Microb Ecol
DOI 10.1007/s00248-012-0089-8
FUNGAL MICROBIOLOGY
Temporal Patterns in Appearance of Sooty Blotch
and Flyspeck Fungi on Apples
J. C. Batzer & A. J. Sisson & T. C. Harrington &
D. A. Mayfield & M. L. Gleason
Received: 20 February 2012 / Accepted: 21 June 2012
# Springer Science+Business Media, LLC 2012
Abstract Sooty blotch and flyspeck (SBFS) is a complex of
about 80 fungal species that blemish the surface of apple fruit
in humid regions worldwide. The dark colonies become visible
in mid- to late summer, reducing the value of fresh fruit.
Although many SBFS species can co-occur in the same orchard and even on the same apple, little is known about
temporal patterns of these species, including the timing of
colony appearance. To test the hypothesis that colonies of
SBFS species appear on apples at characteristic times during
the growing season, 50 apples were monitored weekly at three
Iowa orchards in 2006 and six orchards in 2007 and 2008.
However, a mean of 24.3 apples per orchard was assessed at
harvest because of apple drop throughout the season. Colonies
were marked with colored pens as they appeared. After harvest
and after storage of apples at 2 °C for 3 months, SBFS colonies
on each fruit were counted and classified by morphology, and a
representative subset of colonies was excised from the fruit and
preserved on dried peels for species identification using rDNA.
Seventeen species were identified. Stomiopeltis spp. RS1 and
RS2 appeared on apples 10 to 14 days before other SBFS taxa.
Dissoconium aciculare was generally the last species to appear
on apple fruit, and it continued to appear during postharvest
storage. The most prevalent taxa in Iowa orchards were also
the most abundant. Diversity of SBFS fungi in an orchard was
positively correlated with cumulative hours of surface wetness
hours due to rainfall or dew, which is believed to favor growth
of SBFS fungi. Species-specific information about temporal
patterns of appearance on apple fruit may lead to improved
SBFS management strategies.
J. C. Batzer (*) : A. J. Sisson : T. C. Harrington :
D. A. Mayfield : M. L. Gleason
Department of Plant Pathology and Microbiology,
Iowa State University,
Ames, IA 50011, USA
e-mail: jbatzer@iastate.edu
Introduction
Fungi in the sooty blotch and flyspeck (SBFS) complex
blemish fruit with waxy cuticles, including apple (Malus×
domestica Borkh.), in humid climates worldwide [1]. In
Iowa, the dark colonies of SBFS fungi on apple commonly
appear as fruit begin to ripen in late summer [2], but in the
Southeast U.S. colonies can become visible by early June
[3, 4]. Because apples with SBFS blemishes are generally
unsuitable for fresh-market sale, substantial economic losses
occur when fruit are downgraded to processing use [4, 5].
Ecological studies of SBFS have been limited by reliance
on morphology for species identification, creating uncertainty about which species were being studied [6–9]. Although each SBFS species expresses a consistent mycelial
type on apple, many species share similar colony morphology [10, 11] and few sporulate readily on apples. Therefore,
characterizing morphology on fruit is insufficient to delineate SBFS species.
Over 80 putative species have been associated with SBFS
using rDNA analysis coupled with morphological characterization [1, 11]. Most described members of the SBFS
complex are in the class Dothideomycetes, and about 95 %
of these are in the order Capnodiales [11]. Recent surveys
have identified SBFS fungi by isolating them in pure culture
before extracting DNA, amplifying by polymerase chain
reaction (PCR), and sequencing portions of the rDNA
[10–12]. Few studies involving the biology of the SBFS
complex have been attempted because of the slow growth
and recalcitrant nature of these fungi. For example, the
success rate for obtaining a pure SBFS isolate from a single
colony on an apple ranges from 5 % to 30 %, depending on
the fungal species and condition of the fruit (Batzer, unpublished data).
As an alternative to culturing, mycelia of SBFS colonies
can be scraped from the apple surface and identified to
J. C. Batzer et al.
genus or species within 2 days using extracted DNA and
PCR with restriction fragment length polymorphisms
(RFLP) [2, 13]. Following DNA extraction, PCR is performed using the primer pair ITS-F/Myc1-R, which are
primers specific to fungi and the order Capnodiales, respectively. The restriction enzyme HaeIII is then used to digest
the amplicons into fragments that can be visualized on an
agarose gel, and banding patterns are compared to a library
of 14 previously identified common SBFS genera [2]. This
method can result in a much higher percentage of identifications than agar plate isolation and provides a much faster
way to process the numerous field samples needed to undertake ecological studies.
Knowledge of the ecology of key SBFS species in a
geographic region could pave the way for development of
more cost-effective management strategies that target the
most important species. In a survey of orchards in 14 U.S.
states, Díaz Arias et al. [11] presented evidence that species
composition of the SBFS assemblage in an orchard differs
according to geographic region and fungicide use. However,
there is insufficient information about the phenology of
SBFS species. A study in the Southeast U.S. noted that
SBFS mycelial types appeared on apples at characteristic
times during a growing season, but species were not identified [14]. In the only previous study to identify phenological
behavior of an identified SBFS species, Cooley et al. [15]
used spore traps to determine timing of inoculum of Schizothyrium pomi in Massachusetts apple orchards.
The objectives of this study were to (1) determine whether
there are species-specific patterns in the timing of colony
appearance on apples, (2) characterize species diversity, prevalence, and abundance of fungi in the SBFS complex in Iowa
orchards, and (3) determine the relationship of leaf wetness
duration to taxonomic diversity and severity of SBFS.
the study plots after first cover, but conventional insecticidespray schedules [16] were maintained throughout the season, presumably with negligible effect on SBFS fungi.
Data Collection
Beginning in early July, arbitrarily selected apples (ten per
tree) in each test block were inspected weekly for SBFS
colonies. Newly appearing colonies that were visible to the
naked eye were marked with a ball-point pen, using different colors and outline shapes each week. Premature fruit
drop reduced the number of apples per orchard from an
initial total of 50 to 8 to 39 apples at harvest (Table 1). After
harvest, the apples were stored at 2 °C for 3 months. Leaf
wetness duration (LWD), defined as the length of time per
day that foliage remained wet, and temperature in each
orchard were monitored hourly with a Watchdog Data Logger (Spectrum Technologies, Inc., Plainfield, IL, USA) at
1.5-m height under the tree canopy in the monitored block
from 10 days after petal fall to harvest (Table 1).
