# Development of a Simplified PCR -Nucleic Acid Lateral Flow Immunoassay (PCR - NALFIA) for Cryptostroma corticale, the Causal Agent of Sooty Bark Disease

Annie Dicaire, Isabella Laughton, Ben Drugmand, Jessica Walker, Ciaran

Woods, Taylor Mcnee, Jaime Aguayo, Mireia Gomez-Gallego, Simon

Shamoun, Joey B Tanney, et al.

► To cite this version:

Annie Dicaire, Isabella Laughton, Ben Drugmand, Jessica Walker, Ciaran Woods, et al. Development of a Simplified PCR -Nucleic Acid Lateral Flow Immunoassay (PCR - NALFIA) for Cryptostroma corticale, the Causal Agent of Sooty Bark Disease. Plant Pathology, 2025, 74 (9), pp.2728-2737. 10.1111/ppa.70044. hal-05490424

HAL Id: hal-05490424

https://hal.inrae.fr/hal-05490424v1

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ORIGINAL ARTICLE

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# Development of a Simplified PCR-Nucleic Acid Lateral Flow Immunoassay (PCR-NALFIA) for *Cryptostroma corticale*, the Causal Agent of Sooty Bark Disease

Annie Dicaire<fnref n="1" /> | Isabella Laughton<fnref n="1" /> | Ben Drugmand<fnref n="1" /> | Jessica Walker<fnref n="1" /> | Ciaran Woods<fnref n="1" /> | Taylor McNee² | Jaime Aguayo³
¹D | ²D | ³D | ⁴D

**Correspondence:** Nicolas Feau (nicolas.feau@nrcan-rncan.gc.ca)

**Received:** 10 June 2025 | **Revised:** 22 July 2025 | **Accepted:** 24 July 2025

**Funding:** This work was supported by Canadian Forest Service—ADM Innovation fund.

**Keywords:** dipstick | DNA sensor | lateral flow assay | maple trees | point-of-use PCR | tree pathogen

## ABSTRACT

Sooty bark disease (SBD), caused by the fungus *Cryptostroma corticale*, is causing a widespread outbreak on sycamore maple in Europe and is presently emerging as a threat to urban and native maple trees in the Pacific Northwest. Rising temperatures and prolonged drought conditions probably exacerbate SBD disease severity. Current detection methods for *C. corticale* rely on laboratory-based PCR and morphological identification, which require specialised equipment and are not suited for rapid field deployment. We developed a portable, species-specific PCR-nucleic acid lateral flow immunoassay (PCR-NALFIA) for point-of-care detection of *C. corticale*. The assay can be performed in 90 min by incorporating a 5-min crude DNA extraction using cellulose dipsticks, a species-specific ITS1-targeted PCR with internal probe labelling on a portable PCR machine, and a lateral flow dipstick for visual detection. The method yielded accurate detection from infected wood, bark and conidia, with detection thresholds as low as 10 conidia or 0.1 mg of stroma in 0.5 mL buffer. The PCR-NALFIA achieved 100% specificity when tested on 87 fungal samples, outperforming other available PCR assays. Furthermore, lyophilised reaction mixes were stable for 60 days at room temperature, making the assay viable for field applications. This tool offers a rapid and reliable diagnostic solution for managing SBD in forestry and urban environments.

## 1 | Introduction

Invasive species are increasingly altering habitats across the world, costing the global economy an estimated $1.3 trillion over the last 50 years (Zenni et al. 2021). Biological invasions are a major driver of biodiversity loss, contributing to 25% of plant extinctions and 33% of animal extinctions (Blackburn et al. 2019). Forest trees, which provide critical economic and ecological services and cultural values, are particularly vulnerable to invasive pathogens. Iconic tree species, such as American

chestnut (*Castanea dentata*) and European ash (*Fraxinus excelsior*), have experienced dramatic population declines due to the introduction of the fungal pathogens *Cryphonectria parasitica* and *Hymenoscyphus fraxineus*, respectively (Combes et al. 2024; Rigling and Prospero 2018).

The early detection and identification of tree pathogens is key to prevention and proper disease management (Luchi et al. 2020). Traditional methods of identification for tree pathogens rely on culturing and morphological identification. However, not every

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2025 His Majesty the King in Right of Canada and The Author(s). *Plant Pathology* published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology. Reproduced with the permission of the Minister of Energy and Natural Resources.

