10.6092/issn.2465-311X/5742
Rhizoctonia solani Kühn, è un fungo basidiomicete ad habitat terricolo, diffuso in tutto il mondo su numerose piante coltivate e causa di significative perdite di produzione. Tra le varie colture, il fagiolo è altamente suscettibile alla malattia causata da R. solani. Il controllo del patogeno, così come di altri patogeni terricoli, può essere ottenuto con la disinfezione del suolo. Tuttavia, in Europa, i prodotti chimici per la difesa delle piante dalle malattie sono stati oggetto di restrizioni d’utilizzo che hanno comportato l’esclusione di molti principi attivi contro i patogeni compresi quelli terricoli. Di conseguenza, molti studi sono stati finalizzati alla ricerca di mezzi alternativi a quelli chimici, come ad esempio i microrganismi. Alcuni di questi, definiti “antagonisti”, sono componenti di bioagrofarmaci autorizzati per la difesa delle piante, ma possono anche essere contenuti in prodotti dotati di azione biostimolante per le piante. In questa ricerca, è stata studiata, in esperimenti in cella climatica, l’efficacia contro R. solani e l’azione biostimolante su piante di fagiolo di prodotti microbici contenenti funghi e batteri (EM Bokashi® 2-fi and EM-5 Sutociu® caratterizzati da un’azione biostimolante delle piante), Trichoderma harzianum T22 (un noto biofungicida) e l’antagonista fungino Trichoderma TJ40. EM-5 Sutociu è stato somministrato ai semi (Sut/Se), al substrato (Sut/S) e alle foglie (Sut/L) più volte, EM Bokashi 2-fi al substrato (Bok/S) singolarmente e in combinazione con Sut; T22 and TJ40 sono stati somministrati una volta ciascuno al substrato. Il patogeno è stato inoculato nel substrato al momento della semina (primo esperimento) oppure all’emergenza nella fase di piantina (secondo esperimento). Nelle nostre condizioni sperimentali, Bok/S+Sut/S+Sut/L, Sut/S+Sut/L, Sut/Se+Sut/S+Sut/L e T22, nel primo esperimento, e tutti i trattamenti ad eccezione di Bok/S somministrato da solo, nel secondo esperimento, hanno ridotto significativamente la gravità della malattia, incrementato il peso secco delle piante e l’area fogliare rispetto il controllo infetto. TJ40 ha ridotto sia l’incidenza di malattia sia la gravità solo nel secondo esperimento. Nell’esperimento sull’azione biostimolante, T22, Bok/S+Sut/S+Sut/L, Sut/S+Sut/L e Sut/Se+Sut/S+Sut/L hanno mostrato di aumentare significativamente sia il peso secco che l’area fogliare. E’ stato inoltre studiato in vitro l’effetto diretto di T22, TJ40, Bok e Sut sulla crescita di R. solani con due metodi: “submerged colony” (SC) e “well diffusion” (WD). La crescita della colonia del patogeno è stata completamente inibita da T22 in entrambi i metodi, da TJ40 in un range di 80-50 % nel saggio con il metodo SD, e in un range di 50-30 % nel saggio WD e leggermente inibita o non inibita da Bok and Sut.
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Pamela Leonardi
Italian Journal of Mycology
biocontrol
Biocontrol of <em>Rhizoctonia solani</em> disease and biostimulant effect by microbial products on bean plants
Text.Serial.Journal
https://italianmycology.unibo.it/
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Articles
2015-12-15
2015-12-09
2015-12-15
2018-08-20
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Ital J Mycol
Pamela Leonardi
2015/12/15
10.6092/issn.2465-311X/5742
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University of Bologna
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Biocontrol of <em>Rhizoctonia solani</em> disease and biostimulant effect by microbial products on bean plants
https://italianmycology.unibo.it/article/download/5742/5463
44
R. Roberti, F. Bergonzoni, A. Finestrelli, P. Leonardi          Micologia Italiana vol. 44 (2015) ISSN 2465‐311X
DOI: 10.6092/issn.2465-311X/5742




  Biocontrol of Rhizoctonia solani disease and
  biostimulant effect by microbial products on bean
  plants
  ______________________________________________________________________________

    Roberta Roberti, Federica Bergonzoni, Angela Finestrelli, Pamela Leonardi

  Department of Agricultural Sciences, Alma Mater Studiorum University of Bologna,
  viale G. Fanin, 46, 40127 Bologna, Italy.

  Corresponding Author R. Roberti e-mail: roberta.roberti@unibo.it

  Abstract

    Microbial products containing a mixture of fungi and bacteria (EM Bokashi® 2-fi and EM-5 Sutociu®
    characterised by plant biostimulant activity), Trichoderma harzianum T22 (biofungicide) and the antagonist
    fungus Trichoderma sp. TJ40 were tested for efficacy against R. solani disease and for their biostimulant
    effects on bean plants, in growth chamber experiments, and for their direct effect on the pathogen growth,
    through in vitro experiments. In growth chamber experiments, EM-5 Sutociu was applied to seed (Sut/Se),
    substrate (Sut/S) and leaf (Sut/L) many times, EM Bokashi 2-fi to substrare (Bok/S) once and combined with
    Sut, T22 and TJ40 were applied once to substrate. The pathogen was inoculated to substrate at seeding time
    (first experiment) or at seedling phase (second experiment). Under our experimental conditions,
    Bok/S+Sut/S+Sut/L, Sut/S+Sut/L, Sut/Se+Sut/S+Sut/L and T22, in the first experiment, and all treatments,
    with the exception of Bok/S applied alone in the second experiment, gave significantly disease severity
    reduction and increase of dry weight and leaf area with respect to the infected control. The TJ40 treatment
    reduced both disease incidence and disease severity only in the second experiment. In the experiment on the
    biostimulant effect, T22, Bok/S+Sut/S+Sut/L, Sut/S+Sut/L and Sut/Se+Sut/S+Sut/L showed significantly
    increases of both dry weight and leaf area. The direct effect of the treatment with T22, TJ40, Bok and Sut on
    R. solani growth in vitro was studied with two methods, submerged colony (SC) and well diffusion (WD)
    assays. The pathogen growth was completely inhibited by Trichoderma T22 in both assays, by Trichoderma
    TJ40 in a range of 80-50 % in SD assay, and 50-30 % in WD assay and slightly inhibited or not inhibited by
    Bok and Sut.

