Masahide Yamato

Partial mycoheterotrophy is a nutritional mode in which green plants supplement photosynthetically derived carbon with fungal carbon obtained from associated fungi. In green arbuscular mycorrhizal (AM) plants, however, detecting partial mycoheterotrophy using natural ¹³C abundance is challenging, because the δ¹³C signatures of AM fungi often resemble those of their host plants. To overcome this limitation, we exploited the large natural δ¹³C contrast between C₃ and C₄ plants as an intrinsic tracer and tested for the incorporation of donor-derived carbon into the shoots of Gentiana squarrosa (Gentianaceae). Using an AM fungus commonly associated with G. squarrosa , we conducted coculture experiments with either a C₃ or C₄ companion plant. A compartmentalized U-shaped coculture system was employed, in which the roots of the companion plant were separated from G. squarrosa seedlings by a nylon mesh that permitted only hyphal passage, thereby minimizing alternative carbon transfer pathways, such as indirect transfer via CO₂ released by donor root respiration. Shoot δ¹³C values of G. squarrosa were significantly higher when seedlings were connected to a C₄ companion than when connected to a C₃ companion. Furthermore, among individuals grown with a C₄ companion, shoot dry mass was strongly and positively correlated with shoot δ¹³C values. These results demonstrate the incorporation of donor-derived carbon into G. squarrosa via AM fungal networks, providing robust evidence of partial mycoheterotrophy and suggesting that the mycoheterotrophy contributed to growth in this AM-associated species.

The community of arbuscular mycorrhizal (AM) fungi was examined in Fukiage Garden Forest at the Imperial Palace in Tokyo, Japan. The AM fungal community was clearly distinguished from those of the previously studied Japanese temperate forests. Some of the dominant AM fungi in the Fukiage Garden Forest were species-described fungi, which contrasts with those in the Japanese temperate forests. Since AM fungal species have been described based on spore morphology, the present result suggests that the dominant AM fungi may have relatively higher spore productivity. The Imperial Palace had been the Edo Castle until the latter half of the 1800s, after which, through various vegetation, the Fukiage Garden Forest was developed. Accordingly, it is likely that the AM fungi with higher sporulation in past vegetation may have been retained in the current AM fungal community of the isolated forest.

We collected in Japan five sporocarpic specimens morphologically identical to those of Epigeocarpum japonicum, a recently described Glomeromycota species. Although 18S-ITS-28S nuc rDNA sequences obtained from these sporocarps showed high sequence variability, phylogenetic analyses based on 18S-ITS-28S, the largest subunit of the RNA polymerase II (rpb1) gene, and concatenated sequences of the two loci convincingly demonstrated the identity of these sporocarps to E. japonicum. Importantly, the 18S-ITS-28S+rpb1 analyses highlighted the key role of rpb1 sequences in reconstructing the phylogenies of Glomeromycota taxa with strongly divergent rDNA sequences. Upon inoculation with sporocarpic spores, E. japonicum formed mycorrhiza with arbuscules and vesicles, which was not confirmed in the original description of the species. Comparisons of E. japonicum 18S sequences with Glomeromycota DNA sequences available in a public database indicated that E. japonicum is a cosmopolitan species and is mainly associated with plants in natural habitats such as grasslands, shrublands, and forests. Phylogenetic analyses also confirmed the autonomy of E. crypticum, another known species of the genus Epigeocarpum whose sporocarps were originally found in Brazil.

Due to the loss of photosynthetic ability during evolution, some plant species rely on mycorrhizal fungi for their carbon source, and this nutritional strategy is known as mycoheterotrophy. Mycoheterotrophic plants forming Paris-type arbuscular mycorrhizas (AM) exhibit two distinctive mycorrhizal features: degeneration of fungal materials and specialization towards particular fungal lineages. To explore the possibility that some understory AM plants show partial mycoheterotrophy, i.e., both photosynthetic and mycoheterotrophic nutritional strategies, we investigated 13 green herbaceous plant species collected from five Japanese temperate forests. Following microscopic observation, degenerated hyphal coils were observed in four species: two Colchicaceae species, Disporum sessile and Disporum smilacinum, and two Gentianaceae species, Gentiana scabra and Swertia japonica. Through amplicon sequencing, however, we found that all examined plant species exhibited no specificity toward AM fungi. Several AM fungi were consistently found across most sites and all plant species studied. Because previous studies reported the detection of these AM fungi from various tree species in Japanese temperate forests, our findings suggest the presence of ubiquitous AM fungi in forest ecosystems. If the understory plants showing fungal degeneration exhibit partial mycoheterotrophy, they may obtain carbon compounds indirectly from a wide range of surrounding plants utilizing such ubiquitous AM fungi.

