Progress 02/01/14 to 01/31/17
Outputs
Target Audience:As was indicated in the project initiation section, the present project ensured the participation and training of undergraduate and graduate students. During the funding period, six undergraduate students (Sean Mahaney, Moyinoliwa Natasha Ogunnowo, Sara LeFevre, Esmeralda Flores, Viva Rase, and Sean Byrne) were recruited via work-study or Directed Studies (Bio 422) to work on different aspects of the project. Undergraduate students received training in potato tissue culture, basic laboratory techniques, arbuscular mycorrhizal (AM) fungus propagation, and experimental design. In addition, two M.S. students, Andrew Schoenherr and Eric Rizzo, were recruited to carry out all the experiments that were proposed. Both graduate students received training in plant physiology, plant molecular biology, fungal ecology, and entomology. In addition, graduate students had the opportunity to interact with scientists from other institutions, Dr. Patricia Manosalva (University of California Riverside), Dr. Paul Ode (Colorado State University), and Dr. Whitney Cranshaw (Colorado State University). Collaborator Dr. Manosalva, worked closely with graduate students, and attended our two annual meetings that took place in Greeley, CO. Both graduate students disseminated their research through oral presentations during Academic Excellence Week at the University of Northern Colorado. In addition, an undergraduate student presented a poster during Academic Excellence Week. Dr. S. Karen Gomez (PD) presented a poster at the Project Directors' meeting that took place at the USDA (Washington, DC) on April 28-29, 2015. Both graduate students gave poster presentations at Plant Biology 2016 in Austin, TX, and at the Plant and Animal Genome Conference 2017 in San Diego, California. Changes/Problems: We encountered unforeseen problems initially - malfunctioning of the greenhouse, and thrips infestation of experimental plants. This caused a significant delay because each experiment took at least 100 days from the moment potato clones were propagated to time of harvest. A no-cost extension was requested to compensate for the delay in recruiting key personnel. Different time points were used because the changes in plant gene expression may occur early in the tripartite interaction, whereas, the impact of the AM symbiosis on insect fitness occur later. Comparison between insect-damaged leaves and systemic undamaged leaves were included in our later experiments, which was not originally proposed. We will be making RNA libraries for Illumina sequencing to obtain a more complete view of the early changes in gene expression during tripartite interactions. What opportunities for training and professional development has the project provided?Six Biology undergraduate students (Sean Mahaney, Moyinoliwa Natasha Ogunnowo, Sara LeFevre, Esmeralda Flores, Viva Rase, and Sean Byrne) participated in this project and received training in potato tissue culture, basic laboratory techniques, arbuscular mycorrhizal (AM) fungus propagation, and experimental design. Undergraduate students were initially trained by Dr. Gomez (PD), and subsequently by graduate students providing them with mentoring experience. The two M.S. students, Andrew Schoenherr and Eric Rizzo who were recruited for this project successfully completed their Master's degree program at the University of Northern Colorado. Additionally, graduate students had opportunities to disseminate their work through oral presentations, and an undergraduate student presented a poster during Academic Excellence Week at the University of Northern Colorado. Graduate students attended two national conferences and presented their work at Plant Biology and at the Plant and Animal Genome (PAG XXV) Conference. Andrew received 1st place for his oral presentation representing the College of Natural Sciences (that includes nine schools/departments within the college) for the Research Excellence Award at the University of Northern Colorado. How have the results been disseminated to communities of interest?Results have been disseminated mainly through oral and poster presentations at the University of Northern Colorado, Plant Biology Conference, and Plant and Animal Genome Conference. News Releases about the Project: UNC Faculty Receive Combined $645,000 in USDA Grants - University of Northern Colorado http://www.unco.edu/news/releases.aspx?id=6446 UNC biologists get USDA grants to address cattle, potato issues - Greeley Tribune http://www.greeleytribune.com/news/10579349-113/agriculture-crop-grants-project Rocky Mountain Ag Notebook: Wyo., Colo. among the most 'agribusiness-friendly' states; Water rights bill passes House - The Fence Post http://www.thefencepost.com/news/10611744-113/state-colorado-program-beef 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 nature, plants form multiple interactions with beneficial and detrimental organisms in soil and aboveground. Root