Screening Methods for Isolation of Biocontrol Epiphytic Yeasts against Penicillium digitatum in Lemons.

Martina María Pereyra,Fabricio Fabián Soliz-Santander,Julián Rafael Dib,Rolf Daniel,Anja Poehlein,Friedhelm Meinhardt,Mariana Andrea Díaz

doi:10.3390/jof7030166

Abstract

Worldwide, the green rot caused by Penicillium digitatum is one of the most aggressive postharvest diseases of lemons. Searching for sustainable alternatives to chemical fungicides, epiphytic yeasts as potential biocontrol agents were isolated from citrus fruits using a tailor-made selective medium. For disclosing their antagonistic potential against P. digitatum, obtained isolates were subjected to direct screening methods, both in vitro and in vivo. In the course of the primary in vitro screening that comprised dual culture assays, 43 yeast strains displaying antagonistic activities against the pathogen were selected. Subsequently, such strains were subjected to an in vivo screening that consisted of a microscale test, allowing the selection of six yeast strains for further analysis. In the final screening using macroscale in vivo tests, three strains (AcL2, AgL21, and AgL2) displaying the highest efficiencies to control P. digitatum were identified. The protection efficiencies in lemons were 80 (AcL2), 76.7 (AgL21), and 75% (AgL2). Based on sequence analysis of the PCR amplified D1/D2 domains of the 26S rRNA genes, they were identified as representatives of the species Clavispora lusitaniae. Interestingly, the strains exhibited a broad action spectrum among citrus fruits as they were also able to combat the green mold disease in grapefruit and two orange varieties. The direct screening methods applied in this study favored the recovery of efficient candidates for application as biological control agents to combat fungal infestations of citrus fruits.

Highlights

Argentina is one of the main lemon-fruit-producing and exporting countries, with the Tucumán province being the lemon-producing hub contributing 78% of the total national production [1]
Lemons are frequently exposed to several phytosanitary issues that cause losses of up to 12% of the fresh fruit [1]; the green mold disease caused by Penicillium digitatum is the most important postharvest fungal infestation [2]
Two isolation strategies were adopted: a non-selective method in which the sources of antagonists were the fruit washing station (FWS) and the essential oil extraction station (EOES); and a method whose selection was based on wounds of uninfected lemons, treated first with the foregoing samples and with the pathogen

Summary

Introduction

Argentina is one of the main lemon-fruit-producing and exporting countries, with the Tucumán province being the lemon-producing hub contributing 78% of the total national production [1]. Chemical control by the use of fungicides has been employed to control postharvest decays [3,4,5]. Due to the emergence of fungicide-resistant pathogens in citrusproduction areas [3,6], the upcoming bans on the use of postharvest fungicides [7], and the public demand to reduce and avoid pesticides, are urgent needs to develop sustainable alternatives and safer technologies for controlling postharvest rots. Drawbacks of fungicides have led markets to increase their demands to restrict chemical compounds, thereby promoting organic production of fruits and vegetables [8]. The use of naturally occurring epiphytic antagonists on fruit surfaces as biological control agents against postharvest pathogens is one of the most feasible alternatives to traditional fungicides. Selecting epiphytic antagonists from the environment where they are intended to be applied implies better adaptive advantages of the microorganisms [9,10]

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Conclusion