Trees DOI 10.1007/s00468-015-1350-9

ORIGINAL ARTICLE

Real-time PCR for detection and quantification, and histological characterization of ditissima in trees

1 2 3 4 Marjan Ghasemkhani • Anna Holefors • Salla Marttila • Kerstin Dalman • 1 3 6 5 Anna Zborowska • Mira Rur • Jonathan Rees-George • Hilde Nybom • 6 7 1 Kerry R. Everett • Reiny W. A. Scheper • Larisa Garkava-Gustavsson

Received: 10 June 2015 / Revised: 7 November 2015 / Accepted: 28 December 2015 Ó Springer-Verlag Berlin Heidelberg 2016

Abstract pathogen identification. A quantitative real-time PCR Key message We designed a pair of primers from a (qPCR) assay was developed for both detection and region of the b-tubulin gene to detect and quantify quantification of this pathogen in infected apple cultivars. in wood of some infected apple Several primer sets were designed from regions of the b- cultivars, and optimized light microscopy to study tubulin gene. One primer set passed several validation fungal-plant interactions. tests, and the melting curve confirmed species-specific Abstract Neonectria ditissima, the causal pathogen of amplification of the correct product. In addition, the N. fruit tree canker, is a sordariomycete that affects ditissima biomass could be detected at variable amounts in apple orchards, especially in north-western Europe. To samples from the infection sites of six different cultivars, prevent serious disease epidemics, an accurate, rapid, and with ‘Aroma’ having the lowest amount of N. ditissima sensitive method for detection of N. ditissima is needed for biomass and ‘Elise’ the highest. To complement the qPCR results, tissue from detached shoots and 1-year-old trees of ‘Cox’s Orange Pippin’ (susceptible) and ‘Santana’ (par- Communicated by W. Osswald. tially resistant) was used in a histopathology study. In both Electronic supplementary material The online version of this detached shoots and trees, fungal hyphae were found in article (doi:10.1007/s00468-015-1350-9) contains supplementary cells of all tissues. No qualitative differences in the anat- material, which is available to authorized users. omy of the infected samples were observed between the & Marjan Ghasemkhani cultivars. In the detached shoot experiment, both cultivars [email protected] were affected but differences in the rate of disease pro- gression suggest that the partially resistant cultivar could 1 Department of Plant Breeding, Swedish University of Agricultural Sciences, Box 101, 230 53 Alnarp, Sweden resist the fungus longer. The qPCR assay developed in our 2 study produced reproducible results and can be used for In vitro Plant-tech AB, Geijersgatan 4, 216 18 Limhamn, N. ditissima Sweden detection of in infected trees. 3 Department of Plant Protection Biology, Swedish University Keywords Apple canker Anatomy B-tubulin gene of Agricultural Sciences, Box 102, 230 53 Alnarp, Sweden Á Á Á Neonectria galligena Á Á qPCR 4 Department of Chemistry and Biotechnology, Uppsala Biocenter, Swedish University of Agricultural Sciences, Box 7026, 750 07 Uppsala, Sweden Introduction 5 Department of Plant Breeding, Swedish University of Agricultural Sciences, Balsga˚rd, Fja¨lkestadsva¨gen 459, 29 194 Kristianstad, Sweden Neonectria ditissima (Tul. & C. Tul.) Samuels & Rossman, 6 The New Zealand Institute for Plant and Food Research Ltd, previously known as Neonectria galligena (Bres.) Rossman PB 92169, Mt Albert, Auckland, New Zealand & Samuels, is a fungal pathogen that occurs in almost all 7 The New Zealand Institute for Plant and Food Research Ltd, apple-producing areas of the world, e.g., Europe, Asia, the PB 1401, Havelock North, New Zealand Middle East, North America, South America, and New 123 Trees

