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RESEARCH ARTICLE
Variation in pathogen aggression and cultivar performance against Sclerotinia sclerotiorum
in soybean and dry bean from Brazil and the U.S.
Thomas J. J. Miorini1, Zhian N. Kamvar1, Rebecca Higgins1, Carlos G. Raetano2, James R.
Steadman1, Sydney E. Everhart1
1Department of Plant Pathology, University of Nebraska, Lincoln, NE, USA, 68583
2Departamento de Proteção Vegetal, Faculdade de Ciências Agronômicas, Universidade Estadual
Paulista (UNESP), Botucatu, SP, Brazil
*Corresponding author: Sydney Everhart; e-mail: [email protected]
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Abstract
Sclerotinia sclerotiorum (Lib.) de Bary is an important yield-reducing disease in the United
States and Brazil with a diverse host range, including dry bean and soybean. Characterization of
both the physiological resistance of commercial cultivars to S. sclerotiorum and the range of
aggressiveness among S. sclerotiorum isolates collected from locations where these cultivars are
to be deployed, provides important information for making management recommendations. To
address this in the present study, we first sought to characterize the range of S. sclerotiorum
aggressiveness on soybean and dry bean, using a selection of isolates from Brazil and the U.S.
Our second objective was to evaluate the performance of dry bean and soybean cultivars that
were developed in Brazil. Eighty-seven isolates of S. sclerotiorum were collected from soybean
crops from the U.S. and Brazil and used in this project. U.S. soybean cultivar Dassel and
Brazilian dry bean cultivar IAC Alvorada were evaluated using a detached leaf bioassay (DLB)
with 65 and 28 isolates respectively. For the straw test, U.S. dry bean cultivar G122 and
Brazilian dry bean cultivar IAC Alvorada were inoculated with 32 and 28 isolates respectively.
Results showed a significant difference among isolates (P < 0.001). After evaluating 23 Brazilian
dry bean cultivars, results of the DLB and straw test showed IAC Diplomata and IPR Tangará
were more tolerant to S. sclerotiorum. Among the 11 Brazilian soybean cultivars, M5410 and
M6410 were found to be less susceptible to S. sclerotiorum. Collectively, results of this study are
important for improving our understanding of variation in pathogen aggressiveness from
geographically isolated populations and in identifying cultivars that are likely to have partial
resistance.
Key words: detached leaf bioassay, Glycine max, Phaseolus vulgaris, sclerotinia stem rot, straw
test, white mold
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Introduction
Sclerotinia sclerotiorum (Lib.) de Bary has a wide geographic distribution and a diverse host
range, including many agronomic crops (Hartman et al. 1999). This pathogen infects over 400
species of plants worldwide. S. sclerotiorum causes Sclerotinia stem rot on soybean (Glycine
max (L.) Merr.), which is recognized as an important yield-reducing disease in the United States
(U.S.; Wrather et al. 1997) and Brazil (Meyer et al. 2014, Meyer et al. 2015). In the 2016-2017
growing season, both countries each produced more than 100 million tons (Mt) of soybeans
(U.S., 117 Mt; Brazil, 114 Mt; USDA 2018). In the U.S., estimated annual losses of soybean due
to Sclerotinia stem rot in 28 states ranged between approximately 0.057 to 1.6 Mt from 1996 to
2009 (Koenning and Wrather 2010; Wrather and Koenning 2009). Disease is widespread in the
southern, southeast, west-central, and some areas of the northeast region of Brazil (Juliatti et al.
2013). Prevalence of the disease in Brazil has increased considerably since 2008, with an
estimated 6.8 million hectares infested, which is equivalent to 22.5% of the harvested area in the
2013/2014 growing season (Meyer et al. 2014, Meyer et al. 2015).
This pathogen also infects dry common bean (Phaseolus vulgaris L.) and causes white
mold. Brazil is one of the world’s leading producer and consumer of edible dry beans, producing
3.4 Mt in 2016-2017 season (Conab 2018), whereas production in the U.S. was 1.0 Mt in 2017
season (USDA-NASS, 2017). Several studies have shown losses on dry bean due to S.
sclerotiorum. For example, in a study conducted between 1994 and 2001 in North Dakota, it was
shown that for every percent unit increase in white mold incidence, yield was reduced by 12 kg
ha-1 in pinto bean and by 23 kg ha-1 in navy bean (Del Río et al. 2004). Moreover, without the
use of fungicide control, dry bean yield loss to white mold in Brazil has been reported to vary
from 17 to 54% (Miorini et al. 2017; Vieira et al. 2010).
The disease cycle caused by S. sclerotiorum on soybean and dry bean is similar, starting
with primary infection of senescing flower parts by ascospores, followed by secondary spread of
the pathogen through direct contact with infected plant parts (Abawi et al. 1975). Disease
outbreaks and epidemics tend to occur regionally and environmental conditions strongly
contribute to the amount of yield loss incurred. Sclerotinia stem rot of soybean in the U.S. is
typically a problem in the Northern states with the largest soybean acreage, but is rarely detected
in Southern states (Koenning and Wrather 2010). Sclerotinia stem rot occurrence in the north
central U.S. was more prevalent when yearly temperatures were below normal and increased
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with geographic latitude, however here is a large degree of temporal heterogeneity from one year
to the next (Workneh and Yang 2000).
