UNIVERSIDAD DEL TURABO
A STUDY OF BACTERIAL ENDOPHYTES OF COCCOLOBA UVIFERA AT
CABO ROJO, PUERTO RICO
By
Ivelisse Irizarry Caraballo
BS, Industrial Microbiology, Universidad de Puerto Rico
THESIS
Escuela de Ciencias y Tecnología
Universidad del Turabo
Partial requisite for the degree of
Master of Environmental Sciences
Specialization in Environmental Analysis
(Biology Option)
Gurabo, PR
May, 2010
ii
Universidad del Turabo
A thesis submitted as a partial requisite for the degree of
Master of Environmental Science
A Study of Bacterial Endophytes of Coccoloba uvifera at
Cabo Rojo, Puerto Rico
Ivelisse Irizarry Caraballo
Approved:
____________________
Sharon A Cantrell, PhD
Research Advisor
____________________
José Pérez-Jiménez, PhD
Member
____________________
Paul Bayman, PhD
Member
© Copyright, 2010
Ivelisse Irizarry Caraballo. All Rights Reserved.
iii
Dedication
To my family, especially my parents Ernesto and Rosa, for always giving me their
unconditional support, love, and strength.
iv
Acknowledgements
I would like to thank Sharon Cantrell for giving me the opportunity to work in her
laboratory, for teaching me so many new things, for her patience, suggestions, and
believing in me and my work. To José Pérez-Jiménez for all his valuable advice and
input contributing towards the completion of this study and for presenting me with other
opportunities which have helped me in my development as a scientist. I would also like
to thank Paul Bayman for his suggestions and interest. Thanks to Teresa Lipsett for her
help. I thank Universidad del Turabo for partially funding this study through the
MiniGrant award program.
I would like to thank all the students who carry out their work at the laboratory. I
learned a lot from each of you and have made great friends along the way. I would also
like to thank the laboratory technician Francisco Rivera for his help with materials.
I especially want to thank all my family and friends for giving me support and
encouraging me to believe in myself.
v
Table of Contents
page
List of Tables………………………………………………………………………….…vii
List of Figures…………………………………………………………………….……..viii
Abstract………………………………………………..………………………………....ix
Chapter One. Introduction………………………………………………….………….....1
Chapter Two. Objectives…………………………………………………….………..….4
Chapter Three. Questions and Hypotheses…….………….………….………………..…5
Chapter Four. Literature Review………………………………….……………….……..7
Biodiversity of Bacterial Endophytes………….………………….….…………...8
Benefits and Applications of Bacterial Endophytes………....……….………….11
Description of the host plant Coccoloba uvifera………………….……………..20
Chapter Five. Materials and Methods………………………………………...…...........23
Research area……………………………………………….……………………23
Sampling and leaf processing……………………………………………………23
Growth of Bacteria……………………………………………………………….26
Characterization of Isolates………………………………………………...…….26
Statistical Analysis……………………………………………………………….27
Species Accumulation Curves and Biodiversity and Richness Estimators…..….28
Chapter Six. Results………….……………………………………………….…...….....29
Frequency of Colonization……………………………...………………………..29
Statistical Analysis…………………………………………...…………………..35
Isolates of Bacterial Endophytes Obtained from Coccoloba uvifera…………….35
vi
Bacterial Endophytes Characterized……………………………………………..36
Species Accumulation Curves and Biodiversity and Richness Estimators…...…46
Chapter Seven. Discussion…...……………………………………………….......……..55
Frequency of Colonization………………...……………………………………..55
Biodiversity…………………………………...………………………………….56
Chapter Eight. Conclusion................................................................................................64
Literature Cited……………...……………………………………………….………..…66
vii
List of Tables
page
Table 6.01. Frequency of fragments colonized by bacterial
endophytes from leaves of Coccoloba uvifera
sampled in September, 2008……………………………………..………31
Table 6.02. Frequency of fragments colonized by bacterial
endophytes from leaves of Coccoloba uvifera
sampled in March, 2009………………………………………….………32
Table 6.03. Frequency of leaf fragments colonized by bacterial
endophytes in trees of Coccoloba uvifera at each
season and site sampled...………………………………………………..34
Table 6.04. Number of isolates and different species of endophytic
bacteria recovered from both sampling sites…………………………….36
Table 6.05. Bacterial endophytes characterized from leaf
fragments of Coccoloba uvifera during the wet
and dry seasons……………………………………………………….….39
Table 6.06. Number of isolates from various
common species of endophytes.……………………………………...….43
Table 6.07. Various biodiversity and richness estimators from
each site and season……………………………………………………...54
viii
List of Figures
page
Figure 4.01 Leaf of Coccoloba uvifera.......................................................................21
Figure 5.01. Studied areas in Cabo Rojo, Puerto Rico…………..………………….....24
Figure 5.02. Picture of Playuela in Cabo Rojo, PR where two trees
of Coccoloba uvifera were sampled…………….……………..…..…….25
Figure 5.03. Picture of the Solar Salterns in Cabo Rojo, PR where two
trees of Coccoloba uvifera were sampled……….……….………………25
Figure 6.01. Total percent of colonized leaf fragments with bacterial
endophytes obtained from Coccoloba uvifera sampled
during the wet and dry seasons in Cabo Rojo, PR……………………….30
Figure 6.02. Percent of colonization of bacterial endophytes
recovered from four trees of Coccoloba uvifera
at both seasons…………………………………………………………...34
Figure 6.03 Species accumulation curve of bacterial endophytes
from tree P1 during the wet season……………………………..………..48
Figure 6.04 Species accumulation curve of bacterial endophytes
from tree P2 during the wet season………………………………………49
Figure 6.05 Species accumulation curve of bacterial endophytes
from tree S1 during the wet season…………………………….……...…49
Figure 6.06 Species accumulation curve of bacterial endophytes
from tree S2 during the wet season……………………………..………..50
Figure 6.07 Species accumulation curve of bacterial endophytes
from trees sampled at Playuela site during the wet season……..………..50
Figure 6.08 Species accumulation curve of bacterial endophytes
from trees sampled at the solar saltern site during
the wet season……………………………………………….………..….51
Figure 6.09 Species accumulation curve of bacterial endophytes
from tree P1 during the dry season…………………………..…………..51
ix
Figure 6.10 Species accumulation curve of bacterial endophytes
from tree P2 during the dry season……………………………….…….52
Figure 6.11 Species accumulation curve of bacterial endophytes
from tree S1 during the dry season……………………………….…….52
Figure 6.12 Species accumulation curve of bacterial endophytes
from tree S2 during the dry season……………………………….…….53
Figure 6.13 Species accumulation curve of bacterial endophytes
from trees sampled at Playuela during the dry season………………….53
Figure 6.14 Species accumulation curve of bacterial endophytes
from trees sampled at the solar saltern site during
the dry season……………………………………………………..…….54
x
Abstract
Ivelisse Irizarry Caraballo (MS, Environmental Science, Environmental Analysis)
A study of bacterial endophytes of Coccoloba uvifera at Cabo Rojo, Puerto Rico.
(May, 2010)
Abstract of a master thesis dissertation at the Universidad del Turabo.
Thesis supervised by Professor Sharon A Cantrell
No. of pages in text 75
All plants contain microorganisms called endophytes that live within their tissues
without causing apparent signs of disease. Most of the endophytes that have been studied
are fungi living in grasses and crops of agricultural importance in temperate regions. A
significantly less amount of information is available on bacterial endophytes of
neotropical trees. In the present study, bacterial endophytes of the coastal tree Coccoloba
uvifera (sea grape) were characterized near a hypersaline solar saltern and a beach site in
Cabo Rojo, Puerto Rico in September, 2008 (wet season) and March, 2009 (dry season).
It is hypothesized that the frequency of colonization and bacterial diversity will differ in
both ecosystems sampled between the wet and dry seasons. Two trees were selected at a
site near the solar saltern fed by the Fraternidad lagoon and two trees in Playuela. On
each sampling, four healthy leaves from each tree were obtained. Leaves were surface
sterilized and ten fragments measuring 2mm x 2mm were chosen randomly from each
leaf. The fragments were inoculated on 50% Tryptic Soy Agar (TSA) until colony
growth was observed. Pure isolates were transferred to 50% TSA Petri dishes. Cultures
of bacteria were separated into morphotypes depending on Gram stains and physical
xi
appearance. DNA was extracted from isolates of different morphotypes and
characterized by 16S rDNA sequencing. Sixty-one cultures were sequenced and they
were characterized as 42 different strains within 14 genera of endophytic bacteria.
Endophytes belonging to the Gammaproteobacteria have been found with higher
frequency, but members of Betaproteobacteria and Bacilli have also been encountered in
this study. Some commonly encountered genera are Stenotrophomonas sp, Pseudomonas
sp, Bacillus sp, and Burkholderia sp. Chi-square and Fisher’s exact test demonstrated
that the frequency of colonization did not significantly differ between the trees studied in
the salterns and Playuela on both seasons sampled. However, significant differences in
the colonization frequencies at the Playuela site were observed. This indicates that
seasonality (precipitation) does not affect the frequency of bacterial endophytes
recovered. However, according to the evidence shown here, the communities of bacterial
endophytes do change depending on the season.
1
1
Chapter 1
Introduction
Endophytes are a group of microorganisms associated asymptomatically with
tissues from both terrestrial and aquatic plants (Stone et al. 2000). Some microorganisms
that have been found as endophytes are fungi, bacteria, yeasts and cyanobacteria. These
organisms are only found when studied through histology, isolation of colonies that have
been grown from surface sterilized plant tissues, or direct amplification of DNA. They
can be found colonizing leaves, stems, roots, and the vascular system of plants.
Endophytes have also been found to colonize fruits and seeds. They include soil bacteria
and latent pathogens that could eventually cause disease on their hosts. This diverse
group of organisms has the potential for the production of bioactive compounds of great
importance and utility for humans. They also serve important ecological functions to the
plants they colonize such as protection against pathogens, nitrogen fixation, and plant
growth promotion.
Most endophytes studied to date are microscopic fungi present in grasses or crops
of economic importance. One of the most researched endophytic relationships is between
the fungus Neotyphodium sp. and grasses (Ravel et al. 1997; Saikkonnen et al. 2008;
Malinowski and Belesky 2006). Less information is available on bacterial endophytes in
comparison to fungal endophytes. Bacteria have been isolated from monocotyledonous
and dicotyledonous plants such as woody trees and herbaceous plants (Ryan et al. 2008).
They have been recovered from potato tubers (Sturz et al. 2002), cotton and sweet corn
(McInroy and Kloepper 1995), pepper plants of the species Capsicum annuum L.
(Sziderics et al. 2007), Coffea arabica (Vega et al. 2005), sweet potato plants (Khan and
2
Doty 2009), and in the vascular system of diverse citrus plants in Puerto Rico (Rivera
Rodríguez 2006).
Approximately 200,000 species of vascular plants exist which can serve as
ecological niches for species of endophytic fungi that have not been discovered
(Hawksworth and Rossman 1997). It has also been observed that endophytic fungi have
a higher abundance and diversity in tropical regions in comparison to more temperate
areas (Arnold et al. 2002; Arnold and Lutzoni 2007). The same could be occurring with
bacterial endophytes.
In the present study, the colonization frequency of bacterial endophytes in the
coastal tropical tree Coccoloba uvifera was analyzed at opposing seasons (wet and dry).
Determining the colonization frequency will give an insight into the amount of bacterial
endophytes in the host. It is expected that the colonization frequency in this study will
exceed most studies performed in temperate regions and be similar to infection rates
found on other plants surveyed in the tropics. However, the number of bacterial
endophytes recovered could be lower since Coccoloba uvifera trees are present in areas
of high environmental stress.
The study was carried out in Los Morrillos Reserve located in Cabo Rojo, Puerto
Rico. This site is unique due to the presence of natural salt flats and solar salterns which
harbor diverse microorganisms due to extreme conditions of salinity and ultraviolet
radiation. Preserved beach areas are also present nearby. Coccoloba uvifera
(Polygonaceae) is a woody tree with widespread distribution in the neotropics. These
trees are mostly found in coastal areas and in sand dunes where they are exposed to high
salinity levels, desiccation, ultraviolet radiation, and heat. Adult C. uvifera trees are
3
naturally found in different areas in the reserve such as the solar salterns and beaches.
The extreme conditions present at the sites might naturally select for unique microbial
communities. There is also potential to find bacterial endophytes previously described as
organisms that promote plant growth. The endophytes may increase plant fitness in such
extreme environments.
4
Chapter 2
Goals and Objectives
The main goal of this scientific research was to characterize bacterial endophytes
harbored in leaf fragments of Coccoloba uvifera (sea grape) near a solar saltern and
Playuela beach in Cabo Rojo, Puerto Rico. This is the first study that looks for
endophytic bacteria in Coccoloba uvifera.
The specific objectives of this research were:
1. Recover bacterial endophytes from the leaf fragments of Coccoloba
uvifera and isolate each colony in a pure culture.
2. Characterize the bacterial endophytes present by sequencing the 16S
rRNA gene.
