Vol.: (0123456789) 1 Eur J Plant Pathol (2024) 170:79–89 https://doi.org/10.1007/s10658-024-02883-4 Diversity and abundance of culturable fungal endophytes in leaves of susceptible and resistant alternate hosts of Cronartium pini and C. ribicola Juha Piispanen · Ulrich Bergmann · Jouni Karhu · Tuomas Kauppila · Johanna Witzell · Juha Kaitera Accepted: 13 May 2024 / Published online: 27 May 2024 © The Author(s) 2024 Abstract Cronartium pini and C. ribicola are rust fungi that cause destructive diseases of pines (Pinus spp.). These rusts spread via alternate hosts, among which Melampyrum spp., Veronica spp. and Impa- tiens spp. are important for C. pini and Ribes spp. for C. ribicola. Congeneric alternate hosts vary in their susceptibility to Cronartium rusts, but the reasons for this variation are not clear. To clarify whether internal, endophytic fungi could explain these differences, we investigated the temporal and spatial variation in fungal endophyte composition of C. pini-resistant M. pratense, V. chamaedrys and I. glandulifera, C. pini-susceptible M. sylvaticum, V. longifolia and I. balsamina, C. ribi- cola-resistant R. rubrum and C. ribicola-susceptible R. nigrum. In total, 2695 fungal endophytic isolates were obtained and classified into 37 morphotypes, with 1373 cultures isolated in early summer and 1322 in late summer. Fifty-two isolates were identified to species or genus level. The most common morphotypes were identified as Heterophoma sp. Some variation in the abundance of morphotypes occurred between collec- tion sites, but the same morphotypes dominated across the sites and species. The diversity of morphotypes was higher in early September than in late June in all spe- cies and the same morphotypes dominated in both early and late season. The diversity of fungal endophytes was higher in resistant Veronica and Ribes than in suscepti- ble congeneric species, but the results suggest that the diversity or abundance of culturable fungal endophytes does not explain the differences in the congeneric spe- cies’ susceptibility to rust fungi. Keywords Alternate hosts · Endophytes · Rust resistance · Scots pine blister rust · White-pine blister rust Introduction Cronartium rusts cause severe damage in pine trees (Pinus spp.) in the northern hemisphere (Gäumann, 1959; Ziller, 1974), with C. pini (Willd.) Jørst damag- ing P. sylvestris L. in Europe and Asia (CABI, 2020; Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s10658- 024- 02883-4. J. Piispanen · J. Karhu · J. Kaitera (*)  Natural Resources Institute Finland, Paavo Havaksen tie 3, FI-90570 Oulu, Finland e-mail: juha.kaitera@luke.fi U. Bergmann  Biocenter, University of Oulu, FI-90014 University of Oulu Oulu, Finland T. Kauppila  Botanical Gardens, University of Oulu, FI-90014 University of Oulu Oulu, Finland J. Witzell  Department of Forestry and Wood Technology, Linnaeus University, Georg Lückligs Väg 1, SE-35256 Växjö, Sweden 80 Eur J Plant Pathol (2024) 170:79–89 1 3 Vol:. (1234567890) Kim et  al., 2022). In Scandinavia, this rust caused serious epidemics in the 2000s (Kaitera, 2000; Wulff et al., 2012). It can spread via over 50 alternate hosts (Kaitera et al., 2015). The main susceptible plant gen- era for C. pini are Melampyrum, Pedicularis, Euphra- sia, Veronica and Impatiens (Kaitera, 1999; Kaitera et  al., 1999, 2012, 2015, 2017, 2018). Cronartium ribicola Fisch. kills five-needle pines, especially in North America, but also in Europe and Asia (Kait- era & Nuorteva, 2006; Zambino, 2010). This rust spreads mainly via Ribes and Pedicularis. Previous research has shown that congeneric alternate hosts can differ in their susceptibility to Cronartium rusts. For instance, Melampyrum sylvaticum is more sus- ceptible to C. pini than M. pratense (Kaitera et  al., 1999, 2015; Kaitera, 1999). While the tight connec- tion between the rust inoculum and the surrounding vegetation with alternate hosts is well characterized, we still know very little about the factors potentially influencing the susceptibility of these hosts to rusts. Better understanding of these interactions could help to develop nature-based solutions to suppress the neg- ative effects of Cronartium rusts in Scots pine stands. One biological factor potentially influencing the interactions between alternate host species and rusts is the plant’s internal microbiome, in particular endo- phytic fungi. These fungi grow inside plants as taxo- nomically rich and spatially and temporally dynamic communities, generally without causing any harm to the host under normal conditions (Rajala et al., 2013; Terhonen et  al., 2019). While the specific function of these communities and their members are largely unknown, there is increasing evidence that endo- phytes may positively affect tree resistance to patho- gens (Ganley et  al., 2008; Witzell et  al., 2014; Ter- honen et  al., 2018). Endophytes may, e.g., directly antagonize pathogens or stimulate