The effects of endophytic fungi of NZ terrestrial orchids: developing methods for conservation

<p>Nearly 40% of New Zealand (NZ) orchid species are of conservation concern, some critically endangered, largely due to habitat loss. In NZ, there are currently no propagation programs for terrestrial orchids all of which rely on symbiotic fungi to provide the nutrients required for germination, and little is known about the specific fungal species that might make this possible. To develop an understanding of the fungal interactions affecting recruitment in the field, a survey of endophytic fungal diversity from the roots of Chiloglottis valida, Microtis unifolia, Pterostylis banksii, Spiranthes novae-zelandiae and Thelymitra longifolia was carried out. The identification of fungi was assisted by obtaining sequences of the ITS rDNA gene marker. Seeds of M. unifolia, P. banksii, S. novae-zelandiae and T. longifolia were inoculated with cultured endophytes that were recovered from the roots of conspecific orchids, and their effect on seed germination evaluated. Seed viability using fluorescein diacetate was assayed on all species prior to all experiments and showed moderate to high viability scores for all species. Recovered endophytes belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The effect of the different endophytes on seed germination was variable, with five inoculants exhibiting a positive response. Three inoculants had a consistent negative effect on seed germination. The distribution of orchid symbiotic mycorrhizae in situ was investigated at Otari-Wilton’s Bush, Wellington, NZ. Mesh seed packets containing seed of M. unifolia and T. longifolia were interred for 150 days, along transects (≤ 1 metre) that originated at adult orchids at three sites, and an additional site with no adult orchids was used as a control. No small-scale patterns were detected; however, germination rates were higher at undisturbed sites. Seed viability was considerably reduced to <2% after five months under the soil suggesting M. unifolia and T. longifolia seeds do not persist in the seed bank beyond one growing season. Sequences of ITS rDNA indicate Tulasnella calospora assists in the germination of M. unifolia at this site. Similarly, Tulasnella calospora promoted germination of the Nationally Vulnerable wetland species S. novae-zelandiae. Pelotons were isolated from the roots of S. novae-zelandiae plants from a wild population from the lower north island and cultured in Petri dishes. Germination of this orchid began after 30 days from inoculation when the pelotons are already observed inside the embryo. Chlorophyllus tissue was observed after c. 80 days of inoculation. The phylogenetic relationship of Asian-Pacific Spiranthes species with New Zealand Spiranthes was also investigated using nuclear (ITS) and chloroplast (trnL-trnF) DNA sequences. Phylogenetic analyses supported the recognition of Spiranthes novae-zelandiae ‘Motutangi’ as a distinct taxonomic unit. It was also found that the Asian-Pacific Spiranthes species are in need of taxonomic revision. Methods used and developed in this thesis study could be used to identify potential orchid symbionts and pathogens, assess suitable potential relocation sites, and propagation of NZ orchids using symbiotic fungi for restoration and conservation purposes.</p>

The effects of endophytic fungi of NZ terrestrial orchids: developing methods for conservation

<p>Nearly 40% of New Zealand (NZ) orchid species are of conservation concern, some critically endangered, largely due to habitat loss. In NZ, there are currently no propagation programs for terrestrial orchids all of which rely on symbiotic fungi to provide the nutrients required for germination, and little is known about the specific fungal species that might make this possible. To develop an understanding of the fungal interactions affecting recruitment in the field, a survey of endophytic fungal diversity from the roots of Chiloglottis valida, Microtis unifolia, Pterostylis banksii, Spiranthes novae-zelandiae and Thelymitra longifolia was carried out. The identification of fungi was assisted by obtaining sequences of the ITS rDNA gene marker. Seeds of M. unifolia, P. banksii, S. novae-zelandiae and T. longifolia were inoculated with cultured endophytes that were recovered from the roots of conspecific orchids, and their effect on seed germination evaluated. Seed viability using fluorescein diacetate was assayed on all species prior to all experiments and showed moderate to high viability scores for all species. Recovered endophytes belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The effect of the different endophytes on seed germination was variable, with five inoculants exhibiting a positive response. Three inoculants had a consistent negative effect on seed germination. The distribution of orchid symbiotic mycorrhizae in situ was investigated at Otari-Wilton’s Bush, Wellington, NZ. Mesh seed packets containing seed of M. unifolia and T. longifolia were interred for 150 days, along transects (≤ 1 metre) that originated at adult orchids at three sites, and an additional site with no adult orchids was used as a control. No small-scale patterns were detected; however, germination rates were higher at undisturbed sites. Seed viability was considerably reduced to <2% after five months under the soil suggesting M. unifolia and T. longifolia seeds do not persist in the seed bank beyond one growing season. Sequences of ITS rDNA indicate Tulasnella calospora assists in the germination of M. unifolia at this site. Similarly, Tulasnella calospora promoted germination of the Nationally Vulnerable wetland species S. novae-zelandiae. Pelotons were isolated from the roots of S. novae-zelandiae plants from a wild population from the lower north island and cultured in Petri dishes. Germination of this orchid began after 30 days from inoculation when the pelotons are already observed inside the embryo. Chlorophyllus tissue was observed after c. 80 days of inoculation. The phylogenetic relationship of Asian-Pacific Spiranthes species with New Zealand Spiranthes was also investigated using nuclear (ITS) and chloroplast (trnL-trnF) DNA sequences. Phylogenetic analyses supported the recognition of Spiranthes novae-zelandiae ‘Motutangi’ as a distinct taxonomic unit. It was also found that the Asian-Pacific Spiranthes species are in need of taxonomic revision. Methods used and developed in this thesis study could be used to identify potential orchid symbionts and pathogens, assess suitable potential relocation sites, and propagation of NZ orchids using symbiotic fungi for restoration and conservation purposes.</p>

