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Defoliation of Interior Douglas-fir Elicits Carbon Transfer and Stress Signalling to Neighbouring Ponderosa Pine Trough Ectomycorrhizal Networks

According to Song et al. extensive regions of Interior Douglas-fir (Pseudotsuga menziesii var. glauca, IDF) forests in North America are being damaged by drought and western spruce budworm (Choristoneura occidentalis). Warmer, drier summers associated with climate change are causing this damage. In order to test whether defoliated IDF can transfer directly resources to ponderosa pine (Pinus ponderosae) that is regenerating nearby, thereby aiding in the recovery of the forest, Song et al., examined the transfer of photosynthetic carbon and defence enzyme response. They grew pairs of ectomycorrhizal IDF ‘donor’ and ponderosa pine ‘receiver’ seedlings in pots and isolated pathways by comparing 35 μm, 0.5 μm and no mesh treatments; they then stressed IDF donors either through manual defoliation or infestation by budworm. The found that manual defoliation of IDF donors led to transfer of carbon to neighbouring receivers via mycorrhizal networks, though not trough soil or root pathways. Both manual and insect defoliation of donors led to increased activity of peroxidase, polyphenol oxidase and superoxide dismutase in the ponderosa pine receivers, by a mechanism that was dependent primarily on the mycorrhizal network. It is indicated by these findings that IDF can transfer these resources and stress signals to interspecific neighbours, which suggests that mycorrhizal networks can serve as agents of interspecific communication which facilitates recovery and succession of forests after disturbances.

According to Song et al. plants have evolved the ability to communicate with neighbouring plants for alleviating stresses within communities by the transmission of volatile compounds aboveground, or a variety of organic and inorganic compounds belowground. Mycorrhizal networks, which are comprised of mycorrhizal fungi that connect the roots of multiple plants, are potentially direct pathways for belowground transference of biochemical messages between plants (Van der Heijden & Horton, 2009). Evidence is increasing that mycorrhizal networks can transmit, e.g., herbivore- or pathogen-induced signalling compounds to warn neighbours of infestations by pests (Song et al., 2010; Song et al., 2014; Babikova et al., 2013), kin recognition signalling compounds that involve micronutrients to communicate genetic relationships of neighbours (File, Murphy & Dudley, 2012; Asay., 2013), toxins such as allelochemicals to convey negative interactions to competing neighbours (Barto et al., 2011), and essential resources, such as carbon, nitrogen, phosphorus or water for altering physiology, survival, or growth of conspecific or heterospecific neighbours (Simard et al., 2012). It has also been shown that mycorrhizal networks transfer phosphorus and nitrogen from dying plants to healthy conspecific neighbours (Eason, Newman & Chuba, 1991), thereby providing a conduit for legacy transference across generations. Clipping has similarly prompted transference of labile carbon from stressed to healthy heterospecific neighbours through arbuscular mycorrhizal networks (Fitter et al., 1998). The neighbours that receive messages could potentially then modify their behaviour through altered morphology, physiology or biochemistry, thereby reducing their stress and improving their fitness. The majority of studies that have been conducted so far have focused on herbaceous or grass species that form arbuscular mycorrhizal networks, though there is increasing evidence for interplant communication through mycorrhizal networks. Commutation belowground between trees linked by ectomycorrhizal networks in forests has, however received little attention.

Around the world forests are experiencing increasing stress and tree mortality as climate changes (van Mantgem, 2009; Allen et al., 2010). Climate change is disrupting coevolved host-pest interactions by altering life cycles of forest trees, insects, and fungal pathogens, which causes outbreaks of bark beetles that are well documented, in pine (Pinus) blights and rusts and spruce (Picea) species in North America (Kurz et al., 2008; Heineman et al., 2010; Wood et al., 2010; Sturrock et al., 2011). Extensive regions of interior Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. glauca (Mayr)) forests are also being defoliated by drought, western spruce budworm (Choristoneura occidentalis) and Douglas-fir tussock moth (Orgyia Pseudotsugata) in a direct response to warmer summer temperatures (Woods et al., 2010). Tree mortality that results in sustained severe defoliation makes new growing space available for tree species from warmer locations and helps to facilitate predicted forest vegetation shifts (Wang et al., 2012). These shifts in forest vegetation are facilitated further by salvage logging of trees that are dead and dying (Kurz et al., 2008). Mortality and salvage logging of dry interior Douglas-fir in interior British Columbia that results from climate change should make favourable conditions for upwards and northern  migration of Ponderosa pine (Pinus ponderosa Douglas ex C. Lawson), as predicted by the climate envelope models of Wang et al., (2017).

