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 Brasier, i.e. ssp. novo-ulmi and ssp. americana, previously known as Eurasian (EAN) and North American (NAN) races, respectively (Brasier and Kirk 2001). These ascomycetes are responsible for the ongoing DED pandemic; since the 1970s, they have replaced the less aggressive O. ulmi (Buisman) Nannf. that caused the first DED pandemic at the beginning of the last century (Spierenburg 1921).

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. (Webber and Brasier 1984). Specifically, S. scolytus (F.) and S. multistriatus (Marsham), the large and small elm bark beetles (EBB), respectively, are the most common and important species spreading the pathogen worldwide (Webber and Kirby 1983; Webber and Brasier 1984; Webber and Gibbs 1989; Webber 1990, 2000; Faccoli 2001, 2004). The small EBB is the main vector in the Mediterranean area (Santini and Faccoli 2015). During spring, at the time of beetle flight, host plants are more prone to be infected and temperatures are favourable for fungal growth in plant tissue, enhancing the pathogen’s aggressiveness (Santini and Faccoli 2015). Callow adults, carrying the O. novo-ulmi conidia, feed at the crotches of 1–2 years-old twigs of adult healthy elm trees to complete their sexual development, thus inoculating the pathogen. Once inoculated, the spores germinate into a growing mycelium and reach the xylem, where the fungus moves into the vessels (Webber and Brasier 1984), inducing the formation of tyloses and gels in the xylem vessels (Stipes and Campana 1981; Rioux et al. 1998; Ouellette et al. 2004a, b; Et-Touil et al. 2005) as a defence response. Later, the beetles move to dying elms to lay eggs in the inner bark of the trunks or branches, which provide the optimal environment for larval development (Rudinsky 1962) and fruiting of the pathogen (Webber and Brasier 1984). New contaminated beetles emerge from the bark to complete the cycle.

Recently, it has been highlighted that other organisms also play roles in the DED pathosystem (Pepori et al. 2018). Some fungi of the genus Geosmithia, a monophyletic morphogenus of anamorphic ascomycetes mainly associated with phloem-feeding bark beetles (Kolařík et al. 2004, 2005, 2007, 2008; Kubátová et al. 2004; Kolařík and Jankowiak 2013; McPherson et al. 2013; Jankowiak et al. 2014; Machingambi et al. 2014; Huang et al. 2019; Crous et al. 2022; Meshram et al. 2022), are consistently found in infected elms (Kolařík et al. 2004, 2005, 2007, 2008; Pepori et al. 2015; Huang et al. 2019; Strzałka et al. 2021; Crous et al. 2022).

Geosmithia spp., like O. novo-ulmi, are associated with elm bark beetles (Pepori et al. 2015, 2018) and can similarly be found in beetle larval galleries – thus sharing habitat with O. novo-ulmi – but the ecological niches of these fungi are different. A widespread horizontal gene transfer of the cerato-ulmin gene between O. novo-ulmi and Geosmithia species has been reported (Bettini et al. 2014).

Pepori et al. (2018) demonstrated the existence of a close and stable relationship, which can be classified as mycoparasitism by Geosmithia spp. towards O. novo-ulmi. There are still several gaps in defining the life cycle and lifestyle of elm-related Geosmithia species, especially when they cross and interact with the life cycle of DED fungi.

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 (Webber and Gibbs 1984; Webber and Hedger 1986; Sutherland and Brasier 1995; Brasier 2000; Griffin 2000; Hintz et al. 2013; Ganley and Bulman 2016).

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.

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 (Santini and Faccoli 2015).

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 (Hardinge and Murray 2020) and HTS, are increasingly used to enable accurate and specific detection and quantification from any substrate (Lindahl et al. 2013).

Multiplex qPCR is an increasingly utilised method (Bonants and te Witt 2017; Luchi et al. 2018; Rizzo et al. 2020) allowing simultaneous detection of different microorganisms in the same reaction, thus significantly reducing both the quantity of samples and the overall cost of the analysis. The use of a multiplex assay may prove particularly important to distinguish pathogens that cause similar symptoms, as in the case of Fusarium circinatum and Caliciopsis pinea, which cause comparable symptoms on Pinus radiata (Luchi et al. 2018) or the study of the four European species of Heterobasidion that attack conifers (Ioos et al. 2019).

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 Kolařík et al. (2008), showing extremely high amounts on the EBB female bodies and in maternal gallery frass.

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 (Kolařík et al. 2008, 2017; Pepori et al. 2018; Huang et al. 2019) and highlights that this association is constant throughout the life of the bark beetle and is not only specific to the subcortical developmental stage. Moreover, fungi benefit from this association because the beetles transport them to new host plants (Paine et al. 1997; Six 2003; Six and Wingfield 2011) and prepare a suitable habitat for their growth. In the galleries dug by insects, the fungi become metabolically more active because they have access to a constant supply of nutrients such as decaying wood (Stokland et al. 2012).

The elm bark beetles are generally unable to digest the lignin, cellulose and hemicellulose components that make up xylem tissues (Dadd 1970; Geib et al. 2008) and feed primarily on the phloem. However, for some phloem-feeding beetles, phloem tissues remain relatively low in usable nitrogen and sterols and, thus, the associated fungi can serve as a complementary source of nutrients (Six 2012). It has been observed that symbiotic fungi are able to access nitrogen stored in the sapwood and translocate it into the phloem where the larvae and pupae of bark beetle feed (Stokland et al. 2012). Bark beetles and ambrosia beetles, as reported also by Kolařík and Kirkendall (2010) and Veselská and Kolařík (2015), use these fungi as principal nutritional symbionts and recently new Geosmithia species associated with ambrosia beetles have been described in a tropical forest in Costa Rica (Kolařík and Kirkendall 2010).

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 (Fransen 1939; Webber and Brasier 1984), where they deposit the DED fungal spores. This study shows that these insects carry large quantities of Geosmithia and much less of O. novo-ulmi (Fig. 3). The spores of the latter reach the xylem and move in the vessels through a phase of yeast multiplication (Webber and Brasier 1984), giving rise to the infection process. Geosmithia, at least in this first phase, is not detectable and this could mean that it does not find optimal conditions to spread or it is translocated in other parts of the plant. In fact, although the new insects flicker from the bark of dying elms carrying 99% Geosmithia spores, to the xylem of elm trees experiencing new attacks, we found only the presence of the DED pathogen. These results are in contrast with those reported by Pepori et al. (2018), who found that the artificial inoculation of both fungi in the same elm clone resulted in significantly lower symptoms than single inoculations of O. novo-ulmi. Maybe the reason lies in the fact that artificial inoculations, generally performed in the internodal section of the twig, circumvent the natural plant reaction, while beetles dig their burrow at the twig crotches (Santini and Faccoli 2015).

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 (Pepori et al. 2018).

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 (Kolařík et al. 2007, 2008; Kolařík and Jankowiak 2013).

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 (Bentz and Jönsson 2015), allowing the expansion of the DED epidemic to more northern latitudes (Jürisoo et al. 2019, 2021).

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.