associations with arbuscular mycorrhizal (AM) fungi provide nutrients to the plant, while insect pests are deleterious. This project investigates the commonly occurring tripartite interaction among potato, AM fungi, and insects of two feeding-guilds, the potato aphid (phloem-feeder), and the cabbage looper (chewing insect). The main goal of this Seed Grant was to evaluate the experimental conditions to create a rigorous system to study two tripartite interactions, and to provide insight that can serve to fully explore these systems in the future to manage pests of economic importance in elite potato germplasm. This research fills the gap in knowledge regarding changes in plant gene expression and plant physiology that occur in an economically important vegetable crop during tripartite interactions. Two tripartite interactions were studied: a) AM fungus, potato, aphids and b) AM fungus, potato, cabbage loopers. The research objectives were: 1) to evaluate the conditions to create a rigorous system to study AM fungus-potato-insect interactions, 2) to examine the effects of aboveground insect herbivory and belowground AM fungus colonization on potato plant physiology, and assess organism performance in these tripartite interactions, and 3) to determine the impact of mycorrhiza-induced resistance in potato against insects of two feeding-guilds. Objective 1: 1) Major activities completed / experiments conducted; Preliminary experiments that tested levels of AM fungus colonization, insect number, and insect feeding duration were conducted, which helped with optimization of the experimental conditions. Cabbage loopers (CL)-potato-AM fungi interaction: Potato roots were inoculated with varying amounts of spores of Glomus intraradices to obtain three distinct levels of colonization designated as "low", "medium", and "high". Once the desired fungal density was reached, CL larvae were placed on potato leaves and were allowed to feed for seven days. Potato aphid (PA)-potato-AM fungi interaction: This experiment included two levels (low and high) of soil inoculum composed of a mixed-strain of G. intraradices, and was designed to address objectives 1 and 3. It focused on measuring the changes in potato gene expression at one day and 10 days of aphid feeding, comparing alterations in local, aphid-infested leaves and systemic (non-infested) leaves of the same plant. 2) Data collected; Percent root length colonized by G. intraradices, insect count and weight, shoot fresh and dry weight, root fresh and dry weight, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). 3) Summary statistics and discussion of results; CL-potato-AM fungi interaction: Larvae gained significantly less weight after feeding on mycorrhizal plants at the low level of G. intraradices root colonization compared to those that fed on highly colonized plants. However, there was no statistical difference in weight gain between larvae that fed on non-mycorrhizal plants and mycorrhizal plants regardless of colonization level. Based on these trends, we opted for the low level of G. intraradices root colonization and increased the number of replicates in subsequent experiments. While defense-related genes were upregulated in shoots of insect-damaged mycorrhizal plants, their expression levels were not statistically different compared to insect-damaged non-mycorrhizal plants. PA-potato-AM fungi interaction: In this experiment, we assessed: i) the impact of two levels (low and high) of G. intraradices root colonization on PA herbivory aboveground, and ii) potato gene expression during these interactions. We focused on genes involved in phytohormone biosynthesis and defense responses. Our results indicated that the mycorrhizal level did not significantly impact plant growth or PA population or colony weight. Aphid herbivory itself produced the strongest impact on shoot gene expression irrespective of mycorrhizal level. Leaves of aphid-infested non-mycorrhizal plants (8.2-fold) and aphid-infested low-colonized plants (5.6-fold) exhibited significant upregulation of the jasmonic acid (JA)-regulated transcription factor MYC2 gene relative to non-infested mycorrhizal plants (3.2-fold). Overall, our research reveals that subtle changes in gene expression (of the nine genes tested) are occurring in potato leaves and roots during tripartite interactions. Changes in gene expression after 10 days of feeding and in systemic (non-infested) leaves are currently underway. 