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Theses and Dissertations
2023-04-10
Halophilic Genes that Impact Plant Growth in Saline Soils
Mckay A. Meinzer
Brigham Young University
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Halophilic Genes that Impact Plant Growth
in Saline Soils
McKay A. Meinzer
A thesis submitted to the faculty of
Brigham Young University
in partial fulfillment of the requirements for the degree of
Master of Science
Brent Nielsen, Chair
Joel Griffitts
Brett Pickett
Department of Microbiology and Molecular Biology
Brigham Young University
Copyright © 2023 McKay A. Meinzer
All Rights Reserved
ABSTRACT
Halophilic Genes that Impact Plant Growth
in Saline Soils
McKay A. Meinzer
The Department of Microbiology and Molecular Biology, BYU
Master of Science
Many plants are highly sensitive to salt in the soil, and their growth and yield can be
greatly hindered by as little as less than 1% salt concentration in the soil. Additionally, soil
salinity is a growing issue globally and affects significant areas in Utah. Halophytes are plants
that are adapted to grow in saline soils and have been widely studied for their physiological and
molecular characteristics, but little is known about their associated microbiomes. Bacteria were
isolated from the rhizosphere and as root endophytes of Salicornia rubra, Sarcocornia utahensis,
and Allenrolfea occidentalis, three native Utah halophytes. Several strains of halophilic bacteria
have been isolated and screened for the ability to stimulate plant growth in saline conditions
despite the high salt concentrations. Halomonas, Bacillus, and Kushneria species were
consistently isolated both from the soil and as endophytes from roots of all three plant species at
all collection times. Of the isolates tested for the ability to stimulate growth of alfalfa under
saline conditions, Halomonas and Kushneria strains stimulated plant growth in the presence of
1% NaCl. The same bacteria used in the inoculation were recovered from surface sterilized
alfalfa roots, indicating the ability of the inoculum to become established as an endophyte. This
raises the question of whether these plant associated halophilic isolates contain genes that aid in
plant growth promotion. We are interested in genomic sequencing of our Halomonas and
Kushneria strains and performing genomic analysis to determine if there is a difference in genes
between plant associated and non-plant associated halophilic isolates.
We explored the hypothesis that certain bacterial properties have been selected for to aid
plant growth. This was accomplished by performing whole genome sequencing of 26 Kushneria
and Halomonas strains, both plant and non-plant associated. These strains came from freezer
stocks of previously collected isolates as well as field trips to collect more samples. Halophilic
bacteria were isolated from bulk soil, rhizosphere, and halophyte tissues (root and shoot tissues).
The non-plant associated (bulk soil) halophilic Kushneria and Halomonas strains aided in
determining if there are specific bacterial genes that are expressed in plant associated strains.
Whole genome sequencing of the isolates was performed on the Oxford Nanopore
platform. The sequence data was then assembled and annotated. The genomes were then
included in a genome wide association study was performed. The results from the GWAS show
that there is not a significant difference between plant and non-plant associated isolates,
disproving our hypothesis. The results also show that few known genes for phytohormone
synthesis were present in the pangenome, highlighting the need for further research to determine
how these halophilic isolates aid in plant growth promotion in saline soils.
Keywords: halophyte, glycophyte, Kushneria, Halomonas, salt-stress, m-GWAS
ACKNOWLEDGEMENTS
I would like to thank those who have helped me to progress to this point. Thank you to
Dr. Brent Nielsen for use of his lab and equipment, as well as his great advice throughout the
project. Thank you to my other committee members Dr. Joel Griffitts and Dr. Brett Pickett for
their advice with experiments. I would also like to thank Dr. Jonathan Hill for use of his Oxford
nanopore Minion, his help with the genomic sequencing, and all of his help with data
acquisitionand analysis. I would also like to especially thank the other members of the Nielsen
lab for all their help in procuring the data and isolates that led to this project and helping me on
various experiments. I would also like to thank my friends and family for their support during
this process. This project has been funded by a John A. Widtsoe Grant and the MMBio
Department.
TABLE OF CONTENTS
TITLE .............................................................................................................................................. i
ABSTRACT.................................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................... iii
TABLE OF CONTENTS ............................................................................................................... iv
LIST OF FIGURES ....................................................................................................................... vi
LIST OF TABLES ........................................................................................................................ vii
Introduction ..................................................................................................................................... 1
The Salt-Tolerant Genus Kushneria ........................................................................................... 2
The Salt-Tolerant Genus Halomonas .......................................................................................... 2
Mechanisms that Bacteria Use to Overcome Salt Stress ............................................................ 3
Increasing Salinity of Soils and the Detrimental Effects on Plants. ....................................... 5
Glycophytes and Their Growth Inhibition by Salt.................................................................. 5
Halophytes and Their Ability to Tolerate High Salt. .............................................................. 6
Bacteria and Their Association with Plants ................................................................................ 7
Bacterial Properties Associated with Plant Growth. ............................................................... 9
The Importance of Plant Hormones and Their Role in Aiding Plant Growth ............................ 9
Purpose of the Study ................................................................................................................. 10
Thesis Objectives .......................................................................................................................... 11
Aim 1: Isolation of halophilic Kushneria and Halomonas ....................................................... 11
Creation of Kushneria and Halomonas Library ........................................................................ 12
Comparative Genomic Analysis of Kushneria Isolates ............................................................ 15
Methods......................................................................................................................................... 16
Isolation of Halophilic Bacteria ................................................................................................ 16
Identification of Halophilic Bacteria......................................................................................... 17
Growth Trials using Halophilic Kushneria ............................................................................... 18
Genomic Sequencing Using Oxford Nanopore ........................................................................ 19
Assembly and Annotation of Halophilic Genomes................................................................... 20
Identification of the Core and Accessory Genomes .................................................................. 20
Results ........................................................................................................................................... 21
Early Trials Indicate that Most Halophilic Kushneria are Not Significant in Plant Growth
Promotion.................................................................................................................................. 21
Assemblies of Halophilic Genomes Result in Genomes of Equal Length to Published Genomes
................................................................................................................................................... 26
iv
Roary Results Show Large Numbers of Orthologous Groups Amongst Genomes .................. 29
Phylogenies Show High Level of Similarity Netween Kushneria and Halomonas Genomes .. 29
No Significance in Sequence Differences Between Plant and Non-Plant Associated Isolates . 30
Discussion ..................................................................................................................................... 37
Plant Growth in Saline Soils ..................................................................................................... 37
The Difference and Relatedness Between Kushneria and Halomonas Genomes ..................... 41
Plant vs Non-Plant Associated Bacterial Genomes .................................................................. 44
Phytohormones and Other Plant Stimulating Genes ................................................................. 45
Different Levels of Stimulation Between Halophilic Isolates .................................................. 48
Possible Directions for the Future ............................................................................................. 49
Conclusion ................................................................................................................................ 51
Bibliography ................................................................................................................................. 53
v
LIST OF FIGURES
Figure 1: Satellite Imagery of the Xinjiang province in China that shows increasing salinity over
time.................................................................................................................................................. 4
Figure 2: A graphic depicting the different mechanisms employed by halophytes in salt
tolerance.. ....................................................................................................................................... 7
Figure 3: Shows the microbiome on the leave (phyllosphere), and the relationship between the
rhizosphere and the rhizosphere microbes that form the rhizosphere microbiome........................ 8
Figure 4: Workflow of halophile sampling.................................................................................... 12
Figure 5: Workflow of genome wide association study................................................................. 13
Figure 6: Growth Trial Data. ........................................................................................................ 22
Figure 7: Growth Trial Data. ........................................................................................................ 23
Figure 8: Growth Trial Data. ........................................................................................................ 24
Figure 9: Growth Trial Data. ........................................................................................................ 25
Figure 10: Genome report of Halomonas isolate A9. ................................................................... 25
Figure 11: Output graph from roary. ............................................................................................ 27
Figure 12: Output graph from roary.. ........................................................................................... 28
Figure 13: Phylogenetic tree of Kushneria isolates. ..................................................................... 29
Figure 14: Phylogenetic tree of Halomonas isolates. ................................................................... 30
vi
LIST OF TABLES
Table 1: Properties of Kushneria .................................................................................................... 1
Table 2: Breakdown of potential conserved plant growth stimulating genes ............................... 31
Table 3: List of genes that were significantly associated with both high NaCl (2M) and
Kushneria. ..................................................................................................................................... 31
vii
Introduction
The human population is increasing at a relentless pace. It is estimated that by the year2050 the
population will reach 10 billion people. The increasing human population creates a much
greater need for food production [3]. However, a key barrier to crop production is soil salinity.
Soil salinity increases when there is inadequate drainage of water, contact with highlysaline
ground water, inadequate rainfall/precipitation to wash away soil salts, etc. [4]. My research has
focused on plants and their varying levels of salt toleration, how bacteria can potentially help
with plant growth in saline soils, and the need for further research into how bacteria can
influence plant physiology.
Table 1: Properties of Kushneria
1
The Salt-Tolerant Genus Kushneria
Kushneria is a genus of bacteria in the Halomonadaceae family and are halophiles.
Halophiles are bacteria that can grow in high NaCl concentrations. The genus Kushneria was
formed in 2009 when Halomonas marisflavi along with two other Halomonas strains were
moved into the novel genus. Strains of Kushneria have been isolated from a variety of different
salty environments including a solar saltern, the leaves of black mangroves, sea water, salt
mines, cured meats, and salt fermented foods [5-11]. Many species in this genus are adapted to
hypersaline environments, and different strains have been isolated from the rhizosphere as well
as the endosphere of halophytes [7, 12]. These bacteria exhibit the ability to produce a variety
ofosmolytes, bioactive compounds (including betaine and ectoine that help protect from stress),
and plant growth hormones [7, 12]. Because Kushneria strains have been isolated from both the
endosphere and the rhizosphere of plants and exhibit some ability to produce a variety of plant
hormones they have potential to aid in promoting plant growth stimulation.
