Enhancement of ​Oryza sativa ​Submergence Tolerance Using ​Nelumbo nucifera​ Fungal

Endophytes

R. Jordan Bunayog (10 - Fermi)

Princess Yasmin Caudilla (10 - Newton)

Althea Sofia Espejo (10 - Hertz)

Joanne Claire Gonzales (10 - Maxwell)

Jan Caitlyn Inta (10 - Newton)

Kai Chenaniah Orpilla (10 - Faraday)

Mrs. Aiza Yang

Research Adviser

Makati Science High School

 CHAPTER I

1.1 Background of the Study

Rice is one of the staple foods of the Philippines and is also the primary source of income

for most Filipino farmers. The agricultural sector is one of the country’s pillars for economic

growth. This is proven and supported by statistics as 10% of the Philippines’ Gross Domestic

Product (GDP) comes from the agricultural sector and it also is the third-largest employer in the

labor sector1. However, despite farming being key to development, it still remains to be one of

the lowest paying professions in the country.

Although rice is the most abundant crop in the country, it does not necessarily mean that

it is easy to cultivate. Like all plants, rice requires a certain set-up for it to grow. It is affected by

environmental factors such as heat stress, availability of water, and many more. The optimum

condition that rice requires to thrive is even more challenging to achieve due to the instability of

the Philippines’ overall climate. The country is susceptible to natural calamities such as droughts

and typhoons. Since the agricultural sector relies heavily on desirable environmental conditions,

the aforementioned calamities greatly affect its chances for bountiful harvests.

There are available genetically modified rice varieties in the market today but they are

quite costly and may not always be accessible to regular farmers as they have limited income. It

is also not guaranteed that they will bear great crop at that price so for the meantime fungal

endophytes could be an affordable alternative for these GMOs. So far, there are only rice

varieties created that are only for enhanced drought tolerance, and even these are not yet widely

1
Danielle. 2016. ​Rappler.​ June 27. Accessed June, 21 2017.
​Nakpil,
https://www.rappler.com/move-ph/issues/hunger/137769-filipino-farmer-plea-support-love-adb
1
distributed in the common, local markets in the Philippines as well as overseas. The process for

perfecting and then approving these GMOs are also long and complicated. Hence, it may still

take a while before these rice varieties will be readily accessible to the public.

Fungal endophytes are abundant in nature but are still understudied since they are

asymptomatic to their hosts and their presence seems to show no effect on the plants. However,

there are studies that state that fungal endophytes help their host plants tolerate certain abiotic

stressors. Therefore, in this study, the researchers have decided to confirm whether fungal

​ ould manifest the same benefits for the submergence

endophytes from ​Nelumbo nucifera w

tolerance of its host plant by observing the growth of ​Oryza sativa subjected to overhydration

with the presence of fungal endophytes.

Fungal endophytes are easily accessible with the proper equipment and techniques as

they are present in virtually all plants and already have a background in plant growth promotion.

Certain species isolates can significantly enhance shoot length, root length, and fresh and dry

seedling weight of plants due to the secretion of various concentrations of amino acids 2 . Since

Nelumbo nucifera s​ trives in aquatic environments and studies of fungal endophytes contributing

to the sustenance of the growth of its host plant have been published, ​Nelumbo nucifera ​fungal

endophytes will be used in this study.

Nelumbo nucifera,​ also known as lotus, are plants that thrive on fresh waters. They are

abundant in bodies of water such as ponds and rivers. They have been chosen to be used in this

2
​Radhakrishnan, Ramalingam, Sang-Mo Kang, In-Youl Baek, In-Jung Lee. 2014.
"Characterization of plant growth-promoting traits of Penicillium species against the effects of
high soil salinity and root disease." ​Journal of Plant Interactions​ 9 (1):754-762.

2
study as they will be easily accessible for the use of the researchers and because it, being an

aquatic plant, will test whether fungal endophytes truly aid its host plant’s survival in its habitat.

To effectively observe the effects of the inoculated fungal endophytes in ​Oryza sativa

from Nelumbo nucifera​, the rice variety Swarna had been selected as it had been cited to be

flood-sensitive and has minimal tolerance to submergence of a week or more.3

​1.2 Literature Review

Multiple studies have already aimed to combat the negative effects of long droughts on

agriculture. The relationship of fungal endophytes with the sustainability of plants under heat

stress have been tackled by several researches at present.4 However, the studies aimed to combat

the negative effects of floods utilizing fungal endophytes are virtually non-existent. The only

available researches are those that observe the effects of overhydration on the fungal endophyte

isolate itself and not its effect on the relationships of the endophyte and its host plant.

