Phylogenetic Analysis and Characterization of Plant...

Phylogenetic Analysis and Characterization of Plant,

Environmental, and Clinical Strains of Pantoea

A Thesis

Submitted to the Faculty of Graduate Studies and Research

In Partial Fulfillment of the Requirements

For the Degree of

Master of Science

In

Biology

University of Regina

By

Geetanchaly Nadarasah

Regina, Saskatchewan

March, 2012.

Copyright © 2012: G. Nadarasah

UNIVERSITY OF REGINA

FACULTY OF GRADUATE STUDIES AND RESEARCH

SUPERVISORY AND EXAMINING COMMITTEE

Geetanchaly Nadarasah, candidate for the degree of Master of Science in Biology, has presented a thesis titled, Phylogenetic Analysis and Characterization of Plant, Environmental, and Clinical Strains of Pantoea, in an oral examination held on March 23, 2012. The following committee members have found the thesis acceptable in form and content, and that the candidate demonstrated satisfactory knowledge of the subject material. External Examiner: Dr. Tanya Dahms, Department of Chemistry & Biochemistry

Supervisor: Dr. John Stavrinides, Department of Biology

Committee Member: Dr.Christopher Somers, Department of Biology

Committee Member: Dr. Nick A. Antonishyn, Adjunct

Chair of Defense: Dr. Andrew Wee, Department of Chemistry & Biochemistry *Not present at defense

i

Abstract

Multihost bacterial pathogens are an increasing concern as more bacterial species are

found to cause harm to humans. Pantoea is recognized as a multihost pathogen,

colonizing various hosts including plants, insects, and humans; however it is unknown

how these strains are related, and the extent of their specific host ranges. Multilocus

sequence analysis (MLSA) on six housekeeping genes of Pantoea revealed that some

species are mixed, and contain plant, clinical, and environmental strains, while other

species groups are composed of only plant or only clinical strains. Comparative growth

assays in maize, onion, and fruit flies revealed that all plant, clinical, and environmental

strains are capable of colonizing both plant and animal hosts. Pantoea clinical strains had

an overall greater growth rate within fruit flies in comparison to either plant hosts. The

results of this work have shown that some Pantoea strains have a broad host range, while

others are more host specific. The close relationship of plant and environmental strains to

clinical strains and their ability to colonize plants and fruit flies with equal efficiency,

highlights the potential for these strains to cause human infections. This work also

suggests that strains causing human infections likely originate from the general

environment.

ii

Acknowledgements

I am deeply grateful to my supervisor, Dr. John Stavrinides, whose patience and

kindness, as well as his academic experience, has been invaluable to me. You accepted

me into your lab when others barely gave me a glance. You have taught me to question

science and have also challenged me to be independent and to explore with new

perspectives. You have not only been a great supervisor but you have also become an

inspiring mentor to whom I will always be indebt to and will never forget.

Sincere gratitude and thanks go to my committee members: Dr. Christopher

Somers and Dr. Nick Antonyshn for their time and guidance throughout the thesis

process. Your support and helpful advice has allowed me to successfully accomplish my

master‟s thesis. Further, the donations of various strains from New Zealand Culture of

Plant Pathogens, Regina General Hospital, Dr. Gwyn Beattie, Dr. David Coplin, Dr.

Brion Duffy, Dr. Paul Levett (SDCL), Dr. Steven Lindow, Texas Children‟s Hospital,

and Sunnybrook Hospital are also greatly appreciated, for without the donation of your

strains I would not have been able to conduct my research.

I am also like to acknowledge the Faculty of Graduate Studies and Research for

providing funding during my masters at the University of Regina.

iii

Post Defense Acknowledgement

I would like to sincerely thank Dr. Tanya Dahms for taking the time to be my

external examiner. Your feedback and support throughout this process has been

incomparable.

iv

Dedication

I am the utmost thankful to my parents and brothers who have helped and

encouraged me to follow my heart and accomplish my goals. Without each of you by my

side and your constant support I do not know if I could have achieved the many goals I

had set for myself. I would also like to thank my past and present lab members whom

have been not only a friend to me but have become a part of my family. Your constant

love and support has helped me to accomplish all I have sought to gain and more by

coming to Regina and your constant smiles have always made my days in the lab

enjoyable. Thank you.

v

Table of Contents

Abstract ................................................................................................................................ i

Acknowledgements ............................................................................................................. ii

Post Defense Acknowledgement ....................................................................................... iii

Dedication .......................................................................................................................... iv

List of Tables ..................................................................................................................... vi

List of Figures ................................................................................................................... vii

List of Appendices ........................................................................................................... viii

List of Abbreviations ......................................................................................................... ix

Chapter 1: General Introduction ......................................................................................... 2

Chapter 2: Phylogenetic Analysis and Characterization of Plant, Environmental, and

Clinical Strains of Pantoea ............................................................................................... 12

Abstract..................................................................................................................11

Introduction............................................................................................................12

Materials and Methods...........................................................................................14

Results....................................................................................................................17

Discussion..............................................................................................................30

Concluding Remarks..............................................................................................34

Future Directions...................................................................................................35

List of References..................................................................................................36

Appendices ........................................................................................................................ 44

vi

List of Tables

Table 1 Nucleotide divergence among different species groups............................21

Table 2 Comparison of nucleotide divergence, in percent,

among unique strains within certain phylogenetic

species groups............................................................................................22

Table 3 Properties of bacterial pathogens...............................................................97

Table 4 Summary of cross-kingdom pathogens....................................................149

vii

List of Figures

Figure 1 Neighbour-joining tree based on the concatenated

nucleotide sequences of fusA, leuS, pyrG, rpoB, gyrB,

and 16S rRNA of 128 Pantoea strains........................................................19

Figure 2 Comparative growth assay of clinical strains across

Pantoea agglomerans, Pantoea septica, and Pantoea

brenneri......................................................................................................25

Figure 3 Comparison of growth within hosts among strains of

Pantoea dispersa........................................................................................26

Figure 4 Overall analysis of growth across clinical strains......................................28

Figure 5 Multihost colonization of the 128 plant, clinical,

and environmental strains..........................................................................29

Figure 6 The role of the aphid as both vector and alternative

primary host for the plant pathogen Pseudomonas syringae

pv syringae B728a (B728a).......................................................................98

Figure 7 A bacterium having multiple hosts must achieve a

balance with both its hosts to achieve optimal fitness...............................99

Figure 8 Plant-first model.......................................................................................100

Figure 9 Insect-first model.....................................................................................101

Figure 10 Cycling of cross-kingdom pathogens between plants

and humans..............................................................................................151

viii

List of Appendices

Appendix A Comparative growth of all species

groups............................................................................................43

Appendix B Characterization of mutants……………………………………..56

Appendix C Information regarding the strains used in this thesis.....................57

Appendix D Insects as alternative hosts for phytopathogenic bacteria.............69

Appendix E Insights into cross-kingdom plant pathogenic bacteria................128

ix

List of Abbreviations

µL microlitre

CF cystic fibrosis

CFU colony forming units

CIP calf intestinal alkaline phosphatase

Exo exonuclease 1

LB lysogeny broth

MEGA molecular evolutionary genetics analysis

MgS04 magnesium sulfate

mL millilitre

MLSA multilocus sequence analysis

mM millimolar

PCR polymerase chain reaction

T3SS type III secretion system

U unit

2

Chapter 1: General Introduction

The role of the general environment in the emergence of new diseases has been

the focus of many studies. Many plant pathogenic bacteria have been shown to cause

human infections [1,2], indicating that environmental bacteria can evolve to exploit

humans and animals as hosts. Bacterial genera like Burkholderia, which infects various

plant species including onion, sorghum, rice, and velvet beans [3,4,5], is also a

recognized human pathogen, causing chronic infections in patients with cystic fibrosis

(CF) and granulomatous disease [6]. Other plant pathogenic bacteria that cause human

disease include Serratia, Erwinia, and Pseudomonas, which have been implicated in

pneumonia and septicaemia, chronic illness, and cystic fibrosis, respectively [7,8,9,10].

The reservoir for these pathogens has been shown to be in the rhizosphere, the zone

around the roots of plants, and in the phyllosphere [2,11,12], both of which have a diverse

composition of nutrients. As a result, infection of a human or animal host by these strains

can occur by a scrape, ingestion, or contact with the skin or mucosal membranes [13].

Aside from direct contact, bacteria may also be transported from plants to indoor

or clinical environments through the activities of carriers like flies and ants [14,15],

providing bacteria with the opportunity to colonize a broader range of hosts. Those

strains that thrive in their new environment, including those that are capable of exploiting

a human host, will increase in the population, and may be returned back to the general

environment through one of many routes. One route may be facilitated by the

symptomology caused by the infection, which can promote bacterial shedding from the

host [16]. Bacteria can move from human wastes to natural watersheds, where irrigation

3

of commercially relevant crops would lead to an increase in bacterial titre on plants and

the general environment [17,18]. Insects may play a role in the return of pathogens back

to the general environment. The common house fly, Musca domestica, has been reported

as a carrier of many pathogenic bacteria including Pseudomonas aeruginosa,

Staphylococcus aureus, and Enterococcus faecalis [15], while the Pharoah ant,

Monomorium pharaonis, is known to transport human pathogenic bacteria such as

Streptococcus and Salmonella [19]. The possible association of plant pathogenic bacteria

with humans through the environment could lead to them becoming adapted to humans

over time. Likewise, the ability of these bacteria to return to the general environment, and

possibly associate with plants, may result in the maintenance of the ability to colonize

multiple hosts. The constant cycling of bacterial pathogens between environments and

hosts may have significant implications for host specificity and the emergence of new

infectious diseases.

Host Specificity

Host specificity encompasses the nature and extent of microbial adaptation to one

or more hosts, with specialists and generalists as the two main categories [20]. Microbes

that are generalists infect multiple hosts with little selectivity, while microbes that are

specialists are more selective, being able to colonize one defined host or group of hosts

[20]. The presence of bacteria in the environment, and their use of insects as possible

vectors for transmission, provides a wider range of potential hosts to colonize, where the

constant cycling of bacteria among multiple unrelated hosts in turn can lead to generalism

[21]. To associate with their preferred host and cause disease a large set of pathogenicity

4

factors are often used by microbes, and these are known as host specificity factors [20].

Host-specific factors range from specialized attachment appendages to specialized

secretion systems [22,23], and selectively target a particular feature of a host to initiate an

association [24]. Interestingly, the presence of host specific genes can enhance host

specificity. In the mutualist, Vibrio fischeri, the two-component sensor kinase, RscS,

induces the Syp exopolysaccharide, resulting in the formation of biofilm and the

colonization of the squid light organ [25,26]. The expression of rscS in a Vibrio strain

deficient of the gene enabled host colonization [25], which shows that this gene is a host

specificity factor.

Specialists

Specialists are organisms that have adapted to infecting one particular type of host

or group of hosts. Over time, the repeated colonization of the same host may lead to a

steady refinement of the pathogen genetic repertoire and the ability to exploit the host

more efficiently and effectively [27]. Both plant and animal pathogens use host

specificity factors for associating with their host, such as type III secretion system (T3SS)

effectors [20]. T3SS is a protein appendage found commonly among Gram-negative

bacteria [28]. Through this appendage, bacteria are able to attach to their host and release

effector proteins to promote disease and infection [29]. The human-adapted serovars of

Salmonella enterica use T3SS effector proteins: SseC, SseB, SseD, to facilitate the

attachment of the secretion system to the phagosomal membrane of human cells [30]. The

strong genetic similarities of sseD genes and sseD alleles to human cells show the host-

specific binding of the T3SS and its strong association with humans [30]. GTP-binding

5

proteins, particularly Rho subfamily proteins, have also been implicated in the entry of

many species of bacteria into eukaryotic cells. Rho subfamily proteins participate in

various activities within the cell including endocytosis, apoptosis, and cell transformation

[31]. Rho proteins of eukaryotic cells are known to be ADP-ribosylated by Clostridium

botulinum C3-like transferases and they are also key targets of Escherichia coli cytotoxic

necrotizing factors [31]. Through selective activation of the Rho proteins by bacterial

agents, bacteria initiate an association with their host cells and gain entry into their host

to promote disease [31].

Specialist bacterial pathogens have been proposed to be more evolutionarily

favoured due to higher benefits than costs [32]. In a constant and narrow niche, specialist

bacterial pathogens experience a higher probability of fixing beneficial alleles, a lower

frequency of deleterious alleles exhibiting mutation-selection balance, as well as fewer

deleterious alleles drifting to fixation [33]. More importantly, as a result of co-

evolutionary processes between the bacterial pathogen and its host, specialist bacterial

pathogens are able to achieve optimum virulence while maximizing their transmission

potential [27]. Although specialization to one specific host may result in increased

fitness, it comes at the cost of a lower ability to exploit other hosts.

Generalists

In contrast to specialists, generalists can infect and successfully colonize multiple

hosts, but may not necessarily be able to cross species barriers [32]. However, having a

broad host range comes at a cost. Although generalists are able to colonize a wider range

of hosts, this impedes their maximal performance in any one host [33]. A broad niche

6

decreases the probability of fixing beneficial alleles and increases the frequency of

harmful alleles (reduce mutation load) at mutation-selection equilibrium. On the other

hand the narrow niche of specialists increases the rate of evolutionary response [33].

Despite the costs, many bacterial pathogens are generalists, and capable of exploiting a

wide range of hosts.

Both Burkholderia cepacia and Pseudomonas aeruginosa are two generalist

bacterial pathogens that have been studied extensively for their disease-causing

capabilities. Both are ubiquitous in the environment, and can infect a broad range of

hosts, including humans. P. aeruginosa has been found in soil and is known to cause soft

rot in plants such as Arabidopsis thaliana and lettuce [34], but is also a major human

pathogen that infects CF and immunocompromised patients [35,36]. Its presence in the

general and clinical environments has raised questions about its virulence and pathogenic

potential toward humans. Studies have examined the characteristics of environmental and

clinical isolates of P. aeruginosa, and their virulence in a variety hosts. CF strains have

been shown to be more efficient in attaching to surfaces and forming biofilms in

comparison to environmental strains, but have a lower planktonic cell growth rate,

suggesting that the CF lung may be a more extreme environment [35]. Although clinical

and environmental strains vary in their biofilm-forming capabilities, there is a high level

of conservation in a core set of genes (~97%), which includes all known virulence

factors. The conservation of virulence determinants suggests that selection for the

maintenance of such traits exists in the environment. As a result, it is likely that

environmental strains have the ability to cause human infections despite an expected low

probability of encountering a human host. Environmental strains of P. aeruginosa also

7

have resistance to several antibiotics, with some strains showing ten-fold higher

resistance in comparison to a reference clinical strain [37].

Like P. aeruginosa, B. cepacia is a bacterial pathogen found in soil, and known to

infect both plants and humans [6]. Based on phenotypic and genotypic analyses, B.

cepacia has been divided into nine phylogenetically differentiable species, which together

constitute the B. cepacia complex [6]. Species like Burkholderia pyrrocinia are

composed of a mixture of environmental and clinical isolates, while Burkholderia

ambifaria contained only environmental strains [38]. Burkholderia cenocepacia showed

evolutionarily distinct groups for agricultural and clinical strains [39]. A phenotypic

analysis of the clinical and environmental strains across the B. cepacia complex showed

that all were able to macerate onion tissue and colonize nematodes, albeit at varying

levels [38]. Interestingly, clinical strains were found to have a higher level of antibiotic

resistance than environmental strains [6].

Exploration of the pathogenicity of clinical and environmental strains of

Burkholderia, as well as Pseudomonas, has revealed no difference in physical traits, such

as optimal growth temperature and their ability to overcome phage infection [6,40]. Both

clinical and environmental strains are also able to be both haemolytic and proteolytic to

epithelial cells, encode T3SS genes, and quorum-sensing genes, which could be

indicative of broader virulence potential [37].

Interestingly, analyses on Stenotrophomonas and Aeromonas, which are also

known to infect multiple hosts, revealed similar results found in Pseudomonas and

Burkholderia. Phylogenetic studies showed that there was no evolutionary separation of

clinical and environmental strains, and that they had no difference in antibiotic resistance

8

[41]. Surprisingly, environmental strains of Aeromonas displayed a higher level of

enterotoxicity and hemolysin production than clinical strains [42]; however, these

characteristics could lead to a greater potential for human pathogenesis. Both

Stenotrophomonas and Aeromonas are not known to be plant pathogens.

Pantoea as a model system

To understand the evolution of multihost pathogens and their potential impact on

human health, model pathogen groups must be used, such as those that are known to

naturally colonize a broad range of hosts and cause disease. Pantoea is ubiquitous in

nature, colonizing plants, humans, and being found in the general environment. Many

species of Pantoea are known to be plant pathogenic, and commonly found in diverse

ecological niches including aquatic environments, soil, or sediments [43]. Pantoea

stewartii subsp. stewartii causes Stewart‟s vascular wilt disease of sweet corn, while

Pantoea agglomerans is found to cause crown and root gall disease of gypsophila and

beet [44,45]. Interestingly, the recent emergence of the Pantoea in humans has led to the

realization that this genus represents a group of multihost pathogens with broad

capabilities [43,46,47,48]. Many studies have investigated the pathogenicity of Pantoea

in several plant hosts, and identified the possible pathogenicity factors involved during

disease. The pathogenicity of P. stewartii appears dependent on the hrp/wts gene cluster,

which directs the synthesis of a T3SS [29,46]. Additional work identified the

involvement of a quorum-sensing (QS) system, which allows bacteria to monitor their

population density by utilizing small, diffusible signals to orchestrate the expression of

specialized gene systems for pathogenicity [49,50,51]. The QS system organizes the

9

timing and production level of the exo/capsular polysaccharide, stewartan, which

significantly affects the degree of bacterial adhesion during in vitro biofilm formation and

propagation within the plant host [50].

In recent years, however, Pantoea species have been frequently isolated from

humans suffering from soft tissue or bone/joint infections following penetrating trauma

by vegetation [47]. Given the ubiquity of Pantoea in the general environment, and the

increasing number of human-related cases of Pantoea, several studies have examined

how the different strains are related. Their ubiquity in the environment and their

pathogenicity led to several studies examining their relationships using phylogenetics

[52,53]. These studies have used multilocus sequence analysis (MLSA), a technique that

provides an improved method for establishing relationships, and which involves the use

of multiple protein-coding genes in phylogenetic reconstruction to neutralize the effects

of horizontal gene transfer and homologous recombination [52]. In a recent study, MLSA

was used to determine the identification of various Pantoea strains obtained from diverse

origins. Most strains belonged to P. agglomerans, while others belonged to many diverse

phylogenetic branches such as other Pantoea species and probable novel species [53]. A

survey on the presence of repA, a common gene found on plasmids of plant pathogenic

strains was also conducted, and it was shown that clinical strains also contained the repA

gene [53], suggesting that clinical and plant-associated strains do not form distinct

populations and may have similar virulence characteristics. These studies have pursued

further analyses on phenotypic traits among plant and environmental strains. The

maximal hourly growth rate of clinical strains at 37°C, human body temperature, was

significantly less in comparison to plant strains within P. agglomerans, while at 24°C

10

there was no significant difference in maximal hourly growth rate among plant and

clinical strains [43]. Although clinical strains have a lower maximal hourly growth rate at

37°C, their ability to grow at this temperature suggests their ability to exploit a human

host.

