Fungal endophytes limit pathogen damage in a tropical tree

Abstract

Every plant species examined to date harbors endophytic fungi within its asymptomatic aerial tissues, such that endophytes represent a ubiquitous, yet cryptic, component of terrestrial plant communities. Fungal endophytes associated with leaves of woody angiosperms are especially diverse; yet, fundamental aspects of their interactions with hosts are unknown. In contrast to the relatively species-poor endophytes that are vertically transmitted and act as defensive mutualists of some temperate grasses, the diverse, horizontally transmitted endophytes of woody angiosperms are thought to contribute little to host defense. Here, we document high diversity, spatial structure, and host affinity among foliar endophytes associated with a tropical tree (Theobroma cacao, Malvaceae) across lowland Panama. We then show that inoculation of endophyte-free leaves with endophytes isolated frequently from naturally infected, asymptomatic hosts significantly decreases both leaf necrosis and leaf mortality when T. cacao seedlings are challenged with a major pathogen (Phytophthora sp.). In contrast to reports of fungal inoculation inducing systemic defense, we found that protection was primarily localized to endophyte-infected tissues. Further, endophyte-mediated protection was greater in mature leaves, which bear less intrinsic defense against fungal pathogens than do young leaves. In vitro studies suggest that host affinity is mediated by leaf chemistry, and that protection may be mediated by direct interactions of endophytes with foliar pathogens. Together, these data demonstrate the capacity of diverse, horizontally transmitted endophytes of woody angiosperms to play an important but previously unappreciated role in host defense.

Fig. 1.

Fungal endophytes associated with leaves of T. cacao are horizontally transmitted and accumulate over leaf lifetimes. Cultivable endophytes were not found in surface-sterilized seeds (data from ref. 24) and occurred in <1% of tissue segments of mature leaves of seedlings (100 days old) raised under sterile conditions. Under field conditions, proportions of leaf segments (each 2mm²) containing endophytes increased with leaf age (mean ± SE; data from BT, ND, PNS, and BCI; n = 3 leaves per age class per site).

Fig. 2.

Endophytic fungi associated with T. cacao demonstrate spatial structure. Similarity of endophyte assemblages, defined by the abundance-based MH for endophyte taxa occurring in more than one leaf, decreased as a function of distance between hosts. Points represent MH obtained in 10 pairwise comparisons of endophyte assemblages associated with T. cacao in each of five sites (n = 9 leaves per tree, 3 trees per site; P < 0.0001; JI data are congruent and are not shown).

Fig. 3.

Field surveys indicate host affinity among tropical endophytes. Endophyte assemblages associated with three host species at one site are less similar to one another than are endophytes of conspecific hosts at that site, and conspecific hosts at three sites separated by 20–60 km (P = 0.0356). MH for endophytes associated with T. cacao, Ouratea lucens, and Heisteria concinna at BCI (mean based on nine pairwise comparisons between individuals of each host species chosen randomly and without replacement) was significantly less than similarity among three individuals of T. cacao within each of three sites (BCI, PNS, and ND; n = 9 randomized pairwise comparisons), and among three sites (n = 9 randomized pairwise comparisons). Differing superscripts denote significant differences based on a posteriori Tukey-Kramer HSD tests (α = 0.05).

Fig. 4.

Endophyte colonization reduces leaf mortality due to a foliar pathogen in seedlings of T. cacao. No mortality occurred among control leaves lacking endophytes and the pathogen (E–P–), nor among leaves inoculated only with endophytes (E+P–). In the presence of the pathogen, mortality occurred 2.8 times more frequently among leaves lacking endophytes (E–P+) than among leaves with endophytes (E+P+) (P = 0.0317).

Fig. 5.

Fungal endophytes reduce leaf area lost to a foliar pathogen for young and mature leaves of T. cacao, and the scale of beneficial effects by endophytes is specific to leaf age. For both young and mature leaves, inoculation with endophytes was associated with a decrease in mean proportion of leaf area damaged by Phytophthora (P = 0.0130, P = 0.0241). For young foliage, E–P+ leaves lost ≈3.5% more leaf area than did E+P+ leaves. Mature leaves lacking endophytes lost ≈7.1% more leaf area than did mature leaves with endophytes.