Published online November 9, 2017
RESEARCH
Response of Seashore Paspalum Genotypes
to Two Isolates of Sclerotinia homoeocarpa
Nicole D. Benda,* Norma C. Flor, Phillip F. Harmon, and Kevin E. Kenworthy
ABSTRACT
Seashore paspalum (Paspalum vaginatum
Schwartz) is a warm-season turfgrass noted
for its salt tolerance and is used on golf course
fairways and greens. Dollar spot (Sclerotinia
homoeocarpa F.T. Benn.) is an important disease of seashore paspalum, causing distinct
silver dollar-sized necrotic patches to form on
the turf. Previous research by Flor et al. (2013)
identified host plant resistance as a potential
tool for dollar spot disease management using
a single dollar spot isolate, UF0421. However,
another isolate appeared more virulent in followup experiments. With the intent of developing
stronger selection pressure, disease response
to these two characterized S. homoeocarpa isolates was compared in the current study under
controlled environmental conditions using nine
experimental lines of seashore paspalum and
two commercial cultivars (‘SeaDwarf’ and ‘Sea
Isle Supreme’) as controls. The second, putatively more virulent isolate consistently caused
more damage to all 11 paspalum genotypes.
An isolate genotype interaction was not
observed. Plant genotypes differed significantly
in disease response, and all genotypes but one
were consistent with the previous study. We
successfully identified a more virulent strain
of dollar spot. Using a more virulent strain will
provide stronger selection pressure for development of resistance to dollar spot, allowing for
clearer identification of resistant genotypes.
N.D. Benda, N.C. Flor, K.E. Kenworthy, Dep. of Agronomy, Univ. of
Florida, Gainesville, FL, USA; P.F. Harmon, Dep. of Plant Pathology,
Univ. of Florida, Gainesville, FL, USA. Received 1 June 2016. Accepted
31 May 2017. *Corresponding author (nbenda@ufl.edu). Assigned to
Associate Editor James Murphy.
Abbreviations: ITS, internal transcribed spacer; rDNA SSU, ribosomal
DNA small subunit.
S
eashore paspalum (Paspalum vaginatum Schwartz) is a warmseason turfgrass noted for its tolerance to salinity, poor water
quality, a range of pH, and drought (Skerman and Riveros, 1990;
Duncan and Carrow, 1999). This turf species also has low fertility requirements and a dense, deep root system. Tolerance to low
mowing heights and wear make it appealing for use on golf course
fairways and greens. Management of disease remains a limiting
factor. Dollar spot disease is the most problematic disease of seashore paspalum (Walsh et al., 1999).
Dollar spot affects all warm-season turfgrasses (Walsh et al.,
1999). It is caused by the fungus Sclerotinia homoeocarpa F.T. Bennett. In Florida, the S. homoeocarpa population includes a diverse
group of 18 vegetative compatibility groups identified by Liberti
et al. (2012). Based on cultural morphology, DNA sequence data,
and symptoms, two morphological-types within the species were
described: Floridian type (predominant in Florida) and common
type (present in Florida and northern states) (Liberti et al., 2012).
Isolates collected from both cool- and warm-season turfgrasses
vary significantly by phenotype, mating compatibility, and DNA
sequence (internal transcribed spacer [ITS] regions of the ribosomal DNA small subunit [rDNA SSU] and two mating type
genes (MAT1-1 and MAT1-2) (Liberti et al., 2012).
Dollar spot infection on seashore paspalum appears as tan
lesions with a darker margin on individual leaf blades. White
cottony mycelia may also be visible after extended leaf wetness.
Published in Int. Turfgrass Soc. Res. J. 13:454–458 (2017).
doi: 10.2134/itsrj2016.06.0474
© International Turfgrass Society and ACSESS | 5585 Guilford Rd., Madison, WI
53711 USA
All rights reserved.
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Distinct features of this disease are the sunken, circular, straw-colored patches with red-brown margins, 5 to
7.5 cm wide (Smith, 1955), that develop on closely mowed
turf. These patches can coalesce over time, causing plant
death over larger areas.
Conditions that can lead to severe dollar spot symptoms include hot days coupled with cool nights, nitrogen
deficiency, and abundant dew formation. Adjustments in
nitrogen fertilization, irrigation, and dew removal are
common cultural control tactics for this disease. Thatch
reduction can also reduce disease incidence (Smiley et
al., 1992). Currently, fungicides are the most effective
and commonly used control method. Many fungicide
products are labeled for dollar spot management; however, fungicide-resistant populations of S. homoeocarpa are
widespread (Walsh et al., 1999; Jo et al., 2008).
