Progress 09/01/09 to 08/31/14
Outputs
Target Audience: Graduate and Undergraduate Education. The scientific community. Fruit and vegetable growers; land managers. Efforts: One graduate student and four undergraduates received hands on research experience from this project. Jacquelyn Harth, Matt Ferrari each gave talks at the 2014 Ecological Society of America annual conference, and Andrew Stephenson presented our findings at the autumn 2013 Entomological Society of America Conference and the 2014 Cucurbitaceae conference. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? PI/PDs: Matthew J. Ferrari, PI, Designed experiments, assisted in field and greenhouse experiments; analyzed data; worked on model development, parameterization, and simulations; assisted with manuscript preparation, oversaw and trained grad students and undergraduates. Andrew G. Stephenson, PI, Designed experiments, assisted in field and greenhouse experiments; analyzed data; performed crosses to produce seeds for next season; assisted with manuscript preparation; oversaw and trained grad students and undergraduates. Jacquelyn Harth: Graduate Student, Assisted with field and laboratory experimental design; performed the day to day field work in summer 2014; assisted in training and supervising undergraduates; analyzed data; Assisted in manuscript preparation. Heather E. Simmons: Former Graduate Student, analyzed data; assisted in manuscript preparation. : Undergraduates: Danelle Weakland, Aisling Ryan and Kayla Nowak: Screened seeds for the presence of the transgene using DAS-ELISA, Analyzed data, and assisted in the daily field work. Sean Hardison, and Rebecca Kolstrom, worked as independent study students on this project. They assisted in field and greenhouse experiments related to insect vectored disease transmission. Collaborators: Drs. Mark Mescher, Consuelo De Moraes, and Irmgard Seidl-Adams, Department of Entomology, The Pennsylvania State University, assisted in the studies of insect transmission of the viral and bacterial wilt diseases. How have the results been disseminated to communities of interest? During the 2014 field season, our research was highlighted and included on the tours of The Pennsylvania State University Agriculture Experiment Station that were given in association with PSU Ag Progress Days activities. In addition, we presented our findings at the 2013 Ecological Society of America's annual conference, at the Entomoligical Society of America's annual conference in Austin TX Oct 2013, Cucurbitaceae 2014 meeting held in October 2014. This conference is attended by leaders from industry, academic and government and we gave talks at the Penn State University Department of Plant Pathology and to the Virus Research faculty at PSU. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In 2014 we continued to perform large scale field experiments designed to obtain key data for the model that we developed to predict the spread of the virus resistant transgene upon escape from cultivated squash into wild populations of C. pepo. Our data and preliminary model suggested that when zucchini yellow mosaic virus (ZYMV) spreads through a field containing a mixture of transgenic and non-transgenic wild squash and a second pathogen, Erwinia tracheiphila, the cucumber beetles which vector the Erwinia prefer to eat healthy (not infected with ZYMV) plants thereby concentrating the Erwinia onto the transgenic plants. To test this prediction and to quantify a key parameter in the model (the concentration of beetles effect on fitness of transgenic and non-transgenic plants, we performed a large scale field experiment that is designed to obtain these data. During the summers of 2014, we transplanted 180 total plants: Texas gourds (Cucurbita pepo ssp. Texana) and their backcross 7 (BC7; Texas gourd by Liberator III cultivar, with Texas gourd as the recurrent parent) into each of 4 one acre plots. In all 4 of the plots, 45 of the BC7 were transgenic for resistance to cucumber, watermelon and zucchini yellow mosaic virus (CMV, WMV, ZYMV, respectively and 135 were non-transgenic plants (90 wild type; 45 non-transgenic BC7). In two of the plots, 20 non-transgenic plants were inoculated with ZYMV just prior to transplanting. This experiment was specifically designed to examine the impact of 'early viral disease' in a mixed (transgenic and susceptible) population in which bacterial wilt disease (Erwinia tracheiphila) was also naturally circulating. These are the early ZYMV plots. On the other two fields, located a km away, no plants were inoculated with ZYMV and this disease did not arrive in these fields until mid-August. These are the late ZYMV plots. This experimental design was similar to that used in 2013...so we have two years of data. Not surprisingly, we found that fitness decreases with the number of days with virus during the growing season. However, we also found that, in the early virus fields only, the transgenic plants experienced high levels of mortality due to bacterial wilt disease. The higher level of mortality due to wilt disease on the transgenic plants almost completely negated the fitness benefit of the transgene when virus was present in the population but not in the late virus fields. When simulations with these data were run in our preliminary model, we are left with the surprising (counter-intuitive) conclusion that the fitness benefit of the transgene decreases with an early arrival of virus when bacterial wilt disease