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BACTERIAL ENDOSYMBIONTS OF ENDOPHYTIC FUNGI: DIVERSITY,
PHYLOGENETIC STRUCTURE, AND BIOTIC INTERACTIONS
by
Michele Therese Hoffman
_______________________
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF PLANT SCIENCES
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
WITH A MAJOR IN PLANT PATHOLOGY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2010
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THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation
prepared by Michele T. Hoffman
entitled "Bacterial endosymbionts of endophytic fungi: diversity, phylogenetic structure,
and biotic interactions.”
and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of Philosophy.
A. Elizabeth Arnold
_______________________________________________________ Date:
4/21/10
Judith L. Bronstein ________________________________________________________ Date: 4/21/10
Marc J. Orbach
___________________________________________________________
Date: 4/21/10
Final approval and acceptance of this dissertation is contingent upon the candidate’s
submission of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and
recommend that it be accepted as fulfilling the dissertation requirement.
A. Elizabeth Arnold ____________________________________ Date: 4/21/10
Dissertation Director
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STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an
advanced degree at the University of Arizona and is deposited in the University Library
to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided
that accurate acknowledgement of source is made. Requests for permission for extended
quotation from or reproduction of this manuscript in whole or in part may be granted by
the head of the major department or the Dean of the Graduate College when in his or her
judgment the proposed use of the material is in the interests of scholarship. In all other
instances, however, permission must be obtained from the author.
SIGNED: Michele T. Hoffman
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ACKNOWLEDGMENTS
This research would not have been possible without the assistance of many wonderful
individuals. I wish to first thank my advisor A. Elizabeth (Betsy) Arnold for her support
and guidance these past years. I am forever grateful to have had this opportunity to
participate in this outstanding research program. I appreciated that I was given the
freedom to explore my own research questions while being rigorously challenged as a
scientist. I am also most grateful to the members of my committee, Judie Bronstein for
your thoughtful questions and feedback, Marc Orbach for helping me troubleshoot
research problems and Sandy Pierson, for all things prokaryotic. I have appreciated all
your time, guidance and patience.
I want to thank the School of Plant Sciences and the IGERT program for funding
and the staff members in Plant Pathology and Microbiology for your kindness and good
will.
Numerous friends deserve my thanks and gratitude for their support, cheerful
spirit, and camaraderie. I wish to especially thank Cara Gibson and members of her
phylogenetics lunch club, Jeff Oliver and Chris Schmidt, along with fellow graduate
students and faculty: Fabiola Santos Rodriguez, Anne Estes, David Maddison, Randy
Ryan, David Bentley, Periasamy (Ravi) Chitrampalam, Scott Kroken, Mary Jane Epps,
Jana U’ren, Rhodesia Celoy, Mariana Del Olmo Ruiz, David Jarvis, Gerard White,
Baomin Wang, Mary Shimabukuro, Dylan Grippi, Andrea Woodard, Jacob Russell, Eric
McDowell, and Bridget Barker.
I could not have accomplished this work without the help and guidance of Mali
Gunatilaka. You are a special soul Mali and I owe you one million thanks! Finally, I
wish to thank my incredible friends and family for their love and support.
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DEDICATION
This journey represents the culmination of many years of good intentions, perseverance,
and the unwavering belief that dreams can and do eventually come true. I dedicate this
work to Edie, the person who encouraged me to dream.
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TABLE OF CONTENTS
ABSTRACT.........................................................................................................................7
INTRODUCTION...............................................................................................................9
1.1 Literature review.……………………………….…..................................................9
1.2 Explanation of dissertation format………………………………………………...15
PRESENT STUDY............................................................................................................17
2.1 Geographic locality and host identity shape fungal endophyte communities in
cupressaceous trees……………………………………………………………………....17
2.2 Diverse bacterial endosymbionts inhabit living hyphae of phylogenetically
diverse fungal endophytes …………………………………………………………...….18
2.3 IAA production by endophytic Pestalotiopsis neglecta is enhanced by an
endohyphal bacterium .…………………………………..…………...……………....…18
REFERENCES…………………………………………………………………………..19
APPENDIX A: GEOGRAPHIC LOCALITY AND HOST INDENTITY SHAPE
FUNGAL ENDOPHYTE COMMUNITIES IN CUPRESSACEOUS TREES...............24
APPENDIX B: DIVERSE BACTERIAL ENDOSYMBIONTS INHABIT LIVING
HYPHAE OF PHYLOGENETICALLY DIVERSE FUNGAL ENDOPHYTES ….….49
APPENDIX C: IAA PRODUCTION BY ENDOPHYTIC PESTALOTIOPSIS
NEGLECTA IS ENHANCED BY AN ENDOHYPHAL BACTERIUM..…………….99
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ABSTRACT
This dissertation comprises a series of studies designed to explore the associations
between plants and the endophytic fungi they harbor in their above-ground tissues. By
viewing endophyte diversity in ecologically and economically important hosts through
the lenses of phylogenetic biology, microbiology, and biotechnology, this body of work
links plant ecology with newly discovered symbiotic units comprised of endophytic fungi
and the bacteria that inhabit them.
This work begins with a large-scale survey of endophytic fungi from native and
non-native Cupressaceae in Arizona and North Carolina. After isolating over 400 strains
of endophytes, I inferred the evolutionary relationships among these fungi using both
Bayesian and parsimony analyses. In addition to showing that native and introduced
plants contained different endophytes, I found that the endophytes themselves harbor
additional microbial symbionts, recovering members of the beta- and gammaproteobacterial orders Burkholderiales, Xanthomonadales, and Enterobacteriales and
numerous novel, previously uncultured bacteria. This work finds that phylogenetically
diverse bacterial endosymbionts occur within living hyphae of multiple major lineages of
ascomycetous endophytes.
A focus on 29 fungal/bacterial associations revealed that bacterial and fungal
phylogenies are incongruent with each other and did not reflect the phylogenetic
relationships of host plants. Instead, both endophyte and bacterial assemblages were
strongly structured by geography, consistent with local horizontal transmission.
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Endophytes could be cured of their bacterial endosymbionts using antibiotics, providing a
tractable experimental system for comparisons of growth and metabolite production
under varying conditions. Studies of seven focal fungal/bacterial pairs showed that
bacteria could significantly alter growth of fungi at different nutrient and temperature
levels in vitro, and that different members of the same bacterial lineages interact with
different fungi in different ways.
Focusing on one isolate, I then describe for the first time the production of indole3-acetic acid (IAA) by a non-pathogenic, foliar endophytic fungus (Pestalotiopsis
neglecta), suggesting a potential benefit to the host plant harboring this fungus. I show
that this fungus is inhabited by an endohyphal bacterium (Luteibacter sp.) and
demonstrate that mycelium containing this bacterium produces significantly more IAA in
vitro than the fungus alone. I predict that the general biochemical pathway used by the
fungal-endohyphal complex is L-tryptophan-dependent and measure effects of IAA
production in vivo, focusing on root and shoot growth in tomato seedlings.
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INTRODUCTION
1.1 Literature review
1.1.1 Fungal endophytes
All lineages of land plants surveyed to date harbor living fungi within their healthy,
aboveground tissues (e.g., Carroll, 1988; Petrini, 1996; Davis et al., 2003; Cohen, 2004;
Arnold & Lutzoni, 2007; Kauserud et al., 2008; Arnold et al., 2009). These endophytic
fungi colonize and persist within living tissues such as leaves and stems without causing
overt symptoms of disease (Petrini, 1991; Stone et al., 2000). Fungal endophytes are
highly diverse at the species level (Carroll, 1995; Fröhlich & Hyde, 1999; Lu et al., 2004;
Rodrigues et al., 2004; Arnold, 2007), represent every major lineage of non-lichenized
Pezizomycotina (Higgins et al., 2007; Arnold, 2008), and have arisen multiple times in
the evolution of Fungi (Spatafora et al., 2006; Arnold et al., 2009). In life history they
range from the systemic, vertically transmitted clavicipitaceous endophytes that have coevolved with their cool-season grass hosts (Class 1 endophytes, Rodriguez et al., 2009) to
highly localized, horizontally transmitted endophytes that associate at various levels of
specificity with all major lineages of plants (Class 3 endophytes, Rodriguez et al., 2009).
Among these, the horizontally transmitted, phylogenetically diverse Class 3 endophytes
associated with foliage of long-lived perennial trees are of special interest: individual
trees concurrently harbor dozens to hundreds of endophyte species within their
asymptomatic photosynthetic tissues (Lodge et al., 1996; Müller & Hallaksela, 1998;
Arnold et al., 2003; Göre & Bucak, 2007), raising questions regarding the nature of their
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interactions with hosts. These interactions appear to range from latent pathogenicity to
latent saprotrophy, and encompass a variety of defensive mutualisms and physiological
costs and benefits (Arnold, 2007). Such interactions change frequently over evolutionary
time and may be variable and unpredictable in ecological time frames as well (Arnold,
2008; Arnold et al., 2009).
The mechanisms driving variation in such interactions provide the context for my
dissertation research. Whether variable outcomes reflect flexible genomic architecture in
endophytes or their plant hosts, sensitivity of the interaction or either partner to biotic or
abiotic stress, or additional microbial participants in species interactions is not yet clear.
In this dissertation, I have focused on the third area, with special attention to the
previously unexplored roles of endohyphal bacteria in shaping plant-endophyte
interactions.
1.1.2 Endohyphal bacterial symbionts
In addition to interacting with environmental and ectosymbiotic bacteria, some plantassociated fungi harbor bacteria within their hyphae (first noted as ‘bacteria-like
organisms’ of unknown function by MacDonald and Chandler, 1981). These bacteria,
best known from living hyphae of several species of Glomeromycota and
Mucoromycotina, can alter the fungal phenotype (see Bianciotto et al., 1996; Bertaux et
al., 2003; Bidartondo et al., 2004; de Boer et al., 2005; Waller et al., 2005; Shefferson et
al., 2005). For example, Candidatus Glomeribacter gigasporarum colonizes spores and
hyphae of the arbuscular mycorrhizal (AM) fungus Gigaspora gigasporarum (Bianciotto
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et al., 2000; Levy et al., 2003; Lumini et al., 2007). When cured of the bacterium, fungal
growth and development are suppressed, and the morphology of the cell wall, vacuoles,
and lipid bodies changes noticeably. Recovery of a P-transporter operon in Burkholderia
sp. from Gigaspora margarita (Ruiz-Lozano & Bonfante, 1999), and the discovery of
phosphate-solubilizing bacteria within Glomus mossae spores (Mirabal-Alonso & OrtegaDelgado, 2007), suggest a competitive role in phosphate acquisition and transport by
these bacteria within the AM symbiosis. Recently, Partida-Martinez and Hertweck (2005)
reported that a plant pathogen, Rhizopus microsporus (Mucoromycotina), harbors an
endosymbiotic Burkholderia sp. that produces a phytotoxin (rhizoxin) responsible for the
pathogenicity of the fungus. Each of these suggests a pervasive role of these endohyphal
bacteria not only on their fungal hosts, but on plant-fungus interactions as well. To date,
however, endohyphal bacteria have been studied only in rhizosphere-associated or plantpathogenic fungi. These studies have not encompassed some of the most species-rich
fungal lineages, nor the ubiquitous, avirulent endophytic fungi found in above-ground
plant tissues.
1.1.3 Endohyphal bacteria: hidden drivers of plant-endophyte interactions?
In this dissertation, I demonstrate that foliar endophytes harbor diverse, apparently
horizontally acquired, and facultative bacterial endosymbionts, and provide the first
evidence for their effects on foliar endophytes and plant-endophyte interactions. I have
focused on host plants representing the Cupressaceae (cedars and cypresses), an
economically and ecologically important family of conifers. The family comprises 130-
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140 species of trees and shrubs in approximately 27-30 genera and encompasses the
widest geographic range of any conifer family, with species ranging from 70°N to 55°S.
Well-known species include coastal redwood (Sequoia sempervirens), giant sequoia
(Sequoiadendron giganteum), and bald cypress (Taxodium spp.). In the mid-elevation
woodlands and forests of Arizona and much of the intermountain west, Cupressaceae
form the dominant component of tree communities. Species of Juniperus and Cupressus
are especially common both naturally and as ornamentals, with species such as
Platycladus orientalis (Oriental arbor-vitae) commonly planted as well. Endophytic fungi
have been examined in a small number of cupressaceous hosts previously (Carroll 1978,
1995; Camacho et al., 1997; Deckert, 2000; Hata, 1996; Arnold et al., 2007), but never in
an explicit geographic context, nor with the geographic breadth and depth of the present
work.
I first address the degree to which endophytic fungal associates of trees in the
Cupressaceae differ across the geographic ranges of hosts. In this work I examine the role
of native vs. non-native status in shaping endophyte communities as well. My goal is to
understand the intrinsic variation in fungal communities encountered and acquired by
plants in different geographic areas. Using both genotype-level and phylogenetic
analyses, I show that (1) conspecific trees in widely separated geographic areas
(southwestern vs. southeastern US) harbor different endophyte communities, and (2) that
native and introduced trees in the same geographic areas differ in their endophyte
assemblages. That study yielded a tremendous diversity of previously unknown
endophytic fungi and a new understanding of their phylogenetic relationships.
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Next, I examined the potential for such endophytic fungi to harbor endohyphal
bacteria. For this work, I concentrated on fungi associated with healthy foliage of
Cupressaceae at a smaller geographic scale, with a particular focus on exploring fungal
communities associated with plants on multiple mountain ranges within Arizona. After
extracting total genomic DNA from several axenic endophyte cultures, I discovered the
presence of measurable bacterial DNA within those DNA samples. After ruling out
contamination (Hoffman and Arnold, 2010), I sequenced the 16S ribosomal DNA region
of representative strains and recovered members of the beta- and gamma-proteobacterial
orders Burkholderiales, Xanthomonadales, and Enterobacteriales, including putative
Ralstonia pickettii, Variovorax paradoxus, Luteibacter rhizovicina, Pantoea spp., and
numerous novel, previously uncultured bacteria. I localized these bacteria within hyphae
and confirmed the viability of both the bacteria and the hyphae harboring them using
Molecular Probes Live-Dead stain. Phylogenetic analyses of fungi and their associated
bacteria then provided strong evidence that bacteria can occur within endophytic
members of four classes of Ascomycota. My work suggests that these are facultative
associates of their fungal hosts, and highlights the lack of congruence among bacterial,
fungal, and host plant phylogenies.
After developing methods to cure fungi of their endohyphal bacteria, I examined
the effects of bacterial endosymbionts on endophytic growth rates in vitro. Specifically,
by cultivating fungi on growth media containing the antibiotic ciprofloxacin, I produced
fungal cultivars that were confirmed by microscopy and PCR to lack endohyphal bacteria
(Hoffman and Arnold, 2010). By comparing the growth of replicate clones with and
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without bacteria on various media, at different temperatures and at different pH levels, I
found that these bacteria had variable and sometimes unpredictable effects. One strain,
9143, proved especially interesting because (1) I could identify the both the fungus and
bacterium with reasonable certainty using morphological and phylogenetic methods, (2) I
could separate the fungal and bacterial partners in vitro, and (3) this particular genus
(Pestalotiopsis) is well known for its production of numerous medicinally important
secondary metabolites.
The ability to synthesize phytohormones, including indole-3-acetic acid (IAA),
gibberellins, ethylene, cytokinins, jasmonic acid and abscisic acid, have been documented
in numerous bacteria and fungi (Costacurta and Vanderleyden, 1995; Glick, 1995;
Lindow et al., 1998; Robinson et al., 1998; Koga, 1995; Maor et al., 2004; Sirrenberg et
al., 2007). Some of these microorganisms utilize a L-tryptophan-dependent pathway for
IAA production, whereas others utilize a L-tryptophan independent pathway (Tudzynski
and Sharon, 2002; Buchanan et al., 2005). My investigation of IAA production in
infected endophytes found that some isolates were capable of IAA production when
supplied with L-tryptophan and that the infected endophytes produced significantly
higher levels of IAA than the cured counterpart, under identical growth conditions.
Culture filtrate from the endophyte-bacterium complex significantly increased root
growth in vitro in seedlings of tomato relative both to controls and to filtrate from the
endophyte alone. These data suggest that this newly described microbial partnership can
manipulate plant biochemistry with a positive effect.
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1.2 Explanation of dissertation format
The goals of this dissertation are to explore the interactions of endophytic fungi with
other microorganisms and their plant hosts, and to expand our knowledge of endophyte
diversity, community structure, and ecological roles in nature. I introduce this
dissertation research, methods and results as three appendices.
In Appendix A, I examine the abundance, diversity, and species composition of
endophytic fungi in pairs of related species of Cupressaceae from a mesic forest (central
North Carolina, USA) and from a xeric desert environment (southern Arizona, USA),
Utilizing a culture-based approach, I examined the fungal communities of two native
species (Cupressus arizonica, AZ and Juniperus virginiana, NC) and a co-occurring,
non-native species (Platycladus orientalis in both AZ and NC). My results lay the
groundwork for addressing relative importance of locality, host species, and native/nonnative status in endophyte community structure.
In Appendix B, I examine evidence of endohyphal bacteria associated with these
fungal endophytes and began to elucidate the infection frequency, taxonomy, and
phylogenetic relationships of these cryptic microorganisms. This work reports, for the
first time, the occurrence of endohyphal bacteria in foliar endophytes and highlights their
occurrence in four classes of Pezizomycotina. I recover novel lineages of endohyphal
bacteria relative to previous studies and suggest that the association of these bacteria and
their endophytic hosts is facultative, reflecting (1) loss of bacterial endosymbionts in
culture over time, and (2) incongruent fungal- and bacterial phylogenies. These analyses
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set the stage for experimental work addressing the ecological importance of these
previously unknown symbionts.
The data presented in Appendix C are the results of in vitro and in planta
experiments, conducted in collaboration with Malkanthi K. Gunatilaka, E. M. Kithsiri
Wijeratne, and A. A. Leslie Gunatilaka, measuring phytohormone production in a focal
endophytic fungus. This work demonstrates, for the first time, the ability of a foliar
endophyte to produce indole-3-acetic-acid (IAA), a phytohormone with strong effects on
plant cell growth and elongation. Moreover, we provide the first documentation of a role
of an endohyphal bacterium in enhancing IAA production, and demonstrate a strong
effect on seedling growth. In general, this work begins to answer the question: how do
bacterial endosymbionts influence the outcome of plant-fungal symbioses?
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PRESENT STUDY
The methods, results and conclusions from this research are presented in the
appended manuscripts. The following is a summary of the most important findings from
each.
2.1
Geographic locality and host identity shape fungal endophyte communities in
cupressaceous trees
Fungal endophytes, found in every major lineage of the most species-rich group of fungi
(Ascomycota), are believed to represent a significant proportion of Earth’s fungal
diversity. Using 95% sequence similarity as a proxy for species recognition, I report a
high level of species richness in three species of Cupressaceae, an economically and
ecologically important family of conifers. In addition to isolating over 400 strains of
endophytes, I inferred the evolutionary relationships among these fungi for the first time
using both Bayesian and parsimony analyses. Analyses of the nuclear ribosomal internal
transcribed spacer regions (ITS rDNA) for 109 representative isolates showed that
diversity was two times greater in plants from North Carolina vs. Arizona, and that
endophytes recovered in Arizona were more likely to be host-generalists relative to those
in North Carolina. Overall abundance, diversity, and taxonomic composition of these
endophyte communities differed as a function of host identity, native vs. non-native
status, and locality.
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2.2
Diverse bacterial endosymbionts inhabit living hyphae of phylogenetically
diverse fungal endophytes
Here I report that phylogenetically diverse bacteria occur within living hyphae of
endophytic fungi, including members of multiple major lineages of Ascomycota.
Drawing from surveys of endophytes in healthy photosynthetic tissues of ecologically
and economically important trees in five biogeographic provinces, I show that bacterial
and fungal phylogenies are not congruent and do not reflect the evolutionary relationships
of the host plants they both inhabit. Instead, both endophyte and bacterial endosymbiont
species appear to be horizontally transmitted and are structured more by geography than
by host identity.
2.3 IAA production by endophytic Pestalotiopsis neglecta is enhanced by an
endohyphal bacterium
In this section, I demonstrate the effects of a representative endohyphal bacterium,
identified using phylogenetic analyses as Luteibacter sp., on growth rates of endophytic
Pestalotiopsis neglecta under various nutrient, temperature and pH conditions. I show
that this fungal endophyte is capable of producing the phytohormone indole-3-acetic acid
(IAA) in vitro. I report a significant difference in production of IAA for the infected vs.
cured and that this same compound could positively influence the growth rate of tomato
seedlings.
19
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24
APPENDIX A
GEOGRAPHIC LOCALITY AND HOST INDENTITY SHAPE FUNGAL
ENDOPHYTE COMMUNITIES IN CUPRESSACEOUS TREES
25
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Dear Sir or Madam,
I wish to obtain permission from your journal to reprint the following publication as part
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Michele Hoffman
Hoffman, M. T. and A. E. Arnold. 2008. Geographic locality and host identity shape
fungal endophyte communities in cupressaceous trees. Myco. Res. 112:331â344.
mycological research 112 (2008) 331–344
26
journal homepage: www.elsevier.com/locate/mycres
Geographic locality and host identity shape fungal
endophyte communities in cupressaceous trees
Michele T. HOFFMAN, A. Elizabeth ARNOLD*
Division of Plant Pathology and Microbiology, Department of Plant Sciences, 1140 East South Campus Drive,
University of Arizona, Tucson, AZ 85721, USA
article info
abstract
Article history:
Understanding how fungal endophyte communities differ in abundance, diversity,
Received 13 March 2006
taxonomic composition, and host affinity over the geographic ranges of their hosts is key
Received in revised form
to understanding the ecology and evolutionary context of endophyte–plant associations.
28 August 2007
We examined endophytes associated with healthy photosynthetic tissues of three closely
Accepted 24 October 2007
related tree species in the Cupressaceae (Coniferales): two native species within their natural
Corresponding Editor: Barbara Schulz
ranges [Juniperus virginiana in a mesic semideciduous forest, North Carolina (NC); Cupressus
arizonica, under xeric conditions, Arizona (AZ)], and a non-native species planted in each
Keywords:
site (Platycladus orientalis). Endophytes were recovered from 229 of 960 tissue segments
Ascomycota
and represented at least 35 species of Ascomycota. Isolation frequency was more than three-
Biodiversity
fold greater for plants in NC than in AZ, and was 2.5 (AZ) to four (NC) times greater for
Cupressaceae
non-native Platycladus than for Cupressus or Juniperus. Analyses of ITS rDNA for 109 repre-
Molecular phylogeny
sentative isolates showed that endophyte diversity was more than twofold greater in NC
Species richness
than in AZ, and that endophytes recovered in AZ were more likely to be host-generalists
Symbiosis
relative to those in NC. Different endophyte genera dominated the assemblages of each
host species/locality combination, but in both localities, Platycladus harboured less diverse
and more cosmopolitan endophytes than did either native host. Parsimony and Bayesian
analyses for four classes of Ascomycota (Dothideomycetes, Sordariomycetes, Pezizomycetes,
Eurotiomycetes) based on LSU rDNA data (ca 1.2 kb) showed that well-supported clades of
endophytes frequently contained representatives of a single locality or host species, underscoring the importance of both geography and host identity in shaping a given plant’s
endophyte community. Together, our data show that not only do the abundance, diversity,
and taxonomic composition of endophyte communities differ as a function of host identity
and locality, but that host affinities of those communities are variable as well.
ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction
Recent studies have begun to elucidate the diversity and ubiquity of fungal endophytes associated with terrestrial plants,
providing a framework for understanding the evolutionary
context of these plant–fungus associations (reviewed by Stone
et al. 2000; Schulz & Boyle 2005; Arnold & Lutzoni 2007).
Despite a strong foundation laid by a growing number of surveys, the degree to which endophytes differ in abundance,
diversity, taxonomic composition, and host affinity over the
geographic ranges of particular host lineages remains unclear.
Resolving the importance of locality and host identity in shaping these aspects of endophyte communities is important for
understanding fundamental aspects of endophyte symbioses.
* Corresponding author.
E-mail address: [email protected]
0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2007.10.014
332
Previous studies have indicated that the abundance, diversity, and species composition of endophytes can be strongly
influenced by the locality in which a given plant occurs (e.g.