3. Use statistics to determine if there are significant differences between
the frequency of colonization of bacterial endophytes through time
(season) and space (sampling site).
5
Chapter 3
Questions and Hypothesis
The main questions to be addressed were:
1. What bacterial endophytes are present in the leaf fragments obtained from trees
of Coccoloba uvifera in the solar salterns and Playuela in Cabo Rojo, PR?
2. Are the bacterial endophytes found in the same frequency between dry and wet
seasons on both sites?
3. Do the bacterial endophyte communities differ between the two sampling sites?
Some hypotheses to be explored in this study were:
Null hypothesis 1: The diversity of bacterial endophytes found in trees of Coccoloba
uvifera for both seasons (wet and dry) and on both sites sampled (saltern and Playuela) is
the same.
Alternate hypothesis 1: The diversity of bacterial endophytes found in trees of
Coccoloba uvifera for both seasons (wet and dry) and on both sites sampled (saltern and
Playuela) is different.
Null Hypothesis 2: The colonization frequency of bacterial endophytes is the same for
both seasons (wet and dry) and sites sampled (saltern and Playuela).
Alternate hypothesis 2: The colonization frequency of bacterial endophytes is different
for both seasons (wet and dry) and sites sampled (saltern and Playuela).
Null Hypothesis 3: The colonization frequency of bacterial endophytes in leaf fragments
of C. uvifera during the dry season is less than or equal to the colonization frequency
observed during the wet season.
6
Alternate Hypothesis 3: The colonization frequency of bacterial endophytes in leaf
fragments of C. uvifera during the dry season is greater than the colonization frequency
observed during the wet season.
Null Hypothesis 4: The colonization frequency of bacterial endophytes in leaf fragments
of C. uvifera at the Playuela and solar saltern sites during the dry season is less than or
equal to the colonization frequency observed during the wet season.
Alternate Hypothesis 4: The colonization frequency of bacterial endophytes in leaf
fragments of C. uvifera at Playuela and solar saltern sites during the dry season is greater
than the colonization frequency observed during the wet season.
The most commonly expected genera are those of soil inhabiting bacteria such as
Bacillus and Pseudomonas. Bacteria of these genera were isolated from citric plants in a
previous study carried out in Puerto Rico (Rivera Rodríguez, 2006).
The frequency of colonization of bacterial endophytes should be higher in trees of
Coccoloba uvifera than in hosts found in temperate regions. However, it should be lower
than in tropical hosts not present in extreme environments in Puerto Rico. The extreme
abiotic conditions should have a limiting effect on the total number of endophytes
recovered. In a previous study on citrus plants in São Paulo, Brazil, bacterial endophytes
were found with a colonization frequency of 90%-100% (Lacava et al., 2004).
7
Chapter 4
Literature Review
The word endophyte refers to any microorganism that colonizes plant tissue
asymptomatically without causing apparent signs of disease on the host. This diverse
group of microrganisms is comprised of fungi, bacteria, yeasts, and even cyanobacteria
(Saikkonen et al. 1998; Gai et al. 2009; Krings et al. 2008). These organisms may be
parasites, facultative saprophytes, mutualists, commensalists and even pathogens of the
host plant (Stone et al. 2000).
Endophytic bacteria usually come from soil bacteria present in the rhizosphere
and rhizoplane of plants. They can also come from colonized seeds and fruits. The
bacteria may enter plants through natural openings, such as stoma and wounds. Some
may also produce enzymes which aid their entry into the host (Viswanathan et al. 2003).
Bacterial endophytes are less studied than fungal endophytes because the latter
group is more abundant in plant tissue. However, these organisms have been isolated in
many occasions indicating that there must be an established relationship between the
plant and the bacteria. Defining bacteria encountered asymptomatically in plant tissues
as endophytes has its controversy. Endophytes can be defined as a group of
microorganisms associated asymptomatically with various organs and tissues of
terrestrial and aquatic plants (Stone et al. 2000). The controversy resides in that bacterial
endophytes can affect their hosts without necessarily expressing symptoms that are
obvious to the naked eye. For example, the presence of bacteria in plant tissue can
reduce crop yield or decrease growth (Kobayashi and Palumbo 2000). Also, all bacteria
that colonize plant tissue including pathogens have a latency and incubation period that
8
allows it to colonize the host asymptomatically. A bacterium can be an endophyte or a
pathogen depending on the moment, environmental conditions, and host health when the
organisms in the plant are studied.
Biodiversity of Bacterial Endophytes
Bacterial endophytes are a diverse group of symbionts that are thought to be
found in virtually every plant on Earth. Most endophytes are believed to be soil or
phyllosphere bacteria that have found their way into the interior of plants through natural
entrances or wounds of the plant. Endophytes have also been found to be transported
within seeds of their hosts (Ryan et al. 2008).
Bacterial endophytes from sugarcane have been identified in a previous study.
Stems and leaves from sugarcane plants in Brazil were sampled. Bacterial endophytes
were characterized by sequencing a 300 bp portion of the 16S rRNA gene. Most of the
sequences of endophytes obtained were homologous with Enterobacter sp. and
Pseudomonas sp. Other endophytes encountered were Pantoea sp., Staphylococcus sp.,
Bravibacillus sp., Klebsiella sp., Erwinia sp., and Curtobacterium sp. (Magnani et al.
2010).
Endophytic bacteria of Coffea arabica L. have previously been identified in
various trees sampled in Colombia, Hawaii, and Mexico (Vega et al. 2005). In this study,
87 culturable isolates were obtained which represented a total of 19 genera of endophytic
bacteria. Bacteria were isolated from various tissues such as berries, leaves, stems, and
roots. Endophytes of coffee were Burkholderia cepacia, B. gladioli, B. glathei, and B.
pyrrocinia. Other endophytes found were Stenotrophomonas maltophilia,
Methylobacterium radiotolerans, Pantoea agglomerans, Klebsiella sp., Enterobacter sp.,
9
Curtobacterium sp., Bacillus sp., Cedecea sp., Chromobacterium sp., Clavibacter sp.,
Escherichia vulneris, Micrococcus sp., Pseudomonas sp., Rhodococcus sp., Salmonella
sp., Serratia sp., Vanovorax sp., Xanthomonas sp., and Yersinia sp. (Vega et al. 2005).
A large scale study on endophytic bacteria was carried out on agronomic crops
and prairie plants in the Midwest United States during six years (Zinniel et al. 2002). In
that study, 853 different bacterial strains were isolated from 4 agronomic crop species
and 27 prairie plant species. Endophytes in this study were identified by carbon source
utilization, fatty acid methyl-ester analysis, and 16S rRNA gene sequencing. Some
genera of endophytes recovered were Curtobacterium, Agrobacterium, Bacillus,
Bradyrhizobium, Cellulomonas, Clavibacter, Corynebacterium, Enterobacter, Erwinia,
Escherichia, Klebsiella, Microbacterium, Micrococcus, Pseudomonas, Rothia, and
Xanthomonas (Zinniel et al. 2002).
Bacterial endophytes of cotton and sweet corn were identified in plants sampled
in Alabama, US. They were isolated on Tryptic Soy Agar plates and identified by fatty
acid methyl-ester (FAME) analysis (McInroy and Kloepper 1995). Endophytes isolated
only in cotton were Acinetobacter baumanii, Alcaligenes spp., Cellulomonas spp.,
Comamonas testosteroni, and Erwinia carotovora. Endophytes that were only isolated
from sweet corn were: Citrobacter koseri, Flavomonas oryzihabitans, Microbacterium
spp., and Stenotrophomonas maltophilia. Several endophytes were encountered in both
cotton and sweet corn. These were Agrobacterium radiobacter, Bacillus megaterium, B.
pumilus, B. subtilis, B. thuringiensis, Bacillus spp., Burkholderia cepacia, B. gladioli, B.
picketti, B. solanacearum, Clavibacter spp., Curtobacterium spp., Enterobacter spp.,
10
Pantoea spp., Serratia spp., Klebsiella spp., Escherichia spp., and Rhizobium spp.
(McInroy and Kloepper 1995).
Sweet potatoes have been surveyed for bacterial endophytes. Samples were
obtained from a grocery store in Seattle, WA. Bacteria were characterized by analysis of
16S rRNA sequences (Khan and Doty 2009). Eleven culturable endophytes were
characterized as belonging to the genera Enterobacter, Rahnella, Rhodanobacter,
Pseudomonas, Stenotrophomonas, Xanthomonas, and Phyllobacterium.
Not many studies have been conducted in Puerto Rico regarding bacterial
endophytes. Most studies performed have concentrated on describing fungal endophytes.
However, there are some previous studies on bacterial endophytes. Bacteria in the
vascular system of various citrus trees in Puerto Rico were described (Rivera Rodríguez
2006). They were characterized by their morphology, biochemical characteristics,
BIOLOG® method, and amplification of a 973 bp fragment of the 16S rRNA gene.
Eighty-six isolates were recovered from the vascular system and were grouped into 11
strains. Out of the 11 strains, some were characterized as Bacillus thuringiensis, B.
cereus, Bacillus, Delftia acidovorans, Pseudomonas putida, and Brevundimonas diminuta
(Rivera Rodríguez 2006).
An extensive study exists on prokaryotic endophytes of the sea grass Thalassia
testudinum in various locations in Puerto Rico (Couto-Rodríguez 2009). In this study,
endophytes of sea grass beds were obtained from four areas: Buyé Beach, Los Morrillos
in Cabo Rojo, Cayo Enrique in Lajas, and Puerto de la Libertad in Vieques. A total of
3,240 leaf fragments were processed from 60 plants that were sampled at the previously
mentioned locations. Endophytic prokaryotes were recovered from 1,987 (61%) leaf
11
fragments. Some encountered endophytes were Bacillus, Staphylococcus, Vibrio,
Enterobacter, Halobacillus, Pseudovibrio, Nesterenkonia, Exiguobacterium,
Geobacillus, Pseudovibrio, Pseudomonas, Microbulbifer, Pseudoalteromonas, Vibrio,
and Cobetia.
Benefits and Applications of Bacterial Endophytes
Some bacterial endophytes provide an advantage to the host they colonize over
non-infected plants. These organisms are capable of promoting plant growth both
directly and indirectly. Endophytes can promote growth directly by the production of
plant growth hormones and by nitrogen fixation. They can also promote plant growth
indirectly by alleviating the effect of environmental stressors and the deterrence of
pathogenic organisms. Bacterial endophytes could be used in the future to improve
results of phytoremediation efforts as well as for the use as a biocontrol agent of many
phytopathogenic diseases which can affect crop yield in plants of agricultural importance.
Another very useful application of endophytes is that many are capable of producing
secondary metabolites with antibiotic properties which could someday be useful in
medicine.
Bacterial endophytes can have a direct effect on the hosts they colonize by
synthesizing compounds which are used directly in plant metabolism. Some of these
compounds can be plant hormones, metabolic precursors of necessary compounds, or
provide more nitrogen and phosphorous to the plant. Bacterial endophytes can improve
plant growth by the production of plant hormones. Indole-3-acetic acid is a plant
hormone from the auxin family. It causes the plant to grow apically, mediates growth
towards light, promotes the development of vascular tissue, promotes the activity of
12
secondary meristems, induces the formation of roots, inhibits leaf and fruit loss, and
stimulates the development of the fruit (Graham et al. 2003). It has been found that many
bacterial endophytes are capable of producing indole-3-acetic acid. Some bacteria that
can produce indole-3-acetic acid are Pseudomonas sp., Bacillus sp., Azospirillum sp., and
Rhizobacterium sp., Mesorhizobium sp., Sinorhizobium sp., Brevibacterium sp.,
Bifidobacterium sp., Agrobacterium tumefaciens, among others (Long et al. 2008;
Spaepen et al. 2007; Lodewyckx et al. 2002; Nimnoi and Pongslip 2009). The bacterium
Burkholderia kururiensis isolated from an aquifer environment was found to become
endophytic and colonize rice. This species produces indole-3-acetic acid inside the rice
and therefore has the potential of promoting its growth and rice yield (Mattos et al. 2008).
Bacteria producing this compound inside plants have the potential to have an effect on
the overall plant growth of its host by altering hormone levels (Spaepen et al. 2007).
Indole-3-acetic acid can be produced by bacteria by numerous metabolic pathways.
Some pathways used by bacteria to produce indole-3-acetic acid that have been described
are: indole-3-acetamide pathway, indole-3-pyruvate pathway, tryptamine pathway,
tryptophan side-chain pathway, indole-3-acetonitrile pathway, and tryptophan-
independent pathway (Spaepen et al. 2007).
Other phytohormones that can be affected by endophytes are cytokinins.
Cytokinins are compounds that affect root growth, differentiation of cells, stimulate cell
division and growth, stimulates germination, and retards the aging process in some
organs (Campbell et al. 1999). These compounds are called cytokinins because they
induce the cytokinesis stage of the cell cycle. It has been found in a previous study that
the bacterium Methylobacterium extorquens, an endophyte of Scots pine, is capable of
13
promoting the production of cytokinins indirectly. This bacterium does not produce the
cytokinin itself but it produces an adenine derivative which is used as a precursor by the
plant to produce the final form of the hormone cytokinin which affects the host’s growth
(Pirtilla et al. 2004).