<footnote n="1">¹Pacific Forestry Centre, Natural Resources Canada, Canadian Forest Service, Victoria, British Columbia, Canada | ²Department of Plant Pathology and Puyallup Research and Extension Center, Washington State University, Puyallup, Washington, USA | ³ANSES Plant Health Laboratory, Mycology Unit, USC INRAE 1480, Domaine de Pixérécourt, Malzéville, France | ⁴Université de Lorraine, INRAE, IAM, Nancy, France</footnote>
species is suitable for laboratory-based culturing, and in many
cases with woody tissues, efficiency is reduced by the recovery
of many non-target microorganisms. Morphological identifi-
cation requires specialised knowledge and may be inadequate
for distinguishing, for example, morphologically similar but
phylogenetically distinct species. Nucleic acid-based identifica-
tion techniques, including PCR amplification, real-time PCR
and sequencing of specific loci such as the internal transcribed
spacer (ITS) of nuclear ribosomal DNA (rDNA), still require
extensive laboratory settings and technical expertise (Baldi
and La Porta 2020; Capron et al. 2020). In the last decade, ad-
vances in point-of-care diagnostics have simplified plant patho-
gen detection in the field. For example, dipstick-based DNA
extraction methods eliminate the need for specialised equip-
ment and reagents, while the increasing availability of portable
PCR machines allows for testing in any location (Baldi and La
Porta 2020; Mason and Botella 2020; Zou et al. 2017). Results
can be rapidly visualised and interpreted using a lateral flow de-
vice (LFD), which operates similarly to at-home COVID-19 tests
and is now easily understood by non-specialists (Baldi and La
Porta 2020).

Sooty bark disease (SBD) of maple (*Acer*) trees, caused by the fungal agent *Cryptostroma corticale*, is an emerging disease in the Pacific Northwest region of North America (Brooks et al. 2023; Tanney et al. 2024). The fungus is a saprobe of sugar maple (*Acer saccharum*) and has not been associated with disease within its endemic range in eastern North America (Ellis and Everhart 1889). However, after its first introduction to Europe in 1945 (Gregory and Waller 1951), it has caused repeated disease outbreaks and significant tree mortality in sycamore maple (*Acer pseudoplatanus*), especially in the last two decades (Tanney et al. 2024). This pathogen causes leaf wilt, crown dieback and green to brown wood stains towards the heartwood of the tree. Blisters appear and coalesce on the outer bark, eventually cracking and sloughing off to reveal a thick layer of dark brown to black, soot-like asexual spores (conidia) developing from elongated stroma (Feau et al. 2024; Gregory et al. 1949). Although *C. corticale* can exist for long periods as an asymptomatic endophyte, it has been demonstrated that, under elevated stress from prolonged drought and high summer temperatures, it can turn pathogenic and cause tree death (Muller et al. 2023; Ogris et al. 2021). Sooty bark disease has been noted to particularly affect ornamental sycamore maples in urban forests in the Pacific Northwest region of North America. In this area, disease incidence is also rising among *Acer macrophyllum*, the native bigleaf maple, resulting in an increased risk of the pathogen escaping into natural maple forests (Brooks et al. 2023; Tanney et al. 2024). The removal of SBD-infected trees in urban forests is a potentially hazardous task, as the vast number of allergenic spores released from disturbance has been linked to hypersensitivity pneumonitis, necessitating specialised training and equipment (Braun et al. 2021; Okhowat et al. 2025; Tewksbury et al. 1968). Furthermore, typical tree disposal methods, such as chipping and using the resulting wood chips as mulch in urban environments, are discouraged because of the potential mass release of inoculum and subsequent exposure risk (Burgdorf et al. 2025; Tanney et al. 2024). Proper tree removal and disposal are dependent on the correct identification of *C. corticale*, a task that is complicated by the overlapping

range of lookalike fungi including species of *Biscogniauxia*,
a genus that includes causal agents of diseases like charcoal
canker, and the brittle cinder fungus *Kretzschmaria deusta*.
Besides morphology, current identification methods for *C. cort-
ricale* rely on nested PCR and quantitative real-time PCR
(qPCR) assays that were designed and validated with *C. cor-
ticale* isolates and sister species from Europe (Kelnarová
et al. 2017; Muller et al. 2023). Although these tools have been
extensively used to confirm diagnostics and conduct surveys
(Brooks et al. 2023; Kelnarová et al. 2017; Muller et al. 2023),
they were designed to function in conventional molecular bi-
ology laboratories and are not adapted to field testing. Herein,
we present a new point-of-care test for the rapid identification
of *C. corticale*. This PCR-nucleic acid lateral flow immunoas-
say (PCR-NALFIA) consists of a fast dipstick-based DNA ex-
traction protocol, portable PCR and quick result visualisation
with LFD test strips as a downstream detection method. The
entire process takes less than 2 h and allows for correct identi-
fication for rapid, on-site decision making.

2 | Materials and Methods

## 2.1 | Specimen Collection

We assembled an inclusive collection of 87 samples from North
America and Europe to test and validate the PCR-NALFIA.
Fifty-nine samples were true *C. corticale* positive isolates as iden-
tified by culture morphology and/or sequencing the ITS region
of the nuclear rDNA (see Section 2.2). Of these, 26 were either
conidia, bark with stroma or wood samples from naturally in-
fected trees; six were from wood samples infected in vitro; three
from cultures; and 24 were DNA extracts from cultures from
the United States (Washington State), France, Slovenia, Italy
and the United Kingdom (Table S1). The remaining 28 sam-
ples were true negatives, of which eight were cultures or DNAs
from closely related species within the genus *Biscogniauxia* and
15 were from diseased maple trees showing signs of *K. deusta*,
*Biscogniauxia* sp. or symptoms of decay. Five samples were col-
lected from healthy maple trees of different species (Table S1).

