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|Isolation and Characterization of Epiphytic Orchidaceous Mycorrhizas Associated With the Populations of Rhynchostylis Retusa from Kangra Valley, Himachal Pradesh
|Reddy, M. S.
|Orchidaceae, Mycorrhizae, Rhynchostylis retusa, ITS, Tulasnella, Biochemical characterization, Phylogenetics
|Orchids are most extravagant group of flowering plants and are getting depleted and threatened due to habitat destruction, macroclimatic changes, shifting cultivation, overexploitation and developmental activities. Orchids produce the smallest seeds (nearly microscopic in size) of any flowering plant. These dust-like seeds are produced in great numbers, often over a million seeds per plant and lack an endosperm, resulting in a small embryo covered only by a thin protective wall. This lack of storage tissues for food reserves and protection makes the seeds extremely vulnerable to their environment, resulting in a high mortality rate unless optimum conditions are found for germination. The rate of seed germination in nature is very poor, that is, 2-5%. The symbiotic association between epiphytic orchids and mycorrhizal fungi is vital for essential nutrient supply during germination and seed establishment. Most of the mycorrhizal fungi of orchids fall into a non- sporing group known as Rhizoctonia, Rhizoctonia solani is a common symbiont of the members of Orchidaceae. Rhynchostylis retusa(L.) Bl. is a epiphytic orchid species found in Kangra, Himachal Pradesh, India. The ITS region is highly variable among fungal species, allowing to discrimination between even closely related species-based mycological studies at the sub-generic level and used for fungal species identification. In India till date a liitle work has been done on the mycorrhizal species related to orchids. This study focuses on the Tulasnella specific mycorrhizal interaction with Rhynchostylis retusa, which is an orchid specific mycorrhizal association. The isolated and pure seven fungal strains were amplified with Tulasnella specific primers set: ITSI/ITS4-Tul, out of which five showed positive results. The five fungal isolates were morphologically and microscopically characterized. Media optimazation on MMN media by OFAT method and various enzyme activity tests like acid phosphatase activity, phytase enzyme activity, biomass and pH reduction, cellulose enzyme reduction and phosphate solublization activity were performed and the results were analysed statistically on Graph Pad Prism 5.0 software and 2 Way-ANOVA also was performed to find the possible interactions and the significant statistical values. All of the fungal isolates showed varying significant results positive results. The molecular characterization of the fungal isolates was performed by sequencing the Tulasnella specific amplified PCR products. Out of five fungal species two sequences were able to be sequenced and the other three are under process. The two sequences were analysed and using BLAST search tool to homology of unknown sequences with those present in databases. The results showed that the sequences T1 and T2 were homologous to Fusarium sp. 208f and Leptosphaerulina chartarum respectively. The further studies are to be continued.
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