First report of Colletotrichum godetiae causing bitter rot on ‘Golden Delicious’ apples in the Netherlands

Apple (Malus domestica) is an important fruit crop in the Netherlands, with a total production of 418,000 tons in 2011. Symptoms of apple bitter rot were observed on ‘Golden Delicious’ apples in the Netherlands in July 2013 after 9 months of storage in a packing house at controlled atmosphere. Lesions were round, 1 to 5 cm in diameter, gray and dry with acervuli, producing orange spore masses in concentric rings. Fruit were rinsed with sterile water, and lesions were sprayed with 70% ethanol until droplet runoff. The skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto Potato Dextrose Agar (PDA). The PDA plates were incubated at 20°C in the dark, and single-spore isolates were propagated on PDA. The isolates were identified as Colletotrichum sp. based on culture morphology, having light gray to pale orange mycelium and, when viewed from the reverse side, ranged from pink to reddish orange. The cultures carried yellowish spore masses and dark melanized structures similar to acervuli that oozed orange conidia. Conidia were cylindrical to fusiform, pointed at one or both ends, and measured 8.0 to 17.0 μm × 3.5 to 5.0 μm. Both cultural and morphological characteristics of the pathogen were similar to those described for C. acutatum, causal agent of bitter rot of apple. A representative isolate (PPO-44377) was used for multilocus gene sequencing (Damm et al. 2012). Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the KingFisher method (Waltham, USA) and six loci were amplified and sequenced. Primer pairs ACT-512F + ACT-783R, CHS-354R + CHS-79F, GDF1 + GDR1, CYLH3F + CYLH3R, BT2Fd + BT4R, and ITS1 and ITS4 (White et al. 1990) were used for amplification of parts of the actin (ACT), chitin synthase (CHS-1) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3), beta-tubulin (TUB2) and ITS region of the rDNA gene, respectively. One sequence for each locus has been deposited in GenBank under Accession Nos. KR180290 (ACT), KR180292 (CHS-1), KR180293 (GAPDH), KR180294 (HIS3), KR180295 (TUB2), and KR180296 (ITS). MegaBLAST analysis revealed that the ITS sequences matched with 98.9 to 100% identity to Colletotrichum spp. belonging to C. acutatum species complex (including C. godetiae). The phylogenetic trees constructed using sequences of ACT, CHS-1, GAPDH, HIS3, and TUB2 of our strain (PPO-44377), and available sequences from GenBank confirmed the identity of this strain as C. godetiae. Koch’s postulates were performed on 15 ‘Golden Delicious’ apples. Surface-sterilized fruit were inoculated with 20 μl of a spore suspension (105 conidia/ml) prepared from a 15-day-old PDA culture after wounding with a needle. Inoculated fruits were sealed in a plastic bag and were incubated in darkness at 20°C. Symptoms appeared after 4 to 6 days on 80% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA had morphological characteristics that resembled the original isolate from the infected apples. There are few reports of symptoms associated with C. godetiae on apple in Europe (Baroncelli et al. 2014; Ivic et al. 2013; Munda 2014). This is the first report of bitter rot caused by C. godetiae on apple fruit in the Netherlands. Currently, bitter rot is not an important disease in apples in the Netherlands. However, it is ranges worldwide and is considered one of the most important diseases, causing considerable crop losses, and may become an emerging problem in the Netherlands in the near future