Endophyte Mediated Drought Stress Alleviation in Rice and Nitrogen Fixation by

Endophytes in Rice and Tomatoes

Carolyn Hartman

Abstract

With a changing climate, rising temperatures are predicted to increase frequency and severity of
drought. Along with drought, there is a need to increase crop production, and expanding
agriculture into areas with poor quality soil is one solution. Approximately 78% of the Earths
atmosphere consists of nitrogen; however, plants cannot use nitrogen that is available in the
atmosphere, since it is in the form of dinitrogen gas. The current dominant nitrogen solution in
agriculture is chemical nitrogen fertilizer, which can be harmful to aquatic ecosystems and
pollutes groundwater if used in large quantities. This project focuses on the uses of microbial
endophytes in agriculture. Endophytes are bacteria and fungi that live within plants. Some strains
of endophytes have been shown to increase drought stress tolerance and nitrogen availability. In
Prof. Dotys lab, we have tested how individual and a consortium of endophyte strains mediate
drought stress alleviation in rice by subjecting the plants to a period of drought followed by a
rehydration period. We anticipate the plants that were inoculated with endophytes to survive the
drought period and recover during the rehydration phase. We have also tested one endophyte
strain for nitrogen fixation in rice and tomato plants by inoculating the plants with the wild strain
and nitrogen fixation mutant strain. If the experimental results are as anticipated, endophytes
could potentially be a more sustainable solution for agricultural crops under drought and nutrient
stress.

Introduction

Rice is a vital crop for more than half of the worlds growing population (International Rice
Research Institute). With a changing climate, the rising temperatures are predicted to increase
frequency and intensity of drought. Drought can significantly reduce crop yield and increase the
likelihood of disease and pest infestations (NASA). When a plant does not receive enough water
it can decrease plant growth and vegetation production (Xu, 2010).

Approximately 78% of Earths atmosphere consists of nitrogen; however, plants cannot use the
nitrogen that is available in the atmosphere, since it is in the inert form of dinitrogen gas.
Chemical fertilizer exists in the form of ammonium and nitrate can be harmful to the
environment if too much is applied. Heavy rainfall or excess watering can wash the concentrated
nitrogen that is not used all at once by the plant from an agriculture field into surface water and
groundwater (McMcKague, 2011). Groundwater is polluted mainly by nitrates and is an
important source of drinking water. Fertilization of surface waters can result in eutrophication
and cause algal blooms in aquatic ecosystems, which causes disruptive changes in the ecosystem.
More algae present will consume dissolved oxygen that fish need to survive. This is true for both
inland and coastal waters (Food and Agriculture Administration).

Endophytes are bacteria and fungi that live within plants (Wilson, 1995). Some strains of
endophytes have been shown to increase growth by increasing nutrient availability and hormone
production. They can also increase stress tolerance such as drought and temperature stress in
return for sugars from the plant host (Vandenkoornhuyse, 2015). Previous studies have shown
that diazotrophic (nitrogen fixing) endophytes can improve physical characteristics of rice and
tomatoes, such as root and shoot growth in nitrogen limited conditions (Kandel, 2015 and Khan,
2012). Endophytes have a broad host range, including both monocots, dicots and even
gymnosperms. It is important to study how endophytes behave in both monocots and dicots
because they may have very different requirements, such as phytohormones (Doty, 2009). One
study showed that the WP5 endophyte increased the number of tillers in rice plants when
compared to uninoculated rice plants. Rice tillers are an estimation to crop productivity. The
study also demonstrated that the WP5 endophyte increased overall root and shoot biomass in rice
plants (Kandel, 2015). In another study the effects of the WP5 endophyte was tested with the
Glacier Tomato variety. In this study the WP5 inoculated tomato plants in nitrogen limited
conditions had a significantly higher root and shoot biomass compared to the uninoculated
tomato plants, with root biomass being more pronounced. In this study, the inoculated tomato
plants also flowered earlier (108 flowers) and earlier compared to the uninoculated tomato
plants (65 flowers) at 1.5 months of growth. Tomato plants inoculated with WP5 also produced
more fruit (95 fruits) than uninoculated tomato plants (43 fruits) (Khan, 2012). Another study has
shown that single or multi-strain endophyte inoculum exhibits mutualistic behavior including
drought stress tolerance when added to plants. Some endophytes can benefit plants by increasing
root biomass thus reducing water requirements and increasing survivability during dry seasons.
Biomolecules can be protected during osmotic stress with trehalose, which some endophytes are
able to synthesize. Plants produce a small amount of ethylene normally but production increases
when the plant is exposed to stress, which causes a decrease in growth. Some endophytes are
believed to produce ACC Deaminase, which reduces host stress ethylene. The same study
suggests that when added together there is an even larger effect (Khan, 2016).