Carroll & Carroll 1978; Petrini et al. 1982; Bills & Polishook
1992; Fisher et al. 1994; Hata & Futai 1996; Bayman et al. 1998;
Arnold et al. 2001; Higgins et al. 2007). For example, different
microhabitats vary in inoculum potential, with higher infection frequencies observed beneath forest canopies versus in
forest clearings (Arnold & Herre 2003) or in more mesic microhabitats relative to proximate sites that are more xeric (Petrini
et al. 1982; Petrini 1996). At larger geographic scales, endophytes are frequently more abundant in tropical regions
than in boreal, arctic, or some temperate regions (Arnold &
Lutzoni 2007), although local climate, short-term weather patterns, and some disturbance regimes can override latitudinal
gradients (see Suryanarayanan et al. 2000; Arnold et al. 2007;
Higgins et al. 2007). Similarly, diversity of endophytes differs
at small scales as a function of land use history, vegetation
cover, and other factors. For example, Gamboa & Bayman
(2001) found higher endophyte diversity in leaves of Guarea
guidonia in a forest preserve relative to a disturbed forest
area in Puerto Rico. At larger scales, endophyte diversity
varies as a function of latitude and annual rainfall (Arnold &
Lutzoni 2007), although the contributions of co-varying factors, such as plant diversity, remain to be explored. Finally,
the species composition of endophyte assemblages also differs as a function of localities. For example, Arnold et al.
(2003) found distinctive endophyte communities associated
with Theobroma cacao at multiple sites in Panama. In a study
of endophytes and saprotrophs associated with closely related
palms in Australia and Brunei Darussalam, Fröhlich & Hyde
(1999) found that only 27 of 189 fungal species were shared between localities. Similarly, Fisher et al. (1995) found that only
two of 25 species of endophytes recovered from Dryas octopetala (Rosaceae) in Switzerland also occurred in the same host
in Norway.
By comparing endophyte assemblages in a single host species or genus in multiple sites, these studies have shown that
plants interact with distinctive endophyte communities
across the geographic areas in which they grow. However, it
is unclear whether plants growing in different localities experience not only a distinctive abundance, diversity, and species
assemblage of endophytes, but also a different degree of relative host specificity. Do sympatric plant species differ in terms
of the relative host specificity or host-generalism of the endophytes they harbour? Do different regions or environmental
conditions select for more or less host-specific endophytes?
To our knowledge, these questions have not been addressed
previously.
Most studies examining host affinities of endophytes have
focused on distantly related host species that co-occur within
particular geographic areas, and have yielded conflicting results. For example, a study involving endophytes of mistletoe
and fir established that despite close physical proximity of
these two plant taxa, endophyte communities remained exclusively associated with their particular host (Petrini 1996).
Similarly, Suryanarayanan et al. (2000) found distinctive endophytes in Cuscuta reflexa relative to its host plants, and Arnold
et al. (2000) found in lowland Panama that distantly related
hosts, growing within metres of one another, bore distinctive
27
M. T. Hoffman, A. E. Arnold
endophyte communities. Yet Cannon & Simmons (2002) recovered similar endophyte communities from phylogenetically diverse angiosperm trees in a tropical forest reserve.
Suryanarayanan et al. (2004) recovered the same endophyte
species from diverse plant species in mangroves, dry deciduous forest, and other tropical forest types (Phyllosticta capitalensis), and Mohali et al. (2005) found that Lasiodiplodia theobromae
occurs in multiple hosts and sites at a global scale. Suryanarayanan et al. (2005) found no evidence for host specificity
among endophytes of cacti in the southwestern USA, yet Higgins et al. (2007) found that most genotypes of endophytes recovered from arctic and boreal plants were associated with
only one host species. Thus, research to date remains in conflict with regard to the prevalence of host specificity among
endophytes, and the potential for host specificity of endophyte communities to differ over the range of a plant taxon,
or among species within a particular site, is not clear.
To our knowledge, no study to date has concurrently
assessed the importance of host identity and locality in shaping endophyte abundance, diversity, taxon composition, and
relative host specificity by simultaneously sampling pairs of
closely related plants in geographically distinct sites. Molecular tools are especially useful in this regard, providing a framework for explicitly comparing sterile endophytes and
providing a tool to disentangle potentially cryptic species for
which morphological characters are of limited use (Lacap
et al. 2003; Arnold et al. 2007). In turn, phylogenetic analyses
provide insight into the evolutionary associations of fungal
lineages with hosts and localities, and can provide muchneeded insight into the degree to which particular clades of
fungi are more or less likely to form host-specific associations
with plants. Unfortunately, endophyte surveys are often restricted in terms of molecular phylogenetics due to the cost
associated with sequencing informative loci for numerous
and often phylogenetically diverse isolates (see Arnold et al.
2007; Higgins et al. 2007). In particular, there is a need to examine the ability of particular loci to infer topologies that are consistent with reconstructions provided by multi-locus analyses
(e.g., Lutzoni et al. 2004; James et al. 2006) and to maximize the
phylogenetic power provided by loci that can be sequenced
rapidly and at reasonable cost.
Using pairs of related species of the conifer family Cupressaceae from a mesic forest [central North Carolina (NC)] and
from a xeric desert environment [southern Arizona (AZ)],
we used a culture-based approach to examine the foliar
endophyte communities of two native species (Cupressus
arizonica, AZ and Juniperus virginiana, NC) and a co-occurring,
non-native species (Platycladus orientalis in both AZ and
NC). Our objectives were to: (1) examine the abundance,
diversity, taxonomic composition, and host preferences of
endophytic fungi of these cupressaceous hosts; (2) infer
the relationships of endophytes associated with these trees
in a broad phylogenetic context; (3) evaluate an accelerated
phylogenetic search strategy for rapidly and robustly diagnosing these phylogenetic relationships using the LRORLR7 region of the LSU rDNA; and (4) generate hypotheses
regarding the relative importance of geographic locality
and host identity in structuring the endophyte communities associated with these ecologically and economically
important trees.
28
Endophytes of Cupressaceae
Materials and methods
Host species
The cypress family, Cupressaceae (Coniferales), is widely distributed in warm-temperate regions and contains 110–130 species
of evergreen trees and shrubs in 25–30 genera (Fralish &
Franklin 2002). Within the Cupressoideae, the genera Cupressus
and Juniperus comprise 70–86 species, whereas Platycladus is
monotypic (Platycladus orientalis). Cupressus arizonica (AZ
cypress) is indigenous in the western United States and northern
Mexico (Little 1950; USDA, NRCS 2004). Juniperus virginiana (Eastern red cedar) is distributed from Nova Scotia to Texas, and is the
most widespread conifer in the eastern USA (Fralish & Franklin
2002). Platycladus orientalis is native to Korea, eastern Russia, and
China. It has been extensively cultivated in southeastern Asia
and has become naturalized in Florida (Little 1980; USDA,
NRCS 2004). It is commonly planted as an ornamental in both
AZ and NC.
Sample collection
In March and April 2005, fresh, asymptomatic photosynthetic
tissue was collected from healthy trees at the campus arboretum at the University of AZ (Tucson: Platycladus and Cupressus;
four individuals/species) and at Duke Forest, Duke University
(Durham, NC: Platycladus and Juniperus; four individuals/species). The University of AZ (32.231 N, 110.952 W, elevation
787 m; mean annual temperature ¼ 20.2 C) is located within
the Sonoran Desert bioregion. This site is relatively xeric, receiving an average of 30.5 cm of precipitation annually. Durham, NC (36.001 N, 78.940 W, elevation 97 m; mean annual
temperature ¼ 15.5 C) is relatively mesic, receiving an average of 109.2 cm of precipitation annually. At the University
of AZ, all focal plants were within 250 m of one another, and
were cultivated in mixed, open stands with a variety of native
and non-native ornamental plants. Focal individuals were
fully established and mature, and received minimal supplemental water through infrequent irrigation. At Duke University, all Juniperus individuals were located beneath an open
canopy of Pinus taeda interspersed with Liquidambar styraciflua,
Carya spp., Quercus spp., Platanus occidentalis, and Acer spp. At
that site, all Platycladus individuals were located within
200 m of native J. virginiana and were planted adjacent to the
forest edge.
From each individual, we harvested mature photosynthetic tissues from three branches extending in three randomly chosen cardinal directions. Material was bagged for
transport to the laboratory and was used in endophyte isolations within 4 h of collection.
Endophyte isolations
Photosynthetic tissue from each sample was rinsed in running tap water for 30 s and then cut into 2 mm pieces before
surface-sterilization. Tissue fragments were agitated in 95 %
ethanol for 30 s, 10 % bleach (0.5 % NaOCl) for 2 min, and
70 % ethanol for 2 min (Arnold et al. 2007). Under sterile conditions, tissue segments were allowed to surface-dry before
333
plating on 2 % malt extract agar (MEA). To confirm the efficacy
of surface-sterilization, a subset of tissue segments was
pressed against the medium under sterile conditions for
30 s, and then removed. No mycelial growth was observed
from these surface impressions. A total of 384 tissue segments
(96 per individual tree) were plated for each species from AZ,
and 96 tissue segments (24 per individual tree) were plated
for each species from NC, corresponding to pilot data regarding differences in infection frequency (Arnold & Lutzoni 2007).
Tissue segments were incubated on sealed plates under ambient light/dark conditions at room temperature (ca 21.5 C).
Hyphal growth was monitored over an eight-week period.
Using aseptic technique, fungi were transferred to axenic culture on 2 % MEA in 60 mm Petri plates and photographed after
one week of growth. All samples were archived as living
vouchers in sterile water at the Robert L. Gilbertson Mycological Herbarium at the University of AZ. Based on mycelial
characteristics (Arnold et al. 2000), representatives of all
unique morphotypes were selected for molecular analysis.
Genomic DNA extraction and PCR
Total genomic DNA was extracted directly from pure cultures
following Arnold et al. (2007). PCR was used to amplify the ITS
rDNA and 5.8s gene (ca 600 bp) and LSU rDNA (ca 1.2 kb) following Higgins et al. (2007). Primers included ITS1F or ITS5
and ITS4 for ITS rDNA (White et al. 1990; Vilgalys & Hester
1990; Gardes & Bruns 1993), and LROR and LR7 for LSU rDNA
(Vilgalys, unpubl.; Vilgalys & Hester 1990). Sigma Readymix
REDTaq PCR reaction mix with MgCl2 (St. Louis, MO) was
used for all PCR reactions. The 25 ml reaction mixture included
12.5 ml REDTaq, 1 ml of each primer (10 mM), 1 ml DNA template, and 9.5 ml PCR-quality water. Cycling reactions were
run on an MJ Research PTC200 thermocycler (Waltham,
MA) with the following protocol: 94 C for 3 min; 36 cycles of
94 C for 30 s, 54 C for 30 s, 72 C for 1 min; and 72 C for
10 min. SYBR Green I stain (Molecular Probes, Invitrogen;
Carlsbad, CA) was used to detect DNA bands on a 1 % agarose
gel. All products demonstrated single bands.
Sequencing and analyses
PCR products were cleaned, quantified, and normalized at the
GATC sequencing facility at the University of AZ. Sequencing
was performed on the Applied Biosystems 3730XL DNA
Analyser (Foster City, CA). The software applications phred
and phrap (Ewing & Green 1998; Ewing et al. 1998) were used
to call bases and assemble contigs, with automation provided
by the ChromaSeq package (Maddison & Maddison: http://
mesquiteproject.org) implemented in Mesquite v. 1.06
(Maddison & Maddison: http://mesquiteproject.org). All base
calls were verified by inspection of chromatograms in
Sequencher version 4.5 (Gene Codes, Ann Arbor, MI).
ITS rDNA sequences were obtained for 109 representative
isolates (Supplementary Material Appendix 1). LSU rDNA sequences were obtained for 64 representative isolates (Supplementary Material Appendix 2) based on ITS rDNA genotype
groups (described below). Sequence data generated for this
study are available from GenBank under accession numbers
29
334
EF419892–EF420019 (ITS rDNA sequences) and EF420020–
EF420083 (LSU rDNA sequences).
Endophyte richness, diversity, and similarity
among hosts and sites
ITS rDNA sequence data were used in BLAST searches of
GenBank to provide preliminary identification at higher taxonomic levels and to guide taxon sampling for LSU rDNA analyses. To designate operational taxonomic units (OTU) based
on ITS rDNA data, we used Sequencher to delimit groups corresponding to 90, 95, and 99 % ITS rDNA similarity without
considering differences in sequence length (Arnold et al.
2007). Previous studies have used 90–97 % ITS rDNA sequence
similarity as a proxy for species boundaries in Fungi (e.g. 97 %:
O’Brien et al. 2005). One study has explicitly shown that groups
based on 90 % ITS rDNA similarity are highly congruent with
phylotypes based on a second locus (Arnold et al. 2007). Based
on these studies, we used 90 % ITS rDNA genotype groups as
a highly conservative proxy for species in all subsequent
analyses.
Species accumulation curves and bootstrap estimates of
total species richness were inferred using EstimateS v. 7.5
(Colwell 2005: http://viceroy.eeb.uconn.edu/EstimateS). In
addition to assessing richness (number of species) for each
partition of the data, we also calculated diversity, which takes
into account the abundance of different species. Diversity was
measured using Fisher’s a (Fisher et al. 1943), which is robust
for small sample sizes and thus accommodates a wide range
of sampling effort (Higgins et al. 2007).
All ITS rDNA genotypes (based on 90 % sequence similarity)
that were recovered more than once were compared for all
host-locality combinations. Jaccard’s index was used to assess
similarity on the basis of presence/absence data only (see
Arnold et al. 2000). The Morisita–Horn index was used to assess similarity on the basis of isolation frequency (see Gamboa
& Bayman 2001; Arnold et al. 2003). Similarity indices were
inferred using EstimateS v.7.5 (Colwell 2005: http://viceroy.
eeb.uconn.edu/EstimateS).
M. T. Hoffman, A. E. Arnold
Manual alignment was performed using MacClade 4.08
(Maddison & Maddison 2005). Each alignment was based on
LSU rDNA secondary structure as defined by Saccharomyces cerevisiae (Cannone et al. 2002). All ambiguously aligned regions
were excluded. The alignment for the Dothideomycetes consisted of 4595 characters, of which 1208 were included; for
the Sordariomycetes, 4605 characters, of which 1218 were included; for the Eurotiomycetes, 5443 characters, of which 2056
were included; for the Pezizomycetes, 4595 characters, of which
1208 were included.
For each dataset, initial heuristic searches using parsimony as the optimality criterion were implemented in PAUP
4b10 (Swofford 2002), but these searches were ineffective in
recovering optimal trees after up to five days. To improve
the quality of searches, we initiated 30 sets of 200 searches
for each dataset using PAUPRat (Sikes & Lewis 2001), which
implements the ‘parsimony ratchet’ method (Nixon 1999) to
efficiently recover shortest trees in datasets that are challenging for traditional heuristic searches (Sikes & Lewis 2001). The
‘filter trees’ command in PAUP 4b10 (Swofford 2002) was used
to select all shortest trees from the resulting pools of trees
(6001 trees per data set). The resulting set of shortest trees
was then used to construct a strict consensus tree. Support
was measured using a neighbor-joining bootstrap (1K
replicates).
To verify the quality of the topology given the PAUPRat
approach, we conducted an additional set of analyses for
each dataset using Bayesian MCMCMC. Based on comparisons of negative log likelihood values in Modeltest 3.7
(Posada & Crandall 1998), GTR þ I þ G was chosen as the
best-fitting model for each dataset (Supplementary Material
Appendix 4). Searches were implemented in MrBayes v.
3.1.1 (Huelsenbeck & Ronquist 2001) for 5M generations, initiated with random trees, four chains, and sampling every
500th tree. Extension of each analysis by up to 5M generations
had no significant effect on negative log likelihood values. After elimination of the burn-in for each analysis, a majority
rule consensus based on remaining trees was inferred.
Phylogenetic analyses
Results
Sixty-four LSU rDNA sequences for representative endophytes were organized at the class level on the basis of BLAST
results and integrated into existing core alignments for the
Dothideomycetes, Sordariomycetes, Eurotiomycetes, and Pezizomycetes. Core alignments included representative endophytes
from boreal, temperate, and tropical sites (Arnold et al., in
review; Arnold & Lutzoni 2007; Higgins et al. 2007; Supplementary Material Appendix 2) as well as named species of
Ascomycota obtained from GenBank (Supplementary Material
Appendix 3), and were subjected to preliminary analyses to
ensure that the initial BLAST results were accurate (Higgins
et al. 2007). Taxon sampling for the Dothideomycetes consisted
of 26 named sequences and 78 endophytes (46 from this
study); for the Sordariomycetes, 59 named sequences and 34
endophytes (15 from this study); for the Eurotiomycetes, 19
named sequences and three endophytes (one from this
study); and the Pezizomycetes, 23 named sequences and four
endophytes (two from this study).
From 960 tissue samples, 229 isolates of endophytic fungi
were recovered in culture. Isolation frequency, defined as
the percent of tissue segments bearing cultivable endophytes,
was more than threefold greater for hosts from NC (56.3 %)
than AZ (15.8 %), and was 2.5 to four times greater in Platycladus (AZ, 25.5 %; NC, 80.2 %) than in Cupressus (AZ; 6 %) or
Juniperus (NC; 32.4 %; Table 1).
Among 109 isolates sequenced for ITS rDNA, we recovered 35 unique ITS rDNA genotypes (90 % ITS rDNA sequence
similarity; Fisher’s a ¼ 17.9). A total of 43 and 64 ITS rDNA
genotypes based on 95 % and 99 % sequence similarity was
recovered (Fisher’s a ¼ 26.2, and 65.1, respectively).
Diversity of endophytes differed as a function of locality
and host identity. Diversity of endophytes from hosts in
NC was more than twofold greater than for hosts from AZ
(Table 1). The total diversity of endophytes recovered from
Juniperus and Cupressus was 1.4 times greater than from Platycladus (Table 1). In NC, diversity was ca twofold greater in
30
Endophytes of Cupressaceae
335
Table 1 – Number of tissue segments examined, isolates recovered, and isolates sequenced for ITS rDNA; diversity (Fisher’s
alpha) of endophytes using functional taxonomic units based on 90, 95, and 99 % ITS rDNA sequence similarity; and
number of singletons for each group based on 90 % ITS rDNA sequence similarity
Host taxon/location
Segments
plated
Isolates
recovered
Isolates
sequenced
Cupressus/AZ
Juniperus/NC
Platycladus/AZ
Platycladus/NC
384
96
384
96
23
31
98
77
Cupressus & Juniperus
Platycladus
Cupressus & Platycladus (AZ)
Juniperus & Platycladus (NC)
480
480
768
192
54
175
121
108
Fisher’s alpha
Singletons
(90 % ITS rDNA)
90 %
ITS rDNA
95 % ITS
rDNA
99 % ITS
rDNA
13
18
28
50
3.0
21.1
4.6
8.2
13.0
42.4
6.7
9.2
33.8
42.4
18.4
22.1
1
6
4
9
31
78
41
68
17.9
12.7
7.5
19.1
48.9
15.6
11.2
23.6
75.9
39.5
38.8
43.7
7
13
5
15
Hosts include Cupressus arizonica, Arizona (AZ); Juniperus virginiana, North Carolina (NC), and Platycladus orientalis in AZ and NC.
Juniperus than in Platycladus regardless of the stringency of ITS
rDNA groups. In AZ, diversity in Cupressus was 1.6 times less
than in Platycladus when species boundaries were based on
90 % ITS rDNA similarity. However, when species boundaries
were defined at greater levels of ITS rDNA similarity (i.e. 95
and 99 % ITS rDNA congruence), diversity was ca twofold
greater in Cupressus than in Platycladus (Table 1).
Endophyte species richness also differed as a function of
locality and host identity (Fig 1A–F). Greater species richness
per sampling effort was recovered in NC versus AZ (Fig 1A–
B), in Juniperus and Cupressus versus Platycladus (Fig 1C–D),
and in Juniperus in NC relative to any other species/locality
combination (Fig 1E–F). Overall, observed species richness
fell within the 95 % confidence intervals for estimated
richness, suggesting that our sample was effective in capturing the species richness of these endophyte communities
(Fig 1G). However, the relatively steep slope of the accumulation curve indicates that numerous species of endophytes
are yet to be recovered from these hosts as a whole.
When genotype accumulation curves were compared for
ITS rDNA groups based on different levels of sequence similarity, we found the largest differentiation between the 95 and
99 % groupings, with the most marked difference occurring
at ca 98 % similarity (Fig 1H). This reflects that most of the endophytes recovered in this study are clustered in phylogenetically distinct groups that share high (98 %) ITS rDNA
sequence similarity (see also Figs 2–5).
Relative importance of locality versus host identity
Similarity indices based on both presence/absence data (Jaccard’s index) and isolation frequencies (Morisita–Horn index)
and using 90 % ITS rDNA groupings suggest that endophyte
communities in these hosts are structured more by locality
than by host identity (Table 2). Highest similarity was observed among samples from different host species in the
same site (i.e. Platycladus and Cupressus in AZ; Platycladus
and Juniperus in NC). In contrast, samples from the same
host species in different sites had low similarity values (i.e.
Platycladus in NC and AZ). When compared across study sites,
Juniperus and Cupressus did not share more endophytes with
one another than with Platycladus.
Despite a large number of shared species between hosts
within each site, the dominant endophyte taxon associated
with each host and locality was distinct (Supplementary
Material Appendix 1). In all cases, the most abundant taxa
were Dothideomycetes. Cupressus (AZ) was dominated by an
unidentified dothideomycete (90 % ITS rDNA genotype I).
The highly diverse community of endophytes associated
with Juniperus (NC) was not characterized by a dominant
species, although three isolates (of 18 sequenced) represented
a dothideomycete with phylogenetic affinity to Letendraea
(90 % ITS rDNA genotype E; see below). Platycladus in NC was
dominated by Phyllosticta sp. (90 % ITS rDNA genotype C; 21
of 68 isolates), and in AZ by Aureobasidium sp. and Phoma sp.
(90 % ITS rDNA genotypes F and A, respectively, comprising
ten and nine isolates of 41 sequenced).
Phylogenetic relationships of endophytes
Results of phylogenetic analyses of endophytes from Cupressaceae, representative endophytes from other host species and
sites, and representative Ascomycota for each class are shown
in Figs 2–5. The majority of endophytes recovered in this study
were placed with support in the Dothideomycetes (46 isolates)
and Sordariomycetes (15 isolates). Only one endophyte (9147
from Platycladus in NC) was recovered as a member of the
Eurotiomycetes. No endophytes were recovered among the Leotiomycetes, or from the lichen-dominated clades Lecanoromycetes, Lichinomycetes, and Arthoniomycetes. Two endophytes
from Platycladus in AZ formed a clade within a well-supported
lineage of Pezizomycetes.
Phylogenetic analyses of the Dothideomycetes (Fig 2) using
the parsimony ratchet yielded 780 best trees with tree lengths
of 731 steps. BS support 70 % was observed for 12 nodes.
Bayesian PP values 95 % were observed for 65 nodes, including all nodes with strong BS support. Many endophytes recovered in this study were reconstructed as part of larger clades
of endophytic fungi from diverse hosts and sites that were
strongly supported as sister to Botryosphaeria ribis (clade A),
reconstructed in a clade containing Discosphaerina fagi and
a variety of boreal forest endophytes (clade B), or strongly supported as part of a clade containing Phoma glomerata and
a conifer endophyte from boreal forest (clades E and F). The
31
336
M. T. Hoffman, A. E. Arnold
35
35
A
30
Native & non-native
Arizona
25
25
20
20
15
15
10
10
5
5
0
Native & non-native
North Carolina
0
0
35
10
20
30
C
40
50
60
70
80
90
0
35
All native
AZ & NC
30
ITS rDNA genotypes
B
30
25
20
20
15
15
10
10
5
5
20
D
30
25
10
30
40
50
60
70
80
90
70
80
90
All non-native
AZ & NC
0
0
0
35
10
20
30
40
50
60
70
80
35
E
30
0
90
25
20
20
15
15
Platycladus AZ
10
30
40
50
60
Platycladus NC
Juniperus NC
10
Cupressus AZ
5
20
F
30
25
10
5
0
0
0
50
5
10 15 20 25 30 35 40 45 50
0
70
G
10 15 20 25 30 35 40 45 50
H
60
40
5
99% ITS rDNA genotype
50
95% ITS rDNA genotype
30
40
90% ITS rDNA genotype
30
20
20
10
10
0
0
0
25
50
75
100
125
0
25
50
75
100
125
Isolates
90% ITS rDNA genotype
95% confidence intervals
Bootstrap
Fig 1 – Genotype accumulation curves for fungal endophytes of Cupressus arizonica (AZ), Juniperus virginiana (NC), and
Platycladus orientalis (AZ and NC). (A–G) Genotype groups are based on 90 % ITS rDNA sequence similarity, which is used here
as a conservative proxy for species boundaries (following Arnold et al. 2007). (A) Species accumulation for endophytes of
hosts in AZ. (B) Species accumulation for endophytes of hosts in NC. (C) Species accumulation of endophytes of native hosts:
Cupressus (AZ) and Juniperus (NC). (D) Species accumulation of endophytes from non-native Platycladus (AZ and NC).
