Functional differences of cultivable leaf-associated microorganisms in the native Andean tree Gevuina avellana Mol. (Proteaceae) exposed to atmospheric contamination.

AIMS: This study aimed to evaluate and describe the functional differences of cultivable bacteria and fungi inhabiting the leaves of Gevuina avellana Mol. (Proteaceae) in an urban area with high levels of air pollution and in a native forest in the southern Andes. METHODS AND RESULTS: Phyllosphere microorganisms were isolated from the leaves of G. avellana, their plant growth-promoting capabilities were estimated along with their biocontrol potential and tolerance to metal(loid)s. Notably, plants from the urban area showed contrasting culturable leaf-associated microorganisms compared to those from the native area. The tolerance to metal(loid)s in bacteria range from 15 to 450 mg l-1 of metal(loid)s, while fungal strains showed tolerance from 15 to 625 mg l-1, being especially higher in the isolates from the urban area. Notably, the bacterial strain Curtobacterium flaccumfaciens and the fungal strain Cladosporium sp. exhibited several plant-growth-promoting properties along with the ability to inhibit the growth of phytopathogenic fungi. CONCLUSIONS: Overall, our study provides evidence that culturable taxa in G. avellana leaves is directly influenced by the sampling area. This change is likely due to the presence of atmospheric pollutants and diverse microbial symbionts that can be horizontally acquired from the environment.

Juvenile Plant-Microbe Interactions Modulate the Adaptation and Response of Forest Seedlings to Rapid Climate Change.

The negative impacts of climate change on native forest ecosystems have created challenging conditions for the sustainability of natural forest regeneration. These challenges arise primarily from abiotic stresses that affect the early stages of forest tree development. While there is extensive evidence on the diversity of juvenile microbial symbioses in agricultural and fruit crops, there is a notable lack of reports on native forest plants. This review aims to summarize the critical studies conducted on the diversity of juvenile plant-microbe interactions in forest plants and to highlight the main benefits of beneficial microorganisms in overcoming environmental stresses such as drought, high and low temperatures, metal(loid) toxicity, nutrient deficiency, and salinity. The reviewed studies have consistently demonstrated the positive effects of juvenile plant-microbiota interactions and have highlighted the potential beneficial attributes to improve plantlet development. In addition, this review discusses the beneficial attributes of managing juvenile plant-microbiota symbiosis in the context of native forest restoration, including its impact on plant responses to phytopathogens, promotion of nutrient uptake, facilitation of seedling adaptation, resource exchange through shared hyphal networks, stimulation of native soil microbial communities, and modulation of gene and protein expression to enhance adaptation to adverse environmental conditions.

Exogenous Nitric Oxide Alleviates Water Deficit and Increases the Seed Production of an Endemic Amazonian Canga Grass.

Open pit mining can cause loss in different ecosystems, including damage to habitats of rare and endemic species. Understanding the biology of these species is fundamental for their conservation, and to assist in decision-making. Sporobolus multiramosus is an annual grass endemic to the Amazon canga ecosystems, which comprise rocky outcrop vegetation covering one of the world's largest iron ore reserves. Here, we evaluated whether nitric oxide aids S. multiramosus in coping with water shortages and examined the physiological processes behind these adaptations. nitric oxide application improved the water status, photosynthetic efficiency, biomass production, and seed production and germination of S. multiramosus under water deficit conditions. These enhancements were accompanied by adjustments in leaf and root anatomy, including changes in stomata density and size and root endodermis thickness and vascular cylinder diameter. Proteomic analysis revealed that nitric oxide promoted the activation of several proteins involved in the response to environmental stress and flower and fruit development. Overall, the results suggest that exogenous nitric oxide has the potential to enhance the growth and productivity of S. multiramosus. Enhancements in seed productivity have significant implications for conservation initiatives and can be applied to seed production areas, particularly for the restoration of native ecosystems.

