For the dual cultures of Test 1, 76 taxa with 102 strains were tested. For this test the method of Rigerte et al. (2019) was followed regarding set up and measurements (see Figure 2A). For each strain of C. corticale (CC1-CC3) triplicates per tested strain were set up. The inoculated Petri dishes were kept at room temperature and ambient light, and checked for growth on days three, five, and 10, each measured and marked on the back of the Petri dish. The cultures were measured along the two axes (alpha for the horizontal measurement and beta for the vertical measurement). The measurement stopped once mycelial contact was observed. If an overgrowth of one fungus over the other was observed an “NA” was noted in the measurements for that interaction (Supplementary Table 1). When both fungi met in a deadlock without overgrowth, the same measurements were noted for the following day of measurement since no additional growth was observed. On day 10, pictures were taken with a Nikon D3400 camera of one representative Petri dish out of each three replicates. The picture was later used for visual assessment and placement into the five different types of interaction I—V (Table 3), based on terms defined by Boddy (2000) and Badalyan et al. (2002).

The set-up of Test 1 was repeated after the initial results for all potential antagonists with at least one interaction in type I. The plugs of the potential antagonists were placed on the plate 1 week prior to the inoculation with C. corticale. The goal was to see whether the inhibition at a distance would become more prominent, if the potential antagonist had time to establish itself first.

Measuring data of Test 1 was analyzed using RStudio (V. 4.1.2, R Core Team, 2019; Supplementary Table 2) for statistical analysis following the first part of the script by Rigerte et al. (2019). For the R script analysis, the spherical index (alpha/beta) of the fungal growth was calculated. For this approach, it was assumed that a single pathogen on a Petri dish of medium would have a circular growth, i.e., have the same measurements for both alpha and beta, resulting in a spherical index of 1 (Rigerte et al., 2019). If this is true for single cultures, it follows that if the interaction continues in a dual culture setup, alpha and beta would not have the same value, resulting in a spherical index unequal to 1. An inhibition of the pathogen was detected in the data analysis, if the spherical index for the pathogen was smaller than 1 (growth on the alpha axis smaller than on the beta axis) and the spherical index for the endophyte on the dual assay was bigger than 1 (growth on the alpha axis higher than on the beta axis; Rigerte et al., 2019; Figure 3). In this study the control culture was set up with each tested fungus in a pairing against itself, also prepared in triplicates.

For specific investigation of the fungi which had at least one interaction against C. corticale classified as type I, the set-up of Test 1 was repeated with two different types of media for Test 2, i.e., MYP and PDA at 25°C. For each pairing of C. corticale with the respective antagonist, three replicates were prepared following the procedure of Test 1. The Petri dishes were stored in a Binder KBW240 climate chamber at 25°C and ambient daylight for 10 days, measured three times, as described for Test 1, and photographed on day 10. In total, nine strains which had at least one interaction classified as type I were tested.

The strains tested in Test 3 had at least one interaction classified as type I, II, or III in Test 1 and 2. A total of 22 strains were tested against C. corticale (Table 4). An experimental set up was designed where the C. corticale plug was placed in the middle of the Petri dish, surrounded by six plugs colonized by the potential antagonist (Figure 2B), which were placed at a distance of 3 cm to C. corticale. Strains observed to grow much slower than C. corticale, namely Beauveria pseudobassiana S.A. Rehner & Humber, Cadophora prunicola (strain NW-FVA 6996) Damm & S. Bien, Calosporella innesii (Curr.) J. Schröt., Diaporthe pustulata Sacc., Hypholoma fasciculare (Huds.) P. Kumm., Jackrogersella cohaerens (Pers.) L. Wendt, Kuhnert & M. Stadler, Leptosillia muelleri (Duby) Voglmayr & Jaklitsch, Lophiostoma carpini Andreasen, Jaklitsch & Voglmayr (strain NW-FVA 6993), Neonectria sp., Neosetophoma cf. samarorum, Paracamarosporium cf. fagi, Pezicula sporulosa Verkley, Preussia cf. aemulans, Serpula himantioides (Fr.) P. Karst, Tolypocladium sp. (strain NW-FVA 7010 and 7,191) and a coelomycete (NW-FVA 7709) were placed on the test Petri dish 1 week prior to C. corticale, to give them time to establish themselves before the inoculation of the fast-growing C. corticale. After ten days each interaction was evaluated and again categorized into one of the five interaction types.

