Veteranising Scots pine trees by initiating tree hollowing: Inoculation with the fungal keystone species Porodaedalia pini

Hollow trees are crucial for forest biodiversity but are becoming increasingly rare in many ecosystems, including the Scots pine forests of northern Europe. Here, we inoculated heartwood of live Scots pine trees with the fungal keystone species Porodaedalia pini to initiate tree hollowing. The fungus was inoculated in 50-, 110-and 170-year old stands, using wood dowels containing mycelia. Three different strains were used to test for intraspecific variation. Molecular analysis of samples from inoculated trees seven years after treatment showed that 67% were successfully colonised, with no differences between stands. Fungal strain had no effect on colonisation success. Our findings suggest that inoculation with P. pini has the potential to be an efficient method to restore a key ecological process, tree hollowing, in degraded Scots pine forests. The possibility of initiating the process even in young trees may be a way to accelerate the formation of hollow pines in younger forests.


Introduction
Living trees with cavities and decaying wood are keystone structures in forest ecosystems worldwide and provide crucial habitats for a wide array of biodiversity (Gibbons and Lindenmayer 2002;Cockle et al., 2011;Remm and Lohmus 2011).Unfortunately, such trees are becoming increasingly rare in many ecosystems due to the loss of large, old trees combined with low recruitment of new cavity trees (Lindenmayer et al., 2012).In northern Europe, the vast majority of boreal Scots pine (Pinus sylvestris) forests have been subjected to high-grading or clear cutting, leaving behind a fundamentally transformed forest landscape.Old forests (>200 years) have been almost entirely replaced by younger stands and plantations, resulting in a severe lack of old pine trees and snags with cavities and hollows (Linder and Ostlund 1998).Furthermore, rotation periods are generally shorter than the time required for most cavity-forming processes to occur, which hampers the formation of new hollow pines (Lohmus 2016).A major challenge to forest restoration, therefore, is to develop practices that can be applied to restore these key structures for biodiversity in degraded Scots pine forests.
The most important decayer of living Scots pines in northern Europe is the heart rot fungus Porodaedalia pini.As the main architect behind hollow pine formation, it is considered an 'ecological engineer' and a keystone species for maintaining biodiversity and function of pine forests (Lohmus 2016).By decomposing heartwood, it creates rotting hollows that provide habitat for a wide range of threatened species (Lohmus 2003(Lohmus , 2016;;Ehnström and Holmer 2017).Trees with heartwood softened by P. pini are preferred for cavity excavations by woodpeckers, whose cavities serve as nest sites for hole-nesting birds and microhabitats for fungi and insects (Lohmus 2016;Ehnström and Holmer 2017).Like hollows in oaks, pine hollows are often filled with wood mould, a mixture of rotten wood, mycelia, old bird nests, droppings and dead insects.This distinctive environment supports a unique community of invertebrates, including several beetle species listed on the Swedish red-list (Ehnström and Holmer 2017).Tree hollowing by Porodaedalia pini is a slow process and colonised trees can live for many decades seemingly unaffected.However, over time hollowed trees become structurally weaker and prone to wind-breakage, resulting in chimney-like high stumps.The cavities in the broken tops provide important nest sites for large owls, such as the Ural owl (Strix uralensis) (Saurola 1992).Such chimney snags were previously a characteristic feature of northern European pine forests but are now almost exclusively found in protected forests, so that Ural owls now largely depend on nest boxes for successful nesting (Saurola 1992;Lohmus 2003).It is not known when P. pini naturally colonises trees, but in northern Europe, fruiting bodies rarely appear until trees reach 150-200 years old (Ehnström and Holmer 2017).Due to the decline in old pines, P. pini has also decreased and is now on the Red List of threatened fungi in both Sweden and Norway (SLU Artdatabanken 2020; Artsdatabanken 2021).While rare in managed forests, P. pini remains relatively common in many natural pine forests, where it plays a key role in small-scale deadwood dynamics (Lohmus 2016).Therefore, a potentially effective way to promote P. pini and associated biodiversity in managed forests would be to introduce P. pini in retention trees and set-asides by active management.Additionally, given the general lack of old pine trees for natural colonisation, introducing P. pini into younger trees could be valuable.If so, it may be possible to accelerate the formation of hollow pines in younger forests.
The procedure in which young trees are intentionally damaged to accelerate the formation of structures or properties usually associated with old trees is called veteranisation (Bengtsson et al., 2012).Here we evaluate a method to veteranise living pine trees of different ages by artificially inoculating them with P. pini.Since tree hollowing by P. pini is a very slow process, often taking many decades or even centuries in boreal forests, we focus only on the early colonisation phase, examining the overall colonisation success and establishment in relation to stand age and fungal strain seven years after inoculation.Porodaedalia pini is a resource-unit-restricted fungus, meaning it can only spread by spores, and cannot grow out of resources and spread via mycelia from one tree to another.It is generally accepted that resource-unit-restricted fungi colonise the heartwood of living trees through stem wounds or dead and broken tops or branches (Boddy 2001).Despite young trees generally exhibiting greater vigor and more robust defense mechanisms against stressors, they also tend to have more actively growing tissues, facilitating the efficient compartmentalization and sealing off of wounds compared to older trees.Therefore, we hypothesize that the heartwood of younger trees can be successfully colonized through artificial inoculation with P. pini.

