Research Article |
Corresponding author: Alessia L. Pepori ( alessia.pepori@ipsp.cnr.it ) Academic editor: Christophe Orazio
© 2023 Alessia L. Pepori, Nicola Luchi, Francesco Pecori, Alberto Santini.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Pepori AL, Luchi N, Pecori F, Santini A (2023) Duplex real-time PCR assay for the simultaneous detection of Ophiostoma novo-ulmi and Geosmithia spp. in elm wood and insect vectors. In: Jactel H, Orazio C, Robinet C, Douma JC, Santini A, Battisti A, Branco M, Seehausen L, Kenis M (Eds) Conceptual and technical innovations to better manage invasions of alien pests and pathogens in forests. NeoBiota 84: 247-266. https://doi.org/10.3897/neobiota.84.90843
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Dutch elm disease (DED) is a destructive tracheomycosis caused by Ophiostoma novo-ulmi, an ascomycete probably originating in East-Asia that is devastating natural elm populations throughout Europe, North America and Asia. The fungus is mainly spread by elm bark beetles that complete their life cycle between healthy and diseased elms. Recently, it has been highlighted that some fungi of the genus Geosmithia, which are similarly well associated with bark beetles, seem to also play a role in the DED pathosystem acting as mycoparasites of O. novo-ulmi. Although some relationship between the fungi is clear, the biological cycle of Geosmithia spp. within the DED cycle is still partly unclear, as is the role of Geosmithia spp. in association with the bark beetles. In this work, we tried to clarify these aspects by developing a qPCR duplex TaqMan assay to detect and quantify DNA of both fungi. The assay is extremely sensitive showing a limit of detection as low as 2 fg μl–1 for both fungi. We collected woody samples from healthy and infected elm trees throughout the beetle life cycle. All healthy elm samples were negative for both Geosmithia spp. and O. novo-ulmi DNA. Geosmithia spp. are never present in infected, but living trees, while they are present in frass of elm bark beetles (EBB – Scolytus spp.) and at each stage of the EBB life cycle in much higher quantities than O. novo-ulmi. This work provides a better understanding of the role and interactions occurring amongst the main players of the DED pathosystem.
DNA quantification, duplex qPCR, Dutch Elm Disease, Geosmithia spp. life cycle, Ophiostoma novo-ulmi, Scolytus multistriatus
Dutch elm disease (DED) is a destructive tracheomycosis that has devastated natural elm populations throughout Europe, North America and Asia. The disease is caused by two subspecies of Ophiostoma novo-ulmi ssp. novo-ulmi ssp. americana, previously known as Eurasian (EAN) and North American (NAN) races, respectively (
The fungus is mainly spread by species of elm bark beetles (Coleoptera, Curculionidae, Scolytinae) that complete their life cycle between healthy and diseased elms. Bark beetles belonging to the genus Scolytus Geoffroy are the main vectors of O. ulmi s.l. (
Recently, it has been highlighted that other organisms also play roles in the DED pathosystem (
Geosmithia spp., like O. novo-ulmi, are associated with elm bark beetles (
Previously, several methods of biocontrol of O. novo-ulmi have been investigated and have appeared promising under experimental conditions, although their practical application in the field has been limited (
An accurate description of the life cycle and identification of the key factors that can enhance the attitude of Geosmithia spp. to act as effective biocontrol agents against O. novo-ulmi may be strategic in controlling the further spread of the disease.
In this study, a new, ad hoc duplex real-time PCR assay, based on TaqMan probe chemistry genus-specific for Geosmithia and species-specific for O. novo-ulmi, for the simultaneous quantification of both fungi from different matrices, was developed. Application of this molecular approach will fill the knowledge gaps related to the life cycle of Geosmithia spp. and will uncover the tripartite interactions amongst O. novo-ulmi, Geosmithia spp. and EBBs.
