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Pre- and postharvest application of alternative means to control Alternaria Brown spot of citrus
Accepted Manuscript Pre- and postharvest application of alternative means to control Alternaria Brown spot of citrus Francesca Garganese, Simona Marianna Sanzani, Danila Di Rella, Leonardo Schena, Antonio Ippolito PII:
S0261-2194(19)30094-8
DOI:
https://doi.org/10.1016/j.cropro.2019.03.014
Reference:
JCRP 4766
To appear in:
Crop Protection
Received Date: 21 June 2018 Revised Date:
5 February 2019
Accepted Date: 21 March 2019
Please cite this article as: Garganese, F., Sanzani, S.M., Di Rella, D., Schena, L., Ippolito, A., Pre- and postharvest application of alternative means to control Alternaria Brown spot of citrus, Crop Protection (2019), doi: https://doi.org/10.1016/j.cropro.2019.03.014. 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- AND POSTHARVEST APPLICATION OF ALTERNATIVE MEANS TO CONTROL
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ALTERNARIA BROWN SPOT OF CITRUS
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Francesca Garganese1, Simona Marianna Sanzani1*, Danila Di Rella1, Leonardo Schena2,
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Antonio Ippolito1
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Aldo Moro, Via Amendola 165/A, 70126 Bari Italy.
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Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito,
89122 Reggio Calabria, Italy.
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*Corresponding author: [email protected]
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Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari
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ACCEPTED MANUSCRIPT Abstract
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Alternaria brown spot (ABS) is one of the main diseases of mandarins and their hybrids. Its control
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is based on fungicide applications, but their use is facing difficulties related to the development of
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resistant pathogen strains and adverse effects on animals, humans and the environment. The present
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study was undertaken to find feasible alternative solutions to control ABS, by testing the
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effectiveness of phenolic compounds and alternative formulations present on the market. They were
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tested in vitro and in vivo on a laboratory scale and then in semi-commercial conditions.
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Umbelliferone was the most effective phenolic compound in controlling by 86% ABS on mandarins
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cv. Fortune in semi-commercial conditions. Its effect seemed related to the ability of inducing
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resistance in the host.Whereas among the formulations, chitosan-based HendophytPS and salt-based
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FortisolCaPlus gave the best results in field-scale trials, with a reduction of disease incidence by 59-
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63 and 54-75% on fruit and leaves, respectively, as compared to the control, being the effect similar
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to that obtained by chemical controls. The formulations showed mainly to induce host resistance.
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The application of proposed alternative treatments, singly or in a combined strategy, might
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represent an interesting substitute to synthetic fungicides against ABS.
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Keywords: Alternaria alternata, citrus, salts, chitosan, phenolic compounds.
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1. Introduction
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Alternaria brown spot (ABS) is a highly destructive disease of mandarins (Citrus reticulata Blanco)
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and their hybrids (Akimitsu et al., 2003), whereas, satsumas (Citrus unshiu Mark. Marc.), and
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clementines (Citrus clementina Hort. ex Tan.) seem resistant at some extent. The causal agent is a
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pathotype of Alternaria alternata (Fr.) Keissl., which is prevalent in humid citrus-production areas
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as Florida, Brazil, Argentina, Colombia, Cuba, Peru, and China, but also in Mediterranean areas
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with cool, moist winters and hot, dry summers as Spain, Italy, Greece, Turkey, and Israel (Bassimba
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et al., 2014). In Italy, the disease was first reported by Bella et al. (2001), and recently by Garganese
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ACCEPTED MANUSCRIPT et al. (2016). Temperatures of 25-27ºC with at least 10 h of wetness are optimal for infection.
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Conidia are produced on the lesion surface, and subsequently released by rainfalls (Timmer et al.,
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1998). The pathogen causes brown to black spots on young leaves and fruit, and twig dieback.
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Under optimal conditions, symptoms may appear within 24 h after infection (Timmer et al., 2003).
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In addition to a decrease in plant productivity, ABS symptoms cause loose of commercial value of
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fruit.
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Where allowed, fungicides (e.g. dithiocarbamates, dicarboximides, strobilurins, and conazoles) are
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used for ABS control (Feichtenberger et al., 2005). Copper is also recommended in many countries
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(Timmer et al., 2000; Vicent et al., 2004). However, the management of ABS is particularly
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challenging in presence of recurrent rains and jumps in relative humidity (Timmer et al., 1998). In
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Italy, the containment of the disease is critical, and the number of needed treatments may change
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according to the weather conditions year by year. In a study conducted by Bella et al. (2012) during
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2005-2011, two scheduled fungicide applications (late summer and in the fall) resulted inadequate
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to control ABS. Copper and, from late 2015, mancozeb and pyraclostrobin are the only fungicides
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allowed in Italy in conventional/integrated production, being the sole copper allowed in organic
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agriculture (http://m.bdfup.it/bdfup/). Mancozeb and pyraclostrobin belong to a family of fungicides
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suspected for human health hazard (Bolognesi, 2003; Çayır et al., 2014). Moreover, the use of
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metallic copper in organic agriculture is limited to 6 kg per ha per year (European Commission
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Regulation N. 889/2008), and following the recommended spray schedules for citrus, this threshold
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is easily overcome (Vicent et al., 2009). Therefore, the search for safer and effective alternatives is
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gaining attention. Among them, 2-6% organic and inorganic salts were tested on a wide range of
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crops including citrus (Palou et al., 2008; Romanazzi et al., 2012; Wisniewski et al., 2016). Most of
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them belong to the GRAS (Generally Regarded as Safe) category by the US Food and Drug
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Administration (FDA) (Palou et al., 2016). Salts have been investigated both as pre- (Nigro et al.,
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2006; Teixidó et al., 2010; Youssef et al., 2012) and postharvest treatments (Youssef et al., 2012,
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2014; Montesinos-Herrero et al., 2016). Phosphite salts proved to both induce host resistance and
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ACCEPTED MANUSCRIPT control mycelial growth of oomycetes (Lobato et al., 2008; Thao and Yamakawa, 2009; Olivieri et
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al., 2012). Although marketed as fertilizers and plant strengtheners, they proved to be potential
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alternatives to conventional fungicides (Lobato et al., 2008; Olivieri et al., 2012) including citrus
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(Panabieres et al., 2016). Phosphites can have different application strategies according to the crop
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and the pathogen, but foliar spray is the commonest (Deliopoulos et al., 2010). Salts have been
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introduced in the market as formulations. For instance, FortisolCaPlus, a water-soluble mix of Ca,
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K and P, showed a protection against citrus postharvest disorders and fungicide-related
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phytotoxicity (Parra et al., 2014; Martínez-Hernández et al., 2017). Similarly, the combined pre-
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and postharvest application of DeccoPhosk, a potassium phosphite formulation, was effective
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against green and blue mold of lemons, as well as chilling injury and aging (Strano et al., 2015).
