title: Postharvest application of a novel chitinase cloned from Metschnikowia fructicola and overexpressed in Pichia pastoris to control brown rot of peaches.
abstract: Metschnikowia fructicola strain AP47 is a yeast antagonist against postharvest pathogens of fruits. The yeast was able to produce chitinase enzymes in the presence of pathogen cell wall. A novel chitinase gene MfChi (GenBank accession number HQ113461) was amplified from the genomic DNA of Metschnikowia fructicola AP47. Sequence analysis showed lack of introns, an open reading frame (ORF) of 1098 bp encoding a 365 amino acid protein with a calculated molecular weight of 40.9 kDa and a predicted pI of 5.27. MfChi was highly induced in Metschnikowia fructicola after interaction with Monilinia fructicola cell wall, suggesting a primary role of MfChi chitinase in the antagonistic activity of the yeast. The MfChi gene overexpressed in the heterologous expression system of Pichia pastoris KM71 and the recombinant chitinase showed high endochitinase activity towards 4-Nitrophenyl β-d-N,N',N″-triacetylchitotriose substrate. The antifungal activity of the recombinant chitinase was investigated against Monilinia fructicola and Monilinia laxa in vitro and on peaches. The chitinase significantly controlled the spore germination and the germ tube length of the tested pathogens in PDB medium and the mycelium diameter in PDA. The enzyme, when applied on peaches cv. Redhaven, successfully reduced brown rot severity. This work shows that the chitinase MfChi could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short shelf life, and confirms that P. pastoris KM71 is a suitable microorganism for cost-effective large-scale production of recombinant chitinases.
authors: Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy; DiSAFA - Dept. Agricultural, Forestry and Food Sciences, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy.
authors: Houda
authors: H
authors: Banani
authors: Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy; DiSAFA - Dept. Agricultural, Forestry and Food Sciences, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy. Electronic address: davide.spadaro@unito.it.
authors: Davide
authors: D
authors: Spadaro
authors: Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy.
authors: Dianpeng
authors: D
authors: Zhang
authors: Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy; DiSAFA - Dept. Agricultural, Forestry and Food Sciences, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy.
authors: Slavica
authors: S
authors: Matic
authors: Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy.
authors: Angelo
authors: A
authors: Garibaldi
authors: Centre of Competence for the Innovation in the Agro-environmental Sector - AGROINNOVA, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy; DiSAFA - Dept. Agricultural, Forestry and Food Sciences, University of Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO, Italy.
authors: Maria Lodovica
authors: ML
authors: Gullino
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D001203:Ascomycota / Q000187:drug effects*; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism*; D005516:Food Microbiology; D005638:Fruit / Q000382:microbiology*; D055163:Metschnikowia / Q000201:enzymology* / Q000235:genetics; D025282:Mycelium / Q000187:drug effects; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics; D027861:Prunus / Q000382:microbiology*
chemical_list: D000935:Antifungal Agents; D002688:Chitinases
keywords: Cloning; Monilinia fructicola; Monilinia laxa; Pichia pastoris; Recombinant expression; Reverse transcription quantitative PCR (RT-qPCR)