Elsevier

Biological Control

Volume 150, November 2020, 104246
Biological Control

Semi-commercial testing of regional yeasts selected from North Patagonia Argentina for the biocontrol of pear postharvest decays

https://doi.org/10.1016/j.biocontrol.2020.104246Get rights and content

Highlights

  • P. membranifaciens and V. victoriae proved to be safe for human health.

  • Biomass production (22 L) reached 1 × 1010 CFUmL−1 in a low cost molasses based medium.

  • Antagonist yeasts applied on fruit reached 1 × 107–108 CFU cm−2 after 90 d of cold storage.

  • Yeasts controlled 78–100% of decays on pears stored during 90 d at semi-commercial scale.

  • CaCl2 increased the antagonist effect of V. victoriae.

Abstract

The efficacy of two regional yeasts, Pichia membranifaciens NPCC 1250 and Vishniacozyma victoriae NPCC 1263, for controlling pear postharvest decay was evaluated at large scale (semi-commercial conditions) in two different packing houses. The human safety of the two yeast strains was initially assayed by assessing growth at body temperature, pseudohyphal growth, phospholipases production, and growth in simulated gastric juice. Subsequently, yeasts biomass was produced in a bioreactor (22 L) containing 18 L of culture medium based on sugarcane molasses plus urea. The produced biomass, with/without CaCl2 (2% w/v) addition, was used in biocontrol experiments carried out at semi-commercial scale on Beurre d'Anjou and Packham’s Triumph pears. Vishniacozyma victoriae NPCC 1263 plus CaCl2 showed high performance on controlling decay caused by Penicillium expansum and Botrytis cinerea. On Beurre d'Anjou cv, P. expansum and B. cinerea incidence was reduced by 70% and 100%, respectively, after 90 days of commercial storage. On Packham’s Triumph the incidence of the two decay forms was 63% after 150 days. The two antagonistic yeasts were able to establish and colonize the surface of the fruits, with an estimated increase in the population density of approximately three orders of magnitude during the postharvest period. This work is the first to evaluate and demonstrate the efficacy of local biocontrol experiments with regional yeasts under semi-commercial conditions.

Introduction

Fungal spoilage causes significant losses during fruit storage, affecting up to 25% of the total production in industrialized countries and over 50% in developing countries (Nunes, 2012). The production of pome fruits (apples and pears) is one of the main economic activities in Northern Patagonia (Argentina). Penicillium expansum and Botrytis cinerea are the most important pathogens of pome fruits worldwide (Chand-Goyal and Spotts, 1997, Yu et al., 2007, Nunes, 2012, Usall et al., 2016), although other fungi, e.g. Alternaria sp. and Cladosporium sp., are also relevant fruit pathogens (Sugar and Basile, 2008, Lutz et al., 2017). Postharvest fungal infections have been linked to high moisture levels, excessive nutrients, low pH values, and decreased intrinsic decay resistance with maturity (Droby et al., 2016).

Postharvest pathogens are mainly controlled with chemical fungicides applied in the packing house. However, in recent years the use of synthetic fungicides has been reduced due to: (i) pathogen resistance to many key fungicides; (ii) poor fungicide efficacy; (iii) appearance of new pathogen biotypes; (iv) increased levels of fungicide residues in the fruits; (v) toxicological risk related to human health, and (vi) negative environmental impact (Wisniewski et al., 2016, Dukare et al., 2018). Additionally, the growing interest in organic production has led to the search for alternative methods for controlling postharvest diseases around the world. In this context, biological control using microbial agents represents an exciting research topic. The use of antagonist microorganisms offers some advantages in comparison to chemical control, such as: (a) no residual toxicity; (b) environmental friendliness; (c) safe applicability; (d) easy delivery; and (e) low production costs (Bonaterra et al., 2012, Wisniewski et al., 2016).

