Biological and Molecular Variability of Alfalfa mosaic virus Affecting Alfalfa Crop in Riyadh Region

Abstract

In 2011-2012, sixty nine samples were collected from alfalfa plants showing viral infection symptoms in Riyadh region. Mechanical inoculation with sap prepared from two collected samples out of twenty five possitive for Alfalfa mosaic virus (AMV) by ELISA were produced systemic mosaic on Vigna unguiculata and Nicotiana tabacum, local lesion on Chenopodium amaranticolor and C. quinoa. Vicia faba indicator plants that induce mosaic and mottle with AMV-Sagir isolate and no infection with AMV-Wadi aldawasser isolate. Approximately 700-bp was formed by RT-PCR using AMV coat protein specific primer. Samples from infected alfalfa gave positive results, while healthy plant gave negative result using dot blot hybridization assay. The nucleotide sequences of the Saudi isolates were compared with corresponding viral nucleotide sequences reported in GenBank. The obtained results showed that the AMV from Australia, Brazil, Puglia and China had the highest similarity with AMV-Sajer isolate. While, the AMV from Spain and New Zealaland had the lowest similarity with AMV-Sajer and Wadi aldawasser isolates. The data obtained in this study has been deposited in the GenBank under the accession numbers KC434083 and KC434084 for AMV-Sajer and AMV- Wadialdawasser respectively. This is the first report regarding the gnetic make up of AMV in Saudi Arabia.

Keywords: AMV; RT-PCR; biological variability; hybridization; sequence.

Fig. 1.

Natural infection of AMV: mosaic and mottling symptoms (A and B from Wadi aldawasser and C from Sagir) and D: healthy on alfalfa plants.

Fig. 2.

Reaction of selected indicator plants to mechanical inoculation with the the two isolats of AMV (Wadi aldawasser and Saj):Chorotic local lesion on C. amaranticolor (A) and Ch. quinoa (B), mosaic on N. tabacum (C), V. unguiculata (D) and V. faba (E)

Fig. 3.

Gel electrophoresis on RT-PCR amplification of a fragment from AMV genome using specific primer pair designed to amplify 700 bp fragment of CP gene. Lane M represents Hyper-Laddert II (Bioline), Lane 1–4 (Sample 1–4 from infected alfalfa plants collected from Sajer location), Lane 6–11 (Leaf sample from symptomatic alfalfa plants collected from Wadi aldawasser location), and Lane 5 (healthy alfalfa as a negative control.

Fig. 4.

Evaluation of PCR-labeled probes by agarose gel electrophoresis. Lanes 1 and 5: unlabeled PCR product Lanes 2, 3, and 4: corresponding DIG-labeled PCR products, Lane 6 healthy alfalfa plant,M: Hyperladder DNA marker.

Fig. 5.

Results of dot blot hybridization assay of several infected alfalfa samples collected from Wadi aldawasser (Row A:1, 2, 3, 4 and 5) and Sajer (Row B: 1, 2, 3, 4 and 5) using AMV DIG-cDNA probe. (Row C:1, 2),RT-PCR product DNA as positive control. (No hybridization reaction was observed healthy alfalfa samples (Row C: 3, 4 and 5).

Fig. 6.

A phylogenetic tree constructed from the alignment of nucleotide sequences of the coat protein gene of of AMV Saudi Arabian isolates and 24 AMV isolates obtained from GenBank (Table 1) using DNAMAN based on the nucleotide sequences.