WW12 AND COFFEE J ENDOPHYTE AFFECT ON CORN
AND TOMATO PLANTS
Carolyn Hartman
ESRM 404
INTRODUCTION
It is important to conduct
physiological assays to characterize
endophyte strains. A physiological
assay allows us to observe the
endophyte influences on plant height
and biomass, nutritional health,
stomatal conductance, stomatal
density, and chlorophyll fluorescence.
For example, some strains may help
the plant yield more biomass than
other strains.
Diazotrophic endophytes can provide
them with growth hormones,
solubilize phosphates, produce
siderophores, or fix atmospheric
nitrogen into a form the plant can use.
Nitrogen is 1.5% of total plant
biomass [1], of that 1.5% about 75%
is allocated to the leaves [2], and of
that 75% about 60% is allocated to
photosynthesis apparatuses [3]. This
means plants with an association with
nitrogen-fixing bacteria have more
nitrogen to allocate to chlorophyll and
thus photosynthesize more. Nitrogen
is central to the chlorophyll molecule.
Plant height was a physiology test to
assess growth, which could indicate
that the plant is being supplied with
either growth hormone such as the
auxin IAA, or the plant might also be
receiving adequate fixed nitrogen from
its microbe symbionts.
Chlorophyll content is correlated with
nitrogen content because it is vital to
the chlorophyll molecule. Measuring
chlorophyll content can give us an
idea of how much nitrogen is in the
plant and if the nitrogen-fixing bacteria
have aided, with its main source of
nitrogen being the bacteria.
Chlorophyll fluorescence was
measured as an indication of plant
health and to determine if the plants
were stressed. Stressed plants will
fluoresce at a lower level due to
photosystem II damage.
Stomatal conductance was also
measured as an indication of plant
health or stress. Stomatal
conductance is a measure of gas
exchange between the plant and the
atmosphere. Healthy plants will have a
higher stomatal conductance.
Related, stomatal density is correlated
to stomatal conductance because if
there are more stomata, there can be
more gas exchange, thus stomatal
density is also an indicator of health or
stress in plants. We were also able to
see if the stomata were open or
closed. Closed stomata could be an
indicator of stress because stressed
plants may want to keep their stomata
closed in order to retain as much
water as needed.
MATERIALS AND METHODS
Eight corn seeds and eight tomato
seeds were potted into 4 pots on
January 23rd. The seeds were given
about 3 days to germinate and on
January 26th the plants were
inoculated with WW12 and Coffee J
strains. Later the plants were reduced
to 2 plants per pot, to avoid
competition.
The inoculation solution was
produced by first measuring the
optical density of NL-CCM culture
solution with the OD600. Next the
needed volume of nitrogen free
medium was calculated and pipetted
into a tube with the culture pellet. The
pellet was suspended within the
nitrogen free medium creating a total
of 15 ml of each strain solution. A
total of 1 ml of the nitrogen free
medium and culture solution was
pipetted onto each seedling.
This physiological assay was
conducted in a greenhouse. The
conditions of the greenhouse were on
average 149.4 umol m-2 s-1 for light
intensity, 20.8 degrees Celsius, and
50.9% humidity. The experiment
started in the greenhouse on February
2nd and the plants were harvested on
March 9th.
Nitrogen limited soil and fertilizer were
used because we wanted to test the
strains ability to fix nitrogen and
supply it to the plant. We watered the
plants with 200 ml of Qubit (lacking
nitrogen) solution per pot once per
week. On February 17th we began
watering the corn plants with 500 ml
of Qubit solution per pot.
Plant biomass was measured in
multiple ways. Plant height was
measured in centimeters on a weekly
basis for 6 weeks. When the plants
were harvested, leaf area was
measured with a leaf area meter in
square centimeters. After the plants
were harvested and dried in a drying
oven for 5 days, the weight of dry
roots and dry shoots were measured
in grams. With the dry root and shoot
biomass, we could also calculate the
root to shoot ratio.
Plant height was measured with a
ruler or meter stick. For the height of
corn plants, the leaves were stretched
upwards and the highest point was
measured. For tomato plants, the top
of the highest leaf was measured,
without being stretched.
Chlorophyll content was measured
with a SPAD meter on the newest fully
expanded leaf, approximately 3
inches from the tip of the leaf for corn.
For tomato the newest fully expanded
leaf was used. The SPAD Meter
measures the amount of chlorophyll
present in a leaf, which correlates with
the greenness of the plant leaf and
nutritional condition of the plant,
including nitrogen. It works by
measuring absorbance of the leaf in
the red and near-infrared regions.
SPAD measurements were taken
once per week beginning on February
9th.
Stomatal conductance was measured
with the Porometer. The leaf
Porometer measures stomatal
conductance of leaves. The
Porometer works by comparing the
conductance of a leaf with two known
conductance elements, and
comparing the humidity (water vapor
flux) measurements between them.
By measuring stomatal conductance,
we know how much CO2 and H2O
are going into the plant. Stomatal
conductance was measured on the
newest fully expanded leaf. It only
measured once per plant, on February
23rd.
Stomatal density was measured by
creating an imprint of the underside of
the leaf. To create the imprint, we
painted a coat of clear nail polish on
the underside of a leaf, waited for it to
dry, took it off with a piece of tape,
and placed it on a glass slide. We
then looked at the slide under a
microscope at x40 magnification.
Stomata were counted in a 0.152
mm2 area. These measurements
were also taken from the newest fully
expanded leaf and only once per
plant, on February 23rd.
0.79-0.84. These measurements were
also taken from the newest fully
expanded leaf and only once per
plant, on March 2nd.
We used a leaf area meter to measure
leaf are. The leaf area meter measures
leaf area by measuring the area of the
shadow each leaf creates. We
measured the overall leaf area of each
plant after the plants were harvested
on March 9th.
RESULTS
In both corn and tomato plants, plants
inoculated with Coffee J endophytes
were slightly taller than those
inoculated with WW12 and on
average, both Coffee J and WW12
inoculated plants were taller than the
control plants. However, there was
not a significant difference.