The setup of Test 1 was repeated for Test 4 (see Table 4). Twenty strains with interactions categorized into type I, or II were tested. The aim was to see whether C. corticale hyphae were still viable after the hyphal growth on the Petri dish stopped or if the hyphal tips had died and thus stopped growing. After 10 days two areas of the C. corticale culture were examined by transferring a 2 mm piece of a single hypha from different spots of the interaction zone. One piece was taken from the side of the interaction zone and one from the middle of the interaction zone, permitted the mycelia had no contact (Figure 2C). The taken pieces were transferred onto an additional new medium Petri dish, respectively, according to Brglez et al. (2020). If the fungi had grown into each other, one sample was taken from each of the two side margins of the culture, where no mycelial contact was observed. The hyphae were sampled under a Stereomicroscope (ZEISS Stemi 508) to ensure that only one hyphal tip was sampled.

We considered those strains as potential antagonists that had the majority of their tested interactions with C. corticale placed in type I and the remaining interactions in no less than type II. Potential antagonists, which were considered for further testing were reconsidered after each test. An antagonism was considered “stronger” or “more prominent” when the distance between the two fungal cultures was larger than in a previous test. For some taxa multiple strains were available and tested. Ultimately, fungi which had consistent antagonistic results over all conducted tests were chosen as potential antagonists. The fungi classified as type III were not considered as potential antagonists since the interaction between the two fungi could not be undoubtedly classified.

All 102 tested strains, comprising 76 fungal taxa, were grouped as types based on Test 1. The detailed results for the 39 strains classified as type I-IV can be found in Table 4. The remaining 63 strains, which are not shown in this table had all interactions placed into type V and can be found in Table 2. In total 918 interactions were evaluated. For four of the tested taxa a deadlock without contact was observed against all C. corticale strains, thus classifying them as type I. These were B. pseudobassiana, N. cf. samarorum, P. sporulosa, and Pr. cf. aemulans. Eleven strains were classified as type II, exhibiting signs of inhibition against C. corticale. The strains categorized as type II are Cal. innesii, Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams, D. pustulata, Eutypella quaternata (Pers.) Rappaz, L. muelleri, Lo. carpini (strain NW-FVA 6993), Neonectria sp., Nigrograna cf. norvegica, S. himantioides, and Tolypocladium sp. (strains NW-FVA 7010 and NW-FVA 7191). Three strains were categorized as type III, Coprinellus sp. and two strains of Nectria cinnabarina (Tode) Fr. (strains NW-FVA 6236 and NW-FVA 6239). Eleven strains were categorized as type IV, and 63 strains were categorized as type V (see Table 2).

Four of the tested strains, C. prunicola (strain NW-FVA 6996), H. fasciculare, Pa. cf. fagi, and J. cohaerens showed different interactions with each of the three C. corticale strains and were classified as either type I or type II. The remaining 11 strains had to be placed into more than one category regarding the interactions with the different strains of C. corticale (see Tables 2 and 4). For Cephalotrichum sp. (NW-FVA 6347) the interactions with the three different strains of C. corticale were all placed in different interaction types (CC1 type V, CC2 type I, and CC3 type II).

