A community of unknown, endophytic fungi in western white pine
Abstract
The endophytic fungi of woody plants may be diverse as often claimed, and likewise, they may be functionally novel as demonstrated in a few studies. However, the endophyte taxa that are most frequently reported tend to belong to fungal groups composed of morphologically similar endophytes and parasites. Thus, it is plausible that endophytes are known (i.e., described) parasites in a latent phase within the host. If this null hypothesis were true, endophytes would represent neither additional fungal diversity distinct from parasite diversity nor a symbiont community likely to be novel ecologically. To be synonymous with parasites of the host, endophytes should at least be most closely related to those same parasites. Here we report that seven distinct parasites of Pinus monticola do not occur as endophytes. The majority of endophytes of P. monticola (90% of 2,019 cultures) belonged to one fungal family, the Rhytismataceae. However, not a single rhytismataceous endophyte was found to be most closely related by sequence homology to the three known rhytismataceous parasites of P. monticola. Similarly, neither endophytic Mycosphaerella nor endophytic Rhizosphaera isolates were most closely related to known parasites of P. monticola. Morphologically, the endophytes of P. monticola can be confounded with the parasites of the same host. However, they are actually most closely related to, but distinct from, parasites of other species of Pinus. If endophytes are generally unknown species, then estimates of 1 million endophytes (i.e., approximately 1 in 14 of all species of life) seem reasonable.
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Specialized parasites are not alone in infecting plants. Endophytic fungi also infect plants, although as nonpathogenic colonists. At least in some plants, endophytic fungi perform novel ecological functions (e.g., thermotolerance of plants growing in geothermal soils; ref. 1). Endophytes can influence community biodiversity (2) or even directly enhance plant growth (3). In grasses (4) and other herbaceous plants (5), dominant endophytes are known to produce toxic alkaloids that deter or poison herbivores. In woody plants, endophytes also may function in specific defense roles (6) or more generally function to limit pathogen damage (7). Together with mycorrhizal fungi, endophytes form an integral part of the extended phenotype or symbiotic community of a plant (8). The full range of ecological functions of endophytes of woody plants is poorly understood, but it is likely to be correlated with their species diversity (9).
However, endophyte diversity in woody plants is clouded in ambiguity. In the absence of traditional species delimitations appropriate for endophytes, estimates of their diversity have been based on the “morphospecies” concept or morphological similarity to known species. Unfortunately, endophytes of a given plant are typically similar morphologically to known parasites of that same plant or to those of closely related hosts. Therefore, it is possible that many of these so-called endophytes are actually cryptic or latent, but known, parasites. Thus, these fungi remain inherently ambiguous.
How plausible is it that endophytes could largely be known parasites that have colonized the host in a cryptic or latent manner? Confounding of parasitism and endophytism easily results from the fact that “related fungi frequently infect related hosts” (10). Similarly, many fungi that are found on plants are assumed to be plant pathogens, but without proof of Koch's Postulates, the assumption is questionable. Such assumptions can be problematic, especially in quarantine diagostics when endophyte morphology closely resembles that of pathogenic relatives. This problem was recently highlighted in Citrus in which a ubiquitous endophyte of many woody plant species was confounded with the citrus black spot fungus, Guignardia citricarpa. The nonpathogenic endophyte does not cause citrus black spot, yet it was subject to needless quarantine restrictions (11). Further examples of this kind are summarized in Table 1 to illustrate the general problem that has been insurmountable for morphology-based approaches to endophyte diversity.
Table 1.