Zealand (CPCI 2014). This pathogen causes cankers on hybridization probe, designed to the ITS1 region. A mag- numerous tree and shrub species, and is especially preva- netic capture-hybridization PCR technique (MCH–PCR) lent in commercial apple (Malus 9 domestica) and employing a biotinylated capture oligonucleotide could (Pyrus communis) orchards (Castlebury et al. 2006). The detect fungus in old cankered tissue and in asymptomatic pathogen attacks branches and twigs to cause dieback in tissue either adjacent to a canker or in a newly infected full-grown trees. The lesions often expand to girdle the apple tree whereas PCR without MCH was unsuccessful main trunk and kill all branches above the infected point. In (Langrell and Barbara 2001). However, application of addition, this fungus can cause rotting at the calyx end of MCH-PCR can be limited by the additional cost and rela- fruits (eye rot). Symptoms on fruit are generally expressed tive assay complexity. By comparison, qPCR is technically during prolonged storage causing a serious post-harvest straightforward. problem (Xu and Robinson 2010). During the early stage of qPCR assay development in Since N. ditissima can produce spores throughout the the present study, specific ITS1 region primers Ch1/Ch2 year and infect through suitable entry sites (leaf scars, used by Langrell (2002) for detection of N. ditissima in emerging buds, wounds due to tree ties or pruning), control end-point PCR, were tested in qPCR. The region targeted of this disease is difficult (Webster et al. 2001). Trees can by these primers apparently did not contain a sufficient also become infected during propagation and the infection number of well-positioned polymorphisms to differentiate may remain latent for 3–5 years before symptoms appear between N. ditissima and apple since apple DNA was (McCracken et al. 2003). amplified in samples from tissue-cultured plants. More- Canker symptoms caused by N. ditissima vary depend- over, these primers generated a large product (412 bp) ing on the stage of disease development, age of the infected whereas short amplicons are essential to obtain reasonable wood, and cultivar (Gustavsson 2012). On twigs, cankers amplification efficiencies in qPCR (Kubista et al. 2006). caused by N. ditissima can be confused with those caused Therefore a target DNA region, the b-tubulin gene, was by malicorticis H. S. Jacks. (syn. Cryptospo- instead selected and new primers were designed. Crous riopsis curvispora (Peck) Gremmen) (Braun 1997). Tra- et al. (1999) showed that the b-tubulin gene is more ditionally, microscopic examination of spore size and informative than the ITS regions for distinguishing some shape has been used to differentiate between the canker species of ascomycetous fungi, e.g., Cylindrocladium. The causative pathogens (De Jong et al. 2001; Gariepy et al. b-tubulin gene region is highly conserved in eukaryotes 2003; Kasson and Livingston 2009). and has proved useful for designing species-specific pri- The ability to detect and quantify fungal DNA using mers in previous studies (Nakayama et al. 2013; Yun et al. real-time, or quantitative PCR (qPCR), has been developed 2013). The b-tubulin gene contains an extremely variable and applied to plant pathogens (Catal et al. 2013; Haegi 50 end and a more conserved intron-poor 30 end, and has et al. 2013). qPCR thus provides new research opportuni- been used to determine levels of relatedness in ascomy- ties to study the diagnosis and epidemiology of fruit tree cetes (Landvik et al. 2001). A paralogous copy has been canker (Gadkar and Filion 2014). discovered in ascomycetes (Gold et al. 1991; Panaccione An oligonucleotide primer (ChInt), used in combination and Hanau 1990) but the duplication event has apparently with the universal ITS4 primer, has previously been occurred after the divergence of the designed from the internal transcribed spacer (ITS) 1 (Landvik et al. 2001; Zhao et al. 2014). region of ribosomal DNA (rDNA) of N. ditissima (Brown More information on the plant–pathogen interaction is et al. 1993). However, the application of this primer set is needed for understanding how partial resistance to apple limited since it cannot differentiate between closely related canker can arise in some apple cultivars. In general, host species which co-exist on the same host (Langrell 2002). plants defend themselves by either structural characteristics Langrell (2002) designed an improved pair of primers or biochemical compounds to inhibit pathogens from (Ch1/2) to detect N. ditissima, also from the ITS regions of gaining entrance and spreading within the plant. In a his- the ribosomal RNA gene. This primer set was mostly tological study in Fraxinus mandshurica (Manchurian ash), successful in detecting N. ditissima in cankered wood but, Sakamoto et al. (2004) showed how N. ditissima affected on occasion, the reaction was inhibited by contaminating the stem tissues during canker formation; fungal hyphae substances from lignified wood resulting in false negatives. were found in different cell types, including the vessel By contrast, the fungus could not be detected in symp- lumina of the affected xylem. Previous studies reported tomless tissue immediately adjacent to cankers, probably significant differences between cultivars in the level of because the PCR was not sufficiently sensitive or was resistance to N. ditissima and several cultivars like ‘Cox’s inhibited by contaminating substances. Langrell and Bar- Orange Pippin’, ‘Elise’, ‘James Grieve’ were found to be bara (2001) improved the PCR sensitivity and reliability very susceptible, while ‘Santana’, ‘Aroma’, ‘Florina’ through implementation of a specific complementary showed relatively high level of partial resistance (Garkava- 123 Trees

Gustavsson et al. 2013; Ghasemkhani et al. 2015). How- ITS sequencing of Neonectria ditissima isolates ever, comparative studies on the invasive behavior of the fungus on susceptible versus partially resistant hosts have ITS1F and ITS4 (TAG Copenhagen) were used to amplify not yet been conducted. the internal transcribed spacer (ITS) region of the ribosome In this study, a qPCR assay was applied to identify and encoding genes (White et al. 1990; Ihrmark et al. 2012). quantify N. ditissima in cankered apple trees, and investi- PCRs were performed in a 2720 Thermal Cycler (Applied gate a possible association with levels of partial resistance Biosystems) with 12.5 ng of DNA of the isolates from of the hosts. In addition, changes in woody tissue anatomy Sweden and Belgium in a total volume of 50 ll PCR during fungal infection were documented with light mixture consisting of 7.5 U DreamTaq (Thermo Scientific, microscopy. Maryland, USA), 5 ll DreamTaq Green buffer, 1.25 mM MgCl2, 0.2 mM dNTP, 0.5 mM of each primer. The con- ditions for PCR were as follows: 95 °C for 5 min, 35 Materials and methods cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 7 min. The PCR PCR-based study products were then purified with AgencourtÒ AMPure PCR Purification kit (Agencourt Bioscience Corporation, USA) Source of materials according to the instructions. The PCR products were sequenced (Macrogen Inc, Amsterdam). Then the sequen- All isolates of N. ditissima collected from naturally ces were assembled and trimmed using SeqMan Pro ver- occurring cankers on apple branches, and other fungal sion 12.0.0 (DNASTAR Inc.) and compared for similarity species used in this study are listed in Tables 1 and 2. using BLASTN at NCBI. Out of the top 100 hits, all the Isolates in Table 1 were identified based on morphological hits for N. ditissima/N. galligena (assigned with a species characteristics of spores as well as BLAST ID using the name) were downloaded along with two closely related ITS sequence, while isolates in Table 2 were identified species: Neonectria major and Neonectria neomacrospora, based on morphological characteristics of conidia and and one more distant lugdunensis (to serve as an sequencing the entire ITS region in Sweden and using the outgroup). All sequences were aligned by ClustalW in primers developed by Langrell (2002) in New Zealand. MEGA version 6 (Tamura et al. 2013). Eighteen 1-year-old apple trees grown in pots, with three trees of each cultivar, were used. Three of these cultivars Primer design are regarded as susceptible to N. ditissima: ‘Elise’, ‘Cox’s Orange Pippin’, and ‘Discovery’, while the other three are Specific primers for N. ditissima were designed by aligning partially resistant: ‘Aroma’, ‘Santana’, and ‘Golden Deli- the b-tubulin gene region from Neonectria ditissima with cious’ (Garkava-Gustavsson et al. 2013; Ghasemkhani several closely and more distantly related fungi using CLC et al. 2015). The trees were grown on the rootstock M9 Main Workbench (Qiagen). The sequences used were (Olien and Lakso 1986). retrieved from GenBank at NCBI, and were N. ditissima (AM419113, AM419114, AY297216, and AB237487), DNA extraction Neonectria major (Wollenw.) Castl. & Rossman (DQ789853 and DQ789872), Neonectria punicea (Sch- DNA of N. ditissima from different isolates and from midt:Fr.) Castl. & Rossman comb. nov. (Basionym: freshly infected and healthy woody tissue, respectively, Sphaeria punicea Schmidt in P. Kunze & Schmidt: Fr., was extracted using the Fermentas genomic DNA purifi- Myk. Hefte 1: 61, 1817: System. Mycol. 2: 415, 1823; cation kit (Thermo Scientific, Maryland, USA) and anamorph C. album) (DQ789873 and DQ789824), WizardÒ SV Genomic DNA (Promega, Madison, USA) Neonectria hederae (DQ789895), Neonectria fuckeliana purification system. DNA concentration and purity (260/ (C. Booth) Castl. & Rossman (DQ789871 and DQ789847), 280 nm [1.8) were measured using a NanoDropÒ ND- Fusarium lateritium (AY707153), Ilyonectria radicicola 1000 equipment (Wilmington, USA) and through agarose (Gerlach & L. Nilsson) P. Chaverri & Salgado (AJ877059 gel electrophoresis (2 %). To serve as non-target controls, and AJ877058), rigidiuscula (Berk. & DNA from several fungi, including saprotrophs com- Broome) Rossman & Samuels (HQ008937), Pestalotia monly found in orchards, other apple pathogens, and vaccinii (DQ787844), Phomopsis viticola (GU294725), closely related pathogens of the genus Fusarium from Botryosphaeria obtusa (Schwein.) Shoemaker other hosts (Table 1) was extracted using the WizardÒ SV (DQ458857), B. parva (DQ356366), Neonectria ramula- Genomic DNA (Promega, Madison, USA) purification riae (DQ789863 and DQ789864) and Neonectria coccinea system. (DQ789831, DQ789840 and DQ789835). Primers were 123 Trees

