VTechWorks Repository :: Browsing by Author "Westwood, James H."

Browsing by Author "Westwood, James H."

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Biological watermelon (Citrullus lanatus L.) seed treatments for control of Acidovorax citrulli
Klein, Rachel (Virginia Tech, 2020-06-03)
Acidovorax citrulli is a seedborne pathogen responsible for bacterial fruit blotch (BFB), an economically important disease in melon and watermelon throughout the world. BFB is highly virulent and in affected fields can cause yield reduction of up to 95%, which has resulted in over $100,000 in losses to melon growers in some cases. The efficacy of green tea as an antimicrobial seed treatment against A. citrulli was tested. Watermelon seeds were treated with green tea after inoculation with transgenic A. citrulli expressing green fluorescent protein (GFP). Forty five percent of watermelon seedlings inoculated with a high level (OD600:1.0, ~8 x 108 cells/ml) of A. citrulli displayed GFP in their cotyledons. When these seeds were treated with green tea, only 11.2% displayed GFP in their cotyledons. None of the treated watermelon seedlings inoculated with a low level (OD600:0.001, ~8 x 105 cells/ml) of A. citrulli displayed GFP in their cotyledons. Green tea treatments effectively controlled the disease when administered as a liquid to infected watermelon seeds. Green tea has potential as an effective commercial treatment for pericarp infected seeds that could also be used by growers participating in the National Organic Program.
Biology and Control of Eastern Black Nightshade, Palmer Amaranth, and Common Pokeweed, in No-Till Systems on the Eastern Shore Regions of Virginia and Maryland
Vollmer, Kurt Matthew (Virginia Tech, 2014-12-05)
Eastern black nightshade, Palmer amaranth, and common pokeweed are three hard to control weed species on the Eastern Shore regions of Virginia and Maryland. Herbicide resistance and lack of herbicide efficacy further complicate the job of controlling these weeds. Studies were conducted on each of these weeds in order to determine herbicide efficacy and potential herbicide resistance. In addition, the translocation and metabolism of 14C-glyphosate was studied in common pokeweed. This research identified a population of eastern black nightshade that was differentially sensitive to families of ALS-inhibiting herbicides, with tolerance to members of the sulfonylurea family, but controlled with herbicides of the imidazolinone family. A population of Palmer amaranth was found to be glyphosate-resistant, but herbicide programs were identified that could control this biotype in soybean and corn systems. Experiments on the fate of glyphosate in common pokeweed indicated that glyphosate does not readily translocate from treated foliage to other plant parts, which may contribute to shoot regeneration from taproots following glyphosate treatment. Taken together, this research highlights the important weed control issues, including resistant and perennial weeds in agronomic crops that have arisen in Eastern Shore agriculture. This work will help growers to better assess their particular control issues, and take appropriate steps to mitigate any problems.
Characterization and Management of Acetolactate Synthase Inhibiting Herbicide Resistant Mouse-Ear Cress (Arabidopsis thaliana) in Winter Wheat
Randhawa, Ranjeet Singh (Virginia Tech, 2017-09-20)
The first case of field evolved acetolactate synthase (ALS) inhibiting herbicide resistance in the model plant, mouse-ear cress, was reported in winter wheat fields in Westmoreland County, Virginia. A putative resistant (R) mouse-ear population was assessed for ALS resistance relative to a putative susceptible (S) and a susceptible lab population Columbia (C). Results indicated that the R population needed 23 to >2400 fold rate of thifensulfuron relative to S or C population, and it has evolved cross-resistance to sulfonylureas (SU), triazolopyrimidine sulfonanilides (TP), and sulfonylaminocarbonyltriazolinones (SCT). Further studies sequenced the whole genome for four field populations, representing two locations and two resistance levels (high and low) per location, to characterize the genetic mechanism of ALS resistance. The results revealed that all populations contained mutations in the ALS gene at the Pro197 site, although the Pro was substituted by Phe in one location and Thr in the other. Also, both high- and low-level resistant plants at one location had additional mutations (Trp574Leu or Asp376Glu) known to confer resistance to ALS inhibiting herbicides. Patterns of herbicide cross-resistance also varied among the populations. Additionally, research was conducted to assess preemergent (PRE) and postemergent (POST) alternative herbicide options for control of ALS resistant mouse-ear cress and its interference with winter wheat. Results indicate flumioxazin, pyroxasulfone, and metribuzin can be used for effective PRE control whereas 2,4-D, dicamba, and metribuzin can be effective post control options. No mouse-ear cress interference with winter wheat was observed at density of more than 300 plants m-2.
