effects of herbivore damage and nitrogen fertilizers on the selective benefits of the (medicago...

Effects of Herbivore Damage and Nitrogen Fertilizers on the Selective Benefits of the (Medicago truncatula) legumes —

(Sinorhizobium meliloti) Rhizobia Mutualism.

Shaina Short

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– 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


– 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


In the Rhizobium, flavonoid activates the gene regulator Nod D


The Nod gene is activated as the transcription factor, Nod D, binds tothe Nod Box, a DNA region.


Nod genes are produced. These enzymes are used in a metabolic pathway.


This metabolic pathway creates a Nod factor consisting of a chitinlike substance


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


– 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


– 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


•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


•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 responsestrain inoculations

Herbivore treatments

Method and Materials


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






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 responsestrain inoculations Herbivore treatments




Harvesting

S. meliloti rhizobia Distraction

—Hydrate and sterilize nodules

—Mix in to BYMA buffer solution






Harvesting

S. meliloti rhizobia Distraction

S. meliloti Strain Analysis

—Plate distracted S. meliloti on BYMA with solidified granulated agar

—Incubate plates at 30C

—Check for blue coloration

Mutualistic responsestrain inoculations Herbivore treatments


Results: mutualistic response strain inoculations

Figure 1: Effects of Rhizobia Inoculations and Nitrogen Treatments on Intial M.Truncatula Pod Production

0

5

10

15

20

25

30

ABS7 ABS7/WS480 WS480

Rhizobia Inoculation

Average Time Until First Pod (days)

France Population

France 4 with Nitrogen

Greece 6

Greece 6 with Nitrogen

Spain 9

Spain 9 with Nitrogen

Portugal 10

Portugal 10 with Nitrogen

-Greece and Portugal had earliest initial pod production with combination strain

-Spain had earliest initial pod production with combination strain only without added N2

Figure 2: Effects of S. Meliloti Inoculations and Nitrogen Treatments on Average M. truncatula Pod Mass

0

0.5

1

1.5

2

2.5

3



Average Pod Mass (g)

France Population


Greece 6


Spain 9


Portugal 10



-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 Rhizobia inoculations on Pod Mass p < 0.05

Nitrogen Treatments on pod mass p < 0.01

Figure 3: Effects of Rhizobia Inoculations and Nitrogen Treatments on Average M.Truncatula Pod Number

0

5

10

15

20

25

30



Average Pod Number

France Population


Greece 6


Spain 9


Portugal 10


-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


Figure 4: Effects of Rhizobia Inoculations and Nitrogen Treatments on First M.truncatula Flower

0

5

10

15

20

25

30

35



Average Time until First Flower (days)

France Population


Greece 6


Spain 9


Portugal 10


-All populations produced earliest flowers with combination strain and added N2

-The Spain population without added N2

consistently produced earliest flowers


-Without added N2 all populations had highest nodule number with the combination strain

-Greece, Portugal, and France with added N2 produced most nodules with ABS7

Rhizobia on Nodulation p < 0.05

Population on nodulation p < 0.05

Figure 5: Effects of Rhizobia Inoculation and Nitrogen Treatments on M.truncatula Average Nodule Development

0

20

40

60

80

100

120

140

160

180

200



Average Nodule Number

France Population


Greece 6


Spain 9


Portugal 10



Figure 6: Effects of Rhizobia Inoculation and Nitrogen Treatments on M.truncatula Above-ground Biomass

0

0.5

1

1.5

2

2.5

3



Average Above-ground Biomass

France Population


Greece 6


Spain 9


Portugal 10


-All populations had an increase in above-ground biomass with added nitrogen.

-France and Spain without N2 produced greatest above-ground biomass with the combination strain



Figure 7: Average Vine Length of the France Populations

0

5

10

15

20

25

30

35

0 2 4 6 8

Date of Data Collection

Vine Length in cm

ABS7

ABS7 with nitrogentreatment

ABS7-WS480

ABS7-WS480 with nitrogentreatment

WS480

WS480 with nitrogentreatment

Figure 8: Average Vine Length of the Greece Population

0

10

20

30

40

50

0 2 4 6 8


Vine Length in cm

ABS7

ABS7 with nitrogen treatment

ABS7-WS480

ABS7-WS480 with nitrogentreatment

WS480


Figure 9: Average Vine Length of the Spain Populations

05

10152025303540

0 2 4 6 8


Vine Length in cm

ABS7

ABS7 with nitrogentreatmentABS7-WS480

ABS7-WS480 withnitrogen treatmentWS480


Figure 10: Average Vine Length of the Portugal Populations

05

1015202530354045

0 2 4 6 8


Vine Length in cm

ABS7

ABS7 with nitrogentreatment

ABS7-WS480

ABS7-WS480 withnitrogen treatment

WS480


-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

Figure 11: The Effects of Herbivore Treatments on Nodule Numbers of the Spain and Portugal Populations

0

2

4

6

8

10

12

14

16

1 21 70 1 21 70

Time of Herbivore Treatment (days)

Average Nodule Number

Population 9

Population 10

Imposed Herbivore Treatment on day 3

-Spain increased nodule development with herbivore

Mutualistic response strain inoculations

• The ABS7-WS480 rhizobia strain inoculation proved to be the most powerful symboiant.

Conclusion



• ABS7 proved to produce the stronger symbiotic relationship

Conclusion




• WS480 strain enhanced the strength of the legume-rhizobia relationship when combined with ABS7.

Conclusion




• 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

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

effects of herbivore damage and nitrogen fertilizers on the selective benefits of the (medicago...

Documents

purposes slide

ws480 nitrogen

meliloti rhizobia strains

legume leaflets introduction

herbivore treatments

chitinlike substance

shaina short slide

gene regulator nod d