Phlyctema vagabunda is the main causal agent of apple bull’s eye rot in New Zealand

Bull’s eye rot is a postharvest storage disease of apples. Until now the cause of this disease in New Zealand was not clear. A survey of 6880 apples from five regions of New Zealand over two seasons was conducted. Neofabraea malicorticis and N. perennans were not found. One hundred and seventy-nine isolates were identified as Phlyctema vagabunda by specific polymerase chain reactions and/or sequencing the β-tubulin gene region followed by phylogenetic analysis. Two isolates were identified as N. kienholzii. Previous records of the presence of N. malicorticis and N. perennans in New Zealand were based on spore morphology and presence in pruning wound cankers. There is overlap in spore morphology for N. malicorticis, N. perennans and P. vagabunda, accounting for misidentifications. Based on our results it is likely that previous records were P. vagabunda, which can also infect pruning wounds.


Introduction
Bull's eye rot is a postharvest storage rot of apples. Circular, sunken, brown lesions form on fruit, usually after prolonged cool storage (Creemers 2014). Lesions have a lighter brown or tan centre (Spotts 2014). Concentrically arranged grey or brown acervuli form in the centre of older lesions (Creemers 2014;Spotts 2014). Lesions also occur around lenticels, and around the stem or the calyx (Spotts 2014). Grove (1990) attributed the causal organisms of apple bull's eye rot as Pezicula malicorticis and Neofabraea perennans. Verkley (1999) recognised Neofabraea instead of Pezicula as the correct name for this group of fungi, and proposed three species causing apple fruit rots: N. alba syn. Pe. alba, anamorph Phlyctema vagabunda, N. malicorticis syn. Pe. malicorticis, anamorph Cryptosporiopsis were designed to discriminate these species (Gariepy et al. 2005;Soto-Alvear et al. 2013).
A revision of these and related fungi (Chen et al. 2016) based on multi-locus phylogenetic analysis using ITS, LSU, rpb2 and tub2 gene regions has split Neofabraea alba into a separate clade from these other species (N. perennans, N. malicorticis, N. kienholzii, N. krawtzewii), and to a different genera, and it was renamed Phlyctema vagabunda.
In New Zealand, a storage rot of apples was first noticed in 1923, and named 'Delicious spot' (Cunningham 1925). The causal fungus was not known, and was later morphologically identified by Brien (1932) as N. perennans. This was followed by a report of ripe spot caused by N. malicorticis (Brien 1934). Eight years later ripe spot was reported to occur in all apple growing districts of New Zealand (Taylor and Brien 1943).
The early New Zealand descriptions of N. perennans and N. malicorticis fit the criteria for conidial morphology of Verkley (1999). Rots were caused almost equally by N. perennans and a fungus with curved conidia, but hundreds of cankers yielded a fungus with straight spores and only two a fungus with curved spores (Brook 1957). However, Brook (1957) pointed out that there was overlap in conidial morphology between N. malicorticis and another described species that also causes fruit rots on apples, N. alba (Osterwalder 1907;Wilkinson 1943;Brook 1957) suggested that the only reliable means of distinguishing these two fungi, which both produce curved conidia, in contrast to the straight conidia of N. perennans, was whether they colonise apple branches and twigs to form cankers (Kienholz 1939;Corke 1955). Based on morphology, Brook (1957) concluded that the fungus with curved spores isolated from fruit was N. alba, and goes on to suggest that previous identifications of N. malicorticis (Taylor and Brien 1943) were also probably N. alba.
Phylogenetic analysis of the sequence of the inter-transcribed spacer region (ITS) of ribosomal DNA of cultures from apples deposited in ICMP (International Collection of Microorganisms from Plants, Landcare Research, Auckland) as N. alba and N. malicorticis confirmed the identifications of N. alba, but the single N. malicorticis culture was identical to a fungus from kiwifruit, N. actinidiae (Johnston et al. 2004). Whether it was isolated from apple, or whether it was incorrectly ascribed to apple but was isolated from kiwifruit, is not known. There were no vouchered specimens available for N. perennans isolated from apple in New Zealand. Based on these results and unpublished isolation records, Johnston et al. (2005) proposed that N. malicorticis and N. perennans may not ever have been present in New Zealand.
The present study aims to investigate the causal agent of apple bull's eye rot in New Zealand.

