Contribution of Leaf-Surface Fungi to the Air Spora

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. 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% Results of leaf surface cultures

12. Fungi Identified from Leaf Surfaces

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):

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

23. Leaf Surface Significance • The leaf surface concentrations Oak: 27.00 CFU/cm2 Elm: 33.68 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: 1.35 x 108 CFU Elm: 2.53 x 108 CFU

24. Possible Atmospheric Output on Peak Leaf Surface Days

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.