First Report of Fusarium proliferatum as the Causal Agent of Seed Rot of Hyssopus officinalis in Serbia

Abstract

Fusarium spp. are important soil- and seed-borne pathogens of many field and vegetable crops, including orchards and medicinal plants (Leslie and Summerell 2006). Fusarium proliferatum is the most common pathogen infecting numerous crop plants and occurring in various climatic zones. In Serbia, this species is well-known as a pathogen of wheat, maize, bean, and recently, garlic and onion (Lević et al. 2009; Stanković et al. 2007). Hyssop (Hyssopus officinalis L.) is grown in Serbia as a member of the Lamiaceae family for the needs of pharmaceutical companies and tea production, because of its medicinal and aromatic properties. In Serbia, production takes place on about 500 ha (no official data). During a routine quality control of hyssop seeds collected from Rumenka (Vojvodina Province), in 2018, fungal infection followed by seed rot was noticed on an average of 22%. White mycelium covered infected seeds with violet pigmentation occurring under the seeds. Microscopic observation confirmed the presence of Fusarium spp. Prior to isolation, seeds were surface disinfected in 1% NaOCl for 3 min, rinsed, dried, and plated onto potato dextrose agar (PDA). Plates were incubated at 25°C under ultraviolet light (“black light”) with a 12-h photoperiod (Mathur and Kongsdall 2003). Seven days later, 12 Fusarium spp. isolates (JBL 4003/1 to 4003/12) were single spored and subcultured on both PDA and carnation leaf agar. Pathogenicity testing was performed in vitro using a modified agar slant method in the test tube with PDA amended (Porter et al. 2015). A piece of mycelium of each isolate grown on PDA for 7 days was placed at the bottom of the tube, and 2 cm above the inoculum, dried hyssop seed was carefully placed. After 10 days, fungal mycelia of 12 isolates caused seed rot and seedling decay. All the tested isolates were reisolated and used for further analysis. The isolate JBL 4003/2 formed white, aerial, and abundant colonies, with light violet to brown pigmentation in agar. Within 5 days, microconidia were formed in the aerial mycelium, in long chains or cohering in false heads. Slightly curved rather straight macroconidia were formed, with a distinct foot cell, mostly three to five septate, with average dimensions of 31 to 53 × 3.4 to 4.1 µm. No chlamydospores were observed. Based on the description given by Gerlach and Nirenberg (1982), cultural and morphological characteristics indicated that the isolate JBL 4003/2 belongs to F. proliferatum. To obtain a DNA sequence-based identification, total DNA was extracted directly from the mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Following DNA extraction, the translation elongation factor 1-alpha region was amplified by PCR using the primer pair EF1 and EF2 (Geiser et al. 2004). Sequences were analyzed and BLAST searched against GenBank (http://blast.ncbi.nlm.nih.gov/) and FUSARIUM-ID (http://fusariumdb.org/). Isolate designated as JBL 4003/2 was deposited in the NCBI GenBank database under the accession number MK061541.1. BLASTn queries of GenBank and the Fusarium-ID database showed 100% identity to accession numbers KY801934.1 and MK507798.1, which belong to F. proliferatum (Matsushima) Nirenberg. Based on Koch’s postulates and sequence analysis, to our knowledge this is the first report of F. proliferatum as the causal agent of H. officinalis seed rot in Serbia.