Disease management for Sclerotinia diseases of soybean and dry bean is similar. For both
crops, it is recommend to integrate cultural and chemical control measures. Increasing plant
spacing is an important factor and has been shown previously to reduce disease (Paula Junior et
al. 2009, Vieira et al. 2010) because increased air flow within the plant canopy reduces
microclimatic effects that favor secondary spread of the pathogen. For example, increased row
spacing from 19 to 75 cm showed disease severity was reduced from 25 to 41% (Peachey et al.
2006). White mold incidence and severity may also be reduced by minimizing irrigation and
fertilizer, and use of upright cultivars with an open plant canopy (Ando et al. 2007, Kolkman and
Kelly 2002, Schwartz et al. 1987). However, there are critical limitations to these
recommendations. Increasing row-spacing and decreasing inputs will decrease plant density and
canopy development, which will decrease total yields and will result in a loss to the farmer in
years when disease incidence is low. Moreover, not all producers have machinery capable of
wide row widths, so to adopt this recommendation can come at a major initial cost.
It is recommended to combine these cultural practices with prophylactic application of
fungicides, which are timed to prevent primary infection of susceptible plant parts. In Brazil, a
two year study showed application of fungicides resulted in yields that were 20.2 to 87.4%
greater than the non-treated plots (Miorini et al. 2017). Likewise, application of fungicides in
common bean results in yields that were 29.9 to 118.6% greater than the untreated plots (Vieira
et al. 2010). However, application of fungicides is a direct cost for growers and sustained use of
fungicides may result in negative environmental impact, specifically with respect to the
beneficial soil microbial community and risk to human health.
Evaluation of cultivars in the field using multiple screening sites allows assessment of
field deployment ability across a range of regional environmental conditions. A limitation to
field studies, however, is that conditions favoring disease can be sporadic. Moreover, variation in
the pathogen population could affect results. Pathogen aggressiveness is the relative ability to
colonize the host and cause damage (Shurtleff and Averre 1997), for which it is known that S.
sclerotiorum populations have differences in aggressiveness across multiple screening sites in
the U.S. (Otto-Hanson et al. 2011, Kamvar et al. 2017) Thus, in order to determine whether
isolates used for screening cultivars in the greenhouse are representative of what is expected in
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producer fields, it is important to characterize the variation in isolate aggressiveness where the
cultivar will be, or is already, deployed. Although it is known that pathogen aggressiveness
should be considered prior to cultivar deployment because this may impact cultivar performance
(Kull et al. 2003), this is rarely carried out in practice. Once variation in pathogen aggressiveness
has been assessed from regions where the cultivars will be deployed, that information can be
used to select representative isolates best suited to differentiate cultivars with varying degrees of
partial resistance for that region.
Selection of plant cultivars with partial resistance is important for managing diseases
caused by S. sclerotiorum and to reduce the reliance on chemical control. However, there are a
few partially resistant cultivars to white mold in common bean (Schwartz and Singh 2013).
Similarly, soybean cultivars with high levels of resistance to Sclerotinia stem rot is the most
valuable management practice; however, no cultivars are completely resistant to Sclerotinia stem
rot. Many researchers have reported differences in susceptibility to Sclerotinia stem rot among
soybean cultivars (Chawla et al. 2013, Kim et al. 2000, Kim et al. 1999, McLaren and Craven
2008, Rousseau et al. 2004, Wegulo et al. 1998, Yang et al. 1999). Furthermore, characterization
of partial resistance should be evaluated under controlled conditions to determine physiological
resistance, following which, field evaluations must be performed to determine whether improved
performance is observed. In some cases, the plant shape and size may counteract the benefit of
increased physiological resistance to infection.
The purpose of the present investigation was to characterize variation in pathogen
aggressiveness from the U.S. and Brazil, and to identify cultivars of soybean and dry bean from
Brazil likely to have partial resistance to S. sclerotiorum infection. In the first part of our study,
we sought to characterize the range of S. sclerotiorum aggressiveness on soybean and dry bean,
using a selection of isolates from Brazil and the U.S. In the second part, our objective was to
evaluate the performance of dry bean and soybean cultivars that were developed in Brazil.
Materials and methods
Variation in aggressiveness of isolates of S. sclerotiorum was evaluated using a total of four
separate experiments and performance of cultivars was performed in three separate experiments.
These seven experiments were independently conducted from 2013 to 2016 and therefore, some
of the methods used for each experiment varied.
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Isolate aggressiveness
Aggressiveness of isolates was characterized using a total of 69 isolates from Brazil, 13 from the
U.S., and 5 from Argentina. Isolates were deposited in the culture collection of Dr. James R.
Steadman in the Department of Plant Pathology at the University of Nebraska. Brazilian isolates
were assessed on the Brazilian dry bean cultivar IAC Alvorada, which is characterized as having
intermediate resistance (Ferreira et al. 2014) and a Carioca type bean. Isolates from all countries
were assessed for aggressiveness using both the soybean cultivar Dassel, which is a partially
resistant soybean cultivar (Arahana et al. 2001), and the dry bean cultivar G122—a partially
resistant cultivar of Andean origin that produces cranberry-type beans (Miklas et al. 2001). In
general, dry bean is considered to have a greater level of susceptibility to S. sclerotiorum
infection, thus disease progress over time and/or use of a straw test method is more consistent
than using a detached leaf bioassay (DLB; Kull et al. 2003, Schwartz and Singh 2013).