Characterization of Acetolactate Synthase-Inhibiting Herbicide-Resistant Smooth Pigweed and Corn Weed Management Programs Utilizing Mesotrione in Combinations with Other Herbicides
Whaley, Cory Miller (Virginia Tech, 2005-03-01)
Repeated use of acetolactate synthase (ALS)-inhibiting herbicides in recent years has resulted in the selection of 89 weed species resistant to these herbicides. One management strategy that can eliminate or slow the development of resistance is applying mixtures of herbicides with different modes of action. This research involved the characterization of ALS-inhibiting herbicide-resistant smooth pigweed (Amaranthus hybridus L.), as well as investigations on weed management programs in corn (Zea mays L.) utilizing mesotrione, a triketone, in mixtures with other herbicides. ALS-inhibiting herbicide-resistant smooth pigweed biotypes were collected from fields in Virginia, Delaware, Maryland, and Pennsylvania to evaluate response to ALS-inhibiting herbicides and to determine the molecular mechanisms of resistance. Sequencing of the ALS genes from these biotypes revealed two amino acid substitutions known to confer resistance, Ala₁₂₂ to Thr and Ser₆₅₃ to Asn, and one that has not been previously reported in plants, Asp₃₇₆ to Glu. The smooth pigweed biotype with an Asp₃₇₆ substitution displayed resistance to four classes of ALS-inhibiting herbicides that included imidazolinone (IMI), sulfonylurea (SU), pyrimidinylthiobenzoate (PTB), and triazolopyrimidine sulfonanilide (TP) chemistries. Transformation of this smooth pigweed ALS gene into Arabidopsis thaliana confirmed that the Asp₃₇₆ substitution is responsible for the resistance. Other biotypes that had a substitution at Ala₁₂₂ exhibited resistance to an IMI herbicide, little to no resistance to SU herbicides, and increased sensitivity to a PTB and a TP herbicide, whereas, biotypes that had a substitution at Ser₆₅₃ exhibited high-level resistance to an IMI herbicide and lower resistance to PTB and SU herbicides. Experiments were also conducted to investigate the effectiveness of mesotrione in preemergence (PRE) and postemergence (POST) corn weed management programs in Virginia. Mesotrione applied PRE in mixtures with S-metolachlor and atrazine controlled common lambsquarters (Chenopodium album L.), smooth pigweed, common ragweed (Ambrosia artemisiifolia L.), and morningglory (Ipomoea spp.) species when a timely rainfall followed application. POST applications of mesotrione controlled common lambsquarters and smooth pigweed, but common ragweed and morningglory species were not always controlled. Common ragweed and morningglory species were controlled by mesotrione in a mixture with atrazine POST. Large crabgrass [Digitaria sanguinalis (L.) Scop.] and giant foxtail (Setaria faberi Herrm.) control was generally better when the ALS-inhibiting herbicides nicosulfuron plus rimsulfuron or rimsulfuron plus thifensulfuron plus atrazine were applied in a mixture with mesotrione. Mixtures of mesotrione with other POST herbicides in a total POST program produced corn yields comparable to standard PRE followed by POST weed management programs.
Characterization of Host Plant Defense Responses to Parasitization by Orobanche aegyptiaca
Griffitts, Amanda Aline (Virginia Tech, 2001-05-07)
Orobanche (spp.) are parasitic plants that attack the roots of many important crops. O. aegyptiaca penetrates the host root (aided by digestive enzymes) and forms connections to the host vascular tissue, from which it will withdraw all of its water and nutrient requirements. In order to control this weed, it is important to understand the relationship between the host and the parasite. To investigate how parasitism effects host defense pathways, we are studying the patterns of expression of host genes known to be involved in various aspects of plant defense responses. With respect to local defense responses, two genes of the isoprenoid pathway were studied, one of which is expressed in wounded tissue (hmg1), and another that is induced in response to wounding yet repressed in response to pathogen elicitors (squalene synthase). Genes analyzed that are associated with systemic defense include PR-1, PR-2, and PR-5, all of which are induced in response to pathogen attack as part of the systemic acquired resistance (SAR) response. Plant gene expression was studied using transgenic tomato plants containing hmg1-GUS fusions, and northern hybridization analysis of tobacco and Arabidopsis roots using gene-specific probes. Results indicated that expression of hmg1 is induced, PR-2 and squalene synthase are repressed, and PR-1a, PR-1, and PR-5 are not affected in tissue parasitized by O. aegyptiaca. Together, these results indicate a complex response to the parasite. Whereas hmg1 induction is consistent with O. aegyptiaca inflicting a simple wound-like injury, the repression of squalene synthase is consistent with plant recognition of a pathogen attack. In contrast, the failure of Orobanche to induce SAR- related PR-1 in tobacco and PR-1, PR-2, or PR-5 in Arabidopsis indicates an ability to avoid or perhaps inhibit some defense-related pathways. By comparing the regulation of these defense genes in response to O. aegyptiaca attack, we are able to gain a greater understanding of the host plant response to parasitization and explore potential gene candidates for future engineering strategies to create Orobanche resistant crops.