Table 1 Fungal species Species Host Culture number Origin associated with apple used in this study to verify species- Aureobasidium sp. Malus domestica RS 204 New Zealand specificity of primers Botryosphaeria dothidea Persea americana ICMP 15694 New Zealand Botryosphaeria sp. P. americana JRG 15 New Zealand Botrytis cinerea Cucurbita moschata ICMP 15696 New Zealand Cladosporium sp. P. americana ICMP 15697 New Zealand Colletotrichum acutatum P. americana ICMP 15698 New Zealand Colletotrichum gloeosporioides M. domestica ICMP 12071 New Zealand Cryptosporiopsis actinidiae Actinidia deliciosa ICMP 15699 New Zealand Ilyonectria robusta Vitis vinifera LUPP 980 New Zealand Ilyonectria europaea V. vinifera LUPP 985 New Zealand Ilyonectria liriodendri V. vinifera LUPP 1083 New Zealand Ilyonectria liriodendri V. vinifera LUPP 968 New Zealand Dactylonectria torresensis V. vinifera LUPP 957 New Zealand Dactylonectria novozelandica V. vinifera LUPP 1008 New Zealand Elsinoe pyri M. domestica ICMP 18437 New Zealand Epicoccum nigrum M. domestica ICMP 11503 New Zealand Fusarium acuminatum M. domestica BTH 511 New Zealand Fusarium avenaceum M. domestica RS 27 New Zealand Fusarium culmorum M. domestica RS 163 New Zealand Fusarium lateritium M. domestica RS 162 New Zealand Fusarium sp. M. domestica RS 111 New Zealand Nectria cinnabarina M. domestica RS 211 New Zealand Neonectria fuckeliana Pinus radiata NZFS 3082 New Zealand Neofabraea krawtzewii Populus trichocarpa CBS 102868 Norway Neofabraea alba M. domestica CBS 261.32 Denmark Neofabraea malicorticis M. domestica CBS 122030 USA Neofabraea perennans M. domestica CBS 453.64 United Kingdom Nigrospora sp. P. americana ICMP 15701 New Zealand Penicillium sp. P. americana ICMP 15702 New Zealand Pestalotia sp. P. americana ICMP 15703 New Zealand Phialophora sp. A. deliciosa JRG 309 New Zealand Phlyctema sp. A. deliciosa JRG 24 New Zealand Phoma sp. P. americana ICMP 15705 New Zealand Phoma sp. M. domestica JRG 23 New Zealand Phomopsis sp. P. americana ICMP 15706 New Zealand Sphaceloma perseae P. americana CBS 406.34 USA Stemphylium sp. P. americana ICMP 15707 New Zealand Venturia inaequalis M. domestica ICMP 13258 New Zealand JRG, RS, personal collections of authors (J. Rees-George and R. Scheper) BTH, personal collection of BT Hawthorne, located at The New Zealand Institute for Plant and Food Research, Auckland, New Zealand ICMP International Collection of Micro-organisms from Plants, Manaaki Whenua Landcare Research, Auckland, New Zealand, CBS Centraalbureau voor Schimmelcultures, Uppsalalaan, Netherlands, NZFS National Forestry Culture Collection, Scion, New Zealand, Forest Research Institute Ltd, LUPP Lincoln University Plant Pathology designed from regions conserved within N. ditissima but several regions including SNPs were selected for primer variable in comparison to other species. The robustness of design. We compared several primer combinations by in some SNPs across alleles of the same region was assessed silico analysis. Two primer sets that were specific to N. among the other allelic sequences in the alignment. In total, ditissima, Bt-fw135 (5-CTCCAACACAACAACATTCG-

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Table 2 Isolates of Neonectria ditissima from Sweden and New Zealand, isolated from different apple cultivars, used to verify the amplification of the target region by Bt-fw135/Bt-rw284 primers in genetically diverse material Strain number Culture number Hosta Origin Year Reference