We performed a total of four experiments to characterize isolate aggressiveness, two
using a DLB and two using a straw test assay (both described in detail below). In the first
experiment, a selection of 65 S. sclerotiorum isolates from Argentina (n = 5), the U.S. (n = 9),
and Brazil (n = 51) were reactivated and inoculated onto soybean cultivar Dassel in the U.S.
using the DLB. For each of three experimental replicates, ten leaves were inoculated with each
isolate. Leaves were collected for each experimental replication at 21, 28 and 35 days after
emergence. Lesion areas (cm2) were evaluated 48 hours after inoculation. In the second
experiment, 28 Brazilian isolates were used to inoculate Brazilian dry bean cultivar IAC
Alvorada using the DLB. For each of three experimental replicates, 10 leaves were inoculated
with each isolate. Leaves were collected for each experimental replication at 21, 28 and 35 days
after emergence. For this experiment, evaluations were performed with 24, 30, 36, 42 and 48
hours after inoculation, although subsequent data analysis used measurements made at 48 hours
In the third isolate aggressiveness experiment, 32 isolates from Argentina (n = 2), the
U.S. (n = 11), and Brazil (n = 19), were evaluated on dry bean cultivar G122 using the straw test.
The experiment was arranged using a randomized complete block design with 12 replications.
There were 32 pots per block, each pot received one of the 32 isolates, and evaluations were
performed 8 days after inoculation. In the fourth experiment, 28 isolates were evaluated in IAC
Alvorada using the straw test, and plants were incubated for 8 days post inoculation. A total of
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11 plants were used for each inoculation and the experiment was conducted once. The first and
third experiments were conducted in the U.S. and the second and fourth experiments performed
with cultivar IAC Alvorada were conducted in Brazil.
Cultivar performance
Performance of varieties of soybean and dry bean was conducted in Brazil. Eleven soybean
cultivars were evaluated: Potência, TMX Força, M8349, M8210, M5917, M5410, M8330,
AS3820, M6410, M7110, and CD 2728. Seeds of each cultivar were planted in single rows of
approximately 5 m in length. Leaves were collected once each cultivar had at least 10 fully
expanded leaves present among all plants. Due to poor germination and slow plant growth,
possibly because some cultivars were not optimally adapted to the climate present in Pereiras,
SP, Brazil field site, leaves were collected from plants at 34- and 60-days after emergence.
Evaluations were performed using 10 leaves of each cultivar and inoculating with one isolate
(collected from Rio Grande do Sul) using the DLB with evaluations of lesion size performed at
48 hrs. Twenty-three dry bean cultivars were evaluated: Pérola, BRS Pontal, BRS Cometa, BRS
Requinte, IPR Gralha, IPR Curió, IPR Uirapuru, IPR Tuiuiu, IPR Andorinha, IPR Bem-te-vi,
IPR Tangará, IPR 139, IPR Campos Gerais, IPR Quero-quero, IAPAR 81, IPR Juriti, IPR
Chopim, IAC Imperador, IAC Formoso, IAC Una, IAC Alvorada, IAC Diplomata, and IAC
Kaburé. Leaves were collected at 28 days after emergence for DLB, and the same plants were cut
between fourth and fifth node for the ST. Performance was assessed using the DLB and straw
test with two reactivated S. sclerotiorum isolates (972B and 972D, respectively), collected in
2012 from Pereiras, SP, Brazil. Different isolates were used for each experiment. The
performance of most of dry bean cultivars with respect to resistance to S. sclerotiorum was
unknown. Six and seven repetitions were used for each isolate in each cultivar respectively in the
first experimental repetition and only 972B (more aggressive) for DLB (12 inoculations) and
only 972D (less aggressive) for straw test (15 inoculations in each cultivar) in the second
experimental repetition.
Reactivation of isolates
Sclerotia of S. sclerotiorum were reactivated by surface sterilization and plating on media. Once
collected, the sclerotia were stored in 1.5 mL microcentrifuge tubes in a refrigerator at 4 ºC.
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Sclerotia were rinsed once with 50% Clorox bleach (6% NaOCl, The Clorox Company, Oakland,
CA) for 5-8 min in a vortexer, the bleach solution was discarded, then the sclerotia were
vortexed twice with ddH2O for 5 min, following which, the water was drawn off and sclerotia
were transferred to sterile paper towel to air dry. Sclerotia were transferred to water agar (WA)
plates (Bacto Agar, Becton, Dickinson and Company, Sparks, MD) and 4–5 days later, a 6 mm
plug of mycelia growth was transferred to PDA plates (Difco Potato Dextrose Agar, Becton,
Dickinson and Company, Sparks, MD).
Detached leaf bioassay
The detached leaf bioassay was performed as described previously by Leone and Tonneijck
(1990). In the U.S., soybean seeds were grown in a soil mix: one-third peat moss, one-third sand-
vermiculite (one-half each), and one third topsoil mix. In Brazil, plants for isolate aggressiveness
and dry bean cultivar performance were grown in pots containing a mixture of clay soil, washed
coarse sand and cow manure at a 1:1:1 ratio, supplemented with 0.6 kg ammonium sulphate, 1.7
kg superphosphate, 0.6 kg potassium chloride and 0.8 kg dolomitic lime per m3 of mixture.
These plants were grown in the greenhouse of Plant Protection Department, Botucatu, São Paulo.
The greenhouse temperatures were maintained at 22 ± 2 ºC. Soybean cultivars were sown in the
field at Centro de Pesquisa e Desenvolvimento Agrícola (CPDA) at Arysta Life Science in
Pereiras, São Paulo.
