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.

The plant microbiome may be bottlenecked at the level of endophytes of individual seeds. Strong defense of developing seeds is predicted by optimal defense theory, and we have experimentally demonstrated exclusionary interactions among endophytic microbes infecting individual seeds of Centaurea stoebe. Having found a single, PDA-culturable microbe per seed or none in an exploratory study with Centaurea stoebe, we completed a more extensive survey of an additional 98 plant species representing 39 families. We again found that individual, surface-sterilized seeds of all species hosted only one PDA-culturable bacterial or fungal endophyte per seed, or none. PDA-unculturables were not determined but we expect them to also be bottlenecked in individual seeds, as they too should be governed by exclusionary interactions. If the bottleneck were confirmed with high-throughput sequencing of individual seeds then it would make sense to further investigate the Primary Symbiont Hypothesis (PSH). This includes the prediction that primary symbionts (i.e., the winners of the exclusionary battles among seed endophytes) have strong effects on seedlings depending on symbiont identity.

Summary: Foliar endophytes of Populus do not induce the hypersensitive response associated with major genes for resistance to Melampsora leaf rust. But they could contribute to the quantitative resistance that represents a second line of defense. Quantitative resistance is thought to be determined by suites of minor genes in both host and pathogen that are influenced by the abiotic environment. Here, we determined the relative importance to quantitative resistance of foliar endophytes, one element of the biotic environment. Leaves of six host genotypes differing in genetic resistance to Melampsora × columbiana were inoculated first with one of four foliar endophytes (Stachybotrys sp., Trichoderma atroviride, Ulocladium atrum or Truncatella angustata), and then with Melampsora. These endophytes greatly reduced rust severity within inoculated leaves (i.e. local effects), but they had no systemic effect on rust of leaves not inoculated with endophytes. Differences among endophytes and their controls explained 54% of the total variation in quantitative resistance (i.e. rust severity); the six host/pathogen genotypes explained just 5%. In terms of magnitude of effect on rust severity, Stachybotrys, Trichoderma, Ulocladium and Truncatella were ranked in this order on all host/pathogen genotypes. Endophytes may contribute significantly to quantitative resistance to Melampsora in leaves of Populus.

The recently described rust hybrid Melampsora ×columbiana was discovered as a result of its novel pathogenic variation on Populus trichocarpa × P. deltoides (T×D) hybrid poplar. To characterize this pathogenic variation, 10 commercial T×D clones, all F₁ clones, were chosen as host differentials. Fourteen mononuredinial isolates of Pacific Northwestern field collections of M. ×columbiana, from 1996 to 1998 inclusive, were determined to be 13 distinct pathotypes. In contrast, four Southeastern isolates of M. medusae could not be distinguished on the same T×D host differentials, although they can be distinguished as pathotypes using P. deltoides differentials. The first three pathotypes of M. ×columbiana (Mxc1, Mxc2, and Mxc3) and a Mississippi isolate of M. medusae were inoculated onto a three-generation T×D pedigree, formerly used to characterize the Mmd1 gene for resistance to M. medusae. Resistance to the Mxc3 pathotype mapped to the same linkage group (group Q) as the Mmd1 gene. In contrast, linked genes for resistance to Mxc1 and to Mxc2 were located on linkage group O, and were unlike Mmd1 and Mxc3 in that they were inherited from P. deltoides. The latter two genes resembled Mmd1 and Mxc3 in that infection type was correlated with quantitative traits such as uredinial density and latent period. Pathogenic variation in M. ×columbiana matches resistance genes from both P. trichocarpa and P. deltoides and reveals the vulnerability to hybrid rust of commercial T×D hybrid poplar clones.

Dung fungi, such as Sordaria fimicola, generally reproduce sexually with ascospores discharged from mammalian dung after passage through herbivores. Their life cycle is thought to be obligate to dung, and thus their ascospores in Quaternary sediments have been interpreted as evidence of past mammalian herbivore activity. Reports of dung fungi as endophytes would seem to challenge the view that they are obligate to dung. However, endophyte status is controversial because surface-sterilization protocols could fail to kill dung fungus ascospores stuck to the plant surface. Thus, we first tested the ability of representative isolates of three common genera of dung fungi to affect plant growth and fecundity given that significant effects on plant fitness could not result from ascospores merely stuck to the plant surface. Isolates of S. fimicola, Preussia sp., and Sporormiella sp. reduced growth and fecundity of two of three populations of Bromus tectorum, the host from which they had been isolated. In further work with S. fimicola we showed that inoculations of roots of B. tectorum led to some colonization of aboveground tissues. The same isolate of S. fimicola reproduced sexually on inoculated host plant tissues as well as in dung after passage through sheep, thus demonstrating a facultative rather than an obligate life cycle. Finally, plants inoculated with S. fimicola were not preferred by sheep; preference had been expected if the fungus were obligate to dung. Overall, these findings make us question the assumption that these fungi are obligate to dung.

Temple trees, including the gymnosperm Platycladus orientalis and the angiosperm Styphnolobium japonicum, have been planted in China for thousands of years. Tree age thus varies widely from young to ancient trees. Foliar endophytes of P. orientalis and S. japonicum were surveyed in this exploratory study that was based on isolation into culture and sequencing of fungi from trees varying in age from 10 to 5000 years (P. orientalis) and from 10 to 1700 years (S. japonicum). Sequenced endophytes of P. orientalis and S. japonicum belonged to 24 and 16 fungal genera, respectively. Principal components analysis showed that 14 components were necessary to explain 90% of the variance in endophyte community structure in P. orientalis. In S. japonicum eight components were needed for 90%. It is against that backdrop of complex etiology in community structuring, that the relative frequencies (abundances) of 17 of the 24 endophytes from P. orientalis and 9 of the 16 from S. japonicum were significantly correlated with tree age. There were two major trends. Abundant fungal genera [Fusarium + Alternaria = 74.57% (P. orientalis) and 81.24% (S. japonicum)] tended to decline linearly with tree age. Most of the rare fungal genera, in contrast, increased in relative abundance linearly with tree age. Diversity (H′) and richness (Margalef) of endophyte communities in foliage thus increased as the trees aged. Relative abundances of pathogenic endophytes, or latent pathogens, (Pestalotiopsis funerea and Amyloporia subxantha in P. orientalis; Collectotrichum gloeosporioides and Botryosphaeria dothidea in S. japonicum) also increased linearly as the trees aged.Since leaf age does not vary with tree age in the deciduous S. japonicum, nor in the evergreen P. orientalis, ‘tree age’ currently lacks a mechanistic explanation for its apparent importance among common foliar endophytes.