Browsing by Author "Welbaum, Gregory E."

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    Allyl isothiocyanate reduces Salmonella enterica Michigan and Listeria monocytogenes on the surface of whole cantaloupe (Cucumis melo L.)
    Duckson, Margaret Anne (Virginia Tech, 2014-04-24)
    Since 2006 there have been four Salmonella enterica and one Listeria monocytogenes foodborne outbreaks linked to whole cantaloupe fruit. No post-harvest intervention to reduce potential contamination on cantaloupe currently exists. The complex surface topography of netted cantaloupes aids bacterial attachment. This research evaluates the use of allyl isothiocyanate (AITC; a natural antimicrobial) to reduce populations of S. enterica Michigan and L. monocytogenes on the surface of cantaloupe. Fifty μl of S. Michigan or L. monocytogenes was inoculated onto whole ‗Athena‘ or ‗Hales Best Jumbo‘ (‗HBJ‘) cantaloupe fruit in 22 mm diameter circles and allowed to dry for 90 min. resulting in 6.60 log CFU/g. Cantaloupe received either AITC liquid or vapor, sterile deionized water, 200 ppm sodium hypochlorite per circle, or no treatment. All cantaloupes were stored in separate sealed glass desiccators for 1 or 24 h at 25°C or 35°C. To enumerate the bacteria following treatment, 22 mm sections of the rind were removed, homogenized and plated onto appropriate agar. Headspace analysis using Gas Chromatography-Mass Spectrometry (GC-MS) quantified the concentration of each AITC vapor treatment. The texture quality of the pericarp tissue of whole cantaloupes was evaluated after 24 h treatments, followed by two weeks of storage at 4°C. The concentration of vapor ranged from 3.4 to 19.6 μl AITC/L inside the desiccators. The liquid treatment reduced (P < 0.05) S. Michigan populations on ‗Athena‘ (3 log CFU/g) and L. monocytogenes on ‗HBJ‘ (2.6 log CFU/g). The longer exposure time to the AITC vapor (24 h versus 1 h) resulted in a greater reduction of both S. Michigan and L. monocytogenes on ‗Athena‘ and treatments at 35°C reduced microbial populations up to 4.5 times greater (P < 0.05). The highest vapor concentration reduced (P < 0.05) both pathogens at least 3.0 log CFU/g on ‗Athena‘ at 25°C. Generally, bacterial pathogens from the surface of ‗Athena‘ cantaloupe were reduced more than pathogens inoculated on the surface of ‗HBJ.‘ The application of AITC liquid or vapor is a natural alternative post-harvest treatment to 200 ppm free chlorine to reduce the level of bacterial contamination on cantaloupe surfaces for certified organic production.
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    Amplified fragment length polymorphism (AFLP) analysis of genetic variability in Phalaenopsis
    Chang, Yeun-Kyung (Virginia Tech, 2008-07-23)
    Amplified fragment length polymorphism (AFLP) markers allow a rapid assessment of the level of genetic variation that would be difficult to evaluate using a limited number of morphological markers. AFLP was used to assess the level of genetic variation among 16 different Phalaenopsis species and hybrids. Ten AFLP primer combinations were used for genetic analysis of these Phalaenopsis and 95% of polymorphism in 16 Phalaenopsis species and hybrids was detected. The genetic similarity among Phalaenopsis species and hybrids ranged from 0.298 to 0.774 based on Dice coefficient. The dendrogram derived by UPGMA analysis clustered into two main groups. A significant linear relationship (r² = 0.524, P < 0.0001) was observed between known pedigrees and AFLP-derived genetic similarity for 136 pairwise comparisons of Phalaenopsis species and hybrids. The results indicate that there is an abundance of genetic diversity among within Phalaenopsis and that AFLP can be used to distinguish morphologically similar genotypes. In a second study, the effect of gametophytic selection on genetic diversity in Phalaenopsis was examined by AFLP analysis. Sixteen F1 seedlings resulting from cross-pollination that occurred within high (30 ºC) and low (14 ºC) temperature incubators between two hybrid Phalaenopsis [P. (Taisoco Windian à Sogo Yukidian) by P. hybrid unknown], were subjected to genetic analysis by AFLP. A total of 651 fragments ranging in size from 100 to 350 bp were detected using six primer combinations, of which 387 (59.4%) were polymorphic. Seedlings derived from different temperature treatments exhibited 25.5% to 35.9% polymorphism. The genetic similarity among 16 F1 seedlings ranged from 0.825 to 0.946 based on the Dice coefficient. A dendrogram based on 387 polymorphic markers was derived by UPGMA analysis resulting in three major groups and one subgroup. The dendrogram analysis showed clear clustering in Phalaenopsis hybrids pollinated under different temperature treatments, suggesting that several loci may have been selected during the divergent temperature stress treatments during pollination and early pollen tube growth.