This paper will address endophyte mediated drought stress alleviation in rice and nitrogen
fixation by endophytes in rice and tomatoes.

Methods

Nitrogen Fixation by Endophytes in Tomatoes

WP5 is an endophyte found in black cottonwood and the 16S rRNA sequence has shown that it
is closely related to Rahnella aquatilis. This strain grows best on Nitrogen Free Media (NFM)
agar and has been shown to fix nitrogen in the rhizosphere of maize and wheat (Doty, 2009).

Tomato plants of the Glacier variety (Lycopersicon lycopersicum) were inoculated with WP5 to
investigate the endophyte nitrogen fixing ability. For both crop plants (rice and tomato), there
were six different treatment groups. Each treatment group had ten plants for a total of 60 plants
per crop. The treatment groups included full nitrogen mock (no endophytes), full nitrogen mutant
(no nitrogen fixing (nifH) gene), full nitrogen wild type, no nitrogen mock, no nitrogen mutant,
and no nitrogen wild type.

Tomato plants were grown in nutrient poor soil. Light timers were set for 12 hours of light and
12 hours of dark to mimic spring until week 12. Starting week 12 light timers were set to 16
hours of light and 8 hours of dark, to mimic summer and signal the end of the fruiting season.

For no nitrogen groups, watering media was made by adding 100 ml of 10x NFM and tap water
filled to the 1 L line. For full nitrogen groups, watering media was made by adding 100 ml of
10x NFM and 32 ml of 0.5M Ammonium Nitrate and tap water filled to the 1 L line. The no
nitrogen tomato groups were watered with a mixture of NFM and 1/8th (4 ml per 1 L) the amount
of Ammonium Nitrate as the full nitrogen group until week 4. After week 4 the no nitrogen
group was watered with NFM media only. During weeks 1-5 tomato plants were watered with
100 ml of full nitrogen and no nitrogen media once per week. After week 6 tomato plants were
watered with 100 ml of media twice per week due to increasing non-nitrogen nutrient stress.

Tomato plant measurements were taken monthly and included height, SPAD, and fruit count.
Once the fruit ripened, they were harvested, weighed, and stored in labeled bags in a refrigerator
until all growing fruiting was ripened.

After 120 days the tomato plants were harvested in the order of mock, wild type, and then mutant
in order to decrease the risk of endophyte contamination for genomic testing of the mutant strain.
The roots were separated from the soil and detached from the shoots; roots and shoots were
bagged, dried, and weighed separately.

Due to differences between mutant inoculated and both mock and wild type inoculated tomato
plant growth, a colonization assay was done. The goal of this process was to reisolate the WP5
mutant on Kanamycin (Km) media from the leaves and petioles of mutant inoculated tomato
plants.
Nitrogen Fixation by Endophytes in Rice

Rice seedlings of the M206 variety were surface sterilized in a laminar flow hood with freshly
prepared 3% bleach solution and a drop of Joy detergent for 30 minutes. Seedlings were rinsed 7
times with sterile water and then transferred to sterile vessels. 7 ml of 15N, 14l of Cb, 14 l of
Cf, 35 l of ppm, and 35 l of vc were added to sterile vessels to sterilize the inside of the seed
of all other microbes. The antibiotic concentrations were: Vc 5 ml/L, PPM 5ml/L, Cb 100 ug/ml,
and Cf 500 ug/ml. 15N is used to give the rice plants a trace amount of organic nitrogen and is
used as a stable isotope label. Most dinitrogen gas in the atmosphere is in the form of 14N. If the
endophytes are fixing nitrogen, it will dilute the 15N with 14N fixed by the endophytes.