The duplex qPCR assay was validated using 12 isolates of Geosmithia spp. belonging to nine different species (G. fassiatiae, G. flava, G. funiculosa, G. langdonii, G. lavendula, G. obscura, G. omnicola, G. pallida and G. putterillii) and eight isolates of Ophiostoma from five species (O. himal-ulmi, O. novo-ulmi ssp. novo-ulmi, O. novo-ulmi ssp. americana, O. quercus and O. ulmi). Two ubiquitous species were also included as outgroups (Table
Species | Isolate Code | Host | Origin | Duplex qPCRa (O. novo-ulmi/ Geosmithia spp.) |
---|---|---|---|---|
Geosmithia fassiatiae | CCF3334 | Quercus pubescens | Czech Republic | (-/+) |
G. flava | MK1551 | Pteleobius vittatus (on Ulmus laevis) | Czech Republic | (-/+) |
G. funiculosa | IVV7 | U. minor | Italy | (-/+) |
G. funiculosa | CNR28 | U. minor | Czech Republic | (-/+) |
G. langdonii | MK1643 | Scolytus multistriatus (on U. laevis) | Czech Republic | (-/+) |
G. langdonii | MK1644 | Scolytus multistriatus (on U. laevis) | Czech Republic | (-/+) |
G. lavendula | CCF3394 | Chaetopyelius vestitus (on Pistacia terebinthus) | Croatia | (-/+) |
G. obscura | MK86 | Scolytus intricatus (on Quercus robur) | Czech Republic | (-/+) |
G. omnicola | CNR5 | U. minor | Czech Republic | (-/+) |
G. omnicola | CNR21 | U. minor | Czech Republic | (-/+) |
G. pallida | MK1622 | S. kirschii (on U. minor) | Spain | (-/+) |
G. putterillii | CCF3342 | Scolytus rugulosus (on Prunus sp.) | Czech Republic | (-/+) |
Ophiostoma himal-ulmi | CBS374.67 | U. wallichiana | India | (-/-) |
O. novo-ulmi ssp. novo-ulmi | CKT11 | Ulmus sp. | Iran | (+/-) |
O. novo-ulmi ssp. novo-ulmi | R64 | Ulmus sp. | Romania | (+/-) |
O. novo-ulmi ssp. americana | H172 | Ulmus sp. | USA | (+/-) |
O. novo-ulmi ssp. americana | H363 | Ulmus sp. | Ireland | (+/-) |
O. quercus | CBS722.95 | Quercus sp. | Austria | (-/-) |
O. ulmi | E2 | Ulmus sp. | Netherlands | (-/-) |
O. ulmi | R21 | Ulmus sp. | Romania | (-/-) |
Epiccoccum sp. | F15 | Q. suber | Italy | (-/-) |
Cladosporium sp. | F11 | Q. suber | Italy | (-/-) |
Elm bark beetle (EBB) here means exclusively Scolytus multistriatus (Marsham), as it is the most common, active and effective DED vector in Italy and the only one found during sampling.
A total of 123 samples were collected from: i) wood of healthy elm trees; ii) dying elm trees showing DED symptoms (wood from newly-DED infected tissues, wood from old DED infections, living EBB larvae, living EBB pupae and wood frass from maternal and larval galleries); iii) EBB callow adults in flickering traps; and iv) adult females in galleries after oviposition (Table
Source | N° of collected samples | Species | Sample | Geographic orgin (Lat., Long.) |
---|---|---|---|---|
Healthy trees | 8 | Ulmus minor | Wood | Florence, Italy (43.772402°N, 11.176578°E) |
6 | U. minor | Wood | Sesto Fiorentino, Italy (43.817554°N, 11.188349°E) | |
New DED infection | 7 | U. minor | Wood | Siena, Italy (43.317361°N, 11.306896°E) |
4 | U. minor | Wood | Castelnuovo Berardenga, Italy (43.341865°N, 11.519271°E) | |
3 | U. minor | Wood | Asciano, Italy (43.296617°N, 11.460314°E) | |
Old DED Infections | 6 | U. minor | Wood | Bagno a Ripoli, Italy (43.734871°N, 11.324844°E) |
4 | U. minor | wood | Montelupo Fiorentino, Italy (43.720481°N, 10.988996°E) | |
