Pre-treatment of soybean plants with calcium ...

Accepted Manuscript Pre-treatment of soybean plants with calcium stimulates ROS responses and mitigates infection by Sclerotinia sclerotiorum Arbia Arfaoui, Abdelbasset El Hadrami, Fouad Daayf PII:

S0981-9428(17)30383-2

DOI:

10.1016/j.plaphy.2017.11.014

Reference:

PLAPHY 5054

To appear in:

Plant Physiology and Biochemistry

Received Date: 9 August 2017 Revised Date:

15 November 2017

Accepted Date: 24 November 2017

Please cite this article as: A. Arfaoui, A. El Hadrami, F. Daayf, Pre-treatment of soybean plants with calcium stimulates ROS responses and mitigates infection by Sclerotinia sclerotiorum, Plant Physiology et Biochemistry (2017), doi: 10.1016/j.plaphy.2017.11.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Pre-treatment of soybean plants with calcium stimulates ROS responses

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and mitigates infection by Sclerotinia sclerotiorum

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Arbia Arfaoui1,2, Abdelbasset El Hadrami2, Fouad Daayf*1

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MB, R3T 2N2, Canada

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Department of Plant Science, 222, Agriculture Building, University of Manitoba, Winnipeg,

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OMEX Agriculture Inc. 290 Agri Park Road, Oak Bluff, Manitoba, R4G 0A5, Canada

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Arbia Arfaoui, Ph.D. E-mail: [email protected]

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11 Abdel El Hadrami, Ph.D.

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R&D Director, OMEX Agriculture Inc.

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290 Agri Park Road, Oak Bluff, Manitoba

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R4G 0A5, Canada

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Ph: 1-204-477-4052

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Fax: 1-204-477-4057

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E-mail: [email protected]

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*Corresponding author

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Fouad Daayf, Ph.D.

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Professor & Department Head

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University of Manitoba

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Department of Plant Science

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222, Agriculture Building,

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Winnipeg, MB, R3T 2N2, Canada

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Phone: 204-474-6096

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Fax: 204-474-7528

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E-mail: [email protected]

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Abstract

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Considering the high incidence of white mold caused by Sclerotinia sclerotiorum in a variety

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of field crops and vegetables, different control strategies are needed to keep the disease under

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economical threshold. This study assessed the effect of foliar application of a calcium

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formulation on disease symptoms, oxalic acid production, and on the oxidative stress

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metabolism in soybean plants inoculated with each of two isolates of the pathogen that have

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contrasting aggressiveness (HA, highly-aggressive versus WA, weakly-aggressive). Changes

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in reactive oxygen species (ROS) levels in soybean plants inoculated with S. sclerotiorum

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isolates were assessed at 6, 24, 48 and 72 hours post inoculation (hpi). Generation of ROS

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including hydrogen peroxide (H2O2), anion superoxide (O2-) and hydroxyl radical (OH.) was

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evaluated. Inoculation with the WA isolate resulted in more ROS accumulation compared to

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the HA isolate. Pre-treatment with the calcium formulation restored ROS production in plants

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inoculated with the HA isolate. We also noted a marked decrease in oxalic acid content in the

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leaves inoculated with the HA isolate in presence of calcium, which coincided with an

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increase in plant ROS production. The expression patterns of genes involved in ROS

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detoxification in response to the calcium treatments and/or inoculation with S. Sclerotiorum

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isolates were monitored by RT-qPCR. All of the tested genes showed a higher expression in

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response to inoculation with the WA isolate. The expression of most genes tested peaked at 6

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hpi, which preceded ROS accumulation in the soybean leaves. Overall, these data suggest that

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foliar application of calcium contributes to a decrease in oxalic acid production and disease,

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arguably via modulation of the ROS metabolism.

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Key words: Sclerotinia sclerotiorum, soybean, calcium formulation, oxidative stress markers,

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gene expression, ROS.

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Introduction

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White mold caused by the fungal pathogen Sclerotinia sclerotiorum is a major disease in

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many crops including soybeans. It causes significant yield losses and affects seed quality

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(Hegedus and Rimmer, 2005). Commonly used disease management strategies include crop

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rotation, sclerotia burial, fungicide treatments, along with the use of tolerant varieties. Taken

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either separately or combined, none of these strategies provide a complete control of this

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disease. Alternative control measures, such as those relying on the elicitation of the host’s

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innate defense mechanisms are needed, more than ever, in order to keep the disease under

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economical thresholds.

