White root rot, caused by Rosellinia necatrix Prill, is one of the most important disease on fruit trees in Iran and world wide. Although the range of host comprises is more than 170 plant species, damages resulted from this disease is important in regards of wilting and death of the fruit trees. Control of the white root rot is complicated by its ubiquitarian presence in the soil and by a number of specific characteristics including: resistance to drought, tolerance to a wide range of soil pH, high number of hosts and penetration deep into the soil and resistance to various common fungicides. Therefore, control of white root rot, should be mainly based on the avoidance of the pathogen through the use of certified healthy propagative materials and new plantings in non-infested soils. To achieve a simple and sensitive pathogen detection method in soil and root, this investigation was conducted. Eighteen isolates of R. necatrix were obtained from root of cherry and apple and confirmed using species specific primer, R2 and R8. To study genetic diversity, PCR-RFLP of ITS region using Mbo I, Msp I and Hin fI restriction enzymes, was applied. The result revealed that there was no genetic diversity among isolats using PCR-RFLP. Soil direct culture method, root infected in culture medium, Nested PCR method and pathogen trapping (using twig Bait) were used to determine the best method of pathogen detection. Among used methods, the direct culture method from soil was not suitable and wasn’t sensitive for detection. However, other methods were capable to detect which the Nested PCR was better than other. Relation ship among primary inoculum, time needed for detection and symptoms occurrence, showed that by increasing in primary inoculum, more time is needed for detection and disease symptoms. To verify the accuracy of the used detection method, ITS resion was coloned in pTZ57R/T vector and sequenced. Comparison of the sequenced showed %98.99 similarity to recognized isolate of R. necatrix . In conventional PCR, using primer pairs R2-R8 10 pg ?l ?1 was amplified, however in Nested PCR by R7-R10 primer pairs, 1 fg ?l ?1 was amplified, enable the detection of R. necatrix , gave a detectable amplification product of template DNA in direct PCR and in artificially inoculated soils. The results revealed that NestedPCR method of detection in soils and root is more sensitive and reliable. Key words : Detection, White Root Rot, Rosellinia necatrix , Nested PCR, pathogen
