Occurrence and Partial Characterization of Lettuce big vein associated virus and Mirafiori lettuce big vein virus in Lettuce in Iran

Abstract

Mirafiori lettuce big vein virus (MiLBVV) and lettuce big vein associated virus (LBVaV) were found in association with big vein disease of lettuce in Iran. Analysis of part of the coat protein (CP) gene of Iranian isolates of LBVaV showed 97.1–100 % nucleotide sequence identity with other LBVaV isolates. Iranian isolates of MiLBVV belonged to subgroup A and showed 88.6–98.8 % nucleotide sequence identity with other isolates of this virus when amplified by PCR primer pair MiLV VP. The occurrence of both viruses in lettuce crop was associated with the presence of resting spores and zoosporangia of the fungus Olpidiumbrassicae in lettuce roots under field and greenhouse conditions. Two months after sowing lettuce seed in soil collected from a lettuce field with big vein affected plants, all seedlings were positive for LBVaV and MiLBVV, indicating soil transmission of both viruses.

Lettuce big vein, first described in California in 1934 [3], is a soil-borne disease reported from many parts of the world. It affects all lettuce types grown in soil in the open or under cover, as well as in hydroponic cultures [12]. The disease lowers the quality and quantity of the crop due to the distortion of the foliage, delays in head formation and decrease in head size [16, 18].

The lettuce big vein disease was formerly believed to be caused by a varicosavirus then known as lettuce big vein virus. However, a causative relationship was never established. Roggero et al. [12] presented evidence that the disease is caused by an Ophiovirus later designated as Mirafiori lettuce big vein virus (MiLBVV). However, many studies have demonstrated that plants exhibiting big vein symptoms are often coinfected with both viruses, suggesting that LBVaV may also have a role in the development of the disease [8, 9, 12]. Both LBVaV and MiLBVV have segmented ssRNA genomes. The viral RNA of LBVaV is predominantly negative-sense [19, 20] whereas MiLBVV particles contain approximately equimolar amounts of RNA molecules of both polarities [4, 23]. The LBVaV genome contains two RNA segments. Both viruses are believed to be naturally transmitted by Olpidium species. Big vein disease of lettuce is a soil-borne disease that is spread by the fungus Olpidiumvirulentus (a noncrucifer variant of O.brassicae sensu lato) [6]. Use of the name O.virulentus instead of O.brassicae has been proposed on the basis of recent biological and molecular analyses on Olpidium spp. [2, 5, 17]. The resting spores of the fungus can persist for over 20 years in soil and can retain the ability to transmit the disease for over 15 years [1]. There have been preliminary reports on the detection of LBVaV and MiLBVV in Iran. The present paper deals with partial molecular and biological characterization of MiLBVV and LBVaV and association of the latter with mild and severe symptoms in the Fars province of Iran.

Fifty-seven lettuce samples with mild or no symptoms and 10 lettuce samples with severe symptoms were collected in 2009–2011 in lettuce fields in the vicinity of Shiraz, Zarghan, Zafarabad, Kaftarak and Kazeroun (Fars Province), Tehran (Tehran Province), and Gachsaran (Kohguiluyeh–Boyer Ahmad Province), Iran. The samples were examined for the presence of Olpidium-like structures and assayed by PCR for the presence of LBVaV and MiLBVV.

Modified one-tube RT-PCR [14] was used for MiLBVV and LBVaV detection in plants using the primer pairs MiLBVV RNA4 [2], MiLV VP [8], MiLV CP3/CP4 and LBVaV CP3/CP4 [13] covering partial nucleotide sequence of the coat protein gene of LBVaV and partial nucleotide sequence of the coat protein gene and RNA4 of MiLBVV. Amplification conditions consisted of 30 min reverse transcription at 54 °C using M-MuLV reverse transcriptase (Fermentas), followed by 35 cycles of 94 °C for 30 s, annealing temperature for 45 s, and 72 °C for 1 min, with a final extension at 72 °C for 15 min. Annealing temperature was 50, 54, or 62 °C for primer pairs LBVaV CP3/CP4, MiLBVV RNA4, or MiLV VP, respectively. RT-PCR reaction products were separated by electrophoresis in 1 % agarose gels.

The PCR products were ligated into PTZ57R/T vector (Fermentas) and cloned in Escherichiacoli strain DH5α as described by Sambrook et al. [15]. At least three clones of each PCR fragment were sequenced. Nucleotide and putative amino acid sequences of the CP genes were compared with those available in the GenBank database using NCBI/BLAST to search for related sequences. The Clustal X (1.8), a windows interface for the Clustal W, was used to obtain multiple alignments of nucleotides for the MiLBVV and LBVaV coat protein genes [22]. Phylogenetic and molecular evolutionary analyses were performed using MEGA 4.0 [21].

Soil samples with lettuce root debris were collected from lettuce fields in Fars and Kohguiluyeh–Boyer Ahmad Provinces, where characteristic symptoms of big vein disease were observed. Seed of lettuce were planted in soil samples under greenhouse conditions and the developing seedlings were observed for symptoms and presence of Olpidium-like structures and were tested for MiLBVV and LBVaV by PCR after 2 months. Fungal structures (resting spores and zoosporangia) were visualized by light microscopy of root segments stained with fuchsin.

Field symptoms in 67 lettuce samples ranged from slight swelling to pronounced thickening of veins, severe leaf crinkling, and plant stunting. Symptoms were more pronounced during the cool season (December, January and February). LBVaV was detected in plants with the whole range of symptoms including none. All 57 tested plants with mild or no symptoms from Fars, Tehran and Kohguiluyeh–Boyer Ahmad Provinces were singly infected by LBVaV (Fig. 1d). On the other hand, all 10 tested plants with severe symptoms from Fars Province (Fig. 1a–c) were infected by both MiLBVV and LBVaV.