4) Key outcomes or other accomplishments realized. No changes were observed on PA population and colony weight neither at one nor at 10 days of feeding on mycorrhizal plants. Objectives 2 & 3: 1) Major activities completed / experiments conducted; CL-potato-AM fungi interaction: Based on the results obtained in objective 1, two separate experiments (one for gene expression and one for plant physiology) were designed using low levels of G. intraradices root colonization. We used spores and a soil inoculum of G. intraradices for the gene expression experiment and the plant physiology experiment, respectively. CL fitness was evaluated by comparing larval weight gain after five and eight days of feeding. Gene expression was measured in shoots and roots. Plant physiology was assessed on insect-damaged leaves and undamaged leaves of the same plant after five and eight days of CL feeding. PA-potato-AM fungi interaction: To assess objective 2, a separate three-way interaction experiment was conducted to determine the impact of seven and 14 days of aphid infestation and an established AM symbiosis (high colonization) on plant physiology. A soil inoculum of G. intraradices was used. 2) Data collected; Percent root length colonized by G. intraradices, insect count and weight, fresh and dry weights of shoots and roots, RT-qPCR, water potential, photosynthesis, and relative chlorophyll content. 3) Summary statistics and discussion of results; CL-potato-AM fungi interaction: For the gene expression experiment, the results revealed larvae gained more weight when they fed on non-mycorrhizal plants compared to mycorrhizal plants. We found that genes involved in activating plant defenses in shoots were induced in insect-damaged plants regardless of mycorrhizal status (±G. intraradices). For the plant physiology experiment, our results were consistent, showing that CL larvae that fed on mycorrhizal plants gained significantly less mass than those that fed on non-mycorrhizal plants. Undamaged mycorrhizal plants had more shoot mass compared to non-mycorrhizal plants and insect-damaged plants (±AM fungus) at the eight-day time point. Overall, subtle changes in plant physiology were detected after CL herbivory on mycorrhizal plants and non-mycorrhizal plants. Potato aphid-potato-AM fungi interaction: Our findings demonstrated that high levels of G. intraradices root colonization resulted in enhanced aphid colony weight at seven and 14 days post feeding. Though we did not document enhanced tolerance to aphid herbivory in highly AM fungus-colonized plants according to the physiological metrics of water potential, photosynthetic rate, and chlorophyll content, we did observe a clear trend of enhanced cell death that was restricted to aphid-infested mycorrhizal plants. 4) Key outcomes or other accomplishments realized. It was found that by extending the aphid-feeding period to 14 days, PA population and colony weight increased after feeding on highly colonized mycorrhizal plants, which could be attributed to increased phosphate levels in leaves. On the other hand, CL larval weight was consistently reduced after feeding on low-colonized mycorrhizal plants, and this was in part explained by significant accumulation of defense-related transcripts in shoots.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Poster Conference Presentations:
Rizzo E., P. Manosalva, and S.K. Gomez. 2017. An established arbuscular mycorrhizal symbiosis positively impacts potato aphids, Plant and Animal Genome (PAG XXV) Conference, San Diego, CA, January 14-18, 2017.
Schoenherr A., P. Manosalva, and S.K. Gomez. 2017. The mycorrhizal status of potatoes alters plant physiology and the fitness of a chewing insect pest, Plant and Animal Genome (PAG XXV) Conference, San Diego, CA, January 14-18, 2017.
Rizzo E. and S.K. Gomez. 2016. Menage � Trois: The effects of simultaneous mycorrhizal symbiosis and aphid herbivory on plant physiology, Plant Biology 2016, Austin, TX, July 9-13, 2016.
Schoenherr A. and S.K. Gomez. 2016. Effect of belowground mycorrhizal fungi on aboveground cabbage looper herbivory depends on the extent of root colonization, Plant Biology 2016, Austin, TX, July 9-13, 2016.
Workshop:
*Gomez S.K., E. Rizzo, A. Schoenherr, and P. Manosalva. 2015. How do arbuscular mycorrhizal fungi affect potato-insect interactions?, National Institute of Food and Agriculture (NIFA) Project Director Workshop, Agriculture and Food Research Initiative (AFRI) Plant-Associated Insects and Nematodes Program (U.S. Department of Agriculture), Washington, D.C., April 28-29, 2015. (poster)
Presentations at the University of Northern Colorado:
Schoenherr A., and S.K. Gomez. 2016. Does the extent of belowground mycorrhizal fungus colonization affect aboveground cabbage looper herbivory?, University of Northern Colorado Research Day, Greeley, CO, April 2016.
Byrne S., A. Schoenherr and S.K. Gomez. 2015. Arbuscular mycorrhizal fungi: Help or hindrance during insect attack? Investigation of the tripartite interaction between pest, plant, and fungi, University of Northern Colorado Research Day, Greeley, CO, April 2015. (poster)
Schoenherr A. and S.K. Gomez. 2015. Can belowground symbiosis affect aboveground insect herbivory?, University of Northern Colorado Research Day, Greeley, CO, April 2015.
Rizzo E. and S.K. Gomez. 2015. Tripartite relationships: a model for the study of plant-insect-fungal interactions, University of Northern Colorado Research Day, Greeley, CO, April 2015.