In North and South America Douglas-fir and ponderosa pine, in a replacement series experiment, are host to hundreds of species of ectomycorrhizal fungi, which include many ‘generalist’ fungi that are common to both tree species (Barroetaveña, Cázares & Rajchenberg, 2007) and they can thereby be linked to a mycorrhizal network where the 2 tree species co-occur in nature (Perry et al., 1989; Molina, Massicotte & Trappe, 1992). Douglas-fir and ponderosa pine yielded greater biomass in co-culture than in monoculture when colonised by Laccaria laccata (Scop. Ex Fr.) Bk & Br., where foliar nitrogen and phosphorus that are enhanced transfer through a mycorrhizal network (Perry et al., 1989). It is suggested by these results that interplant transfer of nutrients that are mediated by mycorrhizal network has the potential to influence the distribution of resources and performance of plants within the communities (Selosse et al., 2006; He et al., 2009; Teste et al., 2009). The possibility that mycorrhizal networks can act as a conduit for the transfer of nutrient legacies or stress signals from interior Douglas-fir to healthy ponderosa pine neighbours in response to climate-induced defoliator outbreaks, is also opened by this discovery.

Trees can respond to infection by producing an array of defence compounds that mediate their interactions with the invader (Hare, 2011), as they have coevolved with native insects and pathogens. Trees also coevolved with ectomycorrhizal fungi that are responsible for uptake of nutrients and water in exchange for carbon (Molina, Massicotte & Trappe, 1992). It was also shown by previous research with arbuscular mycorrhizal tomatoes that when plants are connected by a mycorrhizal network, stress signals can transfer from infected plants to conspecific neighbours through this network, thereby increasing activity of defence compounds, which induce defence-related genes, activating the jasmonate pathway, and increasing the resistance to pests in receiver plants (Song et al., 2010; Song et al., 2014). Additionally, Song et al. have shown in other previous studies that interior Douglas-fir can transfer carbon, nitrogen and water through ectomycorrhizal networks to conspecific or heterospecific neighbours, and that this has been associated with increase survival, growth and foliar nutrition of recipient neighbours (Teste et al., 2009; Simard Climate change is disrupting 1997; Philip, Simard & Jones, 2010; Bingham & Simard, 2012). It is suggested that, taken together, transfer of signals and resources transfer from interior Douglas-fir to ponderosa pine through ectomycorrhizal networks could play a role in the facilitating and shaping of the predicted forest vegetation shifts in this region as climate changes.

The objective of this study was to determine whether when interior Douglas-fir are injured by being defoliated by insects or manual defoliation would induce the interspecific transfer of carbon and stress signals to neighbouring healthy Ponderosa pine seedlings through a mycorrhizal network. The first hypothesis of Song et al. was that manual and insect defoliation would cause the export of labile carbon from interior Douglas-fir directly to neighbouring Ponderosa pine through mycorrhizal networks. The expectation was that increasing levels of carbon export with increasing degree of injury. They also expected that the amount of transfer would be reduced by the presence of root competition. Their second hypothesis was that manual and insect defoliation would cause interior Douglas-fir to communicate via organic stress signals with ponderosa pine to increase the defence response. They expected that there would be greater defence response with insect defoliation than with manual defoliation because the trees had coevolved with the insects species. They also expected that there would be greater transfer of the stress signal directly through mycorrhizal networks than indirectly through soil pathways.         

Discussion

Belowground communication with and transfer of legacies from damaged trees to other encroaching tree species may facilitate forest vegetation shifts with climate change. This study demonstrated for the first time that injury to 1 tree species induces substantial belowground transfer of photosynthetic carbon and elicits a rapid defence response of a different tree species, probably through the transfer of stress signals. Moreover, this interspecies communication occurs through mycorrhizal networks, bypassing microbial transformations that can occur along soil pathways. The ectomycorrhizal network of the 4-month old interior Douglas-fir and ponderosa pine was comprised of a single taxon Wilcoxina rehmii (Ascomycota, Pezizales order), an E-strain fungal species (Eggen1996) that is well known as an early coloniser of interior Douglas-fir and ponderosa pine seedlings in forest soils that have been disturbed recently (Egger, 1996; Fujimura et al., 2005; Barker et al., 2013).

