Host Preference and Seasonal Variation in The Colonization of Tubakia s.l. Associated with Evergreen Oak Species in Eastern Japan

doi:10.21203/rs.3.rs-802935/v1

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Host Preference and Seasonal Variation in The Colonization of Tubakia s.l. Associated with Evergreen Oak Species in Eastern Japan

https://doi.org/10.21203/rs.3.rs-802935/v1

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Foliar fungal endophytes are ubiquitous and hyperdiverse, and tend to be host-specific among dominant forest tree species. The genus Tubakia s.l. comprises foliar pathogens and endophytes that exhibit host preference for Quercus and other Fagaceae species. To clarify interspecific differences in ecological characteristics among Tubakia species, we examined the endophyte communities of seven evergreen Quercus spp. at three sites in eastern Japan during summer and winter. We also compared their spore germination rates at different temperatures. Host tree species was the most significant factor affecting endophyte community composition. The Tubakia species found at the study sites were divided into five specialists and three generalists according to their host ranges. Specialists were dominant on their own host in summer, and their dominance declined in winter. In contrast, generalist Tubakia species 3 became dominant in some tree species in winter. This seasonal variation in Tubakia species dominance was consistent with their cold tolerance, which was evaluated in spore germination tests. This is the first study to report ecological differentiation among endophytic fungal species within a genus.

Behavioral Ecology

host specificity

endophyte

Quercus

Cyclobalanopsis

Tubakiaceae

spore germination rate

Endophytes are organisms that inhabit plant organs at some stage during their lifetime, without causing apparent harm to the host [1]. Foliar fungal endophytes are ubiquitous and hyperdiverse [2, 3]. Endophytes of woody plants are transmitted horizontally, with some showing host preference [4, 5]. Because most tree endophytic fungi infect a broad range of tree species at different frequencies, an understanding of host preference is important for interpreting endophytic fungal diversity, as well as their biological roles and the evolutionary lineages of both endophytic fungi and their hosts [6]; this would facilitate evaluations of global biodiversity [7].

Despite the high species diversity of endophytes within and among tree species, endophyte communities in different host species within the same plant family are dominated by closely related endophyte species, which may indicate beneficial co-evolution between endophytes and their hosts [3]. Lophodermium species are dominant foliar endophytes specific to Pinaceae [8–10], their hyperdiversity and intraspecific genetic variation (including cryptic species) has been revealed in recent studies examining the relationships between fungal speciation and host affinity[11, 12].

The host ranges and infection patterns of endophytes within a plant community have been described in various terms including host specificity [13], host preference [14], host affinity [15], host association [16], and host restriction [7], and in accordance with subdivisions of these categories [17]. In this study, we adopt the term host specificity, which was defined by [13] as the state of occupying a limited proportion of hosts in a plant community. Host specificity is generally measured in terms of the number of host species. However, among endophytic fungal communities, rare fungal species are often observed as “accidental tourists”[13, 18]. Specificity can be assessed in terms of the closeness of the relationship between the fungus and host species, or variation in the range of hosts exploited by a fungus across its geographic range [19]. Therefore, a good understanding of the ecology of endophytic fungi, and of structural (abundance and evenness) and phylogenetic (evolutionary relationships) host specificity across their geographical ranges, is needed [13, 19]. Few studies have evaluated the host preference and specificity of endophytic fungi by investigating multiple tree species at the same site. Some studies that focused on multiple tree species indicated that endophytes co-evolve with their hosts (e.g., [20] ), whereas others have not (e.g., [21] ). Because the factors affecting endophyte abundance and composition are often influenced by temporal and spatial variation, comparisons across different fungal endophyte communities are challenging [13].

Tubakia species sensu lato are leaf pathogens that tend to exhibit host preference for Quercus spp. and Castanea crenata [22, 23], but are also endophytes of various evergreen and deciduous species [20, 24, 25]. In our previous studies, we isolated multiple Tubakia species from the leaves of various evergreen Quercus (sect. Cyclobalanopsis) species in Japan [20, 26].