Ethylene is another plant hormone that may be altered by bacterial endophytes
and have a beneficial effect for its host. Ethylene is a gas that controls leaf drop, induces
ripening of fruits, causes senescence of leaves, affects cell specialization, causes aging of
flowers, and can help a plant’s defense against pathogens (Graham et al. 2003). Another
important effect of ethylene is that it opposes or reduces some of the effects the hormone
auxin has on plants (Moore et al. 1995). Bacterial endophytes are organisms with the
capacity of altering ethylene levels in plants, therefore affecting its physiology. Bacteria
can decrease ethylene levels by producing the enzyme 1-aminocyclopropane-1-
carboxylate (ACC) deaminase (Hardoim et al. 2008). Some endophytic bacteria such as
members of the genus Burkholderia have a widespread capacity of producing ACC
deaminase. Eighteen different species of Burkholderia sp. were found to have the acdS
gene and produce enough ACC to be able to have a significant effect on reducing plant
ethylene levels (Onofre-Lemus et al. 2009). The previous authors believe that the
widespread ACC production in various species of Burkholderia sp. and the common
frequency they are found inside plants indicates that these bacteria may be significant
contributors to plant growth in nature.
Plants can benefit by bacteria directly because of their ability to fix nitrogen. The
nitrogen fixing ability of some endophytes has been previously described. One of these
cases is of the bacterium Herbaspirillum sp which was found to colonize wild rice
14
(Elbeltagy et al. 2001). This bacterium was proven to fix nitrogen using the acetylene
reduction assay. Other endophytic diazotrophs found in wild rice were Ideonella sp.,
Enterobacter sp., and Azospirillum sp. Another bacterium that has been studied for its
ability to fix nitrogen within plants is Acetobacter diazotrophicus (Tapia-Hernández et al.
2000). This bacterium was studied in the inner tissues of surface sterilized roots, stems,
and leaves of pineapple plants. It was found with higher frequency in buds that had not
been fertilized with nitrogen and in lower frequencies inside buds previously fertilized
with nitrogen (Tapia-Hernández et al. 2000). This indicates that the plant could be aided
by bacteria that fix nitrogen in cases where nitrogen is depleted.
Endophytes have been described as beneficial for hosts by alleviating the effects
of environmental stressors. Plants are able to tolerate stress by associating themselves
with mutualistic endosymbionts. Endophytic fungi “may confer tolerance to drought,
metals, disease, heat, and herbivory, and/or promote growth and nutrient acquisition”
(Rodríguez and Redman 2008). Endophytes have the ability to improve its host’s fitness
in harsh environments. For example, an endophytic fungus closely related to the fungus
Curvularia sp. was found to confer its host, Dichanthelium lanuginosum, with a wider
range of thermotolerance compared to nonsymbiotic hosts. This plant grows naturally in
geothermal soils. A study was conducted in order to identify any endophytes that may be
aiding these plants to survive under heat stress. Endophyte free plants began to show
signs of deterioration at 50ºC, while plants with the fungus survived temperatures up to
65ºC (Redman et al. 2002).
Endophytes also have the potential of protecting plants present under high
salinity. The bacterial endophytes of the halophytic plant Prosopis strombulifera were
15
studied at El Berbedero saline in Argentina (Sgroy et al. 2009). Cultivable bacterial
endophytes obtained from surface sterilized roots were characterized by sequencing of
the 16S rRNA gene. Twenty-nine strains of bacterial endophytes were isolated and some
were further tested for various plant growth-promoting properties. They were analyzed
for siderophore production, phosphate solubilization, nitrogen fixation, ACC deaminase
production, antifungal activity, protease production, and phytohormone production.
Some strains had DNA sequences that were highly homologous with Lysinibacillus
fusiformis, Bacillus subtilis, Brevibacterium halotolerans, Bacillus licheniformis,
Bacillus pumilus, Achromobacter xylosoxidans, and Pseudomonas putida. Most of the
strains tested produced ACC deaminase, fixed nitrogen, and produced phytohormones
(Sgroy et al. 2009). These characteristics suggest that the endophytic bacteria present in
these halophytic plants may be contributing to their fitness in such harsh environments.
The same could possibly be occurring in trees of Coccoloba uvifera in Cabo Rojo.
Bacterial endophytes can help plant growth indirectly by helping the plant defend
itself against pathogens. It has been previously found that these organisms can form
antagonistic relationships with organisms that are known to cause disease in plants. A
novel plant growth-promoting bacterium called Delftia tsuruhatensis HR4 has proven to
suppress the growth of plant pathogens such as Xanthomonas oryzae pv. oryzae,
Rhizoctonia solani,and Pyricularia oryzae. As well as protecting plants from pathogens,
D. tsuruhatensis H4 has the ability to fix nitrogen (Han et al. 2005). The nitrogen-fixing
activity of this bacterial strain was demonstrated in culture and the nif gene was also
located on its chromosome. The fixation of atmospheric nitrogen benefits the host
directly by aiding providing an essential nutrient to the plant.
16
Red rot is caused by the microfungus Colletotrichum falcatum and has multiple
hosts including sugar cane (Viswanathan et al. 2003). Bacterial strains present in sugar
cane stalks were found to be antagonizing towards C. falcatum suggesting that they play
a role in a plant’s defense. Out of the 51 bacterial endophyte isolates found, 7 were
found to have a strong antagonizing relationship. The 7 isolates belong to Pseudomonas
aeruginosa, Pseudomonas fluorescens, or Pseudomonas putida (Viswanathan et al.
2003).
Bacterial endophyte communities can also help protect their hosts by the
production of antibiotics in specific locations of the plant. Endophytes have been found
to contribute to disease resistance in potato tubers (Sturz et al. 2002). The antibiotic
activity of endophyte communities was studied among various layers of the tuber peel.
Antibiosis was measured against the pathogens Fusarium sambucinum, F. avenaceum, F.
oxysporum, and Phytophthora infestans in all layers studied. The authors determined that
antibiotic activity by endophyte communities in potato tubers was most significant
against these pathogens in isolates recovered from the outermost peel of the potato tubers
(Sturz et al. 2002).
Bacterial endophytes could have many applications with advantages to society
and agriculture. These microorganisms are, in many cases, capable of producing
compounds which are of economic and medical importance for mankind. Researching
organisms that are capable of producing unique compounds at a greater yield could have
drastic effects in the pharmaceutical industry and make novel treatments available for
patients who need them. Endophytes are also capable of being used for bioremediation
and biocontrol purposes. These bacteria can be genetically modified to aid the plant in
17
the removal of toxic compounds and be used to fight off economically devastating
diseases which affect crops of agricultural importance.
Endophytes from the genus Pseudomonas sp. have shown to be active producers
of antimycotic compounds such as pseudomycins and ecomycins. Pseudomonas
syringae, for example, produces the antimycotic compounds pseudomycins (Harrison et
al. 1991). Pseudomycin A, B, C, and D were isolated from liquid cultures of P. syringae.
One of these compounds, Pseudomycin A, demonstrated a strong ability to antagonize the
pathogen Candida albicans (Harrison et al. 1991). Other antimycotic compounds
produced by a Pseudomonas sp. are ecomycins. These peptide antimycotics produced by
Pseudomonas viridiflava showed to have significant bioactivity against various human
and plant pathogenic fungi (Miller et al. 1998). They were effective against Candida
albicans and Cryptococcus neoformans.
Streptomyces sp. is a group of very versatile actinobacteria which are capable of
producing a variety of peptide antibiotics and are active against some plant and human
pathogenic bacteria. They are capable of producing peptide antibiotics such as
coronamycins, munumbicins, and kakadumycins (Ezra et al. 2004; Castillo et al. 2002,
2003). Coronamycin have been isolated from the endophyte Streptomyces sp. (MSU-
2110) and proven effective against some pythiaceous fungi, Streptococcus pneumoniae,
Cryptococcus neoformans, and the protist Plasmodium falciparum (Ezra et al. 2004).
Munumbicins A, B, C, and D are four antibiotic compounds obtained from an endophytic
Streptomyces NRRL 3052 found in the medicinal snakevine plant (Castillo et al. 2002).
Each munumbicin identified demonstrated different antibiotic activities towards certain
pathogens. However, it was found that munumbicins in general are effective against
18
Gram positive bacteria such as Bacillus anthracis. Specifically notable was munumbicin
D which had a significant effect on the malaria parasite Plasmodium falciparum (Castillo
et al. 2002). Kakadumycins are similar to munumbicins in that it is also generally
effective against Gram positive bacteria and Bacillus anthracis (Castillo et al. 2003).
Bacterial endophyes can be used for phytoremediation efforts. Phytoremediation
is the practice of removing contaminants from soil by the use of plants which possess the
ability to degrade or store the contaminant within its tissues. The phytoremediation of
pollutants is a combined effort between the plants and their associated microorganisms
(Lodewyckx et al. 2002). It has been used to treat contaminants such as metals,
petroleum, solvents, explosives, polycyclic aromatic hydrocarbons, and other organic
pollutants (Doty 2008). Successful cases of phytoremediation using endophytic bacteria
have been summarized in a recent review (McGuinness and Dowling 2009).
Bacterial endophytes may naturally possess the ability to remove certain
contaminants from the soil. The presence of the contaminant itself should be sufficient to
promote the growth of bacteria that are resistant to the contaminant’s toxicity and
possibly be capable of using it as a substrate for its growth. In certain cases though, it has
been demonstrated that genetically engineered endophytes are capable of further
improving phytoremediation results (Taghavi et al. 2005). Genetically engineered
endophytes can be produced in order to remove a greater quantity of certain contaminants
from the environment. The bacterium Burkholderia cepacia VM1468 containing the
plasmid pTOM-Bu61, which allows for toluene degradation, was inoculated in Poplar
trees (Taghavi et al. 2005). Even though this bacterium did not become established at a
detectable level within the endophyte community in poplar, it was found that there was
19
horizontal gene transfer of the pTOM-Bu61 plasmid to other endophytes present in the
plant (Taghavi et al. 2005). Engineering plasmids that can carry out processes necessary
for the degradation of a contaminant could be spread among other members in the
community by horizontal gene transfer and increase the amount of bacteria capable of
metabolizing the contaminant. In another case studied, Burkholderia cepacia VM1330
with the pTOM plasmid was found to reduce the amount of toluene evapotranspirated by
50-70% compared to control plants (Barac et al. 2004).
Bacterial endophytes have been researched for their biocontrol abilities. Some
bacterial endophytes are capable of suppressing disease in crops of agricultural
importance. Their use can lead to a significant reduction in crop losses to pathogen
damage and increase crop yield. It could also lead to a reduction in costs incurred in
products used for protecting the crops against pathogens.
Species of Bacillus spp. have been studied for their potential to suppress black rot
disease caused by the pathogen Phytophthora capsici on the cocoa tree Theobroma cacao
(Melnick et al. 2008). In this study the endophytic bacterium Bacillus cereus colonized
cacao trees and caused a significant reduction in the severity of the black rot disease. The
authors also concluded that the endophyte caused an induced systemic resistance to
pathogens since newly formed leaves that were uncolonized by endophytes also
demonstrated disease suppression.
Bacterial endophytes have also been researched for their potential to control
crown gall disease which is caused by the pathogen Rhizobium vitis (Eastwell et al.
2006). Some species studied for their ability to control disease were the endophytes
Pseudomonas fluorescens isolate 1100-6, Bacillus subtilis isolate EN63-1, and Bacillus
20
species isolate EN71-1. All three of these bacteria were studied for crown gall formation
in Nicotiana glauca. It was demonstrated that all three of these endophytes were capable
of reducing crown gall size and population of Rhizobium vitis relative to control plants
(Eastwell et al. 2006).
Another study on biological control of a pathogen by endophytes was carried out
by Shlomi et al. (2006). In this study, bioprospecting of bacterial endophytes capable of
controlling the effect of the pathogen Hemileia vastatrix (coffee rust) was carried out.
Isolates of bacterial endophytes obtained from coffee plants were studied specifically for
their ability to inhibit the germination of urediniospores and control the leaf rust in the
leaves of the coffee plant. After testing 23 of the 40 isolates obtained, the authors
determined the most effective biocontrol agents to be Bacillus lentimorbus, Bacillus
cereus, Clavibacter michiganensis subsp. michiganensis Smith, and Klebsiella
pneumoniae Schroeter.
It has been demonstrated that bacterial endophytes are capable of improving
phytoremediation efforts for the removal of contaminants in the environment. They are
also capable of reducing the effects of disease on various plants including those of
significant agricultural importance. Both these benefits could have a profound impact in
agriculture, if further developed.
Description of the host plant Coccoloba uvifera
Coccoloba uvifera (L.), commonly known as the sea grape, belongs to the family
Polygonaceae. Coccoloba is the largest genus within the family in the neotropics
containing between 120 and 200 species (Smith et al. 2004). Coccoloba uvifera is a
ramified small tree or shrub that has large, round, and simple leaves with an alternate
21
arrangement. This family is characterized by the presence of a membranous structure
called ocrea at the base of the petiole (Smith et al. 2004) (Fig. 4.01)
Figure 4.01. A leaf of Coccoloba uvifera. The arrow indicates
the ocrea structure characteristic of this family.