Development of seashore paspalum cultivars with
resistance to dollar spot disease is a critical component of
an integrated pest management approach. Using a single
S. homoeocarpa isolate, Flor et al. (2013) reported heritable
differences in dollar spot disease severity among seashore
paspalum genotypes, with disease severity ranging from 22
to 41%. In Flor et al. (2013), however, ‘Sea Isle Supreme’
had a moderate disease response after exposure to the S.
homoeocarpa isolate used, whereas a previous study indicated that Sea Isle Supreme was highly susceptibility to
dollar spot (Unruh et al., 2007). More recently, a second
isolate was tentatively assessed as more virulent than the
isolate used by Flor et al. (2013) when disease symptoms
were visually compared in inoculated potted plants. To
identify the best isolate to screen for resistance to the
pathogen and to determine if genotype isolate interactions are a concern, the two isolates were compared for
induction of disease response among several genotypes of
seashore paspalum. The objectives of this study were (i) to
compare disease severity caused by two differing Florida-type isolates of S. homoeocarpa on seashore paspalum
and (ii) to determine if the difference in disease severity
between the two isolates is consistent among nine selected
seashore paspalum genotypes and two cultivar controls.
MATERIALS AND METHODS
Germplasm
Nine genotypes of seashore paspalum from the University
of Florida turfgrass breeding program (BA480-2, BA480-9,
BA511-2, UF07-14, UF15-3, UF19-10, UF19-18, UF22-3,
and UF25-5) were tested in this study. These genotypes were
previously evaluated for genotype variation to dollar spot
susceptibility (Flor et al., 2013). Commercial cultivars Sea
Isle Supreme and ‘SeaDwarf ’ (with low and high dollar spot
resistance, respectively; Unruh et al., 2007) were included as
controls in this study.
Twelve plugs (5 cm) of each seashore paspalum genotype
were collected in January 2015 and again in December 2015 for
Experiments 1 and 2, respectively. Plugs were cut using a sod
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plugger from field plots planted in 2012 in Citra, FL. Plugs were
planted in 10.5-cm pots using Fafard 3B Mix potting mix (Sun
Gro Horticulture) and were grown under greenhouse conditions (ambient light, 10–38°C, 71–90% relative humidity). At
planting, the plugs were treated with chlorothalonil (140 mL
9.1 m−2) (Daconil 2787 Flowable Fungicide, 40.4% a.i., Syngenta Canada) and ammonium nitrate (34–0–0, N–P–K) (5 mL
pot−1) to suppress dollar spot from field inoculum. Plant maintenance included daily morning irrigation, twice weekly hand
trimming (1.5 cm), and weekly fertilization with 100 mL of a
3.5 g L−1 solution of Miracle Gro Water Soluble All Purpose
Plant Food (24–8–16) (Scotts, Miracle-Gro).
Sclerotinia homoeocarpa Isolates
Two S. homoeocarpa isolates, UF0421 and UF0402, were used in
the inoculations. UF0421 was isolated from seashore paspalum
in Santa Rosa County, FL, and is the isolate used by Flor et
al. (2013). UF0402 was isolated from common bermudagrass
(Cynodon dactylon L.) in Collier County, FL. Both isolates were
identified as Floridian-type S. homoeocarpa (Liberti et al., 2012).
The isolates differed in stroma type, vegetative compatibility, response to bromophenol blue acidified media, and DNA
sequence of the ITS region and rDNA SSU. UF0421 showed
stronger virulence than UF0402 by producing larger lesions on
St. Augustinegrass [Stenotaphrum secundatum (Walter) Kuntze]
(Liberti et al., 2012).
Inoculation and Data Collection
UF0421 and UF0402 were maintained on colonized winter
wheat seeds and on colonized filter paper strips (3 1 mm),
respectively, at 4°C. The UF0421 had not yet been transferred
to the longer-term storage technique of colonized filter paper,
but the isolate was still highly viable on the colonized seeds. To
activate the isolates, four wheat seeds or filter paper strips were
transferred to 8.5-cm Petri dishes of potato dextrose agar (39 g
L−1) (Difco, Becton, Dickinson and Company). The dishes
were incubated at 25°C for 3 d. Meanwhile, 40 g of wheat seeds
were soaked in 40 mL tap water for 24 h and then autoclaved for
25 min once daily for 3 d. After 3 d of incubation, five 5-mm
colonized agar plugs were excised from the expanding margin
of the colony in the Petri dishes and transferred to the autoclaved wheat seeds. Seeds were then incubated for 7 d at 25°C.