Tree species that have been described as hosts of Tubakiaceae are widely distributed in Japan; evergreen Quercus (sect. Cyclobalanopsis) are major components of the warm, temperate forests of East Asia [27]. Unlike the cool, temperate deciduous forests of northeastern Japan, which are dominated by Fagus crenata and Quercus crispula, the warm, temperate forests of southwestern Japan are dominated by several evergreen Quercus species. Species differentiation among these evergreen oaks and their endophytic Tubakia species can be used as a model of co-evolution between endophytes and their host trees. These warm, temperate forests have experienced anthropogenic disturbances throughout history, and the remaining natural forests are limited [28, 29]. Thus, the diversity of Tubakia species on evergreen oaks must also be clarified in the context of biodiversity conservation.

In this study, to clarify interspecific differences in the ecological roles of Tubakia species, and to explore their host preference mechanism and its ecological significance, we examined the endophyte communities of seven evergreen Quercus species in three forests of eastern Japan, in summer and winter. We also compared spore germination rates among different temperatures to evaluate their adaptation to cold temperatures in winter.

2.1. Study sites

Sampling was conducted in three natural warm, temperate evergreen forests: Mt. Takao (TKO), the University of Tokyo Chiba Forest (CBF), and the University of Tokyo Arboricultural Research Institute (ARI) (Fig. 1). The climatic data (Supplementary material Fig. S1) were obtained from the nearest automated meteorological data-acquisition system (AMeDAS) weather station and .the University of Tokyo Forest [30].

At the TKO site (35°37'46"N, 139°15'11"E; elevation, 400 m a.s.l.), sampling was conducted in a natural evergreen forest dominated by evergreen Quercus species, particularly Quercus salicina Blume, Quercus acuta Thunb., and Quercus glauca Thunb. [31]. The forests surrounding the sampling site were mainly dominated by Abies firma Siebold et Zucc. Cool, temperate deciduous forests dominated by Fagus crenata Blume were distributed at high altitudes near mountain summits, and secondary forests dominated by Pinus densiflora Siebold et Zucc, Quercus serrata Murray, and Carpinus spp, as well as plantations of Cryptomeria japonica (L.f.) D.Don and Chamaecyparis obtusa (Siebold et Zucc.) Endl. were widely distributed on the mountain slopes. The sampling site included many old trees. Sampling was conducted in an area where leaf endophytic communities have been studied previously[20, 24].

The CBF site (35°10'46"N, 140°6'54"E) is located on the southern tip of the Boso Peninsula and is characterized by a warm, moist oceanic climate. Sampling was conducted in a natural forest composed mainly of Castanopsis sieboldii (Makino) Hatus. ex T.Yamaz. et Mashiba subsp. sieboldi and Quercus spp., mainly Q. salicina and Q acuta. Planted Quercus gilva Blume and Quercus phillyreoides A.Gray were also sampled at this site. The forests surrounding the CBF site included natural conifer forests dominated by Abies firma, secondary deciduous forests of Q. serrata and Zelkova serrata (Thunb.) Makino, and plantations of Cr. japonica and Ch. obtusa [32].

The ARI site (34°41'47"N, 138°50'22"E) is located at the southern tip of the Izu Peninsula and has the warmest temperatures among the three sites. The sampling area was a natural forest of C. sieboldii and Quercus spp., also containing Neolitsea sericea (Blume) Koidz and Camellia japonica L. Conifer plantations of Cr. japonica and Ch. obtuse, and Cinnamomum camphora (L.) J. Presl. and Eucalyptus spp. forests, were distributed around the sampling site.

2.2. Leaf sampling

We selected seven evergreen Quercus (subgen. Cerris) species for endophyte isolation, six of which (Q. acuta, Q. glauca, Q. gilva, Q. salicina, Quercus myrsinifolia Blume, and Quercus sessilifolia Blume) belong to sect. Cyclobalanopsis; the other one (Q. phillyreoides) belongs to sect. Ilex [33]. Sampling was conducted in summer (TKO, July 24; CBF, August 21; ARI, September 2) and winter (TKO, December 4; CBF, December 16; ARI, December 22) in 2015. We selected three to five individuals per species at each site (Supplementary material Table S2), avoiding adjacent conspecific trees except for Q. gilva, which is sparsely distributed in eastern Japan. We selected as many species as possible at the same microsites to minimize variation in abiotic factors among sample trees.

We collected 10 healthy leaves from each tree; the leaves were transported to the laboratory, washed under running tap water for 24 h and then surface-sterilized in 80% ethanol for 1 min, 1% NaOCl for 1 min, and 80% ethanol for 1 min; they were then rinsed twice in sterile water [20] . The leaf surface was allowed to dry, and a 6-mm-φ leaf disk was punched out of each leaf, avoiding the midrib. The leaf disks were placed on 1/2 potato dextrose agar (PDA) plates that contained 19.5 g/L PDA, 9 g/L agarose, and 600 mg/L chloramphenicol, and then incubated at 20°C for about 3 weeks.