Thirteen species exist of the genus Coccoloba in Puerto Rico. These are found on
all forest types and elevations. The species under study, Coccoloba uvifera, is not
endemic to Puerto Rico and has a widespread distribution in the neotropics. The sea
grape is native to the coasts of the south of Florida, Bermuda, the Bahamas, the West
Indies, the north and east of South America, Mexico, Central America, and on the Pacific
Coast of South America up to Peru (Parrota 1994). In Puerto Rico, this tree is found
naturally in coastal habitats and is utilized as an ornamental tree.
22
This small tree or shrub can grow up to 15 meters in height and is very common
in sand dunes around coastal areas. It is easily recognizable by its leaves which are round
and thick, and large racemes of small, round, and edible fruits that look like grapes
(Parrota 1994). This species is important because it is one of the first to colonize coastal
areas. It has the ability to tolerate high levels of salt, ultraviolet radiation, and
temperatures. The extreme environment where some trees of Coccoloba uvifera are
found could select for unique and rare species of endophytes.
23
Chapter 5
Materials and Methods
Research area
The study area is located in the southwest coast of Puerto Rico in the municipality
of Cabo Rojo (17˚56’13.39”N and 66˚11’18.52”W). Trees of Coccoloba uvifera were
sampled in two different sites. The first site is next to a hypersaline solar saltern which is
fed by the Fraternidad Lagoon. The second site is in Playuela located further south from
the salterns. A total of four (4) trees were sampled, two at each site. Various mature
trees of Coccoloba uvifera are found naturally in both areas. The locations of the trees
sampled are shown in Figure 5.01. Photographs of Playuela and the Solar Salterns in
Cabo Rojo where trees were sampled are represented in Figures 5.02 and 5.03.
Sampling and Leaf Processing
Four (4) trees of Coccoloba uvifera were sampled. Two (2) trees were studied
near the salterns and two (2) in Playuela. From each tree, four (4) mature symptomless
leaves were obtained. Leaves were collected, labeled, placed in plastic bags, and stored
inside a cooler until processed. The first sampling was in September, 2008 (wet season)
and the second in March, 2009 (dry season). Leaves were sampled in the same manner
on both sampling periods. Leaves were processed in the laboratory within 48 hours.
Once in the laboratory, they were washed with water to remove excess debris. Leaves
were then surface sterilized by submerging sequentially in 75% ethanol for 1 minute, 5%
sodium hypochlorite for 3 minutes, and 75% ethanol for 30 seconds (Lodge et al. 1996).
24
Figure 5.01. Location and areas studied in Cabo Rojo, Puerto Rico. Cabo Rojo is located
in the southwest of Puerto Rico. Two of the trees studied (S1 and S2) were located near
the hypersaline solar salterns. The other two trees studied (P1 and P2) were located at
Playuela.
Figure 5.02. Picture of Playuela in Cabo Rojo, PR where
two trees of
Figure 5.03. Picture of the Solar Salterns in Cabo Rojo, PR
where two trees of
Figure 5.02. Picture of Playuela in Cabo Rojo, PR where
two trees of Coccoloba uvifera were sampled.
Figure 5.03. Picture of the Solar Salterns in Cabo Rojo, PR
where two trees of Coccoloba uvifera were sampled.
25
26
They were finally rinsed with distilled water and patted dry. Using a sterile
scalpel and scissors, fragments from the entire leaf measuring 2 mm x 2 mm were excised
and placed in a glass beaker. Cutting the leaf fragments in a smaller size could destroy
the tissues of the leaf (Gamboa et al 2002). Fragments from each leaf were selected
randomly by shaking the beaker and extracting a random fragment. From each leaf
obtained, 10 fragments were chosen randomly. A total of 320 leaf fragments of
Coccoloba uvifera were analyzed.
Growth of Bacteria
Leaf fragments were inoculated on 50% TSA media. The plates were incubated
at 25˚C until growth was observed. Once bacterial colonies were observed growing from
the leaf fragments they were isolated in pure culture in 50% TSA. Negative controls for
each leaf were made, before cutting the leaf into fragments, by pressing on TSA plates.
The purpose of a negative control was to make sure bacteria being recovered were
endophytes and not epiphytic organisms.
Characterization of Isolates
Pure cultures were classified according to their physical appearance in culture
(margin, elevation, color, and growth pattern). Gram stains were performed in order to
corroborate and confirm the data obtained by DNA sequencing. The identification of the
bacterial endophytes was done by DNA sequencing of the 16S rRNA gene. DNA
extractions were carried out using PrepMan™ Ultra (Applied Biosystems, Foster City,
CA). Once the appropriate dilution for amplification was obtained, polymerase chain
reaction (PCR) was used to amplify the DNA using the primers 27F (5’-
AGAGTTTGATCMTGGCTCAG-3’) and 1525R (5’-AAGGAGGTGWTCCARCC-3’).
27
Amplicons were verified using 1% agarose gel electrophoresis. Positive amplicons were
then purified using ExoSAP-IT® Reagent Kit (USB Corporation, Cleveland, OH).
Sequencing was performed using Big Dye Sequencing v3.1 Kit (Applied Biosystems,
Foster City, CA) with the same primers mentioned before. The samples were then
alcohol precipitated on a 96-well microplate. They were sequenced in a ABI 3130
Genetic Analyzer (Applied Biosystems, Foster City, CA) and analyzed with the software
Sequencing Analysis v 5.2 (Applied Biosystems, Foster City, CA). The sequences were
assembled using the software AutoAssembler (Applied Biosystems, Foster City, CA) and
homologous sequences were obtained by BLAST analysis in GenBank
(http://blast.nbi.nlm.nih.gov/Blast.cgi; Altschul et al. 1990).
Statistical Analysis
The frequency of colonization of endophytes was carried out as described by
Fisher and Petrini (1987). The mathematical equation is:
Colonization Frequency = (Ni/Nt) x 100
Where Ni is the number of fragments where a species of endophyte was detected and Nt is
the total number of inoculated fragments. The frequencies were then compared by chi-
square statistical analysis. Chi-square statistics were performed using Minitab®
v15.1.30.0 software. It was used to determine significant difference between the
frequencies of leaf fragments colonized by bacterial endophytes. Two statistical analyses
were performed during this study. One was used to compare the frequency of
colonization of bacterial endophytes during the wet and dry seasons on both sites
(Playuela and solar saltern). The second statistical analysis was more specific and tested
for significant difference between the frequencies of colonization of bacterial endophytes
28
on leaf fragments obtained from all four trees of Coccoloba uvifera sampled on both
seasons.
Another statistical analysis, similar to the Chi-square test, was used to analyze the
observed data. Some of the data could be manipulated and placed into 2 x 2 contingency
tables and analyzed using a Fisher’s exact test (Daniel 2005). The Fisher’s exact test
helped determine if the proportion of fragments colonized by bacterial endophytes during
the dry season compared to the data from the wet season. It was also used to compare the
amount of leaf fragments colonized by bacterial endophytes in each of the sites sampled
and between sites.
Species Accumulation Curves and Biodiversity and Richness Estimators
Species accumulation curves were constructed for all trees sampled during both
seasons. They were also constructed for each site and season. These graphs allowed
visualizing the accumulation of bacterial endophyte species in the amount of leaf
fragments of Coccoloba uvifera sampled. Observed species accumulation and expected
species accumulation were graphed against the number of leaf fragments. The software
EstimateS v.8.2.0 was used to calculate Coleman rarefaction values for the expected
species accumulation. Biodiversity indeces were useful for measuring the diversity of
species in a given any sample. These were also calculated using the software EstimateS
v.8.2.0.
29
Chapter 6
Results
Frequency of Colonization
The colonization frequency of bacterial endophytes in leaf fragments of
Coccoloba uvifera was calculated for all the leaves sampled. The total colonization
frequency of bacterial endophytes recovered during the wet and dry seasons is
summarized in Fig. 6.01, Table 6.01 and Table 6.02. The total frequency of colonization
for each season was calculated based on the combined results obtained from the four trees
sampled (P1, P2, S1, and S2). Forty leaf fragments per tree were studied during each
season for a total of 160 fragments. Out of 160 fragments inoculated during the wet
season, 102 of them were colonized by bacterial endophytes. In the case of the dry
season, 96 leaf fragments contained bacterial endophytes. The colonization frequency
was highest during the wet season (63.8%).
The frequencies of leaf fragments containing bacterial endophytes vary between
both sites and seasons sampled. In Figure 6.02 and Table 6.03, the percents of
colonization of leaf fragments by bacterial endophytes are summarized. The highest
frequency of endophytes was recovered during the wet season at tree P2 located at
Playuela where 35 out of a total of 40 fragments (87.5%) contained endophytic bacteria.
The lowest frequency of endophytes was recovered during the wet season as well, but in
tree S1 located at the solar saltern where only 16 out of 40 fragments (40%) contained
endophytes. During the dry season the largest frequency of endophytes was recovered
from tree S2 in the solar salterns with observed growth on 30 of the 40 fragments
inoculated (75%). The lowest frequency of endophytes during the dry season was
30
obtained from the tree S1 with 21 out of 40 fragments displaying bacterial growth
(52.5%).
Figure 6.01. Total percent of colonized leaf fragments with bacterial endophytes
obtained from trees of Coccoloba uvifera sampled during the wet and dry seasons in
Cabo Rojo, Puerto Rico.
63.8%60%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
September, 2008 (wet) March, 2009 (dry)
Percent of Colonization
Sample Season
31
Table 6.01. Frequency of fragments colonized by bacterial endophytes
from leaves of Coccoloba uvifera sampled in September, 2008 (wet season).
Where P = Playuela, S = saltern, and L = leaf. Leaves were numbered 1-4.
Leaf
Frequency of Colonization
(# of colonized fragments/total)
P1L1
5/10 (0.50)
P1L2
5/10 (0.50)
P1L3
6/10 (0.60)
P1L4
8/10 (0.80)
P2L1
10/10 (1.00)
P2L2
5/10 (0.50)
P2L3
10/10 (1.00)
P2L4
10/10 (1.00)
S1L1
4/10 (0.40)
S1L2
5/10 (0.50)
S1L3
6/10 (0.60)
S1L4
1/10 (0.10)
32
Table 6.01 Continued
S2H1
8/10 (0.80)
S2H2
5/10 (0.50)
S2H3
6/10 (0.60)
S2H4
8/10 (0.80)
Table 6.02. Frequency of fragments colonized by bacterial endophytes from
leaves of Coccoloba uvifera sampled in March, 2009 (dry season). Where
P = Playuela, S = saltern, and L = leaf. Leaves were numbered 5-8.
Leaf
Frequency of Colonization
(# of colonized fragments/total)
P1L5
5/10 (0.50)
P1L6
8/10 (0.80)
P1L7
5/10 (0.50)
P1L8
4/10 (0.40)
P2L5
10/10 (1.00)
P2L6
4/10 (0.40)
33
Table 6.02 Continued
P2L7
7/10 (0.70)
P2L8
2/10 (0.20)
S1L5
4/10 (0.40)
S1L6
5/10 (0.50)
S1L7
4/10 (0.40)
S1L8
8/10 (0.80)
S2L5
9/10 (0.90)
S2L6
7/10 (0.70)
S2L7
8/10 (0.80)
S2L8
6/10 (0.60)
34
Table 6.03. Frequency of leaf fragments colonized by bacterial endophytes in trees of C.
uvifera at each season and site sampled. Where P = Playuela and S = solar saltern.
Trees Sampled
September, 2008
(wet)
March, 2009
(dry)
Total
P1
24/40 (0.60)
22/40 (0.55)
46/80 (0.575)
P2
35/40 (0.875)
23/40 (0.575)
58/80 (0.725)
S1
16/40 (0.40)
21/40 (0.525)
37/80 (0.4625)
S2
27/40 (0.675)
30/40 (0.75)
57/80 (0.7125)
Total
102/160 (0.6375)
96/160 (0.60)
198/320 (0.61875)
Figure 6.02. Percent of colonization of bacterial endophytes recovered from four trees of
Coccoloba uvifera at two seasons.
60%
87.5%
40%
67.5%
55% 57.5%52.5%
75%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
P1 P2 S1 S2
Percent of Colonization
Trees Studied
September, 2008 (wet)
March, 2009 (dry)
35
Statistical Analysis
The Chi-square statistical analysis showed that there was not a significant
difference in fragments colonized by bacterial endophytes on both sites and seasons
sampled (DF=1, α=0.05, X2=2.386, p-value=0.122). In this analysis, the difference
between the frequency of colonization of bacterial endophytes between Playuela and
solar saltern site was analyzed.
The same statistical analysis was carried out to compare leaf fragments colonized
on all four trees sampled on separate seasons. Instead of comparing the frequency of
colonization between both sites, this analysis compared the frequency of colonization of
bacterial endophytes in the four trees sampled. The statistical analysis indicated that
there was not a significant difference among the quantities of leaf fragments colonized by
bacterial endophytes on all four trees of Coccoloba uvifera sampled during both seasons
(DF=3, α=0.05, X2= 3.224, p-value=0.358). The colonization frequency of bacterial
endophytes on each leaf sampled during the wet and dry season is summarized on Table
6.01 and Table 6.02.
