Symbiotic germination and development of the myco-heterotroph Monotropa hypopitys in nature and its requirement for locally distributed Tricholoma spp.

• Germination and symbiotic development of the myco-heterotrophic plant Monotropa hypopitys were studied by sequential recovery of packets of seed buried in dune slacks in relation to distance from mature M. hypopitys and presence and absence of shoots of its autotrophic coassociate Salix repens. • Fungal associates of M. hypopitys growing under S. repens in the dune slacks, and under S. caprea and Pinus sylvestris at two other locations in the UK, were identified by molecular analysis. • While the earliest stage of germination could be found in the absence both of mature M. hypopitys, and S. repens, further development was dependent upon mycorrhizal colonisation, which was most common close to these plants. Molecular analysis showed that when growing with Salix, M. hypopitys associated with the Salix-specific ectomycorrhizal fungus Tricholoma cingulatum, whereas under Pinus it was colonised by the closely related, Pinaceae-specific, T. terreum. • We establish the first definitive chronology of development of M. hypopitys and highlight its critical dependence upon, and specificity for, locally distributed Tricholoma species that link the myco-heterotroph to its autotrophic coassociates.

Symbiotic germination and development of myco-heterotrophic plants in nature: ontogeny of Corallorhiza trifida and characterization of its mycorrhizal fungi.

The processes of symbiotic germination and seedling development were analysed in the myco-heterotrophic orchid Corallorhiza trifida, seeds of which were buried in 'packets' either adjacent to or at varying distances from adult plants in defined communities of ectomycorrhizal tree species. Germination occurred within eight months of burial under Betula-Alnus and within seven months under Salix repens. It was always associated with penetration of the suspensor by a clamp-forming mycorrhizal fungus. Four distinct developmental stages were defined and the rates of transition through these stages were plotted. There was no evidence of a relationship between extent of germination or rate of development and the presence of naturally distributed plants of C. trifida at the spatial scale of 1 m. The best germination and the most rapid rate of development of C. trifida seedlings occurred in a Salix repens community located at a considerable distance from any extant C. trifida population. Determination of internal transcribed spacer (ITS) RFLPs and of gene sequences of the fungi involved in symbiotic germination and growth of C. trifida, revealed them to belong exclusively to the Thelephora-Tomentella complex of the Thelephoraceae. These fungi are known also to be ectomycorrhizal associates of trees. It is hypothesized that the rate of growth of the C. trifida seedlings is determined by the ability of the fungal symbionts to transfer carbon from their ectomycorrhizal co-associates.

Symbiotic germination and development of myco-heterotrophic plants in nature: transfer of carbon from ectomycorrhizal Salix repens and Betula pendula to the orchid Corallorhiza trifida through shared hyphal connections.

Seedlings of the myco-heterotrophic orchid Corallorhiza trifida which had been germinated in the field in mesh bags developed hyphal links and mycorrhizas with Betula pendula and Salix repens, but not with Pinus sylvestris, when transplanted into soil microcosms. The fungus connecting the myco-heterotroph to Betula and Salix formed endomycorrhiza in the orchid with typical pelotons, but formed ectomycorrhizas with the autotrophs. The orchid plants, when linked to Betula and Salix by fungal hyphae, gained 6-14% in weight over 25-28 wk. In microcosms supporting P. sylvestris, and in control microcosms which lacked autotrophs, the Corallorhiza plants lost 13% of their weight over the same period. In the course of the 28-wk experimental period new Corallorhiza seedlings, in addition to those added as part of the experiment, appeared in the microcosms containing Salix and Betula but not in the Pinus microcosms. Shoots of Betula and Salix plants grown in association with Corallorhiza were fed with 14 CO2 , and the movement of the isotope was subsequently traced by a combination of digital autoradiography and tissue oxidation. Direct transfer of C from both autotrophs to the myco-heterotroph occurred in all cases where the associates had become connected by a shared fungal symbiont. Orchid seedlings lacking these hyphal connections, introduced to the microcosms as controls immediately before isotope feeding, failed to assimilate significant amounts of C. The results provide the first experimental confirmation that growth of Corallorhiza trifida can be sustained by supply of C received directly from an autotrophic partner through linked fungal mycelia.