MG 1 CBS 139255 Tompkins King Balsga˚rd, Sweden 2013 – (N56°60, E14°90) MG 2 CBS 139256 Baron Gembloux, Belgium 2014 – (N50°330,E4°420) MG 16 CBS 139268 James Grieve Jo¨nko¨ping, Sweden 2013 – (N57°460, E14°90) MG 17 CBS 139269 A˚ kero¨ Julita, Sweden 2013 – (N59°110, E16°10) MG 20 CBS 139272 Aroma Jonstorp, Sweden 2013 – (N56°130, E12°400) MG 22 CBS 139274 Ingrid Marie Bja¨rred, Sweden 2013 – (N55°430, E13°10) MG 25 CBS 139389 Ingrid Marie Kivik, Sweden (N55°410, 2013 – E14°130) MG 27 CBS 139277 Eva-Lotta Stockholm, Sweden 2014 – (N59°190, E18°30) – ICMP 9472 Royal Gala Pukekohe, Waikato 1987 Langrell (2002) and Scheper et al. (2014) (S37°110, E174°580) – ICMP 14415 Royal Gala Waikato, New Zealand 2000 Amponsah et al. (2014) (S38°30, E175°260) – ICMP 14417 Aurora Nelson, New Zealand 2000 Walter et al. (2014) (S41°160, E173°170) – ICMP 5430 Apple (cultivar unknown) Auckland, New Zealand 1965 Langrell (2002) (S36°500, E174°450) RS 324p – Golden Delicious New Plymouth, Taranaki 2009 Scheper et al. (2014, 2015) (S39°30, E174°40) RS 305p – Brookfield Lower Moutere, Nelson 2009 Scheper et al. (2014, 2015) (S41°160, E173°170) MG, RS, personal collections of authors (M. Ghasemkhani and R. Scheper) CBS Centraalbureau voor Schimmelcultures, Uppsalalaan, Netherlands, ICMP International Collection of Micro-organisms from Plants, Man- aaki Whenua Landcare Research, Auckland, New Zealand a Cultivars of Malus domestica

30)/Bt-rw284 (5-AGTATCCCCGCACGTTAGAA-30) and following conditions: an initial step of denaturation for Bt-fw179 (5-AGGAACAAGATGGCTGACAA-30)/Bt- 5 min at 95 °C, followed by up to 35 cycles of denaturation rw306 (5-GTTACCCTATCCACGCATTG-30), were fur- for 15 s at 95 °C, annealing for 30 s at 60 °C, extension for ther evaluated. 15 s at 72 °C, and a post-amplification step of 5 min at 72 °C to extend any incomplete products. The amplified Validation of primer specificity DNA was analyzed by gel electrophoresis, and PCR products were purified with the JETSORB gel extraction The specificity of N. ditissima primers was validated in kit (GENOMED GmbH, Lo¨hne, Germany) according to the three ways. First, gDNA of N. ditissima was amplified and manufacturer’s instructions. The quality and quantity of the sequenced. PCR was performed with the designed primers isolated DNA were analyzed by spectrophotometry and gel in 25 ll reaction mixtures containing 2.5 ll of 10X PCR electrophoresis. The purified DNA was sequenced (UIO, buffer, 0.2 mM dNTP mix, 0.5 lM of each primer, Oslo, Norway) and the sequences were assembled and

1.5 mM MgCl2, 1 Unit of Taq DNA polymerase (Thermo aligned to gDNA sequences of N. ditissima using the CLC Scientific, Maryland, USA), and 10 ng genomic DNA. Main Workbench. Amplifications of each sample were performed in either a Secondly, primer specificity was validated through the Bio-Rad S1000 or a Techne thermal cycler under the absence and presence of a single PCR product after end-