The Salt-Tolerant Genus Halomonas
Halomonas is another genus of halophilic bacteria. Halomonas bacteria are able to grow in high
salt conditions and are able to grow in high pH. Halomonas can also resist contaminationby
other microbes, due to its ability to grow in highly saline and alkali conditions [13].
Additionally, Halomonas spp. have been isolated from the endosphere of different plants,
shrubsand trees. They have been identified as gram-stain-negative, aerobic with yellow
pigmentation, and are rod shaped [14]. Strains of Halomonas have been isolated from a variety
of highly salineenvironments including: salt marshes, the endosphere of halophytes, salt cured
meats, and fermented foods [14-16]. Bacteria from the genus Halomonas have been shown to
produce a plethora of diverse biochemicals and exopolysaccharides (EPSs) [15, 16]. The genus
2
Halomonas used to include bacteria that have since been moved to the genus Kushneria, and as
such sharemany characteristics with bacteria found in that genus.
Mechanisms that Bacteria Use to Overcome Salt Stress
Salt stress is one of the largest abiotic factors that can impact growth of an organism. Saline
environments can cause osmotic stress for the organisms living in those environments.
Halophilic bacteria have multiple mechanisms to counteract the osmotic stress of saline
environments. There are two main types of adaptation mechanisms that halophiles use to
preventdessication in saline environments: accumulation of water soluble organic compounds in
the cytoplasm, and controling the flux of inorganic ions. The main way that bacteria control the
fluxof inorganic ions is by exporting K+ ions, to offset in the influx of Na2+ ions. A variety of
halophiles utilize accumulation of water soluble organic compounds (ectoine, hydroxyectoine,
betaine, and choline) to offset the osmotic stress of highly saline environments. The
accumulation of these compounds, or osmolytes, help to draw water into the bacterial cell
preventing dessication of the cell [13]. Another strategy that is employed by a wide variety of
halophiles is controlling the flux of inorganic ions in the cell. If a cell has an influx of inorganic
ions this can lead to dessication of the cell and eventual death of the organism. One method to
prevent this influx of ions, typically sodium ions, is to actively pump intracellular potassium
outside of the cell. This potassium typically comes in the form of KCl, and the export of KCl
helps to offset the influx of NaCl from the saline environment [13]. While under salinity stress,
aplants production of reactive oxygen species (ROS) increases substantially. These ROS act as
signaling molecules within the plant. But at elevated levels for a long duration, the ROS can be
detrimental to the health of the plant. It has been shown that bacteria isolated from the
rhizosphere and endosphere participate in ROS scavenging and can reduce the concentration of
3
ROS within the plant improving plant health [17, 18]
The Negative Impact of Salt on Plants
1995
Legend
non saline
slightly saline
moderately saline
highly saline
extremely saline
2006
Legend
non saline
slightly saline
moderately saline
highly saline
extremely saline
Figure 1: Satellite Imagery of the Xinjiang province in China that shows
increasing salinity over time.
The images were captured using infrared cameras to look at 7 idfferent
soil attributes and determine which soils are saline. The top picture was
taken in 1985 and the bottom picture from 2006. The image was taken
from Ivushkin et al. (2019). [2].
4
Increasing Salinity of Soils and the Detrimental Effects on Plants. Farmers world-wide
have been experiencing a phenomenon of soil salinization within recent years. The increasing
salt concentrations have dire effects within agriculture. Because most crop plants are
glycophytic, the increase in soil salt concentration is having negative effects on not only plant
harvest, but plant growth and viability as well [19-21]. High levels of salt in the soil cause ionic
stress in plants, and disruption of cellular pathways due to high levels of Na+ ions. Climate
change has exacerbated the issue of saline soils. It is estimated that roughly 700,000 hectares of
arable land are abandoned each year due to salinization. [19-21]. High salinity is one of the most
severe abiotic factors globally. No other substance is asdamaging in plant growth and the plant
life cycle [21]. Because so much of the arable land globally is becoming salinized (Fig. 1), the
ability to grow enough crops to provide for the growing human population is decreasing.
However, there has been recent research into the ability to cultivate plants not only in salty soils,
but using saline water [22]. The ability to use salinized soils could help reclaim many tracts of
land for cultivation. Additionally, the use of saline water would increase farmers’ ability to
provide the necessary water for their plants [23,24].
Glycophytes and Their Growth Inhibition by Salt. Many crops, whether they be for plant
by-products or consumption, are glycophytes. Glycophytes are plants that are sensitive to high
salt concentrations. These plants either cannot survive in salty soils, or their yield is dramatically
reduced. Ionic stress is one of the most important components of salinity stress, and it results
from a high Na+ accumulation [25]. Recent studies on glycophytes sensitivity to high salt
concentrations show that enzymes play a prominent role in a plant’s stress response. It has been
5
shown that medium (50-100 mM) to high (200+ mM) levels of cytosolic NaCl can inhibit
enzymatic functions. With salt concentrations of ~333 mM, protein function decreases by 5070% [4]. This accumulation of cellular Na+ can lead to a multitude of issues within the plant
including imbalance in cellular homeostasis, oxidative stress, increased ROS secretion, nutrient
deficiency, interference with K+ and Ca+ functions, retarded growth, and the eventual death of
the cell. [4, 25]. Salinity can also alter the activity of different enzymes and their selectivity, as
well as gene expression of genes related to metabolism. Increased metabolic demand is, in part,
what leads to decreases in plant yield [26].
Halophytes and Their Ability to Tolerate High Salt. Halophytes are defined as plants that
can complete their entire life cycle in salt concentrations of 200 mM NaCl or greater [26, 27].
Understanding how halophytes survive and thrive in saline soils can be beneficial in increasing
the amount of land that is available for cultivation. One reason why cultivating halophytes seems
so promising is because, intracellularly, halophytic cells typically contain more than 500 mM
NaCl. Extreme halophytes, such as Tecticornia contain NaCl concentration as high as 2000 mM
[21, 28, 29]. There are different categories of halophytes, and their mechanism for dealing with
high salt concentrations varies drastically. The first type of halophytes excrete salt. These plants
have special glandular cells that excrete the excessive salt out of the plant body. The next type of
halophytes are succulents. Succulents use salt bladders, typically located on the leaf surface, that
hold a large amount of water to counteract the osmotic potential of the salt. The last category of
halophytes are obligate halophytes or true halophytes. These plants need salt in order to complete
their life cycle, as they deal with the high salt concentrations by compartmentalizing different
ions in cells as well as the whole plant (Fig. 2) [30]. With soil salinity reaching a salt
concentration of 300-400 mM on average, the exploitation of halophytes and the genes that they
6
possess could help farmers feed the growing population with a dwindling supply of arable land.
The halophytes’ ability to deal with high salt concentrations is dependent on controlled uptake
and compartmentalization of Na+, K+, and Cl- [20, 31]. The ability of halophytes to tolerate
multiple stresses is of intense interest in rehabilitating soils for cultivation [20].
Bacteria and Their Association with Plants
Figure 2: A graphic depicting the different mechanisms employed by halophytes in salt tolerance. The figure
depticts mechanisms at the cellular level, as well as the whole organism level. The image was take from Xu, et
al. (2016) [1].
The relationship between bacteria and plants in the rhizosphere. The rhizosphere is thearea in
soil that immediately surrounds the roots of plants [32, 33]. The rhizosphere contains countless
species and a vast diversity of microorganisms [32, 34]. One important component ofthe
rhizosphere is plant mucilage. Mucilage is excreted by plant root tissue and can serve as a
7
carbon source for microorganisms. The amount and composition of the mucilage can have a
drastic impact of the bacterial species that live in the rhizosphere [33, 35, 36]. By excreting
mucilage, plants can attract beneficial microbes to the rhizosphere and benefit from their ability
to break down sugars, fix nitrogen, suppress pathogens, etc. Mucilage can also play a role in
attracting, and supplying sugars, for halophilic bacteria. These halophilic bacteria could then in
turn aid the plant in saline soils.
Bacteria and Their Endophytic Relationships with Plants
Figure 3: Shows the microbiome on the leave (phyllosphere), and the relationship between the
rhizosphere and the rhizosphere microbes that form the rhizosphere microbiome. The presence of
endosphere associated microbes is also shown.
Species of bacteria and fungi that live within plant tissues without causing harm to the plant are
defined as bacterial and fungal endophytes respectively. There has been increasing interest into
the role of endophytes in plant health and their ability to overcome abiotic stresses.The plantmicrobial symbiosis beneficially affects plant growth and health, and helps to overcome the
adverse impacts of conventional agricultural practices while also improving soil health and
nutrient cycling [37]. The direct effects of endophytes include production of growth regulators,
phosphate solubilization, nitrogen fixation, plant defense responses against disease, biosynthesis
8
of plant hormones, siderophore production, nutrient mobilization, pathogen suppression, indole3-acetic acid (IAA) production, and 1-aminocyclopropane-1-carboxylic acid(ACC) deaminase.
[38]. Microorganisms are known to produce over 20,000 different secondary metabolites [39].
These metabolites can affect the survival and performance of other organisms. Not only do
endophytes produce secondary metabolites which can aid the plant in growth and in disease
response, endophytes also produce novel biomolecules and plant growth promotors [40]. The
ability of endophytic bacteria to wield such a positive effect in plant growth and health make
them ideal candidates in the fight not only against climate change but also in poor soil
conditions.