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Progress 02/01/15 to 01/31/16
Outputs
Target Audience:In the second year of this project, mainly one undergraduate student, Sara LeFevre worked throughout the report period (2-1-2015 to present). Sara's main role was to propagate and maintain mycorrhizal fungal spores and potato plantlets. Two undergraduate students, Esmeralda Flores and Viva Rase, joined the project in 2016. Esmeralda has been working with graduate student Andrew Schoenherr doing mainly potato tissue culture, and she is assisting both graduate students, Andrew and Eric Rizzo with experimental set up. Viva Rase has been assisting both graduate students in sterilization of soil substrates and helping them clean up pots and other items used in the project. Viva is also helping graduate students with harvest of the experiments. Both graduate students gave oral presentations about their project during our Academic Excellence Week (April 7, 2015) at the University of Northern Colorado acknowledging the USDA for funding this project. Dr. Karen Gomez (PD) is planning to attend both the Project Director's meeting that is planned for September 24, 2016, and the International Congress of Entomology's meeting in Orlando, FL.In the second year of this project, mainly one undergraduate student, Sara LeFevre worked throughout the report period (2-1-2015 to present). Sara's main role was to propagate and maintain mycorrhizal fungal spores and potato plantlets. Two undergraduate students, Esmeralda Flores and Viva Rase, joined the project in 2016. Esmeralda has been working with graduate student Andrew Schoenherr doing mainly potato tissue culture, and she is assisting both graduate students, Andrew and Eric Rizzo with experimental set up. Viva Rase has been assisting both graduate students in sterilization of soil substrates and helping them clean up pots and other items used in the project. Viva is also helping graduate students with harvest of the experiments. Both graduate students gave oral presentations about their project during our Academic Excellence Week (April 7, 2015) at the University of Northern Colorado acknowledging the USDA for funding this project. Dr. Karen Gomez (PD) is planning to attend both the Project Director's meeting that is planned for September 24, 2016, and the International Congress of Entomology's meeting in Orlando, FL. Changes/Problems: Two separate experiments for each tripartite interaction were conducted (summer and fall 2015) to identify the "best" level of AM fungus colonization needed to negatively alter cabbage looper and potato aphid fitness. Unfortunately, we encountered unforeseen problems - malfunctioning of the greenhouse, and thrips infestation of experimental plants. This caused a significant delay because each experiment takes at least 100 days from the moment potato clones are propagated to harvest of plant tissue. The experiments were repeated for each tripartite interaction and we obtained promising results for the cabbage looper-potato-AM fungus system. The experiment for the potato aphid-potato-AM fungus system is underway. We increased the number of biological replicates to 8 to reduce variance. Increased the number of cabbage loopers per plant from 3 to 5 (size between 1st to 2nd instar) to cause more damage to insect-infested plants. Increased the number of potato aphids per plant from 5 to 8. Tested 8 reference genes whose nucleotide sequences (or primers) were obtained from the literature in order to identify 2 suitable reference genes for quantitative PCR (qPCR). We now have 2 reference genes (elongation factor 1α and β tubulin) that are suitable for both potato shoot and root samples. We tested and optimized the annealing temperatures for all the primers used for qPCR. A no-cost extension was requested to compensate for the delay in recruiting key personnel. What opportunities for training and professional development has the project provided?Three undergraduate students - Sara LeFevre, Esmeralda Flores, and Viva Rase have been recruited to work on specific aspects of the project. Undergraduate students were trained by Dr. Gomez (PD) and graduate students in areas such as: potato tissue culture, basic laboratory techniques, AM fungus propagation, and experimental design. Two M.S. graduate students, Andrew Schoenherr and Eric Rizzo are also being mentored by Dr. Gomez. Both graduate students recently gave oral presentations about their projects on Research Day (April 7, 2016) at the University of Northern Colorado and will be attending the Plant Biology conference in Austin, TX. Andrew received 1st place for his oral presentation representing the College of Natural Sciences (9 schools/departments within the college) for the Research Excellence Award. The tile of his presentation was 'Does the Extent of Belowground Mycorrhizal Fungus Colonization Affect Aboveground Cabbage Looper Herbivory?". How have the results been disseminated to communities of interest?We anticipate disseminating results in the months ahead; now that we have optimized the system What do you plan to do during the next reporting period to accomplish the goals? We are re-designing the three-way interaction experiments that will be used to assess plant gene expression. We are using the "best" colonization level to negatively alter insect fitness We will continue to use the RNeasy Plant Mini kit for solation of RNA from roots and shoots which have been problematic using Trizol reagent, mainly because of the presence of inhibitors that affect cDNA synthesis. We will take advantage of RNA-seq data from another project involving pea aphid-Medicago-AM fungus interactions to select candidate genes for our potato aphid-potato-AM fungus interaction system. We will design primers for the remaining target genes. After optimizing the conditions for the three-way interaction experiments we will perform the plant physiology experiments (objective 2).
Impacts
What was accomplished under these goals?