123 Trees point PCR on genomic DNA from a) different isolates produce sporodochia. Subsequently the culture media representing a range of fungal species found on apple wood containing sporodochia were kept at 4 °C until further (Table 1), b) fungi closely related to N. ditissima from both usage. Spore suspensions of the fungus (MG1) were pre- apple and other hosts (Table 1) and c) different isolates of pared with distilled water and mixed well. Number of N. ditissima (Table 2), respectively. DNA from purified viable macroconidia was counted using a Fuchs-Rosenthal cultures of N. ditissima (CBS 139255, ICMP 9472, ICMP hemocytometer under a Leica DMLB compound micro- 5430, RS 324p, RS 305p) was used as a positive control scope. Finally, the spore concentration was adjusted to and sterilized water was used as a negative control. To 1 9 105 conidia/ml. confirm the reliability of the reaction, PCR products were visualized by agarose gel electrophoresis (2 %) and Inoculations and sampling ethidium bromide staining, and compared to a PCR ladder marker set (50–1000 bp, Thermo Scientific). Three axillary buds, number 11, 14, and 17 from the apex, Thirdly, primer specificity was confirmed through were removed with a scalpel. Inoculations were carried out melting curve analysis during quantitative PCR, the first by aliquoting 10 ll of above described spore suspension derivative plot of the melting curve should contain a with the aid of a micropipette onto the bud scar wound single peak with correct melting temperature if the primer within 5 min after wounding. Wounds were covered by pair produces a single amplicon (Pagliarani et al. 2013). white petroleum jelly (VaselineÒ ACO, Sweden) after the This method was also used to detect N. ditissima and suspension was completely absorbed into the tissue to explore the relationships between lesion size on infected preserve the moisture. Vaseline was removed 4 days after apple trees and the amount of N. ditissima genomic DNA inoculation using tissue paper. After inoculation, the trees in the tissue. were kept in an unheated greenhouse with frost protection for 2 months. The mean temperature was 9 ± 2 °C with 70 % of the relative humidity. The lesion length was PCR efficiency and limit of detection measured with a plastic caliper. Samples for DNA analysis were taken with a sterile scraper from wood and bark of the The amplification efficiency (E) of the best primer set, Bt- infected areas as well as from surrounding but apparently fw135/Bt-rw284, was estimated using the slope of the line healthy areas, approximately 5 mm above and below derived from the standard curve, according to the equation: infection sites. The first set of samples was taken 1 month E ¼ 10ðÀ1=slopeÞ. The qPCR assay included a standard curve after inoculation before appearance of any symptoms. A in the plate. Each standard curve comprised 5 serial 1:10 second set of samples was taken after 2 months, when dilutions (in triplicate) of the amplicons obtained from a symptoms had developed. For each cultivar, samples were fixed amount of gDNA using designed primers, starting taken from three buds scars (one scar per tree) that had from 100 ng. similar symptoms with approximately the same size of The limit of detection (LOD) of N. ditissima was lesion. Samples were kept frozen individually at -20 °C determined by mixing multiple 10- and 5-fold dilutions of until processing. gDNA extracted from a pure culture of N. ditissima (60 samples: 10 ng, 1 ng, 0.1 ng, 0.01 ng, 1 pg, 0.1 pg, Quantitative real-time PCR amplification 0.01 pg, 1 fg, 0.1 fg, 0.01 fg, and 30 samples: 2.5 ng, 0.25 ng, 25 pg, 2.5 pg, 0.25 pg) with 10 ng apple DNA, The quantitative real-time PCR assay and data analysis which was extracted from healthy woody tissue, in 6 were performed with CFX96 Touch Real-Time PCR replicates. Detection System (BIO-RAD, USA). All reactions were run in a total volume of 20 lL using 0.2 ml 96-well PCR Inoculum preparation for qPCR assays plates. The reaction mix comprised SsoFast EvaGreen supermix (BIO-RAD, USA), 0.6 llof10lM forward and N. ditissima was isolated from naturally infected cankered reverse primers, and 2.5 ll of 10 ng extracted gDNA of the wood with large numbers of sporodochia, collected from infected trees. The quantitative real-time PCR was run at the apple germplasm orchard at Balsga˚rd, southern Sweden 95 °C for 3 min, followed by 40 cycles of 98 °C for 5 s 0 0 (N56°6 , E14°9 ) in autumn 2012. Conidia were transferred and 60 °C for 5 s. Each 96-well plate included a reference from the wood to 1.5 mL Eppendorf tubes with 50 llof sample, which was a mixture of 10 ng purified genomic sterile distilled water and then plated on 1.5 % water agar DNA of N. ditissima with 5 ng purified genomic DNA of media (Difco, USA) at room temperature. The following apple, and a negative control with only sterile water. All day single macroconidia were cultured on water agar media qPCR experiments were carried out with three biological (1.5 %) and incubated at 20–22 °C with daylight to (trees) and three technical replicates per reaction. In total, 123 Trees nine replicates were thus obtained for each cultivar. For 0.1 M Na-phosphate buffer, and then stored at 4 °Cin amplification of N. ditissima and apple DNA, primers buffer until further processed. designed in the present study and ubiquitin gene primers described by Botton et al. (2008) were used, respectively. Plastic embedding To confirm the specificity of the amplification product, melting curve analysis was performed and each melting Fixed and stored samples were washed with distilled water curve was assessed for the presence of a single peak with 3 9 15 min, followed by dehydration with acidified 2, the correct melting temperature. 2-dimethoxypropane (DMP) 2 9 30 min and acetone 2 9 20 min. Fresh Spurr’s resin (low viscosity kit; Ted Statistical analyses Pella, Redding, CA, USA) was prepared according to the manufacturer’s instructions for infiltration with acetone– For each sample, the amplified N. ditissima DNA was resin (3:1) for 3 h, acetone–resin (1:1) 3 h, acetone–resin normalized to the amplified apple DNA and to the (1:3) 24 h, then 100 % resin 24 h, followed by 6 h in fresh above-mentioned reference sample. Data analysis was 100 % resin the next day. All steps were performed under performed using Bio-Rad CFX manager Software v. gentle agitation in a fume hood. Polymerization was per- 3.0.1. The association between lesion size and relative N. formed with fresh resin in polyethylene capsules at 70 °C ditissima DNA quantity was evaluated across the six for 24 h, followed by 48 h at 40 °C, and post-polymer- cultivars by Pearson correlation. Statistical analyses were ization in a fume hood a few days before sectioning. performed using the software R (R Development Core Semithin sections of 1 lm were cut with an ultramicrotome Team 2013). (Ultrotome, LKB, Bromma, Sweden) in transverse with a section thickness of 1 lm and placed on object slides Histopathology study (SuperFrost Plus, Menzel-Gla¨ser, Braunschweig, Ger- many) for light microscopy. Source of materials and inoculation Staining One susceptible cultivar, ‘Cox’s Orange Pippin’ and one partially resistant cultivar, ‘Santana’, were chosen for light Toluidine blue staining (Sigma-Aldrich, Steinheim, Ger- microscopy studies with both detached shoots and 1-year- many) was used for the observation of general tissue old potted trees. structure and the presence of fungal hyphae. The sections Dormant shoots were collected from mature healthy were stained with filtered 1 % Toluidine Blue O in 1 % Na- trees in the apple germplasm orchard at Balsga˚rd, put in tetraborate for 30 s on a hot plate, rinsed with water, air- glass bottles with water and liquid fertilizer (Chrysal, dried, and mounted with Biomount (British Biocell, Cardiff, Naarden, the Netherlands) and placed under a tent in the UK). Structural studies were performed using a Leica greenhouse. These shoots were inoculated with a N. ditis- DMLB light microscope and photographs were taken with a sima spore suspension as described previously (Garkava- Leica DFC450C digital camera (Leica Microsystems). Gustavsson et al. 2013). Samples from the site of infection were taken at 5, 18, 29, and 36 days after inoculation. One-year-old potted trees were kept in an unheated Results greenhouse (9 ± 2 °C and 70 % RH), watered weekly and inoculated as previously described (Garkava-Gustavsson The isolates from Sweden and Belgium were identified as et al. 2013). Samples from these trees were taken 110 days N. ditissima by ITS sequencing and blasting showing after inoculation (dai) when the lesions were clearly visi- 99–100 % identity to N. ditissima/N. galligena. Results of ble. The samples, approximately 1 mm thick slices through alignment showed that only one variable site within the N. the stem, were immediately fixed for further processing for ditissima/N. galligena isolates was found, indicating a high light microscopy. conservation. In addition, there is no resolution in ITS region for the clade N. ditissima/N. galligena/N. neo- Fixation macrospora/N. major (supplementary file 1).