An example of a study explaining only the effects of the host plant’s amount of received

water on the fungal endophyte itself concluded that there is a significant difference between the

Predawn Potential water and Midday Potential water between stressed (trees under cover) and

non-stressed (control group) trees. There were also more endophytes found exclusively in

non-stressed trees (eight), in comparison to stressed trees (three) and majority of the samples

from controlled trees have a greater number of fungal taxa or no significant difference has been

3
​Ramendra
Singh, J.N Reddy, V.N Singh, Uma Shankar Singh. 2009. "Swarna Sub1- A boon for
submergence –prone rainfed lowlands of eastern India." ​SABRAO journal of breeding and
genetics.
4
​Hubbard, M., Germida JJ., Vujanovic V. 2014. "Fungal endophytes enhance wheat heat and
drought tolerance in terms of grain yield and second-generation seed viability." Journal of
Applied Microbiology 116 (1): 109-122.
3
detected when compared to the number of fungal taxa collected from stressed trees.5 These

findings are still relevant to this current study because they provide insight to a fungal

endophytes’ behavior in roots that are subjected to overhydration and optimal hydration.

Studies focusing only on the fungal endophytes’ ability to sustain the growth of plants

under biotic stress such as fungal pathogens also have also been discussed by a study that

isolated endophytic populations from Oryza sativa. Nineteen different fungal taxa, a

Streptomyces sp. and bacterial species were isolated. ​Streptomyces sp., ​Chaetomium globosum,​

Penicillium chrysogenum​, ​Fusarium oxysporum and ​Cladosporium cladosporioides were

dominant endophytes in the study. The study concluded that rice harbors many endophytic

organisms and some of them have antagonistic properties against fungal pathogens.6

On the other hand, the researches that do tackle the relationship of fungal endophytes to

their hosts, have only observed it when the plants have been subjected to a lack of water.

Majority of these studies have similar conclusions, stating that fungal endophytes do improve

their host plants’ drought tolerance.

One particular research states that endophytic association under no stress (NS) and

prolonged heat stress (HS) conditions significantly improved plant growth attributes, such as

plant height, fresh weight, dry weight, and chlorophyll content. The researchers concluded that

endophytic fungi used in the present experiments are capable of producing phytohormones and

5
​Gonthier, Paulo, Massimo Gennaro, and Giovanni Nicolotti. 2006. “Effects of water stress on
the endophytic mycota of Quercus robur.”​ Fungal Diversity ​21: 69-80.
6
​B. ShankarNaik, J.Shashikala, Y.L.Krishnamurthy. 2009. "Study on the diversity of endophytic
communities from rice (Oryza sativa L.) and their antagonistic activities in vitro."
Microbiological Research Volume 164, Issue 3 290-296.
4
bioactive compounds that can promote plant growth, induce abiotic stress tolerance, and prevent

stress damage.7

Another study had confirmed that their tested consortium of endophytes had the ability to

improve wheat agricultural traits under drought or heat stress.8 The results from this study state

that wheat plants cultivated at higher temperatures and colonized by fungal endophytes produced

seeds with a shorter time to 50% germination than uncolonized wheat. Uncolonized plants

exposed to drought stress gave rise to seeds that took an average of 10 days to reach 50%

germination while seeds from plants colonized by fungal endosymbionts took a shorter period of

time to reach the same level.

However there is also a study that instead of supporting the ability of endophytes to

improve the drought tolerance of its hosts, have opposed this with its results. The researchers of

this study have found that the endophyte ​Epichloë festucae d​ id not increase the resistance of

Festuca rubra p​ lants to drought. Instead, differences in biomass production and proline content

in response to water availability occurred between plant lines.9 Despite of this, results showed

that the plant nutrition with endophytes had significantly improved.