Ninety-nine of the tested strains had a slower or similar growth rate as C. corticale and 61 (type V) of them were overgrown by C. corticale. Three species tested in this study had a faster growth than C. corticale. Of the 13 taxa with multiple strains tested, only repeat strains of seven taxa were categorized into the same type for their interactions with all three C. corticale strains. Six of which were categorized as type V, while N. cinnabarina was categorized as type III. For C. prunicola strain NW-FVA 6996 the interaction with CC1 was classified as type II and the CC2 and CC3 interactions as type I. In comparison all interactions of C. prunicola strain NW-FVA 6596 were categorized as type V. Similar results were observed for Lo. carpini strain NW-FVA 6993 and NW-FVA 6994.

The results of the re-tested type I fungi, which were placed on the Petri dish 1 week prior to inoculation with C. corticale (Supplementary Table 3) show a more prominent antagonism at a distance for six of the nine tested strains than those of the original Test 1. For those six strains it is visible, that C. corticale is unable to grow forwards. This is most prominent for P. sporulosa and Pr. cf. aemulans. For Cephalotrichum sp., Neosetophoma cf. samarorum and J. cohaerens no antagonism at a distance could be observed. For H. fasciculare an inhibition at a distance could only be observed for CC2 and CC3. CC1 had mycelial contact with the potential antagonist, though the plate was not fully colonized (type II).

According to the results of the analysis of the spherical indices from Test 1 (Figures 4A–C), CC1 expressed a slower growth than against itself in the pairings with four of the potential antagonists. Of these pairings three were classified as type I, while one was classified as type II. CC2 showed a slower growth than in a pairing against itself against five potential antagonists. Four interactions of these pairings were classified as type I, one interaction was classified as type II. Compared to a pairing against itself, CC3 showed a slower growth when paired against six of the potential antagonists. Out of these only three interactions were classified as type I, the remaining interactions were classified as type II.

In total 19 strains were classified as type I, type II, or a combination of the two, and subsequently as potential antagonists to be investigated in the following tests (Table 4).

In Test 2, the fungi that previously exhibited an interaction classified as type I against at least one C. corticale strain, showed variation between types of media. The antagonistic reaction was more prominent and stronger on PDA than on MYP at a temperature of 25°C for all stains except Cephalotrichum sp. (NW-FVA 6247; see Supplementary Table 3). After the tenth day of incubation at 25°C, differences in growth rate and observed antagonism on MYP could be observed for some of the tested fungi compared to the tests at room temperature (see Table 5 and Supplementary Table 3). Three strains had a visibly slower growth at 25°C than at room temperature against themselves. The interactions between H. fasciculare and CC2 and CC3 were more prominent at 25°C. In both, N. cf. samarorum and Pa. cf. fagi, all combinations had mycelial contact without the Petri dish being fully colonized, so they were classified as type II and not as type I as at room temperature. For Pr. cf. aemulans all interactions were classified as type I, confirming the results of Test 1. However, the C. corticale cultures stopped growing in a straight line at 25°C, while those at room temperature continued to colonize the Petri dish, growing in a half circle, as shown in Table 3 for type I. Cephalotrichum sp. did not show an antagonistic reaction against CC2 in Test 2 on neither of the tested media and interacted according to type V, not confirming the observations from Test 1.

For three out of the four fungi classified as type I, the initially observed interaction type from Test 1 was confirmed in Test 3 (Table 4). For N. cf. samarorum the interaction type could not be confirmed for CC2 in this test, for CC3 the interaction type appears to be confirmed for four of the six mycelium plugs placed. In this interaction, C. corticale was able to advance to mycelial contact with the remaining two mycelium plugs, but was not able to overgrow them during the 10-day observation. Out of the 11 type II fungi the growth challenge was conducted for 10. Nigrograna cf. norvegica was not viable anymore at the time of testing. Out of these 10 the interaction for three strains, S. himantoides, and cf. Tolypocladium sp. (strains NW-FVA 7010 and 7,191), could be classified as type I and the interaction according to type II was confirmed for two other strains (Cal. innesii and D. pustulata). The interaction in the growth challenge was classified into type IV for Clonostachys rosea and E. quaternata, and into type V for Lo. carpini. For the three remaining type II strains tested, the results differed among the three C. corticale strains. C. prunicola strain NW-FVA 6996, H. fasciculare and P. cf. fagi had their interactions in Test 3 classified into type I, respectively, as opposed to a mix of type I and II in Test 1. Strain NW-FVA 7709 (coelomycete), as well as Cephalotrichum sp. had all three interactions of Test 3 were classified into type V, thus not showing any antagonistic interaction. For both N. cinnabarina strains the reaction according to type III was confirmed.