To create the rice inoculum, WP5 and the WP5 nifH mutant were streaked onto MGL agar and
were grown for 2 days. Then two 125 ml flasks with 25 ml of TYC broth and several WP5 and
mutant colonies were incubated on the 30C shaker overnight. After cultures were grown they
were rinsed by transferring cultures to sterile 50 ml conical tubes and centrifuging them at 8K for
10 minutes at 4C. Supernatant was decanted off and replaced with 25 ml of NFM, vortexed, and
centrifuged again. This process was repeated 3 times. Because this process did not rinse away the
nitrogen on the inside that the microbes have not used up, the cultures were transferred to sterile
flasks and incubated in the shaker again overnight. After they were incubated overnight, cell
density of the cultures were tested with the spectrometer (OD600). Next, the amount of culture
necessary to make about 25 ml of OD600 of 0.1 was calculated and poured into a sterile vessel.

In preparation for inoculating the sterilized rice seedlings, 3 sterile vessels were prepared in a
laminar flow hood. One vessel contained 20 germinated seeds and NFM, another with 20
germinated seeds and mutant WP5 inoculum, and the last with 20 germinated seeds and wild
type WP5 inoculum. Germinated seeds were 21 days old. The vessels were prepared in the order
of mock, mutant, and then wild type to reduce contamination.

The seedlings were then planted in autoclaved 2-tier wick vessels containing nutrient poor sand
in the top and the appropriate media in the bottom connected by a wick. One seed was
transferred to each pre-labeled vessel in the order of mock, mutant, wild type. All vessels were
randomized between treatment groups and placed under 16 hour timed lights in the tissue culture
room. Media was changed every two weeks.

After growing in the vessels for 13 days, the rice leaves hit the cap of the vessel and were
bagged. A plastic bag was secured onto each vessel to keep the plants sterile for as long as
possible. After an additional 16 days, they outgrew the bags and holes were added to get them
used to having less than 100% humidity. After 5 additional days, the bags were completely
removed.
Rice plants grew for approximately 3 months. Planned final assessments included wet and dry
weights of roots and shoots separately. If the experiment was a success, total nitrogen and
15N/14N ratio data would be collected on a subset of the plants to determine if nitrogen fixation
by endophytes occurred.

Endophyte Mediated Drought Stress Alleviation in Rice

This experiment tested endophyte mediated drought alleviation in rice of the M206 variety with
the following strains of endophytes. These strains were isolated from poplar and willow trees
growing in stressful environments and were selected based on their plant growth promoting
abilities under drought and nitrogen-limiting conditions on a variety of plants and grasses (Khan,
2016).

Endophyte Strain 16S rRNA Match

WP1 Rhodotorula graminis

WP40 Burkholderia sp.

WPB Burkholderia vietnamiensis

PTD1 Rhizobium tropici

WP19 Acinetobacter calcoaceticus

WP9 Burkholderia sp.

WP5 Rahnella sp.

WW5 Sphingomonas yanoikuyae

WW6 Pseudomonas sp.

WW7 Curtobacterium sp.

WW11 Sphingomonas yanoikuyae

(Khan 2016, 2012 and Kandel)

Rice seeds of the M2016 variety were surface sterilized and given antibiotics with the same
protocol as described previously in the nitrogen rice section. However, these seeds were in 7 ml
of sterile water, rather than 15N during the antibiotic sterilization.
Rice seeds were divided into 13 groups with 3 seeds in each group. The groups are mock (no
endophytes), WP1, WP40, WPB, PTD1, WP9, WP5, WW5, WW6, WW7, WW11, and a
consortia mix of the strains listed.