A batch of the youngest fully expanded trifoliate leaves of soybean and dry bean plants
were cut at the petiole, placed in a labeled, moistened paper towel, bagged, and transported to the
laboratory. Four trifoliates were labeled and assigned to aluminum pans as incomplete blocks
with four units per incomplete block. In each aluminum pan (HFA inc., Wheeling, IL, 527 x 325
x 73 mm; WYDA, Sorocaba, SP, Brazil, 515 x 355 x 73 mm), four folded paper towels were
placed in the bottom. Four glass Petri dishes were placed upside down in each pan on towels to
serve as platforms for detached leaves. Floral water tubes (polypropylene plastic vial and plastic
cap with septa) were used for experiments conducted in the U.S. and a test tube (12 x 75 mm)
with a bung lid that had a central hole were used in for the experiments conducted in Brazil.
These were filled with tap water, capped, and placed in pans with one tube placed under each
Petri dish. Petioles were pushed through the cap/bung septum until the cut end reached the water.
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A mycelial plug from the growing colony edge of each isolate was transferred from WA
to PDA medium and incubated at 25 ºC for 48 h. To inoculate leaves, a mycelial plug 6-mm in
diameter was removed from the advancing mycelial edge of the plate and placed mycelium side
down centered on one side of the middle trifoliolate leaf between the main leaf vein and the leaf
edge, then gently pressed to ensure contact with the leaf surface. In each pan, 300 mL of tap
water was added and then covered with plastic wrap that was 450 mm wide (Alpes - Indústria e
Comércio de Plásticos Ltda., São Paulo, SP, Brazil; Foodservice Film - Distributed by UniPro
Foodservice, Inc., Atlanta, Georgia, U.S) to maintain humidity. Pans containing inoculated
leaves were incubated on a lab bench and maintained at 25 ± 3 ºC. After 48 h, each inoculated
leaf from the first experiment was photographed and the digital images were measured using
ImageJ software (version 1.50i; Wayne Rasband National Institutes of Health, U.S.). Actual size
of each lesion was estimated by pre-calibration of the software on a grid of a known size (1 cm2)
that was included in each photo (Fig. 1A). Additional evaluations at 24, 30, 36, and 42 hours
after inoculations were made for the second experiment in isolate aggressiveness and for dry
bean cultivar performance.
Straw test
This experiment was performed in the greenhouse and temperatures were maintained at 22 ± 2
ºC. Dry bean cultivar G122 was grown in a soil mix in the U.S. comprised of four ingredients:
soil (23.1%), sand (19.2%), vermiculite (19.2%), and Canadian peat moss (38.5%). IAC
Alvorada for isolates aggressiveness and Brazilian dry bean cultivars were grown in potting soil
with the same ingredients that were used for DLB. These plants were grown in greenhouse at
Plant Protection Department, Botucatu, São Paulo. At 28 days after the pre-germinated seeds
were planted, at the age just preceding flowering, plants were inoculated with S. sclerotiorum
mycelia using the straw test, as described previously (Otto-Hanson et al. 2011). A plastic
drinking straw containing a 6 mm PDA plug with fungal mycelium was placed over a cut stem.
The stem of each bean plant was cut about 3 cm above the fourth node (the internode between
the fourth and fifth node). For inoculation, clear drinking straws were cut to 2.5 cm in length and
closed at one end. The open end of the straw was pressed into the reverse side of a 2 days old
thick PDA culture at the advancing edge of the mycelia of each S. sclerotiorum isolate in isolates
aggressiveness experiments and isolates 972B and 972D (for the first experimental repetition)
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and only 972D in dry bean cultivar performance. Plants were watered using capillary trays under
pots in order to prevent washing off the straw with fungal inoculum.
The plants were incubated for 8 days after inoculation and then the lesion was rated using
the Modified Petzoldt and Dickson Scale (Teran et al. 2006). This rating scale is from 1 to 9,
where 1 to 3 is considered resistant (no disease symptoms or symptoms only to the adjacent first
node), 4 to 6 is considered intermediate resistance (white mold symptoms beyond the adjacent
node to the next node), and 7 to 9 is considered susceptible (white mold symptoms past the
second node to plant death). When ST ratings were used to characterize isolate aggressiveness,
we defined ratings of 1 to 3 as low aggressiveness, 4 to 6 as intermediate, and 7 to 9 as high
aggressiveness.
Data analysis
Isolate aggressiveness data were from the DLB at 48 hours post inoculation and ST assessments.
Soybean cultivar performance data were from the DLB at 48 hours post inoculation. Dry bean
cultivar performance data were from the DLB assessed at multiple time points up to 48 hours,
which were used to calculate the area under the disease progress curve (AUDPC) in Microsoft
Excel (Shaner and Finney 1977). Dry bean cultivar performance data also included ST
assessments.
Comparisons were made using an analysis of variance (ANOVA) where experimental
repetition was treated as a fixed effect, and blocks were treated as a random effect with no
interactions. If experimental replicate was shown to be significant, models were re-run on each
experimental replicate separately. These models were implemented in the lme4 (version 1.1-5;
Bates et al. 2015) and lmerTest (version 2.036; Kuznetsova et al. 2017) packages in R (version
3.4.3; R Core Team 2017). We additionally used the agricolae package (version 1.2-8; De
Mendiburu and Simon 2015) to perform post-hoc tests using least significant differences (LSD)
with Bonferroni-adjusted p-values to correct for multiple comparisons. Results were plotted
using the ggplot2 package (version 2.2.1; Wickham 2009) in R and sigmaplot (version 10.0).