2.3. Fungal identification via DNA analysis

Isolated fungi were distinguished morphologically and representative isolates were identified by sequencing the rDNA internal transcribed spacer (ITS) region. Fungal DNA was extracted from cultured fungal mycelia, as described by Izumitsu [34]. Then, the rDNA ITS region was amplified using the ITS5 and ITS4 primers [35]. Polymerase chain reaction (PCR) was conducted using 10-µL reaction volumes containing GoTaq Master Mix (Promega, Madison, WI, USA), DNA extracts, and primer pairs under the following conditions: initial denaturation 94°C for 10 min, followed by 30 cycles of 94°C for 30 s, 51°C for 1 min, 72°C for 1 min, and a final elongation of 72°C for 10 min.

The PCR products were purified using Exo-Sap (GE Healthcare, Chicago, IL, USA) and sequenced using the Big Dye Terminator Cycle Sequencing kit (version 3.1) and a 3130 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA), following the manufacturer’s protocols. Endophyte ITS sequences were BLAST-searched and clustered into operational taxonomic units (OTUs) at a threshold of 97% similarity. Endophyte sequence data were deposited in GenBank under the accession numbers listed in Supplementary material Table S3. Six major OTUs belonging to Tubakia s.l. showed no matches to known species, and were therefore tentatively named Tubakia spp. 1–5, and Tubakia sp. QA-b [24]. We deposited 94 cultures of Tubakia s.l. in the NARO Genebank (Tsukuba, Japan) as MAFF 246300- 246393.

2.4. Spore germination test

To examine the effect of temperature on each Tubakia s.l. species, we performed spore germination tests. Spore suspensions (10⁴ spores/mL) of T. subglobosa, Tubakia sp. QA-b, and Tubakia spp. 1–3 were prepared from pure cultures, and 200 µL of each suspension was applied to a 9-cm PDA plate containing 39 g/L PDA and 600 mg/L chloramphenicol. After incubation at 10, 15, 20, 25, or 30°C for 48 h, the spores were dyed with cotton blue lactophenol and observed under a microscope (BX50; Olympus, Tokyo, Japan) at a magnification of 200×. Germination rates were estimated by observing 100 spores. For each species, three plates were used as replicates.

2.5. Statistical analyses

The isolation frequency (IF, %) of endophyte species at each site was calculated as follows:

IF = (Total number of leaf discs from which endophytic fungi were isolated)⁄ (Total number of leaf discs) × 100.

To evaluate the effects of site, season, and host of dominant Tubakia species, we performed two- or three-way analysis of variance (ANOVA) using the ‘aov’ function of R software (R Development Core Team, Vienna, Austria), depending on the number of Tubakia isolates.

To visualize and compare endophytic fungal compositions, we performed heatmap analysis using the ‘heatmap’ function in the R package vegan, using relative abundance data for all isolates. To visualize seasonal variation of endophytic fungal composition among the three tree species collected from all sites (Q. glauca, Q. salicina, and Q. myrsinifolia), we conducted non-metric multidimensional scaling (NMDS) using the ‘metaMDS’ function in vegan; the number of isolates was applied to the Bray–Curtis dissimilarity matrix. Next, we performed permutational multivariate analysis of variance (PERMANOVA) using the ‘Adonis’ function in vegan to evaluate differences in endophytic fungal composition among host species, sampling sites, and sampling seasons. The Tukey–Kramer test for multiple comparisons was used to compare germination rates at different temperatures using the ‘TukeyHSD’ function in R

We isolated 692 and 623 isolates in summer and winter, respectively. The endophytic fungal species composition and abundance in evergreen oaks at all three sites are shown in Fig. 2 and Supplementary material Table S4. Tubakia species dominated the endophytic fungal communities of all leaves from the tested evergreen oak species at all sites. We also isolated eight Tubakia s.l. species from seven Quercus species, consistent with a previous observation that East and Southeast Asia (including Japan and Thailand) appear to be hotspots of Tubakiaceae genetic diversity [22]. Phomopsis spp., Colletotrichum spp., and Xylaria sp. were also isolated from all tree species. Moreover, these genera were isolated from Fagaceae trees in our previous study [20] and have been found in other tree species over a broad host range.

