1. Contribution of Leaf-Surface Fungi to the Air Spora
Estelle Levetin and Kip Dorsey

2. Air Spora and Leaf Surfaces
Phylloplane fungi considered major contributors to air spora
In Tulsa leaves appear by mid-April and remain on the trees till November
High concentration of airborne spores occur during this same period
Few studies have compared taxa on leaf surfaces with those in the atmosphere

3. Leaf Surfaces
Leaf surfaces colonized by many types of bacteria and fungi
Both saprobic and pathogenic species
Leaf surfaces can also serve as traps for airborne fungi that settle on surface
Both colonizers and settled spores may become entrained (or reentrained) into the atmosphere

4. Purpose of Study
Examine the phylloplane fungi on two common trees in the Tulsa area
Look for patterns in the communities on leaves throughout the growing season
Compare these taxa to those appearing in the atmosphere
Look for possible parallels in changes of air spora and leaf surface fungi

5. Daily Air Sampling
Air sampling was carried out with a Burkard Spore Trap on the roof of biology building on the University of Tulsa campus
Sample drums were changed every 7 days and cut into 24 hour segments
Slides were examined at 1000x using the single longitudinal transverse method

6. Tree Types
Quercus (Oak) and Ulmus (Elm) are two dominant tree types in eastern Oklahoma
Quercus palustris and Ulmus americana were chosen for this studies

7. Leaf Surface Fungi Leaves collected weekly from Apr. 18 - Nov. 23
Three leaves from each tree were aseptically picked, placed in sterile plastic bags and immediately taken to the Aerobiology Lab
Various methods tested for fungal isolation from leaf surfaces
4cm2 areas on both adaxial and abaxial surfaces were separately wiped with sterile cotton swabs that were dampened with sterile distilled water
Swabs were each put in a sterile tube with 1ml of sterile distilled water and vortexed for 30 seconds
Swabs were removed and 0.5ml of each spore suspension was pipetted onto petri dishes containing malt extract agar plus streptomycin

8. Leaf Surface Fungi
12 plates were prepared each week – total 360 plates
Cultures were incubated at room temperature for 5 to 7 days
Colonies were counted and fungi identified to genus level with standard keys

9. Airborne Fungal Spore Concentration

10. 23 Taxa identified: Cladosporium, Ascospores, Basidiospores, and Alternaria Comprised 90% of Total

11. Results of leaf surface cultures
21,624 colonies isolated from leaf surfaces
23 taxa identified
15 taxa appeared on first sample, April 18
Most prevalent taxa (over 90%):
Yeast – 62.3%
Phoma-type – 12.3%
Cladosporium – 10.8%
Alternaria – 4.7%

12. Fungi Identified from Leaf Surfaces
Acremonium
Drechslera
Pithomyces
Alternaria
Epicoccum
Rhizopus
Arthrinum
Fusarium
Seimatosporium
Aspergillus
Geotrichum
Sporothrix
Aureobasidium
Hyalodendron
Trichoderma
Choanephora
Nigrospora
Yeast
Cladosporium
Penicillium
Non-sporulating
Curvularia
Phoma-type

14. Leaf Surface Concentrations
Generally there was a greater number of colonies isolated from the abaxial surface but no significant difference
Generally there was a greater number of colonies isolated from elm leaves but no significant difference between oak and elm
Mean concentrations of leaf surface fungi (CFU/cm2):
Oak Adaxial
Oak Abaxial
Elm Adaxial
22.3
31.7
32.7
34.6

15. Yeasts
A general term to describe unicellular fungi that reproduce by budding
No attempt was made to identify yeast other than Aureobasidium
Several different types of yeasts were routinely isolated based on pigmentation and cell shape
Yeasts generally dispersed by rain splash; however, Taylor recently reported that Aureobasidium was the most abundant taxa identified on Burkard air samples from a southern California location

16. Phoma
A large genus (223 spp) of anamorphic fungi in the Ceolomycetes that are characterized by conidia formation in a pycnidium ~ spore dispersal by rain splash
Many common ascomycetes have a Phoma anamorph including species in
Leptosphaeria
Didymella
Mycosphaerella
Pleospora
Phaeosphaeria
Diaporthe
Phoma - like genera: Phomopsis, Plenodomus, Peyronellaea, etc
Some Epicoccum isolates have a Phoma synanamorph

17. Concentrations of Leaf Surface Fungi and Airborne Spores

18. Correlation of Airborne Spores and Leaf Surface Fungi
When individual genera examined there was no significant correlation between leaf surface fungi and airborne concentration
Phoma did show a significant correlation with airborne ascospore levels
Nine fungal taxa were found on both leaf surface cultures and the airborne samples
The mean concentrations for the season were compared using a Spearman Correlation
There was a significant correlation between leaf surface fungi and airborne fungi (r=0.74, p=0.035)

19. Comparison of Airborne Ascospore Concentration to Leaf Surface Phoma
Positive correlation r = 0.41, p<0.05

20. Correlation with meteorological variables
Temperature was the most important factor of airborne spore levels
Airborne spore concentrations were significantly related to average daily temperature during the April to November study period
Rainfall was the most important factor for leaf surface fungal concentrations
Several taxa showed significant correlations with weekly rainfall totals

21. Comparison of total weekly rainfall and number of colonies from leaf surfaces

22. Correlation of Leaf Surface Fungi and Rainfall
Spearman R
P Value
Total Colonies
0.557
0.001
Yeast
0.554
Phoma
0.481
0.007
Cladosporium
0.131
0.489
Alternaria
-0.232
0.217

23. Leaf Surface Significance
The leaf surface concentrations
Oak: CFU/cm2
Elm: CFU/cm2
Approximate surface area of elm and oak leaves
Oak: 50 cm2
Elm: 23 cm2
Total number of leaves per tree estimated using average branch-to-branchlet technique
Oak: 100,000 leaves
Elm: 325,000 leaves
Surface area estimate for trees:
Oak: ~5.0 x 106 cm2
Elm: ~7.5 x 106 cm2
Estimate of CFU per tree:
Oak: x 108 CFU
Elm: x 108 CFU

24. Possible Atmospheric Output on Peak Leaf Surface Days
Fungi
Peak
Date
Conc.
CFU/cm2
Possible
Oak total
Elm total
Cladosporium
6-Sep
6.67
3.34 x 107
5.00 x 107
Alternaria
13-Nov
3.58
1.79 x 107
2.69 x 107
Curvularia
22-Aug
0.78
3.9 x 106
5.85 x 106
Drechslera
18-Nov
1.00
5.0 x 106
7.5 x 106
Epicoccum
1-Nov
1.04
5.2 x 106
7.8 x 106
Pithomyces
25-Jul
1.08
5.4 x 106
8.1 x 106
Penicillium
1.13
5.65 x 106
8.48 x 106
Aspergillus
18-Aug
0.33
1.65 x 106
2.48 x 106
Nigrospora
3-Oct
0.50
2.5 x 106
3.75 x 106

25. Conclusion
Leaf surface fungi include taxa with airborne dispersal and those with rain splash dispersal
Leaf surface fungi with airborne dispersal can be major contributors to the air spora
Questions remain about
Population changes on leaf surface
Contribution of Phoma-complex and yeast to the air spora

26. Acknowledgments
Assistance of Claudia Owens for help with air sample analysis is greatly appreciated.