Orchids parasitically depend on external nutrients from mycorrhizal fungi for seed germination. Previous findings suggest that orchids utilize a genetic system of mutualistic arbuscular mycorrhizal (AM) symbiosis, in which the plant hormone gibberellin (GA) negatively affects fungal colonization and development, to establish parasitic symbiosis. Although GA generally promotes seed germination in photosynthetic plants, previous studies have reported low sensitivity of GA in seed germination of mycoheterotrophic orchids where mycorrhizal symbiosis occurs concurrently. To elucidate the connecting mechanisms of orchid seed germination and mycorrhizal symbiosis at the molecular level, we investigated the effect of GA on a hyacinth orchid (Bletilla striata) seed germination and mycorrhizal symbiosis using asymbiotic and symbiotic germination methods. Additionally, we compared the transcriptome profiles between asymbiotically and symbiotically germinated seeds. Exogenous GA negatively affected seed germination and fungal colonization, and endogenous bioactive GA was actively converted to the inactive form during seed germination. Transcriptome analysis showed that B. striata shared many of the induced genes between asymbiotically and symbiotically germinated seeds, including GA metabolism- and signaling-related genes and AM-specific marker homologs. Our study suggests that orchids have evolved in a manner that they do not use bioactive GA as a positive regulator of seed germination and instead auto-activate the mycorrhizal symbiosis pathway through GA inactivation to accept the fungal partner immediately during seed germination.

Gentiana zollingeri is an annual photosynthetic plant that employs a mycoheterotrophic growth strategy during its underground seedling stage (initial mycoheterotrophy). Notably, the morphological characteristics of its flowering shoots, such as shoot size, leaf size, and leaf color, are highly variable, and it was hypothesized that these variations may be linked to nutritional mode. The morphological characteristics of G. zollingeri individuals were thus investigated alongside environmental factors, ¹³ C abundance, and diversity of colonizing arbuscular mycorrhizal (AM) fungi. The majority of G. zollingeri flowering individuals were found to exhibit a high affinity for the specific AM fungi that exclusively colonize roots of the mycoheterotrophic seedlings, while other phylogenetically diverse AM fungi could also be detected. The leaves to shoot dry weight ratio (leaf ratio) was negatively correlated with the canopy openness in the habitat, suggesting that leaf development is impeded in sunny conditions. Furthermore, the shoot weight of G. zollingeri was positively correlated with leaf ¹³ C abundance. Given that ¹³ C enrichment can provide indirect evidence of mycoheterotrophy in AM plants, the results suggest that the utilization of carbon obtained through mycoheterotrophy, at least during the underground seedling stage, is crucial for G. zollingeri.

Orchids parasitically depend on external nutrients from mycorrhizal fungi for seed germination. Previous findings suggest that orchids utilize a genetic system of mutualistic arbuscular mycorrhizal (AM) symbiosis to establish parasitic symbiosis. In AM symbiosis, recent studies have revealed that the plant hormone gibberellin (GA) negatively affects fungal colonization and development. Although previous studies imply that GA is important for orchid mycorrhizal symbiosis, the molecular mechanism of seed germination in which mycorrhizal symbiosis co-occurs remains unclear because, in AM plants, GA regulates seed germination and symbiosis positively and negatively, respectively. To elucidate this conflict, we investigated the effect of GA on Bletilla striata seed germination and mycorrhizal symbiosis using asymbiotic and symbiotic germination methods. Additionally, we compared the transcriptome profiles between asymbiotically and symbiotically germinated seeds. Exogenous GA negatively affected seed germination and fungal colonization, and endogenous bioactive GA was actively converted to the inactive form during seed germination. Transcriptome analysis showed that B. striata shared many of the induced genes between asymbiotically and symbiotically germinated seeds, including GA metabolism- and signaling-related genes and AM-specific marker homologs. Our study suggests that orchids have evolved in a manner that they do not use bioactive GA as a positive regulator of seed germination and instead, auto-activate the mycorrhizal symbiosis pathway through GA inactivation to accept the fungal partner immediately during seed germination.

Morphological analyses of three glomoid spore-producing fungi suggested that two of them were undescribed species of Glomeraceae (phylum Glomeromycota), and the third differed slightly from Dominikia glomerocarpica and Epigeocarpum crypticum, recently described in Glomeraceae. The first two fungi originated from the Mediterranean Sea sand dunes of the Peloponnese, Greece, and the third was originally found in a tree plantation in Yokohama City, Japan. Phylogenetic analyses of sequences of the 45S nuc rDNA region and the RPB1 gene showed that (i) the three fungi belonged to Glomeraceae; (ii) the first two represented a new genus, here described as Complexispora gen. nov. with C. multistratosa sp. nov. and C. mediterranea sp. nov. and (iii) the third enlarged the monospecific genus Epigeocarpum, as E. japonicum sp. nov.

Gentiana zollingeri (Gentianaceae) is an initial mycoheterotrophic plant that depends on a specific group of arbuscular mycorrhizal (AM) fungi for carbon source during underground growth after seed germination. In this study, a mycorrhizal fungus dominant in mycoheterotrophic seedlings of G. zollingeri was successfully isolated from a soil core collected from a point close to a flowering G. zollingeri. The AM fungal isolate was identified as conspecific or closely related to Dominikia aurea (Glomeraceae) by spore morphology and molecular phylogeny. Basic Local Alignment Search Tool (BLAST) searches against the MaarjAM database showed that the nuclear small subunit ribosomal DNA sequences of the isolate matched the AM fungal sequences obtained from a wide range of plants in various ecosystems, including several mycoheterotrophs. Thus, it is suggested that the AM fungal isolate is one of the cheating susceptible AM fungi. Furthermore, the sequences corresponded to those of a group of AM fungi dominantly detected in Japanese temperate forests. Accordingly, there is a possibility that mycoheterotrophic plants, including seedlings of G. zollingeri, may target AM fungi with a wide host range and ubiquitous distribution.