    Keywords: Rhizoctonia solani; antagonistic microorganisms; plant biostimulants; biocontrol

  Riassunto

    Rhizoctonia solani Kühn, è un fungo basidiomicete ad habitat terricolo, diffuso in tutto il mondo su
    numerose piante coltivate e causa di significative perdite di produzione. Tra le varie colture, il fagiolo è
    altamente suscettibile alla malattia causata da R. solani. Il controllo del patogeno, così come di altri patogeni
    terricoli, può essere ottenuto con la disinfezione del suolo. Tuttavia, in Europa, i prodotti chimici per la
    difesa delle piante dalle malattie sono stati oggetto di restrizioni d’utilizzo che hanno comportato l’esclusione
    di molti principi attivi contro i patogeni compresi quelli terricoli. Di conseguenza, molti studi sono stati

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R. Roberti, F. Bergonzoni, A. Finestrelli, P. Leonardi              Micologia Italiana vol. 44 (2015) ISSN 2465‐311X
DOI: 10.6092/issn.2465-311X/5742

       finalizzati alla ricerca di mezzi alternativi a quelli chimici, come ad esempio i microrganismi. Alcuni di
       questi, definiti “antagonisti”, sono componenti di bioagrofarmaci autorizzati per la difesa delle piante, ma
       possono anche essere contenuti in prodotti dotati di azione biostimolante per le piante. In questa ricerca, è
       stata studiata, in esperimenti in cella climatica, l’efficacia contro R. solani e l’azione biostimolante su piante
       di fagiolo di prodotti microbici contenenti funghi e batteri (EM Bokashi® 2-fi and EM-5 Sutociu®
       caratterizzati da un’azione biostimolante delle piante), Trichoderma harzianum T22 (un noto biofungicida) e
       l’antagonista fungino Trichoderma TJ40. EM-5 Sutociu è stato somministrato ai semi (Sut/Se), al substrato
       (Sut/S) e alle foglie (Sut/L) più volte, EM Bokashi 2-fi al substrato (Bok/S) singolarmente e in combinazione
       con Sut; T22 and TJ40 sono stati somministrati una volta ciascuno al substrato. Il patogeno è stato inoculato
       nel substrato al momento della semina (primo esperimento) oppure all’emergenza nella fase di piantina
       (secondo esperimento). Nelle nostre condizioni sperimentali, Bok/S+Sut/S+Sut/L, Sut/S+Sut/L,
       Sut/Se+Sut/S+Sut/L e T22, nel primo esperimento, e tutti i trattamenti ad eccezione di Bok/S somministrato
       da solo, nel secondo esperimento, hanno ridotto significativamente la gravità della malattia, incrementato il
       peso secco delle piante e l’area fogliare rispetto il controllo infetto. TJ40 ha ridotto sia l’incidenza di malattia
       sia la gravità solo nel secondo esperimento. Nell’esperimento sull’azione biostimolante, T22,
       Bok/S+Sut/S+Sut/L, Sut/S+Sut/L e Sut/Se+Sut/S+Sut/L hanno mostrato di aumentare significativamente sia
       il peso secco che l’area fogliare. E’ stato inoltre studiato in vitro l’effetto diretto di T22, TJ40, Bok e Sut
       sulla crescita di R. solani con due metodi: “submerged colony” (SC) e “well diffusion” (WD). La crescita
       della colonia del patogeno è stata completamente inibita da T22 in entrambi i metodi, da TJ40 in un range di
       80-50 % nel saggio con il metodo SD, e in un range di 50-30 % nel saggio WD e leggermente inibita o non
       inibita da Bok and Sut.

       Parole chiave: Rhizoctonia solani; microorganismi antagonisti; biostimolanti delle piante; lotta
       biologica

  Introduction

  Rhizoctonia solani Kühn, teleomorph Thanatephorus cucumeris (Frank) Donk, is a soil-borne
  basidiomycete fungus globally-distributed. This fungus survives and overwinters in soil as mycelium or as
  sclerotia in plant debris and can infect plants during the growing season. This pathogen employs a
  necrotrophic lifestyle on numerous cultivated plant species causing significant crop losses (Parmeter,
  1970; Sneh et al., 1991). Among the various crops, bean is highly susceptible to stem, hypocotyl and root
  rot disease by R. solani.
  As for almost all soil-borne pathogens, the control of R. solani could be obtained by soil disinfestation, an
  important practice in horticultural intensive systems. However, nowadays, effective chemical fumigants
  for soil disinfestation, such as methyl bromide, chloropicrin, dazomet and 1,3-dicloropropene, are no
  longer included in the European list of approved substances. In Italy, very few chemicals are allowed for
  use on bean crops; among them are fumigants with some fungicide effect, such as methyl isothiocyanate
  generators, metham sodium and metham potassium, and non-fumigant fungicides for seed disinfestation,
  such as thiram alone or in combination with carboxin. Recently, some microbial products are
  commercialized as “plant protection products” or “pesticides” for soil application. All plant protection
  products in the European Union, and therefore in Italy, are regulated by the Regulation (EC) No
  1107/2009. They are contained in a list of approval under the cited EC Regulation (EU Pesticide database,
  2015). Among these products, those sold in Italy for soil application against plant diseases are based on
  fungi (Trichoderma harzianum, T. asperellum and T. gamsii and Coniothyrium minitans), or bacteria
  (Streptomyces sp.), and can be used both in integrated pest management (IPM), which is mandatory in
  Italy since 1 January 2014, both in organic agriculture. Among fungi, Trichoderma spp. are the most


