Isolating a functionally relevant guild of fungi from the root microbiome of Populus

Abstract

Plant roots interact with a bewilderingly complex community of microbes, including rootassociated fungi that are essential for maintaining plant health. To improve understanding of the diversity of fungi in the rhizobiome of Populus deltoides, P. trichocarpa and co-occuring plant hosts Quercus alba and Pinus taeda, we conducted field and greenhouse studies and sampled, isolated, and characterized the diversity of culturable root-associated fungi on these hosts. Using both general and selective isolation media we obtained more than 1800 fungal isolates from individual surface sterilized root tips. Sequences from the ITS and/or D1-D2 regions of the LSU rDNA were obtained from 1042 of the >1800 pure culture isolates and were compared to accessions in the NCBI nucleotide database and analyzed through phylogenetics for preliminary taxonomic identification. Sequences from these isolates were also compared to 454 sequence datasets obtained directly from the Populus rhizosphere. Although most of the ectomycorrhizal taxa known to associate with Populus evaded isolation, many of the abundant sequence types from rhizosphere and endosphere 454 datasets were isolated, including novel species belonging to the Atractiellales. Isolation and identification of key endorrhizal fungi will enable more targeted study of plant-fungal interactions. Genome sequencing is currently underway for a subset of our culture library with the aim of understanding the mechanisms involved in host-endophyte establishment and function. This diverse culture library of fungal root associates is a valuable resource for metagenomic research, experimentation and further studies on plant-fungal interactions.

Facultative root‐colonizing fungi dominate endophytic assemblages in roots of nonmycorrhizal Microthlaspi species

“…The match between results from both methods is a debated topic (Porras‐Alfaro & Bayman, ). Our result agree with recent studies (Bonito et al ., ; Siddique et al ., ) in describing a core set of endophytes detectable via both methods likely representing the dominant taxa, plus a large diversity of OTUs exclusively detected by direct sequencing. It can be argued that the latter represent fungi with particular nutritional requirements or low saprotrophic potential, such as biotrophs or fungi linked to niches constructed by other microbes (Buser et al ., ).…”

“…Other common nonmycorrhizal root-colonizing fungi are also cultivable, and many are frequent saprotrophs in substrates other than roots, such as soil or plant debris (Rodriguez et al, 2009). So far, little is known about the proportion of microorganisms in plant microbiomes that are easily cultivable vs uncultivable using standard approaches (Porras-Alfaro & Bayman, 2011), although recent studies have begun to tackle this question (Oono et al, 2015;Bonito et al, 2016;Siddique et al, 2017). It also remains unknown whether the distribution of both groups is affected differently by environmental and geographical factors.…”

Isolation and characterization of phosphofungi, and screening of their plant growth-promoting activities

“…The rhizosphere soil was collected from roots by dipping and gentle shaking in sterile distilled water under aseptic conditions, and mixed on a table concentrator for 30 min. The resultant soil solution was then serially diluted up to 10 −6 -fold, and the respective dilutions were plated on Martin agar medium supplemented with 30 μg/mL streptomycin, and incubated at 28 ± 2 °C for 2–3 days (Bonito et al 2016; Mehta and Nautiyal 2001). Colonies showing the predominant morphology were selected at random from each plate; isolates showing various colony morphologies were purified and screened for their phosphate-solubilizing ability.…”

Time to re‐think fungal ecology? Fungal ecological niches are often prejudged

“…Recurrent transitions from saprotrophy to endophytism may explain the dominance of facultative endophytes in roots, which also behave as soil saprotrophs, e.g. in the Brassicaceae Microthlaspi (Glynou et al ., ) or in Populus (Bonito et al ., ). Noteworthy, among endophytes of aerial tissues, the trajectory from neutral or deleterious to mutualistic endophyte (Fig.…”

“…There are additional barcoding reports that Fusarium spp. can be found as endophytes in various other plants (Bonito et al ., ; Glynou et al ., ). Thus, a major disease in introduced grasses in North America emerged from a common endophyte of native grasses, adding another example to the growing list of evidences that fungi which are reputedly ‘phytopathogenic’ can be recovered as endophytes from healthy plant tissue (Rodriguez et al ., ; Hardoim et al ., ; Almario et al ., ).…”