Identification and phylogenetic analysis of Fusarium sp. FIESC3 the causal agent of seed rot in onion (Allium cepa L.)

Abstract

Onion (Allium cepa L.) is one of the most important vegetable crops in Serbia, where it is grown on an approximate surface of 20,000 ha. During the routine quality control analysis of onion seed in 2014, fungal infection was observed in an average of 28% of the seed. The objective of this paper was to isolate, determine, and identify Fusarium sp. based on the pathogen's morphological and molecular characteristics. Onion seed samples were collected from different localities in the region of Vojvodina. To obtain a DNA sequence-based identification, a total DNA of the 25 isolates was extracted directly from the mycelium (100 mg wet weight), with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Following DNA extraction, the translation elongation factor 1-alpha region was amplified by PCR with the primer pair EF1 and EF2. An amplicon of 700 bp was amplified in 25 tested isolates. Identification of one isolate was performed by sequencing the translation elongation factor EF-1α gene, which was deposited in the NCBI GenBank database under accession number KP658211 (Fusarium sp. FIESC3).

Crni luk (Allium cepa L.) spada u najznačajnije povrtarske biljne vrste u Srbiji. Gaji se na površini od oko 20.000 hektara. Tokom rutinske kontrole kvaliteta semena crnog luka u 2014. godini, primećena je pojava Fusarium sp. u visokom procentu - od 28%. Cilj rada bio je izolacija i identifikacija Fusarium sp. na osnovu morfoloških i molekularnih karakteristika patogena. Uzorci semena luka sakupljeni su s različitih lokaliteta i iz velikog broja skladišta u Vojvodini. Nakon izolacije patogena odabrano je i morfološki okarakterisano 25 izolata Fusarium sp. Izolacija DNK izvršena je direktno iz micelija gljive (100 mg), korišćenjem Dneasy Plant Mini Kit (Qiagen, Hilden, Germany). Amplifikacija DNK ciljanog gena (translation elongation factor EF-1α gene) obavljena je pomoću PCR korišćenjem para prajmera EF1 i EF2. U svim proučavanim izolatima formirani su amplikoni veličine 700 bp. Identifikacija jednog odabranog izolata izvršena je sekvenciranjem translacionog faktora EF-1α gena, koji je deponovan u NCBI bazu podataka pod brojem KP658211 (Fusarium sp. FIESC 3).