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First Report of Anthracnose on Sunflower Caused by Colletotrichum destructivum in China

Sun, H. Y., Liang, Y.
Plant disease 2018 v.102 no.1 pp. 245
Colletotrichum destructivum, DNA primers, Helianthus annuus, actin, anthracnose, conidia, culture media, flowering, gardens, genes, glyceraldehyde-3-phosphate dehydrogenase, growth chambers, hulls, internal transcribed spacers, leaves, mycelium, oils, oilseed crops, ornamental plants, photoperiod, phylogeny, ribosomal DNA, seeds, sodium hypochlorite, stems, tubulin, China
Sunflower (Helianthus annuus L.) is one of the most important oilseed crops worldwide. In October 2016, symptoms resembling anthracnose were observed on the mid-lower stems and pitches of most sunflowers growing in a residential garden in Liaoning, China (41.81° N, 123.85° E). These symptoms were present on plants from late flowering to milking stage. Light brown, oval to elongated lesions, approximately 5 to 6 cm in length, with over three lesions per plant, subsequently became dark brown and spread over the stems. During severe infection, plants wilted during the flowering stage. Flower and seeds were not infected but there were fewer matured seeds and more empty hulls compared with healthy plants. For isolations, small pieces were cut from the margins of stem lesions, surface disinfected with 1% sodium hypochlorite for 2 min, rinsed three times in sterilized distilled water, dried, and then placed aseptically onto potato dextrose agar (PDA) medium. Samples were cultured at 25°C under a 12-h light/dark photoperiod for 2 days. Isolates were transferred to fresh PDA medium, and cultured for 7 days. The cultured isolates produced grayish aerial mycelia that covered the entire PDA surface (9 cm in diameter) as well as pale orange conidia. Dark brown structures with straight setae were also observed. Conidia were 13.2 to 20.2 × 3.1 to 5.2 µm (n = 30), hyaline and cylindrical, with rounded ends. Cultural and morphological characteristics were consistent with those described for Colletotrichum destructivum (Damm et al. 2014). Genomic DNA was extracted, after which the rDNA internal transcribed spacer (ITS) region (GenBank accession no. MF178172) was amplified using universal primers (ITS1 and ITS4) and sequenced. Partial sequences of an actin gene (MF346075), glyceraldehyde-3-phosphate dehydrogenase gene (MF346076), and β-tubulin 2 gene (MF351853) were sequenced and used for further characterizations (Weir et al. 2012). Phylogenetic and BLAST analyses revealed the sequenced amplification products were >99% identical to C. destructivum reference sequences (FJ450058, KC843544, KM105561, and GU935894). To satisfy Koch’s postulates, conidial suspensions (1 × 10⁵ conidia/ml) were prepared for each isolate and point-inoculated (20 μl) on three intact and wounded leaves as well as stems of 40-day-old plants while mock inoculations were completed using sterile distilled water. After a 3-day incubation at 25°C in a growth chamber under a 12-h light/dark photoperiod, the symptoms on the inoculated leaves and stems were identical to those observed on the original diseased sunflowers. The mock-inoculated samples were symptomless. Fungal cultures reisolated from the leaf and stem lesions were morphologically identical to the original isolates. To the best of our knowledge, this is the first report of C. destructivum causing anthracnose on sunflower in China. There has been an enormous increase in the sunflower plantation area in northern regions of China due to the relevance for oil production as well as an edible seed. In addition, sunflower is becoming more popular as an ornamental plant. Further investigations on the prevalence of this disease in residential gardens and farmers’ fields will be undertaken to prevent and manage this potential threat.