Isolation, Molecular Identification of Endophytic Fungi in Oryza Sativa and Characterization of Their Secondary Metabolite

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The term endophytic fungi refers to an organism which lives within photosynthetic plant tissue by forming symbiotic relationship with host and has no harmful effect to the host plant. They inhabit plant hosts for all or part of their life cycle. They colonize the internal plant tissues beneath the epidermal cell layers without causing any apparent harm or symptomatic infection to their host, living within the intercellular spaces of the tissues and its seems that they may penetrate the living cells. There are about a million endophytic fungal species present in all plants. These endophytic fungi were involved in defense mechanism and other essential secondary metabolites are produced and were able to induce to enhance the production of plant secondary metabolites which are source of very important chemical constituents for plant defense as well as pharmaceutical industry. Most of the medicinal plants were exploited for the occurrence of endophytic fungi from various parts and were exploited for bio-prospecting purpose. Endophytic fungi protect their host against insects, pests, pathogens and even from herbivores.

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Fungal taxonomy is traditionally based on comparative morphological features. However, special caution should be taken when closely related or morphologically similar endophytes are identified, because the morphological characteristics of some fungi are medium-dependent and cultural conditions can substantially affect vegetative and sexual compatibility. Furthermore, the conventional methods cannot be applied for identifying fungal isolates that fail to sporulate in culture, which are categorized as mycelia sterilia. In contrast, molecular techniques exhibit high sensitivity and specificity for identifying microorganisms and can be used for classifying microbial strains at diverse hierarchical taxonomic levels. Several recent studies have shown that genetic methods can be successfully used in the studies of endophytic fungi. Most of the endophytic fungi were detected and identified by comparative analyses of the ribosomal DNA sequences, especially the ITS region. For example, Harney et al. (1997) identified arbuscular mycorrhizal fungi from Artemisia californica using the ITS region. Also based on ribosomal DNA sequences, Guo et al. (2000) and Lacap et al. (2003) evaluated the endophytic fungal ‘morphotype’ concept concerning mycelia sterilia. Queloz et al. (2005) monitored the spatial and temporal dynamics of the tree-root endophyte Phialocephala fortinii using the restriction fragment length polymorphism (RFLP) analysis.

Study of rice endophytes has been mostly focused on endophytic diazotrophic bacteria, previous work has demonstrated their diversity, nitrogen-fixing potential, the infecting pathways and their distribution and colonization in plants. Several species of fungal endophytes have been found on rice leaves.

In India, diversity of endophytic fungi from rice plant in the South has been studied and the most frequently isolated endophytes were Chaetomium globosum, Penicillium chrysogenum, Clados-porium cladosporioides and Streptomyces sp.. Seed germination and seedling growth of rice are significantly influenced by the endophytic fungi. Endophytic fungi isolated from the Ricinus communis show a great antibacterial activity against 6 human pathogenic bacteria. Further growing those on large scale, modifying culture conditions and supplying some stimulants might help in getting better production of particular bioactive compound. Fungal extracts also exhibits excellent antiproliferative effect against hepatocellular carcinoma cells, the active compounds present in the extracts were identified through GC-MS analysis, which depicts the presence of aspirin and diethyl phthalate as the major constituents.

Aspergillus japonicus isolated from Rhynchosia beddomei, an endemic medicinal plant was characterized at molecular level using fungal specific 18S rRNA primers.

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