Author name:
علاء محمد رشا عبد الامير الرديني
Supervisor name:
محمد عامر فياض | طالب احمد جايد
Abstract:
This study had been carried out at Plant Protection Department and Genetic Engineering Labs of Agricultural College / Basrah University, while pots experiments were carried out at fields of Agricultural College / Basrah University. The present study included a comparison experiments of different Rhizoctonia solani isolates depending on molecular genetic techniques alongside with morphological and physiological parameters, host range and anastomosis groups identification.Thirty two different isolates of R. solani have been isolated from several host plants from several plant families throughout different geographical areas at Basra governorate. Result of pathogenicity test revealed that the majority of R. solani isolates caused a significant reduction in the germination percent of cabbage seeds, the isolates of Rs5, Rs16 and Rs19 were found to be more pathogenic and led to complete inhibition of germination, hence, the other isolates varied in their effect on seed germination percent and reported the range of 3.3 - 66.7 %, while, the isolate of BRs15 (binucleate isolate) did not show any significant effect on seeds germination compared to control (untreated) treatment (100%). According to pathogenicity test results, the most pathogenic isolates (Rs2, Rs5, R9, Rs16, Rs27 and Rs29) were selected for more followed experiments.Morphological analyses of R. solani isolates elucidated a profound difference among these isolates, the colony color was dark brown with the Rs9 isolate, light brown at the Rs16, while it was cream at the Rs2, Rs5, and Rs27, a white color with light brownish color was observed with the isolates of Rs29. The growth patterns at the medium supplemented with Potassium Chlorat showed that the isolates of Rs5, Rs9, Rs27 and Rs29 were sensitive to chlorate, while, the isolates of Rs2 and Rs16 were resistant to Chlorate. Regaling the effect of temperature on the growth of R. solani, it was found that the optimal growth temperature was at 25 °c for all examined isolates, while no growth was observed for all isolates at the temperature of 10 and 40 °c. The best pH values for growth supporting were 6 and 7 for all examined isolates.Anastomosis group analysis revealed the occurrence of anastomosis among the isolates of Rs5, Rs16, Rs27 and Rs29 with the standard isolate AG2, this confirms that these isolates belong to the group AG2, while the anastomosis between the two isolates Rs2 and Rs9 with standard isolate AG2 did not happen, therefore these isolates belong to a different group. Microscopic observation revealed theBoccurrence of more than two nuclei in each cell of examined isolates the average of nuclei number was 4 - 8 nucleus / fungal cell, while the number of binucleate isolate BRs15 exhibition two nuclei / fungal cell.The DNA extraction experiments for R. solani isolates (Rs2, Rs5, Rs9, Rs16, Rs27, Rs27, Rs30 and AG2) revealed the efficiency of CTAB procedure to get a high DNA yield and that it’s a good quality, as indicated results of measuring the concentration of DNA isolates it was good quantity amount (197.9 - 560.4 ng / μl) was obtained by following this procedure. The random amplified polymorphic DNA (RAPD) results showed that all DNA of examined R. solani isolates were amplified by employing different RAPD primers, also each genetic primer succeeded in differentiation among isolates, as showed the polymorphic bands and monomorphic bands.The results of restriction fragment length polymorphism technique (RFLP) using RS1 and RS4 primers showed that the bands size for R. solani isolates obtained between 500 - 522 bp, restriction technique results using enzymes AvaII, HincII, MseI and MunI showed that Rs2 isolate belong to the group AG1 - IB, while Rs5, Rs16, Rs27, Rs29 and the standard isolate (AG2) belong to the group AG2 - 2, while Rs9 isolate belongs to group AG11 so Rs30 isolate belongs to group AG3 - PT.Result of host range of R. solani isolates showed a significant differences in the host preference, the isolates of Rs2 was able to infect all plant hosts except okra plant, while Rs5, Rs16, Rs27 and Rs29 were able to induce the infection on marshmallow, okra, cowpea, cotton and eggplant, no infections were reported on the host of melon and wheat plants, regarding R. solani isolate Rs9 an infection was observed on marshmallow, wheat and cotton, while no infection was noticed on melon, okra, cowpea and eggplant.Results of binucleate R. solani isolates role in protection of cotton plant from the infection with R. solani, revealed their protective role, the pathogenic effect of R. solani was significantly reduced, the germination percents were increased with BRs15 treatment reached 83.3, 86.7, 93.3, 83.3 and 90% at the treatments of Rs2, Rs5, Rs9, Rs16 and Rs27 respectively, without any significant difference with control treatment (93.3%). The highest germination percent was observed at BRs15 treatment with Rs29 and reported the percent of 96.7 %. Similar results were observed with damping - off percent of cotton plant, which proved the significant efficiency of binucleate isolate in their protection against all examined R. solani pathogenic isolates.
