Broomrapes are achlorophyllous holoparasites of many economically important dicotyledonous crops. As weeds, they cause reductions in crop yield, adversely affect crop quality, and result in loss of cultivated land due to reduced crop alternatives. Few effective control measures exist for broomrapes. One of the most promising approaches is the use of low rates of glyphosate in hosts with tolerance to the herbicide. Recently, availability of glyphosate-resistant crops has provided an alternative in broomrape infested areas. Knowledge about the nitrogen status of broomrapes is essential for developing new control strategies. Broomrapes have two potential sources of amino acids. First, the haustorium aids in the translocation of amino acids from the host plant to the parasites. Second, broomrapes may be able to synthesize some amino acids themselves and obtain the rest from the host. However, the relative importance of these two modes of acquiring amino acids by broomrapes is not clear. Osmotic stress has been implicated as a possible reason for inhibition of broomrape germination by nitrogen. To date, there has been no attempt to correlate osmotic potential with nitrogen induced inhibition of broomrape germination. Optimum temperatures for conditioning and germination are different among broomrape species. Although temperature is known to influence germination in broomrape, its effect on subsequent development of the parasitic seedling has not been studied. Studies were conducted to determine the use of glyphosate in controlling broomrape in common vetch that is tolerant to low rates of glyphosate, and to compare this response with broomrape control in oilseed rape that has been genetically engineered for glyphosate resistance. Glyphosate dose response studies using a commercial formulation and patterns of absorption, translocation, and metabolism, using ¹⁴C-glyphosate, were determined for both host crops. Glyphosate significantly reduced the growth of broomrape at 0.18 and 0.36 kg ae ha⁻¹> in common vetch and 0.25 to 0.75 kg ha⁻¹ in oilseed rape. More than 25% of translocated ¹⁴C-glyphosate in both host crops accumulated in broomrape tubercles. Broomrape parasitism caused a redistribution of translocated ¹⁴C-glyphosate in the roots of both host crops. Glyphosate was metabolized up to 25% in common vetch, but remained intact in oilseed rape. Studies were conducted to analyze amino acid composition of both nonparasitized and broomrape-parasitized hosts and associated broomrape after hydrolysis and phenylisothiocyanate derivatization of amino acids. Results indicated that amino acid concentrations of leaves of parasitized carrot plants were lower than those of the leaves of nonparasitized carrot plants. Broomrape tubercles had equal or higher amino acid concentrations compared to those of the leaves of nonparasitized carrot plants. Levels of free alanine and arginine concentrations of broomrape callus were higher than those of any other tissue of either carrot or broomrape. The effect of glyphosate on the host-broomrape interaction regarding amino acid metabolism was examined. Glyphosate generally increased the amino acid concentrations in common vetch and oilseed rape plants, and broomrape attachments. The aromatic amino acids, phenylalanine and tyrosine, did not differ from this pattern. Concentrations of certain amino acids in broomrape were similar to those of parasitized common vetch and parasitized oilseed rape, whereas levels of several others, were higher in broomrape attachments compared to the host plants. <I>In vitro</I> studies were conducted to determine the influence of osmotic potential and temperature on broomrape germination. Osmotic potential significantly affected germination and radicle elongation of broomrapes. No correlation was found between osmotic potential and ammonium-induced inhibition of germination of broomrapes. Temperature significantly influenced germination and radicle elongation of all broomrape species tested. / Ph. D.