is also present in the population. This has direct implications for management of the escaped transgene in wild populations. The predictions of the model were validated by the data in the late virus fields, which showed that fitness of the BC plants in terms of both seed production and number of seeds sired by the pollen, was greater than in the early virus fields. We also made a second surprising finding during the 2013 and 2014 field seasons. Virus infected plants are far more resistant to a third pathogen, powdery mildew (PM). We found that PM came into all of our fields in August, and after establishment on the leaves, it greatly depressed both growth and reproductive output. However, PM became established on virus infected plants approximately 3 weeks later than on healthy plants. In the early virus fields, the healthy plants were almost exclusively transgenic plants. This means that the transgenic plants disproportionately suffered from the adverse effects of PM, when virus is present in the population. This finding was independently verified with a large scale greenhouse experiment involving ZYMV inoculations and exposure to PM infected plants. We are currently revising our model to include the interactions of all three pathogens (ZYMV, PM, Erwinia tracheiphila) and their effects on the fitness of transgenic introgressives. These findings again suggest that the fitness of pathogen resistance transgenes in wild Cucurbita hybrids can only be determined within the context of the full pathosystem and these findings strongly suggest ways to manage the transgene upon escape into wild populations. We performed a series of greenhouse, field and illumina sequencing experiments designed to determine the pattern of movement of ZYMV within plants, to determine which mutations are vertically transmitted via seeds, to determine if ZYMV can be vertically transmitted via pollen. These studies took advantage of plant materials available through this grant. Our results show that (1) there are severe genetic bottlenecks as ZYMV moves systemically through wild squash vines, (2) certain mutations in the 5'UTR of the ZYMV genome is preferentially passed through vertical infection of seeds, and most surprisingly, our preliminary data suggest that ZYMV can be transmitted via pollen and because of this, seeds from transgenic plants can be vertically infected . These studies have very large implications for the commercial seed industry.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Harth, J.E., D.R. Weakland, K.J. Nowak, J.A. Winsor, M.J. Ferrari and A.G. Stephenson. 2014. An assessment of male fitness of an escaped virus resistant transgene from cultivated Cucurbita pepo during introgession into wild Cucurbita pepo. Pp 141-144 In: Cucurbitaceae 2014: Eds. M. Harvey, Y. Weng, B. Day and R. Grumet. American Society of Horticultural Science, Alexandria VA.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Dunham, J.P., H.E. Simmons, E.C. Holmes, and A.G, Stephenson. 2014. Analysis of viral (zucchini yellow mosaic virus) genetic diversity during systemic movement through a Cucurbita pepo vine. Virus Research 191:172-179. http://dx.doi.org/10.1016/j.virusres.2014.07.030
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Simmons, H.E., H.R. Prendeville, J.P. Dunham, M.J. Ferrari, J.D. Earnest, D. Pilson, G.P. Munkvold, E.C. Holmes, and A.G. Stephenson. Submitted. Transgenic virus-resistance in Cucurbita pepo does not prevent vertical transmission of Zucchini yellow mosaic virus. Plant Disease.
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Progress 09/01/12 to 08/31/13
Outputs
Target Audience: Graduate and Undergraduate Education. The scientific community. Fruit and vegetable growers; land managers. Efforts: Two graduate students andfour undergraduates received hands on research experience from this project. Jacquelyn Harth, Matt Ferrari each gave talks at the 2013 Ecological Society of America annual conference, and Andrew Stephenson presented our findings at the 2012 Cucurbitaceae conference. Heather Simmons gave at talk at the University of Nebraska on the data she obtained from these studies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? PI/PDs: Matthew J. Ferrari, PI, Designed experiments, assisted in field and greenhouse experiments; analyzed data; worked on model development, parameterization, and simulations; assisted with manuscript preparation, oversaw and trained grad students and undergraduates. Andrew G. Stephenson, PI, Designed experiments, assisted in field and greenhouse experiments; analyzed data; performed crosses to produce seeds for next season; assisted with manuscript preparation; oversaw and trained grad students and undergraduates. Jacquelyn Harth: Graduate Student, Assisted with field and laboratory experimental design; performed the day to day field work in summer 2013; assisted in training and supervising undergraduates; analyzed data. Heather E. Simmons: Graduate Student, Designed and performed the zucchini yellow mosaic virus transmission studies; assisted in training and supervising undergraduates; analyzed data; assisted in manuscript preparation. : Undergraduates: Danelle Weakland: Screened seeds for the presence of the transgene using DAS-ELISA, Analyzed data, and assisted in the daily field work. Undergraduate Kaity DiOrio: Assisted in field and greenhouse experiments and the daily field work. Sean Hardison, Aisling Ryan, and Rebecca Kolstrom, worked as independent study