# Tree diversity reduces co-infestation of Douglas fir by two exotic pests and pathogens

Alex Stemmelen, Bastien Castagneyrol, Quentin Ponette, Simone Prospero, Gilles San Martin, Salome Schneider, Hervé Jactel

► To cite this version:

Alex Stemmelen, Bastien Castagneyrol, Quentin Ponette, Simone Prospero, Gilles San Martin, et al. Tree diversity reduces co-infestation of Douglas fir by two exotic pests and pathogens. NeoBiota, 2023, 84, pp.397 - 413. 10.3897/neobiota.84.94109. hal-04513412

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# Tree diversity reduces co-infestation of Douglas fir by two exotic pests and pathogens

Alex Stemmelen, Bastien Castagneyrol, Quentin Ponette, Simone Prospero, Gilles San Martin, Salome Schneider, Hervé Jactel

► To cite this version:

Alex Stemmelen, Bastien Castagneyrol, Quentin Ponette, Simone Prospero, Gilles San Martin, et al. Tree diversity reduces co-infestation of Douglas fir by two exotic pests and pathogens. NeoBiota, 2023, 84, pp.397 - 413. 10.3897/neobiota.84.94109. hal-04513412

HAL Id: hal-04513412

https://hal.inrae.fr/hal-04513412v1

Submitted on 20 Mar 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Tree diversity reduces co-infestation of Douglas fir by two exotic pests and pathogens

Alex Stemmelen¹, Bastien Castagneyrol¹, Quentin Ponette², Simone Prospero³, Gilles San Martin⁴, Salome Schneider³, Hervé Jactel¹

1 INRAE, University of Bordeaux, UMR Biogeco, F-33612 Cestas, France 2 UCLouvain – Université catholique de Louvain, Earth & Life Institute, Croix du Sud 2, box L7.05.09, 1348 Louvain-la-Neuve, Belgium 3 Swiss Federal Research Institute WSL, Zürcherstrasse 111, CH-8903 Birmensdorf, Switzerland 4 Walloon Agricultural Research Centre (CRA-W), Gembloux, Belgium

Corresponding author: Alex Stemmelen (alex.stemmelen@inrae.fr)

Academic editor: M. Branco | Received 26 August 2022 | Accepted 11 January 2023 | Published 18 May 2023

**Citation:** Stemmelen A, Castagneyrol B, Ponette Q, Prospero S, San Martin G, Schneider S, Jactel H (2023) Tree diversity reduces co-infestation of Douglas fir by two exotic pests and pathogens. 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: 397–413. https://doi.org/10.3897/neobiota.84.94109

## Abstract

The number of non-native invasive pests and pathogens has increased dramatically in recent years, with disastrous consequences for the health of forests worldwide. Multiple studies have shown that mixed forests may suffer less damage from insect pests than single species forest. This “associational resistance” can be notably explained by the fact that heterospecific neighbours make it more difficult for herbivores to locate and then exploit their host tree. However, the validity of these findings in the case of non-native, invasive pests and pathogens remains to be demonstrated. In this study, we monitored over two hundred Douglas firs in pure and mixed plots of a tree diversity experiment to assess the damage from the non-native gall midge *Contarinia pseudotsugae* and the non-native needle cast *Nothophaecryptopus gaeumannii*. The probability of Swiss needle-cast infection was lower in Douglas fir trees surrounded by heterospecific neighbours. Gall midge damage was lower on Douglas firs surrounded by taller neighbours, consistent with the hypothesis of reduced host Apparency. Douglas fir trees that were more damaged by *C. pseudotsugae* were also more often infected by *N. gaeumannii*. Our study thus provides partial support of the associational resistance hypothesis of mixed forests against exotic pests and pathogens. Promoting forest species diversity at the stand level could, therefore, offer interesting prospects for reducing the impact of biological invasions, especially those involving both pests and pathogens.

## Keywords

Douglas fir, exotic pathogen, exotic pest, resource concentration, tree apparency, tree diversity
# Tree diversity reduces co-infestation of Douglas fir by two exotic pests and pathogens

Alex Stemmelen¹, Bastien Castagneyrol¹, Quentin Ponette², Simone Prospero³, Gilles San Martin⁴, Salome Schneider³, Hervé Jactel¹

1 INRAE, University of Bordeaux, UMR Biogeco, F-33612 Cestas, France 2 UCLouvain – Université catholique de Louvain, Earth & Life Institute, Croix du Sud 2, box L7.05.09, 1348 Louvain-la-Neuve, Belgium 3 Swiss Federal Research Institute WSL, Zürcherstrasse 111, CH-8903 Birmensdorf, Switzerland 4 Walloon Agricultural Research Centre (CRA-W), Gembloux, Belgium

Corresponding author: Alex Stemmelen (alex.stemmelen@inrae.fr)

Academic editor: M. Branco | Received 26 August 2022 | Accepted 11 January 2023 | Published 18 May 2023

**Citation:** Stemmelen A, Castagneyrol B, Ponette Q, Prospero S, San Martin G, Schneider S, Jactel H (2023) Tree diversity reduces co-infestation of Douglas fir by two exotic pests and pathogens. 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: 397–413. https://doi.org/10.3897/neobiota.84.94109

## Abstract

The number of non-native invasive pests and pathogens has increased dramatically in recent years, with disastrous consequences for the health of forests worldwide. Multiple studies have shown that mixed forests may suffer less damage from insect pests than single species forest. This “associational resistance” can be notably explained by the fact that heterospecific neighbours make it more difficult for herbivores to locate and then exploit their host tree. However, the validity of these findings in the case of non-native, invasive pests and pathogens remains to be demonstrated. In this study, we monitored over two hundred Douglas firs in pure and mixed plots of a tree diversity experiment to assess the damage from the non-native gall midge *Contarinia pseudotsugae* and the non-native needle cast *Nothophaecryptopus gaeumannii*. The probability of Swiss needle-cast infection was lower in Douglas fir trees surrounded by heterospecific neighbours. Gall midge damage was lower on Douglas firs surrounded by taller neighbours, consistent with the hypothesis of reduced host Apparency. Douglas fir trees that were more damaged by *C. pseudotsugae* were also more often infected by *N. gaeumannii*. Our study thus provides partial support of the associational resistance hypothesis of mixed forests against exotic pests and pathogens. Promoting forest species diversity at the stand level could, therefore, offer interesting prospects for reducing the impact of biological invasions, especially those involving both pests and pathogens.

## Keywords

Douglas fir, exotic pathogen, exotic pest, resource concentration, tree apparency, tree diversity
**Introduction**

In the last decades, climate change and increasing global trade and travel have led to a dramatic increase in invasion by non-native forest insects and pathogens (Parmesan 2006; Jactel et al. 2020). Many of those invasive alien species, such as the emerald ash borer (Poland and McCullough 2006), Asian longhorn beetle (Haack et al. 2010), pinewood nematode (Kim et al. 2020) or Swiss needle cast (Hansen et al. 2000) are often responsible for tremendous damage in forests around the world. Invasive pests and pathogens are of great concern not only because of the severe economic damage they generate (Aukema et al. 2011; Haight et al. 2011; Zenni et al. 2021), but also because they threaten the integrity and vitality of forests, thereby limiting the provision of important ecosystem services, such as climate change mitigation, habitat for forest species, pollination or biomass production (Brockerhoff et al. 2017; Griscom et al. 2017; Mori et al. 2017). Hence, while there is a need to reduce the rate of introduction of non-native species, it is also essential to improve our knowledge of the mechanisms that can help mitigate their damage in forests.

Studies from agricultural systems have shown that plant diversity can lead to a de-
crease in insect damage (Barbosa et al. 2009). This phenomenon, called “associational
resistance”, has also received consistent support in forests, where trees are less prone to
herbivory when growing in mixtures compared to monocultures (Guyot et al. 2019;
Jactel et al. 2021; Ward et al. 2022). Two non-exclusive hypotheses have been proposed
to explain associational resistance (Jactel et al. 2021): the natural enemies’ hypothesis
(Root 1973; Stemmelen et al. 2022) and the resource concentration hypothesis (Ham-
bäck and Englund 2005). The natural enemies’ hypothesis states that richer plant com-
munities can favour herbivore’s natural enemies by increasing the availability of suit-
able microhabitats and the amount of complementary prey or alternative resources,
such as pollen or nectar and will result in a better control of herbivores. On the other
hand, the resource concentration hypothesis states that herbivores will be more likely
to immigrate to and less likely to emigrate from monospecific forest patches due to
higher resource availability. Another consequence of growing trees in monoculture is
that host trees are more accessible, i.e. more easily located by their herbivores. Follow-
ing the “host apparency” theory (Castagneyrol et al. 2013), host trees surrounded by
non-host trees that are taller or emit repellent volatile organic compounds are less likely
to be colonised by specialist herbivores (Castagneyrol et al. 2013; Haase et al. 2015).

The mechanisms driving associational resistance effects are further complicated
by overlooked horizontal interactions between herbivores and between herbivores and
plant pathogens. For example, a meta-analysis by Fernandez-Conradi et al. (2018a)
reports that, on average, plant infestation with pathogenic fungi alters host foraging
behaviour and reduces insect herbivore performance. Yet, pathogenic fungi are also
influenced by tree diversity, which has been well documented in the case of the in-
teraction between oaks and powdery mildew (Field et al. 2020). It follows that any
effect of tree diversity on fungal pathogens likely alters the strength and direction of
**Introduction**

In the last decades, climate change and increasing global trade and travel have led to a dramatic increase in invasion by non-native forest insects and pathogens (Parmesan 2006; Jactel et al. 2020). Many of those invasive alien species, such as the emerald ash borer (Poland and McCullough 2006), Asian longhorn beetle (Haack et al. 2010), pinewood nematode (Kim et al. 2020) or Swiss needle cast (Hansen et al. 2000) are often responsible for tremendous damage in forests around the world. Invasive pests and pathogens are of great concern not only because of the severe economic damage they generate (Aukema et al. 2011; Haight et al. 2011; Zenni et al. 2021), but also because they threaten the integrity and vitality of forests, thereby limiting the provision of important ecosystem services, such as climate change mitigation, habitat for forest species, pollination or biomass production (Brockerhoff et al. 2017; Griscom et al. 2017; Mori et al. 2017). Hence, while there is a need to reduce the rate of introduction of non-native species, it is also essential to improve our knowledge of the mechanisms that can help mitigate their damage in forests.

Studies from agricultural systems have shown that plant diversity can lead to a de-
crease in insect damage (Barbosa et al. 2009). This phenomenon, called “associational
resistance”, has also received consistent support in forests, where trees are less prone to
herbivory when growing in mixtures compared to monocultures (Guyot et al. 2019;
Jactel et al. 2021; Ward et al. 2022). Two non-exclusive hypotheses have been proposed
to explain associational resistance (Jactel et al. 2021): the natural enemies’ hypothesis
(Root 1973; Stemmelen et al. 2022) and the resource concentration hypothesis (Ham-
bäck and Englund 2005). The natural enemies’ hypothesis states that richer plant com-
munities can favour herbivore’s natural enemies by increasing the availability of suit-
able microhabitats and the amount of complementary prey or alternative resources,
such as pollen or nectar and will result in a better control of herbivores. On the other
hand, the resource concentration hypothesis states that herbivores will be more likely
to immigrate to and less likely to emigrate from monospecific forest patches due to
higher resource availability. Another consequence of growing trees in monoculture is
that host trees are more accessible, i.e. more easily located by their herbivores. Follow-
ing the “host apparency” theory (Castagneyrol et al. 2013), host trees surrounded by
non-host trees that are taller or emit repellent volatile organic compounds are less likely
to be colonised by specialist herbivores (Castagneyrol et al. 2013; Haase et al. 2015).