## 2.2 | DNA Extraction and DNA-Barcoding
of Fungal Specimens

All of the samples included in the specimen collection were
identified by DNA barcoding as described by Feau et al. (2023).
Briefly, DNA was extracted using a Plant Pro kit (Qiagen) fol-
lowing the manufacturer's protocol. An extra incubation at
65°C-70°C for 10 min on a shaking platform at 200 rpm was
added before grinding down the samples. The ITS region was
amplified by PCR in a 50 µL reaction volume containing 0.25
U of Platinum Taq DNA polymerase High Fidelity (Invitrogen),
1x PCR buffer, 0.2µM dNTPs, 1.5 mM MgCl₂ and 0.2µM each
of primers ITS1F (Gardes and Bruns 1993) and ITS4 (White
et al. 1990). PCR conditions were set at 94°C for 2 min; 35 cy-
cles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min; then 72°C
for 10 min. PCR amplicons were then sequenced at the Centre
Hospitalier Universitaire de Québec-Université Laval Research
Center Sanger Sequencing platform in Québec (Canada) and as-
sembled using Geneious Prime (Kearse et al. 2012). Similarity
searches were performed with consensus sequences against the NCBI nucleotide collection (nt) using the BLASTn algorithm (Altschul et al. 1990).

## 2.3 | Crude DNA Extraction Using Cellulose Dipsticks

Dipstick-based crude DNA extraction was performed based on the protocol developed by Zou et al. (2017) and Mason and Botella (2020). Approximately 1–5 mg of fungal or plant material was added to 500 µL of lysis buffer (1.2 mg Tris base, 0.73 mg NaCl, 7.3 mg EDTA, 0.25 mg SDS, 500 µL water; pH 8.0) alongside one 0.635-cm diameter ceramic bead. Fungal spores were preferred when available. Samples were vigorously shaken for 30 s to achieve cell lysis and release DNA. The DNA was absorbed by a 6–10 mm² piece of cellulose paper at the end of a dipstick, which was then transferred to 1.5 mL of washing buffer (1.8 mg Tris

base, 1.5 µL Tween 20, 1.5 mL water; pH 8.0) and dipped 10 times to remove contaminants. The dipstick was placed into a PCR tube containing PCR solution for 30 s to deposit the DNA.

## 2.4 | Development of a Species-Specific PCR Assay for *C. corticale*

A species-specific primer pair targeting a short portion of the ITS1 region in *C. corticale* was designed by considering an alignment of 12 ITS sequences of this pathogen and 23 ITS sequences of closely related species within the order Xylariales downloaded from NCBI (Figure 1A; Table S1). Nine additional *C. corticale* ITS sequences obtained from this study were added to this alignment to increase the representation of strains from western Canada (Figure 1A). Primers and probes were designed using Primer3 software (Untergasser et al. 2012). Compatibility between each primer-primer and primer-probe was tested using

<figure><img src="image_4.png" /><figcaption>**FIGURE 1** | Phylogeny of *Cryptostroma corticale* and closely related species used for primer and probe design for the PCR-nucleic acid lateral flow immunoassay (PCR-NALFIA). (A) Maximum-likelihood phylogeny of the internal transcribed spacer region (ITS) of the nuclear rDNA including sequences from *C. corticale* and related species in Xylariales obtained from the International Nucleotide Sequence Database Collaboration database (INSDC) and specimens sequenced in this study; numbers above or below nodes are statistical support values obtained from 1000 bootstrap samples in maximum likelihood/maximum parsimony; statistical support < 50% is not represented. Scale bars indicate the number of nucleotide substitutions per site; sequence conservation and polymorphisms at the binding locations of the two primers and the internal probe on the ITS1 locus are indicated with sequence logo on the right. (B) Sequences of the forward and reverse primers and the internal probe designed for *C. corticale* detection. Result of compatibility analysis (ΔG free energy) for primer-primer and primer-probe interactions is represented on the right. [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
the OligoAnalyzer Tool (Integrated DNA technologies [IDT]) as
follows: a change in Gibbs free energy (ΔG) with a value below
-9 kcal/mol (corresponding to five or six contiguous complemen-
tary base pairs) was considered the threshold for incompatibil-
ity. Primers and probes were then synthesised by IDT, including
an internal probe biotinylated at the 5' end (SBD-PFC-probe)
and a reverse primer modified with a 6-FAM (fluorescein) mol-
ecule attached to its 5' end (SBD-PFC-R) (Figure 2A–III). PCRs

were carried out using 1× AllTaq Master Mix (Qiagen), 0.1 µM
each of forward and reverse primers and 1 µM of internal probe
in a total volume of 20 µL on a mini8X portable thermocycler
(miniPCR bio). PCR conditions were set at 95°C for 2 min; 35 cy-
cles of 95°C for 30 s, 62°C for 30 s and 72°C for 30 s; then 95°C for
1 min. An additional step of 1 min at 42°C was added to bind the
reverse PCR product strand with the SBD-PFC-probe internal
probe (Figure 2A–III).

