Browsing by Author "Smita Nair"

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    Evidence of 16SrXI group phytoplasma DNA in embryos of root wilt diseased coconut palms
    (2013) Manimekalai, R.; Smita Nair; Soumya, V.P.
    The root wilt disease (RWD) is a major phytoplasma disease of coconut palms in South India. Phytoplasma belonging to the 16SrXI group has been associated with the disease. In our earlier studies we detected phytoplasma from leaves, inflorescence and roots of symptomatic palms in the nested PCR. In this work we examined the embryo from diseased palms for the presence of phytoplasma through semi-nested PCR assay with primers 1F7/7R3 - 1F7/7R2 specific for phytoplasma 16S rRNAgene. Out of 270 embryos collected in four rounds of sampling from two different locations, positive amplification was obtained for 45 (16.67 %) embryos. Sequencing and blastn analysis confirmed the presence of coconut root wilt phytoplasma. To check the potential seed transmission of phytoplasma, mature nuts from diseased palms were germinated in poly bags under disease free conditions and seedlings were sampled for DNA isolation and nested PCR analysis. But phytoplasma DNA could not be detected in any of the seedlings raised in poly bags.
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    Isolation and characterization of partial secA gene from coconut root wilt and arecanut yellow leaf disease phytoplasma and assessment of its possible use for sero diagnosis
    (2015-12) Manimekalai, R.; Smita Nair; Gangaraj, K.P.; Soumya, V.P.; Rao, G.P.
    A 425 bp fragment of phytoplasma secA gene encoding SecA, a component of the Sec protein translocation system, was isolated and characterized from coconut root wilt disease and arecanut yellow leaf disease phytoplasma, both belonging to the 16SrXI·B sub group phytoplasma. The gene showed 99% nucleotide identity with sugarcane grassy shoot phytoplasma secA gene. In the phylogenetic analysis of the translated SecA protein sequence, all the 16SrXI group phytoplasmas clustered together. The three dimensional structure of the protein was predicted using Moddler 9.13. The validation of the predicted structure indicated that 83.6% of the amino acids lie in the favored region indicating that the predicted structure was ideal. The possible antigenic domains in the protein were also predicted using the tool SVM TriP. The antigenicity prediction of the SecA protein fragment showed the presence of 4 possible antigenic domains. The secA gene was expressed in pET vector and the protein was used for the development of polyclonal antiserum in rabbit. Because the titre ot the phytoplasma was very less, the color development in ELISA was less Intense. A technique for isolation of higher number of phytoplasma cells has to be standardized.
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    Molecular characterization identifies 16SrXI-B group phytoplasma (‘Candidatus Phytoplasma oryzae’-related strain) associated with root wilt disease of coconut in India
    (2014) Manimekalai, R.; Soumya, V.P.; Smita Nair; George V. Thomas; Baranwal, V.K.
    The root wilt disease of coconut is a major threat to coconut cultivation in southern India. Here we report the species assignment and 16Sr sub group classification of phytoplasma associated with coconut root wilt disease. Leaf samples were collected from root wilt symptomatic palms in Kayankulam district of Kerala. The phytoplasma 16S rRNA gene was amplified using three sets of primers namely, 1F7/7R3-1F7/7R2, 3Fwd/3Rev-3Fwd/5Rev, and P1/P7-R16F2n/R16R2 producing amplicons of 490, 1300, and 1250 bp respectively. Partial secA gene sequence of 480 bp from root wilt disease phytoplasma was amplified using primers cocsf/cocsr. Sequence characterization and phylogenetic analysis of 16S rRNA and secA genes of root wilt disease phytoplasma grouped it with the rice yellow dwarf group phytoplasmas and identified coconut RWD phytoplasma as ‘Candidatus Phytoplasma oryzae’ related strain. Further, in silico restriction digestion study of phytoplasmal 16S rRNA gene region between primers R16F2n/R16R2 was performed for 16Sr group/sub group classification. The root wilt disease phytoplasma grouped with the 16SrXI group members. In the sub group classification it produced identical restriction profile as the sugarcane white leaf phytoplasma and arecanut yellow leaf disease phytoplasma, and hence, it was placed in the 16SrXI-B sub group.
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    Molecular marker-based genetic variability among Yellow Leaf Disease (YLD) resistant and susceptible arecanut (Areca catechu. L.) genotypes
    (2012-12) Manimekalai, R.; Deeshma, K.P.; Manju, K.P.; Soumya, V.P.; Sunaiba, M.; Smita Nair; Ananda, K.S.
    Yellow Leaf Disease (YLD) is a lethal disease affecting the arecanut palms of south India causing heavy yield loss. Presently, there is no cure for this disease and breeding for resistance is the only solution. Disease-free palms identified in hotspot areas (i.e., heavily disease affected regions), may harbour special gene family. The present study aimed to differentiate YLD resistant areca palms with susceptible palms using three different marker systems, i.e., RAPD, ISSR and resistant gene based markers. Amplification of arecanut genomic DNA using these marker systems yielded a total of 248 fragments, that could be scored, of which 130 were polymorphic between YLD resistant and susceptible individuals. Among these markers, RAPD generated greater polymorphic fragments (61.6%) than ISSR (34.9%) and resistant gene specific markers (40.7%). The primers, UBC 321, OPAF 15, and OPE13 produced distinct banding patterns for resistant and susceptible palms. The average genetic similarity coefficient for pair-wise comparison of individuals ranged from 0.73 to 0.88. The average similarity between the YLD resistant palms was found to be 0.79 and that of susceptible was 0.80. The highest similarity coefficient, 0.88 was observed between the YLD resistant individuals (R1 and R2). It has been found that the resistant and susceptible areca palms show relatively less genetic diversity and are skewed towards their phenotype. The results of this molecular characterization may provide starting points for map-based cloning of the YLD resistant genes.
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    Real-time PCR technique for detection of arecanut yellow leaf disease phytoplasma
    (2014) Smita Nair; Roshna, O.M.; Soumya, V.P.; Vinayaka Hegde; Suresh Kumar, M.; Manimekalai, R.; George V. Thomas
    The Yellow Leaf Disease, with phytoplasmal etiology, is a serious disease affecting arecanut palms and causing substantial yield loss. Phytoplasmas are cell wall less, unculterable, phloem limited plant pathogens generally detected using microscopy, serology and molecular techniques. Here we report a SYBR green based real time PCR approach for detection of arecanut yellow leaf disease phytoplasma.We designed efficient primers for SYBR green based real time PCR to overcome the problems in conventional PCR. Primers QPF2/QPR2, designed from highly conserved phytoplasma 16S rRNA gene was used to amplify DNA preparation from spindle leaf tissues of symptomatic palms using real time PCR. A unique melting peak at 82.3±0.5 °C was observed for symptomatic arecanut samples. The PCR products were further purified, sequenced and analysed using BLASTn. The sequences showed 99 % nucleotide identity with Indian arecanut yellow leaf disease phytoplasma sequences in the database. Two representative sequences were also deposited in the Genbank database. The present study thus devised a platform for rapid and sensitive detection of phytoplasma associated with the arecanut yellow leaf disease.
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    Real-time PCR technique-based detection of coconut root (wilt) phytoplasma
    (2011-11) Manimekalai, R.; Smita Nair; Soumya, V.P.; Roshna, O.M.; George V. Thomas