Specimens were fixed in a mixture of 2 % (w/v) Primer validation paraformaldehyde and 2.5 % (v/v) glutaraldehyde in 0.1 M Na-phosphate buffer, pH 7.2, during gentle agitation for The validation results of specificity of the designed primer 4 h at room temperature and then for 24 h at 4 °C. Sub- pairs showed that first, only the specific target sequences sequently the specimens were rinsed 3 9 15 min with were amplified when the direct sequencing of the 123 Trees amplicons from N. ditissima gDNA after using the two primer sets was performed. In the second validation step performed through end-point PCR with the two primer sets on gDNA of other fungi and different isolates of N. ditis- sima, a single amplicon of the anticipated size (150 bp) was obtained from all isolates of N. ditissima (Fig. 1). Only one primer set, Bt-fw135/rw284, did not produce bands from the other fungi (Table 1). Third, amplification specificity for the primer set Bt-fw135/rw284 was verified by melting curves of N. ditissima with a single specific peak, indicating the absence of non-specific amplifications or primer dimers. When samples of cankered tissue were tested with primer set Bt-fw135/rw284, this peak was detected in qPCR assay (Fig. 2). In silico analysis of the b-tubulin region of the rare Fig. 2 Melting curve analysis of qPCR. The single peak for Bt- apple pathogen Neonectria ramulariae and the ubiquitous fw135/rw284 primer pairs was detected in cankered tissues of six cultivars and shows the presence of only one PCR product pathogen of , N. coccinea (Castlebury et al. 2006) showed no complementary DNA sequence to PCR primers Bt-fw135/rw284, Bt-fw179/rw306 (data not shown). DNA and the Ct values across the standard samples (serial Additionally, the result of in silico analysis of b-tubulin dilutions). Reaction efficiency and coefficient of determi- 2 gene of different isolates of Neonectria ditissima from nation (R ) were 99.6 % and 0.986, respectively. Europe, New Zealand, and USA showed that b-tubulin The limit of detection (LOD) was identified by the last gene region is highly conserved in the region that our dilution when successful qPCR amplification of DNA primers bind and PCR primer pairs, Bt-fw135/rw284, Bt- occurred (indicated by a sigmoid curve to termination) fw179/rw306, detect N. ditissima isolates from other accompanied by a melting curve peak temperature specific countries (supplementary file 2). to N. ditissima. The mean Ct value observed for the limit of detection (LOD) was 37.74 and it was calculated from three Assessment of qPCR efficiency and limit of detection replicates of gDNA of N. ditissima mixed with gDNA of healthy woody tissue. The smallest pathogen DNA con- The standard curve method was selected to determine the centration that produced a positive signal in all replicates amplification efficiency of the primer set Bt-fw135/rw284, was 0.25 pg. which allowed for unambiguous discrimination between the target species and closely related species. Sensitivity of the qPCR assays standard qPCR assay was determined by testing five fold serial dilution from 100 ng to 1 pg of gDNA preparations of The new qPCR assay was applied to wood and bark tissue N. ditissima in triplicate to establish the linearity of the from different apple cultivars and the fungus was not assay. A linear relationship was found between the input detected in tissues without symptoms, i.e., in stem samples

Fig. 1 PCR using specific primers to amplify DNA from several different isolates of Neonectria ditissima. Lanes 1–8 are N. ditissima MG 19, MG 17, MG 16, MG 25, MG 1, MG 22, MG 2 and MG 26, lane 9 negative control, and lane 10 positive control (strain MG 1) 123 Trees taken 1 month after inoculation. The specific primers partially resistant ‘Santana’ 110 days after inoculation detected the fungus at the site of infection after 2 months, (Fig. 4b, c). This may be the first step in the development when visible symptoms had developed. Different levels of of asexual fruiting structures produced by N. ditissima.In N. ditissima biomass were observed in different cultivars; the healthy tissue, cortex parenchyma cells were loosely the susceptible ‘Elise’ had the highest amount and the arranged with large intercellular spaces (Fig. 4d) while in partially resistant ‘Aroma’ had the lowest. Lesion size of the infected samples of both cultivars, the cells were those bud scars that were sampled for qPCR analysis was deformed and collapsed (Fig. 4e, f). A large number of measured 2 months after inoculation, and ‘Elise’ had the hyphae were present in the cortex. Cells in the secondary highest mean value while ‘Aroma’ had the lowest. The phloem and vascular cambium of the infected tissues were mean lesion size for the 3 inoculated bud scars sampled for more heavily colonized by mycelium in the susceptible each cultivar ranged from 7.6 to 26 mm while the mean cultivar (Fig. 4e) when compared with the partially resis- relative N. ditissima DNA quantity ranged from 0.3 to 39.4. tant cultivar (Fig. 4f). The primary phloem fibers were also Mean lesion size was not significantly correlated to mean affected in the infected stems of both cultivars (Fig. 4e, f). relative quantity of N. ditissima gDNA, r = 0.73, Vascular tissue in the wood, primary and secondary xylem P = 0.09, df = 4, when calculated across cultivars and xylem rays retained their shape and form in the (Fig. 3). infected tissues in both cultivars even when fungal hyphae were observed in xylem rays, xylem vessels, and tracheids Histopathology study (Fig. 4g, h, k). Despite the presence of fungi in pith cells, cell shape, and cell walls were normally arranged in both In the histopathology study, the best preservation of stem cultivars (Fig. 4i, m, n). The susceptible cultivar appeared structures was achieved by plastic embedding with long to be more heavily colonized by N. ditissima than the incubation times compared to the less time-consuming partially resistant cultivar except for the cortex colonized paraffin embedding (not shown). For detecting the fungal by the putative sporodochia structures. Signs of gel for- hyphae, toluidine blue staining was superior over Uvitex mation were found in some of the samples but there was no 2B and aniline blue staining (data not shown). clear difference in either frequency or extent between the The tissue and cells were then examined to visualize susceptible and resistant cultivars (data not shown). differences between a susceptible and a partially resistant In the detached shoots (Fig. 5), no anatomical changes cultivar. Fungal hyphae in the stem tissue stained dark were observed in the infected stem tissues until 18 days purple and along with morphological differences, allowed after inoculation (dai), when parenchyma cells in the cortex distinguishing fungal cells from plant cells. started to deform and secondary phloem cells collapsed in The outer bark of a healthy stem compared with an the susceptible ‘Cox’s Orange Pippin’. Fungal hyphae were infected, susceptible stem, and an infected, partially resis- seen in the cortex and in the phloem and xylem cells. At the tant stem of 1-year-old trees is shown in Fig. 4(a–c). A same time point (18 dai), no hyphae were detected in the tangled mass of fungal hyphae was observed under the partially resistant cultivar ‘Santana’. The cortex in both epidermis of the susceptible ‘Cox’s Orange Pippin’ and the cultivars seemed intact while the phloem cells in ‘Cox’s Orange Pippin’ were deformed (Fig. 5a, b). By 29 dai, the cortex and phloem cells had collapsed in both cultivars 3 (Fig. 5c, d). In both cultivars, gel formation could be