Characterizing RNA translocation in the parasitic weed Cuscuta pentagona
LeBlanc, Megan Leanne (Virginia Tech, 2013-06-03)
The obligate stem parasite Cuscuta pentagona is able to take up host plant mRNA through a specialized organ known as the haustorium. Direct cell-to-cell symplastic connections between two different organisms are rare, and the translocation mechanisms and fate of these RNAs in the parasite is not understood. To characterize this phenomenon, mobile Arabidopsis and tomato mRNAs were identified from microarray and transcriptome sequencing projects and quantified in the host-parasite system. Mobile RNAs were quantified using real time (qRT)-PCR and were found to vary substantially in their rate of uptake and distribution in the parasite. Transcripts of tomato Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Protease Inhibitor (SlPI) can be traced over 30-cm of parasite stem. SlPI was abundant in the C. pentagona stem, but the number of copies decreased substantially within the first eight hours post detachment. Additional studies of mobile RNAs from Arabidopsis, Translationally Controlled Tumor Protein (AtTCTP), Auxin Response Factor (AtARF) and a Salt-inducible Zinc Finger Protein (AtSZFP) supported the idea that mRNA molecules differ in their mechanisms of uptake and mobility between host and parasite. Known phloem-mobile RNAs (SlGAI and AtTCTP) have uptake patterns that differ from each other as well as from other RNAs that are not reported to be phloem mobile (SlPI and AtSZF1). The function of RNAs in plants extend beyond protein translation to include post transcriptional gene silencing or long distance signaling, and mobile RNA in C. pentagona systems offers novel insights into this aspect of plant biology. Studies of cell-to-cell trafficking of RNAs and other macromolecules would be facilitated by the ability to manipulate individual cells. To this end, work was initiated to explore alternative approaches to understanding single cell biology using laser-mediated approaches. Optoperforation, or the use of multiphoton processes to form quasi-free electron plasmas to initiate transient pore formation in plasma membranes, has been demonstrated, but not in cells of an intact plant. This work details a protocol for optoperforation of Arabidopsis epidermal cells to allow for uptake of external dye-labeled dextrans and retention for up to 72 hours, and has the potential for transformation and molecular tagging applications.
Comparative Metabolomics of Early Development of the Parasitic Plants Phelipanche aegyptiaca and Triphysaria versicolor
Clermont, Kristen; Wang, Yaxin; Liu, Siming; Yang, Zhenzhen; dePamphilis, Claude W.; Yoder, John I.; Collakova, Eva; Westwood, James H. (MDPI, 2019-06-13)
Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. The connection of the haustorium to the host marks a shift in parasite metabolism from autotrophy to at least partial heterotrophy, depending on the level of parasite dependence. Species within the family Orobanchaceae span the spectrum of host nutrient dependency, yet the diversity of parasitic plant metabolism remains poorly understood, particularly during the key metabolic shift surrounding haustorial attachment. Comparative profiling of major metabolites in the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor before and after attachment to the hosts revealed several metabolic shifts implicating remodeling of energy and amino acid metabolism. After attachment, both parasites showed metabolite profiles that were different from their respective hosts. In P. aegyptiaca, prominent changes in metabolite profiles were also associated with transitioning between different tissue types before and after attachment, with aspartate levels increasing significantly after the attachment. Based on the results from ¹⁵N labeling experiments, asparagine and/or aspartate-rich proteins were enriched in host-derived nitrogen in T. versicolor. These results point to the importance of aspartate and/or asparagine in the early stages of attachment in these plant parasites and provide a rationale for targeting aspartate-family amino acid biosynthesis for disrupting the growth of parasitic weeds.
Cross-Species Translocation of mRNA from Host Plants into the Parasitic Plant Dodder
Flagg, Jeannine K. (Virginia Tech, 2006-04-11)
Dodders (Cuscuta spp.) are parasitic plants that live by tapping into the vascular tissue of a host plant. Contents of the host phloem translocate readily into the parasite, and shared plasmodesmata have been documented between host cortical cells and dodder searching hyphae. Dodder is known to transmit viruses from one host to another, which is consistent with viral ability to traverse plasmodesmata (PD) with the aid of movement proteins (MPs). Plant endogenous mRNAs may also associate with specific proteins to pass through PD and traffic long distances in the phloem, a process that appears to play a role in coordination of development. We have evaluated the hypothesis that dodder is able to accumulate host phloem-mobile mRNAs by assaying lespedeza dodder (C. pentagona) for the presence of host transcripts. Reverse transcriptase PCR (RT-PCR) and tomato microarrays were used to probe RNA from dodder parasitizing tomato. Transcripts from four tomato genes were detected in dodder grown on tomato, but were not detected in control dodder grown on other hosts. Notable among these was LeGAI, a transcript previously shown to be phloem translocated. In addition, RT-PCR of RNA from dodder grown on pumpkin detected three mobile pumpkin mRNAs (CmNACP, CmSUTP1, and CmWRKYP). These results imply the existence of an extraordinary situation in which mobile mRNAs move from one plant into another, and raise questions about the role of this phenomenon in plant development and parasite pathogenicity.