Over the past few decades many antagonistic microorganisms have been selected for the control of postharvest diseases. However, only a few of them have been successfully formulated and commercialized. Recently, Wisniewski et al. (2016) listed commercially available antagonists for pome fruits: Candifruit (Candida sake, Sipcam-Inagra, Spain), Aspire (Candida olephila, Ecogen USA), Nexy (Candida oleophila, Lesaffre Belgium), Yield Plus (Cryptococcus albidus, Lallemand South Africa), Boni Protect (Aureobasidium pullulans, Bioprotect, Germany), and Shemer (Metschnikowia fructicola, Bayer, Germany). Among them, only Nexy, Boni Protect and Shemer have been reported as currently in use.

Prior to commercial formulation of a biological control agent, it is necessary to establish its potential health risks, as many microorganisms, including yeasts, are potential pathogens for humans (de Llanos et al., 2006, Klingberg et al., 2008). Some key methods to test pathogenicity are the ability to grow at body temperature conditions (Antley and Hanzen, 1988, Nadeem et al., 2013), evaluation of in vitro pseudohyphal growth, invasive growth on solid agar, growth in gastrointestinal tract or in simulated gastric juice (Alegre et al., 2013), and secretion of degradative enzymes such as proteinases and phospholipases (Steenbergen and Casadevall, 2003, Cafarchia et al., 2008). Consideration must also be given to the presence of virulence factors in the microorganisms, which can affect their use as fruit biocontrol agents (BCAs).

Recent studies carried out in our laboratory have proposed a strategy to obtain and evaluate potential postharvest BCAs in pears. These were based on in situ assays carried out under cold storage conditions and testing against local isolates of P. expansum and B. cinerea, selected for their high aggressiveness (Lutz et al., 2012, Lutz et al., 2013). As a result, two regional yeast strains, named Pichia membranifaciens NPCC 1250 and Vishniacozyma victoriae NPCC 1263, were selected and their efficacy evaluated at semi-commercial scale.

To obtain high amounts of the selected BCAs to be used in semi-commercial scale assays it is necessary to scale-up the production process using a low-cost culture medium. Some aspects of the fermentation process, such as culture medium components, have been addressed in investigations carried out in shake flasks and/or small bioreactors (not larger than 5 L; laboratory scale) (Abadias et al., 2003, Pelinski et al., 2012). However, some aspects of the scale-up process remain insufficiently considered (Droby et al., 1998, Long et al., 2007, Janisiewicz et al., 2008). The objectives of this work were: (i) to evaluate the safety of P. membranifaciens NPCC 1250 and V. victoriae NPCC 1263 on human health; (ii) to develop a bench-scale fermentation process (20 L) based on cane molasses, a readily-available industrial waste, as a substrate for production of the candidate BCAs; (iii) to evaluate the yeasts biocontrol efficacy against two of the most relevant pear pathogens in semi-commercial packing houses after long cold storage; (iv) to study the effect of spray application on yeast viability and its ability to colonize the fruits after long cold storage.

Section snippets

Antagonistic yeasts

Pichia membranifaciens NPCC 1250 and Vishniacozyma victoriae NPCC 1263 (Gen Bank access numbers MN 848352 and MN 848499), were selected for their antagonistic behavior in situ against P. expansum and B. cinerea (Lutz et al., 2012, Lutz et al., 2013) and used in the semi-commercial assays. The yeasts were preserved in glycerol 20% (v/v) and stored at −20 °C in the North Patagonian Culture Collection (NPCC), Neuquén, Argentina. Fresh yeast culture was prepared by growth on Glucose Peptone

Human safety assays

Negative results were obtained for both Pichia membranifaciens NPCC 1250 and Vishniacozyma victoriae NPCC 1263 in all the human safety assays. No viable yeasts were recovered after exposure to simulated gastric stress or growth at 37 ± 2 °C, nor for different incubation temperatures or sampling times (data not shown). Moreover, the antagonist yeasts were able to produce neither phospholipases nor pseudohyphal growth.