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Furthermore, natural compounds of animal or plant origin showed good control performance of
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postharvest diseases. For example, chitosan, a linear polysaccharide from crustacean shells, proved
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to control numerous pre- and postharvest diseases on several vegetables and fruits (Romanazzi et
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al., 2003; Bautista-Baños et al., 2016; Palou et al., 2016) including green mold on citrus
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(Panebianco et al., 2014). Indeed, chitosan has direct antimicrobial activity, and, at the same time,
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triggers resistance mechanisms (El Hadrami et al., 2010). Among compounds of plant origin,
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phenolic substances are considered of a certain interest from a disease control perspective. They
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occur constitutively or are formed in response to pathogen attack (Jeandet et al., 2013).
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Accumulation of quercetin, umbelliferone, scopoletin, and scoparone in plant tissues, together with
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their antioxidant and antifungal properties (Sourivong et al., 2007; Sanzani et al., 2009, 2014) make
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them good “natural pesticide” candidates to increase the resistance of plants to fungal infections
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(Sanzani et al., 2010).
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The aim of this study was to evaluate the effectiveness of selected phenolic compounds and
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alternative formulations against ABS, by testing their effectiveness in laboratory- and semi-
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commercial conditions.
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2. Materials and methods
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2.1 Phenolic compounds and alternative formulations
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The
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trihydroxyflavanone
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umbelliferone (7-Hydroxycoumarin) with a purity >95% were purchased from Sigma-Aldrich
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(Milan, Italy). Stock solutions of pure standards were prepared in a phosphate buffer (Sanzani et al.,
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2009) at a concentration of 50 mg/ml.
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The tested alternative formulations were Fortisol®CaPlus (water-soluble mix of Ca, K and P,
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Citrosol, Valencia, Spain), DeccoPhosk® (potassium phosphite, Decco Italia, Belpasso, Italy), and
compounds
hesperidin
quercetin
naringin
(4′,5,7-
(3,3',4',5,7-pentahydroxyflavone),
and
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(hesperetin7-rhamnoglucoside),
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phenolic
Hendophyt®PS (chitosan, Iko-Hydro Ltd., Rutigliano, Italy).
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The fungicides Ossiclor (copper oxychloride, 50% w/w, Manica, Rovereto, Italy) and CabrioWG
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(Pyraclostrobin, 20% w/w, BASF, Faenza, Italy) were used as positive controls at 175 g/hl and
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0.06g/hl a.i., respectively.
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2.2 Alternaria alternata conidial suspension
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A. alternata morphotype alternata strain A23, morphologically and molecularly identified
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(Garganese et al., 2016), and deposited in the “Fungal Culture Collection” of the Department of
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Soil, Plant, and Food Sciences, University of Bari Aldo Moro (Italy), was cultured on PDA (Conda,
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Spain) plates for 7 days at 24±1°C. To produce inoculum, colony surface was washed with 5 ml of
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a 0.05% (v/v) Tween 80 aqueous solution. The obtained suspension was filtered through a double
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layer of sterile gauze and counted by a Thoma counting chamber (HGB Henneberg-Sander GmbH,
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Lutzellinden, Germany). A 105 conidia/ml suspension was used for all in vitro and in vivo trials.
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2.3 In vitro assays
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The effect of selected treatments on the mycelial growth of A. alternata was determined on
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amended PDA. Briefly, a solution of each single compound/formulation was 0.22-µm filtered and
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µg/ml (100 and 1000 µg/dish, respectively) for the phenolic compounds, and 0.5, 0.5, and 1% (w/v)
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for Fortisol, DeccoPhosk, and Hendophyt, respectively. Dishes were seeded centrally with a 5 mm
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mycelial plug of the pathogen. Non-amended PDA plates were used as a control. For each
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treatment/concentration, five dishes were used as replicates. Colony growth (average of the two
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orthogonal diameters, mm) was recorded at 7 days post-inoculation (DPI). The experiment was
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repeated twice.
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2.4 Laboratory in vivo assays
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2.4.1 Citrus samples
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Mandarins cv. Fortune, a late ripening variety, were harvested at physiological maturity in a
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conventional farm located in the Bernalda area (Basilicata, Southern Italy). Fruit were uniform in
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size, colour, and ripeness and free of defects or injuries. The selected fruit were randomized,
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surface-sterilized in a 2% sodium hypochlorite solution, washed in tap water and left to dry at room
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temperature.
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2.4.2. Testing of direct antifungal activity
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Fruit were wounded twice (3 mm wide × 3 mm depth) with a sterile nail at opposite sides in the
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area between the peduncle and the equatorial axis. Then, 1000 µg/wound of phenolic solutions, or
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0.5% Fortisol/DeccoPhosk, or 1% Hendophyt were pipetted into each wound. Fruit whose wounds
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were treated with phenolic solving buffer or water served as controls. After drying, the same
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wounds were inoculated by 10 µl of A. alternata conidial suspension. Each treatment had three
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replicates, consisting of six mandarins each (18 fruit/36 wounds per treatment). Replicates were
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incubated at 24±1°C and 95-98% RH for 14 days. Disease incidence (infected wounds, %) and
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severity (lesion diameter, mm) were assessed at 14 DPI. The trial was performed twice.
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2.4.3 Testing of products as resistance inducers
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ACCEPTED MANUSCRIPT Surface-sterilized fruit were wounded and treated as reported above. Fruit amended with phenolic
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solving buffer or water were used as controls. After an incubation of 24 h at 24±1°C and 95-98%
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RH, another round of wound was made on each fruit about 5 mm from the previous one and
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inoculated with A. alternata conidial suspension (10 µl). Each treatment was made of three
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replicates with six fruit each (18 fruit/36 wounds per treatment). Replicates were incubated as
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reported above. Incidence of decay and disease severity were assessed at the end of incubation. The
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entire experiment was repeated twice.
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2.5 Shelf-life experiments
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The phenolic compounds were further tested as postharvest treatments on the freshly harvested
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mandarins cv. Fortune against natural infections by Alternaria spp. Briefly, 1000 µg of each
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selected compound was pipetted into each wound (two per fruit) as reported above. Fruit were
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incubated at 24±1°C and 95-98%. Each treatment comprised three replicates with twelve fruit each
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(36 fruit/72 wounds per treatment). At 14 DPI, disease incidence was assessed as previously
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reported. The entire experiment was repeated twice.
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2.6 In the field experiments
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Field trials with formulates were conducted in 2014 and 2015 by monthly treatments, from May to
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December. The experimental tests were conducted in an orchard of mandarin plants cv. Fortune,
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located in Pisticci area (Basilicata, Southern Italy). The plants were 8-year-old, grafted on Troyer
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citrange, and arranged in a completely randomized block design with 4 plots of 3 plants each. Plants
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were selected for canopy and fruit set homogeneity and lack of disorders. There were two-plant
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buffers between plots to reduce spray drift. A spray lance, powered by a walking-type motor pump,
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was used to apply substances at 20 atm into the canopy at a rate of 5 l/plant (20 hl/ha),
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approximately. The same number of plots was treated by the fungicides Ossiclor and CabrioWG as
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positive controls. Each year, a month after the last treatment, a survey was carried out before
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harvesting to assess the disease on leaves and fruit, randomly choosing 10 twigs and 10 fruit per
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plant, 120 fruit/twigs per treatment, and counting the number of A. alternata lesions on five leaves
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per twig and on each fruit surface, respectively.
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2.7 Data analysis
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The statistical software package Statistics for Windows (StatSoft, Tulsa, OK, USA) was used.
