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Abstract
Usama Samy Fathy Mohamed Elkelany: Controlling of Root-Knot Nematodes In Eggplants Using Genetically Improved Bacteria. Unpublished Ph.D. Thesis, Department of Plant Protection, Faculty of Agriculture, Ain Shams University, 2016.
Meloidogyne javanica is one of the major pest infecting eggplants, the important vegetable crop in Egypt. The world wide control of root-knot nematodes depends on the use of synthetic nematicides. Due to the high cost and toxicity of the chemical nematicides, alternative control measures have been investigated. One plausible alternative is the use of biocontrol strategies which involves the application of antagonistic microbes to nematodes. The most common microorganisms involved in this strategy are soil bacteria named rhizobacteria. The objectives of this study are to improve the nematicidal potential of selected rhizobacterial strains against M. javanica infecting eggplant through biotechnological process i.e. protoplast fusion and gene cloning. The study was started with the screening of sixteen identified rhizobacterial strains Anoxybacillus flavithermus - Bacillus alvea N3- B. cereus Bc6 - B. cereus 6A15- B. cereus I/6 - B. licheniformis N5– B. licheniformis 5A3 -B. pumilus I1 -B. subtilis Ain-shams- B. subtilis D.B100 – B. subtilis Al-Azhar- B. thuringiensis I.B- B. thuringiensis I.A- B. thuringiensis K- Geobacillus stearothermophilus 9A5 and Pseudomonas fluorescens against M. javanica J2s. The highly percentage mortality in M. javanica J2s were 87.5% and 84.09 % resulted from A. flavithermus and B. pumilus, respectively after three days of exposure time. Under laboratory conditions, bioassay tests were carried out to evaluate the egg hatch inhibition and mortality effect on M. javanica J2s due to cell suspension of A. flavithermus, B. pumilus, B. subtilus, B. thuringiensis and P. fluorescens. The greatest inhibition of egg hatch percentage was 87.69% due to A. flavithermus and the highly recorded percentage mortality in M. javanica J2s (86.15%) was due to B. subtilus, as compared to control. The greenhouse experiment using root dip treatment for 20 min in 10 ml (2 × 106cfu/ml) cell suspension for the five aforementioned rhizobacterial strains simultaneously and one week before nematode infection, showed a significant suppression effect for all strains on nematode reproduction with an improvement in eggplant growth parameters, as compared to control. Data revealed that root dipping one week before nematode infection, was more promising than that of treated simultaneously. The pot experiment conducted to study the effect of drenching the soil with 10 ml (2 × 106cfu/ml) cell suspension of the aforementioned rhizobacterial strains using three times of application relatively to nematode infection (simultaneously, one week before and one week after) revealed that, all treatments significantly reduced M. javanica population in soil and roots and improved eggplant growth parameters.
Soil drench either with bacteria one week before nematode infections or simultaneously were more promising than one week after. A split root technique experiment was carried out to confirm the systemic resistance potential of B. pumilus against M. javanica infecting eggplant. Obtained results showed a significant reduction in nematode population in responder half root as compared to untreated control.
A. flavithermus and B. pumilus were selected as parent models to construct a protoplast fusion for their nematicidal, biochemical and physical characters that related to the production of chitinase, gibberellins, protease and thermopiles as well as to their higher efficiencies in biocontrolling nematode in the preliminary study.
Ten fusants resemble to their parent were selected and evaluated for their nematicidal potential against M. javanica J2s mortality and egg hatching. Full nematode mortality was recorded after 72 hrs of exposure due to all fusants except No. 29 and 30 which induced 92.05% and 98.86% mortality, respectively. The maximum egg hatching inhibition 84.09% was resulted from the fusants 65/3 and 68/5. Under greenhouse condition the highly three promising fusants (65/3, 68/3 and 71/4) were investigated in comparison with their parents separately or concomitantly against M. javanica infecting eggplant by root dipping treatment in 10ml (2 × 106cfu/ml) cell suspension for 20 min. and two times of nematode infection (simultaneously and one week before root dip). Data showed that, all fusants were more effective in reducing nematode population either in soil or roots as compared to their parent and control. Also, improved eggplant growth parameters was recorded. To examine the nematicidal effect of the aforementioned fusants as soil drenching treatment using three times of nematode infection (simultaneously, one week before and one week after) a pot experiment was carried out. Results showed that, all fusants were more effective than their parents in reducing nematode population and improved eggplant growth parameters. Another set of pot experiment was conducted to evaluate the effect of drenching the soil with two doses from the aforementioned bacterial strains with one month interval comparing with the only one dose. Results showed that the two doses were more effective than the one dose in reducing nematode population. Enhancement of eggplant growth parameters was achieved. Data also showed that, the one dose from the fusants were more promising than the two doses from their parents. In the pot experiment conducted to compare the nematicidal effect of the three fusants and their parent as root dip or soil drench treatments, results showed that soil drenching was more effective in reducing nematode population than that of root dipping.
A gene encoding chitinase from B. subtilis has been cloned and expressed in E. coli. The supernatants of the transformed E. coli with chitinase gene T.CHI-NRC-4 and T.CHI-NRC-6 were investigated for their inhibitory effect on M. javanica egg hatch under laboratory conditions. Result showed a 96% and 94% inhibition in egg hatching due to the transformed E. coli T.CHI-NRC-6 and T.CHI-NRC-4 respectively as compared to control. A greenhouse experiment was carried out to evaluate the nematicidal effect of the supernatants of the two transformed E. coli T.CHI-NRC-4 and T.CHI-NRC-6 against M. javanica infected eggplant. Obtained results showed a significant reduction in nematode population in soil and roots and enhancement in eggplant growth parameters as compared to control.
Key words: Protoplast fusion - Gene cloning- chitinase -Meloidogyne javanica – Eggplant - Rhizobacteria.