Contribution of Leaf-Surface Contribution of Leaf-Surface Fungi to the Air SporaFungi to the Air Spora

Estelle Levetin and Kip DorseyEstelle Levetin and Kip Dorsey

Air Spora and Leaf SurfacesAir Spora and Leaf Surfaces

Phylloplane fungi considered major Phylloplane fungi considered major contributors to air spora contributors to air spora

In Tulsa leaves appear by mid-April and In Tulsa leaves appear by mid-April and remain on the trees till Novemberremain on the trees till November

High concentration of airborne spores High concentration of airborne spores occur during this same period occur during this same period

Few studies have compared taxa on leaf Few studies have compared taxa on leaf surfaces with those in the atmospheresurfaces with those in the atmosphere

Leaf SurfacesLeaf Surfaces

Leaf surfaces colonized by many types of Leaf surfaces colonized by many types of bacteria and fungibacteria and fungi Both saprobic and pathogenic speciesBoth saprobic and pathogenic species

Leaf surfaces can also serve as traps for Leaf surfaces can also serve as traps for airborne fungi that settle on surface airborne fungi that settle on surface

Both colonizers and settled spores may Both colonizers and settled spores may become entrained (or reentrained) into the become entrained (or reentrained) into the atmosphereatmosphere

Purpose of StudyPurpose of Study

Examine the phylloplane fungi on two Examine the phylloplane fungi on two common trees in the Tulsa areacommon trees in the Tulsa area

Look for patterns in the communities on Look for patterns in the communities on leaves throughout the growing seasonleaves throughout the growing season

Compare these taxa to those appearing in Compare these taxa to those appearing in the atmospherethe atmosphere

Look for possible parallels in changes of Look for possible parallels in changes of air spora and leaf surface fungiair spora and leaf surface fungi

Daily Air SamplingDaily Air Sampling

Air sampling was carried Air sampling was carried out with a Burkard Spore out with a Burkard Spore Trap on the roof of Trap on the roof of biology building on the biology building on the University of Tulsa University of Tulsa campuscampusSample drums were Sample drums were changed every 7 days changed every 7 days and cut into 24 hour and cut into 24 hour segmentssegmentsSlides were examined at Slides were examined at 1000x using the single 1000x using the single longitudinal transverse longitudinal transverse methodmethod

Tree TypesTree Types

QuercusQuercus (Oak) and (Oak) and UlmusUlmus (Elm) are two dominant tree (Elm) are two dominant tree types in eastern Oklahomatypes in eastern OklahomaQuercus palustrisQuercus palustris and and Ulmus americanaUlmus americana were chosen for were chosen for this studiesthis studies

Leaf Surface FungiLeaf Surface Fungi

Leaves collected weekly from Apr. 18 - Nov. 23Leaves collected weekly from Apr. 18 - Nov. 23Three leaves from each tree were aseptically picked, Three leaves from each tree were aseptically picked, placed in sterile plastic bags and immediately taken to placed in sterile plastic bags and immediately taken to the Aerobiology Labthe Aerobiology LabVarious methods tested for fungal isolation from leaf Various methods tested for fungal isolation from leaf surfacessurfaces4cm4cm22 areas on both adaxial and abaxial surfaces were areas on both adaxial and abaxial surfaces were separately wiped with sterile cotton swabs that were separately wiped with sterile cotton swabs that were dampened with sterile distilled waterdampened with sterile distilled waterSwabs were each put in a sterile tube with 1ml of Swabs were each put in a sterile tube with 1ml of sterile distilled water and vortexed for 30 secondssterile distilled water and vortexed for 30 secondsSwabs were removed and 0.5ml of each spore Swabs were removed and 0.5ml of each spore suspension was pipetted onto petri dishes containing suspension was pipetted onto petri dishes containing malt extract agar plus streptomycin malt extract agar plus streptomycin

Leaf Surface FungiLeaf Surface Fungi

12 plates were prepared each week – total 360 plates12 plates were prepared each week – total 360 plates

Cultures were incubated at room temperature for 5 to 7 daysCultures were incubated at room temperature for 5 to 7 days

Colonies were counted and fungi identified to genus level with Colonies were counted and fungi identified to genus level with standard keysstandard keys

Airborne Fungal Spore ConcentrationAirborne Fungal Spore Concentration

0

10,000

20,000

30,000

40,000

50,000

60,000

Sp

ore

s/m

3

0

10,000

20,000

30,000

40,000

50,000

60,000

Sp

ore

s/m

3

23 Taxa identified: 23 Taxa identified: CladosporiumCladosporium, Ascospores, , Ascospores,

Basidiospores, and Basidiospores, and AlternariaAlternaria Comprised 90% of Total Comprised 90% of Total

