Abstract:
Identification of 14 isolates of Trichoderma spp. collected from durian orchards in Chanthaburi Province were done based on morphological characteristics, colony morphology, colony color, growth rate and spore production. Molecular features were examined by PCR to amplify DNA for the ITS region and beta-tubulin gene of the isolates by using ITS1/ITS4 and beta-tubulin (T1/T22)primers, respectively. Sequence analysis of all isolates was determined and identified as Trichoderma asperellum. Each isolate was cultured on PDA with Phytophthora palmivorausing dual culture method. The result showed percent of mycelial growth inhibition to P. palmivora with 63.86-75.90. Responsiveness of T. asperellum to 6 fungicides was evaluated using poisoned food technique at the recommend concentrations and found that all isolate of T. asperellum grew only on PDA mixed with metalaxyl and iprodione. Moreover, mycelial growth of some isolates like S1TK, S2TK, S1ST,S3ST, S1SK, S2SK, S11TK andS22TK on PDA+metalaxyl was greater than PDA (control). Fungicidal sensitivity of both fungicides was evaluated by increasing the concentrations, All isolate of T. asperellum could grow on to PDA+metalaxyl (900 ppm) while some isolates could thrive on PDA+iprodione (1,200 ppm) indicating fungal resistance might be occurred. Later, design of specific primers to histidine kinase nik-1 gene was investigated to confirm that the histidine kinase gene was also found in T. asperellum by using PCR amplified DNA for histidine kinase gene, ITS region and beta-tubulin gene using histidine kinase (H1/H2), ITS1/4 and beta-tubulin (T1/T22)primers, respectively. The result showed that T.asperellum growing on PDA+iprodione could not be amplified DNA of histidine-kinase gene. Whilst DNA amplified using ITS1/4 and beta-tubulin primers one fragment (600 and 1,800 bp, respectively) Inhibition capacity of all isolates growing on PDA+iprodione to P. palmivora was evaluated on PDA+metalaxyl (150ppm) andiprodione (750 ppm) including PDA. The result showed that T. asperellum, isolate S22TK grew faster than P.palmivora on PDA+iprodione, It might be directly impact from iprodione. The highest percentage of P. palmivora mycelial inhibition was 93.10. Furthermore, some avirulent as S1TT, R1ST and Ba2SK were found resistant to iprodione growing on PDA+iprodione with the percentage of P. palmivora mycelial inhibition was 91.37, 84.48 and 89.65, respectively.