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Carolyn Hartman
ESRM 404

auxin IAA, or the plant might also be

INTRODUCTION receiving adequate fixed nitrogen from
It is important to conduct its microbe symbionts.
physiological assays to characterize
endophyte strains. A physiological Chlorophyll content is correlated with
assay allows us to observe the nitrogen content because it is vital to
endophyte influences on plant height the chlorophyll molecule. Measuring
and biomass, nutritional health, chlorophyll content can give us an
stomatal conductance, stomatal idea of how much nitrogen is in the
density, and chlorophyll fluorescence. plant and if the nitrogen-fixing bacteria
For example, some strains may help have aided, with its main source of
the plant yield more biomass than nitrogen being the bacteria.
other strains. Chlorophyll fluorescence was
Diazotrophic endophytes can provide measured as an indication of plant
them with growth hormones, health and to determine if the plants
solubilize phosphates, produce were stressed. Stressed plants will
siderophores, or fix atmospheric fluoresce at a lower level due to
nitrogen into a form the plant can use. photosystem II damage.
Nitrogen is 1.5% of total plant Stomatal conductance was also
biomass [1], of that 1.5% about 75% measured as an indication of plant
is allocated to the leaves [2], and of health or stress. Stomatal
that 75% about 60% is allocated to conductance is a measure of gas
photosynthesis apparatuses [3]. This exchange between the plant and the
means plants with an association with atmosphere. Healthy plants will have a
nitrogen-fixing bacteria have more higher stomatal conductance.
nitrogen to allocate to chlorophyll and Related, stomatal density is correlated
thus photosynthesize more. Nitrogen to stomatal conductance because if
is central to the chlorophyll molecule. there are more stomata, there can be
Plant height was a physiology test to more gas exchange, thus stomatal
assess growth, which could indicate density is also an indicator of health or
that the plant is being supplied with stress in plants. We were also able to
either growth hormone such as the see if the stomata were open or
closed. Closed stomata could be an plants with 200 ml of Qubit (lacking
indicator of stress because stressed nitrogen) solution per pot once per
plants may want to keep their stomata week. On February 17th we began
closed in order to retain as much watering the corn plants with 500 ml
water as needed. of Qubit solution per pot.

MATERIALS AND METHODS Plant biomass was measured in

Eight corn seeds and eight tomato multiple ways. Plant height was
seeds were potted into 4 pots on measured in centimeters on a weekly
January 23rd. The seeds were given basis for 6 weeks. When the plants
about 3 days to germinate and on were harvested, leaf area was
January 26th the plants were measured with a leaf area meter in
inoculated with WW12 and Coffee J square centimeters. After the plants
strains. Later the plants were reduced were harvested and dried in a drying
to 2 plants per pot, to avoid oven for 5 days, the weight of dry
competition. roots and dry shoots were measured
in grams. With the dry root and shoot
The inoculation solution was biomass, we could also calculate the
produced by first measuring the root to shoot ratio.
optical density of NL-CCM culture
solution with the OD600. Next the Plant height was measured with a
needed volume of nitrogen free ruler or meter stick. For the height of
medium was calculated and pipetted corn plants, the leaves were stretched
into a tube with the culture pellet. The upwards and the highest point was
pellet was suspended within the measured. For tomato plants, the top
nitrogen free medium creating a total of the highest leaf was measured,
of 15 ml of each strain solution. A without being stretched.
total of 1 ml of the nitrogen free Chlorophyll content was measured
medium and culture solution was with a SPAD meter on the newest fully
pipetted onto each seedling. expanded leaf, approximately 3
This physiological assay was inches from the tip of the leaf for corn.
conducted in a greenhouse. The For tomato the newest fully expanded
conditions of the greenhouse were on leaf was used. The SPAD Meter
average 149.4 umol m-2 s-1 for light measures the amount of chlorophyll
intensity, 20.8 degrees Celsius, and present in a leaf, which correlates with
50.9% humidity. The experiment the greenness of the plant leaf and
started in the greenhouse on February nutritional condition of the plant,
2nd and the plants were harvested on including nitrogen. It works by
March 9th. measuring absorbance of the leaf in
the red and near-infrared regions.
Nitrogen limited soil and fertilizer were SPAD measurements were taken
used because we wanted to test the once per week beginning on February
strains ability to fix nitrogen and 9th.
supply it to the plant. We watered the
Stomatal conductance was measured 0.79-0.84. These measurements were
with the Porometer. The leaf also taken from the newest fully
Porometer measures stomatal expanded leaf and only once per
conductance of leaves. The plant, on March 2nd.
Porometer works by comparing the
conductance of a leaf with two known We used a leaf area meter to measure
conductance elements, and leaf are. The leaf area meter measures
comparing the humidity (water vapor leaf area by measuring the area of the
flux) measurements between them. shadow each leaf creates. We
By measuring stomatal conductance, measured the overall leaf area of each
we know how much CO2 and H2O plant after the plants were harvested
are going into the plant. Stomatal on March 9th.
conductance was measured on the RESULTS
newest fully expanded leaf. It only
measured once per plant, on February In both corn and tomato plants, plants
23rd. inoculated with Coffee J endophytes
were slightly taller than those
Stomatal density was measured by inoculated with WW12 and on
creating an imprint of the underside of average, both Coffee J and WW12
the leaf. To create the imprint, we inoculated plants were taller than the
painted a coat of clear nail polish on control plants. However, there was
the underside of a leaf, waited for it to not a significant difference.
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.

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


Corn 414.48 74.35 74.05 4.8785 1.406

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
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 variables such as lighting conditions,
In tomato plants, plants inoculated carbon dioxide conditions, and not a
with WW12 endophytes started off large enough sample size.
with significantly lower SPAD values In inoculated corn and tomato plants
and in the last week had significantly stomatal density differed. In corn
higher SPAD values. In corn plants, plants, plants inoculated with WW12
plants inoculated with WW12 had had a higher stomatal density, while
significantly higher stomatal density tomato plants inoculated with Coffee
than control and Coffee J inoculated J had a higher stomatal density. Corn
plants. Plants inoculated with WW12 is a monocot and tomato is a dicot.
also had a significantly lower stomatal
density than the control plants and Overall, Coffee J and WW12 impact
plants inoculated with Coffee J. on plant growth. WW12 did have
positive effects on plant growth as
Coffee J inoculated corn plants also well. Both grew more biomass than
had a significantly lower stomatal the control plants. This difference in
density than control plants and plants stomatal density may be due to the
inoculated with WW12. In tomato parallel veins in corn leaves and the
plants, plants inoculated with Coffee J net veins in tomato leaves. Leaves are
had a significantly higher stomatal where the stomata are located.
density than control plants and plants
inoculated with WW12 endophytes. REFERENCES

Corn and tomato plants inoculated [1] French CS, Briggs WR. 1989.
with WW12 and Coffee J had higher Photosynthesis : proceedings of the
dryweight biomasses and root to C.S. French Symposium on
shoot ratios than control plants. Photosynthesis, held in Stanford,
California, July 17- 23, 1988. New
In tomato plants, stomatal York: A.R. Liss; [2] Poorter H, Remkes
conductance was significantly higher C, Lambers H. 1990. CARBON AND
control plants than in plants NITROGEN ECONOMY OF 24 WILD-
inoculated with Coffee J and WW12. SPECIES DIFFERING IN RELATIVE
The control corn plants had a GROWTH-RATE. Plant Physiology
significantly different fluorescence in 94(2): 621-627; [3] Taiz L, Zeiger E.
comparison to plants inoculated with 2010. Plant physiology. Sunderland,
WW12. MA: Sinauer Associates.

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