Chlorophyll fluorescence was

measured with a Fluorometer. The
Fluorometer measures chlorophyll Figure 1: Corn Height Over Time
fluorescence, which is the amount of
light fluoresced. We took the Fv/Fm
measurements. Fv/Fm measures
maximum quantum efficiency and has
been shown to correlate with carbon
assimilation. It is used for
fluorescence, plant stress detection,
and as an indicator of stress effects
on photosystem II. Healthy plants
have a Fv/Fm value in the range of
 Figure 4: Tomato SPAD Values Over Time
Figure 2: Tomato Height Over Time
In corn plants, plants inoculated with
In corn plants, there was no WW12 had slightly higher rates of
significant difference in SPAD values stomatal conductance, but they were
between the control, Coffee J, and not significant. In tomato plants,
WW12 inoculated plants. In tomato stomatal conductance was
plants, plants inoculated with WW12 significantly higher control plants than
endophytes started off with in plants inoculated with Coffee J and
significantly lower SPAD values and in WW12.
the last week had significantly higher
SPAD values. Control plants and
plants inoculated with Coffee J
endophytes remained relatively similar
throughout the 6 weeks, with the
exception of a significantly higher
value in Coffee J inoculated plants in
week 5.

Figure 5: Corn Stomatal Conductance

Figure 3: Corn SPAD Values Over Time

Figure 6: Tomato Stomatal Conductance

In corn plants, plants inoculated with fluorescence in comparison to plants
WW12 had significantly higher inoculated with WW12.
stomatal density than control and
Coffee J inoculated plants. Coffee J
inoculated plants also had a
significantly lower stomatal density
than control plants and plants
inoculated with WW12. In tomato
plants, plants inoculated with Coffee J
had a significantly higher stomatal
density than control plants and plants
inoculated with WW12 endophytes.
Plants inoculated with WW12 also Figure 9: Corn Chlorophyll Fluorescence

had a significantly lower stomatal

density than the control plants and
plants inoculated with Coffee J.

Figure 10: Tomato Chlorophyll Fluorescence

Overall, plants inoculated with

endophytes had a higher Dryweight
Figure 7: Corn Stomatal Density
mass and slightly higher root to shoot
ratios. (Shown in the table below)

Strain Leaf Root Shoot Total Root/

Area Length Length Dryweight Shoot
(cm2) (cm) (cm) (g) Ratio

Corn 353.45 55.67 74.15 3.3592 0.767

Control

Corn 414.48 74.35 74.05 4.8785 1.406

WW12
Figure 8: Tomato Stomatal Density
Corn 503.94 58 2.2755 4.591 1.012
Coffee J
In tomato and corn plants, the control
plants had the highest chlorophyll Tomato 131.51 33.583 21.9 0.96583 0.326
Control
fluorescence and plants inoculated
with WW12 had the lowest Tomato 166.55 36 25.55 1.7225 0.373
fluorescence. Only the control corn WW12

plants had a significantly different Tomato 164.08 30 25.8 1.734 0.329

Coffee J
DISCUSSION
In tomato plants, plants inoculated
with WW12 endophytes started off
with significantly lower SPAD values
and in the last week had significantly
higher SPAD values. In corn plants,
plants inoculated with WW12 had
significantly higher stomatal density
than control and Coffee J inoculated
plants. Plants inoculated with WW12
also had a significantly lower stomatal
density than the control plants and
plants inoculated with Coffee J.
Coffee J inoculated corn plants also
had a significantly lower stomatal
density than control plants and plants
inoculated with WW12. In tomato
plants, plants inoculated with Coffee J
had a significantly higher stomatal
density than control plants and plants
inoculated with WW12 endophytes.
Corn and tomato plants inoculated
with WW12 and Coffee J had higher
dryweight biomasses and root to
shoot ratios than control plants.
In tomato plants, stomatal
conductance was significantly higher
control plants than in plants
inoculated with Coffee J and WW12.
The control corn plants had a
significantly different fluorescence in
comparison to plants inoculated with
WW12.
These results were interesting
because in the physiological assays,
plants inoculated with Coffee J and
WW12 seemed to produce similar
results, but the microbiology assays
indicated that Coffee J could not fix
nitrogen, but WW12 could. This
connection may be loose due to other
variables such as lighting conditions,
carbon dioxide conditions, and not a
large enough sample size.
In inoculated corn and tomato plants
stomatal density differed. In corn
plants, plants inoculated with WW12
had a higher stomatal density, while
tomato plants inoculated with Coffee
J had a higher stomatal density. Corn
is a monocot and tomato is a dicot.
Overall, Coffee J and WW12 impact
on plant growth. WW12 did have
positive effects on plant growth as
well. Both grew more biomass than
the control plants. This difference in
stomatal density may be due to the
parallel veins in corn leaves and the
net veins in tomato leaves. Leaves are
where the stomata are located.
REFERENCES
[1] French CS, Briggs WR. 1989.
Photosynthesis : proceedings of the
C.S. French Symposium on
Photosynthesis, held in Stanford,
California, July 17- 23, 1988. New
York: A.R. Liss; [2] Poorter H, Remkes
C, Lambers H. 1990. CARBON AND
NITROGEN ECONOMY OF 24 WILD-
SPECIES DIFFERING IN RELATIVE
GROWTH-RATE. Plant Physiology
94(2): 621-627; [3] Taiz L, Zeiger E.
2010. Plant physiology. Sunderland,
MA: Sinauer Associates.