Fig. 1.

Severe symptoms of vein thickening (a) leaf crinkling (b), and irregular growth and stunting (c) in lettuce infected with MiLBVV and LBVaV. Mild big vein symptoms on a lettuce plant infected with LBVaV alone (d)

Discovery of MiLBVV in big vein symptomatic plants, and its later report as the causal agent of big vein, was an important breakthrough for big vein research [2]. Our results agree with previous reports [8, 12] in showing a strong correlation between severe symptoms of big vein and MiLBVV. However our findings also support the view that some field strains of LBVaV may contribute to the development of big vein symptoms, although they may occur in non-symptomatic plants as well [8, 12, 16].

One-tube RT-PCR method was successfully employed for detection of both viruses associated with lettuce big vein disease in infected lettuce plants. Use of primer pairs MiLBVV RNA4, MiLV VP, MiLV CP3/CP4 and LBVaV CP3/CP4 resulted in the amplification of 570, 469, 743 and 570 bp (Fig. 2a, b) products, respectively. The sizes of these products were in accordance with the expected values [2, 8, 13]. One-tube RT-PCR was reliable, fast and less expensive than regular RT-PCR protocol. Similar results were obtained with different MiLBVV primers.

Fig. 2.

Agarose gel electrophoresis pattern of PCR products from big vein affected lettuce samples using a MiLV VP primers (lanes 1, 2, 3) or b MiLBVV RNA4 (lanes 1 and 2), and LBVaV CP3/CP4 (lanes 3 and 4) primers. M, DNA molecular marker

The MiLBVV CP sequences obtained from geographically distinct regions were compared with the Iranian isolate (GenBank Acc. No. JN576419). BLAST search analysis of the cloned CP of MiLBVV at the nucleotide level indicated 99 % identity with the MiLV-ALM2 isolate of MiLBVV [9]. Multiple alignments of the CP nucleotide and amino acid sequences of MiLBVV isolates indicated low sequence diversity, although higher than in the case of LBVaV CP. Multiple sequence alignment showed that Iranian MiLBVV isolate has nucleic acid homologies of 88.6–98.8 with MiLBVV characterized elsewhere.

The MiLBVV isolates form two major groups referred to as subgroups A and B. According to the studies of Navarro et al. [9], isolates of subgroup B are less variable than those of subgroup A. Within subgroup A, there was a cluster of geographically distinct MiLBVV isolates from Spain (MUR1, ALM2, ALM3 and GAL1), Germany (GER1, GER2 and GER3), England (UK1), Australia (AUS1), Denmark (DEN1), and Italy (ITA). Analysis of CP nucleotide sequences indicated that the Iranian isolate was positioned in this subgroup (Fig. 3a). Subgroup B contained 6 MiLBVV isolates from Spain.

Fig. 3.

a Phylogenetic analysis of MiLBVV isolates based on partial nucleotide sequences of coat protein. Dendrogram was constructed by the Neighbor-joining method. The numbers above the nodes indicate the percentage of bootstrap support. The tree was rooted by the CP gene of Citrus psorosis virus (CPsV, AFO36338). b Phylogenetic tree of LBVaV isolates based on partial nucleotide sequences of the coat protein. Dendrogram was constructed by the Neighbor-joining method. The numbers above the nodes indicate the percentage of bootstrap support. The tree was rooted by the CP gene of SYNV

There were two nucleotide insertions (an A in the position 272 and a C in the position 927 of the whole gene) in the CP-encoding region of the Iranian isolate (GenBank Acc. No. JN576418) of MiLBVV. Despite that, there was no major change in the position of the Iranian isolate when the dendrogram was based on amino acid sequence.

The nucleotide sequences of CP gene of six Iranian isolates (Kaftarak (Acc.No. JN400922), Kazeroon (Acc.No. JN400921), Zarghan (Acc.No. JN400919), Zafarabad (Acc.No. JN400920, Gachsaran (Acc.No. JN400923) and Tehran (Acc.No. JN400924)) of LBVaV were compared with those of other isolates present in GenBank. All isolates were quite similar in their CP nucleotide sequences and amino acid deletions and/or insertions were not observed in the CP of any isolates. The Iranian isolates from different geographical regions formed a group with a homology ranging from 97.2–99.7 %. However, they were scattered over the dendrogram when the latter was based on amino acid sequence homology. Phylogenetic analysis of the LBVaV CP gene was done (Fig. 3b) by rooting the tree with the CP gene of Sonchusyellownetvirus (SYNV), a member of the family Rhabdoviridae.

Two months after sowing lettuce seed in infested field soil, the seedlings were tested for the presence of MiLBVV and LBVaV by RT-PCR. All samples were positive for both viruses. However, the seedlings showed no particular symptoms, probably due to the fact that the experiments had been carried out in greenhouse at temperatures above 18 °C. These results agree with those of Roggero et al. [11] who showed that MiLBVV, like LBVaV, appears to be soil borne.

Transmission of lettuce big vein associated viruses with Olpidiumbrassicae zoospores is well established [1, 7]. In the present study Olpidium-like fungal sporangia and resting spores were found in the epidermal cells of lettuce roots both under the field conditions and in the greenhouse. Rasoulpour and Izadpanah [10] have isolated the fungus from the roots of many plants in Iran. Widespread occurrence of LBVaV and MiLBVV in Iran is correlated with the presence of Olpidium sp. in various geographical regions in this country.

In conclusion, both MiLBVV and LBVaV appear to be widespread in Iran. MiLBVV may assume economic importance by deformation of plants and reduction of growth. LBVaV is more prevalent in the fields but causes little, if any, damage to the crop.