Colony Characterization
Materials and Methods
After 3 months of cold storage, all SBFS colonies on each
harvested apple were classified by mycelial type using a
dissecting microscope [1, 10]. Colonies that were not
marked during field inspection were assumed to have developed during cold storage. Three to five representative
colonies of each mycelial type for each time period were
excised from each apple along with the subtending apple
peel. In order to minimize contamination from adjoining
colonies, colonies visually separate from other colonies on
the fruit were selected for analysis. Excised colonies were
pressed between paper towels until dry. Dried peels were
photographed then stored at room temperature in individual
wells of 24-well plastic culture plates for up to 4 weeks prior
to fungal DNA extraction.
Orchard Locations
Genetic Characterization
Three central Iowa orchards were monitored in 2006 and six
in 2007 and 2008 (Table 1; Fig. 1). Trees were cv. Golden
Delicious, except cv. Honeygold at Community Orchard in
2006 and cv. Liberty at Apple Ridge Orchard in 2007. Plots
consisted of a row segment of five contiguous mature trees
located on the edge of the orchard. A standard phenology
driven spray program for apples was used in each orchard
[16]. Plots were sprayed with myclobutanil (a fungicide
with negligible activity against SBFS fungi) from the tight
cluster stage (pre-bloom) through first cover. The first cover
fungicide spray is generally applied 10 days after petal fall
and targets apple scab, fruit rots, cedar apple rust, and
powdery mildew diseases. No fungicides were applied to
Fungal mycelium was scraped from the dried peels and transferred to tubes containing 50 μl of Prepman Ultra Sample
Preparation Reagent (Applied Biosystems, Foster City, CA).
DNA was extracted according to the manufacturer's protocol
and stored at −20 °C until amplification [2]. The ITS region
and a portion of the large subunit (~800 bp) of rDNA was
amplified using the fungal specific primer ITS1-F and the
Capnodiales-specific primer Myc-1R (5′-ACTCGTCC
GAAGGAGCTACG-3′). If the extracted DNA failed to amplify on the first attempt, 5 % DMSO was added to the PCR
master mix and a tenfold dilution was applied to the DNA
extract. If samples still did not amplify in the second PCR, the
amount of DNA extract was doubled (from 1 to 2 μl) in a 50-
Temporal Patterns in Appearance of Sooty Blotch and Flyspeck
Table 1 Number of apples collected, key dates, and cumulative leaf wetness duration (LWD) from days after petal fall until first appearance and
until harvest at seven orchards in Iowa from 2006 to 2008
Year
2006
2007
2008
Orcharda
CG
CO
HRS
AR
BP
CG
DO
HRS
PN
AR
BP
CG
DO
HRS
PN
Cultivarb
Number of apples
JDc of 1st cover spray
JD SBFS first observedd
JD of harvest
LWD at 1st appearancee
LWD at harvest
gd
23
144
227
261
354
771
hg
35
144
237
250
269
NA
gd
39
145
233
268
265
437
lb
22
128
220
250
396
641
gd
29
127
219
250
494
742
gd
23
140
237
250
262
286
gd
25
141
226
253
330
562
gd
22
150
221
253
220
379
gd
24
140
208
251
328
641
gd
22
148
216
251
355
652
Gd
8
148
217
258
540
947
gd
22
148
217
258
440
1071
gd
23
155
216
258
535
888
gd
28
156
213
247
258
460
gd
20
148
217
259
NAf
NA
a
Orchards: AR0Apple Ridge (42°31′N 93°12′W), BP0Berry Patch (41°55′N 93°27′W), CG0Center Grove (41°52′N 93°28′W), CO0Community
Orchard (42°33′N 94°11′W), DO0Deal's Orchard (41°59′N 94°24′W), HRS0Iowa State University Horticultural Research Station (42°06′N 93°35′
W), and PN0Pella Nursery (41°40′N 92°87′W)
b
gd0cv. Golden Delicious, lb0cv. Liberty, hg0cv. Honeygold
c
Julian day, i.e., 1210May 1, 1520June 1, 1820July 1, 2130Aug 1, 2440Sept 1, 2740Oct 1
d
Beginning in July, apples were inspected weekly for the presence of SBFS signs. Dates when first colonies became visible on apples are shown
e
Cumulative hours of leaf wetness duration (LWD) was monitored using a sensor from first cover spray (7 to 10 days after petal fall) to first
appearance of any SBFS colony
f
NA0not available
μl PCR mixture rather than a 100-μl mixture. Samples of the
compact speck mycelial type [10] that did not amplify with
ITS1-F/Myc-1R were subsequently subjected to PCR using a
specific primer pair for the putative species Dothideomycete
sp. CS1 [(3′-TTGCGTCTCCTGTCGGGTC-5′) and (5′ATCCGAGGTCAACCATTAAAG-3′)] using the same master mix and thermocycler conditions [2].
Products that amplified with ITS1-F/Myc-1R were digested
with HaeIII endonuclease; banding patterns on a 2 % agarose
gel were compared to a library of previously identified SBFS
Figure 1 Locations of seven
Iowa orchards sampled for SBFS
in 2006 to 2008: AR0Apple
Ridge; BP0Berry Patch; CO0
Community Orchard; DO0
Deal's Orchard; HRS0Iowa
State University Horticulture
Research Station; and
PN0Pella Nursery
species and genera [2]. As a quality control measure for banding patterns that matched those of previously identified SBFS
taxa [2], the PCR products of two subsamples per orchard were
purified and submitted for sequencing at the DNA Sequencing
and Synthesis Facility of Iowa State University using primers
ITS1-F/Myc-1R. Resulting sequences were compared with
known SBFS sequences using the online search tool BLASTn
(NCBI, Bethesda, MD). When restriction digest gel patterns
did not match those of previously identified SBFS fungi [2],
the PCR product was sequenced using primers ITS1-F and
J. C. Batzer et al.
Myc-1R. To verify the identification of the Dothideomycete
sp. CS1, a subset of PCR products obtained from primer pair
CS1F/CS1R was sequenced using the same primer pair.