These experiments along with the phylogenetic analyses prompted questions as to

the host range and pathogenic capabilities of Pantoea strains. One study conducted a

comparative virulence assay of five plant and five clinical strains using soybean plants

and embryonated hen eggs as hosts. Interestingly, both plant and clinical strains of P.

agglomerans developed stable epiphytic populations on soybean plants, while in the

embryonated hen egg model similar levels of growth were observed [47]. However, the

plant and clinical P. agglomerans strains were significantly less virulent than a

phytopathogenic P. ananatis isolate in the hen egg model [47]. The ability of both plant

and clinical strains to colonize a plant and a eukaryotic host suggests that the bacteria

might possess similar virulence potential, where plant strains may have the capacity to

infect humans. While this study and the several phylogenetic studies described above

have provided some important insight in the pathogenicity of Pantoea, they have focused

primarily on a few select strains, leaving many unanswered questions about the

capabilities of this bacterial group.

11

Objectives

The first objective was to examine how a broad collection of plant, clinical, and

environmental strains of Pantoea are related using a phylogenetic analysis through

multilocus sequence analysis (MLSA) of six housekeeping genes. The clustering pattern

of strains can be used to determine whether there is any evidence of host specialization in

this opportunistic pathogen. The second objective was to analyze the colonization

abilities of the various strains of Pantoea using maize, onion, and fruit flies as candidate

hosts. Results from these growth assays provided a profile for each of the strains,

possibly leading to a host specificity pattern that can be used to identify potential human

pathogens.

12

Chapter 2: Phylogenetic Analysis and Characterization of Plant,

Environmental, and Clinical Strains of Pantoea

Abstract

Strains of Pantoea have been found to colonize many different types of hosts such as

insects, plants, and humans, but it is unclear how they are related and if they are virulent

on hosts dissimilar to their origin of isolation. Phylogenetic analysis using multilocus

sequence analysis (MLSA) on six housekeeping genes of Pantoea showed that some

species within the genus contain a diverse array of plant, clinical, and environmental

strains, while other species are more specific, containing only one type of strain.

However, there is no distinct separation of the plant, clinical, and environmental strains.

Comparative growth assays using three hosts: maize, onion, and fruit flies, showed that

all strains were able to colonize hosts other that from which they were isolated. Some

species groups within the phylogenetic tree had an equal ability to colonize all three

hosts, while other species groups were more specialized. Overall, clinical strains had a

significantly greater growth within fruit flies than in either of the two plant hosts. The

results of this work show that some strains are diverse in their abilities to colonize

different hosts, while others are more dedicated to colonizing one specific type of host.

The close relationship of plant and environmental strains to clinical strains and their

ability to colonize plants and fruit flies with equal efficiency, highlights the potential

harm these strains may cause to humans while in the environment. A better understanding

of the relationships and colonization abilities of the various strains may subsequently lead

to the discovery of Pantoea strains that could be new potential human pathogens.

13

Introduction

Multihost bacterial pathogens colonize a wide range of hosts, with many being

capable of crossing both interspecific and intraspecific boundaries. The frequent isolation

of bacteria from humans following interactions with plants through open wounds or

abrasions [48,54,55,56,57], or consumption of contaminated water [58] indicates a

prominent role for the general environment as a reservoir for human pathogens. Pantoea

is widespread in nature and is commonly known as a plant epiphyte, where it can be

found in many diverse ecological niches such as soil and aquatic environments

[59,60,61]. Pantoea stewartii subsp. stewartii is the causal agent of Stewart‟s vascular

wilt disease of sweet maize and maize [62] while Pantoea citrea is known to cause pink

disease in pineapples [63]. Although common plant epiphytes and plant pathogens,

several strains of Pantoea agglomerans produce antibiotics which are sold as commercial

biological control agents against Erwinia amylovora, the fire blight pathogen of pear and

apple trees [64,65], and Pseudomonas syringae, the basal kernel blight pathogen of barley

[66].

In recent years, however, Pantoea has been frequently identified in human

infections with soft tissue or bone/joint infections following penetrating trauma by

vegetation [48,54,55,56,57]. P. agglomerans bacteremia has also been described in

association with the contamination of intravenous fluid [67], total parenteral nutrition

[68], the anesthetic agent propofol [69] and blood products [70]. The increasing number

of Pantoea species being isolated from plants, the general environment, and most

importantly, humans has led to the reclassification of Pantoea as a group of opportunistic,

ubiquitous, multihost pathogens, with an unknown virulence potential.

14

Previous studies have analyzed the relationships of plant, environmental, and

clinical strains of Pantoea to evaluate their potential virulence potential. Through a

multilocus sequence analysis (MLSA) on the different types of strains of P. agglomerans,

plant, environmental, and clinical strains co-clustered, indicating that all different types

of strains are closely related and may have similar host specificity and virulence [53].

One study used a host assay to evaluate host specificity and virulence, and compared

plant and clinical strains of P. agglomerans to a plant strain of Pantoea ananatis. Both

plant and clinical strains of P. agglomerans developed stable epiphytic populations on

soybean plants, while in the embryonated hen egg model no difference in virulence was

detected [47]. However, the five plant and five clinical P. agglomerans strains were

significantly less virulent than a phytopathogenic P. ananatis isolate in the hen egg model

[47]. Still, only a few strains were evaluated.

In this study, we used phylogenetic approaches and MLSA to evaluate the

relationships between plant, clinical, and environmental strains of Pantoea. We then

tested the colonization abilities of each strain in maize, onion, and fruit flies to determine

their host ranges. Evaluation of the relationships and virulence of the different types of

strains could lead to the detection of new potential human pathogens.

Materials and Methods

Bacterial Strains

A total of 128 Pantoea strains from clinical (56 strains), plant (33 strains), and

environmental (39 strains) sources were analyzed in this study. Clinical strains were

15

obtained from the Regina General Hospital, Dr. Paul Levett at the Saskatchewan Disease

Control Laboratory, St. Boniface General Hospital in Winnipeg, Texas Children‟s

Hospital, and Sunnybrook Hospital. The plant strains were obtained from New Zealand

Culture of Plant Pathogens (NZCPP), Dr. Steven Lindow at Berkeley, and Dr. Gwyn

Beattie at Ohio State. The environmental strains were acquired from sampling different

sources in the environment around Saskatchewan. Each strain was plated onto rifampicin

plates to select for rifampicin-mutant strains to reduce the potential for contamination.

DNA extraction, amplification, and sequencing

Bacterial strains were grown on Luria Bertani (LB) medium for 24 hours at 30

degrees Celsius. Genomic DNA was extracted and purified from 3 mL overnight cultures

in LB medium using the Qiagen genomic DNA purification kit (Mississauga, Ontario).

Polymerase chain reaction (PCR) amplification and sequencing of internal portions of the

six housekeeping genes fusA, leuS, pyrG, rpoB, gyrB, and 16S rRNA were performed

using primers described previously [53]. Each PCR was performed in a total volume of

20 µL using 14.4 μL sterile water, 2.0 μL 10x Standard Taq Buffer, 0.2 μL Taq DNA

polymerase (5 U/μL), 0.2 μL forward primer (50 μM), 0.2 μL reverse primer (50 μM),

2.0 μL dNTPs (200 μM each), and 1 μL (200-500 ng/μL) template DNA. PCR amplicons

were then purified from the PCR mix by cleaning with Calf Intestinal Alkaline

Phosphatase (CIP) and Exonuclease 1 (Exo) and incubated at 37 degrees Celsius for 30

minutes followed by an inactivation step at 85 degrees Celsius for 15 minutes. Primers

were then premixed with clean PCR products and sent off for sequencing to Operon

Sequencing, Huntsville, Alabama.

16

Data and sequence analysis

Sequences were aligned using Clustal W [71] to produce a concatenated tree.

These sequences were compared with a selection of concatenated rpoB and gyrB

sequences of Pantoea species from GenBank, other genera within the Enterobacteriaceae

family, and a representative of Pseudomonas as an outgroup to all strains. Gaps and

errors in sequencing were then corrected using GeneDoc, and the alignment was

transferred into Molecular Evolutionary Genetics Analysis (MEGA) version 4.0 [72] to

create a phylogenetic tree. A Neighbour-joining tree was constructed, with 1000

bootstrap replicates. Nucleotide divergence was determined using DnaSP program

version 5.10 [73].

Plant growth assay

Maize seeds (Golden Bantam) were sown in potting soil, a mixture of soil and

peat moss, and were grown in plant pots, where three seeds were sown per pot and were

watered each morning. Seeds were sown 2-5cm (1-2”) deep in potting soil and were

grown in 16 hour day cycles at 25 degrees Celsius. Plants, 3-4 weeks old plants with

broader leaves, were used for inoculation. Overnight cultures of bacteria grown in

rifampicin-LB broth were used for inoculating the maize plants. Optical densities of the

cultured broths were used to calculate a final concentration of 10-7

colony forming units

(CFU)/mL in 10 mM MgSO4. Bacterial suspensions were then inoculated (1 mL syringe)

into the leaves of the three different plants per pot to provide three replicates. Nine spots

per plant were inoculated on the leaves to provide three leaf tissue samples per replicate

over day 0, day 1, and day 5. Samples of 1 cm radius from the inoculated leaves were

17

obtained right after inoculation (day 0) and on day 1 and 5. Samples were placed in 1.5

mL microcentrifuge tubes containing 300 µL of 10 mM MgSO4 buffer and subsequently

ground to release the bacteria. Homogenates were then serially diluted to 10-2

CFU/mL in

90 µL of 10 mM MgSO4 buffer. Samples were then plated onto rifampicin plates to select

for the microbe of interest, and incubated overnight at 30 degrees Celsius. Bacterial

counts were taken for each replicate, which were then used to determine the number of

CFU per inoculated leaf tissue. Onion growth assays, using yellow onion, were

performed as above in the same manner of maize assays.

Fruit fly growth assay

Optical densities of overnight cultures of bacterial strains grown in LB containing

rifampicin were used to make 2 mL of 10-7

CFU/mL in 20 percent sucrose. Vials

containing a single cotton puff were then soaked with the bacterial solution, and 27 adult

flies were allowed to feed for 16 hours. Following feeding, flies were transferred to vials

containing only Drosophila media. At each time point, three flies were sampled per

replicate, and placed into a 1.5 mL microcentrifuge tube containing 300 µL of 10 mM

MgSO4 buffer. Flies were subsequently crushed and serially diluted to 10-2

CFU/mL in

90 µL of 10 mM MgSO4 buffer. Samples were then plated onto rifampicin plates to

reduce bacterial contamination and incubated overnight at 30 degrees Celsius. Bacterial

counts were used to calculate growth.

Standardization and data analysis of growth assays

Because initial doses varied between strains and across species groups, we

standardized all growth assays to facilitate comparisons across hosts. To do this, we

18

converted all raw values obtained from each growth assay to log base two. The growth

rate was then determined between day 0 and day 1, and day 1 and day 5, and these rates

were then used to standardize all growth assays, assuming an initial dose of 10 CFU.

SPSS version 17.0 was used to perform a two-way analysis of variance (ANOVA) on

values obtained for each respectable growth assay [74]. Differences were found to be

significant if p-values were <0.05.

Results

Genotypical analysis

Phylogenetic reconstruction by Neighbour-joining on the collection of 128 strains

revealed that our collection contains at least ten different species groups, as defined by

the location of marker sequences from type species. These include Pantoea agglomerans,

Pantoea eucalyptii, Pantoea conspicua, Pantoea brenneri, Pantoea anthophila, Pantoea

stewartii, Pantoea ananatis, Pantoea dispersa, Pantoea eucrina, Pantoea calida, and

Pantoea septica. Several strains provided to us as Pantoea fell outside of the genus and

were later identified as species of Erwinia, Cronobacter, Escherichia, and Klebsiella

(Figure 1).

To validate the phylogenetic groupings, an analysis of nucleotide divergence was

conducted to determine if the species groups were valid as predicted by phylogeny (Table

1). The average “within” species group percentage of nucleotide divergence is expected

to be less than 1.5%, while the average “between” species divergence is expected to be

greater than 1.5% [75]. Nucleotide divergence between the predicted species groups was

19

greater than 1.5%, except for P. stewartii and P. ananatis (1.474%) as well as P. stewartii

and P. brenneri (1.136%). In some monophyletic groups, several strains were more

divergent, and formed a sister group to the other strains. To test whether these may

represent additional species, divergence was calculated between these strains and the rest

of the group (Table 2). Interestingly, 17671 within P. ananatis had a nucleotide

divergence of 2.577% and 06868 had a nucleotide divergence of 2.628% within the P.

eucrina species group.

20

Figure 1. Neighbour joining tree based on the concatenated nucleotide sequences of fusA,

leuS, pyrG, rpoB, gyrB, and 16S rRNA of 128 Pantoea strains. Bootstrap values after

1000 replicates are expressed as percentages where only those of 70% and greater are

shown. Pseudomonas was included as an outgroup. (original in color)

SP01230

B015092

SP01220

SP00202

SP03412

G4032547

SP05120

770398

SP03310

SS03231

BB834250

SP01202

SN01201

SP02022

SP00303

SN01080

SP05092

SP03383

SP04011

B026440

TX10

SP05051

SP05052

SP02243

SS02010

SP04010

SP04022

SP05130

EH318

SN01121

SN01122

B016395

SP04021

SP02230

SN01170

B025670

SP05061

SP05091

H42501

P ann

3581

7373

SP00101

12534

12531

13301

17124

788

83

P herb

5565

7612

P agg

1574

308R

240

1512

Pantoea agglomerans

SP04013

299R

B011489

SP03391

F9026

SP02021

SM03214

SP03372

Pantoea eucalyptii

Pantoea conspicua B011017

B024858

091151

B011483

B016381

B014130

Pantoea brenneri

Pantoea anthophila 1373

626

P stPantoea stewartii

17671

26SR6

BRT175

BRT98

15320

M232A

B7

Cit30-11R

Pantoea ananatis

M1657A

M1657B

625

Pantoea dispersa

06868

TX6

TX5

Pantoea eucrina

BB957621A2

BB957621A1

BB957621B1

BB957651C1

BB957621C2

B021323

BB957621B2

Pantoea calida

TX4

TX3

M1517

BB350028B

BB350028A

B016375

VB38951B

VB38951A

M41864

101150

091457A

091457B

G4071105

062465B

062465A

081828

G3271436

G2291404

X44686

Pantoea septica

Erwinia amylovora EA

EHWF18

EHWHL9Erwinia billingiae

B012497

Cronobacter sakazakii 12202

SN01070

07703

TX1

TX2

E. coli / Shigella dysenteriae

10854

Klebsiella B011499

Klebsiella G4061350

Pseudomonas sp100

100

100

100

99

82

88

100

100

100

100

72

100

100

100

100

100

100

70

100

72

90

83

100

100

99

98

98

100

99

99

98

98

100

98

100

100

85

100

100

100

100 100

99

100

100

100

100

100

100

100

97

88

97

100

90

73

98

96

99

100

96

100

98

72

100

99

99

78

72

74

87

21

After phylogenetic reconstruction, we mapped on the origin of isolation (plant,

clinical, or environment) to determine whether there was any obvious association

between phylogeny and origin of isolation. P. agglomerans is a mixed group having 18

plant, 10 clinical, and 29 environmental strains. There are two clear subgroups within this

species, one composed of a mixture of plant, clinical, and environmental strains, while

the other composed of only plant strains.

P. eucalyptii and P. dispersa are also mixed, with P. eucalyptii having plant,

clinical and environmental strains, and P. dispersa having two clinical and one plant

strain from the collection. In contrast, P. conspicua, P. brenneri, P. eucrina, P. calida,

and P. septica all contain only clinical strains, while P. anthophila, P. stewartii, and P.

ananatis are made up of plant strains. There is some correlation between species group,

and location of isolation, with some species being composed of only clinical strains, only

plant strains, or mixtures.

22

Table 1. Nucleotide divergence expressed in percent among the different species groups within the phylogenetic concatenated tree.

Pantoea

agglomerans

Pantoea

eucalyptii

Pantoea

brenneri

Pantoea

stewartii

Pantoea

ananatis

Pantoea

dispersa

Pantoea

eucrina

Pantoea

calida

Pantoea

septica

Erwina

billingiae

Escherichia

coli Klebsiella

Pantoea agglomerans na

Pantoea eucalyptii 3.355 na

Pantoea brenneri 2.789 2.308 na

Pantoea stewartii 2.724 1.864 1.136 na

Pantoea ananatis 2.868 2.563 1.942 1.474 na

Pantoea dispersa 3.957 2.947 2.955 2.131 2.504 na

Pantoea eucrina 4.69 3.374 3.684 3.314 3.397 1.752 na

Pantoea calida 4.515 3.291 4.086 3.291 3.255 3.291 3.464 na

Pantoea septica 3.518 2.697 2.768 2.269 2.725 3.663 4.334 4.021 na

Erwina billingiae 4.651 4.19 4.517 4.119 4.013 3.267 4.214 2.202 4.037 na

Escherichia coli 3.984 2.912 3.239 2.699 3.072 2.462 3.946 4.948 2.527 4.498 na

Klebsiella 5.271 3.8 4.602 3.977 4.634 3.125 4.877 4.995 3.432 4.403 2.024 na

23

Table 2. Comparison of nucleotide divergence, in percent, among unique strains within

certain phylogenetic species groups.

Comparisons Nucleotide Divergence

SP04013 vs. Pantoea eucalyptii 0.61%

17671 vs. Pantoea ananatis 2.58%

06868 vs. Pantoea eucrina 2.68%

BB957621B2 vs. Pantoea calida 0.07%

TX4 and TX3 vs. Pantoea septica 1.43%

24

Growth Assays

Quantitative growth assays were conducted for all plant, clinical, and

environmental strains using maize, onion, and fruit flies as hosts. Strains in the mixed P.

agglomerans and P. eucalyptii groups had no statistical difference in growth in the maize,

fruit fly, and onion hosts. All plant, environmental, and clinical strains in P. agglomerans

grew to approximately 105 CFU by day 5 in all three different hosts. In contrast, plant

strains of the species group P. eucalyptii had a greater growth in maize plants, reaching

approximately 103 CFU by day five, while clinical strains had a greater growth within

fruit flies, reaching approximately 105 CFU by day 5. Interestingly, environmental strains

within P. eucalyptii reached approximately 104 CFU by day 5 in both the maize and fruit

fly hosts.

Species groups containing only one type of strain, like P. stewartii, P. ananatis,

P. eucrina, and P. calida had no significant difference in the ability of their strains to

colonize the three different hosts. P. stewartii and P. ananatis, which contain plant

strains, grew better in maize and onion plants, reaching approximately 105 CFU by day 5

(Refer to Appendix A). Surprisingly, there was no significant difference in their ability to

colonize fruit flies. Similarly, P. calida and P. eucrina, species groups containing only

clinical strains, were able to grow better in fruit flies attaining 104 CFU by day 5, but

were able to colonize plant hosts equally as well (Refer to Appendix A). Interestingly,

both pairs of groups are monophyletic.

