First Report of Dieback Disease on Cedars Caused by Diplodia seriata in China

Cedars (Cedrus deodara (Roxb.) G. Don) are well known as evergreen ornamental trees widely used in horticulture in temperate climates. In March 2013, dieback symptoms were found on cedar trees in different locations (including the campus of Nanyang Normal University) in Nanyang (33°01′ N, 112°29′ E), a southwestern city of Henan Province, China. Characteristic symptoms included needle discoloration and defoliation, canker formation and gummosis on trunks and branches, browning and tissue necrosis under the bark as well as dieback of branches/trunks. Of 873 cedar trees investigated, 139 (16%) were symptomatic. A total of 21 Diplodia sp. isolates were obtained from 102 tissue pieces randomly sampled from the lesion margins of 31 affected trees with a conventional method for isolation of culturable fungal species from plant tissues. Monohyphal cultures were isolated from actively growing edges of colonies to purify the isolates. The purified isolates were grown on 2% water agar with sterilized stems of Foeniculum vulgare to induce their colonies to form pycnidia (4). Unmatured conidia were hyaline, aseptate, and turned light to dark brown with maturity. Mature conidia were aseptate (rarely uniseptate), ovoid with truncated or rounded base and obtuse apex, externally smooth, roughened on the inner surface, and 8 to 11 × 23 to 26 μm (n = 50). These morphological characteristics of the isolates agreed with those of Diplodia seriata (the anamorph of Botryosphaeria obtusa) (5). The rDNA-ITS sequences of two representative isolates (xs-01 and xs-06) were amplified with primers ITS1 and ITS4. PCR products were purified and ligated with PMD-19T vector for sequencing. The rDNA-ITS sequences were submitted to GenBank with accession nos. KJ463386 and KJ549774 for isolates xs-01 and xs-06, respectively, showing 100% identity with multiple isolates of D. seriata (HQ660463, KC461297, and KF535906). Koch's postulates were fulfilled in greenhouse tests on 2-year-old cedar plantlets inoculated by the two isolates. Ten plantlets were used for the inoculation tests for each of the isolates, and their trunks were wounded to a 2 mm depth with a sterilized cork borer (3 mm diameter). The wounds were inoculated by mycelial plugs cut from 7-day-old colonies grown on potato dextrose agar (PDA) plates and wrapped with Parafilm, and those inoculated with pure PDA plugs served as control. Inoculated plantlets were incubated in a greenhouse with alternating cycles of 14 h fluorescent light/10 h darkness under moist conditions for 30 to 60 days at 28°C. Nine of 20 inoculated plantlets developed needle discoloration and shoot blight symptoms similar to those observed on naturally infected cedar trees. The control plantlets remained symptomless during the incubation period. D. seriata cultures were constantly recovered from each diseased plantlet, indicating that the isolated D. seriata isolates were responsible for the disease. D. seriata has been reported as a phytopathogen causing dieback diseases worldwide on multiple woody plant species such as olive (4), mulberry (1), Pinus spp., and Picea glauca (2,3). To our knowledge, this is the first report of D. seriata causing dieback disease on cedars in China. References: (1) M. Arzanlou et al. Arch. Phytopathol. Plant Protect. 46:682, 2013. (2) T. Burgess et al. Appl. Environ. Microbiol. 67:354, 2001. (3) G. Hausner et al. Can. J. Plant Pathol. 21:256, 1999. (4) J. Kaliterna et al. Plant Dis. 96:290, 2012. (5) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007.