Molecular Mechanisms Underlying Mimosa acutistipula Success in Amazonian Rehabilitating Minelands.

Mimosa acutistipula is endemic to Brazil and grows in ferruginous outcrops (canga) in Serra dos Carajás, eastern Amazon, where one of the largest iron ore deposits in the world is located. Plants that develop in these ecosystems are subject to severe environmental conditions and must have adaptive mechanisms to grow and thrive in cangas. Mimosa acutistipula is a native species used to restore biodiversity in post-mining areas in canga. Understanding the molecular mechanisms involved in the adaptation of M. acutistipula in canga is essential to deduce the ability of native species to adapt to possible stressors in rehabilitating minelands over time. In this study, the root proteomic profiles of M. acutistipula grown in a native canga ecosystem and rehabilitating minelands were compared to identify essential proteins involved in the adaptation of this species in its native environment and that should enable its establishment in rehabilitating minelands. The results showed differentially abundant proteins, where 436 proteins with significant values (p < 0.05) and fold change ≥ 2 were more abundant in canga and 145 in roots from the rehabilitating minelands. Among them, a representative amount and diversity of proteins were related to responses to water deficit, heat, and responses to metal ions. Other identified proteins are involved in biocontrol activity against phytopathogens and symbiosis. This research provides insights into proteins involved in M. acutistipula responses to environmental stimuli, suggesting critical mechanisms to support the establishment of native canga plants in rehabilitating minelands over time.

Diversity of Root-Associated Fungi of the Terrestrial Orchids Gavilea lutea and Chloraea collicensis in a Temperate Forest Soil of South-Central Chile.

The diversity of orchid mycorrhizal fungi (OMF) and other beneficial root-associated fungi in temperate forests has scarcely been examined. This study aimed to analyze the diversity of mycorrhizal and rhizosphere-associated fungal communities in the terrestrial orchids Gavilea lutea and Chloraea collicensis growing in high-orchid-population-density areas in the piedmont of the Andes Cordillera with native forest (Nothofagus-Araucaria) and Coastal Cordillera with an exotic plantation (Pinus-Eucalyptus) in south-central Chile. We focused on rhizosphere-inhabiting and peloton-associated OMF in a native forest (Andes Cordillera) and a mixed forest (Coastal Cordillera). The native terrestrial orchids G. lutea and C. collicensis were localized, mycorrhizal root segments were taken to isolate peloton-associated OMF, and rhizosphere soil was taken to perform the metabarcoding approach. The results revealed that Basidiomycota and Ascomycota were the main rhizosphere-inhabiting fungal phyla, showing significant differences in the composition of fungal communities in both sites. Sebacina was the most-abundant OMF genera in the rhizosphere of G. lutea growing in the native forest soil. In contrast, Thanatephorus was the most abundant mycorrhizal taxa growing in the rhizosphere of orchids from the Coastal Cordillera. Besides, other OMF genera such as Inocybe, Tomentella, and Mycena were detected. The diversity of OMF in pelotons differed, being mainly related to Ceratobasidium sp. and Tulasnella sp. These results provide evidence of differences in OMF from pelotons and the rhizosphere soil in G. lutea growing in the Andes Cordillera and a selection of microbial communities in the rhizosphere of C. collicensis in the Coastal Cordillera. This raises questions about the efficiency of propagation strategies based only on mycorrhizal fungi obtained by culture-dependent methods, especially in orchids that depend on non-culturable taxa for seed germination and plantlet development.

Draft Genome Sequence of the Novel, Moderately Thermophilic, Iron- and Sulfur-Oxidizing Firmicute Strain Y002, Isolated from an Extremely Acidic Geothermal Environment.

We report the draft genome sequence of the Firmicute strain Y002, a facultatively anaerobic, acidophilic bacterium that catalyzes the dissimilatory oxidation of iron and sulfur and the reduction of ferric iron. Analysis of the genome (2.9 Mb; G+C content, 46 mol%) provided insights into its ability to grow in extremely acidic geothermal environments.