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**FIGURE 2** | Detection of *Cryptostroma corticale* by a PCR-nucleic acid lateral flow immunoassay (PCR-NALFIA). (A) schematic representation of the assay including (I) sample collection and (II) DNA purification step; (III, IV) PCR and binding of the primer-probe complex on lateral flow dipstick (LFD). (B) *C. corticale* ITS haplotypes 1 (JTCC-1456) and 2 (SBD15) were detected using an electrophoresis assay; an isolate of *Biscogniauxia* sp. (JTCC-1167) was used as a negative control; C-, PCR negative control (water). (C) The same *C. corticale* and *Biscogniauxia* sp. isolates detected using the LFD. [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
## 2.5 | PCR-NALFIA

Following the PCR and probe hybridisation step, a 10-µL aliquot of the solution containing the PCR product-probe complex solution was mixed with 90 µL of HybriDetect assay buffer (MGHD1; Milenia Biotec). The HybriDetect dipstick was then inserted into this solution for 1 min. The 6-FAM and biotin-labelled PCR product-probe complex bound to anti-FAM antibodies labelled with gold ions and migrated along the strip until reaching the streptavidin ligands of the test line (Figure 2A-IV). These bound to the biotin-labelled probe, and the resulting accumulation of probe-DNA-gold ions complexes created a visible red line on the LFD. Excess gold ions continued to the control line where they bound to the anti-rabbit antibodies present to create a second red line (Figure 2A-IV). After removal from the solution, the LFD was dried for 5 min on a paper towel before reading the results. Two clear bands indicated a positive result. If only the control band was present, then the result was negative.

## 2.6 | Detection Thresholds

Initial concentrations of DNA extracted from samples SBD48, SBD51 and SBD52 were measured using the Qubit dsDNA HS Assay Kit on a Qubit 4 fluorometer (ThermoFisher Scientific). Samples were diluted in a seven-step serial 1:10 dilution; then processed with the standard PCR protocol to assess DNA detection thresholds.

Two additional experiments were conducted directly on *C. corticale* tissues (sample SBD51). In the first experiment, conidia concentration was measured using a haemocytometer and diluted to prepare solutions containing 10, 50, 100, 1000, 10⁴, 10⁵ and 10⁶ conidia in 500 µL of lysis buffer (see Section 2.3). These conidial solutions were then tested using the crude DNA extraction protocol with cellulose dipsticks, followed by PCR-NALFIA. In the second experiment, different quantities of stroma (0.1, 0.5, 1.0, 5.0, 10, 20 and 30 mg) were weighed and added to 500 µL of lysis buffer for the same testing procedure. Both experiments were performed in triplicate.

## 2.7 | PCR-NALFIA Performance

Sensitivity, specificity and accuracy of the PCR-NALFIA were evaluated on a panel of DNA extracted from our collection of true positive and true negative samples (Table S1). Results of these detections were then benchmarked with those obtained with the PCR TaqMan assay developed in the ITS region for *C. corticale* by Muller et al. (2023). This assay was run on a subpanel of 33 positives and 20 negatives from the same DNA samples as those tested with the PCR-NALFIA with two technical replicates as described. Real-time PCRs were performed in a 10 µL solution containing 1x PrimeTime Gene Expression Master Mix and 1x PrimeTime Mini qPCR assay probe (IDT) containing primers and the TaqMan probe labelled with fluorescein (6-FAM) at the 5' end and with the quencher Iowa Black FQ (ZEN-IBFQ). Amplification reactions were completed on a QuantStudio 3 Real-Time PCR System instrument (ThermoFisher Scientific) with the following conditions: 95°C, 15 min enzyme activation step, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. For

both tests, sensitivity was defined as the true positive rate or the ability of the assay to correctly identify true positive samples. Specificity or true negative rate was defined as the ability of the assay to correctly identify true negative samples as negative, and accuracy of the assay was the proportion of both positive and negative results that were correct.

## 2.8 | Field-Ready Master Mix

Field-ready reaction mixes were prepared using lyophilised reagents that comprised the PCR mix, primers, and the internal probe, reconstituted in a 20 µL reaction volume, with trehalose added to a final concentration of 5%. The reactions were aliquoted in 100 µL 8-tube strips (ThermoFisher Scientific), frozen at -20°C for 1 h, and then lyophilised for 30 min in a ThermoSavant SpeedVac SPD111V modular concentrator (ThermoFisher Scientific). After lyophilisation, the strips were stored in the dark at room temperature (about 20°C). Stability of the lyophilised reagents was tested at 1, 20, 40 and 60 days by running a PCR followed by the LFD strip test. At each time point tested, the lyophilised reagents were resuspended in a final volume of 20 µL of PCR-grade water, and crude DNA extracted from a *C. corticale* stroma sample was added by following the crude extraction protocol with the cellulose dipstick.

