Sensing Disease in Apple Trees: Potential Signature Compounds for European Canker (Neonectria ditissima) Infection
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Sensing Disease in Apple Trees: Potential Signature Compounds for European Canker (Neonectria ditissima) Infection
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  • Catherine E. Sansom
    Catherine E. Sansom
    The New Zealand Institute for Plant and Food Research Limited, Department of Chemistry, University of Otago, Dunedin 9054, New Zealand
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  • Elaine J. Burgess
    Elaine J. Burgess
    The New Zealand Institute for Plant and Food Research Limited, Department of Chemistry, University of Otago, Dunedin 9054, New Zealand
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  • Lesley Larsen
    Lesley Larsen
    The New Zealand Institute for Plant and Food Research Limited, Department of Chemistry, University of Otago, Dunedin 9054, New Zealand
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  • Nigel I. Joyce
    Nigel I. Joyce
    The New Zealand Institute for Plant and Food Research Limited, Canterbury Agriculture and Science Centre, Lincoln 7608, New Zealand
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  • Peter Jaksons
    Peter Jaksons
    The New Zealand Institute for Plant and Food Research Limited, Canterbury Agriculture and Science Centre, Lincoln 7608, New Zealand
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  • Rebecca E. Campbell
    Rebecca E. Campbell
    The New Zealand Institute for Plant and Food Research Limited, Brooklyn, Motueka 7198, New Zealand
    More by Rebecca E. Campbell
  • Ria S. Rebstock
    Ria S. Rebstock
    The New Zealand Institute for Plant and Food Research Limited, Mt Albert, Auckland 1025, New Zealand
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  • Ian C. Hallett
    Ian C. Hallett
    The New Zealand Institute for Plant and Food Research Limited, Mt Albert, Auckland 1025, New Zealand
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  • Jonathan Rees-George
    Jonathan Rees-George
    The New Zealand Institute for Plant and Food Research Limited, Mt Albert, Auckland 1025, New Zealand
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  • Kerry R. Everett
    Kerry R. Everett
    The New Zealand Institute for Plant and Food Research Limited, Mt Albert, Auckland 1025, New Zealand
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  • Reiny W. A. Scheper
    Reiny W. A. Scheper
    The New Zealand Institute for Plant and Food Research Limited, Havelock North, Hawke’s Bay 4130, New Zealand
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  • Monika Walter
    Monika Walter
    The New Zealand Institute for Plant and Food Research Limited, 412 No 1 Road, RD 2, Te Puke 3182, New Zealand
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  • Nigel B. Perry*
    Nigel B. Perry
    The New Zealand Institute for Plant and Food Research Limited, Department of Chemistry, University of Otago, Dunedin 9054, New Zealand
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0003-3196-3945
Open PDFSupporting Information (1)

Journal of Agricultural and Food Chemistry

Cite this: J. Agric. Food Chem. 2025, 73, 24, 15003–15013
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https://doi.org/10.1021/acs.jafc.4c10597
Published June 10, 2025
Copyright © 2025 The Authors. Published by American Chemical Society
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Abstract

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Neonectria ditissima is the fungal cause of costly European Canker (EC) in apple trees. A range of secondary metabolites were found at higher concentrations in infected twigs than in disease free twigs. Apple trees were then experimentally inoculated with N. ditissima and analyzed periodically until EC symptoms were visible at 12–13 weeks post-inoculation. Established destructive detection methods used were microscopy, which showed extensive hyphal penetration by 8 weeks post inoculation, and qPCR analyses, which confirmed the presence of N. ditissima. Headspace solid-phase microextraction GC-MS data showed significantly higher concentrations of styrene in apple twigs at six weeks after inoculation, and LC-MS data showed phloretin, triterpene acids, and 1-benzoyl β-d-glucose at raised concentrations after this time. Therefore, these compounds could be useful indicators of N. ditissima infection prior to visible canker formation, suitable for nondestructive disease detection development after further research on apple variety, pathogen specificity, and on field detection technology.

Copyright © 2025 The Authors. Published by American Chemical Society

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Introduction

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European Canker (EC) is caused by the fungus Neonectria ditissima (Tul. & C. Tul) Samuels and Rossman (syn. Neonectria galligena, Nectria galligena). This pathogen has a wide host range (1,2) and its nomenclature has varied over time, hosts and continents. (3,4) It is an important pathogen in many apple (Malus spp., Rosaceae) growing regions around the world. (5−7) The fungus can infect wounds all year round whenever spores are present and weather conditions are suitable. (8,9) Wood age, (10) wound age, (11,12) wound size and conidia concentration (13) all affect the likelihood of infection and the incubation period (time from infection to symptom development). As few as three spores are needed for successful infections with incubation periods of weeks to years. (13,14) EC can severely affect newly planted apple orchards because it is possible for symptomless trees with latent infections to be planted. (14) Trees can then develop canker lesions up to three years later, (14) depending on temperature and rainfall frequency, (15) becoming an infection source in the new orchard. There is no cure for EC so prevention is important: wounds need to be protected with pruning paints or fungicides, and inoculum reduced by removing infected/dead branches or even entire trees. (16−20)
Early detection of asymptomatic N. ditissima infection would be a great advance for the worldwide apple industry, particularly the nursery sector. (18,21) Currently the best methods for asymptomatic detection of N. ditissima in apple trees are quantitative polymerase chain reaction (qPCR) (22) or isolation onto low-nutrient agar, (13) used in The Netherlands, New Zealand and other countries. We propose that another approach for rapid, ideally nondestructive, detection of EC in apple trees might be discovering small (molecular weight <1000 Da) signature compounds (SCs) produced during/from infection. These SCs could then be detected before planting out into orchards, e.g. in nursery rootstocks, scion wood and young trees. This “sensing disease” approach not been applied to detection of EC in apple trees, but has been used for Huanglingbong (HLB) citrus greening, with various classes of volatile and nonvolatile compounds found in HLB infected citrus leaves and fruits. (23,24) A recent report used portable gas chromatography–mass spectrometry (GC-MS) for field detection of HLB SCs, using headspace solid-phase microextraction (HS-SPME) sampling of citrus leaves. (25)
There are few reports of N. ditissima metabolites, or of metabolites in EC infected apple trees. An early paper reported benzoic acid produced in apple fruits in response to EC infection. (26) Since then, bioactive metabolites from liquid cultures of N. ditissima have been described, including terpenes and a polyketide. (27) Another study (abstract with limited detail and growing conditions not specified) reports 60 compounds in the fungal body and 45 secreted metabolites from Nectria (species not stated) using GC-MS. (28)
The objective of the current study was to use metabolomic analyses to detect possible SCs from EC-infected apple trees. We then did a time course study using chemical analyses of EC infected samples to determine whether these possible SCs were detectable before visual symptoms developed, comparing with PCR and microscopy detection methods.

