Apple is an important crop in United Kingdom, with a total production of 233,750 tonnes in 2011. Symptoms of apple bitter rot were observed on apple fruits (Malus domestica L.) in the Newcastle area, United Kingdom, in October 2008. Lesions were round, 1 to 5 cm in diameter, brown and dry, with acervuli producing yellowish spore masses in concentric bands. Infected material was sent to the W-HRI (University of Warwick) for identification of the causal agent. Fungal isolates with morphological characteristics similar to those of Colletotrichum acutatum sensu lato were isolated from diseased fruits. Monoconidial isolates were grown on PDA at 25°C with a 12-h light period. The cultures were light gray, with cottony aerial mycelium getting darker with age and with color ranging from whitish to dark gray on the reverse side of the colony. The cultures have yellowish spores masses and dark melanized structures similar to acervuli. Colletotrichum spp. are difficult to identify solely on morphology; therefore, representative isolates were used for multi-locus gene sequencing and characterization (1). Genomic DNA was extracted using a modified Chelex100 protocol. Three loci were amplified and sequenced: the ITS region was amplified and sequenced using the universal primers ITS4 and ITS5. Primers TB5 and TB6 were used for the amplification and sequencing of the variable region of the TUB gene. Primers GDF1 and GDR1 were used to amplify a 200-bp intron region of the GAPDH gene. No differences were found among the strains at any of the loci. One sequence for each locus has been deposited in GenBank under accessions KF834206 (ITS), KF834207 (TUB), and KF834208 (GAPDH). In GenBank, ITS sequences matched with 100% identity to C. higginsianum (EU400147) and to C. gloeosporioides (AJ301931 to 972); and with identity between 99.6 and 99.8% with sequences belonging to C. godetiae (part of C. acutatum species complex). The TUB sequences match with 100% identity to more than 25 sequences belonging to C. godetiae. The GAPDH sequences match with 100% identity to JQ948739 and 35 belonging to C. godetiae strains IMI 381927 and CBS 131331. A multilocus phylogenetic tree (ITS, TUB, and GAPDH) was reconstructed using sequences of reference strains belonging to C. higginsianum, C. gloeosporioides, C. godetiae, and related species. The phylogenetic tree confirmed the identity of the strains isolated from apple as C. godetiae. Koch's postulates were tested with representative isolate by artificial inoculation of 12 healthy fruits of the cv. Golden Delicious. Fruit surfaces were sterilized with 70% ethanol, wounded with a sterile needle, and then inoculated with a plug of actively growing mycelium prepared from a 10-day-old culture grown on PDA. Inoculated fruits were incubated in sterile conditions at 25°C with a 12-h photoperiod. In 83% of fruits, symptoms appeared between 7 and 15 days later. The rot begins as light brown, circular lesion getting darker with orange spore masses. Fungal colonies isolated from the lesions and cultured on PDA have identical morphological characteristics of the isolate used for the pathogenicity assay. To the best of our knowledge, this is the first report of apple bitter rot caused by C. godetiae in the United Kingdom. Apple bitter rot is spread worldwide and in moist, temperate regions it is considered one of the most important diseases causing considerable crop losses. Since the losses are more severe under prolonged warm and wet weather conditions, bitter rot caused by C. acutatum species may become an emerging problem in the United Kingdom in the near future, and may require investigation of management practices to control this new disease.
References: (1) R. Baroncelli. Colletotrichum acutatum sensu lato: From diversity study to genome analysis. Coventry, United Kingdom, PhD thesis, 2012. (2) U. Damm et al. Stud. Mycol. 73:37, 2012.