Accumulation of pathogens in soil microbiome can explain long-term fluctuations of legumes in a grassland community.

All plant populations fluctuate in time. Apart from the dynamics imposed by external forces such as climate, these fluctuations can be driven by endogenous processes taking place within the community. In this study, we aimed to identify potential role of soil-borne microbial communities in driving endogenous fluctuations of plant populations. We combined a unique, 35-yr long abundance data of 11 common plant species from a species-rich mountain meadow with development of their soil microbiome (pathogenic fungi, arbuscular mycorrhizal fungi and oomycetes) observed during 4 yr of experimental cultivation in monocultures. Plant species which abundance fluctuated highly in the field (particularly legumes) accumulated plant pathogens in their soil mycobiome. We also identified increasing proportion of mycoparasitic fungi under highly fluctuating legume species, which may indicate an adaptation of these species to mitigate the detrimental effects of pathogens. Our study documented that long-term fluctuations in the abundance of plant species in grassland communities can be explained by the accumulation of plant pathogens in plant-soil microbiome. By contrast, we found little evidence of the role of mutualists in plant population fluctuations. These findings offer new insights for understanding mechanisms driving both long-term vegetation dynamics and patterns of species coexistence and richness.

Nutrient responses of vascular plants to N2-fixing tree Alnus hirsuta encroachment in a boreal peatland.

The N2-fixing trees Alnus spp. have been widely encroaching into boreal peatlands, but the nutrient responses of native vascular plants remain unclear. Here, we compared nutrient concentrations and isotope signal of six common plants (Betula fruticosa, Salix rosmarinifolia, Vaccinium uliginosum, Rhododendron tomentosum, Chamaedaphne calyculata, and Eriophorum vaginatum) between Alnus hirsuta island and open peatland and assessed plant nutrient responses to A. hirsuta encroachment in boreal peatlands. Alnus hirsuta encroachment increased nitrogen (N) concentration of leaf, branch, and stem. Despite no significant interspecific difference in branch and stem, the increment magnitude of leaf N concentration varied among species, with greatest magnitude for R. tomentosum (55.1% ± 40.7%) and lowest for E. vaginatum (9.80% ± 4.40%) and B. fruticosa (18.4% ± 10.7%). Except for E. vaginatum, the significant increase in δ15N occurred for all organs of shrubs, with interspecific differences in change of leaf δ15N. According to the mass balance equation involving leaf δ15N, R. tomentosum and E. vaginatum, respectively, obtained highest (40.5% ± 19.8%) and lowest proportions (-14.0% ± 30.5%) of N from A. hirsuta. Moreover, the increment magnitudes of leaf N concentration showed a positive linear relationship with the proportion of N from A. hirsuta. In addition, A. hirsuta encroachment reduced leaf phosphorus (P) concentration of deciduous shrubs (i.e., B. fruticosa, S. rosmarinifolia, and V. uliginosum), thus increasing N:P ratio. These findings indicate that Alnus encroachment improves native plant N status and selectively intensifies P limitation of native deciduous shrubs, and highlight that the N acquisition from the symbiotic N2-fixing system regulates plant N responses in boreal peatlands.

Lipids Mediate Arbuscule Development and Senescence in Tomato Roots Colonized by Arbuscular Mycorrhizae Fungus under Drought Stress.

Arbuscular mycorrhizae (AM) symbiosis can enhance plant resistance to drought stress (DS). This study aimed to investigate the DS effects on lipids at different stages of symbiosis and to link lipid profiles to arbuscule dynamics in tomato roots colonized by AM fungi. DS increased mycorrhizal colonization and arbuscule abundance at an early stage but decreased them at a later stage, delayed arbuscule development, and accelerated arbuscule senescence at a later stage. DS decreased the contents of phospholipids (PLs) and saturated neutral lipids (NLs) at the early stage but increased the contents of saturated PLs and unsaturated NLs at the late stage. Specifically, DS inhibited AM-specific PL contents but increased AM-specific NL contents, which was supported by the expression of RAM2, STR/STR2. These data indicate the negative effect of DS on AM symbiosis and arbuscule dynamics with the effect size depending on the symbiosis stage, which highlights the importance of the symbiosis stage under abiotic stress.