Materials and Methods

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Apple Twig Samples for Signature Compound Discovery

One-year old ‘Royal Gala’ and ‘Scifresh’ trees on ‘M9’ rootstock, were sourced from The New Zealand Institute for Plant &and Food Research Ltd. (PFR) Hawke’s Bay, New Zealand (NZ) site and planted on 28 August 2012 at PFR Whakarewa orchard, Motueka, Tasman, NZ. Leaf scar wounds at twig nodes were made by gently removing an autumn-colored leaf on 28 May 2013 or 6 June 2013 (Figure 1A). (16) Four twigs (five leaf scars per twig) were inoculated per tree per inoculation time, with up to 10 trees for each cultivar. Inoculation of leaf scars with spore suspension produced from field-collected lesions, identified as N. ditissima by one of us (MW), was done as described previously: (16) 20–30 μL of N. ditissima spore suspension (2 × 105 conidia/mL) brushed onto the freshly made leaf scar using a Pasteur pipet fitted with a small camel hair brush. Inoculated twigs were marked with flagging tape and leaf scars with paint pen ∼1 cm below the node. Twigs that had developed visible EC infection (Figure 1B) were harvested for HS-SPME-GC-MS and LC-MS analyses on 30 September 2013:12 twigs from seven ‘Royal Gala’ plants and seven twigs from four ‘Scifresh’ plants. Five disease free control twigs from uninoculated, EC-free, potted 1-year-old ‘Royal Gala’ plants without branches, produced in a disease-free environment in a glasshouse at PFR Hawke’s Bay, were harvested for analysis on 30 September 2013.

Figure 1

Figure 1. (A) Apple twig leaf scar, with leaf bud above (image by Tony Corbett, PFR). (B) Apple twig with visible European Canker infection. (C) Cross-section of infected apple twig leaf scar showing Neonectria ditissima symptoms (brown tissue at the top).

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Apple Twig Samples for the Time Course Experiment

Three-year old ‘Royal Gala’ apple trees growing in the Whakarewa research orchard were inoculated as above on 27 May 2014. Five fresh leaf scars (after removal of autumn-colored leaves) on each of four twigs on each of 25 trees, were inoculated by applying 1 × 105 conidia/mL EC spore suspension in a 20–30 μL water droplet with a Pasteur pipet equipped with a camel hair brush. Nontreated control samples (two twigs on each of the same 25 trees) were prepared by removing leaves to form leaf scars as above, but not inoculated. Shoots and leaf scars were also identified with flagging tape and paint pen, respectively, using different colors between inoculated and uninoculated treatments.
The first samples were collected immediately after inoculation, then after 1, 2, 3, 4, 6, 8, 10, 12, and 13 weeks. At each sample time, three control and seven inoculated twigs were collected from trees at random: one control and three inoculated twigs were analyzed by HS-SPME-GC-MS and LC-MS; one control and two inoculated twigs were analyzed using microscopy; and one control and two inoculated shoots were analyzed using qPCR. Each shoot was uniquely labeled, placed in a clean clip-seal bag and shipped (overnight) for chemical analyses, qPCR and microscopy. If any other leaves remained on the shoot, these were removed at sampling time before bagging.

HS-SPME-GC-MS Analyses

All SPME vials were silanized. (29) Twig nodes were freshly ground using a rasp (cleaned between twigs), weighed into 20 mL SPME vials, sealed and stored for up to 7 h at ambient temperature on the autosampler until analysis. Subsamples were stored at −20 °C for LC-MS analyses (see below).
A test solution containing hexanal, hexanol and cis-3-hexen-1-ol (green leaf volatiles, GLVs), β-caryophyllene and germacrene D (sesquiterpenes) and ethyl hexanoate, previously reported from apple twigs (30−32) was used to optimize the analytical conditions and sample size. A 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco, Australia) was conditioned at 270 °C for 60 min before first use and for 30 min at the beginning of each day. The sample was equilibrated at 70 °C for 1 min followed by fiber exposure to headspace for 10 min then desorption immediately in the GC injector at 230 °C.
For GC an Agilent 7890A gas chromatograph with a CTC Analytics PAL system autosampler and an Agilent 5975C inert XL mass sensitive detector (MSD) with triple axis detector (under the control of Enhanced ChemStation software) was used. The injection, at 230 °C, was splitless with purging at 50 mL/min after 1 min. Injections (SPME fiber desorption for 5 min) were made onto a 30 m Agilent HP5 ms column with a 0.25 mm internal diameter (ID) and 0.25 μm film, with H2 carrier gas at 1.5 mL/min. This flow was split using deactivated silica columns between MSD (0.5 m long × 0.1 mm ID) and flame ionization detector (2 m × 0.18 mm ID). The GC oven was heated from 40 to 240 °C at 20 °C/min. Detection was by total ion current mass spectrometry (MS transfer line 270 °C, source 230 °C, quad 150 °C) over 35–350 Da with data collected every 0.05 s.
Total ion current data from the mass spectrometry detector were exported into MS Excel, sorted and corrected for sample weight. Principal components analysis (PCA) was performed using The Unscrambler software (https://www.pharmaceutical-technology.com/products/unscrambler-x/).

