Garello, M.

Schiavon, G.

Spadaro, D.

English

Institutional Research Information System University of Turin

14 October 2023AperTO - Archivio Istituzionale Open Access dell'Università di TorinoOriginal Citation:Efficacy of biofumigation with essential oils in the control of postharvest rots on nectarinesTerms of use:Open Access(Article begins on next page)Anyone can freely access the full text of works made available as "Open Access". Works made availableunder a Creative Commons license can be used according to the terms and conditions of said license. Useof all other works requires consent of the right holder (author or publisher) if not exempted from copyrightprotection by the applicable law.Availability:This is a pre print version of the following article:This version is available http://hdl.handle.net/2318/1891336 since 2023-02-08T19:39:04ZEfficacy of biofumigation with essential oils in the control of postharvest rots on nectarinesMarco Garello1,2, Giada Schiavon1,2, Davide Spadaro1,21 Dept.  Agricultural,  Forest  and  Food  Sciences  (DISAFA),  University  ofTorino, Largo Braccini 2, 10095 Grugliasco (TO), Italy;2 AGROINNOVA –  Centre of  Competence for  the  Innovation  in  the  Agro-environmental  Sector,  University  of  Torino,  Largo  Braccini  2,  10095Grugliasco (TO), Italy.AbstactThe  most  common  postharvest  pathogens  on  nectarines  areMonilinia  fructicola (G.  Winter)  Honey  and  M.  laxa  (Aderh.  &Ruhland)  Honey,  the  agents  of  brown  rot.  The  efficacy  of  fiveessential  oils  (EOs)  at  0.1%,  0.5%  and  1%  concentration  wasevaluated in vitro against M. fructicola strain CVG1539 after 14 daysat  25°C.  Subsequently,  biofumigation  was  realized  with  the  threemost  effective  EOs  (fennel,  basil,  and  lemon),  this  time  at  2%concentration. Disease incidence was evaluated after a longer storage(28 days) at 1°C and after further 5 days shelf-life at 20°C. At the endof  storage,  nectarines  treated  with  EOs  showed  a  significantreduction in rots, which were caused by Monilinia spp. At the end ofshelf-life,  Monilinia  spp.,  Penicillium  spp.,  Botrytis  spp.,  Alternariaspp., and Rhizopus spp. were isolated from the rots. Fruit quality andmicrobiome composition were analysed at harvest, after 28 days ofstorage, and after 5 days of shelf-life.  The firmness in the treatedfruits  with  EOs  was  higher  compared  to  the  untreated  ones.Treatments with biofumigation with EOs are promising tools for thecontrol of postharvest rots.Keywords:  biofumigation,  essential  oils,  Monilinia  spp.,  nectarine,postharvest disease. INTRODUCTIONDespite  a  steady decline  over  the  last  three  decades,  peaches  andnectarines still represent the third most important fruit in Italy, with overone million tonnes in 2020 (FAOSTAT, 2020). Several fungal pathogens areassociated to post-harvest production loss,  such as  Monilinia spp.  (causalagent of brown rot),  Botrytis cinerea  (causal agent of grey mould decay),Penicillium spp.  (causal  agent  of  blue mould  decay),  Rhizophus nigricans(causal agent of  Rhizopus decay), but also  Alternaria  spp.,  Aspergillus  spp.and  Mucor  spp.  (Mari  et  al.,  2019).  Among  all  mentioned  pathogens,Monilinia  spp.  is  the  most  common  and  economically  important,  withproduction  loss  up  to  100%  in  favourable  environmental  conditions(Papavasileiou  et  al.,  2020).  In  recent   years,  several  control  strategiesagainst  post-harvest disease have been proposed as alternatives to fungicidetreatments with fludioxonil, such as hot water dipping (Spadoni et al., 2013)or the use of biocontrol yeasts (Mari  et al., 2012). Another line of researchhas  focused  on  using  essential  oils  (EOs),  which  are  oily  substancesextracted from several plant species belonging to order Lamiales, Asterales,Rutales  and Apiales.  