Progress 08/06/15 to 08/05/20
Outputs
Target Audience:The project has had multiple target audiences. The pirmary audience is the scientific community. For some portions of the project, the target audience was high school students; these students were trained in natural product extraction and evaluation, and presented research results at regional and international competitions. Undergraduate research community was also a target audience. Students presented research results at university wide competitions. Graduate students are a primary target audience, primarily in the final years of the project. Changes/Problems:The research on the elucidation of the role of saponin in resistance of quinoa to Cucumber mosaic virus (CMV) was a major change to this project. When the project was initiated, the technology for the differential gene expression in time-series study of CMV infection was unavailable. Quinoa was chosen because agronomic varieties were available. The epazote used in this study was from natural seed collections and did not support replication of the virus. High-throughput transcriptome sequencing allowed us to compare the differential reaction to the virus in three varieties of quinoa that differed in saponin content. This research provided the first report of integrated RNASeq and sRNASeq data in quinoa-virus interactions and increased knowledge of terpene metabolism in the Family Chenopodiacae in both stress and nonstress conditions. What opportunities for training and professional development has the project provided?This project has served as training for two graduate students, three undergraduate students, and four high school students. Work on this project has proven to be career changing for some students in the early stages of their careers. Four high school students participated in the regional science fair (Southern Appalachian Science and Engineering Fairs); two won the grand prize and competed at the International Science and Engineering Fair. All of these former high school students, except one, have now completed baccalaureate degrees and are in graduate school. Career choices by at least one student were shaped by this research experience. The three undergraduates have or are pursuing degrees in food, agricultural, natural resources, and human sciences. One student (who worked in the program in both high school and undergraduate) has graduated with an Honors degree (4-year program) in Food Science (B.S) and is pursuing a MS degree in that discipline. This program provided her with experience in FANHS, and she made her career decisions based on this early research experience. Two students completed the two-year university-wide honors programs; the research experience provided by this program was central to that program. One student changed his major to plant science with a minor in Entomology and Plant Pathology as a result of the research project. He plans to continue his graduate education in FANHS, focusing on bioinformatics. The graduate student presented research results at three conferences (one international, one national, and one regional), and developed bioinformatics pipelines for terpene synthesis in the Chenopodiaceae. He is working as a postdoctoral associate in Plant Pathology at another university. A second graduate student included the extracts in a separate project on natural products as insecticides. How have the results been disseminated to communities of interest?Dissemination of the results has primarily been through scientific publications and presentations. Scientific community - Six presentations at national and international meetings and two refereed journal articles have resulted from this research. High School Students - Foundational training was provided to three high school students who disseminated their research at several conferences. The students gave two presentations at local high school fairs, and one poster was presented at both the Southern Appalachian Science and Engineering Fair (Grand Champion) and the International Science and Engineering Fair (4th place Plant Science). Undergraduate Research Community - Three undergraduate students have worked in this program. Two students presented at the university-wide Honors research conference, and one was recognized as the top 1794 Scholars Research Fellow. Graduate Students - The graduate student participated in annual meetings of the American Phytopathological Society (APS) and the 2018 International Congress of Plant Pathology. He was active in the APS Graduate Student Committee. The results of the bioassay for activity against fall armyworm were presented at the Tennessee Entomological Society. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. Bioassays. Activity against bacterial and fungi. Rapid bioassays for the evaluation of biopesticidal activities in complex extracts have been developed. Suspended propagules (bacterial cells or fungal spores) are mixed with extracts from plants in the Family Chenopodiaceae (chenopods) in microplates; these are incubated at appropriate growth temperature, and absorbance is monitored at least once every 30 minutes for 24-48 h, depending upon organism. After development for this project, this bioassay has contributed to rapid identification of activity in other systems (e.g., Sciadopitys resins and ant gland secretions), and can be used by others to evaluate relative biopesticidal activity. Activity against nematodes. Bioassays to evaluate antihelmintic activity were developed for this project. Juveniles of the model nematode, Caenorhabditis elegans or the plant parasitic nematode, Meloidogyne incognita, were exposed to extracts, and viability was monitored microscopically. Viability was defined as mobility within a 5 second screen. Nonmobile juveniles were probed, and if the organism still did not move after a second 5 seconds, they were considered immobile. Hatch of plant parasitic nematode eggs was also monitored. Technical knowledge derived from development of these bioassays will allow the scientific community to rapidly evaluate natural products for nematicidal activity. Bioassay development and utilization (Objective 3) served as training exercises for high school and undergraduate students. Objective 2. Chemical composition. A combination of FTIR and PCA provided a fast and reliable method for determining chemical differences among members of the Family Chenopodiaceae. Despite the fact that Dysphania ambrosioides (epazote) extract had no activity in the foaming test, the FTIR spectra had several peaks characteristic of saponins. Extracts from Chenopodium berlandieri that were analyzed extensively exhibited all described peaks except for the peak indicating the carbonyl group of an oleanane-type triterpenoid. Approximately 75% of spectral variation was explained by two principal components. There was no relationship between Principal Component 1 (PC1) and saponin content or biological activity. Blitum bonus-henricus (GKH) is the only extract that was uniformly negative for PC2, and neutral for PC1. Water extracts from D. amrosioides contained little saponin; the foaming index was less than 100, and the extract did not lyze red blood cells. Domestication reduced the number of triterpenes in the extracts. When epazote purchased from a local market (sold for use as an herb) was compared to a variety grown in our greenhouse from commercial native seed, the extract from the herb had fewer detected triterpenes, and these differences were correlated with biological activity. In some tests, estimation of total phenols aligned with biological activity. The methods described allow for the rapid characterization of the extracts. Differences among chenopods in some but not all methods explain the differences in biological activity. Methods developed for this portion of the project can be used to characterize other potential biopesticide sources (e.g. our work on Sciadopitys). Chemical characterization served as training for high school and undergraduate students. Results from these experiments were included in regional, national, and international presentations. Thepresentation by the high school students at the International Science and Engineering Fair was a combination of the FTIR and Caenorhabditis elegans bioassay. Objective 3.Biological activity. Water extracts of D. ambrosioides were active against bacteria and nematodes. In experiments, water extracts from other chenopds with high saponin activity were used as a comparison. Biological activity of epazote extracts against Bacillus cereus was greater for extracts from the domesticated variety of the herb than from plants cultivated from seed in the greenhouse. The loss of activity with domestication was also true for C. berlandieri, a close relative of epazote. In experiments with C. elegans and various chenopods, loss of viability was highest in GKH and epazote. Saponin was 17x greater in GKH than in epazote. When populations of C. elegans were exposed to epazote extracts, viability of juveniles was dose dependent. Since epazote had a low concentration of saponins, it is unlikely that these compounds are the source of activity against C. elegans. However, a synergistic effect with ascaridole should be considered. The activity of GKH extracts may be attributed to saponins. Effect of a saponin standard, Quilla bark saponin, was similar to the effect of GKH on M. incognita juvenile survival and egg hatch. Epazote extracts did not have a significant effect on egg hatch, but did reduce viability of M. incognita juveniles in a dose dependent manner. Objective 