LC-MS Analyses

For the Signature Compound Discovery experiment, duplicate nodes from each twig were cut out as ∼30 mm sections, which were frozen to −80 °C before freeze-drying. Dried nodes were then powdered with a wood rasp, weighed and extracted with 1:1 MeOH:water (1 mL solvent per 100 mg sample) by vortex mixing, soaking overnight at 4 °C, and vortex mixing again. The supernatant extract was recovered by centrifuging, then filtration through a 0.22 μm nylon syringe filter into 12 × 32 mm amber glass vials with PTFE/red rubber septa screw caps.
For the Time Course experiment, frozen rasped samples (subsamples from HS-SPME-GC-MS preparation above) were freeze-dried then stored at −20 °C until analyzed. Subsamples (∼50 mg) in 15 mL Greiner Tubes were extracted with MeOH (1 mL per 50 mg sample) by vortex mixing for 2 h, soaking overnight at 4 °C, and vortex mixing again. The supernatant extract was recovered by centrifuging, then 400 μL of each extract was filtered with a Single Step vial 0.22 μm PVDF filter (Thompson Part No. 65531-200).
For LC, a Thermo Electron Corporation (San Jose, CA, USA) Accela UHPLC pump, Thermo Accela Open Auto sampler (PAL HTC-xt with DLW), Finnigan Surveyor PDA plus detector and a ThermaSphere TS-130 column heater (Phenomenex, Torrance, CA, USA) were used. Each extract was analyzed by two LC methods with two ion formation modes.
An aliquot (2 μL) was analyzed by reverse phase LC using a Kinetex C18 column (2.6 μ, 100 Å, 100 × 2.1 mm, Phenomenex, Torrance, CA, USA) at 30 °C with a flow rate of 250 μL/min. The mobile phase was mixed from 0.1% formic acid in water (mix A) and 0.1% formic acid in acetonitrile (mix B): 0–1 min at 95% A, linear mixing to 0% A at 7 min, 7–10 min at 0% A, linear mixing to 95% A at 11 min, 11–16 min at 95% A.
An aliquot (2 μL) was analyzed by normal phase LC using a zwitterionic ZIC-HILIC stationary phase (3.5 μm, 150 mm × 2.1 mm i.d.; Merck SeQuant, Umea,Sweden, guard HILIC GRACE) at 30 °C with a flow rate of 300 μL/min. The mobile phase was mixed from 0.1% formic acid in acetonitrile (A) and 0.1% formic acid with 5 mM ammonium acetate in water (B): 0–1 min at 95% A, linear mixing to 5% A at 10 min, 10–15 min at 5% A, linear mixing to 95% A at 16 min, 16–20 min at 95% A.
Eluents from each chromatography method were scanned by photodiode array (PDA, 200–600 nm) and electrospray ionization (ESI) MS (LTQ, 2D linear ion-trap, Thermo-Finnigan, San Jose, CA, USA) in negative and positive ion modes. Data were acquired for precursor masses from m/z 110–2000 amu with up to MS3 product spectral tree formation.
Data were processed with the aid of Xcalibur2.20 (Thermo Electron Corporation) and Mass Frontier 7 SR1 (Thermo Electron Corporation), XCMS online (https://xcmsonline.scripps.edu/) for data filtering and statistical feature analysis, Lipid Maps (http://www.lipidmaps.org/), Mass Bank (http://www.massbank.jp) and an in-house PFR database for spectral identification. XCMS online filtering parameters were: step: 0.1; fwhm: 30; profStep: 1; mzwid: 0.05; minfrac: 0.5; bw: 5; Welch t test, posthoc, paired; p-value threshold high: < 0.01, low 0.05; fold change threshold: 1.5; EIC width, seconds: 200.

Microscopy

Leaf scars from 0, 1, 2, 3, 4, 6, 8, 10, 12, and 13 weeks post inoculation were collected for microscopy. One leaf scar was investigated for early time points (0–3 weeks), while four leaf scars were investigated for the six later time points. Leaf scars were sampled by cutting away the twig immediately above the leaf scar (Figure 1C), and then ∼10 mm below the leaf scar. This material was then immersed in a fixative comprising 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and briefly subjected to vacuum, then stored at 4 °C until further processed.
To prepare leaf scars for optical microscopy, a longitudinal hand cut slice was taken from the center of each leaf scar and then divided up into smaller pieces suitable for resin embedding. Material was washed in 0.1 M phosphate buffer and dehydrated in an ethanol series (30–100%), then embedded in LR White resin (London Resin, Reading, UK). (33) Sections (1 μm) were cut using a UCT ultramicrotome (Leica Microscopy Systems Ltd., Heerbrugg, Switzerland), mounted on poly-l-lysine coated slides, dried on a hot plate at 45–50 °C and left overnight. Fluorescence immunolabeling to identify fungal cell walls was carried out using a primary antibody to (1 → 3)-ß-glucan diluted 1:100 in 0.1% bovine serum albumin (BSA-C; Aurion, Wageningen, The Netherlands) and a secondary antibody conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, Oregon, USA), diluted 1:600 in PBS using established techniques. (33) Sections were viewed using a Vanox AHBT3 compound microscope (Olympus Optical Co Ltd., Tokyo, Japan), using an interference blue filter (excitation 495 nm, dichroic mirror 505 nm and excitation ≥ 515 nm). All images were captured using a CoolSnap color digital camera (Roper Scientific Ltd., Tucson, Arizona).
Scanning electron microscopy was carried out following methods by Hallett et al. (34) and samples were imaged with a Quanta 250 scanning electron microscope (FEI Company, Hillsboro, Oregon).