Fungal strains and preparation of dowels
Three strains of P. pini were isolated from fruiting bodies located on different living Scots pine trees in the Norra stadsberget Nature Reserve, close to Sundsvall, central Sweden.Pure cultures were obtained by removing small pieces of the inner part of the fruiting bodies and placing them on Petri dishes containing 2% malt extract medium (MA).Species identity of the strains was confirmed by comparing colour and morphological characters of the mycelia with a sequenced reference culture belonging to the Mid Sweden University Fungi Collection (GeneBank accession no.JQ437410).Isolated strains were maintained on MA plates prior to experimental use.To obtain dowels for the experiment, 5 mm diameter wood cores from Scots pine trees were obtained by drilling living trees with an increment borer.Wood cores were split into 5 cm long sections and soaked in water over night, before being sterilised by autoclaving twice at 120 • C for 30 min.Thereafter they were put on 15 cm diameter, ventilated Petri dishes with actively growing cultures of P. pini and incubated in darkness at room temperature for six months (Fig. 1).Control dowels without fungi were sterilised and incubated, following the same procedure as those inoculated with P. pini.

Field experimental setup
In 2009, three experimental sites were established in Scots pine forest stands within the Stormyran-Lommyran Nature Reserve, central Sweden (O15,742697 N62,38250).These stands are similar in terms of field layer, edaphic conditions, and productivity but differ significantly in average ages, which are approximately 50, 110, and 170 years.The two younger stands are even-aged and have a history of forest management, whereas the oldest stand is semi-natural.The field layer is dominated by a mixture of the dwarf shrubs Vaccinium myrtillus and V. vitis-idea, and the bottom layer mainly consists of bryophytes, predominantly Pleurozium schreberi and Hylocomium splendens.All stands are located within a 1.8 km radius.The experiment was conducted on 18 randomly selected trees in each stand, including three control trees, making a total of 54 trees.Only healthy trees without any visual signs of damage or infections were included.Each tree was drilled at breast height to the heartwood pith with an increment borer with a 5 mm core diameter, resulting in a ca. 10 mm diameter hole.Drillings were performed perpendicular to the stem and the resulting wood core was carefully inspected for any signs of rot that would disqualify the tree from the experiment.However, no signs of rot were found in any tree.A dowel colonised by P. pini mycelium was inserted as far as possible into each hole and the opening was closed with a 3 cm long ca. 10 mm diameter, sterilised wood dowel.To account for intraspecific variation in fungal behaviour, trees were inoculated with one of the three strains of P. pini, resulting in 5 trees per strain and site.Control trees were treated in exactly the same way but were "inoculated" with sterilised dowels without fungi.

