Identification of the causal fungal agent of bull's-eye rot on apple(Neofabraea perennans)imported from US

Abstract: 【Objective】Bull's-eye rot of apple caused by species of Neofabraea can infect a variety of fruit trees and ornamental trees in the family of Rosaceae. This is an important branch and postharvest disease of apples and pears producing cankers on branches and twigs and/or decay on postharvest fruits, which lead to more than 40% postharvest losses in severe year. So far, this disease distributes in Europe, America and Oceania while there is no record of occurrence in China. Up to September 2015, in Guangdong port, decayed fruits of apple with suspicious symptom of bull's-eye rot had already been intercepted from32 shipments of apples imported from Washington state, USA and 46 fungal strains were isolated from decayed fruits. Up to now, four species of the genus Neofabraea are known to be associated with bull's-eye rot of apple, i.e. N. malicorticis, N. perennans, N. alba and N. kienholzii. It was reported that the geographical distribution of the four bull's-eye rot pathogens described above often overlap, morphological an molecular difference between N. malicorticis and N. perennans are considered to be subtle. To identify the species of the causal agent associated with decayed apple fruits intercepted from US and provide scientific basis to assess the risk of the invasion of the pathogen, fungal strains were isolated from fruit samples and subjected to DNA sequence, morphological and cultural analysis.【Methods】Suspicious decayed fruits of apple(Malus domestica)were collected in Guangdong port from imported fruits, the symptoms were photographed and main features of decayed spots were observed under microscope. Isolates were obtained from decayed fruits. After spraying with 75% ethanol, the skin of decayed fruit was peeled from the marginal area between decayed and healthy tissues and small fragments(4×4 mm) were excised and plated on potatodextrose agar(PDA), or mycelium or conidia were picked from the skin surface of decayed fruits onto PDA plate and incubated under the condition of 24 h dark at 20 ℃ until sporulation(about 5-7 days). Single spore subcultures were obtained for each isolate using the procedure described by Goh(1999). 8 representative strains Nep A 0901-Nep A 0908 were selected for morphological and phylogenetic analyses. Acervuli, conidiophores and conidia morphology were examinated under microscope. The growth change of colonies and formation of fruiting body and sporulation of isolates were observed under 24 h dark at 20 ℃. Effect of temperature on radial mycelial growth rate of isolate Nep A 0902 were evaluated on PDA under 24 h dark at 0-30 ℃. In total 10 healthy fruits of each cultivar of USA Red Delicious apple, Shandong Red Fuji apple and Hebei Ya pears were selected for pathogenic test. A cut with the length of 10 mm and the depth of 2 mm was made on the skin of each fruit using a sterile scalpel, 25 μL of 1×104 conidia·m L^-1 suspension were dropped inside the cut. Then the inoculated fruits were moisturizing incubated under 24 h dark at 20 ℃, the incidence of the fruit and the symptom change were observed and recorded at 5 days, 10 days and 30 days after inoculation. The genomic DNA of strains were extracted as PCR templates, the primer pairs ITS1/ITS4(White et al. 1990) and Bt-T2m-Up /Bt-LEV-Lo(O'Donnell, K,1997) were used to amplify the internal transcribed spacer region of the nuclear ribosomal RNA operon(ITS1, 5.8S nr DNA and ITS2) and β-tubulin genes, respectively. The PCR products were verified by staining with gelview on1% agarose electrophoresis gel, sequenced and BLAST. ITS and β-tubulin gene sequences of 8 representative isolates in the study and 31 strains downloaded from the NCBI Gen Bank were aligned using Clustal X 2.0. Phylogenetic analyses were performed using MEGA 6.06 with neighbor-joining method and Kimura 2-parameter model.【Results】The decayed apple(Malus domestica) fruits displaying bull's-eye rot were intercepted in Guangdong port from 32 shipments of apples imported from Washington state of USA. The decayed fruits showed stem-end rot mostly with relatively firm rotted tissues and brown circular spots with a pale center shaping like a bull's eye. Totally, 46 strains were isolated from all decayed Washington apples. On PDA,colonies were rounded, aerial hyphae scarce, white to cream, gradually deepen to amber to dark brown and finally formed concentric rings of light and dark brown. The mycelium of the isolate Nep A 0902 grew at 0-25 ℃ and reached the optimum growth at 1.74 mm·d-1under 15-20 ℃. The isolate was verified as the causal fungal agent of the decayed fruits by Koch's postulates, which could cause American‘Red Delicious' apple, Shandong‘Red Fuji'apple, and Hebei Ya pears to produce bull'seye rot symptoms. Pulp tissue of all fruits inoculated with conidia became soften and brown rot 5 days after inoculation, a small and large amount of milky colloid acervuli could be seen on the surface of decayed tissue 10 days and 30 days after inoculation, respectively. On decayed fruits and PDA as well, the fungus formed colloid cream acervuli, conidiophores phialidic, macroconidia and microconidia cylindrical,straight or slightly bent in the lower part, hyaline with single cell. Phylogenetic analyses inferred from the sequences of ITS showed that two known species N. perennans and N. malicorticis of bull's-eye rot pathogens overlap and clustered in one clade, and the 8 representative Neofabraea isolates Nep A 0901-Nep A0908 clustered in the same clades of N. perennans and N. malicorticis. While phylogenetic analyses in-ferred from the sequences of β-tubulin showed that N. perennans and N. malicorticis were grouped in two separate clade. Moreover, the 8 representative Neofabraea isolates clustered in the same clade as those N.perennans strains originated from north American(USA and Canada), Europe(Germany, the Netherlands,Britain, the Czech republic) and Australia(bootstrap support=99%), with the cluster N. malicorticis adjacent(bootstrap support=89%), with the cluster N. kienholzii and N. alba adjacent secondly and thirdly in turn. Mutation occurred at 424 sites of β-tubulin sequences of the isolates Nep A 0901, Nep A 0903, Nep A 0905, Nep A 0907 and Nep A 0908, therefore, these 5 strains formed a small branch within the clade of N. perennans【.Conclusion】On the basis of morphology, cultural character, pathogenicity test and phylogenetic analyses inferred from the sequences of β-tubulin, it is confirmed that N. perennans Kienholz(1939)(taxonomic status: Fungi, Ascomycota, Pezizomycotina, Leotiomycetes, Leotiomycetidae, Helotiales, Dermataceae)(Anamorph: Cryptosporiopsis perennans [Zeller & Childs) Wollenw. 1939] is associated with bull's-eye rot of intercepted fruits of apple imported from Washington state of USA, which is presently subjected to phytosanitary legislation in China. The pathogen can be introduced through international trade of fresh fruits possessing a safety hazard to fruit production in China.