Total IF values were lower in winter than summer, and IF decreased in winter for most tree species (Fig. 3). Fungal species richness and diversity indices also generally decreased in winter (Fig. 3). Fungal composition and dominant species differed between seasons, with Tubakia spp. generally dominating in summer and Tubakia sp. 3, Colletotrichum spp., and Xylaria sp. Being frequently isolated in winter, when the dominant species varied among tree species.

3.1. Host preference and season and site effects

Tubakia spp. were dominant in five of the six tree species, excluding Q. gilva, from which Tubakia spp. were not isolated (Figs. 2, 4). All Tubakia spp. exhibited the same host preference in all three sites, except for Tubakia sp. QA-b, which was not isolated from Q. acuta at the ARI site. These findings were consistent with those of our previous studies, which showed that Tubakia sp. 1 was dominant among leaf endophytic communities of Q. myrsinifolia in TKO and other regions of eastern Japan (i.e., Chiba and Ibaraki) [20]. Tubakia sp. QA-b was found to be dominant on Q. acuta in TKO and in Miyazaki, western Japan in another study [24] ; samples collected from Q. gilva in Miyazaki in our previous study contained no Tubakia species (unpublished data).

In this study, we divided Tubakia species into specialists and generalists according to their host range and IF (Table 1). Specialists were isolated from only one or two host species, whereas generalists were isolated from three to all host species. We considered T. dryina, which was isolated from three host species, as a generalist because it has been reported to infect deciduous oaks and other Fagaceae trees [23].

Table 1

Natural host range of *Tubakia* spp. at the three study sites. Isolation frequency (IF) was calculated as follows: IF = (Number of trees in which a given *Tubakia* species was found) / (Total number of trees studied). Qa, *Quercus acuta*; Qg, *Quercus glauca*; Qgi, *Quercus gilva*; Qp, *Quercus phillyreoides*; Qs, *Quercus salicina*; Qm, *Quercus myrsinifolia*; Qse, *Quercus sessilifolia*.
Fungal species	Host Preference	Host species (frequency)
Fungal species	Host Preference	Qa	Qg	Qgi	Qp	Qs	Qm	Qse
T. subglobosa	Specialist		19/26
T. sp. 1	Specialist						18/22
T. sp. 5	Specialist				10/10
T. sp. QA-b	Specialist	18/20						3/10
T. sp. 4	Specialist		6/26			4/26
T. dryina	Generalist	4/20	1/26		8/10
T. sp. 2	Generalist	1/20	1/26			5/26	7/22
T. sp. 3	Generalist	16/20	20/26			26/26	14/22	10/10

A few studies have documented the host specificity of endophytic fungal species within a genus by examining the endophytic communities of multiple host species within the same site and/or season. Schlegel et al. reported host specificity in endophytic Venturia spp., which are dominant in European ash (Fraxinus excelsior), flowering ash (Fraxinus ornus), and sycamore maple (Acer pseudoplatanus) [36]. In contrast, a comparison of 11 Japanese Betulaceae species across three climatic regions showed that endophytic communities included a wide range of species such as Muscodor sp., Nemania sp. (Xylariaceae), Glomerella acutata, and two Diaporthales species; the results revealed no host specificity among endophytic fungi [21].

Among trees from which specialists were isolated, the IFs of specialists (T. subglobosa, Tubakia sp. QA-b, Tubakia spp. 1 and 4) on their specific hosts were higher in summer than winter, whereas those of generalists (Tubakia spp. 2 and 3) were higher in winter (Fig. 4 and Table 2). The generalist species Tubakia sp. 3 became dominant in winter in some tree species across all sites. This result suggests that infection phenology differs between specialists and generalists. Our ANOVA results showed that all factors were significant for Tubakia generalists (Table 2), which suggests that they have weak host preference and are more susceptible to other factors than specialists.

