First report of Turnip mosaic virus infecting saffron in Iran

Abstract

Saffron (Crocus sativus) is widely used as an important herbal medicine crop worldwide. Iran is the largest producer and exporter of saffron in the world. Turnip mosaic virus (TuMV) is a species of the genus Potyvirus, which infects a wide range of plants species. During the autumn of 2019, saffron plants with symptoms including colour breaking of the flowers, chlorosis and mild mosaic of leaves were collected from the major saffron cultivation areas of Iran. After the isolation of total RNA from diseased samples, RT-PCR was done using TuMV-specific primers. Amplicons of two selected isolates were purified, cloned, and sequenced. BLASTn of obtained sequences indicated high similarity with TuMV sequences. To the best of our knowledge, this is the first finding of TuMV infection in the saffron plant of Iran.

Saffron (Crocus sativus, Iridaceae) is one of the world’s highest-priced medicinal and aromatic plants and is widely cultivated in different countries including Iran. Saffron yield and quality is often hampered by various diseases [3]. Turnip mosaic virus (TuMV) (family Potyviridae, genus Potyvirus) is a major worldwide pathogen that infects a wide range of plant species, including those of the genus Crocus [4]. During the autumn of 2018–2019, symptoms of colour breaking of the flowers, and chlorosis and mild mosaic of leaves (Fig. 1) were observed in saffron fields in the major saffron cultivation areas of Iran, Razavi Khorasan and South Khorasan. To confirm the presence of the virus, the symptomatic samples of leaves and flower petals from 10 diseased saffron plants were collected. As a negative control, leaf material from the asymptomatic apparently healthy plant was sampled. Total RNA was extracted from 50 mg of fresh tissues (including a mix of leaves and flowers) using Promega SV Total RNA Isolation Kit (USA), according to the manufacturer’s instructions. Reverse-transcription polymerase chain reaction (RT-PCR) was conducted with the specific primer pair TuMVfor1 (5′-CAAGCAATCTTTGAGGATTATG-3′) and TuMVrev1 (5′-TATTTCCCATAAGCGAGAATAC-3′) [6], that amplify a 986-bp band from the 3′-region of TuMV genome (encompassing partial NIb (54 nt), complete CP (867 nt), and partial 3'UTR (65 nt)). The first cDNA strand was synthesized using total RNA, reverse primer TuMVrev1, and MMuLV reverse transcriptase by the method described earlier [5]. PCR amplifications of cDNA were performed in 25-µL reaction mixtures consisting of 12.5 μL of Taq DNA Polymerase Master Mix RED (Ampliqon, Denmark), 1 μl each of forward and reverse primer (10 pmol), 7.5 μl of double distilled water, and finally 3 μL of template cDNA. The PCR thermal profile was 95 °C for 3 min; followed by 35 cycles of 94 °C for 30 s, 54 °C for 30 s, 72 °C for 1 min; and the final extension at 72 °C for 7 min. PCR products and DNA ladder were separated by 1% agarose gel electrophoresis, visualized using DNA Green Viewer staining, and photographed with ultraviolet-illumination. Five plants with symptoms yielded bands of approximately 1000 bp, which were not present in the asymptomatic apparently healthy plant (Fig. 2). Amplified fragments of the expected size of two isolates (IR-SKH4 and IR-SKH6) were purified from the agarose gel using the DENAzist Gel Extraction Kit (DENAzist Asia, Iran). The purified products were ligated into the pTG19-T vector (Vivantis, Malaysia) according to the manufacturer's protocol. Plasmids were then introduced into competent cells of Escherichia coli DH5α by transformation. The transformants were selected on LB-agar plates containing ampicillin, IPTG and X-gal. The positive clones were confirmed by colony-PCR using M13 primers. Bacterial cells harbouring the recombinant plasmids were grown at 37 °C. Recombinant plasmids were purified from bacterial cells using the DENAzist Plasmid DNA isolation Kit and two independent colonies for each isolate were sequenced bidirectionally by Sanger sequencing method (Macrogen Inc. Seoul, South Korea). The consensus sequences were verified using BLASTn and BLASTx searches in the NCBI database. BLAST analysis revealed that both isolates had high similarity to previously determined sequences of TuMV in the GenBank. The compared sequences of the isolate IR-SKH4 shared the highest nucleotide identity (98.58%) with the corresponding sequence of the isolate IRNTP1 (AP017802) from Iran. Subsequently, IR-SKH6 exhibited the highest sequence identity (98.68%) with IRNTuSh18 (AP017808) from Iran. IR-SKH4 and IR-SKH6 shared 98.37% and 98.69% amino acid sequence identity with the Iranian isolates IRNTP1 and IRNTuSh18, respectively. These two obtained sequences shared 98.48% nucleotide sequence identity and 98.37% amino acid sequence identity with one another. The obtained sequences were submitted to the GenBank® under accession numbers OP207886 and OP207887. The infection of TuMV on C. sativus was previously reported from China, New Zealand, France, and Spain [1, 2]. To the best of our knowledge, this is the first scientific report of TuMV infection in saffron producing fields of Iran. In addition to the symptoms mentioned, TuMV-positive specimens were shorter compared to healthy plants. TuMV is naturally transmitted by aphids in a nonpersistent manner. It also can be transmitted through mechanical wound made during farming operations (e.g. tunic removing and corm progeny separation) [1]. Therefore, the use of sanitary practices during planting and harvesting is recommended as important measures to mitigate the virus infection. This finding complements the previous reports of viruses (other than TuMV) associated with saffron mosaic disease in Iran. Further investigation would be desirable to understand the pathogenesis and interaction of saffron infecting viruses and their impact on saffron production to develop disease management strategies.

Fig. 1.

Saffron (Crocus sativus) plants infected with Turnip mosaic virus, collected from Razavi Khorasan, and South Khorasan, Iran. Symptoms including chlorosis and mild mosaic on the leaves and colour breaking on the flowers

Fig. 2.

Agarose gel electrophoresis of RT-PCR products from saffron samples using primers specific for TuMV. Lanes 3 and 4, PCR product from TuMV infected plants; Lane 1, from healthy plant (as a negative control). M: Marker, 1 Kb DNA Ladder (100–10,000 bp)

Declarations

Conflicts of interest

The authors declare that they have no conflict of interest.