Browsing by Author "Kalita, Eeshan"

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Bipartite molecular approach for species delimitation and resolving cryptic speciation of Exobasidium vexans within the Exobasidium genus
(Elsevier B.V., 2021) Chaliha, Chayanika; Kaladhar, V. Chandra; Doley, Robin; Verma, Praveen K.; Kumar, Aditya; Kalita, Eeshan
Exobasidium vexans, a basidiomycete pathogen, is the causal organism of blister blight disease in tea. The molecular identification of the pathogen remains a challenge due to the limited availability of genomic data in sequence repositories and cryptic speciation within its genus Exobasidium. In this study, the nuclear internal transcribed spacer rDNA region (ITS) based DNA barcode was developed for E. vexans, to address the problem of molecular identification within the background of cryptic speciation. The isolation of E. vexans strain was confirmed through morphological studies followed by molecular identification utilizing the developed ITS barcode. Phylogenetic analysis based on Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) confirmed the molecular identification of the pathogen as E. vexans strain. Further, BI analysis using BEAST mediated the estimation of the divergence time and evolutionary relationship of E. vexans within genus Exobasidium. The speciation process followed the Yule diversification model wherein the genus Exobasidium is approximated to have diverged in the Paleozoic era. The study thus sheds light on the molecular barcode-based species delimitation and evolutionary relationship of E. vexans within its genus Exobasidium.
Optimizing in vitro culture conditions for the biotrophic fungi Exobasidium vexans through response surface methodology
(Springer Nature Publishing AG, 2020) Chaliha, Chayanika; Kalita, Eeshan; Verma, Praveen K.
The blister blight disease caused by the fungus, Exobasidium vexans has serious implications on the quality of tea production. The disease however, has been poorly studied and hence there is very limited information on the pathogen and as such the pathogenesis of blister blight infection. One of the major roadblocks in understanding E. vexans is the obligate and biotrophic nature of the fungus which limits the establishment and maintenance of in vitro cultures. To address this issue, a Central Composite Design based Response Surface Methodology (RSM) was adopted to study the modification of three fungal culture media viz. czapek dox, potato dextrose, and v8 juice, and the effect of altered media composition on growth conditions and media compositions were assessed. The response parameter for the RSM experiments was the mycelial biomass produced under different culture conditions. The uni and bi-parametric interactions among the experimental variables provided the basis for the statistically optimized conditions for maximal fungal growth. The study thus presents the recommended modifications of existing media that can lead to the successful establishment and maintenance of E. vexans in vitro cultures.
Rapid and precise detection of cryptic tea pathogen Exobasidium vexans: RealAmp validation of LAMP approach
(Springer Nature Publishing AG, 2023) Chaliha, Chayanika; Srivastava, Richa; Kalita, Eeshan; Sahoo, Lingaraj; Verma, Praveen K.
This work embodies the development of a real time loop mediated isothermal amplification (RealAmp) assay for the rapid detection of the cryptic tea phytopathogen, Exobasidium vexans, the causal organism of blister blight disease. Due to the widespread popularity of tea as a beverage and the associated agro-economy, the rapid detection and management of the fast-spreading blister blight disease have been a longstanding necessity. Loop-mediated isothermal amplification (LAMP) primers were designed targeting the E. vexans ITS rDNA region and the reaction temperature was optimized at 62 °C with a 60 min reaction time. Amplification of the E. vexans isolates in the initial LAMP reactions was confirmed by both agarose gel electrophoresis and SYBR Green I dye based colour change visualization. The specificity of the LAMP primers for E. vexans was validated by negative testing of seven different phytopathogenic test fungi using LAMP and RealAmp assay. The positive findings in RealAmp assay for E. vexans strain were corroborated via detecting fluorescence signals in real-time. Further, the LAMP assays performed with gDNA isolated from infected tea leaves revealed positive amplification for the presence of E. vexans. The results demonstrate that this rapid and precise RealAmp assay has the potential to be applied for field-based detection of E. vexans in real-time.