The mechanisms driving associational resistance effects are further complicated
by overlooked horizontal interactions between herbivores and between herbivores and
plant pathogens. For example, a meta-analysis by Fernandez-Conradi et al. (2018a)
reports that, on average, plant infestation with pathogenic fungi alters host foraging
behaviour and reduces insect herbivore performance. Yet, pathogenic fungi are also
influenced by tree diversity, which has been well documented in the case of the in-
teraction between oaks and powdery mildew (Field et al. 2020). It follows that any
effect of tree diversity on fungal pathogens likely alters the strength and direction of
associational effects on insect herbivores (Field et al. 2020). However, since many plant pathogens are vectored by insects or simply benefit from mechanical injuries made by insect mouthparts to plants, the inverse relationship may also be true. It is unsure whether the same mechanisms that are effective against native herbivores are symmetrically efficient against introduced pests. On the contrary, the fact that introduced species have not co-evolved with trees, herbivores and enemies in their introduced range likely alter the effect of tree diversity (Brockerhoff and Liebhold 2017; Nunez-Mir et al. 2017). For example, biological control can be expected to be enhanced in mixed forests only if native predators or parasitoids are generalist enough to switch to exotic preys (Fernandez-Conradi et al. 2018b). If not, then even an increase in the diversity of herbivores' enemies in mixed stands may not be sufficient to increase biological control of exotic pests. On the other hand, the dilution or diversion effect of host trees by non-host trees in mixed species forests can be expected to be more powerful against exotic herbivores that have not co-evolved with these native tree species.

*Contarinia pseudotsugae* Condrashoff (Cecidomyiidae) is an exotic invasive forest pest in western Europe, originating from North America where it causes damage on Douglas fir (*Pseudotsuga menziesii* Franco), a coniferous tree species also originating from North America (Condrashoff 1961; Roques et al. 2019). It is part of a complex of three species of needle midge, the others being *Contarinia constricta* Condrashoff and *Contarinia cuniculator* Condrashoff (henceforth often referred to as *Contarinia* spp.). In Europe, it was first reported in Belgium and the Netherlands in 2015, from where it spread to France and Germany in 2016 (EPPO 2019; Wilson et al. 2020). Adults lay eggs within opening buds. Few days later, eggs hatch and larvae enter the needles, inducing the formation of galls. Damaged needles tend to bend, gradually change colour and to fall prematurely. Although it is not considered a mortality-inducing factor on its own, heavy infestation by *C. pseudotsugae* can lead to severe defoliation, in association with other pests or pathogens and could threaten the health of Douglas fir trees or impair their growth (EPPO 2019; Ligot et al. 2020; Wilson et al. 2020).

One of the main pathogens frequently co-occurring with *C. pseudotsugae* on Douglas fir is the fungus *Nothophaecryptopus gaeumannii* (T. Rohde) Videira, C. Nakash., which causes a foliar disease called Swiss needle cast. Although discovered in Switzerland in 1925, this pathogen is native to North America – hence its misleading common name – and is nowadays present in Europe and Australasia (Hood and Kimberley 2005; Kimberley et al. 2011). Symptoms develop slowly, but lead to chlorosis, necrosis and premature fall of the infected needles (Black et al. 2010; Wilson et al. 2020). It is well known that insects can act as vectors for fungal pathogens, either directly or indirectly by wounding the plants and promoting the infection (Almeida and Purcell 2005). Larvae of *C. pseudotsugae* induce the production of new plant tissue during the cecidogenous process and might alter the ability of fungi to invade needle tissues. However and despite the fact that *C. pseudotsugae* and *N. gaeumannii* symptoms often co-occur on Douglas fir, no study so far has investigated the potential interaction dynamics between the pest and the pathogen.
associational effects on insect herbivores (Field et al. 2020). However, since many plant pathogens are vectored by insects or simply benefit from mechanical injuries made by insect mouthparts to plants, the inverse relationship may also be true. It is unsure whether the same mechanisms that are effective against native herbivores are symmetrically efficient against introduced pests. On the contrary, the fact that introduced species have not co-evolved with trees, herbivores and enemies in their introduced range likely alter the effect of tree diversity (Brockerhoff and Liebhold 2017; Nunez-Mir et al. 2017). For example, biological control can be expected to be enhanced in mixed forests only if native predators or parasitoids are generalist enough to switch to exotic preys (Fernandez-Conradi et al. 2018b). If not, then even an increase in the diversity of herbivores' enemies in mixed stands may not be sufficient to increase biological control of exotic pests. On the other hand, the dilution or diversion effect of host trees by non-host trees in mixed species forests can be expected to be more powerful against exotic herbivores that have not co-evolved with these native tree species.

*Contarinia pseudotsugae* Condrashoff (Cecidomyiidae) is an exotic invasive forest pest in western Europe, originating from North America where it causes damage on Douglas fir (*Pseudotsuga menziesii* Franco), a coniferous tree species also originating from North America (Condrashoff 1961; Roques et al. 2019). It is part of a complex of three species of needle midge, the others being *Contarinia constricta* Condrashoff and *Contarinia cuniculator* Condrashoff (henceforth often referred to as *Contarinia* spp.). In Europe, it was first reported in Belgium and the Netherlands in 2015, from where it spread to France and Germany in 2016 (EPPO 2019; Wilson et al. 2020). Adults lay eggs within opening buds. Few days later, eggs hatch and larvae enter the needles, inducing the formation of galls. Damaged needles tend to bend, gradually change colour and to fall prematurely. Although it is not considered a mortality-inducing factor on its own, heavy infestation by *C. pseudotsugae* can lead to severe defoliation, in association with other pests or pathogens and could threaten the health of Douglas fir trees or impair their growth (EPPO 2019; Ligot et al. 2020; Wilson et al. 2020).

One of the main pathogens frequently co-occurring with *C. pseudotsugae* on Douglas fir is the fungus *Nothophaecryptopus gaeumannii* (T. Rohde) Videira, C. Nakash., which causes a foliar disease called Swiss needle cast. Although discovered in Switzerland in 1925, this pathogen is native to North America – hence its misleading common name – and is nowadays present in Europe and Australasia (Hood and Kimberley 2005; Kimberley et al. 2011). Symptoms develop slowly, but lead to chlorosis, necrosis and premature fall of the infected needles (Black et al. 2010; Wilson et al. 2020). It is well known that insects can act as vectors for fungal pathogens, either directly or indirectly by wounding the plants and promoting the infection (Almeida and Purcell 2005). Larvae of *C. pseudotsugae* induce the production of new plant tissue during the cecidogenous process and might alter the ability of fungi to invade needle tissues. However and despite the fact that *C. pseudotsugae* and *N. gaeumannii* symptoms often co-occur on Douglas fir, no study so far has investigated the potential interaction dynamics between the pest and the pathogen.
In this study, we assessed the effect of forest mixtures on damage caused to Douglas fir by the exotic pest *C. pseudotsugae* and the exotic pathogen *N. gaeumannii*, as well as the potential effect of *C. pseudotsugae* damage on the co-occurring infection by *N. gaeumannii*. We used a long-term tree diversity experiment to sample 207 Douglas firs of various height, located in plots of increasing tree diversity from monoculture to four species mixtures. We measured needle damage by the Douglas fir needle midge and infection by the Swiss needle cast to test the following hypotheses: (1) Needle damage by *C. pseudotsugae* and *N. gaeumannii* increases with increasing proportion of Douglas fir in forest plot (host concentration hypothesis); (2) Douglas firs taller than neighbouring trees suffer more needle damage than smaller ones (host Apparency hypothesis); (3) Damage by *C. pseudotsugae* increases the probability of infection by *N. gaeumannii*. In doing so, our study aimed at a better understanding of the ecological factors driving primary and subsequent invasion by exotic pests and pathogens in pure vs. mixed forests.

# Materials and methods

## Study site and tree selection

The study was conducted in Belgium, six years after the first detection of *C. pseudotsugae* in the country, in the tree diversity experiment FORBIO (Verheyen et al. 2013), belonging to the larger network TreeDivNet (Paquette et al. 2018). Specifically, the experiment took place on the site of Gedinne, located in the Ardennes (Belgium, 49°59'N, 4°58'E) and consisting of two sub-sites ca. 2 km apart (Gribelle and Gouverneurs). At each sub-site, the same pool of five species was planted: sycamore maple (*Acer pseudoplatanus* L.), European beech (*Fagus silvatica* L.), hybrid larch (*Larix x eurolepis*), sessile oak (*Quercus petraea* Liebl.) and Douglas fir. Each sub-site is composed of 42 (Gribelle) to 44 (Gouverneurs) plots of increasing tree species richness, ranging from 1–4 tree species (Fig. 1), with a total of twenty different compositions: all five monocultures, all five possible four-species combinations and a random selection of five two- and five three-species combinations (For more details on plot composition, see Verheyen et al. (2013)).

Tree species were planted at an equivalent proportion in mixtures (e.g. 50:50 in two-species mixtures). Even in mixture, trees were planted in monospecific patches of 3 × 3 trees, with patches arranged in a checkerboard pattern in the two-species mixtures and randomly distributed in the three- and four-species mixtures. Therefore, individual trees were surrounded by a varying number of conspecific neighbours, even in mixed plots in which the central tree of each monospecific patch was always surrounded by conspecific neighbours. Finally, four subplots were delimited in each plot and consisted in a 4 × 4 tree patches, where tree height and circumference are measured yearly.

In each of the 20 plots containing Douglas fir (10 plots in each sub-site), we randomly selected three of the four sub-plots in which tree height and circumference had been measured in the current year (2021). We sampled every Douglas fir present
In this study, we assessed the effect of forest mixtures on damage caused to Douglas fir by the exotic pest *C. pseudotsugae* and the exotic pathogen *N. gaeumannii*, as well as the potential effect of *C. pseudotsugae* damage on the co-occurring infection by *N. gaeumannii*. We used a long-term tree diversity experiment to sample 207 Douglas firs of various height, located in plots of increasing tree diversity from monoculture to four species mixtures. We measured needle damage by the Douglas fir needle midge and infection by the Swiss needle cast to test the following hypotheses: (1) Needle damage by *C. pseudotsugae* and *N. gaeumannii* increases with increasing proportion of Douglas fir in forest plot (host concentration hypothesis); (2) Douglas firs taller than neighbouring trees suffer more needle damage than smaller ones (host Apparency hypothesis); (3) Damage by *C. pseudotsugae* increases the probability of infection by *N. gaeumannii*. In doing so, our study aimed at a better understanding of the ecological factors driving primary and subsequent invasion by exotic pests and pathogens in pure vs. mixed forests.

# Materials and methods

## Study site and tree selection

The study was conducted in Belgium, six years after the first detection of *C. pseudotsugae* in the country, in the tree diversity experiment FORBIO (Verheyen et al. 2013), be-longing to the larger network TreeDivNet (Paquette et al. 2018). Specifically, the experiment took place on the site of Gedinne, located in the Ardennes (Belgium, 49°59'N, 4°58'E) and consisting of two sub-sites ca. 2 km apart (Gribelle and Gou-verneurs). At each sub-site, the same pool of five species was planted: sycamore maple (*Acer pseudoplatanus* L.), European beech (*Fagus silvatica* L.), hybrid larch (*Larix x eu-rolepis*), sessile oak (*Quercus petraea* Liebl.) and Douglas fir. Each sub-site is composed of 42 (Gribelle) to 44 (Gouverneurs) plots of increasing tree species richness, ranging from 1–4 tree species (Fig. 1), with a total of twenty different compositions: all five monocultures, all five possible four-species combinations and a random selection of five two- and five three-species combinations (For more details on plot composition, see Verheyen et al. (2013)).

Tree species were planted at an equivalent proportion in mixtures (e.g. 50:50 in two-species mixtures). Even in mixture, trees were planted in monospecific patches of 3 × 3 trees, with patches arranged in a checkerboard pattern in the two-species mixtures and randomly distributed in the three- and four-species mixtures. Therefore, individual trees were surrounded by a varying number of conspecific neighbours, even in mixed plots in which the central tree of each monospecific patch was always surrounded by conspecific neighbours. Finally, four subplots were delimited in each plot and consisted in a 4 × 4 tree patches, where tree height and circumference are measured yearly.

