First report of Cryptosphaeria pullmanensis as causal agent of Cryptosphaeria canker of Populus nigra in Iran

Abstract

A diatrypaceous fungus was isolated consistently from cankers on Populus nigra trees showing dieback symptoms in Kohgiluyeh Boyer-Ahmad, Zanjan and Esfahan provinces in Iran. Morphological characteristics and phylogenetic analyses of the ITS region of the rDNA identified the taxon as Cryptosphaeria pullmanensis. Pathogenicity tests conducted in potted 3-month-old cuttings of P. nigra confirmed Koch's postulates and revealed that C. pullmanensis caused canker on this host. This is the first report of C. pullmanensis causing a canker disease on P. nigra in Iran.2 weeks in the dark at 24°C, covering a 60-mm Petri dish after 8 days ( Figure 1i). Teleomorph (sexual stage) and anamorph (asexual stage) morphologies of the isolates from Iran were in close agreement with morphological descriptions of Glawe (1984) and Trouillas et al. (2010). The anamorph of the fungus also resembles the form genus CytosporaEhrenb. with respect to the multichambered conidiomata and allantoid conidia (Glawe, 1984). However, C. pullmanensis can be distinguished from species of Cytospora by having annellides, sympodulate, longer and larger conidia (Glawe, 1984;. A total of 20 strains associated with Cryptosphaeria canker were isolated from P. nigra trees growing in three provinces of Iran. Single isolates of both types of morphs yielded identical colonies, showing that both types of morphs belonged to the same fungus. Three isolates (one from each district surveyed) isolated from cankers and two additional strains (IRAN 2426C and IRAN 2512C) isolated from conidiomata and ascomata on the host tissue were sequenced. Five new ITS sequences were obtained and aligned with 21 reference sequences retrieved from GenBank (Table 1). The length of our sequences was between 530 and 540 bp and was identical among the five isolates. Based on a BLASTn search of NCBI GenBank nucleotide database, the closest sequence to our fungus was C. pullmanensis (GenBank KT428044; identities = 530/531(99%), gaps = 1/531(0%)). Isolates obtained in this study grouped in a well-supported clade (bootstrap value = 100%) with C. pullmanensis (Figure 2). Based on both morphology and ITS sequencing, the occurrence of this pathogen in Iran was confirmed.In the pathogenicity test, cankers with an average size of 1-3 cm in length, 1-2 cm wide and 0.3-0.6 mm depth formed after 5 months and were similar to those occurring on naturally infected stems.Following removal of the bark, the necrotic phloem was exposed and a dark brown discoloration of the wood was visible (Figure 1f-h).The fungus produced asexual fruiting bodies and spore tendrils around the inoculation site after 5 months that were similar to those occurring on infected hosts described above (Figure 1f). The pathogen was re-isolated consistently from the lesions, which confirmed Koch's postulates. Percent fungal recovery for all of three isolates tested was 100%, whereas no fungi were isolated from the control plants. Pathogenicity tests using three isolates of different origin showed no differences in virulence or pathoge...

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