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Percentage data of incidence of decay were arcsine-square-root transformed before ANOVA
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analysis. In case of homogeneity of variance, data from repeated experiments were combined.
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Significant differences (P≤0.05) were identified by the General Linear Model (GLM) procedure
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with the Duncan’s Multiple Range Test (DMRT). A control index (CI) expressed the extent of the
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control exerted by treatments (Sanzani et al., 2009).
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3. Results
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3.1 In vitro assay
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None of the phenolic compounds at both tested concentrations reduced A. alternata growth (Table
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1). Whereas, the three formulations significantly inhibited colony diameter (Table 1). Hendophyt
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provided the best results, inhibiting the growth of the colonies by 93%, followed by Fortisol and
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DeccoPhosk with a reduction of 80 and 77%, respectively.
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3.2 Laboratory in vivo assays
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3.2.1 Testing of direct antifungal activity
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The incidence and severity of the decay on fruit treated by phenolic compounds and inoculated by
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A. alternata in the same wound are reported in Figure 1A. Although none of the tested phenolic
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compounds reduced disease incidence, quercetin and hesperidin proved to decrease disease severity
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by 53 and 32%, respectively, as compared to the control.
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58%) and severity (-45%) as compared to the control (Fig. 1B). Whereas DeccoPhosk reduced only
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disease severity (-57%). Hendophyt did not influence directly the disease development.
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3.2.2. Testing of products as resistance inducers
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In absence of a direct contact between treatment and pathogen, umbelliferone was the only phenolic
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compound able to reduce both disease incidence and severity by 47 and 90%, respectively, as
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compared to the control (Fig. 2A). Whereas, quercetin and naringin reduced only the severity by 74
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and 73%, respectively. Concerning the formulations, Hendophyt and Fortisol reduced disease
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incidence by 40 and 47%, respectively. In Hendophyt-treated fruit even the disease severity was
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reduced by 42% (Fig. 2B).
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3.3 Shelf-life experiments
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Phenolic compounds were tested against Alternaria natural infections on harvested mandarin fruit at
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concentration 1000 µg/wound. Quercetin and umbelliferone reduced disease incidence by 35 and
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86%, respectively as compared to the control (Fig. 3).
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3.4 In the field experiments
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In the field, the incidence of disease was recorded on citrus leaves and fruit treated from May to
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December each year in two consecutive years. Hendophyt and Fortisol were the most effective
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alternative treatments (Fig. 4). Compared to the control, the number of lesions on leaves and fruit
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treated with Hendophyt and Fortisol were reduced by 63 and 75%, and by 59 and 54%, respectively
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(Fig. 4A-B). DeccoPhosk was not effective in controlling lesion appearance on both tissues.
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Furthermore, no phytotoxicity was observed on fruit and leaves treated by the alternative
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formulations. Finally, Hendophyt and Fortisol results were similar to those obtained by fungicide
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treatments, with a reduction ranging between 89 and 92% for fruit, and 88 and 78% for leaves by
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Ossiclor and CabrioWG, respectively.
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4. Discussion
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ABS is a severe disease on susceptible mandarin and mandarin-like cultivars, so that their
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cultivation in absence of an effective control strategy might be unprofitable (Peres and Timmer,
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2006; Vicent et al., 2009). Thus in several citrus-producing countries, chemical fungicide sprays are
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scheduled intensively during the growing season. However, the risks of residues on the product and
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in the environment has increased the demand for alternative substances less harmful for humans and
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animals (Li Destri Nicosia et al., 2016). Last alternative control strategies for pre- and postharvest
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decays are based on products with modes of action different from traditional fungicidal active
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ingredients, but also with multiple activities, e.g. direct effect plus induction of resistance
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(Wisniewski et al., 2016). Recently, the use of elicitors of either biotic or abiotic source to enhance
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host defenses has gained attention as approach of choice in integrated strategies to control
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postharvest diseases (Romanazzi et al., 2016). Following ripening, disease resistance of harvested
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fruit usually declines, increasing host susceptibility to pathogen attack. Thus, resistance inducers
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may be useful to reinstate a suitable level of resistance (Pangallo et al., 2017). Phenolic compounds
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are antimicrobial compounds produced by plants that accumulate or appear de novo in response to
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cellular damage; their production/increase in infected tissues is recognized as one of the main
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resistance mechanisms in plants (Lachhab et al., 2014). Their induction in challenged plant tissue or
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exogenous application have been proposed as alternative control approaches against blue and green
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mold of apple and citrus fruits, respectively (Afek et al., 1999; Sanzani et al., 2009, 2014). In the
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present investigation, a selection of phenolic compounds was applied against ABS. At the tested
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concentrations, they did not show a direct effect in vitro against the growth of Alternaria. Similar
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results were obtained, as far as quercetin and umbelliferone concern, against P. expansum (Sanzani
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et al., 2009) and P. digitatum (Sanzani et al., 2014). Whereas, on Alternaria-inoculated fruit,
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quercetin and hesperidin applied in the same wound inoculated by the pathogen proved to reduce
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disease severity as compared to the control. Overall, tested phenolics seemed to be more efficient in
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ACCEPTED MANUSCRIPT vivo than in vitro, in agreement with Sanzani et al. (2009, 2014). Better disease control results were
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obtained when phenolics were applied as resistance inducers. Indeed, all tested phenolics controlled
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disease severity, with umbelliferone reducing even ABS incidence. Thus, they seemed to exert their
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control effect indirectly. Several possible mechanisms might be involved: activation of host natural
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defenses including biochemical (e.g. PR-proteins) and physical (e.g. lignification) barriers;
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accumulation with endogenous phenolics, thus reaching a fungitoxic concentration; chemical
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interaction with pre-existing compounds, thus forming new toxic molecules (Sanzani et al., 2009).
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Quercetin ability to induce resistance phenomena in apples has been reported (Sanzani et al., 2010)
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as well as that of umbelliferone, which played a role in defense mechanisms of unripe reactive
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grapefruit against wound-pathogens as P. digitatum (Afek et al., 1999). Overall, in the present
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investigation Alternaria seemed less susceptible to treatments than other genera as Penicillium
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(Sanzani et al., 2009, 2014). This finding might be ascribed to the intense melanization of
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Alternaria structure, since melanin contributes to virulence of melanized microorganisms by
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decreasing their susceptibility to host defense mechanisms (Nosanchuk and Casadevall, 2003).
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Umbelliferone confirmed its efficacy even in shelf-life trials, and thus, although further larger scale
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assays are needed, this compound might be considered a good candidate to be applied after harvest
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within an ABS integrated control strategy, made up of pre- and postharvest application of
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alternative means, to prevent losses of citrus fruit.
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Furthermore, in this study some commercial alternative formulations commercialized as citrus
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biostimulants were tested before harvest. In in vitro assays, they all proved to control Alternaria
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growth. However, when applied in vivo on fruit, only Fortisol, formulate based on sodium, calcium,
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and phosphorus salts, reduced disease incidence and severity. Furthermore, it proved to act even as
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resistance inducer, together with Hendophyt, a chitosan-based formulate. When applied preharvest
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monthly on fruit and leaves of mandarin cv. Fortune, these two formulations confirmed their
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reduction of the disease incidence on both organs. Chitosan is known to have multiple activities.