High rate of gene family evolution in proximity to the origin of ectomycorrhizal symbiosis in Inocybaceae.

The genomes of ectomycorrhizal (ECM) fungi have a reduced number of genes encoding Carbohydrate-Active EnZymes (CAZymes), expansions in transposable elements (TEs) and small secreted proteins (SSPs) compared with saprotrophs. Fewer genes for specific peptidases and lipases in ECM fungi are also reported. It is unclear whether these changes occur at the shift to the ECM habit or are more gradual throughout the evolution of ECM lineages. We generated a genomic dataset of 20 species in the ECM lineage Inocybaceae and compared them with six saprotrophic species. Inocybaceae genomes have fewer CAZymes, peptidases, lipases, secondary metabolite clusters and SSPs and higher TE content than their saprotrophic relatives. There was an increase in the rate of gene family evolution along the branch with the transition to the ECM lifestyle. This branch had very high rate of evolution in CAZymes and had the largest number of contractions. Other significant changes along this branch included expansions in transporters, transposons-related genes and communication genes such as fungal kinases. There is a high concentration of changes in proximity to the transition to the ECM lifestyle, which correspond to the identified key changes for the gain of this lifestyle.

Coordinated influence of Funneliformis mosseae and different plant growth-promoting bacteria on growth, root functional traits, and nutrient acquisition by maize.

Rhizospheric interactions among plant roots, arbuscular mycorrhizal fungi, and plant growth-promoting bacteria (PGPB) can enhance plant health by promoting nutrient acquisition and stimulating the plant immune system. This pot experiment, conducted in autoclaved soil, explored the synergistic impacts of the arbuscular mycorrhizal fungus Funneliformis mosseae with four individual bacterial strains, viz.: Cronobacter sp. Rz-7, Serratia sp. 5-D, Pseudomonas sp. ER-20 and Stenotrophomonas sp. RI-4 A on maize growth, root functional traits, root exudates, root colonization, and nutrient uptake. The comprehensive biochemical characterization of these bacterial strains includes assessments of mineral nutrient solubilization, plant hormone production, and drought tolerance. The results showed that all single and interactive treatments of the mycorrhizal fungus and bacterial strains improved maize growth, as compared with the control (no fungus or PGPB). Among single treatments, the application of the mycorrhizal fungus was more effective than the bacterial strains in stimulating maize growth. Within the bacterial treatments, Serratia sp. 5-D and Pseudomonas sp. ER-20 were more effective in enhancing maize growth than Cronobacter sp. Rz-7 and Stenotrophomonas sp. RI-4 A. All bacterial strains were compatible with Funneliformis mosseae to improve root colonization and maize growth. However, the interaction of mycorrhiza and Serratia sp. 5-D (M + 5-D) was the most prominent for maize growth improvement comparatively to all other treatments. We observed that bacterial strains directly enhanced maize growth while indirectly promoting biomass accumulation by facilitating increased mycorrhizal colonization, indicating that these bacteria acted as mycorrhizal helper bacteria.

Optimizing plant growth, nutrient uptake, and yield of onion through the application of phosphorus solubilizing bacteria and endophytic fungi.