the host plant’s defensive mechanisms so that pathogen colonization processes are impeded (Witzell & Martín, 2018). The endophytic communities of alternate hosts of Cronartium rusts have not been systematically studied and thus their potential influence on the tra- jectories of conifer rust epidemics is unknown. The general aim of this study was to provide basic infor- mation about these fungal communities and to detect trends and patterns that could clarify whether the endophytic fungi have a role in the resistance of alternate host plants to Cronartium rusts of pines. A culture-based approach, accompanied by molecular identification of the most common morphotypes, was chosen as the method for this first step. The specific objectives were 1) to characterize the abundance and diversity of culturable fungal endophytes in the leaves of different alternate host plants, 2) to describe the temporal variation in fungal endophytic communi- ties of alternate host plants in different parts of the growing season, 3) to assess site-specific variation in fungal endophytic diversity and abundance, and 4) to compare fungal endophytic communities in rust- susceptible and resistant alternate host species and to evaluate whether certain endophytes are positively or negatively connected to their resistance to rusts. Material and methods Collection of plants Six alternate host species of C. pini and two of C. ribicola were collected for the study. Cronartium pini-resistant species were Melampyrum pratense L., Veronica chamaedrys L. and Impatiens glandulifera Royle, while the susceptible congeneric species were M. sylvaticum L., V. longifolia L. and I. balsamina L. The C. ribicola-resistant species was R. rubrum L., while susceptible species was R. nigrum L. Five plants of each of seven of these species were collected at random from one to three locations in the city area of Oulu in early season (late June 2021) and late season (early September 2020); they were placed in paper bags and transported immediately to the lab- oratory. The early season collection in late June was performed at three locations (sites I-III) for the seven species. The late season collection in early September was performed in one area (site I for M. sylvaticum, M. pratense and R. nigrum), two areas (sites I and II for V. chamaedrys and R. rubrum) or three areas (sites I, II and III for V. longifolia and I. glandulifera). In addition, plants of the eight species, I. balsamina, were grown directly from seeds in a greenhouse and the leaves were collected as with the other plant species in late June and early September 2021. The geographic locations of the sites and the greenhouse are given in Table 1. Isolation of fungal endophytes In the laboratory, five healthy-looking, green leaves were separated aseptically from each plant in a 81Eur J Plant Pathol (2024) 170:79–89 1 3 Vol.: (0123456789) laminar cabinet using a scalpel and tweezers. First, the leaves were surface sterilized as follows: rinsing with sterile water, 70 % ethanol (30 s), 1 % sodium hypochlorite (2 min), 70 % ethanol (30 s) and again with sterile water. Four pieces (about 0.5 × 0.5 cm) were cut from each leaf and placed on water agar (1.5 %) in Petri dishes. After an incubation period of 1-2 weeks at ca. 21.5  ºC, mycelia emerging from the pieces were transferred to fresh malt extract agar (MEA; 13.5 g Bacto agar, 15 g malt extract/1 L water, ø=9 cm Petri dishes), and when necessary, the cul- tures were transferred to new malt extract agar plates until morphologically homogeneous colonies were obtained. The water agar plates with the leaf pieces were also checked 2-4 weeks later to isolate possible slow-growing colonies. The rate (percentage of pieces yielding mycelial growth on each plate) was recorded. Morphotyping of the endophytes The pure cultures were incubated on MEA at 18 °C in a growth chamber (Binder GmbH, Tuttlingen, Ger- many). The isolates were grouped after one month into morphotypes based on colony characteristics, i.e. color, growth rate on MEA and the structure and form of the mycelia. The dominant color of the colony was described as hyaline, light, light brown, dark brown, red brown or green. The secondary (special) charac- teristics in color or form were described as: brownish, irregular, greenish, yellowish, reddish, with brownish spots, blotched, rhizoid, ring-shaped and filamentous. The growth rate was evaluated as either fast or slow, with fast growth rate indicating that the isolate filled the plate within one week whereas the isolates with slow growth rate filled the plate after several weeks to one month. A total of 2695 isolates were grouped into 37 morphotypes (Suppl. Table 1). Genetic identification of the fungal endophytes From each plant species, the isolates representing the dominant morphotypes were selected for DNA sequencing. The isolates, in total 65 and representing 9 morphotypes, were grown on 