Changes in the phyllosphere community of the resurrection fern,
Polypodium polypodioides , associated with rainfall and wetting
Evelyn F. Jackson, Haley L. Echlin & Colin R. Jackson
Department of Biological Sciences, Southeastern Louisiana University, Hammond, LA, USA
Correspondence: Colin R. Jackson,
Department of Biological Sciences, SLU
10736, Southeastern Louisiana University,
Hammond, LA 70402, USA. Tel.: 11 985 549
3444; fax: 11 985 549 3851; e-mail:
[email protected]
Received 13 November 2005; revised 15 March
2006; accepted 19 March 2006.
First published online 8 June 2006.
DOI:10.1111/j.1574-6941.2006.00152.x
Editor: Kornelia Smalla
Keywords
phyllosphere; resurrection fern; 16S rRNA gene;
wetting; enrichment culture; epiphytes.
Abstract
A combination of analyses were used to characterize the changes that occur in a
bacterial community present in the phyllosphere of the epiphytic resurrection fern,
Polypodium polypodioides, as the fern rehydrates from a desiccation-resistant,
physiologically inactive state. Enrichment assays showed an increase in the viable
count of bacteria using labile organic substrates following rainfall. Isolates
obtained from enrichments were predominantly Gram-positive bacteria affiliated
with various groups of the Actinobacteria and Firmicutes. In contrast, sequencing
of 16S rRNA genes clones obtained from whole community DNA revealed that
much of the community was dominated by other taxa, particularly the Alphaproteobacteria. Similar isolates were obtained from both dry and hydrated
P. polypodioides fronds, whereas 16S rRNA gene sequencing of community DNA
revealed different ribotypes on the dry and wet fern, and an overall reduction in
richness following wetting. Wetting also produced changes in phyllosphere extracellular enzyme activity, with an initial burst of activity following rainfall and a
subsequent burst approximately 48 h later. These findings suggest that the resurrection fern harbors a complex phyllosphere community, and that rehydration of the
fern following rainfall may act as an enrichment culture stimulating certain
bacterial populations and changing the overall community structure and activity.
Introduction
The aerial leaf surface or phyllosphere is regarded as a harsh
environment for microorganisms, characterized by longand short-term fluctuations in environmental conditions
such as ultraviolet radiation, nutrient availability, temperature and moisture content (Hirano & Upper, 2000; Lindow
& Brandl, 2003). Historically, many of the studies on phyllosphere microbial communities have focused on bacteria
known to be plant pathogens, such as Pseudomonas syringae
and various species of Erwinia (Lindow & Brandl, 2003).
However, phyllosphere communities have recently been
recognized as containing much higher microbial diversity
and are now being studied with approaches used for other
microbial habitats. Increasing numbers of bacterial species
have been identified from a variety of leaf surfaces using
both traditional enrichment and molecular techniques
(Lindow & Brandl, 2003). In a survey of the bacteria
associated with the leaves of citrus trees that combined
molecular and enrichment approaches, the phyllosphere
community was found to have high species diversity and
the capability of using a variety of carbon sources (Yang
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et al., 2001). More recently, molecular approaches have been
used to show that the bacterial assemblages on maize
respond to changes in ultraviolet radiation (Kadivar &
Stapleton, 2003). Phyllosphere microbial communities have
also been shown to vary both spatially (Kinkel et al., 2000;
Monier & Lindow, 2005) and temporally (Jacques et al.,
1995; Ellis et al., 1999; de Jager et al., 2001) on a variety of
scales. Despite these advances, the vast majority of studies
have focused on plants of economic importance, and very
little is known about the bacterial communities inhabiting
the phyllosphere of nonagricultural plants.
The resurrection fern, Polypodium polypodioides (L), is a
vascular epiphyte that is commonly found growing on the
bark of certain tree species in the southern USA (Pessin,
1925). P. polypodioides and other resurrection plants are
capable of surviving long periods of desiccation, tolerating
almost complete water loss in their vegetative tissues.
Following wetting these plants rehydrate and return to a
fully metabolizing state, typically within 48 h (Scott, 2000;
Proctor & Tuba, 2002). The change in states is readily visible,
as P. polypodioides switches from a curled brown appearance
to lush green growth (Fig. 1). This capability is adaptive for
FEMS Microbiol Ecol 58 (2006) 236–246
237
Changes in the phyllosphere community of the resurrection fern
by P. polypodioides was collected in Ponchatoula, Louisiana,
USA. Live oaks are native to this region of the USA, and are
often colonized by P. polypodioides. There had been no
appreciable rainfall in the area for at least 7 day and the
fronds of P. polypodioides were dry and in the brown, curled
state (Fig. 1). The branch was gently watered with 2.5 cm of
sterile water over a 2 h period, and then exposed to ambient
temperatures and light (but no additional rainfall) for 9 day.