In each of the 20 plots containing Douglas fir (10 plots in each sub-site), we randomly selected three of the four sub-plots in which tree height and circumference had been measured in the current year (2021). We sampled every Douglas fir present
<figure><img src="image_2.png" /></figure>

**Figure 1.** Map of Belgium with the location and experimental set-up of the FORBIO Gedinne sub-sites. The tree species diversity per plot ranged from one species (white) to four species (dark grey).

in those subplots, for a total of 207 trees. Finally, we calculated the mean height difference between each selected Douglas fir and their direct neighbours, regardless of whether they were other Douglas firs or trees of the other three species (henceforth called “Apparency”), setting tree height to zero when neighbours of sampled Douglas fir were missing (dead).

## Needle damage by *Contarinia pseudotsugae*

On each sampled Douglas fir, we selected a branch, at mid-height of the tree crown and collected five current-year shoots, starting with the terminal shoot, then picking up every second lateral shoot down to the base of the branch. Shoots were then placed in paper bags, sealed and sent to Bordeaux (France) for damage assessment by a single observer (AS), blind to treatment (plot) identity.

We estimated “needle damage” as the percentage of needles on a shoot that turned dark-brown because of the presence of *Contarinia pseudotsugae* galls, following the methodology developed by the Walloon Forest Health Observatory (OWSF). We used an ordinal scale of six percentage classes of damage: 0%; 1 – 10%; 11 – 20%; 21 – 40%; 41 – 60%; 61 – 90%; 91 – 100%, assigning each of the five shoots to one damage class. Finally, we estimated needle damage at the level of individual tree by averaging the median values of defoliation class across the five shoots. Five needles per trees were collected and used to detect the presence or absence of *N. gaeumannii*.

## Infection by *Nothophaecryptopus gaeumannii*

For DNA extraction, the collected needles were placed in 2ml Eppendorf Tubes containing a sterile metal bead (4mm diameter). Tubes were closed with an AirPore tape, stored at -20 °C and lyophilised in an Alpha 2-4 LD plus freeze dryer (Christ, Osterode am Harz, Germany) for 1.5 days. After lyophilisation, the needles were crushed in a MM400 Retsch Mill (Retsch, Haan, Germany) at 30 Hz for 2 min. Further steps in DNA extraction were done using the Sbeadex Plant kit (LGC Genomics GmbH, Berlin, Germany). Lysis was performed by adding 300 µl Lysis Buffer PVP
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Map of Belgium with the location and experimental set-up of the FORBIO Gedinne sub-sites. The tree species diversity per plot ranged from one species (white) to four species (dark grey).</figcaption></figure>

in those subplots, for a total of 207 trees. Finally, we calculated the mean height difference between each selected Douglas fir and their direct neighbours, regardless of whether they were other Douglas firs or trees of the other three species (henceforth called “Apparency”), setting tree height to zero when neighbours of sampled Douglas fir were missing (dead).

## Needle damage by *Contarinia pseudotsugae*

On each sampled Douglas fir, we selected a branch, at mid-height of the tree crown and collected five current-year shoots, starting with the terminal shoot, then picking up every second lateral shoot down to the base of the branch. Shoots were then placed in paper bags, sealed and sent to Bordeaux (France) for damage assessment by a single observer (AS), blind to treatment (plot) identity.

We estimated “needle damage” as the percentage of needles on a shoot that turned dark-brown because of the presence of *Contarinia pseudotsugae* galls, following the methodology developed by the Walloon Forest Health Observatory (OWSF). We used an ordinal scale of six percentage classes of damage: 0%; 1 – 10%; 11 – 20%; 21 – 40%; 41 – 60%; 61 – 90%; 91 – 100%, assigning each of the five shoots to one damage class. Finally, we estimated needle damage at the level of individual tree by averaging the median values of defoliation class across the five shoots. Five needles per trees were collected and used to detect the presence or absence of *N. gaeumannii*.

## Infection by *Nothophaecryptopus gaeumannii*

For DNA extraction, the collected needles were placed in 2ml Eppendorf Tubes containing a sterile metal bead (4mm diameter). Tubes were closed with an AirPore tape, stored at -20 °C and lyophilised in an Alpha 2-4 LD plus freeze dryer (Christ, Osterode am Harz, Germany) for 1.5 days. After lyophilisation, the needles were crushed in a MM400 Retsch Mill (Retsch, Haan, Germany) at 30 Hz for 2 min. Further steps in DNA extraction were done using the Sbeadex Plant kit (LGC Genomics GmbH, Berlin, Germany). Lysis was performed by adding 300 µl Lysis Buffer PVP
(LGC Genomics GmbH) containing 40 µl/ml Debris capture beads (LGC Genomics GmbH), 0.3 µg/µl Proteinase K (LGC Genomics GmbH) and 1.3% 1-thioglycol (Sigma-Aldrich, Buchs, Switzerland) to each sample and subsequent mixing until the samples were well suspended. After incubation at 60 °C for 1 hr, samples were centrifuged at 5,700 g for 5 min and 200 µl of lysate added to a 96-well deep-well plate (Thermo Fisher Scientific, Waltham, USA) containing 420 µl Binding Buffer PN (LGC Genomics GmbH) and 10 µl Sbeadex Particles (LGC Genomics GmbH). The following steps were conducted using the automated KingFisherTM Flex Purification 96 System (Thermo Fisher Scientific). After mixing for 30 min, the Sbeadex Particles with the DNA attached were collected and transferred to 400 µl Wash Buffer PN1 (LGC Genomics GmbH) and mixed for 6 min. This step was repeated before the particles were transferred into 400 µl Wash Buffer PN2 (LGC Genomics GmbH). After mixing for 6 min, the particles were transferred to 50 µl Elution Buffer AMP (LGC Genomics GmbH) and mixed for 7 min to elute the DNA attached to the particles. All DNA purification steps were performed at room temperature. The DNA extracts were stored at -20 °C before dilution and further analyses.

To detect *N. gaeumannii*, we performed a multiplex quantitative real-time PCR (qPCR) with a specific primer pair targeting the beta tubulin gene and the corresponding dual-labelled probe (Winton et al. 2002). An universal 18S primer pair with the corresponding dual-labelled probe 18S uni-P (Ioos et al. 2010) was also used, targeting a highly conserved region of the 18S ribosomal rDNA, to assess DNA quality. The concentrations of the universal primers and probes were reduced in the multiplex reaction mixture to avoid any competition for reagents with the other primers and probes. Quantitative PCR conditions established by Winton et al. (2002) were adapted for high-throughput analyses using the qPCR Takyon core kit NO ROX (Eurogentec, Seraing, Belgium). Reaction volumes of 20 µl contained 5 µl of the 1:10 diluted needle DNA extracts, 1× reaction buffer (Eurogentec), 5.5 mM MgCl₂ (Eurogentec), 0.4 mM dNTPs (Eurogentec), 0.1 × ROX reference dye (Invitrogen, Waltham, USA), 0.5 µM of the specific forward and reverse primer, 0.15 µM each of the universal primers, 0.3 µM of the specific probe, 0.05 µM of the universal probe, 0.5 U Takyon enzyme (Eurogentec) and LiChrosolv Water (Merck, Darmstadt, Germany). Cycling conditions were 3 min of initial denaturation at 95 °C, followed by 40 cycles of 10 s of denaturation at 95 °C and 60 s of annealing and extension at 61 °C. All qPCRs were performed in a QuantStudio 5 Real-Time PCR System (Applied Biosystems, Rotkreuz, Switzerland). As a standard in the qPCR assay, a plasmid construct (pUC57 derivate, Eurogentec) containing the sequence obtained by the PGBT primers, was used in a 10-fold serial dilution from 5 × 10⁷ to 5 copies per µl. Five µl of the serial dilution were used for each dilution step and standard curve reactions were performed in triplicate.

## Explanatory variables

To test the hypothesis that taller trees are more damaged by *C. pseudotsugae* or more exposed to infection by *N. gaeumannii*, we used Douglas fir absolute (Height) and relative (Apparency) height. We also included the density of Douglas fir in the plot and
(LGC Genomics GmbH) containing 40 µl/ml Debris capture beads (LGC Genomics GmbH), 0.3 µg/µl Proteinase K (LGC Genomics GmbH) and 1.3% 1-thioglycol (Sigma-Aldrich, Buchs, Switzerland) to each sample and subsequent mixing until the samples were well suspended. After incubation at 60 °C for 1 hr, samples were centrifuged at 5,700 g for 5 min and 200 µl of lysate added to a 96-well deep-well plate (Thermo Fisher Scientific, Waltham, USA) containing 420 µl Binding Buffer PN (LGC Genomics GmbH) and 10 µl Sbeadex Particles (LGC Genomics GmbH). The following steps were conducted using the automated KingFisherTM Flex Purification 96 System (Thermo Fisher Scientific). After mixing for 30 min, the Sbeadex Particles with the DNA attached were collected and transferred to 400 µl Wash Buffer PN1 (LGC Genomics GmbH) and mixed for 6 min. This step was repeated before the particles were transferred into 400 µl Wash Buffer PN2 (LGC Genomics GmbH). After mixing for 6 min, the particles were transferred to 50 µl Elution Buffer AMP (LGC Genomics GmbH) and mixed for 7 min to elute the DNA attached to the particles. All DNA purification steps were performed at room temperature. The DNA extracts were stored at -20 °C before dilution and further analyses.

To detect *N. gaeumannii*, we performed a multiplex quantitative real-time PCR (qPCR) with a specific primer pair targeting the beta tubulin gene and the corresponding dual-labelled probe (Winton et al. 2002). An universal 18S primer pair with the corresponding dual-labelled probe 18S uni-P (Ioos et al. 2010) was also used, targeting a highly conserved region of the 18S ribosomal rDNA, to assess DNA quality. The concentrations of the universal primers and probes were reduced in the multiplex reaction mixture to avoid any competition for reagents with the other primers and probes. Quantitative PCR conditions established by Winton et al. (2002) were adapted for high-throughput analyses using the qPCR Takyon core kit NO ROX (Eurogentec, Seraing, Belgium). Reaction volumes of 20 µl contained 5 µl of the 1:10 diluted needle DNA extracts, 1× reaction buffer (Eurogentec), 5.5 mM MgCl₂ (Eurogentec), 0.4 mM dNTPs (Eurogentec), 0.1 × ROX reference dye (Invitrogen, Waltham, USA), 0.5 µM of the specific forward and reverse primer, 0.15 µM each of the universal primers, 0.3 µM of the specific probe, 0.05 µM of the universal probe, 0.5 U Takyon enzyme (Eurogentec) and LiChrosolv Water (Merck, Darmstadt, Germany). Cycling conditions were 3 min of initial denaturation at 95 °C, followed by 40 cycles of 10 s of denaturation at 95 °C and 60 s of annealing and extension at 61 °C. All qPCRs were performed in a QuantStudio 5 Real-Time PCR System (Applied Biosystems, Rotkreuz, Switzerland). As a standard in the qPCR assay, a plasmid construct (pUC57 derivate, Eurogentec) containing the sequence obtained by the PGBT primers, was used in a 10-fold serial dilution from 5 × 10⁷ to 5 copies per µl. Five µl of the serial dilution were used for each dilution step and standard curve reactions were performed in triplicate.

