INFECTIOUS DISEASE DYNAMICS AND THE DIRECT AND INDIRECT EFFECTS OF AN ESCAPED VIRAL-RESISTANCE TRANSGENE ON PLANT FITNESS IN A WILD SQUASH
Open Access
- Author:
- Sasuclark, Miruna Ariadna
- Graduate Program:
- Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 23, 2010
- Committee Members:
- Andrew George Stephenson, Dissertation Advisor/Co-Advisor
Andrew George Stephenson, Committee Member
Richard Cyr, Committee Chair/Co-Chair
James Landis Rosenberger, Committee Member
Mark C Mescher, Committee Member
James Alan Winsor, Committee Member - Keywords:
- bacterial wilt disease
viral-resistance transgene
infectious disease
Cucurbita pepo
ZYMV
path analysis - Abstract:
- ABSTRACT This dissertation consists of six chapters: an introduction, four data chapters, and a conclusion: in the Introduction, I present background and provide general information about Cucurbita pepo ssp. texana (the wild progenitor of cultivated squashes), the major herbivores that feed on this plant, and the pathogens they transmit. Background on the two major statistical methods that I have used in the dissertation, analysis of variance (ANOVA) and path analysis, is also included in this section. Additionally, the introduction contains a review of previous experiments from Dr. Stephenson’s laboratory that led to my dissertation work. The second chapter was motivated by previous field studies in the Stephenson lab. These studies revealed that selfed (S) wild gourd plants (Cucurbita pepo ssp. texana) experience greater cucumber beetle herbivory than outcrossed (X) plants but outcrossed plants experience a higher incidence of bacterial wilt disease. Subsequent studies showed that both types of plants were equally susceptible to this disease when exposure was similar. Given that the pathogen responsible for bacterial wilt disease (Erwinia tracheiphila) is known to be transmitted when cucumber beetles chew leaves and then defecate onto the open wounds, the difference in incidence rates required explanation. It is known that cucumber beetles aggregate in the flowers to mate and the Stephenson lab had data showing that X plants produced more flowers than S plants. Furthermore, the flowers from X plants produced more of the volatile compounds that are known to attract cucumber beetles. This phenomenon could be explained if E. tracheiphila can be transmitted via the floral nectaries. Therefore, I explored the possibility of transmission of this plant pathogen via the floral nectaries by: (1) Inoculating greenhouse-grown plants by placing a solution containing Erwinia tracheiphila onto the nectaries of the flowers (after removing the nectar), and showing that E. tracheiphila is able to infect plants via the nectaries; (2) Transforming E. tracheiphila cells with a GFP-marker and visually showing the progression of the bacteria through the nectary tissue and into the xylem of the pedicel; (3) Collecting cucumber beetle frass from field collected flowers and showing that more than 50% of flowers contain cucumber beetle frass in or around the nectaries, and (4) Developing E. tracheiphila-specific primers to screen cucumber beetle frass collected from flowers in the field and showing that more that 90% of flowers contained cucumber beetle frass contaminated with the bacteria.The results of these experiments provide strong circumstantial evidence that E. tracheiphila can be transmitted via the floral nectaries in C. pepo ssp. texana. Chapter three contains a series of experiments to determine if the nectar of C. pepo ssp. texana has antimicrobial properties.Having showed that E. tracheiphila cells can to traverse the nectary tissue of C. pepo flowers and infect plants, I asked if the nectar present in the flowers may interfere with the growth of the bacteria inside the floral nectaries. Flowers of many species have nectar that inhibits the growth of microbes and the secondary compounds responsible for this inhibition are thought to function in preventing the degradation of nectar (to maintain its quality as a pollinator reward). However, in the case of C. pepo, nectar could also function to prevent infection with E. tracheiphila, a floral transmitted pathogen. In one experiment, I examined the effect of nectar on the growth of Erwinia tracheiphila and Escherichia coli using disk diffusion assays. To accomplish this, I grew lawns of E. tracheiphila and E. coli on Petri dishes and examined the area cleared by a filter paper disk impregnated with C. pepo nectar, 40% glucose or ampicillin. This study showed that C. pepo nectar inhibits the growth of both E. tracheiphila and E. coli compared to glucose, a negative control. Additionally, the antibiotic effect of nectar was as great as ampicillin for 12 hrs. In a second experiment, I inoculated greenhouse-grown plants via flowers with and without nectar. This experiment showed that plants inoculated through flowers without nectar experienced significantly higher incidence of wilt disease. Together, these findings show that antimicrobial compounds in C. pepo nectar inhibit the growth of E. tracheiphila and can function to retard the pathogen’s transmission through the floral tissues, allowing time for an abscission layer to form between the flower and pedicel. Chapter four examines the direct and indirect effects of a virus-resistance transgene (VRT) on fitness during introgression in C. pepo ssp. texana in the presence of the full range of insect herbivores (vectors) and diseases. Virus-resistant transgenic squash (Cucurbita pepo ssp pepo) are grown throughout the United States and much of Mexico and it is likely