Kenneth C. Stetina

Immunohistochemical investigation of the necrotrophic phase of the fungus Colletotrichum gloeosporioides in the biocontrol of hemp sesbania (Sesbania exaltata; Papilionaceae)

UNLABELLED PREMISE OF THE STUDY Fungal plant pathogens exert much of their effect on plant cells through alterations in the host cell walls. However, obtaining biochemical proof for this change is difficult because of the relatively small number of cells that are affected by the pathogen relative to the bulk of host tissue. In this study, we examined the differences in host wall composition between infected and uninfected areas of seedlings of the weed hemp sesbania (Sesbania exaltata) that were treated with the biocontrol agent Colletotrichum gloeosporioides. • METHODS To determine the changes in cell wall composition, we used semi-thin sections and a battery of antibody probes that recognize components of the cell wall and immunogold-silver cytochemistry to visualize the probes. • KEY RESULTS A loss of specific plant cell wall polysaccharides in the region surrounding the primary fungal infection and the creation of a defensive layer by the plant to limit the fungal invasion were the two most obvious changes noted in this study. At the invasion site, there was significant loss of rhamnogalacturon-1 (RGI) and esterified and de-esterified homogalacturonan (HG)-reactive epitopes from the cell walls. In contrast, boundary tissue between the vascular tissue and the fungal lesion reacted more strongly with antibodies that recognize arabinogalactan proteins (AGPs) and xyloglucans than in unaffected areas. • CONCLUSIONS These data strongly indicate a role of pectinases in the invasion of the biocontrol agent and the importance of extensins, AGPs, and xyloglucans as defense by the host.

Bioherbicidal enhancement and host range expansion of a mycoherbicidal fungus via formulation approaches

ABSTRACT Eastern black nightshade (Solanum ptycanthum; EBN) is a problematic weed partly due to its tolerance or resistance to certain herbicides. We examined the effects of an invert emulsion (IE) on the host range and weed control efficacy of the fungus Colletotrichum coccodes (NRRL strain 15547) for biocontrol of EBN. Greenhouse tests demonstrated that several other solanaceous weeds were also infected and killed, and field tests revealed >90% EBN control and dry weight reduction in plants treated with the fungus-IE formulation. These results demonstrate that this IE formulation can promote the efficacy of this bioherbicidal pathogen.

Interaction of Glufosinate and Colletotrichum truncatum on Ammonia Levels and Glutamine Synthetase Activity in Hemp Sesbania

The use of microbes and microbial products as bioherbicides has been studied for several decades, and combinations of bioherbicides and herbicides have been examined to discover possible synergistic interactions to improve weed control efficacy. Bioassays were conducted to assess possible interactions of the herbicide glufosinate [2-amino-4-(hydroxymethylphosphinyl) butanoic acid] and Colletotrichum truncatum (CT), a fungal bioherbicide to control hemp sesbania (Sesbaniaexaltata)]. Glufosinate acts as a glutamine synthetase (GS) inhibitor that causes elevated ammonia levels, but the mode of action of CT is unknown. GS has also been implicated in plant defense in certain plant-pathogen interactions. The effects of spray applications of glufosinate (1.0 mM) orbioherbicide (8.0 × 104 conidia ml-1), applied alone or in combination were monitored (88 h time-course) on seedling growth, GS activity and ammonia levels in hypocotyl tissues under controlled environmental conditions. Growth (elongation and fresh weight) and extractable GS activity were inhibited in tissues by glufosinate and glufosinate plus CT treatments as early as 16 h, but CT treatment did not cause substantial growth reduction or GS inhibition until after ~40 h. Generally, ammonia levels in hemp sesbania tissues under these various treatments were inversely correlated with GS activity. Localization of hemp sesbania GS activity on electrophoretic gels indicated a lack of activity after 30 h in glufosinate and glufosinate plus CT-treated tissue. Untreated control tissues contained much lower ammonia levels at 24, 64, and 88 h after treatment than treatments with CT, glufosinate or their combination. CT alone caused elevated ammonia levels only after 64 - 88 h. Glufosinate incorporated in agar at 0.25 mM to 2.0 mM, caused a 10% - 45% reduction of CT colony radial growth, compared to fungal growth on agar without glufosinate, and the herbicide also inhibited sporulation of CT. Although no synergistic interactions were found in the combinations of CT and glufosinate at the concentrations used, further insight on the biochemical action of CT and its interactions with this herbicide on hemp sesbania was achieved.