First Report of Anthracnose on Cinnamomum camphora (Camphor Tree) Caused by Colletotrichum fioriniae and Colletotrichum siamense in China

Abstract

Camphor tree (Cinnamomum camphora) is native to east Asia, which could produce pharmaceutical metabolites, such as camphor, linalool, and so on (Chen, Tang et al. 2020). In September 2020, severe anthracnose symptoms were observed on the leaves of camphor trees in Nanchang, and estimated incidences ranged from 30% to 80%, which could inhibit leaf growth and reduce their biomass. The lesions were appeared on the leaves of annual branchlets, which the irregular dead areas appeared on leaf tips or margins (Figure 1 A and B), sometimes moving onto the shoots and small twigs. For pathogen isolation, fifteen leaves with typical symptom were randomly collected in Jiangxi Agricultural University (N28°45'38", E115°50'0.006") and the fungi were isolated from the symptomatic-asymptomatic junction and cultured on potato dextrose agar (PDA) at 25℃ in darkness. A total of 40 isolates were obtained from tissue samples, in which 32 isolates were identified as belonging to Colletotrichum spp. following the published works (Damm, Cannon et al. 2012, Damm, Cannon et al. 2012, Weir, Johnston et al. 2012). Based on the morphologies of conidia, all the 32 isolates were classified into two categories. For further precise identification, the represented isolate YK1 and YK18 were selected to analyzed using morphological characters after 7 days of incubation, and multiple genes including ITS (White, Bruns et al. 1990), ACT, GAPDH, TUB (Damm, Woudenberg et al. 2009) and RPB2 (Réblová, Gams et al. 2011). Sequences were deposited in GenBank with accession numbers from MZ229311 to MZ229326. Conidia of isolate YK1 were aseptate, primarily fusiform and measured 14.07-21.21 µm × 4.99-6.79 µm (n = 51) (Figure 1 L) and acervulus were 60.24 to 113.56 µm × 44.24 to 102.63 µm (n = 6) (Figure 1 K), while that of YK18 were one-celled, cylindric with obtuse ends (Figure 1 N) and measured 13.28-16.51 µm × 4.10-5.82 µm (n = 52) and acervulus were 73.85 to 131.70 µm × 63.93 to 105.66 µm (n = 6) (Figure 1 M). Acervulus of isolate YK1 and YK18 were produced on alfalfa stems 40 days after inoculation and dark brown to black in color. For all the genes showed greater than 99% similarity to multiple C. fioriniae and C. siamense accessions, respectively. The phylogram reconstructed from the combined dataset using W-IQ-TREE (Trifinopoulos, Nguyen et al. 2016) showed that isolate YK1 and YK18 clustered with C. fioriniae and C. siamense, respectively. Pathogenicity of both species was tested in the field by ten inoculating surface-sterilized mature leaves with puncture wound (Figure 1 C and D) and ten non-wounded young leaves with 20 µL of a conidial suspension (105 conidia ml-1) (Figure F and G). Leaves treated with sterilized water under the same conditions served as controls. After 4 to 7 days, the inoculated leaves of camphor tree developed typical dark brown to black lesions, similar to symptoms observed in the field, whereas controls remained symptomless. To fill the Koch’s postulates, C. fioriniae and C. siamense were consistently re-isolated, and confirmed morphologically and molecularly. C. siamense have been found to cause anthracnose on Cinnamomum camphora in China (Xu, 2017). To our knowledge, this is the first report of anthracnose on Cinnamomum camphora with C. fioriniae in China. In addition, this is an indication to the complexes about pathogens to anthracnose on camphor tree, which can pose serious threat to the production of Cinnamomum camphora in China.