students on this project. They assisted in field and greenhouse experiments related to insect vectored disease transmission. Collaborators: Drs. Mark Mescher, Consuelo De Moraes, and Irmgard Seidl-Adams, Department of Entomology, The Pennsylvania State University, assisted in the studies of insect transmission of the viral and bacterial wilt diseases. How have the results been disseminated to communities of interest? During the 2013 field season, our research was highlighted and included on the tours of The Pennsylvania State University Agriculture Experiment Station that were given in association with PSU Ag Progress Days activities. In addition, we presented our findings at the 2013 Ecological Society of America's annual conference in Minneapolis, MN and at Cucurbitaceae 2012 meeting held in October 2012. This conference is attended by leaders from industry, academic and government and we gave talks at the University of Nebraska and the University of Virginia. What do you plan to do during the next reporting period to accomplish the goals? 1. Complete the 2013 field season, analyze the data. 2. Finalize our model, run simulations, present out findings at conferences and publish the model in the most visible journal possible. 3. Analyze the impact of virus disease on the male function; present out findings at conferences and publish our results. 4. Determine whether ZYMV can be transmitted via pollen by germinating seeds from our controlled crosses; use PCR to determine the presence of ZYMV in seeds produced by pollen from ZYMV infected plants; present out findings at conferences and publish our results.
Impacts
What was accomplished under these goals?
Our data and preliminary model suggested that when zucchini yellow mosaic virus (ZYMV) spreads through a field containing a mixture of transgenic and non-transgenic wild squash and a second pathogen, Erwinia tracheiphila, the cucumber beetles which vector the Erwinia prefer to eat healthy (not infected with ZYMV) plants thereby concentrating the Erwinia onto the transgenic plants. To test this prediction and to quantify a key parameter in the model (the concentration of beetles effect on fitness of transgenic and non-transgenic plants, we performed a large scale field experiment that is designed to obtain these data. During the summer of 2013, we transplanted 180 total plants: Texas gourds (Cucurbita pepo ssp. Texana) and their backcross 6 (BC6; Texas gourd by Liberator III cultivar, with Texas gourd as the recurrent parent) into each of 4 one acre plots. In all 4 of the plots, 45 of the BC6 were transgenic for resistance to cucumber, watermelon and zucchini yellow mosaic virus (CMV, WMV, ZYMV, respectively and 135 were non-transgenic plants (90 wild type; 45 non-transgenic BC6). In two of the plots, 20 non-transgenic plants were inoculated with ZYMV just prior to transplanting. These are the early ZYMV plots. On the other two fields, located a km away, no plants were inoculated with ZYMV and this disease did not arrive in these fields until mid-August. These are the late ZYMV plots. We are currently examining the effects of the transgene on plant fitness, herbivory by cucumber beetles, and the incidence of a non-target pathogen (Erwinia tracheiphila, the causative agent of bacterial wilt disease) and the rate of virus transmission in the susceptible plants using DAS-ELISA tests. To determine if the transgene increased in frequency from one generation to the next via the male (pollen function), we will harvest the mature fruits from the Texas gourds and the non-transgenic BC6 and we will score a sample of the progeny for the presence of the transgene using DAS-ELISA during the late autumn and winter. In addition, we are analyzing the herbivory data, the incidence of viral disease, and the reproductive output (male flowers and total mature fruits) from the 2012 field experiment. Data from previous years indicated that ZYMV can be vertically transmitted. To determine if ZYMV is vertically transmitted via pollen or ovules, we are performing a series of controlled pollination experiments and we will screen a large sample of the resulting seeds for the presence of ZYMV. In addition, we performed Illumina sequencing on ZYMV that were vertically and horizontally transmitted to determine if there are differences in the types of mutations that are transmitted vertically and horizontally. Our previous observations suggested that vertically transmitted ZYMV were asymptomatic and could potentially protect plants from horizontal infection. Finally, we have been using the data from previous years to develop and parameterize a model to predict the spread of the escaped transgene into populations with the target diseases, the non-target disease, and both diseases. In previous years, we showed that the beetles prefer to eat healthy plants and, as virus spreads through the susceptible plants in our fields, the beetles and the non-target pathogen they transmit becomes increasingly concentrated onto transgenic plants. From the data obtained during the 2012 field season we were able to quantify the effect of transgene frequency on the concentration of beetle damage. The data we are obtaining in 2013 will allow us to assess the impact of virus spread on the concentration of beetle damage. This is a key parameter in our model. Analysis of previous years data also showed that the beetles are preferentially attracted to the volatile organic compounds produced by the foliage of wilt