Fresh weight analysis of seedling shoots treated with H2O and with Silwet L-77 were not significantly different from each other in either species and plant growth in the MV and MV-Sector BSH treatments were reduced by 20 and 15%, in hemp sesbania and sicklepod,respectively .Previous studies have examined the effects of MV spore preparations on growth and development of hemp sesbania and sicklepod seeds , and were also chosen for studies here. The effects of imbibing weed seeds in mycelial preparations of MV and MV-Sector BSH showed that MV was slightly more toxic to hemp sesbania than to sickle pod and was also slightly more potent than the MV-SectorBSH to the early growth of both species . Comparison of SDS-PAGE protein profiles on gels of MV and the sector cell extracts,demonstrated that many bands were similar with respect to presence and abundance and Figure 5. However, some differences were observed and generally more bands appeared to be in greater relative abundance in the MV-Sector BSH versus the MV parent gel and Figure 5.
Protein bands 1 and 3 were more abundant in MV versus MV-Sector BSH , whereas four bands were more abundant in the sector compared to its parent. Comparison of the protein profiles of the liquid culture supernatants of un-inoculated media with supernatants of the MV and MV-Sector BSH showed that both organisms were able to transform media proteins. Some differences in protein bands in the media culture supernatants of MV and MV-Sector BSH were observed,possibly indicating differential substrate preference of the organisms for proteins supplied by the media, and/or to variance in the production of extracellular proteins .Other strains of Myrothecium verrucaria are known to produce various hydrolyticenzymes including xylanases , pectinases , and proteases . Such enzymes play important roles in determining host range and virulence. The nature of these protein differences will need to be examined using native gels and 2-Dgel electrophoresis to determine any possible role as virulence factors.Early bioherbicidal studies on spore formulations of this parent strain of MV showed that the fungus produced trichothecenes that have mammalian toxicity.Another M. verrucaria isolate from leafy spurge exhibited a dissimilar weed host specificity range, but also produced trichothecenes .
Although MV possessed desirable bioherbicidal traits , production of undesirable mycotoxins remained problematic with regard to the development of a commercial product. Approaches to possibly reduce or eliminate these mycotoxins were considered, and cultural manipulation of carbon and nitrogen source/level , spore clean-up or washing and growth of MV in liquid culture without spore production were found successful. Of these techniques, the mycelial form of MV was found to be the most efficient for further studies .We have also previously shown that MV has produced other mutant sectors that had varying degrees of virulence when tested on kudzu . In those tests a white sector was found, but it exhibited no bioherbicidal efficacy on kudzu and was not tested on other weeds. In these present studies a white mutant was discovered and isolated, and shown to be virulent in several tests on hemp sesbania, sicklepod,kudzu and glyphosate-resistant Palmer amaranth. The fact that this virulent sector is stable, does not produce spores and exhibits bioherbicidal activity on major weeds indicatesit may be a useful stand-alone bioherbicide or as a tool for future comparative studies with MV.
Since this sector reproduces only through mycelial growth, it is assumed that that the production of trichothecenes is also mitigated. However, this will have to be monitored. Future studies can be envisioned, including more extensive host range tests with weeds , interactions with herbicides and tests for efficacy in the field. Since some proteins differed in MV-Sector BSH cells and in culture supernatants compared to those of MV, their identity and possible function in infection and virulence processes should be examined. Heat is a valuable entity that has served a variety of needs throughout human history, but only relatively recently has heat been used for weed control, where it may serve as an alternative to chemical herbicides. Much of the research involved with heat to control weeds has been via four general methods: controlled burning, flaming of weeds, hot water treatment and steam application. Heat injury can cause denaturation/aggregation of cellular proteins and protoplast expansion/rupture, resulting in plant tissue desiccation, potentially leading to death .Depending on the heat treatment exposure time, protein denaturation in plant tissues may be initiated at 45˚C . Temperatures of 55˚C – 95˚C can be lethal to plant leaf and stem tissues. Exposure to a flame for only 0.065 – 0.130s was sufficient to kill leaf tissue and higher temperatures were more effective.For example, cellular structural changes were more pronounced when the cellular temperature changed more rapidly compared to gradual and lower temperature changes .