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For example, it can penetrate the plasma membrane and eventually kill the pathogen cells, as
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ACCEPTED MANUSCRIPT observed for Neurospora crassa (Palma-Guerrero et al., 2009). Furthermore, chitosan forms a
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physical barrier around the infection sites, preventing pathogen spread to healthy tissues (Bittelli et
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al., 2001). In numerous studies chitosan proved to prevent spore germination, germ tube
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elongation, and mycelial growth of pathogens as Botrytis cinerea (Monjil et al., 2013;
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Arasimowicz-Jelonek et al., 2014), Fusarium solani (Li et al., 2014), Rhizopus stolonifer (Monjil
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et al., 2013), Penicillium spp. (Olivieri et al., 2009; Arasimowicz-Jelonek et al., 2014),
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and Sclerotium rolfsii (Li et al., 2014). However, it is frequently used as a potent elicitor for
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controlling plant diseases rather than as antimicrobial agent (Romanazzi et al., 2017). Indeed,
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chitosan can chelate nutrients and minerals (i.e., Fe, Cu), and bind mycotoxins (Bornet et al., 2007),
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which may contribute in reducing damage of host tissues (Sanzani et al., 2012). The control activity
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of salt-based formulate Fortisol was remarkable too. Salts proved to have a multiple mode of action
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based not only on direct activity against fungi (Fallanaj et al., 2016), but also on resistance
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induction in treated tissues (Youssef et al., 2014). The activity of Fortisol and Hendophyt was equal
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or similar to that of chemical fungicides on leaves and fruit, respectively. In our study, the efficacy
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of copper and pyraclostrobin was analogous to that reported in literature (Vicent et al., 2007). The
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monthly application of treatments was scheduled based on data according which copper compounds
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can provide protection for around 1 month under rain-free weather conditions (Vicent et al., 2009).
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However, copper compounds, especially in summertime and as repeated sprays may become
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phytotoxic on citrus fruit (Albrigo et al., 1997). Thus, reducing copper applications would lessen the
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phytotoxicity hazard. To this aim, pyraclostrobin is applied efficiently at beginning of the infection
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season, when fruit and leaves are small. However, since chemical fungicides are not free from
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drawbacks, an integrated pest management system with alternative formulations as Fortisol and
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Hendophyt could maintain a similar level of control without side effects. At this regard, the
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development and validation of predictive models that can estimate the real risk of infection and
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identify the optimal time for treatments according to the phenological stages of crop and climatic
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conditions encountered in the field might be particularly useful (Peres and Timmer, 2006).
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5. Conclusions
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In order to safeguard the quality and commercial value of the productions of mandarins cv. Fortune
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and others mandarins, it is necessary to widen the range of products approved for controlling ABS
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and to develop effective control programs.The good efficacy of alternative treatments, alongside the
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possibility to obtain healthier products, makes them a viable option to fungicides; the use of which,
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currently, has been reduced greatly for issues related to the presence of residues, operator safety,
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environmental pollution, and restrictions provided by the national legislations. The alternative
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products are gaining wide acceptance among consumers and operators, and as such might represent
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a valid mean to be used.
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Afek, U., Orenstein, J., Carmeli, S., Rodov, V., Joseph, M.B., 1999. Umbelliferone, a phytoalexin
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use in timing fungicide applications. Plant Dis. 84, 638-643.
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6.
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Biol. Technol. 87, 61-69.
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ACCEPTED MANUSCRIPT Table 1. Alternaria alternata colony diameter (mm) on PDA amended with phenolic compounds
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(100 and 1000 µg/dish), and 0.5, 0.5, and 1% (w/v) Fortisol, DeccoPhosk, and Hendophyt. Dishes
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were incubated at 24±1°C for 7 days in the dark.
Control Quercetin_1000 Hesperidin_1000 Naringin_1000 Umbelliferone_1000
65.3 ± 5.4 63.0 ± 4.0 63.0 ± 1.9 69.0 ± 3.4 59.5 ± 4.3
Control DeccoPhosk Fortisol Hendophyt
57.9 ± 4.3 13.1 ± 2.3 11.4 ± 1.0 4.8 ± 1.0
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Colony diameter± SEM 48.8 ± 4.5 50.5 ± 1.9 42.5 ± 5.7 53.9 ± 2.4 45.5 ± 5.1
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Treatments Control Quercetin_100 Hesperidin_100 Naringin_100 Umbelliferone_100
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Caption to figure
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Fig.1. Disease incidence (infected wounds, %) and severity (lesion diameter, mm) on harvested
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citrus fruit wound-treated by A) different phenolic compounds (1000 µg/wound) or B)
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DeccoPhosk/Fortisol (0.5%) and Hendophyt (1%), and inoculated by Alternaria alternata. Water-
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or buffer-treated fruit were used as a control. Data were recorded after 14 days of incubation at
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24±1ºC and 95-98% RH. Bars represent the standard error of the mean (SEM). For each parameter,
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columns with different letters are statistically different according to DMRT (p≤ 0.05).
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Fig.2. Disease incidence (infected wounds, %) and severity (lesion diameter, mm) on harvested
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citrus fruit wound-treated by A) different phenolic compounds (1000 µg/wound) or B)
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DeccoPhosk/Fortisol (0.5%) and Hendophyt (1%), and after 24 h inoculated by Alternaria alternata
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in different but close wounds. Water- or buffer-treated fruit were used as a control. Data were
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recorded after 14 days of incubation at 24±1ºC and 95-98% RH. Bars represent the standard error of
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the mean (SEM). For each parameter, columns with different letters are statistically different
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according to DMRT (p≤ 0.05).
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Fig.3. Disease incidence (infected wounds, %) on harvested citrus fruit wound-treated by different
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phenolic compounds (1000 µg/wound) against Alternaria natural infections. Bars represent the
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standard error of the mean (SEM). Columns with different letters are statistically different
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according to DMRT (p≤ 0.05).
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Fig. 4. Disease incidence (number lesions) on citrus leaves (A) and fruit (B) monthly treated in the
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field by alternative products DeccoPhosk and Fortisol (0.5%), and Hendophyt (1%) and two
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chemical fungicides Ossiclor and CabrioWG (commercial dose). Water treated samples were used
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statistically different according to DMRT (p≤ 0.05).
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infected wounds
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ACCEPTED MANUSCRIPT Highlights
• We tested phenolic compounds and alternative formulations against Alternaria brown spot. • In vitro and in vivo tests on a laboratory scale and semi -commercial conditions were performed.
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• Umbelliferone was the most effective phenolic compound; it seemed to induce host resistance. • Hendophyt and Fortisol were the most effective formulations, comparable to chemical controls.
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• These alternatives, singly or in a combined strategy, might be effective against the disease.