1.5% malt extract (see above) on a thin cellophane film. DNA was isolated using PrepManTM Ultra Sample Preparation Reagent (Thermo Fisher, Catalog number 4318930). First, 50 µl PrepMan solution was pipetted into 1.5 ml Eppen- dorf vials and a small amount of mycelium was trans- ferred into the tubes and ground using a glass rod. The samples were then placed in a thermal block at 95 ºC for 10 min. After that, the vials were left to cool at room temperature for ca. 5 minutes and centrifuged at 12000 rpm for 10 min. The supernatant was trans- ferred to a new 1.5 ml Eppendorf vial. The sample was diluted to 1:50 for PCR. The PCR was performed on the samples using ThermoFisher DreamTag Green Master Mix (Cata- log number K1081). The mixture was as follows: DreamTag Green Master mix x 2 12.5 µl, primers ITS1F 25 µM 0.125 µl and ITS4R 25 µM 0.125 µl, sterilized water 11.25 µl and template 1 µl (diluted 1:50). The PCR program included 35 cycles at 95 ºC for 3 min, 95 ºC for 30 s, 55 ºC for 30 s, 72 ºC for 1 min, 72 ºC for 10 min and 4 ºC for ∞. The selected primers, ITS1F and ITS4R, produce PCR product of the fungi that covers ITS1 and ITS2 regions and the 5.8S region between them, which contains high varia- tion between fungi and thus is very suitable for identi- fication purposes. The primers were: Table 1 Geographic sites of the plants Plant species Sites I II III M. sylvaticum and M. pratense 65º2,69N, 25º28,04E 65º1,29N, 25º25,38E 65º1,70N, 25º25,01E V. chamaedrys 65º1,30N, 25º25,96E 65º1,70N, 25º25,01E 65º1,31N, 25º25,97E V. longifolia 65º2,27N, 25º29,16E 65º2,26N, 25º29,12E 65º3,66N, 25º27,67E I.glandulifera 65º3,03N, 25º25,20E 65º3,04N, 25º25,26E 65º3,02N, 25º25,16E I.balsamina 65º3,86N, 25º27,79E R. nigrum 65º3,86N, 25º27,79E 65º2,26N, 25º29,12E 65º1,30N, 25º26,89E R. rubrum 65º3,86N, 25º27,79E 65º3,85N, 25º27,80E 65º1,26N, 25º27,13E 82 Eur J Plant Pathol (2024) 170:79–89 1 3 Vol:. (1234567890) ITS1F Forward CTT GGT CAT TTA GAG GAA GTAA and ITS4R Reverse TAA ACT TCA GGG TGA CCA AAA AAT CA. The PCR products were processed in 1% aga- rose gel. One clear PCR product was obtained from each sample and purified using an Exo-Sap reagent (Applied Biosystems™)[5 µl EXO (20 U/ µl), 100 µl SAP (1 U/ µl), 895 µl water]. For 5 µl of the PCR product, 2 µl of Exo-Sap reagent was used. The puri- fication was performed by incubating at 37 ºC for 15 min and heating at 80 ºC for 15 min. The purified PCR products were sequenced by Macrogen (Amsterdam, the Netherlands) with the primer ITS1F. Poor quality parts of the sequences from the beginning and the end were removed using the Geneious Prime® 2021.0.1 program (https:// www. genei ous. com/ prime). A search for the puri- fied sequences was performed using Blast with NCBI (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi ) and Unite [UNITE (ut.ee)], with over 98% similarity criterion. Results Morphotype abundance and diversity in the alternate host species The colonization rate was 74% for all the leaf material. Among plant species, mycelia emerged from 93% (M. sylvaticum and M. pratense), 80% (V. longifolia), 79% (V. chamaedrys), 75% (R. rubrum), 63% (I. glandulif- era), 52% (R. nigrum) and 47% (I. balsamina) of the leaf pieces. The highest total number of fungal endophyte isolates was recovered from V. longifolia (Table 2). The isolation frequency was relatively high in samples col- lected from both Melampyrym species, I. glandulifera, V. chamaedrys and R. rubrum, whereas clearly fewer isolates were recovered from R. nigrum and the low- est number of fungal endophytes was isolated from the greenhouse-grown I. balsamina (Table 2). The plant species exhibited distinct morphotype abundance profiles, but overall, morphotypes 13 and 19 were found to be common in all alternate hosts, along with morphotypes 2, 3, 31 and 35 (Fig. 1). The abundance of each morphotype was lowest in I. bal- samina, but in this species, morphotypes 13, 19, 31 and 35 were also the most common ones (for the data, see Suppl. Table  1). The diversity of fungal endo- phytes, estimated as the number of distinct morpho- types found per plant species across all sites and time points, was highest in V. chamaedrys and R. rubrum and lowest in R. nigrum and I. balsamina (Table 2). Fifty-two isolates were identified at least to genus- level and represented mainly Ascomycota (Table 3). The most common isolates from Melampyrum species, clas- sified to morphotypes 13 and 19, were identified as Het- erophoma species. However, isolates from R. rubrum that were classified to morphotype 19 were identified as several other species. Several common fungi, including Alternaria alternata (Fr.) Keissl. and Cladosporium spp. were identified among isolates classified to other fre- quently detected morphotypes (2, 31 and 35). Temporal variation in morphotype abundance and diversity The abundance and diversity of culturable fungal endophytes showed some temporal variation (Fig. 