In the interest of open and reproducible science, all data for each of the seven
experiments and corresponding R script implementing the methods described above are
deposited in the Open Science Framework and are available for re-use with attribution
(https://osf.io/2x7fc/; Miorini et al. 2018). To ensure data preservation and computational
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readability, we have additionally converted these data from their Microsoft Excel format to
separate comma-separated value (csv) formatted text files using the readxl (version 1.0.0;
Wickham and Bryan 2017) and readr (version 1.1.1; Wickham et al. 2017) R packages. Some of
the data within this repository was collected during the experiments, but were not used in the
analyses. For the second experiment conducted in Brazil for isolate aggressiveness, the AUDPC
was calculated in Microsoft Excel from accumulated hour counts of size of lesions in leaves per
entry and treatment. These data can therefore be reused for educational purposes and/or meta-
analyses.
Results
A total of 87 isolates were used in this project that were collected from soybean in crop
production regions in U.S., Argentina, and in the Brazilian states of Goiás, Mato Grosso do Sul,
Rio Grande do Sul, Paraná, Bahia and Minas Gerais. Among the 87 isolates, 69 were from
Brazil, 13 from the U.S., and 5 were from Argentina (Table 1). Selections of these isolates were
used in four experiments to assess the range of aggressiveness of isolates from these locations.
Aggressiveness of 65 isolates (9 U.S., 5 Argentina, 51 Brazil) was determined on the
U.S. soybean cultivar Dassel using the DLB. Results showed that aggressiveness was
significantly different by experimental repetition (Fig. 2; F = 212, P < 0.001), thus data were
analyzed separately. The mean necrotic lesion area was larger for experimental replicates one
and two, with a mean lesion area for each experimental replicate of 11.3, 9.12, and 7.13 cm2. The
necrotic area of the 65 isolates using DLB in soybean cultivar Dassel was significantly different
(P < 0.001) when data were analyzed for each experimental replicate separately. The same was
trend was observed for maximum necrotic leaf area with each subsequent replicated experiment,
yet these maximum values were not attributed to the same isolates in each experiment. Isolates
identified as the top 10 most aggressive in each experiment were only moderately consistent,
with just eight isolates identified within the most aggressive isolates in more than one
experimental repetition.
Aggressiveness of 28 isolates from Brazil was also determined using a DLB with
Brazilian dry bean IAC Alvorada. Results showed a significant difference in necrotic area by
experimental replicate (F = 5.84, P = 0.003), however, the mean necrotic area was similar for the
first and second experiment (μ = 13.2 and 13.7 cm2) compared to the third experiment (μ = 14.5
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cm2). Further similarities between experiment one and two were also found with respect to the
rank of the top most aggressive isolates, wherein seven (IDs 975B, 975C, 976D, 977C, 978A,
978C, and 978E) of the top 10 were the same in both experiments 1 and 2. Two isolates in the
top 10 for the first experiment were also in the top ten for the third experiment (978C, 976D).
The largest necrotic lesions were 29.7 cm2 for experiment 2 and 34.1 cm2 for experiment 1.
When necrotic lesion areas within each experiment were separated and compared, each showed
differences between isolates were significant (P < 0.001).
Results of 32 isolates from Argentina (n = 2), the U.S. (n = 11), and Brazil (n = 19)
evaluated on U.S. dry bean cultivar G122 using the straw test showed isolate aggressiveness
varied from 1 to 8 and the average among all isolates was 5.08. There were no isolates that were
considered to have high aggressiveness, which was defined as those having an average straw test
ratings that ranged from 7 to 9, wherein a value of 9 was achieved when the isolate produced
necrotic lesions that progressed past the second node or led to plant death. The majority of the
isolates (31 isolates representing 96.9%), results of the straw test showed isolates had
intermediate aggressiveness, with necrotic lesion progression beyond the adjacent node.
Comparison of aggressiveness ratings showed there was a significant difference between isolates
(F = 5.34, P < 0.001).
Results of isolate aggressiveness using the straw test with IAC Alvorada showed that
average ratings per isolate varied from 1 to 9 and averaged 7.85 among all isolates. The majority
of isolates (25 isolates, representing 89.3% of those evaluated) were considered to have high
aggressiveness (average ST ratings 7 to 9), and two isolates (7.14%) had intermediate
aggressiveness (ST ratings 4 to 6). Comparison of lesion length by isolates showed there was a
significant difference with ANOVA (F = 11.5, P < 0.001). However, the LSD post-hoc test
showed this to be due to a single isolate “972D”, which had a mean score of 3.36. We ran the
same ANOVA model after this isolate was removed and while this still reported a significant
difference (F = 3.87, P < 0.001), the LSD showed no significant difference between isolates at
alpha = 0.05.
There were only 11 isolate shared between the DLB for Dassel and IAC Alvorada (973D,
974B, 974C, 975C, 975E, 976B, 977A, 977B, 977C, 977E, and 978A), and all but 974B were in
the top 10 for at least one experimental replicate (Table 2). Comparison of isolate aggressiveness
determined with the straw test ratings on G122 and IAC Alvorada showed four isolates were
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ranked in the top 10 for both cultivars (976B, 973C, 977B, 975D). When comparing isolates in
the top 10 for IAC Alvorada determined using the DLB and the ST, in common were four
isolates (976C, 973D, 976B, and 975D). No isolates were in the top 10 for all experiments
uniformly. A total of 13 isolates assessed across three or more experiments were found to be in
the top 10 for at least one experimental replicate: 976B, 974C, 973D, 975C, 975E, 977C, 977B,
977A, 978A, 975D, 973C, 977E, and 976C (Fig. 3). Only three other isolates were assessed
across at least three experiments: 974B, 973D, and 974D. The only isolate found to be in the top
10 in at least one experimental replicate across all four experiments was 976B, however, it was
not found to be the most aggressive isolate in any of these experiments. Out of these 13 isolates,
only three were found to be the most aggressive in any of the experimental replicates (974C,
Dassel DLB 28 dae; 975C, IAC Alvorada 21 dae; 973C, G122 and IAC Alvorada ST).