To create the rice inoculum, individual strain wild types were each streaked onto MGL agar and
grown for 3 days. Then four 125 ml flasks with 10 ml of NLCCM broth and several colonies of
each strain were incubated on the 150-rpm shaker overnight. After cultures were grown they
were rinsed by transferring cultures to sterile 50 ml conical tubes and centrifuging them at 8K for
10 minutes at 4C. Supernatant was decanted off and replaced with 10 ml of NFM, vortexed, and
centrifuged again. This process was repeated 3 times. Cell density of the cultures was tested with
the spectrometer (OD600). Next, the amount of culture necessary to make 10 ml of OD600 of
0.1 was calculated and poured into sterile vessels with rice seedlings for each group. For the
consortia, the amount of each culture to make 10 ml of OD600 of 0.01 was calculated and
combined to create an inoculum with a total OD600 of 0.1. The mock vessel was filled with 10
ml of NFM instead of inoculum.

Rice seedlings were planted in pots under timed lights in the plant room. The plants receive 12
hours of light and 12 hours of dark. There are 3 pots per endophyte, consortia, and mock group
for a total of 39 potted rice plants. The pots are rotated twice per week and are bordered by pots
filled with soil receiving the same amount of water in order to reduce any edge effect on potted
plants.

The rice plants grew for one week and received adequate watering. Then they stopped receiving
water in order to put them under drought stress for 3-4 days until the soil became light and dry.
Next, the plants were watered adequately again for another week. This process is repeated until
there are visual differences between the health of the plants, signs of lasting stress or death.
Results

Nitrogen Fixation by Endophytes in Tomatoes

The bar graphs shown above show the average dry biomass for tomato shoots, roots, and total
dry biomass. From this data, there is not a significant difference between mock, mutant, and wild
type dry biomass within each nitrogen treatment group.
The bar graph shown above shows the average fruit mass for tomatoes harvested within a 21 day
period. From this data, there was more tomato fruit mass harvested from mock tomato plants in
both nitrogen treatment groups and only slightly more mutant than wild type in both groups.

The bar graph shown above shows the average height of tomato plants after 3 months of growth.
From this data, tomato plants inoculated with the mutant WP5 endophyte were taller in both
nitrogen treatment groups.
The photo above shows the tomato plants after 120 of growth. Tomato plants in the full nitrogen
treatment groups looked visually more green and healthy than those in the no nitrogen treatment
group.

The results of the mutant colonization assay did not show the presence of the WP5 mutant on
Km media.

Nitrogen Fixation by Endophytes in Rice

Data for this experiment has was not collected due to the death of the majority of the plants.

Endophyte Mediated Drought Stress Alleviation in Rice

All rice plants in the first round of this experiment died due to prolonged exposure to drought.
Data has not been collected for the second round of this experiment yet.

Discussion

Nitrogen Fixation by Endophytes in Tomatoes

Over the course of the tomato experiment there were a few problems encountered. The first was
the realization that one of the bench lights had not been on a 12 hour timer, but a 24 hour timer.
To address this problem, the timer was set to 12 hours. The tomato plants on this bench were
heat and light stressed and their leaves were beginning to curl. The tomatoes were again heat and
light stressed during week 8 due to the growing shoots and close proximity to the lights. To fix
this problem, the lights were moved from 57 cm to 1 meter in height. During week 6 the tomato
plants were noticeably nutrient deficient in phosphorus and magnesium. To address the nutrient
deficiency all NFM was remade and the amount of media administered has increased from 100
ml per week to 100 ml twice per week.

SPAD measurements were taken for the first 3 months; however, they were most likely not
reliable. For example, a SPAD measurement on a very green leave could have read as a 0.4,
while a SPAD reading on a yellow leaf might have read as a 50.

Due to the differences between mutant inoculated and both mock and wild type inoculated
tomato plant growth, a colonization assay was done on the leaves and petioles of mutant
inoculated tomato plants. The results did not show the presence of the WP5 mutant. These results
suggest that either the mutant did not colonize in the tomato plants, or did not colonize in the leaf
and petiole tissues of the plant. Alternatively, this could also mean that the mutant was no longer
resistant to Km and thus did not show up on a Km agar. Another possibility is that the WP5
strain lost its effectiveness in fixing atmospheric nitrogen over time. Perhaps a virus was actually
causing the benefits to the plant and the endophyte was just a host. If this was the case, the virus
would have died when the strain was frozen.