(E) Comparison of species accumulation curves for each host species in AZ. (F) Comparison of species accumulation curves for
each host species in NC. (G) Accumulation of ITS rDNA genotypes over the entire study. (H) Species accumulation and
richness inferred using groups based on 90, 95, and 99 % ITS rDNA similarity.
32
Endophytes of Cupressaceae
100/ 96
96/ -
100/ -
98/ 100/ 100/ 100/ 100/ -
100/ 100/ -
100/ 100
100/ 100/98
100/ -
100/ -
100/ 99
100/ -
100/ -
95/ 100/ 96/ -
100/ 100/ 100/ 98/ -
100/ 100/ 100/ 100/ 100/ -
100/ 100/ 100/ 100
100/ -
95/ 99/ 100/ -
100/87
100/ -
99/ -
100/ -
100/ 100/ 100/ 100/90
100/70
97/ -
100/ -
100/ -
96/ -
100/ 96/ 98/ 100/ 95
99/96
100/ 98/ 100/83
100/ 100/ -
100/ -
98/97
100/ 98/ 100/ -
Dendrographa minor
Botryosphaeria ribis
469 Laetia thamnia - Panama
2834 Dryas integrifolia - Québec
2779 Dryas integrifolia - Québec
3300 Dryas integrifolia - Québec
1182 Magnolia grandiflora - North Carolina
6730 Swartzia simplex - Panama
6731 Swartzia simplex - Panama
435b Laetia thamnia - Panama
9149b Platycladus orientalis - NC Δ C
9130 Platycladus orientalis - NC Δ C
9144 Platycladus orientalis - NC Δ C
9135 Platycladus orientalis - NC Δ C
9134 Platycladus orientalis - NC Δ C
9129 Platycladus orientalis - NC Δ C
Microxyphium citri
Raciborskiomyces longisetosum
9128 Platycladus orientalis - NC Δ U
2712 Huperzia selago - Québec
2722 Huperzia selago - Québec
462 Laetia thamnia - Panama
4140 Dryas integrifolia -Québec
3358A Dryas integrifolia - Québec
4221 Dryas integrifolia - Québec
3326 Picea mariana - Québec
5007 Picea mariana - Québec
Sydowia polyspora
1029 Magnolia grandiflora - North Carolina
4109 Huperzia selago - Québec
Delphinella strobiligena
4947A Picea mariana - Québec
4089 Picea mariana - Québec
Dothidea insculpta
Dothidea sambuci
Dothidea ribesia
Stylodothis puccinioides
9096 Platycladus orientalis - AZ Δ n/a
Discosphaerina fagi
3275 Dryas integrifolia - Québec
3335 Dryas integrifolia - Québec
3353 Dryas integrifolia - Québec
9042 Platycladus orientalis - AZ Δ F
9060 Platycladus orientalis - AZ Δ F
9079 Platycladus orientalis - AZ Δ n/a
9084b Platycladus orientalis - AZ Δ n/a
9002 Platycladus orientalis - AZ Δ F
9009b Platycladus orientalis - AZ Δ F
9028 Platycladus orientalis - AZ Δ F
9031 Platycladus orientalis - AZ Δ F
9036 Platycladus orientalis - AZ Δ F
3329 Picea mariana - Québec
3377 Picea mariana - Québec
Lojkania enalia
5718 Dryas integrifolia - Iqaluit
Trematosphaeria heterospora
5095 Picea mariana - Québec
Westerdykella cylindrica
Preussia terricola
9106 Cupressus arizonica - AZ M
9116 Cupressus arizonica - AZ M
9120 Cupressus arizonica - AZ M
9104 Cupressus arizonica - AZ M
9051 Platycladus orientalis - AZ Δ M
Byssothecium circinans
3323 Dryas integrifolia - Québec
Bimuria novae-zelandiae
Letendraea helminthicola
9203 Juniperus virginiana - NC E
9200 Juniperus virginiana - NC E
9149a Platycladus orientalis - AZ Δ E
9137 Platycladus orientalis - NC Δ E
9145 Platycladus orientalis - NC Δ E
9140 Platycladus orientalis - NC Δ E
Phoma glomerata
9158 Platycladus orientalis - NC Δ A
9054 Platycladus orientalis - NC Δ A
9107 Cupressus arizonica - AZ A
9065 Platycladus orientalis - AZ Δ A
5654 Picea mariana - Québec
9005 Cupressus arizonica - AZ A
9008 Cupressus arizonica - AZ A
9007 Platycladus orientalis - AZ Δ A
9018 Platycladus orientalis - AZ Δ A
9015 Platycladus orientalis - AZ Δ F
9027 Platycladus orientalis - AZ Δ A
9030 Platycladus orientalis - AZ Δ A
Curvularia brachyspora
Cochliobolus heterostrophus
Setosphaeria monoceras
Pyrenophora tritici-repentis
9055 Platycladus orientalis - AZ Δ B
9026 Platycladus orientalis - AZ Δ B
9021 Platycladus orientalis - AZ Δ B
9009a Platycladus orientalis - AZ Δ B
9057 Cupressus arizonica - AZ I
9059 Cupressus arizonica - AZ I
9058 Cupressus arizonica - AZ I
Setomelanomma holmii
Ampelomyces quisqualis
Phaeosphaeria avenaria
2851 Huperzia selago - Québec
2706 Huperzia selago - Québec
5246 Huperzia selago - Québec
337
A
H
B
C
D
E
F
G
Fig 2 – Majority rule consensus tree based on Bayesian analyses of LSU rDNA data for the Dothideomycetes. Numbers above
branches indicate branch support: Bayesian PP values 95 % are shown before the slash; NJ BS values 70 % are shown after
the slash. Branches recovered in strict consensus tree from parsimony analyses are shown with bold black lines. Circles after
endophyte culture numbers indicate native host species; triangles indicate non-native hosts. Letters indicate 90 % ITS rDNA
genotype groups based on sequence similarity (Supplementary Material Appendix 1). Endophytic fungi from other studies
are listed in Supplementary Material Appendix 2. All named taxa and GenBank identification numbers are shown in Supplementary Material Appendix 3. Dendrographa minor was included as an outgroup.
33
100/100
95/ 100/ -
97/ -
100/77
96/100
100/ 100/93
100/ -
97/ -
95/71
- /100
99/ 100/ -
95/ 100/ -
100/ 100/100
100/ 99/ 99/ -
100/100
100/ -
100/100
78/ -
98/90
100/70
100/ -
97/ -
100/100
100/97
100/80
100/ 100/88
97/ -
100/95
100/100
97/ 96/ 100/ -
100/100
100/ 100/73
100/ - 100/83
99/96
100/100
100/100/99
100/77
100/ -
100/ 100/99
100/ -
Leotia lubrica
Lulworthia fucicola
Lulworthia grandispora
Oxydothis frondicola
Microdochium nivale
Hyponectria buxi
4653 Picea mariana - Québec
Arthrinium phaeospermum
Apiospora sinensis
6722 Faramea occidentalis - Panama
9143 Platycladus orientalis - NC J
9089 Platycladus orientalis - AZ
R
9126 Platycladus orientalis - NC
J
9121 Platycladus orientalis - NC
J
Cryptosphaeria eunomia
Fasciatispora petrakii
Cainia graminis
Seynesia erumpens
Hypoxylon fragiforme
256 Laetia thamnia - Panama
2204 Pinus taeda - North Carolina
6717 Gustavia superba - Panama
9202 Juniperus virginiana - NC L
9201 Juniperus virginiana - NC L
Astrocystis cocoes
Rosellinia necatrix
Xylaria acuta
Xylaria hypoxylon
6728 Faramea occidentalis - Panama
Halorosellinia oceanica
Thyridium vestitum
Gaeumannomyces graminis var graminis
6702 Trichilia tuberculata - Panama
Diaporthe phaseolorum
Valsa ambiens
Cryptodiaporthe corni
Cryphonectria cubensis
Cryphonectria havanensis
Melanconis marginalis
Discula destructiva
Gnomonia setacea
Gnomoniella fraxini
Plagiostoma euphorbiae
Cephalotheca sulfurea
Menispora tortuosa
Camarops microspora
Farrowia longicollea
Farrowia seminuda
Neurospora crassa
Sordaria macrospora
Sordaria fimicola
Chaetomium globosum
9038 Platycladus orientalis - AZ
P
9097 Cupressus arizonica - AZ
T
9069 Platycladus orientalis - AZ
H
9093 Platycladus orientalis - AZ
H
9094 Platycladus orientalis - AZ
H
9092 Platycladus orientalis - AZ
S
9085 Platycladus orientalis - AZ
H
Pleurothecium recurvatum
6733 Swartzia simplex - Panama
412 Laetia thamnia - Panama
6756 Gustavia superba - Panama
6741 Theobroma cacao - Panama
6749 Theobroma cacao - Panama
6714 Gustavia superba - Panama
151 Laetia thamnia - Panama
Microascus trigonosporus
Ascosalsum cincinnatulum
Magnisphaera stevemossago
Ascosacculus heteroguttulatus
Natantispora lotica
Halosarpheia marina
Sagaaromyces abonnis
Corollospora maritima
Varicosporina ramulosa
Torrubiella luteorostrata
Hydropisphaera erubescens
Cordyceps khaoyaiensis
425 Laetia thamnia - Panama
4939 Picea mariana - Québec
416 Laetia thamnia - Panama
6708 Gustavia superba - Panama
Balansia henningsiana
Hypocrea citrina
Verticillium epiphytum
Emericellopsis terricola
K
9154 Platycladus orientalis - NC
K
9168 Platycladus orientalis - NC
4780 Dryas integrifolia - Iqaluit
Hypomyces polyporinus
Cordyceps irangiensis
Cordyceps sinensis
A
B
C
D
Fig 3 – Majority rule consensus tree based on Bayesian analyses of LSU rDNA data for the Sordariomycetes. Numbers above
branches indicate branch support: Bayesian PP values 95 % are shown before the slash; NJ BS values 70 % are shown after
the slash. Branches recovered in strict consensus tree from parsimony analyses are shown with bold black lines. Circles after
endophyte culture numbers indicate native host species; triangles indicate non-native hosts. Letters indicate 90 % ITS rDNA
genotype groups based on sequence similarity (Supplementary Material Appendix 1). Endophytic fungi from other studies
are listed in Supplementary Material Appendix 2. All named taxa and GenBank identification numbers are shown in Supplementary Material Appendix 3. Leotia lubrica was included as an outgroup.
34
Peltula umbilicata
Arthroderma curreyi
100/98
Auxarthron zuffianum
Aphanoascus fulvescens
100/88
Coccidioides posadasii
- / 93
Paracoccidioides brasiliensis
100/90
Ascosphaera apis
100/ Eremascus albus
Byssochlamys nivea
100/96
100/ -
Penicillium freii
100/87
9147 Platycladus orientalis - NC Δ J
A
Pyrenula cruenta
Pyrenula pseudobufonia
100/97
4466 Picea mariana - Québec
Verrucaria pachyderma
100/ Ceramothyrium carniolicum
100/ -
Capronia mansonii
99/ -
422 Laetia thamnia - Panama
99/ Exophiala jeanselmei
Glyphium elatum
Capronia pilosella
99/ Dermatocarpon luridum
Fig 4 – Majority rule consensus tree based on Bayesian analyses of LSU rDNA data for the Eurotiomycetes. Numbers above
branches indicate branch support: Bayesian PP values 95 % are shown before the slash; NJ BS values 70 % are shown after
the slash. Branches recovered in strict consensus tree from parsimony analyses are shown with bold black lines. Triangle
indicates non-native host. Letters indicate 90 % ITS rDNA genotype groups based on sequence similarity (Supplementary
Material Appendix 1). Endophytic fungi from other studies are listed in Supplementary Material Appendix 2. All named taxa
and GenBank identification numbers are shown in Supplementary Material Appendix 3. Peltula umbilicata was included as an
outgroup.
35
Candida albicans
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Neolecta vitellina
Taphrina communis
Ascobolus carbonarius
100/100
Ascobolus crenulatus
9098 Platycladus orientalis - AZ Δ N/A
100/91
100/ -
A
9084a Platycladus orientalis - AZ Δ Q
100/100
Sarcosphaera crassa
Peziza succosa
4250 Dryas integrifolia - Iqaluit
Peziza proteana
100/72
Peziza quelepidotia
Sarcoscypha coccinea
95/ -
4245 Dryas integrifolia - Iqaluit
100/ Otidea onotica
97/ Aleuria aurantia
99/ -
97/94
Cheilymenia stercorea
Barssia oregonensis
100/100
Helvella compressa
100/70
Gyromitra californica
100/93
Gyromitra esculenta
100/100
Disciotis venosa
-/99
Verpa conica
100/ 100/100
Morchella elata
Morchella esculenta
Fig 5 – Majority rule consensus tree based on Bayesian analyses of LSU rDNA data for the Pezizomycetes. Numbers above
branches indicate branch support: Bayesian PP values 95 % are shown before the slash; NJ BS values 70 % are shown after
the slash. Branches recovered in strict consensus tree from parsimony analyses are shown with bold black lines. Triangles
indicate non-native hosts. Letters indicate 90 % ITS rDNA genotype groups based on sequence similarity (Supplementary
Material Appendix 1). Endophytic fungi from other studies are listed in Supplementary Material Appendix 2. All named taxa
and GenBank identification numbers are shown in Supplementary Material Appendix 3. The five taxa at the base of the tree
were included as outgroups.
36
Endophytes of Cupressaceae
Table 2 – Similarity of endophyte assemblages with
regard to locality and host identity based on nonsingleton ITS rDNA genotypes (90 % sequence similarity)
Platycladus Platycladus Juniperus Cupressus
NC
AZ
NC
AZ
Platycladus NC
Platycladus AZ
Juniperus NC
Cupressus AZ
0.05
0.09
0.40
0.10
0.09
0.50
0.24
0.12
0.04
0.42
0.09
0.10
Similarity values reflect the Morisita–Horn index (above diagonal:
index based on isolation frequencies) and Jaccard’s index (below diagonal: index based on presence/absence data only). Both indices
range from 0 (no overlap between communities) to 1 (total overlap
between communities). Highest similarities for each comparison
are shown in bold.
AZ, Arizona; NC, North Carolina.
majority of endophytes within the Dothideomycetes were
reconstructed with strong support as members of the wellsupported Pleosporales (clades C–G). One additional endophyte
(H) was reconstructed with strong support as the sister taxon
of Raciborskiomyces longisetosum. Clade C was reconstructed as
sister to Preussia terricola, albeit without strong support. Clade
D was reconstructed as sister to Letendraea helminthicola.
Clades E and F were strongly supported as a clade with Phoma
glomerata and included an endophyte from Picea mariana in
Québec. Clade G was well supported as sister to Pyrenophora
tritici-repentis. Throughout the Dothideomycetes, ITS rDNA genotype groups (based on 90 % ITS rDNA similarity) were
largely congruent with LSU rDNA phylotypes. Phylotypes containing endophytes from cupressaceous hosts often reflected
locality (AZ versus NC; e.g. clades A, B), but within-clade structure based on locality and host identity was suggested in several clades (e.g. clades D–G).
Analyses of the Sordariomycetes (Fig 3) using the parsimony
ratchet yielded 626 best trees with tree lengths of 1289 steps.
BS support 70 % was observed for 25 nodes, and Bayesian
PP 95 % was observed for 53 nodes. Twenty-four nodes
were supported by both methods. Endophytes from cupressaceous hosts were reconstructed within the well-supported
Xylariales (clades A–B), the Sordariales (clade C), and the Hypocreales (clade D). Clades A and B were reconstructed with support as sister to endophytes from Panama. Clade C was
reconstructed as sister to the ubiquitous fungus Chaetomium
globosum, and clade D as sister to Emericellopsis, albeit without
strong support. ITS rDNA genotype groups were consistent
with LSU rDNA phylotypes for the hypocrealean endophytes (clade D) and xylarialean endophytes in clade B.
However, clades A (Xylariales) and C (Sordariales) were characterized by poor matches between ITS rDNA genotype
groups and LSU rDNA phylotypes. Phylotypes reflected clustering of endophytes from different host taxa (clade C) and
sites (clade A) although two well-supported phylotypes
(clades B, D) contained endophytes from a single host species in a single site.
For the Eurotiomycetes (Fig 4), the parsimony ratchet approach yielded 1005 best trees with tree lengths of 387 steps.
BS support 70 % was observed for seven nodes, and Bayesian
PP 95 % was observed for 13 nodes. Six nodes were supported
341
by both measures. Endophyte 9147 was reconstructed with
strong support as sister to Penicillium freii within the wellsupported Eurotiales.
For the Pezizomycetes (Fig 5), the parsimony ratchet approach yielded 827 best trees with tree lengths of 561 steps.
BS support 70 % was observed for 11 nodes; Bayesian PP
95 % was observed for 16 nodes. Ten nodes received significant support from both methods. Endophytes 9098 and
9084a from Platycladus in AZ were reconstructed as sister to
one another with strong support, and are nested within
a strongly supported clade within the Pezizales.
Discussion
We used a culture-based approach, paired with sequencing of
both a fast-evolving locus (ITS rDNA) and a phylogenetically
informative locus (LSU rDNA), to assess the diversity and
taxon composition of fungal endophytes among native and
non-native conifers in two geographically distinct localities.
We found that the abundance, diversity, species composition,
and relative host affinity of endophyte communities differed
as a function of locality and host species. Cultivable endophytes were present in up to 80.2 % of tissue segments examined, but their incidence differed more than 13-fold as
a function of locality and host identity. Despite very conservative estimates of species boundaries, diversity ranged from
low values (Fisher’s a ¼ 3: Cupressus, AZ) to values consistent
with those of angiosperm trees in tropical forests (Fisher’s
a ¼ 21.1: Juniperus, NC; Arnold & Lutzoni 2007). Most endophytes were placed in phylogenetic analyses within the Dothideomycetes and, to a lesser extent, the Sordariomycetes, but
members of both the Eurotiomycetes and Pezizomycetes were
also recovered. Isolates representing the Dothideomycetes
were proportionally more common in AZ than NC (ca 88 versus
ca 75 % of isolates with definitively identified ITS rDNA genotypes, respectively; Supplementary Material Appendix 1, Figs
2–5). The Sordariomycetes were proportionally more common
in NC than AZ (ca 21 versus ca 10 %, respectively). Overall and
for three host/locality combinations (Cupressus AZ, Platycladus
AZ, and Platycladus NC), the majority of definitively identified
isolates were placed in the Dothideomycetes. However, Juniperus
from NC was characterized by a higher incidence of Sordariomycetes (ca 56 % of identified isolates; Supplementary Material
Appendix 1).
Although endophyte genotypes often occurred in more
than one host species (Supplementary Material Appendix 1)
and clades often contained representatives of multiple hosts
and sites (Figs 2–5), the occurrence of numerous singleton species (Table 1, Fig 1) and relatively low similarity among hosts
and localities (Table 2) suggest that each tree species in each
locality has a signature community of endophytic symbionts.
This was underscored by the observation that the dominant
endophyte species associated with each host was distinct
(Supplementary Material Appendix 1). The conservative species concept applied here likely underestimates richness and
diversity (Table 1), and may overestimate similarity of
communities among hosts and sites (Table 2). Diversity of
endophytes in these temperate hosts thus appears to be surprisingly high, with a high degree of geographic turnover
342
and distinctively structured communities in each host and
locality.
Fisher et al. (1993, 1994) found that endophyte assemblages
of trees planted outside their native range are depauperate in
terms of infection frequency, species richness, and prevalence
of host-specific endophytes. Although our study did not examine endophytes of Platycladus within its native range, we
did compare endophytes of Platycladus with those of two
closely related, native hosts in a mesic forest and an arid desert. In both sites, isolation frequency was higher in Platycladus
than in either native host. Moreover, Platycladus in NC
harboured cultivable endophytes in a significantly higher percentage of tissue segments (80.2 %) than did seven representative, native species studied previously in Duke Forest, NC
(mean S.D. ¼ 36.4 % 27 of tissue segments; Wilcoxon
signed-rank test, P ¼ 0.031; Arnold & Lutzoni 2007). In contrast,
Juniperus fell within the range of other native taxa in that
forest (32.3 %; Wilcoxon signed-rank test, P ¼ 0.578).
Elevated frequencies of endophyte infection in Platycladus
relative to closely related, co-occurring plants could reflect
three non-exclusive factors: (1) the presence of both local
and introduced endophytes within these non-native trees;
(2) host-specific rates of infection that are independent of
the native/non-native status of these plants; and/or (3) the
presence of more cosmopolitan/less host-specific endophytes, which might be more readily cultivated than those
with greater host specificity.
Further sampling is needed to address the first two scenarios, such that firm conclusions on this topic cannot be drawn
from this study. However, support for the third scenario comes
from previous studies indicating that opportunistic, hostgeneralist endophytes are especially common in plants outside their native ranges (e.g. Fisher et al. 1994). Under this
scenario, endophytes of Platycladus should be characterized
by a greater proportion of host-generalists than Juniperus and
Cupressus. Even though all species harboured putatively opportunistic, widespread taxa (e.g. Phoma sp., Aureobasidium sp.,
Alternaria sp., Cladosporium sp., Xylaria sp.), these cosmopolitan
genera were nearly five times more common in Platycladus
than either native host (Supplementary Material Appendix 1).
We also found that these cosmopolitan genera were three
times more common in AZ than in NC. We hypothesize that
desert endophytes may be under particularly strong selection
to act as host-generalists: the benefit of opportunistically
infecting hosts is likely high relative to the cost of prolonged
exposure to intense heat, UV radiation, and desiccation typical
of this region. High rates of host generalism among desert endophytes are further supported by similarity indices, which
showed greater similarity in endophyte communities for different host species in AZ than in NC (Table 2). The most common endophytes recovered from our AZ site showed affinity
to Aureobasidium sp., Phoma sp., and Alternaria sp. (Supplementary Material Appendix 1), consistent with a recent survey of
non-host-specific cactus endophytes in AZ (Suryanarayanan
et al. 2005). Thus our data suggest that relative host affinity of
endophyte communities has the potential to change over the
geographic range of host plant lineages, and may differ among
host plant taxa as well.
Arnold et al. (2007) investigated the use of a 600 bp region of
LSU rDNA, paired with a phylogenetic backbone constraint, as
37
M. T. Hoffman, A. E. Arnold
a search strategy for parsimony analyses involving environmental samples (e.g. unknown endophyte cultures).
That study concluded that additional data were needed to
more adequately infer phylogenetic relationships for large
datasets. In this study, we found that ca 1200 bp of LSU
rDNA, coupled with the PAUPRat method, provided the tools
for expediently recovering topologies within four classes of
Ascomycota. These topologies were not in conflict with topologies resulting from Bayesian analysis (Figs 2–5). However,
Bayesian methods consistently yielded greater numbers of
significantly supported nodes than did the BS method
employed here, which relied on NJ to rapidly assess branch
support. Well-supported phylogenies are key to diagnosing
the taxonomic affinity of unknown endophytes and are necessary for adequately addressing ecological questions for these
sterile isolates. Future studies will benefit from multiple, concurrent analysis methods, especially with regard to linking
traditional morphological species concepts to extensive molecular datasets.
The occurrence of clades composed only of endophytes
limits our ability to diagnose species boundaries using the
phylogenetic criteria proposed in other studies (e.g. Arnold
et al. 2007). However, well-supported, endophyte-containing
clades provide a tool for assessing the frequency with which
particular fungal lineages, rather than genotypes, are represented among different host species or sites. The prevalence
of endophyte-only clades suggests that cupressaceous hosts
harbour interesting and perhaps novel taxa of Ascomycota,
but such statements can only be made with caution pending
the population of public databases with additional sequences
for known but unsequenced fungi, and ongoing morphological delimitation of the isolates recovered here. Such clades
also are consistent with the diversification of fungi through
the endophyte symbiosis. Intensive sampling of multiple
host species within a given plant family represents an important but under-explored approach for investigating potential
co-cladogenesis of endophytes and their plant hosts.
Although the designation of ITS rDNA genotype groups
based on percent similarity is a straightforward and rapid
method for designating species boundaries, there is no
threshold value of sequence similarity that is universally useful for distinguishing species of fungi. Percent divergence between species is likely to range widely among and within
major lineages, different biological species of fungi frequently
have identical ITS rDNA sequences (Lieckfeldt & Seifert 2000),
and cryptic species may sometimes only be revealed through
a phylogenetic approach based on other loci (Taylor et al.
2000). We found that our conclusions regarding the relative
diversity of endophytes associated with Cupressus versus Platycladus were reversed when different ITS rDNA genotype similarities were used to designate species boundaries (Table 1).
This observation, coupled with clade-specific differences in
concordance between ITS rDNA genotypes and phylotypes
(Figs 2–5), warrants continued conservatism in using sequence similarity as a proxy for species boundaries.