4. In planta activity. Low saponin (sweet) varieties of quinoa (Chenopodium quinoa) have been developed because seeds high in saponins taste bitter. The aim of this study was to elucidate the role of saponin in resistance of quinoa to Cucumber mosaic virus (CMV). Differential gene expression was studied in time-series study of CMV infection. High-throughput transcriptome sequence data were obtained from 36 samples (3 varieties × +/- CMV × 1 or 4 days after inoculation × 3 replicates). Translation, lipid, nitrogen, amino acid metabolism, and mono- and sesquiterpenoid biosynthesis genes were upregulated in CMV infections. In 'Red Head' (bitter), CMV-induced systemic symptoms were concurrent with downregulation of a key saponin biosynthesis gene, TSARL1, four days after inoculation. In local lesion responses (sweet and semi-sweet), TSARL1 levels remained up-regulated. Known microRNAs (miRNA) (81) from 11 miR families and 876 predicted novel miRNAs were identified. Differentially expressed miRNA and short interfering RNA clusters (24nt) induced by CMV infection are predicted to target genomic and intergenic regions enriched in repetitive elements. This is the first report of integrated RNASeq and sRNASeq data in quinoa-virus interactions and provides comprehensive understanding of involved genes, non-coding regions, and biological pathways in virus resistance. Water-based extracts from chenopods had no impact on the germination of seeds of greenhouse crops and so can be used as seed or soil treatments. Efficacy of chenopod extracts including epazote as a preventative fruit wash against Botrytis cinerea were tested. Although the results for these tests were inconclusive, in the process of developing the assay, undergraduate students developed a new technique for inducing sporulation in isolates that have stopped sporulating in culture. Taken together, the tests performed on these extracts have formed the basis for the continued development of these water extracts as biopesticides. Protection against losses in crop production due to diseases, while reducing negative impacts on consumers, remains a key issue in ensuring a global safe food supply. Sustainability can be incorporated into crop production by expanded use of biopesticides for plant protection. The research sponsored in this project has led to the development of potential biopesticide(s) and the identification of new sources of activity. This portion of the research is continuing and is currently being evaluated for control of greenhouse plant diseases.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Solatani, N. 2019. Genome-enabled analysis of Quercus rubraozone and Chenopodium quinoa-Cucumber mosaic virus interactions. PhD Dissertation, University of Tennesseee, Knoxville
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
Soltani, N., M. Staton, K.D. Gwinn. Integration of transcriptome and small RNA sequencing to decipher molecular interaction of Chenopodium quinoa varieties with cucumber mosaic virus. PLOS One
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Integration of Transcriptome and Small RNA Sequencing to Decipher Molecular Interaction of Chenopodium quinoa Varieties with Cucumber Mosaic Virus. Nourolah Soltani, Margaret Staton, Kimberly D. Gwinn. BioRXiv doi: https://doi.org/10.1101/2020.07.20.212795
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The primary audiences for this research are the scientific community and students. Students are involved in generating the information needed by the scientific community. The ultimate audience for this research will be those producers and growers who wish to use biopesticides. Changes/Problems:Chenopodium quinoa, aclose relative of Dysphania, was added to this project in order toinvestigateadditional chemicalsimportant in host-pathogen interactions; epazote was not susceptible to the virus. Quinoa was chosen because of its high value of the crop and the availability of varieties that differ in saponin levels. What opportunities for training and professional development has the project provided?The research was the basis for a PhD dissertation. How have the results been disseminated to communities of interest?Results of experiments were presented at the American Phytopathological Society, Southern Division and at the International Congress for Plant Pathology. What do you plan to do during the next reporting period to accomplish the goals?Complete research on the objectives. Soltani, N. 2019. Genome-enabled analysis of Quercus rubra-ozone and Chenopodium quinoa-Cucumber mosaic virus interactions. PhD Dissertation. University of Tennessee, Knoxville will be completed in December 2019. Publication of the genome-enabled analysis of the quinoa-CMV interaction.
Impacts
What was accomplished under these goals?