Table 2

Numbers of *Tubakia* isolates by season, site, and host (generalists only) according to two- or three-way analysis of variance (ANOVA).
	Specialist					Generalist
	T. subglobosa	T. sp.1	T. sp.4	T. sp.5	T. sp. QA-b	T. dryina	T. sp.2	T. sp.3
Season	***	***	*	0.60	*	0.18	***	***
Site	0.13	0.09	0.42	0.15	*	***	0.06	***
Host						***	*	***
Season×Site	*	0.06	0.42	0.49	0.79	0.10	*	***
Season×Host						0.37	0.11	***
Site×Host						***	0.12	0.55
Season×Site×Host						0.30	0.17	***
Significance codes: * : P < 0.001; :P < 0.01; *: P < 0.05
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3.2. Fungal endophyte composition

NMDS of fungal endophyte composition on the three host species common to all sites (Q. glauca, Q. salicina, and Q. myrsinifolia) showed that the fungal compositions of different hosts in TKO and CBF were similar in winter (Fig. 5), whereas those in ARI showed similarity in summer, but not in winter. This difference can be explained by decreased fungal diversity among host specialists in winter in TKO and CBF, which did not occur in ARI, perhaps due to higher winter temperatures at this site. PERMANOVA analysis results showed significantly different endophytic fungal compositions among host species (r² = 0.358, P < 0.001), sampling sites (r² = 0.080, P < 0.001), and sampling seasons (r² = 0.077, P < 0.001). At all three sites, endophytic fungal compositions were most strongly affected by tree species.

Our heatmap analysis showed that fungal communities of the same host species belonged to the same cluster in four of the six host species (Q. myrsinifolia, Q. salicina, Q. gilva, and Q. phillyreoides) (Fig. 4). In the two remaining host species, the fungal community of Q. glauca in TKO was dominated by Tubakia sp. 3, but not the specialist T. subglobosa, while the fungal community of Q. acuta in ARI was dominated by T. dryina, but not Tubakia sp. QA-b. This variation may be due to significant site or indirect host effects (Fig. 4).

A survey of fungal symbiont composition among the leaves of nine C3 grass species along replicated elevation gradients in the grasslands of the Colorado Rocky Mountains revealed that the taxonomic richness and composition of foliar fungal symbionts were mainly controlled by plant host phylogeny rather than climate [37]. A study of landscape-scale endophyte beta diversity in the Hawaiian archipelago showed that endophyte community structure was most strongly associated with host plant phylogeny and evapotranspiration [38]. Our results showed that host tree species and their phylogenic distance were the major factors affecting fungal composition [39], followed by temporal and spatial factors.

3.3. Spore germination rates

In summer, Tubakia species compositions were similar across all three sites; the same dominant specialists were the same, whereas in winter they differed significantly among sites. The three sites showed a maximum difference in average temperature of 4.4°C in December (Supplementary material Fig. S1). Changes in endophytic fungal composition are mainly caused by propagule dispersal and/or environmental conditions that favor abundant propagule production [5, 40] or propagule germination and infection. Therefore, we evaluated the effects of winter temperature on Tubakia spore germination.

After incubation for 48 h, Tubakia species showed a dramatic increase in spore germination rates. The effect of temperature on spore germination differed among species (Fig. 6); those of T. subglobosa and Tubakia sp. 1 (which had significantly higher IF values at ARI, the warmest site) decreased significantly as temperature decreased from 15°C to 10°C (P < 0.05), whereas those of Tubakia sp. 3 (which had significantly higher IF values at TKO, the coolest site) remained constant above 50%, even at temperatures as cool as 10°C. The germination rates of specialists decreased significantly at low temperatures, whereas those of generalists remained constant. These differences in cold adaptation between specialists and generalists may be caused by seasonal community changes. These results are consistent with those of previous studies [23, 24, 41], which showed that T. subglobosa, Tubakia sp. QA-b, and Tubakia sp. 1 grew only within a narrow temperature range.

3.4. Japanese evergreen Quercus and Tubakiaceae

High host specificity and temporal turnover within Tubakia clearly demonstrated a strong association between Japanese evergreen oaks and Tubakia s.l.; therefore, we explored the different factors affecting endophytic communities among host tree species.

