DNA barcoding of spider mites (Prostigmata:Tetranychidae) associated with ornamental plants

Abstract

Total area under floriculture in India is estimated to be 0.31 million hectares with a production of 2.08 million tonnes loose flowers and 0.82 million tonnes cut flowers in 2018-19. In Kerala, Thrissur district is considered as the centre of floriculture nursery business. Majority of nurseries in the area do not maintain their own sources of mother plants and hence import planting materials from other countries. Trade of commercial ornamentals has been recognized worldwide as an important invasion pathway for non-native pests, especially insects and mites. Though mites are reported as major pests of commercial ornamental plants from different parts of India, no systematic study has been conducted so far to document the diversity of mites associated with ornamental plants in Kerala. Considering this, the present study, ‘DNA barcoding of spider mites (Prostigmata: Tetranychidae) associated with ornamental plants’ was undertaken with the objectives to generate DNA barcodes for different species of spider mites infesting commercial ornamental plants of central Kerala and to find the genetic variability among them. The study included collection and culturing of spider mites, morphology based identification and molecular characterization of selected accessions. Purposive sampling surveys were carried out in commercial ornamental nurseries and homestead gardens of Thrissur and Ernakulam districts, covering 12 ornamental plants. Mite infested leaf samples were collected, brought to the laboratory and maintained separately as isoline cultures by assigning unique accession numbers. Morphological characterization of 26 isoline cultures revealed the occurrence of six species of spider mites under two genera viz., Tetranychus and Oligonychus. The genus Tetranychus was more diverse with five species viz., Tetranychus truncatus, T. urticae, T. okinawanus, T. neocaledonicus and T. marianae. The genus Oligonychus was represented by only one species, Oligonychus biharensis Hirst. Rose recorded the highest diversity of spider mites with five species. The mite species, T. okinawanus recorded wider host range with eight host plants viz., Adenium, rose, Gerbera, chrysanthemum, orchid, cairo morning glory, marigold and balsam. All the host plants except Adenium are new host records of T. okinawanus from India. The study recorded three new host plants for T. truncatus from India viz., rose, cock’s comb and marigold. In this study, T. marianae was recorded for the first time from Kerala. For molecular characterization, DNA was isolated and COI locus of 868 bp length was amplified using universal primer, specific to COI. Polymerase chain reaction (PCR) products of 12 accessions representing five species were sequenced and in-silico analysis was carried out. Homology analysis of sequences of 11 accessions showed 90-99 per cent similarity with sequences in NCBI database, which were in consensus with morphological identification. The sequence of the accession, Ros1vk (T. marianae) did not show significant similarity with any of the sequences in the NCBI database. In this study, COI sequence of T. marianae was submitted for the first time in GenBank. Barcode gaps among the species were examined by aligning the COI sequences using Clustal Omega tool and species-specific barcodes were identified at different nucleotide positions. Pairwise distance analysis of the sequences showed intraspecific divergence ranging from 0.00 to 0.036 and interspecific divergence ranging from 0.070-0.217. Phylogenetic analysis revealed the monophyly of T. truncatus and T. urticae and the close relationship of T. marianae with T. okinawanus. The study has shown that rose harbours many species of spider mites, indicating the need for imposing strict quarantine regulations for movement of planting materials of rose to avoid entry and invasion of mites into newer areas. The potential of T. okinawanus and T. truncatus to turn invasive in Kerala’s ecosystems is also brought out. The study establishes the reliability of COI locus as a marker for species discrimination in spider mites.