This study compared the phyllospheres of the invasive plant species Oxalis pes caprae and the native plant species Arctostaphylos glauca. Experiments found that O. pes caprae leaves had more bacterial colonies than A. glauca leaves, and the presence of bacteria on O. pes caprae inhibited the growth of fungi. This suggests the more abundant bacteria provides the invasive species with a competitive advantage over the native species in the same environment. Further research is needed to identify the bacteria and fungi species and determine the interactions between them.

1. A Study of the Differences in Phyllospheres
between Oxalis pes caprae and Arctostaphylos
glauca
Richard Leung
Roberto Carlos Segura
Biology 230
Christine Case
May 13, 2016

2. Differences in Phyllospheres 1
Abstract

3. Differences in Phyllospheres 2
Invasive species are one of the largest threats to native wildlife. When alien species are
introduced in native ecosystems, they vastly outperform native species. The purpose of this study
was to observe the phyllosphere of an invasive species, Oxalis pes caprae, and a native one,
Arctostaphylos glauca, in the Bay Area (figure 1). It was predicted that O. pes caprae would
have differences that grants it distinct advantages in its phyllosphere compared to A. glauca.The
differences observed in this study were the bacterial colonies and the growth of fungi in each
species. In order to compare the bacteria growing on the two species, both sides of the leaves of
both species were pressed into EMB and PEA petri plates to test for microbial growth. Following
repeated trials of leaf pressing, new leaves were gather for use in procedure two. In procedure
two dilutions of leaf solution made from sterile water and leaves for each species were made to
test for the growth of bacterial colonies. At the end of the experiment it was concluded that
bacterial colonies on the O. pes caprae vastly outnumbered that of A. glauca, and that the
presence of fungi was greater on A. glauca than on O. pes caprae. It was hypothesized that the

4. Differences in Phyllospheres 3
presence of the bacterial colonies on the invasive species inhibited the growth of pathogenic
fungi which
would allow the
invasive species
to thrive.
Hypothesis
H0: There are
not any
discernable
differences
between the
bacterial colonies of an invasive species and an native one.
H1: If both a native species and an invasive species inhabit the same environment, then the
invasive species will have a distinct advantage over the native species.
H2: The native species, being accustomed to its environment after generations of evolution, will
have an advantage over the invasive species.
Background
The ecosystems of the Bay Area contain one of the few remaining estuaries left on Earth.
The Bay Area is also one of the major economic centers of the United States as millions of
planes and ships come every year bringing tourists and goods. These methods of transportation
introduces invasive species into the Bay area and as a result puts pressure on the natural
ecosystems. Invasive species are species whom are not indigenous to an environment that
possesses the capability to rapidly spread outside their natural habitats and pose a danger to

5. Differences in Phyllospheres 4
environment, economy, or human health (2). When placed in the same environment native
species simply cannot compete with invasive species. There have been studies showing how the
presence of bacteria on the leaves of plants can be beneficial to the growth of the plant, and this
can correlate with how an invasive species gets its competitive edge over the native plants (4).
The extinction of plant species poses a great danger being that plants make up the most of
Earth’s biomass and produce energy into environment through photosynthesis. Weed invasive
species have major impacts and are the subject of intensive and expensive management efforts
(7). Oxalis pes caprae is such a weed and is found all over the world, including Coastal
California. The plant has a reputation for being very hard to exterminate once it has spread over a
large area of land and can threaten the growth of native species such as Arctostaphylos glauca
(3). A. glauca is a large shrub native to California where it grows in the chaparral and woodland
of coastal hills and mountains (5).
Procedures
A total of 5 experiments were conducted over the course of the project. To begin, leaf
presses were prepared in EMB media and incubated at 23 ℃ for one week. Each leaf was pressed
onto a plate for several seconds, then removed. To set negative controls, leaves were washed in a
bottle containing 70% alcohol by shaking by hand for several minutes. For the A. glauca leaves,
one leaf was used per press, and for the O. pes caprae leaves, six leaves were used to
compensate for the difference in surface areas. Surface areas were calculated by tracing a leaf
onto a sheet of graph paper and calculating surface area using the grids. This surface area was
used in all of the following results. The plates were then incubated at 23 ℃ for one full week.
Data collection for all experiments consisted of counting colonies present and classifying unique
members, with anything above the range of 500 being considered too numerous to count. For the