| Genera of fungi isolated as endophytes from leaves: | ||
|---|---|---|
| Woody plant host genus from which endophytes were isolated (ref.) | Includes at least one species that parasitizes the host from which the endophytes were isolated* | Includes at least one parasitic species† but not from the host genus from which the endophytes were isolated |
| Abies [fir] (14) | Leptostroma, Phyllosticta, Tiarosporella | Geniculosporium, Xylaria |
| Alnus [alder] (31) | Diaporthe, Gnomonia, Gnomoniella, Septoria | Cladosporium, Mycosphaerella, Ophiovalsa, Pezicula, Phloeosporella, Phomopsis, Xylaria |
| Betula [birch] (32) | Melanconium, Venturia | Aureobasidium |
| Bruguiera [mangrove] (33) | Colletotrichum, Phyllosticta, Pestalotiopsis | |
| Fagus [Japanese (34) and European beech (35)] | Discula, Phomopsis | |
| Opuntia [prickly pear cacti] (36) | Coniothyrium, Phomopsis | Alternaria, Ascochyta, Cladosporium, Coniella, Epicoccum, Fusarium, Leptosphaeria, Nigrospora, Phoma |
| Picea [spruce] (14) | Leptostroma, Phomopsis | Coryneum, Geniculosporium, Ulocladium |
| Pinus [pines in North America (14) and Europe (37)] | Cenangium, Cyclaneusma, Lophodermium (Leptostroma) | Cladosporium, Cryptodiaporthe |
| Quercus [oak] (38) | Discula | |
Endophytes in genera in which species are known to be parasites of the host (2nd column) could be those same parasites unless ruled out by an explicit, phylogenetic test that includes those parasites. Endophytes in genera known to be parasitic on other hosts (3rd column) require further tests to rule out the possibility that the endophyte host is an occasional, alternate host for a known fungal species.
*
According to the authors, with confirmation in Funk (17), Hansen and Lewis (15), and Farr et al. [Farr, D. F., Rossman, A. Y., Palm, M. E. & McCray, E. B. (n.d.) Fungal Databases, Systematic Botany and Mycology Laboratory, Agricultural Research Service, U.S. Department of Agriculture. Retrieved June 8, 2004, from http://nt.ars-gin.gov/fungaldatabases/]
†
According to Rossman et al. (39)
Sequence-based approaches allow for a partial solution to the problem inasmuch as they provide an opportunity to test the null hypothesis (i.e., that endophytes are merely known fungal pathogens or parasites in a latent phase of their life cycle). If the null hypothesis were true, endophytes should at least be most closely related to those same parasites in phylogenetic analyses. However, sequence-based studies of endophytes are still rare (12, 13), and for many hosts, incomplete knowledge of parasites (e.g., tropical trees) precludes tests of the null hypothesis. Nevertheless, without such tests, the importance of endophytes to estimates of fungal diversity is unclear.
Pinus monticola (western white pine), like other white pines susceptible to white pine blister rust, has been surveyed and scrutinized for the better part of a century by forest pathologists. As a result, fungal parasites and saprobes of P. monticola are better studied than those of most species of forest tree even within the temperate zones. Most endophytes of Pinus tend to belong to the aforementioned Rhytismataceae (14). Within this family, those fungi that are proven parasites tend to be specialized, at least to a single subgenus of Pinus. For example, the only rhytismataceous parasites of P. monticola are Bifusella linearis, Lophodermella arcuata, and Meloderma desmazieresii (15–17). The first is known to parasitize Pinus strobus, P. monticola, Pinus flexilis, and Pinus albicaulis, all white pines in subgenus Strobus. The host range of Lophodermella arcuata is also restricted to subgenus Strobus. Only Meloderma desmazieresii parasitizes both subgenus Strobus and subgenus Pinus. However, there are rhytismataceous fungi that parasitize subgenus Pinus only (e.g., Cyclaneusma minus, Elytroderma deformans, Davisomycella medusa, Lophodermium baculiferum, Lophodermium seditiosum, and others) (15, 18).
Four additional ascomycetous fungi are known to parasitize P. monticola: Mycosphaerella pini, Leptomelanconium allescheri, Linodochium hyalinum, and Rhizosphaera pini (17). If endophytes are just latent parasites, then these four, nonrhytismataceous fungi and the three rhytismataceous species should dominate the endophyte community of P. monticola. Here we report that endophytes that could be confounded with those seven parasites do dominate the community. Those same endophytes are most closely related to, but distinct from, parasites of other species of Pinus. Thus, they are not parasites in a latent phase.