Quinoa, aclose relative of epazote, was added to this project in order to investigateadditional chemicalsimportant in host-pathogen interactions;epazote was not susceptible to the virus. Quinoa isa pseudocereal crop with high nutritional value that has increased in popularity. Quinoa seeds high in saponin taste bitter. Sweet (low saponin) varieties are highly desired because saponin removal increases costs and reduces nutrition. In many plants, saponins are active against plant pathogens, including viruses, and contribute to host resistance. In this study, semi-sweet and sweet varieties had local lesion (resistant) responses following inoculation of Cucumber mosaic virus (CMV), and in this interaction, a gene involved in regulation of saponin biosynthesis (TSARL1)was upregulated. The bitter variety wassusceptible (systemically infected), but TSARL1 was downregulated. Together these provide the first evidence for role of saponin in the quinoa-CMV interaction. Small RNAs, induced by CMV infection, are predicted to target genomic and intergenic regions enriched in repetitive elements; this points to a potential role in RNA interference, an evolutionarily-conserved plant defense mechanism from which viral evasion is a crucial event. This study is the first to provide a comprehensive understanding of the involved genes, non-coding regions, and biological pathways in the quinoa-CMV interaction. Based on our results, we propose that antiviral saponins play a role and are controlled in quinoa-virus interactions. This study provides a change inknowledge of plant-virus interactions at the molecular level; this new knowledge can be used in development of disesase resistant varieties. 1. Develop rapid bioassay for bioassay-guided fractionation. An automated system for impact of extracts on plant parasitic nematodes is in development. Early tests with the system result in rapid identification of toxicity of extracts. 2. Determine chemical composition of water extracts of D. ambrosioides. No progress in this period. 3. Determine biological activity of water extracts of D. ambrosioides. No progress in this period. 4. Determine applicability of water extracts of D. ambrosioides for control of bacterial and fungal diseases of greenhouse cropping systems. In order to determine the role of the saponins associated with chenopods two studies were conducted. The objective of the first study was to determine if infection by Cucumber Mosaic Virus (CMV) differed in the chenopod genera, Chenopodium, Dysphania, and Blitum. Virus titer was insufficient for detection in all varieties of Dysphania and in Chenopodium berlanieri BYU 937. Virus was detected in C. berlanidieri BYU 803, C. quinoa, and in Blitum bonus-henricus, a species with high saponin levels. In a subsequent experiment, three varieties of quinoa that differed in saponin were inoculated with CMV. The high saponin cultivar was the most susceptible to the virus. The objective of the second study was to determine if saponin levels in quinoa predict plant response to root-knot nematode (RKN - Meloidogyne incognita). Eggs (2100 per plant) served as inoculum. Three cultivars (9 plants per variety) were inoculated: 'Jesse' (sweet); 'Redhead' (high saponin); and QQ74 (moderate saponin). After 28 d, roots were washed and weighed, and galls were counted. Numbers of galls/g root were approximately 3.5× greater in 'Jesse' and QQ74 than in 'Redhead' (P<0.05). Root mass was greater for 'Redhead' than the other two varieties, which did not differ. In a separate experiment, mobility of second stage juveniles (J2s) in aqueous extracts from uninoculated plants was tested. Oven-dried roots were autoclaved (15 mg/ml water), stored at room temperature (24 h), filtered, then re-autoclaved. Freshly hatched J2s were more sensitive to extracts from 'Jesse' than to extracts from 'Redhead' or QQ74 (P<0.05). Since extracts did not foam when shaken, it is unlikely that bioactivity is due to saponins; therefore, other anti-nematodal compounds in roots may contribute to RKN tolerance. Further research is needed to fully understand tolerance in quinoa.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Soltani, N.*, E.C. Bernard, K.D. Gwinn. 2019. Response of root-knot nematode (Meloidogyne incognita) and saponin content of quinoa (Chenopodium quinoa). Phytopathology 109:S1.36. Southern Division of the American Phytopathological Society. Orlando, FL. February 7-9.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Soltani, N.*, M.E. Staton, K.D. Gwinn. 2019. RNASeq: virus infection alters terpenoid biosynthesis in Chenopodium quinoa. Phytopathology Phytopathology 109:S2.184. American Phytopathological Society, Cleveland, OH. August 3-7.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audiences for this portion of the project have been high school students, undergradutes students,and the scientific community. Students were trained in natural product extraction and evaluation, and presented research results at regional competitions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?High school students and undergraduates were engaged in testing new bioassays and extracts. A graduate student assisted with the student projects. How have the results been disseminated to communities of interest?The undergraduate students had to blog about their experiences as part of the 1794 Scholars - Research Assistant program. One of the students won the Research Assistant of the Year award. What do you plan to do during the next reporting period to accomplish the goals?Analysis of the water extracts is pivotal to the completion of the project and will be the primary objective this period.Testing of phytotoxicity will be conducted, the appropriate host-pathogen systems will be selected.
Impacts
What was accomplished under these goals?
