Detection, Identification and Molecular Characterization of Phytoplasma Diseases in Uttar Pradesh Region of India
Loading...
Date
2015
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
SHIATS
Abstract
Phytoplasma severely impair productivity and phytochemical content of affected plants. A survey was conducted in Uttar Pradesh during October 2009-July 2012. Several fruit, vegetable and flowering plants and trees samples were collected from Meerut, Bulandshahr and Allahabad districts. Toria and sesame plants with phyllody and witches‟-broom, tomato and eggplant samples with severe symptoms of phyllody, yellowing and curling of leaves, peach with decline, leaf reddening and witches‟-broom, papaya with leaf yellowing, intervenial chlorosis and curl apical necrosis, pot marigold with phyllody and viresence and corn samples with leaf and midrib reddening collected from Meerut. Mango tree samples with phyllody, declines and shortening of internodes collected from Bulandshahr and chrysanthemum flowering plants samples with little leaf and formation of bladder like siliques from Allahabad. Peach samples collected three years from same site. Total DNA was extracted from symptomatic and asymptomatic plants were subjected to direct and nested-PCR assay using phytoplasma 16S rDNA region specific universal primers. A ~1800bp fragment was amplified in toria, peach, sesame, tomato and eggplant and shearing was found in chrysanthemum and pot marigold plants with primer pair P1/P7. A ~1600 bp fragment was amplified in symptoms bearing plants of toria, peach, mango, papaya and corn with P1/Tint primer pair. There was no amplification in all symptomless plants with both primer sets. P1/P7 amplified PCR products were also subjected for nested PCR with universal primer pair R16F2n/R2n, a ~1200bp fragment was amplified in toria, peach, tomato, chrysanthemum and pot marigold PCR samples. The partial 16S rDNA gene sequences of toria and peach were cloned to facilitate sequencing and sesame, tomato and eggplant were sequenced directly. The 16S rDNA sequences of peach collected on 2010, 2012, 2013, toria, sesame, tomato, eggplant associated phytoplasma were deposited in GenBank, accession no. HM988985, AB858361, KF72359, HM988986, JF706215, JX104335 and JX104336 respectively. Only a low titer of phytoplasma was observed in mango, corn, papaya, chrysanthemum and pot marigold infected plants, therefore sequencing was unsuccessful of amplified 16SrDNA sequence.
On the basis of phylogenetic analysis, virtual RFLP patterns and similarity coefficient calculations of the 16S rDNA sequence of phyto-peach1 phytoplasma (GenBank accession no. HM988985), it is concluded that phytoplasma infecting peach in India belongs to group 16SrI „Ca. Phytoplasma asteris‟. Sequencing and BLAST analysis of phyto-peach2 (GenBank accession no. AB858361) and phyto-peach3 (GenBank accession no. KF72359) phytoplasma 16S rDNA gene indicated that phytoplasma associated with peach disease was identical to aster yellows group phytoplasma. Phylogenetic analysis of 16S rDNA peach phytoplasma also grouped it with 16SrI-A. Phyto-toria (GenBank accession no. HM988986) showed the highest sequence identity 99% with „Brassica rapa' phyllody phytoplasma isolate Ludhiana (GenBank accession no. HM559245), member of group 16SrIX „Ca. Phytoplasma phoenicium‟. The sequence analysis of Indian sesame phyllody 16SrDNA (GenBank accession no. JF706215), showing 99% similarity with Mulberry dwarf phytoplasma (GenBank accession no. AB693124) a member of „Ca. Phytoplasma asteris‟ group 16SrI-B. TMT-Meerut sequence analysis showed the highest sequence identity 100% with that of members of group 16SrII „Ca. Phytoplasma aurantifolia‟ including the papaya yellow crinkle phytoplasma strain Y10097 designated as „Ca. Phytoplasma australasiae‟ that belongs to subgroup 16SrII-D. Phyto-eggplant phytoplasma 16S rDNA (GenBank accession no. JX104336) sequence showed the highest sequence identity 99% brinjal little leaf phytoplasma (KC178679), identified as a member of group 16SrVI, „Ca. Phytoplasma trifolii‟. To our knowledge, this is first detection of a witches‟ broom phytoplasma associated with peach and phyllody disease associated to tomato. This study also first time reported phytoplasma association to mango witches‟-broom, pot marigold phyllody and virescence and corn midrib reddening disease in India.
Description
Submitted in partial fulfillment of the requirements for the awards of the degree of Doctor of Philosophy in Biotechnology by Anchal Rani