Of the 102 hyphal samples taken from C. corticale, 74 (73%) were still viable. From five of the 20 potentially antagonistic strains tested, all the examined hyphae of C. corticale were still able to grow after being transferred to a new MYP-Petri dish (Tables 4 and 6). For the interactions between the three C. corticale strains and H. fasciculare only the samples taken from the side were viable. Out of the four type I fungi, all six samples taken from N. cf. samarorum were taken from the side and were still viable. Whereas for both B. pseudobassiana and P. sporulosa one sample from the middle and one sample from the side margin of every Petri dish was taken, while only the samples from CC1 were both still viable. For both of these strains one of the CC2 samples was still viable, for B. pseudobassiana the sample from the side margin and for P. sporulosa the sample taken from the middle. All four samples taken from the interaction with CC3 were not viable anymore. Five out of six samples taken from Pr. cf. aemulans were still viable. Here, also equal samples of middle and side margin were taken. The only sample not viable anymore from this strain was the sample taken from the middle of the interaction with CC1. No samples of C. corticale could be taken from pairings with E. quaternata, L. muelleri and Lo. carpini, because C. corticale had overgrown the potential antagonist.

Test 1 revealed that C. corticale often overgrew potential antagonists due to its rapid growth compared to most fungi. Given that C. corticale has a higher growth rate than the majority of fungi in our study, it appears to have an advantage over these slow-growing fungi in terms of colonizing tissue. Six of the re-tested type I strains, which were placed on the Petri dish 1 week prior to C. corticale, exhibited a more prominent antagonism than observed in the original Test 1. This could indicate that organic compounds were released into the growth media by the potential antagonists, creating a stronger antagonistic reaction (Boddy, 2016). Similar results were demonstrated by Sonnenbichler et al. (1994) and recently shown for antagonism assays between H. fraxineus and an endophyte isolated from F. excelsior by Demir et al. (2023). This information can be used for further investigation into a potential control for C. corticale.

The majority of the fungi tested showed the same response in the three replicates and pairings with the three different C. corticale strains. However, for some tested fungal strains one or more of the replicates showed different results and the results varied strongly between the three C. corticale strains. The various interactions with the different C. corticale strains were therefore categorized into different interaction types, as evident for Cephalotrichum sp. (NW-FVA 6247). It has previously been observed, that the fungus-fungus interaction can vary in some cases between the same fungi, even under seemingly identical conditions (Rayner et al., 1994; Boddy, 2000; Brglez et al., 2020). This might be a factor in the differences observed between the interactions with the individual C. corticale strains.

The statistical analysis of the measurements of Test 1 simply represents the growth of each individual fungus by its respective spherical index. Consequently, the data analysis does not reflect the interaction between the fungi, i.e., replacement or deadlock. The spherical index of the fungal growth does not account for the space left in between the fungi, since it only analyses the growth of one of the two fungi. This aspect was also critically stated by Rigerte et al. (2019) in the original methodology. Observation of distance left between the tested fungi on the last day of measurement was a valuable characterization for detecting potential antagonisms, allowing us to detect 19 potential antagonists (type I and II) from Test 1.

