In order for plants to thrive they must obtain certain nutrients. One of the most important is nitro

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Introduction In order for plants to thrive
they must obtain certain nutrients. One of the
most important is nitrogen. Most plants obtain
nitrogen directly from the soil is via their root
system through active transport of ammonium or
nitrate ions. A small minority of plants have a
different strategy which includes a relationship
with a bacteria, where the bacteria converts
atmospheric nitrogen to ammonia, providing more
available nitrogen directly to the root system.
One of the prokaryotes that possesses this
capability is the filamentous bacteria Frankia.
Frankia fixes nitrogen within specialized root
nodules of the plant, in return the plant
continuously supplies fixed carbon to the nodule
the Frankia resides in, creating a mutualistic
relationship. The nodule is created by the
process of Frankia root infection, which is
accomplished by either root hair infection or
intercellular penetration. With root hair
infection Frankia infects the root by way of a
root hair and inserts itself into the primordial
cell of the nodule while the nodule is still
growing. With intercellular penetration, root
hairs are not needed as Frankia is guided into
the plant via intercellular pectic tubes.
(Frankia Actinorhizal Plants) Frankia as a
genus of bacteria is quite adaptable to many life
styles, including living independently or
associating with some select plant families.
Within each plant family, however, there is a
high specificity of Frankia strains that are
employed. For example, Group I Frankia strains
associate with only the Rosaceae, Rhamnaceae, and
Datiscaceae, Group II associate with only Alnus,
and finally Group III associate with Elaegnaceae.
This study focuses on the Group III strains
infecting Elaeagnus angustifolia, commonly known
as the Russian Olive. Russian olive was
introduced into the United States in the late
1800s, and was neutralized in the state of
Colorado in the 1950s. Russian Olive is a very
tolerant plant to varying conditions with water,
soil, temperature, and salinity lending to its
success in Colorado, which has cause it to be
labeled as a List B Noxious Weed by the Colorado
Department of Agriculture (United States). To be
classified as such the plant must be considered a
quarantine pest under the guidelines set forth
by the International Plant Protection Convention
(IPPC) in the Glossary of Phytosanitary Terms.
Basically this states that the plant, whether not
in an area yet or wide spread, must present an
economic threat and be officially controlled
(Secretariat IPPC). Like the ring produced in
the water after a stone is thrown in, the Russian
Olive is slowly moving across the Coloradan
landscape. One possible reason that allows this
transition to be easier for Elaeagnus is its
close association with Group III Frankia
strains. Why should we care? What effect does
this have on the environment? Elaeagnus
agustifolia was imported from overseas it is not
a native plant to the United States let alone
Colorado, meaning it arrived from an environment
where it had natural enemies to help regulate
growth to an environment where there are no
natural enemies. Remembering that Russian Olive
has a very tolerant nature, add the relationship
with Frankia and no natural enemies, and it all
adds up to a highly competitive plant. Two more
things that also add to its competitive nature
are 1.) birds love the fruit, so seeds have a
high dispersal rate, 2.) Elaeagnus is very
resistant to eradication methods such as mowing
or fire. The Russian Olive is currently
out-competing our native plants for the available
resources in the soil and even for sun. Native
plants are being slowly removed from the
landscape. What is happening to the Frankia? By
displacing native plants and by being associated
with Frankia, Russian Olive may be changing the
native microbial (Frankia) community by selecting
different Frankia strains than the native plants.
To get the most accurate comparison of the
Frankia communities within native and exotic
plants, we examined nodules of Shepherdia
rotundifolia, a native plant that associates with
Group III Frankia and Elaeagnus angustifolia.
Results In this research we had 41 successful
DNA amplifications of nodule DNA out of 78
attempts. Amplification products were 474 base
pairs long as expected using the primer
combination of DB41/DB44. These were added and
aligned to the existing matrix composed of
Frankia sequences. The existing tree was based on
55 DNA sequences with 465 base pairs (Please See
Figure 1). The tree is now 90 sequences with 474
base pairs after the 41 successful amplifications
were added. Reading the tree one can see that
there are three separate clusters represented,
Group one- Rosaceae, Group two- Alnus, Group
three- Elaeqgnus (see Figure 1). Within each
cluster, the nodules that have been sequences are
each labeled and coded from previous studies.
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Discussion Although hoped for, a high level of
diversity was not expected as previous studies
have reported low diversity in Frankia (Clawson
and Benson 1998). As noted in a study conducted
by Vanden Heuvel et. al., there are a few factors
that can contribute to the levels of Frankia
diversity, a) a few strains of Frankia may be
dominating the environment, b) the current plants
could be selecting certain strains thereby
creating a bias in the diversity, and c) there
may have been an evolutionary bottleneck which
greatly altered the diversity (Vanden Heuvel
et.al. 2004). We found a high level of diversity
within the Elaeagnus population, and a relatively
low diversity within the Shepherdia population.