P. brenneri, P. dispersa, and P. septica are composed almost exclusively of

clinical strains, and exhibit a preference for the fruit fly host. P. brenneri grows to 104

CFU by day 5 in flies, but only to 102

in the plant hosts (p=0.001). P. brenneri also

25

contains only clinical strains, which show significantly greater growth in fruit flies than

in either of the two plant hosts (p=0.001) (Figure 2). P. dispersa is a smaller group

comprising two closely related clinical strains, M1657A and M1657B, and the plant

strain, 625. The two clinical strains grow to significantly higher densities in fruit flies

than the plant strain, reaching approximately 105 CFU by day 5 (Figure 3). Surprisingly,

both clinical strains grew equally well in the two plant hosts. This is also seen for P.

septica, which is composed of only clinical strains. P. septica has a greater growth in fruit

flies, reaching 104 CFU by day 5, in comparison to maize and onion plants, 10

3 CFU by

day 5, which is an almost two fold decrease in growth (p<0.001) (Figure 2). Interestingly,

all clinical strains had equal growth in both plant hosts.

Non-Pantoea groups were included within the phylogenetic tree for comparison,

but were also included in our quantitative growth assays. Both Erwinia and Cronobacter

contain only plant strains, while Escherichia and Klebsiella contain only clinical strains.

For Erwinia, a significant difference in growth can be seen in the two hosts (p=0.017),

where plant strains grow better in maize (103 CFU by day 5) than in the fruit fly hosts,

(102

CFU by day 5). Clinical strains of E.coli and Klebsiella grew equally well in all three

different hosts. Both E. coli and Klebsiella were able to colonize the two plant hosts

equally well, but were able to grow to higher densities in fruit flies, reaching

approximately 103 CFU by day 5.

26

Figure 2. Comparative growth assay of clinical strains across Pantoea septica, and

Pantoea brenneri. All clinical strains overall within the species Pantoea septica,

and Pantoea brenneri have a significantly greater growth in fruit flies in

comparison to both maize and onion plants (p<0.05). The growth, number of

colony forming units (CFU), was determined per leaf disk for maize and onion

hosts and per fruit fly for the fruit fly host. (n=33 for maize, n=32 for fruit fly, and

n=31 for onion for all clinical strains, df=2, F=30) (original in color)

27

Figure 3. Comparison of growth within hosts among strains of Pantoea dispersa. The two

clinical strains within Pantoea dispersa had a significant difference in their ability to

colonize maize plants (p=0.000), fruit flies (p=0.053), and onion plants (p=0.001) in

comparison to the plant strain. No significant difference in growth can be seen in the two

clinical strains to colonize both the maize and onion plants (p=1.000). The growth,

number of colony forming units (CFU), was determined per leaf disk for maize and onion

hosts and per fruit fly for the fruit fly host. (n=2 for clinical strains in all three hosts,

while n=1 for plant strain, df=2, F=3.7) (original in color)

28

For most groups within the phylogenetic tree, clinical strains show a greater

ability to colonize fruit flies in comparison to plant and environmental strains. An overall

comparison of clinical strains from all the different groups further affirmed a significantly

greater growth in fruit flies in comparison to maize and onion. To evaluate the ability of

all clinical strains to colonize fruit flies, clinical strains were separated into their

respective origins of isolation: blood, urine, and external. Interestingly, all three clinical

groups: blood, urine, and external, had had an approximate ten-fold greater growth in

fruit flies than in maize and onion plants (p=0.001), with a significant difference of

p=0.000, p=0.004, and p=0.008 for blood, external, and urine respectively (Figure 4).

Conversely, an analysis of all plant and environmental strains in their ability to colonize

the different hosts revealed no difference in growth. Overall, these analyses show that

despite their origin of isolation all plant, environmental, and clinical strains were able to

colonize maize, onion, and fruit flies, and clinical strains are able to colonize fruit flies

more efficiently than plant hosts (Figure 5).

29

Figure 4. Overall analysis of growth across clinical strains. All clinical groups: blood,

external, and urine had an approximately ten fold greater growth (p=0.001) in fruit flies

than in maize and onion plants. The growth, number of colony forming units (CFU), was

determined per leaf disk for maize and onion hosts and per fruit fly for the fruit fly host.

(n=25 for maize, n=24 for fruit flies, and n=24 for onion for blood, external, and urine,

df=4, F=2.65) (original in color)

30

Figure 5. Multihost colonization of the 128 plant, clinical, and environmental strains.

Plant, clinical, and environmental strains show an ability to colonize all three hosts: corn,

fruit fly, and onion. Different levels of growth were achieved by day five but

environmental and plant strains were able to colonize fruit flies while clinical strains were

able to colonize both plants as effectively as the environmental and plant strains studied.

The growth, number of colony forming units (CFU), was determined per leaf disk for

maize and onion hosts and per fruit fly for the fruit fly host. (n=3 for environmental,

clinical, and plant for all three hosts) (original in color)

31

Discussion

MLSA of all 128 strains indicated that the majority of strains belonged to P.

agglomerans, while others belonged to other Pantoea species or other

Enterobacteriaceae.

Both P. agglomerans and P. eucalyptii are mixed groups containing plant,

clinical, and environmental strains, which suggests that plant, clinical, and environmental

strains are closely related and may therefore have similar characteristics and virulence

potential. This could imply that plant and environmental strains may have the ability to

cause human disease, and human strains may have the ability to be pathogenic towards

plants and the environment. Similar results were obtained by previous studies that used

MLSA to evaluate the relationships between plant, clinical, and environmental strains of

P. agglomerans [43]. Interestingly other species groups like P. septica contain only

clinical strains, and may be specialized to colonizing animal or human hosts. It is also

possible that these strains are still capable of infecting other organisms, like plants, but

are more frequently isolated from the clinical environment due to higher persistence in

the environment, or specific characteristics that promote more efficient dispersal.

Species groups established by phylogeny were well supported by our analysis of

nucleotide divergence. Most comparisons between species groups led to nucleotide

divergences greater than 1.5%, suggesting that the species groups predicted by the

phylogeny are supported. The plant pathogenic group, P. stewartii, and the clinical

pathogenic group, P. brenneri, had a nucleotide divergence of less than 1.5%. The low

divergence could reflect horizontal gene transfer between the two species groups,

resulting in a higher overall phylogenetic relatedness. The constant cycling of pathogens

32

in the environment could allow clinical and plant strains to interact and possibly

exchange genetic information, leading to a more generalist lifestyle in the environment.

P. ananatis strain17671 and the P. eucrina strain 06868 both had nucleotide

divergences greater than 1.5%, suggesting that they are not similar to the strains within

their respective species group as shown through the phylogeny. These likely represent

new Pantoea species, but this would have to be validated though biochemical tests.

Growth assays using plants and insects were especially informative in evaluating

the potential pathogenicity of each strain. Strains within the mixed groups, P.

agglomerans and P. eucalyptii, had no significant difference in their ability to colonize all

three hosts. The ability of plant and environmental strains to have similar virulence

potential and close relatedness to clinical strains further affirms the potential of plant and

environmental strains to colonize humans. The equal ability of all plant, clinical, and

environmental strains within P. agglomerans and P. eucalyptii to colonize fruit flies

suggests that they are possible human pathogens. Similarly, in another study, plant and

clinical strains of P. agglomerans were able to colonize soy bean plants and embryonated

hen eggs with equal growth [47]. Interestingly, a plant strain of P. ananatis was able to

attain a significantly higher mortality rate in an embryonated hen egg in comparison to

clinical strains of P. agglomerans [47]. The ability of a plant strain to be more virulent

than a clinical strain indicates that these strains can become problematic if they encounter

a suitable human host.

P. dispersa displayed different virulence potential between its two clinical strains

and one plant strain, with the two clinical strains colonizing the fruit fly better than the

two plant hosts and the plant strain colonizing the two plants hosts more efficiently than

33

the fruit fly host. Both P. brenneri and P. septica had a significantly greater ability to

colonize fruit flies in comparison to either maize or onion plants, which suggest that these

clinical species groups may be more specialized to colonizing animal hosts than plant

hosts. This suggests that some strains within some species groups of Pantoea may have

some level of host specificity, and phylogeny is not a good predictor of host specificity.

Although mammalian models were not used in this study, fruit flies have been

shown to mimic the human immune response [76,77]. Our quantitative growth assays

revealed that despite the assumption that plant strains could colonize only plant hosts and

clinical strains only human hosts, all plant, clinical, and environmental strains can

colonize maize, onion, and fruit flies, albeit at varying levels. Although all strains were

unable to grow to high titres in all three different hosts, the ability of Pantoea strains to

grow to levels higher than the initial dose shows their versatility, and potential to exploit

a wide range of hosts. It is possible that through constant exposure to these hosts, plant,

clinical, and environmental strains might be able evolve the necessary host specific

pathogenicity factors that would enhance their growth in one or more hosts. Pantoea

would have to possesses a wide range of pathogenicity factors that would give it the

ability to establish; however, aside from the plant pathogenicity factors, including

exopolysaccharide production, type III secretion system effectors, toxins, and adhesins,

the pathogenicity determinants that allow it to colonize fruit flies are unknown [78,79].

Fruit flies have closely related innate immune system pathways to humans and similar

mechanisms of recognition of microbial invaders [80]. The ability of plant and

environmental strains to colonize fruit flies as well as they colonize plant hosts shows the

potential for these strains to cause human infections.

34

Phylogenetic analysis reaffirms the idea that plant and environmental strains of

Pantoea retain human pathogenic potential, based on the fact that they are closely related

to clinical strains. Growth assays demonstrate that origin of isolation does not indicate

host range. The ability of plant and environmental strains to have close relationships with

clinical strains and have an equal ability to colonize both plants and fruit flies

demonstrates the versatility of Pantoea, and suggests that the pathogenicity of these

strains is greater than previously assumed.

35

Concluding Remarks

A thorough understanding of the relationship between plant, environmental, and

clinical strains within Pantoea is critical for determining their ability to emerge as serious

human pathogens. By analyzing the relationships of a diverse collection of strains, this

study has increased not only our understanding of how these different types of strains are

related, but also their potential virulence in various hosts. Phylogenetic analysis revealed

that plant, clinical, and environmental strains are closely related and thus may have

similar characteristics and virulence potential. Some species of Pantoea are mixed, and

contain strains isolated from humans, plants, and the general environment while others

are composed of strains isolated from only humans or only plants. Interestingly, analysis

of nucleotide divergence revealed the presence of potentially new species of Pantoea, but

also highlighted cases of possible exchange of genetic information between different

species. The broad scale evaluation of colonization ability of the different species in our

collection showed that growth in fruit flies seems to be good indicators of clinical

relevance. Fruit flies and humans share a high level of conservation of intracellular

pathways within their respective innate immune systems, in addition to having similar

methods to overcome pathogen invasion. The close relatedness of plant and

environmental strains to clinical strains, and their ability to colonize and grow in the fruit

fly affirms that these strains have human pathogenic potential. Lastly, this work sets the

foundation for the identification of host specificity factors and determinants that allow

Pantoea to colonize different hosts.

36

Future Directions

The understanding of the relationships of plant, clinical, and environmental strains

across many species of Pantoea and their virulence in different hosts is clear but the

actual virulence factors involved during pathogenicity are not well known. Investigation

into the specific pathogenicity factors involved during disease will help to understand

how these different types of strains are able to colonize multiple hosts and if there is

further similarities among the different type of strains. A genetic screen using transposon

mutagenesis using fruit fly larvae will assist in achieving the characterization of genes

involved in pathogenesis.

37

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44

Appendices

Appendix A. Comparative growth of all species groups .

Figure 1. Growth of plant, environmental, and clinical strains within Pantoea

agglomerans in maize, fruit fly, and onion hosts. (original in color)

45

Figure 2. Comparative growth assays of plant, environmental, and clinical strains within

Pantoea eucalyptii in maize and fruit fly. (original in color)

46

Figure 3. Comparative growth of clinical strains within Pantoea brenneri in maize, fruit

fly, and onion hosts. (original in color)

47

Figure 4. Comparative growth of plant strains within Pantoea stewartii in maize, fruit fly,

and onion hosts. (original in color)

48

Figure 5. Comparative growth analysis of plant strains within Pantoea ananatis in maize,

fruit fly, and onion. (original in color)

49

Figure 6. Comparative growth of plant and clinical strains within Pantoea dispersa in

maize, fruit fly, and onion hosts.

50

Figure 7. Comparative growth of clinical strains within Pantoea eucrina in maize, fruit

fly, and onion hosts. (original in color)

51

Figure 8. Comparative growth analysis of clinical strains within Pantoea calida in maize,

fruit fly, and onion. (original in color)

52

Figure 9. Growth of clinical strains within Pantoea septica in maize, fruit fly, and onion

hosts. (original in color)

53

Figure 10. Comparative growth of plant strains within Erwinia in maize and fruit fly


54

Figure 11. Growth of clinical strains within Escherichia coli in maize, fruit fly, and onion


55

Figure 12. Comparative growth of clinical strains within Klebsiella in maize, fruit fly,

and onions hosts. (original in color)

56

Appendix B. Characterization of mutants.

Table 1. Characterization of mutants generated through transposon mutagenesis of Pantoea ananatis strain 15320.

Name Characterization E-value Accession Number

0_506 hypothetical protein (Actinobacillus ureae ATCC 25976) 4.1 ZP_08066977.1

0_517 ATPase YhdZ (Pantoea ananatis AJ13355) 6E-135 BAK12886.1

0_587 ATPase YhdZ (Pantoea ananatis AJ13355) 2E-109 BAK12886.1

19_5b yahK gene product, Zn binding alcohol dehydrogenase (Pantoea ananatis LMG 5342) 8E-105 YP_005197581.1

20_7a yaha3 gene product (Pantoea ananatis LMG 5342), sugar:cation sympoter family protein 4E-78 YP_005196650.1

21_11e DegP gene (Pantoea ananatis LMG 20103), htrA gene product 3E-13 YP_003519080.1

22_5b thymidine kinase Tolk (Pantoea ananatis PA 13) 2E-34 AER32518.1

23_5e XylE (Pantoea ananatis LM 20103) 1E-91 YP_003519920.1

24_12d PgI protein product unnamed (Pantoea ananatis LMG 20103), unnamed protein product 0.18 YP_003518619.1

28_4e unnamed protein product (Pantoea ananatis LMG 5342) 4E-15 YP_005196290.1

29_5a carbamoyl transferase of the NodU/CmcH family (Pseudomonas florescens WH6) 3E-84 ZP_07777816.1

http://www.ncbi.nlm.nih.gov/protein/322513899?report=genbank&log$=prottop&blast_rank=1&RID=R6ERXHZP016

http://www.ncbi.nlm.nih.gov/protein/327395464?report=genbank&log$=prottop&blast_rank=1&RID=R6EZ3Y3M01N

http://www.ncbi.nlm.nih.gov/protein/327395464?report=genbank&log$=prottop&blast_rank=1&RID=R6EZ3Y3M01N

http://www.ncbi.nlm.nih.gov/protein/378769106?report=genbank&log$=prottop&blast_rank=1&RID=R67JVRJM013

http://www.ncbi.nlm.nih.gov/protein/378768179?report=genbank&log$=prottop&blast_rank=1&RID=R686YAVE012

http://www.ncbi.nlm.nih.gov/protein/291616338?report=genbank&log$=prottop&blast_rank=1&RID=R68H8CFP01S

http://www.ncbi.nlm.nih.gov/protein/354988394?report=genbank&log$=prottop&blast_rank=1&RID=R6EHZ8ZB01S

http://www.ncbi.nlm.nih.gov/protein/291617178?report=genbank&log$=prottop&blast_rank=1&RID=R68A0V6N016

http://www.ncbi.nlm.nih.gov/protein/291615877?report=genbank&log$=prottop&blast_rank=1&RID=R68MWCVX012

http://www.ncbi.nlm.nih.gov/protein/378767820?report=genbank&log$=prottop&blast_rank=1&RID=R68EAN0A01N

http://www.ncbi.nlm.nih.gov/protein/312963333?report=genbank&log$=prottop&blast_rank=1&RID=R67EN0RU01S

57

Appendix C. Information regarding the strains used in this thesis.

Strain Origin Isolated Host Patient

Information Extra Information

83 Environment Isolated from wheat Received from Maureen Fletcher, Landcare Research, New Zealand

Culture of Plant Pathogens

625 Environment Isolated from grain

sorghum

Received from Maureen Fletcher, Landcare Research, New Zealand

Culture of Plant Pathogens, Isolated in India

626 Environment Isolated from corn



788 Environment Green bean


Culture of Plant Pathogens, Isolated in the United Kingdom

1373 Environment Balsam



1512 Environment Green bean Received from Maureen Fletcher, Landcare Research, New Zealand

Culture of Plant Pathogens

1574 Environment Unidentified


Culture of Plant Pathogens, Isolated in Japan

3581 Environment Oat seed


Culture of Plant Pathogens, Isolated in Scotland

5565 Environment Soybean


Culture of Plant Pathogens, Isolated in New Zealand

7373 Environment Onion


Culture of Plant Pathogens, Isolated in South Africa

7612 Animal Grass grub



6686 Clinical, human Unidentified Headache Received from the St. Boniface

General Hospital, Winnipeg, Manitoba

58



7703 Clinical, human Unidentified Abdominal pain Received from the St. Boniface


10854 Clinical, human Ruptured appendix Received from the St. Boniface


12202 Environment Melon


Culture of Plant Pathogens, Isolated in Brazil

12531 Environment Baby's breath


Culture of Plant Pathogens, Isolated in the Netherlands

12534 Clinical, human Knee laceration


Culture of Plant Pathogens, Isolated in Zimbabwe

13301 Environment Golden delicious

apple



15320 Environment Rice


Culture of Plant Pathogens, Isolated in Australia

17124 Environment Olive


Culture of Plant Pathogens, Isolated in Italy

17671 Environment Rice


Culture of Plant Pathogens, Isolated in Cambodia

81828 Clinical, human Unidentified Post Hemicholectomy Received from St. Boniface General

Hospital, Winnipeg, Manitoba

91151 Clinical, human Unidentified Received from St. Boniface General


101150 Clinical, human Unidentified Received from St. Boniface General


062465A Clinical, human Unidentified Cerebellar CVA

(stroke) Received from St. Boniface General


59



062465B Clinical, human Unidentified Cerebellar CVA

(stroke) Received from St. Boniface General


770398 Clinical, human Blood Female Received from Sunnybrook Hospital,

Toronto, Ontario

091957A Clinical, human Renal failure Received from St. Boniface General


091957B Clinical, human Renal failure Received from St. Boniface General


240R Environment None Received from Dr. Steven Lindow,

Berkeley, California

26SR6 Environment None Received from Dr. Steven Lindow,






B011017 Clinical, human Superficial wound

back 11 year old female

Received from Dr. Paul Levett, Saskatchewan Disease Control

Laboratory, Regina, Saskatchewan

B014130 Clinical, human Superficial wound leg 11 year old male Received from Dr. Paul Levett, Regina,

Saskatchewan

B015092 Clinical, human Urine midstream 9 years old female Received from Dr. Paul Levett, Regina,

Saskatchewan

B016375 Clinical, human Finger 56 year old female Received from Dr. Paul Levett, Regina,

Saskatchewan

B016381 Clinical, human Groin 52 year old female Received from Dr. Paul Levett, Regina,