Bacterial Properties Associated with Plant Growth. Indole-3-acetic acid (IAA) is a plant
hormone that aids in the production of new root and shoot tissues. It has been shown that IAA
produced by bacteria can induce adventitious shoot growth [38, 41-44]. High salinity induces
the utilization of 1-aminocyclopropane-1-carboxylic acid (ACC). ACC is a precursor for
ethylene, a plant hormone that mediates a wide range of essential plant responses. However, at
elevated levels ethylene has a deleterious effect on root and shoot elongation, leaf expansion, and
overall plant health [44]. ACC deaminase in an enzyme that breaks down ACC preventing the
production of ethylene and can alleviate the stress response in plants. ACC deaminase producing
bacteria can help aid the plant when it is under salt stress, and even help promote plant growth
and antioxidant production [44].
The Importance of Plant Hormones and Their Role in Aiding Plant Growth
Plant hormones are involved in a plethora of plant activities including coordinating and
controlling cell division, growth, and differentiation, seed dormancy or germination, plant
growth and overall health, flowering and fruiting, and finally, death [45, 46]. Plant hormones
9
have long been considered one of the most important endogenous molecules and some
symbioticbacteria have been shown to produce important plant hormones [42, 47]. Salt stress
tolerance in plants is mediated by regulating different hormones, biochemical processes,
specific transcription factors, and gene expression. One of the most important hormones in
response to a variety of abiotic stresses is abscisic acid. Abscisic acid has been shown to aid in
the acclimatization to lower water levels by closing stomata and accumulating various proteins
and osmoprotectants [47]. Auxin is another plant hormone that plays a major role in plant
growth andoverall health. Studies have shown that in the presence of high concentrations of salt,
production of auxin is severely limited. Plants grown in salty soils had insufficient auxin levels
and suffered from stunted growth [47]. Bacteria have been shown to induce hormone
production, and supplement plant hormones through miRNAs [42].
Appropriate levels of plant hormones are important for proper growth and development of the
plant. As discussed above, there are a plethora of different phytohormones that are important for
the growth and maturation of the plant. And there are a completely different set ofhormones that
aid in the maturation and ripening of fruits, both dried and fleshy [48]. Plant hormones are an
important and integral part of the lifecycle of a plant. If a plant has insufficienthormones levels,
the growth and development of the plant, as well as the fruit, can suffer and yield can be
significantly decreased.
Purpose of the Study
With soil salinization continuing to be a major issue in agriculture and food production, the
ability to cultivate crops in these saline soils becomes an ever-greater requirement. To produce
enough food for the continuously growing human population strategies to deal with saline soils
becomes ever pressing. The inability of nearly all crops to grow in highly saline soils presents a
10
problem with no easy solution. However, it has been shown that glycophytes grown insalty soils
can have their growth “rescued” with inoculation of halophilic bacteria from the microbiome of
halophytes [49]. The ability of halophilic bacteria to not only produce phytohormones but
sequester salt and aid plants in their growth is an area of research that needs to be expanded.
Additionally, the role of Kushneria and Halomonas in plant microbiomes and their ability to
stimulate plant growth is currently not understood. More research is needed to characterize the
genomes of Kushneria and Halomonas and determine which (if any) of their genes are critical
to the support of glycophytes growing in saline salts.
Thesis Objectives
In this project, we sought to better understand how strains of Kushneria and Halomonas,
common soil and marine halophiles, impact plant growth stimulation in saline soils. We used
genomic analysis to test the hypothesis that specific bacterial genes aid in plant growth while
cultivated in saline soils, and that these genes have been selected for in plant associated
Kushneria and Halomonas. In the first aim, we sought to obtain and sequence different
halophilic Kushneria and Halomonas strains. The second aim focused on annotating, finding
orthologs, and quantifying genomic differences in strains of Kushneria and Halomonas.
Aim 1: Isolation of halophilic Kushneria and Halomonas
The first step in categorizing different Kushneria and Halomonas properties was to determine
which collected isolates are strains of Kushneria and which isolates are strains of Halomonas.
This was performed by growing strains on 2 M NaCl plates and selecting coloniesthat have the
morphology of Kushneria isolates: reddish hue, glossy, motile, and aerobic colonies.
Halomonas isolates are typically a milky white, glossy, can occasionally have a yellowish hue,
aerobic, and are motile. We worked under the hypothesis that strains of Kushneria and
11
Halomonas have a significant difference in their genomes between plant and non-plant
associated strains. This hypothesis was tested by performing whole genome sequencing on 26
strains of both Kushneria and Halomonas.
Creation of Kushneria and Halomonas Library
Figure 4: Workflow of halophile sampling. A workflow depicting the collection of halphilic bacterial samples
from halophytes in Uth. Appropriate collection sites were determined, and samples were taken from
halophyte root and shoot tissues as well as rhizosphere soil. Bulk soil was also collected. Bacteria were
plated out on 2M NaCl plates and colonies that have Kushneria characteristics were selected.
The Kushneria and Halomonas strains were isolated from soil and tissue samples of halophytes
near Goshen, Utah (Fig. 4). Halophiles has been shown to live in a variety of salty
environments, including the endophytic and rhizospheric microbiomes [7]. The initial
identification of halophilic strains was performed by plating bacterial samples on 2M NaCl
plates. 2M NaCl plates aided in finding strains of Kushneria and Halomonas because they both
can tolerate higher salt concentrations than Bacillus. This is important because Bacillus strains
comprise most of the strains isolated from saline environments. Colonies that survived were
thenidentified using PCR using 16S rRNA primers and Sanger sequencing (Fig. 5). This library
of Kushneria and Halomonas isolates were sequenced using whole genome sequencing. The
wholegenome sequencing employed the Oxford Nanopore system provided by the Jonathan Hill
12
lab [50]. The Oxford Nanopore system utilizes protein adapters. These proteins are ligated on to
the genomic DNA. The proteins that are ligated on to the DNA are helicases, and they help to
feed the DNA into the pore while controlling the speed at which the DNA enters the pore.
Whole genome sequencing and annotation allowed for the visualization of the different genes
within thebacterial genome and characterization of said genes. The whole genome sequencing
provided thenecessary data to determine which genes are common amongst strains of Kushneria
and Halomonas.
Aim 2: Genomic Analysis and Identification of Orthologous Genes.
Figure 5: Workflow of genome wide association study. Bacteria isolated are
split into two groups; plant associated and non-plant associated. Whole
genome sequencing is performed on DNA from isolates of both groups.
Bioinformatic analysis of the resulting FASTA files is performed to
determine genomic and genetic differences between the two groups.
To determine whether there are differences between plant and non-plant associated Kushneria
strains, a comparative microbial genomic analysis was performed. This genomic analysis
allowed determination of the presence or absence of gene orthologs amongst the strains.Before
13
the sequence analysis, the genomic data was run through a bioinformatic pipeline that aided in
the annotation of the bacterial genomes. The bioinformatic pipeline utilized the FLYE program
for genome assembly, and the PROKKA program for genome annotation [51]. The FLYE
assembly program takes the raw reads from the Nanopore sequencing and uses the FASTA
files. It also utilizes the GUPPY program, which is the Nanopore base calling program. GUPPY
uses the FASTQ files and outputs FASTA files that the FLYE program uses for genome
assembly. The FLYE program looks at the data for long overlapping reads which it uses for
building the scaffold. The program goes through several rounds of error correction of the input
data to determine if reads that initially didn’t pass the QC can be used to aid in the genome
assembly. The PROKKA program incorporates several things to annotate the genome. It
BLASTs portions of the genome to find predicted gene function and uses genomes of related
species for gene prediction [51]. The validation for the genome assembly and annotation was
done using a genome sequence from Taalin Hoj. She sequenced the genome of a wild-type CRE
(Carbapenem resistant Enterobacteriaceae). After assembly and annotation, the genome that was
produced matched the published CRE genome 99.99%.
After assembly and annotation of genomes, genomic analyses were performed. A genome
analysis will look for synteny amongst the different strains. Synteny is having the same order of
genes on the same chromosome. Synteny is important because it allows for genome to be more
easily aligned. When genomes are aligned it allows for visualization of differences between the
genomes. Another approach utilized the FASTTREE software. The program built a
phylogenetic tree of the genomes. The building of the phylogenetic tree aided in determining
how different the plant and non-plant associated strains are. After annotation of the genomes
andidentification of all orthologs, a genome wide association study (GWAS) was then
14
performed.
The GWAS looked at the genomic data of plant and non-plant associated Kushneria and
Halomonas strains. Microbial Genome Wide Association Studies (mGWAS) look at genetic
variation amongst strains. Each mGWAS looks at genetic variation through SNP’s and INDELs,
gene presence or absence, and copy number variations and sequence inversions. The specific
type of genome wide association study that we performed was a study that looks at all three
categories. It is important to look at all three categories of genetic variation during a novel
mGWAS, especially when the type of genetic variation is unknown [52, 53]. The GWAS aided
in determining if plant associated bacterial strains have SNPs or entire genes that differ from
non- plant associated bacteria (Fig. 3). The specific software that we utilized were the roary and
scoary programs. The roary and scoary programs are specific for pangenome analysis and
validates its data through a post hoc label switching permutation test. Another advantage to
usingthe scoary program is that it does not need large sample sizes, in fact it is hindered by
using largesample sizes. The benefit of performing a mGWAS is that it told us about the genetic
differencesbetween the halophilic Kushneria and Halomonas strains and which genes are
different between plant and non-plant associated strains. This is the main function of a
mGWAS, whereas the FASTTREE software aided in identification of orthologous genes and
creating a phylogeny. Thetwo different approaches are needed in concert, as one makes up for
the analytical shortcomings of the other [54].
Comparative Genomic Analysis of Kushneria Isolates
To determine the level of similarity between the Kushneria isolates a comparative genomic
analysis was performed. This analysis was performed on our Kushneria genomes. Comparative
genomic analysis is a tool that compares the entire genome of one species to that ofanother.