Evaluation of fungal colonisation success
In 2016, seven years after the fungus was inoculated, the presence or absence of P. pini was investigated in each tree using molecular techniques.Heartwood samples were taken 3 cm above and below the inoculation site with a 5 mm increment corer, coring through the bark to the pith.Samples from the same tree were pooled, collected in zip bags, placed in a cooler bag with dry ice, transported to the laboratory, and stored at − 20 • C until processing.The heartwood was separated from the sapwood in each wood core.The heartwood samples from different trees were then ground separately into a fine powder in a sterilised ceramic pestle using a mortar and liquid nitrogen.DNA was subsequently extracted using a PowerSoil DNA Isolation Kit (MoBio, Carsbad, Ca, USA).PCRs were run with a primer pair specific for P. pini, targeting a species-specific sequence in the ITS region; forward primer GCCTTCGTGCTTAATCCACTC and reverse primer AACCTCCAAATC-CAAGCCCC.The theoretical specificity of the primer was confirmed using the BLAST tool (NCBI, GeneBank), and its functionality was tested in the lab using different concentrations of DNA extracted from pure cultures with P. pini.PCRs were then run on all extracted samples according to the manufacturer's recommendations (Kapa Biosystems Robust PCR Kit).PCR-products were visualised with EtBr on 0.8 % agarose gels under UV light, and P. pini was considered to be present in a sample if a successful amplification was detected.
In 2021, twelve years after inoculation all trees were inspected for fruiting bodies.To visually verify colonisation by P. pini and to roughly estimate the mycelial spread in the stem over time, three trees in which colonisation by P. pini had been confirmed by pcr were felled.Only a few trees were felled since we wanted to keep the experiment as intact as possible to be able to follow long-term development.Felled trees were first cut at the location of inoculation.Cut surfaces were carefully inspected for signs of P. pini decay, such as colour changes and softened wood.If signs of decay were found, additional 5 cm cross-sections were cut until no more signs were visible.Decay by P. pini exhibits characteristic signs and was assessed according to the classification given by Szewczyk (2015): stage 1 -early decay indicated by pale pink coloured xylem without structural changes; stage 2 -medium decay indicated by more intensive xylem colouring, from pink to pale cocoa, sometimes associated with certain brittleness and occasionally the presence of the first pockets; stage 3 -late decay indicated by reddish to cocoa brown coloured xylem with visible cracking and pockets filled with white cellulose; stage 4 -hollow rot indicated by empty spaces in the trunk.

Statistical methods
The effect of stand and fungal strain on the presence/absence of P. pini seven years after inoculation was investigated using a binomial generalised linear model (GLM) with a logit link using R (R Core Team 2022).

Presence of fruiting bodies and extension of established mycelia 12 years after inoculation
No fruiting bodies had emerged 12 years after inoculation.However, clear signs of decay by P. pini were found in all three felled trees (Fig. 3).Decay was mostly in stage 1, represented by pale pink coloured wood in parts of the heartwood, especially close to the sapwood border (ring rot).Closer to the inoculation site, moderate decay with wider radial extension was observed, characterized by clearly softened and dark pink wood.No signs of more advanced decay (stages 3 or 4) were found.The fungus' spread in the heartwood fibre direction (vertical) was on average 41 cm, ranging from 30 to 49 cm, which is equivalent to an average extension rate of 3.4 cm per year.

