گزارش نخست ویروس شکستن زعفران وحشی روی Colchicum autumnale از منطقه حفاظت شده Natura2000 در پارک ملی مجارستان

در اواسط آوریل 2018 نوارهای خطی سبز روشن تا زرد سبز بر روی برگ‌های گیاهان زعفران چمنی (Colchicum autumnale) در منطقه محافظت‌شده Natura2000 در پارک ملی دانا-ایپولی مجارستان مشاهده شد. علائم شکستن گل در فصل پاییز نشان‌دهنده عفونت ویروسی بود. آزمایش‌های مولکولی و ایمنی‌شناسی نشان داد وجود ویروس Potyvirus با هویت ۹۳.۹۱٪ مشابه ویروس شکستن زعفران چمنی (MSBV) تایید شده است. این اولین گزارش MSBV در گیاه زعفران چمنی وحشی در این منطقه است. این ویروس از طریق تماس و شته‌ها منتقل می‌شود و گسترش آن ممکن است موجب کاهش جمعیت این گیاه شود.

In mid-April of 2018 light green to greenish yellow linear stripes (Fig. S1.) were observed on the foliage of meadow saffron (Colchicum autumnale) plants – which are native to Hungary – at a strictly protected Natura2000 site maintained by the Duna-Ipoly National Park (DNPI). By autumn, during the flowering season, flower breaking symptoms (Fig. S2.) were noticed, which indicated possible viral infection. With the permit of the Government Office of Pest County and the DNPI, 200 mg leaf sample was collected from one symptomatic plant in spring 2021 and stored at -70 °C until further processing. At the time of the sampling about 2.5 % of the ~ 5000 meadow saffron were symptomatic. Multiplex RT-PCR testing of the sample and an asymptomatic C. autumnale plant for cucumber mosaic virus, tomato spotted wilt virus (Nemes and Salánki 2020) and Nepovirus subgroup-A (Digiaro et al. 2007) gave negative results.

The asymptomatic plant also tested negative for potyviruses (Salamon and Palkovics 2005). The asymptomatic (healthy) C. autumnale plant was inoculated with leaf sap of the sample (0.02M Sörensen’s phosphate buffer pH 7.2 + 2 % PVP-40 (m/v)) resulting in symptoms of flower breaking in autumn of 2021, and linear stripes on the foliage in spring 2022, identical to symptoms on the originally infected plant. ELISA tests were carried out on the source plants in duplicate using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) (Agdia, Elkhart, IN, USA). Absorbance values were 1.519 and 1.530, while the negative controls were 0.003 and 0.007, respectively indicating potyvirus infection of the sample. Molecular tests were carried out on the source and inoculated plant samples in 2022. Total nucleic acid was extracted with the modified CTAB protocol of Xu et al. (2004), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) with poly T2 (5′-CGGGGATCCTCGAGAAGCTTTTTTTTTTTTTTTTT-3′) primer (Salamon and Palkovics 2005). PCR amplification was carried out with poty7941 (5′-GGAATTCCCGCGGNAAYAAYAGYGGNCARCC-3′) and poly T2 primers as described earlier (Salamon and Palkovics 2005). A PCR product of ~ 1.6 kb was obtained in each case (Fig. S3.), cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA) and transformed into E. coli DH5α strain.

The obtained 1642 nucleotide (nt) sequence encompassing the complete coat protein (CP) was determined (Accession No: OP057214). The virus sequence present in the source and inoculated plants shared 100% nt identity. EcoRV digestion of the PCR products yielded two restriction fragments (369/1273 bp), indicating the presence of a single potyvirus in the infected plant tissue (Fig. S3.). BLASTN analysis of the CP cistron revealed highest nt identity (93.91 %) to meadow saffron breaking virus (MSBV) isolate FR GenBank Acc. No.: AY388995. MSBV was first reported in the Alsace region of France at an INRA research station in cultivated meadow saffron plants showing similar symptoms and the disease reached 100% incidence within a year (Poutaraud et al. 2004). Potyviruses are transmitted mechanically and by aphids (Inoue-Nagata et al. 2022). The spread of MSBV could lead to the infection and decline of the population of Colchicum in protected ecosystems. To our knowledge, this is the first report of MSBV on wild meadow saffron plant from a strictly protected Natura2000 site at a Hungarian National Park.