Isolation, characterization and identification of microbial endophytes from fruits of Manilkara zapota (sapodilla fruit)
Manilkara zapota, commonly known as sapodilla or chikoo, is a long-lived, evergreen tree native to southern Mexico, Central America and the Caribbean. It is largely grown in south east asia and mexico. Sapodilla fruit is highly nutritious fruit and is one of the staple fruit used in India.
An endophyte is an embded symbiont, often a bacterium or fungus that lives within a plant for at least part of its life cycle without causing any disease.
Endophytes have been found in most species of plants studied to date; however, in most cases relationships are not well explained. Some endophytes enhance host growth, nutrient accumulation and help the to tolerate abiotic stresses, such as drought, and decrease biotic stresses. Here in this study we are going to focus on sapodilla fruit endophytes, study of various literatures on the web and books suggest that no such study has been done so far on sapodilla fruit.
So here I will try to find out the endophytic microbes diversity in the sapodilla fruit and their probable significance.
Materials and methods
A. Collection of the sample:
Fresh undamaged and healthy fruits will be collected in sterilized plastic bags from different places at different maturity stages.
B. Isolation of endophytic bacteria from sapodilla fruit
i. Surface sterilization
Surface of fresh fruit collected will be washed with distilled water than wiped with 70% ethanol followed by wahing with 0.1% mercuric chloride solution.
ii. Isolation of endophytic Bacteria
The samples of fruits will be cut into small pieces and macerated separately in phosphate buffer of pH 7.2 with a sterile pestle and mortar. Tissue extract will be then prepared for tenfold dilution in sterile saline. Serial dilutions (10-5, 10-6, and 10-7) will be prepared from this extract. For inoculations 0.1ml of the aliquot will be used on Nutrient Agar medium and PDA medium. The inoculations will be done in triplicates. These plates will be incubated at 370
C. Observations were taken after 48 to 72 hrs. Bacterial colonies were differentiated on the basis of morphological colony characters. Bacterial isolates were picked from plates and purified by streaking techniques and incubated at 370C. The isolation process repeated till monocultures were obtained for further experimentations.
D. Characterization of isolates: microscopic morphological study will be performed with proper gram staining; various biochemical tests will be performed for identification of genera.
E. 16s rDNA sequencing for species identification: 16S rRNA gene sequencing will be used for identi?cation and taxonomic classi?cation of bacterial species. Comparison of the bacterial 16S rRNA sequence has been considered as a valuable genetic technique. 16S rRNA gene has hyper variable regions provide species-specific signature sequences useful for bacterial identification. It is also capable of reclassifying bacteria into completely new species, or even genera. The sequencing techniques can be used to describe new species that have never been successfully cultured in laboratories. For performing 16s rDNA sequencing following steps is required.
i. Extraction of DNA:
The extraction methods to efficiently purify DNA from various sources have to be adapted depending on factors such as sample size, the freshness of the sample, and the biochemical content of the cells from which DNA is being extracted.
ii. Polymerase Chain Reaction
PCR should be performed to amplify particular DNA sequence with suitable universal marker for 16s DNA to gain enough quantity of DNA for sequencing.
iii. Agarose Gel Electrophoresis
Electrophoresis will be performed to separate the specific amplified segment of DNA.
iv. Elution of DNA
Extraction of specific bands of DNA from agarose gels in which they are separated through electrophoresis. Highly purified DNA can be stored in Tris-EDTA buffer for further sequence analysis.
v. Sequencing of purified DNA and phylogenetic tree construction by obtained sequence data using bioinformatics tool for confirming species identity.
Possible outcomes and significance of study:
To obtain noble microbial isolates from sapodilla fruit and their molecular identification to explore possible presence of some unknown microbial species.
To study endophytic microbial diversity in sapodilla fruit.
If identified strains are previously known than to establish that whether they are beneficial or harmful strains for plant as well as for consumer.
Possible study for PGPR activity and Probiotic potential of isolates.
This study is significant because till now no endophytic study is done on sapodilla fruit and it will result in some new noble acknowledgement for us.
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