DNA Based Identification of Aphid Species and Vector-Virus Association Analysis of Aphid Borne Luteovirus

Abstract

Aphids (Hemiptera: Aphididae) are globally important crop pests and vectors of viral disease, but their identification is generally difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial cytochrome c oxidase I (COI-5ʹ) gene (DNA barcode) has proven effective for the identification of species in many groups of insect pests and also in aphids. In this research, 728 aphids were collected from various plant hosts in north-central Pakistan, and identified and analyzed at COI barcode. Three species (Periphyllus lyropictus, Aphis nasturtii and Aphis astragalina) were reported for the first time in Pakistan. The Barcode Index Number (BIN) system assigned the 728 sequences to 52 BINs, while the Automatic Barcode Gap Discovery detected 51 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 7.26% sequence divergence (range = 0.2-10.32%) at COI, while intraspecific distances averaged 0.13 (ranged from 0-3.19%). BIN analysis suggested that specimens with intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Aphis gossypii, Sitobion avenae and Aphis craccivora) showed deep intraspecific divergences, providing evidence that each was a cryptic species complex. The study compiled the first barcode reference library for the aphids of Pakistan, and examined global haplotype diversity in six important Luteovirus vector aphid species. This study also examined the aphid-Luteovirus association considering the carrot mottle disease (CMD) which is caused by co-infection of Carrot red leaf virus (CtRLV), Carrot mottle virus (CMoV) and Carrot red leaf virus RNA (CtRLVaRNA), and transmitted by aphid, Cavariella aegopodii in a persistent, non-propagative manner. Myzus persicae is an important vector of many plant viruses but its involvement in the spread of CMD has not been documented. The current study determined the involvement of M. persicae in the transmission of virus complex, responsible for CMD, by transmission studies. M. persicae were exposed to the CMD-infected plants of chervil and then transferred to disease-free plants (chervil, fennel, celery, carrot, cilantro and parsley) of the family Apiaceae. Healthy plants infested with the xiii viruliferous M. persicae developed typical CMD symptoms. The transmission of respective viruses was confirmed through RT-PCR by amplifying the target genes with CtRLV-, CMoV- and CtRLVaRNA-specific primers. Sequence comparisons of the amplified virus genes with those in the GenBank showed high sequence diversity in the three components of the disease complex causing CMD both in amino acids and nucleotides.