Development of novel approaches to study Cuscuta campestris biology
Bernal Galeano, Vivian Angelica (Virginia Tech, 2021-09-16)
Cuscuta campestris is an obligate parasitic plant that lacks expanded leaves and roots and requires a host to complete its lifecycle. Parasite-host connections occur via an haustorium, a unique organ that acts as a bridge for the exchange of water, nutrients, macromolecules like mRNA, microRNA, and proteins, and microorganisms. Studies of Cuscuta spp. are challenging due to its dependence on the host and other host influences on the parasite. Recent research has shown intriguing aspects of Cuscuta biology like exchange genetic material with its hosts and loss of genes involved in processes such as high photosynthetic rates and defense. We developed new tools and methodologies that allow us to explore C. campestris biology in an unprecedent way. Foremost of these is an axenic method to grow C. campestris on an Artificial Host System (AHS). The AHS allows C. campestris to display its entire life cycle in vitro, including seed production. Using the AHS, we studied haustorial function, determining the role of nutrients and phytohormones on parasite haustorium development and growth, and found genes involved in haustorial function. The AHS allowed us to demonstrate the positive effect of light on C. campestris growth in the absence of a photosynthetic host and to investigate carotenoid- and ABA- related processes in the haustorial regions. We also wanted to understand how C. campestris defenses work independently from a plant host, so we studied the parasite responses to the bacterial epitope flg22 and the bacteria Peudomonas syringe. Our findings indicate that C. campestris is able to sense flg22, but its response differs from those observed in other non-parasite plants. Transcriptomic analysis revealed up-regulation of genes related to biotic and abiotic stresses, and downregulation of genes related to cuticle development. Our study contributes to understanding the C. campestris immune response in the absence of a host plant. Taken together, this research contributes novel methodologies that enable insights into C. campestris biology without the interference of a plant host on the parasite.
Differential Response of a Virginia Common Lambsquarters (Chenopodium album) Collection to Glyphosate
Hite, Grace Ann (Virginia Tech, 2008-04-29)
The purpose of this research was to evaluate a common lambsquarters (Chenopodium album) collection from Westmoreland County, Virginia, which exhibited a differential response to glyphosate treatments as compared to most other common lambsquarters. Plants from this site that survived glyphosate applications were collected in both 2002 and 2004. Greenhouse studies were conducted on F1, F2, and F3 progeny from this collection and compared to a wild type collection from Montgomery County, Virginia. Evaluations were conducted on these plants treated with a range of glyphosate rates. F1 progeny of the Westmoreland plants from both 2002 and 2004 collections showed reduced response to glyphosate relative to the Montgomery collection. Vigor reduction of F1 progeny from three 2004 Westmoreland source plants with 0.84 kg ae/ha of glyphosate ranged from 66 to 85% at 28 days after treatment (DAT), compared to 89% for the Montgomery collection. Evaluation of four Westmoreland F2 common lambsquarters lines derived from 2002 collections indicated significant differences in glyphosate sensitivity. Fifteen F2 lines were generated from 2004 collections from each of three Westmoreland source plants and from the Montgomery source. For the least sensitive Westmoreland source, vigor reduction ranged from only 24 to 36% across F2 lines in response to 1.68 kg/ha of glyphosate at 28 DAT, relative to 55 to 100% for the Montgomery source. I50 estimates for fresh weight reduction were 0.91 and 0.32 kg/ha, for these sources, respectively. Sequential treatments of 0.42, 1.26, and 1.68 kg/ha applied at three-week intervals to the least susceptible 2004 Westmoreland F2 line resulted in only 37% vigor reduction and no mortality among 360 treated plants. Growth chamber studies were also conducted on the F2 progeny of these sources to determine if differential growth responses occur in noncompetitive environments and in the absence of glyphosate treatment. Generally, few differences were observed among the Westmoreland and Montgomery collections in growth parameters including height, leaf number, leaf area, leaf size, shoot weight, and reproductive output. However, significant differences were observed with regard to root weight, root length, and root density. In germination studies, it was determined that the Montgomery source had significantly faster and greater seed germination than the Westmoreland source. The susceptibility of F3 seedlings to glyphosate varied significantly with respect to F2 parent line and glyphosate rate. Mortality of 100% was observed in F3 seedlings from the Montgomery source in response to the 3.36 kg ae/ha glyphosate rate, while no mortality was observed in Westmoreland F3 seedlings in response to this glyphosate rate.
