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.

The parasitic angiosperm Orobanche aegyptiaca (Pers.) (Egyptian broomrape) is a root holoparasite that causes severe losses in yield and quality of many crops. Control of Orobanche is extremely challenging, in part because the parasite is hidden underground for most of its life cycle. However, the dependence of the parasite on the host suggests that broomrape-resistant hosts could be an ideal control method. Genetic engineering strategies may facilitate realization of this goal, but require an understanding of host defense responses to parasitism. Previous studies with tobacco indicated that broomrape parasitism induces host genes associated with jasmonic acid (JA)-mediated defenses such as wound responses and localized production of phenylpropanoid and isoprenoid phytoalexins. However, the gene for the pathogenesis-related (PR) protein, PR-1a, was not induced by parasitism in tobacco. Expression of PR-1a is correlated with the salicylic acid (SA)-mediated defense pathway that leads to systemic acquired resistance (SAR). The objective of this research was to extend the characterization of PR gene expression in order to define the scope of host defense response. Analyses of gene expression using RNA hybridization and RT-PCR in broomrape-parasitized Arabidopsis thaliana roots indicated that PR-1, PR-2, PR-5, as well as the JA-associated PDF1.2, were slightly induced by parasitism. Expression of PR-1, PR-5, and PDF1.2 in parasitized roots was not detectable by RNA hybridization analysis, but was demonstrated by RT-PCR. Interestingly, shoots of the parasitized plants showed greater PR gene expression levels than roots, indicating that O. aegyptiaca induced a response in the host that was systemic and amplified in shoots. Microarray analysis of parasitized Arabidopsis roots demonstrated a broad range of host gene expression changes including both defense- and non-defense-related genes. Genes induced were consistent with O. aegyptiaca preferentially stimulating JA-mediated responses. The failure of O. aegyptiaca to elicit SA-mediated defenses in host roots suggested that exogenous induction of this signaling pathway could enhance host resistance to parasitism. Treatment of O. aegyptiaca-inoculated tobacco with BTH, a SA analog that activates SAR, caused a 49% reduction in O. aegyptiaca numbers. Analysis of PR-1a using RNA hybridizations and protein immunoblots in treated plants showed the expected induction in shoots, but not in roots, confirming the organ-specific differences in defense response observed in Arabidopsis. Experiments using a strategy to engineer the hypersensitive response via the gene-for-gene interaction confirmed previous findings that parasite-specific activation of an R/Avr interaction in tobacco reduced parasitism by approximately 50%. This research suggests that approaches to stimulate SAR in susceptible host plants may be useful for reducing Orobanche parasitism / Master of Science

The cyst nematode, Globodera tabacum tabacum Behrens, and the parasitic angiosperm, Egyptian broomrape, Orobanche aegyptiaca Pers., are obligate root parasites that cause severe yield and quality loss of many important crop hosts. Although these represent two diverse classes of parasites, they have significant similarities in the modes of parasitism and complex interactions with their hosts. Conventional control methods have had limited success in controlling these parasites. The overall objective of this research was to engineer resistance to the cyst nematode and Egyptian broomrape by expressing genes encoding parasite specific toxins under the control of parasite-responsive promoters using tobacco (Nicotiana tabacum L. cv. Xanthi). For nematode resistance, an anti-feeding strategy was employed utilizing the tomato proteinase inhibitor I (PI-I) gene as a nematode specific toxin. Transgenic tobacco plants were generated that expressed genes encoding an intracellarly retained or secreted form of tomato PI-I under the control of the nematode-inducible promoter, derived from tomato (Lycopersicon esculentum L.) Hmg2 gene. Our goals were to determine the effectiveness of local PI-I expression on nematode resistance and to determine if intracellular or extracellular PI-I deposition enhances resistance. Two constructs were generated that contained either the coding region of the tomato PI-I gene, lacking the signal sequence (EM1), or the coding region of PI-I including the signal sequence (EM2), fused to the nematode-responsive Hmg2 promoter. Transgenic PI-I plants were inoculated with G. t. tabacum cysts and evaluated for nematode interactions. Our results suggest that local expression of intercellular of PI-I significantly reduced cyst production when compared to the nontransformed controls. For broomrape resistance, a well characterized R/avr gene pair, the tobacco N resistance gene and the tobacco mosaic virus replicase (TMV) gene, was utilized to create novel gene-for-gene resistance via a N gene-mediated hypersensitive response (HR) to limit broomrape parasitism. The bean (Phaselous vulgaris L.) chalcone synthase 8 (CHS8) promoter has been characterized as a broomrape–responsive promoter. We introduced the CHS8:TMV replicase gene construct into tobacco plants that contains an endogenous N gene. Transgenic tobacco plants were inoculated with O. aegyptiaca seeds and monitored for parasite attachment and development. The expression of the TMV replicase leads to a significant reduction in broomrape parasitism. These genetic engineering strategies show promise in enhancing resistance to these destructive parasites. / 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.