With the exception of native fescue grass in AZ, the community structure, ecological roles, and evolution of fungal endophytes have not been well defined in desert environments,
particularly with molecular techniques (Faeth & Sullivan
2003; but also see Suryanarayanan et al. 2005). How such
Endophytes of Cupressaceae
endophytes might benefit or negatively influence their host
plant, particularly in times of severe stress, has not been determined. Similarly, the biotically rich forests of the mesic
southeastern USA remain mostly unexplored in terms of their
endophytic symbionts, and may harbour a diversity of endophytes similar in scale to that in tropical forests (Arnold &
Lutzoni 2007). The relative costs and benefits of endophytic
symbioses for particular plant species remain largely unresolved, and are particularly interesting in an evolutionary context given the ways in which endophyte communities differ in
abundance, diversity, composition, and relative host specificity over the ranges of their host plants.
Acknowledgements
We thank the University of Arizona’s Division of Plant
Pathology and Microbiology and College of Agriculture and
Life Sciences for supporting this work. Additional support
from the National Science Foundation [DEB-0343953 to
A.E.A. (and James W. Dalling); DEB-0200413 to A.E.A.] is gratefully acknowledged. We thank David Maddison for sharing
pre-release versions of MacClade and Mesquite, and for advice with regard to phylogenetic analyses. We are grateful to
Rebecca Porter, Mary Shimabukuro, Eric Janson, and Lindsay
Higgins for technical assistance, and to Cara Gibson, Mali
Gunatilaka, Ming-Min Lee, and especially François Lutzoni for
helpful discussion.
Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.mycres.2007.10.014.
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40
Appendix 1. Endophyte isolates (identification numbers, host taxa, and origin), ITS rDNA genotype groups based
on 90%, 95%, and 99% sequence similarity, and top BLAST matches based on analyses conducted in AugustOctober, 2005. Isolates used for phylogenetic analyses of LSU rDNA data are marked with asterisks. Four isolates
were included in LSUrDNA analyses but are not listed below because ITS rDNA sequences were not obtained
(9079, 9084b, 9096, and 9098). Identification numbers correspond to vouchers maintained as living cultures at the
University of Arizona.
Isolate
Host
Location
ITS rDNA ITS rDNA ITS rDNA
genotype genotype genotype Blast top match (ITS):GenBank accession number, ID, and e-value
(90%)
(95%)
(99%)
*9002 Platycladus
AZ
F
G
3L
gi|30026473|gb|AY225167.1| Aureobasidium pullulans isolate LB... 1053
*9005 Cupressus
AZ
A
2Q
R
gi|28974197|gb|AY183371.1| Phoma glomerata 18S ribosomal RNA ... 993
0.0
*9007 Platycladus
AZ
A
A
J
gi|59783265|gb|AY831558.1| Leptosphaerulina trifolii strain W... 805
*9008 Cupressus
AZ
A
A
A
gi|33302361|gb|AY337712.1| Phoma herbarum 18S ribosomal RNA g... 862
*9009a Platycladus
AZ
B
B
B
gi|32394890|gb|AY154712.1| Alternaria tenuissima strain IA287... 1063
*9009b Platycladus
AZ
F
G
3L
gi|30026473|gb|AY225167.1| Aureobasidium pullulans isolate LB... 1047
*9015 Platycladus
AZ
F
G
S
gi|37786120|gb|AY213639.1| Aureobasidium pullulans strain UWF... 991
*9018 Platycladus
AZ
A
A
A
gi|33302361|gb|AY337712.1| Phoma herbarum 18S ribosomal RNA g... 860
*9021 Platycladus
AZ
B
B
B
gi|32394890|gb|AY154712.1| Alternaria tenuissima strain IA287... 1082
0.0
*9026 Platycladus
AZ
B
B
B
gi|32394890|gb|AY154712.1| Alternaria tenuissima strain IA287... 1112
0.0
*9027 Platycladus
AZ
A
A
J
gi|59783265|gb|AY831558.1| Leptosphaerulina trifolii strain W... 837
*9028 Platycladus
AZ
F
G
K
gi|30026472|gb|AY225166.1| Aureobasidium pullulans isolate SK... 993
*9030 Platycladus
AZ
A
A
J
gi|59783265|gb|AY831558.1| Leptosphaerulina trifolii strain W... 839
*9031 Platycladus
AZ
F
G
K
gi|30026472|gb|AY225166.1| Aureobasidium pullulans isolate SK... 1017
0.0
*9036 Platycladus
AZ
F
G
K
gi|30026472|gb|AY225166.1| Aureobasidium pullulans isolate SK... 918
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
41
*9038 Platycladus
AZ
P
P
T
gi|30089120|emb|AJ458185.1|CNI458185 Chaetomium nigricolor 5.... 714
*9042 Platycladus
AZ
F
G
K
gi|30026472|gb|AY225166.1| Aureobasidium pullulans isolate SK... 993
0.0
*9051 Platycladus
AZ
M
M
U
gi|41058714|gb|AY465446.1| Dothideales sp. GS2N1c 18S ribosom... 646
*9054 Platycladus
AZ
A
2Q
M
gi|28974197|gb|AY183371.1| Phoma glomerata 18S ribosomal RNA ... 950
*9055 Platycladus
AZ
B
B
B
gi|32394890|gb|AY154712.1| Alternaria tenuissima strain IA287... 1114
*9057 Cupressus
AZ
I
H
L
gi|47420271|gb|AY546017.1| Fungal endophyte WMS23 internal tr... 813
0.0
*9058 Cupressus
AZ
I
H
L
gi|47420271|gb|AY546017.1| Fungal endophyte WMS23 internal tr... 813
0.0
*9059 Cupressus
AZ
I
H
L
gi|47420271|gb|AY546017.1| Fungal endophyte WMS23 internal tr... 813
*9060 Platycladus
AZ
F
G
V
gi|30026472|gb|AY225166.1| Aureobasidium pullulans isolate SK... 995
9064 Platycladus
AZ
A
2Q
3K
gi|28974197|gb|AY183371.1| Phoma glomerata 18S ribosomal RNA ... 997
*9065 Platycladus
AZ
A
2Q
M
gi|28974197|gb|AY183371.1| Phoma glomerata 18S ribosomal RNA ... 993
*9069 Platycladus
AZ
H
F
H
gi|29165262|gb|AY219880.1| Lecythophora sp. UBCtra1453C 18S r... 868
*9084a Platycladus
AZ
Q
Q
W
gi|25045988|gb|AF491556.1| Peziza varia KH-97-88 (C) 18S ribo... 1183
0.0
*9085 Platycladus
AZ
H
R
X
gi|28202120|gb|AY141180.1| Aureobasidium pullulans 18S riboso... 821
0.0
*9089 Platycladus
AZ
R
S
Y
gi|22773864|gb|AF405301.1| Bartalinia robillardoides 18S ribo... 854
*9092 Platycladus
AZ
S
T
Z
gi|71725083|dbj|AB231012.1| Lecythophora hoffmannii genes for... 880
*9093 Platycladus
AZ
H
F
H
gi|29165262|gb|AY219880.1| Lecythophora sp. UBCtra1453C 18S r... 868
0.0
*9094 Platycladus
AZ
H
F
2A
gi|29165262|gb|AY219880.1| Lecythophora sp. UBCtra1453C 18S r... 872
0.0
*9097 Cupressus
AZ
T
U
2B
gi|12583572|emb|AJ271575.1|TSU271575 Thielavia subthermophila... 1053
*9104 Cupressus
AZ
M
N
2C
gi|46243857|gb|AY510419.1| Preussia similis strain s19 intern... 942
*9106 Cupressus
AZ
M
N
2D
gi|41058713|gb|AY465445.1| Dothideales sp. GS5N1b 18S ribosom... 890
*9107 Cupressus
AZ
A
2Q
3K
gi|28974197|gb|AY183371.1| Phoma glomerata 18S ribosomal RNA ... 971
9115 Cupressus
AZ
I
V
2E
gi|47420271|gb|AY546017.1| Fungal endophyte WMS23 internal tr... 722
*9116 Cupressus
AZ
M
W
2F
emb|AJ972795.1| Monodictys sp. MA 4647 18S rRNA gene, 5.8S rR... 751
*9120 Cupressus
AZ
M
M
2G
gb|AY465446.1| Dothideales sp. GS2N1c 18S ribosomal RNA gene,... 607
*9121 Platycladus
NC
J
J
2H
gb|AF377289.1| Pestalotiopsis funereoides 18S ribosomal RNA g... 1035
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1e-170
9122 Platycladus
NC
J
J
O
gb|AF377289.1| Pestalotiopsis funereoides 18S ribosomal RNA g... 1039
0.0
*9126 Platycladus
NC
J
J
O
gb|AF377289.1| Pestalotiopsis funereoides 18S ribosomal RNA g... 1037
0.0
*9128 Platycladus
NC
U
X
2I
gb|AF393724.2| Cladosporium tenuissimum strain ATCC 38027 18S... 961
0.0
*9129 Platycladus
NC
C
C
D
0.0
*9130 Platycladus
NC
C
C
D
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1154
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN...
1160 0.0
9133 Platycladus
NC
D
D
E
gb|AY786322.1| Botryosphaeria dothidea strain CBS 116743 18S ... 1041
0.0
42
*9134 Platycladus
NC
C
C
D
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1158
0.0
*9135 Platycladus
NC
C
C
D
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1154
0.0
0.0
9136 Platycladus
NC
C
C
D
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1116
*9137 Platycladus
NC
E
E
F
gb|AY561200.1| Foliar endophyte of Picea glauca sp. P3 intern... 1063
0.0
9138 Platycladus
NC
V
Y
2J
gb|DQ094165.1| Fungal sp. YX-41 internal transcribed spacer 1... 999
0.0
*9140 Platycladus
NC
E
E
F
gb|AY561200.1| Foliar endophyte of Picea glauca sp. P3 intern... 1072
0.0
9142 Platycladus
NC
C
C
C
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1154
*9143 Platycladus
NC
J
J
2K
gb|AF377289.1| Pestalotiopsis funereoides 18S ribosomal RNA g... 1037
*9144 Platycladus
NC
C
C
D
gi|12082793|gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ... 1217
*9145 Platycladus
NC
E
E
F
gb|AY561200.1| Foliar endophyte of Picea glauca sp. P3 intern... 1076
0.0
0.0
0.0
0.0
9146 Platycladus
NC
E
E
I
gb|DQ094168.1| Fungal sp. YX-47 internal transcribed spacer 1... 1041
0.0
*9147 Platycladus
NC
W
Z
2L
gb|AF125942.1| Penicillium sp. NRRL 28148 internal transcribe... 1033
0.0
9148 Platycladus
NC
E
E
I
gb|DQ094168.1| Fungal sp. YX-47 internal transcribed spacer 1... 1017
0.0
*9149a Platycladus
NC
E
E
F
gb|AY561200.1| Foliar endophyte of Picea glauca sp. P3 intern... 1055
0.0
*9149b Platycladus
NC
C
I
N
gi|12082793|gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ... 908
9150 Platycladus
NC
C
C
G
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1122
9151 Platycladus
NC
C
C
G
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1120
9153 Platycladus
NC
X
2A
2M
gb|AY063310.1| Fungal endophyte WMS14 internal transcribed sp... 648
*9154 Platycladus
NC
K
K
2N
gb|AY833034.1| Uncultured mycorrhizal ascomycete isolate 1 in... 387
9157 Platycladus
NC
A
A
2O
gb|AY337712.1| Phoma herbarum 18S ribosomal RNA gene, partial... 825
*9158 Platycladus
NC
A
A
J
gb|AY131201.1| Ascochyta lentis isolate MU AL1 18S ribosomal ... 811
9160 Platycladus
NC
C
C
D
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1164
0.0
9162 Platycladus
NC
C
C
G
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1120
0.0
9163 Platycladus
NC
C
I
2P
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 771
0.0
9165 Platycladus
NC
Y
2B
2Q
gb|AY265334.1| Scolecobasidium humicola strain B3343 internal... 315
8e-83
9167 Platycladus
NC
E
E
I
gb|DQ094168.1| Fungal sp. YX-47 internal transcribed spacer 1... 1033
0.0
*9168 Platycladus
NC
K
K
2R
gb|AY833034.1| Uncultured mycorrhizal ascomycete isolate 1 in... 387
2e-104
9169 Platycladus
NC
K
K
2S
dbj|AB067720.1| Cordyceps sinensis genes for 18S rRNA, ITS1, ... 464
1e-127
9171 Platycladus
NC
Z
2C
2T
emb|AJ390436.1|HAN390436 Nemania serpens 18S rRNA gene (parti... 969
9172 Platycladus
NC
C
C
D
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1158
9173 Platycladus
NC
C
C
D
gi|12082793|gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ... 1078
9174 Cupressus
AZ
F
G
2U
gb|AY225166.1| Aureobasidium pullulans isolate SK3 18S riboso... 934
0.0
0.0
0.0
0.0
2e-104
0.0
0.0
0.0
0.0
0.
0.0
43
9176 Platycladus
NC
C
C
C
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1156
9177 Platycladus
NC
C
C
C
gi|12082793|gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ... 1245
0.0
0.0
9178 Platycladus
NC
C
I
N
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 908
0.0
9179 Platycladus
NC
G
O
Q
gb|AF502622.1| Leaf litter ascomycete strain its031 isolate 1... 820
9183 Juniperus
NC
A
2Q
2V
gi|28974197|gb|AY183371.1| Phoma glomerata 18S ribosomal RNA ... 1049
9184 Platycladus
NC
C
C
G
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1076
0.0
9187 Juniperus
NC
O
2D
2W
dbj|AB107890.1| Phomopsis sp. MAFF 665006 genes for ITS1, 5.8... 1021
0.0
9188 Juniperus
NC
O
2E
2X
gb|AY577815.1| Diaporthe phaseolorum strain E99382 18S riboso... 892
9189 Juniperus
NC
2A
2F
2Y
gb|AY208791.1| Stagonospora sp. Po41 internal transcribed spa... 839
9190 Juniperus
NC
D
D
E
gb|AY786322.1| Botryosphaeria dothidea strain CBS 116743 18S ... 987
9191 Platycladus
NC
2B
2G
2Z
gb|AY213667.1| Paecilomyces lilacinus strain UWFP 674 18S rib... 969
9193 Juniperus
NC
2C
2H
3A
gb|AY063297.1| Fungal endophyte WMS1 internal transcribed spa... 593
9194 Platycladus
NC
G
O
Q
gb|AF502622.1| Leaf litter ascomycete strain its031 isolate 1... 813
9195 Platycladus
NC
C
C
D
gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ribosomal RN... 1154
9198 Juniperus
NC
2A
2I
3B
gb|AY183366.1| Rhizosphaera kalkhoffii 18S ribosomal RNA gene... 829
emb|AJ539482.1|MCR539482 Morchella crassipes 18S rRNA gene (p... 646
0.0
0.0
0.0
0.0
0.0
0.0
2e-166
0.0
0.0
0.0
9199 Juniperus
NC
2D
2J
3C
*9200 Juniperus
NC
E
E
F
gb|AY561200.1| Foliar endophyte of Picea glauca sp. P3 intern... 1067
0.0
*9201 Juniperus
NC
L
L
P
emb|AJ390411.1|HAN390411 Biscogniauxia atropunctata 18S rRNA ... 1057
0.0
*9202 Juniperus
NC
L
L
P
emb|AJ390411.1|HAN390411 Biscogniauxia atropunctata 18S rRNA ... 1017
0.0
*9203 Juniperus
NC
E
E
F
gb|AY561200.1| Foliar endophyte of Picea glauca sp. P3 intern... 1061
9206 Platycladus
NC
2E
2K
3D
gb|AY971711.1| Fungal sp. 4.32 18S ribosomal RNA gene, partia... 607
9208 Juniperus
NC
2F
2L
3E
gb|AY315399.1| Xylariaceae sp. C8 internal transcribed spacer... 410
9209 Juniperus
NC
2G
2M
3F
gb|AF409982.1| Pestalotiopsis sp. EN 7 18S ribosomal RNA gene... 442
9211 Juniperus
NC
2H
2N
3G
gb|AY315396.1| Xylaria sp. F23 internal transcribed spacer 1,... 1322
9217 Juniperus
NC
E
E
F
9220 Juniperus
NC
J
J
3H
gb|AY687871.1| Pestalotia lawsoniae voucher PSH2000I-057 18S ... 1070
0.0
9247 Juniperus
NC
N
2O
3I
gb|AF260224.1|AF260224 Kabatina juniperi 18S ribosomal RNA, p... 1155
0.0
9248 Platycladus
NC
G
O
Q
gb|AF502622.1| Leaf litter ascomycete strain its031 isolate 1... 816
9249 Platycladus
NC
C
C
C
gi|12082793|gb|AF312009.1|AF312009 Phyllosticta spinarum 18S ... 1245
9283 Platycladus
NC
2I
2P
3J
dbj|AB041241.1| Guignardia mangiferae genes for 18S rRNA, ITS... 1274
0.0
0.0
1e-170
1e-111
4e-121
0.0
gb|AY561200.1| Foliar endophyte of Picea glauca sp. P3 intern... 1037
0.0
0.0
0.0
0.0
44
Appendix 2. GenBank accession numbers for LSU rDNA sequences of endophytic fungi
obtained from other studies for phylogenetic analyses.
Endophyte isolate
Accession no.
4245
4250
4466
422
4653
6722
256
2204
6717
6728
6702
6733
412
6756
6741
6749
6714
151
425
4939
416
6708
4780
469
2834
2779
3300
1182
6730
6731
435b
2712
2722
462
4140
3358A
4221
3326
5007
1029
DQ979442
DQ979443
DQ979444
EF420091
DQ979449
EF420100
EF420088
EF420087
EF420099
EF420101
EF420096
EF420104
EF420089
EF420107
EF420105
EF420106
EF420098
EF420086
EF420092
DQ979453
EF420090
EF420097
DQ979452
EF420095
DQ979423
DQ979422
DQ979427
EF420085
EF420102
EF420103
EF420093
DQ979419
DQ979420
EF420094
DQ979440
DQ979433
DQ979441
DQ979429
DQ979455
EF420084
45
4109
4947A
4089
3275
3335
3353
3329
3377
5718
5095
3323
5654
2851
2706
5246
DQ979438
DQ979454
DQ979437
DQ979425
DQ979431
DQ979432
DQ979430
DQ979434
DQ979463
DQ979457
DQ979428
DQ979462
DQ979424
DQ979418
DQ979458
46
Appendix 3. Representative Ascomycota species included in phylogenetic analyses and
GenBank identification numbers. Sequences for Coccidioides posadasii were obtained from
TIGR (http://www.tigr.org).
Taxon
GenBank ID
Aleuria aurantia
Ampelomyces quisqualis
Aphanoascus fulvescens
Apiospora sinensis
Arthrinium phaeospermum
Arthroderma curreyi
Ascobolus carbonarius
Ascobolus crenulatus
Ascosacculus heteroguttulatus
Ascosalsum cincinnatulum
Ascosphaera apis
Astrocystis cocoes
Auxarthron zuffianum
Balansia henningsiana
Barssia oregonensis
Bimuria novae-zelandiae
Botryosphaeria ribis
Byssochlamys nivea
Byssothecium circinans
Cainia graminis
Camarops microspora
Candida albicans
Capronia mansonii
Capronia pilosella
Cephalotheca sulfurea
Ceramothyrium carniolicum
Chaetomium globosum
Cheilymenia stercorea
Coccidioides posadasii
Cochliobolus heterostrophus
Cordyceps irangiensis
Cordyceps khaoyaiensis
Cordyceps sinensis
Corollospora maritima
Cryphonectria cubensis
Cryphonectria havanensis
Cryptodiaporthe corni
Cryptosphaeria eunomia var. eunomia
Curvularia brachyspora
Delphinella strobiligena
Dendrographa leucophaea f. minor
Dermatocarpon luridum
Diaporthe phaseolorum
Disciotis venosa
Discosphaerina fagi
Discula destructiva
Dothidea insculpta
Dothidea ribesia
45775583
31415592
33113349
27447246
27447247
33113367
45775606
45775607
34453082
34453080
11120650
27447238
33113353
45155172
45775581
15808399
11120642
33113391
15808400
20334356
27447236
671812
11120644
12025061
20334357
11120645
13469777
45775590
TIGR222929
45775574
13241779
13241765
29466997
22203655
22671402
22671403
22671407
27447241
12025063
15808401
47499217
52699696
1399144
45775596
15808402
22671422
52699697
15808403
47
Dothidea sambuci
Emericellopsis terricola
Eremascus albus
Exophiala jeanselmei
Fasciatispora petrakii
Farrowia longicollea
Farrowia seminuda
Gaeumannomyces graminis var. graminis
Glyphium elatum
Gnomonia setacea
Gnomoniella fraxini
Gyromitra esculenta
Gyromitra californica
Halorosellinia oceanica
Halosarpheia marina
Helvella compressa
Hydropisphaera erubescens
Hypocrea citrina
Hypomyces polyporinus
Hyponectria buxi
Hypoxylon fragiforme
Leotia lubrica
Letendraea helminthicola
Lojkania enalia
Lulworthia fucicola
Lulworthia grandispora
Magnisphaera stevemossago
Melanconis marginalis
Menispora tortuosa
Microascus trigonosporus
Microdochium nivale
Microxyphium citri
Morchella elata
Morchella esculenta
Natantispora lotica
Neolecta vitellina
Neurospora crassa
Otidea onotica
Oxydothis frondicola
Paracoccidioides brasiliensis
Peltula umbilicata
Penicillium freii
Peziza proteana
Peziza quelepidotia
Peziza succosa
Phaeosphaeria avenaria
Phoma glomerata
Plagiostoma euphorbiae
Pleurothecium recurvatum
Preussia terricola
Pyrenophora tritici-repentis
Pyrenula cruenta
Pyrenula pseudobufonia
Raciborskiomyces longisetosum
Rosellinia necatrix
Saccharomyces cerevisiae
Sagaaromyces abonnis
Sarcoscypha coccinea
Sarcosphaera crassa
Schizosaccharomyces pombe
Setomelanomma holmii
Setosphaeria monoceras
45775610
1698507
11120651
4206347
27447243
13469782
13469784
16565889
17104831
16565895
16565884
52699700
45775602
27447237
34453085
45775584
45155165
45775578
32261014
27447249
27447244
45775573
15808405
15808406
22203665
22203666
34453095
22671436
45775611
1399149
2565289
11120643
45775594
12025081
34453084
12025084
13469785
17154899
27447250
2271524
15216700
52699706
45775588
52699707
17154944
45775613
31415593
22671445
45775614
45775615
45775601
12025090
52699710
15808410
27447239
172409
34453078
45775576
45775597
288696
28144315
15808411
48
Seynesia erumpens
Sordaria fimicola
Sordaria macrospora
Stylodothis puccinioides
Sydowia polyspora
Taphrina communis
Thyridium vestitum
Torrubiella luteorostrata
Trematosphaeria heterospora
Varicosporina ramulosa
Verpa conica
Verrucaria pachyderma
Verticillium epiphytum
Valsa ambiens
Westerdykella cylindrica
Xylaria acuta
Xylaria hypoxylon
12025093
45155203
37992293
11120648
45775604
52699720
45775600
13241778
15808412
22203671
45775595
15216679
8777749
16565896
11120649
45775605
45775577
Appendix 4. Model parameters for Bayesian analyses for each focal class of Ascomycota.
