First Report of Colletotrichum circinans Causing Anthracnose in Allium fistulosum var. giganteum in Gansu Province, China.

Abstract

In September 2018, severe symptoms and high incidence (about 60%) of an onion anthracnose disease attributable to infection by Colletotrichum spp. was observed in production fields of Dingxi city, Gansu Province, China. The mature onion plants near to harvest (Allium fistulosum L. var. giganteum Makino) expressing necrotic symptoms had oval lesions with dark spots that were made up of stromatic masses that had formed beneath the cuticle of the plant base. Twenty symptomatic plants were sampled. Symptomatic tissues were surface sterilized with sodium hypochlorite, transferred aseptically to 20 mL potato dextrose agar in a petri plate and incubated at 30 ± 2 °C. After seven days, cultures produced acervuli and setae, a characteristic sign of Colletotrichum spp. Acervuli were observed with a black, bulbous base and acicular setae while conidia were elliptical (14-25 x 3-6 µm), unicellular hyaline and slightly falcate (Figure 1). DNA was extracted from a 7-day old culture and the ITS region was amplified using primers D1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and D2 (5'-GGTCCGTGTTTCAAGACGG-3') (Kurtzman and Robnett, 1997). The resulting sequence (548 bp) was deposited with NCBI GeneBank under accession number MW127281. The fungus was confirmed as Colletotrichum circinans after conducting a BLAST search with the ITS sequence that reported a highest homology (99% similarity) with MH81329.1 (C. circinans). The speciation was further confirmed by amplifying regions of the TUB2, GAPDH, and ACT genes using primers given in Table S1 and sequences were also submitted to NCBI GeneBank under accession numbers MZ456033, MZ456032 and MZ456031, respectively. The subsequent BLAST results for these three additional gene regions were consistent with the results of the ITS region and fungus was identified as C. circinans. The isolated pathogen was tested for its pathogenicity on onion plants (Allium fistulosum L. var. giganteum Makino). A conidial suspension 30 ml (5 × 105 conidia/ml) was mixed with 1 kg sterilized potted soil in 15 cm diameter plastic pots. Un-inoculated, sterilized soil was used as control. Three green onion plants per pot were planted (Figure S2). The experiment was repeated three times with 15 replications in each experiment. Plants were maintained for 120 days under greenhouse conditions and were monitored for the development of anthracnose symptoms. Symptoms recorded previously on onion plants in field (i.e. necrosis, sunken oval lesions and dark spots) were observed after 30 days on plants grown in inoculated soil while control plants remained asymptomatic (Figure S1). Three samples from symptomatic tissues of each plant were used for re-isolation of the pathogen on PDA, as described above. Cultures grown on PDA were confirmed both on a morphological and molecular basis as Colletotrichum circinans.These morphological, molecular, and pathogenicity tests of the isolated fungus confirm that the anthracnose symptoms observed on onion in Gansu Province, Beijing, China was caused by Colletotrichum circinans. Six different Colletotrichum spp. have been reported to cause diseases on onion worldwide (Rodriguez et al. 2012). C. circinans, which causes smudge, is an occasional onion pathogen was previously recorded as C. dematium (Pers.) Grove f. circinans Arx, which is specific to Allium spp. (von Arx 1970; von Arx 1981). However, Sutton (1992) described C. circinans as a distinct species from C. dematium. The fungus causes smudge or leaf spot of Allium spp. (Farr et al. 1989) and has been reported from Korea, Japan, Argentina, India, the UK and most other regions of the world (Cho & Shin 2004; Kiehr et al. 2012). Smudge may be a disease of concern post-harvest as fungal growth compromises the onion scales and bulb (Walker 1921). In China, this is the first report of C. circinans causing anthracnose in Allium fistulosum L. var. giganteum Makino in Gansu Province.