Saskatchewan

60



B016395 Clinical, human Superficial wound

forearm 83 year old female

Received from Dr. Paul Levett, Regina, Saskatchewan

B021323 Clinical, human Urine midstream 28 year old female Received from Dr. Paul Levett, Regina,

Saskatchewan

B024858 Clinical, human Breast abscess 26 year old female Received from Dr. Paul Levett, Regina,

Saskatchewan

B025670 Clinical, human Superficial wound toe 13 year old male Received from Dr. Paul Levett, Regina,

Saskatchewan

B026440 Clinical, human Superficial wound toe 10 year old male Received from Dr. Paul Levett, Regina,

Saskatchewan

B7 Environment Unidentified Received from Dr. Steven Lindow,


BB350028A Clinical, human Blood culture, fever Female Received from St. Boniface General


BB350028B Clinical, human Blood culture, fever Female Received from St. Boniface General


BB834250 Clinical, human Sputum, aortic

aneurysm Female

Received from St. Boniface General Hospital, Winnipeg, Manitoba

BB957621A1 Clinical, human CAPD dialysate,

peritonitis Male


BB957621A2 Clinical, human CAPD dialysate,

peritonitis Male


BB957621B1 Clinical, human CAPD dialysate,

peritonitis Male


BB957621C1 Clinical, human CAPD dialysate,

peritonitis Male


61



BB957621C2 Clinical, human CAPD dialysate,

peritonitis Male


BRT 98 Environment Unidentified Received from Dr. Steven Lindow,


BRT 175 Environment Unidentified Received from Gwyn Beattie, Iowa

State University, Iowa

Cit30-11R Environment Unidentified Received from Dr. Steven Lindow,


Eh318 Environment Biocontrol of Erwinia

amylovora

Received from Dr. Brion Duffy, Switzerland

EhWHF18 Environment Unidentified Received from Gwyn Beattie, Iowa


EhWHL9 Environment Unidentified Received from Gwyn Beattie, Iowa


G0063668 Clinical, human CSF Received from the General Hospital in


G2291404 Clinical, human Blood Received from the General Hospital in


G3271436 Clinical, human Urine Received from the General Hospital in


G4032547 Clinical, human Ear Received from the General Hospital in


G4061350 Clinical, human Urine Received from the General Hospital in


62



G4071105 Clinical, human Urine

Received from the General Hospital in Regina, Saskatchewan

F9026 Clinical, human Blood Male Received from Sunnybrook Hospital ,

Toronto, Ontario

H42501 Clinical, human Blood Male Received from Sunnybrook Hospital,

Toronto, Ontario

M1517 Clinical, human Blood Female Received from Sunnybrook Hospital,

Toronto, Ontario

M1657A Clinical, human Blood Male Received from Sunnybrook Hospital,

Toronto, Ontario

M1657B Clinical, human Blood Male Received from Sunnybrook Hospital,

Toronto, Ontario

M232A Environment Unidentified

Received from Dr. Steven Lindow, Berkeley, California

M41864 Clinical, human Blood Female Received from Sunnybrook Hospital,

Toronto, Ontario

Pantoea agglomerans Plant Type strain

Received from David Coplin, Columbus, Ohio

Pantoea ananatis Plant Type strain


Pantoea herbicola Plant Type strain


Pantoea stewartii Plant Type strain Received from David Coplin,

Columbus, Ohio

63



SM02150 Environment Water with water

fleas

Isolated in Saskatchewan by summer students of Dr. John Stavrinides

SM03214 Environment Goose feces Isolated in Saskatchewan by summer

students of Dr. John Stavrinides

SN01080 Environment Slug Isolated in Saskatchewan by summer


SN01121 Environment Bee Isolated in Saskatchewan by summer


SN01122 Environment Bee Isolated in Saskatchewan by summer


SN01170 Environment Caterpillar Isolated in Saskatchewan by summer


SP00101 Environment Raspberry bush Isolated in Saskatchewan by summer


SP00202 Environment Apple Isolated in Saskatchewan by summer


SP00303 Environment Raspberry bush Isolated in Saskatchewan by summer


SP01201 Environment Strawberry Isolated in Saskatchewan by summer


SP01202 Environment Strawberry leaf and

stem

Isolated in Saskatchewan by summer students of Dr. John Stavrinides

64



SP01220 Environment Healthy rose bush Isolated in Saskatchewan by summer


SP01230 Environment Virginia creeper leaves and stem

Isolated in Saskatchewan by summer


SP02021 Environment Thistle leaf Isolated in Saskatchewan by summer


SP02230 Environment Unidentified tree Isolated in Saskatchewan by summer






SP03372 Environment Corn Isolated in Saskatchewan by summer




SP03391 Environment Unidentified Isolated in Saskatchewan by summer


SP03412 Environment Bean leaf Isolated in Saskatchewan by summer


SP04010 Environment Tomato leaf Isolated in Saskatchewan by summer


SP04011 Environment Tomato Isolated in Saskatchewan by summer


65



















SP05120 Environment Corn leaf Isolated in Saskatchewan by summer


SP05130 Environment Corn stamen Isolated in Saskatchewan by summer


SS02010 Environment Soil Isolated in Saskatchewan by summer




66



TX1 Clinical, human CF sputum Received from Texas Children`s

Hospital, Houston, Texas



TX3 Clinical, human Blood Received from Texas Children`s


















SP03391 Environment Unidentified Isolated in Saskatchewan by summer


67



SP03412 Environment Bean leaf Isolated in Saskatchewan by summer




SP04011 Environment Tomato Isolated in Saskatchewan by summer


















SP05120 Environment Corn leaf Isolated in Saskatchewan by summer


68



SP05130 Environment Corn stamen Isolated in Saskatchewan by summer






VB38951A Clinical, human Blood culture, sore

throat Female


VB38951B Clinical, human Blood culture, sore

throat Female


X44686 Clinical, human Blood Female Received from Sunnybrook Hospital,

Toronto, Ontario

69

Appendix D. Insects as alternative hosts for phytopathogenic

bacteria.

Note: This appendix has been republished, with permission, from Nadarasah, G., and

Stavrinides, J. Insects as alternative hosts for phytopathogenic bacteria. FEMS

Microbiology Reviews. 35(3): 555-575.

Insects as alternative hosts for phytopathogenic bacteria

Geetanchaly Nadarasah and John Stavrinides

Running Title

Non-plant hosts for plant pathogenic bacteria

Authors‟ Address

Department of Biology


3737 Wascana Parkway

Regina, Saskatchewan, Canada

S4S0A2

Corresponding Author

John Stavrinides



3737 Wascana Parkway


S4S0A2

Ph: (306) 337-8478

Fx: (306) 337-2410

Email: [email protected]

Keywords

plant pathogens, bacteria, host, insects, pathogenicity, transmission, vector, multi-host,

host specificity, evolution

70

Abstract

Phytopathogens have evolved specialized pathogenicity determinants that enable them to

colonize their specific plant hosts and cause disease; but, their intimate associations with

plants also predisposes them to frequent encounters with herbivorous insects, providing

these phytopathogens with ample opportunity to colonize and eventually evolve

alternative associations with insects. Decades of research have revealed that these

associations have resulted in the formation of bacterial-vector relationships, in which the

insect mediates dissemination of the plant pathogen. Emerging research, however, has

highlighted the ability of plant pathogenic bacteria to use insects as alternative hosts,

exploiting them as they would their primary plant host. The identification of specific

bacterial genetic determinants that mediate the interaction between bacterium and insect

suggests that these interactions are not incidental, but have likely arisen following

repeated association of microbes with particular insects over evolutionary time. This

review will address the biology and ecology of phytopathogenic bacteria that interact

with insects, including the traditional role of insects as vectors, as well as the newly

emerging paradigm of insects serving as alternative primary hosts. Also discussed is one

case where an insect serves as both host and vector, which may represent a transitionary

stage in the evolution of insect-phytopathogen associations.

71

Introduction

Plant pathogenic bacteria are responsible for some of the most devastating losses

of major agricultural crops and vital fruit trees, causing millions of dollars in damage

annually. Their agricultural and economic impact has afforded them significant attention

over the last 30 years, resulting in enormous strides in the exploration of their

epidemiology and specialized disease strategies. Research of plant pathogenic bacteria

has not only seeded our understanding of the genetics of disease, epidemiology, and the

factors contributing to emerging infectious diseases, but has also led to the development

of effective control and prevention measures for many plant diseases [81,82]. More

recently, however, there has been a shift in the exploration of the plant pathogens to a

broader community level, which moves beyond the traditional single host-single

pathogen model to a wider and more encompassing view of the evolution and ecology of

plant pathogenic bacteria. Much of this research has expanded the field into a new

direction, and has resulted in the unearthing of the hidden ecology and true pathogenic

potential of many bacteria that have long been considered strict and very dedicated

phytopathogens.

The exploration of phytopathogen life histories is often trumped by the striking

and often contrasting disease symptomology that develops on host plants as a

consequence of disease. Traditionally, this has resulted in an almost exclusive focus on

the biology, ecology, and genetics of specific plant-phytopathogen relationships, often to

the exclusion of other potentially relevant yet presumably less obvious potential

associations. Even the most intimate association between plant pathogen and plant host in

the natural environment, whether occurring at the interface of the phyllosphere or within

72

plant tissues is still subject to incursions by other ecological players. Phytophagous

insects, in particular, which graze frequently and recurrently on plant tissues that may be

colonized by epiphytic or plant pathogenic bacteria, are often neglected as key ecological

players, despite the fact that they are most likely to have repeated encounters and

association with phytopathogenic bacteria that reside in or on their preferred host plants.

There are numerous potential interactions that can result from the association

between a microbe and an insect, all of which are defined by the relative effects on the

fitness of the individual organisms (referred to as symbionts). Mutualisms may form

between the two organisms, where both derive a benefit from their interaction.

Mutualisms may be defensive, where the microbe provides protection to the insect host

[83], or nutritional, where the microbe supplements the diet of the insect host with key

nutrients [84]. Parasitisms may also develop between microbe and insect, where the

microbe benefits by extracting nutrients from its host at a cost to its host. In the case of

the latter, the microbe may cause disease in the insect, usually marked by a condition that

impairs or disrupts normal host functioning and physiology, and is often associated with

specific symptomology. A commensalism describes the association between insect and

microbe where the microbe benefits and the insect is unaffected. Commensalisms likely

characterize many of the interactions that exist in the natural environment, but are most

likely to go unnoticed. Both commensalistic and parasitic symbioses can range from

highly specific to non-specific, with the development of more specific interactions being

favoured in cases where specialist microbes encounter specialist insects recurrently over

long periods of time, and more general interactions in cases where generalist or transient

insects encounter specialist bacterial pathogens (or vice versa).

73

Phytopathogenic bacteria have evolved to harness insects as vectors to effect their

dissemination and delivery directly onto or into their preferred plant hosts. These

partnerships can be either commensalistic or slightly parasitic to the insect, but in either

case, the insect performs as a living carrier that transmits the microbe to its final

(definitive) host. Many of these symbioses are highly specific, and are categorized by the

ability of the bacterium to replicate in and move through its vector. The tendency to

replicate within the insect vector is described as either propagative or non-propagative,

while the tendency of the microbe to move through its vector can be described as

circulative or non-circulative [85]. In circulative non-propagative transmission, the

microbe is ingested by its insect vector as it feeds on the host plant, after which it

migrates into the midgut or hindgut epithelium, and is then released into the haemolymph

of the insect [85]. The microbe then enters into the salivary glands, and can be inoculated

to healthy plant hosts via the saliva while the insect feeds [86,87]. In this case, the

microbe does not replicate in its host vector. In contrast, circulative propagative

transmission occurs when a microbe is able to replicate within its insect vector, and

spread to other organs within the insect. The microbe crosses the membrane to enter into

the haemolymph and is later instigated into the saliva to thereby be released from the

insect to cause new infection in the host plant [88]. Non-circulative and non-propagative

microbes are those which generally form a physical association with the insect, and are

subsequently mechanically transmitted to the plant host (infection by an insect stylet that

is coated with a pathogen, for example [89].

Over the last decade, the exploration of phytopathogenic bacteria and their

interactions with insects has expanded beyond the traditional phytopathogen-vector

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relationship to include cases where phytopathogens exhibit entomopathogenic

associations. Most of these relationships have been characterized only recently, and

represent a new paradigm in bacterial-insect interactions. Certainly, this has not lessened

the focus on the traditional plant-microbe or vector-microbe association, but has provided

additional breadth to the biology, ecology, and evolution of phytopathogenic bacteria.

This review will examine the alternative associations of phytopathogenic bacteria with

insects, focusing on the genetics and ecological relevance of those insects that can serve

as either their primary host or vector. Also examined is one special interaction where the

microbe exploits a single insect as both host and vector, and explore the possibility that

this unusual interaction represents a transitionary phase in the evolution of

phytopathogen-insect interactions.

Insects as vectors for phytopathogenic bacteria

The evolution of effective and stable phytopathogen-insect vector partnerships is

dependent largely on the opportunity for the insect and the microbe to encounter each

other frequently. Generally, the dependence of many insects and phytopathogens on

plants as their primary source of nutrition may lead to an overlap of ecological niche,

providing the necessary conditions for insects to encounter, contact, or ingest

phytopathogenic bacteria. In this section, we describe the best characterized symbioses

between insects and phytopathogens wherein the insect serves as a delivery vessel for the

bacteria.

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Xylella fastidiosa and the sharpshooter

Xylella fastidiosa is xylem-restricted, fastidious phytopathogen that causes citrus

variegated chlorosis and Pierce‟s Disease of grape [90,91]. X. fastidiosa is transmitted

between plant hosts exclusively by xylem-feeding sharpshooter leafhoppers (Hemiptera,

Cicadellidae) and spittlebugs (Hemiptera, Cercopidae) [92,93], which deliver the bacteria

directly into the plant. Leafhoppers use their piercing and sucking mouthparts to penetrate

the water-conducting xylem vessels of host plants to access the xylem sap, and if

infected, extravasate X. fastidiosa through their food canal injecting the bacteria directly

into the xylem vessels of the plant [94]. Once inside, the bacteria multiply and spread

from the site of infection to colonize the xylem and form a biofilm [91,95,96,97]. From

there, the bacteria spread to adjacent uncolonized xylem vessels, possibly through the pit

membrane [91], resulting in the physical obstruction of water flow through plant tissues,

causing leaf, shoot, and eventually, plant death [98].

The infiltration of key insect vectors into important grape and citrus farming areas

of North America prompted a dramatic increase in the exploration of the epidemiology of

X. fastidiosa and the role of insect vectors in pathogen dispersal [91,97,99,100,101]. The

relatively recent introduction of the efficient leafhopper vector, the glassy-winged

sharpshooter, Homalodisca vitripennis, and the blue-green sharpshooter, Graphocephala

atropunctata (Signoret) into California resulted in Pierce‟s Disease becoming a more

aggressive and prevalent disease; however, because X. fastidiosa lacks vector-species

specificity, as seen with many other phytopathogenic bacteria [102], nearly all

sharpshooter species are able to transmit X. fastidiosa, albeit with differing transmission

efficiencies [91]. Although both insect vectors are capable of transmitting X. fastidiosa,

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the glassy-wing sharpshooter is often seen as a more efficient vector than the blue-green

sharpshooter [99]. Transmission efficiency may be linked to feeding site preference since

the blue green sharpshooter is known to have a preference for feeding on young tissue

and leaves, while the glassy-wing prefers both young tissue and mature woody parts of

the plant [100]. Linked to this is the fact that X. fastidiosa is found to be

disproportionately dispersed within symptomatic plants, an attribute that may influence

the acquisition of the pathogen, depending on the tendency of specific insect species to

feed on tissues that may have lower bacterial concentrations [99]. Acquisition efficiency

was significantly higher from plants which had a higher bacterial load, thus implying a

direct correlation among bacterial concentration and vector transmission efficiency

[99,103].

Following ingestion, the bacteria become localized to the insect foregut where

they multiply and grow [104]. The pathogen can be transmitted immediately after

acquisition [91,94,105], indicating that bacterial multiplication in the foregut of the insect

vector is not vital for pathogen transmission, and that X. fastidiosa is a noncirculative

vectored phytopathogen [94,99,105]. Although X. fastidiosa may propagate through non-

circulative propagative transmission, the bacterium cannot be passed from parent to

offspring, as neither transovarial (immature egg to adult) transmission nor transstadial

(mature egg to adult) transmission have been observed for this bacterium [106,107]. In

addition, infected new born nymphs generally lose their infectivity after molting their

foregut cuticular lining [105].

The interaction of X. fastidiosa with its insect vectors appears to be determined by

the rpf locus (regulation of pathogenicity factor) [108]. X. fastidiosa uses cell-to-cell

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signalling mediated by a small diffusible signalling molecule known as DSF (Diffusible

Signalling Factor) [91]. The production of DSF is dependent on the gene rpfF, which has

characteristics similar to long-chain fatty acyl CoA ligases [109,110,111]. Mutations in

rpfF caused a deficiency in the ability of the bacteria to form a biofilm in the insect host,

despite being taken up from the plant [91]. Suprisingly, rpfF mutants are hypervirulent in

grape plants [108]. Likewise, mutation of a second locus, rpfC, does not impair the ability

of X. fastidiosa to colonize the insect, but does alter its ability to be transmitted to new

host plants [91]. It has been proposed that this is due to rpfC mutants being stronger

biofilm formers than the wildtype strain, which reduces the number of planktonic cells

that can be released from the insect during feeding. For plant virulence, mutations in the

rpfC gene cause X. fastidiosa to become deficient in longitudinal migration along the

xylem vessel, resulting in lower growth and spread in grape stems than the wild type

strain [91].

There is early evidence that X. fastidiosa has developed a seemingly specific

relationship with the xylem-feeding sharpshooters and spittlebugs. The identification of

the rpf locus provides a promising beginning to understanding the specific genetic

underpinnings of the interaction between X. fastidiosa and its insect vectors, but many

aspects of this relationship still remain unexplored.

Pantoea stewartii and the flea beetle

Stewart‟s disease (or Stewart‟s wilt) of corn, caused by the bacterium Pantoea

stewartii, (formerly Erwinia stewartii) causes significant yield loss in dent and sweet

corn as a result of leaf blighting [112]. The development of Stewart‟s wilt has two

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distinct symptomologies: wilt and leaf blight. In both cases the manifestation of disease

initially begins once the bacterium has successfully invaded the leaf tissue through

lesions produced by the flea beetle [112]. Upon entry, P. stewartii multiplies within the

leaves producing yellowish, water-soaked lesions or streaks that eventually elongate and

later coalesce along the leaf veins of corn leaves and soon become necrotic [113,114].

The bacterium colonizes the xylem vessels, where their production of large amounts of

bacterial exo/capsular polysaccharide (EPS), also known as stewartan, restricts the flow

of free water, causing wilting, and this can be followed by a general browning and water-

soaking of the stalk tissue [112,115,116].

The successful infection of corn plants by P. stewartii appears dependent on the

hrp/wts gene cluster, which directs the synthesis of a type III secretion system [29,117].