2). Table 2 Isolation frequency, percentage of isolates from all isolates, number of distinct morphotypes (MTs) and their percentage in eight alternate hosts of Cronartium rusts Species Nr isol. % of all isolates Nr of distinct MTs % of all MTs Melampyrum pratense 372 13.8 20 57 Melampyrum sylvaticum 372 13.8 23 62 Veronica chamaedrys 396 14.7 26 70 Veronica longifolia 485 18.0 21 57 Ribes nigrum 210 7.8 18 49 Ribes rubrum 385 14.3 26 70 Impatiens balsamina 94 3.5 10 27 Impatiens glandulifera 381 14.1 22 59 Sum 2695 100 83Eur J Plant Pathol (2024) 170:79–89 1 3 Vol.: (0123456789) The pooled morphotype abundance, shown as violin plots that depict the distributions of the data points (including outliers) for the different morphotypes, indicated higher frequencies for several of the domi- nant morphotypes (13 and 19) in samples collected in June 2021, compared to those collected in September 2020 (Fig. 2). All 37 morphotypes were found in the samples collected in September 2020, whereas only 26 morphotypes were represented in samples col- lected in June 2021 (Suppl. Table  1). In the pooled data, several morphotypes (e.g., 10, 14, 16, 18, 20) that were present in samples collected in September 2020 were missing from the samples collected in June 2021 (Fig 2). Variation in morphotype diversity and abundance between sampling sites Our results suggest that while the same morpho- types tended to dominate the fungal endophytic communities at the different sites, some differences existed (Suppl. Table 1; Fig. 3). Based on the pooled data, site I had the richest diversity: 33 of the 37 mor- photypes were found in samples collected from site I, whereas only 26 of the morphotypes were found in samples collected from site III (Suppl. Table  1; Fig. 3). Fungal endophyte abundance and diversity in congeneric plants showing different resistance and susceptibility to rusts There were no clear patterns in the abundance of fungal endophytes between the congeneric species differing in resistance to Cronartium rusts. However, comparison of pooled data on diversity, estimated as the number of distinct morphotypes present on a species, showed that diversity was higher in the resistant V. chamaedrys and R. rubrum compared to their susceptible congeneric species V. longifolia and R. nigrum (Table 2). Fig. 1 Abundance-profiles of fungal morphotypes in seven plant species (M.pra. = Melampyrum pratense; M. syl. = M. sylvaticum; V. cha. = Veronica chamaedrys; V. lon. = V. longi- folia; I. gla. = Impatiens glandulifera; R. nig. = Ribes nigrum and R. rub. = R. rubrum) that are alternate hosts to Cronartium rusts. Shown is the sum abundance across three sites and two time points 84 Eur J Plant Pathol (2024) 170:79–89 1 3 Vol:. (1234567890) Table 3 Identification of endophytes isolated from leaves of plant species that are alternative hosts for Cronartium rusts of pines (Melampyrum pratense; M. sylvaticum; Veronica chamaedrys; V. longifolia; Impatiens glandulifera; I. balsamina; Ribes nigrum and R. rubrum). Sequences of >98% match to GenBank were listed. Time: 1=Late June 2021, 2=Early September 2020 (for I. balsamina September 2021). The plants were growing on three sites, I-III. MO=Morphotype Plant species Time Site MO Taxa Match (%) M. sylvaticum 2 I 13 Heterophoma sp. 100 M. sylvaticum 2 I 13 Heterophoma sp. 100 M. sylvaticum 2 I 13 Heterophoma sp. 100 M. sylvaticum 1 I 13 Heterophoma sp. 100 M. sylvaticum 1 II 13 Heterophoma sp. 100 M. sylvaticum 1 III 13 Heterophoma sp. 100 M. sylvaticum 2 I 19 Heterophoma sp. 100 M. sylvaticum 2 I 19 Heterophoma sp. 100 M. sylvaticum 1 I 19 Heterophoma sp. 100 M. sylvaticum 1 I 19 Heterophoma sp. 100 M. sylvaticum 1 II 19 Heterophoma sp. 100 M. sylvaticum 1 III 19 Heterophoma sp. 100 M. pratense 2 I 13 Heterophoma sp. 100 M. pratense 2 I 13 Heterophoma sp. 100 M. pratense 2 I 13 Heterophoma sp. 100 M. pratense 1 II 13 Heterophoma sp. 100 M. pratense 1 III 13 Heterophoma sp. 99,8 M. pratense 1 III 13 Heterophoma sp. 100 M. pratense 2 I 19 Heterophoma sp. 100 M. pratense 1 II 19 Heterophoma sp. 100 M. pratense 1 III 19 Heterophoma sp. 100 M. pratense 1 III 19 Heterophoma sp. 100 V. chamaedrys 1 I 3 Diaporthe padi var. padi 99,0 V. chamaedrys 2 I 2 Trichoderma sp. 99,8 V. chamaedrys 2 II 2 Seimatosporium lichenicola 99.6 V. chamaedrys 1 I 2 Sistotrema brinkmannii 100 V. longifolia 2 I 19 Penicillium sp. 100 V. longifolia 2 III 19 Pleosporaceae sp. 99,8 V. longifolia 1 I 19 Phaeosphaeria sp. 100 V. longifolia 1 I 19 Pleosporaceae sp. 100 I. glandulifera 2 I 36 Mycocentrospora acerina 100 I. glandulifera 1 III 35 Rhexocercosporidium sp. 98,9 I. balsamina 2 I 35 Alternaria alternata 100 I. balsamina 2 I 35 Alternaria alternata 100 I. balsamina 2 I 35 Alternaria alternata 100 I. balsamina 2 I 35 Alternaria alternata 100 I. balsamina 2 I 31 Cladosporium sp. 100 I. balsamina 2 I 31 Cladosporium cladosporioides 100 I. balsamina 2 I 31 Penicillium sp. 99,8 R. nigrum 