الفهرس 
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Abstract
Biological control of root – knot nematode, Meloidogyne incognita infecting tomato plants by genetic improvement of lytic bacteria, Serratia marcescens, Pseudomonas fluoresences and Bacillus licheniformis to increase the lytic enzyme, chitinase and protease production about several times than that producing by the wild types, were studied under both laboratory and greenhouse conditions.
The wild types of the lytic bacteria, Serratia marcescens, Pseudomonas fluoresences and Bacillus licheniformis and their ten mutants (overproducing chitinase and alkaline protease via using mutation), namely: mutant SM 190, mutant SM 167, mutant SM 36 and. mutant SM 21 from S. marcescens, mutant BL 3, mutant BL 132, mutant BL21, mutant BL 37 and, mutant BL 40 from B. licheniformis and mutant PF 63 from P. fluoresences which were produced by using mutagenic chemicals (MMS, EMS) and three fusants (overproducing chitinase and alkaline protease via recombinant strains on protoplast fusion technique) and the parents of two strains S. marcescens (mutant No. 18) and B. licheniformis (mutant No. 10), were evaluated against the root – knot nematode, Meloidogyne incognita, as safer and environmental friendly control alternative to chemical nematicides. They were evaluated as lytic bacteria (chitinase and alkaline protease) against juvenile survival of M. incognita under laboratory conditions by using standard (S) or (S /10), and exposure period (24, 48 and 72 hrs).
1- Laboratory tests:
The mutants and fusants were obtained by the following procedures:
a)Evaluated 8 wild types isolates bacterial: two isolates of Serratia spp., two isolates of Bacillus spp., Four isolates of Pseudomonas spp., in production enzymes of each of the alkaline protease and chitinase enzyme. The best isolates are S. marcescens, P. fluoresences and B. licheniformis.
b)Genetic improving by mutation for the previous best three isolates by using methel methan sulfonate (MMS) were isolated and the definition of mutants was achieved and the results were as follows: 209 isolates of S. marcescens, 168 isolates of B. licheniformis and 155 isolates of P. fluorescens (See appendix).
c)Evaluated these mutants in enzymes production of
(protease and chitinase).
dPerforming repeat mutation again by ethyl methan sulfonate (EMS) to the highest isolates from the previous transaction. The definition of mutations resulting from the transaction, were evaluated on the survival and vitality of nematode Meloidogyne incognia juveniles by using (S) and standard (S/ 10), and the period of exposure (24, 48 and 72 hours).Generally, the results indicated that, the three fuasnts and ten mutants achieved the high death of the M. incognia juveniles compared with the wild types of parents. The highest death of M. incognia juveniles was achieved by the use of mutant BL 21and SM 36. On the other hand, The least impact was achieved by using the wild type and parents of bacteria.
Generally, fusants achieved the best effect on juveniles compared to the mutants and their parents. This effect was due to its ability to increase production of enzymes under this study and compilation of the best qualities by protoplast fusion. The numbers of either viable juveniles (VJ) or non viable juveniles (NVJ) clearly were reduced at their two dilutions (S and S/10) of different exposure periods (24, 48 and 72 hr) compared to the untreated control and consequently decreasing the numbers of both (VJ) and (NVJ). It seemed that, most of the dead nematode bodies were degraded and destroyed by these fusants. In this respect, fusant no. 103 achieved the most effective mortality and had the least numbers of juveniles as compared to the bacterial parents and untreated control.
The wild types of the lytic bacteria, S. marcescens, P. fluoresences and B. licheniformis produced ten mutants (overproducing chitinase and alkaline protease via using mutation), namely mutant SM 190, mutant SM 167, mutant SM 36 and. mutant SM 21 from S. marcescens, mutant BL 3, mutant BL 132, mutant BL 21, mutant BL 37 and, mutant BL 40 from B. licheniformis and mutant PF 63 from P. fluorescens by using mutagenic chemicals (MMS, EMS). Three fusants (overproducing chitinase and alkaline protease via recombinant strains on protoplast fusion technique) and the parents of two strains S. marcescens (isolate No. 18) and B. licheniformis (isolate No. 10) were obtained. Altogether, were tested on egg hatching on gelatinous matrix forming the eggmasses of the root – knot nematode, M. incognita.
Results indicated that, all tested lytic bacteria, fusants, mutants and parents obviously achieved high effect on hatchability of M. incognita eggs compared with the wild types and untreated control. The fusant F103 is more effective on the nematode eggs followed by the fusant F88 and then mutant SM 36 as compared to the wild types which achieved the lowest viability of eggs. Also, the tested treatments resulted different efficiency on eggs causing abnormal developmental stages containing decayed egg shells, dead juveniles and degraded juveniles. The highest mortality was found at using fusant F103 followed by the mutant BL 21 or SM 36 followed by the other mutants and finally, the bacterial wild types which caused the lowest effect with some exception of abnormal developmental stages. In addition, there were positive relationships between mortality of eggs and numbers of abnormal developmental stages and both bacteria concentration and enzyme production from the fuasnts and mutants.
2- Greenhouse experiments:
Effect of wild types and different mutants and the parents of two strains S. marcescens (isolate No. 18) and B. licheniformis (isolate No. 10) were tested against nematode development in case of using juveniles as a source of tomato plant infection. Four application methods of adding bacteria in soil were applied as follows:
1-Adding nematodes, M. incognita and after one week adding bacteria.
2-Adding nematodes, M. incognita and bacteria in the same time.
3-Adding bacteria to soil and after one week adding nematodes.
4-Adding nematodes, M. incognita and bacteria together on tomato plant and after one week adding another dose of bacteria.
The best application of bacteria on the infected soil of tomato plant with nematodes, M. incognita was in the same time and after one week from adding another dose of bacteria. In this application the mutant BL 21 sustained the best effect on controlling the juveniles, the galls and the eggmasses formation as compared to the untreated control. These reductions reached 92.15 % juveniles in soil and 77.08 % of galls and 88.43 % of eggmasses followed by the mutant SM 36 which recorded 90.72 % number of juveniles in soil, 70.63 % of galls and 86.50 of eggmasses as compared to the wild type of SM which attained the lowest reduction in either of galls number or eggmasses (26.49 % and 30.85; respectively). These reductions were corresponding to improvements of tomato growth.
Generally, results indicated that, the effect of the fusants and parents of M .incognita juveniles under greenhouse conditions in the case of the use of the best application of the bacteria on the tomato plant in the soil were as follows: F103 fusants achieved the best effect on controlling the juveniles, the galls and the eggmasses formation as compared to the untreated control. These reductions were corresponding to improvements of tomato growth.