Data Analysis
Each SBFS species produces a single characteristic mycelial
type on apple fruit [10], but more than one species can cause
the same mycelial type. Taxa within each mycelial type were
defined using the RFLP match previously shown to delineate
to genus [2]. We estimated the number of colonies per taxon
on a given apple as colonies per apple (CPA) by determining
the proportion of a taxon in a given mycelial type from the
subsamples (3 to 5 colonies per week per orchard as described
above in colony characterization), and extrapolating that proportion to the total colony count of the mycelial type on that
apple (Table 2). Species richness and Shannon's diversity
index [17] were calculated and compared among orchardyears using analysis of variance (PROC GLM) (SAS Inc.,
Durham, NC, USA).
Linear regression was used to test the hypothesis that
prevalence (the number of orchards in which a taxon was
found in a given year) was correlated with severity (CPA of
each taxon). To test the hypothesis that diversity in an orchard
was correlated with severity, the mean CPA of each orchardyear was regressed against the number of species per orchardyear. To determine the effects of cumulative LWD on severity
and diversity, CPA for each orchard-year and number of taxa
per orchard-year, respectively, were regressed against cumulative LWD from 10 days after petal fall to harvest. Precision
of these regression models was evaluated using the coefficient
of determination (R2) and the standard error of the estimate
(SEEy) (Sigma Plot 2011, Systat Software Inc.).
Because many taxa occurred sporadically across orchardyears (Table 3), closely related taxa were grouped into seven
operational taxonomic units (OTUs) based on previous phylogenetic analysis of the ITS and LSU regions of rDNA
sequences and colony morphology on apple fruit [10]. Seven
OTUs were designated as species residing in a putative genus
in monophyletic clades expressing the same mycelial type on
apple (Table 4). Operational taxonomic unit were designated
from genus-mycelial types as follows: “Stomiopeltis-RS” was
comprised of two putative species of Stomiopeltis (RS1 and
RS2) that each expressed the ramose mycelial type on apple,
Table 2 Identification of sooty blotch and flyspeck (SBFS) fungi from colonies on excised apple peels from seven Iowa orchards from 2006 to 2008
Orchard
Apple Ridge
Berry Patch
Center Grove
Community
Deal
HRS
Pella Nursery
Total
Year
Number of colonies
sampleda
Number successfully
amplifiedb
RFLP Matchedc
RFLP not matchedd
Total
Previously identified
rDNA sequence
New rDNA
sequence
Unable to
sequence
2007
2008
2007
2008
2006
2007
2008
2006
2007
2008
2006
2007
2008
82
105
135
83
49
39
91
63
92
136
65
62
107
58
90
120
61
47
26
55
51
62
98
57
51
99
56
83
113
58
45
26
53
49
60
88
49
46
96
0
0
1
1
0
0
0
0
0
9
0
4
0
2
7
0
0
0
0
2
0
1
1
0
0
2
0
0
6
2
2
0
0
2
1
0
8
1
1
2007
2008
15
142
133
1384
123
117
1115
122
117
1061
0
0
15
1
0
16
0
0
23
a
For each set of marked colonies from a weekly orchard visit, three to five colonies of commonly occurring mycelial types and all colonies with
unique mycelial types were sampled
b
rDNA was amplified using specific primers for Capnodiales or Dothideomycetes sp. CS1
c
PCR products from ITS1-F/Myco1were digested with HaeIII and banding patterns from RFLP analysis were compared to previously characterized species of SBFS [24]. Gels with patterns identical to those of previously characterized species were termed “Matched.”
PCR products with RFLP banding patterns that did not match a library of previously characterized species were termed “Not Matched” and were
sequenced with primer pair ITS1-F/ITS4. Sequences were compared to other fungi in GenBank using BLASTn and were verified to be previously identified
SBFS species or were designated as a “New sequence.” Colonies that were unable to be sequenced were found to be mixed colonies on the apple fruit
d
Temporal Patterns in Appearance of Sooty Blotch and Flyspeck
Table 3 Prevalence (frequency of occurrence in 15 orchard-years) and severity (mean colonies per apple) of SBFS taxa identified from apples at
harvest
Taxa identifieda
Representative rDNA
ITS sequence
Mycelia type
on appleb
Number of orchardyears detectedc
Mean colonies
per appled
Stomiopeltis sp. RS1
Stomiopeltis sp. RS2
Dissoconium aciculare
Microcyclosporella spp.
Ramose
Ramose
Discrete speck
Ridged honeycomb
15
15
15
15
4.8
5.2
3.4
3.9
Schizothyrium pomi or Zygophiala cryptogama
Z. wisconsinensis
Colletogloeopsis-like sp. FG2
Phaeothecoidiella spp.
Peltaster fructicola
Peltaster sp. P2.1
Peltaster sp. P2.5
AY598882
AY598883
AY598874
FJ425195, AY598866,
AY598868, FJ425196
AY598851, FJ425208
FJ425209
FJ425193
AY598878, AY598879
AY598887
AY598888
JQ347532
Flyspeck
Flyspeck
Fuliginous
Arborescent punctatee
Punctate
Punctate
Punctate
14
3
13
13
8
12
7
2.4
0.6
1.7
1.1
1.5
2.4
0.6
Pseudoveronaea sp.
Uwebraunia commune
Sporidesmajora pennsylvaniensis
Diatractium-like sp.