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Oxalic acid (OA) is a critical and multifunctional pathogenicity factor that governs the

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infection success of S. sclerotiorum in a variety of crops (Cessna et al. 2000). Several studies

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using OA-deficient mutants suggested that the toxic acid secreted by S. sclerotiorum during

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the first steps of infection weakens the plant cell walls by sequestering Ca2+ and by

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compromising the functions and signaling that are calcium-dependent (Tian et al. 2002; Paula

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Júnior et al. 2009). It also provides the right acidity level in the intercellular space for an

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optimal activity of cell wall-degrading enzymes (CWDEs) such as chitinases, pectinases and

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polygalacturonases (Cotton et al. 2003). OA can affect the programmed opening and closure

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of stomata in plant leaves causing water loss and leaf wilting (Guimarães and Stotz, 2004). In

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addition to its direct role in disease, OA can also indirectly impact pathogenesis by

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manipulating some of the host signaling pathways (Kim et al. 2008, 2011). Recently OA has

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been reported as an elicitor of plant programmed cell death (Da Silava et al. 2011) involving

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increased levels of reactive oxygen species (ROS).

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Reactive oxygen species (ROS), i.e., H2O2 (hydrogen peroxide), O2- (superoxide ion) and OH.

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(hydroxyl radical), play a key role in both plant defense to pathogens (Sharma et al. 2012) as

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well as in pathogenesis (Aguirre et al. 2005; Bailly et al. 2008). Colonization by necrotrophic

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and hemi-biotrophic pathogens such as S. sclerotiorum is thought to be enhanced by ROS

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(Walz et al. 2008) since these pathogens have developed effective mechanisms to cope with

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ROS toxicity (Mayer et al. 2001).

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Plants have also evolved antioxidant protective mechanisms to maintain ROS at their lowest

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level in the cells (Wojtaszek, 1997; El Hadrami et al. 2005), hence lessening damage to the

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cell walls and other organelles and cell constituents. These mechanisms encompass non-

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enzymatic antioxidant protection, which involves ascorbates, glutathione, α-tocopherol,

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ACCEPTED MANUSCRIPT carotenoids, flavonoids, phenolic compounds and alkaloids (Kesheri et al. 2011), as well as an

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enzymatic anti-oxidative machinery (El Hadrami et al. 2005). The latter includes superoxide

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dismutases (SOD), catalases (CAT), peroxidases such as ascorbate and glutathione

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peroxidases (APX, GPX), and peroxiredoxins (Prx). The SOD catalyzes the dismutation of

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the anion O2- into oxygen and hydrogen peroxide. APX plays a major role in detoxifying

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hydrogen peroxide in the chloroplast and supports the activity of both SOD and CAT during

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biotic and abiotic stresses (Shigeoka et al. 2002; El Hadrami et al. 2005). Peroxiredoxins (Prx)

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convert hydrogen peroxide into water and oxygen (Pulido et al. 2010). These enzymes belong

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to a family of proteins with a catalytic oxido-redox cysteine in their active site able to react

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with a variety of peroxide substrates. The cysteine quenches the peroxide substrate and

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reaches an oxidative state as sulfenic acid, which reverts back to a reduced thiol after

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interaction with other proteins such as glutaredoxin or thioredoxin, allowing the Prx to

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proceed to the next catalytic cycle (Trujillo et al. 2007).

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In a previous study, we had demonstrated that pre-treatment of soybeans with calcium

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enhances defense responses with higher accumulation of isoflavone phytoalexins, thereby

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reducing Sclerotinia infection (Arfaoui et al. 2016), and suggesting an indirect effect on the

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pathogen.

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In this study, we examined the effect of foliar calcium application on disease symptoms, on

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oxalic acid production, and on ROS generation and detoxification in soybeans infected with

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SS18 and SSPetri, a weakly- and highly-aggressive isolates of S. sclerotiorum, respectively.

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The response to the oxidative stress caused by inoculation with either isolate was also

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examined at the cellular level through overtime monitoring of changes in transcript levels of

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four genes involved in ROS detoxification (Sodb2, Apx1, Prx2b and Thioredoxin).

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Materials and Methods

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Fungal isolates

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We tested S. sclerotiorum SSPetri and SS18, a highly- (HA) and weakly-aggressive (WA)

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isolates, respectively, as previously described (Arfaoui et al. 2016). Both isolates were grown

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on PDA (Potato Dextrose Agar) at 20°C.

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Plant material

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ACCEPTED MANUSCRIPT We used seeds of soybean cv. Thunder 27005RR2 from Thunder Seed, Canada to grow

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seedlings for 21 days in a growth chamber (20oC, 60-80% RH, and a 16 h photoperiod). For

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each treatment, three pots, filled with soil and sand (1:1), were used, and six seeds were

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planted per pot. Two days before inoculation, the seedlings were divided into two sets. One

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set was sprayed with a 0.1% calcium formulation (P3; OMEX Agriculture Inc., Canada) until

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the leaves were wet. 5 millimiter plugs of 5 day old culture were used to inoculate plants at

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the V2 leaf stage. Plugs with Sclerotinia inoculum were placed in the middle of each leaf.