3 | U. minor | Wood | Florence, Italy (43.811942°N, 11.240917°E) | |
2 | U. minor | Wood | Castagneto Carducci, Italy (43.194141°N, 10.567814°E) | |
2 | U. minor | Wood | Asciano, Italy (43.296617°N, 11.460314°E) | |
2 | U. minor | Wood | Poggibonsi, Italy (43.476425°N, 11.180486°E) | |
2 | U. minor | Wood | Castelnuovo di Val di Cecina, Italy (43.267503°N, 10.960795°E) | |
1 | U. minor | Wood | Asciano, Italy (43.296617°N, 11.460314°E) | |
1 | U. minor ‘CEM187’ | Wood | Bagno a Ripoli, Italy (43.734871°N, 11.324844°E) | |
1 | U. minor ‘CEM370’ | Wood | Bagno a Ripoli, Italy (43.734871°N, 11.324844°E) | |
1 | U. minor | Wood | Chiusdino, Italy (43.163653°N, 11.088422°E) | |
1 | U. minor | Wood | Castelnuovo Berardenga, Italy (43.341865°N, 11.519271°E) | |
Frass from EBB galleries | 4 | U. minor | Wood frass | Poggibonsi, Italy (43.476425°N, 11.180486°E) |
3 | U. minor | Wood frass | Castelnuovo di Val di Cecina, Italy (43.267503°N, 10.960795°E) | |
3 | U. minor | Wood frass | Sesto Fiorentino, Italy (43.817554°N, 11.188349°E) | |
2 | U. minor | Wood frass | Chiusdino, Italy (43.163653°N, 11.088422°E) | |
EBB larvae | 5 | Scolytus multistriatus | Larvae | Montelupo fiorentino, Italy (43.720481°N, 10.988996°E) |
3 | S. multistriatus | Larvae | Castelnuovo di Val di Cecina, Italy (43.267503°N, 10.960795°E) | |
2 | S. multistriatus | Larvae | Chiusdino, Italy (43.163653°N, 11.088422°E) | |
EBB pupae | 4 | S. multistriatus | Pupae | Castelnuovo di Val di Cecina, Italy (43.267503°N, 10.960795°E) |
2 | S. multistriatus | Pupae | Montelupo fiorentino, Italy (43.720481°N, 10.988996°E) | |
2 | S. multistriatus | Pupae | Chiusdino, Italy (43.163653°N, 11.088422°E) | |
EBB in the galleries | 5 | S. multistriatus | Insect | Asciano, Italy (43.296617°N, 11.460314°E) |
2 | S. multistriatus | Insect | Castagneto Carducci, Italy (43.194141°N, 10.567814°E) | |
1 | S. multistriatus | Insect | Florence, Italy (43.811942°N, 11.240917°E) | |
1 | S. multistriatus | Insect | Montelupo fiorentino, Italy (43.720481°N, 10.988996°E) | |
EBB callow adult | 11 | S. multistriatus | Insect | Sesto Fiorentino, Italy (43.817554°N, 11.188349°E) |
6 | S. multistriatus | Insect | Montelupo fiorentino, Italy (43.720481°N, 10.988996°E) | |
4 | S. multistriatus | Insect | Florence, Italy (43.811942°N, 11.240917°E) | |
4 | S. multistriatus | Insect | Castagneto Carducci, Italy (43.194141°N, 10.567814°E) | |
2 | S. multistriatus | Insect | Chiusdino, Italy (43.163653°N, 11.088422°E) | |
1 | S. multistriatus | Insect | Bagno a Ripoli, Italy (43.734871°N, 11.324844°E) | |
1 | S. multistriatus | Insect | Florence, Italy (43.772402°N, 11.176578°E) | |
1 | S. multistriatus | Insect | Vaglia, Italy (43.890112°N, 11.339246°E) |
Each woody sample (approx. 100 mg fresh weight from each collected tree and frass) and each insect sample (approx. 5.4 mg fresh weight –containing up to 4 larvae or pupae collected alive) was transferred into 2-ml microfuge tubes (Sarstedt), each containing two tungsten beads (Qiagen) and ground with a Mixer Mill 300 (Qiagen) (2 min; 20 Hz). DNA extraction was performed by using the E.Z.N.A. Plant DNA Minikit (Omega Bio-tek), according to the manufacturer’s instructions.