Through transposon mutagenesis, Frederick et al. (2001) identified the wtsE gene, which

encodes a 201-kDa protein which is strikingly similar to DspE in E. amylovora and the

protein AvrE found in P. syringae pv. tomato, both of which have been implicated in

virulence [118,119]. Additional work on P. stewartii pathogenesis identified the

involvement of a quorum-sensing (QS) system, which allows bacteria to monitor their

population density by utilizing small, diffusible signals and to orchestrate the expression

of specialized gene systems for pathogenicity [50,51,120]. Studies conducted by

Koutsoudis et al. [50] suggested a possible functional corollary between bacterial biofilm

development and xylem colonization similar to that described for X. fastidiosa infections

of grape vine. From their research they recognized that QS system organized the timing

and level of EPS produced, which significantly affects the degree of bacterial adhesion

during in vitro biofilm formation and propagation within the plant host. Moreover, their

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microscopic studies revealed that P. stewartii colonizes the xylem of corn with spatial

specificity rather than by arbitrary growth to fill the lumen of the xylem, as seen with X.

fastidiosa.

X. fastidiosa is disseminated among suitable host plants via a specific insect

vector – the corn flea beetle, Chaetocnema pulicularia – which acquires the pathogen

while feeding on infected corn plants [113]. The pathogen becomes localized along the

alimentary tract of adult corn flea beetles [121], where it remains for the entire duration

of the insect‟s life [112]. The beetles overwinter in the soil of grassy areas near

agricultural fields for the duration of the winter season, and although colder winter

temperatures reduce beetle survivorship, many beetles still survive to transmit the disease

[112,114]. With the spring thaw, the beetles exit their dormancy stage and begin to feed,

and deposit the pathogen into the feeding wounds via their feces, allowing P. stewartii to

enter the veins of corn leaves and cause disease [112,114].

Beetles that feed on infected tissue acquire the bacterium and promote the spread

of the pathogen throughout the season [112]. The colonization of corn flea beetles by P.

stewartii appears to be mediated by a type III secretion system (T3SS) that is distinct

from that used for colonizing the plant host [122]. Recently, Correa et al. [123] studied a

mutant strain of P. stewartii DC283, which had a mutation in the ysaN gene, a component

of the second T3SS apparatus. They discovered that the beetles were able to acquire both

the mutant and wildtype strains of P. stewartii equally, but the ysaN mutant did not

persist like wildtype, and declined in frequency four days following acquisition. Through

the use of confocal laser scanning microscopy, Correa et al. (2008) demonstrate that P.

stewartii persists in the hindgut lumen of beetles, but does not invade the gut cells. P.

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stewartii is capable, however, of invading cells of Malphigian tubules that protrude from

the gut of beetles. Correa et al. (2008) indicate that this supports previous studies that

indicate that the most likely route of baterial transmission is through insect frass. This is

also supported by observations by Correa et al. (2008), who have observed flea beetles

clustering together in small groups on maize leaves under growth chamber conditions,

which they suggest would promote P. stewartii infiltration into plant tissues.

P. stewartii utilizes the corn flea beetle as a vector to disperse to corn plants, and

this interaction appears to be facilitated at least in part by a T3SS. By extension, this

would implicate the involvement of specific type III secreted effectors, which likely

interact with host substrates to facilitate bacterial colonization of the insect. Although the

actual mechanism of how P. stewartii colonizes their insect vector is not understood

fully, it appears that the phytopathogen has adapted to the beetle, which promotes its

dissemination.

Serratia marcescens and the squash bug

Serratia marcescens is a phloem-resident pathogen that causes cucurbit yellow

vine disease of pumpkin (Cucurbita moschata L.) and squash (Cucurbita pepo L.), which

is characterized by wilting, phloem discoloration, and yellowing foliage [124,125,126].

Recent studies have shown that S. marcescens produces a biofilm along the sides of the

phloem tissues of the plant once inside its host, blocking the transport of water and

nutrients and eventually causing the plant to wilt and die [127,128]. A genetic screen to

identify genes that modulate biofilm formation in S. marcescens revealed the

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involvement of fimbrial genes, as well as an oxyR homolog, a conserved bacterial

transcription factor having a primary role in oxidative stress response [129].

S. marcescens is transmitted by the squash bug, Anasa tristis (DeGreer), which is

commonly found throughout the U.S. as well as between Canada and Central America

[130]. The squash bug feeds on its plant host using piercing-sucking mouthparts, which

penetrate intracellularly through the tissue, and are directed towards the vascular bundles

of the plant vasculature [131]. The visible signs and extent of feeding damage to squash

plants correlate with the number and size of bugs, as well as the amount of time each bug

spends on the plant and on the feeding site [131]. Long term feeding on the fruit leads to

fruit collapse, while leaf feeding induces isolated necrotic lesions [131]. Early

experiments revealed the presence of starch granules in the gut of A. tristis, which are

only found in the cytoplasm of plants, suggesting that the squash bugs ingest the

intracellular contents of plant cells [132]; however, experiments in which squash bugs

were allowed to feed on plants having safranin-stained xylem fluid found the red dye in

the gut of the insects, suggesting that xylem is also a food source for the insects [131].

Surprisingly, squash bug feeding damage extends beyond the xylem vessels and into the

phloem. Areas adjacent to the spongy mesophyll of the leaf and the cells of the palisade

and epidermal layers of leaves also exhibit signs of localized feeding-induced injury

[131,133,134]. Extensive feeding on the stem can damage the vascular tissue of the plant,

thereby resulting in the wilt of the leaf apical to the feeding site, or wilt of the entire plant

if it is a seedling [131,133,135]. Heavy feeding can cause leaves to turn black and soon

become crisp [130].

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Early studies examined the colonization of the squash bug by S. marcescens.

Wayadande et al. [94] initially hypothesized that S. marcescens shared a similar

relationship with its insect vector as seen between X. fastidiosa and sharpshooters, where

the bacteria are localized to the foregut of the insect vector and are released through the

food canal during successive feeding bouts; however, upon examination of the foregut of

adult and nymph squash bugs allowed to feed on bacteria-infiltrated squash cubes, the

foregut cibaria of the infected insects were found to be clear of any bacteria-like

structures. From their results, Wayadande et al. concluded that the ability of A. tristis to

transmit S. marcescens after molting indicated that the hemocoel, and not the gut, acts as

a possible site of retention for the infectious bacteria. This is in contrast to work showing

that S. marcescens is pathogenic once introduced into the haemolymph of A. tristis

[94,136,137].

The incubation time (or latent period) of S. marcescens was shown to be very

short, with some adults being capable of transmitting the bacterium one to two days after

initial acquisition [136]; however, adult squash bugs upon bacteria-infiltrated squash fruit

cubes, were noted to transmit the bacterium only sporadically to squash plants within a

twenty one day testing period [94]. This short latent period coupled with an irregular

transmission patterns are indicative of a noncirculative mode of transmission [105,136].

Despite its noncirculative association, S. marcescens overwinters in the dormant insect

vector, a strategy that protects the pathogen against low winter temperatures, ensuring a

high survival rate and thus successful transmission to plants in the following season

[138].

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Erwinia tracheiphila and the cucumber beetle

Bacterial wilt is a serious threat to commercial melon and cucumber production in

some parts of the world, including parts of North America. Bacterial wilt is caused by the

bacterium, Erwinia tracheiphila, which is transmitted by both the striped cucumber

beetles (Acalymma vittata) and spotted cucumber beetles (Diabrotica undecimpunctata)

[139]. These beetles are attracted to their host by cucurbitacins, a group of secondary

plant metabolites that are commonly found within the plant family Cucurbitaceae [140].

Cucurbitacins are bitter toxic compounds [141], which are known to accumulate in

cucumber beetles and confer protection against predation [142,143], but have detrimental

effects on most invertebrate and vertebrate herbivores [144,145]. The preferred plant

hosts of E. tracheiphila are wild and cultivated cucurbits, including muskmelon,

pumpkin, gourd, and squash, with cucumbers being the most susceptible hosts [146].

Mechanical wounding of the plant tissue is necessary for bacterial infection, since

the bacterium cannot infect the cucumber plant through the normally found openings

(stomates and hydathodes) of a plant [139]. While feeding on infected cucurbits with

their piercing and sucking mouthparts, the cucumber beetle acquires E. tracheiphila,

which then migrates to the insect gut epithelium. While infected beetles feed on healthy

curcurbit plants, bacteria are deposited on the leaves via beetle fecal droppings, which

leach into the lesions created by the feeding beetles [147]. E. tracheiphila can only

migrate toward a wound providing there is a sufficient aqueous film on the leaf surface

[139], although the cucumber beetles‟ stylet can also become infected with the pathogen,

providing a direct, mechanical method of infection [147]. Once inside the plant, E.

tracheiphila spreads to the xylem vessels, multiplies, and infects all parts of the plant. As

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the bacterium multiplies in the xylem, the efficiency of water transport is reduced to less

than one-fifth of normal water flow, resulting in extensive plugging of the vessels and the

subsequent wilt of the plant [146].

Although little is known about the interaction between E. tracheiphila and the

cucumber beetle, there appears to be some level of coevolution. The bacterium is able to

overwinter in the digestive tract of its vector, and is able to escape through the fecal

droppings, without any adverse impact on its insect vector. Although the insect is left

with the burden of carrying the bacterium for the rest of its life, its association with the

pathogen does not drastically affect its lifestyle. The existence of any coevolutionary

processes leading to the formation of the interaction between E. tracheiphila and the

cucumber beetle is still unknown.

Erwinia amylovora and pollinators

Erwinia amylovora is the causal agent of fire blight of apple and pear, a

detrimental bacterial disease of rosaceous plants, infecting primarily significant pear and

apple varieties [148,149]. The effects of E. amylovora on apple and pear tress have been

catastrophic, causing the death of blossoms, shoots, limbs, and at times, entire trees [65].

The primary infection site of the pathogen in fire blight disease is through tree blossoms

[148,150,151], which begins with bacterial colonization of the stigma, reproduction on

the stigmatic surface, migration along the length of the style, and eruption into the host

tissue via the nectarthodes [149,152]. Stigmas, which are borne on the ends of the style,

have been demonstrated to be the principal site of epiphytic colonization by E. amylovora

[65,150,152,153,154,155]. Despite the generalization that aerial surfaces of plants like

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stigmatic surfaces are unreceptive to bacterial growth due to exposure to ultraviolet

radiation and varying osmotic pressure, stigmas provide E. amylovora with a nutrient-

rich, protected, and hydrated environment for growth [65]. Micrographs showing E.

amylovora growing mostly within the large intracellular spaces between the secretory

papillae of stigmas have reaffirmed this [153,154].

E. amylovora has a non-specific association with pollinating insects that travel

from tree to tree collecting nectar [65], including honey bees, Apis mellifera (family

Apidae), which have been shown to be extremely efficient vectors [156]. To investigate

which species of insects were able to transmit E. amylovora, Emmett and Baker (1971)

inoculated various insects with E. amylovora and transferred the insects to apple and pear

blossom trusses and evaluated rates of tree infection. Several insects were able to transmit

the bacterium and induce infections in blossoms and shoots, although it appeared that

larger species of insects, like bees, were more efficient in transmitting the pathogen to

blossoms in comparison to smaller species of insects, such as anthomyiid flies. Larger

insects were able to infect more trusses and more flowers per truss, possibly due to their

ability to carry more inoculum, as well as their larger overall migration distances [156].

There have been no conclusive studies demonstrating that the bacterium enters and

colonizes insects; rather, there is overwhelming evidence that E. amylovora adheres to

the external surfaces of its insect vectors and is subsequently transmitted to healthy plants

mechanically. In one experiment by Hildebrand (2000) Aphis pomi was surface

contaminated with fluorescent E. amylovora through exposure of a thin lawn sprayed

with the bacterium. Over several consecutive days, aphids were crushed, plated,

aliquoted, and bacterial presence analysed by PCR. Results revealed florescent bacteria

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on the legs, cornicles, proboscis, and on the antennae of the aphids [157]. Persistence of

bacteria on insect surfaces has been shown to be at least seventy two hours on Aphis pomi

[158], nine days on the flesh fly Sarcophaga carnaria [159], five days on the green

lacewing (Chrysoperla carnea) [157], and up to twelve days on some aphid species,

likely facilitated by the exopolysaccharide capsule of the bacteria [157].

Because there is no evidence of bacterial internalization by insects, overwintering

of E. amylovora appears to be within the canker on its host plant. Once spring emerges

and temperatures are favourable, bacteria ooze from the cankers, and cause an infestation

of the blossom [160]. This process is contrary to P. stewartii, S. marcescens, and E.

tracheiphila, which overwinter in their specific dormant insect vector to protect

themselves from the harsh winters.

Candidatus Liberibacter and citrus psyllid

Candidatus Liberibacter is a phloem-limited phytopathogenic bacterium that

causes huanglongbing disease (HLB) or citrus greening on citrus fruits around the world

[161,162]. Ca. Liberibacter has a semi-specific symbiotic relationship with two different

psyllid insect vectors: Diaphorina citri Kuwayama [163] and Trioza erytreae (del

Guercio) [164]. D. citri is the principal vector in Asia, Brazil, and Florida, while T.

erytreae transmits Liberibacter in Africa [162]. D. citri has been in existence in Brazil for

over 60 years [165,166] and in Florida since 1998 [167]; however, HLB appeared in both

locations simultaneously. The psyllid has also been reported in areas of Texas in 2001

[168] as well as in several other countries in the Caribbean basin [169].

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HLB has been divided into Asian and African strains based on the influence of

temperature and host symptoms. In Asia the bacterium of HLB has been characterized as

Candidatus Liberibacter asiaticus (Las), where it infects the majority of citrus cultivars

and causes extensive economic loss by limiting the lifespan of infected trees

[170,171,172,173]. Las is heat tolerant, and can produce HLB symptoms at temperatures

above thirty degrees [171,174], whereas the African species, Candidatus Liberibacter

africanus, is heat-sensitive and does not cause symptoms above thirty degrees [171,174].

Recently, a new species of Candidatus Liberibacter was identified, which was unique

from the other three species because it caused disease in solanaceous plants, and was

vectored by a different psyllid species, Bactericera cockerelli [175]. B. cockerelli is a

polyphagous phloem feeder, which can productively reproduce on a wide variety of host

plant species, but has predominantly been a pest of potato (Solanum tuberosum L.) and

tomato (Solanum lycopersicon L.) [175,176,177].

Las-carrying T. erytreae and D. citri psyllids are efficient vectors of HLB, which

carry the bacteria in the haemolymph and salivary glands [178,179]. Work by Hung et

al., (2004) demonstrated that infected nymphs, which are barely mobile, quickly develop

into Las-carrying adults with the capability to fly and transmit the pathogen to other

citrus plants. They show that Las cannot be detected at all in first instars, suggesting that

first instars are incapable of carrying the pathogen. Second instars, however, do carry the

pathogen, but at extremely low titre. Psyllids are therefore able to bear the bacterium in

either adult or nymphal stages, but not as first instars. Hung et al. (2004) also

demonstrate that bacterial titre increases with each instar, suggesting that the pathogen

replicates during vector metamorphosis [171], and can therefore be considered

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propagative [162]. In a separate study, HLB was found to be present at a higher infection

frequency in eggs, first instars, and second instars isolated from potato host plants than

from those isolated from tomato [175]. Psyllids from potato were found to have a fixed

concentration of bacteria from the first instar stage to the adult phase, whereas those

isolated from tomato had very low titres at the egg and first instar phase, which increases

considerably in the second instar stage and becomes fixed at the third instar period [175].

This suggested that Ca. Liberibacter is vertically transmitted, but its rate is dependent on

the host plant from which it was isolated. This is in direct conflict to previous reports that

Las persists in the adult insect vector for twelve weeks and does not directly transmit Las

to its offspring [171].

The interaction between Ca. Liberibacter and its insect permits the pathogen to

disperse, and gain entry into its plant host. The ability of Ca. Liberibacter to be

transmitted by both sharpshooters and spittlebugs suggests that its interaction with these

sap-feeding insects may be semi-specific. Although the genetics of the interaction have

yet to be explored, Ca. Liberibacter may have specific genetic factors that enable insect,

association, colonization and persistence, with the extent of any adaptation or coevolution

with its insect vectors having yet to be determined.

Pectobacterium and the fruit fly

Pectobacterium carotovorum (formerly Erwinia carotovora) is member of the

Enterobacteriaceae [180] and the causal agent of the tuber-borne lethal potato blackleg

disease [181]. The pathogen produces pectolytic enzymes, which target plant cell walls

and lead to their complete destruction [182]. Production of these exoenzymes is

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controlled by a global regulatory mechanism, and more specifically, the expI gene [183].

expI mutants are deficient in exoenzyme production, and are completely avirulent as they

can neither break down the plant tissue nor multiply within potato plants [182,183,184].

expI has a general signalling function, and directs the synthesis of a signal molecule that

is involved in cell density-dependent control of exoenzyme genes in P. carotovorum.

Pirhonen et al. (1993) demonstrated this hypothesis, through extracellular

complementation of the defect in exoenzyme production, where the diffusible signal

molecule produced by ExpI-proficient cells can be recognized by the mutant and

subsequently used to activate exoenzyme gene expression.

In addition to causing disease in potato, P. carotovorum also has another suitable

host: the fruit fly, Drosophila. In 2000, Basset et al. (2000) identified a strain of P.

carotovorum, Ecc15, which provoked a systemic immune response in Drosophila larvae

following natural ingestion [185,186]. Feeding of larvae with living Ecc15 resulted in

them having high expression of antimicrobial peptide genes in their fat body, which is

functionally analogous to mammalian liver [187]. Although this bacterial strain did not

appear to be pathogenic to its insect vector, its ability to induce a systemic immune

response implied that it may have infectious properties that can be recognized by the

Drosophila innate immune system [188]. Out of the sixteen Ecc strains tested by Basset

et al. (2003) [188], only three were found to have the ability to infect Drosophila larvae

by natural infection. Such a result enabled Basset et al. to hypothesize that there may be

specific genes that permitted Ecc15 to associate with its insect vector [185].

Using a genetic screen, Basset et al. in 2003 identified two genes that are required

by P. carotovorum to cause infection in Drosophila. One gene, evf, enabled persistence in

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the host [188], and was controlled by the hor gene – a key regulator capable of conveying

signals from various environments to effectors involved in both plant pathogenesis and

Drosophila infection [188]. Transfer of the evf gene to non-infectious Pectobacterium

strains or to other enterobacteria was found to improve the ability of the bacterium to

survive in the gut of Drosophila and trigger an immune response [188]. The fact that the

gene evf was found in only a few P. carotovorum suggested that this gene had been

acquired recently through horizontal gene transfer [188]. When the evf gene was

overexpressed in P. carotovorum, bacteria were able to colonize the apical side of the gut

epithelium and at times to spread to the body cavity [188].

The expression of the evf gene results in the accretion of bacteria in the anterior

midgut and radically influences gut physiology [189]. It was suggested that evf could

disrupt the peritrophic membrane, which is a chitinous membrane that outlines the insect

vector‟s gut and averts bacteria from coming into the gut cells [188]. Basset et al. (2003)

also proposed that evf could allow the propagation of bacteria in this environment, or

produce a toxin which could disrupt the physiology of the gut cells. Recent crystal

structure data of Evf shows it to be an α/β protein having a novel fold and intricate

topology, with evidence for a palmitoic acid being covalently linked to the 209 cysteine

residue of the Evf protein through an association with a thioester linkage, suggesting that

Evf may be targeted to membranes [190]. Palmitoylation, a post-translational

modification that increases the affinity of soluble proteins for lipid membranes [191,192],

is necessary for biological activity as shown by the abolishment of Evf function following

mutation of the key cysteine residue required for palmitoylation [190]. Surprisingly, Evf

was found to be present in the cytoplasm, not in the periplasm [189], but was shown to

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bind to model membranes and promote aggregation. In subsequent studies, Quevillion-

Cheruel et al. (2009) showed that the overexpression of the Evf protein promoted

bacterial accumulation in the gut in an arrangement typical of an organized community,

as seen in a biofilm, and suggest that the ability of the Evf protein to be able to amass

bacteria may be due to its capacity to interact with and promote aggregation of vesicles.