7
Waqas, Muhammad, Abdul Latif Khan, Raheem Shahzad, Ihsan Ullah, Abdur Rahim Khan,
In-Jung Lee. 2015. "Mutualistic fungal endophytes produce phytohormones and organic acids
that promote japonica rice plant growth under prolonged heat stress." Journal of Zhejiang
University Science B 16 (12): 1011-1018.
8
​Hubbard, M., Germida JJ., Vujanovic V. 2014. "Fungal endophytes enhance wheat heat and
drought tolerance in terms of grain yield and second-generation seed viability." Journal of
Applied Microbiology 116 (1): 109-122.
9
​Vázquez-de-Aldana BR, García-Ciudad A, García-Criado B, Vicente-Tavera S,
Zabalgogeazcoa I. 2013. "Fungal Endophyte (Epichloë festucae) Alters the Nutrient Content of
Festuca rubra Regardless of Water Availability." PLoS ONE

5
 This current disagreement between the results of these existing studies is also one of the

reasons behind this research. Through the experiment, this can potentially determine the ways

the presence of fungal endophytes is beneficial to its plant hosts.

However, even with the aforementioned studies, the knowledge gap regarding the effect

of the fungal endophytes’ presence on its host subjected to overhydration remains undiscussed.

Hence, this research aims to fill that gap and conclude whether fungal endophytes can improve

submergence tolerance or not through the use of Oryza sativa and the fungal endophytes present

in the tissues of the aquatic plant, Eichhornia crassipes.

A study that supports endophyte effectiveness in sustaining or promoting the growth of a

rice plant had results that prove that fungal endophytes, particularly Penicillium isolates,

increased the sesame plant’s growth under high soil salinity and that they all induce an increase

in the production of beneficial amino acids in sesame plants. The NICS01 strain increased the

amount of chlorophylls, proteins, amino acids and liginans while both the external applications

of RDA01 and NICS01 strains significantly increased shoot length and fresh and dry seedling

weight under salt stress conditions.10

For the submergence stage in the experimentation, several articles had been consulted to

better understand the growing conditions and methods of the rice plant. Rice is typically grown

in between temperatures “16°C – 27°C and 100 cm to 200 cm of rainfall is ideal for rice

cultivation for the entirety of its lifetime. Plain lands or gentle slopes are suitable for the

10
​Radhakrishnan, Ramalingam, Sang-Mo Kang, In-Youl Baek, In-Jung Lee. 2014.
"Characterization of plant growth-promoting traits of Penicillium species against the effects of
high soil salinity and root disease." ​Journal of Plant Interactions​ 9 (1):754-762.

6
production of rice because stagnancy of water is a must for the cultivation of rice.11 Therefore, in

​ ill be subjected to artificial partial submergence, with an increase of 3

this study, ​Oryza sativa w

cm in water depth every day, ending with a total of 210 cm of water for a span of two months.

Given ​Oryza sativa’​ s relatively higher requirement for water compared to other plants,

most existing studies only focus on finding means to improve its drought tolerance. Aside from

this there are also a few other researches which have only paid attention the rice plants’ tolerance

to salinity and high temperature.12 13

However, the only existing solutions to increase ​Oryza

sativa’​ s submergence tolerance are biotechnology-related and requires the incorporation of

SUB1 gene into rice.14

The rice variety Swarna has been selected for this study as it had been cited to be

flood-sensitive and is incapable of withstanding submergence for more than a week. This will

allow the possible changes or improvements that the Eichhornia crassipes fungal endophytes will

have on Oryza sativa’s submergence tolerance to be more clearly observable.15

11
​Bose, Anil. 2013. "Geographical Condition for Rice Cultivation." Important India. Accessed
February 19,
2018.https://www.importantindia.com/4401/geographical-condition-for-rice-cultivation/
12
​Reddy, Inja Naga Bheema Lingeswara, Beom-Ki Kim, In-Sun Yoon, Kyung-Hwan Kim,
Tae-Ryoun Kwon. 2017. “Salt Tolerance in Rice: Focus on Mechanisms and Approaches.” ​Rice
Science​ 24 (3): 123-144
13
​Das, S., P. Krishnan, Monalisa Nayak, B., Ramakrishnan. 2014. “High temperature stress
effects on pollens of rice (​Oryza sativa ​L.) genotypes.”​ Environmental and Experimental Botany
101: 36-46
14
​Rice Knowledge Bank. "Climate change-ready rice." Accessed February 19, 2018,
http://www.knowledgebank.irri.org/step-by-step-production/pre-planting/rice-varieties/item/clim
ate-change-ready-rice
15
Ramendra Singh, J.N Reddy, V.N Singh, Uma Shankar Singh. 2009. "Swarna Sub1- A boon
for submergence –prone rainfed lowlands of eastern India." ​SABRAO journal of breeding and
genetics.
7
 C. Significance of the Study

To aid in sustaining the growth of crops under harsh conditions, many genetically

modified organisms and technological advancements have been developed. However, as stated

above, Filipino farmers do not usually receive high incomes that would allow them to afford

such products or devices. Therefore, they have no choice but to use non-modified seeds and

fertilizers that are deficit in nourishing crops under unfavorable conditions.