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DOI: 10.6092/issn.2465-311X/5742


  widely species studied for a long time (Tronsmo & Hjeljord, 1998) and, colonizing roots, can enhances
  root growth and development, crop productivity, resistance to biotic and abiotic stresses and the uptake
  and use of nutrients (Harman et al., 2004; Matsouri et al., 2010), acting as a plant biostimulant.
  Plant biostimulants are defined by the EBIC (European Biostimulants Industry) as follows: “Plant
  biostimulants contain substance(s) and/or microorganisms whose function when applied to plants or the
  rhizosphere is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency,
  tolerance to abiotic stress, and crop quality. Biostimulants have no direct action against pests, and
  therefore do not fall within the regulatory framework of pesticides” (European Biostimulants Industry,
  2012a). Biostimulants also include products with some nutrients, but their effect on plant growth
  promotion is not through direct fertilization. In fact, the EBIC (2012b) cites “Biostimulants operate
  through different mechanisms than fertilisers, regardless of the presence of nutrients in the products”.
  Brown & Saa (2015) hypothesized that biostimulants are beneficial for plant productivity by interacting
  with plant signalling processes thereby reducing negative plant response to stress, and Veronesi et al.
  (2009) demonstrated that some biostimulants are able to enhance the plant defence mechanisms against
  biotic stresses. Moreover, biostimulants can potentially act against plant pathogens since they also contain
  microorganisms such as fungi (e.g. Trichoderma sp.) and bacteria (e.g. Bacillus subtilis). Therefore, based
  on their characteristics, biostimulants may have an important role in the integrated production.
  Based on the above considerations, and also on the absence of commercial cultivars of bean resistant to the
  disease caused by R. solani, the objectives of this study were to investigate ì) the role of microbial
  products on the disease control, and their biostimulant effect on bean in growth chamber experiments, and
  ii) their direct effect on the pathogen growth through in vitro experiments.

  Materials and Methods

  Plant material, pathogen and natural compounds
  Seeds of bean (Phaseolus vulgaris L.) cv. Borlotto (Semencoop a.r.l, Martorano di Cesena, FC, Italy) were
  employed. Before seeding, 200 seeds randomly selected were analysed to verify the absence of R. solani
  natural infection. Seeds were firstly observed visually, to exclude the presence of brown lesions, then
  superficially disinfected with sodium hypochlorite solution (NaOCl, 2% available chlorine), and plated on
  potato dextrose agar (PDA, 3.9 %, Difco, Detroit, MI, USA) added with streptomycin sulphate (100
  mg/L). Plates were incubated at 25 °C in the dark for 10 days.
  The pathogen R. solani (Rs) (Fig. 1) was obtained from bean seedlings showing symptoms of post-
  emergence damping-off and brown lesions on the hypocotyl. Rhizoctonia solani was identified based on
  morphological features by examining hyphal branching septal, pore type and cellular nuclear number close
  to the tips of young hyphae, after 2 days of growth on alkaline water agar plus 100 µg/ml streptomycin
  sulphate (Gutierrez et al., 1997, modified), followed by staining with aniline blue. Colonies of the fungus
  also showed sclerotia of irregular shape and monilioid cells after 21 days growth on PDA. This fungus was
  pathogenic on bean cv. Borlotto in a previous experiment and caused characteristic crown and root rot
  symptoms. The pathogen was kept on PDA at 4°C until use.
  The tested compounds were all based on microorganisms and are listed in Table 1. EM Bokashi®
  2-fi and EM-5 Sutociu® are commercialized as biostimulants, Trianum-P is a biofungicide
  registered by Italian Health Ministry for the application against soil-borne pathogens on various
  crops. Moreover, the Trichoderma sp. J40 strain (Fig. 2), kindly supplied by CRA - Centro di
  Ricerca per le Colture Industriali di Bologna (Italy), was tested.


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DOI: 10.6092/issn.2465-311X/5742




  Fig. 1 - Rhizoctonia solani: colonies grown on PDA for 10 days (a), hyphae with typical ramifications and monilioide
  cells at light microscope (b) and mycelium grown in PDB for 10 days in an orbital shaker (c).
  Rhizoctonia solani: colonie cresciute su PDA per 10 giorni (a), ife con tipiche ramificazioni e cellule monilioidi al
  microscopio ottico (b) e micelio cresciuto in PDB per 10 giorni in un agitatore orbitale (c).




  Fig. 2 - Colony of Trichoderma sp. J40 grown on PDA for 7 days.
  Colonia di Trichoderma sp. J40 cresciuta su PDA per 7 giorni.


  Growth chamber experiments
  All growth chamber experiments were carried out at 25 ± 1°C, 84 88 % relative humidity, 12-h
  photoperiod (5000 lux) using a plant growing substrate constituted by loamy soil, peat moss and sand
  (2:1:1 v:v:v). The substrate was heat-treated at 120°C for 30 min on two consecutive days, and then cooled
  before the use. Before seeding, the bean seeds were first surface-disinfected with NaOCl (2% available
  chlorine) for 5 min, rinsed twice in sterile distilled water and dried on sterile paper in a laminar flow
  cabinet. Seeds were sown in 12 × 10 × 6 cm pot at the rate of 20 seeds per pot. Each pot represented a
  replicate. The experiments were laid out in completely randomized design with three replicates of each
  treatment. Each experiment was repeated twice.




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DOI: 10.6092/issn.2465-311X/5742

  Tab. 1 - Microbial products, active ingredients, dosage, site and symbol, and time of application in greenhouse
  experiments.
  Prodotti a base di microrganismi, principi attivi, dosi, sito d’applicazione e relativo simbolo e momento di
  trattamento.