Samples of P. polypodioides were taken after 0 (prior to
wetting), 1, 3, 8, 26, 48, 72, 96 and 216 h. Sampling consisted
of collecting three 7–10 cm long fern fronds for moisture
content and three similarly sized fronds for enzyme assays.
In each case, randomly selected fern fronds from throughout
the branch were taken. Fronds taken for moisture content
were weighed, dried (75 1C, 48 h), reweighed, and moisture
content of the fern determined by subtraction. Fronds taken
for enzyme assays were processed immediately.
Enzyme assays
Fig. 1. Substantial changes in the appearance of the resurrection fern,
Polypodium polypodioides, that occur during periods of drought (upper
panel) and 48 h after rainfall (lower panel). The fern is growing on the
bark of a branch from a live oak (Quercus virginiana).
growth on hard substrates characterized by intermittent
moisture availability, as is typical of epiphytes (Proctor &
Tuba, 2002). The phyllosphere of non-desiccation-tolerant
plants is considered an extreme environment for microorganisms (Hirano & Upper, 2000; Lindow & Brandl, 2003),
and the phyllosphere of resurrection plants might be considered even more extreme in that the plant essentially shuts
down physiological activity during periods of drought.
Studies on other plants have found increases in bacterial
numbers and metabolic activity on wet leaves compared to
dry leaves (Kinkel et al., 2000; Monier & Lindow, 2004), but
no studies have examined the effects of wetting on the microbial community present on desiccation-tolerant plants.
In this study we use a combination of 16S rRNA molecular
techniques with enrichment and enzymatic approaches to
characterize the changes that occur in a phyllosphere community of P. polypodioides during wetting and rehydration.
Materials and methods
Fern sample collection and artificial wetting
A large (c. approximately 1 m) branch from a live oak
(Quercus virginiana Mill.) tree that was heavily colonized
FEMS Microbiol Ecol 58 (2006) 236–246
Fern samples at different stages of wetting were assayed for
the activities of the microbial extracellular enzymes, b-1,4glucosidase (EC 3.2.1.21), acid phosphatase (EC 3.1.3.2),
and phenol oxidase/laccase (EC 1.10.3.2). Individual pinnae
or leaflets (1–2 cm) were cut from the three larger fern
fronds and pooled together. Four randomly selected pinnae
were transferred into 1.5 mL microcentrifuge tubes (three
replicate tubes per enzyme), weighed, and amended with
300 mL of artificial substrate. The substrates for b-1,4glucosidase and acid phosphatase were 5 mM p-nitrophenyl
(pNP)-b-D-glucopyranoside and pNP-phosphate, respectively. The substrate for phenol oxidase was 5 mM L-3,4dihydroxyphenylalanine (L-DOPA). All substrates were dissolved in pH 5.5 acetate buffer (0.1 M acetic acid, 0.1 M
sodium acetate). Duplicate sample controls for each enzyme
consisted of four pinnae amended with acetate buffer;
duplicate substrate controls consisted of the appropriate
substrate without the fern.
Tubes were shaken gently for 1 h at 22 1C, centrifuged
(5 min, 2000 g), and 100 mL of the supernatant transferred to
a microplate well. Supernatants from b-1,4-glucosidase and
acid phosphatase assays received 10 mL 1 M NaOH and
190 mL of water, and absorbance was determined at 410 nm.
Activity for these enzymes was calculated by dividing the
adjusted absorbance by 16.9 (the absorbance of 1 mmol
p-nitrophenol under these specific assay conditions).
The supernatants for the phenol oxidase assays were mixed
with 200 mL of water, absorbance determined at 460 nm,
and activity determined by dividing the adjusted absorbance
by 2.3 (the absorbance of 1 mmol completely oxidized
L-DOPA under these conditions). Final activity for all three
enzymes was expressed as mmol substrate consumed/h/g dry
weight of fern.
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238
Enrichment cultures and plate counts
Based on the results of the enzyme assays experiment (see
below), we decided to focus on ‘dry fern’ and 48 h postwetting (‘wet fern’) for more detailed molecular and enrichment analyses. Eight 7–10 cm long fronds of dry fern (at
least 7 days since rain) were collected aseptically from a
branch of a live oak tree in the same location as above,
immediately prior to a rain event. A second set of fronds were
collected from the same tree branch 48 h after rainfall. Three
fronds from each sample were placed in 30 mL sterile dilution
buffer and shaken vigorously for 5 min to dislodge attached
bacteria. Samples were serially diluted and plated onto four
types of agar: nutrient agar (Difco), R2A agar (Reasoner &
Geldrech, 1985), cellulose agar (5 g cellulose, 0.05 g MgSO4,
0.05 g NH4NO3, 0.3 g K2HPO4, 0.025 g yeast extract, 15 g
agar, 1 L H2O), and glucose agar (5 g glucose, 0.05 g MgSO4,
0.05 g NH4NO3, 0.3 g K2HPO4, 0.025 g yeast extract, 15 g
agar, 1 L H2O). Plates were incubated for up to 10 d at 22 1C,
and the numbers of colony forming units (CFU) determined.