The use of fungal endophytes as a protection to certain plants from abiotic stressors can

serve as a cheaper and more accessible alternative for the benefit of the country’s agricultural

sector. Not only is it common, but also easily accessible for endophytic bacteria and fungi are

found in the roots, stems and leaves of plants. Also, the process of isolating endophytes and

distributing the isolates in the plants’ growth is more cost-efficient than the process of

genetically modifying crops. The controversy surrounding the currently available genetically

modified rice will also not apply on this new method for tolerance to overhydration as none of

the involved organisms will have their genetic sequences altered or modified.

Common water lotus (​Nelumbo nucifera​) is regarded as an endemic species of plant in

the Philippines as well as other Asian countries. Besides its known function of providing fish and

other aquatic inhabitants with shade and cover from predators, it is highly regarded for its

various uses in the field of medicine, wastewater treatment and liquid chromatography. Sourcing

endophytes from this plant could provide a new insight on possible applications of this organism

and present additional information that may aid in other scholarly articles.

8
 This study aims to promote plant growth under harsh conditions such as overhydration

using the fungal endophytes isolated from ​Nelumbo nucifera a​ nd provide an alternative to GMOs

that will not only directly benefit the agricultural sector and Filipino farmers of the country, but

will also fill the knowledge gap regarding the relationship of fungal endophytes and plants.

Research Framework

The amount of water and the presence of fungal endophytes are two independent

variables that will affect the dependent variable submergence tolerance of rice plants. Under the

said variable are five more specific dependent variables that will be measured to better gauge the

effect of the two independent variables on the rice plant’s submergence tolerance, namely, the

survival rate, number of tillers, chlorophyll content, malondialdehyde content, and dry weight.

9
The amount of water may also affect fungal endophytes’ ability or inability to improve the

submergence tolerance of the plants.

D. Statement of the Problem

This research aims to study the effects of the presence of fungal endophytes of ​Nelumbo

nucifera​ on the tolerance of Swarna to submergence. It will answer the following questions:

● How will the presence of fungal endophytes affect the survival rate, height , chlorophyll

content, and dry weight of the rice plants under:

a. partial submergence, initially having 3 cm of water, with 3 cm increase in water depth

every day starting 24 days after sowing?

b. constant water level of 5 cm until 42 days after sowing?

E. Research Hypothesis

The presence of fungal endophytes on ​Oryza sativa s​ ubjected to overhydration may:

H​1: ​improve the survival rate, height, chlorophyll content, and dry weight that will be attained by

the rice plants, therefore, manifesting an improved tolerance to submergence.

CHAPTER II

A. Research and Sampling Design

This experimental research will be using random sampling since the rice population will

be divided into fourteen (14) groups, and each plant’s height, dry weight, Chlorophyll content,

10
and survival rates were recorded from each group in the experiment. There will be two types of

inoculation for the plants with fungal endophytes: seed and soil. There will be three (3) replicas

for each type with the replicas containing five (5) pots of plants each. Plants without fungal

endophytes will also have three (3) replicas with five pots each.

The controlled variables in this study, which are the general location of the plants, the

temperatures of the set-ups, and the type of soil where the rice seedlings will be planted, are

combinations of the controlled variables aligned with those identified in previous similar studies.

Some researches used the first three mentioned controlled variables.16 17

B. Data Gathering Procedure

a) Gathering of Materials

Before the isolation of fungal endophytes ​Nelumbo nucifera p​ lant w

​ as washed to remove

any dirt. It was then cut into separate samples of Lotus Brown Stalk (LBS), Lotus White Stalk

(LWS), and Lotus Root (LR) before it was cut into smaller pieces and rinsed again. A mixture

with 100 mL of Hypochlorate and 2000 mL of water was combined to be used for surface

16
​Hubbard,
M., Germida JJ., Vujanovic V. 2014. "Fungal endophytes enhance wheat heat and
drought tolerance in terms of grain yield and second-generation seed viability." ​Journal of
Applied Microbiology 1​ 16 (1): 109-122.
17
​Hossain,
Md. Motaher, Farjana Sultana, Mayumi Kubota, Hiroyuki Koyama, Mitsuro
Hyakumachi. 2007. "The Plant Growth-Promoting Fungus ​Penicillium simplicissimum​ GP17-2
Induces Resistance in ​Arabidopsis thaliana​ by Activation of Multiple Defense Signals." ​Plant
and Cell Physiology ​48 (12):​ 1​ 724-1736.