2 I 2 Phlebia tremellosa 99,1 R. nigrum 1 III 2 Phlebiopsis gigantea 100 R. nigrum 1 III 2 Phlebiopsis gigantea 100 R. nigrum 1 I 31 Dothiora ribesia 100 R. nigrum 1 I 31 Cladosporium cladosporioides 100 R. nigrum 2 I 7 Jackrogersella multiformis 99,0 R. nigrum 1 III 7 Microsphaeropsis olivacea or Didymellaceae sp. 99,8 R. nigrum 1 III 7 Microsphaeropsis olivacea or Didymellaceae sp. 100 85Eur J Plant Pathol (2024) 170:79–89 1 3 Vol.: (0123456789) Discussion Our results indicate that fungal endophytic com- munities vary between the plant species that act as alternate hosts to Cronartium rusts. This result is in agreement with previous studies that have shown the importance of host species as a determinant of fungal endophytic communities (Leopold & Busby, 2020; Romeralo et  al., 2022), which may reflect the varying quality of the different hosts as a substrate for fungi. The high colonization rate of Melampyrum and Veronica species indicates that they are suitable hosts for endophytic fungi. The quality of leaves as a habitat for endophytic fungi could not be analyzed in this study, but it has been suggested that second- ary metabolites, such as potentially antifungal phe- nolics could be a factor directing the microbiome composition (Witzell et  al., 2022). In previous stud- ies, some of these compounds have been found to be high in rust-resistant alternate host species (Kaitera & Witzell, 2016; Piispanen et al., 2023). In the plants of this study, chlorogenic acid is found at high lev- els in M. pratense, R. rubrum and R. nigrum (Piis- panen et al., 2023) and quercitrin in I. glandulifera, I. Table 3 (continued) Plant species Time Site MO Taxa Match (%) R. rubrum 1 III 2 Calophoma sandfjordenica 99,8 R. rubrum 1 I 19 Alternaria anthropophila 100 R. rubrum 1 I 19 Didymella tanaceti 99,4 R. rubrum 1 III 19 Colletotrichum dematium 100 R. rubrum 1 III 19 Phaeosphaeria lycopodina 99,4 Fig. 2 Violin plots visualizing the distribution of the abun- dance of fungal morphotypes in June 2021 and in September 2020. The fungi were isolated from leaves of seven plant spe- cies (Melampyrum pratense, M. sylvaticum, Veronica chamae- drys, V. longifolia, Impatiens glandulifera, Ribes nigrum and R. rubrum) that are alternate hosts of Cronartium rusts and were growing at three sites 86 Eur J Plant Pathol (2024) 170:79–89 1 3 Vol:. (1234567890) balsamina, R.rubrum and R. nigrum (Piispanen et al., 2023). Quercitrin is also present at higher levels in rust-resistant V. chamaedrys than rust-susceptible V. longifolia (Piispanen et  al., 2023), while quercitrin and apigenin derivates are higher in rust-resistant M. pratense than rust-susceptible M. sylvaticum (Kaitera & Witzell, 2016; Piispanen et  al., 2023). However, the relationship between endophytic fungi and phe- nolic metabolites may not be straightforward (Witzell et al., 2022), and some fungal endophytes are known to be able to use phenolics as a carbon source (Blu- menstein et al., 2015). Although practical biocontrol applications rely- ing on endophytes are still pending, there is a grow- ing body of evidence demonstrating this potential (Busby et al., 2016). Ganley et al. (2008) found that endophyte-inoculated Pinus monticola Dougl. ex D. Don seedlings survived longer than their endophyte- free counterparts after infection by Cronartium ribi- cola, and they also exhibited a notable reduction in the severity of white pine blister rust disease. Bulling- ton et  al. (2018) provided evidence that endophytes and terpenes simultaneously contribute to C. ribicola resistance in Pinus albicaulis Engelm. and concluded that endophytes have potential as biocontrol agents to protect the trees from C. ribicola infection. Raghaven- dra and Newcombe (2013) found that four foliar endo- phytes accounted for 54% of the variation in quantita- tive resistance among six poplar genotypes exhibiting diverse genetic resistance to virulent isolates of Mela- mpsora, suggesting that foliar endophytes serve as a secondary line of defense, complementing major genes in the resistance against leaf rust. Based on this evidence, we expected that the fungal endophyte diversity or abundance between the congeneric plant species pairs studied would show a distinct pattern, indicating that fungal endophytic communities could contribute to the different patterns of susceptibil- ity to Cronartium pathogens. Indeed, we found that the diversity of fungal endophytes, estimated as the pooled number of distinct morphotypes found per plant species across all sites and time points, was higher in the resistant V. chamaedrys and R. rubrum compared to their susceptible congeneric species, V. longifolia and R. nigrum. However, whether these dif- ferences have functional and biological consequences for the rusts in forest ecosystems remains to be stud- ied. The lack of clear patterns in the abundance of morphotypes suggests that the role of fungal endo- phytes as determinants