Class
Selected model
- Ln likelihood
Dothideomycetes
Sordariomycetes
Eurotiomycetes
Pezizomycetes
GTR+I+G
GTR+I+G
GTR+I+G
GTR+I+G
8463.17251
5924.5000
3613.9267
4519.40130
Proportion of invariant
sites
0.420231
0.565827
0.634904
0.522561
Gamma shape
parameter
0.526946
0.4267
0.55459
0.639625
Estimated Dothideomycetes base frequencies = A:0.266879 C:0.217424 G:0.307280 T:0.208417
Estimated Pezizomycetes base frequencies = A:0.287876 C:0.185319 G:0.288132 T:0.238673
Estimated Eurotiomecetes base frequencies = A:0.273327 C:0.208930 G:0.305662 T:0.212081
Estimated Sordariomycetes base frequencies = A:0.276040 C:0.210782 G:0.300050 T:0.213129
49
APPENDIX B
DIVERSE BACTERIAL ENDOSYMBIONTS INHABIT LIVING HYPHAE OF
PHYLOGENETICALLY DIVERSE FUNGAL ENDOPHYTES
50
51
Diverse bacteria inhabit living hyphae of phylogenetically diverse fungal
endophytes
Michele T. Hoffman and A. Elizabeth Arnold
Division of Plant Pathology and Microbiology
School of Plant Sciences
1140 E. South Campus Drive
University of Arizona
Tucson, AZ 85721 USA
For correspondence: AE Arnold, Tel: 520.621.7212; Fax: 520.621.9290; Email:
[email protected]
Running head: Endohyphal bacteria of endophytic fungi
Contains 3 tables, 5 figures, 4 appendices
52
Abstract
Both the establishment and outcomes of plant-fungal symbioses can be influenced by
abiotic factors, the interplay of fungal and plant genotypes, and additional microbes
associated with fungal mycelia. Recently, bacterial endosymbionts have been
documented in soilborne Glomeromycota and Mucoromycotina, and in at least one
species each of mycorrhizal Basidiomycota and Ascomycota. Here, we show for the
first time that phylogenetically diverse endohyphal bacteria occur in living hyphae of
diverse foliar endophytes, including representatives of four classes of Ascomycota. We
examined 414 isolates of endophytic fungi, isolated from photosynthetic tissues of six
species of cupressaceous trees in five biogeographic provinces, for endohyphal bacteria
using microscopy and molecular techniques. Viable bacteria were observed within
living hyphae of endophytic Pezizomycetes, Dothideomycetes, Eurotiomycetes, and
Sordariomycetes from all tree species and biotic regions surveyed. A focus on 29
fungal/bacterial associations revealed that bacterial and fungal phylogenies were
incongruent with each other and with taxonomic relationships of host plants. Overall,
eight families and 15 distinct genotypes of endohyphal bacteria were recovered; most
were Proteobacteria, but a small number of Bacillaceae also were found, including one
that appears to occur as an endophyte of plants. Frequent loss of bacteria following subculturing suggests a facultative association. Our study recovered distinct lineages of
endohyphal bacteria relative to previous studies, is the first to document their
occurrence in foliar endophytes representing four of the most species-rich classes of
fungi, and highlights for the first time their diversity and phylogenetic relationships
53
with regard both to the endophytes they inhabit and the plants in which these
endophyte-bacterial symbiota occur.
Keywords: Ascomycota, endohyphal bacteria, Bayesian analysis, Burkholderiales,
Cupressaceae, diversity, fungal endophytes, phylogeny, FISH
54
Introduction
Traits related to the establishment and outcome of plant-fungal symbioses can reflect
not only abiotic conditions and the unique interactions of particular fungal and plant
genotypes (50, 51, 57, 60, 63, 68), but also additional microbes that interact intimately
with fungal mycelia (4, 12, 43). For example, mycorrhizosphere-associated
actinomycetes release volatile compounds that influence spore germination in the
arbuscular mycorrhizal (AM) fungus Gigaspora margarita (Glomeromycota) (14).
Levy et al. (35) describe Burkholderia spp. that colonize spores and hyphae of the AM
fungus Gigaspora decipiens and are associated with decreased spore germination.
Diverse "helper" bacteria have been implicated in promoting hyphal growth and the
establishment of ectomycorrhizal symbioses (23, 26, 58, 71). Minerdi et al. (44) found
that a consortium of ectosymbiotic bacteria limited the ability of the pathogen Fusarium
oxysporum to infect and cause vascular wilts in lettuce, with virulence restored to the
pathogen when ectosymbionts were removed.
In addition to interacting with environmental and ectosymbiotic bacteria, some
plant-associated fungi harbor bacteria within their hyphae (first noted as 'bacteria-like
organisms' of unknown function) (39). These bacteria, best known from living hyphae of
several species of Glomeromycota and Mucoromycotina, can alter fungal interactions
with host plants in diverse ways (see 12, 32, 52). For example, the vertically transmitted
bacterium Candidatus Glomeribacter gigasporarum colonizes spores and hyphae of the
arbuscular mycorrhizal (AM) fungus Gigaspora gigasporarum (9, 10). Removal of the
bacterial partner from the fungal spores suppresses fungal growth and development,
55
altering the morphology of the fungal cell wall, vacuoles, and lipid bodies. (38). In turn,
the discovery of phosphate-solubilizing bacteria within Glomus mossae spores (45),
coupled with the recovery of a P-transporter operon in Burkholderia sp. from Gigaspora
margarita (55), suggest a competitive role in phosphate acquisition and transport by these
bacteria within the AM symbiosis. Within the Mucoromycotina, Partida-Martinez and
Hertweck (52) reported that a soilborne plant pathogen, Rhizopus microsporus, harbors
endosymbiotic Burkholderia that produce a phytotoxin (rhizoxin) responsible for the
pathogenicity of the fungus.
These examples, coupled with the discovery of bacteria within hyphae of
ectomycorrhizal Dikarya (Tuber borchii; Ascomycota; Laccaria bicolor and
Piriformospora indica; Basidiomycota) (5, 6, 7, 59), suggest that the capacity to harbor
endohyphal bacteria is widespread among fungi. To date, however, endocellular
bacteria have been documented only from fungi that occur in the soil and rhizosphere
(12, 32). Here we report for the first time that phylogenetically diverse bacteria occur
within living hyphae of foliar endophytic fungi, including members of four classes of
filamentous Ascomycota. We use a combination of light and fluorescent microscopy to
visualize bacterial infections within living hyphae of representative strains. Then,
drawing from surveys of endophytes from asymptomatic foliage of cupressaceous trees
in five biogeographic provinces, we provide a first characterization of the phylogenetic
relationships, host associations, and geographic distributions of endohyphal bacteria
associated with focal fungal endophytes.
56
Methods
Conifers (Pinophyta or Coniferae), comprising ca. 630 species, form the dominant tree
component in major biomes such as boreal forests, temperate tree savannas, and
temperate rainforests (20, 21, 22). The most widely distributed family of conifers, the
Cupressaceae (cypresses and relatives), includes 30 genera and ca. 142 species in seven
proposed subfamilies (25). Eleven genera are placed in the subfamily Cupressoideae,
including three examined in this study (Cupressus, Juniperus, and Platycladus).
We collected foliar endophytes from six species of Cupressoideae, as available,
in five localities (Table 1): semi-deciduous forest in the piedmont of North Carolina,
USA (NC), and four distinct regions of Arizona, USA (AZ) selected on the basis of
distinctive biogeographic histories and the occurrence of desired plant species. These
sites included the Chuska Mountains (CHU) of northeastern AZ, with petran montane
coniferous forest featuring significant elements of the Rocky Mountain flora; the
Mogollon Rim (MOG) and central mountains, including Mingus Mountain and the
Bradshaw Mountains, which lie at the interface of the Great Basin coniferous
woodlands, interior chaparral, and petran montane forest; the Sky Island archipelago
(SKY) of southeastern AZ, including the Santa Catalina and Chiricahua Mountains,
characterized by madrean evergreen forest; and the Campus Arboretum at the
University of Arizona (UA), a semi-urban, cultivated setting within the Arizona upland
province of the Sonoran Desert.
57
Endophyte collection
Fresh, asymptomatic photosynthetic tissue was collected from at least three healthy,
mature individuals of each focal species in each locality during the growing seasons of
2004-2007. From each tree, healthy, mature photosynthetic tissue was collected from
three haphazardly selected branches at the outer canopy ca. 1-2 m above ground.
Material was transferred to the laboratory for processing within 6-12 hours of
collection.
Tissue samples were washed in running tap water and then cut into 2mm
segments, corresponding to infection domains for individual fungi (3). Segments were
surface-sterilized by rinsing in 95% ethanol for 30 seconds, 10% Clorox® (0.6% sodium
hypochlorite) for two minutes, and 70% ethanol for two minutes (1), allowed to surfacedry under sterile conditions, and plated on 2% malt extract agar (MEA), which
encourages growth by a diversity of endophytes (24). In sum, 384 tissue segments (96
per individual) were plated per species at each location, with the exceptions of
Juniperus deppeana in the Chiricahua Mountains (144 segments), and Platycladus
orientalis and Juniperus virginiana from North Carolina (96 segments/species).
Plates were incubated at room temperature and inspected for hyphal growth
daily for 18 weeks. Emerging fungi were isolated on 2% MEA, archived as living
vouchers in sterile water, and deposited at the Robert L. Gilbertson Mycological
Herbarium at the University of Arizona (ARIZ; accession numbers available from MH).
Seven isolates of bacteria obtained directly from surface-sterilized plant tissue (i.e.,
58
bacterial endophytes) were accessioned as sterile cultures and maintained in sterile 80%
glycerol at -80°C.
PCR amplification and sequencing
Total genomic DNA was extracted directly from axenic fungal cultures following
Arnold et al. (2). In addition, DNA was extracted a second time from mycelium of five
isolates using the QIAGEN® DNeasy® Plant Mini kit to ensure that none of our records
of endohyphal bacteria represented contamination of reagents in our standard DNA
extraction protocol. Our two extraction methods were consistent in terms of DNA
extraction quality, sequence quality, and resulting diagnoses of bacterial infection.
For each fungal isolate, we used PCR to amplify the nuclear ribosomal internal
transcribed spacers and 5.8s gene (ITSrDNA), and when possible, the first 600bp of the
large subunit (LSUrDNA) as a single fragment (ca. 1000-1200bps in length) using
primers ITS1F and ITS4 or LR3 (27, 56, 67, 70). Each 25µl reaction included 12.5 µl
of Sigma ReadymixTM REDTaqTM, 1 µl of each primer (10 µM), 9.5 µl of PCR-quality
water, and 1 µl of DNA template. Cycling reactions were run with MJ Research
PTC200TM thermocyclers and consisted of 94°C for 3 minutes; 36 cycles of 94°C for 30
seconds, 54°C for 30 seconds, 72°C for 1 minute; and 72°C for 10 minutes.
The presence or absence of bacteria within the surrounding matrix was
determined initially using light microscopy. Fungal isolates were examined after one
week of growth in pure culture on 2% MEA using a Leica DM4000B with bright field
imaging (400X; NA = 0.75). Once visual examination ruled out non-endohyphal
59
bacteria (i.e., contaminants in the medium or microbes on hyphal surfaces), total
genomic DNA extracted from fresh mycelia was examined using PCR primers specific
to bacterial 16S rDNA: 10F and 1507R (1497 bp) or 27F and 1429R (1402 bp) (34, 46).
PCR reaction mixes and cycling parameters were as described above, except that
annealing temperatures were 58°C (10F and 1507R) or 55°C (27F and 1429R).
SYBR® Green I stain (Molecular Probes, Invitrogen) was used to detect DNA
bands on 1.5% agarose gels. Positive PCR products were cleaned, quantified, and
normalized at the University of Arizona Genomic Analysis and Technology Core
facility (GATC), followed by bidirectional sequencing with PCR primers (5 µM) using
an Applied Biosystems 3730XL DNA Analyzer. PCR products of insufficient
concentration for sequencing were cloned using Agilent Technologies StrataClone™
cloning kits following the manufacturer’s instructions. Water was used in place of
template for negative controls. Positive clones were amplified and sequenced using
primers M13F and M13R.
The software applications phred and phrap (18, 19) were used to call bases and
assemble bidirectional reads into contiguous consensus sequences, with automation
provided by ChromaSeq (40) implemented in Mesquite v. 2.6 (41). Base calls were
verified by inspection of chromatograms in Sequencher™ v 4.5 (Gene Codes Corp.,
Ann Arbor, MI). ITSrDNA and partial LSUrDNA sequence data have been submitted to
GenBank under accession numbers GQ153054-GQ153264 or were published
previously by the authors (28), and bacterial 16S rDNA sequences under accession
numbers HM046622-HM046627 and HM117722-117749.
60
Diversity and taxonomic placement of endophytes
Previous studies have used 90-97% ITS rDNA sequence similarity to estimate species
boundaries for environmental samples of fungi (97%, O’Brien et al. (49); 90%,
Hoffman & Arnold (28); 95%, Arnold et al. (1). Empirical estimates of percent
sequence divergence between sister species for four endophyte-containing
ascomycetous genera (Botryosphaeria, Colletotrichum, Mycosphaerella, and Xylaria)
indicated that groups based on 95% ITS rDNA sequence similarity conservatively
estimate species (66). We used Sequencher™ v 4.5 (Gene Codes Corp., Ann Arbor, MI.;
default settings except for the requirement of 20 character minimum overlap; see
Arnold et al. (3)) to estimate fungal OTU at 95% ITSrDNA sequence similarity, and
bacterial OTU at 97% 16S sequence similarity (62).
Taxonomic placement for fungi at the level of order and above was estimated by
BLAST comparisons with the curated ITSrDNA database for fungi maintained by the
Alaska Fungal Metagenomics Project (FMP; http://www.borealfungi.uaf.edu/) and
phylogenetic analysis (below). Bacterial taxonomy was estimated by BLAST
comparisons with GenBank and the Ribosomal Database Project (RDP; release 9)
classifier program (Naive Bayesian rRNA Classifier Version 2.0, July 2007) (15, 69),
combined with phylogenetic analyses (below). All statistical analyses were performed
in JMP 7.0 (SAS Institute Inc. Cary, NC).
61
Live-Dead stain
Live-Dead stain was used to confirm the presence and viability of endohyphal bacteria
for fungal isolates identified as containing bacteria on the basis of light microscopy and
PCR (above). We treated fresh mycelia in sterile water with 2µl of Molecular Probe
Live-Dead fluorescent stain for 15 min and prepared slides with VECTASHIELD®
HardSet™ 1400 medium (Vector Laboratories, Inc.) to prevent photobleaching. A Leica
DM4000B microscope with Luminera camera and 100W mercury arc lamp was used
for fluorescent imaging with a Chroma Technology filter set 35002 (480nm
excitation/520nm emission) and 1000X APO oil objective (NA=1.40-0.70; 0.13mm
working distance) at room temperature. Visible fluorescence of bacterial nucleic acids
within living hyphae that was not consistent with fungal mitochondrial or nuclear DNA,
coupled with positive PCR results and a lack of extrahyphal or ectosymbiotic bacteria,
provided evidence of viable endohyphal bacteria (47, 64).
Fluorescent in situ hybridization (FISH)
To rule out misinterpretation of fluorescence results from Live-Dead analyses we used
FISH with a probe specific for eubacteria to confirm endocellular infection in two focal
isolates (9084b and 9143). In each case, we examined hyphae from cultures grown on
2% MEA and on 2% MEA amended with antibiotics, which removed evident bacterial
infections (Hoffman and Arnold, in prep.).
Fresh mycelium was harvested and suspended in 1000ul 1X phosphate buffered
saline (PBS). After centrifugation at 8000rpm for 5 minutes, PBS buffer was replaced
62
with fresh 4% formaldehyde solution and incubated for 1.5 hours at 4°C. Samples then
were centrifuged at 8000rpm for 5 minutes and the pellet resuspended in PBS buffer
twice prior to storage in absolute ethanol at -20°C. Fixed mycelium was collected to
0.5 ml tubes and dehydrated in a series of ethanol/PBS solutions beginning with 50%,
70%, and finally 95% ethanol. Dehydrated mycelium was incubated for 90 minutes at
46°C in 10ul of hybridization buffer (HB) using 40% formamide hybridization
stringency (800ul formamide, 800ul DEPC water, 500ul EDTA) with 2ul of EUB338
probe (10uM), a universal 16S rDNA oligonucleotide probe (labeled with 5' TAMRA
fluorochrome tag; 5' GCTGCCTCCCGTAGGAGT 3'; EX559nm/EM583nm; Integrated
DNA Technologies, Inc.). Each sample was rinsed in 100ul wash buffer (460ul 5M
NaCl, 1ml 1M Tris, 50ul SDS, DEPC water, fill to 50ml) and warmed to 46°C twice.
This method was repeated using [1] antibiotic-cured isolates of each strain, which
showed no evidence of endohyphal bacteria; and [2] hybridization buffer and PBS only,
but no EUB338 probe, to control for autoflorescence. Mycelium was mounted on
gelatin-coated glass slides using DAPI as a counter-stain (42) and examined with either
an Olympus BX61 with a mercury arc lamp or a Leica DMI 6000 confocal system
equipped with a 543nm laser (63X oil; 1024x1024 format; xyz acquisition; line average
=1; frame average = 4). Leica software LAS-AF v 1.8.2 was used to capture images.
Phylogenetic analyses
Based on positive 16S rDNA PCR results and the lack of any extrahyphal bacteria in our
axenic cultures, endohyphal bacteria were observed in 75 of 414 fungal isolates. Twenty-
63
one infected isolates representing a broad diversity of Pezizomycotina, and 49 isolates in
which infections were never observed and that represented a broad array of ITS rDNA
genotype groups, were sequenced for 1200 bps of LSU rDNA following Arnold et al. (3)
(primers LROR or LR3R and LR7; protocols as described above). Additional LSU rDNA
sequence data have been submitted to GenBank under accession numbers HM117750HM117756.
These 70 sequences were integrated into core alignments of 157 representative
Ascomycota, partitioned by class into Sordariomycetes, Dothideomycetes, and
Eurotiomycetes (28) using Mesquite v 2.6 (41). Details of taxon sampling are given in
Table 2 and Appendix 1. Because our sample of Pezizomycetes contained only one
isolate with strong evidence of endohyphal bacteria, we did not address phylogenetic
relationships of pezizomycetous endophytes here.
Alignment for each class, based on LSU rDNA secondary structure defined by
Saccharomyces cerevisiae (13), was performed using ClustalW with manual adjustment
in Mesquite. All ambiguously aligned regions were excluded, with matrix details given
in Appendix 2. Alignments are available online at http://arnoldlab.net/alignments.html.
Phylogenetic relationships for members of each class were inferred using
parsimony and Bayesian Metropolis-coupled Markov chain Monte Carlo (MCMCMC)
analyses. For the former, five sets of 200 heuristic searches with random stepwise
addition and tree bisection-reconnection (TBR) were implemented using PAUPRat,
which incorporates the ‘parsimony ratchet’ method (28, 48, 61) in PAUP* 4b10 (65).
The ‘filter trees’ command in PAUP* 4b10 was used to select all shortest trees, which
64
were used to assemble strict consensuses. Bootstrap support was determined using 1000
replicates and the default settings for nonparametric NJ analysis as implemented in
PAUP* 4b10. Clades with ≥70% bootstrap support were considered strongly supported.
Details of parsimony searches are given in Appendix 2.
For Bayesian analyses, Modeltest 3.7 (54) was used to select the appropriate
model of evolution; in each case, GTR+I+G was selected using the Akaike Information
Criterion. Analyses were executed in Mr. Bayes 3.1.2 (31) for four runs of up to 6
million generations each, initiated with random trees, four chains, and sampling every
1000th generation. Likelihoods converged to a stable range for each data set and all trees
prior to convergence were discarded as burn-in. Clades with > 95% posterior
probability values were considered to have significant support.
Twenty-nine bacterial 16S DNA sequences, derived from total genomic DNA
extractions of fungal endophyte mycelia with no evidence of extrahyphal bacteria
(Table 2), and seven 16S sequences from bacterial endophytes obtained directly from
plant material (Table 3), were incorporated into a core alignment of 30 sequences of
named bacterial taxa obtained from the Ribosomal Database Project 9 website (Cole et
al. (15); http://rdp.cme.msu.edu) using seqmatch (type strains, isolates, >1200 bp, good
quality; ca. 1400bp), together with eight additional sequences from GenBank that
represented endosymbionts of fungi isolated in previous studies (Appendix 3) (9, 10,
53). Data were aligned using NAST and screened for chimeric sequences (minimum
sequence length = 300 bp) (http://greengenes.lbl.gov/) (17). Ambiguous regions were
excluded, resulting in a matrix of 6300 characters (http://arnoldlab.net/alignments.html).
65
For the bacterial data set, a heuristic search using maximum parsimony with
random step-wise additions and TBR branch swapping was implemented in PAUP*
4b10, resulting in 167 optimal trees of 3668 steps. Support was assessed using a
nonparametric neighbor-joining bootstrap (1000 replicates). Bayesian MCMCMC
analysis was implemented in MrBayes v. 3.1.2 for 2.5 million generations, initiated
with random trees, four chains, and sampling every 1000th tree, using GTR+I+G based
on evaluation in Modeltest 3.7 (54). After elimination of the first 1,400 trees as burn-in,
the remaining 1,100 trees were used to infer a majority rule consensus. These results
were complemented by phylogenetic inference in ARB v. 7.12.07 (37) using the AxML
maximum likelihood (ML) method (FastDNAML; olsen/felsenstein (F84); no filter: all
positions included in phylogenetic analysis; -ln likelihood = 22430.51). Branch support
was assessed using parametric ML bootstrapping (100 replicates) and Bayesian
posterior probabilities.
Results
Cultivable endophytes were isolated from healthy foliage of all plant species and from
every study site (Table 1), yielding 414 isolates from 4,560 tissue segments. ITS rDNA
data for all isolates were assembled at 95% sequence similarity to yield 113 OTU
(putative species; Fisher’s alpha = 51.2). Representatives of 5 classes and
approximately 10 orders, 13 families, and 28 genera were recovered. The most common
orders differed among host species and sites, but the entire dataset comprised a high
frequency of Pezizales (Pezizomycetes), Capnodiales, Pleosporales, Botryosphaeriales,
66
Dothideales (Dothideomycetes), Helotiales (Leotiomycetes), and Xylariales
(Sordariomycetes) (Appendix 4). Host specificity, diversity, and geographic
distributions of these fungi will be addressed in a forthcoming paper (Hoffman &
Arnold, in prep.).
Endohyphal bacteria
Endohyphal bacteria initially were observed in 75 of 414 endophyte isolates (18%;
determined by positive PCR results using 16S primers and no evidence of extrahyphal
bacteria), including isolates from all four classes of Ascomycota recovered in our
surveys (Pezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes),
isolates from all three host genera (Cupressus, Juniperus, and Platycladus), and isolates
from plants in all biogeographic regions (Table 2). After 47 of these isolates were
subcultured, however, they later were screened as negative for bacteria. In general,
length of time in culture (after ca. 2 weeks) appeared to adversely affect the
detectability or persistence of bacterial infections.
Based on examination with light microscopy and Live-Dead stain, no isolate
scored as positive for endohyphal bacteria had evident extrahyphal or ectosymbiotic
bacteria. Live-Dead stain indicated that bacteria within hyphae were viable and
confirmed viability of the hyphae that housed them (data not shown). Fluorescence in
situ hybridization (FISH) for isolates 9084b and 9143 further confirmed the presence of
endohyphal bacteria within living mycelia. Isolate 9084b is shown with the EUB338
fluorescent probe mounted in anti-fading mount medium only (Fig. 1A). Isolate 9143 is
67
shown with the EUB338 probe and DAPI counter-stain (Fig. 1B), illustrating the
presence of small numbers of bacterial cells in comparison to significant amounts of
total genomic DNA contained in the hyphal structure.
Phylogenetic inferences and taxonomic placement
Phylogenetic analyses confirmed BLAST-level taxonomy at the class level for fungal
endophytes in the Dothideomycetes, Eurotiomycetes, and Sordariomycetes (Figs. 3-5).
No pattern regarding the structure of endophyte lineages as a function of host plant
taxonomy was evident (i.e., the sister relationship of Cupressus and Juniperus, and their
sister relationship to Platycladus, is not reflected in the phylogenetic relationships of
their fungal associates). Isolates containing bacterial associates were spread broadly
across endophyte-containing clades in each class, indicating a phylogenetically
widespread capacity to harbor bacteria.
Taxonomic placement of endohyphal bacteria initially was assessed using
BLAST comparisons in GenBank, typically yielding matches to unidentified or
nameless environmental samples. The RDP classifier (69) placed these bacteria into two
phyla, Proteobacteria (including Alphaproteobacteria, Betaproteobacteria, and
Gammaproteobacteria) and Firmicutes. Overall, we recovered five orders and 10
families, putatively identified as Sphingomonadaceae (Alphaproteobacteria),
Burkholderiaceae, Comamonadaceae, and Oxalobacteraceae (Betaproteobacteria),
Moraxellaceae, Xanthomonadaceae, Pasteurellaceae, and Enterobacteriaceae
(Gammaproteobacteria), and Bacillaceae and Paenibacillaceae (Firmicutes) (Table 2).
68
Based on 97% 16S rDNA identity, these bacteria represented 15 OTU. Of these,
nine were found only once. Three of the remaining six OTU were found in fungi from
multiple genera of Cupressaceae, four were found in fungi from more than one
biogeographic region, and all were found in fungi representing multiple genera. Four
genotypes each were found in fungi representing different classes of Pezizomycotina
(genotypes A, B, C, E) (Table 2).
Proteobacteria were observed in association with fungi of the Dothideomycetes,
Eurotiomycetes, and Sordariomycetes, and in endophytes from all three plant genera
representing hosts in AZ and NC. Firmicutes were found in Eurotiomycetes and one
member of the Pezizomycetes. Nominal logistic analysis provided no evidence for a
significant effect of host genus (Cupressus, Juniperus, or Platycladus), region (AZ vs.
NC), or fungal class on the incidence of major bacterial groups (Alpha-, Beta-, and
Gammaproteobacteria; Firmicutes) (P >0.05 for all effects).
