Stable isotope cellular imaging reveals that both live and degenerating fungal pelotons transfer carbon and nitrogen to orchid protocorms

Abstract

The objective of this study was to elucidate the transfer of nutrient elements in orchid symbiotic protocorms at the cellular level by imaging of stable isotope tracers. We address the long-standing question of whether nutrients move by transport across the symbiotic interface or solely by lysis of fungal pelotons. [U-(13) C]glucose and (15) NH4 (15) NO3 were added to Ceratobasidium sp. hyphae extending from symbiotic protocorms of Spiranthes sinensis. Isotope images were taken from resin-embedded sections of protocorms using ultra-high spatial resolution secondary ion mass spectrometry (SIMS). Analyses of regions of interest were conducted on isotope ratio images for fungal and host structures. Amyloplasts adjacent to young pelotons showed elevated (13) C/(12) C, which indicated that fungal carbon (C) was transferred from live hyphae. Senescent pelotons and their surrounding host cytoplasm showed significantly higher isotope ratios than young pelotons and surrounding host cytoplasm. These results indicate an inflow of C to senescent hyphae, which was then transferred to the host. The findings of this study provide some support for each of the two contradictory hypotheses concerning nutrient exchange in the symbiotic protocorm: the interface between the symbionts is involved before fungal senescence, and peloton degradation also releases a significant amount of C and nitrogen to host cells.