In Test 2, C. corticale appeared to be more competitive growing on MYP at 25°C, its optimal growing temperature (Dickenson, 1980), than at room temperature. For all but one strain the interaction was more prominent on PDA than on MYP at 25°C. As described in several other studies, different types of artificial media affect the growth rates and habitus of fungal cultures (Botella et al., 2016), mycelial interactions between fungi (Brasier and Webber, 2013) as well as the production of antibiotics and a fungus’ response to these antibiotics (Whipps, 1987). These findings account for differences in interaction as well as variance in the morphology of fungi on different artificial media, as observed for J. cohaerens. Furthermore, it was observed that some fungi, which were originally classified as interaction type II in Test 1, were classified into type I in Test 2. On the other hand, a switch from interaction type I to type II was also observed. The different behavior is likely a result of the different temperature, as compared to Test 1.

Test 3 clarified the previous observations made in Test 1 and 2 (Table 4 and Supplementary Table 3). No antagonism was observed for coelomycete (NW-FVA 7709) in Test 3, but interestingly, the culture habitus of C. corticale in this pairing differed from its normal appearance in pure culture. This suggests that there was some kind of interaction between the coelomycete and C. corticale altering the mycelial growth of the latter. Similar observations have been made by Donnelly and Boddy (2001) for H. fasciculare and Agrocybe gibberosa (Fr.) Fay. when placed against Stropharia caerulea (Kriesel). Cephalotrichum sp. (NW-FVA 6247) was the only fungus where all three interactions with the different C. corticale strains were placed into different types in this study, and no antagonism could be observed in Test 3. All interactions from Test 3 for Cephalotrichum sp. were classified as type V, showing no antagonistic properties against any of the tested C. corticale strains. One possible explanation for these inconsistent results observed from Cephalotrichum sp. is that there is a difference between the three C. corticale isolates used in Test 1, which only specifically affects Cephalotrichum sp. Another possible explanation for the change in behavior of Cephalotrichum sp. might be the fact that Test 1 and 2 were conducted the same year the fungi were isolated. Test 3 and 4 were conducted one and a half years later, after the fungi had been stored in the fungal collection in tubes with MYP media at +4°C. As Humber (1997) and Butt and Copping (2000) state, fungi kept on artificial medium, especially after serial transfer, can lose their antagonistic characteristics, as observed for six of the tested strains. To reduce the loss of competitiveness and antagonistic properties of fungi, one solution is to periodically change the growth medium or even periodically transfer the fungus to suitable sterile host material (Marx and Daniel, 1976; Thomson et al., 1993). As a standard procedure conducted in this study, a transfer between different nutrient media was done for each strain after taking them out of the collection. After being stored on MYP the fungi were transferred to Malt-Extract-Agar according to Bußkamp (2018) for a week and then transferred back to MYP to prepare the cultures for the experiment. Nevertheless, this does not exclude the loss or weakening of certain traits (Tables 5, 6).

In Test 4 it was found that 73% of the tested hyphae of C. corticale were capable of growth when re-cultivated on new artificial medium, even though C. corticale no longer showed growth in the original dual culture with the potential antagonist. Similar results were reported by Brglez et al. (2020), who found that after deadlock at distance the sampled hyphae of the tested pathogen, E. quaternata (Pers.) Rappaz, grew out eight out of 10 times. This indicates that although the growth of C. corticale was inhibited by the antagonist, it is still viable and able to colonize fresh nutrient medium, suggesting a fungistatic effect. Possibly, there were inaccuracies in cutting the hyphal pieces from the dual culture when extracting dead hyphal cells next to still living hyphae. However, it is possible that through organic compounds emitted by the potential antagonist, the pathogen might be inhibited in its growth and thus unable to colonize the uncultured media. Baptista et al. (2021), for example, state that many of the secondary metabolites produced by H. fasciculare are known to have antimicrobial effects. Also for P. sporulosa the production of antifungal secondary metabolites is known (McMullin et al., 2017). Some of these secondary metabolites can cause mortality of fungi, as described for a combination of beauvericin and ketoconazole against Candida parapsilosis (Ashford) Langeron & Talice by Wang and Xu (2012).