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After inoculation, all of the plants were kept at 20oC and 90 % RH to provide adequate

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conditions for infection. Leaf samples were collected at 6, 24, 48 and 72 hours post

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inoculation (hpi) and immediately used for ROS production analysis or frozen in liquid

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nitrogen and stored in -80oC, until used for RNA isolation. Leaves were also photographed

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and the percentage of diseased area was calculated using the lesion assay software ASSESS

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2.2.

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Six treatments were compared: (i) water control (Control); (ii) control pre-treated with

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calcium (Control + Ca); (iii) inoculated with the HA isolate (SSPetri); (iv) pre-treated with

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calcium and inoculated with the HA isolate (SSPetri + Ca); (v) inoculated with the WA

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isolate (SS18), and (vi) pre-treated with calcium and inoculated with the WA isolate (SS18 +

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Ca). The experimental design consisted of three biological replicates for each treatment that

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were completely randomized. Leaves from 6 different plants were pooled together to form

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one of three biological replicates. The whole experiment was repeated twice.

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Determination of oxalic acid content in soybean leaves

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Total oxalic acid concentration was determined in soybean leaf tissues using the protocol of

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Xu and Zhang (2000) with a few modifications. Briefly, one g of fresh leaf material was

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extracted in 50 ml of water at 80°C while shaking for 15 min at 120 r.p.m. The suspension

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was centrifuged at 5000 g for 15 min and the supernatant was filtered through a Whatman

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paper before being subjected to an oxalic acid assay. The assay consisted of 50 µl sample (or

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standard OA solution), 27.5 µl of 1 mM bromophenol blue, 49.5 µl of 1M sulfuric acid, 44 µl

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of 100 mM potassium dichromate and 1.2 ml water. The mixture was vortexed and incubated

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in a water bath at 60°C for 10 min. After incubation, the reaction was quenched with 110 µl of

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2 M sodium hydroxide. The absorbance was measured at 600 nm. Oxalic acid concentration

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was expressed as µg.g-1 FW by comparison with an OA standard curve. Assays were

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conducted in triplicate and the whole experiment was independently repeated three times.

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ACCEPTED MANUSCRIPT Oxalic acid released in PDB medium by S. sclerotiorum isolates was also quantified by the

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same protocol of Xu and Zhang (2000). Briefly S. sclerotiorum isolates were grown in 100 ml

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flask containing PDB media amended with 0.1% (v/v) of calcium-based formulation. Flasks

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were statically incubated for 3 and 7 days at room temperature. Cultures filtrates were used

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for oxalic acid determination and mycelial fractions dry weight were determined after drying

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at 60°C for 3 days. Oxalic acid concentration was expressed as µg.mg-1 dry weight mycelium.

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160 161 In situ detection of ROS

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Superoxide ion (O2-) was detected in soybean tissues 48 hpi Leaf tissues were placed for 2

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hours in an aqueous solution in 0.5 mg.ml-1 Nitroblue Tetrazolium (NBT) (10 mM potassium

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phosphate buffer, pH 7.5). After incubation, leaves were rinsed in 70% ethanol and mounted

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in 50% glycerol and photographed (Hernandez et al. 2001).

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Detection of hydrogen peroxide (H2O2) was conducted by staining the tissues in 3,3-

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diaminobenzidine (DAB) as described by Thordal Christensen et al. (1997). H2O2 reacts with

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DAB to form a reddish-brown stain. Leaves were incubated in DAB solution (1 mg.mL-1, pH

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3.8) for 2 hours and vacuum infiltrate at 120 mbar for 1 min. After that, the leaves were

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incubated in plastic boxes for 6-8 hours under high humidity conditions until brown

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precipitate was observed. Stained leaves were fixed in a solution of ethanol: lactic acid:

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glycerol (3:1:1, v/v/v) and photographed.

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Quantification of ROS production

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The production of hydrogen peroxide (H2O2), the anion superoxide (O2-) and hydroxyl radical

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(OH.) were determined in soybeans leaves issued from Ca-treated and untreated plants

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infected with SS18 or SSPetri isolates.

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The production of O2- was quantified based on NBT reduction activity, as described by Doke

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(1983) and El Hadrami et al. (2005). Briefly, leaves were washed using dH2O and placed for 1

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hour in 3 ml of 10 mM potassium phosphate buffer, pH 7.8, containing 0.05% NBT and 10

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mM NaN3. This mixture was heated for 15 min at 85°C and then cooled quickly. The

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reduction activity of the soybean leaf extract was followed by measuring the absorbance at

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580 nm.

