Identification of Penicillium expansum causing postharvest blue mold decay of apple fruit

Abstract

Penicillium expansum (Link) Thom. is one of the most important postharvest pathogens of apple fruit worldwide. It causes blue mold, a decay that can lead to significant economic losses during storage, which can also impact fruit destined for processing due to the production of carcinogenic mycotoxin patulin. Apple fruit cvs. Idared, Golden Delicious and Braeburn with blue mold symptoms were collected from five storage facilities in Serbia and nine fungal isolates were obtained. Pathogenicity of the isolates was tested and proven by artificial inoculation of healthy apples cv. Idared. In order to identify the causal agents of decay, morphological and molecular methods were used. Colony morphology and microscopic features were observed on differential media, and isolates were tested for the production of cyclopiazonic acid. Molecular analysis included PCR amplification with species specific primers for P. expansum based on polygalacturonase gene (Pepg1), universal primers for internal transcribed spacer rDNA region and primers based on β-tubulin gene. All isolates formed compact blue green colonies with characteristic earthy odor. Conidiophores were terverticillate with smooth septate stipes and conidia were smooth, globose to subglobose, born in colums. The average size of conidia was 3.38 +- 0.49 (SD) x 3 +- 0.36 (SD) μm. Using species specific primers PEF/PER the texpected amplicons of ~404 bp were obtained in all nine tested isolates and PCR conducted with the Bt-LEVUp4/ Bt-LEV-Lo1 and universal ITS1/ITS4 primer pairs generated amplicons of the expected sizes of ~800 bp and ~600 bp, respectively. MegaBlast analyses of the 2X consensus of nucleotide sequences of the isolate JP1 partial β-tubulin gene and ITS region showed 99-100% and 100% similarity with several P. expansum sequences of corresponding regions of this species deposited in GenBank. Based on morphological and molecular features, the isolates obtained from decayed apple fruit collected in several storage facilities in Serbia were identified as P. expansum.

Penicillium expansum (Link) Thom. je jedan od najznačajnijih prouzrokovača truleži plodova jabučastog voća u toku čuvanja koji se odlikuje proizvodnjom mikotoksina patulina. U ovom radu je, na osnovu morfoloških i molekularnih osobina, identifikovana vrsta P. expansum kao prouzrokovač truleži plodova jabuke u skladištu. Plodovi jabuke sorti Ajdared, Zlatni delišes i Breburn sa simptomima truleži prikupljene su u skladištima u lokalitetima Pudarci, Suvodol, Čelarevo, Bela Crkva i Svilajnac. Na prikupljenim plodovima uočavale su se svetlosmeđe, meke i udubljene pege u okviru kojih je bila prisutna zelenoplava sporulacija gljive. Izolacija je obavljena standardnim postupkom na podlogu od krompir-dekstroznog agara (KDA), a patogenost izolata proverena je inokulacijom zdravih plodova jabuke. Morfološke odlike kolonija i reproduktivnih struktura izolata praćene su na dijagnostičkim hranljivim podlogama (MEA, CYA i YES). Molekularna identifikacija izolata obavljena je korišćenjem specifičnih prajmera za vrstu P. expansum zasnovanih na genu za poligalakturonazu (Pepg1), univerzalnih prajmera za ITS region i prajmera zasnovanih na genu za β-tubulin. Iz jabuke sa simptomima plave truleži dobijeno je devet izolata čija je patogenost potvrdjena veštačkim inokulacijama zdravih plodova jabuke. Ispitivani izolati su formirali kompaktne zelenoplave kolonije karakterističnog mirisa. Svi izolati su rasli na 5oC, a porasta nije bilo na 37oC. Konidije prosečne veličine 3,38 x 3 μm, bile su okruglaste do ovalne, formirane u dugim nizovima. Konidiofore su bile terverticillata, asimetričnog grananja sa glatkim stipama. Kod svih ispitivanih izolata amplikoni očekivane veličine oko 404 bp dobijeni su korišćenjem specifičnih prajmera za P. expansum PEF/PER, dok su primenom ITS1-ITS4 prajmera amplifikovani produkti veličine oko 600 bp, a primenom prajmera za deo β-tubulin gena, produkti veličine oko 800 bp. MegaBlast analizom dobijenih sekvenci ITS regiona i dela β-tubulin gena izabranog izolata JP1 utvrđena je 100%, odnosno 99-100% sličnost sa sekvencama odgovarajućih regiona vrste P. expansum deponovanim u NCBI bazi podataka. Na osnovu morfoloških i molekularnih osobina dobijenih izolata utvrđeno je da prouzrokovači truleži plodova jabuke u nekoliko skladišta u Srbiji pripadaju vrsti P. expansum.