of rust resistance in alternate Fig. 3 Violin plots visualizing the distribution of the abun- dance of fungal morphotypes in three sites (I-III). Show is the sum abundance of each morphotype in seven plant species (Melampyrum pratense, M. sylvaticum, Veronica chamaedrys, V. longifolia, Impatiens glandulifera, Ribes nigrum and R. rubrum) that are alternate hosts of Cronartium rusts and were sampled at two time points (September 2020 and June 2021) 87Eur J Plant Pathol (2024) 170:79–89 1 3 Vol.: (0123456789) host species may not be critical. Similar results were reported by Moler et al. (2022), who inoculated Pinus albicaulis seedlings with foliar endophytic fungi but found no evidence of a biocontrol effect against C. ribicola. In a recent study, Ata et  al. (2023) charac- terized fungal endophytic communities in the needles of both C. ribicola-resistant and susceptible Pinus flexilis E. James trees. However, they did not find sig- nificant differences in the diversity or richness of the mycobiota and suggested that factors such as host size or site elevation could be more crucial determinants of the fungal endophytic communities. When the data for all species were pooled across the three sites, the diversity of the culturable fungal endo- phytes in leaves of alternate host species seemed to be rather stable, i.e., the same morphotypes tended to dominate the endophyte profiles of the fungal species at different sites. Endophytes infect the plants from the surrounding environment (Gomes et al., 2018) and the dominance of certain fungi in the culturable fraction may thus reflect the high frequency of these fungi in the environmental inoculum in the region of the study area. The fact that the same morphotypes dominated in the samples from the greenhouse-grown I. balsamina supports this view – in greenhouses, the environmental inoculum is limited to spores that are most abundant in the incoming air (Witzell et al., 2022). The morphotype diversity tended to be higher in the late season sampling compared to the early sam- pling, suggesting accumulation of infections during the growing season. Seasonal changes in environmen- tal conditions have been found to change the environ- mental pool of microbe propagules (Collado et  al., 1999). However, the age and developmental stage of the plants or plant parts may also influence the endo- phyte community composition (Nascimento et  al., 2015; Oono et al., 2015). It should be noted that because culture-depend- ent methods like the one we used can only capture a small fraction of the total diversity in fungal com- munities (Fan et  al., 2020; Dos Reis et  al., 2022), further studies with metabarcoding and metagen- omics should be conducted to provide more detailed information about the diversity of fungal communities in these plants. The ITS1 and ITS2 regions typically offer resolution at a within-genus level and often within-species level (Nilsson et al., 2008). While it is used as the fungal DNA barcode, the ITS region may lack the required resolution in certain fungal groups and the use of other or addi- tional markers could thus have improved the reso- lution of our analyses. Several of the fungi identi- fied to species or genus level in our study have also been commonly reported among fungal endophytes in other studies. For instance, Alternaria alternata (Fr.) Keissl. is a cosmopolitan species that has been reported as an endophyte and a pathogen from a wide range of plant hosts (Woudenberg et al., 2013; DeMers, 2022). We found consistent evidence for the association between both Melampyrum species and Heterophoma species. Little is known about the ecology of Heterophoma species in northern forests, but Heterophoma sylvatica (Sacc.) Qian Chen & L. Cai has been reported as a part of the biodiversity of Vaccinium myrtillus L., a common heath species (Gomzhina et  al., 2022). In general, the functional roles of individual endophyte species in hosts are still poorly known. In future, it may be possible to explore the functions using synthetic microbial communities (SynCom), an emerging approach that involves co-culturing multiple taxa under well-defined conditions to mimic the struc- ture and function of a microbiome (de Souza et al., 2020). In conclusion, our results indicate that the cultur- able endophyte communities do not have a straight- forward role in the Cronartium rust resistance or sus- ceptibility of alternate host plants. However, there is generally strong evidence supporting the view that fungal endophytes can influence plant health and pro- ductivity (Witzell & Martin, 2018; Terhonen et  al., 2018), and the community structure captured in cul- tures is known to be limited. Therefore, culture-inde- pendent approaches that allow comparisons of whole communities of fungal endophytes could be used in future studies to further explore the possible role of endophytic fungi in the interaction between heteroe- cious rusts and their hosts. Acknowledgements We thank Timo Mikkonen and Annika Uimonen for helping in culturing the endophytes, and Tuija Hytönen and Suvi Sutela for the genetic identification of the endophytes in the laboratory. The study was financed by the Finnish Academy of Science (grant no. 332811). Funding Open access funding provided by Natural Resources Institute Finland (Luke). 