In the years 2020-2021 as part of the activity of the Campania region hemp fiber project, variety comparison trials were carried out on 7 hemp varieties among those relevant for bast fiber production. During the trials, in particular on the cv. Fibrante, a consistent problem was noted: a noticeable germination failure (80-90%) occurred during the emergence of seedlings. Therefore, experiments were conducted to ascertain the possible presence of seed-borne pathogens. Tests were carried out on 100 seeds that were surface disinfected with 2% sodium hypochlorite solution for 3 min, rinsed in sterile distilled water three times and dried on sterile filter paper. The seeds were plated on potato dextrose agar (PDA Oxoid™) amended with 100 mg L-1 of streptomycin sulphate, kept at 24°C in the dark and observed daily. Growing colonies were subcultured on PDA for 10 days and, subsequently, twenty purified fungal isolates were obtained by single spore isolation. Colonies of these isolates on PDA were initially grayish-white and then turned dark olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these isolates produced light brown and solitary conidiophore with septum. Conidia were obclavate or pyriform, brown, with 1-3 transverse septa and 0-3 longitudinal septa, and measured 12.5 to 28.5 × 5 to 15 µm (n=50). The morphological characteristics observed under the light microscope were consistent with that of Alternaria spp. (Simmons 2007). In order to characterize the representative isolate, total DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and 3 genes were PCR-amplified: the ITS spacer using the primer pair ITS1-ITS4 (White et al., 1990), the transcription elongation factor 1- using the primer pair EF1-983F/ EF1-2218R (Rehner and Buckley., 2005) and the RNA polymerase II second largest subunit (RPB2) using the primer pair RPB2-5F2/fRPB2-7cR (Sung et al 2007; Liu et al 1999). The size-expected amplicons were purified and sequenced at the BMR Genomics (Padova, Italy) and the resulting sequences were deposited in GenBank under the accession numbers ON556507, ON601003, ON601004. BLAST-n analysis revealed 98 to 99% nucleotide identity with some representative isolates of Alternaria rosae E.G. Simmons & C.F. Hill (KU375630.1, XM_046169884.1, XM_046168987.1). To fulfill Koch's postulates, 100 hemp-certified seed were disinfected as mentioned above, left to germinate on the water-agar to discard potentially infected seeds and finally sowed in sterile peat-soil mix (1:1 v/v). The inoculum consisted of 10 mL of 105 conidial suspension obtained by the representative isolate (Ar_H1). Negative control seeds were inoculated with sterile water. After 5-7 days 100% of inoculated seedlings showed weak germinative vigor with yellowing of the epicotyls and dark areas on the root. The tissue narrowed and turned necrotic with abundant white mycelium covering the entire seedling. Small pieces of necrotic roots were plated on PDA and the same Alternaria-like colonies grew in 10 days. DNA sequencing confirmed the presence of A. rosae. Alternaria spp. are fungi that produce a wide range of toxic metabolites, harmful to food safety in the food uses of the seed. This finding further highlights that the quality of the hemp seed must be considered as a priority aspect in the entire hemp supply chain. To the best of our knowledge, this is the first report of A. rosae as seed-borne fungus on hemp.
Keywords: Alternaria rosae; Hemp; damping-off; seed-borne pathogen.