Plants were hand trimmed (1.5 cm) the day before inoculation. Inoculation was performed by transferring 12 infected
wheat seeds to the center of each plant, placing the seeds midway
in the 2.5-cm thatch layer. Five pots of each genotype were
inoculated with each dollar spot isolate. Two additional pots
were mock-inoculated with autoclaved, sterile wheat seeds.
Inoculated plants were placed into 3.7-L resealable clear plastic bags containing a folded paper towel and 30 mL tap water.
Inoculated plants were kept in a walk-in growth chamber with
a 12-h photoperiod for 4 d. Temperature was 25°C ± 2 SD, and
artificial light ranged from 32.6 to 49.0 mmol s−1 m−2 (LI-250
light meter, LI-COR). Pots were then removed from the bags,
returned to the greenhouse, and wheat seeds were removed
from the pots. Pots were watered and fertilized as prior to the
experiment but were not trimmed until all data had been collected (18 d post-inoculation).
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The experiment was a randomized complete block design
with five replications and used a split plot arrangement of treatments, where dollar spot isolates (n = 2) were the main plots
within each block, and seashore paspalum genotypes (g = 11)
the subplots within isolates (Steel et al., 1997). This meant that
each of five blocks was made up of two groups of plants, where
each group had one plant of each of 11 genotypes (22 plants
total in each block), with one group treated with UF0402 and
one group treated with UF0421. Blocks were assigned according to known differences in airflow and light that occur within
the walk-in growth chamber. The entire experiment was conducted twice, beginning on 13 Aug. 2015 and on 23 Feb. 2016.
Each experimental repeat included one untreated block (g = 11,
n = 2), which was subjected to all plant-handling procedures
except that there was no inoculant present. Initial plant conditions (noting any chlorosis or leaf damage) were recorded on
the day of inoculation. Visual observations of disease symptoms
(percentage of all leaves affected by chlorosis and necrosis) were
recorded 4, 7, and 18 d post-inoculation.
Percentage disease severity of infected leaf tissue in each
pot was calculated by adding chlorosis and necrosis percentages.
Statistical Analysis
Individual and interactive effects of genotype and isolate on percentage disease severity at each of the three rating dates—4, 7,
and 18 d—was analyzed using mixed models procedures as
implemented in SAS PROC GLIMMIX (SAS Institute, 2011).
Experiment and blocking factors were treated as random effects,
whereas isolate and genotype and their interaction were treated as
fixed effects. Tukey–Kramer grouping of least squares means was
used to compare percentage disease severity of genotypes within
each rating date. Control plants were used to assess the consistency
of the effect of experimental conditions on symptom development,
but they were not included in the statistical analysis.
RESULTS AND DISCUSSION
Plant genotype and isolate were significant factors affecting percent disease severity at 4 and 7 d post-inoculation
(Table 1). Plant genotype had a significant effect at 18 d
(P < 0.0001), but isolate did not (P = 0.23) (Table 1), possibly because virulence decreased as the plants recovered
(Fig. 1). UF0402 caused a greater disease response than
UF0421 at 4 and 7 d (Fig. 2). This difference was consistent among genotypes (data not shown), as indicated
by the lack of interactive effect between genotypes and
Table 1. Summary of GLIMMIX Type III test results for effects
of seashore paspalum genotypes, dollar spot isolates, and
interactions on percentage disease severity at 4, 7, and 18 d
post-inoculation.
Source
df
4d
F-values
7d
Isolate (I)
1
10.19*
18.81**
18 d
1.55
Genotype (G)
10
8.09**
2.06*
5.49**
IG
10
0.29
1.52
0.51
* Significant at the 0.05 probability level.
isolates for percentage disease severity on the three rating
dates (Table 1, P > 0.13). These observations of increased
disease severity are consistent with the larger lesions on St.
Augustinegrass reported in Liberti et al. (2012).
SeaDwarf was not included in the 2013 study but,
consistent with Unruh et al. (2007), it demonstrated strong
resistance to dollar spot in this study. Comparisons of percentage disease severity at 4 d post-inoculation indicated
that the genotypes initially least affected by dollar spot
inoculation were UF19-18, SeaDwarf, BA480-9, UF223, UF19-10, and UF07-14 (Fig. 1). SeaDwarf, Supreme,
UF19-18, and BA480-2 demonstrated faster recovery at
18 d post-inoculation. Although the GLIMMIX analysis found an effect of genotype at 7 d post-inoculation,
the subsequent Tukey–Kramer analysis did not detect
significant differences between genotypes at this time
point (Fig. 1). These results, similar to those of Flor et al.
