Effects of Herbivore Damage and Nitrogen Fertilizers on the Selective Benefits of the (Medicago truncatula) legumes — (Sinorhizobium meliloti) Rhizobia Mutualism. Shaina Short

–Native Medicago truncatula legume populations Protein based plants France, Greece, Spain, Portugal – Sinorhizobium meliloti rhizobial strains ABS7 and WS480 Nitrogen fixing bacteria ABS7 lab cultured strain, WS480 European strain Introduction: variables investigated

–Native Medicago truncatula legume populations Protein based plants France, Greece, Spain, Portugal – Sinorhizobium meliloti rhizobial strains ABS7 and WS480 Nitrogen fixing bacteria ABS7 lab cultured strain, WS480 European strain – Nitrogen fertilizer treatments Introduction: variables investigated

–Native Medicago truncatula legume populations Protein based plants France, Greece, Spain, Portugal – Sinorhizobium meliloti rhizobial strains ABS7 and WS480 Nitrogen fixing bacteria ABS7 lab cultured strain, WS480 European strain – Nitrogen fertilizer treatments – Imposed herbivore treatments on legume leaflets Introduction: variables investigated

Introduction: Background on nodulation Root releases a signal in the form of a flavonoid detected by only Rhizobia

Introduction: Background on nodulation In the Rhizobium, flavonoid activates the gene regulator Nod D

Introduction: Background on nodulation The Nod gene is activated as the transcription factor, Nod D, binds to the Nod Box, a DNA region.

Introduction: Background on nodulation Nod genes are produced. These enzymes are used in a metabolic pathway.

Introduction: Background on nodulation This metabolic pathway creates a Nod factor consisting of a chitinlike substance

Introduction: Background on nodulation The Nod Factor signals the initiation of the Rhizobia-legume symbiotic relationship by producing an infected thread in the root, ultimately developing nodules

Investigate mutualistic behaviors shared between four M. truncatula legume populations and two S. meliloti rhizobia strains with and without nitrogen and herbivore treatments. Purposes

Investigate mutualistic behaviors shared between four M. truncatula legume populations and two S. meliloti rhizobia strains with and without nitrogen and herbivore treatments. –The study focused on the legume’s mutual benefits by measuring legume fitness levels through pod mass, flowering, growth rates, and above ground biomass. Purposes

Investigate mutualistic behaviors shared between four M. truncatula legume populations and two S. meliloti rhizobia strains with and without nitrogen and herbivore treatments. –The study focused on the legume’s mutual benefits by measuring legume fitness levels through pod mass, flowering, growth rates, and above ground biomass. –The study focused on the mutualistic behavior of rhizobia through nodule development. Purposes

Legumes varying in genomes vary in preference towards rhizobia strain. K. P. Smith et al. Hypotheses

Legumes varying in genomes vary in preference towards rhizobia strain. Herbivore treatments directly disrupt the Krebs cycle and photosynthesis of legumes, making it difficult for the legume to develop extensive root systems in order to obtain carbon. Therefore, the legumes are eager to express mutual behaviors. D. A. Samac et al. J. S. Pate Hypotheses

Legumes varying in genomes vary in preference towards rhizobia strain. Herbivore treatments directly disrupt the Krebs cycle and photosynthesis of legumes, making it difficult for the legume to develop extensive root systems in order to obtain carbon. Therefore, the legumes are eager to express mutual behaviors. Increased nitrogen levels will have negative effects on mutual behaviors of legumes because it will make it easier for the legumes to obtain nitrogen with limited root growth. F.D. Dakora et al. Hypotheses

Preparing and Growing Seeds —Clean seeds —Randomize populations —Record fitness levels (first flowering, leaf number, vine lengths, first pod, pod number) Mutualistic response strain inoculations Herbivore treatments Method and Materials

Preparing and Growing Seeds Legumes Inoculations —Dilute rhizobia strains and combination strain (10^8 cell/ml OD 0.1) —Inject 1 mL of rhizobia strain or combination into surrounding soil Mutualistic response strain inoculations Herbivore treatments Method and Materials