Dual culture tests on artificial media, such as those used in this study, are useful for observing interactions between fungi, but do not consider the complexity of interactions in vivo. The results of antagonism tests have been shown to differ significantly between in vitro and in vivo antagonism tests (Dowson et al., 1988; Woods et al., 2005; Peters et al., 2020). The same applies to the differences between greenhouse and field trials (Heydari and Pessarakli, 2010). Still, in vitro antagonism tests are necessary and useful to gain an insight into fungal interactions previous to in vivo tests. The growing rate in vivo might differ from the growing rate in vitro for both C. corticale and the potential antagonists. The growing rate of C. corticale in vivo is insufficiently researched. The results obtained by Alcock and Wheeler (1983) show though, that the in vivo growth rate of C. corticale appears to depend more on the host factor than the isolate itself. This makes it hard to predict the in vivo interaction, which can differ quite significantly from in vitro observations (e.g., Lundborg and Unestam, 1980; Raziq and Fox, 2003).

One possible explanation for the observed inconsistencies in antagonistic behavior, as described above for, e.g., Cephalotrichum sp., is that most of the tested fungi might utilize a different carbon source than C. corticale and additionally might not naturally be antagonistic. Oliva et al. (2021) showed that the mycobiome of pine trees changes after hail damage and demonstrated that different fungi grow differently on different media, suggesting that they use different carbon sources. Another factor that may be relevant is that C. corticale is not a native fungus of Central Europe. Due to its recent introduction in an evolutionary time frame and the lack of time for coevolution with other fungi (Prospero et al., 2021) or with the host species itself, it is possible that there are no true natural antagonists in Europe despite its presence in Germany since the 1960s. The observation that C. corticale overgrew most of the tested fungi adds to the assumption that it is very competitive, underlining the need for an effective and strong antagonist.

It would be necessary to investigate if H. fasciculare, J. cohaerens, Pa. cf. fagi, P. sporulosa, and Pr. cf. aemulans can cause symptoms on A. pseudoplatanus and, if so, what kind of symptoms are caused. Following the results of such an experiment, it could be decided whether the infection with the potential antagonist is more beneficial than with C. corticale. Considering that H. fasciculare can cause white rot, it is not an ideal candidate for a BCA. As an alternative to using a living organism, which can be unpredictable and could cause symptoms itself, it could be beneficial to get an understanding of the secondary metabolome of the five potential antagonists identified in this study. It is possible that secondary metabolites play a role in the inhibition of C. corticale (Woodward and Boddy, 2008). Such organic compounds with antimicrobial and antifungal properties have been identified for two of the five potential antagonists, namely H. fasciculare and P. sporulosa (McMullin et al., 2017; Baptista et al., 2021). For a wide use as a BCA, it is relevant for the potential antagonist to be effective against any strain of C. corticale, which could be encountered in a plant. This could also be resolved by using secondary metabolites instead of living organisms. Since trees are perennial plants, as opposed to annual crops, it would be beneficial for a biological control measure for trees to be effective over several years. Depending on the pathogen, the development of a disease in woody tissues may take longer than one growing season. Since many pathogens persist in the host for several years (Prospero et al., 2021) they require long-term control. It is also important to consider the time of year a pathogen first infects the plant, the plant tissue the pathogen mainly infects, and how the interaction between pathogen and antagonist develops in time and space (Heydari and Pessarakli, 2010). The time of plant infection is crucial for the successful establishment of the pathogen within the plant (Rodriguez and Redman, 1997). For biological control to be effective, the antagonistic fungus has to establish itself in the plant in order to be effective as a preventative rather than a curative measure (Heydari and Pessarakli, 2010). Several factors have to be considered for a biological control in a forest pathosystem in comparison to the application in agricultural systems, e.g., a more complex anatomy of trees than crops, a longer lifespan of trees, and a lack of regular plant rotation, which can reduce the levels of potential inoculum (Cazorla and Mercado-Blanco, 2016). Treating forest stands with biological control agents could be more challenging than treating trees in urban settings. The number of plants that would need to be treated alone poses a challenge in terms of manpower and the potential for periodic treatment renewal.