Methods used for the study of endophytic fungi: a review on methodologies and challenges, and associated tips

Abstract

Endophytic fungi are microorganisms that colonize the interior of plant tissues (e.g. leaves, seeds, stem, trunk, roots, fruits, flowers) in intracellular and/or extracellular spaces without causing symptoms of disease in host plants. These microorganisms have been isolated from plant species in a wide variety of habitats worldwide, and it is estimated that all terrestrial plants are colonized by one or more species of endophytic fungus. In addition, these microorganisms have been drawing the attention of researchers because of their ability to synthesize a wide range of bioactive molecules with potential for applications in agriculture, medicine and biotechnology. However, several obstacles come up when studying the diversity and chemical potential of endophytic fungi. For example, the usage of an inappropriate surface disinfection method for plant tissue may not eliminate the epiphytic microbiota or may end up interfering with the endophytic mycobiota, which consequently generates erroneous results. Moreover, the composition of the culture medium and the culture conditions can favor the growth of certain species and inhibit others, which generates underestimated results. Other inconsistencies can arise from the fungus misidentification and consequent exploration of its chemical potential. Based on the methodological biases that may occur at all stages of studies dealing with endophytic fungi, the objective of this review is to discuss the main methods employed in these studies as well as highlight the challenges derived from the different approaches. We also report associated tips to help future studies on endophytic fungi as a contribution.

Keywords: Diversity; Endophyte; ITS; Molecular identification; Mycobiome; Symbiosis.

Fig. 1

Flowchart for studying the diversity of endophytic fungi and their potential for producing bioactive compounds

Fig. 2

Biotic and abiotic factors influencing the assembly of the endophytic mycobiota. The community of endophytic fungi within the same plant species is highly dynamic and the richness and diversity of taxa can be influenced and determined by several biotic and abiotic factors. Image generated based on findings by David et al. (2016), Fernandes et al. (2018), Chi et al. (2019), Yao et al. (2019) and Wu et al. (2020)

Fig. 3

Leaf sampling strategy to study endophytic fungi. a The plant microbiome is subject to a mosaic of resource availability and environmental conditions that tend to vary on a microenvironmental scale, and are influenced by biotic and abiotic factors. Therefore, these factors should be considered during the sampling process. The red square refers to the specimen to be sampled; the blue and red arrows indicate the influence of biotic factors (inter- and intra-species interactions) that, for example, result in a lower incidence of sunlight in a specific portion of the tree crown to be sampled; the yellow arrows indicate the influence of abiotic factors (e.g. incidence of sunlight, availability of resources). b Experimental design of selection of individuals to be sampled to obtain a representative sample. c Leaf tissue sampling scheme of the lower, middle and upper leaves. In d, the top view of the sampled specimen is shown, and the black arrows indicate that the sampling should be carried out in different regions. In e, a tissue fragmentation scheme is presented to obtain a representative sample for the study of endophytic fungi. Image designed from the collection strategy by Wu et al. (2020) and Kluting et al. (2019), Denney et al. (2020), and Hurtado-McCormick et al. (2021) on population variations in microenvironments. Created with BioRender.com

Fig. 4

Ribosomal RNA nuclear genes and ITS regions. The ITS region includes the ITS1 and ITS2 sequences separated by the 5.8S rRNA gene, which are located between the 18S rRNA (SSU) and 28S rRNA (LSU) genes (Blackwell et al. 2011). The small arrows observed from the ITS region magnification demonstrate the annealing position of primers used for PCR amplification from this region (White et al. ; Gardes and Hester 1993; O'Donnell, ; Gerrits van den Ende and de Hoog, ; Beguin et al. 2012)

Fig. 5

ITS2 secondary structures of C. coccodes with different life styles showing helixes emerging from a central bulge, A pathogenic, B endophytic and C author’s isolate (MN128230.1). Motifs in the structures are indicated by arrow. Retrieved from Talukdar et al. (2021)