2.5 Elise observed in some of the xylem vessels by 29 dai, as either partially or fully occluded vessels (Fig. 5e–f). No signs of Cox 's Orange 2 tylose formation were found. Fungal hyphae were found in all cell types in ‘Cox’s Orange Pippin’ by 29 dai and in Discovery 1.5 ‘Santana’ by 36 dai (data not shown). The shape of xylem and pith cells had not changed by 36 dai in either cultivar. Golden Delicious 1

Mean lesion size (cm) Santana Aroma 0.5 Discussion

0 A highly specific qPCR assay has been developed for -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 detection and quantification of N. ditissima in apple tissue. Relative quantity of N. ditissima gDNA as log 10 The utilization of the b-tubulin region for designing specific primers allowed detection and quantification of N. Fig. 3 Association between the lesion size and the log10 transformed relative quantity of Neonectria ditissima gDNA, r = 0.73, P = 0.09 ditissima gDNA collected from plant tissues using qPCR 123 Trees

Healthy tissue, ‘Santana’ Infected tissue ‘Cox’s Orange Pippin’ Infected tissue, ‘Santana’ (a) (b) (c)

(d) (e) (f)

(g) (h) (k)

(i) (m) (n)

Fig. 4 Transverse section of 1-year-old apple stem stained with observed under the epidermis of both infected cultivars (b, c). Cortex toluidine blue 110 days after inoculation showing the cortex with cells of both infected cultivars were deformed and collapsed parenchyma cells with intercellular spaces (a–c) and vascular tissue compared with uninoculated control (d, e, f). There was noticeably with phloem and cambium cells (d–f), xylem cells (g–k) and pith more mycelium in the phloem and vascular cambium of the cells (i–n). Healthy tissues, ‘Santana’, are shown in a, d, g, and i as a susceptible (e) compared with the resistant (f) cultivar. Fungal comparison. A susceptible, infected cultivar, ‘Cox’s Orange Pippin’, hyphae in rays, vessels and tracheids (g, h, k). Pith cells invaded by is shown in b, e, h, and m and the resistant cultivar ‘Santana’ in c, f, fungal hyphae (m, n). Fungal hyphae are indicated by arrows. Scale k, and n. Fungal hyphae stained purple. A hyphal aggregation that bar a–f 100 and g–n 20 lm possibly represents an early stage of sporodochial formation was and did not amplify DNA from other fungi or apple tissue, The b-tubulin gene region appears to be highly con- thus demonstrating the usefulness of this region and these served within the N. ditissima species in our study, and this primers. gene region has been shown to be reliable for identification

123 Trees

(a) (b)

CX

PM

CM

(c) (d)

CX CX

PM CM PM

CM

(e) (f)

Fig. 5 Infected tissues of detached shoots observed by light c and d. Cortex cells in the susceptible cultivar are deformed microscopy and stained with toluidine blue. Transverse sections of (a) compared with the resistant cultivar (b). Collapsed cortex and the susceptible ‘Cox’s Orange Pippin’ (a, c, e) and resistant ‘Santana’ phloem cells in both cultivars by 29 dai (c, d). Gel formation in some (b, d, f) after 18 dai (a, b) and 29 dai (c–f). Cortex (CX), phloem (PM) xylem vessels are shown in e–f (arrows). Scale bar a–d 100 and e– and cambium (CM) invaded by fungal mycelium are shown in a, f 50 lm of N. ditissima. The most sensitive and efficient b-tubulin reproducibly amplified a 150 bp PCR product from the primer set for detecting N. ditissima gDNA designed in this targeted pathogen, whereas no products were amplified study was Bt-fw135/Bt-rw284. This primer set from any of the other fungal species isolated from apple

123 Trees trees or from isolates representing some other closely Although the Ch1/Ch2 primers adapted to magnetic related fungi. capture hybridization were used to detect N. ditissima in The six cultivars studied in the qPCR assay differed in symptomless tissue (Langrell and Barbara 2001), these their ability to suppress or withstand fungal development as primers were not suitable for use in qPCR due to cross determined by qPCR quantification of fungal biomass as reactions with apple DNA. Another study has recently well as by measurement of lesion size after inoculation demonstrated that N. ditissima can be isolated also from with macroconidia. Quantification of N. ditissima biomass asymptomatic tissue (Amponsah et al. 2014) using selec- in woody tissue of these six cultivars demonstrated that the tive media. However, our qPCR assay did not amplify N. ditissima biomass increased in the order ‘Aroma’, products from gDNA isolated from apparently healthy ‘Santana’, ‘Cox’s Orange Pippin’, ‘Golden Delicious’, tissue surrounding the infected area. ‘Discovery’, ‘Elise’. Similarly, measurement of lesion size To increase the sensitivity of our primers, other indicated that the most resistant cultivar was ‘Aroma’, approaches are required. As previously shown, the appli- followed by, in order of increasing susceptibility, ‘San- cation of SYBR Green primers and TaqMan probes can tana’, ‘Golden Delicious’, ‘Discovery’, ‘Cox’s Orange increase the sensitivity of primers substantially (Garces Pippin’, ‘Elise’. These findings are in agreement with et al. 2014). It has also been reported that the simultaneous previous studies reporting data from artificial inoculations use of two TaqMan probes labeled with two different of detached shoots (Garkava-Gustavsson et al. 2013; reporter dyes can increase the assay sensitivity and repro- Ghasemkhani et al. 2015). More data are, however, needed ducibility at the low detection range (Yip et al. 2005). to obtain significant results and to determine whether this These and other approaches will be attempted in future qPCR assay can be applied to determine relative resistance research to achieve detection of the pathogen in the tissue in different apple cultivars. Garkava-Gustavsson et al. surrounding the infected areas as well as at earlier stages of (2013) did not report any association between the lesion infection. size and N. ditissima biomass but they analyzed only four Two economically important cultivars, ‘Santana’ and different cultivars. Still, the highly susceptible cultivar ‘Cox’s Orange Pippin’, were selected for the histopathol- ‘James Grieve’ showed a higher amount of fungal DNA ogy study based on results from previous inoculations of compared with the remaining three cultivars, including detached shoots (Garkava-Gustavsson et al. 2013), where ‘Golden Delicious’. In this study our samples were taken in ‘Santana’ was significantly more resistant than ‘Cox’s the central part of the necrosis with the same quantity and Orange Pippin’. Our qPCR study supports these earlier quality of sampled tissues while in Garkava-Gustavsson findings. In our study, extracellular and intracellular et al. (2013) the samples were taken at the edge of the hyphae were observed in all cell types, i.e., cortex, phloem, necrosis in distal and apical directions with two necrosis xylem, pith, and ray cells in both cultivars, and in both samples per shoot to assess the variability within the same detached shoots and 1-year-old trees. Hyphae could easily biological replicate. colonize parenchymal cells and apparently moved from Our study presents the first b-tubulin gene-based qPCR cell to cell through the pits in the secondary cell walls in assay that can detect N. ditissima and determine levels of the xylem and primary phloem fibers as also shown by pathogen biomass in artificially inoculated apple cultivars. Crowdy (1949) in ‘Cox’s Orange Pippin’ and ‘Worcester The present findings are consistent with previous research Pearmain’. Zalasky (1968) reported that N. ditissima pro- on the use of qPCR assays to investigate differences duced sporodochia near the surface of the canker, and a between resistant and susceptible cultivars for several putative first step of sporodochia production was observed pathosystems, such as soybean infected by the root fungus also in this study. pathogen Phytophthora sojae (Catal et al. 2013), and Disease progression in inoculated 1-year-old trees was sugarcane infected by the bacterial pathogen Xanthomonas slower than in inoculated detached shoots. In the detached albilineans (Garces et al. 2014). In apple cultivars, Gus- shoot experiment, the partially resistant cultivar resisted berti et al. (2012) used a housekeeping gene-based qPCR the infection longer than the susceptible cultivar. Similarly assay to differentiate between samples that were suscepti- in the tree experiment, the secondary phloem and vascular ble or resistant to the apple scab agent Venturia inaequalis. cambium were more heavily infected in the susceptible The qPCR assay developed here, following further devel- cultivar than in the partially resistant cultivar. However, in opment and validation, could be included in the molecular both cultivars, the parenchyma cells, phloem, and vascular screening component of a breeding program in combina- cambium became deformed and collapsed when infected, tion with the qPCR assay for V. inaequalis to further regardless of the method used. Similar symptoms were increase the advantages of these methodologies over con- observed when Fraxinus mandshurica var. japonica was ventional screening. infected by N. ditissima (Sakamoto et al. 2004), whereas