Ramularia sp. P5
Dothideomycete sp. CS1
AY598877
AY598876
FJ438379
JQ347531
AY598873
FJ438388
Fuliginous
Fuliginous
Fuliginous
Punctate
Punctate
Compact speck
1
1
1
5
6
9
0.6
1.3
0.1
1.1
1.6
0.5
a
Taxa were delineated using a PCR-RFLP analysis of rDNA [24] and a subset was verified with sequencing
b
Morphology of SBFS colonies on apple surface as described by Batzer et al. [10]
c
Prevalence among orchard-years (out of 15 orchard-years)
d
Mean colonies per apple (CPA) for each taxon. Means were determined across apples per orchard-year, in which that species was present
e
Arborescent punctate mycelia type with distinctive arborescent margins and smaller diameter than other punctate mycelial types [40]
“Microcyclosporella-RH” included four putative species associated with the ridged honeycomb (RH) mycelial type,
“Dissoconium-DS” was comprised of Dissoconium aciculare
associated with the discrete speck (DS) mycelial type,
“Peltaster-PT” included three putative species associated with
the punctate (PT) mycelial type, “Schizothyrium-FS” included
three anamorph Zygophiala species associated with the flyspeck
(FS) mycelial type, “Colletogloeopsis-FG” was associated with
the fuliginous (FG) mycelial type, and “Phaeothecoidiella-AP”
included two species associated with the arborescent punctate
(AP) mycelial type. OTUs detected in one or more orchardyears were included in the analysis of temporal patterns, but
relatively uncommon taxa associated with the punctate, compact
speck, and fuliginous mycelial types were excluded.
To determine the effect of year and orchard on severity of
each OTU, CPA at harvest was compared across orchard-years
using analysis of variance (PROC GLM). The interaction of
orchard, year, and week of appearance for each OTU was
assessed using analysis of variance of the least square mean
CPA (PROC MIXED). To test the hypothesis that timing of
appearance of colonies on the apple differed among OTUs, we
used Friedman's test to compare the mean day of first appearance of OTUs for each orchard-year, then applied a Wilcoxon
signed-rank test to make comparisons between taxa. The percentage of colonies of each OTU that appeared during cold
storage was compared with a Fischer's protected least significant difference test.
Temporal progression of colony appearance was evaluated
for the three most prevalent OTUs using population dynamic
models commonly applied to the study of plant disease epidemics. Preliminary analyses of the linear, monomolecular,
logistical, Gompertz, and exponential models were compared
using curves of CPA vs. time and rate curves (dy/dt) (data not
shown). Based on goodness-of-fit plots (PROC REG), the
monomolecular and exponential models were selected. The
monomolecular (log(1/(1−y)) population dynamic model
describes epidemics for which there is no secondary spread;
that is, there is a fixed amount of initial inoculum. In contrast,
an exponential model (log(y)) describes polycyclic epidemics
that encompass multiple generations of the pathogen and
spread during the growing season [18, 19]. Cumulative weekly
proportions of apples with colonies of the OTU for each
orchard-year were regressed against time (PROC REG) using
the two models. Goodness-of-fit to the linear forms of each
models were compared by examining the F statistic for linearity, the coefficient of determination (R2), the back-transformed
J. C. Batzer et al.
root mean square error (a measure of how well the model
explains a given set of observations), and plots of the residuals
[20]. Curves with fewer than four observations between 0.1
and 0.99 apples with SBFS on the y axis were eliminated
because the number of data points was insufficient.
identical to previously identified isolates of that putative species. The remaining sequences delineated two putative species
that were not previously associated with SBFS: a Diatractium-like sp. and Peltaster sp. P2.5 (ITS sequences JQ347531
and JQ347532, respectively).
Diversity Patterns
Results
At harvest, the mean number of colonies per apple (CPA) of
all SBFS colonies across the 15 orchard-years was 25.9 and
ranged from 11.6 to 55.5 (Table 4), not including colonies that
developed in storage. Premature drops reduced the number of
apples per orchard to an average of 24.3 at harvest (Table 1).
Weather conditions in 2008 were much wetter than in 2007; in
Center Grove Orchard, for example, the cumulative LWD
from 10 days after petal fall until harvest was 286 h in 2007
and 1,071 h in 2008 (Table 1).
Dark colonies on apples were first observed 5 to 7 weeks
before apple harvest and CPA increased weekly until harvest.
Analysis of variance of least square means of weekly CPA of
predominant OTUs showed that neither “Orchard” nor “Year”
affected weekly appearance of new colonies (P00.1592;
P00.1833). However, the significant interaction of “Orchard×Year” (P<0.0001) revealed that these factors could
not be treated independently; thus, further analyses were
based on “Orchard-year.” The assemblage of taxa differed
among orchards, as indicated by the significant interaction
of “Orchard×Taxa” (P<0.0001), but there was no interaction
of “Year×Taxa” (P<0.2447).
Identification of SBFS Colonies
Weekly counts of colony appearance in orchards were used in
all data analyses except for the section addressing the postharvest appearance of SBFS colonies. In total, 17 SBFS taxa
were identified to the genus or species level. Of the 1,384
colonies sampled, 79 % were identified (82 % in 2006, 75 %
in 2007, and 78 % in 2008) (Tables 2 and 3). The primer pair
ITS1-F/Myc1-R produced PCR products for 80 % of the
1,384 subsamples, but 0.36 % of these PCR products were
not digested by HaeIII. To confirm identification, 229 of the
1,061 PCR products with banding patterns that matched previously characterized SBFS species were sequenced. Of 54
samples (5.1 %) yielding RFLP patterns that did not match
previously identified taxa, 23 DNA sequences were unreadable (presumed to be mixtures of PCR products from multiple
species) and 15 DNA sequences matched previously identified SBFS taxa that were not in the library of RFLP patterns
[2]. The latter included Peltaster fructicola and Sporidesmajora pennsylvaniensis, which were newly detected SBFS species for Iowa. Additionally, all 14 PCR products from the
primer pair specific to Dothideomycete sp. CS1 had sequences
Mean species diversity (expressed as the number of species)
was 10.6 for the 15 orchard-years and ranged from 5 to 14
(Table 4). “Year” significantly affected species diversity
(P00.0328), whereas “Orchard” did not (P00.0765). Stomiopeltis-RS had the highest CPA (P<0.0001) in every orchardyear with an overall mean of 10.0 CPA at harvest (Table 4).