Introduction: The application of mineral fertilizers deteriorates soil properties and affects crop yield and nutritional properties. However, plant growth-promoting microorganisms (PGPM- Serendipita indica, phosphorus solubilizing bacteria (PSB), and vesicular arbuscular mycorrhizae (VAM)) have great potential to reduce fertilizers and improve soil fertility, crop yield, and nutrient uptake and mitigate the environmental effect of mineral fertilizers. Material and methods: Hence, a field experiment was conducted involving nine treatments to evaluate the effects of PGPM along with 50% or 100% of the recommended dose of fertilizers on plant growth, soil fertility, nutrient uptake, and onion productivity. Results and discussion: Results indicated that 100% RDF combined with S. indica or PSB led to improved plant growth, and higher nutrient concentrations in both leaves and bulbs of onions compared to RDF alone. Moreover, the application of 100% RDF with S. indica increased total dry matter yield by 11.5% and 7.6% in the 2018-2019 and 2019-2020 seasons, respectively, compared to 100% RDF alone. This treatment also resulted in the highest nutrient uptake, with N uptake increasing by 6.9%-29.9%, P by 13.7%-21.7%, K by 20.0%-23.7%, and S by 18.1%-23.4%. Additionally, the combination of 100% RDF with S. indica inoculation led to a notable increase in bulb yield, with increments of 16.2% and 13.9% observed in 2018-2019 and 2019-2020, respectively, compared to 100% RDF alone. Similarly, the application of 100% RDF along with PSB inoculation resulted in an increase in bulb yield by 7.2% and 9.4% in the respective years. However, VAM did not exhibit satisfactory performance or improvements in the onion crop. Conclusion: Overall, the study suggests that combining 100% RDF with S. indica or PSB can enhance onion productivity and nutrient use efficiency. The present study may open a new avenue of PGPM application in enhancing onion yield and improving the bulb quality as well as soil health. However, field trials across different regions and soil types are necessary to validate these findings for practical adoption by farmers.

Enhancing arbuscular mycorrhiza symbiosis effectiveness through the involvement of the tomato GRAS transcription factor SCL3/SlGRAS18.

Arbuscular mycorrhizal (AM) fungi improve plant growth, nutrition, fitness and stress tolerance while AM fungi obtain carbohydrates and lipids from the host. This whole process of mutual benefit requires substantial alterations in the structural and functional aspects of the host root cells. These modifications ultimately culminate in the formation of arbuscules, which are specialized intraradical and highly branched fungal structures. Arbuscule-containing cells undergo massive reprogramming to hosting arbuscule and members of the GRAS transcription factor family have been characterized as AM inducible genes which play a pivotal role in these process. Here, we show a functional analysis for the GRAS transcription factor SCL3/SlGRAS18 in tomato. SlGRAS18 interacts with SlDELLA, a central regulator of AM formation. Silencing of SlGRAS18 positively impacts arbuscule development and the improvement in symbiotic status, favouring flowering and therefore progress in the formation and development of fruits in SlGRAS18 silenced plants which parallel to a discernible pattern of mineral nutrient redistribution in leaves. Our results advance the knowledge of GRAS transcription factors involved in the formation and establishment of AM symbiosis and provide experimental evidence for how specific genetic alterations can lead to more effective AM symbiosis.

Mechanisms of coexistence: Exploring species sorting and character displacement in woody plants to alleviate belowground competition.

Rarely do we observe competitive exclusion within plant communities, even though plants compete for a limited pool of resources. Thus, our understanding of the mechanisms sustaining plant biodiversity might be limited. In this study, we explore two common ecological strategies, species sorting and character displacement, that promote coexistence by reducing competition. We assess the degree to which woody plants may implement these two strategies to lower belowground competition for nutrients which occurs via nutritional (mostly mycorrhizal) mutualisms. First, we compile data on plant traits and the mycorrhizal association state of woody angiosperms using a global inventory of indigenous flora. Our analysis reveals that species in locations with high mycorrhizal diversity exhibit distinct mean values in leaf area and wood density based on their mycorrhizal type, indicating species sorting. Second, we reanalyse a large dataset on leaf area to demonstrate that in areas with high mycorrhizal diversity, trees maintain divergent leaf area values, showcasing character displacement. Character displacement among plants is considered rare, making our observation significant. In summary, our study uncovers a rare occurrence of character displacement and identifies a common mechanism employed by plants to alleviate competition, shedding light on the complexities of plant coexistence in diverse ecosystems.

Corrigendum: Expression patterns of maize PIP aquaporins in middle or upper leaves correlate with their different physiological responses to drought and mycorrhiza.