A Fisher’s exact test was used to determine if the colonization frequency of
bacterial endophytes in leaf fragments of C. uvifera during the dry season was less than
or equal to the colonization frequency observed during the wet season. The Fisher’s test
indicated that there was not a significant difference in the colonization frequency of
bacterial endophytes between both seasons sampled (α=0.05, p-value=0.2825). A two-
tailed Fisher’s exact test indicated that there was significant difference in colonization
frequency of bacterial endophytes in fragments of C. uvifera leaves sampled at the
Playuela site during the wet season compared to the dry season (α=0.05, p-
36
value=0.0307). Another two-tailed Fisher’s exact test indicated that there was no
significant difference in the frequency of colonization of endophytes in trees sampled at
the solar saltern site at both seasons sampled (α=0.05, p-value=0.2609).
Isolates of Bacterial Endophytes Obtained from Coccoloba uvifera
The number of isolates and the different species of endophytic bacteria recovered
from both sampling sites and seasons is shown on Table 6.04. This was calculated by
adding the number of isolates and species obtained from both trees sampled at the same
site. Data from trees P1 and P2 was combined for the data shown from Playuela and
trees S1 and S2 for data from the solar saltern site.
Table 6.04. Number of isolates and different species of endophytic bacteria recovered
from both sampling sites (Playuela and Solar Saltern) in Cabo Rojo, PR.
Site
Season
Number of Isolates
Number of Species
Playuela
Dry
38
10
Wet
56
10
Solar Saltern
Dry
39
10
Wet
41
13
Total
Dry
77
15
Wet
97
17
37
In total, more isolates were obtained during the wet season from both sites
sampled. Ninety-seven isolates were obtained during the wet season whereas 77 were
recovered during the dry season from both sites. During the dry season, 10 species of
bacterial endophytes were recovered from both sites sampled whereas 13 species were
recovered during the wet season.
The most number of isolates were obtained from both trees at Playuela during the
wet season where 56 isolates were recovered. The lowest number of endophyte isolates
was recovered during the dry season at trees at Playuela where 38 isolates were
recovered. At the solar saltern site, the largest amount of isolates was recovered from the
wet season. However, only two more isolates were obtained during the wet season than
the dry. A difference in number of isolates of bacterial endophytes was more evident in
trees of Coccoloba uvifera at the Playuela sampling site where 18 more isolates were
recovered in the wet season than in the dry.
Bacterial Endophytes Characterized
In total 143 isolates were studied. From these isolates, morphospecies were
selected based on colony appearance and Gram staining for molecular characterization by
DNA sequencing of the 16S rRNA gene. In certain cases where classifying on
morphospecies did not seem to be obvious, bacteria were characterized to confirm their
identity. Duplicates of some morphospecies were also taken. DNA was extracted and
PCR amplifications were carried out successfully. In total, 61 different isolates were
chosen to be characterized which belonged to 40 different strains of bacteria from 14
different genera of endophytic bacteria. The closest homology matches that were
obtained for the cultures are summarized in Table 6.05.
38
The bacterial endophytes characterized during this study belonged to 14 different
genera: Burkholderia sp., Pseudomonas sp., Stenotrophomonas sp., Staphylococcus sp.,
Ralstonia sp., Acinetobacter sp., Proteus sp., Morganella sp., Delftia sp., Achromobacter
sp., Bacillus sp., Providencia sp., Paenibacillus sp., and Lysinibacillus sp.
Of the 61 sequences obtained, 12 could be appropriately matched up to species
level with a significant similarity between sequences submitted and those in the NCBI
BLAST database (>97% homology). These endophytes are: Stenotrophomonas
maltophilia, Staphylococcus epidermidis, Ralstonia picketti, Acinetobacter calcoaceticus,
Proteus mirabilis, Delftia tsuruhatensis, Pseudomonas aeruginosa, Lysinibacillus
sphaericus, Bacillus flexus, Bacillus pumilus, Bacillus cereus, and Bacillus safensis.
Other species were matched, but to a lesser percent of homology were Paenibacillus
thiaminolyticus, Morganella morganii, Providencia rettgeri, and Pseudomonas putida.
In Table 6.06, the number of isolates of various characterized bacterial
endophytes isolated during this study is summarized. It includes the number of isolates
from each tree sampled (P1, P2, S1, S2) during the wet and dry seasons at Playuela (P)
and the solar saltern (S) sites in Cabo Rojo, Puerto Rico.
39
Table 6.05. Bacterial endophytes characterized from leaf fragments of Coccoloba uvifera
during the wet and dry season in Cabo Rojo, Puerto Rico. Where P = Playuela, S =
saltern, and L = leaf. Information was obtained from GenBank.
Sample
GeneBank
No.
Closest Match
% of
Homology
Previous Source
P1L2
P1L3
P2L1
S2L1
S2L2
S2L3
FJ545751.1
Stenotrophomonas sp.
Zsq1
99%
Soil in China
P1L2
EU931549.1
Stenotrophomonas
maltophilia ZFJ-10
100%
Sugar cane roots in Fuzhou,
China
P1L1
EU652101.1
Stenotrophomonas
maltophilia yb6027
96%
Ocean sediment
S2L4
EU543577.1
Stenotrophomonas
maltophilia
99%
Sea water in China
S2L8
P2L5
S1L6
GQ339107.1
Pseudomonas
aeruginosa TNAU
pf32
99%
Aloe vera plant in India
S1L1
EU741085.1
Pseudomonas sp.
13635M
93%
Marine sediment in Costa
Rica
S2L4
EF208895.1
Pseudomonas putida
N6
96%
Aromatic structure
degrader; unknown
40
Table 6.05 continuation
P2L4
EU489564.1
Pseudomonas sp. 471
96%
Marine environment, China.
P1L8
A4486369.1
Pseudomonas
aeruginosa
98%
Midgut of Solenopsis
invicta
P2L6
FJ577677.1
Bacillus safensis B1-1
98%
Unknown
P2L6
EU780103.1
Bacillus pumilus
DT83
97%
Soil in China
P1L2
GQ199590.1
Bacillus cereus DL31
99%
Unknown
P1L6
EF637039.2
Bacillus sp. TD67
94%
Antagonist against plant
pathogenic fungi
P1L8
AF526907.2
Bacillus safensis 51-
3C
99%
Mars Odyssey Orbiter and
encapsulation facility
S2L7
EU741099.1
Bacillus cereus
13651EE
99%
Beach sand, Costa Rica.
P2L3
FJ959366.1
Bacillus cereus
RMLAU1
85%
Treated tannery effluent
S1L8
EU236750.1
Bacillus sp. ZH4
96%
Endophyte of Stipa
purpurea at alpine grassland
S1L8
EF040535.1
Bacillus sp. JS-12
94%
Sea sponge Halichondria
P2L5
DQ870738.1
Bacillus pumilus
JSC_Hp101
96%
Clean room
41
Table 6.05 continued
S1L6
EU195956.1
Bacillus sp. P109
93%
Hydrocarbon polluted
sediment in Vigo, Spain
P2L7
FJ908753.1
Bacillus sp. B121
99%
Endophyteof rice plants in
Guangxi, China
S2L8
EU835567.1
Bacillus sp. HHNB2
97%
Albizia julibrissin (silk tree)
S2L7
FJ948078.1
Bacillus flexus Twd
99%
Plastic degrading consortia
P1L4
FJ392830.1
Burkholderia sp.
SYBC LIP-Y
98%
Soil in China
P1L4
FJ907187.1
Burkholderia cepacia
isolate 4
97%
Melon, China
S2L3
AM910270.1
Lysinibacillus sp. R-
30912
92%
Raw milk
P2L3
CP000817.1
Lysinibacillus
sphaericus C3-41
97%
Biocontrol agent of
mosquitos; soil
P1L4
S2L5
EU661709.1
Acinetobacter
calcoaceticus
NBRAJG93
98%
Water in India
S2L5
FJ975124.1
Acinetobacter sp.
JDC-16
92%
River sludge, China
P2L1
FJ688376.1
Delftia sp. K2-OAIF2
99%
Mosquito Aedes albopictus
42
Table 6.05 continued
P2L1
EF440614.1
Delftia tsuruhatensis
WXZ-1
97%
Unknown
S2L4
FJ378038.1
Delftia sp. JDC-3
100%
China
P2L1
EU880508.1
Delftia sp. PRE5
95%
Pearl River sediment
S2L1
AM942759.1
Proteus mirabilis
H14320
98%
Unknown
S2L1
DQ513315.1
Morganella morganii
VAR-06-2076
96%
Domesticated rabbit
S1L3
EU373416.1
Providencia sp.
YRL09
98%
Endophyte of a radish
P2L5
AJ320490.1
Paenibacillus
thiaminolyticus
DSM726ZT
84%
DSMZ, Germany
S2L7
EU073119.1
Achromobacter sp.
SY8
89%
Arsenic contaminated soil.
S1L5
FJ217188.1
Staphylococcus
epidermidis BBAR7-
13d
99%
Gut of whitefly Bemisia
tabaci
P1L3
DQ908951.1
Ralstonia picketti TA
100%
Soil in Minnesota
43
Table 6.06. Number of isolates from various common species of endophytes.
Species
P1 P2 S1 S2 Total
Dry Wet Dry Wet Dry Wet Dry Wet
Stenotrophomonas
maltophilia 3 8 8 2 4 10 35
Stenotrophomonas sp. 5 2 1 8
Pseudomonas sp. 6 10 15 2 4 2 5 44
Pseudomonas aeruginosa 3 5 8
Burkholderia cepacia 1 1 1 2 1 2 8
Burkholderia sp. 2 3 5
Providencia sp. 2 2
Lysinibacillus sp. 4 2 6
Delftia sp. 3 1 4
Proteus mirabilis 3 3
Pseudomonas putida 1 1
Morganella morganii 1 1
Acinetobacter calcoaceticus 2 2
Acinetobacter sp. 1 1
Ralstonia picketti 2 1 1 2 1 7
Bacillus sp. 6 3 8 8 25
Bacillus cereus 3 3
Bacillus flexus 3 3
Bacillus safensis 1 2 3
Bacillus pumilus 2 2
Paenibacillus thiaminolyticus 1 1
Staphylococcus sp. 2 1 3
Achromobacter sp. 2 2
Total 17 23 20 33 18 15 24 27 177
44
During the wet season, endophytes from 11 different species were obtained from
trees of Coccoloba uvifera at both Playuela and the solar saltern site. In the dry season, 8
separate genera were recovered from leaf fragments sampled. Five of these endophytes
recovered were found on both sites during the wet and dry seasons. These were
Stenotrophomonas maltophilia, Pseudomonas sp., Burkholderia cepacia, Staphylococcus
sp., and Ralstonia picketti. Some endophytes were recovered only during the wet season.
These endophytes were Lysinibacillus sp., Delftia sp., Proteus mirabilis, Morganella
morganii, and Providencia sp. Only 3 endophytes were exclusive to the dry season:
Bacillus sp., Acinetobacter calcoaceticus, and Achromobacter sp.
In trees of Coccoloba uvifera sampled at Playuela (P1, P2) during the wet season,
6 different bacterial endophytes were found. These were Stenotrophomonas maltophilia,
Pseudomonas sp., Burkholderia cepacia, Lysinibacillus sp., Delftia sp., and Ralstonia
picketti. The most frequent endophyte at Playuela during the wet season was S.
maltophilia. This bacterium was isolated on 23 out of a total of 80 fragments sampled
(40 fragments from each tree). The frequency of colonization of S. maltophilia on trees
sampled at Playuela during the wet season was 28.8%. The second most abundant
bacterial endophyte at Playuela during the wet season was Pseudomonas sp., isolated
from 21 fragments out of 80 inoculated. The frequency of colonization was 26.3%. The
other four species were found in significantly lower frequencies. Lysinibacillus sp. was
found at a percent of colonization of 5%, Delftia sp. was found at 3.8%, and Ralstonia
picketti and Burkholderia cepacia were only isolated once.
A similar pattern was observed for Coccoloba uvifera trees sampled at the site
near the solar salterns (S1 and S2). During the wet season, the two most abundant
45
species of bacterial endophytes found were Stenotrophomonas maltophilia and
Pseudomonas sp. Other 7 bacterial endophytes were found at the solar saltern site
including Burkholderia cepacia, Providencia sp., Staphylococcus epidermidis,
Lysinibacillus sp., Delftia sp., Proteus mirabilis, and Morganella morganii.
Stenotrophomonas maltophilia was recovered from 15 leaf fragments out of a total of 80
inoculated from both trees of C. uvifera sampled at the saltern site. The percent of
colonization of S. maltophilia was 18.8%. Pseudomonas sp. was obtained from 10 leaf
fragments and had a frequency of colonization of 12.5%. The other bacterial endophytes
recovered were found at a lower frequency. Burkholderia cepacia was isolated from 6
fragments (7.5%) and Proteus mirabilis from 3 leaf fragments (3.8%). Both Providencia
sp. and Lysinibacillus sp. were isolated from 2 leaf fragments each (2.5%). The
endophytes Staphylococcus epidermidis, Morganella morganii, and Delftia sp. were
recovered from only 1 leaf fragment each during the wet season at trees S1 and S2 from
the solar saltern site.
