Luis C. Mejía

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.

ECOLOGICAL IMPLICATIONS OF ANTI-PATHOGEN EFFECTS OF TROPICAL FUNGAL ENDOPHYTES AND MYCORRHIZAE

We discuss studies of foliar endophytic fungi (FEF) and arbuscular mycorrhizal fungi (AMF) associated with Theobroma cacao in Panama. Direct, experimentally controlled comparisons of endophyte free (E-) and endophyte containing (E+) plant tissues in T. cacao show that foliar endophytes (FEF) that commonly occur in healthy host leaves enhance host defenses against foliar damage due to the pathogen (Phytophthora palmivora). Similarly, root inoculations with commonly occurring AMF also reduce foliar damage due to the same pathogen. These results suggest that endophytic fungi can play a potentially important mutualistic role by augmenting host defensive responses against pathogens. There are two broad classes of potential mechanisms by which endophytes could contribute to host protection: (1) inducing or increasing the expression of intrinsic host defense mechanisms and (2) providing additional sources of defense, extrinsic to those of the host (e.g., endophyte-based chemical antibiosis). The degree to which either of these mechanisms predominates holds distinct consequences for the evolutionary ecology of host-endophyte-pathogen relationships. More generally, the growing recognition that plants are composed of a mosaic of plant and fungal tissues holds a series of implications for the study of plant defense, physiology, and genetics.

Leaf-inhabiting genera of the Gnomoniaceae, Diaporthales

The Gnomoniaceae are characterised by ascomata that are generally immersed, solitary, without a stroma, or aggregated with a rudimentary stroma, in herbaceous plant material especially in leaves, twigs or stems, but also in bark or wood. The ascomata are black, soft-textured, thin-walled, and pseudoparenchymatous with one or more central or eccentric necks. The asci usually have a distinct apical ring. The Gnomoniaceae includes species having ascospores that are small, mostly less than 25 μm long, although some are longer, and range in septation from non-septate to one-septate, rarely multi-septate. Molecular studies of the Gnomoniaceae suggest that the traditional classification of genera based on characteristics of the ascomata such as position of the neck and ascospores such as septation have resulted in genera that are not monophyletic. In this paper the concepts of the leaf-inhabiting genera in the Gnomoniaceae are reevaluated using multiple genes, specifically nrLSU, translation elongation factor 1-alpha (tef1-α), and RNA polymerase II second largest subunit (rpb2) for 64 isolates. ITS sequences were generated for 322 isolates. Six genera of leaf-inhabiting Gnomoniaceae are defined based on placement of their type species within the multigene phylogeny. The new monotypic genus Ambarignomonia is established for an unusual species, A. petiolorum. A key to 59 species of leaf-inhabiting Gnomoniaceae is presented and 22 species of Gnomoniaceae are described and illustrated.

Pervasive effects of a dominant foliar endophytic fungus on host genetic and phenotypic expression in a tropical tree

It is increasingly recognized that macro-organisms (corals, insects, plants, vertebrates) consist of both host tissues and multiple microbial symbionts that play essential roles in their hosts ecological and evolutionary success. Consequently, identifying benefits and costs of symbioses, as well as mechanisms underlying them are research priorities. All plants surveyed under natural conditions harbor foliar endophytic fungi (FEF) in their leaf tissues, often at high densities. Despite producing no visible effects on their hosts, experiments have nonetheless shown that FEF reduce pathogen and herbivore damage. Here, combining results from three genomic, and two physiological experiments, we demonstrate pervasive genetic and phenotypic effects of the apparently asymptomatic endophytes on their hosts. Specifically, inoculation of endophyte-free (E−) Theobroma cacao leaves with Colletotrichum tropicale (E+), the dominant FEF species in healthy T. cacao, induces consistent changes in the expression of hundreds of host genes, including many with known defensive functions. Further, E+ plants exhibited increased lignin and cellulose content, reduced maximum rates of photosynthesis (Amax), and enrichment of nitrogen-15 and carbon-13 isotopes. These phenotypic changes observed in E+ plants correspond to changes in expression of specific functional genes in related pathways. Moreover, a cacao gene (Tc00g04254) highly up-regulated by C. tropicale also confers resistance to pathogen damage in the absence of endophytes or their products in host tissues. Thus, the benefits of increased pathogen resistance in E+ plants are derived in part from up-regulation of intrinsic host defense responses, and appear to be offset by potential costs including reduced photosynthesis, altered host nitrogen metabolism, and endophyte heterotrophy of host tissues. Similar effects are likely in most plant-endophyte interactions, and should be recognized in the design and interpretation of genetic and phenotypic studies of plants.

Phylogenetic placement and taxonomic review of the genus Cryptosporella and its synonyms Ophiovalsa and Winterella (Gnomoniaceae, Diaporthales).

