A leaf is a complex habitat' Leaves of different plants, and sometimes leaves on different parts of

1
Ever Wonder What Lives on a Leaf?
A leaf is many things a beautiful shape, a
colorful tapestry, a photosynthetic powerhouse.
It is also a habitat for microbes. Just like any
other surface on the face of the Earth, a leaf is
typically not sterile. Other organisms grow on
it, viruses, bacteria, fungi, algae, small
animals, often without causing visible damage to
the leaf although some of these can be leaf
pathogens or parasites. In the Hiram College
Microbiology course, we are interested in the
bacterial community that inhabits a leaf habitat,
also called the phyllosphere.
A research project within the Hiram
College Microbiology Course
Cladogram constructed using 16S rRNA sequences
from 2008 cultured isolates of possible strains
of the phylum/kingdom Actinobacteria (dark blue
stars) and publicly available sequences, using
the RDPII aligner (www.rdp.cme.msu.edu) and a
distance matrix (Jukes-Cantor corrected distance
model) method.
A leaf is a complex habitat. Leaves of different
plants, and sometimes leaves on different parts
of the same plant, differ in their shape, their
pigmentation patterns, and their thickness.
They also vary in the thickness of their waxy
cuticles, the presence or absence of secretory
and non-secretory trichomes (leaf hairs),
distribution of stomata (regulated pores that
allow gases to move between the inside of the
leaf and the atmosphere), water content, and
metabolite profile. These characteristics
influence what bacteria are found growing on
leaves and where we find them growing on leaves.
Take a look at some micrographs from Gwyn
Beatties lab at Iowa State University. Bacteria
are found in a patchy distribution across the
leaf surface. Microcolonies of cells, of one
strain or maybe a mixture of strains, can be
found on the waxy cuticle. Some are found in the
crevices where cells join. Others are found
around stomata or trichomes. Some are even
found within the leaf, living in between
cells. www.public.iastate.edu/gbea
ttie/homepage.html We have been sampling
phyllospheres since 2006. Given the timing of
our Microbiology course, we have focused on the
leaves of early spring wildflowers found at the
James H. Barrow Field Station. In 2006 and again
this year, the entire class focused their
efforts on the American Trout Lily (Erythronium
americanum). These ephemeral spring beauties
send up 1-2 leaves each spring, depending on
their age and nutritional status, followed later
by a flower-bearing inflorescence. Our strategy
is to sample the phyllosphere using both
culture-dependent and culture-independent
methods. Culture-dependent methods are exactly
like it sounds. We knock the bacteria off of
the leaves, using sterile technique, and then
coax some of those bacteria to grow on different
media. We then use a variety of techniques to
identify each strain we can get into pure
culture. However, only 1-5 of bacterial
strains on the planet can be cultured using
current techniques, so we need
culture-independent means as well. We isolate
total DNA from the initial bacteria knocked off
the leaf (hopefully without bringing plant cells
along with them) and then amplify out specific
genes or randomly clone some of the total DNA.
Cladogram constructed using 16S rRNA sequences
from 2008 cultured isolates of possible
Flavobacterium strains (green stars) and publicly
available sequences, using the RDPII aligner
(www.rdp.cme.msu.edu) and a distance matrix
(Jukes-Cantor corrected distance model) method.
Actinobacteria
Bacteroidetes
Firmicutes
Cladogram constructed using 16S rRNA sequences
from 2008 cultured isolates of possible Bacillus
strains (light blue stars) and publicly available
sequences, using the RDPII aligner
(www.rdp.cme.msu.edu) and a distance matrix
(Jukes-Cantor corrected distance model) method.
Alpha-Proteobacteria
Beta-Proteobacteria
Gamma-Proteobacteria
Cladogram constructed using publicly available
16S rRNA sequences from most of the known
phyla/kingdoms of the domain Bacteria, using the
RDPII aligner (www.rdp.cme.msu.edu) and a
distance matrix (Jukes-Cantor corrected distance
model) method. This is a rooted cladogram using
the phylum/kingdom Aquificae as the outgroup,
although we acknowledge that there is still
controversy about the deepest branches of the
domain Bacteria. Taxa represented by 2008 Hiram
College cultured isolates, metagenomic PCR
clones, or metagenomic random fragment clones are
indicated by brackets and taxon names.
Cladogram constructed using 16S rRNA sequences
from 2008 metagenomic PCR of strains from the
phylum Beta-Proteobacteria (dark red stars) and
publicly available sequences, using the RDPII
aligner (www.rdp.cme.msu.edu) and a distance
matrix (Jukes-Cantor corrected distance model)
method.
Leaf in tube with buffer to knock off bacteria
Total DNA isolated from uncultured phyllosphere
bacteria
Total DNA used as a target for PCR amplification
of a specific gene
Cladogram constructed using 16S rRNA sequences
from 2008 cultured isolates of possible Erwinia
strains (red stars) and publicly available
sequences, using the RDPII aligner
(www.rdp.cme.msu.edu) and a distance matrix
(Jukes-Cantor corrected distance model) method.
Cladogram constructed using 16S rRNA sequences
from 2008 cultured isolates of possible
Burkholderia strains (dark red stars) and
publicly available sequences, using the RDPII
aligner (www.rdp.cme.msu.edu) and a distance
matrix (Jukes-Cantor corrected distance model)
method.
Phyllosphere bacteria plated onto different media
and identified by cultured-dependent and
independent methods
Total DNA cut and cloned into a plasmid vector to
make a library for sequencing or functional
testing
Cladogram constructed using 16S rRNA sequences
from 2008 cultured isolates of possible
Pseudomonas strains (red stars) and publicly
available sequences, using the RDPII aligner
(www.rdp.cme.msu.edu) and a distance matrix
(Jukes-Cantor corrected distance model) method.
PCR primers used 16S rRNA gene (universal,
domain-specific, phylum- specific,
genus-specific) Agrobacterium biovar 1 telA
gene rpsL/rpsG genes (should be universal)
Acknowledgements. We thank the Howard Hughes
Medical Institute for its support of this
research through a Small College Science
Education Grant. This project is part of the
Hiram Genomics Initiative, a research and
outreach effort within the Center for Deciphering
Lifes Languages at Hiram College.