## Explanatory variables

To test the hypothesis that taller trees are more damaged by *C. pseudotsugae* or more exposed to infection by *N. gaeumannii*, we used Douglas fir absolute (Height) and relative (Apparency) height. We also included the density of Douglas fir in the plot and
in the direct neighbourhood – number of Douglas fir trees present amongst the eight nearest neighbors – of a focal Douglas fir tree to investigate the potential validity of the resource concentration hypothesis. Density of Douglas fir corresponded to the number of Douglas fir either in the plot or in the direct neighbourhood of a focal Douglas fir. Finally, as damage by *C. pseudotsugae* could alter the biology of the needle or weaken Douglas fir prior an infection by the Swiss needle cast, we included *C. pseudotsugae* damage as an additional explanatory variable only in the model built to explain the probability of infection by *N. gaeumannii*.

## Statistical analyses

We built two different models to test for the effect of explanatory variables on needle damage by *C. pseudotsugae* and infection by *P. gaeumannii*.

First, we used a linear mixed model to test the effect of Douglas fir absolute (Height) and relative (Apparency) height and Douglas fir density in the plot (Density plot) and in the direct neighbourhood (Density neigh.) as well as the interaction between height and Apparency on mean *C. pseudotsugae* damage per tree. Subplots (Subplot_ID), nested within Plot (Plot_ID), were included as random factors to account for spatial autocorrelation. Response variable was square-rooted to satisfy model assumptions of normality and homogeneity of residuals and predictors were scaled. Variance inflation factors were checked for every explanatory variables used in the model and were never > 5, the usual cut-off values used to check for multicollinearity issues (Miles 2014). As the interaction between height and Apparency had no significant effect, we removed it from the final model during the model simplification process.

Second, we used a generalised linear model with a binomial error distribution family to analyse sources of variation in the probability of Douglas fir infection by the Swiss needle cast. We used the same model structure as in Eq. 1, adding *C. pseudotsugae*. damage as an additional covariate. Again, the interaction between height and apparen- cy had no significant effect and we removed it from the final model during the model simplification process. Statistical analyses were performed using R software version 4.1.0 (R Core Team 2022), with the package lme4 (Bates et al. 2015).

## Results

The height of the focal Douglas fir studied ranged from 175 to 954 cm, with a mean (± SE) of 559.6 ± 13.0 cm. Mean height (± SE) of European beech, sycamore ma- ple, sessile oak and hybrid larch were 357.8 ± 9.2, 364.0 ± 16.3, 407.6 ± 14.2 and 948.8 ± 13.6 cm, respectively (Supplementary material). Height difference between a Douglas fir and its closest neighbours was on average (± SE) -14.2 ± 13.8 cm, i.e. Douglas firs were on average shorter than their neighbouring trees. Maximum differ- ences were recorded in plots 20 and 22 (Species composition: Beech, maple, larch and Douglas fir, in the Gribelle and Gouverneurs blocks, respectively), where Douglas fir was 400 cm shorter and 520 cm taller than their neighbours, respectively.
in the direct neighbourhood – number of Douglas fir trees present amongst the eight nearest neighbors – of a focal Douglas fir tree to investigate the potential validity of the resource concentration hypothesis. Density of Douglas fir corresponded to the number of Douglas fir either in the plot or in the direct neighbourhood of a focal Douglas fir. Finally, as damage by *C. pseudotsugae* could alter the biology of the needle or weaken Douglas fir prior an infection by the Swiss needle cast, we included *C. pseudotsugae* damage as an additional explanatory variable only in the model built to explain the probability of infection by *N. gaeumannii*.

## Statistical analyses

We built two different models to test for the effect of explanatory variables on needle damage by *C. pseudotsugae* and infection by *P. gaeumannii*.

First, we used a linear mixed model to test the effect of Douglas fir absolute (Height) and relative (Apparency) height and Douglas fir density in the plot (Density plot) and in the direct neighbourhood (Density neigh.) as well as the interaction between height and Apparency on mean *C. pseudotsugae* damage per tree. Subplots (Subplot_ID), nested within Plot (Plot_ID), were included as random factors to account for spatial autocorrelation. Response variable was square-rooted to satisfy model assumptions of normality and homogeneity of residuals and predictors were scaled. Variance inflation factors were checked for every explanatory variables used in the model and were never > 5, the usual cut-off values used to check for multicollinearity issues (Miles 2014). As the interaction between height and Apparency had no significant effect, we removed it from the final model during the model simplification process.

Second, we used a generalised linear model with a binomial error distribution family to analyse sources of variation in the probability of Douglas fir infection by the Swiss needle cast. We used the same model structure as in Eq. 1, adding *C. pseudotsugae*. damage as an additional covariate. Again, the interaction between height and apparen- cy had no significant effect and we removed it from the final model during the model simplification process. Statistical analyses were performed using R software version 4.1.0 (R Core Team 2022), with the package lme4 (Bates et al. 2015).

## Results

The height of the focal Douglas fir studied ranged from 175 to 954 cm, with a mean (± SE) of 559.6 ± 13.0 cm. Mean height (± SE) of European beech, sycamore ma- ple, sessile oak and hybrid larch were 357.8 ± 9.2, 364.0 ± 16.3, 407.6 ± 14.2 and 948.8 ± 13.6 cm, respectively (Supplementary material). Height difference between a Douglas fir and its closest neighbours was on average (± SE) -14.2 ± 13.8 cm, i.e. Douglas firs were on average shorter than their neighbouring trees. Maximum differ- ences were recorded in plots 20 and 22 (Species composition: Beech, maple, larch and Douglas fir, in the Gribelle and Gouverneurs blocks, respectively), where Douglas fir was 400 cm shorter and 520 cm taller than their neighbours, respectively.
Mean needle damage by *C. pseudotsugae* was on average 16.25 ± 1.25% needles attacked, ranging from no damage at all on 12 Douglas firs to 91.5% of damage on the most impacted Douglas fir. Infection by the Swiss needle cast was confirmed on 118 (57%) of the 207 sampled Douglas firs.

Taller Douglas firs (absolute height) suffered significantly more damage by *C. pseudotsugae* than smaller ones (average standardised model coefficient parameter estimate ± standard error: 1.10 ± 0.24, n = 192, Fig. 2, Table 1, model 1). Mean needle damage by *C. pseudotsugae* also significantly increased with Douglas fir Apparency. Douglas firs that were taller than their neighbours suffered from higher damage than Douglas fir that were smaller than their neighbours (0.59 ± 0.22, n = 192, Fig. 3, Table 1, model 1), but the coefficient parameter estimate for this effect was almost twice lower than the one of tree absolute height. Lower Douglas fir Apparency seems to occur more frequently in mixed species plots including *Larix x eurolepis*, the tallest species in the experiment (Supplementary material). None of the other predictors had a significant effect on mean needle damage by *C. pseudotsugae*. Fixed predictors explained 12.1% of the variance in mean needle damage. Fixed plus random predictors explained 19.7% of the variability in mean needle damage by *C. pseudotsugae*.

The probability of presence of *N. gaeumannii* increased significantly with the density of Douglas firs at the neighbouring scale (0.70 ± 0.22, n = 192, Fig. 4B, Table 1, model 2), but not at the plot scale. Additionally, the probability of a Douglas fir being infected by the Swiss needle cast pathogen increased with increasing mean needle damage by *Contarinia* (0.65 ± 0.23, n = 192, Fig. 4A, Table 1, model 2). In this model, fixed predictors and fixed plus random predictors explained 19.6% and 51.8% of the variability in the probability of infection by *N. gaeumannii*, respectively.

<table><caption>**Table 1.** Summary of model coefficients for the two models presented in this study. Bold characters indicate that parameters are significant. R²m and R²c represent R² of fixed and fixed plus random factors, respectively.</caption><thead><tr><th>Models / Parameter</th><th>Estimate</th><th>Standard error</th><th>95% CI</th><th>P value</th><th>R<sup>2</sup>m (R<sup>2</sup>c)</th></tr></thead><tbody><tr><td><strong>Model 1 – Mean needle damage</strong></td><td></td><td></td><td></td><td></td><td>0.121 (0.197)</td></tr><tr><td>(Intercept)</td><td>3.47</td><td>0.19</td><td>(3.10; 3.83)</td><td>&lt; 0.001</td><td></td></tr><tr><td>Apparency</td><td>0.59</td><td>0.22</td><td>(0.16; 1.04)</td><td>0.008</td><td></td></tr><tr><td>Height</td><td>1.10</td><td>0.24</td><td>(0.63; 1.60)</td><td>&lt; 0.001</td><td></td></tr><tr><td>Density neigh.</td><td>-0.01</td><td>0.15</td><td>(-0.31; 0.29)</td><td>0.919</td><td></td></tr><tr><td>Density plot</td><td>0.08</td><td>0.19</td><td>(-0.29; 0.45)</td><td>0.668</td><td></td></tr><tr><td><strong>Model 2 – Infection rate</strong></td><td></td><td></td><td></td><td></td><td>0.196 (0.518)</td></tr><tr><td>(Intercept)</td><td>0.29</td><td>0.36</td><td>(-0.48; 1.09)</td><td>0.420</td><td></td></tr><tr><td>Apparency</td><td>-0.12</td><td>0.34</td><td>(-0.83; 0.53)</td><td>0.711</td><td></td></tr><tr><td>Height</td><td>-0.54</td><td>0.42</td><td>(-1.46; 0.26)</td><td>0.201</td><td></td></tr><tr><td>Density neigh.</td><td>0.70</td><td>0.22</td><td>(0.27; 1.17)</td><td>0.002</td><td></td></tr><tr><td>Density plot</td><td>0.61</td><td>0.37</td><td>(-0.15; 1.45)</td><td>0.105</td><td></td></tr><tr><td>Contarinia damage</td><td>0.65</td><td>0.23</td><td>(0.21; 1.16)</td><td>0.006</td><td></td></tr></tbody></table>
Mean needle damage by *C. pseudotsugae* was on average 16.25 ± 1.25% needles attacked, ranging from no damage at all on 12 Douglas firs to 91.5% of damage on the most impacted Douglas fir. Infection by the Swiss needle cast was confirmed on 118 (57%) of the 207 sampled Douglas firs.

Taller Douglas firs (absolute height) suffered significantly more damage by *C. pseudotsugae* than smaller ones (average standardised model coefficient parameter estimate ± standard error: 1.10 ± 0.24, n = 192, Fig. 2, Table 1, model 1). Mean needle damage by *C. pseudotsugae* also significantly increased with Douglas fir Apparency. Douglas firs that were taller than their neighbours suffered from higher damage than Douglas fir that were smaller than their neighbours (0.59 ± 0.22, n = 192, Fig. 3, Table 1, model 1), but the coefficient parameter estimate for this effect was almost twice lower than the one of tree absolute height. Lower Douglas fir Apparency seems to occur more frequently in mixed species plots including *Larix x eurolepis*, the tallest species in the experiment (Supplementary material). None of the other predictors had a significant effect on mean needle damage by *C. pseudotsugae*. Fixed predictors explained 12.1% of the variance in mean needle damage. Fixed plus random predictors explained 19.7% of the variability in mean needle damage by *C. pseudotsugae*.

The probability of presence of *N. gaeumannii* increased significantly with the density of Douglas firs at the neighbouring scale (0.70 ± 0.22, n = 192, Fig. 4B, Table 1, model 2), but not at the plot scale. Additionally, the probability of a Douglas fir being infected by the Swiss needle cast pathogen increased with increasing mean needle damage by *Contarinia* (0.65 ± 0.23, n = 192, Fig. 4A, Table 1, model 2). In this model, fixed predictors and fixed plus random predictors explained 19.6% and 51.8% of the variability in the probability of infection by *N. gaeumannii*, respectively.

