First Report of Leaf Spot Caused by Pseudocercospora ligustri on Ligustrum japonicum ‘Howardii’ in China

Abstract

‘Howardii’ privet (Ligustrum japonicum ‘Howardii’) is a cultivar of L. japonicum (L.) (Japanese privet) that is native to central and southern Japan and Korea. This cultivar is widely planted as an ornamental in parks and landscapes in China. From 2010 to 2013, a leaf spot disease on L. japonicum ‘Howardii’ was observed in parks in Luoyang, Henan Province, China. A survey revealed that the disease was significant, and more than 50% of leaves in some plantings were infected. Leaf spots initially appeared slightly chlorotic, enlarging slowly, becoming lemon-yellow to purple and finally brown. Fully matured lesions were 5 to 15 mm in diameter, circular to subcircular or irregular, with reddish-purple to black margins. The centers were tan with occasional brown flecks and faintly zonate. Lesions often coalesced to form extensive necrotic areas. A cercosporoid fungus was consistently isolated from diseased leaves. Fungal stromata were well-developed, globular, dark brown, predominantly hypophyllous, and evenly distributed over the leaf spot. Stromata (n = 50) were 22.6 to 58.3 μm in diameter. Conidiophores (n = 50) were densely fasciculate, olivaceous to pale brown, subcylindrical, geniculate-sinuous, 12.8 to 32.3 × 3.0 to 4.2 μm, and aseptate to one septa. The conidiogenous loci were inconspicuous, neither thickened nor darkened. Conidia (n = 50) were pale olivaceous, cylindrical to obclavate, usually slightly curved, sometimes straight, smooth, multiseptate, and 19.3 to 64.1 × 2.0 to 3.9 μm. Three representative isolates of the fungus (JS1201, JS1202, and JS1203) were obtained by single-conidium isolation from fascicles of conidiophores on lesions of diseased plants. Cultures on potato dextrose agar (PDA) were slow growing, 15 to 20 mm diameter after 20 days at 25°C. Colonies were raised, olive gray on topside, black on reverse, and without sporulation. Based upon these morphological and cultural characteristics, the newly collected fungus from China on L. japonicum ‘Howardii’ was similar to Pseudocercospora ligustri (Deighton 1976). For further confirmation, three nuclear gene regions (ITS, ACT, and EF-1α) were amplified and sequenced for multigene phylogenetic analysis. The DNA sequences generated were deposited in GenBank (accession nos. KU255727, KU255728, KU255729, KX429750, KX429751, KX429752, KX397350, KX397351, and KX397352). A BLAST search in GenBank revealed that the sequences showed 99 to 100% identity with several sequences of Pseudocercospora species, including P. lilacis, P. oenotherae, P. marginalis, and others. Phylogenetic analysis was further performed with the concatenated sequence alignment of three genes (ITS, ACT, and EF-1α) using MEGA 6.0 software (Tamura et al. 2013). The neighbor-joining phylogenetic tree showed that the three isolates of P. ligustri from L. japonicum ‘Howardii’ and an isolate of P. lilacis from L. japonicum clustered into one clade (bootstrap value of 100%). In this group, our isolates clustered in one separate subclade with 71% bootstrap support. Pathogenicity testing was conducted on young leaves of 3-year-old potted ‘Howardii’ privet plants with the JS1201 isolate cultured for 2 weeks on PDA plates. Because the cultures did not produce conidia, mycelial discs were used for inoculation (de Miranda et al. 2014). Twenty mycelial discs of P. ligustri, approximately 5 mm in diameter, were placed on the upper leaf surface of the test plants. Control plants were inoculated with agar discs without mycelium. Plants were covered with plastic bags for 48 h after inoculation. Necrotic spots appeared on the inoculated leaves 10 to 12 days after inoculation and were identical to the symptoms observed in the field. P. ligustri was reisolated from symptomatic leaf tissues, confirming Koch’s postulates. No symptoms were observed on control plants. To our knowledge, this is the first report of leaf spot caused by P. ligustri on L. japonicum ‘Howardii’ in China.