  Microbial products
                                 Active ingredients      Dosage          Site       Symbol     Time/application
  (Company)
  EM Bokashi® 2-fi (Punto        Fungi and bacteriaa     20g/L           substrate Bok/S       at seeding by
  EM S.r.l., Sanremo, IM,                                                                      irrigation
  Italia)
  EM-5 Sutociu® (Punto EM Fungi and bacteriab            1 ml/L          seedc      Sut/Se
  S.r.l., Sanremo, IM, Italia)
                                                         5 ml/Ld         substrate Sut/S       at seeding by
                                                                                               irrigation
                                                         5 ml/Le         leaves     Sut/L      twice a week from
                                                                                               emergence by
                                                                                               sprayingf
                                                         20 ml/Lg        leaves     Sut/L      every day by
                                                                                               irrigationh

  Trianum-P (Koppert Italia      Trichoderma             1.5 g/3L        substrate T22         at seeding by
  S.r.l, Bussolengo, VR)         harzianum T22           water/m2                              irrigation
  ‐                              Trichoderma sp. J40 5 × 106 cfu         substrate TJ40        at seeding by
                                                                                               irrigation

  a
   Lactobacillus plantarum (1×106 cfu/ml), L. casei (1×106 cfu/ml) and Streptococcus lactis (1×106 cfu/ml),
  Rhodospeudomonas palustris (1×103 cfu/ml) and Rhodobacter spaeroides (1×103 cfu/ml), Saccharomyces
  cerevisiae (1×105 cfu/ml) and Candida utilis (1×105 cfu/ml), Streptomyces albus (1×104 cfu/ml) and S.
  griseus (1×104 cfu/ml), Aspergillus oryzae (1×103 cfu/ml), Penicillum sp. (1×103 cfu/ml), Mucor hiemalis
  (1×104 cfu/ml), Trichoderma harzianum (1×104 cfu/ml), T. viride (1×103 cfu/ml) added to bran (D. Prisa,
  personal communication); bsame microorganisms of a suspended in liquid substances such as grappa and
  allium extract; ctreated for 12 hrs in liquid suspension; d150 ml per pot; e150 ml per pot, until the
  suspension has dripped over the soil; funtil plants were symptomless; g50 ml per pot, until the suspension
  has dripped over the soil; hfrom disease symptom appearance.
  Biocontrol activity
  The substrate was inoculated with Rs at the rate of 3 g mycelium/L of substrate at sowing time and at
  seedling phase time (6 days after seeding), in order to simulate an early or a late pathogen attack. For the
  inoculum preparation, ten 1-cm2 discs from the actively growing colonies of Rs on PDA were inoculated
  in each 300 ml Potato Dextrose Broth (PDB, 2.4%, Difco, Detroit, MI, USA) conical flask. The flasks
  were gentle shaken at 100 rpm for 7 days at 23- 25 °C in the dark. The mycelial mass (Fig. 1) was
  collected by filtration through Whatman n. 1 filter paper, rinsed with distilled water, weighted and finely
  homogenized in a sterile Ultra-TURRAX® (IKA, Germany) blender for 30 sec.
  Microbial compounds were applied following instruction of manufacturer (Table 1), except for
  Trichoderma sp. J40 that was distributed into substrate as conidial suspension in Ringer’s solution (Sigma-

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DOI: 10.6092/issn.2465-311X/5742


  Aldrich Co, St. Louis, MO, USA)) (5 × 106 cfu/ml). For each substrate and leaf treatment, a volume of
  150 ml of each EM product (20 ml of Sut/L only in presence of disease symptoms) was distributed in each
  pot at the dose and times indicated in Table 1. For seed treatment, seeds were immersed in an aqueous
  suspension of Sut/Se for 12 h. Infected control received the same volume of sterile distilled water.
  Three weeks after pathogen inoculation, the plants were carefully collected and washed under running
  water. The disease incidence (DI) was determined by counting plants with disease symptoms on the total
  of plants. For disease severity (DS), plants were counted and divided into four classes of severity (Fig. 3)
  as follows: 0 = no lesions, 1 = lesions 2.5 mm, 2 = lesions 2.5–5 mm; 3 = lesions > 5 mm and girdling
  plant (modified from Cardoso and Echandi, 1987). Disease severity index was calculated for each pot by
  the following formula: [((plant number in class 1) + 2 (plant number in class 2) + 3 (plant number in class
  3))/total plant number] × 100/3.
  Samples of root and foot 1 2 cm long from plants with Rs disease symptoms were surface disinfected
  with 5% NaOCl, rinsed in sterile distilled water and transferred to Petri dishes containing PDA
  supplemented with 100 mg/L of streptomycin sulphate (Sigma - Aldrich Co.). After incubation at 25°C in
  the dark for 10 days, the presence of Rs was confirmed using a light microscope (Carl Zeiss mod. ZM,
  Germany) at × 300 magnification.
  For each pot, plant leaf area was measured with the LI-3100 Area Meter (LI-COR Bioscience, USA) and
  total plant dry weight was determined after oven drying at 80 °C for 24 h.