During the dry season, 80 fragments of Coccoloba uvifera were also inoculated
from each one of both sites sampled. At Playuela six different endophytic bacteria were
identified. The two most abundant endophytes recovered were Bacillus sp. and
Pseudomonas sp. Bacillus sp. was recovered from 14 out of 80 leaf fragments inoculated
(17.5%) and Pseudomonas sp. was recovered from 13 out of 80 fragments (16.3%). The
endophytes Stenotrophomonas maltophilia, Burkholderia cepacia, and Ralstonia picketti
were isolated from 3 leaf fragments each (3.8%). Acinetobacter calcoaceticus was
isolated from 2 fragments and had a colonization frequency of 2.5%.
46
At the trees sampled at the solar saltern (S1 and S2) site during the dry season,
Bacillus sp. and Pseudomonas sp. were also the two most encountered endophytes.
Bacillus sp. was obtained from 22 out of 80 inoculated fragments (27.5%) while
Pseudomonas sp. was isolated from 6 out of 80 leaf fragments (7.5%). Bacteria from the
genus Staphylococcus sp. were recovered from 3 out of 80 fragments inoculated (3.8%).
Stenotrophomonas maltophilia and Burkholderia cepacia were both isolated from two
leaf fragments each (2.5%). Ralstonia picketti and Acinetobacter calcoaceticus were
each isolated from 1 fragment out of 80 inoculated (1.3%).
Species Accumulation Curves and Biodiversity and Richness Estimators
Coleman rarefaction expected values were obtained and graphed along with the
observed number of bacterial endophytes species against the number of fragments
studied. These values were calculated and graphed for all trees sampled and each site and
season sampled. Figures 6.03, 6.04, 6.05, and 6.06, are the species accumulation curves
for all four trees studied during the wet season (P1, P2, S1, and S2). The species
accumulation curves of bacterial endophytes from the total fragments studied from trees
of C. uvifera at Playuela and solar saltern sites during the wet season are represented in
Figure 6.07 and Figure 6.08, respectively.
It is evident from the species accumulation curves in Figures 6.07 and 6.08 that
there was a greater diversity of bacterial endophytes in trees of Coccoloba uvifera at the
solar saltern site compared to Playuela. The values of expected richness of endophyte
species used were Chao2 and Jack2 estimators in Table 6.07. During the wet season, at
Playuela, between 72%-91% of the expected diversity was recovered. This indicates a
proper sampling effort. Even though the curve of Figure 6.07 does not have an obvious
47
asymptote, it is evident that the curve is beginning to reach the total expected number of
species. Only 52% of the expected diversity was recovered from trees of Coccoloba
uvifera studied at the solar saltern site during the wet season. The shape of the curve in
Figure 6.08 also indicates that the number of species recovered should increase, as the
curve has not yet reached an asymptote. Therefore, the sampling effort at the solar
saltern site during the wet season was not sufficient for recovering most of the
biodiversity expected to be present. The largest values of the Shannon-Wiener and
Simpson’s indexes during the wet season were calculated for the solar saltern site
reinforcing that trees sampled at the solar saltern site have a larger diversity of
endophytes compared to those studied at Playuela. These values are summarized in
Table 6.07.
The species accumulation curves of all trees sampled during the dry season are
represented on Figures 6.09, 6.10, 6.11, and 6.12. Graphs representing the species
accumulation of bacterial endophytes in trees of C. uvifera at Playuela and the solar
salterns during the dry season represent Figure 6.13 and 6.14, respectively. The
sampling effort carried out at both sampling sites during the dry season was sufficient for
recovering most of the expected biodiversity of endophytes. At the Playuela site,
between 83%-96% of the expected species of bacterial endophytes were recovered during
this study. At the solar saltern site between 91%-94% of all biodiversity was obtained.
Both species accumulation curves appear to be reaching an asymptote, as observed in
Figures 6.13 and 6.14. During the dry season the largest Shannon-Wiener and Simpson’s
indexes were calculated for the solar saltern site as shown in Table 6.07.
48
Observing the values of the diversity and richness estimators from both seasons it
could be determined that the biodiversity of bacterial endophytes present in trees of
Coccoloba uvifera at Cabo Rojo was highest at the solar saltern site during the wet
season. The lowest biodiversity indexes were calculated for endophytes at Playuela
during the wet season.
Figure 6.03. Species accumulation curve of bacterial endophytes from tree P1 (Playuela)
during the wet season. N = 40.
0
1
2
3
4
5
6
7
8
1 5 9 13 17 21 25 29 33 37
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
49
Figure 6.04. Species accumulation curve of bacterial endophytes from tree P2 (Playuela)
during the wet season. N = 40.
Figure 6.05. Species accumulation curve of bacterial endophytes from tree S1 (Solar
Saltern) during the wet season. N = 40.
0
1
2
3
4
5
6
7
8
1 5 9 13 17 21 25 29 33 37
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
0
1
2
3
4
5
6
7
1 5 9 13 17 21 25 29 33 37
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
50
Figure 6.06. Species accumulation curve of bacterial endophytes from tree S2 (Solar
Saltern) during the wet season. N = 40.
Figure 6.07. Species accumulation curve of bacterial endophytes from trees sampled at
Playuela site during the wet season (P1 and P2) during the wet season. N = 80.
0
2
4
6
8
10
12
1 5 9 13 17 21 25 29 33 37
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
0
2
4
6
8
10
12
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
51
Figure 6.08. Species accumulation curve of bacterial endophytes from trees sampled at
the solar saltern site during the wet season (S1 and S2) during the wet season. N = 80.
Figure 6.09. Species accumulation curve of bacterial endophytes from tree P1 (Playuela)
during the dry season. N = 40.
0
2
4
6
8
10
12
14
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
0
1
2
3
4
5
6
7
1 5 9 13 17 21 25 29 33 37
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
52
Figure 6.10. Species accumulation curve of bacterial endophytes from tree P2 (Playuela)
during the dry season. N = 40.
Figure 6.11. Species accumulation curve of bacterial endophytes from tree S1 (solar
saltern) during the dry season. N = 40.
0
1
2
3
4
5
6
7
8
1 5 9 13 17 21 25 29 33 37
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
0
1
2
3
4
5
6
7
1 5 9 13 17 21 25 29 33 37
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
53
Figure 6.12. Species accumulation curve of bacterial endophytes from tree S2 (solar
saltern) during the dry season. N = 40.
Figure 6.13. Species accumulation curve of bacterial endophytes from trees sampled at
Playuela site during the dry season (P1 and P2) N = 80.
0
1
2
3
4
5
6
7
8
1 5 9 13 17 21 25 29 33 37
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
0
2
4
6
8
10
12
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
54
Figure 6.14. Species accumulation curve of bacterial endophytes from trees sampled at
the solar saltern site during the dry season (S1 and S2) N = 80.
Table 6.07. Various biodiversity and richness estimators for each site and season.
Site and Season
Shannon-
Wiener
Simpson’s
Chao2
Jack2
Playuela wet season
1.78
4.96
10.98
13.96
Playuela dry season
1.91
5.68
10.32
11.99
Solar saltern wet season
2.04
5.83
24.81
25.25
Solar saltern dry season
1.96
6.31
10.66
11.03
0
2
4
6
8
10
12
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76
Number of Species
Number of Leaf Fragments
Observed No. of Species
Coleman Rarefaction
55
Chapter 7
Discussion
Frequency of Colonization
This is the first study that documents endophytic bacteria in Coccoloba uvifera.
These organisms were successfully cultivated from leaf fragments. In this study, a larger
percent of colonization of endophytes has been observed during the wet season (63.8%)
compared to the dry season (60%). A similar pattern was observed in a previous study
where the isolation frequency of fungal endophytes increased during the wet season in
two separate forests sampled in India (Murali et al. 2007). The frequency of colonization
was lower than that of citrus trees sampled in Sao Paulo, Brazil. In a previous study
bacterial endophytes were found to have between 90%-100% percent of colonization
among asymptomatic and healthy citrus trees (Lacava et al. 2004). The difference in
frequency of colonization is probably due to environmental factors that should be
affecting the microbial communities present in the soil and therefore, bacterial
endophytic communities. High salinity and lower precipitation in the area of Cabo Rojo
may account for a lower frequency of colonization in comparison to previous studies.
During the wet season, a greater percent of colonization was observed in those
trees sampled at Playuela (P1 and P2, 73.8%), than those trees present near the
hypersaline solar saltern (S1 and S2, 53.8%). During the dry season, the opposite is
observed. The trees sampled at the solar saltern site contained a higher frequency of
colonization of bacterial endophytes than those trees sampled at Playuela (63.8% and
66.3%).
56
A greater frequency of colonization of bacterial endophytes was observed in those
trees sampled at the Playuela site compared to those trees sampled near the solar salterns.
This may occur because the increased salinity in the soil surrounding trees at the solar
saltern site could be limiting the amount of bacteria present. However, a greater diversity
of endophytes was recovered from the solar saltern site. More bacterial endophyte
species were recovered from trees sampled at the solar saltern site compared to Playuela.
Both these areas should obtain the same average precipitation because of their
close geographic proximity. Precipitation should not be a factor in the difference of
frequency of colonization of bacterial endophytes between both sites sampled during the
same season. It appears that the environmental abiotic factors that characterize both sites
should have an effect on the amount of endophytes recovered from leaf fragments on
each site. Some of the possible abiotic and biotic factors influencing bacterial endophyte
communities are salinity, precipitation, edaphic properties and microorganisms present.
The effect of seasonality on the amount of bacterial endophytes and isolates recovered
was most evident in the trees sampled at the Playuela site compared to those sampled at
the solar saltern site.
Biodiversity
Of the 143 isolates obtained in culture, 61 of them were characterized by 16S
rDNA sequencing. From these isolates, 14 separate genera were properly characterized
using NCBI BLAST homology analysis. With the characterization of these bacteria, it
was evident that, not only does the frequency of colonization vary seasonally, but that
bacterial endophyte communities change completely as well.
57
During this study, the most encountered endophytes belonged to the
Gammaproteobacteria. This result is expected as the group Gammaproteobacteria
contain many bacteria isolated commonly in soils such as Pseudomonas and
Stenotrophomonas. The same pattern has been observed in a study on endophytes on
Poplar trees where Gammaproteobacteria predominated (Taghavi et al. 2009).
During the wet season the most encountered bacterium was Stenotrophomonas
maltophilia. It was found on 38 fragments out of 160 inoculated from all four trees
sampled from two sites (23.8%). This bacterium has been found as a dominating species
of endophyte in weeds, potato, and rice (Sturz et al. 2001; Garbeva et al. 2001; Sun et al.
2008). It was also found in coffee trees sampled at both Colombia and Hawaii (Vega et
al. 2005). It is interesting to note that during the dry season, the frequency at which
Stenotrophomonas maltophilia was isolated was significantly lower. During the dry
season Stenotrophomonas maltophilia was isolated from 5 out of 160 (3.1%) fragments
inoculated from all trees.
It was also interesting to note that the endophytes from the genera Bacillus was
isolated from 36 out of 160 inoculated fragments (22.5%) during the dry season. Bacillus
endophytes were not recovered during the wet season. This endophytic bacterium in
trees of Coccoloba uvifera sampled was the most affected by seasonality (precipitation).
It is possible that Bacillus bacteria are better suited for the dryer conditions and possible
increase in salinity of the surrounding soils. These bacteria have been reported
previously as moderately halophilic or halotolerant species (Arahal and Ventosa 2002).
Their ability to form endospores should also be a factor allowing them to persist during
the dry season. While endospore formation may confer an advantage to Bacillus species
58
during the dry season, it may be disadvantageous during the rainy season. The absence
of Bacillus species during the wet season was indicative that the pressures of competition
caused other endophytes to displace organisms of this genus. Endospores need to
germinate and become a live cell. During this process of germination, other endophytic
organisms have the ability to colonize the available niches quicker, displacing endospore
forming bacteria.
Species of Pseudomonas were recovered from both sites and seasons sampled.
This was expected since bacteria from this group are ubiquitous to soils and should be
common endophytes. Apparently this endophytic bacterium was the most adapted to
resist changes in season and location in trees of Coccoloba uvifera in Cabo Rojo, Puerto
Rico.
A study on bacterial endophytes was previously carried out on the marine sea
grass Thalassia testudinum at different locations in Puerto Rico (Couto-Rodríguez 2009).
One of the areas studied was in Los Morrillos Reserve in Cabo Rojo. In that study 61%
of the samples were colonized by prokaryotic endophytes. The total frequency of
colonization of bacterial endophytes obtained during the present study was calculated to
be 61.8% (198/320 fragments). Even though Thalassia testudinum is a marine plant and
Coccoloba uvifera is terrestrial, both are subjected to similar environmental
characteristics. Both hosts are exposed to high salinity, temperatures, and ultraviolet
radiation. They are also located on sandy soils. The similarities in abiotic conditions at
the sampling sites may explain why the same frequency of colonization was observed in
both studies.