# 3 | Results

## 3.1 | Assay Design

Maximum-likelihood (ML) and maximum-parsimony (MP) phylogenetic trees, constructed from a 631-bp alignment of 21 *C. corticale* sequences and 23 other Xylariales sequences, displayed one monophyletic clade with all *C. corticale* sequences supported with bootstrap values of 60% in ML and 70% in MP. Seven intraspecific polymorphisms were found within *C. corticale*, resulting in two separate clades of five sequences (bootstrap support [BS] of 67% in ML and 81% in MP) and 16 sequences (BS of 83% in ML and 93% in MP), each corresponding to two ITS haplotypes (Figure 1A). Despite these intraspecific differences, the *C. corticale* isolates shared more than 98.5% nucleotide sequence identity. The comparison between the ITS region of *C. corticale* and the most closely related species, *Biscogniauxia bartholomaei*, revealed 13 fixed sequence polymorphisms, representing 96.3% to 96.6% identity between these two species. Five base variations in the ITS1 between *C. corticale* and *B. bartholomaei* were selected for designing specific primers and an internal probe specific to *C. corticale*. The restricted choice of interspecific polymorphism sites between *C. corticale* and closely related species resulted in the design of the forward primer specific to *C. corticale* over an intraspecific mutation located on the fourth nucleotide after the 5' end of the primer binding site (Figure 1A). Changes in Gibbs free energy of bonding analysis showed that the primers and probes were not likely to form homodimers and heterodimers, with ΔG values higher than -7.5 for every combination (Figure 1B). The amplicon generated using this primer pair was, as expected, 104 bp long and successfully amplified in each ITS haplotype of *C. corticale*, whereas no amplicon was obtained for an unidentified, closely related species in the genus *Biscogniauxia* (Figure 2B; Table S1). Similarly,
after hybridisation of the probe on the reverse complement strand labelled with 6FAM, the PCR-NALFIA was successful in detecting both ITS haplotypes of *C. corticale* (Figure 2C).

## 3.2 | Comparison of PCR-NALFIA and Real-Time PCR Assay Performance

Detection threshold of the PCR-NALFIA was assessed using serial dilutions of three *C. corticale* DNA samples. As little as 1.6 pg of genomic DNA from sample SBD52 was visually detectable with the LFD test strip. The PCR-NALFIA demonstrated 100% test accuracy, with no false-positive or false-negative results when applied to a panel of 77 DNA samples extracted from the specimen collection using Qiagen columns (Table 1). In contrast, even with the developed *C. corticale*-specific primer pair, using the real-time PCR assay on the same DNAs resulted in a false-positive rate of 36.0% caused by the amplification of nine samples of *Biscogniauxia* sp. and *B. bartholomaei* with $C_q$ values ranging from 27.8 to 38.9 (Table 1; Table S1).

## 3.3 | Performance of the Crude DNA Extraction in Combination With the PCR-NALFIA

Conidial dilution testing indicated that as few as 10 spores could be detected by the PCR-NALFIA after crude DNA extraction with the cellulose dipstick. No inhibition of the PCR-NALFIA was observed with a larger number of spores (up to $10^6$ spores) in any of the triplicates (Figure S1A). Similarly, DNA from as little as 0.1 mg of *C. corticale* stroma could be detected with the same method. However, for this tissue, inconsistent inhibitions of the PCR-NALFIA occurred with quantities above 1 mg of stroma (Figure S1B).

Crude DNA extracts obtained with the cellulose dipstick protocol from 35 positive and 15 negative samples from the specimen collection were retested with the PCR-NALFIA. This second test showed an accuracy of 96.0%, with two false negative tests

(Table 1). One of the samples that returned a false negative result was a mouldy and likely poorly preserved piece of bark collected from an infected sycamore maple. The second false negative result was a wood core obtained from the trunk of an infected sycamore maple (SBD16, sycamore maple naturally infected; Table S1). Crude DNA extracts from four wood pieces, each representing different levels of stain and decay from the same tree, were retested with the PCR-NALFIA and yielded positive results (Figure S2).

## 3.4 | Reproducibility of the Dipstick Crude DNA Extraction

The reproducibility of crude DNA extraction with the cellulose dipstick method was further tested by repeating the PCR-NALFIA on three replicates obtained from five *C. corticale* conidia samples and 11 bark samples containing *C. corticale* stroma. For each of the six conidia samples, all three replicate DNA extractions tested positive (Figure 3). Among the stroma samples, only eight out of 12 tested positive with one replicate. With two replicates, two more stroma samples were positive, and after three crude DNA extraction attempts, all of the samples were successfully detected. Adding 5% polyethylene glycol 8000 (PEG) to the lysis buffer drastically improved the crude DNA extraction process, as the 12 *C. corticale* stroma samples were positive after one replicate of the protocol (Figure 3).