S0261-2194(19)30094-8
DOI:
https://doi.org/10.1016/j.cropro.2019.03.014
Reference:
JCRP 4766
To appear in:
Crop Protection
Received Date: 21 June 2018 Revised Date:
5 February 2019
Accepted Date: 21 March 2019
Please cite this article as: Garganese, F., Sanzani, S.M., Di Rella, D., Schena, L., Ippolito, A., Pre- and postharvest application of alternative means to control Alternaria Brown spot of citrus, Crop Protection (2019), doi: https://doi.org/10.1016/j.cropro.2019.03.014. 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- AND POSTHARVEST APPLICATION OF ALTERNATIVE MEANS TO CONTROL
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ALTERNARIA BROWN SPOT OF CITRUS
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Francesca Garganese1, Simona Marianna Sanzani1*, Danila Di Rella1, Leonardo Schena2,
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Antonio Ippolito1
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Aldo Moro, Via Amendola 165/A, 70126 Bari Italy.
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Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito,
89122 Reggio Calabria, Italy.
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*Corresponding author: [email protected]
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Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari
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ACCEPTED MANUSCRIPT Abstract
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Alternaria brown spot (ABS) is one of the main diseases of mandarins and their hybrids. Its control
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is based on fungicide applications, but their use is facing difficulties related to the development of
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resistant pathogen strains and adverse effects on animals, humans and the environment. The present
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study was undertaken to find feasible alternative solutions to control ABS, by testing the
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effectiveness of phenolic compounds and alternative formulations present on the market. They were
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tested in vitro and in vivo on a laboratory scale and then in semi-commercial conditions.
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Umbelliferone was the most effective phenolic compound in controlling by 86% ABS on mandarins
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cv. Fortune in semi-commercial conditions. Its effect seemed related to the ability of inducing
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resistance in the host.Whereas among the formulations, chitosan-based HendophytPS and salt-based
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FortisolCaPlus gave the best results in field-scale trials, with a reduction of disease incidence by 59-
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63 and 54-75% on fruit and leaves, respectively, as compared to the control, being the effect similar
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to that obtained by chemical controls. The formulations showed mainly to induce host resistance.
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The application of proposed alternative treatments, singly or in a combined strategy, might
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represent an interesting substitute to synthetic fungicides against ABS.
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Keywords: Alternaria alternata, citrus, salts, chitosan, phenolic compounds.
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1. Introduction
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Alternaria brown spot (ABS) is a highly destructive disease of mandarins (Citrus reticulata Blanco)
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and their hybrids (Akimitsu et al., 2003), whereas, satsumas (Citrus unshiu Mark. Marc.), and
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clementines (Citrus clementina Hort. ex Tan.) seem resistant at some extent. The causal agent is a
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pathotype of Alternaria alternata (Fr.) Keissl., which is prevalent in humid citrus-production areas
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as Florida, Brazil, Argentina, Colombia, Cuba, Peru, and China, but also in Mediterranean areas
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with cool, moist winters and hot, dry summers as Spain, Italy, Greece, Turkey, and Israel (Bassimba
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et al., 2014). In Italy, the disease was first reported by Bella et al. (2001), and recently by Garganese
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ACCEPTED MANUSCRIPT et al. (2016). Temperatures of 25-27ºC with at least 10 h of wetness are optimal for infection.
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Conidia are produced on the lesion surface, and subsequently released by rainfalls (Timmer et al.,
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1998). The pathogen causes brown to black spots on young leaves and fruit, and twig dieback.
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Under optimal conditions, symptoms may appear within 24 h after infection (Timmer et al., 2003).
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In addition to a decrease in plant productivity, ABS symptoms cause loose of commercial value of
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fruit.
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Where allowed, fungicides (e.g. dithiocarbamates, dicarboximides, strobilurins, and conazoles) are
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used for ABS control (Feichtenberger et al., 2005). Copper is also recommended in many countries
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(Timmer et al., 2000; Vicent et al., 2004). However, the management of ABS is particularly
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challenging in presence of recurrent rains and jumps in relative humidity (Timmer et al., 1998). In
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Italy, the containment of the disease is critical, and the number of needed treatments may change
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according to the weather conditions year by year. In a study conducted by Bella et al. (2012) during
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2005-2011, two scheduled fungicide applications (late summer and in the fall) resulted inadequate
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to control ABS. Copper and, from late 2015, mancozeb and pyraclostrobin are the only fungicides
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allowed in Italy in conventional/integrated production, being the sole copper allowed in organic
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agriculture (http://m.bdfup.it/bdfup/). Mancozeb and pyraclostrobin belong to a family of fungicides
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suspected for human health hazard (Bolognesi, 2003; Çayır et al., 2014). Moreover, the use of
56
metallic copper in organic agriculture is limited to 6 kg per ha per year (European Commission
57
Regulation N. 889/2008), and following the recommended spray schedules for citrus, this threshold
58
is easily overcome (Vicent et al., 2009). Therefore, the search for safer and effective alternatives is
59
gaining attention. Among them, 2-6% organic and inorganic salts were tested on a wide range of
60
crops including citrus (Palou et al., 2008; Romanazzi et al., 2012; Wisniewski et al., 2016). Most of
61
them belong to the GRAS (Generally Regarded as Safe) category by the US Food and Drug
62
Administration (FDA) (Palou et al., 2016). Salts have been investigated both as pre- (Nigro et al.,
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2006; Teixidó et al., 2010; Youssef et al., 2012) and postharvest treatments (Youssef et al., 2012,
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2014; Montesinos-Herrero et al., 2016). Phosphite salts proved to both induce host resistance and
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al., 2012). Although marketed as fertilizers and plant strengtheners, they proved to be potential
67
alternatives to conventional fungicides (Lobato et al., 2008; Olivieri et al., 2012) including citrus
68
(Panabieres et al., 2016). Phosphites can have different application strategies according to the crop
69
and the pathogen, but foliar spray is the commonest (Deliopoulos et al., 2010). Salts have been
70
introduced in the market as formulations. For instance, FortisolCaPlus, a water-soluble mix of Ca,
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K and P, showed a protection against citrus postharvest disorders and fungicide-related
72
phytotoxicity (Parra et al., 2014; Martínez-Hernández et al., 2017). Similarly, the combined pre-
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and postharvest application of DeccoPhosk, a potassium phosphite formulation, was effective
74
against green and blue mold of lemons, as well as chilling injury and aging (Strano et al., 2015).
75
Furthermore, natural compounds of animal or plant origin showed good control performance of
76
postharvest diseases. For example, chitosan, a linear polysaccharide from crustacean shells, proved
77
to control numerous pre- and postharvest diseases on several vegetables and fruits (Romanazzi et
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al., 2003; Bautista-Baños et al., 2016; Palou et al., 2016) including green mold on citrus
79
(Panebianco et al., 2014). Indeed, chitosan has direct antimicrobial activity, and, at the same time,
80
triggers resistance mechanisms (El Hadrami et al., 2010). Among compounds of plant origin,
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phenolic substances are considered of a certain interest from a disease control perspective. They
82
occur constitutively or are formed in response to pathogen attack (Jeandet et al., 2013).
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Accumulation of quercetin, umbelliferone, scopoletin, and scoparone in plant tissues, together with
84
their antioxidant and antifungal properties (Sourivong et al., 2007; Sanzani et al., 2009, 2014) make
85
them good “natural pesticide” candidates to increase the resistance of plants to fungal infections
86
(Sanzani et al., 2010).
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The aim of this study was to evaluate the effectiveness of selected phenolic compounds and
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alternative formulations against ABS, by testing their effectiveness in laboratory- and semi-
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commercial conditions.