11
sterilization of the samples. Afterwards the samples were washed and rinsed with sterilized

distilled water five times to ensure that no mixture was left.

Before cultures were made, Potato Dextrose Agar (PDA) plates were created to be the

growth medium of the fungal endophytes. 200 g of potatoes were sliced into small pieces and

boiled, the collected potato broth was diluted until it reached 1500 mL. Three flasks, each with

8.5 g of agar and 10 g of dextrose, were filled with 500 mL of the diluted potato broth. The

mixtures were autoclaved at 121⁰C for five minutes for sterilization and streptomycin was added

after cooling down to prevent bacteria from growing. Thirty plates of PDA were created from

these flasks.

Samples of LBS, LWS, and LR were cut into 1-2 cm pieces and placed into empty petri

dishes and soaked in 70% Ethanol (EtOH) for two minutes in its own beaker and then washed

three times with sterilized distilled water. The pieces were blot dry and four pieces were placed

in each petri dish which was then incubated.

The plates with mycelial growth were labelled by part-plate number-fungal endophyte

number-replica number. Nine prominent fungal growths were observed, namely, LBS-P1-1-1,

LBS-P2-1-1, LBS-P2-2-1, LBS-P3-1-1, LBS-P4-1-1, LBS-P4-2-1, LBS-P5-1-1, LBS-P5-2-2,

and LWS-P4-1-1. Three isolates of different physical characteristics were chosen out of the nine

isolates: LBS-P2-2-1, LBS-P4-2-1, and, LBS-P5-1-1. Small, identical, rectangular mycelial mats

were scraped off the PDA and transferred to flasks with distilled water and were then macerated

to form a suspension. The suspensions were transferred to flasks with Potato Dextrose Broth

with the use of a pipette. The broth cultures were placed in a shaking incubator for three days.

12
 50 grams of Swarna IR 24 seeds were disinfected with 20 ml of bleach diluted in water,

forming a 500 ml mixture. Seeds were soaked and shook for three minutes then drained. Finally,

the seeds were washed thrice with distilled water.

Two days after being removed from incubation, the broth cultures were blended on the

fourth setting for 30 seconds, the fifth setting for 15 seconds and the sixth setting for 15 seconds.

The blended broths were transferred to small bottles and labelled. The volumes of each bottle

were measured to find the heaviest. Water was added to the other bottles to match the weight of

the heaviest container so that the centrifuge would be balanced. The bottles were then placed

inside a centrifuge to separate the cultures and the excess liquid. After removing the excess

liquid, 2000 ml of distilled water was transferred to each of the bottles.

b) Preparation of set-up

Following the disinfection of the Swarna IR 24 seeds, some were soaked in the liquid

culture for a maximum of two days. These seeds were used as the seed-inoculated plants for the

flooded and non-flooded setups in the experiment. Afterwards both normal and inoculated seeds

were sown in transparent, rectangular plastic containers. The soil where non-inoculated seeds

will be sown were mixed with liquid fungal endophyte cultures for soil inoculation. Each

container had 100 seeds arranged in 10 rows and columns. 18

After 24 days, the plants were transferred in individual parts for the second part of the

experimentation.

18
​Waqas,
Muhammad, Abdul Latif Khan, Raheem Shahzad, Ihsan Ullah, Abdur Rahim Khan,
In-Jung Lee. 2015. "Mutualistic fungal endophytes produce phytohormones and organic acids
that promote japonica rice plant growth under prolonged heat stress." ​Journal of Zhejiang
University Science B​ 16 (12): 1011-1018
13
 c) Experimentation

For 24 days, all groups of plants were grown under identical and optimum conditions.

They were planted under the same general location, receiving the equal amounts of water under

the same light source.