Quevillion-Cheruel et al. (2009) concluded that the function of the Evf protein must be

related to post-translational modification, where the biological function of the evf gene

may be more directed towards membrane anchoring of the protein.

One Ecc strain, Ecc1401, which did not possess the evf gene, was able to induce

an immune response in Drosophila, suggesting there may be additional factors involved

in host association. P. carotovorum can effectively spread from plant to plant via

Drosophila, and although Drosophila may not be its intended carrier, there is evidence

for adaptation of the bacterium to this host that results in efficient bacterial association,

retention, and ultimately, dispersal.

Insects as primary hosts for phytopathogenic bacteria

New research has highlighted several instances of phytopathogenic bacteria

exploiting insects as primary hosts, with experimental evidence pointing to the ability of

many phytopathogens to invade and colonize insects as they would their plant hosts.

These interactions exhibit pathologies similar to those seen between phytopathogens and

their plant hosts, including rapid bacterial growth and the manifestation of disease. In this

section, we describe three distinct cases involving three phytopathogens exploiting an

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insect host. Surprisingly, the pea aphid, Acyrthosiphon pisum, is the target insect host in

all three cases.

Dickeya dadantii and the Pea Aphid

Dickeya dadantii (formerly Erwinia chrysanthemi) is a member of the

Enterobacteriaceae and the agent of soft rot disease of a wide range of economically

important crops, including potatoes and maize [193]. Disease develops following

movement of the pathogen from the stem base throughout the tissues, producing a brown

staining of the vascular tissues, and occasionally necrosis and hallowing of the stem

[194]. D. dadantii causes the rapid disruption of parenchymatous tissues, principally

induced by the use of its pectic enzymes, and accelerates the disease process with

cellulases, iron assimilation, a type III secretion system, exopolysaccharides, motility,

and proteins involved in resistance to plant defences [195,196,197,198,199]. Despite its

long history as a typified plant pathogen, D. dadantii strain 3937 was shown to be a

pathogen of the pea aphid, Acyrthosiphon pisum [195]. Grenier and colleagues (2006)

determined that A. pisum aphids that had ingested D. dadantii eventually succumbed to

their infection, with the minimum infectious dose of D. dadantii being calculated as

fewer than ten bacterial cells [195]. Recent genome sequencing of the D. dadantii strain

3937 revealed the presence of four genes encoding homologues of insecticidal toxins,

which were hypothesized to contribute to the pathogenicity of the bacterium in the aphid

[195,200]. These homologues were later found to be able to complement the cyt family of

genes from Bacillus thuringiensis, which encode haemolytic toxins [201]. Gut proteases

were hypothesized to cleave and activate the D. dadantii Cyt toxins in the aphid, resulting

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in pore formation in the insect gut membrane, and leading to bacterial invasion of the

aphid and eventual death; however, the ∆cyt mutant retained virulence, suggesting that

other virulence genes or factors are involved [195,202]. The cyt-like toxins may therefore

be involved in the early colonization of the aphid digestive tract, which is consistent with

what is known for the B. thuringiensis homologues [203].

In a later study, Costechareyre and colleagues [204] found that the four

coregulated cyt genes are expressed in response to high osmolarity. They suggest that this

is because D. dadantii is commonly found in the low osmolarity intercellular fluids of its

host plant, where toxin synthesis is likely not necessary; however, a high concentration of

sucrose is prevalent in the phloem sap, which would trigger toxin production if bacteria

are internalized in a phloem-feeding insect gut, like that of aphids. Further exploration

revealed that cyt gene expression is repressed by both hns (histone-like nucleoid

structuring protein) [204] and vfmE, a regulator of plant cell wall-degrading enzymes

[205], since both hns and vfmE mutants retained the pathogenicity of wildtype. PecS, a

regulator of pectinases and cellulases [206] appeared to regulate cyt gene expression

since pecS mutants were found to be non-pathogenic when ingested by the aphid.

Mutants of the GacA, OmpR and PhoP regulators, which are involved in plant

pathogenesis [207,208,209] and do not appear to affect Cyt toxin production, had reduced

virulence in the aphid. The Cyt toxins, which are expressed under very specific

conditions, are therefore only part of the suite of virulence factors used by D. dadantii for

causing disease in aphids.

The relatively low minimum infectious dose of D. dadantii required for aphid

infection could suggest high infectious rates for aphids overall, as this low density can be

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easily acquired from plant surfaces, or from feeding on contaminated vascular tissues

[204,210,211]. It is unclear whether D. dadantii is transmitted readily to healthy plants by

the aphids, or whether this represents a more opportunistic or general association.

Erwinia aphidicola and the Pea Aphid

Erwinia aphidicola, a member of the Enterobacteriaceae, has been identified as

the causal agent of leaf spot disease of common bean (Phaseolus vulgaris), and chlorosis

and necrosis of pea (Pisum sativum cv. Tirabeque) [212,213]. In addition to causing plant

disease, E. aphidicola also exhibits pathogenicity toward the pea aphid, A. pisum. Harada

et al. (1997) [214] initiated the study of a mysterious bacterium, called bacterium X,

which was found to infect the gut of insects that had been kept aseptically. The

bacterium, which was later identified as E. aphidicola, could grow productively in the

aphid gut, inhibiting post-final ecdysis and resulting in mortality of the adult insect.

Harada and Ishikawa (1997) were able to note that the bacterium produced extracellular

polysaccharides when they were left to grow in a medium containing sucrose, trehalose

or their component monosaccharides. This capsule may not be essential to cellular

function, but it may allow certain saprophytes to attach to areas where there is an

abundance of nutrients, or allow certain pathogens to avoid engulfment by phagocytes

[215]. The capsule layer may have been contributing to the attachment and colonization

of the pathogen in the aphid gut. The cause of aphid mortality following colonization was

proposed to be due to the aseptic conditions of the aphid gut, since normal gut colonizers

would help maintain E. aphidicola densities in check [215]. Still there are likely many

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genetic factors that contribute to the colonization of the gut, yet these still remain

unexplored.

Pantoea stewartii and the Pea Aphid

P. stewartii, the Stewart‟s wilt pathogen, which normally associates with its flea

beetle vector, was recently found to exploit the pea aphid as a host. In 2010, a study by

Stavrinides et al. (2010) showed that P. stewartii DC283 (DC283) was pathogenic toward

the pea aphid, as aphids fed a single dose of DC283 began to accumulate bacteria in their

gut, with titres reaching 5 x 108 cfu, and aphid death following within 72 hours. To

identify the specific genetic determinants that are involved in pathogenesis, and more

specifically, those that contribute to lethality of the aphid, a transposons mutagenesis

screen was conducted. A single locus, which was termed ucp1 (you cannot pass), was

identified that appeared to be essential for aggregation and pathogenicity of DC283.

ucp1-proficient bacteria formed aggregates in the crop and hindgut, whereas the ucp1

mutant did not. Aggregates of ucp1-expressing bacteria were suggested to be more

resilient, accumulating in the crop and hindgut until the point of barricading the flow of

honeydew. This result coincides with the structural and functional features of ucp1,

which included several predicted transmembrane domains, suggesting membrane

localization and possible substrate or matrix binding capabilities.

Six potential homologs of Ucp1 were identified in the draft genome of DC283, all

of which were found to share a highly conserved N terminus, but an entirely non-

homologous C terminus [216]. The conserved N terminus contains the transmembrane

domains, and prediction of protein localization places the hypervariable C terminus

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facing the extracellular environment. Based on this, ucp1 was proposed to function as a

Microbial Surface Component Recognizing Adhesive Matrix Molecules (MSCRAMM) -

a family of adhesion proteins utilized by animal pathogens to bind to proteinaceous

components of the eukaryotic host cell to permit pathogenesis [217]. To lend credence to

this hypothesis, all seven related genes were expressed in E. coli, and each line fed to

aphids. Only ucp1 was necessary and sufficient for pathogenicity in the aphid, whereas

the other lines were avirulent like control lines. In addition, E. coli lines expressing the

protein exhibited the same aggregation phenotype as seen for wildtype DC283,

suggesting that this protein was necessary and sufficient for this phenotype. It was

unclear, however, if this protein was involved in direct binding to an aphid gut receptor,

and whether the other six related proteins could bind to other matrix molecules in

different hosts. The drastic variability seen in the potentially exposed C terminus of all

seven proteins could be the direct result of genetic shuffling or pathoadaptation imposed

by host immune pressures [216,218,219,220]. Alternatively, it was proposed that the C

terminus of Ucp1 does not bind to eukaryotic proteins, but instead to other exposed Ucp

C termini of nearby cells, thereby promoting the linking of the structures to produce a

bacterial matrix. Although its precise function is unclear, the ucp1 locus appears essential

for the pathogenicity of P. stewartii in pea aphids.

One insect for all occasions

Many insects are efficient vectors of phytopathogenic bacteria, transporting them

to candidate host plants in the environment. In these associations, the bacterium is not

often considered to harm its vector, although there may be a reduction in the fitness of the

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insect as a result of carriage [216]. These interactions can be characterized as

commensalistic or slightly parasitic depending on the specific insect-bacterium

association. In contrast, some insects have been shown to function as a primary host for

bacteria, and are exploited by these pathogens as equivalents to their plant hosts. In cases

where the insect is exploited as a primary host, the association is effectively parasitic,

with the fitness of the bacteria increasing at a cost to the insect. These bacteria exhibit

entomopathogenic characteristics, utilizing specific virulence factors to overcome insect

host defences, propagate, and disperse. But, what if an insect can function as both a

primary host and a vector for a given phytopathogen?

Although there is a tendency for us to categorize the interactions among

organisms into discrete groups, the general biology and ecology of many phytopathogens

and their interactions with other organisms in the environment are still rather nebulous.

More recent studies of phytopathogens have uncovered unique interactions with insects,

including one case where the insect appears to serve as a suitable primary host, as well as

a vector. This particular association raises several interesting issues, including the

difficulty of reconciling the commensalistic and pathogenic life stages of the pathogen.

Pathogens are known to reach a virulence optimum, which maximizes their

aggressiveness and transmission potential [210]. High levels of aggressiveness may result

in the death of the host insect before the bacterium is able to disperse to other hosts; thus,

natural selection will favour a reduction in the aggressiveness of the pathogen to permit

dispersal of the bacterium and thereby increase bacterial fitness [221]. To overcome this

trade-off, the bacterium can exploit its insect host maximally by replicating rapidly, yet

this would come into direct conflict with the dynamics of association between a typical

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phytopathogen and vector. For this last example, the application of the fundamental

concepts of host-microbe associations is complicated by the fact that the insect can

function as both a host and vector. It is an especially exciting interaction for the simple

idea that it may represent a transitional stage in the evolution of phytopathogen-vector

and phytopathogen-host associations.

Pseudomonas syringae and the Pea Aphid

Pseudomonas syringae is a phytopathogenic bacterium noted for its diverse

interactions with different plant species. Although many strains are known to cause

disease on various plants, many epiphytic strains have also been identified [222]. P.

syringae propagation onto and between host plants involves rain splash-mediated

inoculation from infected to uninfected plants, facilitated by the aggressive epiphytic and

aggregation capabilities of P. syringae. P. syringae has also been shown to disperse via

precipitation [223].

P. syringae was considered a very strict phytopathogen, capable of infecting a

variety of different plants [224]; however, a recent study by Stavrinides et al. (2009)

demonstrated that some strains of this species have entomopathogenic potential. The bean

strain, P. syringae pv. syringae B728a (B728a), which is an aggressive epiphyte and

pathogen of bean, was shown to exploit the pea aphid as a suitable alternative primary

host. Within 36-48 hours of ingesting B728a, aphids succumb to infection with growth of

up to 3x106 colony forming units (cfu) per aphid. In contrast, ingestion of the tomato

strain P. syringae pv. tomato DC3000 (DC3000) by aphids, results in bacterial titres of

1x109 cfu/aphid, with no evidence of disease, and aphid survivorship remaining

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unaffected beyond 72 hours. This suggests the presence of strain-specific virulence

factors contribute to the colonization of the aphid by B728a.

Whole genome comparisons of DC3000 and B728a identified toxin complex (tc)

genes in both strains whose homologues have been implicated in insect association [225].

The tc genes present in DC3000 appeared degenerate, with mobile genetic elements and

deletions disrupting the reading frame, whereas the orthologs in B728a were intact.

These genes were strong candidates for explaining the virulence of B728a and the

avirulence of DC3000. Mutation of two of the B728a tc genes does not attenuate

virulence, indicating that these genes were not the primary virulence determinants for

B728a in the aphid. To identify those genetic factors that were involved in aphid

colonization, Stavrinides et al. (2009) performed a mutagenesis screen to identify mutants

that had reduced or abolished virulence. Multiple hypovirulent B728a mutants were

recovered, including one that was defective in the fliL gene, which is required for

flagellar formation. The fliL mutant was completely avirulent, growing to tires of 4x107

cfu/aphid, which were not lethal to the aphid, much like the avirulent DC3000 wildtype

strain. To identify the phenotypic effects of the fliL mutation, various motility assays

were undertaken. A swarming assay revealed that the fliL mutant was incapable of

swarming, a type of movement commonly seen in bacteria that allows for coordinated

movement over a solid or semi-solid surface. It is unclear, however, if it is swarming

specifically that is required for virulence in the aphid, or whether there are pleiotropic

effects, where motility regulates virulence factors.

In exploring the pathogenicity of B728a toward the aphid, Stavrinides et al.

(2009) noted that infected aphids exhibited some very unusual behaviors. After the onset

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of severe disease, aphids would discontinue feeding and commence to wander around,

depositing and moving honeydew behind them. Stavrinides et al. (2009) hypothesized

that the honeydew that was passing through the aphids contained B728a. Using a simple

culturing method, they found that viable B728a was present in the deposited honeydew at

incredibly high doses, suggesting that the epiphyte propagates in the aphid, and then is

redeposited back onto plant surfaces; however, since many of these feeding experiments

were performed under artificial conditions, Stavrinides et al. (2009) attempted to

demonstrate that the aphid could indeed become infected by feeding on plants that were

colonized epiphytically by B728a. Aphids were introduced onto plants that had been

surface-inoculated with B728a, and after a feeding period, aphids were harvested and

screened for the presence of B728a. Aphids were shown to acquire the bacteria, which

likely colonized the digestive tract, multiplied, and were then excreted in the aphid

honeydew. The acquisition of the bacterium by the aphids most probably occurs via

stylet-mediated plant host probing, which occurs when aphids land on a plant and attempt

to determine if it is a suitable plant host [210,226,227]. Infection by epiphytic bacteria

may occur during this process, where aphids repeatedly push their stylet through the host

tissue, pushing down any surface bacteria, and then ingesting those bacteria while

sampling plant fluids. Under this model, aphids acquire the pathogen during probing of

an epiphytically-colonized plant host, with the ingested bacteria subsequently colonizing

and propagating within the aphid. The bacteria escape from the aphid via the honeydew,

and are deposited back onto the plant surface where they are given an opportunity to

reassociate with their plant host. At this point, the aphid functions as a vector.

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To determine the amount of inoculum deposited on the plant surface by infected

aphids, Stavrinides et al. (2009) introduced infected aphids onto host plants, and bacteria

densities quantified following a feeding period. The phyllosphere was shown to be

inoculated with up to 2x107 phytopathogenic bacteria per square centimeter per aphid,

suggesting that the aphid is an excellent culturing vessel for this phytopathogen. They

propose that because honeydew is carbohydrate rich, the deposition of bacteria in a

suspension of nutrients may enable P. syringae to enhance its survival and perseverance

on the surface of the leaf. Certainly, because B728a is pathogenic to the aphid, successful

deposition onto the leaf would have to occur quickly, and before the death of the aphid.

In the case of DC3000, however, the bacteria do not kill the aphid, making this

association more consistent with a true vectoring relationship.

P. syringae shows a very high level of aggressiveness in the pea aphid, which

results in the death of the aphid in only a few days; however, since the bacteria have a

direct and continual route of escape from their host, it has the opportunity to replicate

maximally without the tradeoff of prematurely killing the host due to high

aggressiveness. Such an interaction provides the opportunity to study the dynamics of a

unique relationship between a phytopathogen and an insect that can be used not only as

an alternative primary host, but also as a vector that can provide an active dispersal

mechanism to other plant hosts (Figure 1).

Aphids and Insect Defenses

It is particularly interesting that many of the interactions between insects and

bacteria described above have involved the aphid – one of the most destructive

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agricultural insect pests[215]. These insects are able to incapacitate plants as sap feeders,

pollutive excreters, toxifiers, and as well indirectly as vectors of viral diseases [215].

Their close association with a variety of plants predisposes them to a diversity of

epiphytic and phytopathogenic bacteria [216]. Several studies have highlighted the

general affinity of the members of the Enterobacteriaceae for aphids, many of which

colonize the aphid gut [215,228]. So are aphids more susceptible to pathogen attack than

other insects?

Insects have evolved specific behaviours that allow them to avoid predation,

environmental stressors, and pathogens, but when these stressors bypass the defensive

behaviours, insects must rely on the physical defences, such as those provided by their

protective cuticle or gut pH level for defense [229,230,231,232,233]. If these barriers are

also breached, immunological defence mechanisms such as clotting, encapsulation,

phagocytosis, and the synthesis of antimicrobial substances come into play

[233,234,235]. Analysis of the recently sequenced genome of the pea aphid has revealed

that aphids do have defense mechanisms found universally in other arthropods, including

the JAK/STAT and Toll signalling pathways, which are involved in both development

and immunity; however, several essential genes involved in innate immunity of

arthropods are absent from the genome, including the IMD signalling pathway, c-type

lysozymes, defensins, and PGRPs. The absence of these genes may be due to an inability

to locate homologues due to the large evolutionary distance between aphids and the taxa

from where such genes are well studied [233], or due to aphids possessing an alternative,

yet equally effective immune response. There is little evidence for the latter. It was also

suggested that unlike Drosophila whose source of food is constantly contaminated with a

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diverse array of microbes, aphids would only encounter entomopathogens and bacteria in

the phloem sap of plants very rarely, therefore not requiring a more developed defense

arsenal [236]; however, through probing of plants, aphids have been shown to contact and

ingest a diversity of epiphytic bacteria, both pathogenic and non-pathogenic

[210,216,233]. Another possibility is that aphids invest in terminal reproduction when

faced with an immune challenge in contrast to spending extensive amounts of energy

attempting to defend themselves [233,236]. Indeed, stabbed aphids generated more

offspring than those who were untreated [236], although this is also seen in crickets,

[237], waterfleas [238], and snails [239,240], which appear to have more developed

immune systems [233]. Interestingly, the secondary endosymbionts such as Hamiltonella

defensa, which provides protection against the parasitoid wasp, Aphidius ervi, and

Regiella insecticola, which protects against fungal pathogens [233], persist within the

haemolymph and are detected and managed by the aphid immune system.