Phylogenetic relationships of endohyphal bacteria from fungal endophytes were
not congruent with those of the endophytes they inhabited (Figs. 2-5), nor with
relationships among host plants (data not shown). The bacterial lineages recovered here
were distinct from those recognized previously as endosymbionts of fungi (Fig. 2).
Comparison of bacterial endophytes and endohyphal bacteria
Only one bacterial genotype was represented both among bacterial endophytes (isolated
from surface-sterilized plant tissue only) and putative endohyphal bacteria (genotype A,
Bacillales; Tables 2 and 3). This genotype was isolated directly from foliage of J.
69
osteosperma (Chuska Mountains) and C. arizonica and J. deppeana (Sky Islands), and
was amplified from genomic DNA from a fungal culture recovered from J. deppeana
(Sky Islands). Two additional endophytes recovered here also were Bacillaceae,
representing genotypes that never were found as endohyphal bacteria (genotypes H, J)
(Tables 2, 3). One bacterial endophyte represented the Enterobacteriaceae (putative
Erwinia) and was not found as a bacterial endosymbiont within fungal hyphae.
Discussion
Recent studies have highlighted the potential of endosymbiotic microbes, including
bacteria and viruses, to shape the ecological roles of fungi (e.g., 43, 52). Endohyphal
bacteria have been found previously in living hyphae of plant-associated
Glomeromycota, Mucoromycotina, and up to three taxa of mycorrhizal Dikarya. Our
study is the first to compare their phylogenetic relationships with diverse fungal hosts,
and the first to document their occurrence in ascomycetous endophytes of foliage
representing four of the most species-rich classes of Ascomycota (Pezizomycetes,
Sordariomycetes, Dothideomycetes, and Eurotiomycetes), which together include
numerous plant pathogens, saprotrophs, and pathogens and parasites of animals (31).
Coupled with previous studies demonstrating the presence of bacteria in living mycelia of
mycorrhizal or soil-borne fungi (6, 7, 9, 52), our data provide strong evidence that the
ability of fungi to harbor endohyphal bacteria is phylogenetically widespread.
Our survey data show that endohyphal bacteria-endophyte associations occur in
regions with divergent biogeographic histories and markedly different environmental
70
conditions (e.g., diverse, aridland montane systems vs. piedmont forest in southeastern
North America) and in fungi associated with three genera of plants. Screening of a
small number of isolates obtained in previous studies (Figs. 3-5) confirmed that
endohyphal bacteria occur in endophytes from additional biogeographic provinces and
host plant lineages: infections were observed in endophytes isolated from Pinaceae in
boreal forest (endophyte 4466 from Picea mariana in Canada) and two families of
angiosperms from a tropical forest (endophyte 6722 from Faramea occidentalis,
Rubiaceae, and endophyte 6731 from Swartzia simplex, Fabaceae, recovered at Barro
Colorado Island, Panama). Because infections either were lost or became more difficult
to detect following subculturing, and because of potential limitations of our microscopy,
screening methods and primer selection, our results likely underestimate the incidence
of endohyphal bacteria in these plant-associated fungi.
Examination with Live-Dead stain provided evidence that bacteria within fungal
hyphae were viable, and showed that such bacteria occur within living fungal tissues.
FISH confirmed these results by diagnosing the presence of bacteria using 16S rDNA
specific tags (Fig. 1). In turn, our phylogenetic inferences show that endohyphal
bacteria include a greater phylogenetic diversity – both within and beyond the
Proteobacteria – than was previously known (23, 33, 53; Fig. 2).
In contrast to previous studies, which have focused primarily on bacteria
associated with a particular fungal species or closely related group of species, we
addressed the diversity of bacteria associated with a phylogenetically broad sample of
ecologically similar fungi. Our analyses provide no evidence of co-cladogenesis: fungal
71
and bacterial trees were obviously incongruent, and neither matched the phylogenetic
relationships among the plant species surveyed here. Our phylogenetic results are
consistent with the hypothesis of horizontal transmission both for fungal endophytes, as
expected (1, 28), and for the bacterial symbionts examined here (Figs. 2-5).
Using cultivation media infused with antibiotics we have found that many of
these fungi can be cured of their endosymbionts (Hoffman & Arnold, in prep.), and we
currently are investigating methods to re-infect cured mycelia. These attempts will shed
light on our hypotheses regarding the facultative nature of these associations, their
transmission modes, and their costs and benefits with regard to the fungi they inhabit.
Notably, some endophytes were unable to grow when transferred to media containing
antibiotics (Hoffman & Arnold, in prep.), suggesting that the relationship may be more
intimate or tend toward greater obligacy in some pairs of fungi/bacteria than others. For
one isolate (9143), we have successfully isolated the bacterial partner from the fungus
and confirmed its identity using 16S rDNA sequencing (Hoffman et al., in prep.).
Attempts to reinfect the endophyte with this bacterium have been ineffective to date.
Discovering the diversity and ecological roles of fungal endophytes
encompasses a trove of future research that will be important for understanding plant
ecology and evolution. So too does elucidation of the incidence, diversity, and
ecological importance of their endohyphal bacteria, which appear to be common but
previously overlooked inhabitants of plant-symbiotic fungi. Evidence from a variety of
studies (reviewed by 12, 32) indicates that endohyphal bacteria have the capacity to
alter the outcome of plant-fungal interactions in a phylogenetically diverse array of
72
symbioses. Experimental assessment of such effects will provide key evidence to
understanding the degree to which bacterial associates influence the nature of
endophytic symbioses. In turn, incorporating ancestral state reconstructions and further
sampling of both endohyphal bacteria (of fungi) and bacterial endophytes (of plants)
will elucidate the degree to which bacteria have transitioned from endophytic to fungalendosymbiotic, or the reverse – or have followed a distinctive evolutionary trajectory.
Acknowledgments
We thank the Division of Plant Pathology and Microbiology, School of Plant Sciences,
and College of Agriculture and Life Sciences at the University of Arizona, and the
Arizona-Nevada Academy of Sciences, for supporting this work. Additional support from
the National Science Foundation (NSF-0626520 and 0702825 to AEA; NSF-IGERT to
MTH) is gratefully acknowledged. We thank D.R. Maddison for sharing pre-release
versions of Mesquite and Chromaseq; M. Gunatilaka, M. Shimabukuro, D. Grippi, M. J.
Epps, and C. Weeks-Galindo for lab assistance and helpful discussion; J. U’Ren, F.
Lutzoni, E. Gaya, J. Miadlikowska, and F. Santos-Rodriguez for isolation of samples
from the Chiricahua Mountains; and B. Klein and undergraduate students at Diné College
for isolation of samples from the Chuska Mountains on the Navajo Nation. We are
especially grateful to H. Van Etten and R. Palanivelu for access to microscopy facilities,
A. Estes for EUB338 probes and helpful discussion about FISH, and J. L. Bronstein for
comments on the manuscript. This paper represents a portion of the doctoral dissertation
research of M.H. in Plant Pathology and Microbiology at the University of Arizona.
73
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Table 1. Locations and characteristics of sampling sites, and plant species surveyed for endophytes in each location.
Habitats
Latitude,
Longitude
Elevation
(m)
Mean annual
temperaturea
(°C)
Mean annual
precipitation
a
(mm)
Temperate mixed forest
36.00°N, 78.94°W
97
15.5
1092
J. virginiana L., P. orientalis (L.) Franco
Ponderosa pine forest
36.16°N, 108.91°W
2200
10.4
253
J. osteosperma (Torr.) Little, J. scopulorum Sarg.
Iron Springs, AZ (IS)
Pinyon-juniper woodland
34.58°N, 112.60°W
1851
11.8
482
C. arizonicab Greene, J. deppeana Stead
Mingus Mtn, AZ (MM)
Ponderosa pine-pinyon-juniper woodland
34.70°N, 112.13°W
2354
15.3
483
J. deppeana Stead
Strawberry, AZ (ST)
Ponderosa pine-pinyon-juniper woodland
34.40°N, 111.49°W
1707
13.2
541
J. deppeana Stead
Mt Lemmon, AZ (MTL)
Madrean evergreen woodland
32.44°N, 110.78°W
1600
9.2
751
C. arizonicab Greene, J. deppeana Stead
Chiricahua Mtns AZ (CHI)
Madrean evergreen woodland
32.03°N, 109.38°W
1371
11.7
549
J. deppeana Stead
Cultivated trees, semi-urban setting
32.23°N, 110.95°W
787
20.2
305
C. arizonicab Greene, J. virginiana L., P. orientalis (L.) Franco
Site
Plant species
Piedmont, Central NC (DUKE)
Duke Forest, NC (NC)
Petran forest, NE AZ (CHU)
Chuska Mtns, AZ (CHU)
Mogollon Rim, Central AZ (MOG)
Sky Islands , SE AZ (SKY)
Campus Arboretum, SE AZ (UA)
Univ. of Arizona (UA)
a
b
Climate information obtained from http://www.wrcc.dri.edu/coopmap/
Recently designated change from Cupressus arizonica Greene to Callitropsis arizonica (Greene) is acknowledged (36).
81
Table 2. Taxonomic classification for 29 fungal endophytes and associated endohyphal bacteria. Bacterial taxonomic classifications
are based on BLAST results and Ribosomal Database project (RDP) classifier program (Naive Bayesian rRNA Classifier Version 2.0,
July 2007) (69). Fifteen distinct 16S rDNA OTU, based on 97% sequence similarity, are designated in groups A-R. Asterisks indicate
one genotype found also as a bacterial endophyte in plant tissue, such that its status as only an endosymbiont of fungi is not clear
(Genotype A; see Table 3). Fungal identifications were based on comparisons with the FMP curated database in March 2009 and are
approximate at the genus and species levels, but strongly supported at the class level based on phylogenetic analyses (Figs. 3-5).
Fungal class
16S
genotype
Top 16S
BLAST/GenBank acc.
Bacterial lineage (based on RDP classifier)
Dothidea sambuci AY930108.1
Dothideomycetes
K
AZ: UA
Alternaria mali AY154683.1
Dothideomycetes
L
P. orientalis
AZ: UA
Phoma glomerata AY183371.1
Dothideomycetes
M
9055
P. orientalis
AZ: UA
Alternaria mali AY154683.1
Dothideomycetes
C
9060
P. orientalis
AZ: UA
Aureobasidium pullulans AY225166.1
Dothideomycetes
E
9094
P. orientalis
AZ: UA
Lecythophora sp. AY219880.1
Sordariomycetes
E
9096
P. orientalis
AZ: UA
Hormonema sp. AF182378.1
Dothideomycetes
N
9106
C. arizonica
AZ: UA
Preussia africana DQ865095.1
Dothideomycetes
C
9107
C. arizonica
AZ: UA
Phoma glomerata AY183371.1
Dothideomycetes
F
9112
C. arizonica
AZ: UA
Lecythophora sp. AY219880.1
Sordariomycetes
P
9120
C. arizonica
AZ: UA
Monodictys sp. AJ972795.1
Sordariomycetes
E
9122
P. orientalis
NC: Duke
Pestalotiopsis caudata EF055188.1
Sordariomycetes
C
Acinetobacter sp.
FJ422393
Acinetobacter junii
NR_026208
Uncultured Sphingomonas
EU341143
Uncultured bacterium
clone DQ984599
Uncultured bacterium
clone EU236303
Uncultured bacterium
clone EU236303
Oxalobacteraceae sp.
DQ490310
Uncultured bacterium
clone DQ984599
Uncultured Haemophilus
sp. EU071476
Variovorax paradoxus
DQ257419
Uncultured bacterium
clone EU236303
Uncultured bacterium
clone DQ984599
Gammaproteobacteria, Pseudomonadales,
Moraxellaceae
Gammaproteobacteria, Pseudomonadales,
Moraxellaceae
Alphaproteobacteria, Sphingomonadales,
Sphingomonadaceae
Gammaproteobacteria, Xanthomonadales,
Xanthomonadaceae
Betaproteobacteria, Burkholderiales,
Burkholderiaceae
Betaproteobacteria, Burkholderiales,
Burkholderiaceae
Betaproteobacteria, Burkholderiales,
Oxalobacteraceae
Gammaproteobacteria, Xanthomonadales,
Xanthomonadaceae
Gammaproteobacteria, Pasteurellales,
Pasteurellaceae
Betaproteobacteria, Burkholderiales,
Comamonadaceae
Betaproteobacteria, Burkholderiales,
Burkholderiaceae
Gammaproteobacteria, Xanthomonadales,
Xanthomonadaceae
Isolate
Host plant
Site
2611
J. scopulorum
AZ: CHU
9026
P. orientalis
9054
Top BLAST match
82
9128
P. orientalis
NC: Duke
Cladosporium oxysporum AJ300332.1
Dothideomycetes
E
9133
P. orientalis
NC: Duke
Botryosphaeria dothidea AY640254.1
Dothideomycetes
C
9135
P. orientalis
NC: Duke
Phyllosticta spinarum AF312009.1
Dothideomycetes
C
9140
P. orientalis
NC: Duke
Microdiplodia sp. EF432267.1
Dothideomycetes
R
9143
P. orientalis
NC: Duke
Pestalotiopsis caudata EF055188.1
Sordariomycetes
C
9145
P. orientalis
NC: Duke
Microdiplodia sp. EF432267.1
Dothideomycetes
D
9147
P. orientalis
NC: Duke
Penicillium citreoni AY373908.1
Eurotiomycetes
E
11123
J. virginiana
AZ: UA
Pithya cupressina U66009.1
Pezizomycetes
G
11164
J. deppeana
AZ: MTL
Eurotiomycetes
A*
11259
J. deppeana
AZ: MTL
Sordariomycetes
B
11272
J. deppeana
AZ: MOG
Eurotiomycetes
B
11035
J. deppeana
AZ: MTL
Phaeomoniella chlamydospora
AY772236.1
Biscogniauxia. mediterranea
AJ390413.1
Phaeomoniella chlamydospora
AB278179.1
Pestalotiopsis besseyi AY687313.1
Sordariomycetes
A*
9084b
P. orientalis
AZ: UA
Aureobasidium pullulans DQ680686.1
Dothideomycetes
F
9106 clA
C. arizonica
AZ: UA
Preussia africana DQ865095.1
Dothideomycetes
E
9106 clB
C. arizonica
AZ: UA
Preussia africana DQ865095.1
Dothideomycetes
O
9126 clB
P. orientalis
NC: Duke
Pestalotiopsis caudata EF055188.1
Sordariomycetes
Q
9149b
P. orientalis
NC: Duke
Phyllosticta spinarum AF312009.1
Dothideomycetes
D
Uncultured bacterium
clone EU236303
Uncultured bacterium
clone DQ984599
Uncultured bacterium
clone DQ984599
Pantoea agglomerans
FJ756346.1
Uncultured bacterium
clone DQ984599
Pantoea oleae
AF130967
Uncultured bacterium
clone EU236303
Bacillus foraminis
AJ717382
Bacillus subtilis
FJ549011
Paenibacillus sp.
AM906086
Paenibacillus sp.
AM906086
Bacillus atrophaeus
FJ194961
Uncultured bacterium
clone EF509196
Uncultured bacterium
clone EU236303
Acinetobacter sp.
FJ267573
Pantoea agglomerans
EU598802.1
Pantoea oleae
AF130967
Betaproteobacteria, Burkholderiales,
Burkholderiaceae
Gammaproteobacteria, Xanthomonadales,
Xanthomonadaceae
Gammaproteobacteria, Xanthomonadales,
Xanthomonadaceae
Gammaproteobacteria, Enterobacteriales,
Enterobacteriaceae
Gammaproteobacteria, Xanthomonadales,
Xanthomonadaceae
Gammaproteobacteria, Enterobacteriales,
Enterobacteriaceae
Betaproteobacteria, Burkholderiales,
Burkholderiaceae
Firmicutes, "Bacilli", Bacillales,
Bacillaceae
Firmicutes, "Bacilli", Bacillales,
Paenibacillaceae
Firmicutes, "Bacilli", Bacillales,
Paenibacillaceae
Firmicutes, "Bacilli", Bacillales,
Paenibacillaceae
Firmicutes, "Bacilli", Bacillales,
Paenibacillaceae
Gammaproteobacteria, Pasteurellales,
Pasteurellaceae
Betaproteobacteria, Burkholderiales,
Burkholderiaceae
Gammaproteobacteria, Pseudomonadales,
Moraxellaceae
Gammaproteobacteria, Enterobacteriales,
Enterobacteriaceae
Gammaproteobacteria, Enterobacteriales,
Enterobacteriaceae
83
Table 3. Bacterial endophytes isolated from foliage of focal species of Cupressaceae, collection sites, 16S rDNA OTU based on 97%
sequence similarity when compared among themselves and with the endohyphal bacteria listed in Table 2, top RDP classifier match
and accession information, and bacterial lineage.
Isolate
Plant species
Location
16S genotype
RDP Classifier match
Bacterial lineage
11124
J. deppeana
AZ: MTL
H
Bacillus megaterium FJ527650
Firmicutes, "Bacilli", Bacillales, Bacillaceae
11136
C. arizonica
AZ: MTL
A
Bacillus subtilis FJ549011
Firmicutes, "Bacilli", Bacillales, Bacillaceae
11180
J. osteosperma
AZ: CHU
A
Bacillus sp. FJ514812
Firmicutes, "Bacilli", Bacillales, Bacillaceae
11219
C. arizonica
AZ: MTL
A
Bacillus subtilis NR_024931
Firmicutes, "Bacilli", Bacillales, Bacillaceae
11290
J. deppeana
AZ: MTL
A
Bacillus subtilis FJ549011
Firmicutes, "Bacilli", Bacillales, Bacillaceae
11351
C. arizonica
AZ: MOG
I
Erwinia persicina EU681952
Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae
11394
J. virginiana
AZ: UA
J
Bacillus licheniformis EU071553
Firmicutes, "Bacilli", Bacillales, Bacillaceae
84
Figure legends
Figure 1. Fluorescent in situ hybridization (FISH) microscopy showing hyphae of two
isolates of endophytic fungi harboring endohyphal bacteria. A (isolate 9084b,
Dothideomycetes) shows TAMRA fluorophore at site of internal structure in hyphal cells.
B (isolate 9143, Sordariomycetes) shows TAMRA fluorophore with DAPI counterstain
(blue), highlighting the fungal nuclear and mitochondrial DNA in addition to the bacterial
16S rDNA (yellow/green). Scale bars: A, 10µm for image A and 25µm for image B.
Figure 2. Phylogenetic relationships of 29 endohyphal bacteria, 7 bacterial endophytes
(paired circles), and 38 named taxa based on Bayesian analysis of 16S ribosomal DNA
sequences. Branch support values indicate parsimony bootstrap bootstrap proportions
(>70%; before slash) and Bayesian posterior probabilities (>95%; after slash). Branches
in bold indicate >70% neighbor-joining bootstrap proportions. Named bacterial
sequences and GenBank accession numbers are listed in Appendix 3; genotype groups for
fungi are indicated by letters. Sequence data representing previously described bacterial
endosymbionts of Glomeromycota and Mucoromycotina (9, 10, 52) are marked with
solid squares.
Figure 3. Phylogenetic relationships of fungal endophytes with representative
Dothideomycetes based on majority rule consensus tree from Bayesian analysis. Branch
support values indicate parsimony bootstrap proportions (>70%; before slash) and
Bayesian posterior probabilities (>95%; after slash). Taxon labels in bold indicate
endophytes isolated in this study and screened for bacterial symbionts. Isolate names are
85
followed by plant species and collection site. Endophytes in which endohyphal bacteria
were observed indicate the GenBank accession number for the top 16S BLAST match,
taxonomic placement, and 16S rDNA genotype group. Closed circles indicate infections
that were observed initially but were lost during cultivation. The open circle (isolate
6731) represents an infected endophyte from another study (1).
Figure 4. Phylogenetic relationships of endophytes with representative Eurotiomycetes
based on majority rule consensus tree from Bayesian analysis of 25 LSU rDNA
sequences. Branch support values indicate parsimony bootstrap proportions (>70%;
before slash) and Bayesian posterior probabilities (>95%; after slash). Taxon labels in
bold indicate endophytes isolated in this study and screened for bacterial symbionts.
Isolate names are followed by plant species and collection site. Endophytes in which
endohyphal bacteria were observed indicate the GenBank accession number for the top
16S BLAST match, taxonomic placement, and 16S rDNA genotype group. The closed
circle (isolate 11164) indicates an infection was lost during cultivation. The open circle
(isolate 4466) represents an infected endophyte from another study (1).
Figure 5. Phylogenetic relationships of endophytes with representative Sordariomycetes
based on majority rule consensus tree from Bayesian analysis of 95 LSU rDNA
sequences. Branch support values indicate parsimony bootstrap proportions (>70%;
before slash) and Bayesian posterior probabilities (>95%; after slash). Taxon labels in
bold indicate endophytes isolated in this study and screened for bacterial symbionts.
Isolate names are followed by plant species and collection site. Endophytes in which
86
endohyphal bacteria were observed indicate the GenBank accession number for the top
16S BLAST match, taxonomic placement, and 16S rDNA genotype group. Closed circles
indicate infection was lost during cultivation. The open circle (isolate 6722) represents
infected endophyte from another study (1).
87
Figure 1.
100/98
Figure 2.
96/99
91/100
90/-
0.10
Candidatus Glomeribacter gigasporarum * AJ251633.1 n
Candidatus Glomeribacter gigasporarum * X897272 n
100/Candidatus Glomeribacter gigasporarum * AJ251634.1 n
100/Candidatus Glomeribacter gigasporarum * AJ251635.1 n
98/100 Burkholderia sp 20577 * AJ938141.1 n
100/- Burkholderia rhizoxinica * AJ938142.1 n
100/98
Burkholderia sp 30887 * AJ938143.1 n
100/Burkholderia sp 69968 * AJ938144.1 n
97/100
Burkholderia fungorum * AF215705
98/100
Burkholderia sordidicola * AF512827
Burkholderia cepacia ATCC25416T * AB334766
99/100 99/100
Burkholderia pseudomallei ATCC23343 * CP000573
96/100
Ralstonia solanacearum ATCC11696 * EF016361
Ralstonia mannitolilytica * AJ270258
100/9147 Penicillium * P. orientalis NC * EU236303 * E
99/100
9094 Lecythophora * P. orientalis UA * EU236303 * E
100/100
9060 Aureobasidium * P. orientalis UA * EU236303 * E
100/100/9106clA Preussia * C. arizonica UA * EU236303 * E
100/9120 Monodictys * C. arizonica UA * EU236303 * E
98/9128 Cladosporium * P. orientalis NC * EU236303 *E
99/9096 Hormonema * P. orientalis UA * DQ490310 * N
94/100
9112 Lecythophora * C. arizonica UA * DQ257419 * P
98/100
Nitrosomonas sp. BF16c52 * AF386747
96/100
Xanthomonas campestris LMG568T * AM920689
97/100
Xanthomonas oryzae LMG5047 * NR_026319
100/9133 Botryosphaeria * P. orientalis NC * DQ984599 * C
94/100
9135 Phyllosticta * P. orientalis NC * DQ984599 * C
100/9106 Preussia * C. arizonica UA * DQ984599 * C
100/- 9143 Pestalotiopsis * P. orientalis NC * DQ984599 * C
100/- 9055 Alternaria * P. orientalis UA * DQ984599 * C
98/9122 Pestalotiopsis * P. orientalis NC * DQ984599 * C
99/Pseudomonas syringae * AJ308316
93/9026 Alternaria * P. orientalis UA * FJ596644 * L
99/2611 Dothidea * J. scopulorum CHU * FJ422393 * K
95/9106clB Preussia * C. arizonica UA * FJ267573 * O
91/100
9084b Aureobasidium * P.orientalis UA * EF509196 * F
95/9107 Phoma* C. arizonica UA * EU071476 * F
98/100 9145 Microdiplodia * P. orientalis NC * AF130967 * D
98/9149b Phyllosticta * P. orientalis NC * AF130967 * D
100/Escherichia coli ATCC11775T * NR_024570
100/Erwinia amylovora ATCC15580 * U80195
99/11351 C. arizona IS * EU681952 * I ==
100/Pantoea agglomerans ATCC27155 * FJ357815
9126clb Pestalotiopsis * P. orientalis NC * EU598802.1 * Q
98/9140 Microdiplodia * P. orientalis * NC EF432267.1 * R
100/100
9054 Phoma * P. orientalis UA * EU341143 * M
100/100
Sphingomonas mali IFO10550 * NR_026374
91/100
Caulobacter leidyia ATCC15260 * NR_025324
98/98
Sphingomonas phyllosphaerae * AY563441
96/100
Howardella ureilytica * DQ925472
96/100
Lactovum miscens * AJ439543
98/100
11259 Biscogniauxia * J. deppeana MTL * AM906086 * B
98/11272 Phaeomoniella * J. deppeana IS * AM906086 * B
98/100
Paenibacillus graminis * AJ223987
97/Cohnella phaseoli * EU014872
96/100
11123 Pithya * J. virginiana UA * AJ717382 * G
99/99
11124 J. deppeana MTL * FJ542812 * H ==
100/- Bacillus anthracis * AM747220
98/100
Bacillus cereus ATCC31293 * FJ601653
99/99
100/100 Bacillus mycoides * AF155956
11394 J. virginiana UA * EU071553 * J ==
100/100
Bacillus licheniformis * AM913932
98/100
11180 J. osteosperma CHU * FJ514812 * A ==
100/100
11035 Pestalotiopsis * J. deppeana MTL * FJ194961 * A
100/99
11219 C. arizonica MTL * NR_024931 * A ==
100/99
11136 C. arizonica MTL * FJ549011 * A ==
100/- 11164 Phaeomoniella * J. deppeana MTL * FJ549011 * A
11290 J. deppeana MTL * FJ549011 * A ==
90/100
Desulfomicrobium orale * NR_025378
Desulfomicrobium norvegicum * NR_025407
86/100
Campylobacter helveticus * NR_025948
Campylobacter jejuni subsp. jejuni * CP000768
Bacteroides ovatus ATCC8483T * X83952
Bacteroides fragilis ATCC25285T * CR626927
94/100
88
Figure 3.