Differences in CPA of Stomiopeltis-RS were associated primarily with “Year” (P00.0163). In contrast, for Dissoconium-DS
and Schizothyrium-FS, CPA differences among orchard-years
were associated with orchard location (P 00.0330 and
P00.0002, respectively). The effects of “Orchard” or “Year”
were not significant for the CPA of the other four OTUs.
The SBFS taxa with higher CPA were also detected in more
orchard-years. For the 17 SBFS species, there was a significant
(P00.0003) positive relationship between mean CPA of a taxon
and its prevalence (the number of orchard-years in which it was
detected) (Fig. 2a). Prevalence of a given taxon explained 59 %
(R2 00.5890) of the variation in predicted CPA of that taxon
within about 1 % (SEEy01.023). The mean CPA for taxa
detected in fewer than 10 orchard-years was ≤1.6, whereas
more prevalent taxa averaged 1.1 to 5.2 CPA (Table 3). Three
taxa (Uwebraunia commune, Pseudoveronaea obclavata, and
S. pennsylvaniensis) were detected in only a single orchardyear (Table 3). Species diversity in an orchard was weakly, but
not significantly, associated with total CPA (P00.079, R2 0
0.2180, SEEy012.32) (Fig. 2b). Therefore, severity of SBFS
was not a reliable indicator of species diversity.
There was no relationship between cumulative LWD in
an orchard and CPA of SBFS (P00.4347) (Fig. 3a). In
contrast, there was a significant (P00.012) linear relationship between cumulative LWD and number of SBFS taxa
per orchard-year (Fig. 3b), for which LWD explained nearly
half of the variation in the number of taxa (R2 00.4493) and
predicted species diversity within about 2 % (SEEy01.96).
Timing of Newly Appearing SBFS Colonies
The cumulative number of SBFS colonies over time was approximately linear for the three most common OTUs (StomiopeltisRS, Dissoconium-DS, and Microcyclosporella-RH) at Center
Grove and Iowa State University HRS orchards (Fig. 4). To
examine the temporal rate of appearance of these OTUs, disease
progress curves were constructed by plotting the proportion of
apples colonized by the taxon versus day of year at each orchard.
Preliminary analysis suggested that the curves would best fit
16.3
0. 4
4.2
0.1
0.8
2.7
0.1
Stomiopeltis-RS
Microcyclosporella-RH
Dissoconium-DS
Peltaster-PT
Schizothyrium-FS
Colletogloeopsis-FG
Phaeothecoidiella-AP
16.4
1.9
0.2
0.2
1.4
1.97
0.9
11
2.93
24.1
12.1
2.3
4.8
0.6
1.9
1. 9
2.0
10
2.71
26.5
3.5
2.0
0.7
0.1
0.6
0.9
0.1
12
2.84
11.6
12.0
4.8
4.0
2. 9
4.5
0
1.7
12
2.51
43.0
BP
4. 5
0.1
0. 6
0
0.3
0
0
5
2.15
12.7
CG
1.5
0.2
0.6
1.2
4.2
1.7
1.5
9
2.77
12.4
DO
8.2
1.2
1.2
0
0.5
0.9
0.5
7
2.08
15.2
HRS
4.7
3.0
6.3
1.9
4.1
3.5
0.9
11
3.01
30.0
PN
7.4
29.3
4.5
2.8
0.6
2.9
1.0
13
2.51
55.5
AR
2008
9.6
1.2
1.8
2.0
4.1
0.9
0
14
3.12
22.0
BP
7.5
1.1
4.0
1.1
0.7
0.6
0.9
12
2.90
16.4
CG
3.5
3.0
0.7
18.0
3.3
2.3
4.2
11
2.39
42.2
DO
7.1
0.8
1.9
0.3
0.6
1.5
1.0
11
2.55
13.1
HRS
6.4
4.1
11.3
6.1
5.1
0.3
0.4
13
2.74
36.9
PN
10.0
3.5
3.0
2.3
2.0
1.4
0.9
10.6
2.60
25.9
Mean
0.0836
0.3964
*0.0330
0.3443
*0.0002
0.6450
0.6492
0.0765
0.7581
0.6103
Orchard
P valuec
*0.0163
0.5650
0.1323
0.3214
0.1997
0.3895
0.2405
*0.0328
0.8193
0.4026
Year
Orchards: AR0Apple Ridge, BP0Berry Patch, CG0Center Grove, CO0Community Orchard, DO0Deal's Orchard, HRS0Iowa State University Horticultural Research Station, PN0Pella Nursery
Analysis of variance was performed across orchard and year for each dependant variable in the left column
*P values showing significant differences among orchards and years for each OTU
c
b
Operational taxonomic unit were designated from genus-mycelial types as follows: Stomiopeltis includes two putative species associated with the ramose (RS) mycelial type, Microcyclosporella
includes four putative species associated with the ridged honeycomb (RH) mycelial type, Dissoconium aciculare is associated with the discrete speck (DS) mycelial type, Peltaster includes three
putative species associated with the punctate (PT) mycelial type, Schizothyrium includes three species associated with the flyspeck (FS) mycelial type, Colletogloeopsis-like sp. FG2 is associated
with the fuliginous (FG) mycelial type, Phaeothecoidiella includes two species associated with the arborescent punctate (AP) mycelial type
a
8
2.15
24.5
AR
HRS
CG
CO
2007
2006
Species richness
Shannon's diversity index
All SBFS colonies
Year orchardb
Table 4 Species richness, Shannon's diversity index, and mean number of colonies per apple at time of harvest of the most common operational taxonomic units (OTU)a of SBFS fungi in seven
orchards in 2006, 2007, and 2008 and P values for variation among orchards or years (within row)