88 Eur J Plant Pathol (2024) 170:79–89 1 3 Vol:. (1234567890) Data availability The data that support the findings of this study are available from the corresponding author upon reason- able request. Declarations The authors bear all the ethical responsibilities of this manuscript. They declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest and that it does not include any animal and/or human trials. Open Access This article is licensed under a Creative Com- mons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Crea- tive Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Ata, J. P., Schoettle, A. W., Sitz, R. A., Ibarra Caballero, J. R., Holtz, C. T., Abdo, Z., & Stewart, J. E. (2023). Characteri- zation of foliar fungal endophyte communities from white pine blister rust resistant and susceptible Pinus flexilis in natural stands in the Southern Rocky Mountains. Phytobi- omes Journal, 7, 259–269. Blumenstein, K., Albrectsen, B., Martín, J. A., Hultberg, M., Sieber, T., Helander, M., & Witzell, J. (2015). Nutritional niche overlap potentiates the use of endophytes in biocon- trol of a tree disease. BioControl, 60, 655–667. https:// doi. org/ 10. 1007/ s10526- 015- 9668-1 Bullington, L. S., Lekberg, Y., Sniezko, R., & Larkin, B. (2018). The influence of genetics, defensive chemistry and the fungal microbiome on disease outcome in whitebark pine trees. Molecular Plant Pathology, 19, 1847–1858. Busby, P. E., Ridout, M., & Newcombe, G. (2016). Fungal endophytes: Modifiers of plant disease. Plant Molecular Biology, 90, 645–655. CABI. (2020). Cronartium flaccidum (Scots pine blister rust) in: Invasive Species Compendium. CAB International. Retrieved 21 May 2021 from https:// www. cabi. org/ isc/ datas heet/ 16148 Collado, J., Platas, G., Gonzalez, I., & Pelaez, F. (1999). Geo- graphical and seasonal influences on the distribution of fun- gal endophytes in Quercus ilex. New Phytologist, 144, 525– 532. https:// doi. org/ 10. 1046/j. 1469- 8137. 1999. 00533.x de Souza, R. S. C., Armanhi, J. S. L., & Arruda, P. (2020). From Microbiome to Traits: Designing Synthetic Microbial Communities for Improved Crop Resiliency. Front Plant Science, 11, 1179. DeMers, M. (2022). Alternaria alternata as endophyte and pathogen. Microbiology, 168, 001153. https:// doi. org/ 10. 1099/ mic.0. 001153 Dos Reis, J. B. A., Lorenzi, A. S., & do Vale, H. M. M. (2022). Methods used for the study of endophytic fungi: a review on methodologies and challenges, and associated tips. Archives of Microbiology, 204(11), 675. https:// doi. org/ 10. 1007/ s00203- 022- 03283-0 Fan, S., Miao, L., Li, H., Lin, A., Song, F., & Zhang, P. (2020). Illumina-based analysis yields new insights into the diver- sity and composition of endophytic fungi in cultivated Huperzia serrata. PLoS ONE, 15(11), e0242258. https:// doi. org/ 10. 1371/ journ al. pone. 02422 58 Ganley, R. J., Sniezko, R. A., & Newcombe, G. (2008). Endo- phyte-mediated resistance against white pine blister rust in Pinus monticola. Forest Ecology and Management, 255, 2751–2760. Gäumann, E. (1959). Die Rostpilze Mitteleuropas. Beiträge zur Kryptogamenflora der Schweiz, 12, 1–1407. Gomes, T., Pereira, J. A., Benhadi, J., Lino-Neto, T., & Bap- tista, P. (2018). Endophytic and epiphytic phyllosphere fungal communities are shaped by different environmental factors in a Mediterranean ecosystem. Microbial Ecology, 76, 668–679. https:// doi. org/ 10. 1007/ s00248- 018- 1161-9 Gomzhina, M. M., Gasich, E. L., Gagkaeva, T. Y., & Ganni- bal, P. B. (2022). Biodiversity of Fungi Inhabiting Euro- pean Blueberry in North-Western Russia and in Finland. Doklady Biological Science, 507(1), 441–455. https:// doi. org/ 10. 1134/ S0012 49662 20600 47 Kaitera, J. (1999). Cronartium flaccidum fruitbody produc- tion on Melampyrum spp. and some important alternate hosts to pine. European Journal of Forest Pathology, 29, 391–398. Kaitera, J. (2000). Analysis of Cronartium flaccidum lesion devel- opment on pole-stage Scots pines. Silva Fennica, 34, 21–27. Kaitera, J., Hiltunen, R., & Hantula, J. (2015). Cronartium rust sporulation on hemiparasitic plants. Plant Pathology, 64, 738–747. Kaitera, J., Hiltunen, R., & Samils, B. (2012). Alternate host ranges of Cronartium flaccidum and C. ribicola in north- ern Europe. Botany, 90, 694–703. Kaitera, J., Kalleinen, L., Mikkilä, J., & Hantula, J. (2017). Cronartium flaccidum sporulates on new Euphrasia spe- cies in natural habitats in Finland. Forest Pathology, 47(5), e12349. Kaitera, J., Kauppila, T., & Hantula, J. (2018). New alternate hosts for Cronartium spp.: Odontites, Euphrasia Rhinan- thus and Papaver. Forest Pathology, 48, e12466. Kaitera, J., & Nuorteva, H. (2006). Susceptibility of Ribes spp. to pine stem rusts in Finland. Forest Pathology, 36, 225–246. Kaitera, J., Seitamäki, L., Hantula, J., Jalkanen, R., & Kurkela, T. (1999). Inoculation of known and potential alternate hosts with Peridermium pini and Cronartium flaccidum aeciospores. Mycological Research, 103, 235–241. Kaitera, J., & Witzell, J. (2016). Phenolic profiles of two Mela- mpyrum species differing in susceptibility to Cronar- tium rust. European Journal of Plant Pathology, 144(1), 133–140. 