This project investigates the interaction among potato, arbuscular mycorrhizal (AM) fungi, and insects of two feeding-guilds, the potato aphid (phloem-feeder), and the cabbage looper (chewing insect). The main goal of this project was to evaluate the experimental conditions to create a rigorous system to study tripartite interactions, and to provide insight that can serve to fully explore this system in the future to manage pests of economic importance in elite potato germplasm. This research could potentially lead to the implementation of management practices that balance an efficient AM symbiosis and limit insect outbreaks. In year 2, we studied two tripartite interactions: a) AM fungus, potato, aphids and b) AM fungus, potato, cabbage loopers. The research objectives were: 1) to evaluate the conditions to create a rigorous system to study AM fungus-potato-insect interactions, 2) to examine the effects of aboveground insect herbivory and belowground AM fungus colonization on potato plant physiology, and assess organism performance in these tripartite interactions, and 3) to determine the impact of mycorrhiza-induced resistance (MIR) in potato against insects of two feeding-guilds. Our work in year 2 has focused mainly on part of objective 1 and objective 3. We decided to postpone the plant physiology experiments (objective 2) until we further optimize the conditions for the three-way interactions. Objective 1: 1) Major activities completed / experiments conducted; Decided to revisit objective 1 (Aim 1a) to determine the conditions for successful potato root colonization by AM fungi. The School of Biological Sciences recently acquired two plant growth chambers, so we tested how many days it takes to reach the desired levels of AM fungus colonization under a more controlled environment. Previously, every experiment took around 100 days from the moment potato clones were propagated to harvest of plant tissue. Carried out an experiment using three levels of AM fungus colonization in our recently acquired growth chamber. Isolated total RNA from potato shoots (leaves and stems) and roots Tested 8 reference genes whose sequences were obtained from the literature in order to use 2 reference genes for quantitative PCR (qPCR). We now have two reference genes (elongation factor 1α and β tubulin) that can be used for potato shoot and root samples Carried out quantitative PCR (qPCR) analyses. 2) Data collected; Percent root length colonized by G. intraradices, insect count and weight, shoot fresh and dry weight, root fresh and dry weight, semi-quantitative PCR, and qPCR. 3) Summary statistics and discussion of results; AM fungus-potato interactions: We did not reduce the number of days for every experiment by growing plants in the growth chamber. This experiment took 128 days from the moment potato clones were propagated to harvest of plant tissues. The length of each experiment was mostly dependent on how fast the AM fungus is able to colonize roots. The goal of this experiment was to obtain three different levels of root colonization by AM fungi. We successfully obtained three levels of AM fungus colonization (25%, 39%, and 50%) that were significantly different (P=0.0001). However, these levels of AM fungus colonization did not alter fresh weights of shoots (P=0.1042), roots (P=0.0593) or tubers (P=0.2323). 4) Key outcomes or other accomplishments realized. No major accomplishments to report at this time. Objectives 2 & 3: 1) Major activities completed / experiments conducted; Given that we did not find statistical significance in both of the three-way interaction experiments (objective 3), we proposed a few changes (described in the 1st year report) to our future experiments. One of these changes involved testing different levels of root colonization by Glomus intraradices (low, medium, and high). Two separate experiments were conducted to identify the "best" levels of AM fungus colonization needed to alter cabbage looper and potato aphid fitness. After repeating these experiments twice (summer and fall 2015) we did not obtain reliable results because of unforeseen problems. We carried two three-way interaction experiments using three levels of AM fungus colonization. One experiment was completed and one experiment is ongoing. 2) Data collected; Percent root length colonized by G. intraradices, potato aphid count and weight, weight of cabbage loopers pre- and post-feeding on potato, fresh and dry weights of shoots and roots. 3) Summary statistics and discussion of results; AM fungus-potato-cabbage looper interactions: To test the effect of root colonization by G. intraradices on cabbage looper's larval growth, we performed a three-way interaction experiment (non-mycorrhizal control; low, medium and high AM fungus level; all + insects) for a feeding period of 7 days. We increased the number of replicates to 8 potted-plants per treatment, and used 5 caterpillars (2nd instar) per plant. Insects were added when plants reached the three levels of colonization desired. We found that cabbage looper weight gain was statistically significant among treatments (P=0.069). Cabbage loopers that fed on mycorrhizal plants that had low levels of AM fungus colonization showed the least weight gain compared to cabbage loopers that fed on highly mycorrhizal plants (P=0.0224). Cabbage loopers that fed on non-mycorrhizal plants compared to mycorrhizal plants with low levels of AM fungus colonization showed slightly less weight gain (P=0.0682) but was not significant. Based on these promising results, we are conducting an experiment including all four treatments (± low level AM fungus; ±insects), 8 replicates, and 