## 3.5 | Field-Ready Lyophilised Kits

Because lyophilised PCR reagents must be prepared in advance for point-of-care use, evaluating the long-term stability without refrigeration was key in assessing the practicality of this approach. The strips were stored at room temperature in the dark and tested four times over the course of 60 days, running the assay in duplicate and including a positive control containing lyophilised DNA of *C. corticale* and a negative control without target DNA. Amplification products were

<table><caption>**TABLE 1** | Accuracy of the PCR-nucleic acid lateral flow immunoassay (PCR-NALFIA) and real-time PCR assays used to detect *Cryptostroma corticale* DNA obtained from two DNA extraction methods.</caption><thead><tr><th rowspan="2">Test</th><th rowspan="2">Classification and accuracy</th><th colspan="2">DNA extraction method</th></tr><tr><th>Qiagen column</th><th>Cellulose dipstick</th></tr></thead><tbody><tr><td rowspan="5">PCR-NALFIA</td><td>True positive</td><td>52/52 (100.0%)</td><td>33/35 (94.3%)</td></tr><tr><td>False positive</td><td>0/25 (0.0%)</td><td>0/15 (0.0%)</td></tr><tr><td>True negative</td><td>25/25 (100.0%)</td><td>15/15 (100.0%)</td></tr><tr><td>False negative</td><td>0/52 (0.0%)</td><td>2/35 (5.7%)</td></tr><tr><td>Accuracy</td><td>100.0%</td><td>96.0%</td></tr><tr><td rowspan="5">Real-time PCR assay</td><td>True positive</td><td>46/46 (100.0%)</td><td>—</td></tr><tr><td>False positive</td><td>9/25 (36.0%)</td><td>—</td></tr><tr><td>True negative</td><td>15/25 (60.0%)</td><td>—</td></tr><tr><td>False negative</td><td>0/46 (0.0%)</td><td>—</td></tr><tr><td>Accuracy</td><td>87.3%</td><td>—</td></tr></tbody></table>
<figure><img src="image_8.png" /><figcaption>**FIGURE 3** | Testing crude DNA extraction replicates for two types of *Cryptostroma corticale* tissues (six conidia samples and 12 stroma samples) with the dipstick DNA extraction protocol and the PCR-nucleic acid lateral flow immunoassay (PCR-NALFIA). Dark grey boxes indicate positive detection for *C. corticale*; light grey boxes indicate failure to detect *C. corticale*. For each sample, three separate crude DNA extractions were performed to assess detection consistency. PEG, 5% polyethylene glycol 8000. [Colour figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>

detected for all time points for the two duplicates and for the positive control. A video demonstrating the test procedure is available at https://youtu.be/0d8xpkUjZa8?si=hzgAPm3M3Q FajV2v (PCR Test for Field Detection of Sooty Bark Disease), providing a visual overview of its field application. The estimated cost per reaction for the PCR-NALFIA assay was 8.75 CAD. This is primarily driven by the high price of the commercial lateral flow dipstick used for the visual detection of the PCR product. However, this cost remains comparable to the PCR TaqMan assay when DNA extraction using a Qiagen column is included, and technician time is excluded (Table 2; Table S2).

**4 | Discussion**

SBD, caused by *C. corticale*, is rapidly emerging into new territories, causing the death of ornamental and native maple trees. As disease severity is linked to drought events and high temperatures, it is reasonable to assume that the range and severity of this disease will increase under most projected climate change scenarios (Garbelotto 2024; Muller et al. 2023). Identifying and removing infected trees is key to controlling the spread of invasive diseases. Given the human health risks associated with this fungus, a reliable diagnostic is also essential to guide decisions

on worker training and use of safety gear. However, controlling SBD is challenging due to its endophytic phase and the difficulty in identifying its causal agent. *C. corticale* shares overlapping traits and host tissues with other maple-infecting fungi, making specialised and rapid identification methods necessary. To answer this demand, we successfully developed a new and simple point-of-care method for the identification of this pathogen. With its simple and effective design, this tool shows promise in facilitating the early detection of *C. corticale* and helping with decision-making processes for SBD management.