Temporal Patterns in Appearance of Sooty Blotch and Flyspeck
J. C. Batzer et al.
6
A
4
3
2
1
0
Mean colonies per apple in the orchard-year
0
60
3
6
9
12
15
Number of orchard-years each SBFS taxa was detected
B
50
40
A
60
30
20
10
0
0
3
6
9
12
Number of species in the orchard-year
15
Figure 2 a Number of colonies per apple (CPA) for 17 SBFS species
and the number of orchard-years in which a species was detected during a
3-year study of seven Iowa orchards (15 maximum) (P00.0003, R2 0
0.589, SEEy01.023). b CPA in15 orchard-years and species diversity in
each orchard-year (P00.079, R2 00.2180, SEEy012.32)
Mean number of SBFS colonies per apple
Mean colonies per apple
5
To account for large variations among orchards and years,
we determined the Julian day (JD) of the first appearance of
each OTU in each orchard-year, and the order of OTU appearance was ranked for each orchard-year (Table 6). The
Friedman's test detected a significant difference (P00.0001)
among OTUs in time of first appearance. Paired tests (P<0.05)
indicated that Stomiopeltis-RS appeared earliest in the growing
season, with a mean date of 18 August (JD0230), ranging
from 10 August (2008) to 31 August (2007) and a mean
accumulation of 428 h of LWD from 10 days after petal fall
(Table 6). Dissoconium-DS was generally the last OTU to
appear in Iowa orchards. It appeared after Schizothyrium-FS,
Microcyclosporella-RH, and Colletogloeopsis-FG, with a
mean date of 1 September, ranging from 11 August (2008) to
20 September (2007) and a mean accumulation of 557 h of
2006
50
2007
2008
40
30
20
10
0
0
16
Mean number of SBFS taxa per orchard
either the monomolecular or the exponential model (Fig. 5), so the
data for these three OTUs in all orchard-years were fit to these
models. The monomolecular model had the higher F statistic for
linearity in each of the 12 orchard-years for Stomiopeltis-RS, 11 of
12 orchard-years for Dissoconium-DS, and each of the 13
orchard-years for Microcyclosporella-RH (Table 5). The backtransformed root mean square error and plots of the residual were
also used in model selection (data not shown), and these analyses
also suggested that the monomolecular model fit better than the
exponential model in describing disease progress.
Stomiopeltis-RS was the first group to appear on the fruit.
The increase in CPA and proportion of fruit colonized
occurred rapidly, usually within 1 or 2 weeks of first appearance (Figs. 4 and 5). The timing of first appearance of
colonies of Dissoconium-DS varied among orchard-years by
up to 30 days. The CPA of Dissoconium-DS increased
gradually during the late summer and continued to increase
after harvest. The time of first appearance for Microcyclosporella-RH varied by only 1 week among orchard-years,
and CPA increased steadily until harvest (Fig. 4).
200
400
600
800
1000
1200
Leaf wetness hours from 10 d after petal fall to harvest
B
14
12
10
8
6
4
2
0
0
200
400
600
800
1000
1200
Leaf wetness hours from 10 d after petal fall to harvest
Figure 3 The relationship of cumulative leaf wetness duration hours
during the growing season to a mean number of colonies of SBFS per
apple (P00.4247) and b species richness (P00.012, R2 00.449) in 13
orchard-years
Temporal Patterns in Appearance of Sooty Blotch and Flyspeck
18
18
A
2006
14
14
2007
12
12
2008
10
10
8
8
6
6
4
4
2
2
0
0
200
Colonies per apple
5
220
240
200
260
5
C
4
4
3
3
2
2
1
1
220
240
260
220
240
260
D
0
0
200
3
Colonies per apple
B
16
16
Colonies per apple
Figure 4 Cumulative number
of colonies per apple vs. day of
year for SBFS operational
taxonomic units Stomiopeltisramose (a, b), Dissoconiumdiscrete speck (c, d), and
Microcyclosporella-ridged
honeycomb (e, f) in Center
Grove Orchard (a, c, e) and the
ISU Horticultural Farm Orchard
(b, d, f) from 2006 to 2008.
Weekly data were taken from
the time SBFS first appeared in
the orchard until harvest
220
240
200
260
3
E
2
2
1
1
0
F
0
200
220
240
Day of Year
LWD from 10 days after petal fall. No differences in rank of
appearance were observed among the remaining OTUs; mean
JD differed by 3 days (26 August to 29 August) and mean
LWD ranged from 504 to 588 h.
Post-Harvest Appearance of SBFS Colonies
Some SBFS colonies became visible to the unaided eye
only after cold storage for 3 months. When the total
number of colonies on the apples was compared to the
number that was marked before or during harvest, a
significant (P<0.0001) proportion of the total number
of colonies was found to have developed in storage.
Most (60 %) of the Dissoconium-DS colonies appeared
after storage (Table 6). Smaller, but still noteworthy,
proportions, ranging from 4.2 % to 38 %, of the colonies
of other OTUs, developed in storage.
260
200
220
240
260
Day of Year
Discussion
Our findings suggest that apple growing regions have characteristic dominant SBFS taxa that contribute the largest
portion of colonies on apples. We found year-to-year persistence of a predominant SBFS taxon, Stomiopeltis-RS, in
Iowa orchards. To date, Stomiopeltis spp. RS1 and RS2 have
been detected only in Iowa and Missouri [10]. However, 9
of the 17 species detected in the current study have been
found on multiple continents [1, 6, 12, 21]. This finding
builds on a previous study of 39 orchards in the eastern half
of the U.S., in which 9 of the 60 species were found in over
35 % of the orchards but 45 of 60 species were detected in
less than 13 % of the orchards [11].