   Fig. 3 – Bean plants with typical symptoms of Rhizoctonia solani disease and severity disease classes.
   Piante di fagiolo con i tipici sintomi di malattia di Rhizoctonia solani e classi di gravità di malattia.
  Biostimulant activity
  The experiment on biostimulant activity on bean plants was carried out with the same products described
  above and in absence of the pathogen. The effect of treatments were evaluated by measuring leaf area and
  total dry weight as previously specified.
  In vitro assays
  To evaluate the ability of microbial compounds to inhibit the colony growth of R. solani, plate tests were
  performed on PDA. Bok, Sut, T22 and TJ40 were separately tested at 20 g/L, 5 ml/L, 0.6 g/L and 5 × 106
  cfu/ml, respectively. Two experiments were carried out following two methods: submerged colony (SC)
  and well diffusion (WD). In the first a 5 cm diameter plug removed from the edge of the active growing

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DOI: 10.6092/issn.2465-311X/5742


  colony of Rs was submerged in 2 ml water suspension of each product (Pane et al., 2013). After an
  overnight incubation at 25 °C, the plug was transferred in the centre of a PDA Petri plate (diameter 90
  mm), then incubated at 25 °C for 4 days in the dark. In the second experiment, a modified procedure of
  well diffusion (WD) assay (Magaldi et al., 2003) was used. Once the agarized medium had solidified, three
  wells, each 5 mm in diameter, were cut out of the medium equidistantly from the centre from each PDA
  plate, and 100 µl of the product suspension were placed into each well. A plug (5 mm diameter) removed
  from the edge of the growing pathogen colony was inoculated in the centre of the plate, and then the plates
  were incubated at 25 °C for 4 days in the dark. The inhibition of colony pathogen growth by the
  compounds were indicated as follow: 100 - 80 % (++++), 80 - 50 % (+++), 50 – 30 % (++), < 30 % (+)
  and not inhibited (-), compared to control. Both experiments were repeated two times with 5 replicates for
  each treatment.
  Statistical analysis
  Percentage data were arcsine transformed before statistical analysis and back transformed data are reported
  in the tables. Data were submitted to one-way analysis of variance (ANOVA univariate, Statgraphic Plus
  Version 2.1; Statistical Graphics Corp., USA 1996), and the means were compared by using LSD multiple
  range test (P < 0.05).

  Results

  Growth chamber experiments
  No natural infection by Rs or other seed-borne pathogens was observed on seeds examined before seeding.

       Tab. 2 - Effect of microbial products on disease incidence and disease severity index (0–100) of bean
       plants three weeks after Rhizoctonia solani inoculation to the substrate at seeding time.
       Effetto di prodotti a base di microrganismi sull’incidenza e indice di gravità della malattia (0-100) su
       piante di fagiolo tre settimane dopo l’inoculazione di Rhizoctonia solani nel substrato al momento della
       semina.


        Treatment                           Disease incidence (%)              Disease severity index (%)

        Infected control                    100.0 ± 0.0 c                      91.1 ± 6.2 d
        T22                                 97.9 ± 3.6 bc                      75.0 ± 2.7 ab
        TJ40                                97.4 ± 4.4 bc                      80.8 ± 1.5 bc
        Bok/S                               100.0 ± 0.0 c                      85.2 ± 5.1 cd
        Bok/S + Sut/S +Sut/L                88.6 ± 4.4 ab                      69.6 ± 2.6 a
        Sut/S + Sut/L                       86.2 ± 8.2 a                       68.7 ± 4.7 a
        Sut/Se + Sut/S + Sut/L              89.3 ± 4.9 ab                      68.7 ± 7.4 a

  Means (± SD) followed by the same letter in a column are not significantly different according to LSD test (P <
  0.05).

  Data on the effect of treatments on DI and DS of bean plants inoculated with Rs at seeding time are
  reported in Table 2. The pathogen was highly pathogenic for beans plants as showed by the values of DI
  (100%) and DS (91.1 ± 6.2 %). The combined treatment Bok/S+Sut/S+Sut/L and the treatments

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R. Roberti, F. Bergonzoni, A. Finestrelli, P. Leonardi              Micologia Italiana vol. 44 (2015) ISSN 2465‐311X
DOI: 10.6092/issn.2465-311X/5742


  Sut/S+Sut/L or Sut/Se+Sut/S+Sut/L significantly (P < 0.05) decreased both DI and DS compared with the
  control. These treatments were more efficient in reducing DS (23.6 % - 24.6 %) than DI (10.7 % - 13.8 %).
  Trianum (T22) and TJ40 reduced DS by 17.7 % and 11.3 %, respectively, compared to the control, and
  T22 gave statistically similar DS reduction to Bok/S+Sut/S+Sut/L, Sut/S+Sut/L or Sut/Se+Sut/S+Sut/L.
  In Table 3 the effect of treatments on total plant dry weight (DW) and on total leaf area (LA) of bean
  plants inoculated with Rs at seeding time is reported. Bok/S+Sut/S+Sut/L, Sut/S+Sut/L,
  Sut/Se+Sut/S+Sut/L and T22 significantly (P < 0.05) increased both DW and LA with respect to the
  control. These treatments increased DW from 30,0 % to 72.5 % and LA from 54.6 to 171.1 %. The highest
  increases of DW was obtained with Bok/S+Sut/S+Sut/L and Sut/Se+Sut/S+Sut/L (55.0 % and 72.5 %,
  respectively), and the highest increase of LA was achieved by Bok/S+Sut/S+Sut/L (171.1 % compared to
  the control). Both DW and LA obtained with T22 treatment did not differ from Sut/S+Sut/L or
  Sut/Se+Sut/S+Sut/L.

       Tab. 3 - Effect of microbial products on dry weight and leaf area of bean plants three weeks after Rhizoctonia
       solani inoculation to the substrate at seeding time.
       Effetto di prodotti a base di microrganismi sul peso secco e l’area fogliare di piante di fagiolo tre settimane
       dopo l’inoculazione di Rhizoctonia solani nel substrato al momento della semina.


        Treatment                            Total planta dry weight (mg)          Total leaf area (mm2)

        Infected control                     4.0 ± 1.0 a                           644.2 ± 160.5 a
        T22                                  5.9 ± 0.5 bc                          1090.0 ± 265.0 b
        TJ40                                 4.6 ± 0.8 a                           775.9 ± 1910.8 ab
        Bok/S                                4.4 ± 0.8 a                           795.5 ± 126.2 ab
        Bok/S + Sut/S +Sut/L                 6.2 ± 0.8 c                           1746.8 ± 367.4 c
        Sut/S + Sut/L                        5.2 ± 0.4 b                           1059.0 ± 107.7 b
        Sut/Se + Sut/S + Sut/L               6.9 ± 0.4 c                           995.7 ± 103.9 b

  a
   plants were three weeks old.
  Means (± SD) followed by the same letter in a column are not significantly different according to LSD test (P <
  0.05).