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    Anatomical Developments and the Role of Carbohydrate or Mineral Nutrient Deficiency in Bud Necrosis of 'Riesling' grapevines (Vitis Vinifera L.)
    Vasudevan, Lakshmi (Virginia Tech, 1997-02-26)
    Bud necrosis (BN) is observed as an abortion and death of one or more primordia of the developing compound winter bud. Anatomical developments during the onset of BN in 'Riesling' and 'Chardonnay' grapevines were characterized. Examination of ultrathin (1micro m) sections of 'Riesling' buds under a light microscope revealed a zone of compressed cells immediately beneath the primary bud axis within 60 days after budbreak. Cell rupture occurred in that zone within 90 days after budbreak. Scanning electron microscopy revealed a similar pattern of tissue destruction. Based on the hypothesis that BN was caused by essential substrate deficiency, localized carbohydrate deprivaton was attempted by shading of 'Riesling' grapevines and by shoot tip removal. In one experiment, 92% shade was applied for a three-week period at 20, 40, or 60 days after budbreak in one vineyard and at 40 days after budbreak in another vineyard. In another experiment, 92% shade was applied for a 40-day period at 25 or 65 days after budbreak. Shade reduced photosynthetic photon flux (PPF) in the fruit zone of canopies to <2% of ambient PPF. The first experiment did not increase BN. However, the second experiment increased BN in the distal nodes of the shaded vines compared to the control vines. Shoot vigor, measured as shoot diameter and internode length at season1s end, was positively correlated with BN in shaded as well as unshaded vines. The frequency of necrotic buds was greater at nodes 5 through 16 than at nodes 1 to 4 in both shaded and unshaded vines. Levels of total nonstructural carbohydrates (TNC) measured spectrophotometrically, were not significantly affected by shade treatment. Levels of sucrose, glucose, fructose, and starch in bud, leaf, and stem tissues determined by HPLC, were lower in shaded vines at the point of shade removal than in unshaded vines. Therefore, although three-week periods of shade did not affect BN in 'Riesling', 40-day periods of shade increased BN in distal nodes. Shoot tip removal increased BN at nodes distal to node 12. Bud tissues of shoot-tipped vines had lower levels of sucrose, glucose, fructose, and starch than did the control vines. Carbohydrate analysis of bud, leaf, and stem tissues indicated that 'Riesling' vines (BN-prone) had lower levels of sucrose compared to 'Chardonnay' vines (BN-insensitive). Role of mineral nutrient deprivation was examined in 'Riesling' and 'Chardonnay' buds and the results indicated that BN is unlikely caused by essential nutrient deficiency. 'Chardonnay', the BN-insensitive cultivar had greater levels of starch deposits at 50, 60, 70, and 80 days after budbreak than did the BN-susceptible cultivars, 'Riesling1', Syrah', and 'Viognier'. Starch deposits in grape buds were negatively correlated with BN incidence. From these experiments it can be concluded that a negative correlation between carbohydrate levels of grape buds and BN incidence exists.