Two things are immediately interesting about this
result. First, Elaeagnus appears to be able to
associate with many different native strains of
Frankia, and second, there is very little overlap
between the native Shepherdia and exotic
Elaeagnus signaling that Elaeagnus may be
changing the Frankia community. Our Shepherdia
samples were selected from a site where Elaeagnus
has not penetrated, giving us a sample of what
Frankia population might have been before
Elaeagnus had been introduced to Colorado. In
the neighbor joining tree constructed from our
results, one can see that the Frankia strains
taken from Shepherdia while they are related are
nearly independent of the strains taken from
Elaeagnus. By showing Shepherdia and Elaeagnus
use similar strains but ones that are not
dependant this leads one to accept that there
might be a transition phase of microbial
community changing. Elaeagnus may be using a
broader set of Frankia diversity than the native
plants, thereby increasing Frankia diversity in
the soil.
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Works Cited Benson, D. R., and W. B. Silvester.
1993. Biology of Frankia strains, actinomycete
symbionts of actinorhizal plants. Microbiol. Rev.
57293-319. Clawson, M.L., Caru, M., and Benson,
D.R. 1998. Diversity of Frankia strains in root
nodules of plants from the families Elaeagnaceae
and Rhamnaceae. Appl. Environ. Microbiol. 63
3539-3543. Collins, Emily, . (2002). Introduced
Species Summary Project . Retrieved 24 July 2006,
from Danoff-Burg, James, A. Website
http//www.columbia.edu/itc/cerc/danoff-burg/invas
ion_bio/inv_spp_summ/Elaeagnus_angustifolia.htm.
Frankia Actinorhizal Plants. Molecular and Cell
Biology Dept., University of Connecticut. 31May
2006. http//web.uconn.edu/mcbstaff/benson/F
rankia/FrankiaHome.htmgt. Frankia Actinorhizal
Plants. Molecular and Cell Biology Dept.,
University of Connecticut. 31 May 2006.
lthttp//web.uconn.edu/mcbstaff/benson/Frankia/Fran
kiaHome.htmgt. Secretariat of the International
Plant Protection Convention. International Plant
Protection Convention. ISPM No. 5 Glossary Of
Phytosanitary Terms. 31 May 2005. 03 May 2006.
lthttps//www.ippc.int/servlet/CDSServlet?status
ND0xMzM5OSY2PWVuJjMzPSomMzc9a29zgt. State of
Colorado. Department of Agriculture. Rules
Pertaining To The Administration And Enforcement
Of The Colorado Noxious Weed Act. 31 May 2006. 2
May 2005. lthttp//www.ag.state.co.us/CSD/Weeds/sta
tutes/weedrules.pdfgt. United States. United
State Department of Agriculture. Guide To The
Listing Process For Federal Noxious Weeds. 31 May
2006. 13 June 2006. lthttp//www.aphis.usda.gov/pp
q/weeds/listingguide.pdfgt. Vanden Heuvel, B. D.
et. al. 2004. Low genetic diversity among Frankia
spp. Strains nodulating sympatric populations of
actinorhizal species of Rosaceae, Ceanothus
(Rhamnaceae) and Datisca glomerata (Datiscaceae)
west of the Sierra Nevada (California).
Microbiol. Rev. 50 989-1000.
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Materials and Methods Elaeagnus angustifolia
nodules were collected from an established
population along Fountain Creek in Pueblo,
Colorado. The Shepherdia nodules were collected
from San Isabel Natonal Forest, near Rye.
Nodules were placed in sterile vials with 90
ethanol and transported to the lab at
CSU-Pueblo. At the lab, nodules were cleaned for
DNA extraction. To clean the nodule, individual
lobes were excised and the epidermis removed
using a dissecting scope, clean forceps, half a
Petri dish, and distilled water. After cleaning,
the nodule lobe was then placed into a sterile
1.5mL tube and DNA was extracted employing a
DNeasy Plant Mini kit from Qiagen. The DNA is
then ready to receive the mixture of primers for
the PCR amplification. Using a pre-made
cocktail, forward primer (DB41), reverse primer
(DB44), Taq, and double distilled water, the DNA
solution was amplified using PCR. This entails
each tube being placed in an thermocycler
programmed to the following cycles Cycle 1- 5
minutes at 95 C Cycle 2- 1 minute at 94 C Cycle
3- 1 minute at 50 C Cycle 4- 1 minute at 72
C Cycle 5 through cycle 40 are repeating cycles
2 through cycle 4 Cycle 6- 7 minutes at 72 C.
After the PCR cycle is complete, an agarose
gel is prepared to receive the DNA. Each well
received approximately 10uL of DNA and run with
at 100 volts. Successful amplifications were
viewed on a ultra-violet light table and a
picture is taken to show the DNA bands. DNA bands
are then cut out of the gel and purified using a
QIAgen kit and then sent for sequencing to
Macrogen in South Korea.
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Cor.arb
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Figure 1 A Neighbor joining tree created from an
aligned DNA matrix composed of 58 sequences and a
length of 465 base pairs. This phenogram was
rooted using the sequence from Acidothermus. Each
major cluster is labeled Group 1 consists of
strains infective on the Rosaceae, Group 2
consists of strains infective on Alnus, and Group
3 consists of strains infective on the
Elaeagnaceae. The Shepherdia strains added to
this tree in this study are highlighted in red
text with arrows, the Elaeagnus strains collected
in Pueblo are highlighted in blue.