The subjection to flooding stress of the experimental groups began 24 days after sowing

(DAS) the Swarna IR24 seeds. There were seven (7) groups under flooding stress namely, the

seed-inoculated and soil-inoculated groups of the LBS P2-2-1 isolate, the LBS P4-2-1 isolate,

and the LBS-P5-1-1 isolate, and one group not inoculated with fungal endophytes. Each group

had three (3) plots where five (5) plants will be grown. Another seven (7) groups of plants had

not be subjected to flooding stress, namely, the seed-inoculated and soil-inoculated groups of the

LBS P2-2-1 isolate, the LBS P4-2-1 isolate, and the LBS-P5-1-1 isolate, and one group not

inoculated with fungal endophytes. Again, each group had three (3) plots where five (5) plants

will be grown.

On DAS 24, the experimental groups received water with depth of 3 cm after moving the

individual pots in a crop water tank which can emulate flooded conditions. For the following

days after DAS 24, the experimental groups began undergoing partial submergence. Every day

the water depths on each plot were increased by 3 cm. By DAS 42, the final water depth on each

plot in the experimental groups reached 51 cm.

Except for the varying amounts of water that the groups will receive, all other variables

will not be changed.

14
 d) Data Gathering

​Due to time constraints the plant growth had only been observed for a duration of one and

a half months (42 days). On DAS 23, before moving the experimental groups to the water tank

for flooding stress, the heights of each plant in both control and experimental groups were

recorded for initial results. This was repeated on DAS 39 for additional data on growth progress.

For the final day of observation, the number of surviving rice plants were recorded to

compute for the samples’ survival rates. The following formula was used: (number of remaining

plants/initial number of plants)*100.

After these measurements, for the final day of observation, the final heights of all the

plants were recorded. The plants were then washed and transferred into paper bags for oven

drying. 84 plants, the first two plants from each replicate, were freeze dried for gathering data for

the Chlorophyll content.

e) Data Analysis

Since each treatment, LBS P2-2-1, LBS P4-2-1, or LBS P5-1-1 is compared to the

control treatment, t-test will be the statistical analysis used in the study. The analysis for seed and

soil inoculation were separated; as well as the analysis for normal and flooded conditions.

Plotting and comparing the box plots were done by R, a programming language for statistics

using RStudio for writing the script.

15
 CHAPTER III

3.1 Results

Table 1: SEED (Heights, Weights)

SHOOT ROOT
TREATMENT CONDITION REPLICA HEIGHT
WEIGHT WEIGHT

NF 1 56.06 5.08 1.10

LBS P2-2-1 NF 2 57.92 6.03 1.44

NF 3 61.30 4.16 1.68

NF 1 58.32 4.17 0.82

LBS P4-2-1 NF 2 60.32 4.51 0.81

NF 3 57.16 3.62 0.89

NF 1 55.06 3.63 0.73

LBS P5-1-1 NF 2 57.02 2.57 0.56

NF 3 59.42 2.07 0.60

NF 1 58.38 5.04 1.42

NONE NF 2 61.44 4.40 1.32

NF 3 59.46 5.76 1.45

F 1 88.18 2.14 0.21

LBS P2-2-1 F 2 92.18 2.13 0.46

F 3 89.04 1.85 0.35

F 1 90.70 2.33 0.44

LBS P4-2-1 F 2 90.00 3.33 0.74

16
 F 3 87.04 2.67 0.55

F 1 87.28 2.19 0.49

LBS P5-1-1 F 2 88.22 1.03 0.11

F 3 89.28 3.13 0.64

F 1 91.74 3.15 0.82

NONE F 2 89.30 2.21 0.31

F 3 96.64 2.71 0.55

This table shows the plant heights and shoot and root weights of the seed-inoculated

samples. There are four (4) treatments for this group namely the first endophyte inoculated

(P2-2-1), the second endophyte inoculated (P4-2-1), the third endophyte inoculated (P5-1-1), and

the non-inoculated. Each treatment is either under flooded or non-flooded conditions. For each

replicate, there are five (5) plants. The table only shows the average data of the five (5) plants of

every replicate.