Materials and Methods
Fungal Isolates and Sequences. Symptomless P. monticola needles were collected from seven sites in part of the range of the species in the northern Rocky Mountains of the United States. Endophyte cultures were obtained from mycelial outgrowth from surface-sterilized needles plated on potato dextrose agar. Identification of a representative subset of endophyte cultures as rhytismataceous was initially based on high sequence similarity of the internal transcribed spacer (ITS) region to GenBank sequences of known members of the Rhytismataceae. Subsequently, similarity of culture morphology was used to identify the remainder of the endophytes isolated. In addition, fresh fruiting bodies of the following taxa were collected and sequenced: Rhytisma salacinum [taxon representative of the type genus of the family], Lophodermium species, Lophodermium nitens (1) [Idaho], L. nitens (2) [L. nitens from its type location in the Muskoka region of Ontario, Canada], Lophodermella arcuata, D. medusa, and B. linearis. The latter three fungi were all collected in the northern Rocky Mountains. The Mycosphaerella endophytes and the Rhizosphaera endophyte, also isolated from surface-sterilized P. monticola needles, were identified based on high similarity of the ITS region and culture morphology to members of Mycospharella and Rhizosphaera, respectively. The remainder of the sequences used in this study were obtained from the GenBank database.
GenBank accession numbers for endophytes and known species collected for this study are as follows for rhytismataceous endophytes. Clade 1: endophyte 1 (AY465451); clade 2: endophytes 2 (AY465440), 3 (AY465439), 19 (AY465436), 20 (AY465437), 21 (AY465438); clade 5: endophyte 4 (AY465473); clade 6: endophyes 5 (AY465485), 6 (AY465484), 22 (AY465487), 23 (AY465486), 24 (AY465475); clade 7: endophytes 7 (AY465477), 8 (AY465480), 9 (AY465483), 10 (AY465474), 11 (AY465495), 12 (AY465496), 25 (AY465497), 26 (AY465498), 27 (AY465489), 28 (AY465500), 29 (AY465494), 30 (AY465490); clade 8: endophytes 13 (AY465488), 14 (AY465499), 15 (AY465491), 16 (AY465493), 31 (AY465476), 32 (AY465482), 33 (AY465481), 34 (AY465479), 35 (AY465478). Mycosphaerella endophytes: 17 (AY465456), 18 (AY465457). Rhizosphaera endophyte: 19 (AY465472). Known rhytismataceous parasites and saprobes: B. linearis (AY465527), D. medusa (AY465525), Lophodermella arcuata (AY465518), L. nitens (1) (AY465519), L. nitens (2) (AY465520), Lophodermium species (AY465524), Rhytisma salacinum (AY465515).
GenBank accession numbers for species obtained from the GenBank database are as follows for rhytismataceous taxa: C. minus (AF013222), Cyclaneusma niveum (AF013223), E. deformans (AF203469), Colpoma quercinum (AJ293879), Lirula macrospora (1) (AF462441), Lirula macrospora (2) (AF203472), Lophodermium actinothyrium (AY100663), Lophodermium agathidis (AY100661), Lophodermium australe (1) (AY100647), L. australe (2) (U92308), L. baculiferum (1) (AY100658), L. baculiferum (2) (AY100653), L. baculiferum (3) (AY100655), L. baculiferum (4) (AY100656), Lophodermium conigenum (1) (AY100646), L. conigenum (2) (AF473559), Lophodermium indianum (AY100641), Lophodermium macci (AF540559), Lophodermium minor (AY100665), Lophodermium molitoris (AY100659), L. nitens (3) (AY100640), L. nitens (4) (AF426058), Lophodermium piceae (AF203471), Lophodermium pinastri (1) (AY100649), L. pinastri (2) (AF462434), L. seditiosum (AF473550), Meloderma desmazieresii (AF426056), Spathularia flavida (AF433154), Tryblidiopsis pinastri (U92307). Mycosphaerella taxa: Cladosporium colocasiae (AF393693), Cladosporium fulvum (AF393701), Mycosphaerella africana (AF173314), Mycosphaerella aurantia (AY150331), Mycosphaerella berkeleyi (AY266147), Mycosphaerella brassicicola (AF297236), Mycosphaerella confusa (AF362058), Mycosphaerella dearnessii (1) (AF260817), M. dearnessii (2) (AF211194), M. dearnessii (3) (AF362070), Mycosphaerella ellipsoidea (AF309592), Mycospha-erella keniensis (AF173300), Mycosphaerella lupini (AF362050), Mycosphaerella marasasii (AF309591), Mycosphaerella parkii (AF309590), M. pini (AF013227), Mycovellosiella vaginae (AF222832), Passalora ampelopsidis (AF362053), Passalora arachidicola (AY266154), Passalora fulva (AY251069), Passalora henningsii (AF284389), Passalora loranthi (AY348311), Phaeophleospora destructans (AF309614), Phaeophleospora eugeniae (AF309613), Phaeoramularia dissiliens (AF222835). Rhizosphaera taxa: Rhizosphaera kalkhoffii (AF013231), Rhizosphaera kobayashii (AF462432), Rhizosphaera macrospora (AF462431), Rhizosphaera oudemansii (AF462430), R. pini (AY183365).
