Genetic diversity and molecular characterization of Cucumber mosaic cucumovirus (CMV) subgroup II infecting Spinach (Spinacia oleracea) and Pea (Pisum sativum) in Pothwar region of Pakistan.

M Ahsan,M Ashfaq,Z Khan,M Z Hamza,H Riaz,Z Asad

doi:10.1590/1519-6984.245865

Abstract

Cucumber mosaic virus (CMV) is a tremendous threat to vegetables across the globe, including in Pakistan. The present work was conducted to investigate the genetic variability of CMV isolates infecting pea and spinach vegetables in the Pothwar region of Pakistan. Serological-based surveys during 2016-2017 revealed 31.70% overall CMV disease incidence from pea and spinach crops. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) revealed that all the positive isolates belong to CMV subgroup II. Two selected cDNA from ELISA-positive samples representing each pea and spinach crops were PCR-amplified (ca.1100 bp) and sequenced corresponding to the CMV CP gene which shared 93.7% nucleotide identity with each other. Both the sequences of CMV pea (AAHAP) and spinach (AARS) isolates from Pakistan were submitted to GenBank as accession nos. MH119071 and MH119073, respectively. BLAST analysis revealed 93.4% sequence identity of AAHAP isolate with SpK (KC763473) from Iran while AARS isolate shared maximum identity (94.5%) with the strain 241 (AJ585519) from Australia and clustered with some reference isolates of CMV subgroup II from UK (Z12818) and USA (AF127976) in a Neighbour-joining phylogenetic reconstruction. A total of 59 polymorphic (segregating) sites (S) with nucleotide diversity (π) of 0.06218 was evident while no INDEL event was observed in Pakistani isolates. The evolutionary distance of Pakistani CMV isolates was recorded as 0.0657 with each other and 0.0574-0.2964 with other CMV isolates reported elsewhere in the world. A frequent gene flow (Fst = 0.30478 <0.33) was observed between Pakistani and earlier reported CMV isolates. In genetic differentiation analysis, the value of three permutation-based statistical tests viz; Z (84.3011), Snn (0.82456), and Ks* (4.04042) were non-significant. The statistical analysis revealed the values 2.02535, 0.01468, and 0.71862 of Tajima's D, Fu, & Li's F* and D* respectively, demonstrating that the CMV population is under balancing selection.

Highlights

The Pothwar region, covering 22, 254 Km2 area of northern Punjab province lies in the north-eastern part of Pakistan with diverse vegetation comprising fruits, ornamentals, vegetables as well as cereal crops, and small forests (Ashfaq et al, 2017; Riaz et al, 2022)
The genes perform multiple functions where host specificity, symptoms induction, long-distance movement, interviral recombination and virulence determination are controlled by open reading frames (ORFs) 1, 2a and 2b located on RNA1 and RNA2 while RNA 3 encodes for capsid protein (CP; translated from subgenomic RNA 4) and movement protein (MP) responsible for movement of virions from cell to cell, involved in the transmission of Cucumber mosaic virus (CMV) from infected to healthy plants through aphids (Shi et al, 2008; Nouri et al, 2014; Ohshima et al, 2016)
Survey results revealed that CMV is well established in Pothwar region as it was found infecting both the understudied vegetables, i.e. spinach and pea with an overall 31.70% disease incidence during 2016-17

Summary

Introduction

The Pothwar region, (longitude 71°10-73°55E; latitude 32°10-34°9N), covering 22, 254 Km2 area of northern Punjab province lies in the north-eastern part of Pakistan with diverse vegetation comprising fruits, ornamentals, vegetables as well as cereal crops, and small forests (Ashfaq et al, 2017; Riaz et al, 2022). Spinach (Spinacia oleracea) and Pea (Pisum sativum) are among the most important rabbi vegetables which covers 0.035 million hectares (MH) with an average production of 1.57 MT (Pakistan, 2018). Notably Cucumber mosaic virus (CMV) is known to cause devastating losses and reduce crop quality and quantity and a serious threat to hamper the regional vegetable production (Rehman et al, 2015). The genes perform multiple functions where host specificity, symptoms induction, long-distance movement, interviral recombination and virulence determination are controlled by ORF 1, 2a and 2b located on RNA1 and RNA2 while RNA 3 encodes for capsid protein (CP; translated from subgenomic RNA 4) and movement protein (MP) responsible for movement of virions from cell to cell, involved in the transmission of CMV from infected to healthy plants through aphids (Shi et al, 2008; Nouri et al, 2014; Ohshima et al, 2016)

Methods

Results

Discussion

Conclusion