اولین گزارش Phomopsis amygdali عامل شانکر درختان فندق در ایران

    The common hazel (Corylus avellana) is a species native to the northwest of Iran, with over 12500 ha grown in the province of Guilan. In the autumn of 2011, twigs and branch galls of Corylus avellana were frequently observed on common hazel trees in Eshkevarat orchards in the province of Guilan. On hazel trees the galls appear as large nodules initiated on twigs in which tissues developed more widely than normal twigs. The galls varied in size from 2-4 cm (Fig1). When cut open it consists of woody tissue that was disorganized in comparison to the normal wood. At first bacterial diagnostic assays were done on infected samples to find the probable bacterial agent, after getting negative results, the galls tissues were surface-sterilized in 1% NaOCl for 1 min before drying under a laminar flow hood. Small pieces of internal tissue were plated onto 2% potato dextrose agar (PDA) and malt extract agar; the PDA plates were incubated at 25°C in the dark to promote mycelium growth. Single conidial isolations were made onto fresh PDA plates from sporulating colonies. In order to induce sporulation, isolates were cultured on PDA plates and were incubated at 25°C under near ultraviolet (nuv) light in a 12 h light - darkness regime for 2-3 weeks. To determine the optimum temperatures for growth, radial mycelium growth was measured for isolates plated onto PDA and incubated for 5 days at seven different temperatures ranging from 5-35 ºC in 5 ºC intervals. Culture colours of isolates were described after seven days incubation at 25 ºC. A representative isolate was deposited at the CBS-KNAW Fungal Biodiversity Centre, the Netherlands, The CBS accession number assigned to the strain is CBS 134017. For molecular identification, DNA sequence data were generated for the ITS1-5.8S-ITS2 region (ITS) of the rDNA, and a fragment of the translation elongation factor 1-alpha (TEF) gene region (Van Niekerk et al. 2005). These sequences were compared to other sequences of P. amygdali in GenBank, which confirmed the morphological observations. Sequences were submitted to GenBank under the accession numbers KC609755 for ITS, and KC609756 for TEF.  Pathogenicity tests were conducted using two representative isolates of P. amygdali isolated from infected Corylus avellana twigs, both in the greenhouse and laboratory on 6-month-old common hazel plants and detached hazel twigs respectively. In the laboratory, common hazel detached twigs, 20-25 cm in length provided from a hazel orchard in spring, were wounded, and 5-mm mycelial plugs from the edge of 7-day-old colonies on PDA were placed under the bark of twig wounds and sealed with Parafilm with 10 replicates. Ten control detached twigs were inoculated with uncolonized PDA plugs. All twigs were incubated at 24 to 26°C for 15 days in the dark. Greenhouse pathogenicity tests were conducted with 15 replicates using 6-months-old hazel plants inoculated with the same method as was used for the detached twigs; uncolonized PDA plugs were used for the ten control plants. The inoculated and control plants were kept in a greenhouse for nine months at 20±5°C. Culture characteristics on PDA at 25°C was cottony with white to dark mycelium with inverse dark brownish, the margin of the colony was deeply lobbed and zonate, the isolates grew best at 25°C (colony diameter 45-48 mm after 5 days) on PDA. The isolates produced conidiomata in culture and dark-pigmented pycnidia were produced over the surface of potato dextrose agar (Fig 2). Conidiomata were eustromatic, superficial, scattered, black, and somewhat spherical to irregular, uniloculate formed on PDA had a conidiomatal wall including several cells thick with the outer layers brown to black, ostiole was single, circular to irregular in shape, and some uniloculate conidiomata had a rostrate beak. Conidiophores were short with 1 or 2 septa or multiseptate and branched. Conidia were of two types, alpha conidia were hyaline, fusiform, and straight, guttulate, aseptate and the mean of conidial dimensions were 6.9 × 2.8 µm, beta conidia were hyaline, filiform, straight or curved, eguttulate, aseptate.The ITS sequence results indicated that the sequenced strain (Gen Bank KC609755) differed with 3 nucleotides and 1 gap from Phomopsis amygdali [GenBank AY485752, strain STE-U 5151(Van Niekerk et al. 2005)] and numerous other strains of this species available on GenBank. The fragment of the translation elongation factor 1-alpha (TEF) gene region was identical to Phomopsis amygdali [(Gen Bank GQ250339 (Santes et al. 2010)].Pathogenicity was proved when the brown-black lesions developed and the same fungus was reisolated from all inoculated twigs in the laboratory. Young inoculated plants were decayed after two months and new shoots were grown from their feet. On vigorous infected plants the gall symptoms developed on the inoculation site of the stem after growing nine months.To our knowledge, this is the first report of the occurrence of P. amygdali causing galls on hazel trees in Iran. Phomopsis amygdali (Delacr.) is a known pathogen of peach in the world (Farr et al. 1999) and one of the most virulence Phomopsis species as the causal agents of cane, leaf spot and Phomopsis swollen arm on grapevine. Phomopsis galls of trees and shrub are caused by one or more species of Phomopsis and were found also on oak, maple and elm. The species was recently epitypified as part of a report on a disease survey on almond in Portugal (Diogo et al. 2010). The disease cycle on hazel trees has not been studied yet.