that the VRT has been introduced into wild populations of Cucurbita repeatedly. A three-year, large-scale field study (using wild gourds, transgenic and non-transgenic introgressive plants of various generations) examined reproductive output, herbivory by the primary herbivore (cucumber beetles), and incidence of both the target diseases (viruses transmitted by aphids) and a non-target disease (bacterial wilt disease) on wild gourds and transgenic and non-transgenic introgressives. These studies revealed that (1) Each year, viral diseases became established in mid-July and spread rapidly through the fields until the end of the season when most non-transgenic (susceptible) plants showed symptoms. This also showed that the introgressed transgene was truly effective against viral diseases. (2) The wild gourds, non-transgenic introgressives and transgenic introgressives did not differ in the amount of beetle damage or wilt disease incidence prior to the spread of viral diseases. (3) Once the viral diseases spread through the fields, cucumber beetles preferred to feed on healthy (transgenic) plants and the transgenic plants experienced increased wilt disease incidence. (4) A series of path analysis models showed that the VRT had a direct beneficial effect on reproductive output (transgenic plants are more fit) but this beneficial effect was mitigated by the indirect effects of concentrated cucumber beetle herbivory and increased wilt disease incidence once viral diseases spread thorough the fields. These indirect effects of the VRT on fitness were only apparent in the full context of the Cucurbita pathosystem: thus we can conclude that all target and non-target components are potentially important when examining ecological phenomena. Since chapter four detailed the direct and indirect effects of the VRT when virus is present in all the fields, the next step in chapter five was to determine the costs and benefits of those effects. Therefore, chapter five focuses on the costs and benefits of the VRT during introgression into the wild gourd in the presence and absence of viral diseases, as well as the costs and benefits of cultivar genes during introgression. In this study we planted four one-acre fields with wild gourds, transgenic and non-transgenic introgressives and recorded reproductive output, herbivory by the primary herbivore (cucumber beetles), and incidence of both the target diseases (viruses transmitted by aphids) and a non-target disease (bacterial wilt disease) on the plants in each field. In two fields we allowed viral diseases to spread, while the other two fields were sprayed with Endeavor®, an aphid-specific insecticide that has no effect on cucumber beetles or other chewing insects. The insecticide deterred viral transmission by controlling aphid vectors but had no effect on the vectors of bacterial wilt disease. In the unsprayed (virus) fields the data showed that: (1) Viral diseases became established in mid-July and spread rapidly through the fields and by late August most of the susceptible plants showed virus symptoms. (2) The VRT effectively deters viral disease infection in transgenic introgressives. (3) Cucumber beetle herbivory and wilt disease incidence was higher in transgenic introgressives after the virus spread through the fields. (4) Reproductive output was higher in transgenic introgressives. However, on the sprayed (no-virus) fields the data show that the transgenic introgressives also had greater reproductive output than wild gourds and non-transgenic introgressives but the benefit was not as great as in the unsprayed (virus) fields. The benefit of the VRT in the sprayed fields seems to be due to the introduction of the target virus into the fields in August (end of the season) and a benefit of cultivar genes. The optimal growing conditions for wild gourds are hot and dry and because the summer of 2009 was wet and cool, both the transgenic and non-transgenic introgressives had greater reproductive output than wild gourd plants, regardless of disease pressure. The parental cultivar had been bred to grow and reproduce well in the northeastern US while the wild gourd originated in Texas. These findings also show that there are indirect effects of the VRT on a non-target herbivore (cucumber beetles) and the pathogen it transmits (E. tracheiphila) when viral diseases are allowed to spread through the fields. These findings suggest that yearly variations in environmental variables such as temperature, rainfall and time of virus introduction, can impact the fitness of the VRT during introgression. In addition, it is important to note that our studies were performed north of the native range of wild gourds and that different populations of organisms (insects, predators, pathogens) as well as competition (both inter and intra-specific) among plants may also play roles in the fitness of the VRT during introgression. In summary, my dissertation explores the transmission of an important bacterial pathogen (bacterial wilt disease) through floral tissues of Cucurbita pepo ssp. texana and the effect of floral nectar on the transmission of the pathogen, as well as the direct and indirect effects of a virus-resistance transgene (VRT) on plant fitness, cucumber beetle herbivory, and the incidence of bacterial wilt disease and viral diseases as the VRT introgresses into wild Cucurbita pepo. As a unit, my research suggests that it is important to study the individual components of a pathosystem and their interactions when assessing the costs and benefits of resistance genes (both natural and transgenic).