Macrophomina phaseolina (Tassi) Goid. causes a variety of diseases, including charcoal rot, crown rot, seed rot, wilt, and many others in over 500 commercially-important plants, including soybeans, trees, vegetables and ornamentals. M. phaseolina primarily infects plants from the soil reservoir through the roots, although it may also be seed-borne. M. phaseolina has been reported to produce several secondary metabolites, including asperlin, phomenone, phomalactone, phaseolinone, phaseolinic acid, patulin, and (-)-botryodiplodin, one or more of which may play a role in facilitating the root infection process. A previously-developed in-culture colorimetric assay for the mycotoxin (-)-botryodiplodin was used to screen M. phaseolina isolates and other soybean fungal pathogens for their ability to produce the toxin. Isolates of M. phaseolina negative for (-)-botryodiplodin production in this assay (20-25%) were cultured in potato dextrose broth medium for 7 days, then the culture medium filter-sterilized, mixed 1:1 with hydroponic medium and tested for 72 hours for root toxicity with soybean seedlings in hydroponic culture. 50% of (-)-botryodiplodin-negative isolates exhibited potent phytotoxic responses ranging from prevention of lateral root production to complete seedling death. Culture media from phytotoxic isolates are being examined further by bioassay-guided fractionation and LC/MS, GC/MS, and HP/LC analysis to identify production of secondary metabolites that may play a role in facilitating soybean root infection by M. phaseolina and other fungi.