Preparing and Growing Seeds Legumes Inoculations Harvesting —Remove legumes from the surrounding soil —Count nodules —Remove and dehydrate nodules from roots —Dry and store above and below-ground biomasses —Dehydrate the nodules in silicon filled Eppendorf tube Mutualistic response strain inoculations Herbivore treatments Method and Materials

Preparing and Growing Seeds Legumes Inoculations Harvesting S. meliloti rhizobia Distraction —Hydrate and sterilize nodules —Mix in to BYMA buffer solution Mutualistic response strain inoculations Herbivore treatments Method and Materials

Preparing and Growing Seeds Legumes Inoculations Harvesting S. meliloti rhizobia Distraction S. meliloti Strain Analysis —Plate distracted S. meliloti on BYMA with solidified granulated agar —Incubate plates at 30  C —Check for blue coloration Mutualistic response strain inoculations Herbivore treatments Method and Materials

Results: mutualistic response strain inoculations -Greece and Portugal had earliest initial pod production with combination strain -Spain had earliest initial pod production with combination strain only without added N 2

Results: mutualistic response strain inoculations -N 2 treated France and Greece produced greater pod mass than those without added N 2 -Spain produced a greater pod mass without added N 2 and the combination strains Rhizobia inoculations on Pod Mass p < 0.05 Nitrogen Treatments on pod mass p < 0.01

-Average Pod Number was greatest for Spain and Portugal with the combination strain and without added N 2 -Average pod number was greatest for France and Greece with added N 2 Results: mutualistic response strain inoculations

-All populations produced earliest flowers with combination strain and added N 2 -The Spain population without added N 2 consistently produced earliest flowers Results: mutualistic response strain inoculations

-Without added N 2 all populations had highest nodule number with the combination strain -Greece, Portugal, and France with added N 2 produced most nodules with ABS7 Rhizobia on Nodulation p < 0.05 Population on nodulation p < 0.05 Results: mutualistic response strain inoculations

-All populations had an increase in above- ground biomass with added nitrogen. -France and Spain without N 2 produced greatest above- ground biomass with the combination strain Results: mutualistic response strain inoculations

-Figures 7-10 show populations with nitrogen produced longer vine lengths. -Figures 7 and 8, the average vine lengths with ABS7+WS480 was intermediate to the vine length of legumes inoculated with ABS7 and WS480. (p < 0.05)

Results: herbivore treatments -Spain increased nodule development with herbivore

Mutualistic response strain inoculations The ABS7-WS480 rhizobia strain inoculation proved to be the most powerful symboiant. Conclusion

Mutualistic response strain inoculations The ABS7-WS480 rhizobia strain inoculation proved to be the most powerful symboiant. ABS7 proved to produce the stronger symbiotic relationship Conclusion

Mutualistic response strain inoculations The ABS7-WS480 rhizobia strain inoculation proved to be the most powerful symboiant. ABS7 proved to produce the stronger symbiotic relationship WS480 strain enhanced the strength of the legume-rhizobia relationship when combined with ABS7. Conclusion

Mutualistic response strain inoculations The ABS7-WS480 rhizobia strain inoculation proved to be the most powerful symboiant. ABS7 proved to produce the stronger symbiotic relationship WS480 strain enhanced the strength of the legume-rhizobia relationship when combined with ABS7. Herbivore Treatment Herbivore treatments increased the strength of the symbiotic relationship Conclusion

Practical Implication To learn how to induce this sugar and nitrogen uptake symbiotic relationship for similar protein-based crops. –How much nitrogen is beneficial

Practical Implication To learn how to induce this sugar and nitrogen uptake symbiotic relationship for similar protein-based crops. –How much nitrogen is beneficial To understand evolutions patterns of legume populations and rhizobia strains in order increase the efficiency of nitrogen fixation, ultimately improving crop growth. –To find the ideal strain for specific populations of legume.

Peter Tiffin Katy Heath Lois Fruen Breck research team Acknowledgements

Effects of Herbivore Damage and Nitrogen Fertilizers on the Selective Benefits of the (Medicago truncatula) legumes — (Sinorhizobium meliloti) Rhizobia Mutualism. Shaina Short