<table><caption>**Table 1.** Summary of model coefficients for the two models presented in this study. Bold characters indicate that parameters are significant. R²m and R²c represent R² of fixed and fixed plus random factors, respectively.</caption><thead><tr><th>Models / Parameter</th><th>Estimate</th><th>Standard error</th><th>95% CI</th><th>P value</th><th>R<sup>2</sup>m (R<sup>2</sup>c)</th></tr></thead><tbody><tr><td><strong>Model 1 – Mean needle damage</strong></td><td></td><td></td><td></td><td></td><td>0.121 (0.197)</td></tr><tr><td>(Intercept)</td><td>3.47</td><td>0.19</td><td>(3.10; 3.83)</td><td>&lt; 0.001</td><td></td></tr><tr><td>Apparency</td><td>0.59</td><td>0.22</td><td>(0.16; 1.04)</td><td>0.008</td><td></td></tr><tr><td>Height</td><td>1.10</td><td>0.24</td><td>(0.63; 1.60)</td><td>&lt; 0.001</td><td></td></tr><tr><td>Density neigh.</td><td>-0.01</td><td>0.15</td><td>(-0.31; 0.29)</td><td>0.919</td><td></td></tr><tr><td>Density plot</td><td>0.08</td><td>0.19</td><td>(-0.29; 0.45)</td><td>0.668</td><td></td></tr><tr><td><strong>Model 2 – Infection rate</strong></td><td></td><td></td><td></td><td></td><td>0.196 (0.518)</td></tr><tr><td>(Intercept)</td><td>0.29</td><td>0.36</td><td>(-0.48; 1.09)</td><td>0.420</td><td></td></tr><tr><td>Apparency</td><td>-0.12</td><td>0.34</td><td>(-0.83; 0.53)</td><td>0.711</td><td></td></tr><tr><td>Height</td><td>-0.54</td><td>0.42</td><td>(-1.46; 0.26)</td><td>0.201</td><td></td></tr><tr><td>Density neigh.</td><td>0.70</td><td>0.22</td><td>(0.27; 1.17)</td><td>0.002</td><td></td></tr><tr><td>Density plot</td><td>0.61</td><td>0.37</td><td>(-0.15; 1.45)</td><td>0.105</td><td></td></tr><tr><td>Contarinia damage</td><td>0.65</td><td>0.23</td><td>(0.21; 1.16)</td><td>0.006</td><td></td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Effect of tree height of a focal Douglas fir on mean damage by *C. pseudotsugae*. Green dots represent individual trees. Solid and dashed lines represent prediction and adjusted standard error of model 1, with values of other model variables set at average (Table 1).</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Effect of Douglas fir apparent on the mean damage by *C. pseudotsugae*. Apparenty is the mean height difference between a focal Douglas fir and its direct neighbours. Dots at the right and at the left of the vertical dashed line are Douglas fir trees that are taller and smaller than their neighbours, respectively. Solid and dashed lines represent prediction and adjusted standard error of model 1, with values of other model parameters set at average (Table 1).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Effect of tree height of a focal Douglas fir on mean damage by *C. pseudotsugae*. Green dots represent individual trees. Solid and dashed lines represent prediction and adjusted standard error of model 1, with values of other model variables set at average (Table 1).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Effect of Douglas fir apparent on the mean damage by *C. pseudotsugae*. Apparenty is the mean
height difference between a focal Douglas fir and its direct neighbours. Dots at the right and at the left of
the vertical dashed line are Douglas fir trees that are taller and smaller than their neighbours, respectively.
Solid and dashed lines represent prediction and adjusted standard error of model 1, with values of other
model parameters set at average (Table 1).</figcaption></figure>
<figure><img src="image_5.png" /></figure>

**Figure 4.** Effects of (A) the density of Douglas fir in the direct neighbourhood of a focal Douglas fir and (B) of mean damage by *C. pseudotsugae* on the probability of a Douglas fir being infected by *Nothophaeocryptopus gaeumannii*, the causal agent of Swiss needle cast. Solid and dashed lines represent prediction and adjusted standard error of model 2 (Table 1). Dots at probability 0 and 1 represent Douglas fir that were (1) or were not (0) infected.

Discussion

Our study shows that tree height and Apparency were two main drivers of needle midge *C. pseudotsugae* on Douglas fir. Douglas firs taller than their neighbours exhibited more needle damage. We found no effect of Douglas fir concentration, nor at the plot level, nor at the direct neighbours' scale, on damage by *C. pseudotsugae*. However, there was a significant, positive effect of Douglas concentration at the local scale on the probability of infection by the Swiss needle cast fungus *N. gaeumannii*. Douglas fir suffering more needle damage by *C. pseudotsugae* also had higher probability of being infected by the fungal pathogen.

Tree Apparency is a component of tree diversity. The concept was initially coined
to describe the probability of a plant being identified by its herbivores (Feeny 1970;
Endara and Coley 2011; Strauss et al. 2015). This definition was then refined to include
not only the plant's own characteristics (size, colour and odour), but also those of its
neighbours, ultimately determining how much a plant is prone to be found by herbi-
vores (Castagneyrol et al. 2013). In this study, we found that more apparent Douglas
firs suffered from more needle damage by the Douglas fir needle midge *C. pseudotsugae*
than less apparent trees. Taller, non-host neighbouring trees are known to contribute
to the disruption of visual (Dulaurent et al. 2012) or olfactory cues (Jactel et al. 2011)
used by herbivorous insect. Although no studies so far have been done to identify the
mechanisms used by *C. pseudotsugae* to locate Douglas fir trees, it has been shown that
midge of the same *Cecidomyiidae* family are using both visual and olfactory cues to find
potential hosts (Harris and Rose 1990; Sharma and Franzmann 2001). It could be then
possible that taller neighbours would have made Douglas fir trees more difficult to locate
by the Douglas fir needle midge, thus leading to a lower level of attacks. Those find-
ings would be consistent with other studies having reported lower herbivore damage on
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Effects of (A) the density of Douglas fir in the direct neighbourhood of a focal Douglas fir and (B) of mean damage by *C. pseudotsugae* on the probability of a Douglas fir being infected by *Nothophaeocryptopus gaeumannii*, the causal agent of Swiss needle cast. Solid and dashed lines represent prediction and adjusted standard error of model 2 (Table 1). Dots at probability 0 and 1 represent Douglas fir that were (1) or were not (0) infected.</figcaption></figure>

Discussion

Our study shows that tree height and Apparency were two main drivers of needle midge *C. pseudotsugae* on Douglas fir. Douglas firs taller than their neighbours exhibited more needle damage. We found no effect of Douglas fir concentration, nor at the plot level, nor at the direct neighbours' scale, on damage by *C. pseudotsugae*. However, there was a significant, positive effect of Douglas concentration at the local scale on the probability of infection by the Swiss needle cast fungus *N. gaeumannii*. Douglas fir suffering more needle damage by *C. pseudotsugae* also had higher probability of being infected by the fungal pathogen.

Tree Apparency is a component of tree diversity. The concept was initially coined
to describe the probability of a plant being identified by its herbivores (Feeny 1970;
Endara and Coley 2011; Strauss et al. 2015). This definition was then refined to include
not only the plant's own characteristics (size, colour and odour), but also those of its
neighbours, ultimately determining how much a plant is prone to be found by herbi-
vores (Castagneyrol et al. 2013). In this study, we found that more apparent Douglas
firs suffered from more needle damage by the Douglas fir needle midge *C. pseudotsugae*
than less apparent trees. Taller, non-host neighbouring trees are known to contribute
to the disruption of visual (Dulaurent et al. 2012) or olfactory cues (Jactel et al. 2011)
used by herbivorous insect. Although no studies so far have been done to identify the
mechanisms used by *C. pseudotsugae* to locate Douglas fir trees, it has been shown that
midge of the same *Cecidomyiidae* family are using both visual and olfactory cues to find
potential hosts (Harris and Rose 1990; Sharma and Franzmann 2001). It could be then
possible that taller neighbours would have made Douglas fir trees more difficult to locate
by the Douglas fir needle midge, thus leading to a lower level of attacks. Those find-
ings would be consistent with other studies having reported lower herbivore damage on
trees concealed by non-host neighbours (Floater and Zalucki 2000; Hughes 2012). The Apparency of Douglas fir trees was calculated, based on the height of their neighbours, regardless of their species. It should be noted, however, that the reduction in this Apparency was more often achieved in the presence of larch trees next to Douglas-firs, as *Larix x eurolepis* grows in height faster than Douglas fir at the juvenile stage (Supplementary material). This suggests that mixed stands of Douglas fir and larch might be less attacked by the midge, although not explicitly tested in this study and will be consistent with the associational resistance hypothesis. Conversely, Douglas fir was consistently more apparent in mixtures with slow-growing oaks and could have resulted in more gall midge damage, i.e. associative susceptibility. This leads us to suggest that it might not be species richness per se that confers greater resistance to mixed-species forests, but the identity and functional traits of the associated species included in the mixture (Jactel et al. 2021).

In addition to the effect of tree Apparency, we also found a positive effect of absolute tree height, with taller Douglas firs suffering from more needle damage than smaller ones. This result confirms the outcome of a study conducted by Castagneyrol et al. (2013) in a similar tree diversity experiment in France, showing that both tree Apparency and absolute tree height were positively correlated with damage by herbivorous insects on oaks. A simple explanation of this finding could be that taller trees are more likely to intercept insects moving in the environment than smaller trees. This effect will add to the increase Apparency of taller Douglas firs giving more visual cues than smaller ones. Another, although more speculative explanation of this pattern is related to the growth-differentiation balance hypothesis (Herms and Mattson 1992; Glynn et al. 2007), which suggests a physiological trade-off between growth and production of secondary metabolites in trees. Secondary metabolites in plants are often involved in defence mechanisms and, as such, fast growing trees are expected to produce fewer secondary metabolites, henceforth having lower defence and exhibiting higher level of herbivory (Lerdau et al. 1994; Massad 2013). Since all trees on the FORBIO tree diversity have the same age, it could be then possible that taller, fast-growing Douglas firs, had a lower amount of chemical defences and, thus, represented more suitable hosts for *C. pseudotsugae*. On the other hand, the Douglas fir midge belongs to the guild of gall makers that are known to manipulate the flow of photosynthetic products in the tree for their own benefit. Thus, the largest and, therefore, most vigorous Douglas-fir trees may be the best hosts for *C. pseudotsugae* as a primary pest.

Unexpectedly, we did not find any effect of Douglas fir density on mean damage by *C. pseudotsugae*. Plots with more abundant resource for the invasive pest were not more infested than plots with a lower concentration of host trees, contradicting the resource concentration hypothesis. This might be due to the small size of the plots in the FORBIO experiment (42 m × 42 m) (Bommarco and Banks 2003), the low level of active dispersion behaviour of the Douglas fir needle midge, or the overriding effect of Douglas fir Apparency that depends more on the species composition of the plots than on Douglas-fir density.

By contrast, the probability of a Douglas fir to be infected by *N. gaeumannii* was positively correlated with the density of Douglas fir in its direct neighbourhood. This result is consistent with recent studies that linked host tree proportion and pathogen transmission amongst neighbouring individuals (Pautasso et al. 2005; Field et
trees concealed by non-host neighbours (Floater and Zalucki 2000; Hughes 2012). The Apparency of Douglas fir trees was calculated, based on the height of their neighbours, regardless of their species. It should be noted, however, that the reduction in this Apparency was more often achieved in the presence of larch trees next to Douglas-firs, as *Larix x eurolepis* grows in height faster than Douglas fir at the juvenile stage (Supplementary material). This suggests that mixed stands of Douglas fir and larch might be less attacked by the midge, although not explicitly tested in this study and will be consistent with the associational resistance hypothesis. Conversely, Douglas fir was consistently more apparent in mixtures with slow-growing oaks and could have resulted in more gall midge damage, i.e. associative susceptibility. This leads us to suggest that it might not be species richness per se that confers greater resistance to mixed-species forests, but the identity and functional traits of the associated species included in the mixture (Jactel et al. 2021).

