Effects of Herbivore Damage and Nitrogen Fertilizers on
the Selective Benefits of the (Medicago truncatula) legumes —
(Sinorhizobium meliloti) Rhizobia Mutualism.
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Shaina Short
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
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: 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
Purposes
• 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.
Hypotheses
•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.
Method and Materials
Mutualistic response
strain inoculations
Herbivore treatments
Preparing and Growing Seeds
—Clean seeds
—Randomize populations
—Record fitness levels (first flowering, leaf number,
vine lengths, first pod, pod number)
Method and Materials
Mutualistic response
strain inoculations
Herbivore treatments
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
Method and Materials
Herbivore treatments
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
Method and Materials
Mutualistic response
strain inoculations
Herbivore treatments
Preparing and Growing Seeds
Legumes Inoculations
Harvesting
S. meliloti rhizobia Distraction
—Hydrate and sterilize nodules
—Mix in to BYMA buffer solution
Method and Materials
Mutualistic response
strain inoculations
Herbivore treatments
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
Results: mutualistic response strain inoculations
Figure 1: Effects of Rhizobia Inoculations and Nitrogen Treatments
on Intial M.Truncatula Pod Production
30
Average Time Until First Pod (days)
25
20
France Population
France 4 with Nitrogen
Greece 6
Greece 6 with Nitrogen
Spain 9
Spain 9 with Nitrogen
Portugal 10
Portugal 10 with Nitrogen
15
10
5
0
ABS7
ABS7/WS480
Rhizobia Inoculation
WS480
-Greece and Portugal
had earliest initial pod
production with
combination strain
-Spain had earliest
initial pod production
with combination strain
only without added N2
Results: mutualistic response strain inoculations
Figure 2: Effects ofS. Meliloti Inoculations and Nitrogen
Treatments on AverageM. truncatula Pod Mass
3
Average Pod Mass (g)
2.5
2
France Population
France 4 with Nitrogen
Greece 6
Greece 6 with Nitrogen
Spain 9
Spain 9 with Nitrogen
Portugal 10
Portugal 10 with Nitrogen
1.5
1
-N2 treated France and
Greece produced
greater pod mass than
those without added
N2
-Spain produced a
greater pod mass
without added N2 and
the combination
strains
0.5
Rhizobia inoculations
on Pod Mass p < 0.05
0
ABS7
ABS7/WS480
Rhizobia Inoculation
WS480
Nitrogen Treatments
on pod mass p < 0.01
Results: mutualistic response strain inoculations
Figure 3: Effects of Rhizobia Inoculations and Nitrogen Treatments
on Average M.Truncatula Pod Number
30
Average Pod Number
25
20
France Population
France 4 with Nitrogen
Greece 6
Greece 6 with Nitrogen
Spain 9
Spain 9 with Nitrogen
Portugal 10
Portugal 10 with Nitrogen
15
10
5
0
ABS7
ABS7/WS480
Rhizobia Inoculation
WS480
-Average Pod
Number was greatest
for Spain and
Portugal with the
combination strain
and without added
N2
-Average pod
number was greatest
for France and
Greece with added
N2
Results: mutualistic response strain inoculations
Figure 4: Effects of Rhizobia Inoculations and Nitrogen Treatments
on First M.truncatula Flower
35
Average Time until First Flower (days)
30
-All populations
produced earliest
flowers with
combination strain
and added N2
25
France Population
France 4 with Nitrogen
Greece 6
Greece 6 with Nitrogen
Spain 9
Spain 9 with Nitrogen
Portugal 10
Portugal 10 with Nitrogen
20
15
10
5
0
ABS7
ABS7/WS480
Rhizobia Inoculation
WS480
-The Spain
population without
added N2
consistently
produced earliest
flowers
Results: mutualistic response strain inoculations
Figure 5: Effects of Rhizobia Inoculation and Nitrogen Treatments
on M.truncatula Average Nodule Development
200
180
Average Nodule Number
160
-Without added N2
all populations had
highest nodule
number with the
combination strain
140
France Population
France 4 with Nitrogen
Greece 6
Greece 6 with Nitrogen
Spain 9
Spain 9 with Nitrogen
Portugal 10
Portugal 10 with Nitrogen
120
100
80
60
40
20
0
ABS7
ABS7/WS480
WS480
-Greece, Portugal,
and France with
added N2 produced
most nodules with
ABS7
Rhizobia on
Nodulation p < 0.05
Rhizobia Inoculation
Population on
nodulation p < 0.05
Results: mutualistic response strain inoculations
Figure 6: Effects of Rhizobia Inoculation and Nitrogen Treatments
on M.truncatula Above-ground Biomass
3
Average Above-ground Biomass
2.5
2
France Population
France 4 with Nitrogen
Greece 6
Greece 6 with Nitrogen
Spain 9
Spain 9 with Nitrogen
Portugal 10
Portugal 10 with Nitrogen
1.5
1
0.5
0
ABS7
ABS7/WS480
Rhizobia Inoculation
WS480
-All populations had
an increase in aboveground biomass with
added nitrogen.
-France and Spain
without N2 produced
greatest aboveground biomass with
the combination
strain
Results: mutualistic response strain inoculations
Figure 8: Average Vine Length of the
Greece Population
ABS7
35
30
ABS7 with nitrogen
treatment
ABS7- WS480
25
20
15
ABS7- WS480 with nitrogen
treatment
WS480
10
5
0
0
2
4
6
8
Vine Length in cm
Vine Length in cm
Figure 7: Average Vine Length of the France
Populations
40
ABS7 with nitrogen treatment
30
ABS7-WS480
20
ABS7-WS480 with nitrogen
treatment
10
WS480
0
0
WS480 with nitrogen
treatment
Date of Data Collection
ABS7
50
2
4
6
8
WS480 with nitrogen
treatment
Date of Data Collection
-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)
ABS7
ABS7 with nitrogen
treatment
ABS7- WS480
40
35
30
25
20
15
10
5
0
ABS7- WS480 with
nitrogen treatment
WS480
0
2
4
Date of Data Collection
6
8
Figure 10: Average Vine Length of the
Portugal Populations
WS480 with nitrogen
treatment
Vine Length in cm
Vine Length in cm
Figure 9: Average Vine Length of the
Spain Populations
45
40
35
30
25
20
15
10
5
0
ABS7
ABS7 with nitrogen
treatment
ABS7-WS480
ABS7-WS480 with
nitrogen treatment
WS480
0
2
4
Date of Data Collection
6
8
WS480 with nitrogen
treatment
Results: herbivore treatments
Figure 11: The Effects of Herbivore Treatments on
Nodule Numbers of the Spain and Portugal Populations
-Spain increased
nodule development
with herbivore
16
14
Average Nodule Number
12
10
8
Population 9
Population 10
6
4
2
0
1
21
70
1
Time of Herbivore Treatment (days)
21
70
I mpos ed H erbivore T reatment on day 3
Conclusion
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
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.
Acknowledgements
Peter Tiffin
Katy Heath
Lois Fruen
Breck research team
Effects of Herbivore Damage and Nitrogen Fertilizers on
the Selective Benefits of the (Medicago truncatula) legumes —
(Sinorhizobium meliloti) Rhizobia Mutualism.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Shaina Short