  • Application of Fourier Transform Infrared Spectroscopy for Detection of Bacterial Fruit Blotch Disease
    Margenot, Andrew J.; Parikh, Sanjai J.; Zhao, Bingyu Y.; Walcott, Ronald R.; Welbaum, Gregory E. (2017-09-13)
    Bacterial fruit blotch (BFB), caused by Acidovorax citrulli affects the production of cucurbits worldwide, and causes substantial economic losses. Since cucurbit seeds are the most important source of inoculum for BFB outbreaks, seed health testing is an important component of disease management. Currently, there are no nondestructive assays that are sensitive enough to reliably detect A. citrulli-infected seeds. Fourier transform infrared spectroscopy (FTIR) may provide the sensitivity necessary to detect A. citrulli-infected seeds. Attenuated total reflectance (ATR) FTIR was evaluated for the detection of watermelon seeds infected with A. citrulli by pistil inoculation. A. citrulli cells produced a unique signature at a detection limit of approximately 105 cfu/mL. Infected, dry watermelon seeds whose embryos were infected with A. citrulli produced a different spectral profile compared to non-infected seeds. Spectral subtractions between infected and non-infected seeds suggest the potential for indirect detection of A. citrulli by altered ester C-O absorbance bands. Principal component analysis (PCA) of seeds infected with bacterial concentrations ranging from 0.001 – 0.1 OD demonstrated potential for multivariate detection of infected seeds at intermediate contamination levels (0.01 OD) relative to non-infected seeds. This separation was driven by high loading of ester C-O absorbance at frequencies ranging from 1120 - 1000 cm-1, though absorbances identified in pure A. citrulli culture were not observed. These results suggest that FTIR can be used to nondestructively detect seeds infected with moderate levels (105 cfu/mL) of A. citrulli infection.
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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.
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    Cetylpyridinium chloride direct spray treatments reduce Salmonella on cantaloupe rough surfaces
    Saucedo-Alderete, Raúl O.; Eifert, Joseph D.; Boyer, Renee R.; Williams, Robert C.; Welbaum, Gregory E. (2018-08)
    Cetylpyridinium chloride (CPC) solutions (0, 0.5, or 1.0%) were applied to cantaloupe (Athena and Hale's Best Jumbo cultivars) rind plugs, either before or after inoculation with a broth culture of Salmonella Michigan (10(9) CFU/mL) and held at 37 degrees C for 1 or 24 hr. Rind plugs were diluted, shaken, and sonicated, and solutions were enumerated. Texture quality and color were evaluated over 14 days storage at 4 degrees C after 0 and 1% CPC spray applications. A 0.5 or 1.0% (vol/vol) application of CPC after Salmonella reduced the pathogen levels between 2.34 log CFU/mL and 5.16 log CFU/mL in comparison to the control (p<.01). No differences were observed in the firmness and color of 1% CPC treated cantaloupes. Salmonella concentrations on cantaloupes, treated with 1.0% CPC, were lower after 1 hr storage as compared to 24 hr. And, Salmonella on Athena surfaces were more susceptible to CPC spray treatments than on Hale's Best Jumbo. Practical applicationsCetylpyridinium chloride (CPC) is the active ingredient of some antiseptic oral mouth rinses, and has a broad antimicrobial spectrum with a rapid bactericidal effect on gram-positive pathogens. The spray application of CPC solutions to cantaloupe may reduce the level of Salmonella surface contamination during production from irrigation water and manure fertilizers and, during food processing by contaminated equipment and food handlers. Since the surfaces of cantaloupes are highly rough or irregular, bacteria can easily attach to these surfaces and become difficult to remove. Appropriate postharvest washing and sanitizing procedures are needed that can help control Salmonella and other pathogens on melons, especially on cantaloupes with nested surfaces. A direct surface spray application of CPC may be an alternative antimicrobial postharvest treatment to reduce pathogen contamination of cantaloupe melons, while providing an alternative to chlorine-based solutions.
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    Characterization of Chitinase Activity and Gene Expression in Muskmelon Seeds
    Zou, Xiaohong (Virginia Tech, 2000-11-16)
    Chitinase has been suggested to play a role in defense mechanisms. In this study, the activity and expression of chitinase in muskmelon seeds were investigated. Multiple chitinase isoforms were detected in muskmelon seeds from early development through radicle emergence. One acidic and three basic chitinase isoforms were detected in developing seeds at 40 days after anthesis (DAA). Both acidic and basic chitinase isoforms were detected in endosperm tissue during seed imbibition and after radicle emergence. Basic chitinase isoforms, but not acidic isoforms, were detected in embryo tissue. Basic chitinase isoforms were also detected in the embryonic axis or radicle tissue. Taken together, these observations indicate that chitinases are regulated developmentally and in a tissue-specific manner in muskmelon seeds. Therefore the potential function of chitinases in muskmelon seeds is discussed. Two complete cDNAs, Cmchi1 and Cmchi2, and a partial genomic clone of Cmchi2 have been isolated from muskmelon seeds. Cmchi2 gene has two introns in the coding region while Cmchi1 is intronless. Cmchi1 cDNA encodes a class III chitinase while Cmchi2 cDNA encodes a class II chitinase. Cmchi1 and Cmchi2 proteins might be targeted to secretory pathways because they possess signal peptides. Southern blotting suggested that there is at least one additional gene similar to Cmchi1 in the muskmelon seed genome, while there is only one copy of Cmchi2. Northern blotting analysis showed that both Cmchi1 and Cmchi2 are expressed in the radicle tissue at the time of radicle emergence. This indicates that the expression is regulated developmentally and in a tissue-specific manner. Salicylic acid (SA) and benzothiadiazole (BTH) stimulated the expression of Cmchi1 but not Cmchi2 in seeds after radicle emergence, indicating that SA might be involved in inducing the expression of Cmchi1, while a different signal might be involved in triggering the expression of Cmchi2. The protein encoded by Cmchi1cDNA was expressed in E.coli. It did not show any enzymatic activity. Western blotting using an antibody raised against the class III chitinase protein in cucumber was inconclusive, as this antibody recognized the purified Cmchi1 fusion protein and other unknown proteins isolated from the embryonic axis or the radicle tissue.
