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 curvispora and N. perennans syn. Pe. perennans, anamorph Cryptosporiopsis perennans. The fourth species (N. krawtzewii syn. Pe. krawtzewii anamorph Cryptosporiopsis sp.) is a saprotroph of poplars.

A later molecular investigation showed that when the β-tubulin region was used, five clades were separated: N. perennans, N. malicorticis, N. alba, N. krawtzewii, and an unnamed clade (de Jong et al. 2001). This clade was later assigned the name Cryptosporiopsis kienholzii (Spotts et al. 2009), and finally Neofabraea kienholzii (Seifert 2013; Chen et al. 2016). There was some doubt expressed as to whether there was sufficient difference in morphology and sequence between N. perennans and N. malicorticis to justify the assignment of species names, as these two species have identical ITS sequence. De Jong et al. (2001) argued that, because there were no instances of vegetative compatibility between the two species and there were no intermediate types, that separate species names should be assigned. N. kienholzii is separate from N. alba and N. malicorticis/perennans clades when the ITS region was used for phylogenetic analysis (de Jong et al. 2001).

The division of the Neofabraea fungi into five species was accepted in subsequent literature in which polymerase chain reaction (PCR) primers based on the β-tubulin gene 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 mm2) 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 (Qiagen©) 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.

Initial PCR screening was conducted for 98 isolates in 2013. Reactions were in a total volume of 25 µL/well, consisting of 2 µL of 10 ng DNA, 2.5 µL Taq polymerase buffer, 0.5 µL 10 mM dNTP, 0.75 µL 50mM Mg Cl2, 0.5 µM of each forward and reverse primers (Neo alba-up and Neo_ alba-loTub-439; Gariepy et al. (2003); Soto-Alvear et al. (2013)), 0.125 µL Platinum Taq polymerase (Invitrogen) and 18.875 µL Milli-Q® purified water. PCR reactions were conducted in a thermocycler (Techne®) under the following conditions: initial denaturation at 96 °C for 3 min, 35 cycles of 96 °C for 3 min., 60 °C for 1 min. and 72 °C for 2 min., followed by final elongation at 72 °C for 10 min. PCR products (10 µL) were loaded onto a 1% agarose (w/v) gel in 0.5xTBE buffer. The gel was electrophoresed at 100 V for 90 min, stained in 0.5 µL/mL ethidium bromide in Milli-Q® purified water for 10 min, then visualised using a GelDoc Go imaging system (Bio-Rad, USA).

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.

Table 1 Isolate names, country of origin, host and GenBank code used in phylogenetic analysis of the β-tubulin gene region
Full size table

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).

Fig. 1
figure 1

Phylogenetic tree of maximum likelihood analyses of β-tubulin data of 99 New Zealand single-spore isolates compared with seven known species of Phlyctema vagabunda (P. vaga. CBS 304.62, P. vaga. CBS 109875), Neofabraea actinidiae (N.a. CBS 121403), N. kienholzii (N.k. CBS 126461), N. malicorticis (N.m. CBS 102863), Neofabraea perennans (N.p. CBS275.29), and Neofabraea vincetoxici (N.v. CBS 123727) from GenBank (National Centre for Biological Information). Numbers at nodes are percentage bootstrap values based on 1000 replicates for values over 60%. Infundichalara microchona (I.m. CBS 175.74) was used as the outgroup

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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 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 rots only N. malicorticis (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 (Spadaro et al. 2020). P. vagabunda is also found in Australia (Cunnington 2004), Chile (Henriquez 2005; Soto-Alvear et al. 2013), Japan (Sato et al. 2021), New Zealand (Johnston et al. 2005) and South Africa (Den Breeyen et al. 2020).

Since the discovery of a new species of Neofabraea from apples in the Pacific North West USA (de Jong et al. 2001; Spotts et al. 2009), N. kienholzii has been reported from apples from Australia (Cunnington 2004), Canada (de Jong et al. 2001), the Czech Republic (Pesicova et al. 2017), Ecuador (Valdez Tenezaca 2020), Italy (Neri et al. 2018), the Netherlands (Wenneker et al. 2017, 2018), Poland (Michalecka et al. 2016), Portugal (Lin et al. 2018), Taiwan (Zhang et al. 2019) and the United Kingdom (Kingsnorth et al. 2017). N. kienholzii has also been reported from olives in California (Trouillas et al. 2019), and from grapes in Hungary (Lengyel et al. 2020).

Based on re-examination of the literature, isolations, PCR tests, sequencing the β–tubulin gene region, and phylogenetic comparisons with known strains, there is no evidence that any species other than N. kienholzii and P. vagabunda have ever been present in New Zealand causing bull’s eye rot on apples, and that P. vagabunda is most common.