Tulasnella calospora (UAMH 9824) retains its effectiveness at facilitating orchid symbiotic germination in vitro after two decades of subculturing

Background The technique of symbiotic germination—using mycorrhizal fungi to propagate orchids from seed in vitro—has been used as one method to cultivate orchids in North America and abroad for > 30 years. A long-held assumption is that mycorrhizal fungi used for this purpose lose their effectiveness at germinating seeds over time with repeated subculturing. Results We provide evidence for the lingering efficacy of one particular strain of Tulasnella calospora (266; UAMH 9824) to stimulate seed germination exemplified by the North American terrestrial orchid, Spiranthes cernua, as a case study. This fungus was originally acquired from roots from Spiranthes brevilabris in 1999 and sub-cultured during the two decades since. Seeds inoculated with the fungus in vitro developed to an advanced protocorm stage after 16 days, and leaf elongation was pronounced after 42 days. In a pilot study, seedlings co-cultured with Tulasnella calospora 266 were deflasked after 331 days and later transferred to soil under greenhouse conditions where they eventually initiated anthesis. During the course of two decades, seeds of 39 orchid species, cultivars and hybrids spanning 21 genera, germinated in vitro co-cultured with Tulasnella calospora 266. These orchids included temperate terrestrials and tropical epiphytes alike. Conclusions The sustained effectiveness of this fungus is noteworthy because it argues against the concept of mycorrhizal fungi losing their symbiotic capability through prolonged subculturing. This study serves as an example of why in situ habitat preservation is essential for the conservation of orchids as a source of potentially useful mycorrhizal fungi.

The Dark Side of Orchid Symbiosis: Can Tulasnella calospora Decompose Host Tissues?

Photosynthetic orchids associate with mycorrhizal fungi that can be mostly ascribed to the “rhizoctonia” species complex. Rhizoctonias’ phylogenetic diversity covers a variety of ecological/nutritional strategies that include, beside the symbiosis establishment with host plants, endophytic and pathogenic associations with non-orchid plants or saprotrophic soil colonization. In addition, orchid mycorrhizal fungi (OMF) that establish a symbiotic relationship with an orchid host can later proliferate in browning and rotting orchid tissues. Environmental triggers and molecular mechanisms governing the switch leading to either a saprotrophic or a mycorrhizal behavior in OMF remain unclear. As the sequenced OMF genomes feature a wide range of genes putatively involved in the degradation of plant cell wall (PCW) components, we tested if these transitions may be correlated with a change in the expression of some PCW degrading enzymes. Regulation of several genes encoding PCW degrading enzymes was evaluated during saprotrophic growth of the OMF Tulasnella calospora on different substrates and under successful and unsuccessful mycorrhizal symbioses. Fungal gene expression in planta was investigated in two orchid species, the terrestrial Mediterranean Serapias vomeracea and the epiphytic tropical Cattleya purpurata. Although we only tested a subset of the CAZyme genes identified in the T. calospora genome, and we cannot exclude therefore a role for different CAZyme families or members inside a family, the results showed that the degradative potential of T. calospora is finely regulated during saprotrophic growth and in symbiosis, often with a different regulation in the two orchid species. These data pose novel questions about the role of fungal PCW degrading enzymes in the development of unsuccessful and successful interactions.

Shallow Genome Sequencing for Phylogenomics of Mycorrhizal Fungi from Endangered Orchids

ABSTRACTMost plant species form symbioses with mycorrhizal fungi and this relationship is especially important for orchids. Fungi in the genera Tulasnella, Ceratobasidium, and Serendipita are critically important for orchid germination, growth and development. The goals of this study are to understand the phylogenetic relationships of mycorrhizal fungi and to improve the taxonomic resources for these groups. We identified 32 fungal isolates with the internal transcribed spacer region and used shallow genome sequencing to functionally annotate these isolates. We constructed phylogenetic trees from 408 orthologous nuclear genes for 50 taxa representing 14 genera, 11 families, and five orders in Agaricomycotina. While confirming relationships among the orders Cantharellales, Sebacinales, and Auriculariales, our results suggest novel relationships between families in the Cantharellales. Consistent with previous studies, we found the genera Ceratobasidium and Thanatephorus of Cerabotasidiaceae to not be monophyletic. Within the monophyletic genus Tulasnella, we found strong phylogenetic signals that suggest a potentially new species and a revision of current species boundaries (e.g. Tulasnella calospora); however it is premature to make taxonomic revisions without further sampling and morphological descriptions. There is low resolution of Serendipita isolates collected. More sampling is needed from areas around the world before making evolutionary-informed changes in taxonomy. Our study adds value to an important living collection of fungi isolated from endangered orchid species, but also informs future investigations of the evolution of orchid mycorrhizal fungi.