The type species of Cryptosporella, C. hypodermia, and Ophiovalsa, O. suffusa, as well as closely related species were studied using morphological, cultural, and DNA sequence characteristics. DNA sequence data from three different loci (ITS, LSU, and RPB2) suggest that C. hypodermia and O. suffusa are congeneric within the Gnomoniaceae (Diaporthales). This result is supported by similarities in perithecial, ascal and ascospore morphology, and lifestyles characterized as initially endophytic, becoming saprobic as plant tissues die. Furthermore, both type species produce Disculina anamorphs. A review of the literature indicates that the generic name Cryptosporella has priority over Ophiovalsa and its synonym Winterella sensu Reid & Booth (1987). A redescription of the genus Cryptosporella is included, as well as a description of C. hypodermia, C. suffusa, the type species of Ophiovalsa, a brief account of the other seven species accepted in Cryptosporella, and a key to species of Cryptosporella. Eight new combinations are established: C. alnicola (Fr.) L.C. Mejía & Castleb., comb. nov.; C. betulae (Tul. & C. Tul.) L.C. Mejía & Castleb., comb. nov.; C. confusa (Reid & Booth) L.C. Mejía & Castleb., comb. nov.; C. corylina (Tul. & C. Tul.) L.C. Mejía & Castleb., comb. nov.; C. femoralis (Peck) L.C. Mejía & Castleb., comb. nov.; C. suffusa (Fr.) L.C. Mejía & Castleb., comb. nov.; C. tiliae (Tul. & C. Tul.) L.C. Mejía & Castleb., comb. nov.; and C. wehmeyeriana (Reid & Booth) L.C. Mejía & Castleb., comb. nov.

A systematic account of the genus Plagiostoma (Gnomoniaceae, Diaporthales) based on morphology, host-associations, and a four-gene phylogeny.

Members of the genus Plagiostoma inhabit leaves, stems, twigs, and branches of woody and herbaceous plants predominantly in the temperate Northern Hemisphere. An account of all known species of Plagiostoma including Cryptodiaporthe is presented based on analyses of morphological, cultural, and DNA sequence data. Multigene phylogenetic analyses of DNA sequences from four genes (β-tubulin, ITS, rpb2, and tef1-α) revealed eight previously undescribed phylogenetic species and an association between a clade composed of 11 species of Plagiostoma and the host family Salicaceae. In this paper these eight new species of Plagiostoma are described, four species are redescribed, and four new combinations are proposed. A key to the 25 accepted species of Plagiostoma based on host, shape, and size of perithecia, perithecial arrangement in the host, and microscopic characteristics of the asci and ascospores is provided. Disposition of additional names in Cryptodiaporthe and Plagiostoma is also discussed.

Recommendations of generic names in Diaporthales competing for protection or use.

In advancing to one name for fungi, this paper treats generic names competing for use in the order Diaporthales (Ascomycota, Sordariomycetes) and makes a recommendation for the use or protection of one generic name among synonymous names that may be either sexually or asexually typified. A table is presented that summarizes these recommendations. Among the genera most commonly encountered in this order, Cytospora is recommended over Valsa and Diaporthe over Phomopsis. New combinations are introduced for the oldest epithet of important species in the recommended genus. These include Amphiporthe tiliae, Coryneum lanciforme, Cytospora brevispora, C. ceratosperma, C. cinereostroma, C. eugeniae, C. fallax, C. myrtagena, Diaporthe amaranthophila, D. annonacearum, D. bougainvilleicola, D. caricae-papayae, D. cocoina, D. cucurbitae, D. juniperivora, D. leptostromiformis, D. pterophila, D. theae, D. vitimegaspora, Mastigosporella georgiana, Pilidiella angustispora, P. calamicola, P. pseudogranati, P. stromatica, and P. terminaliae.

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.

New species, phylogeny, host-associations and geographic distribution of genus Cryptosporella (Gnomoniaceae, Diaporthales)

The phylogeny of Cryptosporella is revised to include recently discovered species. Eight species new to science are described and two new combinations are proposed, raising the total number of species accepted in Cryptosporella to 19. The species delimitation and phylogeny for Cryptosporella are determined based on analyses of DNA sequences from three genes (β-tubulin, ITS and tef1-α), comparative morphology of sexual structures on their host substrate, and host associations. The inferred phylogeny suggests that Cryptosporella has speciated primarily on Betulaceae with 16 species occurring on hosts in that plant family. The host range of most species seems to be narrow with nine species reported from a single host species or subspecies and seven species occurring on plants within a single host genus. A key to species is provided. The known distribution of Cryptosporella is expanded to mountain cloud forests of the provinces of Chiriquí in Panama and Tucumán in Argentina.

Imaging mass spectrometry and MS/MS molecular networking reveals chemical interactions among cuticular bacteria and pathogenic fungi associated with fungus-growing ants

The fungus-growing ant-microbe symbiosis is an ideal system to study chemistry-based microbial interactions due to the wealth of microbial interactions described, and the lack of information on the molecules involved therein. In this study, we employed a combination of MALDI imaging mass spectrometry (MALDI-IMS) and MS/MS molecular networking to study chemistry-based microbial interactions in this system. MALDI IMS was used to visualize the distribution of antimicrobials at the inhibition zone between bacteria associated to the ant Acromyrmex echinatior and the fungal pathogen Escovopsis sp. MS/MS molecular networking was used for the dereplication of compounds found at the inhibition zones. We identified the antibiotics actinomycins D, X2 and X0β, produced by the bacterium Streptomyces CBR38; and the macrolides elaiophylin, efomycin A and efomycin G, produced by the bacterium Streptomyces CBR53.These metabolites were found at the inhibition zones using MALDI IMS and were identified using MS/MS molecular networking. Additionally, three shearinines D, F, and J produced by the fungal pathogen Escovopsis TZ49 were detected. This is the first report of elaiophylins, actinomycin X0β and shearinines in the fungus-growing ant symbiotic system. These results suggest a secondary prophylactic use of these antibiotics by A. echinatior because of their permanent production by the bacteria.