Diversity of Mycorrhizal Fungi in Temperate Orchid Species: Comparison of Culture-Dependent and Culture-Independent Methods

Abstract

Many orchid species are endangered due to anthropogenic pressures such as habitat destruction and overharvesting, meanwhile, all orchids rely on orchid mycorrhizal fungi (OMF) for seed germination and seedling growth. Therefore, a better understanding of this intimate association is crucial for orchid conservation. Isolation and identification of OMF remain challenging as many fungi are unculturable. In our study, we tested the efficiency of both culture-dependent and culture-independent methods to describe OMF diversity in multiple temperate orchids and assessed any phylogenetic patterns in cultivability. The culture-dependent method involved the cultivation and identification of single pelotons (intracellular hyphal coils), while the culture-independent method used next-generation sequencing (NGS) to identify root-associated fungal communities. We found that most orchid species were associated with multiple fungi, and the orchid host had a greater impact than locality on the variability in fungal communities. The culture-independent method revealed greater fungal diversity than the culture-dependent one, but despite the lower detection, the isolated fungal strains were the most abundant OMF in adult roots. Additionally, the abundance of NGS reads of cultured OTUs was correlated with the extent of mycorrhizal root colonization in orchid plants. Finally, this limited-scale study tentatively suggests that the cultivability character of OMF may be randomly distributed along the phylogenetic trees of the rhizoctonian families.

Keywords: Ceratobasidiaceae; Orchidaceae; Serendipitaceae; Tulasnellaceae; culture-independent and -dependent methods; fungal phylogeny; metabarcoding; mycorrhizal fungi.

Figure 1

Rhizoctonian OTU diversity and relative frequencies of reads among the 19 studied orchid species. The numbers above the barplots indicate the mean fungal richness per site and orchid species. Rhizoctonian families are colour-coded: Ceratobasidiaceae (in green), Serendipitaceae (in blue) and Tulasnellaceae (in orange). Orchid species: Anacamptis morio (AM), A. pyramidalis (AP), Dactylorhiza sambucina (DS), D. viridis (DV), Gymnadenia conopsea diploid (GCd), G. conopsea tetraploid (GCt), G. densiflora (GD), Neottia ovata (NO), Neotinea ustulata (NU), Ophrys apifera (OPA), Op. holubyana (OPH), Op. sphegodes subsp. passionis (OPP), Orchis anthropophora (OA), O. mascula (OMA), O. militaris (OMI), O. purpurea (OPU), O. simia (OSI), Platanthera bifolia (PB) and Platanthera spp. (PL).

Figure 2

Distance-based redundance analysis comparing the fungal communities in plants of 11 orchid species that occurred at least in two sites. The circles in the diagram represent plants from different orchid species, while the arrows depict explanatory variables: locality (in blue) and orchid host identity (in black). The sites are represented by colours: French sites FR1 (brown) and FR2 (light green), Czech sites CZ1 (dark blue) and CZ2 (light blue). Orchid species: Anacamptis morio (AM); A. pyramidalis (AP); Dactylorhiza sambucina (DS); Gymnadenia conopsea diploid (GCd); Ophrys sphegodes subsp. passionis (OPP); Orchis mascula (OMA); O. militaris (OMI); O. purpurea (OPU); Neottia ovata (NO); Neotinea ustulata (NU); and Platanthera bifolia (PB).

Figure 3

Diversity and dissimilarity indices in OMF community composition of 11 orchid species occurring at multiple sites. The X-axis represents intersite dissimilarity indices within the same orchid species across different occurrence sites. The Y-axis illustrates interspecies dissimilarity indices between various orchid species coexisting within the same site. Each circle symbolizes a pairwise comparison of fungal dissimilarity indices in the same orchid species at two sites, denoted by the colour of circles, either within or between regions. The size of each circle is proportional to the mean Shannon diversity index of the orchid species at both sites. Orchid species include Anacamptis morio (AM); A. pyramidalis (AP); Dactylorhiza sambucina (DS); Gymnadenia conopsea diploid (GCd); Neottia ovata (NO); Neotinea ustulata (NU); Ophrys sphegodes subsp. passionis (OPP); Orchis mascula (OMA); O. militaris (OMI); O. purpurea (OPU); and Platanthera bifolia (PB).

Figure 4

Phylogenetic trees combining the fungal OTUs obtained by culture-dependent and -independent methods. Rhizoctonian families include Tulasnellaceae (two lineages, clades A (red section) and B (green section)) (A), Ceratobasidiaceae (B) and Sebacinales order (two families, Serendipitaceae (orange section) and Sebacinaceae (blue section)) (C). In the illustration, the symbol of DNA helix refers to the fungal OTUs obtained with NGS method, while the black circle represents the fungal OTUs cultivated from pelotons. Additional information on fungal strains includes details about the orchid host and the site of occurrence, indicated as France (FR) or the Czech Republic (CZ) within the tree. Orchid species include Anacamptis morio; A. pyramidalis; Dactylorhiza sambucina; D. viridis; D. majalis; Gymnadenia conopsea diploid; G. conopsea tetraploid; Neottia ovata; Neotinea ustulata; Ophrys apifera; Op. holubyana; Op. sphegodes subsp. passionis; Orchis mascula; and Platanthera bifolia.