According to their phylogenetic relationships, Japanese Quercus subgen. Cerris sect. Cyclobalanopsis species were grouped into three DNA types based on single-nucleotide polymorphisms. The first comprised Q. acuta, Q. sessilifolia, and Q. salicina, the second Q. myrsinaefolia and Q. glauca, and the third Q. gilva [42]. Q. acuta and Q. sessilifolia; they all shared the same host-specific endophyte species (Tubakia sp. QA-b). The heatmap also clustered endophyte communities of Q. acuta, Q. sessilifolia, and Q. salicina together, reflecting their close phylogenetic relationships. In terms of ecological distribution, the six evergreen oak species can be divided into two distinct groups, with Q. acuta, Q. salicina, Q. myrsinaefolia, and Q. sessilifolia found at high elevations, and Q. gilva and Q. glauca at low elevations. Q. glauca is a pioneer species and Q salicina has a wide habitat range [43]. The heatmap showed that endophytes of Q. gilva and Q. glauca were clustered within the same clade, consistent with the altitudinal ranges of both species. Quercus salicina hosted only Tubakia sp. 3, a generalist, perhaps indicating wide environmental variation in suitable host habitat for this species. We were unable to isolate any Tubakia species from Q. gilva in this study, perhaps due to its unusually high trichome density, which protects plant organs from biotic and abiotic stresses [44] including pathogen infection [45].

The ecological significance of these strong associations between evergreen Quercus and Tubakia should be further explored in terms of their benefits to the pathogen and/or host.

In this study, we identified interspecific differences in the basic ecology of Tubakia species on Japanese evergreen oaks. Our findings demonstrated that Tubakia species can be divided into specialists and generalists, and that generalists are more cold-tolerant than specialists. Host phylogeny and ecology were also found to be significantly related to endophyte structure.

Acknowledgments

We thank Dr. N. Matsushita of The University of Tokyo for assistance with sampling.

Funding This work was supported by JSPS KAKENHI Grant Number JP16H06737.

Conflict of Interest The authors declare that there is no conflict of interest.

Availability of data and material Data supporting the findings of this study are available from the corresponding author upon request．

Authors' contributions All authors contributed to the study conception and design. Sample collection was performed by Kenta Morinaga. Laboratory work and data analysis were conducted by Kenta Morinaga and Emi Matsumura. The first draft of the manuscript was written by Kenta Morinaga and Emi Matsumura. Emi Matsumura and Kenji Fukuda reviewed and edited the manuscript. All authors read and approved the final manuscript.

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Ethics approval (Not applicable)

Consent to participate (Not applicable)

Consent for publication (Not applicable)

1. Petrini O (1991) Fungal endophytes of tree leaves. In: Andrews JH., Hirano SS (eds) Microbial ecology of leaves. Springer-Verlag, New York, pp 179–197

2. Arnold A (2007) Understanding the diversity of foliar endophytic fungi: Progress, challenges, and frontiers. Fungal Biol Rev 21:51–66. https://doi.org/10.1016/j.fbr.2007.05.003

3. Sieber T (2007) Endophytic fungi in forest trees: are they mutualists? Fungal Biol Rev 21:75–89. https://doi.org/10.1016/j.fbr.2007.05.004

4. Wilson D (1996) Manipulation of infection levels of horizontally transmitted fungal endophytes in the field. Mycol Res 100:827–830. https://doi.org/10.1016/S0953-7562(96)80029-3

5. Kaneko R, Kaneko S (2004) The effect of bagging branches on levels of endophytic fungal infection in Japanese beech leaves. For Pathol 34:65–78. https://doi.org/10.1111/j.1439-0329.2004.00350.x

6. Hoffman MT, Arnold A (2008) Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees. Mycol Res 112:331–344. https://doi.org/10.1016/j.mycres.2007.10.014

7. Suryanarayanan TS, Murali TS, Thirunavukkarasu N, et al (2011) Endophytic fungal communities in woody perennials of three tropical forest types of the Western Ghats, southern India. Biodivers Conserv 20:913–928. https://doi.org/10.1007/s10531-011-0004-5

8. Carroll G, Carroll FE (1978) Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Can J Bot 3034–3043

9. Hata K, Futai K, Tsuda M (1998) Seasonal and needle age-dependent changes of the endophytic mycobiota in Pinus thunbergii and Pinus densiflora needles. Can J Bot 76:245–250

10. Ortiz-García S, Gernandt DS, Stone JK, et al (2003) Phylogenetics of Lophodermium from pine. Mycologia 95:846–859. https://doi.org/10.1080/15572536.2004.11833044

11. Salas-Lizana R, Santini NS, Miranda-Pérez A, Piñero DI (2012) The Pleistocene glacial cycles shaped the historical demography and phylogeography of a pine fungal endophyte. Mycol Prog 11:569–581. https://doi.org/10.1007/s11557-011-0774-x