The antimicrobial  efficacy  of  EOs is  associated withseveral  molecular  mechanisms,  which  include  alteration  of  cell  wall  andmembrane  permeability,  impairment  of  quorum sensing  mechanisms  andchanges  in  gene  expression  patterns,  including  those  of  mycotoxinassociated genes (Maurya et al., 2021). Moreover, EOs have been shown toact  indirectly by priming host  defence mechanisms  (Banani  et  al.,  2018).Despite  this,  practical  application  of  these  substances  is  hampered  byphytotoxicity phenomena even at moderate EO concentration  (Lopez-Reyeset al., 2013). One of the possible solutions for this problem is biofumigation,where EOs are slowly released in the atmosphere from dispensers. In thisstudy, we first analyzed the in vitro efficacy of five essential oils against M.fructicola,  previously  considered  a  quarantine  pathogen  in  Europe(Pellegrino  et  al.,  2009)  and  now  the  main  postharvest  pathogen  onnectarines in Italy (Franco Ortega  et al.,  2019). We then chose the threemost  effective  ones  and  run  an  in  vivo  biofumigation  test  on  naturallyinoculated  nectarines.  Our  aim  was  to  find  suitable  candidates  for  thetreatment  of  nectarines  against  post-harvest  rots, to  validate  ourbiofumigation protocol and to quantify the impact of the treatment on fruitquality parameters.MATERIALS AND METHODSPreparation of EOs platesEssential oils plates for the in vitro inhibition test were prepared bypouring a slightly modified PDA medium added with essential oils in petridishes. In particular, 18.5 g of standard PDA formulate were added to 499 mlof  deionized  water  and  1  ml  of  Tween-20,  which  were  then  autoclaved.Afterwards,  media  with 0.1%,  0.5% and 1% EO content  were  created byadding 0.5 ml, 2.5 ml and 5 ml of essential oils to 499.5 ml, 497.5 ml and 495ml of PDA medium, respectively. Selected oil for this step were thyme, basil,oregano, fennel and lemon (Flora, Lorenzana, Italy).Inoculum preparation and in vitro inhibition testM. fructicola strain CVG1539 was grown on MEA medium for 21 days.Mycelia plugs were removed and transferred in new MEA plates. Each MEAplate was then joined face-to-face with an essential oil plate in a sandwichconfiguration and closed with parafilm. Plates were incubated at 25°C for 14days and subsequently the growth diameter of the pathogen was measuredfor the different treatments. Results were then compared to the untreatedcontrols  prepared  in  the  same  way  using  PDA.  For  each  treatment,  5biological replicates were measured.In vivo inhibition test of nectarinesFor  the  in  vivo  test,  fennel,  basil  and  lemon  essential  oils  wereselected. EO dispenser plates were prepared as the in vitro inhibition testplates, but with a 2% EO. For each treatment, 3 replicates consisting of 30uninoculated  nectarines  ‘Big  Top’,  were  set  up  in  plastic  boxes  inside  acabinet. Treatment was applied by placing 6 EO dispensers in each cabinet.In addition to these treatments, an untreated control and a chemical controltreated  with  Switch  (Syngenta  Italia  S.p.A.,  cyprodinil,  a.i.:  37.5%;fludioxonil, a.i.: 25%) formed by 3 boxes of 30 fruit each, were also set up.All the treatments were kept inside the cabinets at 1 ± 1 °C for 28 days andthen transitioned to shelf-life conditions (20 ± 5 °C) for 5 days. Rot incidencewas measured at the end of storage and the end of shelf-life.Quality analysesQuality analyses were performed at harvest (t0 ), at the end of storage(t0+28  ) and at the end of shelf-life (t0+33  ). Selected quality parameters werefruit firmness, total soluble solids and titratable acidity. Fruit firmness wasmeasured using a Fruit Texture Analyser penetrometer (FTA, Turoni, Italy)with  a  8  mm  tip.  For  each  treatment,  5  fruit  were  measured  twice  onopposite  sides  of  their  equatorial  region  and  the  results  mediated.  Totalsoluble  solids,  in  particular  sugars,  were  measured  using  a  NR151refractometer (Rose Scientific Ltd, Canada). For each treatment, the juice of3 groups of 3 fruit (one per replication) was extracted and a 1 ml aliquot wastransferred on the instrument for the measurement. Finally, titratable aciditywas measured using 6 g of each replication juice, which were added to 50 mlof water. Titration was carried out adding a 0.1 N NaOH solution up to a 8.2pH endpoint, as measured by a FP20-Std-Kit FiveEasy Plus pHmeter (MettlerToledo,  Italy).  