Basidiospores

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500

1000

1500

2000

2500

3000

3500

4000

4500

5000

1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1

spor

es/m

3

Ascospores

0

2000

4000

6000

8000

10000

12000

1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1

spo

res/

m3

Cladosporium

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1

sp

ore

s/m

3

Alternaria

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500

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2500

1/1 2/1 3/1 4/1 5/1 6/1 7/1 8/1 9/1 10/1 11/1 12/1

spor

es/m

3

Results of leaf surface culturesResults of leaf surface cultures

21,624 colonies isolated from leaf 21,624 colonies isolated from leaf surfacessurfaces23 taxa identified23 taxa identified15 taxa appeared on first sample, April 1815 taxa appeared on first sample, April 18Most prevalent taxa (over 90%): Most prevalent taxa (over 90%): Yeast – 62.3%Yeast – 62.3% Phoma-Phoma-type – 12.3%type – 12.3% Cladosporium – Cladosporium – 10.8%10.8% AlternariaAlternaria – 4.7% – 4.7%

Fungi Identified from Leaf SurfacesFungi Identified from Leaf Surfaces

AcremoniumAcremonium DrechsleraDrechslera PithomycesPithomyces

AlternariaAlternaria EpicoccumEpicoccum RhizopusRhizopus

ArthrinumArthrinum FusariumFusarium SeimatosporiumSeimatosporium

AspergillusAspergillus GeotrichumGeotrichum SporothrixSporothrix

AureobasidiumAureobasidium HyalodendronHyalodendron TrichodermaTrichoderma

ChoanephoraChoanephora NigrosporaNigrospora YeastYeast

CladosporiumCladosporium PenicilliumPenicillium Non-sporulatingNon-sporulating

CurvulariaCurvularia PhomaPhoma-type-type

0

5

10

15

20

CF

U/c

m2

April-18

May-2

May-16

May-30

June-14

June-27

July-11

July-25

Aug-18

Sept-6

Sept-20

Oct-3

Oct-25

Nov-7

Nov-18

Oak

Elm

Leaf Surface Concentration of Phoma spp.

0

1

2

3

4

5

CF

U/c

m2

April-18

May-9

May-30

June20

July-11

Aug-2

Sept-6

Sept-26

Oct-25

Nov-13

Oak

Elm

Leaf Surface Isolates of Alternariaspp.

0

20

40

60

80

100

120

CF

U/c

m2

April-18

May-2

May-16

May-30

June-14

June-27

July-11

July-25

Aug-18

Sept-6

Sept-20

Oct-3

Oct-25

Nov-7

Nov-18

Oak

Elm

Leaf Surface Cocentration of Yeast

0

5

10

15

CF

U/c

m2

April-18

May-9

May-30

June20

July-11

Aug-2Sept-6

Sept-26

Oct-25

Nov-13

Oak

Elm

Leaf Surface Concentration of Cladosporium spp.

Leaf Surface ConcentrationsLeaf Surface Concentrations

Generally there was a greater number of colonies Generally there was a greater number of colonies isolated from the abaxial surface but no significant isolated from the abaxial surface but no significant differencedifferenceGenerally there was a greater number of colonies Generally there was a greater number of colonies isolated from elm leaves but no significant difference isolated from elm leaves but no significant difference between oak and elmbetween oak and elm

Mean concentrations of leaf surface fungi (CFU/cmMean concentrations of leaf surface fungi (CFU/cm22):):

22.322.3 31.731.7 32.732.7 34.634.6

Oak Oak AdaxialAdaxial

Oak Oak AbaxialAbaxial

Elm Elm AdaxialAdaxial

Elm Elm AdaxialAdaxial

YeastsYeasts

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

PhomaPhoma

A large genus (223 spp) of anamorphic fungi in the A large genus (223 spp) of anamorphic fungi in the Ceolomycetes that are characterized by conidia formation Ceolomycetes that are characterized by conidia formation in a pycnidium ~ spore dispersal by rain splashin a pycnidium ~ spore dispersal by rain splashMany common ascomycetes have a Many common ascomycetes have a Phoma Phoma anamorph anamorph including species inincluding species in

LeptosphaeriaLeptosphaeria DidymellaDidymella MycosphaerellaMycosphaerella PleosporaPleospora PhaeosphaeriaPhaeosphaeria DiaportheDiaporthe

PhomaPhoma - like genera: - like genera: Phomopsis, Plenodomus, Phomopsis, Plenodomus, Peyronellaea, Peyronellaea, etcetcSome Some EpicoccumEpicoccum isolates have a isolates have a PhomaPhoma synanamorph synanamorph

Concentrations of Leaf Surface Concentrations of Leaf Surface Fungi and Airborne SporesFungi and Airborne Spores

Weekly Concentrations of Fungi minus All Yeast and Phoma

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

April

-18

April

-25

May

-2M

ay-9

May

-16

May

-23

May

-30

June

-6Ju

ne-1

4Ju

ne-2

0

June

-27

July

-5Ju

ly-1

1Ju

ly-1

8Ju

ly-2

5Au

g-2

Aug-

18Au

g-22

Sept

-6Se

pt-1

3

Sept

-20

Sept

-26

Oct

-3O

ct-1

1O

ct-2

5No

v-1

Nov-

7No

v-13

Nov-

18No

v-23

CFU/

cm2

0

10,000

20,000

30,000

40,000

50,000

60,000

1-A

pr

15

-Ap

r

29

-Ap

r

13

-Ma

y

27

-Ma

y

10

-Ju

n

24

-Ju

n

8-J

ul

22

-Ju

l

5-A

ug

19

-Au

g

2-S

ep

16

-Se

p

30

-Se

p

14

-Oct

28

-Oct

11

-No

v

25

-No

v

Correlation of Airborne Spores and Correlation of Airborne Spores and Leaf Surface FungiLeaf Surface Fungi