15
Like roary above, the comparative genomic analysis will allow for the visualization of the
pangenome. The pangenome is the list of all the genes present amongst all the different
genomes. The pangenome consists of the core and the accessory genomes. The core genome is
the list of the genes that are present within all the genomes. Typically, these genes are necessary
for life. These genes could include polymerase genes, proteins for different parts of the Krebs
cycle, and amino acid synthesis. The accessory genome is comprised of all the genes that aren’t
in the core genome and, therefore, aren’t present in all the genomes. Within the accessory
genome there could be genes present in multiple genomes, or a single genome.
The creation of the pangenome, core, and accessory genomes aids in determining how similar
species are to each other. If the core genome makes up a large portion of the pangenomeone
could reliably assume that the species included in the study have high similarity. But if the core
genome comprises only a tiny fraction of the pangenome then the species involved have a low
degree of similarity. The creation of the Kushneria pangenome helped supplement the data from
creating the phylogeny. In addition to creating the pangenome of just our Kushneria isolates, we
created a pangenome of our Halomonas genomes. The two pangenomes were then compared to
determine the level of similarity. By comparing the two pangenomes we were ableto determine
how similar the Kushneria genomes are to the Halomonas genomes. This visualization was
surface level. It aided in seeing how many genes comprised each of the pangenomes, how many
core genes are in each of the pangenomes, and the number of unique genes.
Methods
Isolation of Halophilic Bacteria
A site for sample collection was selected south of Utah Lake at a nature preserve near thecity of
Goshen, UT [55]. The site was chosen due to high soil salinity and the growth of native
16
halophytes. Halophytic plants, Salicomia rubra, Sarcocomia utahensis, and Allenrolfea
occidentalis, were sampled by removing the entire plant and placing portions of each plant in a
sterile 50 mL conical tube. The rhizospheric soil was kept on the roots. Bulk soil was obtained
by placing a small trowel of dirt into a small sterile plastic bag. The plant samples were labeled
with the type of halophytic plant that was sampled and the location in the nature preserve. The
soil samples were similarly labeled with the location within the nature preserve. These samples
were then opened within the lab. For the soil samples 1 gram of soil was placed in a
microcentrifuge tube and 1ml of 1x PBS was added. The soil sample was then vortexed to get
homogenization of the sample. 1 mL of the homogenized soil solution was then plated on LB
agar + 2M NaCl plates to select for extreme halophiles. The plant samples that were obtained
were opened in the lab and a section of the shoot tissues roughly 2 cm long was placed in a
sterile microcentrifuge tube. This plant sample was then washed with 70% EtOH twice, and
twice with sterile H2O. After the washings the shoot sample was then washed with 1x PBS.
Using a small pestle, the shoot tissue was ground and then 1 mL of the resulting solution was
plated on LB agar + 2M NaCl plates. The soil and shoot tissue plates were then incubated at
30Cfor 24 hours.
Identification of Halophilic Bacteria
After the LB + 2M NaCl plates have been incubated each morphologically different colony was
streaked to singles on a new LB + 2M NaCl plate. These new plates were then incubated at 30C
for 24 hours. After incubating for 24 hours a pure colony was taken and used toinoculate LB
broth + .25M NaCl and incubated for 48 hours. After 48 hours 1 mL of the turbid culture was
placed in a sterile microcentrifuge tube. The culture was then centrifuged at 10,000 rpm for 1
minute and the supernatant was poured off. The pellet was then resuspended in 600 µLTE
17
buffer. The solution is then centrifuged at 10,000 rpm for another 2 minutes and 30 seconds.
The supernatant is poured off and the process is repeated with 600 µL TE buffer. After washing
with TE buffer twice the pellet is resuspended in 500 µL of bacterial lysis buffer and incubated
at37 C for 1 hour. After 1 hour 500 µL of bacterial digestion buffer is then added, and the
solution is vortexed to ensure homogenization. The tube is then incubated at 56 C for 1 hour.
After 1 hourthe solution is incubated at 95 C for 10 minutes to inactivate the proteinase K. The
extracted DNA is then used in PCR. 0.5 µL of the extracted DNA is added to 1 µL of the
universal forward and reverse primer and 7.5 µL of OneTaq Quick-Load 2x MM w/Std Buffer
and then placed in a thermocycler. The parameters of the thermocycler are 94 C for 30 seconds,
48 C for 30 seconds, then 72 C for 1 minute. This is repeated 30x and then held at 4 C. A
portion of the resulting PCR product was run on a 1% agarose gel to ensure that the 16S rRNA
gene was amplified. Once gene amplification is confirmed the remaining PCR product
undergoes PCR clean up using the PCR Cleanup Protocol of the Gel/ PCR DNA Fragments
Extraction Kit from IBI Scientific (Cat. No. IB47030). 5 µL of the cleaned-up PCR product is
then added to 1 µL of the forward primer and sent to the BYU Sequencing Center for Sanger
sequencing performed. The resulting DNA sequence is then submitted for BLAST analysis and
used to determine thegenera of the isolate.
Growth Trials using Halophilic Kushneria
The freezer stocks of each Kushneria isolate were used to inoculate 5 mL of LB broth + 0.25M
NaCl and incubated for 48 hours. The alfalfa seeds were surfaced sterilized by placing ~100
seeds in a conical tube and submerging in 50% bleach. The conical tube was then placed ina
rotating incubator and incubated for 60 minutes. The bleach was then poured off and the seeds
were rinsed 3x with ddH2O. After washing the seeds were then submerged in 70% EtOH and
18
incubated on the rotating incubator for 15 minutes. The EtOH was then poured off and the seeds
were washed 3x with ddH2O. After the last washing the seeds were poured into a sterile petri
dish with sterile filter paper placed at the bottom. The filter paper was wetted with ddH2O. Once
the seeds had been poured out onto the filter paper, the seeds were placed in a dark drawer for
24hours. The soil used for the growth trial was put into small pots. Each pot with an adjoining
lid was then autoclaved to sterilize the soil. 1 mL of the turbid culture was then added to 100
mL of 0.5% NaCl ½ strength Hoagland’s solution, and then poured over the autoclaved soil,
with two pots for each experimental condition. The sprouted seeds were then placed 1cm below
the surface with five seeds to a pot. The negative salt control had no cultures added, and the soil
waswatered with ½ strength Hoagland’s solution without salt. The positive control was watered
with 0.5% NaCl ½ strength Hoagland’s solution, but the bacterial culture was omitted. Once all
of the pots had been watered with Hoagland’s solution and the appropriate culture lids were
placed on the pots that had enough space for the alfalfa shoots to grow and allowed for
appropriate airflow.The pots were labeled with the experimental condition and placed in a plant
growth chamber at 23 degrees Celsius (16 hr light/8 hr dark). The plants were allowed to grow
for four weeks and were watered with 25mL of either .5% NaCl ½ strength Hoagland’s solution
or ½ strength Hoagland’s solution once a week.
Genomic Sequencing Using Oxford Nanopore
Freezer stocks of each Kushneria and Halomonas isolate were streaked to singles on LB agar +
2M NaCl Plates and incubated for 24. These pure colonies were then used to inoculate LBbroth
+ .25M NaCl and incubated for 48 hours. After 48 hours genomic DNA was isolated usingthe
DNeasy PowerLyzer Microbial Kit (Cat. No. 12255-50). The extracted genomic DNA was
quantified using a nanodrop and for any isolate that had less than 50 ng/µL the process was
19
repeated. The genomic DNA for the first 6 isolates were sequenced using the Ligation
sequencing gDNA (SQK-LSK110) protocol on the Oxford Nanopore system using the flongle
flow cell. The next 25 isolates were sequenced using the Ligation sequencing gDNA - native
barcoding (SQK-NBD112.96) protocol on the Oxford Nanopore system using the MinIon flow
cell.
Assembly and Annotation of Halophilic Genomes
The reads from the Nanopore were obtained and uploaded to R studio. A bioinformatic pipeline
was then used which employed FlyE to assemble the reads into a genome and then Prokka was
used to annotate the genomes. A genome report was run on all the genomes to ensureproper
assembly and coverage of the genomes. Any isolates that did not meet QC and did not
circularize were then resequenced.
Identification of the Core and Accessory Genomes
The GFF files that were created by the program PROKKA after the assembly of the genomes
were then taken and uploaded to the supercomputer on campus (MaryLou). Roary is then run to
perform a large-scale pan-genome analysis. The pan-genome is the list of the entire set of genes
from all the strains within a clade. It is then used to determine the genes that comprise the core
genome, as well as the accessory genome. The core genome is a list of gene families shared by
all organisms in a list. The accessory genome contains genes families shared by two or more
organisms and strain specific genes, but not the entirety of the list. Roary can dothis by
extracting coding regions and converting these regions to protein sequences. An all- against-all
comparison is then performed with BlastP [56]. The output from Roary is a
gene_presence_absence.csv file. A traits.csv is then created stating which strains contain which
phenotypes. The gene_presence_absence.csv and the traits.csv are then used as the input for the
20
program Scoary. Scoary scores the components of the pan-genome for associations to observed
phenotypic traits while accounting for population stratification, with minimal assumptions about
evolutionary processes. The process utilized in Scoary is distinctly different from traditional,
single nucleotide polymorphism (SNP)-based GWAS [57].