Total DNA from each adult S. multistriatus beetle collected from flickering traps, as well as in mother and larval galleries, was extracted singly or in batches of four when it came to the beetles collected in the multi-funnel trap. No surface sterilisation was carried out. Beetles were ground by using Mixer Mill 300 (Qiagen) and DNA from the insect’s body was extracted by using the E.Z.N.A. Insect DNA Minikit (Omega Bio-tek), following the manufacturer’s instructions.
Total DNA was checked by agarose gel electrophoresis and was quantified using the Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies). The quality of DNA extracted from elm woody tissue was checked using a SYBR-Green real-time PCR endogenous control for the actin gene, following
Two sets of primers and TaqMan minor groove binding (MGB) probes were newly designed to obtain genus-specific Geosmithia and species-specific Ophiostoma novo-ulmi qPCR markers.
The recently-described G. funiculosa (
Primer and TaqMan MGB probes were designed using Primer Express Software 3.0 (Applied Biosystems Foster City, CA, USA), on the basis of the internal transcribed spacer (ITS2) region of Geosmithia funiculosa (accession n. KR229885 – isolate CNR28) and ITS1 region for O. novo-ulmi ssp. americana (accession n. EF429091 – isolate 182E). The TaqMan MGB probes were labelled with the reporter dyes 6-carboxyfluorescein (FAM) and VIC at the 5’ end and a minor groove binder non-fluorescent quencher (MGBNFQ) at the 3’ end. Primers and probes sequences were reported in Table
Target | Primers and probes | Sequences (5’-3’)a | Amplicon length (bp) | Tm (°C) b |
---|---|---|---|---|
Ophiostoma novo-ulmi | OphF (Forward primer) | GCCGCCCGAACCTTTT | 60 | 58 58 68 |
OphR (Reverse primer) | TGGCTGTTTTTGTTTGTTTCTCA | |||
OphPr (TaqMan MGB probe) | VIC-AAACCAGTAACGAAACGT-MGBNFQ | |||
Geosmithia spp. | GeoF (Forward primer) | CGCCGTAAAACCCCAACTT | 61 | 59 58 69 |
GeoR (Reverse primer) | GTTCAGCGGGTATTCCTACTTGA | |||
GeoPr (TaqMan MGB probe) | FAM-ACCAAGGTTGACCTCG-MGBNFQ |
Homology of the amplicon sequence (both for Geosmithia spp. and Ophiostoma novo-ulmi) with the sequences of other species in the NCBI database was performed using standard nucleotide BLAST (BLASTn) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Primers were synthesised by Eurofins Genomics (Ebersberg, Germany) and probes by Applied Biosystems (Foster City, CA, USA). Specificity of the primers and probes was also tested by qPCR on DNA from axenic cultures (Table
Real-time PCR was assayed in MicroAmp Fast 96-well Reaction Plates (0.1 ml) closed with optical adhesive and using the StepOnePlus Real-Time PCR System (Applied Biosystems, Life Science, Foster City, CA, USA). Singleplex and duplex qPCR mixtures and thermocycler conditions were tested in this study (data not shown) in order to determine optimal qPCR conditions for the two target pathogens, which were finally set up as follows.
Duplex qPCR was performed in a 25 μl final volume containing: 12.5 μl TaqMan Universal Master Mix (Applied Biosystems,), 300 nM each forward primer (OphF and GeoF), 300 nM each reverse primer (OphR and GeoR), 200 nM each TaqMan MGB probe (OphPr and GeoPr) and 5μl genomic DNA. Each DNA sample was assayed in three replicates. Three wells, each containing 5 μl of sterile water, were used as the no-template control (NTC). For singleplex qPCR assay, only one primer set and one TaqMan MGB probe were used and sterile ddH2O was added to reach the final volume (25 µl). The PCR protocol was 50 °C (2 min), 95 °C (10 min), 45 cycles of 95 °C (30 s) and 60 °C (1 min).