Visibly different colonies were transferred to new plates of the
appropriate agar, isolated, and characterized. Initial characterizations consisted of Gram stains and spore stains to
determine basic microscopic structure. Isolates from both
wet and dry fern samples that appeared to be different based
on colony morphology or microscopy were identified more
thoroughly by 16S rRNA gene sequencing.
Molecular analysis of whole community DNA
and isolates
The remaining five fronds collected from the dry and wet
fern were placed in 13.5 mL of a high salt DNA extraction
buffer (Zhou et al., 1996), and frozen for 14 days prior to
analysis. Samples were thawed, vigorously shaken (5 min),
amended with lysozyme to 15 mg mL 1, and gently shaken
for 30 min at 37 1C. After addition of 100 mL of Proteinase K
solution (10 mg mL 1) the extraction was incubated (37 1C,
30 min) a second time. 1.5 mL 20% SDS solution was added,
and the extraction incubated for 2 h at 65 1C. Following this
third incubation step, the samples were centrifuged (6000 g,
10 min) and the supernatant collected, cleaned by chloroform extraction, and DNA recovered by isopropanol precipitation. DNA was extracted from isolates obtained from
plate counts of dry and wet fern samples following the same
protocol save that extractions were carried out on a smaller
scale (1/20 volume for all reagents).
DNA from both the whole community extracts and
individual isolates was the template in PCR amplifications
using 16S rRNA primers Bac8f and Univ1492 (Amann et al.,
1995; Jackson et al., 2001) under amplification conditions
that have been previously described (Jackson et al., 2001).
PCR products from the two community DNA extracts were
cloned in to artificial plasmid vectors (TA TOPO Cloning,
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E.F. Jackson et al.
Invitrogen, Carlsbad, CA), and clone libraries established for
the dry and wet fern communities. The insert from each
clone was amplified using M13 primers (Invitrogen, Carlsbad, CA), and the same PCR conditions as the original 16S
rRNA amplification. PCR products were screened using
three different restriction digests (EcoRI, RsaI, and HaeIII;
2 h, 37 1C) and clones showing the same restriction pattern
for all three enzymes grouped as a single ribotype (Jackson
et al., 2001). The frequency of ribotypes in each clone library
was used to estimate overall bacterial diversity in each
phyllosphere community using the non-parametric abundance-based estimator SChao1, and the proportion of those
bacteria that were actually recovered in the clone libraries
was determined as coverage (Kemp & Aller, 2004a, b).
DNA sequencing and phylogenetic analysis
A portion of the amplified 16S rRNA gene of each ribotype
in the two clone libraries, and each of the different isolates
obtained in the enrichment study, was sequenced using the
original Bac8f primer. The sequencing protocol yielded
sequence information for the 50–600 bp region of the 16S
rRNA gene. Sequences were compared to those in GenBank
(BLAST search) to identify closest relatives. Each sequence,
and its closest GenBank match, were imported into the ARB
software package (Ludwig et al., 2004) running the ARB 16S
rRNA database (Hugenholtz, 2002) available from the
Ribosomal Database Project-II (Cole et al., 2005). Sequences
were aligned automatically and each alignment checked
manually. The ‘Quick add by parsimony’ function was used
to incorporate our sequences into an existing phylogenetic
tree of 8600 16S rRNA gene sequences in the ARB database
(Hugenholtz, 2002). 16S rRNA gene sequences in this
existing tree that were unrelated to those obtained in this
study were subsequently removed without altering tree
topology, so that phylogenetic trees showing only lineages
relevant to this study were generated. This treeing procedure
should minimize distortions to established bacterial phylogenies that might arise from a phylogenetic analysis of
partial, divergent 16S rRNA gene sequences.