Cultivar performance
For Brazilian soybean cultivars, the experimental repetitions were significantly different (F =
93.6, P < 0.001) and are shown separately in Fig. 4. For the experimental repetitions, soybean
leaves were collected in the same plants but in different days, and results showed that older
leaves were less susceptible to infection by S. sclerotiorum (μ = 0.98 cm2 lesion) than younger
leaves (μ = 2.55 cm2 lesion). Necrotic area was significantly different for soybean cultivars. The
most susceptible cultivar to S. sclerotiorum was M8330, wherein the necrotic area was higher in
both experimental repetitions. The cultivars M5410 and M6410 were less susceptible to S.
sclerotiorum. While the difference between cultivars was significantly different for the pooled
model (F = 2.42, P = 0.010), this was shown to be due to the effect of experiment (first
experiment, F = 1.63, P = 0.110; second experiment F = 2.25; P = 0.022).
DLB and STs were used for Brazilian dry bean cultivars (Fig. 5). Straw test experiments
were conducted with the least aggressive isolate collected from dry bean (972D). This isolate
showed the lowest disease ratings in both methods—DLB and straw test—when inoculated in
the Brazilian dry bean cultivar IAC Alvorada (μ = 2.85 cm2 lesion for DLB at 48 hours and μ =
3.36 score for ST). For the DLB, isolate 972B was used because it was collected from dry bean
and showed greater aggressiveness in our previous test (μ = 13.1 cm2 lesion size in the DLB).
AUDPC calculated from the DLB ratings at 24, 30, 36, 42, and 48 hours after mycelial
inoculations and showed a significant difference between cultivars (Fig. 5, F = 4.10, P < 0.001)
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and experiment (F = 49.8, P < 0.001). When the experiments were analyzed separately, however,
the experiments showed differing results (experiment 1, F = 7.23, P < 0.001; experiment 2, F =
1.15, P = 0.306). Results of straw test were not significantly different among the experimental
repetitions (Fig. 5, F = 1.08, P = 0.299) but it was significant among cultivars (F = 5.64, P <
0.001). There were no data for ST in four dry bean cultivars.
There were 19 dry bean cultivars that were tested with both DLB and ST. Of these 19,
eight cultivars were consistently found to be among the 10 least susceptible in both tests. These
eight dry bean cultivars were: Pérola, IPR Uirapuru, IPR Tangará, IPR Juriti, IAC Imperador,
IAC Alvorada, IAC, Diplomata, and IAC Kaburé. Moreover, IAC Diplomata and IPR Tangará
were the least susceptible cultivars for S. sclerotiorum in DLB (ranked in first and second place,
respectively) and in ST (ranked in third and second place, respectively).
Discussion
The overarching goal of this project was to assess the variation in pathogen aggressiveness that is
representative of the regions where dry bean and soybean are cultivated in the U.S. and Brazil.
We also sought to provide new information about physiological resistance of various Brazilian
dry bean and soybean cultivars. Through a set of four experiments conducted in the U.S. and
Brazil, our work collectively provides knowledge about the pathogen and Brazilian cultivars that
highlight the importance of cultivar selection in management of white mold and sclerotinia stem
rot.
Results of the present study showed that there was a significant difference in the
aggressiveness of isolates regardless of the evaluation method or plant host used. However, we
also identified a significant difference in aggressiveness ratings by experiment. This is likely
driven by the use of soybean cultivar leaves that were collected from the same plants at 21, 28,
and 35 days after emergence and that older leaves were less susceptible to infection by S.
sclerotiorum. Although changes in host susceptibility may occur with increasing plant age and
phenological stage, there is a general expectation that resistance to biotrophic pathogens
increases with plant age and resistance to necrotrophic pathogens decreases with plant age
(Farber and Mundt 2017). Since S. sclerotiorum is traditionally considered to be a necrotrophic
pathogen, our current results and those reported in two other papers that noted increased
resistance of older plants are contrary to the expectation for a necrotroph (Augusto and
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Brenneman 2012; Miorini et al. 2017). Authors in the previous studies hypothesized that the
observed increased resistance was the result of fungicide applications and an accumulation of
fungicide residue (Augusto and Brenneman 2012; Miorini et al. 2017). In the current study,
however, no fungicides were applied, consequently, we are able to conclude that the increase in
resistance that we observed is more likely due to physiological changes in the plant that occur
with age, such as thickness of plant membranes, waxes, distance of translocation, and velocity of
xylem translocation (Augusto and Brenneman 2012). Moreover, recent molecular genetic work
investigating the intimate interactions between host plant and pathogen during the infection
process suggest that a hemibiotrophic strategy is the more appropriate description of the
mechanisms that S. sclerotiroum employs (Kabbage et al. 2015). Thus, our observation that host
susceptibility decreases with plant age may be a result of the brief biotrophic period prior to
necrotrophic invasion, providing further support for the notion that we should refer to S.
sclerotiorum as a hemibiotrophic pathogen.
In assessments of isolate aggressiveness, it is important to note that performing assays on
soybean and dry bean yielded similar results (Fig. 3), suggesting that variation in S. sclerotiorum
isolate aggressiveness is not strongly affected by differences between these two plant species.
