First Report of Red-Fleshed Apple Anthracnose Caused by Colletotrichum siamense

The red-fleshed apple (Malus niedzwetzkyana) produces a colored fruit and rich anthocyanins and it has become popular among consumers in Shandong (Yang et al 2020). In recent years, anthracnose diseases have been reported in red-fleshed apple orchards and nurseries in Shandong province, China. The incidence of anthracnose in the red-fleshed apple plantings ranges from 50-90%. Initially, anthracnose lesions on fruit begin as sub-circular shaped, sunken, pale brown. Over time black lesions enlarged and coalesced into large necrotic areas. The sunken centers of mature lesion became filled with slimy pink sporulation. In September 2015, fifteen fruit with anthracnose symptoms and sporulation were collected, and 11 single-spore isolates were obtained. Three representative isolates (JNTW11, JNTW2, JNTW33) were used for morphological and molecular characterization. On PDA, the colonies were initially white and turned into pale brown in three days. Orange-brown pigmentation was produced near the center on the reverse. Aerial mycelium was cottony, dense, pale white to pale gray. Acervuli developed visible orange-pink conidial masses. Conidiophores were colorless, septate, not branched or branched at the base. Conidia were 1-celled, hyaline, subcylindrical, oblong, attenuated with blunt ends, and the average size was 16.7 ± 1.5 × 6.1 ± 0.9 μm (n = 50). Appressoria were brown, obovoid or irregular, 9.2 ± 1.6 × 8.0 ± 1.8 μm (n = 20). The morphological characters matched the descriptions of Colletotrichum gloeosporioides sensu lato (Cannon et al. 2008). Isolates JNTW11, JNTW2, and JNTW33 were subject to bioinformatic characterization by partial sequencing of 6 genetic loci including the ribosomal internal transcribed spacer (ITS), actin (ACT), beta-tub2 (TUB2), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Weir et al, 2012). The ITS (MT577037, MT577040, MT577042), ACT (MT767712, MT767715, MT767717), TUB2 (MT767723, MT767726, MT767728), CAL (MT767689, MT767692, MT767694), CHS-1(MT767700, MT767703, MT767705), and GAPDH (MT767734, MT767737, MT767739) sequences were deposited in GenBank. The six sets of sequence data were concatenated "ITS-GAPDH-ACT-CHS-1-TUB2-CAL", and the aligned sequences (2,007 bp) had 99.0% similarity to ex-type C. siamense ICMP18578. In a maximum likelihood phylogenetic tree, the highest log likelihood was -9148.55, and the isolates tested were in the C. siamense cluster with 96 % bootstrap support. Thus, the isolates were identified as C. siamense on the basis of multilocus phylogenetic analyses and morphological characters. To complete Koch's postulates, several healthy red-fleshed apple fruit ('Jiuhong', 1 month prior to harvest) were inoculated using colonized and uncolonized hyphal plugs and a blank agar as a control. All inoculated fruit were placed in sterile tissue culture bottles containing 2 layers of wet paper towels at 28 °C under a 12 h light/dark cycle. All fruit developed anthracnose symptoms in 7 days while the controls did not develop any symptoms. The symptoms were similar to those collected from fruit in the field, and same fungus was re-isolated from the lesions. Presently it was known that C. acutatum, C. asianum, C. chrysophilum, C. cuscutae, C. fioriniae, C. fragariae, C. fructicola, C. gloeosporioides, C. godetiae, C. kahawae, C. karstii, C. limetticola, C. melonis, C. noveboracense, C. nymphaeae, C. paranaense, C. rhombiforme, C. salicis, and C. theobromicola could infect M. coronaria, M. domestica, M. prunifolia, M. pumila, and M. sylvestris worldwide. To our knowledge, this is the first report of C. siamense as a pathogen of M. niedzwetzkyana. This finding provides crucial information for the management of anthracnose disease in China.