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2018 - 201912020 - 20221
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Subject: Microbiology × Author: Antil, Sonam × Start date: 1992 × Type: Thesis ×
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    ThesisItemOpen Access
    Studies on antagonistic activity of bacteria against root knot nematode(s)
    (CCSHAU Hisar, 2022-09) Antil, Sonam; Pathak, D.V.
    Root-knot nematodes under the genus Meloidogyne are world widely distributed plant parasites that cause serious damages to many important agricultural crops such as potato, cotton, tomato, brinjal, etc. Bacteria associated with roots and rhizosphere of many plant species have been extensively tested for the control of various soil borne pathogens including plant parasitic nematodes. Four bacterial isolates- KMT-4, KMT-5, KMT-8 and KMS-6 were originally isolated from nematode infested rhizospheric soil belonging to research field of Department of Nematology, CCS HAU, Hisar and have demonstrated potential nematicidal activity against root-knot nematode Meloidogyne javanica. All the isolates showed HCN and siderophore production but chitinase activity was exhibited by KMT-5 only. The bacteria were identified based on 16S rRNA gene sequence analysis as: Bacillus aryabhattai (KMT-4), B. cereus (KMT-5), B. megaterium (KMT-8), B. altitudinis (KMS-6). For in vitro bioassays, M. javanica eggs were isolated from infected roots and juveniles (J2) were obtained through MBFT. Effect of bacterial strains on egg hatching and J2 mortality was checked by preparing the extracellular and intracellular extracts of all four bacterial strains and tested their activity on M. javanica eggs and J2. The larval mortality and egg-hatching inhibition rates of M. javanica were increased with the rising concentration of extracellular extracts of all the bacterial strains and the duration of exposure. A pot experiment was also planned in spring 2021 on tomato to check the genetic stability of the bacterial strains. All four bacteria reduced nematode infection and promoted plant growth far better than control and chemical treatment as well. The nematicidal bacterial extracts were subjected to crude extraction from extracellular and intracellular extracts for proteinaceous as well as non-proteinaceous nematicidal compound(s). Crude extraction done for extracellular non-proteinaceous was found potent of all the bacterial strains when tested against M. javanica eggs and J2 with B. cereus KMT-5 showing maximum activity. Thus, this study aimed to separate, purify, and identify nematicidal compound from B. cereus KMT-5 and to validate its anti-M. javanica activities. Compound was purified through silica gel column chromatography using ethyl acetate: methanol as mobile phase with increasing polarity gradient and fractions obtained were checked via thin-layer chromatography. Similar fractions were combined and tested against M. javanica eggs and J2. Out of five subfractions, subfraction III (FIII) showed maximum activity. The nematicidal compound from FIII was extracted via prepTLC and subjected to HPLC. Structural identification was conducted through H1-nuclear magnetic resonance (NMR) spectroscopy. The nematicidal compound was identified as phytosphingosine based on H1-NMR shifts and structure was in silico verified using MestReNova 14.2 software. Purified nematicidal compound phytosphingosine from B. cereus KMT-5 was then tested against M. javanica eggs and J2. Only 5.6 J2 were obtained after 96h of treatment with phytosphingosine whereas in control there were 98.0 J2 showing a significant difference in hatching rate. The mortality rate of M. javanica J2 reached to 94.6% after 72h of exposure to phytosphingosine.
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    ThesisItemOpen Access
    Biocontrol of root-knot nematode (Meloidogyne javanica) in brinjal (Solanum melongena L.) using plant growth promoting rhizospheric bacteria
    (CCSHAU, 2018) Antil, Sonam; Rakesh Kumar
    Egg plant (Solanum melongena L.) or brinjal is a nightshade species belonging to family Solanaceae and is the second most important vegetable crop after tomato and shows high susceptibility to infection by the root knot nematode Meloidogyne javanica. Management of the root-knot nematodes via biological methods such as PGPR is a novel technology emerging for sustainable agriculture. In this context, a total of 63 isolates were isolated from nematode affected rhizospheric soil of brinjal, tomato crops and earthworm culture media. All the 63 isolates were characterized for various plant growth promoting traits. Out of 63, fifteen isolates produced siderophores and maximum production was observed by isolate LBB-1and KMT-2. Chitinase activity was observed in 14 isolates and isolates KMT-2 and KMT-8 showed maximum chitinase activity. Sixteen isolates were found to be positive for HCN production and maximum production was seen in isolates NAE-1, KMB-3, KMT-2 and KMS-6. Fifty seven isolates were capable of excreting ammonia and maximum ammonia excretion was observed in KMT-2 (4.8μg/ml) and LBB-1 (4.7 μg/ml). All the bacterial isolates except one were producing IAA and high level of IAA production was observed in KMT-1 (45.9 μg/ml), KMB-2 (38.8 μg/ml), KMB-4 (27.7μg/ml) and KMT-5 (26.4μg/ml). A total of 39 isolates solubilized phosphate and highest P-SI was observed in isolate KMT-1(2.0) and LBB-4 (1.7). Twelve isolates- NAB-4, NAS-1, NAE-1, LBB-1, LBS-1, KMB-3, KMT-2, KMT-4, KMT-5, KMT-8, KMS-3 and KMS-6 resulted in minimum hatching in egg masses and higher mortality rate in J2 of M. javanica (upto 92%). Based on all the screening tests, a total of 12 were selected for pot house experiment on brinjal crop. Rhizospheric microbial count increased in all treatments from 0 DAP to 45 DAP. Under pot house conditions, T14 (RDF +KMT-4), T15 (RDF +KMT-5), T16 (RDF + KMT-8) and T17 (RDF +KMS-6) showed maximum plant growth, reduced galls and eggs in roots, and final nematode population in soil.