qPCR Analyses

Twigs sampled 6, 8, 10, 12, and 13 weeks after inoculation were surface sterilized by immersion in 70% ethanol for 1 min, washed with tap water for 3 min, then surface water was evaporated by placement on sterile paper tissues in a laminar flow cabinet. Five 2 mm thick discs were cut from twig segments across each of the inoculated or control leaf scars and each disc cut into quarters. DNA was extracted from one-quarter of each of the five discs that included the leaf scar.
These dissected quarters were chopped with a scalpel then placed in 2 mL Eppendorf safelock tubes with one 7 mm stainless steel bead and cooled overnight at −80 °C. Samples were disrupted in Tissue Lyser II (Qiagen) for two runs at 30 Hz for 30 s, stored at −80 °C, then processed in batches of 24 using an M-N Nucleospin Plant II DNA extraction kit. Resultant DNA was diluted (1:5 v/v) with Gibco water and aliquoted to a 48 well microtiter plate and analyzed using an ECO qPCR machine. PCR primers F179/R306 designed to detect N. ditissima were used. (35) Reaction conditions were one cycle at 98 °C for 2 min, 45 cycles of 98 °C for 5 s and 60 °C for 5 s, followed by one cycle of 95 °C for 15 s, 55 °C for 15 s and 95 °C for 15 s. Each qPCR plate had a dilution series of N. ditissima DNA and three nontemplate controls (water). One uninoculated control twig was processed from the week 13 harvest. N. ditissima was regarded as detected when the Ct (threshold cycle) value was less than 35 with a diagnostic Tm (melting temperature) of 84.1–84.4 °C.

Statistical Analyses

A mixed model was fitted to test for the difference in GC- and LC-MS compound peak areas between twigs inoculated with N. ditissima and uninoculated control twigs in the time course experiment. Time, treatment (inoculation/control) and the interaction between treatment and time were included in the model as fixed effects. Twig was included as a random effect and a heterogeneous variance at each time point was estimated. Because most peaks were below the detection limits for both the controls and the inoculated twigs, the model was refitted using data from week 4 onward. Model convergence was achieved, but residual plots indicated that both the assumption of constant variability as well as normality distribution of the residuals were likely violated, perhaps because an unknown number of twigs that were inoculated with EC never developed the disease. This caused a wide range in peak values for the inoculated twigs which made it very difficult fit an appropriate model, so care should be taken when interpreting p-values as these could be less accurate. The p-values were obtained by the conditional Wald test, (36) for the interaction between time and treatment.
The results per volatile compound were visualized by the raw data per time point, colored per treatment. Additionally, a local polynomial spline curve was added, together with 95% error bounds per treatment type.

Compound Synthesis for Identification

1-Benzoyl β-d-glucose was prepared by a published method. (37)

Compound Isolation for Identification: Triterpene Acids and Phloridzin

The N. ditissima inoculated leaf scars of the ‘Royal Gala’ apple twigs were ground into <4 mm pieces before freeze-drying followed by extraction in MeOH (2 × 100 g in 1 L). The two extracts were filtered (Whatman No 1 paper) and combined, then a dried subsample (1.2 g) was preabsorbed onto C18 silica before separating over C18 silica eluting with water then 20, 50, 80 and 100% MeOH.
Triterpenes (200 mg) eluted with 80–100% MeOH were further separated by silica gel column chromatography eluting with petroleum ether and ethyl acetate. A fraction containing mainly one compound eluted with 40% ethyl acetate was purified using preparative LC (see below) to give 2-oxopomolic acid (Registry Number [54963-52-9]): retention time (RT) with 90% MeCN 4.2 min, NMR matching published data. (38) A second fraction eluting with 60% ethyl acetate was purified by LC to give pomaceic acid [2243304-67-6]: RT 5.4 min with 80% MeCN, NMR matching published data. (39)
Phloridzin was eluted from the first C18 column with 50% MeOH and was identified by NMR. (40)

Preparative HPLC

An Agilent HP1260, controlled with Agilent OpenLab software was used, with a C18 column (Phenomenex Luna C18(2) 250 × 10 mm) with a 10 × 4 mm C18 guard column, at 30 °C. Peaks were detected at 206 nm. The mobile phase was isocratic 80 or 90% acetonitrile in water, both with 0.1% formic acid. The flow rate was 5.0 mL/min, with injection volumes of 100 μL.

Results and Discussion

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Potential signature compounds (SCs) were first sought by comparing volatile and nonvolatile compounds from healthy apple twigs with compounds from twigs visibly infected with N. ditissima. We then monitored the time course of production of these potential SCs by apple twigs inoculated with N. ditissima, from the time of inoculation until EC symptoms were visible.

Potential Volatile Signature Compounds

Infected and healthy control (uninoculated) twigs of two varieties of apple, ‘Royal Gala’ and ‘Scifresh’, were analyzed by HS-SPME-GC-MS. Initial HS-SPME-GC-MS analyses (Supporting Information Figure S1) showed the presence of both green leaf volatiles (GLV) and sesquiterpenes in all apple twigs. Four common types of SPME fibers (41) were tested for absorption efficiency for these compound classes. Polydimethylsiloxane (PDMS) fibers were good for sesquiterpenes and polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibers were good for GLV, but neither absorbed both classes of volatiles well simultaneously. Fibers with divinylbenzene/carboxen on polydimethylsiloxane (DVB/CAR/PDMS) and with carboxen/polydimethylsiloxane (CAR/PDMS) did absorb both types of compounds together, but DVB/CAR/PDMS fibers were chosen as being easiest to clean (by heating in a second injector) between samples.
PCA of GC data (Figure 2A) showed all the control samples clustered together regardless of their apple variety or growing region. The EC infected twig samples were mostly separated from the control samples along PC1, which accounted for 71% of the total variation. PC1 was positively loaded for the peaks of GLVs, ethylbenzene and styrene, and negatively loaded for sesquiterpene peaks (Figure 2B).

Figure 2

Figure 2. (A) Two-dimensional projection of principal components analysis of headspace solid-phase micro-extraction GC-MS data of apple twigs showing the variability between different treatments. Red triangles = ‘Scifresh’ inoculated, purple squares = ‘Scifresh’ control, green triangles = ‘Royal Gala’ inoculated, blue squares = ‘Royal Gala’ control from Motueka, light blue squares = ‘Royal Gala’ control from Hawke’s Bay. (B) Loading plot for PC-1, showing positive loadings corresponding to peaks for hexanol and styrene, and negative loadings for sesquiterpenes. The x-axis numbers correspond to GC retention time (min).