This means that isolates found to have greater aggressiveness on soybean are likely also going to
have greater aggressiveness on dry bean. However, our straw test results did show a difference in
discriminatory power between dry bean cultivars IAC Alvorada and G122. Our data showed the
fewest number pairwise differences between isolate aggressiveness were obtained from
evaluations performed using dry bean cultivar IAC Alvorada, which also showed greater
susceptibility to S. sclerotiorum than dry bean cultivar G122. It is not unexpected that a more
susceptible cultivar would identify fewer differences in aggression between S. sclerotiorum
isolates. Greater susceptibility increases the rate of pathogen ingress into the green tissue, which
makes it more difficult to discern very minor differences in lesion length and also makes this
bioassay more temporally sensitive to small deviations in assessment times. For this reason, the
U.S. dry bean cultivar G122 is the standard used in pathogen aggression assays that we have
used in our previous studies that were conducted in the U.S. (Kamvar et al. 2017, Otto-Hanson et
al. 2011). Results of the current study using dry bean IAC Alvorada suggest that it may not be
the optimal choice for differentiating isolate aggressiveness and that a cultivar with greater
resistance may be a better choice for future studies. This cultivar was also used in our DLB,
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which allowed some comparisons between these methods in terms of ability to identify isolates
determined to be the most aggressive. Although the coefficient of variation (CV) can be used for
comparing results of different methods, it is considered questionable to compare methodologies
with different scales of measurement (Kull et al. 2003), thus, we did not compare results of the
DLB and straw test.
As the performance of most of the cultivars in regards to S. sclerotiorum resistance was
unknown, straw test experiments were conducted with the least aggressive isolate collected from
dry bean (972D). This isolate showed the lowest values in both methods—DLB and straw test.
When we used another, more aggressive isolate, the ST scores for almost all dry bean cultivars
was close to 9 after 8 days of incubation (μ = 8.57, data not shown), which classified all cultivars
as susceptible. However, using the most aggressive isolate (972D) in the DLB generated small
necrotic areas, with an average area at 48 hours post inoculation of 3.00 cm2 (data not shown).
Thus, our recommendation is that studies evaluating soybean and dry bean cultivars using the
DLB should select isolates with high aggressiveness and studies evaluating dry bean cultivars
using the ST method should select isolates with low aggressiveness.
Both methods to assess aggressiveness showed that dry bean IAC Alvorada generated
greater average lesion sizes and lengths compared to the soybean cultivar Dassel and the dry
bean G122, which are considered partially resistant cultivars to S. sclerotiorum and commonly
used as standards in field trial evaluations in the U.S. Thus, we conclude that IAC Alvorada does
not possess the same level of physiological resistance and does not represent a partially resistant
cultivar for S. sclerotiorum control. There were eight dry bean cultivars evaluated in our study
that showed greater resistance to S. sclerotiorum than IAC Alvorada, with IAC Diplomata and
IPR Tangará having the smallest leaf lesions in the DLB and classified as intermediate resistant
to resistant, respectively, when evaluated with the ST method. Although we could not compare
results of these methods directly, the observed consistency between methods allows us to
conclude that either method can be used to evaluate susceptibility/resistance for dry bean
cultivars to S. sclerotiorum. Among the soybean cultivars evaluated, were identified one that was
considered most susceptible (M8330) and two others that may possess resistance (M5410 and
M6410), however, a limitation in our assay was that we did not include a soybean cultivar
considered to have resistance, thus further assays using these cultivars is warranted.
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Collectively, results of our study showed we were able to identify cultivars of dry bean
and soybean that performed the best in our assays and likely possess resistance to S.
sclerotiorum, however physiological resistance to S. sclerotiorum is not the only factor that
should be considered. For example, although the dry bean cultivar G122 possesses a desirable
level of physiological resistance to white mold, other characteristics make this an unfavorable
bean, such as lower yields and bean size, color, and shape. It is recommended that future studies
evaluate the performance of these bean cultivars across several field sites in regions of Brazil
where dry bean is grown so that the field performance of these plants can be evaluated in an
environment where they may be exposed to a wide range of S. sclerotiorum isolates with varying
degrees of aggressiveness, such as those evaluated in the present study.
Ultimately, our goal is to identify cultivars with resistance to S. sclerotiorum infection so
that these can be recommended for integration into disease management programs, which is
especially important for common bean because it exhibits only low levels or partial resistance to
white mold (Schwartz and Singh 2013). Both controlled laboratory experiments and
complementary field experiments should be used in order to have complete data. Moreover,
cultivar performance should be evaluated at multiple locations, because white mold may cause
100% yield loss for susceptible common bean cultivars under favorable weather conditions
(Schwartz and Singh 2013). Collectively, results of this study are important for improving our
understanding of variation in pathogen aggressiveness from geographically isolated populations
and in identifying cultivars that are likely to have partial resistance, which represent important
tools for management of potentially devastating diseases caused by S. sclerotiorum.
Acknowledgements
The authors acknowledge the institutions IAPAR (Instituto Agronômico do Paraná), IAC
(Instituto Agronômico), Embrapa (Empresa Brasileira de Pesquisa Agropecuária), and the
company Monsanto that provided the dry bean and soybean cultivars seeds to conduct the
cultivar performance experiment. Also, the authors thank the support provided by Arysta
LifeScience, in special thanks to Dorival Boer Júnior and Ângelo Stavieski. We also thank Lucky
Mehra for advice on statistical analysis.
