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Morphological and molecular diversity of endophytic Colletotrichum gloeosporioides from tea plant, Camellia sinensis (L.) O. Kuntze of Assam, India.

ABSTRACT: Thirty isolates of endophytic fungi were isolated from healthy asymptomatic leaves of tea plant (Camellia sinensis) and identified morphologically based on colony morphology, spore shape and size, growth and sporulation rate. Internal transcribed spacer r-DNA sequence analysis supported for molecular identification of all the isolates. Based on morphological and molecular characteristics the isolates were identified as Colletotrichum gloeosporioides. Variations on colony morphology which included the production of conidial masses, led to divide the isolates into different groups. Variations on spore size, growth rate and sporulation rate were exhibited by all the isolates. With RAPD molecular markers, genetic variations among the thirty isolates were observed. Genetic variations and relatedness among the thirty isolates were analyzed with UPGMA phylogram using NTSYS program. Two major groups were obtained among the thirty isolates. Group I comprised of 16 isolates which included three sub groups (Ia, Ib and Ic) and Group II constituted fourteen isolates and it also had three sub groups (IIa, IIb and IIc). A partial co-relationship among the isolates was established on the basis of morphological and molecular based clustering.

SUBMITTER: Rabha AJ

PROVIDER: S-EPMC6299854 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Project description:BackgroundTea is the most popular beverage worldwide second only to water. Its demand is tremendously rising due to increased awareness of its medicinal importance. The quality and uses of tea depend on the tea-types which are mainly three types including China, Assam and Cambod type having distinct compositions of secondary metabolites. Huge variation in secondary metabolites in different tea-types and cultivars limited the successful application of various approaches used for its trait improvement. The efficiency of a protocol for isolation of protoplast is specific to the types and cultivars of tea plants. The existing tea protoplast-isolation protocols [which were optimized for tea-types (China and Assam type) and Chinese cultivars grown in China] were found ineffective on types/cultivars grown in India due to type/cultivar variability. Therefore, optimization of protoplast-isolation protocol is essential for tea-types/cultivars grown in India, as it is the second largest producer of tea and the largest producer of black tea. Here, efforts were made to develop an efficient protoplast-isolation protocol from all major types of tea (China, Assam and Cambod types) grown in India and also from three types of tender leaves obtained from field-grown, hydroponically-grown and tissue culture-grown tea plants.ResultsDeveloped protoplast-isolation protocol was effective for different types of leaf tissue obtained from the tender leaves of field-grown, hydroponically-grown and tissue culture-grown tea plants. Moreover, optimized protocol effectively worked on all three types of tea including China, Assam and Cambod types cultivated in India. The digestion of leaves with 3% cellulase R-10, 0.6% macerozyme, 1% hemicellulase and 4% polyvinylpyrrolidone for 12 h at 28ºC yielded approximately 3.8-4.6 × 107 protoplasts per gram fresh tissue and 80-95% viability in selected tea cultivars, and tissue culture plant material was found most appropriate for protoplast isolation.ConclusionsIn conclusion, we reported an efficient protocol for isolation of protoplasts from tender tea leaves of all major tea-types (China, Assam and Cambod) grown in India. Moreover, the protocol is also effective for tender-leaf tissue of field-grown, hydroponically-grown and tissue culture-grown tea plants. The findings are expected to contribute to the genetic improvement of tea traits widely.

Project description:Anthracnose disease is caused by Colletotrichum gloeosporioides, and is common in leaves of the tea plant (Camellia sinensis). MicroRNAs (miRNAs) have been known as key modulators of gene expression in response to environmental stresses, disease resistance, defense responses, and plant immunity. However, the role of miRNAs in responses to C. gloeosporioides remains unexplored in tea plant. Therefore, in the present study, six miRNA sequencing data sets and two degradome data sets were generated from C. gloeosporioides-inoculated and control tea leaves. A total of 485 conserved and 761 novel miRNAs were identified. Of those, 239 known and 369 novel miRNAs exhibited significantly differential expression under C. gloeosporioides stress. One thousand one hundred thirty-four and 596 mRNAs were identified as targets of 389 conserved and 299 novel miRNAs by degradome analysis, respectively. Based on degradome analysis, most of the predicted targets are negatively correlated with their corresponding conserved and novel miRNAs. The expression levels of 12 miRNAs and their targets were validated by quantitative real-time PCR. A negative correlation between expression profiles of five miRNAs (PC-5p-80764_22, csn-miR160c, csn-miR828a, csn-miR164a, and csn-miR169e) and their targets (WRKY, ARF, MYB75, NAC, and NFY transcription factor) was observed. The predicted targets of five interesting miRNAs were further validated through 5'RLM-RACE. Furthermore, Gene Ontology and metabolism pathway analysis revealed that most of the target genes were involved in the regulation of auxin pathway, ROS scavenging pathway, salicylic acid mediated pathway, receptor kinases, and transcription factors for plant growth and development as well as stress responses in tea plant against C. gloeosporioides stress. This study enriches the resources of stress-responsive miRNAs and their targets in C. sinensis and thus provides novel insights into the miRNA-mediated regulatory mechanisms, which could contribute to the enhanced susceptibility of C. gloeosporioides in tea plant.

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Morphological and molecular diversity of endophytic Colletotrichum gloeosporioides from tea plant, Camellia sinensis (L.) O. Kuntze of Assam, India.

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