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