59
Various bacterial endophytes obtained previously from Thalassia testudinum were
isolated from Coccoloba uvifera trees sampled at the present study. The most common
bacterial endophyte isolated from Thalassia testudinum plants studied at los Morrillos
site were from the genus Bacillus (Couto-Rodríguez 2009). Some species of Bacillus that
were found on both studies were Bacillus cereus, B. pumilus, and B. safensis. Other
endophytes were Staphylococcus and Pseudomonas. Finding these endophytes in both
hosts might suggest that in Los Morrillos Reserve in Cabo Rojo bacteria of the genus
Bacillus, Pseudomonas, and Staphylococcus have a widespread distribution. There are
endophytes found on both studies which are unique for the host and/or the environment.
For example, Stenotrophomonas maltophilia, Burkholderia cepacia, Ralstonia picketti,
Lysinibacillus sp., Delftia sp., Proteus mirabilis, Morganella morganii, Paenibacillus sp.,
Achromobacter sp., Providencia sp., and Acinetobacter calcoaceticus were all found in
Coccoloba uvifera but were not isolated from Thalassia testudinum.
Many of the species of endophytic bacteria recovered during this study have been
previously described as plant growth promoting bacteria (PGPB) in other hosts. Trees of
Coccoloba uvifera are often found in areas where there is high environmental stress.
The presence of these bacteria in trees of Coccoloba uvifera suggests that they may be
contributing to the overall fitness of the trees sampled at both Playuela and solar saltern
site.
The endophyte Stenotrophomonas maltophilia, previously called Pseudomonas
maltophilia and Xanthomonas maltophilia, has displayed plant growth promoting
characteristics on other hosts. One of the most encountered isolates sequenced during the
wet season (September, 2008) belonged to the group Stenotrophomonas sp. Various
60
strains were isolated, including a clone with a 100% homology with S. maltophilia. In a
previous study, a strain of this bacterium helped improve the fitness of the grass Festuca
arundinacea by being able to suppress leaf spot, which is a disease caused by Bipolaris
sorokiniana (Zhang and Yuen 1999). Stenotrophomonas maltophilia inhibited the
germination of conidia and reduced lesions in the surface of the leaves. This bacterium
was found in Coccoloba uvifera leaves during both seasons and sampling sites.
However, it was most abundant during the wet season where it was found on both sites.
During the wet season there is a greater threat of colonization by fungal phytopathogens
whose conidia are being dispersed by water droplets. It is quite possible that the high
frequency at which S. maltophilia was isolated during the wet season from trees on both
sites, may have a protective effect on trees of Coccoloba uvifera sampled. Besides
possibly being a pathogen supressor, S. maltophilia may be contributing to the plants
fitness directly by the production of nutrients necessary for plant growth. This bacterium
has been previously identified as an endophyte able to produce indole-3-acetic acid and
fix nitrogen (Park et al. 2005). These characteristics make S. maltophilia an ideal plant
symbiont and justifies its widespread presence in studies of bacterial endophytes.
Various specimens with a high percent of homology to members of the genus
Bacillus sp. were recovered during this study in trees of Coccoloba uvifera. Some
species have been previously isolated as endophytes and associated with benefiting their
hosts. Many strains of Bacillus and Paenibacillus suppress pest and pathogen damage
while promoting plant growth (McSpadden 2004). In another previous study, clones that
were homologous to Bacillus sp. and Paenibacillus sp. suppressed diseases caused by the
61
pathogens Rhizoctonia bataticola, Macrophomina phaseolina, and Fusarium udom.
Some also inhibited Sclerotium rolfsii (Senthilkumar et al. 2009).
Some species of the bacterium Pseudomonas sp. have antagonistic properties
against the red rot pathogen Colletotrichum falcatum (Viswanathan et al. 2003). Three
species of Pseudomonas sp. were found to have the largest inhibition zones against the
pathogen: Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas
putida. In the present study various strains of Pseudomonas sp. were recovered from leaf
fragments of the tree Coccoloba uvifera in Cabo Rojo, Puerto Rico. Two strains obtained
had a high percent of homology with Pseudomonas aeruginosa (99%) and another strain
was 96% similar to Pseudomonas putida. Pseudomonad species were frequently isolated
during the dry season (March, 2009). Some pseudomonads are known for producing
antibiotic compounds (Haas and Keel 2003). Their presence could be involved in
protecting Coccoloba uvifera trees from fungal pathogens.
A strain of the bacterium Delftia tsuruhatensis has been previously described as a
PGPB from the rhizoplane of rice. This strain was also identified as a diazotroph capable
of inhibiting various plant pathogens such as Rhizoctonia solani, Fusarium oxysporum,
Verticillium sp., and Xanthomonas oryzae (Han et al. 2005).
In the future, it would be interesting to study plant growth promoting properties of
bacterial endophytes present in C. uvifera trees in these locations. Many of the bacteria
isolated during this study have been previously described as PGPB and have the
possibility of producing compounds of biomedical or ecological importance. Bacteria
present inside plants located in such environments are probably involved in the fitness of
their hosts
62
Chi-square distribution and the Fisher’s exact test help determine that there was
no significant difference or strong effect between seasonality and the total of leaf
fragments colonized by bacterial endophytes. However, seasonality did affect
significantly the frequency at which bacterial endophytes were encountered in leaf
fragments of Coccoloba uvifera at the Playuela sampling site. This was not observed at
the solar saltern site where there was no significance or strong relationship between
seasonality and amounts of endophytes recovered.
The species accumulation curves provided a way of determining whether or not
the sampling effort carried out during this experiment was sufficient to be able to
describe most of the endophytic bacterial species present in the leaf fragments studied.
All of the observed species accumulation curves intersected with the expected species
accumulation curves but not all reached the asymptote. The species accumulation curves
of the solar saltern site and Playuela during the wet season indicate that there are still
species of endophytes to be detected. A greater sampling effort should be performed at
the solar saltern site during the wet season. In Figure 6.06 it is illustrated how the graph
continues to increase and 10 species were found. However, according to the Chao2 and
Jack2 values in Table 6.07, it was expected to find around 25 species of endophytes.
During the dry season it appears that the species accumulation curves have begun to
reach the asymptote as the graphed values are not increasing steeply. This indicates that
the sampling effort carried out during the dry season was appropriate for the amount of
bacterial endophyte species that should have been recovered.
This experiment possesses a bias against culturable organisms. The endophytic
organisms that may not have been isolated could have been fastidious or unculturable
63
microorganisms. It is also interesting to note that the highest values of Chao2 and Jack2
were calculated at the solar saltern site during the wet season. The lowest values of
Chao2 and Jack2 are also at the solar saltern site but during the dry season. It appears
that biodiversity (not frequency) changes most at the solar saltern site according to the
season sampled.
64
Chapter 8
Conclusion
In the present study bacterial endophytes were successfully obtained and isolated
from leaf fragments of Coccoloba uvifera. The frequency of bacterial endophytes on leaf
fragments from all four trees sampled during the wet and dry seasons at both sites was
calculated. The data demonstrated how the frequency of colonization of these
microorganisms varies among sites and seasons sampled. In terms of the total frequency
of colonization, it could not be determined statistically that there was a significant
difference between both sites sampled. The same was also determined when comparing
the data by seasons. Therefore, this hypothesis could not be rejected. This indicates that
both location and seasonality do not have a significant effect on the total amount of
bacterial endophytes that are present in Coccoloba uvifera trees present in Cabo Rojo,
Puerto Rico. However, statistics did determine that there was a significant difference
between the amount of endophytes recovered during wet and dry seasons at the Playuela
site. This implies that while the total amount of endophytes recovered in Cabo Rojo does
not change significantly with the season, it does change when the data of Playuela is only
observed. Therefore, this site was the most affected by seasonality.
Isolates of bacterial endophytes were characterized by DNA sequencing of the
16S rRNA gene. The results from the BLAST homology analysis of the 61 cultures
studied reflected a large diversity of bacteria belonging to 14 separate genera. The most
frequent encountered endophytes during this study were Pseudomonas sp.,
Stenotrophomonas maltophilia and Bacillus sp. Even though a statistical analysis
determined that location and seasonality do not have an effect on the amount of
65
endophytes recovered, it became evident from the data that the species of endophytic
bacteria recovered in both seasons were different. One of the hypotheses established at
the beginning of this study was clearly rejected. It was rejected that the diversity of
bacterial endophytes found in the trees of C. uvifera was the same in both seasons
sampled. Even though some species were shared among seasons, the predominant
endophytes changed with the season. Species of Bacillus were only isolated during the
dry season and the number of isolated Stenotrophomonas maltophilia decreased during
the dry season.
66
Literature Cited
Altschul SF, Gish W, Miller W, Myers, EW, Lipman, DJ. 1990 "Basic local alignment
search tool." J Mol Biol 215:403-410.
Arahal D and Ventosa A. 2002. Moderately Halophilic and Halotolerant Species of
Bacillus and Related Genera. In: Berkeley R, Heyndrickx M, Logan N, De Vos
P. eds. Applications and Systematics of Bacillus and Relatives. Blackwell
Publishing.
Arnold AE, Maynard Z, Gilbert GS, Coley PD, Kursar TA. 2002. Are tropical fungal
endophytes hyperdiverse? Ecology Letters 3(4):267-274.
Arnold AE and Lutzoni F. 2007. Diversity and host range of foliar fungal endophytes:
are tropical leaves biodiversity hotspots? Ecology 88(3):541-549.
Barac T, Taghavi S, Borremans B, Provoost A, Oeyen L, Colpaert J, Vagronsveld J, van
der Lelie D. 2004. Engineered endophytic bacteria improve phytoremediation of
water-soluble, volatile, organic pollutants. Nature Biotechnology 22(5): 583-
588.
Camatti-Sartori V, da Silva-Ribeiro RT, Valdebenito-Sanhueza RM, Pagnocca FC,
Echeverrigaray S, Azevedo JL. 2005. Endophytic yeasts and filamentous fungi
associated with southern Brazilian apple (Malus domestica) orchards to
conventional, integrated or organic cultivation.
Campbell N, Reece J, Mitchell L. 1999. Biology. Fifth Edition. Benjamin/Cummings.
California, USA. p756
Castillo U, Strobel G, Ford E, Hess W, Porter H, Jensen J, Albert H, Robison R, Condron
M, Teplow D, Stevens D, Yaver D. 2002. Munumbicins, wide-spectrum
67
antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia
nigriscans. Microbiology 148:2675-2685.
Castillo U, Harper JK, Strobel GA, Sears J, Alesi K, Ford E, Lin J, Hunter M, Maranta
M, Ge H, Yaver D, Jensen JB, Porter H, Robison R, Millar D, Hess WM,
Condron M, Teplow D. 2003. Kakadumicins, novel antibiotics from
Streptomyces sp. NRRL 30566, and endophyte of Grevillea pteridifolia. FEMS
Microbiology Letters 224(2):183-190.
Colwell R, Mao CX, Chang J. 2004. Interpolating, Extrapolating, and Comparing
Incidence-Based Species Accumulation Curves. Ecology 85(10):2717-2727.
Couto-Rodríguez M. 2009. Endophytic Prokaryotic Diversity Associated with Sea Grass
Beds of Thalassia testudinum from Cabo Rojo, Lajas, and Vieques, Puerto Rico.
Daniel W. 2005. Biostatistics. 8th
ed. John Wiley & Sons.
Doty SL. 2008. Enhancing phytoremediation through the use of transgenics and
endophytes. New Phytologist 179:318-333.
Eastwell K, Sholberg P, Sayler R. 2006. Characterizing potential bacterial control
agents for suppression of Rhizobium vitis, causal agent of crown gall disease in
grapevines. Crop Protection 25(11):1191-1200.
Elbeltagy A, Nishioka K, Sato T, Suzuki H, Ye B, Hamada T, Isawa T, Mitsui H,
Minamisawa K. 2001. Endophytic colonization and in planta nitrogen fixation
by a Herbaspirillum sp. isolated from wild rice species. Applied and
Environmental Microbiology 67(11):5285-5293.
Ezra D, Castillo U, Strobel G, Hess W, Porter H, Jensen J, Condron M, Teplow D, Sears
J, Maranta M, Hunter M, Weber B, Yaver D. 2004. Coronamycins, peptide
68
antibiotics produced by a verticillate Streptomyces sp. (MSU-2110) endophytic
on Monstera sp. Microbiology 150:785-793.
Fisher PJ, Petrini O. 1987. Location of fungal endophytes in tissues of Suaeda fruticosa:
a preliminary study. Trans Br Mycol Soc 89:246–249.
Gai CS, Lacava PT, Maccheroni W Jr., Glienke C, Araújo WL, Miller TA, Azevedo JL.
2009. Diversity of endophytic yeasts from sweet orange and their location by
scanning electron microscopy. Journal of Basic Microbiology 49(5):441-451.
Gamboa M, Laureano S, Bayman P. 2002. Measuring diversity of endophytic fungi in
leaf fragments: Does leaf size matter? Mycopathologia 156:41-45.