Materials and methods
A sample of 1879 rotten apples from long term stored (0.5 ± 0.5 °C) industry library trays from the North (1096) and South Island (799) apple growing regions of New Zealand were transported to Mt Albert Research Centre (Auckland) and placed in a cool store. Apples were inspected for bull's eye rots and isolations made between 26/9/2013 and 16/10/2013.
Five thousand one hundred apples harvested from three commercial orchards in the North Island were placed in the cool store (0.5 ± 0.5 °C) during the 2018/19 season. Apples were inspected and isolations from 100 apples showing symptoms of bull's eye rot were made between 28/6/2019 and 28/10/2019.
The external surface of apples were wiped with 70% ethanol and small tissue pieces from rots were excised from the margin between rotten and healthy tissue and placed on Difco® potato dextrose agar (PDA) in Petri plates under fluorescent lights at 20 °C, 12 h light/12 h dark. After 2-3 weeks' growth on PDA at room temperature, isolated fungi were identified as P. vagabunda by colony and spore morphology. In 2013 a selection of 98 isolates and in 2019 a further selection of 83 isolates identified by morphology as P. vagabunda were single-spore cultured onto PDA. Resultant cultures were stored as agar plugs (5 mm 2 ) in 50% glycerol in cryotubes at -80 °C until required.
Cultures retrieved from agar plugs stored in the − 80 °C were grown for 21 days on PDA in Petri plates at room temperature under lights (12:12 day/night cycle). Mycelium was scraped from the surface using a sterile bent glass rod and re-suspended in 250 µL Wizard™ SV Lysis buffer in a 2 mL safe-lock micro-centrifuge tube. Three 0.2 mm silicon beads (Qiagen©) were added to each sample, then tubes were placed evenly into the TissueLyser Adapter II (Qia-gen©) set and homogenised for 1.5 min at 1800 oscillations/ min after which the rest of the Promega Wizard™ Genomic DNA Purification Kit (Thermo Fisher Scientific, USA) protocol was followed. Purified DNA concentration was determined using a NanoDrop® ND-1000 spectro-photometer (Biolab, Victoria, Australia) and stored at -20 °C.
The β-tubulin region was amplified for 99 isolates (83 isolates from 2019 and the remainder representative isolates from 2013) using PCR reaction conditions and the Br-LEV-Lo1 and Br-LEV-UP4 primers of de Jong et al. (2001). The PCR products were purified by excising the band produced by gel electrophoresis (1% agarose) followed by purification with the Promega Wizard™ CV Gel and PCR cleanup system (Thermo Fisher Scientific, USA). DNA was sequenced in both directions (Macrogen, Korea). Eight sequences from the National Centre of Biological Information (NCBI) were included in phylogenetic analyses: Infundichalara microchona (CBS 175.74) (outgroup), N. actinidiae (CBS 121403), N. kienholzii (CBS 126461), N. malicorticis (CBS 102863), N. perennans (CBS 275.29), N. vincetoxici (CBS 123727), and P. vagabunda (CBS 109875, CBS 304.62) ( Table 1). A Maximum Likelihood tree (Saitou and Nei 1987) was created using MEGAX (Kumar et al. 2018) with 1000 bootstrap replications and the Tamura-Nei model (Tamura and Nei 1993). The tree with the highest likelihood is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites. This analysis involved 107 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding using the partial deletion model. There were a total of 741 positions in the final dataset.

Results
Results showed a product of the expected size (358 bp) amplified using the Neo alba-up and Neo_ alba-loTub-439 primers from the DNA extracted from 89 of the 98 isolates in 2013.
Phylogenetic analysis of the β-tubulin gene region of 99 isolates showed that all except two isolates were in the same clade as P. vagabunda. These two isolates were in the N. kienholzii clade ( Fig. 1; Table 1). No sequences were in the same clade as N. malicorticis or N. perennans (Fig. 1).
Three small clades were separated at > 60% level of probability, but BLAST analysis did not find any named fungal species more similar to these sequences than P. vagabunda.

Discussion
These results suggests that Brook (1957) was correct when he stated that the isolates with curved spores resulting from a survey of New Zealand apples were not N. malicorticis, but were instead P. vagabunda. Similarly Johnston et al. (2005) also stated that N. malicorticis was probably not ever present in New Zealand, and that all isolates were probably P. vagabunda. Brook (1957) distinguished N. perennans from N. malicorticis on the basis of spore curvature, assuming straight spores were N. perennans and curved spores were N. malicorticis. The cankers from which N. perennans were reported in Brook (1957) were predominantly at pruning wounds and damaged fruit spurs, of which the former has also been shown to be a characteristic of P. vagabunda (Creemers 2014). N. perennans macro-conidia are described  (Taylor and Brien 1943) was isolated, which was probably P. vagabunda based on the present results. P. vagabunda was first recorded in 1847 from France (Desmazières 1847), and it was first reported from North America c. 150 years later (Gariepy et al. 2005), suggesting that its origin was Europe. Indeed, the most common bull's eye rot pathogen found in Continental Europe is Phlyctema vagabunda, but it is a minor pathogen in North America by Verkley (1999) as straight or weakly curved, those of P. vagabunda by Chen et al. (2016) as mostly straight and by Verkley (1999) as weakly to strongly curved. It is possible that P. vagabunda could be confused with N. perennans if conidial morphology and isolation from pruning wounds were the major criteria for identification as there is some overlap in spore morphology from these descriptions. Therefore it is possible that N. perennans was also never present in New Zealand. During a later survey of apple fruit Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.