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The ethylbenzene, styrene (Figure 3) and GLV peaks were identified using pure standards analyzed by GC-MS. However, HS-SPME-GC-MS analyses of these same pure standards showed that the DVB/CAR/PDMS fibers used with H2 carrier gas gave previously unprecedented alkene hydrogenations. (42) Hexanol was produced from cis-3-hexen-1-ol and ethylbenzene from styrene. HS-SPME-GC-MS analyses of cis-3-hexen-1-ol and styrene standards with DVB/CAR/PDMS fibers and He carrier gas showed no hexanol or ethylbenzene. We carried on using DVB/CAR/PDMS fibers with H2 carrier gas as this was the most sensitive combination for the HS-SPME-GC-MS analyses of the time course experiment reported below. Ethylbenzene and styrene had approximately the same MS detector response so the total of these two peaks is given as styrene in the results below.

Figure 3

Figure 3. Structures of potential signature compounds for Neonectria ditissima infection of apple twigs.

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The few previous papers on the volatiles of apple tree twigs (30−32) and of N. ditissima. (26−28) did not report styrene. However, styrene has been reported from stored apple fruits as a potential noninvasive biomarker to track shelf life and nutritional changes. (43) We could not find any reports of antifungal activity of styrene, but this simple volatile (Figure 3) is an active component in leaves of pear trees (Pyrus spp.) resistant to damaging insects. (44) Therefore, styrene was a possible volatile SC for N. ditissima infection.

Potential Nonvolatile Signature Compounds

Infected and control twigs were solvent extracted and analyzed by a general rapid LC-MS screening method to obtain a large amount of data within a short time frame for statistical analysis. Untargeted metabolomics data were processed by comparing uninfected controls with infected apple twig extracts based on split groupings of variety (‘Royal Gala’ vs ‘Scifresh’), chromatography (Reverse Phase vs Normal Phase LC) and ion formation technique (positive vs negative), producing eight sets of results. This protocol allowed for a wide range of solubilities and polarities of small and large molecules to be considered as possible SCs.
LC-MS data sets were first processed by untargeted statistical analysis based on LC retention time, pseudomolecular ion m/z and ion intensity utilizing XCMS online (see details above). Discriminant analysis data plots indicated up and down regulation of compounds in infected twigs relative to control twigs with ions showing >5 log2fold-change most likely to be from suitable SCs (e.g., Figure 4). Triterpene acids (TAs) and a benzoic acid glycoside were at higher concentrations in infected twigs, while concentrations of the typical apple dihydrochalcone phloretin and of citric acid were markedly lower in infected twigs (Supporting Information Table S1). The major compounds differentiating infected twigs from control twigs were generally the same for both apple varieties.

Figure 4

Figure 4. LC-MS differential plots for all component mass features that changed between infected and control ‘Royal Gala’ apple twigs by reverse phase liquid chromatography with negative ion (A) and positive ion (B) MS. Plot x-axis log2fold change (negative values indicate down regulation and positive values up regulation due to infection); y-axis ion species mass features as m/z (compounds represented by one or more signal ions M, 2M, M+ adduct, etc.); marker dimension is the maximum area response for an ion species mass feature; color represents Welch’s t-stat as negative (red, down regulation) with a gradient through zero (yellow) to positive values (dark green, up regulation).

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Triterpene acids (TAs), tentatively identified as derivatives of ursolic acids, were upregulated in infected woody tissue (Table S1). Triterpenes in apple fruit have been previously identified by NMR spectroscopy (45−47) and the triterpene acids in skins of several NZ apple varieties have been determined by LC-MS. (48,49) The tentative identifications in Table S1 are based on MS characteristics shown by these reported compounds: ursane triterpenes typically show neutral losses from their parent ions: M-18 (H2O), −36 (H2O)2, −44 (CO2), −46 (HCO2H), −64 (HCO2H, H2O), −82 (HCO2H, (H2O)2).
Two of the main ursane TAs were isolated from a MeOH extract of N. ditissima infected ‘Royal Gala’ apple twigs, purified by several steps of reversed phase C18 and silica gel chromatography. High resolution ESI-MS of the major compound supported the molecular formula C30H46O5 and the NMR spectra matched those reported for 2-oxopomolic acid (Figure 3). (45) The second TA isolated from infected apple twigs had one more oxygen than 2-oxopomolic acid i.e. C30H46O6. 13C NMR spectra showed no ketone signal, but there was a quaternary acetal signal (99.1 ppm) and a C–CH2–O signal instead of one of the C–CH3 signals of 2-oxopomolic acid. Searching the Chemical Abstracts using molecular formula and NMR criteria retrieved pomaceic acid (Figure 3) with matching NMR data. (39) Our 2D NMR data also supported this structure, which has only been reported twice before: from apple pomace (39) and from apple peels. (50)
The previous literature on TAs shows that these potential SCs could be phytoalexins i.e. antifungal components produced by plant tissue in response to N. ditissima infection. We could only find one report of a TA as an apple phytoalexin: 2-oxopomolic acid as induced in the wood of Malus pumila Mill. infected with Chondrostereum purpureum. (38) C. purpureum is a fungal pathogen that causes silverleaf of trees in the Rosaceae, and 2-oxopomolic acid inhibited its growth in vitro. (38) There are several reports of TAs as phytoalexins from other crops, all oxygenated derivatives of either ursolic or oleanolic acids. (51−54) For example, arjunolic acid was found in cocoa stems after inoculation with wilt-causing Verticillium dahlia, (51) first appearing 3 days after infection and subsequently present at concentrations well above those required for fungal toxicity. (51)
LC-MS data showed significantly higher concentrations (62 fold, Table S1) of a benzoic acid glycoside in N. ditissima infected apple twigs compared to controls. MS showed a formic adduct ion with m/z 329 [M-H+46]− and product ions including hexose with m/z 121. The identity of this compound as 1-benzoyl β-d-glucose (Figure 3) was shown by synthesis and LC-MS matching. 1-Benzoyl β-d-glucose has been found in peels of “scalded” apple fruits damaged by storage (55) and in undamaged apple leaves. (56) This compound was up-regulated in tobacco plants in response to a fungal elicitor. (57)
Phloridzin, a characteristic apple secondary metabolite, (58) and its aglycone dihydrochalcone phloretin (Figure 3) were both detected in all the apple twig extracts and were found at lower concentrations in N. ditissima infected twigs (Table S1) than in controls. The phloridzin LC-MS peak was confirmed by purifying this compound from apple twigs and checking its NMR spectra. (40) Phloridzin has been found to be important in pathogen resistance but its concentration has not always correlated to disease resistance. (58,59)
The primary metabolite, citric acid, was at lower concentration in N. ditissima infected woody tissue. This seems to be a new finding, but we did find one reference to citric acid and fungal infection, on pH changes in apple fruits with Penicillium infection. (60)

