Molecular, serological and biological variation among chickpea chlorotic stunt virus isolates from five countries of North Africa and West Asia

Abstract

Chickpea chlorotic stunt virus (CpCSV), a proposed new member of the genus Polerovirus (family Luteoviridae), has been reported only from Ethiopia. In attempts to determine the geographical distribution and variability of CpCSV, a pair of degenerate primers derived from conserved domains of the luteovirus coat protein (CP) gene was used for RT-PCR analysis of various legume samples originating from five countries and containing unidentified luteoviruses. Sequencing of the amplicons provided evidence for the occurrence of CpCSV also in Egypt, Morocco, Sudan, and Syria. Phylogenetic analysis of the CP nucleotide sequences of 18 samples from the five countries revealed the existence of two geographic groups of CpCSV isolates differing in CP sequences by 8-10%. Group I included isolates from Ethiopia and Sudan, while group II comprised those from Egypt, Morocco and Syria. For distinguishing these two groups, a simple RFLP test using HindIII and/or PvuII for cleavage of CP-gene-derived PCR products was developed. In ELISA and immunoelectron microscopy, however, isolates from these two groups could not be distinguished with rabbit antisera raised against a group-I isolate from Ethiopia (CpCSV-Eth) and a group-II isolate from Syria (CpCSV-Sy). Since none of the ten monoclonal antibodies (MAbs) that had been produced earlier against CpCSV-Eth reacted with group-II isolates, further MAbs were produced. Of the seven MAbs raised against CpCSV-Sy, two reacted only with CpCSV-Sy and two others with both CpCSV-Sy and -Eth. This indicated that there are group I- and II-specific and common (species-specific) epitopes on the CpCSV CP and that the corresponding MAbs are suitable for specific detection and discrimination of CpCSV isolates. Moreover, CpCSV-Sy (group II) caused more severe stunting and yellowing in faba bean than CpCSV-Eth (group I). In conclusion, our data indicate the existence of a geographically associated variation in the molecular, serological and presumably biological properties of CpCSV.

Fig. 1

Unrooted tree showing the phylogenetic relationship among the coat protein nucleotide sequences of 18 CpCSV isolates from five countries. The numbers refer to the serial number of each isolate as listed in Table 1. Groundnut rosette assistor virus (GRAV) sequence (acc. no. AF195828) was used as an outgroup. The sequences were aligned and neighbor-joining trees were constructed using the CLUSTAL_X program and viewed in TreeView. The number (in italics) at the major node is the bootstrap score for the division of the two groups of CpCSV isolates. The bar indicates the number of nt substitutions per site

Fig. 2

Alignment of the deduced coat protein amino acid sequence of a group-I and a group-II isolate of CpCSV from Ethiopia (CpCSV-Eth; acc. no. AY956384) and Syria (CpCSV-Sy; acc. no. EU541270), respectively, with that of groundnut rosette assistor virus (GRAV; acc. no. AF195828). Amino acid residues identical to, and different from, those of CpCSV-Eth are indicated by a dash and a letter, respectively. Single dots denote gaps. Identical amino acid residues specific for the pairs CpCSV-Eth/GRAV and CpCSV-Sy/GRAV are boxed using solid and dotted lines, respectively

Fig. 3

Restriction fragment length polymorphisms of RT-PCR products from the coat protein gene of selected group-I and group-II isolates of CpCSV from five countries, digested with enzymes PvuII (a) and HindIII (b). Lane M shows the size markers, and the other lanes contain the digested PCR products from isolates from Ethiopia (1–6), Sudan (7), Morocco (8–12), Egypt (13) and Syria (14). The approximate sizes of the marker bands and the digested products are indicated at the left and right side of the figure, respectively

Fig. 4

Variation in plant stunting (a) and leaf yellowing (b) caused by the Syrian isolate Sy-fb1-03 (CpCSV-Sy; left) and the Ethiopian isolate Et-fb-am (CpCSV-Eth; middle) in the faba bean cultivar Condor under glasshouse conditions. An uninoculated control plant and leaf are shown on the right. Photographs were taken ~6 weeks after inoculation