2 time-points for insect feeding (6 and 12 days). We also decided to use cabbage loopers that are between 1st and 2nd instar. There was difference only in shoot dry weight accumulation (P=0.0003). Highly mycorrhizal plants showed increased shoot dry weight compared to non-mycorrhizal plants and to mycorrhizal plants with medium level of AM fungus colonization. AM fungus-potato-potato aphid interactions: We are currently performing a three-way interaction experiment (non-mycorrhizal control; low, medium and high AM fungus level; all + insects) to test the effect of root colonization by G. intraradices on potato aphid abundance using 2 insect-feeding times (10 and 15 days). We increased the number of replicates to 6 potted-plants per treatment, and used 8 potato aphids per plant. We are conducting an experiment including all four treatments (± low level AM fungus; ±insects), 6 replicates, and 3 time-points for insect feeding (1, 10 and 15 days). 4) Key outcomes or other accomplishments realized. Preliminary results suggest that the level of AM fungus colonization affects plant physiology in a way that alters insect fitness
Publications
|
Progress 02/01/14 to 01/31/15
Outputs
Target Audience: This project ensured the participation and training of undergraduate and graduate students as was indicated in the Project Initiation. Three undergraduate students - Sean Mahaney, Moyinoliwa Natasha Ogunnowo, and Sara LeFevre, were recruited to work on different aspects of the project. Undergraduate students received training in areas such as: potato tissue culture, basic laboratory techniques, arbuscular mycorrhizal (AM) fungus propagation, and experimental design. Currently, one undergraduate student, Sara LeFevre, has been working as a lab technician to maintain potato plantlets and AM fungal spores. Two graduate students, Andrew Schoenherr and Eric Rizzo, were recruited in June and August of 2014 to carry out all the experiments that were proposed in this 2-year project. Both graduate students have been provided with hands-on training in plant physiology, plant molecular biology, fungal ecology, and entomology. In addition, graduate students had the opportunity to interact with scientists from other institutions, Dr. Patricia Manosalva (now at University of California Riverside), Dr. Paul Ode (Colorado State University), and Dr. Whitney Cranshaw (Colorado State University). Dr. Manosalva traveled to Greeley, Colorado (October 1-4, 2015) to attend our proposed planning meeting in year 1. She worked closely with both graduate students providing them with bioinformatics training to find target potato sequences needed for primer design and gene expression analyses. Dr. Ode has provided us with cabbage loopers from his colony, and Dr. Cranshaw has provided us recently with green peach aphids. Both graduate students gave oral presentations about their projects during our Academic Excellence Week (April 9, 2015) at the University of Northern Colorado acknowledging the USDA for funding this project. Dr. S. Karen Gomez (PD) will be presenting a poster at the Project Directors' meeting that will take place at the USDA (Washington, DC) on April 28-29, 2015. Changes/Problems: We did not obtain significant damage on potato plants by using 3 cabbage looper larvae per plant, so we will increase the number of larvae per plant (from 3 to ≥5), and will continue to use larvae of approximately 5 mm in length. We will test the impact of different levels of root colonization by G. intraradices (low, medium, and high) on insect fitness. We expect that the levels of root colonization by G. intraradices needed to alter cabbage looper and potato aphid fitness will be different, so two separate experiments will be conducted to identify the best level of colonization for each three-way interaction. Additional "test" pots containing mycorrhizal plants are being used to assess the level of G. intraradices colonization prior to adding the insects to the plants. We are encountering difficulties rearing potato aphids in the lab, so we obtained green peach aphids that will be tested in our future experiments. Dr. Cranshaw (collaborator) will be looking for potato aphids in the field this summer. We are testing a commercial inoculant that contains a mixture of spores from different species of AM fungi because it is taking about 6 months for G. intraradices to produce spores that can be used in our experiments. We are trying to propagate a different AM fungus (Diversispora epigaea) as back-up. We will test commercial kits used for the isolation of RNA from potato roots which has been problematic because of the presence of inhibitors that affect cDNA synthesis. We are using a new enzyme for cDNA synthesis that is more resistant to the presence of inhibitors in the samples and has proven effective using RNA from shoot samples. A no-cost extension will be requested to compensate for the delay in recruiting key personnel. What opportunities for training and professional development has the project provided? Three undergraduate students - Sean Mahaney, Moyinoliwa Natasha Ogunnowo, and Sara LeFevre, were recruited to work on specific aspects of the project. All three undergraduate students were trained by Dr. Gomez (PD) in areas such as: potato tissue culture, basic laboratory techniques, AM fungus propagation, and experimental design. Two M.S. graduate students, Andrew Schoenherr and Eric Rizzo are also being mentored by Dr. Gomez. Both graduate students were provided with hands-on training in plant physiology, plant molecular biology, fungal ecology, and entomology. Graduate students had the opportunity to interact with Dr. Manosalva