Time Course of Neonectria ditissima Infection and Production of Potential Signature Compounds

Two methods are currently used for the presymptomatic detection of EC infection due to N. ditissima: detection of fungal DNA by qPCR; (22) and microscopy to visually detect the presence of hyphae within the tissue either by staining or fluorescence labeling of (1,3)-β-glucans in fungal cell walls. (61) The aim of this time course experiment was to determine whether the proposed SCs (above) could be detected before visible symptoms, comparing with qPCR and microscopy techniques.
‘Royal Gala’ apple trees growing in an orchard were N. ditissima inoculated at leaf scars in the autumn, with controls sampled at the same time. Three batches of twigs were taken immediately and after 1, 2, 3, 4, 6, 8, 10, 12, and 13 weeks for qPCR, microscopy and chemistry analyses. Note that separate twigs were examined by the three separate techniques.
No external infection symptoms were seen until 12 weeks, when minor symptoms were visible on five of the 15 nodes sent for chemistry analyses, with only one of the three inoculated twigs free of symptoms. At 13 weeks, symptoms of EC infection were visible on all three inoculated twigs, at 11 of the 15 nodes. Of the 32 inoculated twigs remaining after the week 13 sampling, 94% of the inoculated nodes on these had visible symptoms and 81% of these twigs had all five inoculated sites with visible symptoms. This symptom development was much faster than reported after N. ditissima inoculation of leaf scar wounds in past experiments. For example, in nonurea treated ‘Royal Gala’, ‘Braeburn’ and ‘Scifresh’ less than 10% of leaf scars developed EC symptoms within nine months of inoculation, whereas in urea treated plants over 30% of inoculated leaf scars showed disease in this time (62) (elevated nitrogen not only increased apple tree growth but also the growth rate of N. ditissima cankers (63)).
Inoculated twigs were analyzed using fluorescence immunolabeling to determine the spread of hyphae penetration from the inoculation site. Before week 4, hyphae were only observed on the surface of the leaf scar, or to a depth of no more than 100 μm. Four weeks post inoculation, hyphae were observed in three of the four samples and were recorded up to a depth of 0.25 mm from the leaf scar (Figure 5 and Table 1). Hyphal penetration was slow between 4 and 8 weeks post inoculation, with hyphal maximum depth of 0.35 mm by week 6 and 0.75 mm by week 8 (average depths 0.27 mm and 0.47 mm respectively). By 10 weeks post inoculation, hyphae had grown more into the woody tissue, with an average depth of 1.6 mm from the leaf scar and a maximum depth of nearly 2 mm. Week 12 was the first time that hyphae were recorded in all samples, although the penetration was not extensive in one sample (0.3 mm). At 13 weeks post inoculation, hyphal penetration was extensive, ranging between 2.7 and 6 mm in all samples (Figure 5 and Table 1), by which time EC symptoms, such as browning of the woody tissue (Figure 1), were clearly visible.

Figure 5

Figure 5. Fluorescence immunolabeling of fungal hyphae (green) using a (1,3)-β-glucan antibody in tissue sections of inoculated apple twigs. (A) Four weeks post-inoculation: hyphae are mainly visible on the outside of the apple leaf scar but some are showing penetration into the woody tissue (arrows). (B) Thirteen weeks post-inoculation: large areas of hyphal infection inside the woody apple tissue (stars).

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Table 1. Maximum Distances (mm) of Fungal Hyphae Were Recorded from Inoculated Apple Leaf Scars (n = 4 per sampling week), Determined by Fluorescence Microscopy to Detect (1,3)-β-Glucan in Fungal Cell Walls and Scanning Electron Microscopy
weeks post-inoculationmean (mm)range (mm)
40.230–0.25
60.270–0.35
80.470–0.75
101.580–1.95
121.580.3–3
133.032.75–6
As the (1,3)-β-glucan detected by this fluorescence immunolabeling is a component of many fungal cell walls, the presence of N. ditissima was confirmed by PCR analyses. Six weeks after inoculation N. ditissima was only detected in tissue at inoculation sites. The extent of detectable N. ditissima DNA steadily increased with time, with some detection at 8–10 mm from inoculation sites after 13 weeks (Table 2). No N. ditissima DNA was detected from control samples.
Table 2. Numbers of Twig Samples (Out of Five That Are Analyzed at Each Date) in Which Neonectria ditissima Was Detected By qPCR Using F179/R306 Primers
 distance from inoculation site (mm)
weeks post-inoculation0–22–44–66–88–10
650000
852100
1054000
1254410
1354522
13 uninoculated control00000
Therefore, both fluorescence immunolabeling microscopy and qPCR methods showed slight infection in most samples after 6 weeks, but 100% detection of N. ditissima infection away from the inoculation site (0–2 mm) was not achieved until 12 weeks post inoculation, at which stage there were also visible EC symptoms on some samples.
Sixteen volatile compounds were analyzed by HS-SPME-GC-MS in apple twig leaf nodes in this time course experiment. Styrene (Figure 3) was the main volatile compound that showed significant increases (p < 0.001, Wald test) with time from inoculation compared with the controls (Figure 6). The styrene signal from the inoculated samples was significantly elevated compared to the control samples from week 6, 6 weeks before visual symptoms were observed. Significantincreases with time from inoculation were also observed for 4-ethylphenol (p = 0.019) (Supporting Information Figure S2) and benzaldehyde (p = 0.004) (Figure S3) but their maximum signal intensities were much lower than for styrene. 4-Ethylphenol is assumed to be a hydrogenation artifact from the natural product p-hydroxystyrene (Figure 3). Both p-hydroxystyrene and styrene are active components in leaves of pear (Pyrus spp.) varieties resistant to damaging insects. (44) The same authors proposed a biosynthetic pathway to these volatiles, and isolated and characterized a pear phenolic acid decarboxylase which catalyzed the decarboxylation of p-coumaric acid to p-hydroxystyrene. (44) Styrene and 4-ethylphenol signal intensities across the apple twig node samples were positively correlated (R2 = 0.70) suggesting related formation pathways, but styrene and benzaldehyde signals correlated less strongly (R2 = 0.46).