  The effect of treatments on DI and DS of bean plants inoculated with Rs at seedling stage time is reported
  in Table 4. The pathogen caused very high DI (100 %) and 74.5 ± 2.1 % of DS. All treatments, except
  Bok/S, significantly (P < 0.05) reduced values of both disease parameters. The DI was reduced from 25.3
  % (TJ40) to 41.4 % (Bok/S+Sut/S+Sut/L). The DS was reduced from 20.3 % (TJ40) to 38.7 %
  (Bok/S+Sut/S+Sut/L). Treatment with Bok/S+Sut/S+Sut/L and Sut/S+Sut/L showed the lowest DS values,
  45.7 ± 7.7 % and 48.6 ± 9.0 % respectively, compared to 74.5 ± 2.1 % of control.
  In Table 5, the effect of treatments on total plant dry weight (DW) and on total leaf area (LA) of bean
  plants inoculated with Rs at seedling emergence is reported. All treatments, except Bok/S, significantly (P
  < 0.05) increased both DW and LA values. The highest increase of DW with respect to the infected control
  was obtained with Sut/Se+Sut/S+Sut/L (98.1 %). With the exception of Bok/S, values of LA showed by all
  treatments were significantly (P < 0.05) similar. These effective treatments increased LA from 21.9 %
  (T22) to 29.3 % (Bok/S+Sut/S+Sut/L) with respect to the control.

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DOI: 10.6092/issn.2465-311X/5742



       Tab. 4 - Effect of microbial products on disease incidence and disease severity index (0–100) of bean plants
       three weeks after Rhizoctonia solani inoculation to substrate at seedling stage time.
       Effetto di prodotti a base di microrganismi sull’incidenza e indice di gravità della malattia (0-100) su piante
       di fagiolo tre settimane dopo l’inoculazione di Rhizoctonia solani nel substrato allo stadio di piantina.


       Treatment                            Disease incidence (%)                Disease severity index (%)

       Infected control                     100.0 ± 0.0 b                        74.5 ± 2.1 d
       T22                                  63.9 ± 6.2 a                         53.3 ± 2.9 ab
       TJ40                                 74.7 ± 4.3 a                         59.4 ± 5.0 bc
       Bok/S                                93.3 ± 11.5 b                        65.2 ± 3.1 cd
       Bok/S + Sut/S +Sut/L                 58.6 ± 15.8 a                        45.7 ± 7.7 a
       Sut/S + Sut/L                        60.0 ± 11.9 a                        48.6 ± 9.0 a
       Sut/Se + Sut/S + Sut/L               70.4 ± 2.6 a                         54.2 ± 2.1 ab

  Means (± SD) followed by the same letter in a column are not significantly different according to LSD test (P <
  0.05).

         Tab. 5 - Effect of microbial products on total dry weight and leaf area of bean plants three weeks after
         Rhizoctonia solani inoculation to substrate at seedling stage time.
         Effetto di prodotti a base di microrganismi sul peso secco e l’area fogliare di piante di fagiolo tre
         settimane dopo l’inoculazione di Rhizoctonia solani nel substrato allo stadio di piantina.


         Treatment                           Total planta dry weight (mg)        Total leaf area (mm2)

         Infected control                    5.2 ± 0.6 a                         785.8 ± 87.8 a
         T22                                 8.7 ± 0.8 c                         958.3 ± 43.5 bc
         TJ40                                7.1 ± 0.9 bc                        980.8 ± 43.2 c
         Bok/S                               5.2 ± 0.9 a                         793.3 ± 87.8 a
         Bok/S + Sut/S +Sut/L                6.9 ± 1.1 b                         1015.8 ± 87.8 c
         Sut/S + Sut/L                       7.9 ± 1.4 bc                        947.5 ± 33.2 bc
         Sut/Se + Sut/S + Sut/L              10.3 ± 0.9 d                        977.5 ± 57.5 c

  a
   plants were four weeks old.
  Means (± SD) followed by the same letter in a column are not significantly different according to LSD test (P <
  0.05).




       Tab. 6 - Biostimulant effect of the microbial products on four weeks-old bean plants.
       Effetto biostimolante di prodotti a base di microrganismi su piante di fagiolo di quattro settimane.


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DOI: 10.6092/issn.2465-311X/5742



         Treatment                           Total plant dry weight (mg)            Total leaf area (mm2)

         Untreated control                   4.8 ± 0.8 a                            732.4 ± 57.0 a
         T22                                 6.6 ± 1.3 cd                           1155.6 ± 292.2 bc
         TJ40                                5.1 ± 0.3 ab                           899.0 ± 132.6 ab
         Bok/S                               5.7 ± 0.6 abc                          1254.6 ± 328.3 bc
         Bok/S + Sut/S +Sut/L                6.6 ± 0.2 cd                           1669.4 ± 223.2 d
         Sut/S + Sut/L                       6.3 ± 0.7 bc                           1267.5 ± 253.8 bc
         Sut/Se + Sut/S + Sut/L              7.7 ± 1.1 d                            1309.3 ± 114.6 cd

  Means (± SD) followed by the same letter in a column are not significantly different according to LSD test (P <
  0.05).