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The production of H2O2 was estimated as described by Tiedemann (1997) and El Hadrami et

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al. (2005). Soybean leaves were incubated for 2 hours in the dark at room temperature in 2 ml

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ACCEPTED MANUSCRIPT of a reagent mixture containing 50 mM phosphate buffer pH 7.0, 0.05% guaicol and

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peroxidase. The release of H2O2 was monitored by measuring absorbance at 450 nm.

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The production of OH. was estimated as described by Tiedemann (1997) and El Hadrami et

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al. (2005). Leaves were incubated in 1 ml of 1 mM 2-Deoxyglucose then incubated for 45 min

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in the dark at room temperature. Five hundred microliters of this solution were added to 500

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µl of 1% (w/v) thiobarbituric acid and 1 ml of 2.8% (w/v) trichloroacetic acid. The mixture

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was boiled for 10 min and immediately cooled in ice for 10 min. The production of the

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hydroxyl radical was followed by measuring absorbance at 540 nm.

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195 RNA extraction and RT-qPCR

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RNA was extracted from the leaf tissues using the RNeasy plant mini kit (QIAGEN) and

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treated

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recommendations. The RNA was quantified at 260 nm using a NanoDrop ND-2000

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spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and its quality checked on 1%

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agarose gel.

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The first cDNA strand was synthesized using the total RNA with a Promega reverse

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transcription kit (Promega, Madison, WI, USA) as recommended by the manufacturer. QRT-

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PCR was carried out for both the target and reference (actin) genes using CFX96TM Real

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Time System (C1000TM Thermal Cycler, Biorad) and SsoFast EvaGreen Supermix (Biorad,

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Hercules, CA, USA) as recommended by the manufacturer. The amplification was carried out

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using 95°C for 2 min to activate the hot-start recombinant Taq DNA polymerase, followed by

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39 cycles of amplification at 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and 78°C for 11 s to

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avoid primer-dimer formation. Following amplification, a melting curve program (55-95°C

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with a heating rate of 0.5°C.s-1) at 95°C for 0.05 s, 65°C for 0.05 s, and 95°C for 0.5 s was

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performed to ensure that only a single product was generated at the end of the assay. All

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samples were run in parallel with the housekeeping gene actin to normalize cDNA loading.

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Three biological replicates were used per run including the reference gene. Gene expression

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results were analyzed using the 2−∆∆Ct method after verification that the primers amplified

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with an efficiency of approximately 100% as described by Livak and Schmittgen (2001).

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Specific primers were designed based on the soybeans mRNA sequences available and

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GenScript

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bin/tools/primer_genscript.cgi) to test the expression of Sodb2, Apx 1, Prx2b and Thio genes

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(Table 1). The constitutively expressed gene actin was used for normalization of each target

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gene

RNase-free

DNase

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(Ambion)

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Real-time

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Software

(http://www.genscript.com/cgi-

ACCEPTED MANUSCRIPT Statistical analysis

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All data sets from OA content, ROS accumulation and RT-qPCR were analyzed by ANOVA

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using Statistica Software (Statsoft, Tulsa, OK, USA). The data sets were from three

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independent replicates and were tested for variance homogeneity and significance of the

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“experiment” effect. In the absence of significant “experiment” effect, data were pooled and

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presented as averages. Differences among the means were assessed based on the Duncan

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Multi-range test at P < 0.05.

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Results

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Disease assessment

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Five days post inoculation, soybean leaves inoculated with the HA isolate SSPetri displayed a

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lesion of 40% of the leaf area. In contrast, inoculation with the WA isolate SS18 resulted in

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limited infection of 8% (Fig.1). Application of calcium onto the leaves 48 h prior to

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inoculation significantly restricted the lesion development, especially by the HA isolate

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SSPetri with more than 50 % reduction in lesion size (Fig. 1)

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236 Oxalic acid content

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The HA isolate SSPetri produced more oxalic acid in the infected soybean leaves than the

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WA isolate SS18 (Fig. 2). Pre-treatment with calcium onto the leaves 48 hours prior to

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inoculation significantly reduced the production of oxalic acid in planta, especially by the HA

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isolate SSPetri. At 3 and 7 days post inoculation, OA content was significantly lower (30%) in

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the pre-treated than the non-treated leaves in response to inoculation with the HA isolate

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SSPetri.

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The amount of oxalic acid released by SSPetri (286.8µg) in the PDB medium was much

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higher than that released by SS18 (163.6 µg) (Table 2). Adding calcium to the PDB medium

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at a concentration of 0.1% decreased the level of the OA released by both isolates by 10%

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(Table 2).