Proteomic Profiling and Rhizosphere-Associated Microbial Communities Reveal Adaptive Mechanisms of Dioclea apurensis Kunth in Eastern Amazon's Rehabilitating Minelands.

Dioclea apurensis Kunth is native to ferruginous rocky outcrops (known as canga) in the eastern Amazon. Native cangas are considered hotspots of biological diversity and have one of the largest iron ore deposits in the world. There, D. apurensis can grow in post-mining areas where molecular mechanisms and rhizospheric interactions with soil microorganisms are expected to contribute to their establishment in rehabilitating minelands (RM). In this study, we compare the root proteomic profile and rhizosphere-associated bacterial and fungal communities of D. apurensis growing in canga and RM to characterize the main mechanisms that allow the growth and establishment in post-mining areas. The results showed that proteins involved in response to oxidative stress, drought, excess of iron, and phosphorus deficiency showed higher levels in canga and, therefore, helped explain its high establishment rates in RM. Rhizospheric selectivity of microorganisms was more evident in canga. The microbial community structure was mostly different between the two habitats, denoting that despite having its preferences, D. apurensis can associate with beneficial soil microorganisms without specificity. Therefore, its good performance in RM can also be improved or attributed to its ability to cope with beneficial soil-borne microorganisms. Native plants with such adaptations must be used to enhance the rehabilitation process.

Unraveling the plant diversity of the Amazonian canga through DNA barcoding.

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF-atpH, psbK-psbI, trnH-psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.

Glucosidase Inhibitors Screening in Microalgae and Cyanobacteria Isolated from the Amazon and Proteomic Analysis of Inhibitor Producing Synechococcus sp. GFB01.

Microalgae and cyanobacteria are good sources for prospecting metabolites of biotechnological interest, including glucosidase inhibitors. These inhibitors act on enzymes related to various biochemical processes; they are involved in metabolic diseases, such as diabetes and Gaucher disease, tumors and viral infections, thus, they are interesting hubs for the development of new drugs and therapies. In this work, the screening of 63 environmental samples collected in the Brazilian Amazon found activity against ß-glucosidase, of at least 60 min, in 13.85% of the tested extracts, with Synechococcus sp. GFB01 showing inhibitory activity of 90.2% for α-glucosidase and 96.9% against ß-glucosidase. It was found that the nutritional limitation due to a reduction in the concentration of sodium nitrate, despite not being sufficient to cause changes in cell growth and photosynthetic apparatus, resulted in reduced production of α and ß-glucosidase inhibitors and differential protein expression. The proteomic analysis of cyanobacteria isolated from the Amazon is unprecedented, with this being the first work to evaluate the protein expression of Synechococcus sp. GFB01 subjected to nutritional stress. This evaluation helps to better understand the metabolic responses of this organism, especially related to the production of inhibitors, adding knowledge to the industrial potential of these cyanobacterial compounds.

Culture of Mycobacterium smegmatis in Different Carbon Sources to Induce In Vitro Cholesterol Consumption Leads to Alterations in the Host Cells after Infection: A Macrophage Proteomics Analysis.

During tuberculosis, Mycobacterium uses host macrophage cholesterol as a carbon and energy source. To mimic these conditions, Mycobacterium smegmatis can be cultured in minimal medium (MM) to induce cholesterol consumption in vitro. During cultivation, M. smegmatis consumes MM cholesterol and changes the accumulation of cell wall compounds, such as PIMs, LM, and LAM, which plays an important role in its pathogenicity. These changes lead to cell surface hydrophobicity modifications and H2O2 susceptibility. Furthermore, when M. smegmatis infects J774A.1 macrophages, it induces granuloma-like structure formation. The present study aims to assess macrophage molecular disturbances caused by M. smegmatis after cholesterol consumption, using proteomics analyses. Proteins that showed changes in expression levels were analyzed in silico using OmicsBox and String analysis to investigate the canonical pathways and functional networks involved in infection. Our results demonstrate that, after cholesterol consumption, M. smegmatis can induce deregulation of protein expression in macrophages. Many of these proteins are related to cytoskeleton remodeling, immune response, the ubiquitination pathway, mRNA processing, and immunometabolism. The identification of these proteins sheds light on the biochemical pathways involved in the mechanisms of action of mycobacteria infection, and may suggest novel protein targets for the development of new and improved treatments.

