Nitrogen Fixation by Endophytes

in Rice and Tomatoes and

Endophyte Mediated Drought
Stress Alleviation in Rice
Carolyn Hartman & Evora Glenn
 Context and Purpose
Fertilizer use
Nitrogen fertilizer is
sometimes overused
Leaches into
groundwater
Damages aquatic
ecosystems
Rice
One of the most important
crops in the world
About of the
worlds population
relies on rice for of
their daily calorie
intake
Monocot and Dicot
Interaction may vary

(Kandel 2015; http://sciencebitz.com/?page_id=597)

Nitrogen Fixation by Endophytes
in Tomatoes
Previous Research
WP5 in Glacier Variety Tomatoes
Visually healthier and taller
Significant difference in root and shoot
biomass
Increased fruit yield
95 fruits in inoculated
43 in uninoculated
Flowered earlier (counts at 1.5 months)
108 in inoculated
65 in uninoculated
No endophyte reisolated from fruit or
seed
Root weight of tomato-Glacier variety at 2.5 months

(Khan 2012)
Methods
1. Glacier Tomato - Lycopersicon lycopersicum
2. Inoculation - WP5
3. Planting
a. 10 plants for each group (6 groups)
i. Full nitrogen mock, mutant (-nifH), wild type
ii. No nitrogen mock, mutant, wild type
4. Growth period
a. Given 100 ml nutrient media twice per week (once per week before
week 6)
b. 12 hour spring lighting, 16 hour summer lighting starting at ~3
months
c. Randomize location weekly
d. 120 days of growth
5. Monthly measurements
a. Height and SPAD
6. Final assessments
a. Dry weight of roots and shoots
b. Tomato count and mass
c. Attempt to reisolate mutant WP5 endophyte from leaves and petioles
Results

No WP5 endophytes reisolated from leaves and petioles

Results
Discussion
WP5
May not have been resistant to Km anymore
May have not colonized the tomato plants, or just not leaves and petioles
WP5 may have lost effectiveness over time
Tomato plants
Not healthy
Nitrogen Fixation by
Endophytes in Rice
Previous Research
Endophyte inoculation increases root and
shoot biomass in rice
Significantly more tillers at 3 months
Nitrogenase (nifH) gene
Can fix atmospheric nitrogen
Phytohormone production root + shoot, root, shoot biomass - multiple strains
Promotes growth
Other nutrient availability
P, Fe
ACC Deaminase
Decreased ethylene
Causes plants to age and die

(Kandel 2015)
High CFU in roots (left); mock vs. WP5 inoculated biomass (right)
Methods
1. Rice - M206
2. Surface and antibiotic sterilization
a. Antibiotics mixed with 15N
i. Stable isotope label
3. Inoculation
a. Plated, liquid culture, washed, incubated overnight in NFM,
measured at an OD of 0.1, inoculated with 25 ml
4. Planting
a. 10 plants for each group (6 groups)
i. Full nitrogen mock, mutant (-nifH), wild type
ii. No nitrogen mock, mutant, wild type
b. Mock planted first
5. Growth period
a. Grown in sterile (autoclaved twice) 2-tier vessels
b. Media changed every 2 weeks
c. Bagged when outgrown caps
i. Bag eventually removed
6. Measurements
a. Weekly visual assessment
Results

Rice plants after ~3 months of growth

Discussion
Length of experiment might have been too long
Sterile vessels might not have been a suitable growing environment
Contamination
of vessels needed to be remade before planting
WP5 might not have colonized, like with the tomatoes
WP5 may have lost viability over time
Endophyte Mediated Drought
Stress Alleviation in Rice
 Context/background
Importance of Rice
Rice is one of the most important crops in the
world
About of the worlds population relies on rice
for of their daily calorie intake (Kandel et al.
2015)

Climate Change
Rising temperatures are predicted to increase
frequency and severity of drought

Drought
Reduces crop yield
Increase the likelihood of disease and pests
ws/hot-topics/rice-and-climate-change)
Hypothesis/Previous research
Published Works:
Hypothesis:
the endophyte-inoculated rye grass had more Consortia inoculated M206 rice plants
will survive pulses of drought,
root mass, greater average weights, longer
showing more resilience than
roots, taller shoots and survived longer when uninoculated controls.
water stressed [than non-inoculated, no stress
control] (Khan et al. 2012). Purpose: To identify which individual
strains confer the most drought stress
Inoculated rice plants have been seen to alleviation.
develop greater shoot and root biomass than
uninoculated controls in nutrient limiting
conditions (Kandel et al. 2015).
 Methods
Surface sterilization:
3 % bleach, 1 drop detergent soak
Internal sterilization: Soak 2-3 days in Antibiotic solution
Inoculate seeds: Streak, liquid culture, measure OD, add
liguid culture to NFM, let soak 2-3 days
Plant seeds: Sunshine mix #4
Growth period:
Reduced edge effects
Watered to soil saturation
Randomized pot position
14.5 hrs light/day (summer conditions)
Drought period: Various, current exp.
3 days
Recommenced watering: duration 7 days
Recommenced drought: 4 days
Recommenced watering: 3/6/17
Results
Trial 1:

Zero resilience
Possible endophyte mixing
Extended growing period

Trial 2:

Zero resilience
Visual signs of drought stress vs.
expected drought stress (dryness of
soil)
Trial 3: In Progress

100% survival of germinated plants (WW11 and consortia #10 did not germinate)
Recommenced drought periods
Apparent differences in water usage after first drought period.
 Discussion
Going Forward:
Watering method disturbing seeds gravitropism.
Not all seeds germinated before planting
Delayed above ground visual benefits may be due to an allocation of photosynthates to
below ground biomass that may result in advantageous nutrient acquisition over time
(Kandel et al. 2015).
Future experiments: Rice
Purpose: Identifying a smaller number of effective strains on drought stress alleviation
with which to make mutations and subsequent assays to gauge the mechanism by which
endophytes provide drought stress alleviation.

Possible Mechanisms:
Endophyte scavenging or preventing production of ROS (reactive oxygen species)
during drought stress.
Endophytes provide fixed nitrogen and phytohormones that help plants preserve
chlorophyll molecules in leaves, increasing adaptation to low water environment
Endophyte produced abscisic acid (ABA) could trigger stomatal closure.
Additionally, endophyte respired CO2 could be used by plants.
Endophyte produced ACC deaminase to reduce ethylene production (inhibits shoot
growth, stomatal closing, etc. )
(Khan et. al 2016)
Future Experiments: Tomato
Unexpected Results: Extracts of WP5 nifH mutant inoculated plants did not grow when
plated on media with kanamycin, and wild-type WP5 did not show benefits to the plant.

WP5 mutant unable to colonize?

To test we could inoculate seeds with wild type and mutant endophytes and plate out
the extracts.
WP5 Wild-type unable to colonize as well? Lost activity?
Prof. Doty is passaging the strain back through poplar tissue cultures to see if it can
regain its activity