Figure 6

Figure 6. Styrene signals (by HS-SPME-GC-MS) from leaf nodes (ground) of apple twigs inoculated with Neonectria ditissima (at zero weeks) and uninoculated control leaf nodes of apple twigs. Peak values are rescaled to values between zero and one by dividing each peak area by the maximum observed peak area for this compound. A local polynomial spline curve was fitted through the data, with 95% error bounds giving an indication of the time point after which the control and inoculated twigs became different in their volatile compound production. The dashed red line indicates the maximum observed peak area for the uninoculated controls during the study.

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The GLVs hexanal, cis-3-hexen-1-ol and cis-3-hexenyl acetate were detected from most of the apple twig samples, but none of these compounds showed significant increases with time from inoculation (respective p-values of 0.672, 0.750 and 0.108). The sesquiterpenes α-cis-bergamotene, β-caryophyllene, aromadendrene, germacrene D, γ-cadinene and δ-amorphene, identified by mass spectral library and retention index matching, were also detected from most of the samples, but none of these compounds showed significant increases with time from inoculation (respective p-values of 0.454, 0.318, 0.375, 0452, 0.772 and 0.051).
LC-MS analyses for nonvolatile compounds in apple twig leaf nodes from this time course experiment used the same conditions as the control versus infected experiment above. The most differentially expressed compounds in terms of LC-MS peak intensity were phloretin > pomaceic acid >1-benzoyl β-d-glucose >2-oxopomolic acid > phloretin-cinnamic-hexosides.
Phloretin (Figure 3) showed significant increases (p = 0.002) with time from inoculation compared with the controls (Figure 7). Phloridzin (phloretin glucoside) was present, but without any differential expression in inoculated samples.Phloretin exhibits strong broad-range bactericidal and fungicidal activity, (64) and the genes governing its release from phloridzin (phlorizin) in response to biotic stresses have been identified. (59)

Figure 7

Figure 7. Phloretin signals (by LC-MS, C18, m/z [M-H]−) from leaf nodes (ground) inoculated with Neonectria ditissima (at zero weeks) and uninoculated control leaf nodes of apple twigs.

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Four phloretin-cinnamic or ferulic -hexosides, tentatively identified by MS product ion neutral losses of-162 (hexose), −130 (cinnamic acid) or −176 (ferulic acid), also showed increases in concentration (p = 0.002) in inoculated apple twigs (Figures S7 and S8). A Chemical Abstract search retrieved only one possible candidate structure, a cinnamate synthesized from phloridzin. (65)
Three of the other potential nonvolatile SCs identified above showed significant increases (p < 0.001) with time from inoculation compared with the controls: 1-benzoyl β-d-glucose, and the triterpene acids (TAs) pomaceic acid and 2-oxopomolic acid (Figures S4–S6). As noted above, these compounds could be functional phytoalexins produced in apple twigs in response to N. ditissima infection.

Summary and Future Work

We have discovered several distinct classes of secondary metabolites that are at significantly elevated concentrations in apple wood infected with N. ditissima (Figure 3): volatile styrene, and nonvolatile triterpene acids (including the rarely reported pomaceic acid) and 1-benzoyl β-d-glucose. The triterpene acids and the benzoyl glycoside may be phytoalexins produced by the apple tissue in response to the fungal infection. Transcriptome analysis of a time-course of infection, concentrating on both host apple and fungal pathogen, is currently underway to ascertain the source of styrene. These compounds were all significantly elevated in concentration with apple tissue before the external symptoms of EC appeared and therefore could be SCs for nondestructive disease detection. However, several questions about these potential SCs remain to be explored in depth in future research. Are they apple variety specific? Are they apple disease specific? Are they localized at the site of infection, or systematically produced in (or circulated to) other parts of a tree in response to infection? Is the volatile styrene released from tissue into surrounding air before symptoms appear? How weather dependent is the time between significant increase in potential SCs in infected wood and symptoms appearing? These questions must be answered before nondestructive disease detection technology using one or more proven SC could be developed to support the apple industry. One such technology might use portable HS-SPME-GC-MS, as reported for sampling of citrus leaves for field detection of HLB SCs, (25) if this could be developed for reliable and economically feasible disease detection.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c10597.