12. Oono R, Lutzoni F, Arnold AE, et al (2014) Genetic variation in horizontally transmitted fungal endophytes of pine needles reveals population structure in cryptic species. Am J Bot 101:1362–1374. https://doi.org/10.3732/ajb.1400141

13. Apigo A, Oono R (2018) Dimensions of host specificity in foliar fungal endophytes. In: Pirttilä AM, Frank AC (eds) Endophytes of Forest Trees: Biology and Applications. Springer International Publishing, pp 15–42

14. Cannon PF, Simmons CM (2002) Diversity and host preference of leaf endophytic fungi in the Iwokrama Forest Reserve, Guyana. Mycologia 94:210–20

15. Higgins KL, Arnold a. E, Coley PD, Kursar T a. (2014) Communities of fungal endophytes in tropical forest grasses: Highly diverse host- and habitat generalists characterized by strong spatial structure. Fungal Ecol 8:1–11. https://doi.org/10.1016/j.funeco.2013.12.005

16. Vincent J, Weiblen G, Ecology GM-M, 2016 U (2016) Host associations and beta diversity of fungal endophyte communities in New Guinea rainforest trees. Wiley Online Libr 25:825–841. https://doi.org/10.1111/mec.13510

17. Cohen SD (2006) Host selectivity and genetic variation of Discula umbrinella isolates from two oak species: Analyses of intergenic spacer region sequences of ribosomal DNA. Microb Ecol 52:463–469. https://doi.org/10.1007/s00248-006-9073-5

18. Vega FE, Simpkins A, Aime MC, et al (2010) Fungal endophyte diversity in coffee plants from Colombia, Hawai’i, Mexico and Puerto Rico. Fungal Ecol 3:122–138. https://doi.org/10.1016/j.funeco.2009.07.002

19. Poulin R, Krasnov BR, Mouillot D (2011) Host specificity in phylogenetic and geographic space. Trends Parasitol 27:355–361. https://doi.org/10.1016/j.pt.2011.05.003

20. Matsumura E, Fukuda K (2013) A comparison of fungal endophytic community diversity in tree leaves of rural and urban temperate forests of Kanto district, eastern Japan. Fungal Biol 117:191–201. https://doi.org/10.1016/j.funbio.2013.01.007

21. Osono T, Masuya H (2012) Endophytic fungi associated with leaves of Betulaceae in Japan. Can J Microbiol 58:507–515. https://doi.org/10.1139/W2012-018

22. Braun U, Nakashima C, Crous PW, et al (2018) Phylogeny and taxonomy of the genus Tubakia s. lat. Fungal Syst Evol 1:41–99. https://doi.org/10.3114/fuse.2018.01.04

23. Yokoyama T, Tubaki K (1971) Cultural and taxonomical studies on the genus Actinopelte. IFO Res Commun 5:43–77

24. Hashizume Y, Sahashi N, Fukuda K (2008) The influence of altitude on endophytic mycobiota in Quercus acuta leaves collected in two areas 1000 km apart. For Pathol 38:218–226. https://doi.org/10.1111/j.1439-0329.2008.00547.x

25. Shirouzu T, Hirose D, Fukasawa Y, Tokumasu S (2009) Fungal succession associated with the decay of leaves of an evergreen oak, Quercus myrsinaefolia. Fungal Divers 34:87–107

26. Matsumura E, Takahashi Y, Fukuda K (2015) Occurrence of endophytic Tubakia spp.in leaves of sympatric evergreen Quercus spp. on Mt. Takao. Tree For Heal 19:167–168. (In Japanese)

27. Murata G (1977) Phytogeographical consideration on the flora and vegetation of Japan. Acta Phytotaxon Geobot 28:65–83. https://doi.org/10.18942/bunruichiri.KJ00003217442. (In Japanese)

28. Suzuki T (1960) The forest location with the center focus on Japanese oak,Quercus gilva. Japan Soc For Environ 2:1–6. (In Japanese)

29. Sugiura N, Tang D, Kurokochi H, et al (2015) Genetic structure of Quercus gilva Blume in Japan as revealed by chloroplast DNA sequences. Botany 93:873–880. https://doi.org/10.1139/cjb-2015-0025

30. Meteorology division fundamental data development committee The University of Tokyo Forests (2017) Annual report of meteorological observations in the University of Tokyo Forests, The University of Tokyo (Jan. 2015 - Dec. 2015). 59:255–283. (In Japanese)