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2. Materials and methods
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2.1 Phenolic compounds and alternative formulations
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The
94
trihydroxyflavanone
95
umbelliferone (7-Hydroxycoumarin) with a purity >95% were purchased from Sigma-Aldrich
96
(Milan, Italy). Stock solutions of pure standards were prepared in a phosphate buffer (Sanzani et al.,
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2009) at a concentration of 50 mg/ml.
98
The tested alternative formulations were Fortisol®CaPlus (water-soluble mix of Ca, K and P,
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Citrosol, Valencia, Spain), DeccoPhosk® (potassium phosphite, Decco Italia, Belpasso, Italy), and
compounds
hesperidin
quercetin
naringin
(4′,5,7-
(3,3',4',5,7-pentahydroxyflavone),
and
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(hesperetin7-rhamnoglucoside),
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phenolic
Hendophyt®PS (chitosan, Iko-Hydro Ltd., Rutigliano, Italy).
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The fungicides Ossiclor (copper oxychloride, 50% w/w, Manica, Rovereto, Italy) and CabrioWG
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(Pyraclostrobin, 20% w/w, BASF, Faenza, Italy) were used as positive controls at 175 g/hl and
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0.06g/hl a.i., respectively.
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2.2 Alternaria alternata conidial suspension
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A. alternata morphotype alternata strain A23, morphologically and molecularly identified
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(Garganese et al., 2016), and deposited in the “Fungal Culture Collection” of the Department of
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Soil, Plant, and Food Sciences, University of Bari Aldo Moro (Italy), was cultured on PDA (Conda,
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Spain) plates for 7 days at 24±1°C. To produce inoculum, colony surface was washed with 5 ml of
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a 0.05% (v/v) Tween 80 aqueous solution. The obtained suspension was filtered through a double
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layer of sterile gauze and counted by a Thoma counting chamber (HGB Henneberg-Sander GmbH,
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Lutzellinden, Germany). A 105 conidia/ml suspension was used for all in vitro and in vivo trials.
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2.3 In vitro assays
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The effect of selected treatments on the mycelial growth of A. alternata was determined on
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amended PDA. Briefly, a solution of each single compound/formulation was 0.22-µm filtered and
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µg/ml (100 and 1000 µg/dish, respectively) for the phenolic compounds, and 0.5, 0.5, and 1% (w/v)
119
for Fortisol, DeccoPhosk, and Hendophyt, respectively. Dishes were seeded centrally with a 5 mm
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mycelial plug of the pathogen. Non-amended PDA plates were used as a control. For each
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treatment/concentration, five dishes were used as replicates. Colony growth (average of the two
122
orthogonal diameters, mm) was recorded at 7 days post-inoculation (DPI). The experiment was
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repeated twice.
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2.4 Laboratory in vivo assays
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2.4.1 Citrus samples
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Mandarins cv. Fortune, a late ripening variety, were harvested at physiological maturity in a
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conventional farm located in the Bernalda area (Basilicata, Southern Italy). Fruit were uniform in
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size, colour, and ripeness and free of defects or injuries. The selected fruit were randomized,
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surface-sterilized in a 2% sodium hypochlorite solution, washed in tap water and left to dry at room
131
temperature.
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2.4.2. Testing of direct antifungal activity
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Fruit were wounded twice (3 mm wide × 3 mm depth) with a sterile nail at opposite sides in the
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area between the peduncle and the equatorial axis. Then, 1000 µg/wound of phenolic solutions, or
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0.5% Fortisol/DeccoPhosk, or 1% Hendophyt were pipetted into each wound. Fruit whose wounds
136
were treated with phenolic solving buffer or water served as controls. After drying, the same
137
wounds were inoculated by 10 µl of A. alternata conidial suspension. Each treatment had three
138
replicates, consisting of six mandarins each (18 fruit/36 wounds per treatment). Replicates were
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incubated at 24±1°C and 95-98% RH for 14 days. Disease incidence (infected wounds, %) and
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severity (lesion diameter, mm) were assessed at 14 DPI. The trial was performed twice.
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2.4.3 Testing of products as resistance inducers
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solving buffer or water were used as controls. After an incubation of 24 h at 24±1°C and 95-98%
144
RH, another round of wound was made on each fruit about 5 mm from the previous one and
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inoculated with A. alternata conidial suspension (10 µl). Each treatment was made of three
146
replicates with six fruit each (18 fruit/36 wounds per treatment). Replicates were incubated as
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reported above. Incidence of decay and disease severity were assessed at the end of incubation. The
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entire experiment was repeated twice.
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2.5 Shelf-life experiments
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The phenolic compounds were further tested as postharvest treatments on the freshly harvested
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mandarins cv. Fortune against natural infections by Alternaria spp. Briefly, 1000 µg of each
153
selected compound was pipetted into each wound (two per fruit) as reported above. Fruit were
154
incubated at 24±1°C and 95-98%. Each treatment comprised three replicates with twelve fruit each
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(36 fruit/72 wounds per treatment). At 14 DPI, disease incidence was assessed as previously
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reported. The entire experiment was repeated twice.
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Field trials with formulates were conducted in 2014 and 2015 by monthly treatments, from May to
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December. The experimental tests were conducted in an orchard of mandarin plants cv. Fortune,
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located in Pisticci area (Basilicata, Southern Italy). The plants were 8-year-old, grafted on Troyer
162
citrange, and arranged in a completely randomized block design with 4 plots of 3 plants each. Plants
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were selected for canopy and fruit set homogeneity and lack of disorders. There were two-plant
164
buffers between plots to reduce spray drift. A spray lance, powered by a walking-type motor pump,
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was used to apply substances at 20 atm into the canopy at a rate of 5 l/plant (20 hl/ha),
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approximately. The same number of plots was treated by the fungicides Ossiclor and CabrioWG as
167
positive controls. Each year, a month after the last treatment, a survey was carried out before
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harvesting to assess the disease on leaves and fruit, randomly choosing 10 twigs and 10 fruit per
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plant, 120 fruit/twigs per treatment, and counting the number of A. alternata lesions on five leaves
170
per twig and on each fruit surface, respectively.
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2.7 Data analysis
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The statistical software package Statistics for Windows (StatSoft, Tulsa, OK, USA) was used.
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Percentage data of incidence of decay were arcsine-square-root transformed before ANOVA
175
analysis. In case of homogeneity of variance, data from repeated experiments were combined.
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Significant differences (P≤0.05) were identified by the General Linear Model (GLM) procedure
177
with the Duncan’s Multiple Range Test (DMRT). A control index (CI) expressed the extent of the
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control exerted by treatments (Sanzani et al., 2009).
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3. Results
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3.1 In vitro assay
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None of the phenolic compounds at both tested concentrations reduced A. alternata growth (Table
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1). Whereas, the three formulations significantly inhibited colony diameter (Table 1). Hendophyt
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provided the best results, inhibiting the growth of the colonies by 93%, followed by Fortisol and
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DeccoPhosk with a reduction of 80 and 77%, respectively.
