Exploring the Symbiosis of Frankia Bacteria with the Introduction

Exploring the Symbiosis of Frankia Bacteria with the
Invasive Shrub, Autumn Olive
Andrew DeVries, Dr David Dornbos, Dr John Wertz, Calvin College
Elaeagnus umbellata, Autumn Olive, is a deciduous,
actinorhizal shrub and invasive in and non-native to
Michigan. Autumn Olive, a Japan native, was introduced
specifically to re-vegetate the low pH coal mine spoils in
southern Illinois and Indiana and to reduce the erosion along
roadsides after construction. It now thrives in soils
particularly low in nitrogen: forest understories, prairies,
and abandoned farm fields. Its advantage in nitrogen-poor
soil is likely derived from nitrogen reduced by the
symbiotic relationship with a bacteria species, Frankia.
In 1978, a team of researchers at Harvard discovered
the slow growing bacteria, Frankia, was responsible for the
nitrogen fixing properties of sweet fern (another
actinorhizal plant). Frankia is a nitrogen-fixing bacteria
(has a nifH gene that allows for coding the nitrogenase
enzyme) that takes nitrogen in its atmospheric form (N2) to
ammonium (NH4+), which provides the plant with
sufficient nitrogen, a growth limiting factor. This allows for
Autumn Olive and similar actinorhizal plants to have a
significant advantage, providing at least partial explanation
for their ability to thrive in a nutrient-poor ecosystems.
Our initial goal was to compare the Frankia in the
Autumn Olive nodules among different geographic
locations and among different environments due to
morphological differences among plants in these areas.
However, Frankia was not successfully cultured this
summer, so the goal changed. The new goal was to compare
the bacterial communities among different ecosystems
(forest and meadow) and to identify secondary bacterial
symbionts capable of reducing nitrogen by culturing
bacteria and cloning vectors.
Given that plant genetic composition is the same for
meadow and forest Autumn Olive, I hypothesize that the
bacterial communities found within the root nodules will
also be the same. Because biological systems are incredibly
complex, I hypothesize that secondary symbionts are
possible, if not likely, to be at work in this symbiosis.
Noncultivated Bacteria Isolates
Collection of Root Nodules:
The root nodules were collected at Pierce Cedar Creek
Institute (PCCI) and Wittenbach/Wege Environmental Center. The
plants were dug out, placed into pots and taken directly to Calvin.
Preparation of Root Nodules:
The nodules were removed from the roots, washed with
sterile water, sterilized in 70% ethanol (killing any surface soil
bacteria), chopped with a sterile knife, and then homogenized.
The homogenizer was rinsed with sterile water and then ethanol
between each nodule.
PCR Amplification: 16S and nifH:
The extracted DNA was placed into a 96 well plate.
General Bacteria and Frankia 16S rRNA-specific and
Frankia-specific nifH primers were added to the extracted
DNA along with GoTaq polymerase and dNTP’s. The 16S
rRNA and/or nifH genes were amplified in PCR reactions.
DNA Sequencing:
In preparation for DNA sequencing, the PCR products
were cleaned with ExoSAP-it, and the appropriate forward
primers used in the PCR reactions were added. The cleaned
products were then sent to Michigan State University for
Plating and Isolating the Bacteria:
Plating the bacteria was done via spread method with
dilutions ranging from 10-1 to 10-3. Later in the summer, the 10^-3
dilution was cut out to not waste plates (no bacteria would grow
on this dilution). Bacteria were plated either with Novobiocin, an
antibiotic, or without to prevent from contamination.
DNA Extraction:
One loop of isolated bacteria was placed in 500 microliters
of sterile nanopure water, heated in a dry bath at 90 degrees
Celcius for fifteen to thirty minutes, then frozen at -20 degrees
Non-Cultivation Based Microbial Analysis of Root Nodules:
The nodules were removed from the roots, and
homogenized as described in “Preparation of Root Nodules.”
The total DNA from the root nodules was extracted and PCR
amplified with the same primers as described above. PCR
products were Topo-TA cloned into Top10 E.coli. After bluewhite screening, the E. coli were cultured in 96-well deep
plates. Each clone was PCR amplified with the same primers
as above, the PCR products cleaned, and sent to Michigan
State University for sequencing.
Types of Bacteria Found in AO Nodules Located in the
Types of Bacteria Found in Autum n Olive Nodules Located in the
Gamma Proteobacteria
Gamma Proteobacteria
Beta Proteobacteria
Beta Proteobacteria
Alpha Proteobacteria
Alpha Proteobacteria
David Dornbos, John Wertz
Lori Keen
Calvin College
Michigan State University
Piece Cedar Creek Institute
Flat Iron Lake Preserve
Wittenbach/Wege Environmental Center
Figure 1. Cultivation-Based Analyses of Bacterial
These pie charts show the differences in the
bacterial classes of Autumn Olive root nodules between
the forest ecosystem and the meadow ecosystem. The
forest has a heavy amount of alpha proteobacteria, with
very few bacilli species. The meadow system is majorly
Gammaproteobacteria, with very few Actinomycetales. A
Chi Squared Test was used to compare the two data sets,
and it showed a significant difference in the communities
(n = 94, p =0.0005)
Figure 2. Detection of Putative Secondary Symbionts:
Throughout the summer several bacteria were found that
are closely related to organisms found to be important to the
nitrogen economy of plants. Among the cultured bacteria, several
nitrogen fixing bacteria were isolated including: Mesorhizobium,
Bradyrhizobium, Sinorhizobium, and Herbaspirrillus
seropedicae. Non-cultivation based bacterial identification,
Sphingobium sp. and Variovorax paradoxus were found (along
with Frankia). These species were also cultured, and have been
shown to reduce nitrogen compounds.
Variovorax Paradoxus
Shingobium sp
Ralstonia syzygii
Figure 3. NifH-Based Detection of Putative Nitrogen
Fixing Bacteria in Autumn Olive Root Nodules. Noncultivation based approaches showed high amounts of
Frankia-related nifH as well as nifH from several other
bacterial species.
Cultivation-based methods yielded no Frankia species from
Autumn Olive root nodules. However, when non-cultivation
based methods were used, Frankia-specific nifH genes were
detected. A lack of cultivation success could be due to the
fact that Frankia is a slow grower and it was either
outcompeted by fast-growing bacteria or killed on plates that
contained novobiocin (not all species of Frankia are resistant
to this antibiotic).
The cultivated bacterial communities of nodules in the forest
and nodules in the meadow were statistically different. These
differences in the Autumn Olive nodules depending on the
ecosystem leads to some new, interesting questions: Are the
nodules dependent on the bacterial soil compositions in a
given ecosystem? What could the difference in bacterial
communities mean to the growth of the Autumn Olive?
This research raises the possibility that there may be
secondary symbionts that assist in the Autumn Olive/Frankia
symbiosis. Some Sphingobium are found to reduce nitrite to
nitrate, an important step in the nitrogen cycle for plants.
Variovorax paradoxus is known for promoting plant growth,
and has been shown to reduce nitrite to ammonium. While
this research gives evidence that they may be secondary
symbionts, only through future experimentation can we
confidently state that these bacteria are.
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