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
0
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
0
500
1000
1500
2000
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
0
2000
4000
6000
8000
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
f C
olo
nie
s
0
10
20
30
40
50
60
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.