  Table 6 reports data on the biostimulant effect of the treatments on DW and LA. Four treatments, T22,
  Bok/S+Sut/S+Sut/L, Sut/S+Sut/L and Sut/Se+Sut/S+Sut/L showed DW values significantly (P < 0.05)
  higher than the untreated control. The increase obtained with these treatments varied in a range from 31.2
  % (Sut/S + Sut/L) to 60.4 % (Sut/Se+Sut/S+Sut/L). Leaf area was significantly (P < 0.05) enhanced by
  almost all treatments, except for TJ40. The treatment that showed the highest LA value was
  Bok/S+Sut/S+Sut/L that increased LA by 127.9 %.
  In vitro assays
  Almost all products showed to reduce Rs growth in both experiments carried out with different methods
  (Tab. 7). Trichoderma T22 completely inhibited the pathogen growth in SC and WD experiments (Fig. 4).
  Trichoderma TJ40 inhibited Rs growth in a range of 80-50 % in SD assay), and 50-30 % in WD assay.
  Among EM products, Bok reduced pathogen growth less than 30 % in both assays, while Sut slightly
  reduced Rs growth in WD assay only.

       Tab. 7 - Effect of treatments on mycelial growth of Rhizoctonia solani in
       the two in vitro assay.
       Effetto di trattamenti con prodotti a base di microrganismi sulla crescita
       miceliare di Rhizoctonia solani nei due saggi in vitro.

       Treatment                 Submerged colony            Well diffusion

       Control                   -                           -
       T22                       ++++                        ++++
       TJ40                      +++                         ++
       Bok                       +                           +
       Sut                       -                           +

  Inhibition range: 80-100 % (++++), 80 - 50 % (+++), 50 – 30 % (++), < 30 % (+) significant reduction compared
  with control. Not inhibited (-).



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DOI: 10.6092/issn.2465-311X/5742




  Fig. 4 - Effect of T22 on mycelial growth of Rhizoctonia solani after 7 days of incubation on PDA in the submerged
  colony assay (a-d) and in well diffusion assay (e-f), control plates (d, g). Top (a, d, e, g) and bottom view of plates (b,
  f).
   Effetto di T22 sulla crescita miceliare di Rhizoctonia solani dopo 7 giorni d’incubazione su PDA nel saggio
  “submerged colony” (a-d) e nel saggio “well diffusion” (e-f); piastre controllo (d, g); lato superiore (a, d, e, g) e lato
  inferiore (b, f) delle piastre.

  Conclusions

  Rhizoctonia solani is a significant threat to global food security and agro-forestry industries (Hane et al.,
  2014). Chemical control methods may not be feasible nor economical for the control of many soil-borne
  pathogens including R. solani (Paulitz, 2006). Agronomic methods, such as crop rotation are not always
  effective, since many crops are susceptible to this pathogen. Moreover, conventional breeding strategies
  are scarcely employed, since only low or moderate levels of genetic resistance have been found (Bradley et
  al., 2001; Kluth & Varrelmann, 2010). Microbial compounds based on antagonistic microorganisms
  represent an environmental friendly method to control numerous soil-borne diseases on various crops

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DOI: 10.6092/issn.2465-311X/5742


  (Harman, 2000; Haas & Defago, 2005). Antagonistic microorganisms are components of specific plant
  protection products; however, microorganisms, which can potentially act as antagonists, can be contained
  in plant biostimulant compounds. European agricultural and food safety policies have integrated
  environmental considerations and are promoting the safe use of agricultural inputs. This applies to
  alternative solutions including the use of plant biostimulants and biopesticides that are used under the
  integrated crop management schemes (Report for the European Commission, 2014).
  As far as we know, no scientific studies have compared the effect of antagonistic microorganisms
  contained in specific products with that of plant biostimulant compounds in the control of soil-borne
  disease. The present study showed that R. solani disease of bean can be significantly reduced both by
  antagonistic microorganisms and by compounds which their primary function is the promotion of plant
  growth. Our findings were obtained in high inoculation pressure disease that has been much greater than
  that found in typical greenhouse conditions or in the field. Even in these conditions, the products showed
  various levels of efficacy.
  Under our experimental condition, the EM-5 Sutociu treatment alone or combined with EM Bokashi 2-fi,
  applied as recommended by manufacturer, showed almost always the same disease control as the single
  treatment with Trianum-P (Trichoderma harzianum T22), which is a well-known biocontrol agent. EM
  Bokashi 2-fi applied singly and only once to substrate, did not reduced the disease. Considering that EM-5
  Sutociu contains the same microorganisms in the same concentrations of EM Bokashi 2-fi, we can explain
  this result by assuming that they are differently formulated; EM-5 Sutociu contains microorganisms
  suspended in substances such as grappa and allium extract, and EM Bokashi 2-fi is based on bran enriched
  with microorganisms. The treatment TJ40 applied once to substrate was as effective as T22 in reducing
  disease severity. The efficacy of TJ40 in reducing R. solani disease confirmed what demonstrated in in
  vitro assay by Leonardi et al., (2014).
  The biostimulant product EM-5 Sutociu, the biofungicide T22 and Trichoderma sp. J40 probably reduced
  the disease by different modes of action, as demonstrated by the in vitro assays. In fact, as expected, T.
  harzianum T22 and Trichoderma sp. J40, even if at different levels, were able to reduce R. solani colony
  growth more actively then EM-5 Sutociu. Hence, this biostimulant exerted a disease control probably
  mainly by the enhancement of plant defence responses and by very slightly antagonism towards the
  pathogen. This is in accordance with Calvo et al. (2014) who stated by that biostimulants are not
  pesticides, because they do not act directly against pathogens, being the plant growth promotion their
  primary function. Veronesi et al., (2009), have also showed the involvement of plant defence mechanisms
  against biotic stresses.
  The biostimulant effect of EM-5 Sutociu and EM Bokashi 2-fi was confirmed also in the case of beans; in
  particular, they both increased the leaf area. Trianum-P also showed a biofertilization effect, consistently
  to what demonstrated by other authors (Windham et al., 1986; Harman et al., 2004, Matsouri et al., 2010).
  In conclusion, this study shows that the application of biostimulant products, in particular EM-5 Sutociu,
  as well the biocontrol agent T22, could simultaneously increase bean plant growth and reduce R. solani
  disease, although further studies are necessary to verify these effects under different management
  practices.