1. Develop rapid bioassay for bioassay-guided fractionation.Bioscreen C is routinely used for testing of antibacterial activity of extracts. The objective of an undergraduateresearchproject was to design a method to test impact of extracts on fungal disease using organic grape tomatoes purchased from local grocery stores. Tomatoes were treated with water extracts, then spores from Botrytis cinerea were placed on the fruit and a shallow punch was made with a syringe needle. In preliminary tests, the method appeared to work well for extract evaluation, but the method was discarded after a species of Fusarium was routinely isolated with the B. cinerea. 2. Determine chemical composition of water extracts ofD. ambrosioides.In generalD. ambrosioidesextracts did not contain high levels ofsaponin as tested by the foaming tests.Water extracts were submitted to the Small Molecule Center and in water extracts, but the center was unable to reproducibly determine the composition of the water extract. Based on an analysis using GC/MS, the leaves contained p-cymene, limonene, and ascaridole. 3. Determine biological activity of water extracts of D. ambrosioides. Two lines ofD. ambrosioideswere obtained from the US National Germplasm System. Water extracts were prepared and evaluated using Bioscreen C. Plants were grown in controlled conditions in the UT Phytotron. A high school student performed preliminary tests based to test the hypothesis that domesticated plantswould have less antimicrobial activity than those collected from the wild or those grown from seeds collected from the wild. He compared the impact ofwater extracts of plants grown from seed obtained from the National Germplasm Center (Ames 28030) and epazote purchased from a local Mexican market on growth ofBacillus cereus. Bacterial growth was inhibited more by extract from the Ames 28030 than from the purchased epazote. Water extracts were also tested for anti-nematodal activity using the same methods previously developed for Caenorhabditis elegans. Second-stage juveniles (J2s) of root-knot nematodes were exposed to water extracts derived from two plants grown from seed obtained from the National Germplasm Center (Ames 28030 and PI 604781). The J2s were not mobile after 24 h in extracts from the two D. ambrosioides lines. 4. Determine applicability of water extracts of D. ambrosioides for control of bacterial and fungal diseases of greenhouse cropping systems. No progress.
Publications
- Type:
Books
Status:
Published
Year Published:
2018
Citation:
Gwinn, K. D. 2018. Bioactive natural products in plant disease control. Studies in Natural Product Chemistry 56:229-246. https://doi.org/10.1016/B978-0-444-64058-1.00007-8.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Soltani, N., Staton, M., Huff, M. and Gwinn, K.D. 2018. Analysis of Chenopodium-virus interactions using Chenopodium quinoa reference genome. Phytopathology 108: S1.110.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Results were presented to scientific audience at the annual meeting of the American Phytopathological Society in San Antonio, TX (August 5 -9, 2017). Data that demonstrated that exposure of Caenorhabditis elegans to extracts from plants in the Family Chenopodiaceae (chenopods) reduced survival were presented. Nematicidal activity was highest in two chenopods - Blitum bonus-henricus (GKH) and Dysphania ambrosioides (epazote). Results were compared to commerically available commercial Quillaja bark saponin (QBS). elegans. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A high school student (current junior) worked during the summer and the fall semester on developing the Bioscreen technique described above. An undergraduate student maintained plants and did preliminary testing. How have the results been disseminated to communities of interest?Results were presented to scientific audience at the annual meeting of the American Phytopathological Society in San Antonio, TX (August 5 -9, 2017). Data that demonstrated that exposure of Caenorhabditis elegans to extracts from plants in the Family Chenopodiaceae (chenopods) reduced survival were presented. Nematicidal activity was highest in two chenopods - Blitum bonus-henricus (GKH) and Dysphania ambrosioides (epazote). Results were compared to commercially available commercial Quillaja bark saponin (QBS). elegans. What do you plan to do during the next reporting period to accomplish the goals?1. Develop rapid bioassay for bioassay-guided fractionation. Two undergraduate students were awarded 1794 Research Scholarships to examine methods of monitoring antifungal activity. They will compare several methods to determine which are applicable to chemical identification. They will present their research at a university-wide competition. 2. Determine chemical composition of water extracts of D. ambrosioides. A PhD student is beginning a study on terpene metabolism in the Family Chenopodiacae. He will compare terpene synthesis in epazote to that of other chenopods. 3. Determine biological activity of water extracts of D. ambrosioides. The high school student will present his research at the Southern Appalachian Science and Engineering Fair. The objectives of his project are to compare bioactivies and chemical properties of epazote available as an herb in Mexican market(s) to plants grown from seed collected by the National Plant Germplasm System. 4. Determine applicability of water extracts of D. ambrosioides for control of bacterial and fungal diseases of greenhouse cropping systems. Extracts that are inhibitory will be tested in the greenhouse on pathogens of tomatoes and other transplant crops.
