Morphological and Molecular Identification of Cytospora germanica Causing Canker on Populus spp. in China

Species of Cytospora Ehrenb. cause canker and dieback on many genera of hardwoods. During surveys of forest trees in 2004 and 2008, some hardwoods such as Populus spp. with symptoms of canker and dieback were found in Liaoning and Xinjiang provinces, respectively. In these trees, the canker pathogen discolored the sapwood. During wet weather, the conidia were exuded from the fruiting bodies in gelatinous matrices, usually as yellow or orange tendrils. In the spring, the affected trees were wilting and the diffuse cankers spread rapidly and extensively during the period when trees began active spring growth. For saplings, sometimes the death rate of the pathogen has exceeded 50%. Conidiomatal stromata immersed in bark, prominent, circular to ovoid, 1.10 ± 0.23 mm in diameter (n = 10). Discs were white, nearly flat, circular, 0.44 ± 0.04 mm in diameter (n = 10), with one or two ostioles per disc. Ostioles were gray. Locules were subdivided by invaginations into chambers. Conidia were hyaline and lelongate-allantoid shaped, 4 to 5.4 μm long and 1 to 1.4 μm wide. Pieces (5 × 5 mm²) of the junction of affected and healthy tissues were surface sterilized with 1% NaOCl for 30 s and then rinsed twice in sterile distilled water. The fragments were placed on potato dextrose agar (PDA) plates incubated at 25°C for 7 days. The obtained isolates were cultured on PDA at 25°C in diffuse fluorescent light for 30 days, and then were deposited in the culture collection of the Chinese Academy of Forestry. According to these morphological features we initially thought that this pathogen was Cytospora chrysosperma (Pers.) Fr. However, BLAST analysis showed 98% and 99% homology with ITS sequence of isolate CBS118560 (GenBank Accession No. DQ243793), 99% and 100% homology with LSU gene of isolate AR3427 (AF362561) when we amplified ITS-rDNA gene and LSU gene of the isolates. The sequences were submitted to GenBank with the following accession numbers: JQ086563, JQ086564, JX524617, and JX524618. Pathogenicity tests were conducted in the greenhouse by inoculating 20 disinfected (70% ethanol) Populus tomentosa cuttings. Another two cuttings were treated with water agar as controls. The cuttings were incubated at 25°C for 30 days. In 18 of the 20 cuttings, the cambium became brown and appeared water soaked 20 days later, whereas controls did not show any symptoms. C. germanica was reisolated from symptomatic tissues. Inoculations were later repeated two times with similar results. Hubbes (1) and Spielman (2) considered V. germanica (teleomorph of C. germanica) a synonym of V. sordida (teleomorph of C. chrysosperma) based on morphological studies. We almost regarded these isolates as C. chrysosperma too. Morphological identification has been a weak and often inadequate means of identifying Cytospora species (1,2); the present study based on ITS-rDNA gene and LSU gene has substantially elevated identifications. To our knowledge, this is the first report of C. germanica on Populus spp. in China. Populus species are economically important trees in forests of North China and C. germanica has the potential to cause significant economic losses. References: (1) M. Hubbes. Phytopathol. Z. 39:65, 1960. (2) L. J. Spielman. Can. J. Bot. 63:1355, 1985.