Dissection of Regulatory Networks Mediating Resistance and Susceptibility of Arabidopsis thaliana to the Downy Mildew Pathogen Hyaloperonospora parasitica
Hoff, Troy Colston (Virginia Tech, 2008-12-19)
Plants and pathogenic microorganisms are in constant conflict with each other. Understanding the molecular networks that trigger resistance, along with the molecular networks that pathogens might co-opt to infect susceptible plants, is important for developing the integrated, holistic perspective that is necessary for innovative development of engineered resistance to current and emerging pathogens. The first objective of the dissertation was to increase the understanding of mechanisms by which plants recognize pathogen attack and mount an appropriate defense response. These experiments focused on resistance triggered by the Arabidopsis thaliana R gene, RPP7, which encodes a coiled-coil nucleotide binding-leucine-rich repeat (CC-NB-LRR) protein that activates race-specific resistance to the downy mildew pathogen, Hyaloperonospora parasitica (Hpa). Previously-published genetic epistasis tests have established that RPP7 activates defense responses through a signaling mechanism that does not require accumulation of salicylic acid (SA), or components of the ethylene and jasmonate response pathways. Furthermore, RPP7 is not strongly compromised by mutations in genes associated with defense signal transduction (PAD4, NDR1, NPR1, RAR1). Double mutant combinations of these signal transduction components were analyzed to detect additive or functionally-redundant contributions to RPP7-dependent resistance. Most of the double mutants support an enhanced level of asexual sporulation compared to the single mutant parental lines. Time-course experiments with histochemical stains revealed that these double mutants delay, but do not suppress, the oxidative burst and the hypersensitive response. These results suggest that RPP7 activates multiple signaling pathways, each of which makes incremental contributions to the timing of defense activation. The second objective of the dissertation was to investigate the role that auxin plays in enabling virulent H. parasitica to colonize Arabidopsis. Transcript profiling revealed induction of auxin-associated genes in response to infection of Arabidopsis thaliana by virulent strains of the oÃ¶mycete pathogen, H. parasitica. Experiments with the DR5
Egyptian Broomrape (Orobanche aegyptiaca Pers.) and Small Broomrape (Orobanche minor Sm.) Parasitism of Red Clover (Trifolium pratense L.) in Vitro
Morozov, Ivan Vladimirovitch (Virginia Tech, 1998-05-13)
Broomrapes, Orobanche 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. Orobanche 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 Rhizobium, 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 Orobanche spp. attack and Rhizobium nodulation in legumes. Seed germination, number of parasitic attachments, as well as the morphology of two broomrape species, small broomrape (Orobanche minor Sm.) and Egyptian broomrape (Orobanche aegyptiaca Pers.), were studied. O. minor showed a greater percent seed germination, and formed a greater number of attachments on red clover (Trifolium pratense L.) inoculated with Rhizobium leguminosarum bv. trifolii in comparison with non-inoculated plants. However, the addition of the inoculum did not appear to enhance O. aegyptiaca seed germination or the number of its attachments on the host roots compared with the controls. Morphological observations of O. minor 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.
Engineering Resistance to Orobanche aegyptiaca: Evidence of Sarcotoxin IA as an Anti-Parasite Protein and Macromolecule Movement From Host to Parasite
Hamamouch, Noureddine (Virginia Tech, 2004-01-13)
Orobanche 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 Orobanche 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 (Sarcophaga peregrina). 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 O. aegyptiaca. Observations of green fluorescence in O. aegyptiaca 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 (Nicotiana tabacum L. cv. Xanthi) plants expressing the sarcotoxin IA transgene under control of the parasite-inducible HMG2 promoter. In soil experiments, transgenic tobacco plants had greater height and biomass, and showed up to 90% reduction in O. ramosa parasitism as measured by the fresh weight of parasite tubercles. In a semi-hydroponic growth system, where Orobanche tubercles can be visualized at early stages of growth, O. aegyptiaca 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-Orobanche interactions, this research used an antibiotic peptide to engineer partial Orobanche resistance into a highly susceptible crop. This strategy has broad implications for the control of other parasitic weeds.
Epigenetic Responses of Arabidopsis to Abiotic Stress
Laliberte, Suzanne Rae (Virginia Tech, 2023-03-17)
Weed resistance to control measures, particularly herbicides, is a growing problem in agriculture. In the case of herbicides, resistance is sometimes connected to genetic changes that directly affect the target site of the herbicide. Other cases are less straightforward where resistance arises without such a clear-cut mechanism. Understanding the genetic and gene regulatory mechanisms that may lead to the rapid evolution of resistance in weedy species is critical to securing our food supply. To study this phenomenon, we exposed young Arabidopsis plants to sublethal levels of one of four weed management stressors, glyphosate herbicide, trifloxysulfuron herbicide, mechanical clipping, and shading. To evaluate responses to these stressors we collected data on gene expression and regulation via epigenetic modification (methylation) and small RNA (sRNA). For all of the treatments except shade, the stress was limited in duration, and the plants were allowed to recover until flowering, to identify changes that persist to reproduction. At flowering, DNA for methylation bisulfite sequencing, RNA, and sRNA were extracted from newly formed rosette leaf tissue. Analyzing the individual datasets revealed many differential responses when compared to the untreated control for gene expression, methylation, and sRNA expression. All three measures showed increases in differential abundance that were unique to each stressor, with very little overlap between stressors. Herbicide treatments tended to exhibit the largest number of significant differential responses, with glyphosate treatment most often associated with the greatest differences and contributing to overlap. To evaluate how large datasets from methylation, gene expression, and sRNA analyses could be connected and mined to link regulatory information with changes in gene expression, the information from each dataset and for each gene was united in a single large matrix and mined with classification algorithms. Although our models were able to differentiate patterns in a set of simulated data, the raw datasets were too noisy for the models to consistently identify differentially expressed genes. However, by focusing on responses at a local level, we identified several genes with differential expression, differential sRNA, and differential methylation. While further studies will be needed to determine whether these epigenetic changes truly influence gene expression at these sites, the changes detected at the treatment level could prime the plants for future incidents of stress, including herbicides.