A Transcriptomic Approach Provides Insights on the Mycorrhizal Symbiosis of the Mediterranean Orchid Limodorum abortivum in Nature.

The study of orchid mycorrhizal interactions is particularly complex because of the peculiar life cycle of these plants and their diverse trophic strategies. Here, transcriptomics has been applied to investigate gene expression in the mycorrhizal roots of Limodorum abortivum, a terrestrial mixotrophic orchid that associates with ectomycorrhizal fungi in the genus Russula. Our results provide new insights into the mechanisms underlying plant-fungus interactions in adult orchids in nature and in particular into the plant responses to the mycorrhizal symbiont(s) in the roots of mixotrophic orchids. Our results indicate that amino acids may represent the main nitrogen source in mycorrhizal roots of L. abortivum, as already suggested for orchid protocorms and other orchid species. The upregulation, in mycorrhizal L. abortivum roots, of some symbiotic molecular marker genes identified in mycorrhizal roots from other orchids as well as in arbuscular mycorrhiza, may mirror a common core of plant genes involved in endomycorrhizal symbioses. Further efforts will be required to understand whether the specificities of orchid mycorrhiza depend on fine-tuned regulation of these common components, or whether specific additional genes are involved.

Accelerating microbial iron cycling promotes re-cementation of surface crusts in iron ore regions.

Accelerating microbial iron cycling is an innovative environmentally responsible strategy for mine remediation. In the present study, we extend the application of microbial iron cycling in environmental remediation, to include biocementation for the aggregation and stabilization of mine wastes. Microbial iron reduction was promoted monthly for 10 months in crushed canga (a by-product from iron ore mining, dominated by crystalline iron oxides) in 1 m3 containers. Ferrous iron concentrations reached 445 ppm in treatments and diverse lineages of the candidate phyla radiation dominated pore waters, implicating them in fermentation and/or metal cycling in this system. After a 6-month evaporation period, iron-rich cements had formed between grains and 20-cm aggregates were recoverable from treatments throughout the 1-m depth profile, while material from untreated and water-only controls remained unconsolidated. Canga-adapted plants seeded into one of the treatments germinated and grew well. Therefore, application of this geobiotechnology offers promise for stabilization of mine wastes, as well as re-formation of surface crusts that underpin unique and threatened plant ecosystems in iron ore regions.

Proteomic and Transcriptomic Analyses Indicate Metabolic Changes and Reduced Defense Responses in Mycorrhizal Roots of Oeceoclades maculata (Orchidaceae) Collected in Nature.

Orchids form endomycorrhizal associations with fungi mainly belonging to basidiomycetes. The molecular events taking place in orchid mycorrhiza are poorly understood, although the cellular changes necessary to accommodate the fungus and to control nutrient exchanges imply a modulation of gene expression. Here, we used proteomics and transcriptomics to identify changes in the steady-state levels of proteins and transcripts in the roots of the green terrestrial orchid Oeceoclades maculata. When mycorrhizal and non-mycorrhizal roots from the same individuals were compared, 94 proteins showed differential accumulation using the label-free protein quantitation approach, 86 using isobaric tagging and 60 using 2D-differential electrophoresis. After de novo assembly of transcriptomic data, 11,179 plant transcripts were found to be differentially expressed, and 2175 were successfully annotated. The annotated plant transcripts allowed the identification of up- and down-regulated metabolic pathways. Overall, proteomics and transcriptomics revealed, in mycorrhizal roots, increased levels of transcription factors and nutrient transporters, as well as ethylene-related proteins. The expression pattern of proteins and transcripts involved in plant defense responses suggested that plant defense was reduced in O. maculata mycorrhizal roots sampled in nature. These results expand our current knowledge towards a better understanding of the orchid mycorrhizal symbiosis in adult plants under natural conditions.