Nitrogen Fixation by Endophytes in Rice

The majority of the rice plants in this experiment did not survive, therefore the experiment was
ended. This may have been due to a sudden change in humidity when the bags were removed,
fungal or bacterial contamination, or a lack of functionality in the rope that connects the vessel
with the nutrient media to the sand that the plants grow in.

Endophyte Mediated Drought Stress Alleviation in Rice

During the first round of the drought experiment all rice plants died after the drought and
rehydration periods. This might have been due to a drought period that lasted too long. Plants
stopped receiving water after they began to show signs of stress, such as turning brown and
wilting.

For the second round of the drought experiment, the rice plants stopped receiving water once the
soil was dry and light and before the rice plants showed symptoms of stress. The rice plants
received an adequate amount of water for another week, then another drought period
commenced. This process will continue until there are visual differences between the health of
the plants, signs of lasting stress or death.
References

Doty, Sharon L. "Using Natural Microbial Symbionts of Trees to Remove Pollutants, Increase
Plant Growth, and Produce Biochemicals." 17 Nov. 2016. Lecture.

Doty, Sharon L., Brian Oakley, Gang Xin, Jun Won Kang, Glenda Singleton, Zareen Khan, Azra
Vajzovic, and James T. Staley. "Diazotrophic Endophytes of Native Black Cottonwood
and Willow." Symbiosis 47.1 (2009): 23-33.

Food and Agriculture Administration of the United Nations. "Fertilizers as Water Pollutants."
Corporate Document Repository (2017).

International Rice Research Institute. "Rice and Climate Change." IRRI (2017).

Kandel, Shyam L., Andrea Firrincieli, Natalie D. Leston, Kendra M. McGeorge, Patricia
Okubara, Giuseppe Scarascia-Mugnozza, Antoine Harfouche, Soo Hyung Kim, and
Sharon L. Doty. "In Vitro Bio-control and Plant Growth Promotion Potential of
Salicaceae Endophytes." Frontiers in Microbiology (in press).

Kandel, S. L., N. Herschberger, S.h. Kim, and S. L. Doty. "Diazotrophic Endophytes of Poplar
and Willow for Growth Promotion of Rice Plants in Nitrogen-Limited Conditions." Crop
Science 55.4 (2015).

Khan, Zareen, Grant Guelich, Ha Phan, Regina Redman, and Sharon Doty. "Bacterial and Yeast
Endophytes from Poplar and Willow Promote Growth in Crop Plants and Grasses." ISRN
Agronomy (2012).

Khan, Zareen, Hyungmin Rho, Andrea Firrincieli, Shang Han Hung, Virginia Luna, Oscar
Masciarelli, Soo-Hyung Kim, and Sharon L. Doty. "Growth Enhancement and Drought
Tolerance of Hybrid Poplar upon Inoculation with Endophyte Consortia." Current Plant
Biology 6 (2016).

Knoth, Jenny L., Soo-Hyung Kim, Gregory J. Ettl, and Sharon L. Doty. "Biological Nitrogen
Fixation and Biomass Accumulation within Poplar Clones as a Result of Inoculations
with Diazotrophic Endophyte Consortia." New Phytologist 201.2 (2013).

McMcKague, Kevin. "Environmental Impacts of Nitrogen Use in Agriculture." Environmental

Impacts of Nitrogen Use in Agriculture (2011).
National Aeronautics and Space Administration (NASA). "The Consequences of Climate
Change." NASA (2017).

Vandenkoornhuyse, Philippe, Achim Quaiser, Marie Duhamel, Amandine Le Van, and Alexis
Dufresne. "The Importance of the Microbiome of the Plant Holobiont." New Phytologist
206.4 (2015).

Wilson, Dennis. "Endophyte: The Evolution of a Term, and Clarification of Its Use and
Definition." Oikos 73.2 (1995).

Xu, Zhenzhu, Guangsheng Zhou, and Hideyuki Shimizu. "Plant Responses to Drought and
Rewatering." Plant Signaling & Behavior 5.6 (2010).