diseased plants but they avoid the flowers of both virus and wilt diseased plants. They prefer the flowers of healthy plants where they transmit the Erwinia pathogen to the plant. As virus spreads through the susceptible plants, the beetles become increasingly concentrated into the flowers of healthy (most transgenic plants. These findings again suggest that the fitness of pathogen resistance transgenes in wild Cucurbita hybrids can only be determined within the context of the full pathosystem. Our preliminary model has suggested that it is possible to focus Erwinia on transgenic plants, in a mixed population of transgenic and susceptible plants and that the timing of the spread of ZYMV is key to understanding the concentration of beetle damage and the Erwinia bacteria they transmit. We also found, using Illumina sequencing that some mutations of the ZYMV that is aphid transmitted in our fields increase dramatically over the course of the growing season suggesting that it may be possible to identify virulence factors that are specific to squash. Our 2013 Illumina sequencing suggests that certain mutations in the 5’UTR of the ZYMV genome is preferentially passed through vertical infection of seeds. This may have profound implications for the commercial seed industry.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2012
Citation:
Harth, J.E., T.W. Deveney, J.A. Winsor, M.J. Ferrari and A.G. Stephenson. 2012. Background and development of a model to predict the fate of an escaped virus resistant transgene from Cucurbita pepo. Pp. 230-238. In: Cucurbitaceae 2012: Proceedings of the Xth Eucarpia Meeting on Genetics and Breeding of Cucurbitaceae. Cukurova University Press, Antalya, Turkey.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Simmons, H.E., J.P. Dunham, K.E. Zinn, G.P. Munkvold, E.C. Holmes and A.G. Stephenson. 2013. Zucchini yellow mosaic virus (ZYMV, Potyvirus): Vertical transmission, seed infection and cryptic infections. Virus Research 176:259-254.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: During the summer of 2012, we transplanted 180 total plants: Texas gourds (Cucurbita pepo ssp. Texana) and their backcross 5 (BC5; Texas gourd by Liberator III cultivar, with Texas gourd as the recurrent parent) into each of 4 one acre plots. In two of the plots 30 of the BC5 were transgenic for resistance to cucumber, watermelon and zucchini yellow mosaic virus (CMV, WMV, ZYMV, respectively and 30 were non-transgenic plants. This is the low transgene frequency plot. The other two plots were the high transgene frequency plots (120 transgenic BC5 plants per plot). In mid-June we inoculated two non-transgenic plants in the middle of each plot with ZYMV. We are currently examining the effects of the transgene on plant fitness, herbivory by cucumber beetles, and the incidence of a non-target pathogen (Erwinia tracheiphila, the causative agent of bacterial wilt disease) and the rate of virus transmission in the susceptible plants using DAS-ELISA tests. To determine if the transgene increased in frequency from one generation to the next via the male (pollen function), we will harvest the mature fruits from the Texas gourds and the non-transgenic BC5 and we will score a sample of the progeny for the presence of the transgene using DAS-ELISA during the late autumn and winter. In addition, we are analyzing the herbivory data, the incidence of viral disease, and the reproductive output (male flowers and total mature fruits) from the 2011 field experiment. Data from previous years indicated that ZYMV can be vertically transmitted. To determine if ZYMV is vertically transmitted via pollen or ovules, we are performing a series of controlled pollination experiments and we will screen a large sample of the resulting seeds for the presence of ZYMV. Finally, we have been using the data from previous years to develop and parameterize a model to predict the spread of the escaped transgene into populations with the target diseases, the non-target disease, and both diseases. EVENTS: During the 2012 field season, our research was highlighted and included on the tours of The Pennsylvania State University Agriculture Experiment Station that were given in association with PSU Ag Progress Days activities. In addition, we presented our findings at the 2012 Ecological Society of America's annual conference in Portland OR and we are scheduled to present our findings at Cucurbitaceae 2012 in October 2012. This conference is attended by leaders from industry, academic and government and we gave talks at Iowa State University, Ames IA. PRODUCTS: Ms. Jacquelyn Harth, a graduate student involved in the project successfully passed her candidacy exam for the admittance into the PhD program. Mr. Troy Deveney, an undergrad student involved in both the field and lab research, received an Honor's degree from Penn State in May 2012. PARTICIPANTS: PI/PDs: Matthew J. Ferrari, PI, Designed experiments, assisted in field and greenhouse experiments; analyzed data; worked on model development, parameterization, and simulations; assisted with manuscript preparation, oversaw and trained grad students and undergraduates. Andrew G. Stephenon, PI, Designed experiments, assisted in field and greenhouse experiments; analyzed data; performed crosses to produce seeds for next season; assisted with manuscript preparation; oversaw and trained grad students and undergraduates. Jacquelyn Harth: Graduate Student, Assisted with field and laboratory experimental