Results
Early Trials Indicate that Most Halophilic Kushneria are Not Significant in Plant Growth
Promotion
Alfalfa plants were grown in the presence of absence of 0.5% NaCl in 0.5X Hoagland’s solution
and in the presence or absence of inoculation with six different isolates of Kushneria. After four
weeks of growth, the plants were removed from the pots and all soil was removed from the
roots. The total plant, root, and shoot length were measured, and the total plant, root, and shoot
weight were also measured. A two-tailed, unpaired t-test was then performed on the data. The
results show that there is a significant difference between the data obtained from the negative
salt and positive salt controls (Fig.6). This illustrates the fact that salt has a profound effect on
the growth of plants. Additionally, for most of the isolates, the growth between the negative salt
control and different isolates shows that while there is a pattern of growth stimulation there was
not a statistical difference in growth in saline conditions. However, the E4isolate showed
statistical difference in growth compared to the positive salt control (Fig. 6). TheB5 isolate had
previously been shown to be the best promoter of plant growth in earlier trials.
21
Figure 6: Growth Trial Data. Figures are box and whisker plots showing the shoot and root length.
Data from alfalfa inoculated with 6 different Kushneria isolates.
22
Figure 7: Growth Trial Data. Figures are box and whisker plots showing the total length data and the
shoot weigh data from alfalfa inoculated with 6 different Kushneria isolates.
23
Figure 8: Growth Trial Data. Figures are box and whisker plots showing the root and weight and the
total weight data from alfalfa inoculated with 6 different Kushneria isolates.
24
Figure 9: Growth Trial Data. Figure is a box and whisker plot comparing the weight and height data from
alfalfa inoculated with 6 different Kushneria isolates.
Figure 10: Genome report of Halomonas isolate A9. The circular genome that was created for Halomonas
isolate A9 after de novo assembly.
25
Assemblies of Halophilic Genomes Result in Genomes of Equal Length to Published
Genomes
The de novo assemblies were generated using Oxford Nanopore NGS sequencing. Each genome
was sequenced with ~100x coverage of the genome yielding 300-500 Gigabases of data per
strain. The sequencing reads were processed and assembled using FlyE and Prokka, and the
contigs with poor support from mapped reads were removed from analysis. An example of one
of the circular genomes generated is shown in Fig. 10. As a result, total lengths of the final
assemblies of 26 strains ranged from 3.0 to 5.3 Mb. Kushneria genomes are estimated to be
around 3.6 Mb, while Halomonas strains have been shown to be anywhere from 3.5 Mb to 5.0
Mb [5, 58-61]. Thus, the results that we found are consistent with the ranges seen in the
literature. Some of the differences observed in Halomonas genome size comes from the broad
range of environments that these species have been isolated from. One such example is the
species Halomonas sp. MT13, which was isolated from deep sea vents. The genome of MT13
has large portions dedicated to cold-shock response as well as deep-sea environmental
adaptations [58]. These assemblies produced between 1 to 50 contigs, depending on the quality
of the sequencing data, with contig N50 ranging from 10 to 15 kb. Any isolates with more than
5contigs and none of the contigs circularized were re-sequenced. Several of the isolates had
multiple contigs that circularized which denotes the presence of at least one plasmid within
thesebacterial isolates.
There were ten isolates that had multiple contigs circularize. The size of these different
circularized contigs ranged from 6 kb to 200 kb. The contigs that have less than 10 kb are small,
and the circularization of the contig might be a mistake of the annotation and assembly process
or they could be plasmids. These circularized contigs are present in Kushneria and Halomonas
26
isolates, and in plant associated and non-plant associated isolates.
Figure 11: Output graph from roary. A graph that shows the breakdown of core genes,unique genes, and
total genes from each of the 12 Halomonas genomes. There are 20 genes that make up the core genome,
the average gene count is 4440.5, and the average number of unique genes is 455.
27
Figure 12: Output graph from roary. A graph that shows the breakdown of core genes, unique genes, and total
genes from each of the 6 Kushneria genomes. There are 331 genesthat make up the core genomes, the
average gene count is 6486, and the average number of unique genes is 1416.
28
Roary Results Show Large Numbers of Orthologous Groups Amongst Genomes
The processed and annotated genomes were then run through roary to create a core genome
alignment from the pan-genome. As stated previously, the core genome consists of genes that
are present in all the strains and aren’t changing. Typically, the core genome consists of
necessary housekeeping genes and other genes necessary for the health of the organism. The
accessory genome is the list of genes within the pangenome that aren’t shared amongst all of the
genomes. The output from roary was a gene_presence_absence.csv. This file was then used as
the input, along with a traits.csv, for scoary. The results from roary show that there are a lot of
genes within each of the genomes (Figs. 11-12). The average gene count for the genomes is
4294. Additionally, there is a low number of unique genes across all the genomes. Furthermore,
there is a high level of orthologous groups across the genomes. The results from roary are
summarized in Figs. 11 and 12.
Phylogenies Show High Level of Similarity Netween Kushneria and Halomonas Genomes
Figure 13: Phylogenetic tree of Kushneria isolates. The tree scale is to show evolutionary difference. The
branch length of 0.01 shows the number of nucleotide changes per site. 0.01 correlates to 1 change for
every 100 nucleotide sites.
A maximum likelihood tree was calculated for both the Kushneria and the Halomonas isolates.
The Kushneria phylogeny shows that there is a high degree of relatedness between almost all
the Kushneria genomes (Fig. 13). However, there is one major branch that is formedwith
Kushneria isolates OD8 and B2. Similar to Kushneria phylogeny, the phylogeny created with
the Halomonas isolates show a high degree of similarity between almost all the genomes. And
29
again, like the Kushneria phylogeny, the phylogenetic tree created from the Halomonas isolates
show that there is only one branch formed from Halomonas isolates OD3 and OD5 (Fig.14).
Figure 14: Phylogenetic tree of Halomonas isolates. The tree scale is to show evolutionary difference.
The branch length of 0.01 shows the number of nucleotide changes per site. 0.01 correlates to 1 change
for every 100 nucleotide sites.
No Significance in Sequence Differences Between Plant and Non-Plant Associated
Isolates
Scoary compared the genes in the pan-genome to the traits listed in the traits.csv file. There was
no significance associated with plant or with low salt. But there was significance between the
genomes of Kushneria and the genomes of Halomonas. There was also significant difference
between the isolates that could tolerate high NaCl (2M) and those that could not. This
significance was not the result of chance, but rather evolution caused these significant
differences (Tables 2 and 3). The significant differences observed between Kushneria and
Halomonas include many genes that code for metabolism or biosynthesis of different amino
acids, and genes for osmolyte production. We have observed in our lab that Kushneria isolates
are able to grow on LB agar plates with 4M NaCl. Whereas Halomonas can tolerate growth on
LB agar plates with 3M NaCl, and growth was retarded on plates containing 4M NaCl. This
difference in these phenotypes can be explained by the presence of genes coding for different
osmolytes and stress proteins. Similar differences in gene presence are observed between strains
30
that can tolerate high salt (2M NaCl) and strains that cannot (1M NaCl).
Table 2: Breakdown of potential conserved plant growth stimulating genes
Table 3: List of genes that were significantly associated with both high NaCl (2M) and
Kushneria.
