title: Quantitative evaluation of protocorm growth and fungal colonization in Bletilla striata (Orchidaceae) reveals less-productive symbiosis with a non-native symbiotic fungus.
abstract: In nature, orchid plants depend completely on symbiotic fungi for their nutrition at the germination and the subsequent seedling (protocorm) stages. However, only limited quantitative methods for evaluating the orchid-fungus interactions at the protocorm stage are currently available, which greatly constrains our understanding of the symbiosis. Here, we aimed to improve and integrate quantitative evaluations of the growth and fungal colonization in the protocorms of a terrestrial orchid, Blettila striata, growing on a plate medium.
  
  
  
  We achieved both symbiotic and asymbiotic germinations for the terrestrial orchid B. striata. The protocorms produced by the two germination methods grew almost synchronously for the first three weeks. At week four, however, the length was significantly lower in the symbiotic protocorms. Interestingly, the dry weight of symbiotic protocorms did not significantly change during the growth period, which implies that there was only limited transfer of carbon compounds from the fungus to the protocorms in this relationship. Next, to evaluate the orchid-fungus interactions, we developed an ink-staining method to observe the hyphal coils in protocorms without preparing thin sections. Crushing the protocorm under the coverglass enables us to observe all hyphal coils in the protocorms with high resolution. For this observation, we established a criterion to categorize the stages of hyphal coils, depending on development and degradation. By counting the symbiotic cells within each stage, it was possible to quantitatively evaluate the orchid-fungus symbiosis.
  
  
  
  We describe a method for quantitative evaluation of orchid-fungus symbiosis by integrating the measurements of plant growth and fungal colonization. The current study revealed that although fungal colonization was observed in the symbiotic protocorms, the weight of the protocorm did not significantly increase, which is probably due to the incompatibility of the fungus in this symbiosis. These results suggest that fungal colonization and nutrition transfer can be differentially regulated in the symbiosis. The evaluation methods developed in this study can be used to study various quantitative aspects of the orchid-fungus symbiosis.
authors.affiliation: Graduate School of Agriculture, Tottori University, Tottori, Japan.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Faculty of Agriculture, Tottori University, Tottori, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Miura
authors.affiliation: Faculty of Agriculture, Tottori University, Tottori, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Fuji
authors.affiliation: Graduate School of Agriculture, Tottori University, Tottori, Japan.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Nagata
authors.affiliation: Graduate School of Agriculture, Tottori University, Tottori, Japan.
authors.forename: Yuria
authors.initials: Y
authors.lastname: Otani
authors.affiliation: Tsukuba Botanical Garden, National Museum of Nature and Science, Tsukuba, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yagame
authors.affiliation: Faculty of Education, Chiba University, Chiba, Japan.
authors.forename: Masahide
authors.initials: M
authors.lastname: Yamato
authors.affiliation: Faculty of Agriculture, Tottori University, Tottori, Japan. kaminaka@muses.tottori-u.ac.jp.
authors.forename: Hironori
authors.initials: H
authors.lastname: Kaminaka
mesh_terms: D004271:DNA, Fungal / Q000302:isolation & purification; D018525:Germination; D038821:Mycorrhizae / Q000235:genetics / Q000502:physiology*; D029595:Orchidaceae / Q000254:growth & development / Q000382:microbiology*; D013559:Symbiosis
chemical_list: D004271:DNA, Fungal
keywords: Bletilla striata; Germination; Mycorrhizal symbiosis; Orchid; Quantitative evaluation