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ROS accumulation

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H2O2 production was discernable as a reddish-brown stain by DAB. It was more noticeable in

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SS18-infected plants than with SSPetri. A clear zone free of DAB staining was observed in

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the cells directly adjacent to the inoculation site (Fig. 3). A significant change was perceived

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ACCEPTED MANUSCRIPT in the Ca-treated soybean tissues inoculated with either isolate but the response was stronger

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in response to the HA isolate.

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Quantification of H2O2 showed an early response to inoculation with S. sclerotiorum. The

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WA isolate SS18 induced an increase in H2O2 content as early as 6 hpi while the H2O2 content

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in the tissues challenged with the HA isolate SSPetri was similar to that of the untreated, non-

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challenged tissues (Fig. 4 a,b). After pre-treatment with calcium, the concentration of H2O2

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was higher in response to the HA SSPetri (SSPetri+Ca) than to the WA isolate SS18 (SS18-

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Ca). The maximum induction was observed at 72 hpi with 40% increase in response to

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SSPetri+Ca as compared to SS18-Ca. No significant change was observed in the control

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plants whether untreated or pre-treated with calcium.

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An inhibition of O2- production was observed at the site of inoculation with the HA isolate,

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with absence of a blue color around the lesions (Fig. 3). A noticeable production of O2- was,

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on the other hand, observed in the tissues inoculated with SS18. The application of calcium

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prior to inoculation restored the production of O2- in the leaves inoculated with the HA isolate

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SSPetri. The content in O2- in the leaves inoculated with the WA isolate reached its maximum

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at 72 hpi (Fig. 4 c,d).

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The highest OH. production occurred 24 hpi in response to the WA isolate SS18. With the HA

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isolate SSPetri, the level was similar to that of the untreated and non-challenged control. On

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the other hand, pre-treatment with calcium restored the OH. production in the leaves

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inoculated with the HA isolate SSPetri. The maximum production in response to SSPetri+Ca,

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which was three times higher than in response to SS18-Ca, was observed 24 hpi (Fig. 4 e,f).

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Expression patterns of genes controlling anti-oxidative enzymes

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All of the tested genes that control anti-oxidative enzymes showed higher expression in

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response to inoculation with the WA isolate SS18. Most of them peaked as early as 6 hpi,

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which precedes ROS accumulation in the soybean leaves (Fig. 5).

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Sodb2 transcripts showed more abundance in response to the WA isolate SS18 than with the

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HA isolate SSPetri (Fig. 5 a,b). Pre-treatment with calcium caused a drop in sodB2 transcripts

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in response to both isolates.

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Both SS18 and SSPetri isolates induced the expression of Apx1. This expression was stronger

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and peaking almost 10-fold higher at 24 hpi in response to the WA isolate SS18 as compared

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to the HA isolate SSPetri. Pre-treatment of soybeans with calcium increased the level of

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expression of the Apx1 gene in response to inoculation with the HA isolate SSPetri (Fig. 5

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c,d).

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ACCEPTED MANUSCRIPT Prx2b gene had a higher expression in the leaves inoculated with SS18 than with SSPetri.

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Accumulation of transcripts in response to SSPetri was stable over time and similar to the

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untreated control. Pre-treatment with calcium induced a slight increase in this gene’s

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expression in response to inoculation with the HA isolate SSPetri (Fig. 5 e,f).

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The thioredoxin-encoding gene showed a gradual increase in expression over time, with a

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peak at 48 hpi. Its transcripts’ level was 3-fold higher in response to the WA isolate SS18 than

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to the HA isolate SSPetri. Pre-treatment with calcium induced a 2-fold higher expression of

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this gene in response to the HA isolate SSPetri as early as 6 hpi (Fig. 5 g,h).

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Discussion

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Sclerotinia sclerotiorum is considered an atypical necrotroph fungus. It colonizes plant tissues

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according to several distinct phases dictated by a precise reprogramming of the pathogen

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transcriptional and physiological machinery (Kabbage et al. 2013, 2015). Our results indicate

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that adding calcium to the growth medium reduce the amount of OA released by both isolates.

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This is not surprising since OA is intrinsically linked to low pH and adding calcium to the

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medium will reduce the acidity and increase the pH, which impact the OA production. Pre-

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treatment of soybeans with calcium significantly reduced the amount of oxalic acid produced

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in planta, leading to a substantial decrease in disease progress. The response was more

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pronounced with the highly aggressive (HA) isolate SSPetri than with the weakly aggressive

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(WA) isolate SS18. The HA isolate SSPetri produces more oxalic acid and causes more

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disease severity (40%) than the WA isolate SS18 (8.0%). This corroborates other findings

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showing a positive correlation between the rate of production of oxalic acid and the virulence

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of S. sclerotiorum isolates (Durman et al. 2003; 2005). Therefore, chelating produced oxalic

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acid by applying exogenous calcium could likely attenuate the pathogen ability to infect and

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progress on/in the tissues, with consequences on pathogenesis and defense responses.

