The study investigated the rhizosphere microbial community of white clover (Trifolium repens) to identify bacteria with nitrogen-fixing, cellulolytic, and antibiotic functions. Soil was collected from the rhizosphere and metagenomic DNA was extracted. Six bacterial isolates were discovered to produce cellulase enzymes, fix nitrogen, and synthesize antimicrobials. Analysis identified the isolates as Streptomyces species. Metagenomic data showed the rhizosphere had high biodiversity and the highest concentrations of relevant functional bacteria. This supports the unique ability of white clover to colonize nutrient-poor soils.

1. Investigation of Rhizosphere Microbial Community of Trifolium repens for Bacteria with Nitrogen-
Fixing, Cellulolytic, and Antibiotic Activity
Nell Malone1
, Chau Nguyen1
, Priya Sahdev1
, Philip Spencer1
, Emma Goodwin, and Giorgia Pirino
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90024
Isolate Identification
Soil
Collec on
Metagenomic
DNA Isola on
PCR
Ampli ca on
Pyrosequencing
Metagenomic
Analysis
Soil
Enrichment
Wet Mounts and
Gram Stains
Nitrogen Fixing
Assays
DF Media
JMV Media
Cellulase Assay
An bio c
Produc on Assay
16S rDNA PCR
Ampli ca on of
Isolates
BLAST
Analysis
Phylogene c
Tree
Metagenome Composition
Abstract
Introduction
Experimental Procedure
Functional Assay Results
Biodiversity
Conclusion
Future Directions
References
Acknowledgements
Nitrogen Fixation
Cellulase Production
Antibiotic Production
InCalifornia,severedroughthasmadegrowthofmanytraditionalplantspeciesbotheconomically
and environmentally unsustainable. By studying the rhizosphere microbial interactions of
Trifolium repens, Gram Neutral hopes to determine the microbial interactions that improve soil
conditions and help T. repens grow in nutrient poor environments. T. repens has been specifically
noted for its association with nitrogen fixing bacteria; six bacterial isolates from the rhizosphere
of T. repens were also discovered to produce cellulase enzymes and antimicrobial compounds.
These bacterial isolates and the extracted metagenomic DNA of the rhizosphere soil sample
were then sequenced for further analysis. Using BLAST analysis, several of the isolates were
identified to belong to the Streptomyces genus, consistent with the functional assay results for
antibiotic production, nitrogen-fixation, and cellulase production. In addition, comparison of
the metagenomic data from the rhizosphere of T. repens with other noted drought-resistant,
fast-growing, or antibiotic-producing plants revealed that the biodiversity of the rhizosphere of
T. repens was the highest. This metagenomic analysis also revealed that the soil composition
from the T. repens’ rhizosphere contained the highest or second highest concentrations of
nitrogen-fixing, cellulase producing, and antibiotic producing bacteria of the plants studied.
These findings corroborate known properties of T. repens as an effective colonizing organism
capableofflourishinginandimprovingnutrientpoorsoilmeritingfurtherexperimentationofthe
composition of its rhizosphere for bacterial candidates with potential to be biological fertilizers.
Although the earth’s atmosphere is 80% nitrogen, most plants cannot utilize this elemental
nitrogen; plants need nitrogen that has been reduced to ammonia, or NH3
. Nitrogen fertilizers
therefore provide plants with a usable form of nitrogen and have been proven to be one of the
primary requisites for high yield agriculture. However, the synthesis and use of fertilizers are
both economically and environmentally costly and have been shown to deplete concentrations
of nitrogen in the soil (Mulvaney et al., 2009). Because of these detrimental effects, research is
now being conducted on natural alternatives or additives that harness beneficial properties of
microbes (such as the ability to convert elemental nitrogen to ammonia). Trifolium repens is a
leguminous plant associated with nitrogen-fixing (Elgersma and Hassink, 1997) microbes and is
currently a promising candidate for “living mulch”: “living mulch” is used in agriculture to combat
soil erosion, suppress weed growth, increase biomass, and contribute to carbon and nitrogen
cycling. T. repens has also been noted for its ability to rapidly colonize, thrive in, and improve
nutrient-poor soils (Mytton et al., 1993). Due to these notable properties, T. repens was selected
for further research regarding the composition of its soil rhizosphere microbial community, with
hopes of finding specific microbes responsible for the aforementioned plant growth-promoting
functions. If this is achieved, the isolated microbes may be subjected to further experimentation
as additives or alternatives to traditional fertilizers.
