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Leaf Blight of Buxus sempervirens in Northern Forests of Iran Caused by Calonectria pseudonaviculata

Buxus sempervirens subsp. hyrcana (Pojark.) Takht. (boxwood) is an evergreen shrub/tree in Caspian hyrcanian forests covering the Alborz mountain range of northern Iran. During the summer of 2012, a sudden leaf and twig blight disease of boxwood was observed throughout the northern forests of Iran. Disease symptoms included circular dark spots on leaves leading to defoliation, and longitudinal brown-black streaks on the shoots. Diseased plant material was collected from the Guilan and Mazandaran areas, placed in moist chambers, and incubated at 20°C to induce sporulation. Single conidia were plated onto half-strength potato dextrose agar supplemented with 250 mg/L streptomycin and incubated at 25°C under near-ultraviolet light. Isolates were transferred to carnation leaf agar and incubated at 25°C under near-ultraviolet for morphological characterization, and representative isolates were deposited into the culture collection of the CBS-KNAW Fungal Biodiversity Centre under accession numbers CBS 134431 and CBS 134432. Gross morphological characters were determined by mounting fungal structures in lactic acid and 50 measurements at 1,000× magnification were made for all taxonomically informative characters. The observed macroconidiophores consisted of a stipe bearing a penicillate suite of reproductive branches and a stipe extension, terminating in a naviculate vesicle. The stipe extensions were septate, hyaline (85 to 160 × 2 to 4 μm), terminating in a naviculate vesicle, 6 to 11 μm in diam. Conidia were cylindrical, rounded at both ends, straight, with one septum (55 to 68 × 4 to 6 μm). These morphological observations agreed to those provided for C. pseudonaviculata Lombard, M. J. Wingf. & Crous (1,2). To confirm morphological identification, DNA sequence data were generated for the ITS1-5.8S-ITS2 region of the rDNA, and a fragment of the translation elongation factor 1-alpha gene region (3). These sequences were compared to other sequences of C. pseudonaviculata in GenBank (100% similarity for both loci), which confirmed the morphological observations. Sequences were submitted to GenBank under the accession numbers KC736850 and KC736851 for ITS, and KC736852 and KC736853 for TEF. Koch's postulates were proven by spraying a 3 × 10⁶ conidia/ml conidial suspension of isolate CBS 134431 onto 1-year-old B. sempervirens subsp. hyrcana plants until run-off, and covering them for 24 h with a plastic bag to maintain high humidity. Control plants were sprayed with sterile water. Ten plants were used for each treatment and maintained in a greenhouse at 20 to 22°C with 95% relative humidity. Symptoms similar to those observed in nature developed within 4 days of inoculation and the test fungus was successfully reisolated from the inoculated plants. No symptoms were observed on the control plants. Boxwood blight caused by C. pseudonaviculata, was first reported in the United Kingdom in the mid-1990s and has since become widespread, causing epidemics globally (1,2,4). To our knowledge, this study represents the first report of boxwood blight in its native environment and in Iran.

References: (1) P. W. Crous et al. Sydowia 54:23, 2002. (2) B. Henricot and A. Culham. Mycologia 94:980, 2002. (3) L. Lombard et al. Stud. Mycol. 66:31, 2010. (4) M. R. Saracchi et al. J. Plant Pathol. 90:581, 2009.