Garbeva P, van Overbeek LS, van Vuurde JW, van Elsas J. 2001. Analysis of
endophytic bacterial communities of potato by plating and denaturing gradient gel
electrophoresis (DGGE) of 16S rDNA based PCR fragments. Microbial Ecology
41(4):369-383.
Graham L, Graham J, Wilcox L. 2003. Plant Biology. First edition. Pearson Education.
New Jersey.
Gutiérrez Mañero FJ, Ramos Solano B, Probanza A, Mehouachi J, Tadeo F, Talon M.
2001. The plant-growth-promoting rhozobacteria Bacillus pumilus and Bacillus
licheniformis produce high amounts of physiologically active gibberellins.
Physiologia Plantarum 111:206-211.
Haas D and Keel C. 2003. Regulation of antibiotic production in root-colonizing
Pseudomonas spp. and relevance for biological control of plant disease. Annual
Review of Phytopathology 41:117-153.
69
Han J, Sun L, Dong X, Cai Z, Sun X, Yang Hm, Wang Y, Song W. 2005.
Characterization of a novel plant growth-promoting bacteria strain Delftia
tsuruhatensis HR4 both as a diazotroph and a potential biocontrol agent against
various plant pathogens. Systematic and Applied Microbiology 28:66-76.
Hardoim P, van Overbeek L, van Elsas JD. 2008. Properties of bacterial endophytes and
their proposed role in plant growth. Trends in Microbiology 16(10):463-471.
Harrison L, Teplow DB, Rinaldi M, Strobel G. 1991. Pseudomycins, a family of novel
peptides from Pseudomonas syringae possessing broad-spectrum antifungal
activity. Journal of General Microbiology 137(12):2857-2865.
Hawksworth D and Rossman A. 1997. Where are all the undescribed fungi?
Phytopathology 87(9):888-891.
Khan Z and Doty S. 2009. Characterization of bacterial endophytes of sweet potato
plants. Plant and Soil 322(1-2):197-207.
Kobayashi DY, Palumbo JD. 2000. Bacterial Endophytes and Their Effects on Plants
and Uses in Agriculture. In: Bacon CW, White JF, eds. Microbial endophytes.
New York: Jr. Marcel Dekker. p 199-233
Krings M, Hass H, Kerp H, Taylor T, Agerer R, Dotzler N. 2009. Endophytic
cyanobacteria in a 400-million-yr-old land plant: A scenario for the origin of
symbiosis? Review of Paleobotany and Palynology 153(1-2): 62-69.
Lacava PT, Araújo WL, Marcon J, Maccheroni W, Azevedo JL. 2004. Interaction
between endophytic bacteria from citrus plants and the phytopathogenic bacteria
Xylella fastidiosa, causal agent of citrus-variegated chlorosis. Letters in Applied
Microbiology 39:55-59.
70
Lodewyckx C, Vangronsveld J, Porteous F, Moore E, Taghavi S, Mezgeay M, van der
Lelie D. 2002. Endophytic Bacteria and Their Potential Applications. Critical
Reviews in Plant Sciences 21(6):583-606.
Lodge D, Fisher P, Sutton B. 1996. Endophytic fungi of Manilkara bidentata leaves in
Puerto Rico. Mycologia 88(5):733-738.
Long H, Schmidt D, Baldwin I. 2008. Native bacterial endophytes promote host growth
in a species-specific manner; phytohormone manipulations do not result in
common growth responses. PLoS ONE 3(7):e2702.
Magnani GS, Didonet CM, Cruz LM, Picheth CF, Pedrosa FO, Souza EM. 2010.
Diversity of endophytic bacteria in Brazilian sugarcane. Genetics and Molecular
Research 9(1):250-258.
Mahaffee WF and Kloepper JW. 1997. Temporal changes in the bacterial communities
of soil, rhizosphere, and endorhiza associated with field grown cucumber
(Cucumis sativus L.). Microbial Ecology 34:210-223.
Malinowski D and Belesky D. 2006. Ecological importance of Neotyphodium spp. grass
endophytes in agroecosystems. Grassland Science 52(1):1-14.
Mattos K, Pádua V, Romero A, Hallack L, Neves B, Ulisses T, Barros C, Todeschini A,
Previato J, Mendonça-Previato L. 2008. Endophytic colonization of rice (Oryza
sativa L.) by the diazotrophic bacterium Burkholderia kururiensis and its ability
to enhance growth. Anais da Academia Brasileira de Ciencias 80(3):477-493.
McGuiness M and Dowling D. 2009. Plant-Associated Bacterial Degradation of Toxic
Organic Compounds in Soil. International Journal of Research and Public Health
6:2226-2247
71
McInroy J and Kloepper J. 1995. Survey of indigenous bacterial endophytes from cotton
and sweet corn. Plant and Soil 173:337-342.
McSpadden B. 2004. Ecology of Bacillus and Paenibacillus spp. In Agricultural
Systems. Symposium: The Nature and Application of Biocontrol Microbes:
Bacillus spp. Phytopathology 94(11):1252-1258.
Melnick R, Zidack N, Bailey B, Maximora S, Guiltinan M, Backman P. 2008. Bacterial
endophytes: Bacillus spp. From annual crops as potential biological control
agents of black pod rot of cacao. Biological Control 46(1):46-56.
Miller CM, Miller RV, Garton-Kenny D, Redgrave B, Sears J, Condron MM, Teplow
DB, Strobel GA. 1998. Ecomycins, unique antibiotics from Pseudomonas
viridiflava. Journal of Applied Microbiology 84(6):937-944.
Moore R, Clark WD, Sterm K. 1995. Botany. Wm. C. Brown Communications.
Dubuque, IA.
Nimnoi P, Pongslip N. 2009. Genetic diversity and plant-growth promoting ability of
the indole-3-acetic acid (IAA) synthetic bacteria isolatd from agricultural soil as
well as rhizosphere, rhizoplane and root tissue of Ficus religiosa L., Leucaena
leucocephala, Piper sarmentosum Roxb. Research Journal of Agricultural and
Biological Sciences 5(1):29-41.
Myoungsu Park, Chungwoo Kim, Jinchul Yang, Hyoungseok Lee, Shin Wansik, Kim
Seunghwan Kim, Sa Tongmin. 2005. Isolation and characterization of
diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of
Korea. Microbiological Research 160(2):127-133.
72
Onofre-Lemus J, Hernández-Lucas I, Girard L, Caballero-Mellado J. 2009. ACC (1-
aminocyclopropane-1-carboxylate) deaminase activity, a widespread trait in
Burkholderia species, and its growth-promoting effect on tomato plants. Applied
Environmental Microbiology 75(20):6581-6590.
Parrota J. 1994. Coccoloba uvifera (L.) L. Sea grape, uva de playa. SO-ITF-SM-74.
New Orleans, LA: US Department of Agriculture, Forest Service, Southern Forest
Experiment Station. 5p.
Pirttila A, Joensuu P, Pospiech H, Jalonen J, Hohtola A. 2004. Bud endophytes of Scots
pine produce adenine derivatives and other compounds that affect morphology
and mitigate browning of callus cultures. Physiologia Plantarum 121(2):305-
312.
Ravel C, Courty C, Coudret A, Charmet G. 1997. Beneficial effects of Neotyphodium
lolii on the growth and the water status in perennial ryegrass cultivated under
nitrogen deficiency or drought stress. Agronomie 17:173-181.
Redman R, Sheehan K, Stout R, Rodríguez R, Henson J. 2002. Thermotolerance
Generated by Plant/Fungus Symbiosis. Science 298:1581.
Rivera Rodríguez, Glenda. 2006. Bacterias presentes en el sistema vascular de algunos
cítricos en Puerto Rico. Tesis de Maestría. Universidad de Puerto Rico,
Mayagüez. pp. 94
Rodríguez R and Redman R. 2008. More than 400 million years of evolution and some
plants still can’t make it on their own: plant stress tolerance via fungal symbiosis.
Journal of Experimental Botany 59(5):1109-1114.
73
Ryan R, Germaine K, Franks A, Ryan D, Dowling D. 2008. Bacterial endophytes:
recent developments and applications. FEMS Microbiology Letters 278(1):1-9.
Ryan R, Monchy S, Cardinale M, Taghavi S, Crossman L, Avison M, Berg G, van der
Lelie D, Dow J. 2009. The versatility and adaptation of bacteria from the genus
Stenotrophomonas. Natural Reviews in Microbiology 7:514-525.
Saikkonen K, Faeth S, Helander M, Sullivan T. 1998. Fungal endophytes: A Continuum
of Interactions with Host Plants. Annual Review of Ecology and Systematics
29:319-343.
Saikkonen K, Ahlholm J, Helander M, Lehtimäki S, Niemeläinen O. 2008. Endophytic
fungi in wild and cultivated grasses in Finland. Ecography 23(3):360-366.
Schlomi H, Alves H, Soares de Melo I, Vieira F, Bettiol W. 2006. Bioprospecting
endophytic bacteria for biological control of coffee leaf rust. Sci. Agric 63(1):
32-39.
Senthilkumar M, Swarnalakshmi K, Govindasamy V Kerun Lee Y, Annapurna K. 2009.
Biocontrol Potential of Soybean Bacterial Endophytes Against Charcoal Rot
Fungus, Rhizoctonia bataticola. Current Microbiology 58:288-293.
Sgroy V, Cassán F, Masciarelli O, Del Papa M, Lagares A, Luna V. 2009. Isolation and
characterization of plant growth-promoting (PGPB) or stress homeostasis-
regulating (PSHB) bacteria associated to the halophyte Prosopis strombulifera.
Applied Microbiology and Biotechnology 85(2):371-381.
Smith N, Mori S, Henderson A, Stevenson D, Heald S. Flowering Plants of the
Neotropics. Princeton University Press. © 2004
74
Spaepen S, Vanderleyden J, Remans R. 2007. Indole-3-acetic acid in microbial and
microorganism-plant signaling. FEMS Microbiology Reviews 31:425-448.
Stone J, Bacon C, White J. 2000. An Overview of Endophytic Microbes. Microbial In:
Bacon CW, White JF, eds. Microbial endophytes. New York: Jr. Marcel
Dekker.
Sturz AV, Matheson BG, Arsenault W, Kimpinski J, Christie BP. 2001. Weeds as a
source of plant growth promoting rhizobacteria in agricultural soils. Canadian
Journal of Microbiology 47(11):1013-1024.
Sturz, Christie, Matheson, Arsenault, and Buchanan. 2002. Endophytic bacterial
communities in the periderm of potato tubers and their potential to improve
resistance to soil-borne plant pathogens. Plant Pathology 48(3):360-369.
Sun L, Qiu F, Zhang X, Dai X, Dong X, Song W. 2008. Endophytic bacterial diversity
in rice (Oryza sativa L.) roots estimated by 16S rDNA sequence analysis.
Microbial Ecology 55(3):415-424.
Sziderics AH, Rasche F, Trognitz F, Sessitsch A, Wilhelm E. 2007. Bacterial
endophytes contribute to abiotic stress adaptation in pepper plants (Capsicum
annuum L.). Canadian Journal of Microbiology 53(11):1195-1202.
Taghavi S, Barac T, Greenberg B, Borremans B, Vangronsveld J, van der Lelie D. 2005.
Horizontal Gene Transfer to Endogenous Endophytic Bacteria from Poplar
Improves Phytoremediation of Toluene. Applied and Environmental
Microbiology 71(12):8500-8505.
Taghavi S, Garafola C, Monchy S, Newman L, Hoffman A, Weyens N, Barac T,
Vagronsveld J, van der Lelie Daniel. 2009. Genome Survey and Characterization
75
of Endophytic Bacteria Exhibiting a Beneficial Effect on Growth and
Development of Poplar Trees. Applied and Environmental Microbiology 75(3):
748-757.
Tapia-Hernández A, Bustillos-Cristales MR, Jiménez-Salgado T, Caballero-Mellado J,
Fuentes-Ramírez LE. 2000. Natural occurrence of Acetobacter diazotrophicus in
Pineapple Plants. Microbial Ecology 39:49-55.
Vega FE, Pava-Ripoll M, Posada F, Buyer JS. 2005. Endophytic bacteria in Coffea
arabica L. Journal of Basic Microbiology 45(5):371-380.
Viswanathan R, Rajtha R, Ramesh S, Ramamoorthy V. 2003. Isolation and
identification of endophytic bacterial strains from sugarcane stalks and their in
vitro antagonism against the red rot pathogen. Sugar Tech 5(1-2):25-29.
Zinniel D, Lambrecht P, Harris NP, Feng Z, Kuczmarski D, Higley P, Ishimaru C,
Arunakumari A, Barletta R, Vidaver A. 2002. Isolation and Characterization of
Endophytic Colonizing Bacteria from Agronomic Crops and Prairie Plants.
Applied and Environmental Microbiology 68(5):2198-2208.
Zhang Z and Yuen GY. 1999. Biological control of Bipolaris sorokiniana on tall fescue
by Stenotrophomonas maltophilia strain C3. Phytopathology 89:817-822.