First Report of Twig Anthracnose of Acacia melanoxylon Caused by Colletotrichum siamense in China.

Abstract

Acacia melanoxylon R. Br. native to Australia, is a high-quality timber tree with wide genetic and phenotypic diversity. In recent years, A. melanoxylon has been extensively cultivated in some provinces in southern China. In December 2019, anthracnose-like symptoms were observed on twigs of A. melanoxylon in China. In certain valleys in south China, the disease incidence on plants and shoots was 60-75% and 80-90%, respectively. The wither rate of disease branches was 30-40% in dry seasons from September to November. The appearance of symptoms occurred in a humid and warm valley. Symptoms were initially observed on the young branches as brown spindle shaped sunken spots. At later stages, the disease spots girdled the whole branch, which became wilted and its leaves turned reddish-brown. For pathogen isolation, diseased branches were sampled and 55 pieces (5× 5 mm) of these branches section were surface-sterilized in 75% ethanol for 30 seconds, followed by 0.5% NaClO for 5 min and then were rinsed three times in distilled water. After drying with sterilized filter paper, the surface-sterilized sections were transferred to potato dextrose agar medium (PDA) and incubated at 25 °C for 7 days in the dark. Three isolates were obtained as representatives for morphological characterization and were labeled as 1A912, 1B912, and 1C912. These specimens were deposited in the Guangdong Province Key Laboratory of Microbial Signals and Disease Control at the South China Agricultural University (China). Purified isolates were initially white, cottony and with dense aerial mycelium on PDA at 25 ℃, ten days later their colonies turned grayish white with orange conidial masses. Conidia were one-celled, hyaline, straight, cylindrical, with round obtuse ends, and measured 11.0 to16.3× 4.0 to 6.0 μm (n= 100), appressoria were 5.86 to 9.07 × 3.55 to 6.96 μm (n= 100). Morphological characteristics of selected isolates matched the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For further identification, the internal transcribed spacer (ITS) region, and partial sequences of the actin (ACT), beta-tubulin (TUB2), and glycerol dehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced, using primer pairs ITS1/ ITS4 (White et al. 1990), Bt2a/ Bt2b (Donaldson and Glass 1995), ACT512F/ ACT783R, GDF1/ GDR1(Weir et al. 2012). The sequences were deposited in GenBank (ITS: MW228101-MW228103; TUB2: MW250346, MW320707, MW320708; ACT: MW250347, MW320703, MW320704; GAPDH: MW250348, MW320705, MW320706). The multilocus phylogenetic analysis distinguished the isolates 1A912, 1B912, and 1C912 as C. siamense. Pathogenicity of those three isolates of C. siamense was tested on healthy twigs of the one clone of A. melanoxylon. 27 young twigs of nine 1-year-old plants were inoculated with the mycelium of the 7 days-old isolates 1A912, 1B912, and 1C912(Each isolate infected three plants and each infected three young twigs) through an artificial wound. The same nine plants were inoculated with PDA medium alone (each infected three young twigs) as a negative control. Five days after inoculation, brown spindle spots similar to the field disease symptoms were observed on the twigs. No symptoms were observed on the control plants. The experiment was repeated twice. The fungus was successfully reisolated from the symptomatic plants, and had identical morphological and molecular characteristics to the initial isolates, fulfilling Koch´s postulates. To our knowledge, this is the first report of anthracnose caused by C. siamense on A. melanoxylon in China. Twig anthracnose can reduce the growth of A. melanoxylon. Further research on management options for this disease is required.