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    Characterization of chitinase activity and gene expression in muskmelon seeds
    Witmer, Xiaohong; Nonogaki, Hiroyuki; Beers, Eric P.; Bradford, Kent J.; Welbaum, Gregory E. (Cambridge University Press, 2003-05)
    Chitinase is often produced in higher plants as a general defence response after wounding or pathogenic attack. Since germinating seeds are exposed to soil pathogens, the activity and expression of chitinase in muskmelon (Cucumis melo L.) seeds was investigated. One acidic and three basic chitinase isoforms were detected, beginning 40 d after anthesis in developing and fully mature seeds. Both acidic and basic chitinase isoforms were found in endosperm tissue during imbibition and after radicle emergence. Basic chitinase isoforms, but not acidic isoforms, were detected in the embryonic axes of imbibed seeds and in seeds before germination, indicating that chitinases are developmentally regulated in specific seed tissues. Two complete cDNAs, Cmchi1 and Cmchi2, were cloned from germinated muskmelon seeds and are predicted to encode chitinases that show 95% identity to a class III chitinase from cucumber (Cucumis sativus L.) and 61% identity to a class II chitinase from soybean (Glycine max L.), respectively. Southern blotting indicated that Cmchi2 was present only once in the muskmelon genome, while Cmchi1 may be present in one or two copies. Cmchi1 and Cmchi2 mRNAs were only detected in radicles of germinating seeds and in roots of mature plants, so additional genes other than Cmchi1 and Cmchi2 must be responsible for the chitinase activity in developing seeds. Salicylic acid and benzothiadiazole stimulated the expression of Cmchi1, but not Cmchi2, after radicle emergence. A putative role for chitinase in muskmelon seeds is defence against fungal pathogens.
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    Characterization of Effector Genes in Acidovorax citrulli the Causing Agent of Bacteria Fruit Blotch Disease of Cucurbits
    Traore, Sy M. (Virginia Tech, 2014-08-08)
    Bacterial fruit blotch (BFB) of cucurbits is caused by Acidovorax citrulli, a Gram-negative seedborne bacterium that can cause up to 100% fruit yield losses in the field. Currently, BFB is a major problem for the cucurbits industry worldwide. Thus far, attempts to identify resistance in cucurbit germplasm for controlling BFB have been unsuccessful. Despite the importance of the disease, little is known about the molecular mechanisms of A. citrulli pathogenicity, due to a lack of molecular tools for studying the A. citrulli/cucurbit interaction. The genomic sequence of A. citrulli strain AAC00-1 has been determined, and the components of type III secretion system have been identified. The goal of this research was to develop molecular tools for studying the BFB disease. Nineteen putative type III effector genes were cloned from two representative A. citrulli strains (AAC00-1 and M6). The distribution of 19 type III effectors among A. citrulli strains, collected worldwide, was studied. A novel Gateway-compatible binary vector was developed for transient expression of A. citrulli type III effectors genes in planta. A set of modified vectors for marker-exchange mutagenesis in A. citrulli were constructed. The model plant species Nicotiana benthamiana was found to be susceptible to A. citrulli, while Nicotiana tabacum was resistance to A. citrulli, so therefore could carry nonhost resistance genes. Two T3S effectors, Aave1548 and Aave2166, triggered water soaking-like cell death in N. benthamiana, but HR-like cell death in N. tabacum. Bacterial mutagenesis and in planta disease assay confirmed that both Aave1548 and Aave2166 have significant virulence contributions to A. citrulli in N. benthamiana plant and melon seeds. Aave2166 encodes a putative acetyltransferase that belongs to the YopJ super family, which is conserved in both animal and plant pathogenic bacteria. Wild type but not the putative catalytic mutant (C232A) of Aave2166 can trigger cell death phenotype in N. benthamiana and N. tabacum. N. benthamiana yeast two-hybrid cDNA library screening using Aave2166 identified six N. benthamiana proteins/peptides which specifically interacted with Aave2166. Further characterization of these Aave2166 interactors may allow us to understand the virulence mechanism provided by Aave2166. The identification of nonhost resistance genes that can recognize Aave2166 and other type III effectors may help to develop novel strategies to control BFB disease of cucurbit.