[This corrects the article DOI: 10.3389/fpls.2022.1056992.].

Arbuscular mycorrhizal fungi regulate amino acid metabolism, phytohormones and glycolysis pathway to promote the growth of Suaeda salsa under combined Cd and NaCl stresses.

The use of halophytes in conjunction with arbuscular mycorrhizal (AM) fungi has been found to enhance the removal efficacy of heavy metals and salts in heavy metals contaminated saline soil. The mechanisms of AM fungi on promoting halophyte growth and regulating metabolism remain unclear. In this study, combinations of 0 g kg-1 NaCl and 3 mg kg-1 Cd (S0Cd3), 6 g kg-1 NaCl and 3 mg kg-1 Cd (S6Cd3), and 12 g kg-1 NaCl and 3 mg kg-1 Cd (S12Cd3) were employed to explore the impact of Funneliformis mosseae on the growth and metabolism of Suaeda salsa. The results showed that AM fungi increased the biomass and the P, K+, Ca2+, and Mg2+ accumulations, reduced the Cd and Na+ concentrations in S0Cd3 and S6Cd3, and increased the Cd concentrations in S12Cd3. AM fungi inoculation reduced the Cd and Na+ transfer factors and increased the Cd and Na+ accumulations in S6Cd3. The metabolomics of S6Cd3 showed that AM fungi upregulated the expression of 5-hydroxy-L-tryptophan and 3-indoleacid acid in tryptophan metabolism, potentially acting as crucial antioxidants enabling plants to actively cope with abiotic stresses. AM fungi upregulated the expression of arbutin in glycolysis process, enhancing the plants' osmoregulation capacity. AM fungi upregulated the expression of 2-hydroxycinnamic acid in phenylalanine metabolism and dopaquinone in tyrosine metabolism. These two metabolites help effectively remove reactive oxygen species. Correspondingly, AM fungi decreased MDA content and increased soluble sugar content. These results indicate that AM fungi improve the stress resistance of S. salsa by increasing nutrient uptake and regulating physiological and metabolic changes.

Adapted molecular methods to unravel the recalcitrant mycorrhizal associations of Aucoumea klaineana Pierre.

Understanding the role of root microbiota is crucial in sustainable forest management but remains challenging, especially for tropical trees. We developed an efficient and low-toxicity method to extract and amplify the fungal DNA associated with Aucoumea klaineana Pierre fine roots. To improve DNA quality, we optimized a commercial extraction kit by incorporating activated charcoal and modifying incubation periods. This enhanced protocol, combined with bovine serum albumin during PCR, effectively mitigated inhibitors present in A. klaineana tree root samples. This approach opens new perspectives for studying the microbiota of tropical trees.

Testing the ectomycorrhizal-dominance hypothesis for ecosystem multifunctionality in a subtropical mountain forest.

Mycorrhizal associations are key mutualisms that shape the structure of forest communities and multiple ecosystem functions. However, we lack a framework for predicting the varying dominance of distinct mycorrhizal associations in an integrated proxy of multifunctionality across ecosystems. Here, we used the datasets containing diversity of mycorrhizal associations and 18 ecosystem processes related to supporting, provisioning, and regulating services to examine how the dominance of ectomycorrhiza (EcM) associations affects ecosystem multifunctionality in subtropical mountain forests in Southwest China. Meanwhile, we synthesized the prevalence of EcM-dominant effects on ecosystem functioning in forest biomes. Our results demonstrated that elevation significantly modified the distributions of EcM trees and fungal dominance, which in turn influenced multiple functions simultaneously. Multifunctionality increased with increasing proportion of EcM associations, supporting the ectomycorrhizal-dominance hypothesis. Meanwhile, we observed that the impacts of EcM dominance on individual ecosystem functions exhibited different relationships among forest biomes. Our findings highlight the importance of ectomycorrhizal dominance in regulating multifunctionality in subtropical forests. However, this ectomycorrhizal feedback in shaping ecosystem functions cannot necessarily be generalized across forests. Therefore, we argue that the predictions for ecosystem multifunctionality in response to the shifts of mycorrhizal composition could vary across space and time.

