Divine Tree Neem Tree Essay
Chapter – 1
INTRODUCTION
INTRODUCTION
1.1NEEM TREE
The divine tree neem (Azadirachta indica A. Juss) is a tropical evergreen plant native to east India and Burma. It grows in much of Southeast Asia and West Africa (Varma et al., 2006). Neem is an omnipotent tree and a sacred gift of nature. Neem tree is mainly cultivated in the Indian subcontinent. Neem is a member of the Mahogany family of Meliaceae. Today it is known by its botanical name Azadirachta indica (A. indica) A. Juss. Neem has been used extensively by humans to treat various ailments since Vedic periods (Venugopalan et al., 2013).
Figure 1.1: Endophytes isolated from neem tree located at the Botanical Garden, U.
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After sterilization, media allowed to cool for few minutes and then poured to 20 ml big Petri- plates in the laminar air flow (LAF) cabinet.
4.5.2 Inoculation of bacteria:
The bacteria were inoculated in LB broth prepared by dissolving 20g Luria broth powder in 1000ml of distilled water. The bacteria were allowed to grow for 2 days and then inoculated on LBA media around the plate and then Magnoporthe mycelia was inoculated at centre of the plate and incubated at 28°Cand allowed it to grow for 5-6 days.
4.5.3 Recovery of fungal pathogen:
The stability (survival) test was carried out check the survival ability of the pathogen Magnoporthe oryzae after inhibition of neem bacterial and fungal endophytes. After 7 days of incubation, the Magnaporthe was taken from the bioassay plate which was inhibited by the bacterial and fungal entophytes on LB/PDA including control.
4.6. Bioassay of fungal endophytes against fungal pathogen (Magnaporthe oryzae). 4.6.1 Inoculation of fungal
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5.3. DNA Fingerprinting of fungal endophytes.
5.3.1 Genomic DNA isolation.
The genomic DNA was isolated in accordance with protocol explained in Materials and Methods. The DNA was isolated from all the inhibiting fungal endophytes samples. The quality of genomic DNA was found to in between OD 260/280=1.8-2.0 which indicates the good quality of genomic DNA. Figure5.10: Genomic DNA isolation of various neem endophytes (inhibiting Magnaporthe) isolated from the mother plant in UAS botanical garden, GKVK Bangalore, Karnataka on 1% agarose gel.
5.3.2 DNA amplification by 18S - rRNA ITS primers
The PCR was done to amplify DNA using 18S- rRNA ITS-primers forward and reverse primers for the 6 fungal endophyte samples and Magnaporthe control. Figure 5.11: 18S-rRNA ITS PCR profile of various neem endophytes.
5.3.3 PCR clean up of amplified DNA.
Figure 5.12: Gel eluted profile for the 18S- rRNA ITS amplified productson 1% agarose gel.
5.3.4 Sequencing of the purified
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