First report of branch blight caused by Diaporthe ueckerae on Eucalyptus citriodora in China.

Abstract

Eucalyptus citriodora is a wood and oil dual-purpose tree with strong growth adaptability and high ornamental value. Recent years, it has been widely planted in Guangxi in China. In Nov. 2021, branch blight was found to be widespread on E. citriodora in Qinzhou in China (21°57'57"N, 108°42'6"E). The occurrence area was over 7000 m2, and the disease incidence was 23% (23/100). Twigs were withered in most of infected plants, and only top branch died in few plants. The lesions started from branch tips, then expanded and caused 5-20 cm branches died in final. The lesions were tiny and brown at early stage, then turned dark brown or black. Ten diseased branches were sampled randomly in field and were cut into 1 cm pieces. After surface disinfecting with 75% ethanol for 3 min, 0.1% HgCl2 for 5 min, washing with sterile water three times, samples were placed onto Potato Dextrose Agar (PDA) medium. Hyphae appeared after incubating for 5 days at 28 ℃. The diameter of colonies reached 64-71 mm after 7 days incubation. The colonies were white and felt-like, and then turned yellowish gradually with flourish aerial hyphae. Two weeks later, pycnidia appeared, which were nearly spherical, initial pale yellow and later black. Sometimes secretions overflowed from the aperture on pycnidia. There were 2 types of conidia (α and β type), which were unicellular and hyaline. The α type was spindle to ellipse and had 1-2 oil globule, with 5.56±0.50 × 2.67±0.39 μm (n=100) in size. The β type was linear and one end bent in hook shape, with 17.10±2.54 × 1.55±0.32 μm (n=50) in size. Three isolates (LEQZ01, LEQZ02, LEQZ03) were selected for further study. The internal transcribed spacers (ITS) region of rDNA, translation elongation factor 1-α (tef1 α) and β-tubulin (tub2) genes were amplified using primer pairs ITS1/ITS4 (White et al. 1990), EF1-983F/EF1-1567R (Rehner et al. 2005) and Tub1/ Tub2 (Chauhan et al. 2007), respectively. BLASTn searches showed that the ITS (OM339849, ON075781, ON075782), tef1 α (ON093807-ON093809) and tub2 (ON093810-ON093812) sequences had the highest similarity with Diaporthe ueckerae strains, with 99% (543/549, 544/549, 544/549) identity for ITS (NR 147543.1), with 99% (350/351, 353/353, 349/350) identity for tef1 α (KY569388.1), with 99% (754/754, 753/754, 754/754) identity for tub2 (MW514128.1). A neighbor-joining tree constructed by combining 3 sequenced loci. Three isolates clustered in the D. ueckerae clade with 100% bootstrap support. Based on morphological (Yi et al. 2018) and molecular evidences, the pathogen was identified as D. ueckerae. In a pathogenicity test, 20 healthy E. citriodora seedlings were divided into 4 groups. Before inoculation, twigs were surface disinfected with 75% ethanol followed by washing 3 times using sterile water. Tiny artificial wounds at 5 cm below the seedling top were inoculated by hyphae taken from colonies incubated for 7 days at 25 ℃ in the dark, and covered with damp cotton in 1-3 groups (Yi et al. 2018). Yet the wounds were covered with damp cotton in control group. Two days later, wounds started to turn brown in test groups, and symptoms similar to field were obtained after 10 days. But no lesion emerged in control group. Then germs were re-isolated from symptomatic twigs and identified as D. ueckerae following the methods above. To our knowledge, this is the first report of branch blight caused by D. ueckerae on E. citriodora in China. Further researches on disease epidemiology would help to prevent spread to more locations.