Figure 4

Phylogenetic trees combining the fungal OTUs obtained by culture-dependent and -independent methods. Rhizoctonian families include Tulasnellaceae (two lineages, clades A (red section) and B (green section)) (A), Ceratobasidiaceae (B) and Sebacinales order (two families, Serendipitaceae (orange section) and Sebacinaceae (blue section)) (C). In the illustration, the symbol of DNA helix refers to the fungal OTUs obtained with NGS method, while the black circle represents the fungal OTUs cultivated from pelotons. Additional information on fungal strains includes details about the orchid host and the site of occurrence, indicated as France (FR) or the Czech Republic (CZ) within the tree. Orchid species include Anacamptis morio; A. pyramidalis; Dactylorhiza sambucina; D. viridis; D. majalis; Gymnadenia conopsea diploid; G. conopsea tetraploid; Neottia ovata; Neotinea ustulata; Ophrys apifera; Op. holubyana; Op. sphegodes subsp. passionis; Orchis mascula; and Platanthera bifolia.

Figure 4

Phylogenetic trees combining the fungal OTUs obtained by culture-dependent and -independent methods. Rhizoctonian families include Tulasnellaceae (two lineages, clades A (red section) and B (green section)) (A), Ceratobasidiaceae (B) and Sebacinales order (two families, Serendipitaceae (orange section) and Sebacinaceae (blue section)) (C). In the illustration, the symbol of DNA helix refers to the fungal OTUs obtained with NGS method, while the black circle represents the fungal OTUs cultivated from pelotons. Additional information on fungal strains includes details about the orchid host and the site of occurrence, indicated as France (FR) or the Czech Republic (CZ) within the tree. Orchid species include Anacamptis morio; A. pyramidalis; Dactylorhiza sambucina; D. viridis; D. majalis; Gymnadenia conopsea diploid; G. conopsea tetraploid; Neottia ovata; Neotinea ustulata; Ophrys apifera; Op. holubyana; Op. sphegodes subsp. passionis; Orchis mascula; and Platanthera bifolia.

Figure 5

Rhizoctonia richness of orchid species obtained by culture-dependent and -independent methods. Barplots are ranked according to the number of sites of occurrence (1 to 3 sites). The colours indicate the proportion of OTUs detected using different methods: solely through culture-independent methods (in blue), exclusively through culture-dependent methods (in green) and through both methods (in pink). The numbers within the bars correspond to the count of rhizoctonia OTUs. Orchid species include Anacamptis morio (AM); A. pyramidalis (AP); Dactylorhiza sambucina (DS); D. viridis (DV); Gymnadenia conopsea diploid (GCd); G. conopsea tetraploid (GCt); G. densiflora (GD); Neottia ovata (NO); Neotinea ustulata (NU); Ophrys apifera (OPA); Op. holubyana (OPH); Op. sphegodes subsp. passionis (OPP); Orchis anthropophora (OA); O. mascula (OMA); O. militaris (OMI); O. purpurea (OPU); O. simia (OSI); and Platanthera bifolia (PB); Platanthera spp. (PL).

Figure 6

Mean relative frequencies of sequence reads of rhizoctonian fungal OTUs categorized by their cultivability under laboratory conditions (‘cultured’ or ‘uncultured’). The box plots represent median, upper and lower quartiles with the whiskers showing minimum and maximum values, outliers as circles. Different colours and symbols denote abundant OTUs that failed in cultivation from some orchid hosts: Gymnadenia densiflora (in green); Orchis purpurea (in purple); O. militaris (in orange); O. anthropophora (in yellow); Dactylorhiza sambucina (in blue); and other species (in black). The symbols indicate the fungal family: circle, square and triangle for Tulasnellaceae, Ceratobasidiaceae and Serendipitaceae OTUs, respectively. The symbol (***) indicates a significant difference in the relative frequencies of fungal OTUs between the cultivability status (‘cultured’ versus ‘uncultured’) of the strains, with p values ≤ 0.001 based on ANOVA test.

Figure 7

Correlation between the relative frequencies of sequence reads of the cultivable OTUs and their proportion in corresponding mycorrhizal roots. Each dot represents a cultivable OTU’s isolate detected through both culture-dependent and -independent methods across a total of 108 plants. The circles are proportional to the number of isolates. The blue line corresponds to the linear regression model.