89
99/100
100/100
98/-
79/100
87/100
100/100
100/-
100/100
100/100
100/-
100/-
100/100
100/-
83/99/100
98/100
100/100
100/-
100/100
100/100
100/-
100/100
100/100
97/-
78/99/-
100/81/100
90/-
98/-
87/-
-/100
97/-
99/-
-/-
97/-
-/-
90/100/-
100/100
-/-
100/-/-
100/-
100/-
88/-
-/-
100/100
100/100
94/98
99/98
100/100
-/98
99/-
100/100
99/-
100/100
100/-
-/-
100/100
98/77/-
-/-
100/100
100/89/-
100/100
94/100/100
100/-
100/-
82/-
-/-
85/71/-
100/82/73/-
98/100/100
76/-
-/-
99/100
100/100
97/100
100/99
100/99
100/100
100/100
97/100
-/-
-/100/100
99/-
-/-
72/-
100/-
Dendrographa minor
Botryosphaeria ribis
9133 Platycladus orientalis NC * DQ984599 (Xanthomonadaceae) * B
469 Laetia thamnia - Panama
2834 Dryas integrifolia - Quebec
2779 Dryas integrifolia - Quebec
3300 Dryas integrifolia - Quebec
1182 Magnolia grandiflora - North Carolina
6730 Swartzia simplex - Panama
6731 Swartzia simplex - Panama * O
435b Laetia thamnia - Panama
9149b Platycladus orientalis UA * AF130967 (Enterobacteriaceae) * C
9130 Platycladus orientalis NC
9144 Platycladus orientalis NC
9135 Platycladus orientalis NC * DQ984599 (Xanthomonadaceae) * B
9134 Platycladus orientalis NC
9129 Platycladus orientalis NC
Microxyphium citri
Raciborskiomyces longisetosum
9128 Platycladus orientalis NC * EU236303 (Burkholderiaceae) * D
2712 Huperzia selago - Quebec
2722 Huperzia selago - Quebec
462 Laetia thamnia - Panama
4140 Dryas integrifolia - Quebec
3358A Dryas integrifolia - Quebec
4221 Dryas integrifolia - Quebec
3326 Picea mariana - Quebec
5007 Picea mariana - Quebec
Sydowia polyspora
1029 Magnolia grandiflora - North Carolina
DC2611 Juniperus scopulorum CHU * FJ422393 (Moraxellaceae) * H *
4109 Huperzia selago - Quebec
Delphinella strobiligena
4947A Picea mariana - Quebec
4089 Picea mariana - Quebec
Dothidea insculpa
Dothidea sambuci
Dothidea ribesia
Stylodothis puccinioides
9096 Platycladus orientalis UA * DQ490310 (Oxalobacteraceae) * K
Discosphaerina fagi
3275 Dryas integrifolia - Quebec
3335 Dryas integrifolia - Quebec
3353 Dryas integrifolia - Quebec
9042 Platycladus orientalis UA
9060 Platycladus orientalis UA * EU236303 (Burkholderiaceae) * D
9079 Platycladus orientalis UA
9084b Platycladus orientalis UA * EF509196 (Pasteurellaceae) * F
9002 Platycladus orientalis UA
9009b Platycladus orientalis UA
9028 Platycladus orientalis UA
9031 Platycladus orientalis UA
9036 Platycladus orientalis UA
3329 Picea mariana - Quebec
3377 Picea mariana - Quebec
Lojkania enalia
Trematosphaeria heterospora
5095 Picea mariana - Quebec
Westerdykella cylindrica
Preussia terricola
9106 Cupressus arizonica UA * DQ984599 (Xanthomonadaceae) * B
9116 Cupressus arizonica UA
9120 Cupressus arizonica UA * EU236303 (Burkholderiaceae) * D
9104 Cupressus arizonica UA
9051 Platycladus orientalis UA
5718 Dryas integrifolia - Quebec
Byssothecium circinans
3323 Dryas integrifolia - Quebec
Bimuria novae zelandiae
Letendraea helminthicola
9203 Juniperus virginiana NC
9200 Juniperus virginiana NC
11251 Cupressus arizonica MTL
9149a Platycladus orientalis UA
9145 Platycladus orientalis NC * AF130967 (Enterobacteriaceae) *C
9140 Platycladus orientalis NC * FJ756346.1 (Enterobacteriaceae) * O
9137 Platycladus orientalis NC
Phoma glomerata
9158 Platycladus orientalis NC
9054 Platycladus orientalis NC * EU341143 (Sphingomonadaceae) * J
9107 Cupressus arizonica UA * EU071476 (Pasteurellaceae) * F
9065 Platycladus orientalis UA
5654 Picea mariana - Quebec
9005 Cupressus arizonica UA
9008 Cupressus arizonica UA
9007 Platycladus orientalis UA
9018 Platycladus orientalis UA
9015 Platycladus orientalis UA
9027 Platycladus orientalis UA
9030 Platycladus orientalis UA
Curvularia brachyspora
Cochliobolus heterostrophus
Setoshaeria monoceras
Pyrenophora tritici repentis
9055 Platycladus orientalis UA * DQ984599 (Xanthomonadaceae) * B
9026 Platycladus orientalis UA * NR_026208 (Moraxellaceae) * I
9021 Platycladus orientalis UA
9009a Platycladus orientalis UA
9057 Cupressus arizonica UA
9059 Cupressus arizonica UA
9058 Cupressus arizonica UA
Setomelanomma holmii
Ampelomyces quisqualis
Phaeosphaeria avenaria
2851 Huperzia selago - Quebec
2706 Huperzia selago - Quebec
5246 Huperzia selago - Quebec
90
Figure 4.
Peltula umbilicata
Arthroderma curreyi
100/100
Auxarthron zuffianum
100/-
Aphanoascus fulvescens
100/100
Coccidioides posadasii
100/-
Paracoccidioides brasiliensis
100/100
Ascosphaera apis
100/100
Eremascus albus
100/-
Byssochlamys nivea
100/100
100/100
Penicillium freii
9147 P. orientalis NC * EU236303 (Burkholderiaceae) * C
Pyrenula pseudobufonia
Pyrenula cruenta
100/-
4466 Picea mariana * Quebec * O
100/100
11247 Juniperus deppeana MIN
100/100/100
-/-
11272 J. deppeana IS * AM906086 (Paenibacillaceae) * A
11164 J. deppeana MTL* FJ549011 (Paenibacillaceae) * E
100/-
Dermatocarpon luridum
100/-
Verrucaria pachyderma
-/-
Capronia mansonii
422 Laetia thamnia * Panama
100/-
100/-
Exophiala jeanselmei
100/-
Capronia pilosella
100/100/-
Ceramothyrium carniolicum
Glyphium elatum
Figure 5.
91
100/100
100/100
-/99
100/100
-/-
98/98
-/100
-/100
-/95
100/100
-/95
100/100
100/100
-/-/100
-/-
-/99
-/100
100/100
-/95
100/100
-/-/100
100/100
-/99
-/100
-/-
-/-
-/-
-/-/100
100/100
100/100
-/100
-/-
100/96
100/100
100/100
100/99
-/-
-/-
-/100/100
-/100
-/-
-/100
-/96
-/100
100/100
-/-
100/100
-/99
100/99
-/-/100
-/100/94
100/100
-/-/-
-/100
100/100
-/100
99/-
100/100
-/99
100/100
-/-
100/100
-/-/-
100/100
-/-
-/-/100
-/100/100
-/-
-/95
-/100
89/-
Leotia lubrica
Lulworthia fucicola
Lulworthia grandispora
Thyridium vestutum
Gaeumannomyces graminis var graminis
6702 Trichilia tuberculata - Panama
Diaporthe phaseolorum
Valsa ambiens
Cryptodiaporthe corni
Cryphonectria cubensis
Cryphonectria havanensis
Melanconis marginalis
Discula destructive
Gnomonia setacea
Gnomoniella fraxini
Plagiostoma euphorbiae
Cephalotheca sulfurea
Menispora tortuosa
Camarops microspora
Farrowia longicollea
Farrowia seminude
Chaetomium globosum
Neurospora crassa
Sordaria macrospora
Sordaria fimicola
9038 Platycladus orientalis UA
9097 Cupressus arizonica UA *
9069 Platycladus orientalis UA
9093 Platycladus orientalis UA
9094 Platycladus orientalis UA * EU236303 (Burkholderiaceae) * D
9092 Platycladus orientalis UA
9085 Platycladus orientalis UA
Oxydothis frondicola
Microdochium nivale
Hyponectria buxi
4653 Picea mariana - Quebec
Arthrinium phaeospermum
Apiospora sinensis
6722 Faramea occidentalis - Panama * O
9143 Platycladus orientalis NC * DQ984599 (Xanthomonadaceae) * B
9126 Platycladus orientalis NC * DQ984599 (Xanthomonadaceae) * N
9121 Platycladus orientalis NC
9089 Platycladus orientalis UA
11035 Juniperus deppeana SKY * FJ194961 (Paenibacillaceae) * E
Cryptosphaeria eunomia
Fasciatispora petrakii
Cainia graminis
Seynesia erumpens
Hypoxylon fragiforme
256 Laetia thamnia - Panama
2204 Pinus taeda - NC
6717 Gustavia superba - Panama
11122 Cupressus arizonica SKY
9202 Juniperus virginiana NC
9201 Juniperus virginiana NC
Astrocystis cocoes
Rosellinia necatrix
Xylaria acuta
Xylaria hypoxylon
6728 Faramea occidentalis - Panama
Halorosellinia oceanica
Pleurothecium recurvatum
6756 Gustavia superba - Panama
6741 Theobroma cacao - Panama
6733 Swartzia simplex - Panama
412 Laetia thamnia - Panama
6749 Theobroma cacao - Panama
6714 Gustavia superba - Panama
151 Laetia thamnia - Panama
Microascus trigonosporus
Ascosalsum cincinnatulum
Magnisphaera stevemossago
Sagaaromyces abonnis
Corollospora maritima
Varicosporina ramulosa
Ascosacculus heteroguttulatus
Natantispora lotica
Halosarpheia marina
425 Laetia thamnia - Panama
Torrubiella luteorostrata
416 Laetia thamnia - Panama
6708 Gustavia superba - Panama
4939 Picea marina - Quebec
Balansia henningsiana
Hydropisphaera erubescens
Cordyceps khaoyaiensis
4780 Dryas integrifolia - Iqaluit
Emericellopsis terricola
9154 Platycladus orientalis NC
9168 Platycladus orientalis NC
Hypocrea citrina
Verticillium epiphytum
Hypomyces polyporinus
Cordyceps irangiensis
Cordyceps sinensis
92
Appendix 1A. GenBank accession numbers for LSU rDNA sequences of endophytic fungi for
phylogenetic analyses. All endophyte sequences were obtained and published by Higgins et al.,
2007.
Endophyte isolate
Accession no.
151
256
412
416
422
425
435b
462
469
1029
1182
2204
2706
2712
2722
2779
2834
2851
3275
3300
3323
3326
3329
3335
3353
3358A
3377
4089
4109
4140
4221
4466
4653
4780
4939
4947A
5007
5095
5246
EF420086
EF420088
EF420089
EF420090
EF420091
EF420092
EF420093
EF420094
EF420095
EF420084
EF420085
EF420087
DQ979418
DQ979419
DQ979420
DQ979422
DQ979423
DQ979424
DQ979425
DQ979427
DQ979428
DQ979429
DQ979430
DQ979431
DQ979432
DQ979433
DQ979434
DQ979437
DQ979438
DQ979440
DQ979441
DQ979444
DQ979449
DQ979452
DQ979453
DQ979454
DQ979455
DQ979457
DQ979458
93
5654
5718
6702
6708
6714
6717
6722
6728
6730
6731
6733
6741
6749
6756
DQ979462
DQ979463
EF420096
EF420097
EF420098
EF420099
EF420100
EF420101
EF420102
EF420103
EF420104
EF420105
EF420106
EF420107
94
Appendix 1B. Representative Ascomycota species used in phylogenetic analyses with
GenBank identification numbers. Sequences for Coccidioides posadasii were obtained from
TIGR (http://www.tigr.org).
Taxon
Ampelomyces quisqualis
Aphanoascus fulvescens
Apiospora sinensis
Arthrinium phaeospermum
Arthroderma curreyi
Ascosacculus heteroguttulatus
Ascosalsum cincinnatulum
Ascosphaera apis
Astrocystis cocoes
Auxarthron zuffianum
Balansia henningsiana
Bimuria novae-zelandiae
Botryosphaeria ribis
Byssochlamys nivea
Byssothecium circinans
Cainia graminis
Camarops microspora
Capronia mansonii
Capronia pilosella
Cephalotheca sulfurea
Ceramothyrium carniolicum
Chaetomium globosum
Coccidioides posadasii
Cochliobolus heterostrophus
Cordyceps irangiensis
Cordyceps khaoyaiensis
Cordyceps sinensis
Corollospora maritima
Cryphonectria cubensis
Cryphonectria havanensis
Cryptodiaporthe corni
Cryptosphaeria eunomia var. eunomia
Curvularia brachyspora
Delphinella strobiligena
Dendrographa leucophaea f. minor
Dermatocarpon luridum
Diaporthe phaseolorum
Discosphaerina fagi
Discula destructiva
Dothidea insculpta
Dothidea ribesia
Dothidea sambuci
Emericellopsis terricola
Eremascus albus
Exophiala jeanselmei
Accession no.
31415592
33113349
27447246
27447247
33113367
34453082
34453080
11120650
27447238
33113353
45155172
15808399
11120642
33113391
15808400
20334356
27447236
11120644
12025061
20334357
11120645
13469777
TIGR222929
45775574
13241779
13241765
29466997
22203655
22671402
22671403
22671407
27447241
12025063
15808401
47499217
52699696
1399144
15808402
22671422
52699697
15808403
45775610
1698507
11120651
4206347
95
Fasciatispora petrakii
Farrowia longicollea
Farrowia seminuda
Gaeumannomyces graminis var. graminis
Glyphium elatum
Gnomonia setacea
Gnomoniella fraxini
Halorosellinia oceanica
Halosarpheia marina
Hydropisphaera erubescens
Hypocrea citrina
Hypomyces polyporinus
Hyponectria buxi
Hypoxylon fragiforme
Leotia lubrica
Letendraea helminthicola
Lojkania enalia
Lulworthia fucicola
Lulworthia grandispora
Magnisphaera stevemossago
Melanconis marginalis
Menispora tortuosa
Microascus trigonosporus
Microdochium nivale
Microxyphium citri
Natantispora lotica
Neurospora crassa
Oxydothis frondicola
Paracoccidioides brasiliensis
Peltula umbilicata
Penicillium freii
Phaeosphaeria avenaria
Phoma glomerata
Plagiostoma euphorbiae
Pleurothecium recurvatum
Preussia terricola
Pyrenophora tritici-repentis
Pyrenula cruenta
Pyrenula pseudobufonia
Raciborskiomyces longisetosum
Rosellinia necatrix
Sagaaromyces abonnis
Setomelanomma holmii
Setosphaeria monoceras
Seynesia erumpens
Sordaria fimicola
Sordaria macrospora
Stylodothis puccinioides
Sydowia polyspora
Thyridium vestitum
Torrubiella luteorostrata
Trematosphaeria heterospora
Varicosporina ramulosa
27447243
13469782
13469784
16565889
17104831
16565895
16565884
27447237
34453085
45155165
45775578
32261014
27447249
27447244
45775573
15808405
15808406
22203665
22203666
34453095
22671436
45775611
1399149
2565289
11120643
34453084
13469785
27447250
2271524
15216700
52699706
45775613
31415593
22671445
45775614
45775615
45775601
12025090
52699710
15808410
27447239
34453078
28144315
15808411
12025093
45155203
37992293
11120648
45775604
45775600
13241778
15808412
22203671
96
Verrucaria pachyderma
Verticillium epiphytum
Valsa ambiens
Westerdykella cylindrica
Xylaria acuta
Xylaria hypoxylon
15216679
8777749
16565896
11120649
45775605
45775577
Appendix 2. Details of phylogenetic analyses for three classes of fungi.
Parsimony
analyses
Bayesian
analyses
Class
Sequences
(this study/
named taxa)
Included
characters
(total)
Length of
shortest trees
Trees excluded
as burn-in
Trees in
majority rule
consensus
Dothideomycetes
107 (49/58)
1208 (4595)
761
3585
5415
Eurotiomycetes
25 (4/21)
1056 (4443)
418
3000
3400
Sordariomycetes
95 (17/78)
1218 (4605)
1313
5550
8890
97
Appendix 3. Representative species of bacteria used in phylogenetic analyses with GenBank
identification numbers.
Taxon
Bacillus anthracis
Bacillus cereus sp.
Bacillus sp. L164
Bacillus mycoides
Bacteroides fragilis
Bacteroides ovatus ATCC 8483T
Burkholderia cepacia ATCC 25416T
Burkholderia fungorum
Burkholderia pseudomallei ATCC 23343
Burkholderia rhizoxinica
Burkholderia sordicola
Burkholderia sp 20577
Burkholderia sp 30887
Burkholderia sp 69968
Campylobacter helveticus
Campylobacter jejuni subsp jejuni
Candidatus Glomeribacter gigasporarum
Candidatus Glomeribacter gigasporarum
Candidatus Glomeribacter gigasporarum
Candidatus Glomeribacter gigasporarum
Caulobacter leidyia strain ATCC 15260
Cohnella phaseoli
Desulfomicrobium norvegicum
Desulfomicrobium orale
Erwinia amylovora ATCC15580
Escherichia coli ATCC11775T
Howardella ureilytica strain
Lactovum miscens
Nitrosomonas sp. BF16c52
Paenibacillus graminis
Pantoea agglomerans ATCC27155
Pseudomonas syringae
Ralstonia mannitolilytica
Ralstonia solanacearum ATCC 11696
Sphingomonas mali IFO10550
Sphingomonas phyllosphaerae
Xanthomonas campestris pv. campestris
Xanthomonas oryzae strain LMG 5047
GenBank
Accession
AM747220
FJ601653
AM913932
AF155956
CR626927
X83952
AB334766
AF215705
CP000573
AJ938142.1
AF215705
AJ938141.1
AJ938143.1
AJ938144.1
NR_025948
CP000768
X897272
AJ251633.1
AJ251634.1
AJ251635.1
NR_025324
EU014872
NR_025407
NR_025378
U80195
NR_024570
DQ925472
AJ439543
AF386747
AJ223987
FJ357815
AJ308316
AJ270258
EF016361
NR_026374
AY563441
AM920689
NR_026319
98
Appendix 4. Percentages of taxonomic orders of Pezizomycotina obtained in each biogeographic
region based on FMP curated ITS database results. Remaining taxa not shown here include
mitosporic fungi, fungi of uncertain placement, unidentifiable fungi, and a small number of isolates
from lineages other than the Pezizomycotina, such that columns may not sum to 100%.
Percentage of isolates by
taxonomic orders
SKY
MOG
CHU
UA
DUKE
Botryosphaeriales
Capnodiales
Chaetothyriales
Coniochaetales
Diaporthales
Dothideales
Eurotiales
Halosphaeriales
Helotiales
Hypocreales
Myriangiales
Pezizales
Pleosporales
Sordariales
Xylariales
11.1%
11.1%
0
0.7%
0.7%
7.4%
0
0
5.9%
0
0
25.2%
5.2%
0.7%
18.5%
9.9%
1.4%
1.4%
4.2%
0
8.5%
0
0
5.6%
0
0
21.1%
11.3%
1.4%
12.7%
1.6%
1.6%
0
3.2%
3.2%
4.8%
9.7%
3.2%
12.9%
3.2%
0
27.4%
8.1%
0
8.1%
0
11.3%
1.6%
6.5%
0
12.9%
0
3.2%
0
0
0
4.8%
35.5%
3.2%
1.6%
44.0%
1.2%
0
0
2.4%
0
1.2%
0
2.4%
4.8%
1.2%
1.2%
4.8%
0
17.9%
99
APPENDIX C
IAA PRODUCTION BY ENDOPHYTIC PESTALOTIOPSIS NEGLECTA IS
ENHANCED BY AN ENDOHYPHAL BACTERIUM
100
IAA production by endophytic Pestalotiopsis neglecta is enhanced by an endohyphal
bacterium
Michele T. Hoffman1, Malkanthi K. Gunatilaka1, E. M. Kithsiri Wijeratne2, A. A. Leslie
Gunatilaka2 and A. Elizabeth Arnold1
1. College of Agriculture and Life Sciences
Division of Plant Pathology and Microbiology
School of Plant Sciences
1140 E. South Campus Drive, Forbes 303
University of Arizona
Tucson, AZ 85721 USA
2. Southwest Center for Natural Products Research and Commercialization
Office of Arid Lands Studies
250 E. Valencia Road
University of Arizona
Tucson, Arizona 85706 USA
For correspondence: AE Arnold, Tel: 520.621.7212; Fax: 520.621.9290; Email:
[email protected]
Running head: Endohyphal bacterium enhances IAA production by fungal endophyte
Contains 5 figures, and 2 appendices
101
Abstract
A variety of plant-associated microbes produce phytohormones with pervasive effects on
plant growth. Specifically, indole-3-acetic acid (IAA) production has been recorded
among diverse plant pathogens, mycorrhizal and rhizosphere endophytes, and in
endophytic bacteria and yeasts from healthy, above-ground plant tissues. Here, we show
for the first time that a filamentous foliar endophyte, Pestalotiopsis neglecta, produces
IAA, and that its production is significantly enhanced when the endophyte hosts an
endohyphal bacterium (Luteibacter sp.). When cultivated axenically, the bacterium does
not produce IAA in culture. Both P. neglecta and the P. neglecta/Luteibacter complex
appear to rely on L-tryptophan for IAA synthesis. Culture filtrate from the endophytebacterium complex significantly increased root growth in vitro in seedlings of tomato
relative both to controls and to filtrate from the endophyte alone. Our results reveal that
this endophyte, recovered from a coniferous host (Platycladus orientalis), has the
potential to strongly influence growth of a distantly related plant, demonstrate the
previously unknown role of an endohyphal bacterium in enhancing phytohormone
production by a plant-symbiotic fungus, and provide a new lens with which to view the
considerable genotypic, species-level, and functional diversity of foliar endophytic fungi.
Keywords: bacterial endosymbionts, IAA, auxins, Pestalotiopsis, Luteibacter,
Xanthomonadales, fungal endophytes, phylogeny
102
Introduction
Benefits conferred to plants by mycorrhizal fungi, including enhanced uptake of nutrient
and water, are well-documented and generally represent an intuitively straightforward
alignment of plant- and fungal fitness (Parniske, 2008; Bonfante and Anca, 2009;
Kobayashi and Crouch, 2009; Hoeksema et al., 2010). In contrast, the potential
contributions of foliar endophytes to plant physiology are both less obvious and less
thoroughly studied. Such contributions are especially difficult to resolve in the case of
endophytes associated with woody plants, which frequently comprise an exceptional
richness of highly localized infections at the scale of only a few millimeters of leaf area
(Carroll and Petrini, 1983; Lodge et al., 1996; Arnold et al., 2000; Arnold, 2007). These
Class 3 endophytes – a term used to refer to the species-rich, phylogenetically diverse,
horizontally transmitted, tissue-specific, and localized endophytes known from all major
lineages of land plants (Rodriguez et al., 2009) – are a fundamental feature of plant
biology, occurring in plants from the tundra to tropical rainforests (Higgins et al., 2007;
Arnold and Lutzoni, 2007). The benefits or costs they extend to their hosts, the nature of
their interactions with host tissues on the intimate scales of cells and molecules, and the
mechanisms by which they escape, tolerate, or prevent induction of host defenses
represent three major questions in the study of these ubiquitous plant symbionts (Arnold
et al., 2003; Waller et al., 2005; Arnold and Engelbrecht, 2007).