In addition to the effect of tree Apparency, we also found a positive effect of absolute tree height, with taller Douglas firs suffering from more needle damage than smaller ones. This result confirms the outcome of a study conducted by Castagneyrol et al. (2013) in a similar tree diversity experiment in France, showing that both tree Apparency and absolute tree height were positively correlated with damage by herbivorous insects on oaks. A simple explanation of this finding could be that taller trees are more likely to intercept insects moving in the environment than smaller trees. This effect will add to the increase Apparency of taller Douglas firs giving more visual cues than smaller ones. Another, although more speculative explanation of this pattern is related to the growth-differentiation balance hypothesis (Herms and Mattson 1992; Glynn et al. 2007), which suggests a physiological trade-off between growth and production of secondary metabolites in trees. Secondary metabolites in plants are often involved in defence mechanisms and, as such, fast growing trees are expected to produce fewer secondary metabolites, henceforth having lower defence and exhibiting higher level of herbivory (Lerdau et al. 1994; Massad 2013). Since all trees on the FORBIO tree diversity have the same age, it could be then possible that taller, fast-growing Douglas firs, had a lower amount of chemical defences and, thus, represented more suitable hosts for *C. pseudotsugae*. On the other hand, the Douglas fir midge belongs to the guild of gall makers that are known to manipulate the flow of photosynthetic products in the tree for their own benefit. Thus, the largest and, therefore, most vigorous Douglas-fir trees may be the best hosts for *C. pseudotsugae* as a primary pest.

Unexpectedly, we did not find any effect of Douglas fir density on mean damage by *C. pseudotsugae*. Plots with more abundant resource for the invasive pest were not more infested than plots with a lower concentration of host trees, contradicting the resource concentration hypothesis. This might be due to the small size of the plots in the FORBIO experiment (42 m × 42 m) (Bommarco and Banks 2003), the low level of active dispersion behaviour of the Douglas fir needle midge, or the overriding effect of Douglas fir Apparency that depends more on the species composition of the plots than on Douglas-fir density.

By contrast, the probability of a Douglas fir to be infected by *N. gaeumannii* was positively correlated with the density of Douglas fir in its direct neighbourhood. This result is consistent with recent studies that linked host tree proportion and pathogen transmission amongst neighbouring individuals (Pautasso et al. 2005; Field et
al. 2020). Increased resource availability has been theorised to be a key factor in the augmentation of infection by fungal pathogens amongst co-occurring, neighbouring, host trees (García-Guzmán and Dirzo 2004; Keesing et al. 2006, but see Hantsch et al. 2014)). For example, Hantsch et al. (2013) found in a tree diversity experiment in Germany that pathogen loads of several biotrophic fungi of European beech were higher when host trees were surrounded by conspecific neighbours. Ascospores of the Swiss needle cast are wind and water-splash dispersed (Ritóková et al. 2016). As such, it is likely that the transmission of the pathogen was facilitated by the shorter distances between infected and recipient neighbouring Douglas firs in pure plots.

Finally, we found that damage by *C. pseudotsugae* increased the probability of an infection by *N. gaeumannii*. Interactions between two or more invasive species in an area can result in invasion meltdown (Simberloff and Von Holle 1999; O'Loughlin and Green 2017), where a non-native species may facilitate the arrival or establishment of a subsequent invader. It is possible that the presence of *C. pseudotsugae* had indirect effects on the probability of infection by the fungal pathogen, by making Douglas fir more susceptible to colonisation. For example, Meyer et al. (2015) showed that abandoned galls of the chestnut gall wasp *Dryocosmus kuriphilus* could be colonised by the virulent form of the chestnut blight fungus *Cryphonectria parasitica*, which could lead to increased load of virulent inoculum in forests. Gossner et al. (2021) also showed that feeding damage by the weevil *Orchestes fagi* led to higher infection rates of beech leaves by the pathogenic fungus *Petrakia liobae*, by providing entry ports for fungal colonisation. In line with these studies, Douglas firs with a higher amount of galls could be more prone to a subsequent infection by the Swiss needle cast fungus *N. gaeumannii*. However, the opposite may also occur, with the Douglas fir needle midge causing more damage on Douglas firs infected by *N. gaeumannii*. Indeed, numerous studies have already shown that infections by pathogens can lead, directly or indirectly, to a greater susceptibility of trees to insect damage (Stout et al. 2006; Fernandez-Conradi, Jactel et al. 2018b; Moreira et al. 2018). Furthermore, we cannot rule out the possibility that both damage by the Douglas fir needle midge and infection by the pathogen responded to common tree traits that were not measured in this study, but could explain the co-linearity between the two variables. Finally, since the experimental design of this study did not allow us to determine precisely which of the pest or the pathogen initially affect focal Douglas fir trees, it would be interesting to better investigate the temporal dynamic behind the invasion by those two pests and pathogens and the effect that tree diversity could have on the mitigation of invasion meltdown.

## Conclusions

Our study provides indirect support of the associational resistance hypothesis of mixed forests against exotic pests and pathogens. In the case of Douglas-fir Swiss needle cast, the effect was mediated by host dilution amongst heterospecific, non-host tree neighbours. In the case of Douglas-fir midge, the resistance effect was due to a reduction in host tree Apparency by taller neighbours. It should be noted, however, that the reduction of infestations by these exotic organisms was not general in mixed stands and might
al. 2020). Increased resource availability has been theorised to be a key factor in the augmentation of infection by fungal pathogens amongst co-occurring, neighbouring, host trees (García-Guzmán and Dirzo 2004; Keesing et al. 2006, but see Hantsch et al. (2014)). For example, Hantsch et al. (2013) found in a tree diversity experiment in Germany that pathogen loads of several biotrophic fungi of European beech were higher when host trees were surrounded by conspecific neighbours. Ascospores of the Swiss needle cast are wind and water-splash dispersed (Ritóková et al. 2016). As such, it is likely that the transmission of the pathogen was facilitated by the shorter distances between infected and recipient neighbouring Douglas firs in pure plots.

Finally, we found that damage by *C. pseudotsugae* increased the probability of an infection by *N. gaeumannii*. Interactions between two or more invasive species in an area can result in invasion meltdown (Simberloff and Von Holle 1999; O'Loughlin and Green 2017), where a non-native species may facilitate the arrival or establishment of a subsequent invader. It is possible that the presence of *C. pseudotsugae* had indirect effects on the probability of infection by the fungal pathogen, by making Douglas fir more susceptible to colonisation. For example, Meyer et al. (2015) showed that abandoned galls of the chestnut gall wasp *Dryocosmus kuriphilus* could be colonised by the virulent form of the chestnut blight fungus *Cryptонectria parasitica*, which could lead to increased load of virulent inoculum in forests. Gossner et al. (2021) also showed that feeding damage by the weevil *Orchestes fagi* led to higher infection rates of beech leaves by the pathogenic fungus *Petrakia liobae*, by providing entry ports for fungal colonisation. In line with these studies, Douglas firs with a higher amount of galls could be more prone to a subsequent infection by the Swiss needle cast fungus *N. gaeumannii*. However, the opposite may also occur, with the Douglas fir needle midge causing more damage on Douglas firs infected by *N. gaeumannii*. Indeed, numerous studies have already shown that infections by pathogens can lead, directly or indirectly, to a greater susceptibility of trees to insect damage (Stout et al. 2006; Fernandez-Conradi, Jactel et al. 2018b; Moreira et al. 2018). Furthermore, we cannot rule out the possibility that both damage by the Douglas fir needle midge and infection by the pathogen responded to common tree traits that were not measured in this study, but could explain the co-linearity between the two variables. Finally, since the experimental design of this study did not allow us to determine precisely which of the pest or the pathogen initially affect focal Douglas fir trees, it would be interesting to better investigate the temporal dynamic behind the invasion by those two pests and pathogens and the effect that tree diversity could have on the mitigation of invasion meltdown.

## Conclusions

Our study provides indirect support of the associational resistance hypothesis of mixed forests against exotic pests and pathogens. In the case of Douglas-fir Swiss needle cast, the effect was mediated by host dilution amongst heterospecific, non-host tree neighbours. In the case of Douglas-fir midge, the resistance effect was due to a reduction in host tree Apparency by taller neighbours. It should be noted, however, that the reduction of infestations by these exotic organisms was not general in mixed stands and might
mainly depend on the species composition of the mixtures. In particular, the need to incorporate tree species growing faster than Douglas-fir, such as hybrid larch, could be investigated as a mean of reducing pest damage. We also demonstrated for the first time a positive interaction between Swiss needle cast and Douglas fir needle midge. This underlines the necessity to develop a holistic approach to the problem of biological invasions in forests and, in particular, to look for generic prevention methods, amongst which the increase of forest diversity appears to be promising.

## References

Almeida RPP, Purcell AH (2003) Transmission of *Xylella fastidiosa* to Grapevines by *Homalodisca coagulata* (Hemiptera: Cicadellidae). Journal of Economic Entomology 96: 264–271.
https://doi.org/10.1603/0022-0493-96.2.264

Aukema JE, Leung B, Kovacs K, Chivers C, Britton KO, Englin J, Frankel SJ, Haight RG, Holmes TP, Liebhold AM, McCullough DG, Holle BV (2011) Economic impacts of non-native forest insects in the continental United States. PLoS ONE 6(9): e24587. https://doi.org/10.1371/journal.pone.0024587

Barbosa P, Hines J, Kaplan I, Martinson H, Szczepaniec A, Szendrei Z (2009) Associational resistance and associational susceptibility: Having right or wrong neighbors. Annual Review of Ecology, Evolution, and Systematics 40(1): 1–20. https://doi.org/10.1146/annurev.ecolsys.110308.120242

Bates D, Mächler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. Journal of Statistical Software 67(1): 1–48. https://doi.org/10.18637/jss.v067.i01

Black BA, Shaw DC, Stone JK (2010) Impacts of Swiss needle cast on overstory Douglas-fir forests of the western Oregon Coast Range. Forest Ecology and Management 259: 1673–1680. https://doi.org/10.1016/j.foreco.2010.01.047

Bommarco R, Banks J (2003) Scale as modifier in vegetation diversity experiments: Effects on herbivores and predators. Oikos 102(2): 440–448. https://doi.org/10.1034/j.1600-0579.2003.12578.x

Brockerhoff EG, Barbaro L, Castagneyrol B, Forrester DI, Gardiner B, González-Olabarria JR, Lyver PO, Meurisse N, Oxbrough A, Taki H, Thompson ID, van der Plas F, Jactel H (2017) Forest biodiversity, ecosystem functioning and the provision of ecosystem services. Biodiversity and Conservation 26(13): 3005–3035. https://doi.org/10.1007/s10531-017-1453-2

Brockerhoff EG, Liebhold AM (2017) Ecology of forest insect invasions. Biological Invasions 19: 3141–3159. https://doi.org/10.1007/s10530-017-1514-1

Castagneyrol B, Giffard B, Péré C, Jactel H (2013) Plant apparency, an overlooked driver of associational resistance to insect herbivory. Journal of Ecology 101(2): 418–429.
https://doi.org/10.1111/1365-2745.12055

Condrashoff SF (1961) Description and morphology of the immature stages of three closely related species of *Contarinia* Rond. (Diptera: Cecidomyiidae) from galls on Douglas-fir Nee-dles1. Canadian Entomologist 93(10): 833–851. https://doi.org/10.4039/Ent93833-10

Dulaurent A-M, Porté AJ, van Halder I, Vétillard F, Menassieu P, Jactel H (2012) Hide and seek in forests: Colonization by the pine processionary moth is impeded by the presence of
mainly depend on the species composition of the mixtures. In particular, the need to incorporate tree species growing faster than Douglas-fir, such as hybrid larch, could be investigated as a mean of reducing pest damage. We also demonstrated for the first time a positive interaction between Swiss needle cast and Douglas fir needle midge. This underlines the necessity to develop a holistic approach to the problem of biological invasions in forests and, in particular, to look for generic prevention methods, amongst which the increase of forest diversity appears to be promising.