Based  on  consumed  NaOH  volume,  starting  acidity  wascalculated using the formula:VNaOH∗0.0067∗1006where VNaOH are the millilitres of NaOH, 0.0067 is the acidity factor ofmalic acid and 6 is the grams of juice.RESULTS AND DISCUSSIONResults for the in vitro inhibition test (table 1) show that, with theexception of 0.1% lemon, all considered EOs caused a significant reductionin M. fructicola growth compared to the untreated control, with the highestreduction associated to thyme, basil, fennel, and oregano. These results arein accordance with previous studies on essential oil carried out both in vitroand in vivo (Carovic-Stanko et al., 2012; Lopez-Reyes et al., 2013; Santoro etal., 2018; Fontana et al., 2021).Table 1: percentage reduction in colony diameter for different treatments, compared to the untreated control. Significance groups were determined by one-way ANOVA followed by a Duncan post-hoc test, both with alpha < 0.05 .Treatment Diameter reductioncompared to control (%)Significance group(Duncan test)Thyme 1% - 100 aThyme 0.5% - 100 aThyme 0.1% - 100 aBasil 1% - 100 aBasil 0.5% - 96 aBasil 0.1% - 56 bOregano 1% - 100 aOregano 0.5% - 100 aOregano 0.1% - 100 aFennel 1% - 100 aFennel 0.5% - 100 aFennel  0.1% - 28 cLemon 1% - 34 cLemon 0.5% - 16 dLemon 0.1% -9 deControl / eIn the in vivo experiment on nectarines (table 2), all essential oil treatmentsshowed an efficacy comparable with the chemical treatment in reducing rotincidence during storage. By the end of shelf-life, this efficacy was lost, withrot  incidence  values  comparable  to  the  untreated  control.  A  possibleexplanation for this phenomenon is that the shelf-life was spent outside thecabinets, without atmosphere rich in essential oils. For this reason, we couldhypothesize a fungistatic more than fungicidal activity. Table 2: rot incidence values measured at the end of storage and at the end of shelf-life  for nectarines treated with essential oils, as well as an untreated control and a chemical control. Significance groups were determined by one-way ANOVA followed by a Duncan post-hoc test, both with alpha < 0.05Treatment Rot incidence atthe end ofstorage (%)Rot incidence atthe end of shelf-life (%)Untreatedcontrol9%b 11%aChemicalcontrol1%a 10%aLemon EO(2%)3%a 11%aFennel EO(2%)2%a 12%aBasil EO (2%) 4%a 6%aThe most frequently isolated genus from rotten fruit was  Monilinia  (84%),followed by  Penicillium  (7%),  Botrytis (4%),  Alternaria (2%) and  Rhizopus(2%).Fruit  quality  parameters  (figure 1,  figure 2 and  figure 3)  were  almostunaffected  by  EO  treatments.  No  statistically  significant  difference  wasfound in any time point for either titratable acidity or total soluble solids,whereas a statistically significant difference was found at the end of shelf-life,  where  treatments  with  all  essential  oils  resulted  in  increased  fruitfirmness compared to the untreated control. Previous studies showed thatapplication of essential oils through biofumigation in peaches and nectarinesnot only preserves fruit quality, but can also result in increased nutritionalqualities, such as an higher title of vitamin C (Santoro et al., 2018).Figure 1: fruit firmness values measured for the three essential oil treatments, as well as forthe untreated control and chemical control, at harvest, after 28 days of storage and after 5days of shelf-life. The asterisk indicates the presence of a statistically significant difference, asmeasured by a one-way ANOVA followed by a Duncan posthoc test, both with alpha<0.05 .Figure 2: total soluble solids values measured for the three essential oil treatments, as well asfor the untreated control and chemical control, at harvest, after 28 days of storage and after 5days of shelf-life. No statistically significant difference was found, based on a one-way ANOVAwith alpha<0.05 .Figure 3: titratable acidity values measured for the three essential oil treatments, as well as forthe untreated control and chemical control, at harvest, after 28 days of storage and after 5 days of shelf-life. No statistically significant difference was found, based on a one-way ANOVA with alpha<0.05 .CONCLUSIONSFive  essential  oils  (thyme,  basil,  oregano,  fennel  and  lemon)  wereselected to test in vitro efficacy against M. fructicola growth. Three of them(lemon, fennel and basil) were then chosen to test biofumigation efficacy inpreventing  natural  M.  fructicola  inoculum  development  in  nectarines.Results  demonstrate  that  post-harvest  control  of  M.  fructicola can  beachieved  by  biofumigation  with  essential  oils.  