Gene
Annotation
tmrB
Tunicamycin resistance protein
lytB
Amidase enhancer
khtT_1
K(+)/H(+) antiporter subunit KhtT
group_422 Putative multidrug export ATP0
binding/permease protein
cwlC_1
Sporulation-specific N-acetylmuramoylL-alanine amidase
group_114 Putative competence-damage inducible
29
protein
ohrA
Organic hydroperoxide resistance protein
OhrA
arsB_4
Arsenical pump membrane protein
srfAB_1
Surfactin synthase subunit 2
ctc
General stress protein CTC
lytB
Amidase enhancer
group_121 Copper-exporting P-type ATPase
72
mrpG
Na(+)/H(+) antiporter subunit G
mntD
pbpI_3
ohrR
Manganese transport system membrane
protein MntD
Penicillin-binding protein 4B
Organic hydroperoxide resistance
transcriptional regulator
Sensitivity Specific
ity
83.333333 100
33
83.333333 100
33
100
100
100
95.2381
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
100
100
100
100
100
100
100
100
100
100
100
100
Benjamini_
H_p
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
31
gdnD_1
putative guanidinium efflux system
subunit GdnD
group_143 putative iron export permease protein
96
FetB
mdtG_1
Multidrug resistance protein MdtG
ymfD_1
Bacillibactin exporter
pbpF_2
Penicillin-binding protein 1F
group_145 Putative multidrug resistance protein
06
MdtD
yfmC
Fe(3+)-citrate-binding protein YfmC
group_146
44
group_146
51
group_146
70
mdtD_1
General stress protein 39
Copper transport protein YcnJ
efeU_1
Hydrogen peroxide-inducible genes
activator
Putative multidrug resistance protein
MdtD
Iron-uptake system permease protein
FeuB
Ferrous iron permease EfeU
mdtG_2
Multidrug resistance protein MdtG
bslA
Biofilm-surface layer protein A
group_151
04
group_151
05
napA_2
Manganese transport system ATP-binding
protein MntB
Manganese transport system ATP-binding
protein MntB
Periplasmic nitrate reductase
copA_3
Copper-exporting P-type ATPase
ymfD_2
Bacillibactin exporter
pnpB
p-benzoquinone reductase
emrA
Colistin resistance protein EmrA
lnrL_1
Linearmycin resistance ATP-binding
protein LnrL
feuB_3
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
32
corA_1
Cobalt/magnesium transport protein CorA
chaA_1
Ca(2+)/H(+) antiporter ChaA
group_180 Manganese efflux system protein MneS
31
mntH_2
Divalent metal cation transporter MntH
mntH_1
Divalent metal cation transporter MntH
group_180 High-affinity zinc uptake system ATP93
binding protein ZnuC
efeM_2
putative iron uptake system component
EfeM
group_182 putative manganese efflux pump MntP
91
ywrO
General stress protein 14
lnrL_3
ebrA
Linearmycin resistance ATP-binding
protein LnrL
Multidrug resistance protein EbrA
group_226 Multidrug efflux system ATP-binding
61
protein
ydaD_3
General stress protein 39
yflT
General stress protein 17M
mneS_1
Manganese efflux system protein MneS
mneS_2
Manganese efflux system protein MneS
ydbD_1
putative manganese catalase
mneP
Manganese efflux system protein MneP
feuB_2
Iron-uptake system permease protein
FeuB
Stress response protein YvgO
yvgO
mrgA
Metalloregulation DNA-binding stress
protein
group_230 Na(+)/H(+) antiporter subunit B
35
bceA
Bacitracin export ATP-binding protein
BceA
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
33
khtT_2
K(+)/H(+) antiporter subunit KhtT
pbpI_4
Penicillin-binding protein 4B
pbpI_1
Penicillin-binding protein 4B
group_231 Zinc transporter ZitB
31
corA_2
Magnesium transport protein CorA
group_231 Malate-2H(+)/Na(+)-lactate antiporter
76
yocK
General stress protein 16O
group_233 putative siderophore transport system
46
permease protein YfiZ
ebrB
Multidrug resistance protein EbrB
mgtE
Magnesium transporter MgtE
khtS
K(+)/H(+) antiporter modulator KhtS
nhaX
Stress response protein NhaX
lnrL_2
Linearmycin resistance ATP-binding
protein LnrL
General stress protein 18
yfkM
bmrA_3
bmr3_3
Multidrug resistance ABC transporter
ATP-binding/permease protein BmrA
Multidrug resistance protein 3
yceD_2
General stress protein 16U
yceD_1
General stress protein 16U
salA_1
Iron-sulfur cluster carrier protein
efeM_1
bslB
putative iron uptake system component
EfeM
putative biofilm-surface layer protein B
csbD
Stress response protein CsbD
csoR
Copper-sensing transcriptional repressor
CsoR
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
83.333333
33
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
34
copZ
Copper chaperone CopZ
83.333333
33
cadA_2
Cadmium, zinc and cobalt-transporting
83.333333
33
ATPase
fhuD_2
Iron(3+)-hydroxamate-binding protein
83.333333
33
FhuD
fhuD_1
83.333333
Iron(3+)-hydroxamate-binding protein
33
FhuD
mrpF
Na(+)/H(+) antiporter subunit F
83.333333
33
ktrA_2
Ktr system potassium uptake protein A
83.333333
33
bmrR
Multidrug-efflux transporter 1 regulator
83.333333
33
nhaC
Na(+)/H(+) antiporter NhaC
83.333333
33
mepA
Multidrug export protein MepA
83.333333
33
yheH_1
putative multidrug resistance ABC
83.333333
transporter ATP-binding/permease protein 33
YheH
83.333333
group_566 Putative multidrug export ATP33
4
binding/permease protein
tasA_1
Major biofilm matrix component
83.333333
33
yocM
Salt stress-responsive protein YocM
83.333333
33
ydaD_1
General stress protein 39
83.333333
33
bmr3_4
Multidrug resistance protein 3
83.333333
33
83.333333
yxaB_2
General stress protein 30
33
kimA_1
Potassium transporter KimA
83.333333
33
group_150 Na(+)/H(+) antiporter subunit A
100
74
bioY_1
putative biotin transporter BioY
66.666666
67
mrpA_2
Na(+)/H(+) antiporter subunit A
66.666666
67
66.666666
group_158 Copper-exporting P-type ATPase
67
8
group_159 Na(+)/H(+) antiporter subunit D
66.666666
67
3
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
90.4761
9
100
100
100
100
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00197831
4
0.00247914
6
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
35
group_159
4
group_267
18
group_267
26
bmrA_1
Na(+)/H(+) antiporter subunit C
Manganese efflux system protein MneS
General stress protein 26
Multidrug resistance ABC transporter
ATP-binding/permease protein BmrA
group_274 General stress protein 13
70
khtU_1
K(+)/H(+) antiporter subunit KhtU
citM_1
tasA_2
Mg(2+)/citrate complex secondary
transporter
Major biofilm matrix component
gspA_1
General stress protein A
group_751 Sodium, potassium, lithium and
2
rubidium/H(+) antiporter
nhaK_3
Sodium, potassium, lithium and
rubidium/H(+) antiporter
group_764 Sodium-lithium/proton antiporter
9
ktrA_1
Ktr system potassium uptake protein A
yfhA_1
putative siderophore transport system
permease protein YfhA
66.666666
67
66.666666
67
66.666666
67
66.666666
67
66.666666
67
66.666666
67
66.666666
67
66.666666
67
66.666666
67
66.666666
67
66.666666
67
66.666666
67
83.333333
33
83.333333
33
100
100
100
100
100
100
100
100
100
100
100
100
90.4761
9
90.4761
9
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
0.01496677
6
0.02414979
6
0.02414979
6
The last column in Table 3 is the Benjamini-Hochberg correction. The Benjamini- Hochberg
correction controls the False Discovery Rate (FDR) using sequential modified Bonferroni
correction for multiple hypothesis testing. There was no significant difference in genes between
those associated with Kushneria and genes associated with high NaCl (2M). Additionally, there
was no significant gene differences between plant associated isolates and non-plant associated
isolates. There were, however, 121 genes that were significant between Kushneria and
Halomonas, as well as between low or no salt and high salt, that could serve as plant growth
promoting genes (Table 3). These potential growth promoting genes include knownor potential
36
phytohormones, siderophore or iron sequestration genes, genes dealing with the transport of
ions, genes associated with transport of heavy or toxic metals, stress proteins, genes related to
the regulation of biofilms, and genes associated with antibiotic resistance proteins and
production of antibiotics.
Discussion
The results from the above experiments show that while there are some halophilic Kushneria
isolates that can aid in plant growth, the data show that there is not a significant difference
between plants that were inoculated with Kushneria and the positive salt control plants. The
roary and scoary analysis showed that there are many genes present within each of the isolate
genomes, and few of these genes are unique among the genomes, while there is a large set of
orthologous groups in the pangenome. Additionally, there is a significant difference in the genes
between Kushneria and Halomonas isolates, as well as between the isolates that cantolerate high
NaCl (2M) and those that cannot.
Plant Growth in Saline Soils
Relatively few species of Halomonadaceae family relevant to agriculture have been studied at
the whole genome level as compared to clinically important genera e.g., Mycobacterium,
Propionibacterium, etc. Hence, in the present study we have developed de novoassemblies for
26 Kushneria and Halomonas strains [62]. We have chosen to perform de novo assemblies on
Kushneria and Halomonas isolates due to their ability to stimulate plant growth insaline
conditions. Prior to this experiment only one other study has looked into the plant growth
promoting effects of Kushneria [63]. This study utilized Kushneria avicenniae, a strain of
Kushneria that has been shown to produce auxin. This study differs from our study, because our
study is looking at plant growth promotion in saline conditions. The results from the study
37
aboveshow that strains that produce auxin can help promote growth in regular soils, with no
consideration for saline conditions.
In our study we aimed to supplement the results from previous studies looking at plant growth
promoting bacteria by adding the condition of saline soils. What our results show is that
different isolates of Kushneria can aid in the stimulation of plant growth despite growing the
plants at a concentration of NaCl that is deleterious to plant growth. However, the results were
not as conclusive as we would have hoped. What the data show is that several different isolates
of Kushneria can stimulate plant growth, but the level to which the isolate does so varies
substantially. Using the data from this experiment we aimed to determine why the levels of
plantgrowth stimulation varied from isolate to isolate. We wondered about the presence or
absence ofgenes for phytohormones or other known genes that aid in plant growth such as
siderophore production, phosphate solubilization, etc. The data from our growth trial is
consistent with the data procured from other labs and their experiments using bacteria to help
facilitate promotion ofplant growth.
Our data is similar to the data of other researchers in the way that levels of plant growthchanged
from the control without the bacteria to the plants inoculated with the bacteria.
Additionally, another similarity that our experiment shared with others is that experiments with
plants are never as straight forward as experiments with other organisms. Plants are very
complex organisms, and most have very large and complex genomes. With either diploid,
triploid, tetraploid, or even larger polyploidy the way that a species of plant responds to a
stimulus can vary from one experiment to another [64]. One way that we tried to circumvent
thisissue is by using a large sample population and averaging the data obtained from each
experimental condition. However, despite doing this we noticed that within a particular
38
experimental condition, within one pot the plants would be growing extremely well, and in
another pot of the same experimental condition the plants would be growing extremely poorly.
We tried to overcome these challenges by controlling for as many variables as we could. We
autoclaved the soil to rid the experiment of possible contaminating microorganisms. We utilized
a closed pot system to retain water content and prevent contamination from microbes in the
environment of the growth chamber. And we tried to account for seed dormancy by sprouting
theseeds prior to implantation within the soil. However, despite these efforts and actions we still
saw a wide range of plant growth among experimental conditions.
