First report of DNA barcoding, phylogenetic analysis and transmission study of Medicago sativa phytoplasma (16Sr-II-D) and associated insect vectors in Pakistan

Abstract

Medicago sativa is a good fodder crop in Pakistan. Its continuous cultivation and sustainable production is important for the better growth and yield of animals. Phytoplasma is an important well spread disease worldwide which has been reporting from more than 150 years ago on different plants including Lucerne or Alfalfa (Medicago sativa). The study was conducted to identify phytoplasma and potential insect vectors responsible for its transmission. The sampling of infected plants and sucking insects was done from Multan, RY Khan and Faisalabad, Punjab during 2017–2020. The 50 symptomatic samples were observed for the detection of phytoplasma initially through staining and electron microscope. It was further confirmed by Nested Polymerase Chain Reaction (Nested-PCR), Sequencing and Phylogenetic Analysis. The results indicated the presence of phytoplasma bodies in sieve tube cells of the infected plants that were further confirmed by the amplification of 1.8 kb and 1.2 kb fragment of 16 S rRNA gene using the primer pairs P1/P7 and RI6F2n/R2 respectively. Restriction fragment length polymorphism (RFLP) analysis also showed a similar pattern of bands formation associating with 16 S rRNA of 16SrII-D subgroup linked with sesame phyllody (16SrIID) group. The obtained DNA sequences of Pakistani isolates were submitted on NCBI (MT614018.1 and MT614019.1). The phylogenetic analysis using Clustal W and MEGA6 software showed that submitted sequences have > 99% nucleotide identity with phytoplasma strain “Ca. P. australasia” of 16SrII-D subgroup. The potential insect vectors, Orosius orientalis, Orosius argentatus, and Laudelphax striatellus captured from infected plants were detected positive as well as transmission study confirmed their vector status for alfalfa phyllody diseases transmission. To our information, this is first detection of phytoplasma infestation and its insect vectors associated with Medicago sativa in Pakistan. The 16Sr-II D group of phytoplasma is spreading widely in many crops so, control is essential to stop it into other economically important crops.