(2013), further emphasize the strength of these genotypes
as promising new lines for further breeding development
of dollar spot-resistant seashore paspalum.
Genotype rankings were similar for disease severity
at 4 and 18 d post-inoculation (Fig. 1), with a few exceptions. At 18 d post-inoculation, Supreme and BA480-2
had recovered more quickly than the other lower-ranking
lines, increasing their rank relative to 4 d post-inoculation, whereas BA480-9 had not recovered as much and
so went down in rank. Although a consistently resistant
genotype is ideal, a speedy recovery can compensate for
initial disease damage. Recovery is a prominent aspect
of the disease progression data, showing disease severity
ranging from 32 to 66% after 4 d and dropping quickly to
20 to 30% after only 7 d post-inoculation (which was also
3 d after removal of the inoculum) (Fig. 1). Low disease
severity at initial onset of disease, such as that exhibited
by SeaDwarf and UF19-18, is important to reduce the
need to apply fungicides; however, a better rate of recovery (i.e., Seas Isle Supreme and SeaDwarf ) could equate
to differences in growth rate and possibly persistence of a
genotype, reducing the need for subsequent applications
of fungicides.
Disease severity for each genotype was comparable
with the values reported by Flor et al. (2013), with the
exception of one genotype; BA511-2 was among the most
resistant in the 2013 study, but was the most susceptible
genotype in the current study. This difference is not
explained by the inclusion of the more virulent UF0402
isolate and differences between replicate, experiment date,
or initial plant conditions. This genotype will require
additional testing to clarify this difference of results.
This study, conducted under more controlled environmental conditions in growth room and greenhouse
settings, although with only two isolates, corroborates the
results of field trials conducted by Steketee et al. (2016) in
that we also found no interactive effect between seashore
** Significant at the 0.01 probability level.
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Fig. 1. Least squares means of dollar spot disease severity on seashore paspalum, averaged across isolates at 4, 7, and 18 d postinoculation with Sclerotinia homoeocarpa. Genotypes with the same letter above the bar are not statistically different within date (uppercase
letters for 4 d, lowercase for 7 d, and uppercase bold for 18 d), according to a Tukey–Kramer grouping of least squares means at a = 0.05.
Fig. 2. Least squares means of dollar spot disease severity on seashore paspalum by isolate at 4, 7, and 18 d post-inoculation with
Sclerotinia homoeocarpa. Pairs of bars without an asterisk above are not statistically significant, according to a mixed model analysis
using SAS PROC GLIMMIX at a = 0.05.
paspalum genotypes and dollar spot isolates. Chakraborty
et al. (2006) found no interaction between genotypes of
creeping bentgrass (Agrostis stolonifera L.) and dollar spot
isolates. This suggests that a single virulent isolate could
be used to screen seashore paspalum genotypes for dollar
spot disease resistance.
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Although the two isolates tested in the current study
are both part of the Floridian group, they differ in phenotype, mating compatibility, and DNA regions (Liberti et
al., 2012), which may account for differences in virulence.
Interestingly, the more virulent isolate in the current study,
UF0402, was collected from common bermudagrass, not
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from seashore paspalum. In contrast, the most virulent isolate in the Steketee et al. (2016) study was collected from
seashore paspalum, relative to four other isolates collected
from another seashore paspalum, zoysiagrass (Zoysia japonica
Steudel), common bermudagrass, and creeping bentgrass.
Subtracting the percentage disease severity of the control pots did not change the significance of isolate and
genotype effects on disease severity at 4, 7, or 18 d postinoculation (results not shown).
These results verify our preliminary data on the higher
virulence of isolate UF0402 compared with UF0421.
This isolate is a promising inoculum to use in screening
germplasm, as it exhibited consistently high virulence in
all genotypes.
Conflict of Interest
The authors declare that there is no conflict of interest.
Acknowledgments
The authors greatly appreciate statistical advice from Drs. James
Colee and Edzard Van Santen of the Statistical Consulting Unit,
Institute of Food and Agricultural Sciences, and greenhouse
assistance from Aaron Solley, Cody Darling, and Troy Black.
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Nicole Benda