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    Characterization of two genes, trehalose-6-phosphate synthase/phosphatase and nucleotide binding protein, shown to be differentially regulated in roots of Cypripedium parviflorum var. pubescens grown with a mycorrhizal fungus Thanatephorus pennatus
    Watkinson, Jonathan I. (Virginia Tech, 2002-04-23)
    The analysis of gene changes associated with formation of the mycorrhizal symbiosis between orchid and fungi could have broad implications for plant pathogen interactions. Fungi associated with North American terrestrial orchids were once included in the pathogenic genus Rhizoctonia. This suggests that orchids are able to overcome or utilize normally pathogenic pathways to establish symbioses. A differential display technique was employed to analyze gene changes in orchid in response to a fungus. Samples of RNA from roots of Cypripedium parviflorum var. pubescens (CyPP) grown in the presence or absence of a mycorrhizal fungus; Thanatephorus pennatus, were analyzed using AFLP differential display. Forty-four fragments were selected out of 5000 as being differentially expressed, but only 15 sequences were obtained. Most showed homology to ribosomal genes. Two represented genes believed to be regulated by the mycorrhizal interaction: trehalose-6-phosphate synthase/phosphatase (Tps), which showed down-regulation and nucleotide binding protein (NuBP), which showed up-regulation. The Tps partial clone identifies 2100 bp at the 3' end of the gene and encodes a protein of 667 amino acids. The NuBP gene is approximately 1200bp in length and encodes a protein of 352 amino acids. The Tps gene exists in multiple copies with high expression in roots and low expression in rhizomes and leaves. The NuBP gene exists as a single copy and has a low level of expression in rhizomes and leaves. Expression of Tps is induced by sucrose, but reduced by trehalose. Cultivation of CyPP with non-mycorrhizal fungi did not affect expression of Tps or NuBP. Trehalose induced NuBP expression whereas sucrose did not. A second species of mycorrhizal fungi induced expression of NuBP but reduced expression of Tps. Analysis of Tps expression in Arabidopsis was done using promoter:GUS fusions. The Tps promoter:GUS plants revealed that Tps expression is constitutive in roots. Regulation of Tps driven GUS is expressed throughout seedlings. GUS was not detected in leaves of older plants but was detected in anthers and stigmatic surfaces of flowers. Expression of GUS driven by Tps showed a strong wound response and was present in the junction between siliques and pedicels.