Results
Changes in P. polypodioides appearance and
enzyme activities following wetting
The dry P. polypodioides samples showed a typical curled
brown appearance (Fig. 1) and water content accounted for
less than 20% of their total weight. Following wetting there
was an immediate increase in moisture content to 50% after
1 h, and 65–70% after 3 h, and the fern remained in this state
for 3 days (Fig. 2a). The green, uncurled appearance (Fig. 1)
lasted from approximately 12 h postwetting through 4 days,
when the fern began to dry out. Enzyme activities also
FEMS Microbiol Ecol 58 (2006) 236–246
239
Changes in the phyllosphere community of the resurrection fern
(a)
60
40
20
0
0
β-glucosidase activity
(µmol h−1 gDW−1)
10
2
4
6
8
10
(b)
8
6
4
Viable counts of bacteria associated with dry
and wet P. polypodioides and enrichment and
identification of isolates
2
0
Acid phosphatase activity
(µmol h−1 gDW−1)
0
20
2
4
6
8
10
2
4
6
8
10
(c)
16
12
8
4
0
0
Phenol oxidase
activity (µmol h−1 gDW−1)
event. Thus, there were two peaks in the activity of these
enzymes: the first within hours of wetting, the second
2–3 days later (Fig. 2b, c). Following this second peak, the
activity of b-1,4-glucosidase and acid phosphatase gradually
declined as the fern dried out. Phenol oxidase activity
showed a contrasting pattern in that its activity appeared to
be higher on the dry fern samples, and initially declined with
1–3 h of wetting (Fig. 2d). This was followed by a small
increase in activity after 8 h, and a subsequent decline over
1–2 days. However, as with the hydrolytic enzymes, phenol
oxidase activity peaked again after 3 days, although it
declined again before increasing to its original activities as
the fern dried out.
10
(d)
8
6
Viable counts of bacteria associated with dry fern that were
capable of growth on nutrient agar, R2A, cellulose agar and
glucose agar, were very similar at approximately
2 10 5 CFU g 1 fern dry weight (Fig. 3). The number of
CFUs doubled within 48 h of wetting on nutrient agar and
R2A, and more than tripled on glucose agar. In contrast,
there was no difference in the number of viable bacteria
utilizing cellulose agar from dry fern to wet (Fig. 3). Eightysix colonies (40 from dry fern, 46 wet fern) were isolated
from the different fern samples and media types and selected
for further study. Following basic microscopy and further
observation of colony morphology, the number of clearly
different isolates was much lower and 16 apparently different isolates obtained from the dry fern sample and 19 from
4
8
2
0
0
2
4
6
Time after wetting (d)
8
10
Fig. 2. Physiological changes in Polypodium polypodioides and its phyllosphere microbial community that occur following wetting. Changes
occur in the overall water content of the fern (a), and in the activity of
three extracellular enzymes on the leaf surface: b-glucosidase (b), acid
phosphatase (c), and phenol oxidase (d). Values for enzyme activities are
the means (with standard error) of three separate assays for each sample
point.
showed a rapid response to wetting although the patterns
were more complex.
b-1,4-glucosidase and acid phosphatase activities were
low on the dry fern but increased on wetting, almost
doubling within 3 h. However, activity declined over the
next day, but increased again 2–3 days after the artificial rain
FEMS Microbiol Ecol 58 (2006) 236–246
Viable plate count (105/g fern)
Water content
(% fern weight)
80
6
4
2
0
Nutrient
R2A
Cellulose Glucose
Fig. 3. Viable counts of bacteria in the phyllosphere of Polypodium
polypodioides in it’s dry, inactive (open bars) and wet, physiologically
active (shaded bars) states. Counts were obtained by shaking P. polypodioides in buffer to dislodge cells and plating the extract onto nutrient
agar, R2A agar, and agar containing either cellulose or glucose as the sole
carbon source. Numbers are expressed per gram dry weight of fern to
correct for increases in moisture content of the fern following rehydration. Error bars represent the standard error of three plates.
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240
the wet fern were selected for further characterization by
molecular methods.
Partial 16S rRNA gene sequencing revealed that the
majority (25/35) of the isolates from the wet and dry fern
samples were affiliated with the two lineages of Grampositive bacteria, the Actinobacteria and the Firmicutes
(Fig. 4). Many of these isolates were very closely related to
known bacteria, including various Bacillus sp., as well as
species of Arthrobacter and Curtobacterium. Other Grampositive isolates were less closely related to known bacteria
based on partial 16S rRNA gene sequencing (Fig. 4). The
other isolates included five that were affiliated with the
Methylobacteriaceae in the Alphaproteobacteria (Fig. 5), four
that were related to pseudomonads in the Gammaproteobacteria (Fig. 6), and a single isolate that was closely related
to Sphingomonas (Alphaproteobacteria, Fig. 5).
