RESEARCH ARTICLE Variation in pathogen aggression and ... › preprints › 26622.pdf · important for improving our understanding of variation in pathogen aggressiveness from geographically

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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]

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.26622v1 | CC BY 4.0 Open Access | rec: 5 Mar 2018, publ: 5 Mar 2018

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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


https://osf.io/2x7fc/

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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)


Page 14 of 29

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


Page 15 of 29

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,


Page 16 of 29

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.


Page 17 of 29

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.


Page 18 of 29

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.


Page 24 of 29

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


Page 25 of 29

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.


Page 26 of 29

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.


Page 27 of 29

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.


Page 28 of 29

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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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.


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