Characterization of the bacteria associated with alder angular leaf spot in Iran

Abstract

Alnus subcordata (Caucasian Alder, Betulaceae) is known as one of the most important tree species in wood industry with high nitrogen fixation capability and high rate of biomass production. In recent years, a disease symptom with the characteristics of alder bacterial leaf spot has been reported from the forests in Mazandaran province, Iran. To characterize the leaf spot causal agent, samplings were conducted from infected trees in different regions of Mazandaran forests. Characterization of the bacteria isolated from symptomatic tissues was performed based on classical and molecular methods. Twenty colonies were purified and tested by PCR using primers X1 and X2 specific for the genus Xanthomonas and biochemical analyses as well. Based on molecular and phenotypic identifications, the isolates were identified as genus Xanthomonas. Phylogenetic analysis was carried out based on PCR amplification and sequencing of gyrB genes for 14 selected isolates. Consequently, according to the molecular data all recovered isolates were determined as X. arboricola. Furthermore, multilucus sequence analysis using gyrB, fyuA, dnaK and rpoD for various pathovars of X. arboricola showed that our isolates clustered in a single clade sister to X. arboricola pv. juglandis. The results of pathogenicity tests on alder leaves seedlings confirmed that all the isolates were pathogenic and caused leaf spot on A. subcordata. To the best of our knowledge, this is the first comprehensive study on the molecular identification of the alder bacterial leaf spot disease in Iran.