Molecular characterization of whole community
DNA obtained from dry and wet
P. polypodioides
Whole community DNA from the communities associated
with the wet and dry fern samples was amplified and used to
create clone libraries, and individual clones grouped into
ribotypes by restriction enzyme patterns or partial 16S
rRNA genes sequencing. Total diversity estimates (SChao1)
calculated by repeated random sampling from each library
were 69 ribotypes for the dry phyllosphere sample and 54 for
the wet sample. In each case, predictions of SChao1 approached a stable asymptotic with increased library size
(Fig. 7). Coverage, or the proportion of the community that
appeared to have been sampled as part of the cloning
analyses, was greater for the wet phyllosphere sample (0.82)
than the dry (0.71) sample.
The ribotypes detected in both libraries represented
various bacterial lineages (Figs 4–6). The most common
ribotypes in the clone library from the dry fern sample were
members of the Methylobacteriaceae (Clone DC01; Fig. 5)
and Acidobacteria (Clone DC02; Fig. 6). For the wet fern, the
dominant ribotypes were affiliated with the Methylobacteriaceae (Clones WC01 and WC09) or the Beijerinckiaceae
(Clone WC04), both in the Alphaproteobacteria (Fig. 5).
There was little overlap in the 16S rRNA sequences in the
two clone libraries. Only two pairs of sequences (clones
WC18 and DC11 in the Planctomycetes, and clones WC07
and DC02 in the Acidobacteria; Fig. 6) were similar enough
to suggest that they represented the same bacterial species in
both the wet and dry samples. There was also little overlap
between the 16S rRNA sequences obtained from whole
community DNA extractions and those obtained from the
enrichment isolates. Only two different 16S rRNA sequences
were obtained from the whole community DNA analyses
that were affiliated with Gram-positive taxa, those repre2006 Federation of European Microbiological Societies
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E.F. Jackson et al.
sented by clones DC07 and DC17 from the dry fern
community, and neither was closely related to any isolate
(Fig. 4). Similarly, only one cloned ribotype (DC09) was
affiliated with the Gammaproteobacteria, another lineage
that was well represented in our isolates (Fig. 6). While
representatives of the Methylobacteriaceae (Alphaproteobacteria) were identified in both the clone libraries and as
isolates, the 16S rRNA sequences from the different sources
fell into distinct groups (Fig. 5).
Discussion
Functional changes in the microbial
phyllosphere community associated with
P. polypodioides during wetting
Activity of phosphatase and b-1,4-glucosidase on the fern
surface rapidly increased following wetting. This pattern is
characteristic of environments that show dry–wet periods
(Birch, 1958; Lund & Goksoyr, 1980), and is a response of
the community to conditions more suitable for growth.
Following this initial increase, activities declined but increased once more when the fern had fully greened out after
2 days. Wave-like changes in microbial activity also occur in
soils, and represent a succession of different microbial
populations (Lund & Goksoyr, 1980). Thus, wetting initially
stimulates the activity of the existing community, but as the
fern becomes greener different microbial populations replace the others, leading to a second burst in activity. Phenol
oxidase activity decreased following wetting, but then fluctuated, with the highest activity being associated with the
dry fern at the start and end of the experiment. Phenol
oxidases are involved in lignin degradation (Ander &
Eriksson, 1976; Faure et al., 1996), and the higher activities
associated with the dry, inactive fern suggests that the
phyllosphere community in that state might be using
recalcitrant substrates. However, phenol oxidase activity
can also result from enzymes involved in melanin production or cellular morphogenesis (Endo et al., 2002; Claus,
2003), so the patterns may not necessarily represent substrate availability. It’s also possible that activity of any of the
enzymes could be from the resurrection fern itself, although
this plant has not been previously shown to produce these
enzymes.