design; performed the day to day field work in summer 2012; asssisted in training and supervising undergraduates; analyzed data. Heather E. Simmons: Graduate Student, Designed and performed the zucchini yellow mosaic virus transmission studies; asssisted in training and supervising undergraduates; analyzed data; assisted in manuscript preparation. : Undergraduate. Troy Deveney: Undergraduate: Screened seeds for the presence of the transgene using DAS-ELISA. Analyzed data. Undergraduate Kaity DiOrio: . Assisted in field and greenhouse experiments. Collaborators: Drs. Mark Mescher, Consuelo De Moraes, and Irmgard Seidl-Adams, Department of Entomology, The Pennsylvania State University, assisted in the studies of insect transmission of the viral and bacterial wilt diseases. Training and Professional Development: Christopher Balough, Undergraduate, worked as independent study students on this project. They assisted in field and greenhouse experiments related to insect vectored disease transmission. TARGET AUDIENCES: Target Audiences: Graduate and Undergraduate Education. The scientific community. Fruit and vegetable growers; land managers. Efforts: Two graduate students and three undergraduates received hands on research experience from this project. Jacquelyn Harth, Matt Ferrari, and Andrew Stephenson will present our findings at the 2012 Cucurbitaceae conference. Heather Simmons gave at talk at Iowa State University on the data she obtained from these studies in November 2012. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In previous years, we showed that the beetles prefer to eat healthy plants and, as virus spreads through the susceptible plants in our fields, the beetles and the non-target pathogen they transmit becomes increasingly concentrated onto transgenic plants. From the data obtained during the 2011 field season we were able to quantify the effect of transgene frequency on the concentration of beetle damage. This is a key parameter in our model. Analysis of previous years data also showed that the beetles are preferentially attracted to the volatile organic compounds produced by the foliage of wilt diseased plants but they avoid the flowers of both virus and wilt diseased plants. They prefer the flowers of healthy plants where they transmit the Erwinia pathogen to the plant. As virus spreads through the susceptible plants, the beetles become increasingly concentrated into the flowers of healthy (most transgenic plants. These findings again suggest that the fitness of pathogen resistance transgenes in wild Cucurbita hybrids can only be determined within the context of the full pathosystem. We also found, using Illumina sequencing that some mutation of the ZYMV that is aphid transmitted in our fields increase dramatically over the course of the growing season suggesting that it may be possible to identify virulence factors that are specific to squash.
Publications
- Stephenson, A.G. 2012. Safe sex in plants. New Phytologist 193:827-829.
- Simmons, H.E., J. Dunham, J. Stack, B. Dickins, I. Pagan, E.C. Holmes and A.G. Stephenson. 2012. Deep sequencing reveals persistence of intra- and inter-host genetic diversity in natural and greenhouse populations of Zucchini yellow mosaic virus. Journal of General Virology 93:1831-1840. doi:10.1099/vir.0.042622-0
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: ACTIVITIES: During the summer of 2011, we transplanted 180 total plants: Texas gourds (Cucurbita pepo ssp. Texana) and their backcross 5 (BC5; Texas gourd by Liberator III cultivar, with Texas gourd as the recurrent parent) into each of 4 one acre plots. In two of the plots 30 of the BC5 were transgenic for resistance to cucumber, watermelon and zucchini yellow mosaic virus (CMV, WMV, ZYMV, respectively and 30 were non-transgenic plants. This is the low transgene frequency plot. The other two plots were the high transgene frequency plots (120 transgenic BC5 plants per plot). In mid June we inoculated two non-transgenic plants in the middle of each plot with ZYMV. We examined the effects of the transgene on plant fitness, herbivory by cucumber beetles, and the incidence of a non-target pathogen (Erwinia tracheiphila, the causative agent of bacterial wilt disease) in the presence and absence of viral pathogens in each plant. To determine if the transgene increased in frequency from one generation to the next via the male (pollen function), we harvested the mature fruits from the Texas gourds and the non-transgenic BC5 and we will score a sample of the progeny for the presence of the transgene using DAS-ELISA during the late autumn and winter. In addition, we are analyzing the herbivory data, the incidence of viral disease, and the reproductive output (male flowers and total mature fruits) from the field experiment. Finally, we screened the seeds produced by ZYMV infected plants during the 2010 field season using PCR and ZYMV specific primers to determine if ZYMV can be vertically transmitted via the seeds. We are currently wrapping up this field season and beginning to analyze the resulting data. In fall of 2010 we initiated a collaboration with Diana Pilson and Holly Prendenville, who have been conducting field experiments with mixed populations of transgenic and wild-type C. pepo spp. texana at the University of Nebraska- Lincoln. We have analyzed a sample of fruits from their experiment to quantify the change in the frequency of the transgene and to determine if ZYMV can be transmitted via