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Funding Sources
The authors acknowledge at the Coordination for the Improvement of Higher Education
Personnel (CAPES) for the financial support provided to T. J. J. Miorini’s PhD program for
execution of the experimental phase of this study. This project is based on research that was
partially supported by the Nebraska Agricultural Experiment Station with funding from the
Hatch Act (Accession Number 1007272) through the USDA National Institute of Food and
Agriculture. This work was funded in part by grant #58-5442-2-209 from the USDA-ARS
National Sclerotinia Initiative to JRS/SEE and start-up funds from the University of Nebraska-
Lincoln (UNL) to SEE. Funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
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TABLES AND FIGURES
Table 1 Origin and number of Sclerotinia sclerotiorum isolates from the U.S., Argentina and
Brazil used in four experiments to characterize isolate aggressiveness.
DLB; Dassel1 DLB; IAC
Alvorada2
ST; G1223 ST; IAC
Alvorada4
U.S. (13) 9 11
Argentina (5) 5 2
Brazil (69) 51 28 19 28
Total 65 28 32 28
1S. sclerotiorum isolates were inoculated onto soybean cultivar Dassel in the U.S. using
Detached leaf bioassay (DLB). Each isolate was inoculated in 10 leaves in each of three-leaf
collections (21, 28 and 35 days-old after emergence). Lesion areas were evaluated 48 hours after
inoculation. 2Brazilian isolates were used to inoculate dry bean of seed class Carioca, cultivar
IAC Alvorada using the DLB. Each isolate was inoculated in 10 leaves in each of three-leaf
collections (21, 28 and 35 days-old after emergence), and evaluations were performed with 48
hours after inoculation. 3Isolates were characterized on dry bean cultivar G122 using the straw
test (ST), with 12 replications. The evaluations were performed 8 days after inoculation. 4Isolates
were evaluated in IAC Alvorada using the straw test. These isolates were incubated for 8 days
after inoculation. We used a total of eleven plants for each inoculation and the experiment was
conducted once.
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Table 2 Number of experimental replicates (out of three) in which each of the 11 Brazilian
isolates listed below ranked in the top 10 most aggressive isolates evaluated using the
detached leaf bioassay in using soybean cultivar Dassel and dry bean cultivar IAC Alvorada.
Isolate Dassel1 IAC Alvorada2
974C 3 1
976B 2 1
973D 1 1
975C 0 2
977A 0 2
977C 0 2
978A 0 2
975E 0 1
977B 1 0
977E 0 1
974B 0 0
1 Out of 65 isolates 2 Out of 28 isolates
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Fig. 1 Bioassays used to evaluate Sclerotinia sclerotiorum isolate aggressiveness and
performance of soybean and dry bean cultivars inoculated with S. sclerotiorum. Necrotic area is
measured after Sclerotinia sclerotiorum mycelia inoculation and compared to standardized rating
scales. A) Detached leaf bioassay, wherein necrotic area was evaluated 48 hours after soybean
leaf inoculation using ImageJ software. Necrotic area of this leaf was high (30.124 cm2), and it is
considered unusual, because the mean of all experiments was 11.5 cm2. B) Straw test, wherein
length of the necrotic lesion down the stem was measured 8 days after inoculation.
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Fig. 2 Mean results of aggressiveness evaluations of Sclerotinia sclerotiorum isolates collected
from Brazil, the USA, and Argentina using a detached leaf bioassay and straw test. A) Two
detached leaf bioassay experiments were conducted with 65 and 28 isolates inoculated onto U.S.
soybean cultivar Dassel and Brazilian dry bean cultivar ‘IAC Alvorada’, respectively; wherein
leaves were collected at 21, 28 and 35 days after emergence. B) Thirty-two and 24 isolates were
used in a straw test with U.S. dry bean cultivars G122 and IAC Alvorada, respectively.
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Fig. 3 Stacked barplot of mean of necrotic lesion area from the DLB and mean score from the
straw test for Sclerotinia sclerotiorum isolates that were evaluated in at least three of four
experiments. Colors of each bar represent experiment and replicate. Average isolate ratings that
ranked in the top ten most aggressive within a given experimental replicate are outlined with a
black border. Isolates are ordered from left-to-right according to the number of times each was in
the top 10 most aggressive and ranked based on cumulative mean.
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Fig. 4 Results of the detached leaf bioassay on 11 Brazilian soybean cultivars evaluated using
one isolate of Sclerotinia sclerotiorum. Each point represents the average necrotic leaf area (cm2)
and bars are standard error, where filled circles are results from the first experimental repetition
that used leaves collected from plants grown at the Pereiras-SP field site at 34 days after
emergence and open circles are the means for the second experimental repetition with leaves
collected 60 dae. Due to a significant difference in lesion size in the repeated experiment (P <
.0001), data were not combined from the two evaluations. 1dae = days after emergence
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Page 29 of 29
Fig. 5 Results of the area under disease progress curve (AUDPC) from the detached leaf
bioassay (DLB) and lesion length (cm) from the straw test on 23 Brazilian dry bean cultivars
inoculated with one isolate of Sclerotinia sclerotiorum. Each point is the average AUDPC and
bars are standard error, where filled circles are results from the first experimental repetition that
used 6 leaves and open circles are the means for the second experimental repetition with 12
leaves collected. Due to a significant difference in AUDPC in the repeated experiment, data were
not combined. However, the cultivars were sorted by the average of AUDPC of the two
experimental repetitions. Each open square is the average lesion length of 22 measurements and
bars are standard error.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.26622v1 | CC BY 4.0 Open Access | rec: 5 Mar 2018, publ: 5 Mar 2018