Additional  studies  will  benecessary to improve the efficacy of this strategy and simplify the process, inparticular regarding the necessity of a controlled atmosphere.ACKNOWLEDGEMENTSThis  work  was  conducted  within  the  framework  of  the  PRIMAStopMedWaste project, which is funded by PRIMA, a programme supportedby the European Union.Literature citedBanani, H., Olivieri, L., Santoro, K., Garibaldi, A., Gullino, M. L., & Spadaro,D.  (2018).  Thyme  and  Savory  Essential  Oil  Efficacy  and  Induction  ofResistance against Botrytis cinerea through Priming of Defense Responses inApple. Foods, 7(2), 11. https://doi.org/10.3390/foods7020011Carović-Stanko,  K.,  Fruk,  G.,  Satovic,  Z.,  Ivić,  D.,  Politeo,  O.,  Sever,  Z.,Grdiša, M., Strikić, F., & Jemrić, T. (2013). Effects of Ocimum spp. essentialoil on Monilinia laxa in vitro, Journal of Essential Oil Research, 25 (2), 143-148. https://doi.org/10.1080/10412905.2012.751057.FAO.  FAOSTAT.  License:  CC  BY-NC-SA  3.0  IGO.  Extracted  from:https://www.fao.org/faostat/en/#data. Data of Access: 27-07-2022Fontana, D. C., Dourado-Neto, D., Milani Pretto, M., Mariotto, A. B., Caron,B. O., Kulczynski, S. M., & Schmidt, D. (2021). Using essential oils to controldiseases  in  strawberries  and  peaches.  International  Journal  of  FoodMicrobiology,  338:  108980.https://doi.org/10.1016/j.ijfoodmicro.2020.108980Franco Ortega, S., Bustos Lopez, M.P., Nari, L., Boonham, N., Gullino, M. L.,& Spadaro D. (2019).  Rapid detection of Monilinia fructicolaand Monilinialaxa  on  peaches  and  nectarines  using  loop-mediated  isothermalamplification.  Plant Disease,  103: 2305-2314. https://doi.org/10.1094/PDIS-01-19-0035-RELopez-Reyes, J.  G.,  Spadaro, D., Prelle, A.,  Garibaldi, A.,  & Gullino, M. L.(2013). Efficacy of plant essential oils on postharvest control of rots causedby fungi on different stone fruits in vivo.  Journal of Food Protection,  76(4),631–639. https://doi.org/10.4315/0362-028X.JFP-12-342Mari,  M.,  Martini,  C.,  Guidarelli,  M.,  &  Neri,  F.  (2012).  Postharvestbiocontrol of Monilinia laxa, Monilinia fructicola and Monilinia fructigena onstone fruit  by two  Aureobasidium pullulans strains.  Biological Control,  60(2): 132-140. https://doi.org/10.1016/j.biocontrol.2011.10.013.Mari, M., Spadaro, D., Casals, C., Collina, M., De Cal, A., & Usall, J. (2019).Postharvest  Diseases  of  Stone  Fruits.  In:  Postharvest  pathology  of  freshhorticultural produce (Palou L., Smilanick J.L., eds.), CRC Press, Boca Raton,FL, USA, 111-140.Maurya, A., Prasad, J., Das, S., & Dwivedy, A. K. (2021) Essential Oils andTheir Application in Food Safety. Frontiers in Sustainable Food Systems 5 :653420. https://doi.org/10.3389/fsufs.2021.653420. Pellegrino,  C.,  Gullino,  M.  L.,  Garibaldi,  A.,  &  Spadaro,  D.  (2009).  Firstreport of  brown rot of  stone fruit  caused by  Monilinia fructicola in Italy.Plant Disease, 93: 668.https://doi.org/10.1094/PDIS-93-6-0668APapavasileiou, A., Tanou, G., Samaras, A., Samiotaki, M., Molassiotis, A., &Karaoglanidis, G. (2020). Proteomic analysis upon peach fruit infection withMonilinia fructicola and M. laxa identify responses contributing to brown rotresistance.  Scientific  reports,  10(1):  7807.  https://doi.org/10.1038/s41598-020-64864-xSantoro,  K.,  Maghenzani,  M.,  Chiabrando,  V.,  Bosio,  P.,  Gullino,  M.  L.,Spadaro, D., & Giacalone, G. (2018). Thyme and Savory Essential Oil VaporTreatments Control Brown Rot and Improve the Storage Quality of Peachesand  Nectarines,  but  Could  Favor  Gray  Mold.  Foods,  7(1):  7.https://doi.org/10.3390/foods7010007Spadoni, A., Neri, F., Bertolini, P., & Mari, M. (2013).  Control of Moniliniarots on fruit naturally infected by hot water treatment in commercial trials.Postharvest  Biology  and  Technology,  86:  280-284.https://doi.org/10.1016/j.postharvbio.2013.07.011.

Efficacy of biofumigation with essential oils in the control of postharvest rots on nectarines

https://iris.unito.it/bitstream/2318/1891336/1/Garello%20et%20al.%202022%20-%20preprint%20AH.pdf

Efficacy of biofumigation with essential oils in the control of postharvest rots on nectarines

Abstract

Similar works

Full text

Available Versions

Institutional Research Information System University of Turin