Table 2: SOIL (Heights, Weights)

SHOOT ROOT
TREATMENT CONDITION REPLICA HEIGHT
WEIGHT WEIGHT

NF 1 59.68 2.59 0.92

LBS P2-2-1 NF 2 59.12 3.50 0.89

NF 3 58.02 3.76 0.75

NF 1 62.02 2.97 0.93

LBS P4-2-1
NF 2 58.68 3.51 1.14

NF 3 59.70 4.23 1.33

17
 NF 1 60.16 3.53 0.73

LBS P5-1-1 NF 2 59.38 4.44 0.65

NF 3 60.02 4.02 0.48

NF 1 58.38 4.64 1.42

NONE NF 2 61.44 4.40 1.32

NF 3 59.46 5.83 1.45

F 1 87.02 1.42 0.20

LBS P2-2-1 F 2 86.32 1.35 0.35

F 3 85.46 1.64 0.60

F 1 87.06 1.74 0.40

LBS P4-2-1 F 2 89.00 1.49 0.29

F 3 87.92 1.72 0.41

F 1 78.24 0.74 0.11

LBS P5-1-1 F 2 76.14 0.49 0.13

F 3 81.16 0.66 0.17

F 1 91.74 3.03 0.82

NONE F 2 89.30 2.11 0.31

F 3 96.64 2.71 0.55

This table shows the plant heights and shoot and root weights of the soil-inoculated

samples. There are four (4) treatments for this group namely, the first endophyte inoculated

(P2-2-1), the second endophyte inoculated (P4-2-1), the third endophyte inoculated (P5-1-1), and

the non-inoculated. Each treatment is either under flooded or non-flooded conditions. For each

18
replicate, there are five (5) plants. The table only shows the average data of the five (5) plants of

every replicate.

Table 3: SEED (Chlorophyll Content)

TREATMENT CONDITION REPLICA CHL A CHL B

NF 1 5.35292 4.141904

LBS P2-2-1 NF 2 8.26817 6.101777

NF 3 14.02505 10.5830605

NF 1 11.457133 8.0073875

LBS P4-2-1 NF 2 8.9524885 5.9723755

NF 3 10.75172 8.6558435

NF 1 13.97782 9.650102

LBS P5-1-1 NF 2 21.031205 14.36113

NF 3 26.690675 18.3603235

NF 1 5.751066 4.6001955

NONE NF 2 2.9404905 2.2761325

NF 3 3.0499925 2.3645465

F 1 0.797446 1.996179

LBS P2-2-1 F 2 0.669141 1.75048

F 3 5.1250685 6.746578

F 1 1.2747205 3.2302905

LBS P4-2-1 F 2 0.445736 1.154148

F 3 0.3063515 0.79054

19
 F 1 1.507341 3.7827255

LBS P5-1-1 F 2 1.429906 3.3176515

F 3 7.2347475 7.251803

F 1 0.3227985 0.8814735

NONE F 2 1.4889065 4.0657905

F 3 0.569782 1.48681
This table shows the Chlorophyll A and B contents of the seed-inoculated group. There

are still four (4) same treatments which are under either flooded or non-flooded conditions. The

number of replicates is still the same, however, only two plants out of five in each replicate had

been chosen for the Chlorophyll content test. The table only shows the average data of the two

(2) plants of every replicate.

Table 4: SOIL (Chlorophyll Content)

TREATMENT CONDITION REPLICA CHL A CHL B

NF 1 5.35275 4.02905

LBS P2-2-1 NF 2 6.103905 5.560215

NF 3 3.73733 3.07079

NF 1 7.977535 6.037905

LBS P4-2-1 NF 2 3.78034 2.87622

NF 3 6.59476 4.79968

NF 1 5.652965 3.850395

LBS P5-1-1 NF 2 4.790325 3.366875

NF 3 6.00821 4.13403

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 NF 1 4.07883 3.29609

NONE NF 2 2.338215 1.787945

NF 3 1.75729 1.38727

F 1 0.111785 0.326055

LBS P2-2-1 F 2 0.116975 0.298625

F 3 0.107315 0.251445

F 1 4.15035 4.48985

LBS P4-2-1 F 2 2.1754 2.321

F 3 0.108805 0.276315

F 1 0.122165 0.271195

LBS P5-1-1 F 2 0.12625 0.28235

F 3 2.05444 2.46912

F 1 0.142975 0.390425

NONE F 2 0.216505 0.591215

F 3 0.182335 0.477105
This table shows the Chlorophyll A and B contents of the soil-inoculated group. There

are still four (4) same treatments which are under either flooded or non-flooded conditions. The

number of replicates is still the same, however, only two plants out of five in each replicate had

been chosen for the Chlorophyll content test. The table only shows the average data of the two

(2) plants of every replicate.

