Source: UNIVERSITY OF NEBRASKA submitted to
TRANSGENIC VIRUS RESISTANT SQUASH: ECOLOGICAL EFFECT TEN YEARS AFTER COMMERCIAL RELEASE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0204924
Grant No.
2005-33522-16396
Project No.
NEBW-2005-03806
Proposal No.
2005-03806
Multistate No.
(N/A)
Program Code
HX
Project Start Date
Sep 1, 2005
Project End Date
Aug 31, 2010
Grant Year
2005
Project Director
Pilson, D.
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
(N/A)
Non Technical Summary
Transgenic virus resistant squash was commercially released in 1994 with little examination of potential ecological effects. In this project we will survey wild squash populations for virus infection and transgenic virus resistance derived from crop squash. We will also do experiments and develop population models that will allow us to predict the effect of transgenic virus resistance on the population dynamics of wild squash populations.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121429107050%
2121429110150%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1429 - Cucurbits, other;

Field Of Science
1070 - Ecology; 1101 - Virology;
Goals / Objectives
Transgenic crops now account for more than 50% of global crop production, with the US growing over half of this total (>105 million acres in 2003). Despite their widespread use, few studies have examined the potential ecological effects of transgenes moving from crops into wild relatives. This is surprising because the escape of transgenes into wild populations is one of the major ecological risks associated with the release of transgenic crops. Moreover, screening for transgene movement into wild populations and evaluating the consequences of such movement is a high priority of the USDA Biotechnology Risk Assessment Grants (BRAG) Program and the Ecological Society of America. The goal of the work we propose here is to screen wild squash (Cucurbita pepo) populations for the presence of transgenes conferring virus resistance, as well as to evaluate the fitness effects and potential ecological consequences of these genes in wild populations. The work we propose here will provide the most comprehensive available evaluation of the potential ecological effects associated with the release of a transgenic virus resistant crop. In particular, we will 1) survey wild squash populations for the presence of transgenes derived from cultivated squash, 2) survey wild squash populations for the incidence of viral infection, 3) quantify the effect of virus infection on the fitness of wild plants, 4) quantify the fitness benefits associated with the expression of transgenic virus resistance in wild plants, and 5) use stochastic demographic modeling methods to estimate the effects of transgenic resistance on population growth rates in wild squash populations. We suggest that the protocols proposed here could become a model for the evaluation of potential ecological effects of other virus resistant crops in the future. Since more than 80 field test permits have been granted in the last 3 years for 17 crops in the US alone, the need for efficient evaluation of ecological effects is significant.
Project Methods
Objective 1: Survey wild squash populations for transgenic virus resistance. In each of the three years of this project we will collect ~1000 leaf samples from wild squash populations adjacent to squash production fields near the West Tennessee Experiment Station in Jackson, Tennessee. To identify transgenic-wild plants we extract DNA from leaf tissue and use PCR to amplify portions of the transgenes found in each commercially available transgenic squash variety. These data will allow us to determine if crop-wild hybridization has led to the establishment of transgenic virus resistance in wild populations near one crop production area. Objective 2: Survey wild squash populations for infection by five viruses that commonly infect cucurbits. In each of the three years of this project we will collect ~1000 leaf samples from wild squash populations near Jackson, Tennessee. The presence of five common viruses (ZYMV, WMV, CMV, PRSV, SqMV) will be detected using reverse transcription-PCR following extraction of RNA from leaf tissues. Estimates of virus frequency are critical for evaluating potential ecological effects of transgenic virus resistance. Objectives 3: Quantify the effect of virus infection on the fitness of wild plants. In each of two years we will establish common garden populations of wild squash in riparian and agricultural habitats at the West Tennessee Experiment Station. Plants will be inoculated with one of the three viruses for which transgenic virus resistance is available (ZYMV, WMV, CMV) or will remain virus free as a control. Survival, male and female flower production, and fruit production will be used to estimate male and female fitness for each plant, and data will be analyzed by ANOVA. These data will allow us to estimate the potential evolutionary advantage of virus resistance in wild populations. Objective 4: Evaluate the fitness benefits in wild plants of expressing transgenic virus resistance. In the greenhouse we will create BC2 populations that segregate for transgenic virus resistance. We will use these BC2 seeds in a 2x2 factorial field experiment in which transgenic virus resistance and virus inoculation are the experimental factors Survivorship and plant fitness will be estimated. Fitness costs and benefits of transgenic resistance will be analyzed by ANOVA. Objective 5: Use stochastic demographic modeling methods to estimate the effects of transgenic resistance on population growth rate in wild squash populations. These will allow us to determine which life cycle transitions limit the population size of wild squash. Then we will consider how transgenic virus resistance might alter demographic transitions. Our models will be parameterized using data collected in our experiments and surveys. If our models suggest that transgenic virus resistance could lead to higher population growth rates, then wild squash could become a more aggressive weed in agricultural habitats or more abundant in wild habitats. Comparing estimates of population growth rate in wild and transgenic-wild populations is fundamental to assessing the potential ecological consequences of transgene escape into wild populations.