Evolution of alternative associations

Bacterial phytopathogens have been, up to now, considered just that – bacteria

that are capable of colonizing, reproducing, and disseminating from only plant hosts;

however, it is now very evident that these plant pathogenic bacteria have the ability to

exploit insects with which they share an overlapping niche as alternative primary hosts.

Many interesting questions arise from this including those relating to general ecology,

pathogenic potential, and host-specific virulence factors. For example, the ability of a

microbe to associate intimately with two hosts across two different kingdoms likely

compels an evolutionary struggle for the microbe, which must evolve host-specific

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strategies for associating with each of its hosts. A phytopathogen will undergo adaptation

of its overall aggressiveness toward its plant host in order to achieve its fitness optimum,

but this optimum may be different in the insect vector or host, requiring the pathogen to

achieve an intermediate multi-host optimum (Figure 2). The X. fastidiosa rpfF gene,

which is required for insect association causes a reduction in plant virulence [91,108],

illustrating that there are tradeoffs associated with multi-host associations.

Aside from the complexities of multi-host associations, the directionality of host

association is also an interesting paradigm. In the majority of interactions described

above, insects serve as either vectors or alternative hosts for phytopathogenic bacteria,

and in cases where the insect presently serves as a vector, the phytopathogen uses the

insect cavity as a transport vehicle for moving to its next plant host. But how did these

relationships develop? The association of phytopathogens and insects may have begun

with insects feeding transiently on plant tissues colonized by phytopathogens.

Internalized bacteria survived the conditions of the insect cavity as well as

immunological defenses to be dispersed successfully to a new host (Figure 3). The

reiteration of this process over evolutionary time would have selected for those bacterial

variants whose fitness increased as a result of this interaction – namely, those that were

capable of surviving in the insect, were less immunogenic, and/or had higher replication

and dispersal as a result of associating with the insect. This would have resulted in many

of the interactions that exist today between phytopathogens and their vectors; however,

did this association begin so pleasantly? Phytopathogens may have first evolved

entomopathogenicity and began colonizing insects as alternative hosts following

recurrent encounters over the course of evolution. These interactions may have then

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converted to a more benign association, where the entomopathogen became substantially

reduced in virulence, but capable of maintaining its association with specific insect hosts

long enough to ensure its dispersal. In any host-microbe relationship, the specific

tradeoffs endured by each partner will dictate the strength and overall success of the

association. In the associations where phytopathogens are transmitted by vectors, by

definition, there needs to be a very low cost to the insect for carrying the bacterium;

however, there may often be a slight cost to carriage that can destabilize the success of

the association [241].

It is interesting to note that many of the phytopathogens shown to have alternative

insect associations are in the Enterobacteriaceae [242] – a group that generally associates

with animal and insect hosts. So, did these phytopathogens evolve entomopathogenicity,

or were they, in fact, insect-associated microbes that evolved phytopathogenicity, but still

retain their ancestral insect-association lifestyle? If these bacteria were once insect-

associated, either entomopathogens or insect commensals, they may have evolved

phytopathogenic capabilities after repeated deposition on plants over evolutionary time

(Figure 4). An increase in fitness that result from repeated encounters with their own

insect hosts or other insects in the environment would have contributed to the

maintenance of the determinants necessary for insect association. Many of the enteric

plant pathogens described here seem to retain their ancestral gut-associating capabilities.

Phytopathogenic Enterobacteriaceae including, Erwinia, Dickeya, Serratia, and Pantoea,

retain relatively tight pathogenic and non-pathogenic associations with herbivorous- and

plant-associated insects [215,243,244,245]. For example, Erwinia amylovora is

pathogenic to the olive fly and Western flower thrips [243,246], but survives 12 days on

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aphids, and at least 5 days in association with the green lacewing [157], while D.

dadantii, P. stewartii, and E. aphidicola have been shown to be pathogenic to the pea

aphid, colonizing the gut and causing death [195,215,216]. Similarly, the colonization of

Drosophila by P. carotovorum results in a host defence response, characterized by the

production of antimicrobials [185]; however, bacterial persistence is enabled by the

bacterial gene, evf, which enhances survivorship of the bacteria in the gut by preventing

insect excretion [188]. This gene was suggested to be acquired recently through

horizontal gene transfer, possibly suggesting that insect persistence is an acquired and not

an ancestral capability. Certainly, there may be issues of host specificity that also come

into play, where there may be another true host of P. carotovorum in which the bacteria

can persist without the evf gene. In contrast, genomic comparisons of the enteric

phytopathogen Pectobacterium atrosepticum and several enteric animal pathogens

revealed the acquisition of many different plant-associated pathogenicity islands by P.

atrosepticum, including a T3SS, and genes for agglutination, adhesion, and phytotoxin

biosynthesis [211]. These islands share homology to genes from other plant-associated

bacteria, suggesting acquisition through horizontal gene transfer from phytopathogens.

The P. atrosepticum genome does not show obvious signatures of having undergone new

niche adaptation, suggesting that it has only gained new capabilities through incremental

gene loss and gain. The identification of interactions between these phytopathogens and

plant-associated insects could indicate that their ancestral gut associations have remained

an integral component of their lifecycle, and the evolution of plant pathogenicity may

have followed from frequent insect-mediated deposition on plants (Figure 4).

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In the association between P. syringae and the pea aphid, the strain B728a

exhibits pathogenicity toward the pea aphid, with infection resulting in aphid death in less

than 36 hours. The pathogen replicates in the aphid, and is then deposited onto the plant

via the aphid honeydew, making the aphid an efficient vector for the phytopathogen. In

most microbe-vector associations, the bacterium is not pathogenic toward the vector,

since this would reduce the likelihood of being transmitted to the next plant host;

however, since the aphid is already plant associated, the microbe can replicate maximally

without having to offset the cost of killing the host. In this somewhat atypical interaction,

the aphid can be used as both a primary host and vector (Figure 1), which raises

interesting questions about the evolutionary directionality of host association. Could this

interaction represent a transitionary state in the evolution of insect-microbe interactions,

where B728a began as being only vectored by the aphid, but not causing its death, and

gradually moved toward entomopathogenicity? Or is it attenuating in virulence as it is

becoming more adapted to the aphid, perhaps to a strict vectoring association? The ability

of the related tomato pathogen P. syringae pv. tomato DC3000 to replicate within, but not

cause death of the pea aphid, would suggest that B728a has moved toward

entomopathogenicity (Figure 5). The pea aphid is not known to feed on tomato plants,

and would therefore be unlikely to encounter DC3000, supporting the idea that the

entomopathogenicity of B728a is an acquired trait. This exciting prospect lends itself to

further exploration of this interaction, including the identification and characterization of

the specific genetic determinants required for this relationship. The analysis of the

genetics of this interaction is presently underway, and will yield important insight into the

evolution and ecological relevance of these alternative associations.

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Conclusions and future developments

Our knowledge of the general ecology of phytopathogenic bacteria has begun to

expand beyond their immediate interactions with plants, to encompass the other

ecological players in the environment. There is increasing evidence that plant pathogenic

bacteria have evolved specific and non-specific associations with insects, which they

exploit as delivery vehicles or as primary alternative hosts. Specific bacterial genetic

determinants have been identified that lend credence to the notion that these associations

are not incidental, but have evolved with recurrent encounters, followed by natural

selection. While many of these studies have provided an incredible wealth of information

on the genetics and pathology of bacterial association, their ecological relevance remains

ambiguous. It is certain, however, that a better understanding of phytopathogen

epidemiology will require a better understanding of the nature of specific interactions and

associations with other organisms in the environment.

Acknowledgements

We would like to thank Morgan Kirzinger and Taylor Duda for their helpful

comments and feedback on the manuscript. John Stavrinides is supported by a Discovery

Grant from the Natural Sciences and Engineering Council of Canada.

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Tables

Table 1. Properties of bacterial pathogens.

Pathogen Family Genome size

(MB) Plant hosts Insect hosts

Nature of insect

association

Plant pathogenicity

factors

Insect pathogenicity

factors

Candidatus Liberibacter Rhizobiaceae 1.2 Citrus Psyllid Vector - -

Dickeya dadantii Enterobacteriaceae 4.8 Potato, maize Pea aphid Host hrp/hrc cyt

Erwinia amylovora Enterobacteriaceae 3.8 Apple, pear Pollinating insects Vector amylovoran, levan,

hrp/hrc -

Erwinia aphidicola Enterobacteriaceae ~4 Bean, pea Pea aphid Host - -

Erwinia tracheiphila Enterobacteriaceae ~4 Cucumber, melon Cucumber beetle Vector - -

Pantoea stewartii Enterobacteriaceae 5.0 Maize Flea beetle, aphid Vector/ host hrp/wts ysa(ysaN), ucp1

Pectobacterium

carotovorum Enterobacteriaceae 5.0 Potato Fruit fly Vector expl/hor evf/hor

Pseudomonas syringae Pseudomonadaceae 5.6 Bean Pea aphid Vector/host hrp/hrc flilL

Serratia marcescens Xanthomonadaceae 4.6 Pumpkin, squash Squash bug Vector oxyR -

Xylella fastidiosa Enterobacteriaceae 2.1 Citrus, grape Sharpshooter,

spittlebug Vector rpfC rpfF

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Figures

Figure 1. The role of the aphid as both vector and alternative primary host for the plant

pathogen Pseudomonas syringae pv syringae B728a (B728a). Acquisition of B728a by

aphids occurs through feeding on plants colonized epiphytically by bacteria (yellow

dots). The plant pathogenic bacteria replicate within infected aphids, and are excreted in

globules of honeydew, which fall onto the plant surface. Infected aphids may wander to

other plant hosts, vectoring the bacteria in the process. Shortly after infection with B728a,

the host aphid, which has been used as a mass replication vessel by the bacteria,

succumbs to sepsis. Adapted from Stavrinides et al (2009). (original in color)

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Figure 2. A bacterium having multiple hosts must achieve a balance with both its hosts

to achieve optimal fitness. The lifecycles of some phytopathogens include insect vectors,

which function to transmit the bacteria to new plant hosts (middle circle). During their

association with the plant (top), phytopathogens utilize plant-specific strategies for

colonizing and causing disease in plant tissues, eventually reaching a virulence optimum

that maximizes its fitness. This optimum, however, may conflict with the strategies used

for colonizing, persisting, and being transmitted from the insect vector, necessitating a

balance that maximizes the fitness of the pathogen with both hosts. (original in color)

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Figure 3. Plant-first model. Phytopathogenic bacteria may have evolved alternative

associations with insects following either transient interactions with generalists (left),

which may move to an unsuitable plant host for the pathogen, or through interactions

with specialized insects that feed on a limited set or subset of plants that overlap with the

preferred hosts of the pathogen. (original in color)

113

Figure 4. Insect-first model. Present-day phytopathogens that associate with insects may

have had ancestral associations with insects, but following deposition onto plant hosts via

their plant-associating insect hosts, genetic exchange with other plant pathogens found

within the phyllosphere or rhizosphere may have led to the evolution of

phytopathogenicity. (original in color)

114

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Appendix E. Insights into cross-kingdom plant pathogenic bacteria.

Note: This appendix has been republished, with permission, from Kirzinger, M.W.B.,

Nadarasah, G., and Stavrinides, J. Insights into cross-kingdom plant pathogenic bacteria.

Genes. 2(4): 980-997.

Insights into cross-kingdom plant pathogenic bacteria

Morgan W. B. Kirzinger1, Geetanchaly Nadarasah

1, and John Stavrinides

Department of Biology, University of Regina, 3737 Wascana Parkway, Regina,

Saskatchewan, Canada, S4S0A2

1Equal Contribution

Corresponding Author:

John Stavrinides




S4S 0A2

Tel: (306) 337-8478

Fax: (306) 337-2410

[email protected]

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Abstract

Plant and human pathogens have evolved pathogenicity and virulence factors to

successfully exploit their respective hosts. Phytopathogens utilize specific determinants

that help to breach reinforced cell walls and manipulate plant physiology to facilitate the

disease process, while human pathogens use determinants for exploiting mammalian

physiology and overcoming highly developed adaptive immune responses. Emerging

research, however, has highlighted the ability of seemingly dedicated human and plant

pathogens to cause plant and human disease, respectively. Such microbes represent an

interesting paradigm in the evolution of cross-kingdom pathogenicity, and the benefits

and tradeoffs of exploiting multiple hosts with drastically different structures and

physiologies. This review will examine common phytopathogens that exhibit human

disease, and human pathogens that have phytopathogenic capabilities, with discussion on

the evolution of cross-kingdom pathogenicity. We illustrate that while cross-kingdom

pathogenicity appears to be maintained, the directionality of host association (plant to

human, or human to plant) is difficult to determine. Cross-kingdom pathogens have

important implications for the emergence of infectious diseases, and the role of plants as

potential pathogen reservoirs.

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Introduction

Plant pathogenic bacteria are responsible for major economical losses in

agricultural and farming industries worldwide, prompting massive research efforts to

understand their ecology, pathology and epidemiology. Studies of many agriculturally

relevant pathogens like Pseudomonas syringae, Xylella fastidiosa, Erwinia amylovora,

and Xanthomonas campestris [247,248,249,250] have revealed an extensive

specialization to plant systems, including a wealth of plant-specific pathogenicity

determinants such as type III secretion systems, plant hormone analogs, and enzymes that

target plant-specific cell wall components [247,251,252,253]. Despite this, we often

neglect the fact that the interactions of phytopathogenic bacteria are not confined to

plants, and may include other organisms in the environment. Recent studies have begun

to show that many plant pathogens have the capacity to colonize other hosts outside of

the plant kingdom, including insects, animals, and humans [254] [255].

Much like our perceptions of plant pathogens, human pathogens are studied

primarily for their detrimental impact on human health. Pathogens such as Escherichia

coli, Listeria monocytogenes, and Campylobacter jejuni possess a diverse set of genetic

factors that enable pathogenicity, ranging from specialized secretion systems to toxins

and adhesins, all of which are involved in manipulating or circumventing the human

immune system [256,257,258]. We often consider these human pathogenic bacteria to be

exclusive animal pathogens, causing disease and epidemics, yet the constant interaction

of human carriers with their environment predisposes these pathogens to alternative

niches, which includes free-living states, as well as the potential association with non-

human hosts. Not surprisingly, we have begun to discover that some animal pathogens,

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which are known to cause serious human diseases, also have plant pathogenic

capabilities. The epidemiology and disease strategies of these pathogens, whether

considered primarily a plant or human pathogen, are of particular interest from the

perspectives of both the biology and evolution of cross-kingdom pathogenesis.

The disease strategies used by cross-kingdom pathogens to infect unrelated hosts

are of particular interest since plant and animal hosts have distinctive physical barriers

and defense responses. While specialists may have evolved dedicated factors for

overcoming the physical barriers and innate defenses in a certain host, a cross-kingdom

pathogen would require a diverse library of genes and disease strategies to overcome the

specific obstacles of each of its hosts. This would either necessitate a suite of

pathogenicity factors to enable attachment, disease development, and dispersal for each

host, or a universal disease strategy in which similar subsets of pathogenicity factors are

used for all hosts. Although signatures of coevolution may not be as pronounced for

cross-kingdom pathogens, host adaptation will still proceed through incremental

nucleotide substitutions, genetic rearrangements, or acquisition of genetic elements

involved in virulence or host specificity [259]. The identification of these genetic

determinants in cross-kingdom pathogens with both human- and plant-pathogenic

potential can provide a better understanding of the evolution of phytopathogenicity, as

well as the role of plants as potential reservoirs for clinically relevant bacteria.

This review will examine various bacterial pathogens that exhibit cross-kingdom

pathogenicity, where humans and plants are both potential hosts. Pantoea, Burkholderia,

Salmonella, Serratia and Enterobacter, and Enterococcus are six genera that have

brought insight into cross-kingdom pathogenicity, and the versatility of bacteria (Table

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1). First, we examine the pathogenicity of Pantoea and Burkholderia, which are

commonly regarded as plant pathogens, but have been shown to cause human disease.

We then examine what is known about the disease strategies of the human pathogens

Salmonella, Serratia, Enterobacter and Enterococcus, which have been shown to cause

plant disease. We highlight what is known about the disease determinants and strategies

for each pathogen in each of its hosts, and identify those examples where specific

determinants are used in multiple hosts. Finally, we address the evolutionary means by

which plant pathogens may evolve to be pathogenic to humans, as well as the possible

routes of microbes that can lead to the evolution and maintenance of cross-kingdom

pathogenicity.

Plant pathogens that infect humans

Plant pathogens have evolved a repertoire of pathogenicity factors that are used to

invade plant host cells, facilitate disease development, and eventually promote pathogen

dispersal [260]. Surprisingly, some of these plant pathogens cause disease in humans, and

are frequently isolated from human infections in the nosocomial environment. The genus

Pantoea, for example, currently includes seven species (Pantoea agglomerans, Pantoea

ananatis, Pantoea citrea, Pantoea dispersa, Pantoea punctata, Pantoea stewartii, and

Pantoea terra) [261,262,263,264,265,266,267] that are known to cause plant disease. P.

agglomerans causes crown and root gall disease of gypsophila and beet [261,266], P.

ananatis causes bacterial blight and dieback of Eucalyptus [262], brown stalk rot of

maize [268], and stem necrosis of rice [269], and P. citrea is the causal agent of pink

disease in pineapple [267]. The type III secretion system (T3SS) appears to be an

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essential plant pathogenicity factor for many species of Pantoea, enabling efficient

colonization and onset of disease through the injection of bacterial effector proteins that

disrupt defense signalling in host cells [270]. Pantoea agglomerans pvs. gypsophilae and

betae are known for using a T3SS for causing galls on their host plants [271]. While P.

agglomerans pv. betae can induce gall formation on gypsophila and beet plants, P.

agglomerans pv. gypsophilae is only capable of causing gall formation on gypsophila.

Host range is determined in part by the type III effector protein, PthG, which is

recognized by the beet plant resulting in a defense response, but functions as a virulence

factor in gypsophila [271]. Studies with transgenic Nicotiana tabacum plants expressing

PthG suggest that the effector may interfere in the plant auxin signalling pathways,

resulting in higher observed auxin and ethylene levels, and subsequent blockage of root

and shoot development [271]. The manipulation of plant-specific hormones is likely

directly responsible for callus/gall formation, and highlights the specialization of this

pathogen to plant systems.

Despite this specialization to plants, species of Pantoea have also been discovered

to be pathogenic to humans. Now classified as an opportunistic human pathogen, P.

agglomerans was implicated in a large U.S. and Canadian outbreak of septicaemia caused

by contaminated closures on infusion fluid bottles [272]. P. agglomerans has since been

associated in the contamination of intravenous fluid, parenteral nutrition, blood products,

propofol, and transference tubes, causing illness and even death [273,274,275,276,277].

P. agglomerans has also been obtained from joint fluids of patients with synovitis,

osteomyelitis, and arthritis [263], where infection often occurs following injuries with

wood slivers, plant thorns, or wooden splinters [54,263,278,279]. This is also true for P.

