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 Nothophaeocryptopus 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.

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

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 decrease 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 (Hambäck and Englund 2005). The natural enemies’ hypothesis states that richer plant communities can favour herbivore’s natural enemies by increasing the availability of suitable 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. Following 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 interaction 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 Nothophaeocryptopus 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.

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 maple, 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 differences 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.

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).

Figure 3.

Effect of Douglas fir apparency on the mean damage by C. pseudotsugae. Apparency 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).

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.

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.

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 herbivores (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 findings 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 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.

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.