First Report of Neonectria punicea Causing Preharvest Fruit Rot of Apple in Sichuan, China.

Abstract

Apple (Malus domestica Borkh.) is an economic backbone driving poverty alleviation and rural revitalization in remote rural areas of China. Monitoring of pathogens associated with apple is particularly important for the apple industry. In 2019, survey for preharvest fruit diseases of apple was conducted in Yanyuan County, which lies on the southern edge of the Qinghai-Tibet Plateau, Sichuan, China. In one commercial orchard (27°26'49.8"N, 101°39'38.9"E), an uncharacterized fruit rot disease was observed on 'Red Fuji Nagafu No. 2' apple with prevalence of approximately 3%. Symptoms on the fruits occurred as light brown spots about 3 millimeters in diameter. With the progression of the disease, the originally infected sites turned deep brown, lesions expanded and decayed tissues became spongy. Ultimately, multiple lesions coalesced together, severely infected fruits became sunken, soft, wrinkled, and decayed completely. Twenty samples with typical symptoms were collected. Symptomatic apple tissues from the lesion margin were cut into 5 × 5 mm pieces, washed two times with 70% ethanol, disinfected with 0.5% NaClO for 5 min, rinsed twice with sterile double-distilled water, and plated on potato dextrose agar (PDA) medium at 20 °C in darkness for 5 days. Forty-five colonies were isolated from symptomatic tissues on PDA after 3 days and they shared similar morphology. Five single-spore isolates were obtained and a representative isolate designated ABT12-1 was characterized further (CCTCC accession no. M2021680) (Chaverri et al. 2011). The fungal colonies produced radial, abundant cottony aerial mycelium, white-saffron to saffron-ochraceous in color, and the reverse colony was ochraceous to sienna with yellow pigmentation in the agar. Conidiophores were observed developing laterally from aerial hyphae, irregularly branched or in fascicles, 1-4 branched, 21.5 to 87.1 μm long by 3.5 to 7.8 μm wide (n=20). Phialides were generally long and cylindrical or somewhat flask-shaped. Microconidia were generally ellipsoidal, with 0-1 septations and measured 3.2 to 16.6 × 2.5 to 7.2 μm (n=20). Macroconidia were ellipsoidal, oblong to long cylindrical, hyaline, smooth, slightly curved and round at both ends, with 3-7 septations, and averaged 45 × 4 μm (n=20). The fungus was initially identified as a member of the genus Neonectria based on the morphological observations (Lombard et al. 2015). Molecular identification was performed to determine fungal species by sequencing partial actin (Act), internal transcribed spacer (ITS), large subunit of ribosomal DNA (LSU), RNA polymerase II subunit one (Rpb1), translation elongation factor 1-α (Tef1), and the beta-tubulin (Tub) genes, which were amplified with primers Tact1/Tact2, ITS5/ITS4, LR5/LROR, crpb1a/rpb1c, tef1-728/tef1-986 and Btub-T1/Btub-T2, respectively (Carbone and Kohn 1999; Castlebury et al. 2006; O'Donnell and Cigelnik 1997; Schoch et al. 2012; White et al. 1990). The resulting sequences were deposited in GenBank (accession no. MW538899 for Act; MW534396 for ITS; MW534401 for LSU; MW538900 for Rpb1; MW538901 for Tef1; and MW538902 for Tub). Multilocus phylogenetic analysis with neighbor-joining methods based on the above 6 gene sequences showed that the fungal isolate clustered with Neonectria punicea (J.C. Schmidt) Castl. & Rossman. To test the pathogenicity, ten freshly picked 'Red Fuji' apple fruits were wound-inoculated with 2 µl of conidial suspension (1 × 105 conidia/ml) of the isolate ABT12-1; sterile water was used as control. The inoculated samples were placed in sterilized plastic boxes to maintain relative humidity and incubated at 20 °C for seven days. Pathogenicity experiments were conducted four times. After three days, the wound sites inoculated with conidial suspension showed visible diseased spots. As lesions progressively dilated, symptoms similar to those of the decayed fruit in the field were observed, whereas the control fruits remained symptomless. The pathogen was reisolated from symptomatic lesions, thus fulfilling Koch's postulates. Neonectria punicea causes stem and bark canker diseases on a variety of trees (Hirooka et al. 2013; Karadžić et al. 2020; Salgado-Salazar and Crouch 2019). To our knowledge, N. punicea has never been reported causing apple fruit rot in China.