Quantitative evaluation of protocorm growth and fungal colonization in Bletilla striata (Orchidaceae) reveals less-productive symbiosis with a non-native symbiotic fungus

Abstract

BackgroundIn 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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1002-x) contains supplementary material, which is available to authorized users.

Bletilla striata (Orchidaceae) Seed Coat Restricts the Invasion of Fungal Hyphae at the Initial Stage of Fungal Colonization

“…Seeds of B. striata ‘Murasakishikibu’ used as the plant material in this study were prepared as described previously [24]. Collected seeds were stored at 4 °C until use.…”

“…In this study, we aimed to better understand the process of recruitment of symbiotic fungi at the symbiosis initiation stage, focusing on the orchid seed coat since the seed coat is the first point of fungal attachment. To test this, we used an experimental system for a symbiotic germination of a terrestrial orchid, Bletilla striata (subfamily Epidendroideae, tribe Arethuseae), on an oatmeal medium [24]. The seed material was chosen because it has a relatively large air space, leading to easy handling.…”

“…B. striata , is usually found on sunshiny moist grassland and known as a common garden plant in Japan, which produces numerous dust-like seeds (approximately 0.2 mm-long embryo and 1.0 mm-long seed coat). When the symbiotic germination occurs, the embryo swells, epidermal hairs develop within a week, and then the shoot apical meristem of most seeds emerges within two weeks, after the seed coat has ruptured [24]. Since B. striata cells containing pelotons are present in one-week-old protocorms [24], the fungal hyphae are assumed to reach the surface of the embryo through the seed coat in a few days.…”

“…When the symbiotic germination occurs, the embryo swells, epidermal hairs develop within a week, and then the shoot apical meristem of most seeds emerges within two weeks, after the seed coat has ruptured [24]. Since B. striata cells containing pelotons are present in one-week-old protocorms [24], the fungal hyphae are assumed to reach the surface of the embryo through the seed coat in a few days. Consequently, we examined the role of the seed coat in the initial stage of the interaction with symbiotic and pathogenic fungi by comparing intact B. striata seeds with seeds from which the seed coat had been removed under a stereomicroscope.…”

Molecular Identification of Endophytic Fungi and Their Pathogenicity Evaluation Against Dendrobium nobile and Dendrobium officinale

“…In the environment, orchids are chiefly dependent on these fungal endophytes for their nourishment and propagation along with the succeeding seedling (protocorm) phases. However, only imperfect quantitative approaches for assessing the Dendrobium-fungus connections at the protocorm and seedling phase are available at present, which places major constraints on understanding the host-endophyte relationships [16].…”