Data results were analysed using the software SDS 1.9 Sequence Detection System (Applied Biosystems) after manual adjustment of the baseline and fluorescence threshold.
The specificity of primers and probes (genus-specific for Geosmithia and species-specific for Ophiostoma novo-ulmi) were tested both in singleplex and duplex qPCRs using DNA (at final concentration of 5 ng μl–1) from axenic cultures of other strains and species of the target organisms, as well as of closely-related species associated with elm and ubiquitous species (Table
The standard curve was generated using DNA from strain CNR28 (G. funiculosa) and strain 182E (O. novo-ulmi ssp. americana) as standards. For each target species, standard points (ranging from 5 ng μl–1 to 2 fg μl–1) were made using ten 1:5 serial dilutions of standard DNA of both target fungi. Each standard curve was built with standards run in both singleplex and duplex qPCR. The minimum amount of template DNA (limit of detection, LOD) that yielded 100% positive results with the singleplex and duplex assay (qPCR sensitivity) was determined. Three replicates of each dilution were analysed and reactions were repeated at least twice. Quantification of both fungal species DNA in unknown samples was made by interpolation from standard curves generated with O. novo-ulmi and G. funiculosa DNA standards that were amplified in the same PCR run. Reproducibility of the qPCR assay was assessed by computing the coefficient of variation (CV) amongst the mean values in eight independent assays. PCR efficiency was calculated against the slope of the standard curve (Eff = 10 -1/slope -1) (
To evaluate the possible interference of plant DNA extract in the newly-designed qPCR assay, the same ten 1:5 serial dilutions (ranging from 5 ng μl–1 to 2 fg μl–1) of fungal DNA (O. novo-ulmi or Geosmithia spp.) were mixed with DNA extracted from healthy elm woody tissue (at 20 ng/tube final concentration) and run on the same qPCR plate of the standard curve (fungal DNA diluted in sterile ddH2O). All samples were run in triplicate as previously described.
To test the linearity and the sensitivity of each qPCR TaqMan protocol, two different ascospore serial dilutions were obtained from mycelium of axenic culture of Ophiostoma novo-ulmi (strain 182E) and Geosmithia funiculosa (strain CNR28). Fungal isolates were grown on MEA media and, after five days, the presence of the ascospores was observed using a Zeiss Axioskop 50 optical microscope. Each ascospore suspension was obtained by scraping the surface of mycelium with a sterile scalpel and then placing it in 1 ml of sterile water. The number of ascospores per ml was determined in a Burker Chamber and, for each pathogen, six 1:10 serial dilutions (1:1 O. novo-ulmi 1.3 × 107 ascospores per ml; 1:1 G. funiculosa 5.6 × 106 ascospores per ml) were prepared. All suspension dilutions were centrifuged for 3 min at 12,000 rpm, the excess water was removed and the ascospore pellets were ground in a 1.5-ml Eppendorf tube using a micropestle (Eppendorf, Hamburg, Germany) in 500 µl of lysis buffer AP1 (EZNA Plant DNA, Omega Bio-tek) and DNA extraction continued with the recommended protocol provided by EZNA Plant DNA kit (Omega Bio-tek, Inc). For each ascospore dilution, 2.5 µl of extracted DNA was assayed using the StepOnePlus Real-Time PCR System (Applied Biosystems) as previously described.
For each fungal pathogen (Ophiostoma novo-ulmi and Geosmithia spp.), pairwise comparison of Cq values of standard points was conducted between duplex and singleplex using the chi-square (χ2) test. The Bland-Altman plot was used to determinate the agreement between the two assays (
BLAST search in NCBI showed 95–100% homology between the designed amplicon sequences and the sequence of Geosmithia species and Ophiostoma novo-ulmi deposited in GenBank.