C transfer and partitioning

The first hypothesis of Song et al. is partially supported by the manual defoliation, though not the defoliation by insects, caused the interior Douglas-fir to transfer labile carbon directly to neighbouring ponderosa pine through mycorrhizal networks. The lack of response to insect treatment may be explained by the unexpected minimal levels of defoliation by western spruce budworm compared with the response to manual excision, as was evidenced by donor isotope contents in the 35 μm mesh treatment, most likely that the 2 insects were too few or immature at the 3^rd instar stage for vigorous feeding. The study also found that the transfer of carbon to the shoots and roots of ponderosa pine increased with the declining donor Douglas-fir isotope content which suggests that increasing defoliation (which caused lowered isotope uptake) stimulated the belowground flush to networked pine. When the interior Douglas-fir were defoliated manually carbon compounds were exported to roots, a behavioural strategy that is known for helping trees survive subsequent defoliations (Vanderklein & Reich, 1999). The belowground pulses of labile carbon to roots were then transported to extrametrical mycorrhizal network, as indicated by the significant transfer of carbon to the ponderosa pine receivers. Together, the difference in severity between the 2 methods of defoliation and the negative relationship between donor and receiver isotope content, supported the 2^nd expectation of Song et al. that the export of carbon would increase with defoliation injury. They predict that severe sustained insect defoliation would elicit that transfer of carbon of similar magnitude as manual defoliation that was found in this study, though insect treatment was not enough to elicit a response, but future research is still needed in order to quantify these effects in the greenhouse and in situ.

Carbon transfer belowground in the manual defoliations treatment could have occurred by way of 3 alternative pathways: soils, mycorrhizal networks, or roots. The possibility for aboveground communication via volatiles (Karban et al., 2006) was excluded by covering the donors with airtight plastic bags during defoliation, thereby isolating belowground communication pathways (Dicke & Dijkman, 2001). If root exudates were transferred by the soil pathway isotope in the receivers would have been detected in both 0.5 μm mesh and no mesh treatments. If transfer had occurred via roots, Fraser & Lieffers (Fraser, Lieffers & Landhausser, 2006) in Pinus contorta Douglas, isotope would have been detected in receivers in the no mesh treatment. Instead, no transfer was found when mycorrhizal networks were excluded with a 0.5 μm mesh, or permitted root-root contact in the no mesh treatment. It was suggested by these results that the transfer of carbon only occurred though mycorrhizal networks.

As was expected, based on previous research by Bingham & Simard (Bingham & Simard, 2012), the presence of root competition reduced or masked the mycorrhizal transfer of carbon to ponderosa pine. According to Song et al. it is possible that any movement of carbon through the intact mycorrhizal network in the no mesh treatment was bidirectional, as was shown in (Simard et al., 1997) & (Philip et al., 2010), but the interior Douglas-fir was a stronger competitor than the ponderosa pine for this carbon pool. The reduced germination and growth rates of ponderosa pine in the no mesh treatment versus mesh treatments, where interior Douglas-fir roots could mingle freely with those of ponderosa pine, supported the strong competitive ability of the roots of interior Douglas-fir to acquire belowground carbon. It is indicated by its smaller root structure than ponderosa pine that competitive effects of interior Douglas-fir were driven primarily by belowground rather than aboveground processes, which precluded interior Douglas-fir pre-empting light from ponderosa pine.

A ‘mesh effect’ is not supported by the data from this study, where restricted access to grazing invertebrates or greater water retention in the 35 μm bags may have benefited the growth of ponderosa pine, and therefore its sink strength for transferred carbon. High transfer rates of carbon would have been found in the 0.5 μm mesh as well as the 35 μm mesh bags if there was such a mesh effect, but no such effect was found. Also, the presence of the effect of mesh or mesh size on the availability of water to interior Douglas-fir or ponderosa pine within a pot was not found. Moreover, native soil that was used to fill the mesh bags was likely to have contained as many invertebrate grazers as soil in the remainder of the pot. The ability to detect transfer of carbon to ponderosa pine in 35 μm mesh but not in the no-mesh treatment was, therefore, not due to artefacts that were caused by the presence of mesh itself, but is explained most plausibly by the absence of root competition from interior Douglas-fir.