Impacts
What was accomplished under these goals?
1. Develop rapid bioassay for bioassay-guided fractionation. Cell growth maintains a reduced environment which converts resazurin to resorufin (red). If growth is inhibited, an oxidized environment is maintained and resazurin remains unchanged (blue). In our studies, resazurin was added to a Bioscreen C screening system and decrease in absorbance at 600 nm was monitored as an indication of bacterial growth. Incubation temperature was in the optimal temperature range of the bacterium. Plates were incubated with constant shaking. After 2 h incubation, maximum conversion to resorfun was usually achieved. Relative activity of extracts was determined. 2. Determine chemical composition of water extracts of D. ambrosioides. Chenopodium oil from epazote is an effective, but potentially harmful, treatment for reduction of parasites in human and animals. Water extracts of epazote used in this study had the characteristic smell of ascaridole, the anti-helminthic compound in epazote. In this study and a previous study, epazote extracts were active against juveniles of C. elegans. Since previous reports suggested that anti-helmintic properties were retained when ascaridole was removed from extracts and saponins are well known nematicidal compounds, the saponin content of epazote and other extracts was determined. Saponin content of chenopod extracts was determined by foaming index and hemolytic activity. Aqueous suspensions of dried plant material (1.5 g/50 ml) were autoclaved and filtered; filtrate was autoclaved. Extracts of Blitum bonus-henricus (hemolytic) had much higher foaming indices than epazote (non-hemolytic). Extracts from other chenopods were intermediate. Since epazote had a low concentration of saponins, it is unlikely that these compounds are the source of activity against C. elegans. However, a synergistic effect with ascaridole should be considered. 3. Determine biological activity of water extracts of D. ambrosioides. In order to determine if sapoinins were active against C. elegans egg hatch, we tested impact of commercial Quillaja bark saponin (QBS) on egg hatch and survival of first stage juveniles (L1) of C. elegans. Three strains (N2, MT7735, ZD500) were cultured using standard procedures. Eggs were collected from 2-day-old cultures and added to QBS, S basal salt, and water mixtures. Experiments were designed as replicated 3 x 3 factorials [strain x QBS (0, 1, or 10 mg/ml)] with three trials. Eggs and L1s were counted at 8 h; mobility was monitored at 24 and 120 h. Percentage hatch and mobility were reduced in high QBS treatments (P < 0.001). Hatch in ZD500 (defective nose touch response) was higher than either N2 or MT7735 (P = 0.02). Effect of QBS on mobility of L1s varied. At 24 h, percentage of mobile L1s was greater in controls than in QBS treatments (P < 0.0001); this was reversed at 48 h for MT7735 (apoptosis mutant). Similar tests were performed with extracts of epazote and B. bonus-henricus. Egg hatch was generally unaffected by dose of extract, but low doses of Blitum extracts (P=0.0036) stimulated early hatch of eggs from MT7735. Epazote extracts did not impact egg hatch at 8h, but numbers of mobile juveniles were reduced by 37.5% for low and moderate doses and by 62.5% for high doses. There was no effect of nematode strain on percentage of mobile juveniles for either chenopod extract. Based on these studies, saponins may play a role in activity against C. elegans.