Evaluating methiozolin programs for golf putting greens and investigating potential modes of action
Venner, Katelyn (Virginia Tech, 2015-10-06)
Annual bluegrass is a winter annual grass that is problematic on golf putting greens due to its light green color, prolific seedhead production and intolerance to stress. On creeping bentgrass putting greens, herbicides for annual bluegrass control are limited. A new herbicide, methiozolin, developed by Moghu Research Center, LLC, in Daejeon, South Korea, safely and selectively controls annual bluegrass in creeping bentgrass and several other turfgrass species. Methiozolin typically controls annual bluegrass over several weeks, allowing desirable turfgrass time to grow into areas previously infested by annual bluegrass with little surface disruption. The mode of action of methiozolin is unknown, but has been proposed to act as either a cell wall biosynthesis inhibitor (CBI) or an inhibitor of tyrosine aminotransferase (TAT). Field studies were conducted at Virginia Tech to investigate strategies promoting surface recovery on putting greens following atypically rapid annual bluegrass loss resulting from methiozolin application, intensive core-cultivation as well as potential interactions with plant growth regulators (PGR's), like ethephon. In the rapid annual bluegrass removal study, all treatments receiving additional fertility via synthetic fertilizer with or without trinexapac-ethyl or biostimulant recovered 1 to 3 weeks more quickly than treatments that did not include additional fertility. Addition of the PGR trinexapac-ethyl inconsistently regulated speed of canopy recovery, both increasing and decreasing recovery speed. Under normal maintenance conditions, methiozolin does not negatively influence putting green recovery, however, if the putting green is exposed to droughty conditions, methiozolin can reduce recovery time by several weeks. Core-cultivation should be avoided in conjunction with methiozolin and ethephon applications because when this procedure was conducted on the same day as herbicide application it significantly damaged creeping bentgrass, reducing cover to 19% at 2000 g ai ha⁻¹, compared to the non-treated at 62%. Regarding the question of methiozoling mode of action, laboratory studies supported the claim that addition of exogenous 4-hydroxyphenylpyruvate (4-HPP) alleviates symptoms of methiozolin exposure in lesser duckweed, a model monocot species, but feeding various turfgrass species and annual bluegrass exogenous 4-HPP did not alleviate symptoms. Creeping bentgrass secondary root length and density was not affected by methiozolin, although annual bluegrass, Kentucky bluegrass and perennial ryegrass secondary root lengths were reduced. Based on these data, it does not appear that TAT inhibition is a primary mode of action of methiozolin in turfgrass. Studies were conducted to determine if methiozolin inhibited cell wall biosynthesis in desirable turfgrass species and annual bluegrass. All species exhibited decreased enrichment of ¹³C in cell-wall sugars form ¹³C-glucose in response to methiozolin and a known cell wall biosynthesis inhibitor, indaziflam. Indaziflam and methiozolin at 0.01 µM inhibited ¹³C enrichment of all sugars less than methiozolin at 1.0 µM, for xylose, arabinose and glucose, but not galactose. Addition of 4-HPP increased incorporation of ¹³C into xylose, but had no other influence on ¹³C incorporation into other cell wall sugars. Lack of species specific response indicates that cell wall biosynthesis inhibition is probably not the source of interspecific species responses observed in the field.
Evaluation of Seed Impact Mills for Harvest Weed Seed Control in Soybean and Wheat in the Eastern United States
Russell, Eli Carnley (Virginia Tech, 2024-11-11)
Harvest Weed Seed Control (HWSC) concentrates, removes, or destroys weed seeds as they pass through the combine. Seed impact mills are modifications that are mounted directly to the back of a combine and are one way to implement HWSC. Seed impact mills kill weed seeds during harvest, preventing seeds from being added to the soil seedbank. Mills like the Redekop Seed Control Unit (SCU) and the integrated Harrington Seed Destructor (iHSD) could be used in soybean and wheat production in the eastern United States. Understanding the effectiveness and limitations of these mills is important for grower adoption. So, the aim of this research was to evaluate the efficacy of two seed impact mills, the Redekop SCU and the iHSD, in soybean and wheat. The first objective tested general seed kill of problematic species in soybean and wheat and seed kill in adverse conditions, such as high chaff flow rate into the mill and high chaff moisture. Results from objective one indicate that both the Redekop SCU and iHSD killed >98% and >91% of problematic weed seeds in soybean and wheat, respectively. Increases in chaff flow rate and chaff moisture resulted in a decrease in seed kill for specific species depending on the mill. But even at