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The reduction of OA is important in crosslinking calcium ions bound to pectins, which protect

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host cell walls from fungi (Brisson et al. 1994). Gosh et al. (2016), suggested that calcium

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may have a role in low oxalate-mediated cell wall rearrangement towards induced plant

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defense.

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ROS production is an integral part of the recognition process between S. sclerotiorum and its

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host (Bolton et al. 2006; Perchepied et al. 2010). It is also a trigger for the signaling cascade

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that allows plants to set defense responses remotely from the infection site. However,

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pathogens such as S. sclerotiorum have evolved mechanisms to temporarily impede this

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ACCEPTED MANUSCRIPT oxidative burst, thereby allowing them to evade the effect of defense molecules such as

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phytoalexins. The mechanisms underlying this early inhibition of the oxidative burst remain

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unknown in S. sclerotiorum. In the present study, we assessed ROS content produced at the

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site of inoculation and showed that soybean plants respond strongly to the infection by

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releasing higher ROS levels in response to the WA isolate SS18. Inversely, the content of

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ROS in the leaves inoculated with the HA isolate SSPetri was as low as that of untreated and

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non-inoculated tissues. This reduction in ROS accumulation compromises the defence

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responses and leads to a higher infection rate and disease severity. Similar results were

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reported by Cessna et al. (2000) showing an inhibition of the production of H2O2 and a

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suppression of the oxidative burst in tobacco and soybean cell cultures challenged by the

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oxalic acid produced by a virulent isolate of S. sclerotiorum. Other studies found that the

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reducing conditions that S. sclerotiorum engenders during the initial stages of colonization,

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suppress host defense responses, including the oxidative burst and callose deposition

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(Williams et al. 2011). Once infection is established, ROS production is restored leading to

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oxidative burst and programmed cell death (Williams et al. 2011). This could be associated

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with the trophic behavior of S. sclerotiorum as a necrotroph at start, then turning into a

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hemibiotroph at a later stage.

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Calcium pre-treatment induced an increase in ROS accumulation a few hours after inoculation

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with the HA isolate SSPetri. This led to the restoration of the suppressed oxidative burst able

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to elicit back the host defense responses. Similar results were reported in response to the pre-

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treatment with thiamine or biological control agents in response to infection with the wild-

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type of S. sclerotiorum (Jain et al. 2013; Zhou et al. 2013). This is not surprising since

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calcium metabolism and ROS signaling are closely related. Ca2+ flux is known to operate both

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upstream and downstream ROS production. In addition, Ca2+ influx is indispensable for the

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initial ROS accumulation while ROS production is essential for later additional calcium

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fluxes in the cell subsequent to pathogen elicitation (Levine et al. 1996; Blume et al. 2000).

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In plants and fungi, oxalates play an important role in calcium regulation (Bush, 1995;

347

Franceschi et al. 2005) and their role in S. sclerotiorum pathogenesis is well established.

348

Calcium is a key factor acting as signal in many processes of plants, including growth,

349

response to both biotic and abiotic stress (Bush, 1995). In fungi, calcium gradients regulate

350

growth and the uptake of ions from their environment (Dutton and Evans, 1996; Lew, 2011).

351

However, free calcium may become a toxic cellular compound at high concentrations,

352

because it can build complexes with proteins, membranes, and organic acids. As a result of

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ACCEPTED MANUSCRIPT fungal infection, calcium concentrations usually increase due to cell wall lytic activities by the

354

growing hyphae and host cells’ autolysis.

355

Several genes encoding for ROS-scavenging enzymes are often up-regulated hours after

356

infection to protect the host cells from the damage due to elevated ROS content (Levine et al.

357

1994). This was the case in the current study, where most tested genes were up-regulated 6 to

358

24 hpi. Their profile of expression was differential among the WA and HA isolates. Higher

359

transcripts levels were recorded early in response the WA isolate SS18 while those recorded

360

in response to HA isolate SSPetri were delayed and weaker. This suggests a negative

361

correlation between the ROS-scavenging capacity of the host and the success of the pathogen

362

in invading the issues. It also highlights the ability of HA isolate SSPetri to suppress ROS

363

accumulation at levels high enough to induce the synthesis/accumulation of ROS-scavenging

364

transcripts. Sod is one of the main constituents of the ROS scavenging machinery of the plant

365

defense system (Bowler et al. 1992; El Hadrami et al. 2005). Numerous reports revealed an

366

increase in SOD activity in plants under oxidative stress (El Hadrami et al. 2005; Nanda et al.