when she attended our 1st annual meeting in Greeley, CO. She provided training in bioinformatics to find target potato sequences needed for primer design and gene expression analyses (qPCR). Both graduate students recently gave oral presentations about their projects on Research Day (April 9, 2015) at the University of Northern Colorado. Andrew Schoenherr has served as mentor to an undergraduate student, Sean Byrne, who is enrolled in Directed Studies (Bio 422). Both of them conducted a small three-way interaction experiment (±AM fungus; +insects) and prepared a poster for Research Day that was presented by Sean. How have the results been disseminated to communities of interest? News Releases about the Project: UNC Faculty Receive Combined $645,000 in USDA Grants - University of Northern Colorado http://www.unco.edu/news/releases.aspx?id=6446 UNC biologists get USDA grants to address cattle, potato issues - Greeley Tribune http://www.greeleytribune.com/news/10579349-113/agriculture-crop-grants-project Rocky Mountain Ag Notebook: Wyo., Colo. among the most 'agribusiness-friendly' states; Water rights bill passes House - The Fence Post http://www.thefencepost.com/news/10611744-113/state-colorado-program-beef What do you plan to do during the next reporting period to accomplish the goals? Given that we did not find statistical significance in both of the three-way interaction experiments, we are considering the following changes to our future experiments: We will increase the number of cabbage looper larvae per plant (from 3 to ≥5), and will continue using larvae of approximately 5 mm in length. 3rd instar larvae as we initially proposed caused excessive defoliation especially in the later time points. We will test different levels of root colonization by G. intraradices (low, medium, and high) because it was reported in another system (pea aphid-broad bean-AM fungi) that aphid abundance differs based on the level of AM fungi colonization. We also found similar results in our pea aphid-barrel medic-AM fungus system. Two separate experiments will be conducted to identify the best level of AM fungus colonization needed to alter cabbage looper and potato aphid fitness. Additional "test" pots containing mycorrhizal plants are being used in each experiment to assess the level of G. intraradices colonization prior to adding the insects to the experimental plants. We are encountering difficulties rearing potato aphids in the lab, so we obtained green peach aphids that will be used in future experiments. It is taking about 6 months for G. intraradices to produce spores in our sterile cultures, so we are testing a commercial inoculant that contains a mixture of spores from different species of AM fungi using barrel medic plants. We will test commercial kits used for the isolation of RNA from roots which has been problematic using our current method, mainly because of the presence of inhibitors that affect cDNA synthesis. We will design primers for the remaining target genes. After optimizing the conditions for the three-way interaction experiments we will perform the plant physiology experiments (objective 2). Because the start date of the project was after the spring semester had already started (February 1st, 2014), the PD was not able to hire M.S. graduate students until the fall semester (August, 2014). Only one of the graduate students was able to start working in June of 2014. We will be requesting a no-cost extension to compensate for the delay in recruiting key personnel.
Impacts
What was accomplished under these goals?
In nature, plants form beneficial or detrimental interactions with organisms in soil and aboveground. Root associations with arbuscular mycorrhizal (AM) fungi provide nutrients to the plant, while insect pests are deleterious. This project investigates the commonly occurring interaction among potato, AM fungi, and insects of two feeding-guilds, the potato aphid (phloem-feeder), and the cabbage looper (chewing insect). The main goal of this project was to evaluate the experimental conditions to create a rigorous system to study tripartite interactions, and to provide insight that can serve to fully explore this system in the future to manage pathogens and pests of economic importance in elite potato germplasm. This research could potentially lead to the implementation of management practices that balance an efficient AM symbiosis and limit insect outbreaks. In year 1, we studied two tripartite interactions: a) AM fungus-potato-aphids and b) AM fungus-potato-cabbage loopers. The research objectives were: 1) to evaluate the conditions to create a rigorous system to study AM fungus-potato-insect interactions, 2) to examine the effects of aboveground insect herbivory and belowground AM fungus colonization on potato plant physiology, and assess organism performance in these tripartite interactions, and 3) to determine the impact of mycorrhiza-induced resistance (MIR) in potato against insects of two feeding-guilds. Our initial work has focused on objective 1 and part of objectives 2 & 3. We decided to postpone the plant physiology experiments (objective 2) until we further optimize the conditions for the three-way interactions. Objective 1: 1) Major activities completed / experiments conducted; Evaluated soil substrates that promoted potato root colonization by the AM fungus Glomus intraradices. Carried out an experiment to study AM fungus-potato interactions. Conducted two separate experiments to study cabbage looper-potato interactions, and potato aphid-potato interactions. Organized the 1st annual meeting among participants. Carried out bioinformatics analysis to retrieve potato sequences that will be used to design primers for the gene expression analyses. Isolated total RNA from potato shoots (leaves and stems) and roots Carried out semi-quantitative PCR and quantitative PCR (qPCR) analyses. 