First report of leaf blight on Eucalyptus cloeziana caused by Coniella quercicola in China.

Abstract

Eucalypt species are among the most important for timber production worldwide. Eucalyptus cloeziana is increasingly culticated due to its desirable structural properties. Leaf blight is one of the most devastating diseases of E. cloeziana in China. In May 2019, leaf blight samples were collected from E. cloeziana in Chongzuo, Guangxi, China (22°20'37.70"N, 107°49'29.29"E). Lesions began at the leaf margin and extended to 1/4-3/4 of the total leaf surface area. Lesions (26.76±12.64 mm diameter) were round, yellow, and withered in appearance, and sometimes many black, round pycnidia were observed. Leaves with blight were collected randomly from 10 E. cloeziana plants. Tissue blocks (3 mm×3 mm) were sampled from diseased and healthy leaf portions, then surface disinfected with 75% ethanol for 20 s and 0.1% HgCl2 for 3 min. After washing with sterile water three times, dry tissue blocks were placed on potato dextrose agar (PDA) medium and incubated at 28°C for 5 days. Hyphae were milky white or whitish, and sparse. The colonies had petal-shaped edges and the conidiophores were clustered, branched and transparent. Spore-forming cells were solitary and smooth; conidia were smooth, fusiform or oblong, transparent, blunt-based, mostly erect, and 16.54±2.19 × 3.38±0.77 μm (n=100 in each isolate) in size. Three representative isolates (AB-6, AB-9, AB-16) were selected for further study. For molecular identification, the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (TEF1), and large subunit ribosomal RNA (LSU) were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-983F/EF1-1567R (Rehner and Buckleyet al. 2005), and LR0R/LR5 (Vilgalys and Hesteret al. 1990), respectively. BLASTn searches showed that the ITS (OM280456, ON026088-89), TEF1 (ON055278-80) and LSU (OM281346, ON026097-98) sequences had the highest similarity to Coniella quercicola strains with: 99% (600/605, 600/605, 600/604) identity for ITS (MH859478.1); 98% (326/333, 327/334, 325/332) identity for TEF1 (KX833698.1); 99% (870/872, 833/834, 830/831) identity for LSU (MH871258.1) of ex-type CBS 904.69. A Neighbor-Joining phylogenetic tree was constructed by combining 3 sequenced loci. Three isolates clustered in the C. quercicola clade with 100% bootstrap support. Thus, based on morphological (Maas et al. 1979; Wang and Lin et al. 2004) and molecular characteristics, the pathogen was identified as C. quercicola. In a pathogenicity test, 20 healthy E. cloeziana seedlings with at least 5 leaves were divided into 4 groups: groups 1-3 were used to inoculate three isolates respectively, and the fourth group acted as control. After surface disinfection with 75% ethanol and wiping with sterile water, tiny wounds were maked made by inoculation needle on each leaf. Fungal culture plugsblocks cut from 3 isolates were placed on wounds in groups 1-3 respectively,. withWarter- agar blockplugs served as control in group 4. The leaves were covered with wet cotton and sealed in airtight bags to retain moisture at room temperature with natural light. After 3 days, light brown lesions were observed in groups 1-3, with no symptoms present in the control group. The pathogenicity test was confirmed by repeating in triplicate and fungi re-isolated from symptomatic leaves were identified as C. quercicola. To our knowledge, this is the first report of leaf blight on E. cloeziana caused by C. quercicola in China. This study increases our understanding of E. cloeziana leaf blight and future research may allow the development of targeted prevention methods for more effective disease controls.