Sequencing and Data Analysis. DNA extractions and sequencing were performed according to Newcombe (19). Sequences obtained were aligned by eye to other ITS sequences from GenBank in the data editor of the software package paup* 4.0b10 (20). Phylogenetic analysis was performed under maximum parsimony, with heuristic search, by using paup*. The search used the stepwise addition option and was repeated 10 times from different starting points with tree-bisection-reconnection (TBR) branch swapping. All characters were equally weighted and unordered. Alignment gaps were treated as missing data. Confidence in specific clades from the resulting topology was tested by bootstrap analysis with 1,000 replicates with a 50% majority rule. Initially, 75 Rhytismataceae and 33 rhytismataceous endophyte sequences, 123 Mycosphaerella and 2 Mycosphaerella endophyte sequences, and 9 Rhizosphaera and 1 Rhizosphaera endophyte were used for phylogenetic analyses. However, because of the magnitude of the phylogenetic trees generated and the repetitiveness of certain taxa, the number of sequences used was reduced to 35 nonendophyte and 16 endophyte, rhytismataceous taxa, 25 Mycosphaerella and 2 Mycosphaerella endophyte taxa, and 5 Rhizosphaera and 1 Rhizosphaera endophyte taxa. One hundred equally most-parsimonious trees were retained from the 591 nucleotide Rhytismataceae data matrix (length = 736, CI = 0.458, RI = 0.628), of which 153 characters were excluded because of problems with alignment, and from the 507 nucleotide Mycosphaerella data matrix (length = 361, CI = 0.690, RI = 0.787). Four equally most-parsimonous trees were obtained from the 544 nucleotide Rhizosphaera data matrix (length = 119, CI = 0.958, RI = 0.773). In addition to the maximum parsimony analysis, Bayesian phylogenetic inference was also performed by using mrbayes 3.0 (21). The Bayesian analysis was run with uniform priors for 1,000,000 generations and sampled every 100 generations. Posterior nodal probabilities were obtained from the Markov Chain Monte Carlo results, summarized by generating a majority rule consensus tree by using paup*.
Results
Using a sequence-based approach, we set out to determine whether the endophytes of P. monticola are known but latent parasites or are novel fungi that represent additional biodiversity and that are thus likely to play nonparasitic roles within the host and its symbiont community. Endophytes of P. monticola were isolated from surface-sterilized needles from part of its host range in the northwestern United States. Although 10 orders of fungi were represented in the original 2,019 cultures, we focused on those isolates that could be confounded with five of the seven known parasites of P. monticola: Meloderma desmazieresii, B. linearis, Lophodermella arcuata, R. pini, and M. pini. No endophytes that could be confounded with the sixth and seventh parasites, Leptomelanconium allescheri and Linodochium hyalinum, were found. Of the original 2,019 cultures, 1,832 were identified as rhytismataceous based on culture morphology and sequence similarity of the ITS region to known rhytismataceous taxa present in GenBank. This large group of endophytes was thus possibly confounded with Meloderma desmazieresii, B. linearis, and Lophodermella arcuata, the three rhytismataceous parasites. Sequence analysis of 79 of the rhytismataceous subset revealed 33 phylotypes with sequence divergence ranging from 0.2% to 20.2%. Phylogenetic analyses were then conducted on the endophyte ITS phylotypes along with ITS sequences for rhytismataceous taxa representing parasites and saprobes of both P. monticola, and of other hosts. Maximum parsimony and Bayesian analysis generated similar tree topologies that only differed in their ability to resolve certain relationships. The fungal endophytes were distributed throughout the tree topology and were present in six of the eight clades delineated predominantly by both Bayesian and parsimony methods (Fig. 1).