Publications
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2017
Citation:
Gwinn, K.D., Batson, E.K., Kouser, T. and Dee, M.M. 2017. Saponins in chenopods and potential for control of nematodes. Phytopathology
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2017
Citation:
Gwinn, K.D. 2017. Bioactive natural products in plant disease control. Chapter 7. In: Studies in Natural Products Chemistry ISBN: 978-0-444-64058-1.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audiences for this portion of the project have been high school students and the scientific community. Students were trained in natural product extraction and evaluation, and presented research results at regional and international competitions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project was used to train high school students in research methods. One student studied the impact of the plant extracts on the nematode. One student studied the chemistry of plant extracts. A third student studied the the impact of extracts on seed germination and seedling health. How have the results been disseminated to communities of interest?Two of the three students competed in a regional science fair and won the opportunity to compete at the International Science and Engineering Fair. They won 4th place in Plant Sciences. A journal article is in preparation. What do you plan to do during the next reporting period to accomplish the goals?An undergraduate researcher will begin studies the effects of epazote extracts on bacterial and fungal activity in January. Research results will be presented at national meetings and university-wide competitions.
Impacts
What was accomplished under these goals?
Epazote was compared with other chenopods using C. elegans jueveniles as a bioassay. All plant extract had significant negative e?ects on C. elegans juveniles in both a 24 and 48 hour time period. Data from each extract had good fit for regression models, and regression equations were used to calculate EC50 (based on nematodes reduction of mobility) and LC50 (based on nematode death) values. Percentages of nematodes affected and killed correlated with saponin content of some extracts. Epazote had low saponin content, but it was the second most active with EC50 and LC50 values of 10.89 and 20.57, indicating that saponins are not the sole substance responsible for nematicidal activity. Lyophilized extracts were analyzed by FT-IR and spectra were analyzed using Unscrambler. The two principal components explained 75% of variation.All extracts with high saponin content were negative or neutral for Principal Component 2; there was no relationship between Principal Component 1 and saponin content or biological activity.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Batson, E.K. and Kouser, T. 2016. Chemical and Biological Activity of Plants with Saponins. Southern Appalachian Science and Engineering Fair. Knoxville, TN. April 4-7/
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Batson, E.K. and Kouser, T. 2016. Nematicidal Activity and Saponin Concentration of Chenopod Extracts. International Science and Engineering Fair. Phoenix, Ar. May 8-13, 2016.
- Type:
Book Chapters
Status:
Accepted
Year Published:
2016
Citation:
Gwinn, K.D. 2017. Bioactive Natural Products in Plant Disease Control. IN: Studies in Natural Product Chemistry
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Progress 08/06/15 to 09/30/15
Outputs
Target Audience:This project has been used to train high school students in natural product extraction and evaluation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Research mentoring - Two advanced high school students and one undergraduate have been testing epazoate extracts. How have the results been disseminated to communities of interest?One high school student participated in a competitive science project exhibition at his school. What do you plan to do during the next reporting period to accomplish the goals? Objective 1. Rapid bioassay identification. Concentrated extracts will be tested for activity against plant pathogenic bacteria. If active, assays will be used to develop a rapid bioassay for bioactivity of epazote extracts. If extracts are not antibacterial, then antifungal activity will be assessed. Objective 2. Bioassay-guided chemical characterization. 1. Bioassay-guided fractionation will be used to classify the chemical characteristics of epazote extracts. Extracts will be fractionated with a series of organic solvents. Initial fractionation will be performed with liquid:liquid partitioning. When classification of active ingredients are identified, appropriate analytical techniques will be used to further characterize activity. Objective 3. Biological activity identification. Antibacterial activity, antifungal activity, and phytotoxicity of unfractionated extracts will be determined. In addition, as bioactive fractions are identified in the rapid bioassay, the bioactivity in the active fractions will be characterized more fully for bacteria and fungi. A. Antibacterial activity. If sensitivity to epazaote extract is identified in Objective 1, additional assays (e.g., Bioscreen testing, micro-dilution plating; Alamar blue based micro-titer plate assays) will be used to further quantify effects on plant pathogenic bacteria.
Impacts
What was accomplished under these goals?
The project was approved in August 2015. Objective 2. Water extracts of D. ambrosiodes contained very low concentrations of saponins. The foaming index was less than 100 and the extract did not lyze red blood cells. Water extracts have been prepared and lyophilized for further extraction. Objective 3. Water extracts were not phytotoxic when applied to geranium, vinca, tomato, and pepper.
Publications
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