The ITS locus is the primary target for species identification, phylogenetic analyses and the design of species-specific detection assays in fungi, especially when well-established genetic resources are not available (Feau et al. 2011; Martinelli et al. 2015; Schoch et al. 2012). Key advantages of using this region for PCR-based detection are its high copy number in fungal cells, which increases the sensitivity of detection assays, and the presence of a 'barcoding gap', where sequences show sufficient divergence between species while maintaining relatively low intraspecific variation (Feau et al. 2023). Conversely, low interspecific variation among closely related taxa and intragenomic polymorphisms can limit the utility of ITS for species-level resolution and reduce its phylogenetic signal (Cedeño-Sanchez et al. 2024; Lücking et al. 2020). Primer design for *C. corticale*
<table><caption>**TABLE 2** | Comparison of advantages and disadvantages of the PCR-nucleic acid lateral flow immunoassay (PCR-NALFIA) and real-time PCR assay used in this study to detect *Cryptostroma corticale* DNA.</caption><thead><tr><th></th><th>Real-time PCR assay</th><th>PCR-NALFIA</th></tr></thead><tbody><tr><th>Advantages</th><td><p><strong>Accuracy:</strong> High sensitivity.</p><p><strong>Cost:</strong> Overall cost remains low if DNA extraction is performed without the Qiagen column.</p><p><strong>Time:</strong> Approximately 90 min.</p></td><td><p><strong>Accuracy:</strong> High sensitivity and specificity.</p><p><strong>Portability:</strong> Suitable for point-of-care testing.</p><p><strong>Ease of use:</strong> Portable and simple to perform using pre-lyophilised reagents; requires minimal equipment and limited technical expertise.</p></td></tr><tr><th>Disadvantages</th><td><p><strong>Cost:</strong> DNA extraction with the Qiagen column is the primary cost driver, representing 79.2% of the total assay expense.</p><p><strong>Portability:</strong> Non portable; requires specialised equipment (real-time PCR machine).</p><p><strong>Ease of use:</strong> Requires trained personnel.</p><p><strong>Accuracy:</strong> Limited specificity; potential cross-reactivity with closely related species in the <em>Biscogniauxia</em> genus.</p></td><td><p><strong>Cost:</strong> The lateral flow dipstick is the main cost driver, accounting for 61.4% of the assay's total expense.</p><p><strong>Time:</strong> Comparable to real-time PCR (approximately 90 min); no time-saving advantage.</p></td></tr></tbody></table>

was constrained by the presence of six single-nucleotide polymorphisms (SNPs) and one indel distinguishing two haplotypes within the species, and by the high degree of ITS sequence similarity with closely related species in the Xylariales family. To minimise the risk of cross-amplification with related species, we designed our assay to target a high density of interspecific mutations within the ITS1 region. However, as a trade-off, the forward primer covered an intraspecific SNP within *C. corticale*. To increase specificity, we also opted for the addition of an internal probe overlapping one or two polymorphisms with the closest known relatives to *C. corticale*, *B. bartholomaei* and *Graphostroma platystomum* (Koukol et al. 2015). Consistent with this specificity, our assay did not cross-amplify with the panel of 33 other non-*Cryptostroma* specimens tested, and the presence of the intraspecific polymorphism located four bases before the 5' end of the primer did not preclude the detection of *C. corticale* isolates carrying this mutation. In contrast, the real-time PCR-based detection assay used for *C. corticale* showed some cross-reactivity among some of the *Biscogniauxia* species tested. This assay uses a different pair of primers than those used in the PCR-NALFIA assay of this study. The primers of the real-time PCR assay were originally designed for a nested-PCR assay targeting the same region of ITS1 and were validated exclusively on positive and negative samples from Europe (Kelnarová et al. 2017; Muller et al. 2023). Recently, Tanney et al. (2024) pointed out that the cross-reactivity of these two assays with taxa that are phylogenetically related to *C. corticale* should be verified. For instance, *B. bartholomaei* is currently known only from the Pacific Northwest and the Russian Far East, and European monographs of the *Biscogniauxia* genus do not include this species (Callan 2000; IUCN (International Union for Conservation of Nature) 2025). ITS sequences from this species and closely related species within Xylariales were probably not considered during the design of the two *C. corticale* assays developed in European laboratories (Kelnarová et al. 2017; Muller et al. 2023).

Using an internal probe requires an additional hybridisation step at the end of the PCR cycles, which reduces the risk of false positives caused by primer dimers in LFD detection (Ren

et al. 2023). In standard LFD-based PCR detection, both PCR primers are labelled with a different biomolecule (e.g., 6FAM and biotin). However, this approach can lead to false positives if some primer dimers are generated during the PCR and then captured by the LFD. An alternative method consists of labelling only one primer and tagging an internal probe with the second biomolecule. In our approach, the internal probe (tagged with biotin) facilitates immobilisation, while the primer labelled with 6FAM is detected by an antibody conjugated to a reporter (e.g., gold nanoparticles) for optical readout. To prevent false positives from probe-primer complex formation, we minimised the likelihood of self-polymerisation between the probe and primers by ensuring probe-primer compatibility (evaluated via Gibbs free energy changes) and designing the probe with a lower melting temperature (Tm) than the hybridisation temperatures used in PCR cycling. This approach avoided the formation of a probe-primer complex and therefore avoided false positives in LFD detection.