Fig. 1.
For most of the endophytes a closest relative was determined. None of the rhytismataceous endophytes was most closely related to parasites of P. monticola itself (i.e., Meloderma desmazieresii in clade 4, B. linearis, and Lophodermella arcuata). Instead, 12 of the 33 rhytismataceous endophytes were most closely related to parasites of congeners of P. monticola (pairwise distances: C. minus = 15.9%; E. deformans = 0.2–1.4%; Lophodermium baculiferum = 0.7–8.2%). In clade 8, the nine rhytismataceous endophytes were most closely related to L. nitens (pairwise distance = 0–0.8%), a saprobe of P. monticola and other species in subgenus Strobus (22). However, a parasite of subgenus Pinus, D. medusa, is basal in clade 8, and therefore, even the nine endophytes, and L. nitens itself, appear to have descended from a parasite of a congener in subgenus Pinus (Fig. 1). The remaining 12 endophytes from clade 7 showed no specific phylogenetic relationship to any sequenced taxa of the family.
Secondly, endophytes belonging to Mycosphaerella (family Mycosphaerellaceae) and Rhizosphaera (mitosporic Ascomycetes), which were isolated from surface-sterilized P. monticola needles, followed the same pattern as the rhytismataceous endophytes. The Mycosphaerella endophytes were more closely related to M. dearnessii (pairwise distances ranging from 3.4% to 3.7%), a known parasite of subgenus Pinus, than to M. pini, which parasitizes both subgenera, including P. monticola (Fig. 2). Furthermore, the Mycosphaerella endophytes were found to be morphologically distinct from M. dearnessii (Fig. 3). Likewise, the Rhizosphaera endophyte was more closely related to R. kobayashii (pairwise distance = 13.0%) than R. pini, a parasite of P. monticola (Fig. 4). Again, the Rhizosphaera endophyte was morphologically distinct from its nearest relative, R. kobayashii.
Fig. 2.
Fig. 3.
Fig. 4.
Discussion
Estimates of the global diversity of endophytic fungi have run from minimal (23) to a million species (i.e., the maximal estimate based on four endophytes per each of 250,000 plant species; refs. 24 and 25). Although all of the assumptions upon which the maximal estimate rests are not explicit, it is implicit that woody plants are likely to host more endophytes per species than are herbaceous plants. Presumably, almost all of the million would currently be undescribed. This huge range in estimates from minimal to maximal, is a direct consequence of the ambiguity engendered by the possibility that endophytes could be latent parasites of the host. In this study the symbiont community of a temperate forest tree could have been dominated by endophytes that were most closely related to, and hence possibly synonymous with, any one of seven ascomycetous parasites of P. monticola (17). Instead, we found no evidence that any endophytes of P. monticola are merely cryptic or latent versions of known parasites of this same tree species. Although there was no support for the null hypothesis, our results could still be construed as equivocal with respect to estimates of endophyte diversity, given a second layer of possible confounding represented by the last column in Table 1. In this hypothetical scenario, endophytes are known, delimited fungi that occasionally parasitize plants other than the host itself. In other words, P. monticola could be a secondary or alternate host for parasites of related, sympatric plants.
This second layer of ambiguity cannot be totally dismissed, but the general trend in our findings runs counter to the alternate-host hypothesis. Although all of the endophytes were mostly closely related to, or descended from, parasites of congeners of P. monticola, species novelty of endophytes is suggested by any one or all of the following: morphological distinctiveness, extent of sequence divergence in the ITS region, or allopatry of the closest relative. For example, the Mycosphaerella and Rhizosphaera endophytes are easily distinguished from their closest relatives by discontinuous morphological variation indicative of separate species that remain undescribed for now. Further, sequence divergences of 3.4–3.7% and 13%, respectively, are not known to occur within species in these fungal groups. Thirdly, R. kobayashii is a parasite of the Siberian dwarf pine, Pinus pumila, an allopatric congener of P. monticola. P. pumila extends into western Beringia, but there is no current range overlap because P. monticola is not distributed in eastern Beringia. Given range separation or allopatry, P. monticola could not be an occasional, alternate host for R. kobayashii. A few endophytes have been formally described in recent years (25–27); this has yet to be done for the Mycosphaerella and Rhizospaera endophytes.