We selected 2 days postwetting as the time that most likely
represented a change in the phyllosphere community, and
examined numerical differences in culturable phyllosphere
bacteria between dry and wet fern. Plate counts were of the
order of 105 CFU g 1 of fern, comparable to numbers
reported for other plants (O’Brien & Lindow, 1989; Hirano
& Upper, 1990; Lindow & Brandl 2003). Following wetting,
the viable count of bacteria on glucose, nutrient agar and
R2A agar increased. As the fern becomes greener, it’s likely
FEMS Microbiol Ecol 58 (2006) 236–246
241
Changes in the phyllosphere community of the resurrection fern
Isolate W08
Isolate W06
Isolate D06
Arthrobacter protophormiae
Micrococcaceae
Isolate D13
Arthrobacter nicotianae
Isolate W10
Isolate W07
Isolate W09
Isolate W12
Microbacteriaceae
Earthworm cast AY154610
Clone DC17
Agrococcus jenensis
Isolate D16
Actinobacteria
Isolate W15
Curtobacterium luteum
Curtobacterium citreum
Isolate D07
Microbacterium oxydans
Isolate W04
Isolate D14
Cellumonadaceae
Oerskovia enterophila
Oerskovia paurometabola
Isolate D02
Rhodococcus fascians
Nocardiaceae
Isolate D04
Volcanic deposit AY917655
Acidimicrobiaceae
Clone DC07 (2)
Acidimicrobium ferrooxidans
Bacillus sphaericus
Isolate D08
Bacillus fusiformis
Bacillus megaterium
Isolate D03
Bacillus simplex
Isolate W02
Bacilliaceae
Firmicutes
Isolate W05
Bacillus thuringiensis
Isolate D15
Isolate W13
Isolate D11
Isolate W03
Bacillus cereus
Staphylococcus sciuri
Staphylococcaceae
Isolate D09
Staphylococcus lentus
Paenibacillus alvei
Paenibacillaceae
Paenibacillus macerans
0.10
Isolate W01
Fig. 4. Phylogenetic tree of partial 16S rRNA gene sequences obtained from the phyllosphere of Polypodium polypodioides that were affiliated with
Gram-positive taxa. Sequences were obtained by cloning 16S gene rRNA amplifications of DNA extracts from the whole community (designated
‘Clone’) or from isolates obtained from the phyllosphere (‘Isolate’). Samples were taken from both dry (‘D’ for an isolate and ‘DC’ for a cloned sequence)
and wet ferns (‘W’ for an isolate and ‘WC’ for a cloned sequence). For cloned sequences, the number of clones containing each sequence are shown in
parentheses when 41. Sequences of the most closely related environmental clones or cultured organisms are shown for comparison (number indicates
GenBank accession number where applicable). Sequences obtained in this study correspond to GenBank accession numbers DQ268656–DQ268682
(from top to bottom of page).
FEMS Microbiol Ecol 58 (2006) 236–246
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242
E.F. Jackson et al.
Methylobacterium sp. G296-5
Clone DC26
Methylobacterium radiotolerans
Clone DC03 (5)
Oxic rice field AY360555
Methylobacterium hispanicum
Isolate W14
Isolate W16
Isolate D12
Isolate W17
Isolate W18
Methylobacteriaceae
Methylobacterium aquaticum
Clone DC18
Salmon gill AY494633
Salmon gill AY494634
Clone WC09 (10)
Clone WC01 (12)
Clone WC02 (6)
Clone WC22
Clone DC01 (16)
Soil AJ292841
Clone WC12 (2)
Clone WC04 (12)
Soil AJ233469
Clone DC08 (2)
Soil AJ292815
Clone DC05 (4)
Beijerinckiaceae
Clone DC15 (2)
Clone WC06 (4)
Alphaproteobacteria
Clone DC20
Reservoir sediment AJ518150
Beijerinckia indica
Soil microcosm AF358002
Clone WC20 (2)
Sphingomonas phyllosphaerae
Isolate W19
Sphingomonas adhaesiva
Rice plant AB114614
Clone WC15 (3)
Sphingomonas sp. Y57
Sphingomonadales
Sphingomonas sp.
Clone DC22
Clone DC21
Sphingomonas sp.
Clone WC08 (3)
Clone DC12 (2)
Volcanic deposit AY917880
Caulobacter crescentus
Gluconacetobacter xylinus
Clone DC23
Clone WC10 (4)
0.10
Caulobacterales
Acetobacteraceae
Hot spring AY162829
Fig. 5. Phylogenetic tree of partial 16S rRNA gene sequences obtained from the phyllosphere of Polypodium polypodioides that were affiliated with the
Alphaproteobacteria. Sequences were obtained by cloning 16S rRNA amplifications of DNA extracts from the whole community (designated ‘Clone’) or
from isolates obtained from the phyllosphere (‘Isolate’). Samples were taken from both dry (‘D’ for an isolate and ‘DC’ for a cloned sequence) and wet
ferns (‘W’ for an isolate and ‘WC’ for a cloned sequence). For cloned sequences, the number of clones containing each sequence are shown in
parentheses when 41. Sequences of the most closely related environmental clones or cultured organisms are shown for comparison (number indicates
GenBank accession number where applicable). Sequences obtained in this study correspond to GenBank accession numbers DQ268683–DQ268711
(from top to bottom of page).