367

2010; Malencic et al. 2010; Morita et al. 2012). Our results showed a peak of accumulation of

368

Sodb2 transcripts as early as 6 hpi in response to infection with either SS18 or SSPetri,

369

followed by a decrease almost within 24 hpi after infection, which reflects the enzymes

370

involvement in the detoxification of O2-. This was confirmed with a positive correlation with

371

the O2- accumulation in the tissues. Inversely, lower levels of expression of Sodb2 along with

372

low amounts of O2- were detected in response to SSPetri, indicative of a suppression of the

373

oxidative burst and a surreptitiousness behavior of this HA isolate. Calcium pre-treatment did

374

not significantly induce any changes in sodb2 expression after infection with the HA isolate

375

SSPetri. These results suggest that the ROS scavenging mechanism of the HA isolate would

376

have been countered by one or more toxic metabolites produced by S. sclerotiorum during

377

pathogen invasion. Comparable observations were reported in B. napus exogenously supplied

378

with oxalic acid and showing a suppressed SOD activity (Liang et al. 2009).

379

The activation of Apx1 and Prx2b keeps the balance of cellular H2O2 in response to fungal

380

infection. Both genes were induced after infection with WA isolate SS18. However, the

381

expression of the Apx1 gene was stronger, suggesting that Apx1 was more efficient in

382

destroying H2O2 than Prx2b. Increase in Apx1 level was seen after the increase of Sodb2 (24

383

hpi) in response to both tested isolates whereas the increase in Prx2b transcripts was recorded

384

in response to WA isolate SS18 as early as 6 hpi and at the same time as Sod. These results

385

indicate that these two enzymes have different affinities for H2O2, confirming their belonging

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ACCEPTED MANUSCRIPT to separate classes of H2O2-scavenging enzymes (Mittler et al. 2004; Dietz et al. 2006; Foyer

387

and shigeoka, 2010; Maruta et al. 2012, Arfaoui et al. 2013, 2014).

388

Pre-treatment with calcium increased the transcription of Apx1, Prx2b and Thioredoxin in

389

plants infected with the HA isolate SSPetri. This may be explained by the fact that the

390

calcium provided the plants with better elicitation of stress response. These results corroborate

391

reports showing the involvement of three biological control agents Pseudomonas aeruginosa,

392

Trichoderma harzianum and Bacillus subtilis in inducing changes in H2O2 and antioxidant

393

metabolism of peas challenged by S. sclerotiorum (Jain et al. 2013).

394

In light of the results of the present study, pre-treatment with calcium enhances plant

395

protection against oxalic acid toxicity and triggers oxidative stress and the antioxidant

396

systems in a balanced form to keep the alert system active without causing structural damage

397

to the tissues or the cell components. We demonstrated that intervening for the establishment

398

of an initial “reducing” status is critical in hampering the pathogenesis of S. sclerotiorum. At

399

the early stage of infection, calcium could effectively restore the suppressed oxidative burst,

400

essential to the signaling cascades to alert healthy tissues remotely from the infection site.

401

Further research on the mode of action of calcium and mechanisms of interaction with oxalic

402

acid would greatly assist the control of the white mold disease in many crops including

403

soybeans. Intensive efforts using fungal mutants that are compromised in Ca2+ efflux or

404

virulence factors and plant genotypes with various degrees of resistance to white mold will

405

help understanding the basis of calcium-plant-pathogen interactions. Given the importance of

406

oxalic acid and its interaction with calcium, these results suggest that foliar application of

407

calcium may constitute a new approach to control white/soft mold in field crops and

408

vegetables.

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Acknowledgement

411

This research was supported by funding from Manitoba Rural Adaptation Council (MRAC)

412

and OMEX to Dr. Abdelbasset El Hadrami (MRAC-CAAP-MB0396) and from NSERC to

413

Fouad Daayf.