All DNA from Geosmithia spp. isolates (Table
Ophiostoma novo-ulmi-specific assay successfully amplified DNA from all the O. novo-ulmi strains and it did not generate any amplicon DNA with other Ophiostoma species tested, including O. ulmi, Geosmithia spp. or any of other fungal species tested (Table
The standard curves generated with the singleplex and duplex assays did not significantly differ for Geosmithia spp. (χ2 = 0.612; df = 1; P = 0.43) or for O. novo-ulmi (χ2 = 0.167; df = 1; P = 0.68) (Fig.
Comparison between singleplex and duplex qPCR A standard curve of Geosmithia spp. and B Ophiostoma novo-ulmi generated with the singleplex (blue dots) and duplex (red dots). For each targeted gene, ten different 1:5 serial dilutions (ranging from 5 ng μl–1 to 2 fg μl–1) of Geosmithia spp. and O. novo-ulmi standard DNA were assayed in triplicate. Standard curves were generated by plotting the threshold quantification cycle value (Cq value) versus the logarithmic genomic DNA concentration of each dilution series. The Bland-Altman plot for Geosmithia spp. (C) and O. novo-ulmi (D) are shown for the same serial dilutions. The Cq difference between the two methods (ΔCqD-S) is plotted against the average of both methods (x-axis) for every individual pair of measurements. The interval of the mean of the difference ± 1.96 times the standard deviation (SD) defines the 95% interval of the limits of agreement.
The amplification efficiency of duplex qPCR assay was calculated from the slope value of the standard curves according to the equation previously described (
Efficiency, linear correlation and assay precision of duplex qPCR assay for the detection of Geosmithia spp. and O. novo-ulmi.
Fungi and variability experiment | Efficiency (%) | Linear correlation (R2) | Coefficient of variation % |
---|---|---|---|
Geosmithia spp. | |||
Intra assay | 95.3 | 0.999 | 1.18 ± 0.13 |
Inter assay | 92.8 | 0.999 | 1.3 ± 1.07 |
Ophiostoma novo-ulmi | |||
Intra assay | 96.8 | 0.999 | 1.19 ± 0.01 |
Inter assay | 92.3 | 0.999 | 1.06 ± 0.66 |
The limit of detection (LOD) of both duplex and singleplex qPCR assays were as low as 2 fg µl-1 for both Geosmithia spp. and O. novo-ulmi.
The duplex assay revealed no amplification difference between pure fungal DNA (Geosmithia spp. or O. novo-ulmi) in sterile water and the same amounts diluted in a mixture containing DNAs of different organisms (Geosmithia spp., O. novo-ulmi and DNAs from elm wood and insect).
All DNA samples were analysed by duplex qPCR for the quantification of Geosmithia spp. and O. novo-ulmi. No DNA of Geosmithia spp. or O. novo-ulmi was detected in any of the healthy elm samples analysed. Elm samples with recent or previous seasons’ infections showed the exclusive presence of O. novo-ulmi, with increasing amounts of the pathogen according to the stage of infection (from 18 pg DNA⁄μg total DNA in recent infections to 140 pg DNA⁄μg total DNA in older infections) (Fig.
Fungal DNA of O. novo-ulmi and Geosmithia spp. on analysed samples by using duplex qPCR assay A Mean of fungal DNA (pg DNA/μg total DNA) ± SEM (Standard Error of the Mean) B percentage presence of O. novo-ulmi and Geosmithia spp. DNA in plant tissues and EBB samples analysed.
Duplex qPCR results revealed the presence of both fungi in all EBB samples, collected in different stages of their biological cycle (including samples from frass collected in the galleries). In particular, significantly higher quantities of Geosmithia spp. DNA compared to O. novo-ulmi were found on female EBB collected after ovideposition (p < 0.0001, Fig.
DNA extracted from ascospore serial dilution showed a linear relationship for O. novo-ulmi (R2 = 0.999) and Geosmithia spp. (R2 = 0.999) (Fig.