high chaff flow rates, seed kill remained >98% and >77% in soybean and wheat, respectively. At high chaff moisture, seed kill remained >98% and >74% in soybean and wheat, respectively. The second objective evaluated the percentage of weed seeds that bypassed the seed impact mill by exiting the combine in the straw fraction and the percentage of weed seeds that were killed when they entered the seed impact mill during harvest with a commercial combine. Results at field scale indicated that <5% of weed seeds bypassed the seed impact mill by exiting the combine in the straw fraction during harvest in soybean and wheat. Additionally, during a commercial harvest, the seed impact mills killed >99% and >89% of seeds in soybean and wheat, respectively. The third objective monitored population density changes for common ragweed (Ambrosia artemisiifolia) in soybean and Italian ryegrass (Lolium perenne ssp. multiflorum) in wheat following a harvest with a seed impact mill. Results from objective three indicated that in the growing season following a harvest with a seed impact mill, common ragweed density was reduced by 26% and 77% in the spring and fall, respectively, in soybean, and Italian ryegrass density was reduced by 48% in wheat. The fourth objective evaluated Palmer amaranth (Amaranthus palmeri) and its ability to shift its flowering timing in response to HWSC. If weeds flower earlier, they could shatter seeds earlier, and those seeds would bypass HWSC. Through selective breeding, two populations of Palmer amaranth experienced a shift in flowering timing such that the third generations flowered 54.7 and 41.0 days sooner in the greenhouse than the initial generations. In a common garden experiment, the second generations flowered 5.5 and 8.9 days sooner than the initial generations. These results indicate that seed impact mills, like the Redekop SCU and iHSD, can deliver high seed kill rates to a range of weed species at commercial scale in both soybean and wheat. Even in adverse conditions, the mills still killed >74% of seed from tested species. However, weed species can adapt to HWSC selection pressures, resulting in a loss of HWSC efficacy. Overall, this research indicates that seed impact mills are a good tool that growers can implement to reduce the number of weed seeds being returned to the soil seedbank.
Evolution of a horizontally acquired legume gene, albumin 1, in the parasitic plant Phelipanche aegyptiaca and related species
Zhang, Yeting; Fernández-Aparicio, Mónica; Wafula, Eric K.; Das, Malay; Jiao, Yuannian; Wickett, Norman J.; Honaas, Loren A.; Ralph, Paula E.; Wojciechowski, Martin F.; Timko, Michael P.; Yoder, John I.; Westwood, James H.; dePamphilis, Claude W. (2013-02-20)
Background Parasitic plants, represented by several thousand species of angiosperms, use modified structures known as haustoria to tap into photosynthetic host plants and extract nutrients and water. As a result of their direct plant-plant connections with their host plant, parasitic plants have special opportunities for horizontal gene transfer, the nonsexual transmission of genetic material across species boundaries. There is increasing evidence that parasitic plants have served as recipients and donors of horizontal gene transfer (HGT), but the long-term impacts of eukaryotic HGT in parasitic plants are largely unknown. Results Here we show that a gene encoding albumin 1 KNOTTIN-like protein, closely related to the albumin 1 genes only known from papilionoid legumes, where they serve dual roles as food storage and insect toxin, was found in Phelipanche aegyptiaca and related parasitic species of family Orobanchaceae, and was likely acquired by a Phelipanche ancestor via HGT from a legume host based on phylogenetic analyses. The KNOTTINs are well known for their unique “disulfide through disulfide knot” structure and have been extensively studied in various contexts, including drug design. Genomic sequences from nine related parasite species were obtained, and 3D protein structure simulation tests and evolutionary constraint analyses were performed. The parasite gene we identified here retains the intron structure, six highly conserved cysteine residues necessary to form a KNOTTIN protein, and displays levels of purifying selection like those seen in legumes. The albumin 1 xenogene has evolved through >150 speciation events over ca. 16 million years, forming a small family of differentially expressed genes that may confer novel functions in the parasites. Moreover, further data show that a distantly related parasitic plant, Cuscuta, obtained two copies of albumin 1 KNOTTIN-like genes from legumes through a separate HGT event, suggesting that legume KNOTTIN structures have been repeatedly co-opted by parasitic plants. Conclusions The HGT-derived albumins in Phelipanche represent a novel example of how plants can acquire genes from other plants via HGT that then go on to duplicate, evolve, and retain the specialized features required to perform a unique host-derived function.