Biomass production

Biomass production of the two yeast strains was carried out in a 20 L

Discussion

One of the key steps in the selection of yeast strains as BCAs is the determination of their effectiveness in pilot semi-commercial and/or large-scale tests, considering all the factors intervening in the system (Nunes, 2012). Scaling-up the trials requires large amounts of yeast biomass, which must be produced in bioreactors using cost-effective culture media to obtain high yields while simultaneously preserving the antagonistic activity of the candidate bioagent (Abadias et al., 2003). To

CRediT authorship contribution statement

María Cecilia Lutz: Conceptualization, Methodology, Investigation, Visualization, Writing - review & editing, Writing - original draft. Christian Ariel Lopes: Conceptualization, Methodology, Investigation, Visualization, Writing - review & editing. María Cristina Sosa: Conceptualization, Methodology, Investigation, Visualization, Writing - review & editing. Marcela Paula Sangorrín: Conceptualization, Methodology, Investigation, Visualization, Writing - review & editing, Resources, Supervision,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We would like to thank La Deliciosa S.A. (Centenario) and Moño Azul S.A. (El Chañar), who kindly provided us with fruit, packing materials, staff assistance, and cold storage units to carry out this work.

Formatting of funding sources

This work was supported by funds from Universidad Nacional del Comahue, ANPCyT PICT 2014-2780, and CONICET PUE0067.

Ethical approval

This article does not contain any studies involving human participants or animals.

References (51)

  • Y. Shao et al.

    The chemical treatments combined with antagonistic yeast control anthracnose and maintain the quality of postharvest mango fruit

    J. Integrat. Agricult.

    (2019)
  • J.N. Steenbergen et al.

    The origin and maintenance of virulence for the human pathogenic fungus Cryptococcus neoformans

    Microbes Infect.

    (2003)
  • D. Sugar et al.

    Timing and sequence of postharvest fungicide and biocontrol agent applications for control of pear decay

    Postharvest Biol. Technol.

    (2008)
  • J. Usall et al.

    Biological control of postharvest diseases on fruit: a suitable alternative?

    Curr. Op. Food Sci.

    (2016)
  • I. Viñas et al.

    Biological control of major postharvest pathogens on apple with Candida sake

    Int. J. Food Microbiol.

    (1998)
  • M. Wisniewski et al.

    Alternative management technologies for postharvest disease control: The journey from simplicity to complexity

    Postharvest Biol. Technol.

    (2016)
  • T. Yu et al.

    Biocontrol of blue and gray mold diseases of pear fruit by integration of antagonistic yeast with salicylic acid

    Int. J. Food Microbiol.

    (2007)
  • T. Yu et al.

    Integrated control of blue mold in pear fruit by combined application of chitosan, a biocontrol yeast and calcium chloride

    Postharvest Biol. Technol.

    (2012)
  • M. Abadias et al.

    Optimization of growth conditions of postharvest biocontrol agent Candida sake CPA-1 in a lab-scale fermenter

    Int. J. Appl. Microbiol.

    (2003)
  • P. Antley et al.

    Role of yeast cell growth temperature on Candida albicans virulence in mice

    Infection and Immunity

    (1988)
  • A. Bonaterra et al.

    Prospects and limitations of microbial pesticides for control of bacterial and fungal pomefruit tree diseases

    Trees

    (2012)
  • C. Cafarchia et al.

    Phospholipase activity of yeasts from wild birds and possible implications for human disease

    Med Mycol.

    (2008)
  • S. Carmona-Hernandez et al.

    Biocontrol of postharvest fruit fungal diseases by bacterial antagonists: a review

    Agronomy

    (2019)
  • Conway, W., Gross, K.C., Boyer, C. D. dnd Sams, C. E. 1988. Inhibition of Penicillium expansum polygalacturonase...
  • P. Creemers

    Lutte contre les maladies de conservation: situation actuelle et perspectives nouvelles

    Le Fruit Belge.

    (1998)
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    Present address: Centro de Investigación en Toxicología Ambiental y Agrobiotecnología del Comahue (CITAAC, CONICET-UNCo); subsede Instituto de Biotecnología Agropecuaria del Comahue (IBAC) Facultad de Ciencias Agrarias, UNCo. Km 11, 5 Ruta 151, Cinco Saltos, Río Negro, Argentina.

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