Host affinity of endophytic fungi and the potential for reciprocal interactions involving host secondary chemistry.

PREMISE: Interactions between fungal endophytes and their host plants present useful systems for identifying important factors affecting assembly of host-associated microbiomes. Here we investigated the role of secondary chemistry in mediating host affinity of asymptomatic foliar endophytic fungi using Psychotria spp. and Theobroma cacao (cacao) as hosts. METHODS: First, we surveyed endophytic communities in Psychotria species in a natural common garden using culture-based methods. Then we compared differences in endophytic community composition with differences in foliar secondary chemistry in the same host species, determined by liquid chromatography-tandem mass spectrometry. Finally, we tested how inoculation with live and heat-killed endophytes affected the cacao chemical profile. RESULTS: Despite sharing a common environment and source pool for endophyte spores, different Psychotria host species harbored strikingly different endophytic communities that reflected intrinsic differences in their leaf chemical profiles. In T. cacao, inoculation with live and heat-killed endophytes produced distinct cacao chemical profiles not found in uninoculated plants or pure fungal cultures, suggesting that endophytes, like pathogens, induce changes in secondary chemical profiles of their host plant. CONCLUSIONS: Collectively our results suggest at least two potential processes: (1) Plant secondary chemistry influences assembly and composition of fungal endophytic communities, and (2) host colonization by endophytes subsequently induces changes in the host chemical landscape. We propose a series of testable predictions based on the possibility that reciprocal chemical interactions are a general property of plant-endophyte interactions.

Colletotrichum gloeosporioides s.l. associated with Theobroma cacao and other plants in Panama: multilocus phylogenies distinguish host-associated pathogens from asymptomatic endophytes.

Colletotrichum interacts with numerous plant species overtly as symptomatic pathogens and cryptically as asymptomatic endophytes. It is not known whether these contrasting ecological modes are optional strategies expressed by individual Colletotrichum species or whether a species' ecology is explicitly pathogenic or endophytic. We explored this question by inferring relationships among 77 C. gloeosporioides s.l. strains isolated from asymptomatic leaves and from anthracnose lesions on leaves and fruits of Theobroma cacao (cacao) and other plants from Panamá. ITS and 5'-tef1 were used to assess diversity and to delineate operational taxonomic units for multilocus phylogenetic analysis. The ITS and 5'-tef1 screens concordantly resolved four strongly supported lineages, clades A-D: Clade A includes the ex type of C. gloeosporioides, clade B includes the ex type ITS sequence of C. boninense, and clades C and D are unidentified. The ITS yielded limited resolution and support within all clades, in particular the C. gloeosporioides clade (A), the focal lineage dealt with in this study. In contrast the 5'-tef1 screen differentiated nine distinctive haplotype subgroups within the C. gloeosporioides clade that were concordant with phylogenetic terminals resolved in a five-locus nuclear phylogeny. Among these were two phylogenetic species associated with symptomatic infections specific to either cacao or mango and five phylogenetic species isolated principally as asymptomatic infections from cacao and other plant hosts. We formally describe two new species, C. tropicale and C. ignotum, that are frequent asymptomatic associates of cacao and other Neotropical plant species, and epitypify C. theobromicola, which is associated with foliar and fruit anthracnose lesions of cacao. Asymptomatic Colletotrichum strains isolated from cacao plants grown in China included six distinct C. gloeosporioides clade taxa, only one of which is known to occur in the Neotropics.

Two fungal symbioses collide: endophytic fungi are not welcome in leaf-cutting ant gardens.

Interactions among the component members of different symbioses are not well studied. For example, leaf-cutting ants maintain an obligate symbiosis with their fungal garden, while the leaf material they provide to their garden is usually filled with endophytic fungi. The ants and their cultivar may interact with hundreds of endophytic fungal species, yet little is known about these interactions. Experimental manipulations showed that (i) ants spend more time cutting leaves from a tropical vine, Merremia umbellata, with high versus low endophyte densities, (ii) ants reduce the amount of endophytic fungi in leaves before planting them in their gardens, (iii) the ants' fungal cultivar inhibits the growth of most endophytes tested. Moreover, the inhibition by the ants' cultivar was relatively greater for more rapidly growing endophyte strains that could potentially out-compete or overtake the garden. Our results suggest that endophytes are not welcome in the garden, and that the ants and their cultivar combine ant hygiene behaviour with fungal inhibition to reduce endophyte activity in the nest.

Endomelanconiopsis, a new anamorph genus in the Botryosphaeriaceae.

A new lineage is discovered within the Botryosphaeriaceae (Ascomycetes, Dothideomycetes, incertae sedis). Consistent with current practice of providing generic names for independent lineages, this lineage is described as Endomelanconiopsis gen. nov., with the anamorphic species E. endophytica sp. nov. and E. microspora comb. nov. (= Endomelanconium microsporum). Endomelanconiopsis is characterized by eustromatic conidiomata and holoblastically produced, brown, nonapiculate, unicellular conidia, each with a longitudinal germ slit. Phylogenetic analysis of partial sequences of LSU, ITS and translation elongation factor 1 alpha (tef1) indicate that E. endophytica is sister of E. microspora and that they are nested within the Botryosphaeriaceae. However because there is no support for the "backbone" of the Botryosphaeriacae we are not able to see the interrelationships among the many genera in the family. Neither species is known to have a teleomorph. Endomelanconiopsis differs from Endomelanconium because conidia of the type species of Endomelanconium, E. pini, are papillate at the base, conidiogenous cells proliferate sympodially and the pycnidial wall is thinner; we postulate that the teleomorph of E. pini as yet unknown is an inoperculate discomycete. Endomelanconiopsis endophytica was isolated as an endophyte from healthy leaves of Theobroma cacao (cacao, Malvaceae) and Heisteria concinna (Erythroplaceae) in Panama. Endomelanconiopsis microspora was isolated from soil in Europe.

Plants use macronutrients accumulated in leaf-cutting ant nests.

Leaf-cutting ants (Atta spp.) are known for their extensive defoliation in neo-tropical forests and savannahs. Debate about the costs and benefits of their activities has been largely dominated by their detrimental effects on agriculture and agroforestry. However, the large accumulation of nutrients and changes in soil properties near their nests might benefit plants growing near them. Here, we test whether trees use nutrients that accumulate in debris piles near, or refuse chambers within, leaf-cutting ant nests. At two tropical sites (a moist tropical forest site in Panama and a savannah site in Brazil), we fed leaves labelled with the stable isotope 15N to two species of leaf-cutting ants (Atta colombica and Atta laevigata) and traced the stable isotope label in plants surrounding the two nests. Thus, we show that plants in both sites access resources associated with Atta nests. In addition, leaf tissue of trees near the nests labelled with 15N had significantly higher calcium concentrations than those of distal, unlabelled conspecifics. It has been documented that calcium is a limiting macronutrient in tropical forests and savannahs. Atta may thus play an important ecological role through their long-distance transport, redistribution and concentration of critical macronutrients.

Fungal endophytes limit pathogen damage in a tropical tree.

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.