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    Characterization of water stress during cold storage and establishment for Acer platanoides and Crataegus phaenopyrum
    Bates, Ricky Martin (Virginia Tech, 1994-08-01)
    This study examined the affects of desiccation during and after cold storage on the physiology, growth, and marketability of bare-root Acer platanoides (Norway maple), Crataegus phaenopyrum (Washington hawthorn) and Prunus x yedoensis (Yoshino cherry). Histological examination of Acer and Crataegus stems was also conducted. Maple and cherry trees were transplanted into pine bark-filled containers and subjected to mist or non-mist treatments. Xylem water potential increased (became less negative) for misted maple and cherry trees. Water potential increased for non-misted maple and decreased for non-misted cherry trees. Maple and hawthorn seedlings were subjected to cold storage durations of 2, 4, 6, 8, 10, and 12 weeks and storage treatments: whole plant covered, shoots exposed, roots exposed and whole plant exposed. Shoot (Ψs) and root (Ψr) water potentials for all treatments and both species decreased during storage. For maple, (Ψs) and (Ψr) of the exposed shoot treatment were the same as the whole plant covered treatment. In contrast, hawthorn (Ψs) and (Ψr) of the exposed shoot treatment were lower (more negative) than for the whole plant covered treatment. Root hydraulic conductivity was the same for both species and decreased with increased storage duration and for treatments with exposed roots. For the root covered treatments, maple root growth potential (RGP) increased while hawthorn RGP decreased with increased cold storage duration. RGP for both species remained low throughout storage for treatments exposing roots. Days to bud break for Acer and Crataegus seedlings decreased with increased storage time for the whole plant covered treatments but increased for both species when stored with exposed roots. Maple marketability, percent of trees with ≤ 10% shoot dieback, for root covered treatments was high for most storage durations. Hawthorn marketability was generally low except for the whole plant covered treatment during the first six weeks of storage. There was a high positive correlation between RGP and marketability for both maple and hawthorn. Histological examination revealed that Acer stems had a highly suberized periderm, and a uniform cuticle with few disruptions. Periderm suberization of Crataegus stems was variable and extensive peridermal cracking was evident. Cuticle wax decreased with increasing distance from the stem apex for both species. Collectively, results indicated that hawthorn stems had more pathways for water loss than maple shoots. While protection of roots of all bare-root stock is important, desiccation sensitive species such as Washington hawthorn require both root and shoot protection during storage and at transplanting to minimize water loss.
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    Climate Change and Foodborne Illness: Implications for Policy
    Hutchins, Franya (Virginia Tech, 2014-12)
    Research report, lecture notes, and course outline for a freshman-level “First Year Seminar” on the topic of climate change and foodborne illness, intended to contribute to the satisfaction of the General Education requirements at Appalachian State University.
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    Cultivation and Nutritional Constituents of Virginia Grown Edamame
    Carson, Luther C. (Virginia Tech, 2010-04-28)
    Edamame's (Glycine max L. Merrill) consumption in the US has also been growing due to purported health benefits. Edamame grows well around the US, but few have measured the growth and yield in the mid-Atlantic region. The objective of these studies were to determine the potential yield of edamame, determine how yield components change with planting date and cultivar, and to measure total protein, lipids, antioxidant activity and isoflavone concentrations at harvest.. The five cultivars (BeSweet 292, BeSweet 2015, BeSweet 2001, Midori Giant and Sunrise) used in the cultivar evaluation trial and for nutritional constituents analysis were grown in Painter, Virginia in 2008 and 2009. The cultivar evaluation trial yielded between 5,600 and 8,400 kg per ha. Percent marketable pods ranged from 74.3 and 85.6% in the cultivar evaluation trial. There were significant differences in average seed size across cultivars in both years. Cultivar lipid content followed the same patterns in both years with 2009 having lower overall concentrations than 2008. Protein contents were similar in 2008 and 2009. Both years, "BeSweet 2015" and "BeSweet 2001" had high radical scavenging ability and Midori Giant had the lowest. In 2008, there were no significant differences in the ORAC assay. "BeSweet 292" had significantly more reducing activity in the DPPH assay than Sunrise in 2009. Isoflavones were measured in 2008 and 2009 at harvest and temporally in 2009. Of all isoflavones, Malonyl genistin had the highest concentration. Edamame is a suitable crop for cultivation in Virginia, and is high in nutritional quality.
