First Report of Dieback of Salix acmophylla Caused by Paecilomyces formosus in Iran.

Salix acmophylla Boss. (Brook willow), is native to Iran and mainly grows in natural riparian landscapes. It has excellent potential to use in erosion and phytoremediation projects. Since 2018, dieback has been observed on S. acmophylla at the Gilan-e Gharb forest (34°05'59.9"N, 46°00'52.6"E) in Kermanshah province, Iran. Affected trees showed wilting and yellowing of leaves, cankers, branch dieback, and decline (Fig. 1A, B). Small pieces (5 mm2) from the canker margins of 10 affected tree branches were surface sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, samples were rinsed three times with sterile H2O, plated on potato dextrose agar (PDA) supplemented with 100 mg/l streptomycin sulfate and 25 μg/mL chloramphenicole, and incubated at 25°C for 5 days. The same fungus was isolated from all samples. Pure cultures were obtained by monosporic isolation. A representative isolate, RU-PaMa-S1, was used for morphological and molecular characterization and deposited in the Fungal Reference Collection of the Ministry of Jihad-e Agriculture at the Iranian Research Institute of Plant Protection, Tehran, Iran. Radial growth of colonies on PDA and Czapek yeast extract agar (CYA) were 30 and 25 mm after seven days at 25 °C in the dark, respectively. Fungal colonies grew more rapidly on malt extract agar (MEA), and reaches a diameter of about 70 mm within 7 days at 25 °C in the dark. Color of colony on MEA, PDA, and CYA was light yellow to yellow-brown, which gets darker upon aging. In biochemical assay, our isolate grow and produced acid in culture on creatine sucrose agar medium. The conidiophore was irregularly branched out with one to seven phialides. The phialides were in whorls or solitary, flask shaped with cylindrical bases, and narrow apices. Conidia were one-celled, hyaline to yellow brown in mass, smooth, and ellipsoidal to cylindrical with truncate ends. Conidia were 3.0-9.6 (4.5) × 1.7-4.0 (2.6) µm (n = 50). Chlamydospores were present in two week old cultures. The morphology matched the description of Paecilomyces formosus (Sakag., May., Inoue and Tada) Houbraken and Samson (Samson et al., 2009). For an accurate identification, genomic DNA of RU-PaMa-S1 was extracted to amplify ITS regions, and β-tub gene with ITS1/ITS4 (White et al. 1990), and βt2a/βt2b (Glass and Donaldson 1995) primers, respectively. The sequences were deposited in GenBank (ITS: OL891502, β-tub OL944475). A BLAST search of GenBank showed that the ITS, and β-tub sequences of RU-PaMa-S1 were similar to those of Paecilomyces formosus. Baed on the multigene phylogeny and morphology, RU-PaMa-S1 isolate was identified as Paecilomyces formosus (Fig. 2). Molecular analyses based on sequencing of the ITS region, and parts of the β-tubulin and calmodulin genes, showed that P. formosus may be composed of three species, including P. maximus, P. lecythidis and P. formosus. However, the three species could be distinguished by only molecular data and not by microscopical examination (Samson et al. 2009). To fulfill Koch's postulates, 10 detached and 10 attached healthy branches from two 20-year-old S. acmophylla plants at the Gilan-e Gharb forest in Kermanshah province, Iran, were inoculated with 5-mm mycelial plugs of 5-day-old culture of RU-PaMa-S1 isolate (Fig. 1 C). Control branches were treated with agar plugs. The detached inoculated branches were stored in sterilized glass jar containing moist sponge and incubated at 25-30°C for 35 days until the appearance of the canker symptoms. After 35 days, inoculated branches were cut from the trees and transferred to the laboratory to check disease development and re-isolation of the pathogen. All inoculated points on detached and intact branches showed lesions similar to those observed on naturally infected trees, whereas controls were symptomless (Fig. 1 D, E). The same fungus was re-isolated from these lesions. P. formosus has been reported to infect a broad range of hosts, including Amygdalus scoparia, Malus domestica, Pistatia vera, and Querqus brantii (Sabbagh and Khosravi Moghaddam 2016; Sabernasab et al. 2019; Azizi et al. 2020). This is the first report of P. formosus on S. acmophylla in Iran and in the world. This finding provides crucial information on this high-risk disease to S. acmophylla and other forest trees and basis for identifying management strategies.