UNIVERSIDAD DEL TURABO - UAGM...Endophytes have also been found to colonize fruits and seeds. They include soil bacteria ... (Polygonaceae) is a woody tree with widespread distribution

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


from tree P2 during the wet season………………………………………49


from tree S1 during the wet season…………………………….……...…49


from tree S2 during the wet season……………………………..………..50


from trees sampled at Playuela site during the wet season……..………..50


from trees sampled at the solar saltern site during

the wet season……………………………………………….………..….51


from tree P1 during the dry season…………………………..…………..51

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from tree P2 during the dry season……………………………….…….52


from tree S1 during the dry season……………………………….…….52


from tree S2 during the dry season……………………………….…….53


from trees sampled at Playuela during the dry season………………….53


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.

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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


(# 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


(# 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


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



Coleman Rarefaction

0

1

2

3

4

5

6

7

1 5 9 13 17 21 25 29 33 37

Number 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



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



Coleman Rarefaction

51


the solar saltern site during the wet season (S1 and S2) during the wet season. N = 80.


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



Coleman Rarefaction

0

1

2

3

4

5

6

7

1 5 9 13 17 21 25 29 33 37

Number of Species



Coleman Rarefaction

52


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



Coleman Rarefaction

0

1

2

3

4

5

6

7

1 5 9 13 17 21 25 29 33 37

Number 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.


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



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



Coleman Rarefaction

54


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



Coleman Rarefaction

55

Chapter 7

Discussion


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

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UNIVERSIDAD DEL TURABO - UAGM...Endophytes have also been found to colonize fruits and seeds. They include soil bacteria ... (Polygonaceae) is a woody tree with widespread distribution