Discussion
A significant challenge to forest restoration is that many target habitats result from very long-term ecological processes.In the conifer forests of northern Europe this is particularly evident in Scots pine forests, where declining key structures, such as hollow pines, need many decades or even centuries to develop.Hence, restoration practices that initiate and ideally accelerate these processes are important to bridge future gaps in habitat availability.It is important, however, to consider that rapid development of tree microhabitats may negatively impact their persistence (Korkjas et al., 2021).Additionally, a prerequisite for such practises to be efficient is that they initiate or mimic the natural process that produce the desired habitat or structure under natural conditions (Lindenmayer et al., 2006).Here we successfully initiated hollow formation in Scots pine by inoculating the keystone species P. pini into the heartwood of live trees.We also demonstrated that it is possible to initiate hollow formation in younger stands (50 years old), which opens up the possibility of introducing P. pini and potentially shortening the time required for hollow pines to develop.Consequently, this approach could serve as an important conservation tool to address the scarcity of such trees in younger stands or in pine forests degraded by practices like high grading.
Heartwood fungi have been artificially inoculated into living trees since the late 19th century, with varying purposes and species involved (Wainhouse and Boddy 2022).Several studies have inoculated P. pini,  but only two have focused on Scots pine, and not in the boreal region as in the present study.German forest pathologists pioneered this work in the 19th century by inoculating P. pini into healthy temperate Scots pine trees using heartwood from infected trees (Hartig 1874;Möller 1904).However, their small sample sizes prevent general conclusions.In North America, P. pini sensu lato has been inoculated into various tree species using fungus-colonised wood dowels, generally achieving lower success rates than our 67% colonisation.Conner and Locke (1983) reported 53% colonisation in Loblolly pines (Pinus taeda), Basham (1975) found 36% in Jack pine (Pinus banksiana), and Filip et al. (2011) achieved 60% in Douglas fir (Pseudotsuga menziesii) and Ponderosa pine (Pinus ponderosa).Higher success rates were reported in Eastern White pine (Pinus strobus), with Linzon (1962) achieving 87.5% and Silverborg and Larsen (1967) achieving 85% using fungal-colonised wood dowels.
Although largely successful, about one third of our inoculations failed according to the PCR-analysis.The reason is unknown, but dowels could possibly have been destroyed by resins.When drilling through the sapwood, it is inevitable that resin ducts are sometimes accidentally hit, releasing resin into the hole.In the present study, holes were drilled perpendicular to the stem, not at an angle.It is therefore possible that wood dowels became soaked with resin when resin ducts where hit, which inhibited fungal growth.Some authors have reported that holes were drilled at a slight upward angle to prevent entrance of rainwater and resins from flowing into the hole (Basham 1975, Conner et al., 1983).Another way to increase the probability of successful colonisation of a tree is, of course, to use more than one dowel (Filip et al., 2011).
The mycelial extension by inoculated P. pini in wood was not the scope of this study, and only three inoculated trees were examined.Nonetheless, it is worth mentioning that the average vertical extension of 3.4 cm per year is clearly lower than found in other studies.For example, In Scots pine, Hartig (1874) and Möller (1904) reported rates ranging from a few cm to 25 cm per year.Similarly, in White pine, Linzon (1962) and Silverborg and Larsen (1967) found average extension rates of ca. 25 cm per year, though they also noted substantial variation between trees.DNA from dead fungal tissue can remain detectable in soil several months post-mortem (Gordon and van Norman, 2021).How long it remains in wood is unknown, but since we did not culture any fungi from the colonized wood, we cannot entirely rule out detecting dead mycelia from rapidly extending colonizations that subsequently died off.Given the limited number of samples in our study, no definitive conclusions can be drawn about the extension rate in boreal Scots pine until more trees are thoroughly investigated.