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3.2 Discussion

For the seed-inoculated groups, the heights of the plants without fungal endophytes were

taller than those with fungal endophytes regardless of the condition (flooded or non-flooded) for

the plants. The case is the same for the root and shoot weights. However, for the Chlorophyll A

content, the non-flooded plants with fungal endophytes showed statistically significant higher

differences compared to those without fungal endophytes. This also true for the Chlorophyll B

content, however, the plant inoculated with endophyte P5-1-1 under flooded conditions also

showed statistically significant higher differences in addition to the other samples inoculated

with the three different fungal endophytes under non-flooded conditions.

For the soil-inoculated groups, the heights of plants without fungal endophytes were still

greater than those that have been inoculated. However, the difference in heights were only

statistically significant in the plants under flooded conditions. The shoot weights of all inoculated

plants were significantly lower than the shoot weights of those without fungal endophytes except

for the P5-1-1-inoculated samples under non-flooded conditions which does not show significant

differences the shoot weights of the non-inoculated group. P5-1-1-inoculated and

P2-2-1-inoculated under non-flooded conditions and the P5-1-1-inoculated under flooded

conditions had root weights which were significantly less than those without fungal endophytes.

Similar to the seed-inoculated groups, the chlorophyll contents of the plants with fungal

endophytes were significantly higher than those without fungal endophytes. Both the

Chlorophyll A and B contents of the P5-1-1-inoculated plants under non-flooded conditions were

significantly greater than those of the non-inoculated. The P4-2-1-inoculated plants under

27
non-flooded conditions, however, only showed significantly greater difference in Chlorophyll B

content. The only exceptions to are the P2-2-1-inoculated plants which showed significantly

lower Chlorophyll A and B contents.

Based on the gathered data, particularly for the isolate P5-1-1, plants inoculated with this

endophyte manifested significantly higher levels of Chlorophyll content but also significantly

lower heights and root and shoot weights.

CHAPTER IV

4.1 Summary of the Study

Rice is regarded as one of the staple crops that grow and support the Philippine’s

economy. On the other hand, fungal endophytes are organisms that are asymptomatic to their

host plants and are said to aid in plants survival especially in resistance to abiotic stressors. One

common problem for rice crops is the occurrence of flood. The ability of endophytes to affect

submergence tolerance is studied knowing that endophytes aid in drought tolerance. Height, dry

weight, chlorophyll content, and survival rates of rice plants inoculated with three different

fungal endophytes were measured to observe if the organisms had any significant effect on the

plants. Statistical analysis shows that the endophyte LBS P5-1-1 can aid in the rice plant’s

photosynthesis and survival rate even though it is not reflected in its physical properties.

4.2 Limitations of the Study

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 The researchers are not trying to replace the available or current GMO rice varieties but it

may be used as a possible precaution since the Philippines is susceptible to floods.

Additionally, ​Oryza sativa ​requires at least four (4) months to fully mature or ripen,

however, due to time constraints for this study, the rice plant’s final observation will be after one

and a half months of growing in the greenhouse therefore we will not be able to observe the

effects of the fungal endophytes during the final stages of the rice plant growth. The differences

among the gathered data for the dependent variables will not be recorded for the mature rice

plant.

4.3 Conclusions

In conclusion, some fungal endophytes, particularly the P5-1-1 isolate in this study, aided

in increasing the Chlorophyll contents of its host plants. Their heights and weights may be

significantly lower than those without fungal endophytes, but the results show that they are still

able to survive in prolonged submergence.

This means that the P5-1-1 isolate has positive effect on its host plant’s overall

photosynthetic efficiency, hence the increase in Chlorophyll content. However, it is possible that

the fungal endophyte isolate may hinder growth, hence the significantly lower plant heights and

root and shoot weights.

The high Chlorophyll contents among the inoculated plants indicate that they are able to

photosynthesize without necessarily gaining height and additional weight. Despite of being

relatively shorter than non-inoculated plants, the inoculated plants are able to trap light and

execute photosynthesis without having to be taller than the depth of submergence.

29
4.4 Recommendations

The seed inoculation method of fungal endophytes to Oryza sativa is recommended as it

yields superior qualities under normal and stressed conditions. However it is recommended to

assign separate plots to plants with different endophytes to prevent transfer of endophytes. Also

tests with longer period of submergence should still be conducted to better observe the possible

effects the endophytes may bring. Different results may also be seen if the submergence was

administered at a different phase of the plant.

30
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