  References

  BRADLEY C.A., HARTMAN G.L., NELSON R.L., MUELLER D.S., PEDERSEN W.L. (2001) Response of
    ancestral soybean lines and commercial cultivars to Rhizoctonia root and hypocotyl rot. Plant Disease,
    85, 1091–1095.


  60
R. Roberti, F. Bergonzoni, A. Finestrelli, P. Leonardi      Micologia Italiana vol. 44 (2015) ISSN 2465‐311X
DOI: 10.6092/issn.2465-311X/5742

  BROWN P., SAA S. (2015) Biostimulants in agriculture. Frontiers in Plant Science, 6, art. 671, doi:
    10.3389/fpls.2015.00671.
  CALVO P., NELSON L., KLOEPPER J.W. (2014) Agricultural uses of plant biostimulants. Plant Soil, 383, 3–
    41.
  CARDOSO J.E., ECHANDI E. (1987). Biological control of Rhizoctonia root rot of snap bean with binucleate
    Rhizoctonia-like fungi. Plant Disease, 71, 167–170.
  EROPEAN Pesticide Database (2015). http://ec.europa.eu/food/plant/pesticides/eu-pesticides-
     database/public/?event=homepage&language=EN.
  EUROPEAN BIOSTIMULANTS INDUSTRY Council (2012a). EBIC: Promoting the biostimulant industry and
    the role of plant biostimulants in making agriculture more sustainable. http://www.biostimulants.eu/.
  EUROPEAN       BIOSTIMULANTS         INDUSTRY       Council     (2012b).     What     are    biostimulants?
    http://www.biostimulants.eu/about/what-are-biostimulants-benefits/.
  GUTIERREZ W.A., SHEW H.D., MELTON T.A. (1997). Sources of inoculum and management for
    Rhizoctonia solani damping-off on tobacco transplants under greenhouse conditions. Plant Disease, 81,
    604-606.
  HAAS D., DÉFAGO G. (2005). Biological control of soil-borne pathogens by fluorescent pseudomonads.
    Nature Reviews Microbiology, 1-10, doi: 10.1038/nrmicro1129.
  HANE J.K., ANDERSON J.P., WILLIAMS A.H., SPERSCHNEIDER J., SINGH K.B. (2014). Genome Sequencing
    and Comparative Genomics of the Broad Host-Range Pathogen Rhizoctonia solani AG8. PLoS
    Genetics 10(5): e1004281, doi:10.1371/journal.pgen.1004281.
  HARMAN G.E. (2000). Myths and dogmas in biocontrol: Changes in perceptions derived from research on
    Trichoderma harzianum T-22. Plant Disease, 84, 377 393.
  HARMAN G.E., HOWELL C.R., VITERBO A., CHET I., LORITO M. (2004). Trichoderma species-
    opportunistic, avirulent plant symbionts. Nature Reviews Microbiology, 2, 43–56.
  KLUTH C., VARRELMANN M. (2010). Maize genotype susceptibility to Rhizoctonia solani and its effect on
    sugar beet crop rotations. Crop Protection, 29, 230–238.
  LEONARDI P., RIGHINI H., ROBERTI R. (2014). Attività cellulosolitica di specie di Trichoderma e
    interazione pianta-patogeno. Micologia italiana, XLIII (1-2-3), 58-67.
  MAGALDI S., MATA-ESSAYAG S., HARTUNG DE CAPRILES C., PEREZ C., COLELLA M.T., CAROLINA
    OLAIZOLA C., ONTIVEROS Y. (2004). Well diffusion for antifungal susceptibility testing. International
    Journal of Infectious Diseases, 8, 39-45.
  MATSOURI F., BJÖRKMAN T., HARMAN G.E. (2010). Seed treatment with Trichoderma harzianum
    alleviates biotic, abiotic and physiological stresses in germinating seeds and seedlings. Phytopathology,
    100, 1213-1221.
  PANE C., VILLECCO D., ROSCIGNO G., DE FALCO E., ZACCARDELLI M. (2013). Screening of plant-derived
    antifungal substances useful for the ontrol of seedborne pathogens. Archives of Phytopathology and
    Plant Protection, doi:10.1080/03235408.2013.771458.
  PARMETER J.R.JR. editor (1970). Rhizoctonia solani, Biology and Pathology. University of California
    Press, Berkeley, Los Angeles and London, 255 pp.
  PAULITZ T.C. (2006). Low input no-till cereal production in the Pacific Northwest of the US: The
    challenges of root diseases. European Journal of Plant Pathology, 115, 271–281.
  Report for the European Commission, 2014. A Legal Framework for Plant Biostimulants and Agronomic
    Fertiliser Additives in the EU, 115 pp.
  SNEH B., BURPEE L., OGOSHI A. (1991). Identification of Rhizoctonia species. American
    Phytopathological Society Press, St. Paul, MN, USA, 133 pp.
  TRONSMO A., HJELJORD L.G. (1998). Biological control with Trichoderma species. In: Plant-Microbe
    Interactions and Biological Control (Boland G.J., Kuykendall L.D., eds.), Marcel Dekker Inc., New
    York (USA), 111-126.
  VERONESI A., PORTILLO I., ROBERTI R., COLLINA M., BRUNELLI A. (2009). Evaluation of induced
    resistance by biostimulants on tomato against Phytophthora infestans, Petria, 19, suppl. 1, 60-62.
  WINDHAM M.T., ELAD Y., BAKER R. (1986). Mechanism for increased plant growth induced by
    Trichoderma spp. Phytopathology, 76, 518-521.




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