Olive Tree Belowground Microbiota: Plant Growth-Promoting Bacteria and Fungi.

The olive tree is one of the most significant crops in the Mediterranean region. Its remarkable adaptability to various environments has facilitated olive cultivation across diverse regions and agricultural scenarios. The rising global demand for olive products, coupled with climate challenges, is driving changes in cultivation methods. These changes are altering the traditional landscape and may potentially reshape the structure and composition of orchard microbial communities, which can impact productivity and stress tolerance. Bacterial and fungal communities naturally associated with plants have long been recognized as crucial for plant growth and health, serving as a vital component of sustainable agriculture. In this review, we aim to highlight the significance of olive cultivation and the impact of abiotic stresses. We update the current knowledge on the profiles of rhizosphere and root fungal and bacterial communities in olive orchards and examine how (a)biotic factors influence these communities. Additionally, we explore the potential of plant growth-promoting bacteria and fungi in enhancing olive physiological performance and stress tolerance. We identify knowledge gaps and emphasize the need for implementing new strategies. A comprehensive understanding of olive-associated microbiota will aid in developing sustainable agronomic practices to address climatic challenges and meet the growing demand for olive products.

Genome-Wide Identification of the Sulfate Transporters Gene Family in Blueberry (Vaccinium spp.) and Its Response to Ericoid Mycorrhizal Fungi.

Sulfur metabolism plays a major role in plant growth and development, environmental adaptation, and material synthesis, and the sulfate transporters are the beginning of sulfur metabolism. We identified 37 potential VcSULTR genes in the blueberry genome, encoding peptides with 534 to 766 amino acids. The genes were grouped into four subfamilies in an evolutionary analysis. The 37 putative VcSULTR proteins ranged in size from 60.03 to 83.87 kDa. These proteins were predicted to be hydrophobic and mostly localize to the plasma membrane. The VcSULTR genes were distributed on 30 chromosomes; VcSULTR3;5b and VcSULTR3;5c were the only tandemly repeated genes. The VcSULTR promoters contained cis-acting elements related to the fungal symbiosis and stress responses. The transcript levels of the VcSULTRs differed among blueberry organs and changed in response to ericoid mycorrhizal fungi and sulfate treatments. A subcellular localization analysis showed that VcSULTR2;1c localized to, and functioned in, the plasma membrane and chloroplast. The virus-induced gene knock-down of VcSULTR2;1c resulted in a significantly decreased endogenous sulfate content, and an up-regulation of genes encoding key enzymes in sulfur metabolism (VcATPS2 and VcSiR1). These findings enhance our understanding of mycorrhizal-fungi-mediated sulfate transport in blueberry, and lay the foundation for further research on blueberry-mycorrhizal symbiosis.

Differential root and cell regulation of maize aquaporins by the arbuscular mycorrhizal symbiosis highlights its role in plant water relations.

This study aims to elucidate if the regulation of plant aquaporins by the arbuscular mycorrhizal (AM) symbiosis occurs only in roots or cells colonized by the fungus or at whole root system. Maize plants were cultivated in a split-root system, with half of the root system inoculated with the AM fungus and the other half uninoculated. Plant growth and hydraulic parameters were measured and aquaporin gene expression was determined in each root fraction and in microdissected cells. Under well-watered conditions, the non-colonized root fractions of AM plants grew more than the colonized root fraction. Total osmotic and hydrostatic root hydraulic conductivities (Lo and Lpr) were higher in AM plants than in non-mycorrhizal plants. The expression of most maize aquaporin genes analysed was different in the mycorrhizal root fraction than in the non-mycorrhizal root fraction of AM plants. At the cellular level, differential aquaporin expression in AM-colonized cells and in uncolonized cells was also observed. Results indicate the existence of both, local and systemic regulation of plant aquaporins by the AM symbiosis and suggest that such regulation is related to the availability of water taken up by fungal hyphae in each root fraction and to the plant need of water mobilization.