Dutch Elm Disease is still causing massive damage in Europe and the death of elms is still catastrophic in ecological and economical terms through the loss of genetic diversity and trees lost from urban and natural forest stands (
The detection of fungi by traditional methods, such as isolation from plant tissues and insect bodies, may be sometimes challenging and time-consuming, seriously impairing our knowledge of their biological cycles. In addition, these methods do not allow quantification of the target organism. DNA sequence-based molecular tools, such as real-time PCR, digital PCR or, even if indirectly, LAMP (
Multiplex qPCR is an increasingly utilised method (
In this study, the developed and validated duplex qPCR assay was able to detect and quantify the presence of Geosmithia spp. and O. novo-ulmi from different matrices (frass and plant tissue; adults, larvae and pupae of bark beetles) collected from healthy and DED-symptomatic elms.
This duplex qPCR assay showed high reproducibility and specificity for both genus-specific Geosmithia spp. and species-specific O. novo-ulmi and high sensitivity (LODs 2 fg μl-1, for both fungi). This assay allowed the detection in elm trees of O. novo-ulmi infections before symptoms had fully developed, as well as the presence of Geosmithia spp. in different host tissues and on the insect body.
Our results confirm that Geosmithia is closely associated to EBB galleries, as also reported by
Our observations indicate that the humidity and temperature conditions within the subcortical galleries seem to promote the fitness of the fungi studied here, particularly Geosmithia. In addition, the results show that Geosmithia is always present in beetle galleries along the studied period, but the detected DNA quantity decreases significantly as the insect’s maturation progresses, i.e. from the time of ovideposition until the callow adults flicker.
This study confirms the association between bark beetles and Geosmithia, as also reported by other studies (
The elm bark beetles are generally unable to digest the lignin, cellulose and hemicellulose components that make up xylem tissues (
EBBs are the main vectors of Geosmithia spores on their body and maybe use the fungus as a complement to their nutrition, especially during the larval and pupal stages of their life cycle that takes place within the galleries under the elm bark. However, more studies are needed to confirm this hypothesis.
The callow adults complete their maturation over a few days by digging short feeding burrows in the phloem of the twig and sapwood of healthy elms (
None of the target DNAs was detected in healthy elm tissues and only O. novo-ulmi DNA was detected in DED-symptomatic plants, confirming that Geosmithia does not adapt to the conditions of living plants tissues or even in xylem of plants with early DED symptoms (
These findings show that this fungus is not an endophyte, at least in elm. Instead, Geosmithia was detected in abundance on EBB bodies and in EBB tunnels in decaying plants. Our analyses suggest that the presence of this fungus is mostly associated with the breeding activity of the vector insect on elm trees as already observed in other studies (
In conclusion, the duplex qPCR technique developed in this work is extremely sensitive and able to specifically identify and quantify the presence of both O. novo-ulmi and Geosmithia spp. in plants with different levels of DED symptoms, on EBB larvae, pupae and wood frass from maternal and larval galleries and on the body of callow adult insects, providing better insight into the dynamics of this complex fungus-fungus association mediated by S. multistriatus. This work provided solid data on the actual DNA quantity of the two fungi at the different steps of the DED cycle, thus gaining a better understanding of the role and interactions occurring amongst all the pathosystem players.
Dutch elm disease continues to be extremely damaging on planted and natural elm stands in Europe. Critical thresholds comparable to those that led to the decline of the first epidemic do not appear to have been reached and the current disease dynamic seems likely to continue.
Moreover, an increasingly warming climate could have a great influence on beetle epidemics, their aggression, population dynamics and migration (
Several aspects of O. novo-ulmi-Geosmithia-Scolytus interactions within the DED pathosystem need to be further studied and more in-depth information on the biological cycle of Geosmithia spp. during the flickering period of new generations will be essential to use this fungus as a biocontrol agent of DED and finally allow European elms to re-populate the landscape.
This study was supported by the HOMED project (http://homed-project.eu/), which received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 771271. We thank Dr. Miroslav Kolařík for providing Geosmithia spp. isolates from Czech Republic, Croatia and Spain. Authors wish to warmly thank Dr. Matthew Duncan Haworth for English language editing. Authors wish to thank the anonymous referees who helped to significantly improve the manuscript.