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    Delmopinol hydrochloride reduces Salmonella on cantaloupe surfaces
    Saucedo-Alderete, Raúl O.; Eifert, Joseph D.; Boyer, Renee R.; Williams, Robert C.; Welbaum, Gregory E. (Wiley, 2017-11-15)
    Since the surfaces of cantaloupes are highly rough or irregular, bacteria can easily attach and become difficult to remove. Appropriate postharvest washing and sanitizing procedures can help control Salmonella and other pathogens on cantaloupe or other melons during postharvest operations. Delmopinol hydrochloride (delmopinol) is a cationic surfactant that is effective for treating and preventing gingivitis and periodontitis. The application of delmopinol to two cantaloupe cultivars was evaluated for reducing the level of inoculated Salmonella. Athena and Hale’s Best Jumbo (HBJ) cantaloupe rind plugs (2.5 cm. dia.) were inoculated with nalidixic acid-resistant Salmonella Michigan (approx. 1.0 × 109 CFU/ml). After 15 min, rind plugs were sprayed with 10 ml of a delmopinol spray solution (0% or 1.0% vol/vol) and held at 35°C for 1 hr or 24 hr. Rind plugs were diluted with Butterfield’s phosphate buffer, shaken and sonicated, and solutions were enumerated on 50 ppm nalidixic acid-tryptic soy agar. The texture quality and color of additional cantaloupes were evaluated, after 1% delmopinol spray treatment, over 14-day storage at 4°C. A 1.0% application of delmopinol after 1 hr reduced Salmonella concentration by ~3.1 log CFU/ml for both “HBJ” skin rind plugs and “Athena” stem scar rind plugs in comparison to the control (p < .05). No differences were observed in the texture and color (L*, a*, b* values) of 1% delmopinol-treated cantaloupes as compared to control. Storage of cantaloupes treated with 1.0% delmopinol solution for 1 hr had a greater effect on reducing concentration of Salmonella compared to 24-hr treatment. A surface spray application of 1% delmopinol on cantaloupes could be an alternative antimicrobial postharvest treatment that could make surface bacteria more susceptible to sanitizers or physical removal.
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    Detection of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch Disease of Cucurbits, Prevention via Seed Treatments and Disease Resistance Genes
    Kiremit, Merve (Virginia Tech, 2021-04-02)
    Melon (Cucumis melo L.) and watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai) belong to the family Cucurbitaceae. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies are very limited and include: seed production field sanitation, greenhouse transplant sanitation, copper-based bactericide sprays, crop rotation, disease-free healthy seeds, isolating diseased plants, and peroxyacetic acid seed treatments. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. No nondestructive assays are commercially available to detect BFB in seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate NB-LRR genes for disease resistance, and optimization of virus induced gene silencing for melon and watermelon crops. The potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds were evaluated. It was possible to detect BFB in seeds that were pistil inoculated via x-ray imaging and pericarp inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea (Camellia sinensis) and tea polyphenols as seed treatments to sanitize seeds infected with A. citrulli. Green tea unlike black tea inhibited growth of A. citrulli because of polyphenols. Eighty one melon and forty four watermelon NB-LRR genes were reidentified, and genes that have potential resistance against A. citrulli on melon plants were screened based on host selectivity of the pathogen. Finally, the virus-induced, gene-silencing method was optimized for melon and watermelon for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate R genes were identified that might confer stable resistance in the right genetic background.
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    Development of a Greenhouse Tobacco Seedling Performance Index
    Clarke, Jodie Johnson (Virginia Tech, 2001-05-02)
    Tobacco seed performance is traditionally measured as percentage germination at 14 d under controlled laboratory conditions. However, under greenhouse conditions, seed lots with equal 14-d germination may exhibit substantial differences in uniformity of early seedling growth and spiral root incidence that impact the number of usable transplants. A seedling performance index (SPI) was developed to quantitatively describe greenhouse tobacco seedling performance. The 14-d emergence, relative leaf area uniformity, and seedling leaf area determined by computer image analysis were used to calculate the index. Greenhouse tobacco seed trials demonstrated that seed with the Rickard pellet had higher emergence, but the higher spiral root incidence associated with the Rickard pellet lowered the SPI compared to the Cross Creek pellet. Primed seed lots of flue-cured cultivars (NC 72 and NC 71) had a significantly higher SPI than the nonprimed seed lots at one location but not at a second location. Seed lots sown in Premier Pro-Mix TA commercial medium had a higher spiral root incidence, which resulted in a lower SPI compared to Carolina Choice, Carolina Gold, and Sunshine LP5 commercial media. The index quantitatively determined differences in seedling performance under greenhouse conditions not reflected by standard germination tests. Significant differences in the SPI were observed among seed lots with certified 90% germination. The SPI is a simple method to describe seedling performance because the data used to calculate the SPI is obtained from one seed tray image. In contrast, frequent counting and seedling evaluations are involved with standard germination and vigor tests.