In addition to the challenges of using plants as our organism, the data we obtained from our
growth trials is incomplete. Because we were utilizing a growth chamber, closed pots, and
controlling for any confounding element, the data we obtained isn’t complete which makes it
difficult to implement our findings in the real world in large production level fields. However, it
can be amended by performing open pot trials to supplement the data that we obtained. It can do
this because in open pot trials the soil is not autoclaved and so the interplay between the
inoculant and “native” soil microbes is better maintained. It can be determined if the inoculant
has a greater effect than native microorganisms. However, because salt can be so detrimental
andlethal to a wide variety of organisms, and the concentration of NaCl that we are using is
sublethalbut still inhibitory, the halophilic isolates that we inoculate with have a higher chance
of outcompeting the other soil microbes. Additionally, open pot trials would be beneficial to
perform to understand how evaporation can contribute to plant health and the nature of the
experiment. Another way to supplement the data that we obtained with our closed pot
experiments would be performing growth trials out of growth chambers, and even in large
production fields. By performing growth trials in production fields, it better encapsulates and
39
simulates real worldconditions experienced by farmers. One potential drawback to our closed
pot, growth chamber studies is that the type of soil that we used differs from the soil found in
different agricultural fields. We tried to simulate the types of soil found in production fields by
changing the clay, sand, vermiculite, and other organic material concentrations. But it is hard to
replicate such a wide variety of different soil conditions. By performing large growth trials in
production fields, we would be able to get a clearer picture of how the inoculant responds to
environmental conditions, how the soil make-up impacts soil salinity, and with large
populations of plants in each experimental condition we would be better able to get an accurate
picture of how much plant growth is promoted by the individual inoculant. Additionally,
because the aim of this research is to not only help fill holes in the current research of plant
growth promoting bacteria, but also to help aid farmers in the fight against salinizing soils, the
utilization of large open fieldtrials will better mimic real-world conditions. And the data that is
obtained through these field trials will better aid farmers in their decisions.
Additionally, our data is incomplete because our growth trials have focused only on usingone
isolate of Kushneria as the inoculant for each plant. Recent data from other researchers have
shown that inoculating with a consortium of isolates can help improve plant growth more than a
inoculating with a single isolate [65]. By using a consortium of halophilic PGPR the effects of
different isolates can act synergistically and potentially promote plant growth substantially more
than by using a single isolate. However, the experiment needs to be done with these isolates to
determine if a synergistic effect is present with these isolates in these conditions. Additionally, it
has been previously shown that employing a consortium of different genera can aid in plant
growth [66]. One of the reasons for this might be that the effect of one genus can be
supplemented by and enhanced by the genes of a different genus. Such studies are being carried
40
out currently by other members of our research group.
In the current experiment utilizing halophiles to support glycophyte growth in saline soilsit
might be beneficial to create a consortium of halophiles from different genera. Our current data
show that Kushneria isolates help to stimulate plant growth and help to improve crop yield by
close to 20% when in saline soils. Our preliminary data also show that Halomonas isolates help
to rescue plant growth in saline soils. One genus that our study neglects is Bacillus. Different
Bacillus strains have been isolated from each of our collection sites. However, we have selected
against Bacillus isolates in our current selection process because they are not as halophilic as
Kushneria and Halomonas isolates. In a previous study from our lab a Bacillus isolate was
shown to stimulate growth of alfalfa in salty soil [55] Despite not being as halophilic, one
advantage that Bacillus strains have over those of Kushneria and Halomonas is that Bacillus
strains can create spores and are gram positive, which could prove useful as an inoculant. A
consortium of Kushneria, Halomonas, and Bacillus should be tested to see its effect on plant
growth in saline soils.
The Difference and Relatedness Between Kushneria and Halomonas Genomes
There are many different molecular and physiological methods that Plant Growth Promoting
Endophytes (PGPE) and Plant Growth Promoting Rhizobacteria (PGPR) utilize to stimulate
plant growth. Despite knowing these difference mechanisms, no study has looked at the
genomes of isolates from Halomonadaceae to determine if there are any known genes that
promote plant growth. This is the hole that we aimed to fill with this study. We wanted to look
at the genomes of isolates from Kushneria and Halomonas that are unexplored novel PGPR
strainsand try to expand our knowledge by acquiring a better understanding of the PGPR-plant
interaction with halophytes and glycophytes [67].
41
The data obtained from the scoary analysis show that there is a significant difference between
the genomes of Kushneria and Halomonas. This is to be expected because Halomonas and
Kushneria are two separate clades of bacteria. However, there is still significant overlap
between these two genera of bacteria. The overlap is caused by the fact that Kushneria
specifically Kushneria marisflavi used to be categorized as a species of Halomonas. Kushneria
marisflavi used to be known as Halomonas marisflavi. It was then recategorized at Kushneria
marisflavi. Because Kushneria and Halomonas share so many similarities their genomes are
verysimilar but have distinct differences. One such difference was the DNA-DNA relatedness.
Kushneria marisflavi had lower DNA-DNA relatedness to other Halomonas species.
Additionally, a phylogenetic tree showed that Kushneria marisflavi and Kushneria indalinina
formed a separate branch to the other branches [68]. However, their similarities are what we
focused on when choosing both genera for our experiments. The fact that they are both
halophilicand isolated from a plethora of different saline conditions, including halophytes, made
them idealfor our study.
Despite their similarities the differences cannot be overlooked. Based on data that we have
obtained from initial growth trials, Kushneria isolates stimulate plant growth to a larger extent
than Halomonas isolates. We wanted to pursue this line of inquiry and see if there were
differences in the genomes between the two clades that would explain the differences observed
in overall plant growth. It was for this cause that we ran our roary and scoary analysis. The data
obtained from these analyses indicate that there is significant difference between the genomes of
42
the two clades. The differences are mainly in the presence of genes related to amino acid
production, vitamin synthesis, and ion secretion in the presence of high salt. But the results from
the scoary analysis do not highlight any genes that are known plant growth promoters, except
forpossible siderophore production.
In addition to the results obtained from our roary and scoary analyses, the results from our
comparative genomic analysis of the Kushneria and Halomonas genomes show that there is a
large difference between the core genome of our Kushneria isolates compared to that of our
Halomonas genomes. Figures 11 and 12 show how many genes comprise the core genome of
theKushneria and Halomonas pangenomes respectively. The Halomonas core genome is
comprised of 20 genes. This number is surprisingly low. One reason that the core genome is
made up of so few genes could be that a lot of the genes shared amongst the Halomonas
genomes haven’t been annotated yet and, therefore, were not included in the analysis. However,
despite this low number of core genes, the phylogenetic tree calculated from the Halomonas
genomes show that there is a high level of similarity between the Halomonas isolates. There is
only one major branch in the phylogeny that is created with isolates OD3 and OD5. This branch
anomaly could be caused because the OD3 and OD5 isolates might be the same species, or they
were isolated from the same bulk soil/plant tissue sample, and thus diverged from the rest of the
Halomonas isolates relatively early on.
The Kushneria core genome is comprised of 331 genes. This shows that there is a high degree
of similarity between the Kushneria isolates. This was to be expected as all the isolates were
obtained from a physical space of about 1 km2. This proximity could mean that all the
Kushneria isolates are evolutionally very close together. This is corroborated by the
phylogenetictree that was calculated using the Kushneria isolates (Fig. 13). In the phylogeny all
43
the isolates have very short branches between each other. This trend, however, is broken with
the branch thatis formed with isolated OD8 and B2. One reason for this might be that OD8 and
B2 were obtained from the same physical space and might be the same strain of Kushneria.
Another difference between the Kushneria and Halomonas pangenomes are the total amounts of
genes present. The Kushneria pangenome have an average gene count that is nearly 2,000 genes
higherthan that of the Halomonas pangenomes. This increase in average gene count also is
shown is thenumber of unique genes. The Kushneria pangenome has roughly 1,000 more
unique genes than the Halomonas pangenome (Figs. 11-12).
Plant vs Non-Plant Associated Bacterial Genomes
One of the larger premises of our study was that there would be a significant difference between
the genomes of plant associated versus non-plant associated isolates. This hypothesis was made
because from our initial batch of halophilic isolates, the isolates that had the greatest effect on
plant growth were bacteria that were isolated from the endosphere and rhizosphere of
halophytes. The assumption was that there would be specific genes within the genome of plant
associated isolates that weren’t present in the genomes of non-plant associated isolates, and that
this difference could explain why the plant associated isolates had such a profound effect on
plant growth while the other isolates did not. However, after our analyses and running scoary,
theresults show that there is not a significant difference between plant and non-plant associated
genomes.
One possible explanation for this is that the different bacteria were sampled from a relatively
small geographical area. At our collection site in Goshen, Utah, the bacteria were sampled in
two different main areas and all the samples were collected within a 50-meter radiusin each
sample site. It is possible that the bacteria, both Kushneria and Halomonas, collected within this
44
relatively small geographical area were all related. Or at least evolved from a common ancestor.
This could help explain that while we collected halophilic from bulk soil and from root and
shoot tissues there wasn’t a significant different in the genomes of the two populations. One
way that this could be rectified in future studies is to sample halophilic bacteriafrom a wide
range of collection sites. Including multiple sites across Utah, and even from different places
world-wide.
Phytohormones and Other Plant Stimulating Genes
Our initial hypothesis was that these halophilic isolates had many genes coding for known
phytohormones or other genes that have been shown to stimulate plant growth [64-66].
However, after running our analysis and after going through the data from running scoary it
seems that there is not a large presence of these types of genes. The only genes that were
highlighted in the data were genes for siderophore production. As discussed above there is not a
significant difference between plant and non-plant associated genomes. But there is a significant
difference in the genomes between bacteria that can grow in media containing 1M NaCl and
those that can grow in media containing 2M NaCl. These data make sense because the bacteria
that can survive while growing in media containing a higher concentration of salt will need the
necessary genes to survive higher levels of osmotic stress. Genes that code for osmolytes or
efflux pumps would be found in greater number. We found that there are genes that code for ion
transport proteins, as well as genes that code for multiple stress proteins and osmolytes such as:
General stress protein 26, General stress protein 13, and Salt stress-responsive protein YocM.
