Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds.

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

The Role of Non-Mycorrhizal Fungi in Germination of the Mycoheterotrophic Orchid Pogoniopsis schenckii Cogn.

Endophytic fungi are those that inhabit within organs and tissues without causing damage, while mycorrhizal fungi develop hyphal complexes called pelotons within cortical cells of orchid roots. Although abundant and frequent in all plant organs, the role of endophytic fungi has been neglected in relation to orchid's early development. Pogoniopsis schenckii Cogn. is an aclorophyllated and mycoheterotrophic (MH) orchid. This study aimed at i) investigating the endophytic fungal community in organs of P. schenckii and its mycorrhizal fungi associated; ii) evaluating the ability of isolated fungus in the in vitro germination of the seeds of the species, and iii) describing the development of P. schenckii protocorm, analyzing the ultrastructure of the infected cells. Six genera of fungi were isolated and identified through the partial sequencing of the internal transcribed spacer region, all belonging to the phylum Ascomycota. Also, Tulasnellaceae was identified through uncultured technique as potentially mycorrhizal in this MH orchid. Some isolates of the genera Trichoderma, Fusarium, and especially Clonostachys presented germinative potential on P. schenckii seeds, causing rupture of the external tegument. The protocorms showed complete absence of peloton formation, but fungal hyphae were clearly observed within living cells. This is the first report of germination of a MH and aclorophyllated orchid species stimulated by the presence of non-mycorrhizal endophytic fungi isolated from fruits and roots of the same species.