Broomrapes, <i>Orobanche</i> spp., are holoparasites that affect the growth of a variety of broadleaf crops. One of the distinct characteristics of the family Orobanchaceae is the lack of chlorophyll, and hence inability to synthesize their own food. Broomrapes subsist on the roots of the host plant from which they derive the carbon, water, and nutrients needed for further growth. Parasitism as such leads to yield reductions, and in case of heavy infestations, complete crop failure. Among other plants parasitized by broomrapes are several legumes, some of which are also the world's most economically important crops. As part of their unique biology, legumes provide an ecological niche for diazotropic soil bacteria, which belong to the family Rhizobiaceae. In return, the host plant receives fixed nitrogen from the nodules, specialized structures produced on the roots of most legume plants upon inoculation with bacteria. <i>Orobanche</i> spp. germination depends on the presence of chemical stimulant in host root exudates. It has been reported that inoculation of some legumes resulted in greater infestation by parasitic weeds. In addition, bacterial nodules were assumed to provide a place for broomrape invasion of host legume. Furthermore, infestations were observed to be more intense in aerobic conditions when rhizobia are most active. It is possible that production of the stimulant could be correlated with the infection of roots with <i>Rhizobium</i>, and that the nodules formed on the roots could play a role in broomrape parasitism; however, others have not confirmed this. Studies were conducted to investigate the possibility of interaction between <i>Orobanche</i> spp. attack and <i>Rhizobium</i> nodulation in legumes. Seed germination, number of parasitic attachments, as well as the morphology of two broomrape species, small broomrape (<i>Orobanche minor</i> Sm.) and Egyptian broomrape (<i>Orobanche aegyptiaca</i> Pers.), were studied. <i>O. minor</i> showed a greater percent seed germination, and formed a greater number of attachments on red clover (<i>Trifolium pratense</i> L.) inoculated with <i>Rhizobium leguminosarum</i> bv. <i>trifolii</i> in comparison with non-inoculated plants. However, the addition of the inoculum did not appear to enhance <i>O. aegyptiaca</i> seed germination or the number of its attachments on the host roots compared with the controls. Morphological observations of <i>O. minor</i> attachments on red clover suggest that parasitic attachments were not situated over the bacterial nodules, but perhaps involve parasite-induced enzymatic degradation followed by mechanical protrusion of host plant root cortex, possibly utilizing host plant-rhizobacteria interactions as well. / Master of Science

<i>Orobanche</i> species are parasitic weeds that subsist on the roots of many dicotyledonous plants. These parasites form symplastic and apoplastic connections with their hosts and act as strong sinks for the uptake of water, minerals, and photosynthates, often causing severe damage to the hosts. Although the uptake of small molecules such as sugars and herbicides by <i>Orobanche</i> has been documented, movement of macromolecules between host and parasite has not been characterized. The objectives of this research were to 1) determine whether, and by what route, host macromolecules can be translocated to the parasite, and 2) engineer host resistance based on inducible expression of sarcotoxin IA, an anti-microbial peptide from the flesh fly (<i>Sarcophaga peregrina</i>). To address the first objective, transgenic plants expressing GFP localized to either the host cell cytosol (symplast) or secreted to the extra-cellular space (apoplast) were parasitized by <i>O. aegyptiaca</i>. Observations of green fluorescence in <i>O. aegyptiaca</i> tubercles growing on these plants indicate that the 27 kDa GFP molecule was translocated to the parasite via both symplastic and apoplastic routes. This work was supported by studies with xylem- and phloem-specific dyes, which showed that fluorescent dextrans as large as 70 kDa moved into the parasite through xylem connections. The second objective was addressed using tobacco (<i>Nicotiana tabacum</i> L. cv. Xanthi) plants expressing the sarcotoxin IA transgene under control of the parasite-inducible <i>HMG2</i> promoter. In soil experiments, transgenic tobacco plants had greater height and biomass, and showed up to 90% reduction in <i>O. ramosa</i> parasitism as measured by the fresh weight of parasite tubercles. In a semi-hydroponic growth system, where <i>Orobanche</i> tubercles can be visualized at early stages of growth, <i>O. aegyptiaca</i> parasites growing on plants expressing sarcotoxin IA were smaller and had an increased number of senescent tubercles compared to those growing on non-transformed plants. Considering the relatively small size of sarcotoxin IA (4 kDa), it is likely that this peptide moves from host to the parasite, where it accumulates to phytotoxic concentrations. In addition to increasing our knowledge of host-<i>Orobanche</i> interactions, this research used an antibiotic peptide to engineer partial <i>Orobanche</i> resistance into a highly susceptible crop. This strategy has broad implications for the control of other parasitic weeds. / Ph. D.