Over recent decades, a large number of studies have documented the ability of
diverse microbes to synthesize phytohormones such as gibberellins, ethylene, cytokinins,
jasmonic acid, abscisic acid, and especially indole-3-acetic acid (IAA), often with
profound effects on plant growth, tissue differentiation, and reproduction (Costacurta and
103
Vanderleyden, 1995; Glick, 1995; Lindow et al., 1998; Robinson et al., 1998; Koga,
1995; Maor et al., 2004; Sirrenberg et al., 2007; Spaepen et al., 2007). Most of these
studies have focused on plant-pathogenic microbes, mycorrhizal fungi, rhizosphere
endophytes, and endophytic bacteria and yeasts from above-ground tissues (Gruin, 1959;
Sosa-Morales et al., 1997; Nassar et al., 2005; van der Lelie et al., 2009; Chagué et al.,
2009; Xin et al., 2009). Together, they reveal that microbial production of IAA is
phylogenetically widespread, encompassing both bacteria (e.g., Erwinia herbicola; Yang
et al., 2006; Pantoea agglomerans; Barash and Manulis-Sasson, 2009) and diverse
lineages of fungi (e.g., Mucoromycotina, Basidiomycota, and some Ascomycota; Strack
et al., 2003; Bajo et al., 2008; Reineke et al., 2008).
In many cases, IAA production by plant-associated microbes strongly influences
plant physiology in ways that are relevant to plant-microbe interactions. For example,
production of IAA by some mycorrhizal fungi stimulates production of plant biomass and
promotes disease resistance (Bonfante and Anca, 2009). Sirrenberg et al. (2007) noted an
increase in Arabidopsis root growth following exposure to diffusible IAA in filtrate from
liquid cultures of the rhizosphere fungus Piriformospora indica (Sebacinales,
Basidiomycota). Microbial IAA production can inhibit plant hypersensitive responses,
limiting the production of plant-defense compounds such as chitinase and glucanase
enzymes (Tudzynski and Sharon, 2002).
Here, we show for the first time that Pestalotiopsis neglecta, a filamentous foliar
endophyte recovered from Platycladus orientalis (Cupressaceae), produces IAA in vitro,
and that its production is enhanced significantly when the endophyte hosts an endohyphal
bacterium (Luteibacter sp.). Culture filtrate from the endophyte-bacterium complex
104
significantly increased root growth in seedlings of a distantly related plant (tomato)
relative both to controls and to filtrate from the endophyte alone. Our results highlight the
previously unknown role of an endohyphal bacterium in enhancing phytohormone
production by a plant-symbiotic fungus and provide a new lens with which to view the
remarkable genotypic, species-level, and functional diversity of foliar endophytic fungi.
Methods
Identification of fungal endophyte 9143
As part of a larger study, an endophytic fungus (9143) was collected from surfacesterilized foliage of a mature, healthy individual of Platycladus orientalis (Cupressaceae)
in Durham, NC (Hoffman and Arnold, 2008). The isolate was archived as a living
voucher in sterile water at the Robert L. Gilbertson Mycological Herbarium (ARIZ) at the
University of Arizona. Previous phylogenetic analyses (Hoffman and Arnold, 2010)
confirmed ordinal placement of this isolate (Xylariales) but were insufficient to identify it
to species. After observing its bacterial endosymbiont and IAA production (below), we
identified the isolate on the basis of conidial morphology (Steyaert, 1953) and then
confirmed our designation using phylogenetic analyses.
For morphological characterization, we examined conidia of cultures grown on
2% malt extract agar media for 7 days (Appendix 1). For phylogenetic analyses, we
examined ITS rDNA sequence data for 9143 (obtained previously; see Hoffman and
Arnold, 2008) in the context of 59 publicly available ITS rDNA sequences for
Pestalotiopsis spp. (Jeewon et al, 2003, 2004; Wei et al., 2007; Watanabe et al, 2010).
We manually aligned these 60 sequences using Mesquite version 1.06 (Maddison and
105
Maddison, 2005). Taxon sampling included the breadth of close relatives of P. neglecta,
representing our estimation of taxonomic placement based on morphology.
Phylogenetic relationships were inferred using parsimony and Bayesian
Metropolis-coupled Markov chain Monte Carlo (MCMCMC) methods. For maximum
parsimony (MP), a heuristic search with random stepwise addition and tree bisectionreconnection (TBR) was implemented in PAUP* 4b10 (Swofford, 2002). Support was
assessed using parsimony bootstrap (1000 replicates). Bayesian MCMCMC analysis was
implemented in MrBayes v. 3.1.2 (Huelsenbeck and Ronquist; 2001) for 2 sets of 3
million generations each, initiated with random trees, four chains, and sampling every
1000th tree, using GTR+I+G based on sequence evaluation in Modeltest 3.7 (Posada and
Crandall, 1998). After elimination of the first 500 trees as burn-in, the remaining 5002
trees were used to infer a majority rule consensus. Sequence data for this isolate are
deposited in GenBank (ITS data used for species-level identification, as well as
LSUrDNA data from Hoffman and Arnold, 2010; EF419899.1, EF420070.1).
Identification of endohyphal bacterium
Using light microscopy and PCR with 16S rDNA primers, we determined that endophyte
9143 harbored an endohyphal bacterium, which we identified previously only as a
member of the Gammaproteobacteria (Hoffman and Arnold, 2010). To place this
bacterium at the species level, we sampled more densely within the phylum, with a
special focus on Xanthomonaceae, using 36 16S rDNA sequences obtained by BLAST
matches to our 16S rDNA sequence (obtained using 10F/1507R primers; HM117737).
Manual alignment was conducted in Mesquite v. 1.06 (Maddison and Maddison, 2005)
106
and the resulting data set analyzed using parsimony and Bayesian MCMCMC methods.
Relationships were inferred using Felsenstein non-parametric distance methods, saving
10 best trees in each replicate set of 100 in PAUP* 4b10 (Swofford, 2002). Using
maximum parsimony (MP), a heuristic search with random stepwise addition and tree
bisection-reconnection (TBR) was implemented in PAUP*. Support was then assessed
using parsimony bootstrap (1000 replicates). Bayesian analysis was implemented in
MrBayes v. 3.1.2 (Huelsenbeck and Ronquist, 2001) on the CIPRES teragrid portal
(Miller et al., 2009; http://www.phylo.org/sub_sections/portal) for 2 runs of 10 million
generations, initiated with random trees, four chains, and sampling every 1000th tree,
using GTR+I+G based on evaluation in Modeltest 3.7 (Posada and Crandall, 1998).
After elimination of the first 440 trees as burn-in, the remaining 19,122 trees were used to
infer a majority rule consensus.
Production of bacterium-free strain
To eliminate the bacterium from the endophyte, we grew isolate 9143 on 2% MEA
amended with 40µg/ml of antibiotic ciprofloxacin. Infection status (infected, cured)
was confirmed using light microscopy (400X), which ruled out external contaminants,
followed by DNA extraction and 16S rDNA PCR (Hoffman and Arnold, 2010).
Infected and cured cultures of 9143 were stored at -80°C in 80% glycerol. We found no
difference between infected and cured isolates of 9143 in terms of growth rate on 2%
MEA or water agar at 22°C, nor with regard to pH of the growth medium (Appendix 2).
Measurement of indole compound production in vitro
107
Small samples of mycelium from infected and cured isolates were plated on 2% MEA
and allowed to grow for 7 d. Small pieces were removed from the resulting cultures
using a 0.5mm cork borer and used to inoculate three sterile flasks containing 80ml of
Czapek Dox broth (CDB; Hymedia; pH 7.2) augmented with L-tryptophan (5mM). For
each treatment set, one additional flask containing only CDB was used as a negative
control. All flasks were checked for contamination (turbidity) after agitating at 120rpm
at 26°C for 24 h. Contaminated cultures were discarded and replaced.
After 72 hours of agitation, three 1ml aliquots of broth were removed from each
flask and treated with colorimetric Salkowski reagent (0.01 M Fe (NO3)3; 7.0 M
HCIO4) prior to standard spectophotometric measurements at 530nm (Salkowski, 1885;
Ehmann, 1977; Glickmann and Dessaux, 1995). Each 1ml extract was combined with
500µl of Salkowski reagent, incubated at room temperature for 30 minutes, and then
measured once on a Whatman spectrophotometer (530nm). CDB was used as a blank
between readings. The mean of the three readings was calculated for each culture,
concentrations inferred using an IAA standard curve (below), and analyzed with a t-test
in JMP® 8.0 (SAS Institute Inc., Cary, NC.).
The IAA standard curve was inferred using commercial IAA (MW 175.19,
Sigma; 1mM), prepared in a volumetric flask using 95% molecular grade methanol.
Fourteen 1.5ml Eppendorf™ tubes were prepared in proportional ratios with 25-1950µl
of IAA and 50-1975µl of reagent. After incubating in the dark for 30 minutes, each
sample was measured twice. A blank calibration control using 1ml water plus 500µl of
reagent was as a blank between readings.
108
Identification of the indole compound as IAA using chromatography
We used thin layer chromatography (TLC) and high performance liquid chromatography
(HPLC) to identify the indole compound that was observed on the basis of colorimetric
reaction with Salkowski reagent. For chromatographic analysis, two cultures each of
infected and cured mycelia were grown for two weeks in 500mL of sterile CDB
supplemented with 5mM L-tryptophan. One additional culture (9143-infected) was
grown in sterile CDB without L-tryptophan (1.0 L) to determine whether fungus 9143
uses a tryptophan-dependent pathway for IAA production. Each culture was filtered
using Whatman No.1 filter paper and the filtrate (1.0 L) extracted three times with
500mL of ethyl acetate (EtOAc). The combined EtOAc layer was washed three times
with 500mL of H2O, dried over anhydrous Na2SO4, and evaporated under reduced
pressure to yield the EtOAc extract.
Normal phase TLC analysis of the EtOAc extract was performed on aluminumbacked plates coated with 0.20 mm layer of silica gel 60 F254 (E. Merck, Darmstadt).
Spots were visualized by inspection of plates under UV light (254 nm) and after spraying
with Van Urk-Salkowski reagent (Ehmann, 1977) followed by heating. Commercially
available IAA (Sigma) was used as an authentic sample (Eluant: 8% MeOH in
dichloromethane, Rf 0.3). Analytical reversed phase TLC investigations were performed
on aluminum-backed plates coated with 0.20 mm layer of silica gel 60 RP-18 F254S (E.
Merck, Darmstadt; Eluant: 20% MeCN in H2O, Rf 0.5).
Analytical HPLC analysis of the EtOAc extract was performed using a Kromasil
5µm C-18 column (4.6 x 250 mm) on a Shimadzu HPLC system equipped with DGU14A degasser, LC-10ADvp pump, SPD-M10Avp diode array detector and SCL-10Avp
109
system controller utilizing Shimadzu LC-MS solution software. Samples were
redissolved in MeOH (2.0 mg/mL) and injections (10µL) were made with Shimadzu SIL10ADvp auto injector. The mobile phase consisted of H2O/ MeCN/HCOOH
(69.75:30:0.25) with a flow rate of 1.2 mL min-1.
Seedling assays
Cured and infected isolates of 9143 were agitated for 15 days in 200ml CDB + 5µM Ltryptophan. Cultures then were vacuum-filtered (0.44 mm nylon filter) and the liquid
retained in sterile 50ml tubes. The pH was measured from an aliquot of each (pH 5.7 for
9143-infected; pH 4.2 for 9143-cured; pH 7.1 for CDB alone; Denver Instruments pH
meter) and the solutions brought to pH 7.0-7.1, as needed, by amendment with 0.5M
NaOH. Approximately 25ml of this filtrate was further filter-sterilized using 0.2mm
syringe filters into a sterile container and used in seedling assays.
Tomato seeds (ACE 55; The Home Depot®) were surface-sterilized by agitating
in 50% bleach for 12-15 minutes, rinsed in sterile water, and placed on sterile filter paper
in sterile 60mm Petri dishes (ca. 20 seeds/dish). Three milliliters of sterile water was
added to each dish before sealing with Parafilm®. Seeds were allowed to germinate at
25°C for 5 days. Sets of 10 apparently healthy seedlings were chosen haphazardly and
transferred under sterile conditions to new 60mm plates containing sterile filter paper.
Each set of seedlings was watered with 3.5ml of sterile water. Four plates/treatment (40
seedlings/treatment) then received 50µl per seedling of one of five treatments: (a) CDB
from 9143-infected; (b) CDB from 9143-cured; (c) CDB + 5µM L-tryptophan; (d) CDB
+ IAA (0.1mg/ml; pH 7.0); or (e) sterile water.
110
Plates were sealed with Parafilm® and incubated under fluorescent lights at room
temperature for 5 d (12 h light/dark cycle). Seedlings then were harvested and stretched
to full length by mounting to paper with transparent cellophane tape. Shoot and root
lengths to longest points were measured for each seedling using calipers. Data were
analyzed in JMP® 8.0 using ANOVA after normalizing all measurements to the results of
the CDB + IAA treatment (treatment d).
Isolation of the endohyphal bacterium
Incubating the isolate 9143 on 2% MEA at 36°C for 7 d (Appendix 1) resulted in
emergence of bacterial growth from the apparently axenic endophyte culture. We
extracted DNA from the bacterium following Hoffman and Arnold (2010) and sequenced
an 1100bp portion of the 16SrDNA region of using primers 27F /1429R (Lane 1991).
The sequence of this bacterial isolate (BAC182) was identical to that of the bacterium
sequenced directly from endophyte 9143. BAC182 was grown overnight in sterile LB
broth and vouchered in sterile glycerol at -80°C. Using the above methods we found no
evidence of IAA production by axenic cultures of this bacterium (data not shown).
Results
Phylogenetic analyses were used in conjunction with morphology to positively identify
endophyte 9143 as Pestalotiopsis neglecta (Fig. 1, Appendix 1). Multisetulate conidia are
fusiform shaped concolorous cells, lacking knobbed appendages. Phylogenetic analysis
placed this fungal isolate as sister to one of three identified P. neglecta strains from
Watanabe et al. (2010). Our results are congruent with their analysis, placing two other
putative “P. neglecta” sequences in a separate subclade. Reclassification of the
111
taxonomic placement of Pestalotiopsis spp. has been recommended (see Jeewon et al.,
2004; Hawksworth 2005; Wei et al., 2007), with greater emphasis placed on
morphological characteristics combined with rigorous phylogenetic methods.
Phylogenetic analyses of 16S rDNA confirmed placement of fungal endophyte
9143 endohyphal bacterium in the Xanthomonadaceae, with strong support as a member
of the genus Luteibacter (Luteibacter sp.; Fig. 2).
Chromogenic testing indicated that isolate 9143 produced an indole compound in
vitro, with enhanced production in the presence of the endohyphal bacterium (Fig. 3). At
the end of the 14 d cultivation period, mean concentration of the indole compound in
broth from 9143-infected (104.8µg/ml) was 78µg/ml greater than that produced by 9143cured (Fig. 3). Strong differences in indole compound concentration were observed
throughout the 14 d cultivation period (repeated measures ANOVA, F1, 4 = 358.7; p <
0.0001; Fig. 3).
TLC and HPLC identified the compound as indole-3-acetic acid (IAA) and
confirmed that endophyte 9143 produced significantly more IAA when the endohyphal
bacterium was present vs. absent (Fig. 4). No IAA was produced when 9143-infected and
cured isolates were grown in CDB without tryptophan, suggesting a tryptophandependent pathway for IAA production (data not shown).
Growth of tomato seedlings, measured as total seedling length, shoot length, root
length, and root:shoot ratio, differed significantly as a function of treatment (Fig. 5).
Seedlings treated with filtrate from 9143-cured did not differ in any of these measures
relative to the CDB control (Fig. 5, post-hoc Tukey-Kramer test, alpha = 0.05). Seedling
112
length was significantly greater following treatment with 9143-infected, reflecting a
significant enhancement of root elongation (Fig. 5, Tukey-Kramer tests, alpha = 0.05).
Discussion
Endohyphal bacteria have been found previously in living hyphae of plant-associated
Glomeromycota, Mucoromycotina, and several ectomycorrhizal Dikarya (Tuber borchii;
Ascomycota; Laccaria bicolor and Piriformospora indica; Basidiomycota) (Bianciotto et
al., 1996; Barbieri et al., 2000; Bertaux et al., 2003; Levy et al., 2003; Partida-Martinez
and Hertweck, 2005). Recently, we reported their presence in foliar endophytic fungi
representing four classes of Pezizomycotina, and demonstrated that they are both
geographically widespread and phylogenetically diverse (Hoffman and Arnold, 2010).
The present study is the first to (1) definitively identify the partners in a close ecological
relationship between a foliar endophyte and an endohyphal bacterium, (2) show that a
species of Pestalotiopsis (P. neglecta) is capable of producing a phytohormone, (3)
demonstrate that a filamentous foliar endophyte can produce indole-3-acetic-acid (IAA),
(4) show that IAA produced by this endophyte is dependent on the L-tryptophan
pathway, and (5) demonstrate that an endohyphal bacterium (Luteibacter sp.) enhances
this IAA production, with exogenous culture filtrate of the endophyte with its endohyphal
symbiont yielding significantly enhanced growth in roots of a model plant. Together, our
results reveal that products of an endophyte from a coniferous host has the potential to
strongly influence growth of a distantly related plant (see also Rodriguez et al., 2009),
demonstrate the previously unknown role of an endohyphal bacterium in altering
phytohormone production by a plant-symbiotic fungus, and provide a new lens with
113
which to view the considerable genotypic, species-level, and functional diversity of foliar
endophytic fungi.
In the presence of endohyphal Luteibacter sp. and L-tryptophan, P. neglecta
cultivated in CDB produced ca. 100µg/ml of IAA. This amount is likely biologically
significant in the host, as demonstrated both by our seedling assays and by previous
studies with plant-pathogenic and rhizosphere fungi. IAA production observed here was
within the range produced by Ustilago maydis mutant strains (75-262µg/ml) as measured
using the same colorimetric reagents (Sosa-Morales et al., 1997). Using more precise
methods (GC-MS and HPLC-ESI_MS/MS), culture filtrate from Piriformospora indica
measured ca. 0.16µmol of IAA after 4.5 weeks of growth (Sirrenberg et al., 2007),
exceeding that demonstrated here. Isolates of Colletotrichum were found to generate 232µg/ml of IAA using TLC and GC-MS (Robinson et al., 1998; Chagué et al., 2009).
Although our study does not yet address bacterial-fungal-plant interactions during
the endophyte symbiosis per se, it provides a first estimation of one way in which an
apparently facultative bacterial endosymbiosis can influence interactions between an
endophytic fungus and its host. Further experiments involving the establishment of this
endophytic fungus within host tissues, and exploration of relative benefits for hosts more
closely related to that from which the fungus was first isolated, represent important future
directions. The strong response we observed in root growth as a function of filtrate from
cultures containing both the endophyte and its endohyphal symbionts suggests that we
should evaluate the potential for this foliar endophyte to colonize root tissues, and/or
examine how fungi localized in foliage might influence growth in other tissues. We also
are interested to discover whether IAA production by the endophyte or
114
endophyte/endohyphal bacteria complex camouflages the fungus within the host, making
it less detectable by plants and thus enhancing its ability to grow without inducing a
hypersensitive response.
Recent work has shown that even closely related or conspecific fungi can harbor
different endohyphal bacteria (Hoffman and Arnold, 2010). These facultative
associations are by definition flexible and appear to be gained and lost readily (Hoffman
and Arnold, 2010). Our study provides the first evidence that the differences in bacterial
associates of endophytes might play a key role in determining the nature of endophytehost associations, and may account for some of the diversity and ecological plasticity
observed in endophyte-plant interactions (Arnold et al., 2009). More generally,
increasing but still limited exploration of ectohyphal bacteria, endohyphal bacteria, and
mycoviruses have begun to illustrate the powerful but often overlooked ways in which
microbes associated with fungal hyphae can influence the outcome of plant-fungus
interactions (Marquez et al., 2007, Bonfante and Anca, 2009; Kobayashi and Crouch,
2009). Advancing our knowledge of endophyte interactions with plant hosts, other
extrinsic microorganisms, and most recently, diverse endohyphal bacteria, will help
define the functional biology of these diverse and ubiquitous symbionts.
115
Acknowledgments
We thank the Division of Plant Pathology and Microbiology, the Department of Plant
Sciences, the College of Agriculture and Life Sciences at the University of Arizona.
Additional support from the National Science Foundation (NSF-0626520 and 0702825 to
AEA; NSF-IGERT to MTH) is gratefully acknowledged. We thank D.R. Maddison for
sharing pre-release versions of Mesquite and Chromaseq; R. Ryan, M. J. Epps, J. U’Ren,
M. del Olmo Ruiz and A. Woodard for lab assistance and helpful discussion. We are
especially grateful to H. VanEtten for access to chemistry facilities and J. L. Bronstein, L.
S. Pierson, III, and M. J. Orbach for comments on the manuscript. This paper represents a
portion of the doctoral dissertation research of MTH in Plant Pathology and
Microbiology at the University of Arizona.
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Figure legends
Figure 1. Majority rule consensus tree based on Bayesian analyses of 60 ITSrDNA
sequences representing Pestalotiopsis (with Seiridium as outgroup). Support value before
the slash represents maximum-parsimony bootstrap (>70%); number after the slash
represents Bayesian branch support > 90%.
Figure 2. Majority rule consensus tree (using P. syringae as outgroup) based on Bayesian
analyses of 37 proteobacterial 16S rDNA sequences with affinity for Luteibacter spp. in
BLAST analyses. Support value before the slash represents maximum-parsimony
bootstrap >70%, number after the slash represents Bayesian branch support > 90.
Branches in bold indicate neighbor-joining bootstrap support >70%.
Figure 3. Spectrophotometric absorbances at 530nm following treatment of supernatant
from CDB cultures of 9143-infected and 9143-cured with Salkowski reagent, indicating
production of an indole compound (identified as IAA; Fig. 4). Each point represents the
average of three readings (two-tailed t4 = 20.99; p < 0.0001), converted to concentration
amounts using the IAA standard curve (y =0.0108x-0.0049). Error bars indicate standard
deviation.
Figure 4. HPLC analysis (retention time and co-injection) results for (A) IAA standard,
and extracts from (B) 9143-cured and (C) 9143-infected. Elution time was 12.54 minutes
for the IAA standard, 12.89 for 9143-cured, and 12.95 by 9143-infected sample. , IAA
124
comprised 16% of the total sample from 9143-cured, and 29% from 9143-infected (Fig.
2C) based on the area ratio under the peak.
Figure 5. Growth responses of tomato seedlings to pH-consistent supernatant from 9143infected cultures in CDB; supernatant from 9143-cured in CDB; and control treatments
(CDB alone, water). (A) Total seedling length, (B) root length, (C) root to shoot ratio, (D)
shoot length. Data were normalized to measurements for plants that received CDB +
0.1mg/ml IAA treatment (data not shown). Different superscripts within each panel
indicate statistically different means.
125
Figure 1.
126
Figure 2.
127
Figure 3.
9143+
9143-
IAA concentration
(µg/ml)
Days
128
Figure 4.
(A) Indole-3-acetic
acid standard
(B) 9143-cured
(C) 9143-infected
129
Figure 5.
A.
a
B.
F3,156 = 30.2; p< 0.0001
F3,156 = 32.8; p< 0.0001
a
b
b
b
b
c
9143+
9143-
CDB
c
H2 O
9143+
9143-
CDB
H2 O
Seedling length
(mm)
C.
D.
F3,156 = 3.2; p< 0.0001
a
ab
F3,156 = 19.3; p< 0.0001
a
ab
ab
b
c
9143+
9143-
CDB
b
H2 O
Treatments
9143+
9143-
CDB
H2 O
130
Appendix I
Pestalotiopsis spore morphology
Pestalotiopsis neglecta asexual spore morphology description coincides with the morphology of
conidia from endophyte 9143 (Fig A). These spores are fusiform, four-septate, a fuliginous
brown in color, with end cells hyaline. The apical end is short with two or three spreading
setulae, approximately 22um long. The basal end contains a pedicel about 4-7um long
(Steyaert, 1953).
Figure A.
Conidia from 9143-uninfected culture (400X) showing fusiform cells with 4 septae.
Figure B.
Results of growth assays over 14 days for 9143-infected (black diamond) and 9143-cured (open
square) on 2% MEA at pH4.5 (panel A), pH8.0 (panel B), and pH=6.8 (standard 2% MEA;
131
panel C) at 22°C. Panel D shows growth on water agar at 22°C. 9143 did not grow at 36°C.
Error bars indicate standard error of the three replicates performed for all experiments.
A.
B.
C.
D.