Although free moisture is required for conidia to germinate and colonies to grow [22, 23], we did not observe a
positive correlation between CPA at harvest and cumulative
Proportion of apples with colonies
Proportion of apples with colonies
Figure 5 Cumulative
proportion of apples with
colonies of SBFS versus day
of year for SBFS operational
taxonomic units Stomiopeltisramose (a, b), Dissoconiumdiscrete speck (c, d), and
Microcyclosporella-ridged
honeycomb (e, f) in Center
Grove Orchard (a, c, e) and
the Iowa State University
Horticultural Research
Station (b, d, f) from 2006
to 2008. Weekly data were
taken from the time SBFS
first appeared in the orchard
until harvest
Proportion of apples with colonies
J. C. Batzer et al.
1
A
0.8
B
1
0.8
2006
2007
0.6
0.6
2008
0.4
0.4
0.2
0.2
0
200
1
0
220
240
260
200
1
C
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
240
260
220
240
260
220
240
260
D
0
0
200
1
220
220
240
200
260
1
E
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
200
0
220
240
Day of Year
LWD hours. A possible explanation could be that our data
mostly reflected the biology of Stomiopeltis-RS, which
comprised 38 % of all CPA. A similar pattern to Fig. 3a
was observed when CPA of Stomiopeltis-RS was plotted
against cumulative LWD hours (data not shown). Thus, after
initial colonization by Stomiopeltis-RS, cumulative LWD
did not explain differences in CPA. Another explanation is
that the first cover fungicide spray may have been applied
by the cooperating growers from several days after petal fall
until 14 days after petal fall; this relatively arbitrary biofix
may have introduced variability in the accumulated wetness
data. A third explanation is that LWD may not be the most
accurate weather variable for predicting SBFS activity in the
Midwest [24]. In contrast, species diversity was positively
associated with cumulative LWD hours. Although incidence
of the common SBFS fungi may not be dependent on
cumulative LWD to develop, it is reasonable to assume that
F
260
200
Day of Year
activity of other less common SBFS species was favored by
longer periods of dew and rain.
Other factors may also influence SBFS species diversity.
For example, the assortment of plant hosts of SBFS fungi
outside and inside the orchard may act as sources of inoculum [25–28]. Factors such as apple position in the canopy,
fruit age and cuticle condition [29, 30], and competition for
resources on the fruit surface may also play a part in species
diversity of SBFS fungi [31].
This is the first study to clearly identify taxon-specific
patterns in timing of appearance of SBFS colonies on apple.
Early development of Stomiopeltis-RS on apples in Iowa
may give it a competitive advantage over later-colonizing
SBFS fungi by co-opting space and nutrients on the apple
surface. Density-dependent antagonism has been demonstrated for fungal leaf saprotrophs [3, 32, 33] and may be
an additional competitive advantage for early appearing,
Temporal Patterns in Appearance of Sooty Blotch and Flyspeck
Table 5 Comparison of monomolecular (monocyclic) and exponential (polycyclic) disease
progress models (proportion of
apples colonized) of three operational taxonomic units (OTUs)
among orchard-years
OTUa
Stomiopeltis-RS
a
Stomiopeltis-RS includes two
putative species associated with
the ramose mycelia type; Dissoconium-DS includes D. aciculare
and is associated with the discrete
speck (DS) mycelia type; Microcyclosporella-RH includes four
putative species associated with
the ridged honeycomb (RH) mycelia type
Dissoconium-DS
b
For each observation date, the
proportion of apples with colonies
of a given OTU in an orchard-year
was transformed to a monomolecular model using (log(1/(1−y)) and
regressed over time
c
Temporal progress models were
assessed for each orchard-year using linear regression. Orchard
years with less than 4 data points
between 0.01 and 0.99 were eliminated from the analysis. * denotes
the model with the higher F statistics for linearity and R2 for the
orchard-year
d
Coefficient of determination is
the proportion of variability in the
data set that is accounted for by the
model
e
For each observation date, the
proportion of apples with colonies
of a given OTU in an orchard-year
was transformed to exponential
model using (log(y)) and regressed
over time
Microcyclosporella-RH
Orchard
Year
Monomolecularb
Exponentiale
Fc
R2d
F
R2
AR
BP
BP
CG
CG
DO
DO
HRS
HRS
HRS
2007
2007
2008
2006
2008
2007
2008
2006
2007
2008
*30.10
*14.16
*6.48
*920
*7083
*114.72
*574
*6.35
*36.05
20.29
0.858
0.825
0.519
0.697
0.922
0.983
0.657
0.560
0.900
0.835
13.77
3.51
3.52
2.82
5.02
4.31
3.28
2.96
3.39
3.10
0.734
0.539
0.370
0.413
0.456
0.682
0.522
0.372
0.459
0.437
PN
PN
AR
BP
CG
CG
CG
DO
DO
HRS
HRS
HRS
PN
PN
AR
AR
BP
BP
CG
2007
2008
2008
2007
2006
2007
2008
2007
2008
2006
2007
2008
2007
2008
2007
2008
2007
2008
2006
*10.78
*251.44
*13.10
10.38
*52.14
*27.32
*16.19
*26.45
*30.78
*31.82
*37.40
*12.56
*38.72
*109.85
*50.92
*10.11
*37.05
*20.73
*28.99
0.782
0.977
0.724
0.722
0.929
0.932
0.802
0.930
0.837
0.883
0.949
0.807
0.866
0.973
0.962
0.771
0.903
0.775
0.878
3.39
6.83
10.06
*10.48
3.68
4.90
6.16
5.60
9.45
4.95
6.77
11.62
3.96
5.92
4.89
2.75
8.24
7.04
3.89
0.531
0.532
0.668
0.724
0.479
0.710
0.606
0.737
0.612
0.553
0.772
0.795
0.397
0.664
0.710
0.438
0.673
0.540
0.493
CG
DO
DO
HRS
HRS
HRS
PN
PN
2008
2007
2008
2006
2007
2008
2007
2008
*32.28
*49.41
*41.70
*87.18
*8.63
*15.57
*55.39
*39.93
0.865
0.961
0.874
0.946
0.742
0.886
0.949
0.869
7.80
7.41
9.59
5.57
6.55
4.07
6.85
4.11
0.609
0.787
0.615
0.527
0.686
0.671
0.695
0.406
predominant SBFS taxa. Spolti et al. [9] suggested that lateseason fruit rots competed for space and nutrients with
SBFS based on findings that incidence of two late-season
apple diseases, bitter rot (Glomerella cingulata), and bull's
eye rot (Neofabraea spp.), were negatively correlated with
SBFS in a southern Brazil orchard.
SBFS species that appear later in the season may take
advantage of increased levels of sugars and amino acids that
become available on the epicuticle as apples ripen [34].
Dothideomycete sp. CS1 and D. aciculare are particularly
sensitive to nutrient levels in vitro [35] and only became
visible on apples close to harvest in the present study. As a
low-temperature tolerant species [35], D. aciculare could
grow on apples stored at 2 °C, as well as continue to grow
on fallen fruit during the declining temperatures of late
autumn.
