monospecific stands. However, a split application of C. coccodes and bentazon significantly reduced the aboveground biomass and reproductive output of A. theophrasti plants when grown in competition with soybean. The frequency distributions of A. theophrasti plant height, aboveground biomass, and stem diameter were positively skewed (L-shaped) when competing with soybean. However, A. theophrasti plant height and stem diameters were negatively skewed (J-shaped) and the aboveground biomass was positively skewed (L-shaped) in monospecific stands. The allometric relationships of A. theophrasti aboveground biomass and stem diameter in comparison with plant height were curvilinear when grown alone and when in the presence of soybean. However, aboveground biomass and stem diameter showed a simple linear relationship on a log-log scale in both monospecific stands and in competition with soybean.

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