When individual genera examined there was no When individual genera examined there was no significant correlation between leaf surface fungi significant correlation between leaf surface fungi and airborne concentrationand airborne concentrationPhomaPhoma did show a significant correlation with did show a significant correlation with airborne ascospore levelsairborne ascospore levelsNine fungal taxa were found on both leaf surface Nine fungal taxa were found on both leaf surface cultures and the airborne samplescultures and the airborne samples The mean concentrations for the season were The mean concentrations for the season were

compared using a Spearman Correlationcompared using a Spearman Correlation There was a significant correlation between leaf There was a significant correlation between leaf

surface fungi and airborne fungi (r=0.74, p=0.035)surface fungi and airborne fungi (r=0.74, p=0.035)

Comparison of Airborne Ascospore Comparison of Airborne Ascospore

Concentration to Leaf Surface Concentration to Leaf Surface PhomaPhoma

0

2

4

6

8

10

12

14

A M J J A S O N

Ph

om

a C

on

cen

trat

ion

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2000

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10000

12000

Air

bo

rne

Asc

osp

ore

C

on

cen

trat

ion

Positive correlation r = 0.41, p<0.05

Correlation with meteorological variablesCorrelation with meteorological variables

Temperature was the most important Temperature was the most important factor of airborne spore levelsfactor of airborne spore levels Airborne spore concentrations were Airborne spore concentrations were

significantly related to average daily significantly related to average daily temperature during the April to November temperature during the April to November study periodstudy period

Rainfall was the most important factor for Rainfall was the most important factor for leaf surface fungal concentrationsleaf surface fungal concentrations Several taxa showed significant correlations Several taxa showed significant correlations

with weekly rainfall totalswith weekly rainfall totals

Comparison of total weekly rainfall and Comparison of total weekly rainfall and

number of colonies from leaf surfacesnumber of colonies from leaf surfaces

0

500

1000

1500

2000

2500

April-18 May-9 May-30 June20 July-11 Aug-2 Sept-6 Sept-26 Oct-25 Nov-13

Nu

mb

er o

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olo

nie

s

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10

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70

80

mm

Rai

nfa

ll

number of colonies

weekly rain total

Correlation of Leaf Surface Fungi Correlation of Leaf Surface Fungi and Rainfalland Rainfall

Spearman RSpearman R P ValueP Value

Total ColoniesTotal Colonies 0.5570.557 0.0010.001

YeastYeast 0.5540.554 0.0010.001

PhomaPhoma 0.4810.481 0.0070.007

CladosporiumCladosporium 0.1310.131 0.4890.489

AlternariaAlternaria -0.232-0.232 0.2170.217

Leaf Surface SignificanceLeaf Surface SignificanceThe leaf surface concentrationsThe leaf surface concentrations Oak: Oak: 27.00 CFU/cm27.00 CFU/cm22

Elm: Elm: 33.68 CFU/cm33.68 CFU/cm22

Approximate surface area of elm and oak leavesApproximate surface area of elm and oak leaves Oak: Oak: 50 cm50 cm22

Elm: Elm: 23 cm23 cm22

Total number of leaves per tree estimated using average Total number of leaves per tree estimated using average branch-to-branchlet techniquebranch-to-branchlet technique

Oak: Oak: 100,000 leaves100,000 leaves Elm: Elm: 325,000 leaves325,000 leavesSurface area estimate for trees:Surface area estimate for trees:

Oak: Oak: ~5.0 x 10~5.0 x 1066 cm cm22

Elm: Elm: ~7.5 x 10~7.5 x 1066 cm cm22

Estimate of CFU per tree:Estimate of CFU per tree: Oak: Oak: 1.35 x 101.35 x 1088 CFU CFU Elm: Elm: 2.53 x 102.53 x 1088 CFU CFU

Possible Atmospheric Output on Possible Atmospheric Output on Peak Leaf Surface DaysPeak Leaf Surface Days

FungiPeakDate

Conc.CFU/cm2

PossibleOak total

Possible 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 22-Aug 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

ConclusionConclusion

Leaf surface fungi include taxa with Leaf surface fungi include taxa with airborne dispersal and those with rain airborne dispersal and those with rain splash dispersalsplash dispersalLeaf surface fungi with airborne dispersal Leaf surface fungi with airborne dispersal can be major contributors to the air sporacan be major contributors to the air sporaQuestions remain aboutQuestions remain about Population changes on leaf surfacePopulation changes on leaf surface Contribution of Contribution of Phoma-complex Phoma-complex and yeast to and yeast to

the air sporathe air spora

Acknowledgments Acknowledgments

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