123 Trees dead and empty cortex cells were seen in advanced infec- inoculation-based resistance of apple cultivars, and could tion of peach bark by N. ditissima (Zalasky 1968). probably become useful also for detection of symptoms in On the opposite side of the necrotic tissue and longitu- the field with an appropriate sampling strategy. The dinally distant from the necrosis, cells were arranged nor- described qPCR-based detection assay is quick, cost-ef- mally. The hyphae were abundant in the necrotic tissues fective, time-saving, and reliable and therefore, meets all but became less frequent further away from the center of criteria mentioned by Sutton et al. (2014) for an effective necrosis similar to infection of Fraxinus by the same fun- diagnostic tool for fruit tree canker caused by N. ditissima. gus (Sakamoto et al. 2004) and of ‘Cox’s Orange Pippin’ However, the method is apparently not sufficiently sensi- and ‘Worcester Pearmain’ (Crowdy 1949). Crowdy tive to detect N. ditissima in asymptomatic tissue. Other (1949) observed that the infection was relatively local and procedures, such as the TaqMan assay should be investi- the fungus was prevalent only in the necrotic areas. gated to improve the sensitivity. The goal of this study was to identify possible anatomical differences between susceptible and partially Author contribution statement MG conducted histopathology experiment, qPCR assay, primer validation, statistical analyses, and resistant cultivars, such as cell wall thickenings, barrier wrote the manuscript. AH designed the primers and supervised qPCR formations or gels, and tyloses around infected areas that assay. SM supervised the histopathology experiment. KD sequenced could at least partly explain partial resistance to infection. ITS region of the isolates. AZ, MR, JRG, and KRE participated in the We found some occasional gel formation in both the validation of primers. JRG, KRE, and RWAS isolated fungal cultures and provided DNA of isolates. The results of qPCR assay were partially resistant and the susceptible cultivar, but a sys- evaluated by MG, AH, LG-G, and AZ. MG, LG-G, AH, RWAS, and tematic barrier formation could not be observed in the SM designed jointly the study in consensus with the other co-authors. infected samples. No tyloses were found in the infected LG-G provided overall coordination of the study. HN, KRE, SM, and cultivars, which is in agreement with a previous study LG-G helped to develop the concept of the paper and assisted in manuscript writing. (Crowdy 1949). This means that the cultivars did not manage to prevent spreading of the hyphae by blocking Acknowledgments Financial assistance was received from Swedish xylem vessels. Additionally, there may have been some Farmer’s organization and Partnership Alnarp to Larisa Garkava- cell wall thickening in the pith cells in both cultivars but Gustavsson, from the Nordic Ministries of Food and Agriculture there was also some variation in cell wall thickness through the Nordic collaboration on Public–Private Partnership for pre-breeding, administered by NordGen, to Hilde Nybom and the depending on the position of the cells in the pith. Thus, New Zealand Institute for Plant & Food Research Ltd. The authors no major anatomical differences were detected between gratefully acknowledge the help of Kerstin Brismar with microscopy the susceptible and the partially resistant cultivar. In our samples, Prof. Erland Liljeroth for suggestions with regard to the study, the observed difference between the two cultivars isolation of Neonectria ditissima and useful discussions, Marlene Jaspers and Margaret Dick for providing LUPP and NZFS isolates was probably caused by effects on pathogen colonization, and Helle Turesson for help with qPCR analyses. e.g., release of inhibitors such as phytoalexins, low molecular weight antimicrobial compounds that can act as Compliance with ethical standards toxins to pathogens. Degree of host resistance and the Conflict of interest No potential conflict of interest was reported by level of phytoalexin accumulation are often strongly the authors. correlated (Hall et al. 2011). Cell cultures of the apple scab-resistant cultivar ‘Liberty’ produced malusfuran, References which is toxic to Venturia inaequalis at millimolar con- centrations (Hrazdina et al. 1997). 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