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3.2 Laboratory in vivo assays
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3.2.1 Testing of direct antifungal activity
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The incidence and severity of the decay on fruit treated by phenolic compounds and inoculated by
190
A. alternata in the same wound are reported in Figure 1A. Although none of the tested phenolic
191
compounds reduced disease incidence, quercetin and hesperidin proved to decrease disease severity
192
by 53 and 32%, respectively, as compared to the control.
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194
58%) and severity (-45%) as compared to the control (Fig. 1B). Whereas DeccoPhosk reduced only
195
disease severity (-57%). Hendophyt did not influence directly the disease development.
196
3.2.2. Testing of products as resistance inducers
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In absence of a direct contact between treatment and pathogen, umbelliferone was the only phenolic
198
compound able to reduce both disease incidence and severity by 47 and 90%, respectively, as
199
compared to the control (Fig. 2A). Whereas, quercetin and naringin reduced only the severity by 74
200
and 73%, respectively. Concerning the formulations, Hendophyt and Fortisol reduced disease
201
incidence by 40 and 47%, respectively. In Hendophyt-treated fruit even the disease severity was
202
reduced by 42% (Fig. 2B).
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3.3 Shelf-life experiments
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Phenolic compounds were tested against Alternaria natural infections on harvested mandarin fruit at
206
concentration 1000 µg/wound. Quercetin and umbelliferone reduced disease incidence by 35 and
207
86%, respectively as compared to the control (Fig. 3).
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In the field, the incidence of disease was recorded on citrus leaves and fruit treated from May to
211
December each year in two consecutive years. Hendophyt and Fortisol were the most effective
212
alternative treatments (Fig. 4). Compared to the control, the number of lesions on leaves and fruit
213
treated with Hendophyt and Fortisol were reduced by 63 and 75%, and by 59 and 54%, respectively
214
(Fig. 4A-B). DeccoPhosk was not effective in controlling lesion appearance on both tissues.
215
Furthermore, no phytotoxicity was observed on fruit and leaves treated by the alternative
216
formulations. Finally, Hendophyt and Fortisol results were similar to those obtained by fungicide
217
treatments, with a reduction ranging between 89 and 92% for fruit, and 88 and 78% for leaves by
218
Ossiclor and CabrioWG, respectively.
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4. Discussion
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ABS is a severe disease on susceptible mandarin and mandarin-like cultivars, so that their
222
cultivation in absence of an effective control strategy might be unprofitable (Peres and Timmer,
223
2006; Vicent et al., 2009). Thus in several citrus-producing countries, chemical fungicide sprays are
224
scheduled intensively during the growing season. However, the risks of residues on the product and
225
in the environment has increased the demand for alternative substances less harmful for humans and
226
animals (Li Destri Nicosia et al., 2016). Last alternative control strategies for pre- and postharvest
227
decays are based on products with modes of action different from traditional fungicidal active
228
ingredients, but also with multiple activities, e.g. direct effect plus induction of resistance
229
(Wisniewski et al., 2016). Recently, the use of elicitors of either biotic or abiotic source to enhance
230
host defenses has gained attention as approach of choice in integrated strategies to control
231
postharvest diseases (Romanazzi et al., 2016). Following ripening, disease resistance of harvested
232
fruit usually declines, increasing host susceptibility to pathogen attack. Thus, resistance inducers
233
may be useful to reinstate a suitable level of resistance (Pangallo et al., 2017). Phenolic compounds
234
are antimicrobial compounds produced by plants that accumulate or appear de novo in response to
235
cellular damage; their production/increase in infected tissues is recognized as one of the main
236
resistance mechanisms in plants (Lachhab et al., 2014). Their induction in challenged plant tissue or
237
exogenous application have been proposed as alternative control approaches against blue and green
238
mold of apple and citrus fruits, respectively (Afek et al., 1999; Sanzani et al., 2009, 2014). In the
239
present investigation, a selection of phenolic compounds was applied against ABS. At the tested
240
concentrations, they did not show a direct effect in vitro against the growth of Alternaria. Similar
241
results were obtained, as far as quercetin and umbelliferone concern, against P. expansum (Sanzani
242
et al., 2009) and P. digitatum (Sanzani et al., 2014). Whereas, on Alternaria-inoculated fruit,
243
quercetin and hesperidin applied in the same wound inoculated by the pathogen proved to reduce
244
disease severity as compared to the control. Overall, tested phenolics seemed to be more efficient in
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ACCEPTED MANUSCRIPT vivo than in vitro, in agreement with Sanzani et al. (2009, 2014). Better disease control results were
246
obtained when phenolics were applied as resistance inducers. Indeed, all tested phenolics controlled
247
disease severity, with umbelliferone reducing even ABS incidence. Thus, they seemed to exert their
248
control effect indirectly. Several possible mechanisms might be involved: activation of host natural
249
defenses including biochemical (e.g. PR-proteins) and physical (e.g. lignification) barriers;
250
accumulation with endogenous phenolics, thus reaching a fungitoxic concentration; chemical
251
interaction with pre-existing compounds, thus forming new toxic molecules (Sanzani et al., 2009).
252
Quercetin ability to induce resistance phenomena in apples has been reported (Sanzani et al., 2010)
253
as well as that of umbelliferone, which played a role in defense mechanisms of unripe reactive
254
grapefruit against wound-pathogens as P. digitatum (Afek et al., 1999). Overall, in the present
255
investigation Alternaria seemed less susceptible to treatments than other genera as Penicillium
256
(Sanzani et al., 2009, 2014). This finding might be ascribed to the intense melanization of
257
Alternaria structure, since melanin contributes to virulence of melanized microorganisms by
258
decreasing their susceptibility to host defense mechanisms (Nosanchuk and Casadevall, 2003).
259
Umbelliferone confirmed its efficacy even in shelf-life trials, and thus, although further larger scale
260
assays are needed, this compound might be considered a good candidate to be applied after harvest
261
within an ABS integrated control strategy, made up of pre- and postharvest application of
262
alternative means, to prevent losses of citrus fruit.
263
Furthermore, in this study some commercial alternative formulations commercialized as citrus
264
biostimulants were tested before harvest. In in vitro assays, they all proved to control Alternaria
265
growth. However, when applied in vivo on fruit, only Fortisol, formulate based on sodium, calcium,
266
and phosphorus salts, reduced disease incidence and severity. Furthermore, it proved to act even as
267
resistance inducer, together with Hendophyt, a chitosan-based formulate. When applied preharvest
268
monthly on fruit and leaves of mandarin cv. Fortune, these two formulations confirmed their
269
reduction of the disease incidence on both organs. Chitosan is known to have multiple activities.