Exploring the genetic basis of germination specificity in the parasitic plants Orobanche cernua and O. cumana
Larose, Hailey Lee Ann (Virginia Tech, 2018-04-17)
Seeds of the root parasitic plants of the genus Orobanche germinate specifically in response to host-derived germination signals, which enables parasites to detect and attack preferred hosts. The best characterized class of germination stimulants is the strigolactones (SLs), although some species respond to non-SL compounds, such as dehydrocostus lactone (DCL). Recent work indicates that SLs are perceived by members of the KARRIKIN-INSENSITIVE2 (KAI2) gene family, and suggests that within parasitic Orobanchaceae the KAI2 genes have undergone duplication and specialization. The "diverged" clade of these genes, termed KAI2d, has been shown to bind SL germination stimulants in model system assays, but the precise role for KAI2d in regulating germination specificity in a parasitic plant has not been demonstrated. To address this issue, we used genetic and genomic approaches involving two closely related species, Orobanche cernua and O. cumana, which differ primarily in host range and stimulant preference. Orobanche cernua parasitizes tomato (and other Solanaceous crops) and responds to orobanchol, the major SL from tomato roots, whereas O. cumana specifically parasitizes sunflower and responds to DCL. Crosses between O. cernua and O. cumana produced hybrid populations that segregate for stimulant specificity, creating a tractable genetic system. Orobanche cernua contains four KAI2d genes (numbered OrceKAI2d1-4), while O. cumana contains six genes (OrcuKAI2d1-6). The DNA from 94 F2 hybrids was genotyped to identify the KAI2d gene composition and these were correlated with germination phenotype. The pattern of segregation indicated that the KAI2d genes are linked, but pointed to OrceKAI2d2 as a likely orobanchol receptor. Response to DCL was associated with inheritance of all O. cumana KAI2d genes together. Each KAI2d gene was expressed in the Arabidopsis thaliana kai2 mutant background and tested for ability to recover the mutant phenotype when exposed to SLs (including orobanchol, 5-deoxystrigol and GR24) or DCL. One O. cernua gene, OrceKAI2d2, responded to all SLs, but not DCL in this system. No DCL-specific KAI2 genes were identified. In summary, we have identified the likely SL receptor in O. cernua, and show evidence that the DCL receptor is either not a KAI2d protein, or uses KAI2d in combination with other signaling pathway components.
Flowering Gene Homologs Regulate Seasonal Growth Changes in Poplar
Sheng, Xiaoyan (Virginia Tech, 2018-10-19)
The adaptation of trees to temperate and boreal climates depends on their ability to respond to environmental signals that are markers of seasonal changes in order to survive winter and maximize growth. The genus Populus (poplars) is a model system for identifying the genes and molecular mechanisms that regulate growth and dormancy transitions. Photoperiod and temperature regulate both vegetative and floral phenology. FLOWERING LOCUS T (FT) and FLOWERING LOCUS D (FD) are key regulators of flowering time in Arabidopsis and other plants. The distinct functions of three poplar FD-LIKE (FDL) genes and two FTs were explored through gain-of-function, dominant repression, and CRISPR/CAS9-mediated gene editing. We studied trees in controlled environments, including manipulation of daylength and temperature to mimic an annual seasonal growth and dormancy cycle. Our studies showed that the FDL proteins share less than complete functional equivalency. Among the three paralogs, only FDL2.2 promoted precocious flowering, whereas FDL1 and FDL3 appear to have distinct roles in vegetative growth and phenology. Whereas overexpression of any FDL gene delays short day-induced growth cessation and bud set, only FDL3 coordinately altered leaf development and the transition to secondary growth in a photoperiod-dependent manner. For the first time, we demonstrate distinct functions of the two FT paralogs in vegetative phenology. Study of ft1ft2 double mutants and ft1-specific mutants showed that FT1 promotes dormancy release, whereas FT2 is necessary to sustain growth. Collectively, our results reveal that poplar FTs and FDLs have distinct roles in controlling different aspects of vegetative phenology and woody shoot development.
Functional characterization of NAC-domain transcription factors implicated in control of vascular cell differentiation in Arabidopsis and Populus
Grant, Emily H. (Virginia Tech, 2008-12-10)
Wood has a wide variety of uses and is arguably the most important renewable raw material. The composition of xylem cell types in wood determines the utility of different types of wood for distinct commercial applications. Using expression profiling and phylogenetic analysis, we identified many xylem-associated regulatory genes that may control the differentiation of cells involved in wood formation in Arabidopsis and poplar. Prominent among these are NAC-domain transcription factors (NACs). In addition to their roles as regulators of xylem differentiation, NACs are regulators of meristem development, organ elongation and separation. We studied a subset of Populus and Arabidopsis NACs with putative involvement in xylem cell expansion and elongation (XND1/ANAC104, PopNAC118, PopNAC122, PopNAC128, PopNAC129), and secondary cell wall synthesis (ANAC073, PopNAC105, PopNAC154, PopNAC156, PopNAC157). Using quantitative Real-Time PCR, we evaluated expression of the selected Populus NACs in a developmental gradient and in response to bending stress. We prepared transgenic Arabidopsis and Populus plants with increased or decreased expression of select NAC genes. For dominant repression of target gene expression, we evaluated transgenic plants expressing translational fusions of NAC-EAR (ERF amphiphilic repressor) chimeras through chimeric repressor silencing-technology (CRES-T). XND1 overexpression in Populus and Arabidopsis resulted in severe stunting and suppression of xylem differentiation. Overexpression of PopNAC122, an XND1 ortholog, yielded an analogous phenotype in Arabidopsis. Populus XND1 overexpressors lacked phloem fibers and showed a reduction in cell size and number, vessel number and frequency of rays. Knowledge gained through characterization of these wood-associated regulatory genes can be used to optimize molecular breeding and genetic engineering strategies for improved wood quality and increased biomass.

Browsing by Author "Westwood, James H."

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