Analysis of the Fungal Community Composition in Endemic Orchids with Terrestrial Habitat in Subtropical Regions.

Habenaria and Liparis are well-known orchid genera that grow in terrestrial habitats in the tropics, subtropics or temperate zones. Three species have been found in subtropical regions of China, inhabiting terrestrial to epiphytic habitats. This study focuses on three species, H. dentata (distributed in Asia), H. yachangensis, and L. gigantea. For H. yachangensis and L. gigantea, there is no information about the mycorrhizal community in these species. This study aims to conduct the fungal community screening of Chinese ground orchids from subtropical regions. We performed a comparative analysis of the fungal community among H. dentata, H. yachangensis, and L. gigantea, determining their ITS regions using NGS paired-end sequences. The results clarified the diversity and the predominance of fungal genera. Ascomycota was abundant compared to Basidiomycota or other fungi groups in all communities, with a high dominance in all populations, especially for L. gigantea. At different root spatial locations, the fungal community diversity and richness were higher in the soil than in the rhizosphere or inner root. However, the results suggest that L. gigantea has a different fungal community compared to Habenaria species. In this order, the subtropical terrestrial orchids have a different fungal network compared to the northern terrestrial orchids. Also, there is a high probability of co-existence and co-evolution of endophytic fungi in these terrestrial orchids, indicating the potential role of host plants in selecting an endophytic fungal community. Furthermore, our results highlight the need to elucidate the microbe interactions of these unique orchids for long-term purposes, such as isolating indigenous fungi for suitable inoculants for further orchid propagation, restoration, and conservation.

Two novel Archaeorhizomyces species isolated from ericoid mycorrhizal roots and their association with ericaceous plants in vitro.

Archaeorhizomyces is a diverse and ubiquitous genus of the subphylum Taphrinomycotina, which contains soil-inhabiting/root-associated fungi. Although ecological importance and root-associating lifestyles of Archaeorhizomyces can be postulated, morphological aspects of fungal body and root colonization are largely unknown due to the scarcity of cultures. We obtained three unidentified Archaeorhizomyces isolates from ericoid mycorrhizal (ErM) roots of Rhododendron scabrum and Rhododendron × obtusum collected in Japan. To advance our understanding of lifestyle of the genus, we investigated their general morphology, phylogeny, and in vitro root-colonizing ability in ericoid mycorrhizal hosts, Vaccinium virgatum and Rhododendron kaempferi. Some morphological characteristics, such as slow glowing white-to-creamy-colored colonies and formation of yeast-like or chlamydospore-like cells, were shared between our strains and two described species, Archaeorhizomycesfinlayi and Archaeorhizomyces borealis, but they were phylogenetically distant. Our strains were clearly distinguished as two undescribed species based on morphology and phylogenetic relationship. As seen in typical ErM fungi, both species frequently formed hyphal coils within vital rhizodermal cells of ErM plants in vitro. The morphology of hyphal coils was also different between species. Consequently, two novel species, Archaeorhizomyces notokirishimae sp. nov. and Archaeorhizomyces ryukyuensis sp. nov., were described.

The mechanism of arbuscular mycorrhizal fungi-alleviated manganese toxicity in plants: A review.

The development of the mining industry and the overuse of inorganic fertilizers have led to an excess of manganese (Mn) in the soil, thereby, contaminating the soil environment and people's health. On heavy metal-contaminated soils, the combined arbuscular mycorrhizal fungi (AMF)-phytoremediation technique becomes a hotspot because of its environmentally friendly, in situ remediation. AMF inoculation often leads to a decrease in host Mn acquisition, which provides a basis for its application in phytoremediation of contaminated soils. Moreover, the utilization value of native AMF is greater than that of exotic AMF, because native AMF can adapt better to Mn-contaminated soils. In addition to the fact that AMF enhance plant Mn tolerance responses such as regionalization, organic matter chelation, limiting uptake and efflux, and so on, AMF also develop plant-independent fungal pathways such as direct biosorption of Mn by mycorrhizal hyphae, fungal Mn transporter genes, and sequestration of Mn by mycorrhizal hyphae, glomalin, and arbuscule-containing root cortical cells, which together mitigate excessive Mn toxicity to plants. Clarifying AMF-plant interactions under Mn stress will provide support for utilizing AMF as a phytoremediation in Mn-contaminated soils. The review reveals in detail how AMF develop its own mechanisms for responding to excess Mn and how AMF enhance plant Mn tolerance, accompanied by perspectives for future research.