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FEMS Microbiol Ecol 58 (2006) 236–246
243
Changes in the phyllosphere community of the resurrection fern
Erwinia amylovora
Pantoea ananatis
Isolate W11
Pseudomonas fuscovaginae
Isolate D01
Pseudomonas pavonaceae
Gammaproteobacteria
Isolate D10
Pseudomonas fulva
Isolate D05
Sand dune plant isolate, AY822570
Clone DC09 (2)
Xanthomonas campestris
Clone WC28
Clone DC24
Deltaproteobacteria
Freshwater DQ064842
Desulfuromonas acetoxidans
Sphingobacterium heparinum
Sand dune plant isolate, AY822537
Clone DC04 (4)
Bacteroidetes
Sphingobacterium multivorum
Volcanic deposit AY917755
Clone WC13 (3)
Clone DC16
Volcanic deposit AY425773
Volcanic deposit AY917758
Clone WC14 (2)
Clone WC05 (5)
Clone DC13 (2)
Volcanic deposit AY917831
Acidobacteria
Clone DC02 (9)
Clone WC07 (4)
Soil AJ292587
Clone WC19 (2)
Hot spring AY145650
Clone DC10 (2)
Termite gut isolate, AY587229
Clone WC26
Soil AJ233582
Acidobacterium capsulatum
Clone WC18 (2)
Clone DC11 (3)
Soil isolate, AY673410
Planctomycetes
Soil AJ292685
Planctomyces limnophilus
Soil isolate, AY960770
Clone DC14 (2)
Verrucomicrobia
Verrucomicrobium spinosum
Clone DC25
Earthworm cast AY037737
OP10 group
Hot spring AF027092
0.10
Fig. 6. Phylogenetic tree of partial 16S rRNA gene sequences obtained from the phyllosphere of Polypodium polypodioides that were affiliated with
various lineages of bacteria other than the Actinobacteria, Firmicutes, or Alphaproteobacteria. Sequences were obtained by cloning 16S rRNA
amplifications of DNA extracts from the whole community (designated ‘Clone’) or from isolates obtained from the phyllosphere (‘Isolate’). Samples
were taken from both dry (‘D’ for an isolate and ‘DC’ for a cloned sequence) and wet ferns (‘W’ for an isolate and ‘WC’ for a cloned sequence). For
cloned sequences, the number of clones containing each sequence are shown in parentheses when 41. Sequences of the most closely related
environmental clones or cultured organisms are shown for comparison (number indicates GenBank accession number where applicable). Sequences
obtained in this study correspond to GenBank accession numbers DQ268712–DQ268733 (from top to bottom of page).
FEMS Microbiol Ecol 58 (2006) 236–246
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Published by Blackwell Publishing Ltd. All rights reserved
c
244
E.F. Jackson et al.
Predicted diversity
100
80
60
40
20
0
0
20
0
20
40
60
80
100
40
60
80
Clone library size
100
Predicted diversity
100
80
60
40
20
0
Fig. 7. Predicted species diversity as determined from SChao1 analyses of
16S rRNA gene clone libraries for phyllosphere bacterial communities
associated with dry and wet resurrection fern, Polypodium polypodioides. Both libraries approached asymptotic values and gave diversity
estimates of 69 and 54 for dry and wet fern, respectively. Each library
was generated from DNA obtained from five fern fronds obtained from
the same tree branch.
that additional organic matter is present in the phyllosphere,
increasing some bacterial populations. Leaf exudates include
sugars and amino acids, and the concentration of these
substrates varies with environmental and physiological conditions (Tukey, 1970; Wildman & Parkinson, 1981; Fiala
et al., 1990). Wetting also increases the availability of organic
matter on the plant surface (Mercier & Lindow, 2000). The
number of cellulose-degrading bacteria did not differ between dry and wet fern. Thus, while wetting stimulated
bacterial populations that utilize sugars and other labile
substrates, it had much less effect on populations that
utilize more recalcitrant, less soluble polysaccharides such
as cellulose.
Structural changes in the bacterial phyllosphere
community associated with P. polypodioides
following wetting
More diverse 16S rRNA gene sequences were obtained from
the community DNA extraction of the dry fern sample than
that of the wet fern. Sequences obtained from the dry fern
were affiliated with nine major lineages of bacteria compared to just four lineages for fern fronds collected after
wetting. Most ribotypes were Alphaproteobacteria, and as
well as simplifying the overall community, wetting increased
the dominance of this group. They accounted for 75% of the
clones sequenced in the wet fern library compared to 55%
for the dry fern. While Alphaproteobacteria were the most
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Published by Blackwell Publishing Ltd. All rights reserved
c
common ribotypes in the wet and dry phyllosphere, no
individual sequence was found in both samples. Some pairs
of sequences were similar (e.g. clones DC02 and WC07,
clones DC11 and WC18, and clones DC12 and WC08) but,
in general, different sequences were detected in the wet and
dry phyllosphere. This is unlikely to be due to inadequate
sampling, as coverage scores suggested that much of the
community (70–80%) was sampled in each case.