According to Song et al. the transfer of carbon from interior Douglas-fir to ponderosa pine through the mycorrhizal network may have occurred along a carbon or foliar nutrient source-sink gradient (Francis & Read, 1984). A carbon source-sink strength alone could possibly have played a role in regulating the transfer of carbon. It is likely that defoliation stimulated interior Douglas-fir to export rapidly labile carbon from enriched roots to the mycorrhizal network (Vanderklein & Reich, 1999), thereby increasing the source strength, while the rapid growth rate of the ponderosa pine would at the same time have developed a large sink strength. The high amount of carbon that was transferred to receiver shoots in this case would have moved through the xylem or transpiration stream as carbohydrates, drawn by high rates of transpiration of the ponderosa pine shoots (1989). The preferential movement of labelled carbon to receiver shoots that was found in this study has also been found by others (Simard et al., 2012; Deslippe & Simard, 2011; Lerat et al., 2012), including cases in which sink strength shifts over growing seasons (Simard et al., 2012; Deslippe & Simard, 2011; Lerat et al., 2012), which suggests that in ponderosa pine sink strength was an important driver of carbon transfer through the mycorrhizal network. Alternatively, carbon may have been transferred along a foliar nutrient gradient, where carbon was transferred with nutrient elements as free amino acids across the Hartig net of donors and receivers. An urgent nutritional demand of the fast growing pine would have been met by the rapid transfer of these nutrient elements in amino acids as they are essential for enzyme complexes involved in photosynthesis and synthesis of proteins.

Very high potential for carbon transfer belowground and/or sequestration by these dry forest trees by about half of the total carbon that was fixed in this study was partitioned to belowground pools. In the manual defoliation treatment, a large portion (6-8%) was transferred to the Ponderosa pine, though most of the ¹³C remained in the donor seedlings. It is suggested that the carbon that was transferred contributed significantly to biosynthesis in the receiver, by this rate of interspecific transfer though mycorrhizal networks being approximately equivalent to the carbon costs of reproduction (Reekie & Bazzaz, 1987). The rate of carbon transfer that was observed in this study was similar to that found through mycorrhizal networks between ectomycorrhizal Betula payrifera Marsh and Douglas-fir by Simard et al., (Simard et al., 1997) and Philip et al., (Philip, Simard & Jones, 2010), and between conspecific Betula nana pairs in the Arctic tundra by Deslippe & Simard (Deslippe & Simard, 2011), but is greater than carbon transfer between conspecific interior Douglas-fir pairs that were found in temperate forests by Teste et al., (Teste et al., 2009), or Bingham & Simard (Bingham & Simard, 2012). It is suggested by these comparisons that strong source-sink gradients that result from species physiology or harsh environmental conditions drive higher rates of carbon transfer than occur between conspecifics in favourable environments.

Stress signalling

In the interior Douglas-fir and the ponderosa pine receivers the significance of defensive enzyme activities following either manual or insect defoliation of interior Douglas-fir supports the 2^nd hypothesis of Song et al. that interspecific communication of stress signals would increase the defence response of the receiver. In agreement with the expectations of this study, based on Song et al., (Song et al., 2010; Song et al., 2014), it was found that the activity of all 3 defence enzymes in receivers increased more in 35 μm mesh treatment than 0.5 μm mesh or no mesh treatments, which indicates that belowground stress signalling occurred predominantly through mycorrhizal networks. It is suggested by the much smaller increases in enzyme activities in receivers in the 0.5 μm and no mesh treatments, that a lesser amount of stress signal is transmitted through the soil pathway. Stress signals that enter the soil pathway would be subjected to the same microbial degradation as carbon exudates, which is consistent with the observations of this study. In the non-defoliated controls enzyme activities were always lowest, which indicates that the mycorrhization per se did not prime the enzymatic defence response (i.e., mycorrhiza-induced resistance), as was discussed by Cameron et al., 2013). Song et al. concluded therefore that mycorrhizal networks transmitted chemical signals that elicited the defence response of ponderosa pine, which supports recent studies in arbuscular mycorrhizal systems by Song et al., (Song et al., , 2010; Song et al., 2014) and Babikova et al., (Babikova et al., 2013). The Song et al. expectation that defoliation by insects would elicit a greater defensive response than manual defoliation because of coevolution between tree and insect species was not generally met. The transfer of carbon was likely to be due to the .low feeding efficacy of western spruce budworms used in the research. There expectation was, however, supported by a single exception. The POD activity in donors that were defoliated by insects peaked 24 hours after it did so in donors that were defoliated manually, but it then remained higher in the donors than were defoliated by insects. Moreover, in spite of a lower extent of defoliation by the insect versus manually, in both donors and receivers the defence responses were of similar magnitude. It was demonstrated by these results that the light level of insect feeding on donors can elicit a strong and rapid response in receivers. They support the idea that regulation of the production of plant defence compounds is attuned slightly to attack by herbivores, as was expected by from long standing relationships that were coevolved between forest trees and herbivores. It was also shown by the results of this study that an additional interaction that was coevolved, the mycorrhizal symbiosis, and its ability to integrate the plant and fungal community in a network, influences the secondary chemistry of the conifers.