6. Differences in Phyllospheres 5
second experiment, the first was repeated with some modification; to improve the consistency of
the controls, the leaves were washed for a longer period of time. They were also incubated for
only three days compared to a week used last time to get a better measurement on the starting
rate of growth of the cultures. The third experiment consisted of a repetition of the second but
used PEA plates instead of EMB to study the bacterial growth without the growth of fungi. The
plates were also incubated for 5 days. The fourth experiment was a dilution of A. glauca leaf
solution, which began by shaking a cut up leaf in distilled water to loosen bacterium from the
surface, and this original solution was made into a 1:101 solution, from which a 1:102 through
1:106 solutions were made. These were then spread onto solid, prepared PEA plates and
incubated at 30 ℃for 3 days. A negative control was made in a similar fashion to the past
experiments, where a leaf was washed in alcohol before starting the procedure. Lastly, the fifth
experiment consisted of a repeat of the fourth, but using the O. pes caprae leaves instead of the
A. glauca leaves.
Results
From Experiment one and two, it was observed that the fungi and bacterial colonies grew
in large numbers on the A. glauca leaves and on the O. pes caprae leaves. The A. glauca leaves
had more fungi than bacteria, and the O. pes caprae leaves had more bacteria than fungi. It was
also noted that the bacteria and fungi grew evenly across the leaves, showing little preference for
location. There were 2 types of fungi present: one that was green, identified as Penicillium, and
another that was white, identified as Botrytis. There was only one type of bacteria observed, and
due to limitations, the bacterium was not identified. There was also an observed interaction
between the bacteria and fungi; wherever the fungi grew near a bacteria colony, there was a zone
of inhibition separating the bacteria from the fungi. This suggests that the bacterium are

7. Differences in Phyllospheres 6
producing secondary metabolites that are detrimental to fungal growth. The results from
experiment 3 confirmed these results; O. pes caprae had bacteria colonies that were close to
double the numbers
present on the A. glauca
leaves, despite there being
a difference in available
surface area (figure 1).
The fourth and fifth
experiments gave results
that slightly confirmed the
above trend. The tree
plates had bacteria, but the
control plate had
additional red colonies despite being doused in alcohol prior to plate preparation. Similarly,
bacteria growth was observed on the control plates for the weeds, but without the red bacteria.
The weed plates did have more growth per mL*cm2, but only by a small margin (figure 2).
Discussion
The intent of
this project was to find
how an invasive weed
can have a distinct
advantage over a
native shrub. Initially,

8. Differences in Phyllospheres 7
it was believed that the deciding factor was the bacteria; the assumption was that the bacteria
would be pathogenic, and the invasive species would have less bacteria present than the native
species. After the first two experiments, it was observed that there was another factor to include,
the presence of fungal growth. The results from these experiments suggested that the bacteria
plays a beneficial role in the relation between them and the host plant, due to the bacteria
seeming to produce a secondary metabolite that inhibits the growth of the fungi. The results also
prove that the bacteria are more beneficial than harmful. If the bacteria was a harmful agent, it
would grow near the nutrient rich parts of the plant to take advantage of the nutrients available
(6). However, the tests show the bacteria grew evenly on the leaves without concentrating
growth near veins or glandular trichomes, suggesting that they are not harmful (6). Along with
this, the bacteria have the ability to deter fungal growth, which is a documented plant pathogen,
making it more likely that it is beneficial for the plant (1). The fungal colonies followed the
opposite of the bacteria, growing near the center of leaves or near the center vein of the leaves,
implying that they are harmful to the plant. Once this finding was confirmed, the goal became to
study the amount of bacteria present, with more bacteria being the desirable trait. The third,
fourth, and fifth experiments confirmed that the invasive weed hosted more bacteria than the tree
did. The fourth experiment also exposed a possible second bacterium growing on the leaves.
They were only present on the control plates, implying that they are resistant to alcohol. The
contribution of this bacterium to the interaction between bacteria and fungi is currently unknown.
Conclusions and Further Study
From these results, this study illustrates the importance of further studies on the
phyllosphere. The phyllosphere is one of the largest microbial environments on Earth and have
had little studies conducted on it. The study concluded that the presence of bacteria in the

9. Differences in Phyllospheres 8
phyllosphere is beneficial to the plant if a pathologic fungi is present, supporting hypothesis one.
Should similar experiments be conducted, in order to expand on this body of knowledge, more
tests should be done to confirm the secondary metabolite of the bacterium, to confirm the
pathologic nature of the fungi, and to test for the significance of the observed red bacterium from
experiment 4. As invasive species continue to rapidly expand their range all over the world,
further studies should aim at increasing the knowledge of the phyllosphere to help preserve
biodiversity.
Works Cited

10. Differences in Phyllospheres 9
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Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and
Pathogenicity.” APS Journals. 28:232-248.
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neighbors: a mechanism for exotic invasion.” Science 290, 521–523. doi:
10.1126/science.290.5491.521
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Buttercup. Davis CA: UC Statewide Integrated Pest Management Program
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effect on strawberry phyllosphere microbiota under open field and protection conditions.”
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5. Howard, Janet L. 1993. “Arctostaphylos glauca. In: Fire Effects Information System”.
U.S. Department of Agriculture, Forest Service, Rocky Mountain Research Station, Fire
Sciences Laboratory.
6. Monier, J.-M., and S. E. Lindow. “Frequency, Size, and Localization of Bacterial Aggregates
on Bean Leaf Surfaces.” Applied and Environmental Microbiology 70.1 (2004): 346–
355. PMC. Web. 12 May 2016.
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Ecological Mechanisms and Human Responses, eds U. Starfinger, K. Edwards, I.
Kowarik, M. Williamson (Leiden: Backhuys Publishers), 33–42.