Detection and characterization of verticillium dahliae kleb. isolates from grafted eggplanst grown in the province of Antalya by molecular methods

Verticillium wilt caused by soil-borne pathogen Verticillium dahliae is one of the most destructive and economically important disease of eggplants in Antalya province. Growing eggplants varieties grafted onto resistant rootstocks is a common and efficient practice to control the disease in the greenhouses of the region. However, the disease has become serious and caused serious losses in the greenhouse-grown grafted eggplants. In this thesis research, the population and race structure of V. dahliae isolates from grafted eggplants were determined by molecular methods. The fungus were isolated from eggplants collected from 54 greenhouses at 20 different sites in Aksu, Alanya, Finike, Gazipaşa Konyaaltı, Kumluca, Manavgat districts. The pathogen were detected in 30 greenhouses in 11sites from all districts except Manavgat. The isolates were obtained from 9 different combination of eggplant root stock and scion. DNA were extracted from one single spore isolate representing each greenhouse. DNA were also extracted from 4 tomato isolates representing ırk1and ır2 from Japan. V. dahliae specific marker genomic (SSMG) and intergenic spacer (IGS) regions of the isolates were amplified and sequenced. While Amplifications of V. dahliae specific genomic region of the isolates with primers DB19 and DB22 produced PCR products of 520 to 550 bp, amplifications with with IGS regions with primers VdIGSFI/VdIGSR1 yielded products of 1800-2000 bp. The sequence analysis of V. dahliae SSMG regions revealed that the isolates from grafted eggplants had sequences of 526 and 542bp lengths and formed two different population groups named group1 and group2 respectively. The sequences of these groups were compared with the sequences of V. dahliae sequence groups previously described in V. dahliae isolates from different plants in the world and available in the genbank. Comparisons showed that twenty four V. dahliae isolates from eggplant had a sequence of 526 and shared % 100 sequence similarity with seq2 isolates from olive. Six isolates had a sequence of 542 and were % 100 similar to seq4 isolates from cotton. Group 1 eggplant isolates displayed % 100 seqeunce similarity with two race-2 and one race-1 tomato isolates from Japan. The similarity of the group2 isolates to race-1 and race-2 isolates were less than 98%. The IGS sequences analysis also divided grafted eggplants isolates into two groups named group A and group B. The sequence similarity between these two groups was % 87. The isolates in the group A shared more sequence homology (% 95) with race-2 tomato isolates than group B (% 79). Phylogenetic analysis of SSMG sequences from 30 eggplant, 4 tomato and 48 genbank sequences of the isolates also revealed that isolates from eggplants fall into 2 different groups. Group 1 isolates were found to be more closely related to race-2 tomato isolates and other isolates from vegetables. On the other hand group 2 isolates were iv phylogenetically distant from race-1 and race-2 tomato isolates. Phylogenetic analysis of IGS sequences were performed with the sequences of ten grafted eggplant, two race-2 and one race-1 tomato and 42 genbank isolates from different region and different plants around the world. The analysis showed again that graftedd eggplant isolates grouped in two different groups previosuly determined in sequence analysis as group A and group B. Group A isolates grouped together with tomato race-2 and other vegetable isolates. Group B isolates were in phylogenetically distant group with race-1 and race-2 tomato isolates. PCR analysis with V. dahliae race-1 specific primers VdTr1 ve VdTr2 didn't produce any PCR products with any of the 30 isolates from grafted eggplants. This showed that eggplant isolates didn't belong to race-1 isolates. The overall results of this thesis study indicated that there were two genetically different population and isolate groups of V. dahliae in the greenhouses of Antalya province. When sequence and phylogenetic analysis coupled with race-specific analysis, it can be assumed that the eggplant isolates especially first group most probably belonged to race-2 and second group to race-2 or an identified race group.