Exploring the Extracellular Macromolecular Composition of Crude Extracts of Penicillium rubens Strain 212 for Elucidation Its Mode of Action as a Biocontrol Agent

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Penicillium rubens strain 212 (PO212) acts as an inducer of systemic resistance in tomato plants. The effect of crude extracellular extracts of PO212 on the soil-borne pathogen Fusarium oxysporum f. sp. lycopersici has been evaluated. Evidence of the involvement of soluble, thermo-labile, and proteinase-inactivated macromolecules present in PO212 crude extracts in the control of Fusarium vascular disease in tomato plants was found. Proteomic techniques and the availability of the access to the PO212 genome database have allowed the identification of glycosyl hydrolases, oxidases, and peptidases in these extracellular extracts. Furthermore, a bioassay-guided fractionation of PO212 crude extracellular extracts using an integrated membrane/solid phase extraction process was set up. This method enabled the separation of a PO212 crude extracellular extract of seven days of growth into four fractions of different molecular sizes and polarities: high molecular mass protein fraction >5 kDa, middle molecular mass protein fraction 5–1 kDa, low molecular mass metabolite fraction, and nutrients from culture medium (mainly glucose and minerals). The high and middle molecular mass protein fractions retained disease control activity in a way similar to that of the control extracts. Proteomic techniques have allowed the identification of nine putatively secreted proteins in the high molecular mass protein fraction matching those identified in the total crude extracts. Therefore, these enzymes are considered to be potentially responsible of the crude extracellular extract-induced resistance in tomato plants against F. oxysporum f. sp. lycopersici. Further studies are required to establish which of the identified proteins participate in the PO212’s action mode as a biocontrol agent.
This study was supported by funds from RTA2013-00060-C050-00, RTA2017-00019-C03-01 and RTA2017-00019-C03-02 (Plan Nacional de I+D, MINECO and MCINNU, Spain), to IL and grants BFU2015-66806-R (MINECO) and RTI2018-094263-B-100 (MICIU/AEI) to EAE both partially supported by FEDER/EU. M. Carreras received a scholarship from SGIT-INIA. Alba Gutiérrez and Silvia Moreno thank to Consejería de Educación e Investigación from the Madrid’s Community for the financial support of contracts PEJ-2018-AI/BIO-11720 and PEJD-2018-POST/BIO-8797, respectively.