  • Figure S1: Headspace solid-phase micro-extraction gas chromatography–mass spectroscopy total ion current chromatograms (from first experiment) of (top) an EC infected ‘Scifresh’ apple twig node; Figure S2: 4-ethylphenol (artifact from p-hydroxystyrene) signals (by HS-SPME-GC-MS) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S3: benzaldehyde signals (by HS-SPME-GC-MS) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S4: 1-benzoyl β-d-glucose signals (by LC-MS, C18, m/z [M-H]−) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S5: pomaceic acid signals (by LC-MS, C18, m/z [M + H]+) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S6: 2-oxopomolic acid signals (by LC-MS, C18, m/z [M + H]+) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S7: phloretin-cinnamic-hexoside-A signals (by LC-MS, C18, m/z [M-H]−) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Table S1: identification of significant compounds differentially detected in extracts of Neonectria ditissima-infected and control apple twigs (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Catherine E. Sansom - The New Zealand Institute for Plant and Food Research Limited, Department of Chemistry, University of Otago, Dunedin 9054, New Zealand
    • Elaine J. Burgess - The New Zealand Institute for Plant and Food Research Limited, Department of Chemistry, University of Otago, Dunedin 9054, New Zealand
    • Lesley Larsen - The New Zealand Institute for Plant and Food Research Limited, Department of Chemistry, University of Otago, Dunedin 9054, New Zealand
    • Nigel I. Joyce - The New Zealand Institute for Plant and Food Research Limited, Canterbury Agriculture and Science Centre, Lincoln 7608, New Zealand
    • Peter Jaksons - The New Zealand Institute for Plant and Food Research Limited, Canterbury Agriculture and Science Centre, Lincoln 7608, New Zealand
    • Rebecca E. Campbell - The New Zealand Institute for Plant and Food Research Limited, Brooklyn, Motueka 7198, New Zealand
    • Ria S. Rebstock - The New Zealand Institute for Plant and Food Research Limited, Mt Albert, Auckland 1025, New Zealand
    • Ian C. Hallett - The New Zealand Institute for Plant and Food Research Limited, Mt Albert, Auckland 1025, New Zealand
    • Jonathan Rees-George - The New Zealand Institute for Plant and Food Research Limited, Mt Albert, Auckland 1025, New Zealand
    • Kerry R. Everett - The New Zealand Institute for Plant and Food Research Limited, Mt Albert, Auckland 1025, New Zealand
    • Reiny W. A. Scheper - The New Zealand Institute for Plant and Food Research Limited, Havelock North, Hawke’s Bay 4130, New Zealand
    • Monika Walter - The New Zealand Institute for Plant and Food Research Limited, 412 No 1 Road, RD 2, Te Puke 3182, New Zealand
  • Funding

    This work was supported by the New Zealand Institute for Plant and Food Research Limited.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank I. Stewart for NMR spectra and R. Atkinson for suggestions.

References

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This article references 65 other publications.

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  • Abstract

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    Figure 1

    Figure 1. (A) Apple twig leaf scar, with leaf bud above (image by Tony Corbett, PFR). (B) Apple twig with visible European Canker infection. (C) Cross-section of infected apple twig leaf scar showing Neonectria ditissima symptoms (brown tissue at the top).

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    Figure 2

    Figure 2. (A) Two-dimensional projection of principal components analysis of headspace solid-phase micro-extraction GC-MS data of apple twigs showing the variability between different treatments. Red triangles = ‘Scifresh’ inoculated, purple squares = ‘Scifresh’ control, green triangles = ‘Royal Gala’ inoculated, blue squares = ‘Royal Gala’ control from Motueka, light blue squares = ‘Royal Gala’ control from Hawke’s Bay. (B) Loading plot for PC-1, showing positive loadings corresponding to peaks for hexanol and styrene, and negative loadings for sesquiterpenes. The x-axis numbers correspond to GC retention time (min).

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    Figure 3

    Figure 3. Structures of potential signature compounds for Neonectria ditissima infection of apple twigs.

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    Figure 4

    Figure 4. LC-MS differential plots for all component mass features that changed between infected and control ‘Royal Gala’ apple twigs by reverse phase liquid chromatography with negative ion (A) and positive ion (B) MS. Plot x-axis log2fold change (negative values indicate down regulation and positive values up regulation due to infection); y-axis ion species mass features as m/z (compounds represented by one or more signal ions M, 2M, M+ adduct, etc.); marker dimension is the maximum area response for an ion species mass feature; color represents Welch’s t-stat as negative (red, down regulation) with a gradient through zero (yellow) to positive values (dark green, up regulation).

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    Figure 5

    Figure 5. Fluorescence immunolabeling of fungal hyphae (green) using a (1,3)-β-glucan antibody in tissue sections of inoculated apple twigs. (A) Four weeks post-inoculation: hyphae are mainly visible on the outside of the apple leaf scar but some are showing penetration into the woody tissue (arrows). (B) Thirteen weeks post-inoculation: large areas of hyphal infection inside the woody apple tissue (stars).

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    Figure 6

    Figure 6. Styrene signals (by HS-SPME-GC-MS) from leaf nodes (ground) of apple twigs inoculated with Neonectria ditissima (at zero weeks) and uninoculated control leaf nodes of apple twigs. Peak values are rescaled to values between zero and one by dividing each peak area by the maximum observed peak area for this compound. A local polynomial spline curve was fitted through the data, with 95% error bounds giving an indication of the time point after which the control and inoculated twigs became different in their volatile compound production. The dashed red line indicates the maximum observed peak area for the uninoculated controls during the study.

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    Figure 7

    Figure 7. Phloretin signals (by LC-MS, C18, m/z [M-H]−) from leaf nodes (ground) inoculated with Neonectria ditissima (at zero weeks) and uninoculated control leaf nodes of apple twigs.

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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c10597.

    • Figure S1: Headspace solid-phase micro-extraction gas chromatography–mass spectroscopy total ion current chromatograms (from first experiment) of (top) an EC infected ‘Scifresh’ apple twig node; Figure S2: 4-ethylphenol (artifact from p-hydroxystyrene) signals (by HS-SPME-GC-MS) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S3: benzaldehyde signals (by HS-SPME-GC-MS) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S4: 1-benzoyl β-d-glucose signals (by LC-MS, C18, m/z [M-H]−) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S5: pomaceic acid signals (by LC-MS, C18, m/z [M + H]+) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S6: 2-oxopomolic acid signals (by LC-MS, C18, m/z [M + H]+) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Figure S7: phloretin-cinnamic-hexoside-A signals (by LC-MS, C18, m/z [M-H]−) from leaf nodes (ground) of apple twigs inoculated with European Canker (at time 0 weeks) and controls; Table S1: identification of significant compounds differentially detected in extracts of Neonectria ditissima-infected and control apple twigs (PDF)


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