Mycorrhizal associations modify tree diversity-productivity relationships across experimental tree plantations.

Decades of studies have demonstrated links between biodiversity and ecosystem functioning, yet the generality of the relationships and the underlying mechanisms remain unclear, especially for forest ecosystems. Using 11 tree-diversity experiments, we tested tree species richness-community productivity relationships and the role of arbuscular (AM) or ectomycorrhizal (ECM) fungal-associated tree species in these relationships. Tree species richness had a positive effect on community productivity across experiments, modified by the diversity of tree mycorrhizal associations. In communities with both AM and ECM trees, species richness showed positive effects on community productivity, which could have resulted from complementarity between AM and ECM trees. Moreover, both AM and ECM trees were more productive in mixed communities with both AM and ECM trees than in communities assembled by their own mycorrhizal type of trees. In communities containing only ECM trees, species richness had a significant positive effect on productivity, whereas species richness did not show any significant effects on productivity in communities containing only AM trees. Our study provides novel explanations for variations in diversity-productivity relationships by suggesting that tree-mycorrhiza interactions can shape productivity in mixed-species forest ecosystems.

Mode of carbon gain and fungal associations of Neuwiedia malipoensis within the evolutionary early diverging orchid subfamily Apostasioideae.

BACKGROUND AND AIMS: The earliest diverging orchid lineage Apostasioideae consists only of two genera: Apostasia and Neuwiedia. Previous report of Apostasia nipponica indicated a symbiotic association with an ectomycorrhiza-forming Ceratobasidiaceae clade and partial utilization of fungal carbon during the adult stage. However, the trophic strategy of Neuwiedia throughout its development remains unidentified. To further improve our understanding of mycoheterotrophy in the Apostasioideae, this study focused on Neuwiedia malipoensis examining both the mycorrhizal association and the physiological ecology of this orchid species across various development stages. METHODS: We identified the major mycorrhizal fungi of N. malipoensis protocorm, leafy seedling and adult stages using molecular barcoding. To reveal nutritional resources utilized by N. malipoensis, we compared stable isotope natural abundance (δ13C, δ15N, δ2H, δ18O) of different developmental stages to autotrophic reference plants. KEY RESULTS: Protocorms exhibited an association with saprotrophic Ceratobasidiaceae rather than ectomycorrhiza-forming Ceratobasidiaceae and 13C signature was characteristic of their fully mycoheterotrophic nutrition.Seedlings and adults predominantly associated with saprotrophic fungi belonging to the Tulasnellaceae. While 13C and 2H stable isotope data revealed partial mycoheterotrophy of seedlings, it is unclear to what extent the fungal carbon supply is reduced in adult N. malipoensis. However, the 15N enrichment of mature N. malipoensis suggests partially mycoheterotrophic nutrition.Our data indicated a transition in mycorrhizal partners during ontogenetic development with decreasing dependency of N. malipoensis on fungal nitrogen and carbon. CONCLUSIONS: The divergence in mycorrhizal partners between N. malipoensis and A. nipponica indicates different resource acquisition strategies and allows for various habitat options in the earliest diverging orchid lineage Apostasioideae. While A. nipponica relies on the heterotrophic C gain from its ectomycorrhizal fungal partner and thus on forest habitats, N. malipoensis rather relies on own photosynthetic C gain as adult allowing it to establish in habitats as widely distributed as those where Rhizoctonia fungi occur.