Endophytes are microorganisms that live within plant tissues without causing symptoms of disease. They are important components of plant microbiomes. Endophytes interact with, and overlap in function with, other core microbial groups that colonize plant tissues, e.g., mycorrhizal fungi, pathogens, epiphytes, and saprotrophs. Some fungal endophytes affect plant growth and plant responses to pathogens, herbivores, and environmental change; others produce useful or interesting secondary metabolites. Here, we focus on new techniques and approaches that can provide an integrative understanding of the role of fungal endophytes in the plant microbiome. Clavicipitaceous endophytes of grasses are not considered because they have unique properties distinct from other endophytes. Hidden from view and often overlooked, endophytes are emerging as their diversity, importance for plant growth and survival, and interactions with other organisms are revealed.

All orchids have an obligate relationship with mycorrhizal symbionts. Most orchid mycorrhizal fungi are classified in the form-genus Rhizoctonia. This group includes anamorphs of Tulasnella, Ceratobasidium, and Thanatephorus. Rhizoctonia can be classified according to the number of nuclei in young cells (multi-, bi-, and uninucleate). From nine Puerto Rican orchids we isolated 108 Rhizoctonia-like fungi. Our isolates were either bi- or uninucleate, the first report of uninucleate Rhizoctonia-like fungi as orchid endophytes. We sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 26 isolates and identified four fungal lineages, all related to Ceratobasidium spp. from temperate regions. Most orchid species hosted more than one lineage, demonstrating considerable variation in mycorrhizal associations even among related orchid species. The uninucleate condition was not a good phylogenetic character in mycorrhizal fungi from Puerto Rico. All four lineages were represented by fungi from Tolumnia variegata, but only one lineage included fungi from Ionopsis utricularioides. Tropical epiphytic orchids appear to vary in degree of specificity in their mycorrhizal interactions more than previously thought.

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Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30°C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 g/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 g/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.

Orchids parasitize their mycorrhizal fungi and are dependent on them for seed germination. Controversy reigns over how specific the mycorrhizal association is in tropical species. Although there is little experimental evidence to support any viewpoint, some variation is known to exist. We compared mycorrhizal specificity and performance in two phylogenetically related epiphytic orchids from Puerto Rico, Tolumnia variegata and Ionopsis utricularioides (Oncidiinae) by integrating two techniques: phylogenetic analysis of mycorrhizal fungi based on nuclear ribosomal internal transcribed spacer (ITS) sequences, and symbiotic seed germination experiments. Most of the mycorrhizal isolates from T. variegata fell into four different clades of Ceratobasidium, while most of those from I. utricularioides were restricted to a single clade of the same genus. Seeds of T. variegata germinated equally well with fungi from both T. variegata and I. utricularioides, but seeds of I. utricularioides germinated significantly better with its own isolates. Seeds of I. utricularioides germinated and developed faster than those of T. variegata. Both the molecular phylogeny and the seed germination experiments showed that T. variegata is a generalist in its association with fungal symbionts. In contrast, I. utricularioides is more specialized and more effective at exploiting a specific fungal clade. Our data are consistent with the theoretical trade-offs between specialized and generalized interactions.

Plants harbor diverse communities of fungi and other microorganisms. Fungi are known to occur both on plant surfaces (epiphytes) and inside plant tissues (endophytes), but the two communities have rarely been compared. We compared epiphytic and endophytic fungal communities associated with leaves of coffee (Coffea arabica) in Puerto Rico. We asked whether the dominant fungi are the same in both communities, whether endophyte and epiphyte communities are equally diverse, and whether epiphytes and endophytes exhibit similar patterns of spatial heterogeneity among sites. Leaves of naturalized coffee plants were collected from six sites in Puerto Rico. Epiphytic and endophytic fungi were isolated by placing leaf pieces on potato dextrose agar without and with surface sterilization, respectively. A total of 821 colonies were isolated and grouped into 131 morphospecies. The taxonomic affinities of the four most common nonsporulating fungi were determined by sequencing the nuclear ribosomal internal transcribed spacer (ITS) region: two grouped with Xylaria and one each with Botryosphaeria and Guignardia. Of the most common genera, Pestalotia and Botryosphaeria were significantly more common as epiphytes; Colletotrichum, Xylaria, and Guignardia were significantly more common as endophytes. Suprisingly, more morphospecies occurred as endophytes than as epiphytes. Differences among sites in number of fungi per plant were significant. Thus epiphytic and endophytic communities differed greatly on a single leaf, despite living only millimeters apart, and both communities differed from site to site. Significant correlations between occurrence of fungal morphospecies suggested that fungi may have positive or negative effects on their neighbors. This is the first quantitative comparison of epiphytic and endophytic fungal floras in any plant, and the first to examine endophytic fungi or epiphytic fungi in leaves of coffee, one of the world's most valuable crops.

Aspergillus flavus was isolated from soil from a single Arizona cotton field in 1987, 1988, and 1989. Isolates from infected cotton bolls were collected from the same field in 1988. Isolates were assigned to vegetative compatibility groups via complementation tests between nitrate-nonutilizing mutants. Sixty-one of 105 isolates composed 13 vegetative compatibility groups; the remaining 44 isolates could not be assigned to groups. Forty-three isolates from other fields in Arizona composed 21 groups, 6 of which were also found in the test field. Distribution of vegetative compatibility groups in and outside the field was significantly different, based on a G-test. One vegetative compatibility group included 20% of all isolates from the test field, but was not found elsewhere. It was common in the test field in 1987 and 1988, but was not found in 1989. Boll and soil populations from 1988 were not significantly different. Single infected boll locules and 25-g soil samples often contained A. flavus individuals from more than one group. These results suggest that although many vegetative compatibility groups are widely distributed, a single field may have a unique population profile that changes significantly from year to year. Key words: Aspergillus flavus, vegetative compatibility, Nit−, imperfect fungi.

Ochratoxins have been overshadowed by better-known mycotoxins, but they are gaining importance. Here we consider ochratoxins in the context of aflatoxins, which are better understood than ochratoxins on many levels. We review recent work on taxonomic distribution, contamination of commodities, biosynthesis, toxicity and regulatory aspects of ochratoxins. We focus on ochratoxins in coffee, since coffee is becoming a key commodity in ochratoxin research and regulation.