270
For example, it can penetrate the plasma membrane and eventually kill the pathogen cells, as
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272
physical barrier around the infection sites, preventing pathogen spread to healthy tissues (Bittelli et
273
al., 2001). In numerous studies chitosan proved to prevent spore germination, germ tube
274
elongation, and mycelial growth of pathogens as Botrytis cinerea (Monjil et al., 2013;
275
Arasimowicz-Jelonek et al., 2014), Fusarium solani (Li et al., 2014), Rhizopus stolonifer (Monjil
276
et al., 2013), Penicillium spp. (Olivieri et al., 2009; Arasimowicz-Jelonek et al., 2014),
277
and Sclerotium rolfsii (Li et al., 2014). However, it is frequently used as a potent elicitor for
278
controlling plant diseases rather than as antimicrobial agent (Romanazzi et al., 2017). Indeed,
279
chitosan can chelate nutrients and minerals (i.e., Fe, Cu), and bind mycotoxins (Bornet et al., 2007),
280
which may contribute in reducing damage of host tissues (Sanzani et al., 2012). The control activity
281
of salt-based formulate Fortisol was remarkable too. Salts proved to have a multiple mode of action
282
based not only on direct activity against fungi (Fallanaj et al., 2016), but also on resistance
283
induction in treated tissues (Youssef et al., 2014). The activity of Fortisol and Hendophyt was equal
284
or similar to that of chemical fungicides on leaves and fruit, respectively. In our study, the efficacy
285
of copper and pyraclostrobin was analogous to that reported in literature (Vicent et al., 2007). The
286
monthly application of treatments was scheduled based on data according which copper compounds
287
can provide protection for around 1 month under rain-free weather conditions (Vicent et al., 2009).
288
However, copper compounds, especially in summertime and as repeated sprays may become
289
phytotoxic on citrus fruit (Albrigo et al., 1997). Thus, reducing copper applications would lessen the
290
phytotoxicity hazard. To this aim, pyraclostrobin is applied efficiently at beginning of the infection
291
season, when fruit and leaves are small. However, since chemical fungicides are not free from
292
drawbacks, an integrated pest management system with alternative formulations as Fortisol and
293
Hendophyt could maintain a similar level of control without side effects. At this regard, the
294
development and validation of predictive models that can estimate the real risk of infection and
295
identify the optimal time for treatments according to the phenological stages of crop and climatic
296
conditions encountered in the field might be particularly useful (Peres and Timmer, 2006).
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5. Conclusions
299
In order to safeguard the quality and commercial value of the productions of mandarins cv. Fortune
300
and others mandarins, it is necessary to widen the range of products approved for controlling ABS
301
and to develop effective control programs.The good efficacy of alternative treatments, alongside the
302
possibility to obtain healthier products, makes them a viable option to fungicides; the use of which,
303
currently, has been reduced greatly for issues related to the presence of residues, operator safety,
304
environmental pollution, and restrictions provided by the national legislations. The alternative
305
products are gaining wide acceptance among consumers and operators, and as such might represent
306
a valid mean to be used.
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ACCEPTED MANUSCRIPT Table 1. Alternaria alternata colony diameter (mm) on PDA amended with phenolic compounds
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(100 and 1000 µg/dish), and 0.5, 0.5, and 1% (w/v) Fortisol, DeccoPhosk, and Hendophyt. Dishes
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were incubated at 24±1°C for 7 days in the dark.
Control Quercetin_1000 Hesperidin_1000 Naringin_1000 Umbelliferone_1000
65.3 ± 5.4 63.0 ± 4.0 63.0 ± 1.9 69.0 ± 3.4 59.5 ± 4.3
Control DeccoPhosk Fortisol Hendophyt
57.9 ± 4.3 13.1 ± 2.3 11.4 ± 1.0 4.8 ± 1.0
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Colony diameter± SEM 48.8 ± 4.5 50.5 ± 1.9 42.5 ± 5.7 53.9 ± 2.4 45.5 ± 5.1
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Treatments Control Quercetin_100 Hesperidin_100 Naringin_100 Umbelliferone_100
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Caption to figure
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Fig.1. Disease incidence (infected wounds, %) and severity (lesion diameter, mm) on harvested
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citrus fruit wound-treated by A) different phenolic compounds (1000 µg/wound) or B)
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DeccoPhosk/Fortisol (0.5%) and Hendophyt (1%), and inoculated by Alternaria alternata. Water-
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or buffer-treated fruit were used as a control. Data were recorded after 14 days of incubation at
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24±1ºC and 95-98% RH. Bars represent the standard error of the mean (SEM). For each parameter,
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columns with different letters are statistically different according to DMRT (p≤ 0.05).
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Fig.2. Disease incidence (infected wounds, %) and severity (lesion diameter, mm) on harvested
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citrus fruit wound-treated by A) different phenolic compounds (1000 µg/wound) or B)
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DeccoPhosk/Fortisol (0.5%) and Hendophyt (1%), and after 24 h inoculated by Alternaria alternata
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in different but close wounds. Water- or buffer-treated fruit were used as a control. Data were
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recorded after 14 days of incubation at 24±1ºC and 95-98% RH. Bars represent the standard error of
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the mean (SEM). For each parameter, columns with different letters are statistically different
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according to DMRT (p≤ 0.05).
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Fig.3. Disease incidence (infected wounds, %) on harvested citrus fruit wound-treated by different
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phenolic compounds (1000 µg/wound) against Alternaria natural infections. Bars represent the
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standard error of the mean (SEM). Columns with different letters are statistically different
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according to DMRT (p≤ 0.05).
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Fig. 4. Disease incidence (number lesions) on citrus leaves (A) and fruit (B) monthly treated in the
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field by alternative products DeccoPhosk and Fortisol (0.5%), and Hendophyt (1%) and two
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chemical fungicides Ossiclor and CabrioWG (commercial dose). Water treated samples were used
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statistically different according to DMRT (p≤ 0.05).
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infected wounds
lesion diameter
a
a
60
30
a a
b
40
a
b 20
10
0
0
Quercetin
100
Hesperidin
Naringin
infected wounds
Umbelliferone
lesion diameter
B
a
a
a b b
40
20
b
EP
a a
AC C
Disease incidence (%)
80
30
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Control
60
20
0
25 20 15 10 5 0
Control
DeccoPhosk
Fortisol
Hendophyt
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40
a
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a
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Disease incidence (%)
a 80
Disease severity (mm)
A
50
Disease severity (mm)
100
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infected wounds
a a
lesion diameter
40
a
a a
60
a
b
30
b
b
40
b
20 0 Hesperidin
Naringin
infected wounds
lesion diameter
30 25
a 60
a a
b a
EP
a
40 20
20
b
AC C
Disease incidence (%)
Umbelliferone
TE D
Quercetin
100 80
10 0
Control
B
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b
0
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Control
DeccoPhosk
Fortisol
Hendophyt
SC
80
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Disease severity (mm)
100
Disease severity (mm)
Disease incidence (%)
A
SC
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a a
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ab
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b
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c
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Control
AC C
Infected wounds (%)
12
Quercetin
Hesperidin
Naringin
Umbelliferone
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A
0.4
a
Number lesion/leaf
0.2
b b
0.1
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0 Control
Fortisol
DeccoPhosk
Copper
a a
3
b
b
2
1
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Pyraclostrobin
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AC C
Number lesions/fruit
B
Hendophyt
c
c
Copper
Pyraclostrobin
0 Control
Hendophyt
Fortisol
DeccoPhosk
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ACCEPTED MANUSCRIPT Highlights
• We tested phenolic compounds and alternative formulations against Alternaria brown spot. • In vitro and in vivo tests on a laboratory scale and semi -commercial conditions were performed.
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• Umbelliferone was the most effective phenolic compound; it seemed to induce host resistance. • Hendophyt and Fortisol were the most effective formulations, comparable to chemical controls.
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• These alternatives, singly or in a combined strategy, might be effective against the disease.