2) Data collected; Percent root length colonized by G. intraradices, insect count and weight, shoot fresh weight, root fresh weight, semi-quantitative PCR, and qPCR. 3) Summary statistics and discussion of results; AM fungus-potato interactions: We found that a 7-day acclimation period of potato plantlets in soil prior to inoculation with AM fungi helped plants get more colonized by G. intraradices. We observed more consistent levels of root colonization by G. intraradices at 42 days post inoculation (dpi) compared to 28 dpi. As observed in other plant species, potato plants forming symbiosis with G. intraradices started to exhibit mycorrhiza-induced shoot growth compared to non-mycorrhizal potato both at 28 dpi (p=0.051) and 42 dpi (p=0.053). Cabbage looper-potato interactions: Because of the fast defoliation caused by cabbage looper feeding in our 1st experiment (±insects), we made the following modifications to the 2nd experiment (±insects): used smaller size of larvae, reduced the number of larvae to 3 per plant, increased the number of biological replicates, and adjusted the insect feeding periods (1, 5, and 10 days). In this experiment, insects were added at 53 days post transplanting (dpt). We found that shoot and root fresh weights were not significantly (p>0.05) affected by insect feeding. In potato shoots, the levels of transcripts of allene oxide synthase 1 (AOS1) and 12-oxophytodienoate reductase 3 (OPR3) - two jasmonic acid (JA) biosynthesis genes, and the MYC2 transcription factor - a JA signaling gene, were measured using qPCR. We found a modest increase in transcript levels of AOS1, OPR3 and MYC2 in insect-damaged leaves. These data indicated that we need to further optimize the size and number of larvae per plant, and the insect feeding periods to cause more damage to plants in future experiments. Potato aphid-potato interactions: Based on the cabbage looper-potato experiments, we also reduced the number of potato aphids to 5 per plant but we maintained the feeding periods (1, 10 and 15 days) that were initially proposed. In this experiment (±insects), potato aphids were added to plants at 50 dpt. Potato aphid-infested plants exhibited an adequate amount of damage that permitted us to successfully isolate RNA from shoots. We found that both shoot and root fresh weights were significantly (p<0.05) reduced in potato aphid-infested plants after 15 days of feeding. We also found a significant decrease in potato aphid abundance (p=0.035), and increased potato aphid weight (p=0.019) at 15 days. These results indicated that there was a problem with the aphids at 15 days, so we decided to confirm the results by repeating the experiment. 4) Key outcomes or other accomplishments realized. No major accomplishments to report at this time. Objectives 2 & 3: 1) Major activities completed / experiments conducted; Conducted two separate experiments to study AM fungus-potato-cabbage looper interactions (1 time-point for insect feeding) and AM fungus-potato-aphid interactions (3 time-points). 2) Data collected; Percent root length colonized by G. intraradices, potato aphid count and weight, weight of cabbage loopers pre- and post-feeding on potato, shoot fresh weight, and root fresh weight. 3) Summary statistics and discussion of results; AM fungus-potato-cabbage looper interactions: To test the effect of root colonization by G. intraradices on cabbage looper's larval growth, we performed a small three-way interaction experiment (±AM fungus; +insects) for a feeding period of 5 days. Insects were added to plants at 53 dpi with G. intraradices. Although the average fresh weight of larvae per plant was slightly reduced when they fed on mycorrhizal potato plants (30% G. intraradices colonization) compared to non-mycorrhizal potato plants, this difference was not statistically significant (p=0.153). This experiment is being conducted including all four treatments (±AM fungus; ±insects) and 3 time-points for insect feeding (1, 6, and 12 days). AM fungus-potato-potato aphid interactions: We performed a three-way interaction experiment (±AM fungus; ±insects) to test the effect of root colonization by G. intraradices on potato aphid abundance using three insect feeding times (5, 10, and 15 days). Potato aphids were added to plants at 49 dpi with G. intraradices. We found no significant difference (p>0.05) in aphid abundance and individual aphid weight when they fed either on mycorrhizal potato plants or non-mycorrhizal potato plants in the three feeding times. In addition, the levels of root colonization by G. intraradices were not significantly different (p>0.05) between aphid-infested mycorrhizal plants (32 & 47% colonization) and non-infested mycorrhizal plants (48 & 50% colonization) after 5 and 10 days of feeding, indicating that potato aphids did not hinder AM fungus colonization. Shoot and root fresh weight were not significantly different among the four treatments (p>0.05), except for shoot weight after 5 days of feeding (p=0.068), but this was caused by an outlier. Overall, these data indicated that the level of AM fungus colonization used in both experiments was not optimal to detect any differences between mycorrhizal and non-mycorrhizal plants. Future experiments will test different levels of AM fungus colonization. 4) Key outcomes or other accomplishments realized. No major accomplishments to report at this time.
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