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3261-3272

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ACCEPTED MANUSCRIPT 585 Table 1. Primers used in RT–qPCR for selectively amplifying ROS genes in soybean. Target gene and

Forward and reverse primer sequence (5′–

PCR

Primer

Genbank accession

primer name

3′)

product

Tm

No. for target gene

size (bp)

(ºC)†

66

62.18

Β-Actin

F: 5′-CAATCCCAAGGCCAACAGA-3′ R: 5′-ATGGCAGGCACATTGAAAGTC-3′

Sodb2

F: 5'-GCAACACAATTTGGTTCAGG-3'

62.23

82

R: 5'-AAGGAGGATTTGCTGCATTT-3' Apx1

F: 5'-TCACGGAGTTGTTGAGTGGT-3'

141

R: 5'-GTTCCTGAGCCAGGAGAAAG-3' Thioredoxin

F: 5'-CAAATTCATAGAGCCAGCGA-3'

587

o

†Tm: primers pair melting temperature ( C)

588 589 590

594 595 596 597 598 599 600 601 602

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603 604 605 606 607 19

NM_001250972.1

NM_001250856.1

58.67

99

59.08

AF145348.1

59.01

115

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R: 5'-CGCCTCCACATTAAACTCCT-3'

59.15

SC

F: 5'-TACAAGGAATCATGCCCTCA-3'

59.02

58.79

R: 5'-CCTCAGCGTAATCAGCAAAG-3’ Prx2b

AW350943

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586

59.35

59.05

NM_001250511.1

ACCEPTED MANUSCRIPT 608 609

Table 2. Oxalic acid concentration released by Seclerotinia isolates in PDB medium amended

610

or not with 0.1% (V/V) of calcium. Oxalic acid (µg oxalic acid mg-1 dry weight mycelium)

Treatments

7 days

SSPetri

247.69a±5.3

286.8a±15.7

SS18

126.80c±9.4

163.69c±8.0

SSPetri+Ca

213.24b±9.4

254.80b±10.3

SS18+Ca

110.36c±8.3

136.13c±7.0

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3 days

Each data point represents the average for three independent experiments with error bars

612

representing the standard error to the mean. Letters indicate significant differences among

613

treatments according to Duncan test at P < 0.05.

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ACCEPTED MANUSCRIPT 636 Figure captions

638

Fig. 1 Aggressiveness of the weakly (SS18) and the highly (SSPetri) aggressive isolates of S.

639

sclerotiorum in absence/presence of calcium.

640

(A) Lesion development on soybean leaves. Photographs were taken at 120 hours post-

641

inoculation.

642

(B) Relative lesion area on soybean leaves. Lesions area values were analyzed using the

643

lesion assay software ASSESS 2.2. Each data point represents the average for three

644

independent experiments with error bars representing the standard error to the mean. Letters

645

indicate significant differences among treatments according to Duncan test at P < 0.05.

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Fig. 2 Oxalic acid content in soybean leaves. Soybean leaves were inoculated with a PDA

648

plug (5 mm in diameter) colonized with a weakly aggressive isolate (SS18) or a highly

649

aggressive isolate (SSPetri) of S. sclerotiorum in either absence or presence of calcium.

650

Each data point represents the average for three independent experiments with error bars

651

representing the standard error to the mean. Letters indicate significant differences among

652

treatments according to Duncan test at P < 0.05.

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Fig. 3 DAB and NBT staining to assess H2O2 and O2- production (arrows) in soybean leaves

655

in the different treatments: Control; SS18, SSPetri, Control+Ca, SS18+Ca and SSPetri+Ca.

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Fig. 4 Time course of generation of H2O2, O-2 and OH. during infection of soybeans leaves

658

with the weakly (SS18) or the highly (SSPetri) aggressive S. sclerotiorum isolates in absence

659

or presence of calcium. Letters indicate significant differences among treatments according to

660

Duncan test at P < 0.05. Each data point represents the average for three independent

661

replicates with error bars representing the standard errors to the means.

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Fig. 5 Relative gene expression of target genes during the infection of soybean leaves with the

664

weakly (SS18) or the highly (SSPetri) aggressive S. sclerotiorum isolates in absence or

665

presence of calcium. Letters indicate significant differences among treatments according to

666

Duncan test at P < 0.05. Each data point represents the average for three independent

667

replicates with error bars representing the standard errors to the means.

668 669 21

ACCEPTED MANUSCRIPT

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Fig. 3

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Fig. 5

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Highlights Foliar application of calcium contributes to disease reduction. In presence of calcium, OA decreased in leaves inoculated with S. sclerotiorum. Soybean inoculation with an aggressive isolate of S. sclerotiorum induced less ROS

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accumulation.

Pre-treatment with calcium restored ROS production in plants inoculated with S. sclerotiorum. Expression of genes involved in ROS detoxification increased in response to a weakly

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aggressive isolate of S. sclerotiorum.

ACCEPTED MANUSCRIPT

Contributions

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All three authors contributed to the planning and experimental design of the studies. AA run the experiments, collected the data, and performed analysis and interpretation. AE and FD also contributed to the data analysis and interpretation. All three authors contributed to the writing of the manuscript. AE and FD co-supervised the work.