Progress 09/01/05 to 08/31/10

Outputs
OUTPUTS: 1. We performed experiments evaluating the effects of transgenic virus resistance and virus infection on pollinator behavior. 2. We surveyed wild squash populations over four years in Mississippi, Arkansas, Louisiana, Missouri, and Oklahoma for infection by cucumber mosaic virus, squash mosaic virus, papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus. 3. We surveyed these same populations for the presence of transgenic virus resistance. 4. We performed field experiments over two years to evaluate the effect of virus infection and introgressed transgenic virus resistance on wild squash populations. 5. We developed a population matrix model to examine the effect of virus infection and environmental variation on wild squash population dynamics. 6. We developed a population matrix model to examine the effect of transgenic virus resistance in the presence and absence of virus infection on wild squash population dynamics. Results from this work have been presented at several national and regional meetings and will be, or have been, published in the peer-reviewed literature. PARTICIPANTS: Principal Investigator: Diana Pilson, University of Nebraska-Lincoln Co-principal Investigator: T. Jack Morris, University of Nebraska-Lincoln Graduate Student: Dr. Holly Prendeville, University of Nebraska-Lincoln (Ph.D. completed December 2010, primarily supported by this project). Technicians: Beth Barry, formerly University of Nebraska, currently unknown; Xiaohong Ye, University of Nebraska. Collaborators/contacts: Roy French, USDA/University of Nebraska. Undergraduate students serving as field and laboratory assistants (received training in research protocols): 8 individuals. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
1. Transgenic virus resistance in cultivated squash has several pleiotropic effects on floral morphology and nectar reward. In addition, squash bees and honey bees respond differently to transgenic vs. conventional cultivated squash. However, these results appear to vary across years, probably due to environmental differences among years. 2. In 28 published studies 56 of 117 tested plant species were infected with virus; infection rates in infected populations ranged from 0.01-100%. Results of our field survey were comparable. In 21 populations sampled from 2004-2007 virus prevalence varied (from 0-74%) among populations, years, and virus species. In samples analyzed by both ELISA and RT-PCR, RT-PCR detected 6-44% more infections (depending on virus species) than did ELISA. Most published studies used ELISA, suggesting that virus prevalence is higher that is typically reported. 80% of infections in wild squash were asymptomatic. 3. The virus-resistance transgene was not present in any of our samples. 4. Virus infection reduces both plant survival and seed production in surviving plants that do not have transgenic resistance. In contrast, plants into which transgenic resistance has been introgressed (backcross 2 generation; BC2) are not affected by virus infection. 5. Our population matrix model predicts that the population growth rate of wild squash populations can be reduced by virus infection; however the magnitude of effect varies among virus species, years, and plant populations. 6. Virus infection reduced the population growth rate of BC2 plants without the transgene, but had no effect on population growth of trangenic BC2 plants. However, the magnitude of these effects depended on both virus species and plant population.

Publications

  • Prendeville, H.R. and D. Pilson. 2009. Transgenic virus resistance in cultivated squash affects pollinator behavior. J. Appl. Ecology 46:1088-1096.
  • Prendeville, H.R., X. Ye, T.J. Morris, and D. Pilson. 2011. Virus infections in wild plant populations are both frequent and often asymptomatic. submitted to Am. J. Botany
  • Prendeville, H.R., D. Pilson, and B. Tenhumberg. 2011. Environment and virus affect wild squash population dynamics. in preparation.
  • Prendeville, H.R., D. Pilson, and B. Tenhumberg. 2011. The idiosyncratic effects of the virus-resistance transgene and virus infection on wild squash populations. in preparation.


Progress 09/01/05 to 08/31/06

Outputs
OUTPUTS: First year of field work underway (completed in fall 2006). PARTICIPANTS: Worked on grant: Diana Pilson - PI Jack Morris - PI Holly Prendeville - graduate research assistant (provided training) Beth Barry - technician several undergraduate students - laboratory help (provided training) Collaborators and contacts: Roy French (USDA/University of Nebraska) Guillermo Orti (University of Nebraska) Partner Organizations: Delta Conservation Demonstration Center, Metcalfe, MS(location of field experiments) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
no results; first year of field work not yet complete.

Publications

  • No publications reported this period