الفهرس 
Chapter I: Introduction and Aim of the workOnly 14 pages are availabe for public view
 |  | from | 137 |  |  |
| | | from | 137 | | |
Abstract
Root knot nematodes are parasites of a multitude of host plants. Estimated yearly crop losses are approximately 5% worldwide, although the damage inflicted in certain regions of developing countries exceeds this level. Two polymerase chain reaction (PCR) methods for discriminating genera and species of collecting nematode samples were used. First, the amplification of internal transcribed spacer (ITS) of rDNA was used to distinguish between different major genera of root knot nematodes. DNA fragments containing the ITS were amplified from total DNA of 20 geographic isolates that infested most vegetable crops in El Fayoum and Bani-Sweef governorates. When primers 5367 and 5368 were used for amplification of ITS region, all isolates gave one major product of approximately 760 bp, this is specific for all nematodes of genus Meloidogyne . Second, the amplification of mitochondrial DNA was used to distinguish different species of root knot nematodes of genus Meloidogyne. Single juveniles were ruptured in a DROP of water and added directly to a PCR reaction mixture in a microcentrifuge tube. Primer annealing sites were located in the 3 portion of the mitochondrial gene coding for cytochrome oxidase subunit II and the 16S rRNA gene. Following PCR amplification, fragment of size 1700 bp specific for M. incognita and M. javanica was produced. Digestion of the amplified product with restriction endonucleases allowed discrimination among species with identically sized amplification products. Hinf I digestion of the 1700 bp fragment produced a two banded pattern in M. javanica, versus a three banded pattern in M. incognita.
The random amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Meloidogyne species isolates. It was generated by the amplification of random DNA fragments with single primers of arbitrary nucleotide sequence. Populations of each of M. incognita and M. javanica were distinguished by differences in fragment patterns with any of the ten RAPD primers used. from analysis of RAPD fingerprint of all tested populations, two RAPD markers were detected, one was specific for M. incognita populations with primer OPK-2 at fragment size 1000 bp and the second was specific for M. javanica populations with primer OPB-3 at size 1100 bp. These two RAPD markers were converted into SCAR (specific characterized amplified region) markers, which were sequenced and two PCR primer pairs were designed for each of M. incognita (MIE-for and MIE-rev) and M. javanica (MJE-for and MJE-rev). These primers can be used to detect these nematode species in the fields.