89Eur J Plant Pathol (2024) 170:79–89 1 3 Vol.: (0123456789) Kim, Mee-Sook., Hantula, J., Kaitera, J., Zambino, P. J., Woodward, S., Richardson, B. A., Stewart, J. E., Spaine, P., Shaw, D. C., Takeuchi, Y., & Klopfenstein, N. B. (2022). Recovery plan for Scots pine blister rust caused by Cronartium pini. Plant Health Progress, 23, 105–130. Leopold, D. R., & Busby, P. E. (2020). Host genotype and col- onist arrival order jointly govern plant microbiome com- position and function. Current Biology, 30, 3260–3266. https:// doi. org/ 10. 1016/j. cub. 2020. 06. 011 Moler, E. R. V., Reinhardt, K., Sniezko, R. A., & Aho, K. (2022). First report of fungal endophyte communities and non-defen- sive phytochemistry of biocontrol-inoculated whitebark pine seedlings in a restoration planting. Forests, 13, 824. Nilsson, R. H., Kristiansson, E., Ryberg, M., Hallenberg, N., & Larsson, K.-H. (2008). Intraspecific ITS variability in the kingdom Fungi as expressed in the international sequence databases and its implications for molecular species identi- fication. Evolutionary Bioinformatics, 4, 193–201. Nascimento, T. L., Oki, Y., Lima, D. M. M., Almeida-Cortez, J. S., Fernandes, G. W., & Souza-Motta, C. M. (2015). Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity. Fun- gal Ecology, 14, 79–86. Oono, R., Lefèvre, E., Simha, A., & Lutzoni, F. (2015). A com- parison of the community diversity of foliar fungal endo- phytes between seedling and adult loblolly pines (Pinus taeda). Fungal Biology, 119, 917–28. Piispanen, J., Bergmann, U., Karhu, J., Kauppila, T., & Kaitera, J. (2023). Variation of compounds in leaves of susceptible and resistant alternate hosts of Cronartium pini and C. ribicola. European Journal of Plant Pathology, 165(4), 677–692. Raghavendra, A. K. H., & Newcombe, G. (2013). The contri- bution of foliar endophytes to quantitative resistance to Melampsora rust. New Phytologist, 197, 909–918. Rajala, T., Velmala, S. M., Tuomivirta, T., Haapanen, M., Mül- ler, M., & Pennanen, T. (2013). Endophyte communities vary in the needles of Norway spruce clones. Fungal Biol- ogy, 117, 182–190. Romeralo, C., Martin-Garcia, J., Martinez-Alvarez, P., Jordan Munoz-Adalia, E., Goncalves, D. R., Esteban, T., Witzell, J., & Diez, J. J. (2022). Pine species determine fungal microbiome composition in a common garden experi- ment. Fungal Ecology, 56, 101137. https:// doi. org/ 10. 1016/j. funeco. 2021. 101137 Terhonen, E., Blumenstein, K., Kovalchuk, A., & Asiegbu, F. O. (2019). Forest tree microbiomes and associated fungal endophytes: Functional roles and impact on forest health. Forests, 10(1), 42. https:// doi. org/ 10. 3390/ f1001 0042 Terhonen, E., Kovalchuk, A., Zarsav, A., & Asiegbu, F. O. (2018). Biocontrol potential of forest tree endophytes. Endophytes of Forest Trees (pp. 283–318). Springer. Witzell, J., Decker, V. H., Agostinelli, M., Romeralo, C., Cleary, M., & Albrectsen, B. (2022). Aspen leaves as a “chemical landscape” for fungal endophyte diversity – can nitrogen and herbivory drive the community composition? Frontiers in Microbiology. https:// doi. org/ 10. 3389/ fmicb. 2022. 846208 Witzell, J., & Martín, J. A. (2018). Endophytes and forest health. In A. M. Pirttilä & A. C. Frank (Eds.), Endophytes of Forest Trees, Forestry Sciences 86 (pp. 261–282). Springer International Publishing AG. Witzell, J., Martín, J. A., & Blumenstein, K. (2014). Ecological aspects of endophyte-based biocontrol of forest diseases. In V. C. Verma & A. C. Gange (Eds.), Advances in Endo- phytic Research (pp. 321–333). Springer India. https:// doi. org/ 10. 1007/ 978- 81- 322- 1575-2_ 17 Woudenberg, J. H. C., Groenewald, J. Z., Binder, M., & Crous, P. W. (2013). Alternaria redefined. Studia Mycologica, 75, 171–212. https:// doi. org/ 10. 3114/ sim00 15 Wulff, S., Lindelöw, Å., Lundin, L., Hansson, P., Axelsson, A.-L., Barklund, P., Wijk, S., & Ståhl, G. (2012). Adapt- ing forest health assessments to changing perspectives on threats – a case example from Sweden. Environmental Monitoring Assessments, 184, 2453–2464. Zambino, P. J. (2010). Biology and pathology of Ribes and their implications for management of white pine blister rust. Forest Pathology, 40, 264–291. Ziller, W. G. (1974). The tree rusts of western Canada. Cana- dian Forest Service Publications, 1329, 1–272.