In both donors and receivers production of defence enzymes occurred after injury, which suggests that stress signals were exported rapidly to the mycorrhizal networks. Therefore, as defoliated interior Douglas-fir was exporting carbon to ponderosa pine through the mycorrhizal network, it was also transferring defence signals. A defence response could then be induced by the healthy ponderosa pine to protect itself against a possible attack. It was expected that signal transduction would be much faster than the rate of carbon transfer because the molecules of the signal are smaller than amino acids or carbohydrates (Park et al., 2007), thereby enabling them to move more quickly within hyphal networks via cytoplasmic streaming. Though in this study carbon transfer was measured only once after 6 days, a previous study that used ¹⁴C autoradiography shows that it takes at least 3 days for the transfer of carbon from donors to receivers through ectomycorrhizal networks (Wu, Nara & Hogetsu, 2001). In contrast to this, it was found by Song et al., (Song et al., 2014) that the signal molecule jasmonate travelled through the arbuscular mycorrhizal network within 6 hours of infestation by insects based on the accumulation of jasmonate and jasmonate response gene transcripts in receiver tomato plants. The possibility is also raised, however, by recent research on electrical signals produced by pants in response to mechanical and insect chewing damage that the defence was electrically induced via membrane depolarisation events (Brand & Gow, 2009; Salvador-Recatala et al., 2014).

Host specificity

The communication that was observed by Song et al. between interior Douglas-fir and ponderosa pine in response to mechanical and insect defoliation of interior Douglas-fir suggests that the damage elicited a general response. According to Song et al. the networking fungus may have acted to protect its net carbon source by allocating carbon and signals to the healthy, more reliable ponderosa pine. The mycorrhizal network may therefore benefit from transferring carbon and defence signals interspecifically, thereby favouring hosts that are able to provide more carbon (Kiers et al., 2011). Song et al. suggest it is possible, therefore that transfer and signals that are mycorrhizal network-based may evolve to be more generic in stressful environments. The response of the ponderosa pine to a stress signal from interior Douglas-fir may have a large cost and little benefit if the damaging agent is host specific, but if the damage is nonspecific, such as manual defoliation, it may be worth investing in constitutive defence enzymes. It is intriguing that ponderosa pine mounted a defence response to the attack on its interspecific neighbour, though western spruce budworm is a herbivore of interior Douglas-fir, and to a lesser degree Larix, Picea and Abies species. It is suggested by the defence response occurring in response to host-specific and host-generalist damage that the defence signal itself was a generic signal (e.g., jasmonate). It is also possible that ponderosa pine responds particularly well to abiotic damage and broad herbivore taxa (Bidart-Bouzat & Kliebenstein, 2011). In this study the decoupling of carbon and defence signal transfer, which was evident in the differential manual versus insect defoliation effect, suggests that interspecific transfer of carbon and defence signal occurred with host generalist damage (i.e. mechanical damage that can occur in response to abiotic stresses such as wind or drought), though that interspecific signal transduction was possible, even with the host-specific damage. Because of the differences in the foliar treatments, carbon that was transferred was therefore not likely to be a constituent of the defence signal. Song et al. suggest further research is needed to understand the compounds, mechanisms, specificity and fitness consequences of communication through mycorrhizal networks.

Conclusions

It was found by Song et al. that physiologically significant levels of photosynthate derived carbon was transferred by mycorrhizal networks and they transmitted interspecific stress signals that elicited defence responses in ponderosa pine following manual and insect defoliation of interior Douglas-fir. Mycorrhizal networks were shown by these results to be mediators of interactions among trees of different species and defoliators and, therefore, are likely to play a critical role in the defence response and recovery of forests from ether abiotic or insect outbreaks. Shifts in forest composition that are predicted with climate change may be facilitated by the direct pathway and stress signal transfer trough mycorrhizal networks to interspecific plant targets.

Insect pest epidemics and summer droughts in many forests in western North America that are exacerbated by climate change are leaving vast areas of dead trees. These forests are expected to undergo domain shifts to new, hopefully productive stable states of different forest vegetation composition as these forests regenerate. Mycorrhizal networks have been shown by this study to be positioned to play an important role in facilitation of regeneration of migrant species that have adapted better to warmer climates and to be primed for resistance to infestation of insect pests. The importance of conservation practices maintain all of the parts and processes of these highly interconnected forest ecosystems in order to help them deal with new stresses that are brought by the changing climate have been pointed to by these results.

Song, Y., et al. (2015). "Defoliation of interior Douglas-fir elicits carbon transfer and stress signalling to ponderosa pine neighbors through ectomycorrhizal networks." Scientific Reports 5: 8495.