Finally, DNA extraction is a crucial step in PCR detection, as the purity, integrity and yield of the extracted DNA directly influence the sensitivity and specificity of downstream amplification and detection (Kuhn et al. 2017). To allow for rapid and inexpensive processing of field samples, we used a three-step crude DNA extraction method combined with PCR in a portable machine and a downstream detection method based on LFD. The crude DNA extraction method combined an efficient lysis step followed by DNA binding on cellulose and a purification step. Its efficiency was confirmed by testing three different types of fungal and tree tissues infected with *C. corticale*. The extraction of DNA for identification purposes can be difficult for fungi due to chitinous cell walls, high melanin content and low pathogen-to-host ratio (Karakousis et al. 2006). In addition, many factors affect DNA extraction efficiency and introduce contaminants in the DNA extracts, which inhibit PCR amplification; loss of DNA by degradation or absorption has proven to be the major limitations (Schrader et al. 2012). The original method developed by Mason and Botella (2020) successfully processed all the spore samples tested with a single crude DNA extraction attempt. Slight modifications to this protocol, such as adding PEG, helped with challenging
samples taken from decayed wood and stroma, which con-
tained highly melanised, dense and compact mycelium mass.
PEG is widely used for DNA purification and separation
from other contaminants (Nargessi and Pourfarzaneh 2002).
This molecule acts as a crowding agent, promoting DNA ag-
gregation while keeping proteins and other contaminants in
solution and probably enhancing the capture of DNA by the
cellulose dipstick. By incorporating PEG into the extraction
protocol, we improved DNA recovery from challenging fungal
samples and reduced false negatives in the crude assay, mak-
ing this method more robust for field applications. Infected
maple wood with as little as 10 spores and a tissue quantity of
0.1 mg of C. corticale stroma still yielded positive detections,
highlighting the sensitivity and versatility of the crude DNA
extraction method coupled with PCR-NALFIA.

The PCR-NALFIA developed in this study enables accurate
detection of *C. corticale* using crude DNA extracts, a portable,
low-cost thermocycler and lateral flow dipsticks, making it well-
suited for on-site diagnostics in low-resource or field settings.
While the assay has a similar processing time to the real-time
PCR assay available for this pathogen (90min) and the lateral
flow dipstick is a significant cost driver, its portability, simplicity
and high specificity offer clear advantages for field use. This ap-
proach facilitates the timely and reliable identification of maple
trees affected by SBD, a critical requirement for the safe man-
agement of infected trees given the risks associated with aller-
genic spore dispersal. Importantly, this assay serves as a proof
of concept for the development of field-deployable molecular di-
agnostics targeting other forest pathogens, providing a scalable
framework to support early detection and disease surveillance
in forest health programmes.

Author Contributions

**Annie Dicaire:** conceptualization, investigation, writing – original draft. **Isabella Laughton:** methodology, validation. **Ben Drugmand:** methodology, validation, resources. **Jessica Walker:** methodology, validation. **Ciaran Woods:** methodology, validation. **Taylor Mcnee:** writing – review and editing, resources. **Jaime Aguayo:** writing – review and editing, resources. **Mireia Gomez-Gallego:** writing – review and editing, resources. **Simon Shamoun:** funding acquisition. **Joey B. Tanney:** conceptualization, funding acquisition, writing – review and editing, resources. **Nicolas Feau:** conceptualization, investigation, funding acquisition, writing – review and editing, project administration, resources, supervision.

Acknowledgements

N.F., J.B.T. and S.S. gratefully acknowledge financial support from the
Canadian Forest Service—ADM Innovation Fund (Round 9). We also
thank all the collectors listed in Table S1 for their assistance with sam-
pling and for providing *C. corticale* specimens. Open Access funding
provided by the Natural Resources Canada library.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that supports the findings of this study are available in the
**Supporting Information** of this article.

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## Supporting Information

Additional supporting information can be found online in the Supporting Information section. **Figure S1:** Dilutions of *Cryptostroma corticale* (SBD51) conidia (A) and stroma (B) tested with the crude DNA extraction cellulose dipstick and PCR-nucleic acid lateral flow immunoassay (PCR-NALFIA). **Figure S2:** Detection of *Cryptostroma corticale* from naturally infected wood samples by a polymerase-chain reaction-nucleic acid lateral flow immunoassay (PCR-NALFIA) following crude DNA extraction with the cellulose dipstick method. **Table S1:** Positive and negative samples tested with the PCR-NALFIA. **Table S2:** (A) Comparison of cost per reaction between the PCR-nucleic acid lateral flow immunoassay (PCR-NALFIA) developed in this study and the real-time PCR assay developed by Muller et al. (2023). Costs were estimated based on catalogue prices displayed in (B). (B) Catalogue prices (in Canadian dollars [CAD]) of materials and reagents used in the PCR-NALFIA developed in this study and the real-time PCR assay of Muller et al. (2023).