جداسازی و شناسایی قارچ های همراه با بیماری های شاخه و تنه درختان گردو (Juglans regia L.) در استان کرمان

Walnut (Juglans regia L.) is an important fruit trees in Iran. This woody plant is affected by various fungal trunk pathogens that reduce crop yields and increase annual production costs. In the last 10 years, English walnut trees with decline symptoms have been observed and reported in several areas in Kerman province. In the most cases a brown to black wood discoloration of internal wood tissues was observed in cross-sections of affected branches and trunks. Therefore, the objective of this study was to identify fungal species associated with these symptoms based on molecular and morphological methods. For this purpose, several field surveys were conducted on walnut trees in different areas of Kerman province between May 2015 and September 2017. Wood samples were collected from trunk and branches of trees showing disease symptoms including yellowing, branch and trunk cankers, dieback and internal wood necrosis. Symptomatic wood tissues were cut (4×4 mm) and surface disinfested with sodium hypochlorite solution for two min, washed twice with sterile distilled water and were plated on potato dextrose agar (PDA) plates. PDA plates were incubated at 25 °C in the dark and all fungal colonies were transferred to a fresh medium. Pure fungal colonies were obtained for all isolates prior to morphological and molecular identification. Isolates were first identified based on colony characteristics and microscopic observations. Total genomic DNA from representative isolates of each group was extracted and morphological identifications to species level were confirmed by sequence analysis of the internal transcribed spacer (ITS) nrDNA region using the primers ITS1 and ITS4. Based on morphological characteristics and DNA sequence comparisons, five fungal species including Eutypella vitis (six isolates), Cryptosphaeria pulmanesis (six isolates), Sporendocladia bactrospora (10 isolates), Cosmospora viridescens (20 isolates) and Paraconiothyrum hakeae (three isolates) were isolated from symptomatic walnut trees. To our knowledge this study represents the first report of E. vitis, S. bactrospora, C. viridescens and P. hakeae on English walnut trees in the world.