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ananatis, P. septica, and P. dispersa, which are known for causing disease in onion and

sugar cane plants, but have also been implicated in bacteremic infections, septicaemia,

and leukemia, respectively [280,281,282]. Phylogenetic studies examining the

relationship between the plant isolates and the clinical isolates have shown that they are

indistinguishable, and their potential pathogenicity in plant and animal hosts is unclear

[47].

Much like Pantoea, species of Burkholderia are also recognized phytopathogens,

but are ubiquitous in the terrestrial and aquatic environment. Burkholderia species,

including Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia ambifaria, and

Burkholderia pyrrocinia were initially identified as inhabitants of agricultural soil, with

some, like Burkholderia glathei being found in fossil soils in Germany [283]. Given their

ubiquity in the terrestrial environment, it is not surprising that some strains, like

Burkholderia plantarii and Burkholderia gladioli have been shown to be pathogenic to

onion, rice, gladiolus and iris [283]. Similarly, Burkholderia glumae, the most important

bacterial pathogen of rice in Japan, Korea and Taiwan, was shown to carry a plant T3SS,

which is essential for its ability to cause plant disease [284]. An analysis of the proteins

regulated by HrpB, which activates the expression of T3SS genes, identified 34

extracellular proteins that were secreted, with 21 of 34 having putative HrpB-binding

sequences in their upstream regulatory regions [285]. Another set of 16 proteins were

recognized to be secreted via a type II protein secretion system (T2SS) [285]. Mutants

lacking either the T2SS or the T3SS produced toxoflavin, but were less virulent to rice

panicles, indicating the importance of these proteins in plant pathogenicity [285].

Likewise, Burkholderia pseudomallei possesses multiple T3SS gene clusters, one of

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which, T3SS2, showed high similarity to the T3SSs in phytopathogenic Xanthomonas

spp. and Ralstonia solanacearum [286]. Arabinose-negative strains of B. pseudomallei

are more virulent in tomato plants, and these strains appear to carry a T3SS, suggesting

that these two phenotypes are linked to virulence in plants [287]. Other species, like B.

cepacia, have strains that are specialized to plant roots and have been found in the maize

rhizosphere [288], while others are pathogenic to onion, and cause severe rots using a

cocktail of plant-specific enzymes following biofilm formation [289].

Interestingly, B. cepacia that causes onion rot can also cause life-treating

pulmonary infections in individuals with chronic granulomatous disease, or cystic

fibrosis (CF) [290]. Between 1980 and 1990, B. cepacia was associated with a number of

cases of “cepacia syndrome” in CF treatment centres and social gatherings, where CF

patients became infected by other B. cepacia-carrying individuals, with many of the

infections being fatal [283,291,292,293,294]. Recently, fatal infections were also

observed in non-CF patients in reanimation wards in Europe and North America [295].

Investigation into the ability of various strains of B. cepacia to penetrate airway barriers

revealed that all three of the B. cepacia strains tested circumvented the mechanical

barriers of mucus and ciliary transport to penetrate the airway epithelium [296]. Different

strains of B. cepacia use distinct invasion pathways and virulence determinants, which

may account for differences in virulence strains [296].

Several of the pathogenicity determinants for different species of Burkholderia in

human hosts have been determined. For example, B. pseudomallei, which infects tomato

plants, contains a complete pqsA–pqsE operon, which is highly similar to the genes

responsible for the synthesis of the virulence-associated signalling molecule 2-heptyl-3-

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hydroxy-4(1H)-quinolone (PQS) found in Pseudomonas aeruginosa [297]. Introduction

of the B. pseudomallei hhqA and hhqE genes into P. aeruginosa pqsA and pqsE mutants,

promoted the restoration of PQS production and virulence. Likewise, the presence of a

capsular polysaccharide has been implicated in the pathogenicity of Burkholderia toward

humans. Parental strains of B. mallei with the capsule were highly virulent in hamsters

and mice, while capsule mutants were avirulent in both animal models [298].

Human pathogens that infect plants

Our human-centric view of disease often leads to sweeping assumptions that

human pathogenic bacteria are devoted to human hosts. Species of Salmonella, Serratia,

Enterobacter, and Enterococcus are commonly considered problematic human pathogens

that are frequently found in the nosocomial environment, and which cause food

poisoning, general infections, and septicaemia [299,300,301,302] (Table 1). Relatively

recent studies, however, have begun to uncover that these human-pathogenic bacterial

species are also accomplished phytopathogens that are capable of causing disease in a

wide variety of plant hosts.

Salmonella is the causal agent of many human, animal, and bird diseases

worldwide, including gastroenteritis and typhoid fever, and results in approximately 1.4

million human illnesses and 600 deaths annually in the United States [300]. Successful

host infection by Salmonella appears dependent on many pathogenicity determinants,

including two T3SSs and a large suite of secreted effectors that function to mediate both

intercellular and intracellular survival in the host [303]. Other virulence factors include

the flhD gene, which regulates the production of the flagellum, is essential for the full

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invasive potential of the bacterium [304]. Other virulence factors like flavohemoglobin

protect against nitric oxides, and mediate bacterial survival in macrophages after

phagocytosis [305].

Despite its clear adaptation to survival in human hosts, Salmonella has also been

isolated from the phyllosphere of tomato crops [306]. S. enterica levels on wild tomato

(Solanum pimpinellifolium) were lower than on domesticated tomato cultivars (Solanum

lycopersicum), with S. enterica preferentially colonizing type 1 trichomes. Plants

irrigated with contaminated water had larger S. enterica populations than plants grown

from seeds planted in infected soil; however, both routes of contamination resulted in

detectable S. enterica populations in the phyllosphere, suggesting that S. enterica has the

ability to associate with plants and has adapted to survive in the phyllosphere. agfA and

agfB were identified as being involved in enabling S. enterica to associate with plants

[307]. agfA mutants were unaffected in their ability to attach or colonize alfalfa sprouts,

whereas agfB mutants showed reduced colonization ability. This suggests that agfB alone

plays a role in plant attachment [307].

Salmonella is not only capable of colonizing the phyllosphere; it is also capable of

infecting the plant Arabidopsis, and causing death of plant organs [308,309]. Inoculation

of Salmonella in Arabidopsis via shoot or root tissues resulted in chlorosis, wilting, and

eventually death of the infected tissues within seven days [308]. The specific virulence

factors involved are not yet known; however, its plant pathogenicity, much like its human

pathogenicity appears dependent, in part, on the flagellum. Fili, a protein needed for

flagellar assembly has been shown to be essential for plant pathogenesis, possibly

through its involvement in a specialized protein export pathway [310]. The Fili protein is

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similar to the HrpB6 protein of the rice pathogen Xanthomonas, which mediates

interactions between Xanthomonas and its host [310]. Secretion of proteins may therefore

be integral for both human and plant association for Salmonella, although whether the

same proteins are used for pathogenesis, is still unknown.

Serratia marcescens is a human pathogen commonly found in the respiratory and

urinary tracts of humans, and is responsible for approximately 1.4% of nosocomial

infections in the United States [301]. Through transposon mutagenesis, genes involved in

lipopolysaccharide (LPS) biosynthesis, iron uptake, and hemolysin production were

discovered to be essential for bacterial virulence in the host Caenorhabditis elegans

[311]. Further studies have shown that purified protease proteins of S. marcescens

administered to the lung tissue of guinea pigs and mice produced pneumonia symptoms

and haemorrhaging, which was similar to those animals with acute Serratia pneumonia

[312]. Yet, despite its animal virulence factors, Serratia has also been found to be a

common phytopathogen. S. marcescens is recognized as a phloem-resident pathogen that

causes cucurbit yellow vine disease of pumpkin (Cucurbita moschata L.) and squash

(Cucurbita pepo L.), which is marked by wilting, phloem discoloration, and yellowing of

foliage [124,125,126]. Recent studies have shown that S. marcescens produces a biofilm

along the sides of the phloem vessels, blocking the transport of nutrients and eventually

causing the plant to wilt and die [127,128]. A genetic screen to identify genes that

modulate biofilm formation in S. marcescens revealed the involvement of fimbrial genes,

as well as an oxyR homolog – a conserved bacterial transcription factor having a primary

role in oxidative stress response [129]. The involvement of these plant-specific genes in

human pathogenicity has not been determined.

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Another bacterium that has also shown cross-kingdom pathogenesis is the Gram-

negative bacterium Enterobacter cloacae, an important nosocomial pathogen responsible

for bacteremia, lower respiratory tract infections, skin and soft-tissue infections, as well

as urinary tract infections [299]. Recently, an E. cloacae infection of the bloodstream was

traced back to contaminated human albumin [313]. E. cloacae synthesize a Shiga-like

toxin II-related cytotoxin, which was implicated in an infant case of haemolytic-uremic

syndrome [314]. Studies have shown that the concentration of the outer membrane

protein (OmpX) produced by E. cloacae during infection influenced the ability of the

bacterium to invade rabbit intestinal tissue [315]. Overproduction of OmpX led to a 10-

fold increase in the invasiveness of E. cloacae in rabbit intestinal enterocytes in situ,

whereas the mutant and wildtype strain were unable to effectively invade the same tissue

[315]. Interestingly, OmpX shares a high amino acid similarity to the virulence proteins

PagC and Rck of Salmonella typhimurium as well as the virulence-associated Ail protein

of Yersinia enterocolitica. Although there is evidence that E. cloacae has evolved to

colonize the human host, it has also been identified as a plant pathogen of macadamia

(Macadamia integrifolia), and the causal agent of grey kernel disease [316]. The onset of

grey kernel disease affects not only the quality of the kernels produced by the plant, but

results in grey discoloration and a foul odour [316]. E. cloacae also causes bacterial soft

rot disease in dragon fruit (Hylocereus spp.) [264], bacterial leaf rot in Odontioda orchids

[317], and is also responsible for internal yellowing disease in papaya [316]. Again, the

specificity of the virulence factors used in each of these hosts is not known.

Cross-kingdom pathogenesis is not limited to Gram-negative bacteria.

Enterococci are part of the normal intestinal flora of humans and animals, but are also

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important pathogens responsible for serious infections, especially in

immunocompromised patients [302]. With increasing antibiotic resistance, enterococci

are recognized as nosocomial pathogens that can be challenging to treat. The genus

Enterococcus includes more than 17 species, but only a few can cause local or systemic

clinical infections including urinary tract and abdominal infections, wound infections,

bacteremia, and endocarditis [318]. Clinical isolates of Enterococcus faecalis have been

found to produce hemolysin – a virulence factor that leads to the lysis of red blood cells

[319]. Hemolytic strains exhibit multiple drug resistance more frequently than non-

hemolytic strains, while strains isolated from fecal specimens of healthy individuals

display a low (17%) incidence of hemolysin production [319]. The salB gene of E.

faecalis has also been shown to be a virulence factor, since it increases bacterial

adherence to extracellular matrix (ECM) proteins and promotes biofilm formation during

infection [320]. The surface protein Esp was shown to contribute to the colonization and

persistence of the bacterium in the urinary tract [321]. E. faecalis has also been shown to

exhibit pathogenicity toward insects in addition to human hosts, where the extracellular

gelatinase of E. faecalis was found to destroy the host defence system through the

degradation of inducible antimicrobial peptides in insect hemolymph and in human serum

[322]. Similarly, a putative quorum-sensing system gene (fsrB) and a serine protease

(sprE) were shown to play an important role in mammalian and nematode models of

infection [302].

E. faecalis is not only capable of infecting mammalian and nematode hosts; it also

can infect the leaves and root tissue of Arabidopsis thaliana, causing plant death seven

days after inoculation [302]. The manifestation of disease initially begins once the

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bacterium has successfully attached itself to the leaf surface, where upon entry of the leaf

tissue through the stomata or wounds, E. faecalis multiplies and colonizes the

intercellular spaces of the plant host and causes rotting and disruption of the plant cell

wall and membrane structures. The phytopathogenicity of E. faecalis appears to involve

some of the same genetic determinants involved in animal pathogenesis, including the

quorum sensing system (fsrB) and a serine protease (sprE) [302]. The quorum-sensing

mutant (ΔfsrB) exhibited an attenuated virulence in the A. thaliana root model, since

fewer bacteria were able to attach to the root surfaces, and biofilm formation was greatly

reduced. Similarly, the serine protease mutant (ΔsprE) no longer caused plant death,

suggesting that this gene plays an important role in E. faecalis plant pathogenesis [302].

E. faecalis clearly uses a general disease strategy that allows it to use the same virulence

factors for exploiting two different hosts.

Evolutionary models

Cross-kingdom pathogenicity represents an intriguing balance between the costs

and benefits of bacterial generalization versus specialization. While there are benefits to

specializing on one host, and evolving to exploit its subtle vulnerabilities, the availability

of this host would limit the success of the pathogen, thereby favouring host

diversification. Conversely, a broad host range would ensure host availability for the

pathogen, but at the significant cost of having to utilize a universal, and likely suboptimal

infection strategy for exploiting that host. When the alternative host is from a different

kingdom, the dynamics become even more intriguing, since the disease strategy is likely

to be quite different. The evolution of cross-kingdom pathogenicity and its origins can be

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described using several simplistic models. In the context of the examples described here,

one model describes the evolution of a successful animal pathogen that acquires the

ability to cause disease in a plant host (human to plant). If we presuppose that human

pathogens are readily acquired from the environment and are already adapted to

mammalian hosts [323], there are several ways in which these animal pathogens may

acquire plant-pathogenic potential. Deposition of animal pathogens back into the

environment, such that they are introduced near or onto plants recurrently may facilitate

the exchange genetic information with other organisms in the environment, and the gain

of a selective advantage that enables persistence. One primary route to this general

environment is through the natural strategies used by bacteria to ensure dissemination

from their infected host. Symptoms such as diarrhea that result from an infection provide

the bacteria with an escape route from the human host into the environment [324] (Figure

1). Released bacteria move from human wastes to natural watersheds, with subsequent

reuse of these sources for irrigation of commercially relevant crops increasing bacterial

titres in the phyllosphere [308,309]. But, the movement of human pathogens to the

general environment may also occur through indirect routes. The Pharoah ant

(Monomorium pharaonis) is able to transport human pathogenic bacteria including

Salmonella, Staphylococcus, and Streptococcus within a nosocomial setting [325], and

there have been reports of the common house fly, Musca domestica, carrying

Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus [326]

(Figure 1). These insects function as transports for human pathogenic bacteria, moving

them from the clinical setting to the general environment [255], and likely onto plants.

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Acclimation to a plant host would occur over extended periods of time, and would be

accelerated with repeated cycling of pathogens from humans to the general environment.

The second model that can account for the evolution of the cross-kingdom

pathogens has plant pathogenicity as the ancestral state [327], with established plant

pathogens having evolved the ability to exploit humans as alternative hosts. The simplest

mode of transmission is direct contact with an epiphytically colonized or diseased plant

by humans, whether by ingestion, contact with the skin or other mucosal membranes,

scrapes or abrasions, providing the bacteria with a direct route to a potential host. Several

plant pathogens that cause opportunistic human infections are associated with

commercially relevant crop plants. Species of Pantoea are found on beets [261,266],

maize [268], rice [269], and pineapple [267]. In the case of Pantoea, human infections

occur frequently following abrasions caused by rose thorns, splinters, and arthritis,

suggesting that these plant-associated strains can lead to human infection directly (Figure

1) [54,263,278,279]. Similarly, Burkholderia infects various plant species including

onion, rice, sorghum and velvet beans [283,291,328], and may also have a direct route to

humans. Indirect transfer to humans or even to the nosocomial environment may be

mediated by other organisms like ants and flies, which have been implicated as carriers of

many bacterial species [325,326], and may provide environmental isolates with a direct

route to other environments and hosts.

Although there is evidence to support both models of evolution, it is difficult to

establish evolutionary directionality for each pathogen. The ability of human pathogens

to exploit plants as alternative hosts is of particular significance from the perspective of

host jumps and host-specific adaptation, since plants would ultimately serve as an

144

extensive reservoir for clinically relevant bacteria. Some plant endophytes, which are

organisms that live inside of plants often asymptomatically, have been shown to exhibit

human pathogenic potential. Species like Morganella morganii, Klebsiella pneumoniae,

Pantoea agglomerans and Streptomyces sp., have clinical relevance, but have been more

closely investigated for their association with plants [329,330]. M. morganii is a

phosphate solubilising bacterium [331], K. pneumoniae a nitrogen-fixing endophyte

[332], P. agglomerans a gall-forming phytopathogen, and Streptomyces sp. a plant

endophyte and plant pathogen [333,334,335]. Many of these animal-pathogenic

endophytes have been shown to be latent plant pathogens, where after establishing a

symbiotic mutualistic relationship with their plant host, the bacterium infects and causes

severe disease within the plant host possibly due to environmental changes [336]. It has

also been observed that an organism that is an endophyte on one plant species may have

the potential to cause disease in a different plant species [337]. Yet, many human

pathogens have been found to occur naturally in the rhizosphere [329], and it has been

suggested that the mechanisms necessary for surviving in the rhizosphere are similar to

those necessary for causing human infection [2]. Contributing to the maintenance of these

cross-kingdom pathogens may be anthropogenic factors. Certain species with human and

plant pathogenic potential, such as Pantoea and Burkholderia cepacia are also commonly

used as biocontrol agents against Erwinia amylovora and Rhizoctonia solani, respectively

[338,339]. The extensive use of these bacteria as biocontrol agents on commercially

relevant crops can lead to broad dispersal in the environment, increasing their populations

dramatically and providing them with greater opportunity for interaction with other

microbes. Horizontal gene transfer with other plant and human pathogenic bacteria in the

145

environment can lead to extensive exchange of host-specific virulence factors or niche-

specific determinants, creating rapidly-evolving populations that may exhibit oscillations

between host-associative and free-living states.

Conclusion

Advances in molecular genetics coupled with the exploration of the pathogenic

potential of seemingly dedicated pathogens is beginning to reveal that many

phytopathogenic bacteria are capable of exploiting human hosts, and many human

pathogens are capable of exploiting plant hosts. The constant cycling of pathogens from

the general environment to human environments may help to maintain cross-kingdom

pathogenicity, with plants serving as intermediate hosts or reservoirs for human

pathogens. The ability of these cross-kingdom pathogens to maintain their population

levels in a variety of environments increases their pan genome and evolutionary potential,

ultimately making these pathogens significant from the perspective of emerging and

remerging infectious diseases.

Acknowledgements

We would like to thank Taylor Duda, April Sefton, and Lucas Robinson, and

Kollin Schmalenberg for their helpful suggestions and comments. J.S. is supported by

funding from the NSERC Discovery Grant Program, and the Faculty of Science at the

University of Regina.

146

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Figures

Figure 1. Cycling of cross-kingdom pathogens between plants and humans. Movement

of human-associated bacteria (Salmonella, Serratia, Enterobacter, Enterococcus) into the

general environment may occur through wastewater, with additional microbial input

coming from agricultural and livestock runoff. Irrigation using contaminated water,

along with vectoring by insects can lead to inoculation of plant surfaces or plant soils.

Movement of potential human pathogenic bacteria from plants is also likely facilitated by

insects, which can disperse bacteria into the general environment. The route back to

humans is less clear, although some pathogens like Burkholderia and Pantoea can cause

direct infections following cutaneous lesions or injuries from thorns or splinters. (original

in color)

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Figure 1

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