## References

Almeida RPP, Purcell AH (2003) Transmission of *Xylella fastidiosa* to Grapevines by *Homalodisca coagulata* (Hemiptera: Cicadellidae). Journal of Economic Entomology 96: 264–271.
https://doi.org/10.1603/0022-0493-96.2.264

Aukema JE, Leung B, Kovacs K, Chivers C, Britton KO, Englin J, Frankel SJ, Haight RG, Holmes TP, Liebhold AM, McCullough DG, Holle BV (2011) Economic impacts of non-native forest insects in the continental United States. PLoS ONE 6(9): e24587. https://doi.org/10.1371/journal.pone.0024587

Barbosa P, Hines J, Kaplan I, Martinson H, Szczepaniec A, Szendrei Z (2009) Associational resistance and associational susceptibility: Having right or wrong neighbors. Annual Review of Ecology, Evolution, and Systematics 40(1): 1–20. https://doi.org/10.1146/annurev.ecolsys.110308.120242

Bates D, Mächler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. Journal of Statistical Software 67(1): 1–48. https://doi.org/10.18637/jss.v067.i01

Black BA, Shaw DC, Stone JK (2010) Impacts of Swiss needle cast on overstory Douglas-fir forests of the western Oregon Coast Range. Forest Ecology and Management 259: 1673–1680. https://doi.org/10.1016/j.foreco.2010.01.047

Bommarco R, Banks J (2003) Scale as modifier in vegetation diversity experiments: Effects on herbivores and predators. Oikos 102(2): 440–448. https://doi.org/10.1034/j.1600-0579.2003.12578.x

Brockerhoff EG, Barbaro L, Castagneyrol B, Forrester DI, Gardiner B, González-Olabarria JR, Lyver PO, Meurisse N, Oxbrough A, Taki H, Thompson ID, van der Plas F, Jactel H (2017) Forest biodiversity, ecosystem functioning and the provision of ecosystem services. Biodiversity and Conservation 26(13): 3005–3035. https://doi.org/10.1007/s10531-017-1453-2

Brockerhoff EG, Liebhold AM (2017) Ecology of forest insect invasions. Biological Invasions 19: 3141–3159. https://doi.org/10.1007/s10530-017-1514-1

Castagneyrol B, Giffard B, Péré C, Jactel H (2013) Plant apparency, an overlooked driver of associational resistance to insect herbivory. Journal of Ecology 101(2): 418–429.
https://doi.org/10.1111/1365-2745.12055

Condrashoff SF (1961) Description and morphology of the immature stages of three closely related species of *Contarinia* Rond. (Diptera: Cecidomyiidae) from galls on Douglas-fir Nee-dles1. Canadian Entomologist 93(10): 833–851. https://doi.org/10.4039/Ent93833-10

Dulaurent A-M, Porté AJ, van Halder I, Vétillard F, Menassieu P, Jactel H (2012) Hide and seek in forests: Colonization by the pine processionary moth is impeded by the presence of
nonhost trees. Agricultural and Forest Entomology 14(1): 19–27. https://doi.org/10.1111/j.1461-9563.2011.00549.x

EPPO Global Database (2023) *Contarinia pseudotsugae* (CONTPS). [Overview] https://gd.eppo.int/taxon/CONTPS [January 23, 2023]

Endara M-J, Coley PD (2011) The resource availability hypothesis revisited: A meta-analysis. Functional Ecology 25(2): 389–398. https://doi.org/10.1111/j.1365-2435.2010.01803.x

Feeny P (1970) Seasonal changes in oak leaf tannins and nutrients as a cause of spring feeding by winter moth caterpillars. Ecology 51(4): 565–581. https://doi.org/10.2307/1934037

Fernandez-Conradi P, Borowiec N, Capdevielle X, Castagneyrol B, Maltoni A, Robin C, Selvi F, van Halder I, Vetillard F, Jactel H (2018a) Plant neighbour identity and invasive pathogen infection affect associational resistance to an invasive gall wasp. Biological Invasions 20(6): 1459–1473. https://doi.org/10.1007/s10530-017-1637-4

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Field E, Castagneyrol B, Gibbs M, Jactel H, Barsoum N, Schönrogge K, Hector A (2020) Associational resistance to both insect and pathogen damage in mixed forests is modulated by tree neighbour identity and drought. Journal of Ecology 108(4): 1511–1522. https://doi.org/10.1111/1365-2745.13397

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EPPO Global Database (2023) *Contarinia pseudotsugae* (CONTPS). [Overview] https://gd.eppo.int/taxon/CONTPS [January 23, 2023]

Endara M-J, Coley PD (2011) The resource availability hypothesis revisited: A meta-analysis. Functional Ecology 25(2): 389–398. https://doi.org/10.1111/j.1365-2435.2010.01803.x

Feeny P (1970) Seasonal changes in oak leaf tannins and nutrients as a cause of spring feeding by winter moth caterpillars. Ecology 51(4): 565–581. https://doi.org/10.2307/1934037

Fernandez-Conradi P, Borowiec N, Capdevielle X, Castagneyrol B, Maltoni A, Robin C, Selvi F, van Halder I, Vetillard F, Jactel H (2018a) Plant neighbour identity and invasive pathogen infection affect associational resistance to an invasive gall wasp. Biological Invasions 20(6): 1459–1473. https://doi.org/10.1007/s10530-017-1637-4

Fernandez-Conradi P, Jactel H, Robin C, Tack AJM, Castagneyrol B (2018b) Fungi reduce preference and performance of insect herbivores on challenged plants. Ecology 99: 300–311. https://doi.org/10.1002/ecy.2044

Field E, Castagneyrol B, Gibbs M, Jactel H, Barsoum N, Schönrogge K, Hector A (2020) Associational resistance to both insect and pathogen damage in mixed forests is modulated by tree neighbour identity and drought. Journal of Ecology 108(4): 1511–1522. https://doi.org/10.1111/1365-2745.13397

Floater GJ, Zalucki MP (2000) Habitat structure and egg distributions in the processionary caterpillar *Ochrogaster lunifer*: Lessons for conservation and pest management. Journal of Applied Ecology 37(1): 87–99. https://doi.org/10.1046/j.1365-2664.2000.00468.x

García-Guzmán G, Dirzo R (2004) Incidence of leaf pathogens in the canopy of a Mexican tropical wet forest. Plant Ecology 172(1): 41–50. https://doi.org/10.1023/B:VEGE.0000026034.24664.73

Glynn C, Herms DA, Orians CM, Hansen RC, Larsson S (2007) Testing the growth-differentiation balance hypothesis: Dynamic responses of willows to nutrient availability. The New Phytologist 176(3): 623–634. https://doi.org/10.1111/j.1469-8137.2007.02203.x

Gossner MM, Beenken L, Arend K, Begerow D, Peršoh D (2021) Insect herbivory facilitates the establishment of an invasive plant pathogen. ISME Communications 1(1): 1–8. https://doi.org/10.1038/s43705-021-00004-4

Griscom BW, Adams J, Ellis PW, Houghton RA, Lomax G, Miteva DA, Schlesinger WH, Shoch D, Siikamäki JV, Smith P, Woodbury P, Zganjar C, Blackman A, Campari J, Conant RT, Delgado C, Elias P, Gopalakrishna T, Hamsik MR, Herrero M, Kiesecker J, Landis E, Laestadius L, Leavitt SM, Minnemeyer S, Polasky S, Potapov P, Putz FE, Sanderman J, Silvius M, Wollenberg E, Fargione J (2017) Natural climate solutions. Proceedings of the National Academy of Sciences of the United States of America 114(44): 11645–11650. https://doi.org/10.1073/pnas.1710465114

Guyot V, Jactel H, Imbaud B, Burnel L, Castagneyrol B, Heinz W, Deconchat M, Vialatte A (2019) Tree diversity drives associational resistance to herbivory at both forest edge and interior. Ecology and Evolution 9(16): 9040–9051. https://doi.org/10.1002/ece3.5450

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Ioos R, Fabre B, Saurat C, Fourrier C, Frey P, Marçais B (2010) Development, comparison, and validation of real-time and conventional PCR tools for the detection of the fungal pathogens causing brown spot and red band needle blights of pine. Phytopathology 100(1): 105-114. https://doi.org/10.1094/PHYTO-100-1-0105

Jactel H, Birgersson G, Andersson S, Schlyter F (2011) Non-host volatiles mediate associational resistance to the pine processionary moth. Oecologia 166(3): 703-711. https://doi.org/10.1007/s00442-011-1918-z

Jactel H, Desprez-Loustau M-L, Battisti A, Brockerhoff E, Santini A, Stenlid J, Björkman C, Branco M, Dehnen-Schmutz K, Douma JC, Drakulic J, Drizou F, Eschen R, Franco JC, Gossner MM, Green S, Kenis M, Klapwijk MJ, Liebhold AM, Orazio C, Prospero S, Robinet C, Schroeder M, Slippers B, Stoev P, Sun J, van den Dool R, Wingfield MJ, Zalucki MP (2020) Pathologists and entomologists must join forces against forest pest and pathogen invasions. NeoBiota 58: 107-127. https://doi.org/10.3897/neobiota.58.54389

Jactel H, Moreira X, Castagneyrol B (2021) Tree diversity and forest resistance to insect pests: Patterns, mechanisms, and prospects. Annual Review of Entomology 66(1): 277-296. https://doi.org/10.1146/annurev-ento-041720-075234
Keesing F, Holt RD, Ostfeld RS (2006) Effects of species diversity on disease risk. Ecology Letters 9(4): 485–498. https://doi.org/10.1111/j.1461-0248.2006.00885.x

Kim B-N, Kim JH, Ahn J-Y, Kim S, Cho B-K, Kim Y-H, Min J (2020) A short review of the pinewood nematode, *Bursaphelenchus xylophilus*. Toxicology and Environmental Health Sciences 12(4): 297–304. https://doi.org/10.1007/s13530-020-00068-0

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Ligot G, Balandier P, Schmitz S, Claessens H (2020) Transforming even-aged coniferous stands to multi-aged stands: An opportunity to increase tree species diversity? Forestry. Forestry 93(5): 616–629. https://doi.org/10.1093/forestry/cpaa004

Massad TJ (2013) Ontogenetic differences of herbivory on woody and herbaceous plants: A meta-analysis demonstrating unique effects of herbivory on the young and the old, the slow and the fast. Oecologia 172(1): 1–10. https://doi.org/10.1007/s00442-012-2470-1

Meyer JB, Gallien L, Prospero S, Anderson I (2015) Interaction between two invasive organisms on the European chestnut: Does the chestnut blight fungus benefit from the presence of the gall wasp? FEMS Microbiology Ecology 91(11): fiv122. https://doi.org/10.1093/femsec/fiv122

Miles J (2014) Tolerance and variance inflation factor. Wiley StatsRef: Statistics Reference Online, John Wiley & Sons, Ltd. https://doi.org/10.1002/9781118445112.stat06593

Moreira X, Abdala-Roberts L, Castagneyrol B (2018) Interactions between plant defence signalling pathways: Evidence from bioassays with insect herbivores and plant pathogens. Journal of Ecology 106(6): 2353–2364. https://doi.org/10.1111/1365-2745.12987

Mori AS, Lertzman KP, Gustafsson L (2017) Biodiversity and ecosystem services in forest ecosystems: A research agenda for applied forest ecology. Journal of Applied Ecology 54(1): 12–27. https://doi.org/10.1111/1365-2664.12669

Nunez-Mir GC, Liebhold AM, Guo Q, Brockerhoff EG, Jo I, Ordonez K, Fei S (2017) Biotic resistance to exotic invasions: its role in forest ecosystems, confounding artifacts, and future directions. Biological Invasions 19: 3287–3299. https://doi.org/10.1007/s10530-017-1413-5

O'Loughlin LS, Green PT (2017) Secondary invasion: When invasion success is contingent on other invaders altering the properties of recipient ecosystems. Ecology and Evolution 7(19): 7628–7637. https://doi.org/10.1002/ece3.3315