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    Development of intermonoploid somatic hybrids of potato and their molecular analysis based on polymorphism for retroelement Tst1
    Lightbourn, Gordon James (Virginia Tech, 2004-08-12)
    Inbred lines for hybrid crop production have been a mainstay of plant breeding. Biotechnological approaches to hasten the process are available including anther culture to halve the genome and protoplast fusion to create hybrids between incompatible partners. We applied these techniques to potato to evaluate their potential for breeding highly heterozygous, cross-pollinating species. Four families of monoploids (2n=1x=12), developed from diploid hybrids with diverse genomic constitutions but heavily favoring Solanum phureja, a primitive cultivated potato, were used in electrofusion experiments to create intermonoploid somatic hybrids (SH). The "monoploid sieve" results in the survival of only those gametes free of lethal and deleterious genes but generates sterile sporophytes, necessitating protoplast fusion for SH development. From six intermonoploid electrofusion combinations, 276 plants were regenerated over 6-9 months. Fusion conditions were optimized. Ploidy was determined by flow-cytometry and SH confirmed by microsatellite analysis. Field evaluations over three years revealed that intermonoploid SH were inferior to cultivars. Dihaploids derived by anther culture of a tetraploid intermonoploid SH were reduced in vigor with an increase in homozygosity, while 2x X 2x sexually derived populations had better yield than the SH, suggesting that producing SH introduced or eliminated factors required for productivity. Molecular analysis of the SH was conducted to examine genomic stability through protoplast isolation and plant regeneration. Sequence specific amplified polymorphism (S-SAP) represents a hybrid system incorporating amplified fragment length polymorphism (AFLP) technology in conjunction with the use of a defined genomic sequence, e.g., retrotransposon display (RD) when the defined sequence is anchored into a consensus sequence of a retrotransposon such as the long terminal repeat (LTR) sequence of Tst1. Parental monoploids, SH and various Solanaceae were evaluated by RD. Fluorescently-labeled retrotransposon-based primers were used in the ALFexpress automated fragment analyzer system. Eleven probes from RD were created for Southern blot analysis and used to verify taxonomic relationships between selected Solanaceae. Blots of intermonoploid somatic hybrids confirmed hybridity and occasional loss of genomic fragments. No activation or replication of retrotransposons was detected. Sequencing of inter-retrotransposon amplified polymorphism (IRAP) and S-SAP fragments revealed that all fragments had the expected Tst1 retroelement and/or the AFLP adaptor sequence. BLAST analysis identified 4 of the 17 fragments sequenced as part of the chloroplast genome, a tobacco anther-specific gene, repetitive DNA, and the phytochrome F gene.
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    Discovery of New Protein-DNA and Protein-Protein Interactions Associated With Wood Development in Populus trichocarpa
    Petzold, Herman E. III (Virginia Tech, 2017-11-09)
    The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield.
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    Does Position in Cantaloupe (Cucumis melo L.) Fruit Affect Seed Quality?
    Mueller, Kale E.; Welbaum, Gregory E.; Samtani, Jayesh B.; Lavis, Cathie (Virginia Tech, 2021-05-17)
    Effects of the location of seed development inside cantaloupe (Cucumis melo L.) fruit on seed germination percentage and vigor was compared for two Western Shipper cantaloupe cultivars (‘Ropey King’ and ‘Expedition’). Mean time to germination (MTG), as (NiTi )/ (Ni) where Ni is the number of newly germinated seeds at time Ti after imbibition was calculated as a measure of seed vigor. Fruit was grown in Woodland, CA, in a randomized complete block design, consisting of 4 blocks (i.e., replicates). Melons were harvested at the full slip stage of maturity and were measured and sliced into six equal sections (blossom end top, blossom end bottom, middle top, middle bottom, stem end top, and stem end bottom). Harvested seed was equally divided for germination testing without drying immediately after harvest (no seed storage, NSS), and after 6 months of seed storage (6MSS) of dried seeds. 6MSS was stored in a cold temperature-controlled (3.33°C, 35% relative humidity) refrigerator in sealed containers for six months, so moisture content did not fluctuate. Both cultivars showed improved germination percentage after 6MSS. Moreover, the 6MSS of both cultivars resulted in significantly higher (P ≤ 0.05) germination percentages in the stem end and middle sections of both cultivars. In contrast, MTG increased after 6MSS compared to NSS possibly because of differences in hydration. However, after 6MSS seeds from the stem end and middle fruit sections of both cultivars germinated faster (P ≤ 0.05) compared to seed from the blossom end indicating the most vigorous seeds developed in those sections.