Hypotheses:
• If root nodulation and nitrogen-fixing activity is due to microbial interactions, bacteria should
be present in T. repens’ rhizosphere that are capable of growing on nitrogen depleted media
and possess genes associated with nitrogen fixation.
• Cellulases are essential to the formation of root nodules and the breakdown and cycling
of organic materials. Since T. repens has been associated with nitrogen fixation and carbon
cycling, microbes capable of producing carbohydrate polymer degrading enzymes should be
present in the rhizosphere.
• If T. repens ability to outcompete other plant species and suppress weed growth is due to the
productionofantimicrobialcompounds,bacteriacapableofsynthesizingantibioticcompounds
should be present in the rhizosphere.
• Since T. repens is capable of rapidly colonizing new habitats and outcompeting other species,
the microbiome of the rhizosphere associated with T. repens should contain higher than
average biodiversity which supports multifunctionality and sustainability in ecosystems.
F15UCLA109ALGNRDM3:
Prediction: Sinorhizobium meliloti
Functional Proteins: Nitrogenase and Iron proteins
F15UCLA109ALGNRDM4:
Prediction: Streptomyces lavendulae
Antibiotics: Streptothricin
F15UCLA109ALGNRDM18:
Prediction: Streptomyces griseus
Antibiotics: Streptomycin (strFGHIK gene)
Enzymes: Cellulase and Endoglucanase
Both culture-dependent and culture independent experiments contributed to an understanding
of the rhizosphere of Trifloium repens as a rich reservoir of functionally active bacteria. The high
concentrations of bacteria contributing to nitrogen cycling, carbohydrate polymer degradation,
and antimicrobial production all support T. repens’ unique ability to colonize and improve
nutrient poor soils. Furthermore, analysis of the metagenome of the rhizospheric sample
revealed trends of bacterial community composition and biodiversity consistent with that of
invasive plant species. While many invasive species are detrimental when introduced to non-
native environments, much can be learned from these species’ ability to rapidly colonize and
dominate diverse habitats. That T. repens shares similar community composition trends with
these species is consistent with its ability to thrive in and improve nutrient-depleted soils. The
properties of T. repens’ rhizosphere should be further examined and tested as additions to or
alternatives to traditional fertilizers in promoting plant growth in nutrient poor environments.
Isolates should be screened for the nifH gene which encodes for the beta subunit of nitrogenase
reductase.StrongexpressionofnifHindicatesproductionofnitrogenasereductaseandtherefore
capability of fixing atmospheric nitrogen.
Isolates should also be inoculated with young T. repens plants, other common nitrogen fixing
plants, and other plants not known for nitrogen fixing. Nitrogen levels in the soil should then
be analyzed and compared between the sets of plants to see if nitrogen-fixing correlates more
strongly to the specific types of bacteria present in the soil or rather the interactions between
the microbes and the specific plant host. These experiments could have strong implications
for the agricultural business because it could be suggest a good alternative to using artificial
fertilizers determine whether or not specific microbes would serve as beneficial additives or
replacements in traditional fertilizers.
Additionally, since T. repens has been noted to withstand exposure to polycyclic aromatic
hydrocarbons (PAHs) and potentially contribute to their degradation (McGuinness and Dowling,
2009), T. repens isolates should be further examined focusing on degradation properties. A
PAH degradation assay should be performed by exposing the isolates to coal tar extract which
containshighlevelsofPAHs.Theintentofthisassaywouldbetorevealifthebacterialcommunity
has the capability to degrade PAHs. The team hypothesizes that if the isolates are capable of
degrading PAHs, the microbes may use a similar mechanism to also degrade carbohydrate
polymers. If true this could serve to be extremely beneficial for bioremediation technology,
where microorganisms are used to degrade soil’s toxic contaminants into non-toxic substances
(Mcguinness and Dowling, 2009). Bacteria that may be present in T. repens community that are
known to degrade PAHs are Mycobacterium vanbaalenii and Burkholderia xenovorans, both of
which are present in our metagenomic analysis.
Elgersma A, Hassink J, 1997. Effects of white clover (Trifolium repens L.) on plant and soil
nitrogen and soil organism matter in mixture with perennial ryegrass (Lolium perenne L.). Plant
and Soil [Internet]. [cited 2015 Dec 2]; 197: 177-186.