Despite there being a difference between bacteria that can survive 1M and those that cansurvive
2M NaCl, we find a lack of genes that code for known phytohormones, or other genes related to
stimulating plant growth. As discussed previously there are many genes that code for general
45
stress proteins and different osmolytes. These genes could be helpful when these bacteriaare
endophytes or are present in the rhizosphere. They could potentially be excreted by the bacteria
and taken up by the plants, which in turn would help the plants battle the osmotic stress from
the high salt concentration. Additionally, there are many genes that code for antibiotics, as well
as antimicrobial resistance (AMR), present in the pangenome. While these genes may not
expressly contribute to the plant’s ability to withstand higher salt concentrations, it could benefit
the bacteria in outcompeting other rhizobacteria and bacteria in the soil so that they can be taken
up and act as endophytes [69, 70].
Within the pangenome there are a few genes for siderophore production. These siderophores aid
the bacteria in the sequestration and accumulation of necessary iron from the environment. All
organisms need iron, and similar to above, these siderophores could be excreted by the PGPE
and PGPR and aid the plant in acquiring vital iron from the environment.Which in turn would
aid plant growth because the plant then has the needed iron to grow.
However, despite the presence of genes for siderophore production, there are no genes that code
for phosphate solubilization or utilization of other necessary compounds and elements from the
environment such as nitrogen fixation.
Additionally, there is a lack of genes that code for phytohormones. It is known that there is a
plethora of different secondary metabolites secreted by PGPR and PGPE that are structurallyand
chemically very similar to native plant hormones [42, 47, 64]. However, despite this knowledge
within the literature, these genes were not present in the list of genes that were significantly
associated in the pan-genome between Kushneria and Halomonas. We can infer two things
from this: that there might be genes within the pan-genome that code for phytohormones, but
they are just structurally different from genes within the databases and the
46
literature; the other thing that we can infer is there might be genes that help to stimulate plant
growth that haven’t been discovered yet. Both items are equally likely to have occurred within
the pangenome of our halophilic isolates.
Despite there not being many genes present in the data that are known to aid in plant growth
stimulation, there are genes that could help benefit plants under salt stress. These includegenes
that deal with the transport of ions and the transport of toxic or heavy metals, as well as genes
for stress proteins and regulation of biofilms. Transport of ions is vital in helping bacteria
survive saline conditions. As stated previously, one of the major methods that halophilic
bacteriacan tolerate salt and osmotic stress is by transporting ions across the cell membrane. By
transporting ions, specifically potassium, outside of the cell it helps to offset the influx of NaCl
into the cell. Another way that bacteria cope with salt stress is to produce osmolytes that help to
draw water into the bacterial cell, thus lowering the concentration of salt within the cell. One
way that bacteria could aid plants growing in saline conditions is by sequestering the salt within
their cells. By sequestering the salt, the bacterial cell can offset the negative impacts of the
osmotic stress, while removing the salt from the environment and reducing the amount of
osmotic stress that the plants experience. Within the pangenome there are several genes not only
for regulation of inorganic ions (Table 3), but there are several genes that code for production of
osmolytes such as ectoine, hydroxyectoine, betaine, and choline (Table 3).
In addition to the potential benefits to plant growth from ion regulation and osmolyte
production, the pangenome showed several genes that code for stress proteins. Stress proteins
arevital for the survival of bacteria in a wide range of environmental stressors. These stress
proteins are widely conserved amongst bacterial clades, and can cause the bacteria to survive in
severe external stress [71]. The knowledge about the function of bacterial stress proteins is
47
infantile. However, these stress proteins could have a profound effect on host cells, and cause
gene expression changes within the host. A future study about the interplay between bacterial
stressproteins and host gene expression changes could aid in the elucidation of the effect of
stress proteins on plant growth stimulation.
One last category of potential genes that aid in plant growth stimulation are genes that form and
regulate biofilms. There are several genes (Table 3) related to biofilm production and regulation
within the pangenome. Bacteria form biofilms when their population reaches a certain
threshold. The formation of biofilms aid the bacteria by securing them to a substrate, and
provided a vehicle for intercellular communication [72]. Additionally, biofilms are created with
avariety of different materials creating different extracellular matrices. These biofilms can be
RNA based, DNA based, or protein based. Many halophilic biofilms are protein based and
entrapdifferent salts within the matrix [73]. Additionally, some species form biofilms, and then
use potassium as the molecule for intercellular communication [72]. With the formation of
biofilms by halophilic rhizobacteria, the salt present in the environment can be sequestered and
kept from entering the plant and different plant tissues. This would result in a decrease in the
osmotic stresshelping to improve plant growth in saline soils.
Different Levels of Stimulation Between Halophilic Isolates
As previously discussed, there was an observed difference in the levels of plant growth insalty
conditions when inoculated with different Kushneria isolates. It is currently unknown as to why
there is a difference in growth stimulation between the different isolates. Further study is
needed to determine the cause for the differences between the isolates. However, we can
hypothesize that some of the differences could come from genes present within the genome and
the differences in hypothetical proteins. Because of the sheer number of hypothetical proteins
48
within the pan-genome, we didn’t have adequate time or resources to experimentally ascertain
the function of the numerous hypothetical proteins. Additionally, it was beyond the scope of this
study to do so. However, it would be beneficial to learn the function of these hypothetical
proteins for future study and utilization.
Another avenue that these isolates might exploit while promoting plant growth is gene dosage.
Because there weren’t significant differences in genes between plant and non-plant associated
isolates, one potential reason for the differences that were observed in plant growth between the
isolates could be related to gene dosage. In future studies it could be beneficial to perform
RNA-seq experiments using these isolates. By performing RNA-seq, we would be able to
experimentally detect changes in RNA transcripts between the isolates, that could be the cause
for the differences observed between the isolates.
Another route that could be utilized to ascertain why different isolates cause different levels of
plant growth stimulation would be repeating the microbial-Genome Wide Association Study
with different parameters than the ones used within this study. In the current study, the mGWAS that was performed selected for the following traits: if the isolates were Kushneria, if the
isolates were Halomonas, if they could grow on 1M NaCl media, if they could grow on 2M
NaCl, and if they were plant associated or not. Future m-GWAS could utilize the traits of
different levels of plant growth stimulation. By specifically using traits in the m-GWAS for
different levels of stimulation we might be able to ascertain if there are genes present in the pangenome that led to different levels of growth stimulation.
Possible Directions for the Future
Despite not having the genes present for known phytohormones such as ACC deaminase,indole3-acetic acid, etc. or genes for utilization of environmental compounds such as phosphate
49
solubilization or nitrogen fixation, these halophilic isolates have been shown to promote growth
of glycophytes in saline soils. Because these isolates can stimulate growth of plants without any
of the genes previously discussed it gives us cause to wonder how these isolates promote plant
growth. Further research is needed to understand how these isolate aid plants growing in saline
soils.
One possible route for understanding how these isolates aid plant growth in saline soils isby
performing biochemical assays. By utilizing a plethora of biochemical assays, we would be able
to determine if these isolates have different properties (encoded by genes) from those previously
studied and published and shown to promote plant growth. If would be helpful to perform
biochemical assays that determine if the isolates can solubilize phosphate, secrete siderophores,
fix nitrogen, secrete ACC deaminase or indole-3-acetic acid, mobilize nutrients, and suppress
potential plant pathogens. Depending on the results of such studies, it would give cause to take a
closer look at the genomes of these isolates. With the potential of finding genes currently
unknown in function that aid in stimulation of plant growth. Within the pan-genome there were
significant numbers of hypothetical proteins. It is a possibility that some of these hypothetical
proteins cause some of the aforementioned items to occur.
Furthermore, it would be beneficial in a future study to perform growth trials using a larger
number of our halophilic isolates. In our study we only performed growth trials using our
Kushneria isolates. We performed our study this way because our preliminary growth trials
showed that strains of Kushneria stimulated plant growth the best, and with Kushneria there
were larger differences between the inoculated plant and the plus salt control. It would be
advantageous to perform growth trials using our Halomonas isolates, and even using our
numerous isolates of Bacillus. Additionally, it could prove useful to perform growth trials by
50
using permutations of combinations of halophilic isolates. By doing such it would aid us in
determining if there are certain conditions that the isolates meet to promote plant growth.
Additionally, further research should be performed to ascertain which method of inoculating
plants with PGPR and PGPE yield the best results. In the current study we inoculatedthe seeds
at time of implantation within the soil with a small concentration of the designated halophilic
isolate. Previous studies have performed their inoculating by soaking the seeds in a microbial
solution, immersing the roots of sprouted seeds in the inoculum, or coating seeds with a
microbial concoction [64, 74-76]. Studies determining which route of inoculation has the
highest efficacy is crucial for understanding how PGPR and PGPE interact with the host. And it
would aid in any utilization of these technologies within agriculture.
Conclusion
This study aimed to better understand how halophilic isolates helped to rescue the growthof
glycophytes when grown in saline soils. We were able to show experimentally that by
inoculating seeds with halophilic isolates, the growth of alfalfa was enhanced somewhat when
grown in saline conditions compared to the growth of alfalfa plants that were not inoculated. By
obtaining these data we were then able to perform whole genome sequencing on our halophilic
isolates for the purpose of performing a microbial-Genome Wide Association Study (mGWAS).Our analysis showed that there were no significant differences in the genomes of plant
associatedand non-plant associated isolates. This is contrary to our hypothesis that plant
associated isolates would have significantly different genomes compared to non-plant
associated genomes. And thatthe genomes of plant associated isolates would have genes that
code for known phytohormones as well as phosphate solubilization, siderophore production,
and mobilization of other essential molecules. What our m-GWAS did show was that there was
51
a significant difference between the genomes of Kushneria and Halomonas isolates.
Additionally, there were significant differences between isolates that could grow in 1M NaCl
and those that could grow in 2M NaCl. The data obtained during our study show that there is
still a hole within the understanding of how Plant Growth Promoting Rhizobacteria and Plant
Growth Promoting Endophytes aid plant growth in amultitude of conditions. Further research is
required to fill the gap that persists.
52
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