the seeds. We also grew plants that were vertically infected with ZYMV and collected and scored their seeds using PCR and ZYMV specific primers to determine if vertically infected plants can pass ZYMV to their seeds. EVENTS: During the 2011 field season, our research was highlighted and included on the tours of The Pennsylvania State University Agriculture Experiment Station that were given in association with PSU Ag Progress Days activities. In addition, we presented our findings in a keynote address to the 2011 Ecological Society of Mexico conference in Vera Cruz, Mexico and we gave talks at the Ohio Agricultural Research and Development Center. Wooster, Ohio, and to the Plant Pathology Department at Penn State University. PRODUCTS: Ms. Heather Simmons, a graduate student involved with the project, successfully defended her dissertation in Biology and will graduate in December 2011. Ms. Jacquelyn Harth, a graduate student involved in the project successfully passed her candidacy exam for the admittance into the PhD program. PARTICIPANTS: Individuals: PI/PDs: Matthew J. Ferrari, PI, Designed experiments, assisted in field and greenhouse experiments; analyzed data; worked on model development, parameterization, and simulations; assisted with manuscript preparation, oversaw and trained grad students and undergraduates. Andrew G. Stephenon, PI, Designed experiments, assisted in field and greenhouse experiments; analyzed data; performed crosses to produce seeds for next season; assisted with manuscript preparation; oversaw and trained grad students and undergraduates. Jacquelyn Harth: Graduate Student, Assisted with field and laboratory experimental design; performed the day to day field work in summer 2011; asssisted in training and supervising undergraduates; analyzed data. Heather E. Simmons: Graduate Student, Designed and performed the zucchini yellow mosaic virus transmission studies; asssisted in training and supervising undergraduates; analyzed data; assisted in manuscript preparation. : Undergraduate. Katie Zinn assisted in field and greenhouse experiments; assisted in the laboratory portion of the virus transmission studies. Troy Deveney: Undergraduate. Assisted in field and greenhouse experiments. Screened seeds for the presence of the transgene using DAS-ELISA. Collaborators: Drs. Mark Mescher, Consuelo De Moraes, and Irmgard Seidl-Adams, Department of Entomology, The Pennsylvania State University, assisted in the studies of insect transmission of the viral and bacterial wilt diseases. Training and Professional Development: Christopher Balough and Katie Zinn, Undergraduates, worked as independent study students on this project. They assisted in field and greenhouse experiments related to insect vectored disease transmission. TARGET AUDIENCES: Target Audiences: Graduate and Undergraduate Education. The scientific community. Fruit and vegetable growers; land managers. Efforts: Two graduate students and three undergraduates received hands on research experience from this project. Matt Ferrari and Andrew Stephenson presented our findings in talks presented at the 2011 Ecological Society of Mexico annual meetings in April 2011, at the International Symposium on Insect-Plant interactions in August 2011, at the Ohio Agricultural Research and Development Center. Wooster, Ohio, in March 2011; and to the Plant Pathology Department at Penn State University in February 2011. PROJECT MODIFICATIONS: No major changes to report.
Impacts
From the 2010 field season we found that there were no significant differences in the proportion of seeds sired by the transgenic plants in sprayed and unsprayed fields (although in both fields the frequency of the transgene is predicted to increase from one generation to the next). This incongruous result is partially explained by increased herbivory by cucumber beetles and higher incidence of the deadly bacterial wilt disease they vector on transgenic plants. That is, as viral diseases spread through the unsprayed fields, the cucumber beetles that vector the bacterial wilt disease prefer to feed upon the healthy (mostly transgenic) plants and increase the exposure of transgenic plants to a deadly non-target pathogen. These findings suggest that the fitness of pathogen resistance transgenes in wild Cucurbita hybrids can only be determined within the context of the full pathosystem. We found that approximately 2.0% of the seeds produced by vertically infected ZYMV plants are also infected with ZYMV. Moreover, we found that the transgenic plants from our fields, which show no symptoms of ZYMV infection and test negative for ZYMV infection produce seeds with a vertical infection rate that is very similar to the non-transgenic plants growing in the same fields. This strongly suggests that ZYMV can be transmitted from generation to generation via pollen and that the vertical infections that we observed in previous years was due to pollen (rather than ovule) transmission to the seeds. CHANGE IN ACTIONS: The field results will assist us in parameterizing the model that we proposed for the fitness of the transgene during introgression in presence of target and non-target pathogens. The model will, when fully parameterized, assist in the management of the escaped virus resistant transgene. Our studies of vertical ZYMV infections strongly suggest that ZYMV epidemics can be initiated via seeds and that vertical ZYMV infections occur via pollen transmission rather than via ovule transmission.