Elucidating the seasonal patterns of colony appearance is
important not only for ecological understanding of this little-
J. C. Batzer et al.
Table 6 Prevalence, chronology, and development after placement in storage of seven operational taxonomic units (OTUs) of sooty blotch and
flyspeck (SBFS)
OTUa
Orchard-years
detectedb
DOYc Ranking of
appearanced
LWDe Mean colonies per
apple
At
harvest
After
storagef
15
15
230
238
1.3 ah
3.5 b
Microcyclosporella- 15
RH
Colletogloeopsis13
FG
Peltaster-PT
13
240
4.1 b
550 b
3.5
4.6
18.5 b
242
4.2 b
552 b
1.4
1.7
10.2 ab
241
4.5 bc
588 b
2.3
3.6
38.0 c
PhaeothecoidiellaAP
Dissoconium-DS
13
241
4.5 bc
523 b
0.9
1.4
30.1 c
15
244
5.1 c
557 b
3.0
7.0
60.0 d
Stomiopeltis-RS
Schizothyrium-FS
428 ah 10.0
505 b 2.0
Percent colonies developed during
storageg
13.4
2.2
3.4 ah
4.2 a
a
Operational taxonomic unit were: Stomiopeltis includes two putative species associated with the ramose (RS) mycelial type, Schizothyrium
includes three species associated with the flyspeck (FS) mycelial type, Microcyclosporella includes four putative species associated with the ridged
honeycomb (RH) mycelial type, Colletogloeopsis-like sp. FG2 is associated with the fuliginous (FG) mycelial type, Peltaster includes three
putative species associated with the punctate (PT) mycelial type, Phaeothecoidiella includes two species associated with the arborescent punctate
(AP) mycelial type, and Dissoconium aciculare is the associated with the discrete speck (DS) mycelial type
b
Presence within an orchard-year (15 orchard-years)
c
Mean day of year of each orchard-year at first appearance. Day of year, i.e., 230=August 16, 244=September 1. First appearance was determined
by taking the average day of first appearance of each apple sampled for each orchard-year
d
Mean order of appearance for each orchard-year; 1=first, 7=last
e
Mean leaf wetness duration (hour) accumulated from first cover to first appearance on apple
f
3 months storage at 2 °C
g
Mean percent colonies that developed in storage was derived by subtracting mean CPA at harvest from the mean CPA after storage and dividing
the difference by the CPA at the end of storage for each orchard-year
h
Numbers in column followed by the same letter are not significantly different (P<0.05)
studied group of fungi, but also for disease management,
since early appearing and predominant species are the key
species on which SBFS warning systems should focus.
Disease-warning systems are decision aids that can help
growers reduce their costs by targeting fungicide sprays to
periods when there is a tangible threat of economic loss
from a disease [18]. A SBFS warning system developed
for the Upper Midwest [24] incorporated data from several
of the same orchards used in the present study. Thus, it is
likely that this relative humidity driven warning system
predicted primarily the appearance of Stomiopeltis-RS, the
most abundant SBFS species. In contrast, in the Northeast
U.S., where S. pomi is reported to be the predominant SBFS
species, the most widely used SBFS warning system uses
different weather inputs: LWD and rainfall [15, 36–38]. As
our ecological understanding of regional SBFS complexes
expands, we may be able to further customize warning
systems to control SBFS more efficiently and sustainably.
Our study is the first attempt to characterize the temporal
dynamics of SBFS species infections on fruit. The three
most prevalent and abundant OTUs in the central Iowa
SBFS complex were monocyclic, which suggests that there
was minimal spread of the taxa from apple to apple during
the growing season. Monocyclic epidemics, for which there
is no secondary spread of the pathogen from plant to plant
during the season, best fit a linear or monomolecular model,
where disease progress is a function of only initial inoculum
level [18, 19]. The initial inoculum of SBFS fungi may
come from other plants outside of the orchard. Lack of
exponential increase of colonized apples over time suggests
that colonies of the most common OTUs did not frequently
proliferate on the apple surface through secondary sporulation; however, the high variability among apples and
orchard-years prevented drawing firm conclusions.
Our evidence that SBFS epidemics in Iowa orchards are
monocyclic supports similar findings of SBFS in orchards in
the Netherlands and Southern Brazil [9, 39]. However,
regional differences in climate type and prevalent SBFS
species could influence fungal spread on and among apples.
In the southeastern U.S., for example, SBFS blemishes
commonly exhibit vertical streak patterns, presumably from
transport of secondary spores in water droplets during rain
events [11, 23]. Furthermore, the most prevalent taxa in the
Iowa SBFS complex differ from those in other geographic
Temporal Patterns in Appearance of Sooty Blotch and Flyspeck
regions [6, 11, 12, 21]. Because the dominant taxa in Iowa
(i.e. Stomiopeltis spp., D. aciculare, S. pomi) produce discrete colonies on apple and no yeast-like budding in culture,
they may not readily generate abundant secondary conidia
on the apple [35, 36]. Instances of SBFS spread from apple
to apple have been associated with species such as Geastrumia polystigmatis and P. fructicola, which produce abundant secondary inoculum on apple [4, 23]. It is possible that
the latter species exhibit polycyclic epidemics in climates
with high summer rainfall, but this has not been verified
experimentally. Furthermore, the present study indicated
that SBFS fungi caused monomolecular epidemics on apple
fruit in the orchard, but it is not known whether these fungi
produce secondary infections on reservoir hosts around the
orchards that serve as sources of inoculum.
Acknowledgments We thank the Leopold Center for Sustainable
Agriculture and the North Central Region Sustainable Agriculture
Research and Education (SARE) program for project funding, Philip
Dixon for statistical consulting, Forrest Nutter and Sharon Eggenberger
for epidemiology guidance, Katie Penick, Khushboo Hemnani, and
Katie Waxman for technical assistance, and the Iowa apple growers
who kindly cooperated on this project.
11.
12.
13.
14.
15.
16.
17.
18.
19.
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