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•  Motor vehicle transportation contributes to air pollution in the fine particle (PM 2.5) range, which is associated with adverse health effects and mortality

•  A pollution removal strategy by the City of Phoenix is planting trees near roadsides

•  Microorganisms inhabiting the surfaces of plant tissues (i.e. the phyllosphere) contribute to biogeochemical cycling, but little is known of their ability mitigate air pollution

1)  Characterize the composition and biogeochemical function of

bacterial communities inhabiting the urban phyllosphere of city trees in Phoenix

2)  Evaluate phyllosphere community variation due to differences in local air quality and host tree species.

Introduc>on    Phoenix  City  Trees   Methods:  Extrac>ng  phyllosphere  DNA    

I use real-time PCR of the DNA from leaf washing samples to quantify the number of bacteria in each sample. Real-time PCR records a fluorescent signal for each copy of a DNA molecule present in a sample. The leaf washings have DNA from bacteria, chlorophyll, and mitochondria are present, so specific primers must be used.

Quan>fica>on  of  Bacterial  Density  

Future  Research  The aforementioned techniques will provide estimates of how many bacteria inhabit a single tree. Ultimately this study also aims to understand the function of phyllosphere bacteria. Metagenomic analysis of functional genes and taxonomic community composition of phyllosphere bacterial colonizers are the next steps of this research.

Literature  Referenced  

Acknowldgements  •  CAP-LTER for funding this research with a CAP-LTER Graduate Grant

and providing logistical and strategic resources •  Dr. Daniel Childers and the Wetland Ecosystem Ecology Lab (WEEL)

for intellectual development and resources •  Dr. Ferran Garcia-Pichel and his lab for guidance and resources •  Arianne Cease for strategy advising •  Steven Earl for assisting with all requests made of CAP-LTER.

Host tree phylogeny selects for phyllosphere bacteria colonizers. A comparison of several tree species will illuminate differences in density, community composition, and function due to host species. Each of the species chosen for sampling are among the ten most common city trees Collectively they comprise almost 50% of all city trees.

Arizona  State  University  School  of  Sustainability  Benjamin  MacNeille,  Daniel  L.  Childers  

Exploring  Phoenix’s  Urban  Phyllosphere  

Pollu>on  or  Food?  Phyllosphere bacteria are carbon-limited, and carbonaceous aerosols are the dominant species in fine particles (PM 2.5) found on roadsides. If bacterial communities can utilize atmospheric carbon from pollution sources, a higher density and different community composition of bacteria may be supported near roadsides. I sample trees of varied distances to the closest roadside to test the affect of pollution on bacterial communities.

Each sampled tree is photographed from two angles and the color digital image processing method (CD) is used to estimate total leaf area.

Figure 4. Satellite images of Phoenix. Many city trees are planted along the transportation grid. Each city tree is represented by a green dot.

Scaling  up  

Forward  Primer  534  (5ʹ′-­‐CCAGCAGCCGCGGTAAT-­‐3ʹ′)  Reverse  Primer  783  (5ʹ′-­‐ACCMGGGTATCTAATCCKG-­‐3ʹ′)  

Oligonucleo/de  sequences  that  anneal  with  bacterial  rRNA  and  also  plant  mitochondrial  DNA.  

Forward  Primer  1345  (5ʹ′-­‐AGTTTTTGGCCTTATCTTG-­‐  3ʹ′)  Reverse  Primer  1430  (5ʹ′-­‐AAACCCCACTACGTACCACACCAC-­‐3ʹ′)  Oligonucleo/de  sequences  that  anneal  with  plant  mitochondrial  DNA.  

Pollu>on  Removal  by  Trees  

Leaves from city trees are extracted and washed to remove surface microorganisms. Each wash is filtered and its DNA isolated and the total leaf surface area is measured.

City trees are strategically planted near roads because vehicles are pollution sources; air pollution is removed by trees through passive interception.

Figure 3. Satellite image of Washington St. and Gateway, where city trees line the streets. Each city tree is represented by a green dot.

According to the City of Phoenix’s Tree and Shade Master Plan, the city’s vision is to achieve 25% canopy coverage by 2030, doubling the current canopy coverage of 12%, comprised of 92,000 city trees. This will increase the surface area available to phyllosphere bacteria, which colonize leaves in densities of up to 107 cells per cm2.

Figure 2. Electron microscope image of microbial phyllosphere colonizers.

Figure 1. Roadside trees passively intercept motor vehicle pollution

Figure 6. PM 2.5 molecules associated with motor vehicle sources: (left to right) stearic acid, phthalic acid, and nonacosane.

Table 1. Ten most common city trees in Phoenix

Figure 5. Larger trees with more leaf area confer greater benefits, and also provide more phyllosphere area for microorganisms. Trees in Phoenix have varied canopy sizes.

Figure 7. DNA isolated from leaf washes include chlorophyll, mitochondria, and bacteria.