Heartwood formation in Scots pine starts at the age of 20-40 years in Scandinavia (Fries and Ericsson 1998;Bjorklund 1999;Uusitalo 2004).The successful artificial colonisation in a 50-year-old stand clearly shows that P. pini thrives even in younger pine trees, although their heartwood is less developed than in older trees.Thus, most Scots pine trees are probably susceptible to inoculation when they reach an age of 45-50 years.This is further supported by Hartig (1874) who, in a small study, failed to inoculate 30-40-year-old pines, while succeeding with all inoculations of older pines (60-70 years).Furthermore, somewhat similar to our result, Bednarz et al. (2013) found no difference in colonisation success of inoculated Fomitopsis pinicola in 50-and 70-year-old Douglas-fir and Western Hemlock in Washington, USA.Our findings suggest that P. pini, and perhaps resource-unit restricted heart-rot fungi in general, are largely absent from younger trees due to lack of colonisation pathways into the heartwood, such as stem wounds or broken branches.However, we cannot rule out the possibility that P. pini does colonise young trees, although fruiting bodies are not produced until the tree is old.In this study, no fruiting bodies were formed, and decay was limited after 12 years, indicating that extensive decay and fruit body formation by P. pini require very long times in Scots pine, at least in the boreal region.
It is important for restoration practitioners to be aware that inoculation of decay fungi is by no means uncontroversial and should only be undertaken after careful consideration of potential risks.Most importantly, artificial inoculation and translocation may affect the genetic variation of local species populations, with introduced fungal genets potentially outcompeting local strains and leading to a loss of local adaptations (Norden et al., 2020).To mitigate this risk, it is recommended to inoculate only a small proportion of trees and to use strains of local origin, as done in our study.However, there can be significant intraspecific variation in fungal traits, which may impact the overall success of an inoculation project.While our study found no difference in colonisation success between strains, it remains unclear if they differ in other important traits such as mycelial extension or wood decay rate (Lee et al., 2008).Even small intraspecific differences in these traits could significantly affect the timeline for hollow formation in Scots pine.Additionally, it is essential to emphasize that fungal inoculation should never serve as justification for removing stands with naturally existing hollow trees as part of forest exploitation practices, such as using it as a method for ecological compensation.
Porodaedalia pini is generally viewed as the most important heartwood decayer of live Scots pine trees in boreal Scandinavia.Other heartrot fungi, such as Phaeolos schweinitzii and Coniophora spp., are also present, but seem less frequent based on the occurrence of fruiting bodies.Additionally, these fungi typically cause butt or root rot, making trees prone to windthrow, The height at which the fungus enters the tree is crucial for the kind of stump created (Lännenpää et al., 2008).In this study, P. pini was inoculated at breast height.However, for creating high stumps, it may be more effective to introduce the fungus higher up, as the stem is likely to break where decomposition has progressed the longest.Another question that remains to be investigated, related to hollow formation in Scots pine, is how efficient it is to initiate hollow rot just by creating colonisation pathways into the heartwood, without fungal inoculation.Carved hollows are likely to facilitate heart-rot over time, although no long-term studies have yet been reported (Wainhouse and Boddy 2022).

Conclusions
Restoration measures are increasingly implemented to restore natural dynamics in northern European boreal forests.Alongside widely applied conservation practices such as restoration fire and girdling, we show that artificial inoculation with P. pini could be a way to restore natural small-scale dynamics and potentially accelerate the formation of important tree habitats.Inoculation of P. pini could also be a way for forest companies to enhance the quality of forest set asides, retention patches and "eternity trees" left after clear cutting.However, practitioners need to consider the long delivery time and the challenges of scaling up inoculation efforts.Additionally, any reintroduction programme involving P. pini must address the potential risk of negatively influencing the population genetics of the existing, regional P. pini population.Therefore, using inoculation as one veteranisation measure alongside others that depend on the stochastic arrival of propagules is recommended.

Fig. 1 .
Fig. 1.Representative example of wood dowels colonised by Porodaedalia pini six months after inoculation on a MA plate with actively growing mycelium.

Fig. 3 .
Fig. 3. Representative examples of caused by inoculated P. pini 12 years after inoculation.The rot is shown as darker areas with pink to reddish colour in the heartwood centre and close to the sapwood border.The darker, greyish-blackish area in the bottom of the picture (left panel) is resin-soaked sapwood in response to coring.