For the rhytismataceous endophytes that formed the bulk of the symbiont community of P. monticola, separation from their closest parasitic relatives was more problematic because of overlapping variation in cultural or morphological characters. However, in the absence of morphological distinctiveness, extent of genetic divergence again ran counter to the alternatehost hypothesis. The one endophyte in clade 1 (Fig. 1) was so distant (i.e., 15.9%) from C. minus that an interpretation based on intraspecific variation would be unprecedented. Clades 5–7 include 18 endophytes that are most closely related to, or descended from, Lophodermium baculiferum. Again, some or all of the 18 are likely to be reproductively isolated from L. baculiferum if genetic distance is a suitable proxy for reproductive isolation. Only in clade 8 is divergence limited to such an extent (i.e., 0–0.8%) that the alternate-host hypothesis remains plausible. Ironically, the endophytes of clade 8 were most closely related to L. nitens, a nonparasitic saprobe (17). Given the fact that morphologically and biologically distinct congeners of at least some fungi have sometimes diverged surprisingly little in ITS sequences (28), it is possible that even clade 8 represents additional species novelty.
By showing that endophytic fungi are neither latent phases of known, delimited parasites of the host nor likely to be conspecific with parasites found in nonhost plants, we have demonstrated that endophytes are likely to represent substantial, unknown biodiversity in woody plants. The null and alternate-host hypotheses entirely or largely failed to explain the presence of 82 sequenced endophytes (79 rhytismataceous, 1 Rhizosphaera, and 2 Mycosphaerella isolates) in the foliar tissues of just one temperate-zone tree (P. monticola). These hypotheses have yet to be tested for additional endophytes among the 2,019 isolates that together composed a total of 10 orders of fungi. Further, the 2,019 isolates were only drawn from a sample of trees from a portion of the range of the host. In view of these findings, it seems reasonable to propose that even the maximal estimates of total endophyte diversity (i.e., four endophytes per plant species) have been conservative. However, even if there were only 1 million endophytic fungi in plants (25), they would still account for 1 in 14 (9) or even 1 in 10 (29) species of life.
Although a range of diverse ecological functions have been demonstrated for endophytes, the full amplitude is still unclear. In this study, the repeating pattern of descent of endophytes from parasites of congeners rather than from the host itself is intriguing. Could endophytes play a role in nonhost resistance? P. monticola, like all other pines in subgenus Strobus, is resistant to the Elytroderma disease of pines of subgenus Pinus (15). That disease is caused by E. deformans, the closest relative of an endophyte found in P. monticola. That pattern is repeated for the majority of rhytismataceous endophytes, and all of the Mycosphaerella and Rhizosphaera endophytes. Nonhost resistance has invariably been theorized to be nonspecific with respect to parasites (30), but the selective force that maintains nonhost resistance in all plants is currently unknown. As descendants of parasites of congeners, the endophytes of woody plants merit further consideration as the missing force.
Notes
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AY465436–AY465440, AY465451, AY465456, AY465457, AY465472–AY465491, AY465493–AY465500, AY465515, AY465518–AY465520, AY465524, AY465525, and AY465527).
Acknowledgments
We thank J. M. Staley for collections and identification of rhytismataceous parasites of P. monticola; R. M. Van Aelst-Bouma for collections in Montana; A. R. D. Ganley for critical review; and McIntire-Stennis for support.
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Copyright © 2004, The National Academy of Sciences.
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Received: March 3, 2004
Accepted: May 17, 2004
Published online: June 25, 2004
Published in issue: July 6, 2004
Acknowledgments
We thank J. M. Staley for collections and identification of rhytismataceous parasites of P. monticola; R. M. Van Aelst-Bouma for collections in Montana; A. R. D. Ganley for critical review; and McIntire-Stennis for support.
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