Association of cucurbit aphid-borne yellows virus with cucumber plants in India

Abstract

Symptoms like bright yellowing, puckering of the leaf, vein banding, and vein thickening were observed on different cucurbit hosts at the experimental farm of Indian Agricultural Research Institute, New Delhi during Kharif 2019. Leaf-dip electron microscopy of the symptomatic leaves revealed the association of isometric virus particles measuring ~ 25 nm with bitter gourd and cucumber samples. The RT-PCR assay using polerovirus generic primers covering the partial RdRp, intergenic region, and partial CP region was resulted the amplicons of ~ 1.1 kb. Subsequent cloning, sequencing, and sequence analysis revealed the association of cucurbit aphid-borne yellows virus (CABYV) with bitter gourd (Momordica charantia) and cucumber (Cucumis sativus) plants. These results constitute the first report of CABYV infection on cucumber plants from India.

Supplementary information

The online version of this article (10.1007/s13337-020-00645-4) contains supplementary material, which is available to authorized users.

Cucurbit aphid-borne yellows virus (CABYV), belonging to the genus Polerovirus and family Luteoviridae consisting of monopartite positive-sense ssRNA genome, was first described from France in 1992 [3]. Since then, it has been reported worldwide including Asia, Africa America, and Europe [1]. In India, CABYV was first reported from cucurbits growing regions of Andhra Pradesh, on the bitter gourd and teasel gourd crops up to 98% incidence [5]. The virus was subsequently reported on bitter gourd from Tamil Nadu [4]. In this study, symptomatic bitter gourd and cucumber samples were examined to detect the natural infection of polerovirus(es).

During Kharif 2019, some bitter gourd (Momordica charantia; n = 16) and cucumber (Cucumis sativus; n = 4) plants exhibiting the symptoms such as bright yellowing, puckering, vein banding, and vein thickening on the leaf were noticed in the experimental fields of the Division of Vegetable Sciences, Indian Agricultural Research Institute, New Delhi. Leaf-dip electron microscopy revealed the association of isometric virus particles measuring ~ 25 nm (data not shown) with the bitter gourd (n = 6) and cucumber (n = 2) samples.

To ascertain the polerovirus association with the EM positive samples, the total RNA isolated using PureLink™ RNA Mini Kit (Invitrogen) and was subjected to cDNA synthesis followed by PCR amplification using generic primers (Pol-G-F GAYTGCTCYGGYTTYGACTGGAG and Pol-G-R GATYTTATAYTCATGGTAGGCCT TGAG) [2] targeting the genomic region covering the partial RNA-dependent RNA polymerase (RdRp) gene, intergenic region and partial coat protein (CP) gene of poleroviruses using IMPROM-II™ Reverse Transcription system and GoTaq Flexi DNA polymerase kits (Promega, Madison, WI, USA) respectively. The known CABYV infected sample [4] borrowed from Dr. Renukadevi (TNAU, Tamil Nadu) was used as a positive control. The amplicons of (~ 1100) bp derived from bitter gourd (New Delhi and Tamil Nadu isolates) and cucumber (New Delhi isolate) in RT-PCR were gel-purified, cloned into a pGEM-T easy vector (Promega, Madison, WI), and sequenced (n = 1 sample each). Based on the NCBI BLAST analyses, the resultant nucleotide sequences were deposited into the NCBI GenBank database as CABYV isolates with the accession numbers MN843966 & MN843967 for bitter gourd samples (Tamil Nadu and New Delhi isolates respectively) and MN862007 for cucumber sample (New Delhi isolate).

The sequence identity and phylogenetic analyses were performed by comparing with the respective genomic region sequences of other poleroviruses (one from each species) retrieved from the NCBI GenBank database to confirm the associated polerovirus species. The bitter gourd and cucumber isolates from the current study shared 87% nucleotide sequence identity with CABYV isolate originated from Japan (GQ221224) by forming one cluster (Fig. 1 and Supplementary Table 1) suggesting the associated polerovirus species as CABYV, followed by 58 to 85% sequence identities with the respective sequences of other poleroviruses (Supplementary Fig. 1). Among poleroviruses, melon aphid-borne yellows virus (MABYV) was found to be the nearest member of polerovirus with the CABYV isolates of this study by sharing 85% nucleotide sequence identity, while ScYLV was the farthest member with the least identity of about 58%.

Fig. 1.

Phylogenetic (Neighbor-Joining) analysis of the bitter gourd and cucumber associated polerovirus isolates of this study based on partial RdRp, IR, and partial CP coding sequences, in comparison with the other polerovirus isolates retrieved from the NCBI GenBank database. The isolates cloned and sequenced in the current study are marked with an asterisk

CABYV is an emerging polerovirus across the globe including India. Its occurrence has recently been reported from the cucurbits growing regions of India viz., Andhra Pradesh, Chhattisgarh, Karnataka, Orissa, and Tamil Nadu on the bitter gourd and teasel gourd crops [4, 5]. The present study not only confirms the association of CABYV with the bitter gourd (already known host) from New Delhi but also with cucumber, a new host originating from New Delhi, suggesting that the virus is spreading across the country on different hosts. To the best of our knowledge, this is the first report of natural infection of cucumber by CABYV in India.

Supplementary information

Below is the link to the supplementary information.

Funding

Funding was provided by the Indian Council of Agricultural Research (IN), New Delhi (India).