Halsall D. M., Gibson A. H. Cellulose Decomposition and Associated Nitrogen Fixation by Mixed
Cultures of Cellulomonas gelida and Azospirillum Species or Bacillus macerans. Appl Environ
Microbiol [Internet]. [cited 2015 Dec 2]; 50(4): 1021-1026.
McGuinness M, Dowling D. Plant-Associated Bacterial Degradation of Toxic Organic
Compounds in Soil. International Journal of Environmental Research and Public Health.
2009;6(8):2226-2247.
Mulvaney, R. L.; Khan, S. A.; Ellsworth, T.R. 2009. Synthetic Nitrogen Fertilizers Deplete Soil
Nitrogen: A GLobal Dilemma for Sustainable Cereal Production. American Society of Agronomy
[Internet]. [cited 2016 February 24]; 38: 6.
Mytton L.R., Cresswell A, Colbourn P. 1993. Improvement in soil structure associated with white
clover. Grass and Forage Science [Internet]. [cited 2015 Dec 2]; 48: 84-90.
Rodrigues RR, Pineda RP, Barney JN, Nilsen ET, Barrett JE, Williams MA. Plant Invasions
Associated with Change in Root-Zone Microbial Community Structure and Diversity. Liu J, ed.
PLoS ONE. 2015;10(10):e0141424.
Vance C. P. 1997. Enhanced agricultural sustainability through biological nitrogen fixation.
Department of Agronomy and Plant Genetics [Internet]. [cited 2015 Dec 2]; 39: 179-186.
We would like to acknowledge the Department of Microbiology, Immunology, and Molecular Genetics of
UCLA for its support on this project. Furthermore, we would like to personally thank Jimmy Bazzy for his
suggestions on poster design and Jiem Ronas for his discussions about soil community analysis. And finally, we
would like to thank the members of Bac Attack, Bac Streak Girls, Bruin Bacteria, Metagenome World Peace,
MicroMonsters, Rhiz Khalifa, and Wingardium Rhizobia for the use of the data they gathered from their own
metagenomic DNA extraction.
1
All four team members contributed equally to the research and analyses included in this project.
The Shannon-Wiener
Index (H), which takes
into account genus
richness, as well as the
abundance of each genus
in the community, was
used to test biodiversity
in the metagenomic
data from each plant
studied in the class.
When compared with the
other plants tested, the
rhizosphere community
of the White Clover was
the most biodiverse. To
contextualize this finding,
the diversity results were
organized by soil pH;
the resulting correlation
showed a peak in
biodiversity at pH=5.955.
Using known common antibiotic producing
bacterial species, the metagenomic data of each
organism’s rhizosphere was sorted to predict
the abundance of antibiotic producer species.
T. repens showed comparable concentrations of
antibiotic producers with the English Yew, a plant
known to have antibiotic producing bacteria.
Antibiotic production ability was confirmed
through clearings around the bacterial
isolates F15UCLA109ALGN30N17 and
F15UCLA109ALGN30M4 on K. rhizophila lawn
plates.
Cellulase is commonly used by soil bacteria
to break down plant roots and to form root
nodulations. This nodulation process is
characteristic of a symbiotic relationship with
nitrogen-fixing bacteria which would help
bolster the soil nutrient levels. The soil of T.
repens showed one of the highest predicted
values of cellulase activity; 20% of our soil
bacteria composition showed a potential
cellulase activity.
Though it is hard to visualize due to the color
of the dye, three of our isolates showed
cellulase activity: F15UCLA109ALGN30M3,
F15UCLA109ALGN30M18, and
F15UCLA109ALGN30M20.
Trifolium repens is known to be associated
with nitrogen fixing bacteria. As expected,
the soil bacterial composition predicted
to have nitrogenase was the highest for
T. repens at 25% as compared to the next
highest at 18%.
Only the isolates that showed significant
growth results in the DF and N2-BAP were
tested for growth in JMV semi-solid media
with positive (A) and negative (C) controls.
Of the isolates selected for sequencing,
isolates F15UCLA109ALGN30M3 (B) and
F15UCLA109ALGN30M5 showed positive
results as shown as blooming along the
route of inoculation.
A B C
Soil Collection
Metagenomic
DNA Isolation
PCR
Amplification
Pyrosequencing
Metagenomic
Analysis
Soil
Enrichment
Wet Mounts
and Gram
Stains
Nitrogen
Fixing Assays
DF Media
JMV Media
Cellulase Assay
Antibiotic
Production
Assay
16S rDNA PCR
Amplification
of Isolates
BLAST Analysis
Phylogenetic
Tree