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.

Fungal rots are one of the main causes of large economic losses and deterioration in the quality and nutrient composition of fruits during the postharvest stage. The yeast Clavispora lusitaniae 146 has previously been shown to efficiently protect lemons from green mold caused by Penicillium digitatum. In this work, the effect of yeast concentration and exposure time on biocontrol efficiency was assessed; the protection of various citrus fruits against P. digitatum by C. lusitaniae 146 was evaluated; the ability of strain 146 to degrade mycotoxin patulin was tested; and the effect of the treatment on the sensory properties of fruits was determined. An efficient protection of lemons was achieved after minimum exposure to a relatively low yeast cell concentration. Apart from lemons, the yeast prevented green mold in grapefruits, mandarins, oranges, and tangerines, implying that it can be used as a broad-range biocontrol agent in citrus. The ability to degrade patulin indicated that strain 146 may be suitable for the control of further Penicillium species. Yeast treatment did not alter the sensory perception of the aroma of fruits. These results corroborate the potential of C. lusitaniae 146 for the control of postharvest diseases of citrus fruits and indicate its suitability for industrial-scale fruit processing.