An Uncultured Bacterium Associated with Infection in Capsicum annuum in India

Article No.: 111413963 DOI: 10.15580/GJAS.2013.12.111413963 We make an extensive survey of Gorakhpur district of Eastern U.P. region of India from 2011-2013, for vegetable plant samples showing visible symptoms of possible phytoplasmal infection. We observed symptoms such as reduction in leaf size, curling of leaf, etc. in infected chilli plants (Capsicum annuum L., Family Solanaceae), growing in the fields of Gorakhpur district. By using universal phytoplasma specific P1 and Tint primers, we perform their PCR analysis. The PCR product of size 1600 bp was generated which were further purified and sequenced. Nucleotide sequence obtained in present study was deposited in GenBank through accession number KF298062 and named as “Uncultured Pseudomonas sp.' with/ clone=RCGKP1”. BLAST analysis and Phylogenetic tree established their association with ‘Pseudomonas’ group. In our knowledge, this is a new uncultured bacterium (359 bp), associated with infection in Capsicum annuum plant in Gorakhpur district of Eastern U.P. region of India. Submitted: 14/11/2013 Accepted: 22/12/2013 Published: 29/12/2013

Due to advancement in molecular techniques, several phytoplasma has been identified from several diseased plants (Bertaccini, 2007;Bertaccini and Duduk, 2009).
Chilli (Capsicum annuum L.) belonging to Solanaceae family, is a very frequent, delicious vegetable and spice of food menu.Chilli is a rich source of vitamin C and Capsaicin.Capsaicin is useful for treatment of pain, arthritis, blood sugar levels, cancer and several others (http://en.wikipedia.org/wiki/Capsaicin).
During survey period, we have find that infection in chilli plants were very common in Gorakhpur district of Eastern U.P. region of India and this also causes vast loss of chilli productions.
In India, Singh and Singh (2000) reported that chilli little leaf disease is associated with a phytoplasma and Khan and Raj, (2006) identified an Aster yellows phytoplasma ('Candidatus Phytoplasma asteris') (Accession No. DQ343288), infecting chilli plant in India.
In our knowledge, little work has been done for characterization and ultimate identification of phytoplasmas responsible for infection in chilli plants of Gorakhpur district of U.P., India.
So in present work, we make an attempt to identify and characterize the phytoplasma responsible for infection in chilli plants of Gorakhpur district of U.P., India.Ahrens and Seemüller (1992) and include a phytoplasma enrichment step.

MATERIALS AND METHODS
An amount of 1.5 g of infected plant material was incubated for 10 min in 8 ml of Phytoplasma Grinding Buffer in a mortar maintained on ice, and then finely crushed with a pestle, adding 5 ml more of PGB.The homogenate was then centrifuged for 5 min at 2,500 g.The supernatant of each sample was transferred to clean tubes and centrifuged for 25 min at 18,000 g.The pellet was dissolved in 1 ml CTAB buffer.After one-hour incubation at 60°C, the nucleic acids were purified by chloroform-isoamyl alcohol (24:1), and centrifuge at 12,000 g for 10 min.An equal volume of cold isopropanol was added to the drawn aqueous phase, and then incubated in ice for 1 hour.Then centrifuge at 12,000 g for 10 min.After centrifugation, add 1 ml 70% ethanol and centrifuge at 12,000 g for 10 min.Decant supernatant and dry the pellet at 37°C for 30 min.Dissolve DNA in 30 µl of sterile water.

RESULTS AND DISCUSSION
(A) Survey: During the course of survey of phytoplasmal infection in vegetable plants of different locations of Gorakhpur District, a variety of suspected symptoms like reduction in leaf size, leaf curling, leaf twisting, witches' broom, etc. were observed in fields.
Suspected samples of chilli plant showing symptoms of reduction in leaf size, leaf curling was collected and used for further identification and characterization of their causative phytoplasma.• Representative data for PCR amplification: 1% Agarose (w/v) gel electrophoresis of 16S rRNA gene PCR products.
(A) Plant samples: Infected Chilli (C.annuum L.) plant samples showing reduction in leaf size, curling of leaf, etc. (Figure1B) and healthy chilli plant samples (Figure1A), were collected from Gorakhpur district, during the course of survey in their growing seasons2011-2012  and 2012-2013.

Figure 1 A
Figure 1 A: Healthy leaf of Chilli plant.

Figure 1 B
Figure 1 B: Infected leaf of Chilli plant.
CTAB buffer: 2% CTAB; 100 mM Tris pH 8; 1.4 M NaCl; 20 mM EDTA.(C) Primers used in the study: The primers used for PCR were specific to phytoplasma and for sequencing we also used gene specific sequencing primers.P1 forward primer: AAGAGTTTGATCCTGGCTCAGGATT Tint reverse primer: TCAGGCGTGTGCTCTAACCAGC Target gene: 16s-23s rRNA spacer regions.

(
B) PCR analysis: PCR analysis with P1 and tint primers produced 1600 bp PCR products (Figure-3), which were further purified and then sequenced.