Evaluation of genetic variation of the causal agent of leaf spot of citrus (Alternaria sp.) isolates based on RFLP-IGS

 Nowadays RFLP test from IGS region is a used for determination of interspecies diversities. The aim of this study was to determine pathogenic species of Alternaria from leaf spots of citrus in Mamasani region based on morphological and ITS1 sequencing and to determine genetic variation of isolates based on RFLP-IGS.
This descriptive study was performed on 70 leaf spotted samples collected from different citruses species and cultivars of Mamasany orchards including orange (Valencia, Navel and leaf spoon Cv.), lime and sweet lemon (Wikova and local Cv.) during In autumn 2011. The isolates were cultured on PDA medium and purified by using single spore method and their species were identified based on morphological characters. The pathogenicity of the isolates was evaluated by based on Koch’s rules through exposure of isolates to leaf. DNA was extracted from mycelia by CTAB and the ITS1 and IGS regions were amplified by using specific primers. The ITS1 region was sequenced and the IGS region was digested by HinfI, BsnI, RsaI, BssmI and AluI restricted enzymes.
All isolates were identified as Alternaria alternata based on morphological characters and sequencing of ITS1 region. Constructed dendrogram based on RFLP patterns showed existence of three divergent groups in 51% similarity. Orange, lime and sweet lemon isolates grouped in divergent groups. Also Wikova isolates grouped in different clade.
The obtained results clearly showed that the enzyme patterns of IGS can reveal the classification defects of Alternaria alternata form citrus hosts.