Publications
- Simmons, H.E., E.C. Holmes, F.E. Gildow, M.A. Bothe-Goralczyk, and A.G. Stephenson. 2011. Experimental verification of seed transmission in zucchini yellow mosaic virus. Plant Disease 95:751-754.
- Simmons, H.E., E.C. Holmes, A.G. Stephenson. 2011. Rapid turnover of intra-host genetic diversity in zucchini yellow mosaic virus. Virus Research 155:389-396.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: ACTIVITIES: During the summer of 2009 (using pre-award costs), we transplanted 18 Texas gourds (Cucurbita pepo ssp. Texana), 18 backcross 4 (BC4; Texas gourd by Liberator III cultivar, with Texas gourd as the recurrent parent) from each of 5 maternal families into each of 4 one acre plots (180 plants per plot). Nine of the BC4 were transgenic for resistance to cucumber, watermelon and zucchini yellow mosaic virus (CMV, WMV, ZYMV, respectively). The other 9 BC4 plants were non-transgenic siblings. Two of the plots were sprayed on 15 June, 5 July, and 31 July with Pymetrozine (Endeavor 50 WDG) that is effective against aphids (sucking insects) but not effective against cucumber beetles. The other two fields were allowed to become naturally infected (which occurred in mid- to late July). We examined the effects of the transgene on plant fitness, herbivory by the primary herbivores (cucumber beetles), and the incidence of a non-target pathogen (Erwinia tracheiphila, the causative agent of bacterial wilt disease) in the presence and absence of viral pathogens during introgression into wild Cucurbita pepo. To determine if the transgene increased in frequency from one generation to the next via the male (pollen function), we harvested the mature fruits from the Texas gourds and the non-transgenic BC4 and scored a sample of the progeny for the presence of the transgene using DAS-ELISA during the autumn and winter. In addition, we analyzed the herbivory data, the incidence of viral disease, and the reproductive output (male flowers and total mature fruits) from the field experiment. Finally, we screened the seeds produced by ZYMV infected plants using PCR and ZYMV specific primers to determine if ZYMV can be vertically transmitted via the seeds. During the summer of 2010, we repeated the above experiment except that we initiated an epidemic by hand inoculating two plants in the unsprayed fields with ZYMV ten days after transplanting.. We are currently wrapping up this field season and beginning to analyze the resulting data. In fall of 2010 we initiated a collaboration with Diana Pilson and Holly Prendenville, who have been conducting field experiments with mixed populations of transgenic and wild-type C. pepo spp. texana at the University of Nebraska- Lincoln. We have arranged to analyze a sample of fruits from their experiment to quantify the change in the frequency of the transgene. EVENTS: During both field seasons, our research was highlighted and included on the tours of The Pennsylvania State University Agriculture Experiment Station that were given in association with PSU Ag Progress Days activities. In addition, we presented our findings in a talk at the February 2010 Mid Atlantic Fruit and Vegetable Convention in Hershey PA, the April meeting of the International Association of Landscape Ecology, and the Penn State Ecology Fall seminar series, and posters at the Ecology and Evolution of Infectious Disease Symposium in May 2010 and Plant Virus Symposium at Cornell in June 2010. PRODUCTS: Ms. Miruna Sasu, a graduate student involved with the project, received her Ph.D. in Plant Biology in May 2010. PARTICIPANTS: Individuals: PI/PDs: Matthew J. Ferrari, PI, Designed experiments,assisted in field and greenhouse experiments; analyzed data; worked on model development, parameterization, and simulations; assisted with manuscript preparation, oversaw and trained grad students and undergraduates. Andrew G. Stephenon, PI, Designed experiments,assisted in field and greenhouse experiments; analyzed data; performed crosses to produce seeds for next season; assisted with manuscript preparation; oversaw and trained grad students and undergraduates. Miruna A. Sasu: Graduate Student, Assisted with field and laboratory experimental design; performed the day to day field work in summer 2009; asssisted in training and supervising undergraduates; analyzed data, assisted in manuscript preparation. Heather E. Simmons: Graduate Student, Designed and performed the zucchini yellow mosaic virus transmission studies; asssisted in training and supervising undergraduates; analyzed data; assisted in manuscript preparation. Melinda Bothe: Undergraduate. Assisted in field and greenhouse experiments; assisted in the laboratory portion of the virus