Paquette A, Hector A, Castagneyrol B, Vanhellemont M, Koricheva J, Scherer-Lorenzen M, Verheyen K (2018) A million and more trees for science. Nature Ecology & Evolution 2: 763–766. https://doi.org/10.1038/s41559-018-0544-0

Parmesan C (2006) Ecological and evolutionary responses to recent climate change. Annual Review of Ecology, Evolution, and Systematics 37(1): 637–669. https://doi.org/10.1146/annurev.ecolsys.37.091305.110100

Pautasso M, Holdenrieder O, Stenlid J (2005) Susceptibility to fungal pathogens of forests differing in tree diversity. In: Scherer-Lorenzen M, Körner C, Schulze E-D (Eds) Forest Diversity and Function: Temperate and Boreal Systems. Ecological Studies. Springer, Berlin, Heidelberg, 263–289. https://doi.org/10.1007/3-540-26599-6_13
Keesing F, Holt RD, Ostfeld RS (2006) Effects of species diversity on disease risk. Ecology Letters 9(4): 485–498. https://doi.org/10.1111/j.1461-0248.2006.00885.x

Kim B-N, Kim JH, Ahn J-Y, Kim S, Cho B-K, Kim Y-H, Min J (2020) A short review of the pinewood nematode, *Bursaphelenchus xylophilus*. Toxicology and Environmental Health Sciences 12(4): 297–304. https://doi.org/10.1007/s13530-020-00068-0

Kimberley MO, Hood IA, Knowles RL (2011) Impact of Swiss needle-cast on growth of Douglas-fir. Phytopathology 101(5): 583–593. https://doi.org/10.1094/PHYTO-05-10-0129

Lerdau M, Litvak M, Monson R (1994) Plant chemical defense: Monoterpenes and the growth-differentiation balance hypothesis. Trends in Ecology & Evolution 9(2): 58–61. https://doi.org/10.1016/0169-5347(94)90269-0

Ligot G, Balandier P, Schmitz S, Claessens H (2020) Transforming even-aged coniferous stands to multi-aged stands: An opportunity to increase tree species diversity? Forestry. Forestry 93(5): 616–629. https://doi.org/10.1093/forestry/cpaa004

Massad TJ (2013) Ontogenetic differences of herbivory on woody and herbaceous plants: A meta-analysis demonstrating unique effects of herbivory on the young and the old, the slow and the fast. Oecologia 172(1): 1–10. https://doi.org/10.1007/s00442-012-2470-1

Meyer JB, Gallien L, Prospero S, Anderson I (2015) Interaction between two invasive organisms on the European chestnut: Does the chestnut blight fungus benefit from the presence of the gall wasp? FEMS Microbiology Ecology 91(11): fiv122. https://doi.org/10.1093/femsec/fiv122

Miles J (2014) Tolerance and variance inflation factor. Wiley StatsRef: Statistics Reference Online, John Wiley & Sons, Ltd. https://doi.org/10.1002/9781118445112.stat06593

Moreira X, Abdala-Roberts L, Castagneyrol B (2018) Interactions between plant defence signalling pathways: Evidence from bioassays with insect herbivores and plant pathogens. Journal of Ecology 106(6): 2353–2364. https://doi.org/10.1111/1365-2745.12987

Mori AS, Lertzman KP, Gustafsson L (2017) Biodiversity and ecosystem services in forest ecosystems: A research agenda for applied forest ecology. Journal of Applied Ecology 54(1): 12–27. https://doi.org/10.1111/1365-2664.12669

Nunez-Mir GC, Liebhold AM, Guo Q, Brockerhoff EG, Jo I, Ordonez K, Fei S (2017) Biotic resistance to exotic invasions: its role in forest ecosystems, confounding artifacts, and future directions. Biological Invasions 19: 3287–3299. https://doi.org/10.1007/s10530-017-1413-5

O'Loughlin LS, Green PT (2017) Secondary invasion: When invasion success is contingent on other invaders altering the properties of recipient ecosystems. Ecology and Evolution 7(19): 7628–7637. https://doi.org/10.1002/ece3.3315

Paquette A, Hector A, Castagneyrol B, Vanhellemont M, Koricheva J, Scherer-Lorenzen M, Verheyen K (2018) A million and more trees for science. Nature Ecology & Evolution 2: 763–766. https://doi.org/10.1038/s41559-018-0544-0

Parmesan C (2006) Ecological and evolutionary responses to recent climate change. Annual Review of Ecology, Evolution, and Systematics 37(1): 637–669. https://doi.org/10.1146/annurev.ecolsys.37.091305.110100

Pautasso M, Holdenrieder O, Stenlid J (2005) Susceptibility to fungal pathogens of forests differing in tree diversity. In: Scherer-Lorenzen M, Körner C, Schulze E-D (Eds) Forest Diversity and Function: Temperate and Boreal Systems. Ecological Studies. Springer, Berlin, Heidelberg, 263–289. https://doi.org/10.1007/3-540-26599-6_13
Poland TM, McCullough DG (2006) Emerald ash borer: Invasion of the urban forest and the threat to North America's ash resource. Journal of Forestry (April-May, 2006): 118-124. https://www.fs.usda.gov/research/treesearch/15034 [January 18, 2023]

Ritóková G, Shaw DC, Filip G, Kanaskie A, Browning J, Norlander D (2016) Swiss needle cast in Western Oregon Douglas-Fir plantations: 20-Year monitoring results. Forests 7(12): 155. https://doi.org/10.3390/f7080155

R Core Team (2022) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. https://www.R-project.org/

Root RB (1973) Organization of a plant-arthropod association in simple and diverse habitats: The fauna of collards (Brassica oleracea). Ecological Monographs 43(1): 95-124. https://doi.org/10.2307/1942161

Roques A, Auger-Rozenberg M-A, Capretti P, Sauvard D, La Porta N, Santini A (2019) Pests and diseases in the native and European range of Douglas-fir. 3.5, 1-125.

Sharma HC, Franzmann BA (2001) Orientation of Sorghum midge, Stenodiplosis sorghicola, females (Diptera: Cecidomyiidae) to color and host-odor stimuli. Journal of Agricultural and Urban Entomology 18: 237-248.

Simberloff D, Von Holle B (1999) Positive interactions of nonindigenous species: Invasional meltdown? Biological Invasions 1(1): 21-32. https://doi.org/10.1023/A:1010086329619

Stemmelen A, Jactel H, Brockerhoff E, Castagneyrol B (2022) Meta-analysis of tree diversity effects on the abundance, diversity and activity of herbivores' enemies. Basic and Applied Ecology 58: 130-138. https://doi.org/10.1016/j.baae.2021.12.003

Stout MJ, Thaler JS, Thomma BPHJ (2006) Plant-mediated interactions between pathogenic microorganisms and herbivorous arthropods. Annual Review of Entomology 51(1): 663-689. https://doi.org/10.1146/annurev.ento.51.110104.151117

Strauss SY, Cacho NI, Schwartz MW, Schwartz AC, Burns KC (2015) Apparency revisited. Entomologia Experimentalis et Applicata 157(1): 74-85. https://doi.org/10.1111/eea.12347

Verheyen K, Ceunen K, Ampoorter E, Baeten L, Bosman B, Branquart E, Carnol M, De Wandeler H, Grégoire J-C, Lhoir P, Muys B, Setiawan NN, Vanhellemont M, Ponette Q (2013) Assessment of the functional role of tree diversity: The multi-site FORBIO experiment. Plant Ecology and Evolution 146(1): 26-35. https://doi.org/10.5091/plecevo.2013.803

Ward SF, Liebhold AM, Fei S (2022) Variable effects of forest diversity on invasions by non-native insects and pathogens. Biodiversity and Conservation 31(11): 2575-2586. https://doi.org/10.1007/s10531-022-02443-4

Wilson E, San Martin G, Ligot G (2020) The Douglas fir needle midge (Contarinia pseudotsugae): a potential threat to Douglas fir in the United Kingdom and Ireland? Quarterly Journal of Forestry 114: 244-250.

Winton LM, Stone JK, Watrud LS, Hansen EM (2002) Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction. Phytopathology 92(1): 112-116. https://doi.org/10.1094/PHYTO.2002.92.1.112

Zenni RD, Essl F, García-Berthou E, McDermott SM (2021) The economic costs of biological invasions around the world. NeoBiota 67: 1-9. https://doi.org/10.3897/neobiota.67.69971
Poland TM, McCullough DG (2006) Emerald ash borer: Invasion of the urban forest and the threat to North America's ash resource. Journal of Forestry (April-May, 2006): 118-124. https://www.fs.usda.gov/research/treesearch/15034 [January 18, 2023]

Ritóková G, Shaw DC, Filip G, Kanaskie A, Browning J, Norlander D (2016) Swiss needle cast in Western Oregon Douglas-Fir plantations: 20-Year monitoring results. Forests 7(12): 155. https://doi.org/10.3390/f7080155

R Core Team (2022) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. https://www.R-project.org/

Root RB (1973) Organization of a plant-arthropod association in simple and diverse habitats: The fauna of collards (Brassica oleracea). Ecological Monographs 43(1): 95-124. https://doi.org/10.2307/1942161

Roques A, Auger-Rozenberg M-A, Capretti P, Sauvard D, La Porta N, Santini A (2019) Pests and diseases in the native and European range of Douglas-fir. 3.5, 1-125.

Sharma HC, Franzmann BA (2001) Orientation of Sorghum midge, Stenodiplosis sorghicola, females (Diptera: Cecidomyiidae) to color and host-odor stimuli. Journal of Agricultural and Urban Entomology 18: 237-248.

Simberloff D, Von Holle B (1999) Positive interactions of nonindigenous species: Invasional meltdown? Biological Invasions 1(1): 21-32. https://doi.org/10.1023/A:1010086329619

Stemmelen A, Jactel H, Brockerhoff E, Castagneyrol B (2022) Meta-analysis of tree diversity effects on the abundance, diversity and activity of herbivores' enemies. Basic and Applied Ecology 58: 130-138. https://doi.org/10.1016/j.baae.2021.12.003

Stout MJ, Thaler JS, Thomma BPHJ (2006) Plant-mediated interactions between pathogenic microorganisms and herbivorous arthropods. Annual Review of Entomology 51(1): 663-689. https://doi.org/10.1146/annurev.ento.51.110104.151117

Strauss SY, Cacho NI, Schwartz MW, Schwartz AC, Burns KC (2015) Apparency revisited. Entomologia Experimentalis et Applicata 157(1): 74-85. https://doi.org/10.1111/eea.12347

Verheyen K, Ceunen K, Ampoorter E, Baeten L, Bosman B, Branquart E, Carnol M, De Wandeler H, Grégoire J-C, Lhoir P, Muys B, Setiawan NN, Vanhellemont M, Ponette Q (2013) Assessment of the functional role of tree diversity: The multi-site FORBIO experiment. Plant Ecology and Evolution 146(1): 26-35. https://doi.org/10.5091/plecevo.2013.803

Ward SF, Liebhold AM, Fei S (2022) Variable effects of forest diversity on invasions by non-native insects and pathogens. Biodiversity and Conservation 31(11): 2575-2586. https://doi.org/10.1007/s10531-022-02443-4

Wilson E, San Martin G, Ligot G (2020) The Douglas fir needle midge (Contarinia pseudotsugae): a potential threat to Douglas fir in the United Kingdom and Ireland? Quarterly Journal of Forestry 114: 244-250.

Winton LM, Stone JK, Watrud LS, Hansen EM (2002) Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction. Phytopathology 92(1): 112-116. https://doi.org/10.1094/PHYTO.2002.92.1.112

Zenni RD, Essl F, García-Berthou E, McDermott SM (2021) The economic costs of biological invasions around the world. NeoBiota 67: 1-9. https://doi.org/10.3897/neobiota.67.69971