None of the 16S rRNA gene sequences obtained in this
study were closely related to those detected in other phyllosphere communities, although some were related to sequences obtained from environments that undergo similar
fluctuations. A number of sequences were related to those
detected in soils (e.g. clones DC02, DC03, DC08, DC17,
DC25, WC04, WC07, and WC12) or volcanic deposits
(clones DC13, DC16, WC05, WC13, and WC14), environments that are subject to similar wet and dry conditions as
the phyllosphere. That none of the ribotypes had previously
been detected in phyllosphere environments is likely due to
the small number of molecular-based studies of phyllosphere communities, and those few studies have also detected bacterial species that were not identified in culturedependent approaches (Yang et al., 2001; Kadivar & Stapleton, 2003).
While the molecular approach detected ribotypes of
various bacterial lineages, most isolates were Actinobacteria
or Firmicutes, which were not at all dominant in the clone
libraries. Members of the Methylobacteriaceae (Alphaproteobacteria) were detected as both isolates and as ribotypes in
the clone libraries, but the ribotypes and isolates were
different species. The presence of Methylobacterium-like
sequences and isolates in the phyllosphere is not surprising
as these bacteria have been found associated with various
plant species (Romanovskaia et al., 2001; Jaftha et al., 2002;
Van Aken et al., 2004). Many other isolates were related to
known plant-associated bacteria, including Bacillus cereus/
Bacillus thuringiensis and Arthrobacter sp. Some species of
Arthrobacter can tolerate desiccation (Labeda et al., 1976)
and the presence of these bacteria on both wet and dry fern
suggests that the isolates obtained in this study might have
similar capabilities. Other isolates related to known plant
symbionts included species of Curtobacterium and Pseudomonas. Thus, in contrast to the molecular analyses that
identified few sequences that had previously been found in
phyllosphere environments, the isolates obtained in this
study were generally related to those obtained from other
plants.
There were only minor differences between the isolates
obtained from the dry and wet phyllosphere. Many showed
high 16S rRNA gene sequence similarity, and might represent different populations of the same species, with certain
populations being isolated from dry fern and others being
found on wet fern (e.g. various B. cereus/B. thuringiensis
FEMS Microbiol Ecol 58 (2006) 236–246
245
Changes in the phyllosphere community of the resurrection fern
isolates). This may simply represent 16S rRNA gene sequence variation within a species, but the possibility that
some populations are adapted to drier conditions and others
to wetter environments is intriguing.
phyllosphere such studies become possible, and the plant
surface could serve as an ideal system to ask questions about
how microbial communities vary both spatially and with
fluctuating environmental conditions.
The phyllosphere community of P.
polypodioides and its response to wetting
Acknowledgements
We characterized the changes that occur in the bacterial
community on P. polypodioides, as the fern changes from a
dry state to a lush, green state following wetting. Following
rainfall the fern becomes photosynthetically active, which
likely results in the release of organic compounds from the
plant surface and increased availability of existing organics.
More CFU using labile substrates were detected on wet P.
polypodioides fronds compared to dry, which supports this
concept. Wetting is analogous to enrichment, in that increased availability of organic substrates leads to the dominance of certain bacteria and a reduction in species
diversity, an idea that is supported by the molecular analyses. Wetting led to an initial burst in enzyme activity from
the existing microbial community. Activities showed a
second increase after a few days, which may indicate increased production of organic substrates by the plant or the
emergence of a different phyllosphere community. The
effects of environmental conditions such as rainfall on
phyllosphere communities are rarely determined, and have
never been reported for resurrection-type plants that undergo drastic physiological changes. Our observations show
that changes in the phyllosphere do occur following rainfall,
and P. polypodioides may be an interesting system to study
these patterns.
We focused our sampling effort on P. polypodioides that
were growing on a single tree branch, and randomly
sampled and pooled individual fronds. Pooling leaves into
a single sample has often been used in phyllosphere studies
(Ercolani, 1991; de Jager et al., 2001; Yang et al., 2001) but
prevents the determination of spatial variability in community structure. Because the focus of this study was the effects
of wetting (a temporal effect), we felt that the loss of spatial
data and finer scale replication was acceptable. However, the
P. polypodioides system would certainly be amenable to
spatial studies. Determining the variability in community
structure at different spatial scales (e.g. between fronds on
the same branch, between different branches of the same
tree, between different trees, and even between different
pinnae on an individual frond) would be interesting from
both an ecological and evolutionary perspective. Such
studies are rare in phyllosphere microbial ecology, and have
focused on differences in the size of individual microbial
populations (Kinkel et al., 1995; Woody et al., 2003) as
oppose to differences in community composition. With the
increased use of molecular approaches to examine the
FEMS Microbiol Ecol 58 (2006) 236–246
Funding for this work was provided by a Southeastern
Louisiana University OSCAR award to H. E., and by
Louisiana BORSF award (2002-5)-RD-A-25 and an EPA
award through SLU’s West Lake Pontchartrain Research
Program to C. R. J.
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