Chlorophyll Mitochondria Bacteria

Figure 8. Subtracting the quantity of mitochondria from the bacteria and mitochondria mix elucidates the actual number of bacteria in the sample.

hWp://nac.unl.edu  

hWp://www2.daveytreekeeper.com/ufrm/index.html  

hWp://www2.daveytreekeeper.com/ufrm/index.html  hWp://www2.daveytreekeeper.com/ufrm/index.html  

hWp://organicsoiltechnology.com  

www.apnursery.com  

hWp://www.urbantreealliance.org  

Mesquite  Arizona  Ash  hWp://briarpatchdesertplants.com/  hWp://www.dirtdoctor.com/  

Mexican  Fan  Palm  

Aleppo  Pine  hWp://www.public.asu.edu/  

hWp://blog.growingwithscience.com/  

Blue  Palo  Verde  

Shoestring  Acacia  hWp://deserthorizonnursery.com/  

Indian  Rosewood  hWp://www.easybloom.com/  

Araya,T.  R.,  Flocchini,R.,  MoralesSegura,  R.G.E.,  &  LeivaGuzmán,M.a.(2014).  Carbonaceous  aerosols  in  fine  par/culate  maWer  of            San/ago  Metropolitan  Area,  Chile.  The  Scien)fic  World  Journal,  2014,  794590.  doi:10.1155/2014/794590      City  of  Phoenix.  (2010).  Tree  and  shade  master  plan.  Retrieved  from  hWps://www.phoenix.gov/parkssite/Documents/T%20and%20A%202010.pdf      DelmoWe,  N.,  Knief,  C.,  Chaffron,  S.,  Innerebner,  G.,  Roschitzki,  B.,  Schlapbach,  R.,  Vorholt,  J.  a.  (2009).  Community  proteogenomics  reveals  insights  into  the  physiology  of  phyllosphere  bacteria.  Proceedings  of  the  Na/onal  Academy  of  Sciences  of  the  United  States  of  America,  106(38),  16428–33.  doi:10.1073/pnas.0905240106      Heo,  J.,  Dulger,  M.,  Olson,  M.  R.,  McGinnis,  J.  E.,  Shelton,  B.  R.,  Matsunaga,  A.,  ...  Schauer,  J.  J.  (2013).  Source  appor/onments  of  PM2.5  organic  carbon  using  molecular  marker  Posi/ve  Matrix  Factoriza/on  and  comparison  of  results  from  different  receptor  models.  Atmospheric  Environment,  73,  51–61.  doi:10.1016/j.atmosenv.2013.03.004      Kinkel,  L.  L.,  Wilson,  M.,  &  Lindow,  S.  E.  (2000).  Plant  species  and  plant  incuba/on  condi/ons  influence  variability  in  epiphy/c  bacterial  popula/on  size.  Microbial  ecology,  39(1),  1-­‐11.      Laden,  F.,  Schwartz,  J.,  Speizer,  F.  E.,  &  Dockery,  D.  W.  (2006).  Reduc/on  in  fine  par/culate  air  pollu/on  and  mortality:  Extended  follow-­‐up  of  the  Harvard  Six  Ci/es  study.  American  Journal  of  Respiratory  and  Cri)cal  Care  Medicine,  173(6),  667–72.  doi:10.1164/rccm.200503-­‐443OC      Lee,  S.  C.,  Cheng,  Y.,  Ho,  K.  F.,  Cao,  J.  J.,  Louie,  P.  K.-­‐K.,  Chow,  J.  C.,  &  Watson,  J.  G.  (2006).  PM  1.0  and  PM  2.5  Characteris/cs  in  the  Roadside  Environment  of  Hong  Kong.  Aerosol  Science  and  Technology,  40(3),  157–165.  doi:10.1080/027868205004945441    Lindow,  S.  E.,  &  Brandl,  M.  T.  (2003).  Microbiology  of  the  Phyllosphere  MINIREVIEW  Microbiology  of  the  Phyllosphere,  69(4).  doi:10.1128/AEM.69.4.1875      Mar,  T.  F.,  Norris,  G.  a.,  Koenig,  J.  Q.,  &  Larson,  T.  V.  (2000).  Associa/ons  between  Air  Pollu/on  and  Mortality  in  Phoenix,  1995-­‐1997.  Environmental  Health  Perspec)ves,  108(4),  347–  353.  doi:10.1289/ehp.00108347      Nowak,  D.  J.,  Crane,  D.  E.,  &  Stevens,  J.  C.  (2006).  Air  pollu/on  removal  by  urban  trees  and  shrubs  in  the  United  States.  Urban  Forestry  &  Urban  Greening,  4(3-­‐4),  115–123.  doi:  10.1016/j.ufug.2006.01.007      Peper,  P.  J.,  &  McPherson,  E.  G.  (2003).  Evalua/on  of  four  methods  for  es/ma/ng  leaf  area  of  isolated  trees.  Urban  Forestry  &  Urban  Greening,  2(1),  19-­‐29.    Rastogi,  G.,  Tech,  J.  J.,  Coaker,  G.  L.,  &  Leveau,  J.  H.  J.  (2010).  A  PCR-­‐based  toolbox  for  the  culture-­‐independent  quan/fica/on  of  total  bacterial  abundances  in  plant  environments.  Journal  of  Microbiological  Methods,  83(2),  127–32.  doi:10.1016/j.mimet.2010.08.006    Toro  Araya,  R.,  Flocchini,  R.,  Morales  Segura,  R.  G.  E.,  &  Leiva  Guzmán,  M.  a.  (2014).  Carbonaceous  aerosols  in  fine  par/culate  maWer  of  San/ago  Metropolitan  Area,  Chile.  The  Scien)fic  World  Journal,  2014,  794590.  doi:10.1155/2014/794590    

www.wikipedia.com  

Leaf  Area  =  SA  x  CFA    

Silhoue]e  Area  (SA):    Crown  Area/Photo  Frame  Area  

   

Crown  Framed  Area  (CFA):    Height  x  Width  of  the  canopy    

Figure 9. Photoshop and ImageJ are used to estimate the leaf area of photographed trees.

Figure 10. TonB receptor uptake of Vitamin B12. TonB receptors are responsible for the uptake of many different molecules from outside the cell and are commonly found in phyllosphere bacteria.

Objec>ves  

hWp://www.cell.com/