31. Hayashi Y, Koyama Y, Kobayashi Y, et al (1966) Ecological and floristic studies in the Takao mountains. Bull Gov For Exp Stn 196:1–167. (In Japanese)

32. Watanabe R, Miyai S (1978) Studies on vegetation in Kiyosumi region: I. Numerical classification of forest vegetation. Japanese J Ecol 28:281–290. https://doi.org/10.18960/seitai.28.4_281

33. Denk T, Grimm GW, Manos PS, et al (2017) An updated infrageneric classification of the oaks: Review of previous taxonomic schemes and synthesis of evolutionary patterns. In: Gil-Pelegrín E, Peguero-Pina J, Sancho-Knapik D (eds) Oaks Physiological Ecology: Exploring the Functional Diversity of Genus Quercus L. Tree Physiology. Springer, Cham, pp 13–38. https:// doi.org/ 10.1007/978-3-319-69099-5_2.

34. Izumitsu K, Hatoh K, Sumita T, et al (2012) Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR. Mycoscience 53:396–401. https://doi.org/10.1007/S10267-012-0182-3

35. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols. Academic Press Inc., pp 315–322

36. Schlegel M, Queloz V, Sieber TN (2018) The endophytic mycobiome of European ash and sycamore maple leaves - Geographic patterns, host specificity and influence of ash dieback. Front Microbiol 9:1–20. https://doi.org/10.3389/fmicb.2018.02345

37. Kivlin SN, Kazenel MR, Lynn JS, et al (2019) Plant identity influences foliar fungal symbionts more than elevation in the Colorado Rocky Mountains. Microb Ecol 78:688–698. https://doi.org/10.1007/s00248-019-01336-4

38. Darcy JL, Swift SOI, Cobian GM, et al (2020) Fungal communities living within leaves of native Hawaiian dicots are structured by landscape‐scale variables as well as by host plants. Mol Ecol 29:3102–3115. https://doi.org/10.1111/mec.15544

39. Oita S, Carey J, Kline I, et al (2021) Methodological approaches frame insights into endophyte richness and community composition. Microb Ecol. https://doi.org/10.1007/s00248-020-01654-y

40. Jumpponen, A. and Jones, K. L. (2010) Seasonally dynamic fungal communities in the Quercus macrocarpa phyllosphere differ between urban and nonurban environments. New Phytologist 186:496–513. https://doi.org/10.1111/j.1469-8137.2010.03197.x

41. Matsumura, Emi, Fukuda K (2015) Fungal endophytic communities in leaves of Quercus myrsinifolia and Eurya japonica-Their within-leaf distributions and antagonism. Tree For Heal 19:195–204

42. Ohyama M, Baba K, Itoh T (1999) Possibility of grouping of Cyclobalanopsis species (Fagaceae) grown in Japan based on an analysis of several regions of chloroplast DNA. J Wood Sci 45:498–501

43. Ito S, Ohtsuka K, Yamashita T (2007) Ecological distribution of seven evergreen Quercus species in southern and eastern Kyushu, Japan. Veg Sci 24:53–63

44. Karabourniotis G, Liakopoulos G, Dimosthenis Nikolopoulos ·, Bresta P (2020) Protective and defensive roles of non-glandular trichomes against multiple stresses: Structure-function coordination. J For Res 31:1–12. https://doi.org/10.1007/s11676-019-01034-4

45. Valkama E, Koricheva J, Salminen J-P, et al (2004) Leaf surface traits: overlooked determinants of birch resistance to herbivores and foliar micro-fungi? Trees 19:191–197. https://doi.org/10.1007/s00468-004-0380-5

MorinagaSD.docx
Supplementary material Fig. S1 Monthly mean temperatures at each site in 2015. Supplementary material Table S2 Number of leaf disks used for Tubakia spp. isolation in each season. Supplementary material Table S3 BLAST search results. Supplementary material Table S4 Variation in fungal isolation frequency among host species, sites, and seasons.

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You are reading this latest preprint version

Host Preference and Seasonal Variation in The Colonization of Tubakia s.l. Associated with Evergreen Oak Species in Eastern Japan

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Abstract

Figures

1. Introduction

2. Materials And Methods

3. Results And Discussion

3.1. Host preference and season and site effects

3.2. Fungal endophyte composition

3.3. Spore germination rates

3.4. Japanese evergreen Quercus and Tubakiaceae

4. Conclusion

Declarations

References

Supplementary Files

Status:

Version 1