transmission studies. Sarah Scanlon: Undergraduate. Assisted in field and greenhouse experiments. Troy Deveney: Undergraduate. Assisted in field and greenhouse experiments. Screened seeds for the presence of the transgene using DAS-ELISA. Collaborators: Drs. Mark Mescher, Consuelo De Moraes, and Irmgard Seidl-Adams, Department of Entomology, The Pennsylvania State University, assisted in the studies of insect transmission of the viral and bacterial wilt diseases. Training and Professional Development: Christopher Balough and Kelly Wall, Undergraduates, worked as independent study students on this project. They assisted in field and greenhouse experiments related to insect vectored disease transmission. TARGET AUDIENCES: Target Audiences: Graduate and Undergraduate Education. The scientific community. Fruit and vegetable growers; land managers. Efforts: Two graduate students and five undergraduates received hands on research experience from this project. One of the graduate students (Miruna Sasu) was recently hired by the Food BioSafety and Security division of USDA. Matt Ferrari and Andrew Stephenson presented our findings in a talk at the February 2010 Mid Atlantic Fruit and Vegetable Convention in Hershey PA, the April meeting of the International Association of Landscape Ecology, and the Penn State Ecology Fall seminar series, and posters at the Ecology and Evolution of Infectious Disease Symposium in May 2010 and Plant Virus Symposium at Cornell in June 2010. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
CHANGE IN KNOWLEDGE: Our 2009 field season data found that the introgressed transgene effectively deterred viral (ZYMV) infection in our no spray plots. Moreover, the Pymetrozine spray effectively deterred virus from all plants until mid-august. In the absence of the virus (sprayed fields), reproductive output of transgenic introgressive plants did not differ from non-transgenic introgressive plants and they did not have greater herbivory or a higher incidence of bacterial wilt disease. In the presence of the viral pathogen (unsprayed fields), both the transgenic and non-transgenic introgressives had lower reproductive output despite the resistance of the transgenic introgressives to viral infection. In addition, we found that there were no significant differences in the proportion of seeds sired by the transgenic plants in sprayed and unsprayed fields (although in both fields the frequency of the transgene is predicted to increase from one generation to the next). This incongruous result is partially explained by increased herbivory by cucumber beetles and higher incidence of the deadly bacterial wilt disease they vector on transgenic plants. That is, as viral diseases spread through the unsprayed fields, the cucumber beetles that vector the bacterial wilt disease prefer to feed upon the healthy (mostly transgenic) plants and increase the exposure of transgenic plants to a deadly non-target pathogen. These findings suggest that the fitness of pathogen resistance transgenes in wild Cucurbita hybrids can only be determined within the context of the full pathosystem. We found that approximately 1.5% of the seeds produced by ZYMV infected plants are also infected with ZYMV and that these vertically infected seeds, upon germination, can transmit ZYMV horizontally via aphids. CHANGE IN ACTIONS: These findings will assist us in parameterizing the model that we proposed for the fitness of the transgene during introgression in presence of target and non-target pathogens. The model will, when fully parameterized, assist in the management of the escaped virus resistant transgene. Our studies of vertical ZYMV infections strongly suggest that ZYMV epidemics can be initiated via seeds.
Publications
- Stephenson, A.G. 2010. Nectar, bees, beetles and wilt disease: How they interact and influence pest management. Proceedings of the Mid Atlantic Fruit and Vegetable Convention. Pg. 130.
- Sasu, M.A., Wall, K.L. and Stephenson, A.G. 2010. Antimicrobial nectar inhibits a floral transmitted pathogen of a wild Cucurbita pepo (Cucurbitaceae). American Journal of Botany 97:1025-1030. doi:10.3732/ajb.0900381
- Sasu, M.A., Ferrari, M.J. and Stephenson, A.G.. 2010. Inter-relationships among a virus resistance transgene, herbivory, and a bacterial disease in wild Cucurbita. International Journal of Plant Sciences (in press).
- Stephenson, A.G., Shapiro, L. Sasu M.A., DeMoraes C.M., and Mescher, M.C.. 2010. Factors that influence exposure rates and transmission of Erwinia tracheiphila in Cucurbita pepo. In: Cucurbitaceae 2010, J.A. Thies (ed.). North Carolina State University Press, Raleigh, NC. Pp. (in press).
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