Rhizosphere microbiological processes and eucalypt nutrition: Synthesis and conceptualization.

In this review, we present the state of art regarding rhizosphere effects on eucalypt plantations. It provides a greater understanding of carbon (C) and nitrogen (N) turnover in forest soils. P organic hydrolysis, soil mineral solubilization, indoleacetic acid, gibberellin, resistance factors, and production of siderophores by rhizosphere microbial populations help to explain the tolerance of Eucalyptus plants to biotic and abiotic stresses and the apparent steady-state condition of C and N soil stocks in many planted forests. This work aims to present the main findings on Eucalyptus rhizosphere processes and highlights their importance for trees nutrition, especially for N mineralization triggered by microbial activation or microbial community structure changes regarding the so-called rhizosphere priming effect and N fixation. Furthermore, we present an explanatory conceptual model of the steady-state condition for soil organic matter (SOM) stocks and its relation with fertilization based on a nutrient balance model. This review also considers the main experimental and modeling studies that demonstrate the quantitative importance of rhizosphere processes to Eucalyptus genus and their shortcomings. This provides a framework for process modeling under scenarios of global climate change. A better understanding of rhizosphere microbiological processes may allow improvements in Eucalyptus nutrition and production, as well as in accurate long-term estimates of SOM stocks and C-CO2 exchanges between forest soils and the atmosphere.

Mycorrhizal Fungi Isolated from Native Terrestrial Orchids from Region of La Araucanía, Southern Chile.

Mycorrhizal interactions of orchids are influenced by several environmental conditions. Hence, knowledge of mycorrhizal fungi associated with orchids inhabiting different ecosystems is essential to designing recovery strategies for threatened species. This study analyzes the mycorrhizal associations of terrestrial orchids colonizing grassland and understory in native ecosystems of the region of La Araucanía in southern Chile. Mycorrhizal fungi were isolated from peloton-containing roots and identified based on the sequence of the ITS region. Their capacities for seed germination were also investigated. We detected Tulasnella spp. and Ceratobasidium spp. in the pelotons of the analyzed orchids. Additionally, we showed that some Ceratobasidium isolates effectively induce seed germination to differing degrees, unlike Tulasnella spp., which, in most cases, fail to achieve protocorm growth. This process may underline a critical step in the life cycle of Tulasnella-associated orchids, whereas the Ceratobasidium-associated orchids were less specific for fungi and were effectively germinated with mycorrhizal fungi isolated from adult roots.

Coupling physiological analysis with proteomic profile to understand the photosynthetic responses of young Euterpe oleracea palms to drought.

This study examined whether drought sensitivity in açaí (Euterpe oleracea Mart.) is associated with reductions in photosynthesis and increasing oxidative stress in response to down-regulation of proteins related to photosynthetic reactions, photorespiration, and antioxidant system. Well-watered (Control) and drought-stressed plants were compared when leaf water potential in stressed plants reached around - 1.5 and - 3.0 MPa, representing moderate and severe drought. Drought caused 84 and 96% decreases in net photosynthetic rate (Pn) and stomatal conductance. Stress-mediated changes in maximum quantum efficiency of photosystem II (PSII) photochemistry were unobserved, but drought decreased photochemical quenching, actual quantum yield of PSII electron transport, and apparent electron transport rate (ETR). Moderate and severe drought induced, respectively, decreases and increases in non-photochemical quenching (NPQ) and 74 and 273% increases in ETR/Pn. Moderate drought down-regulated PSII protein D2, chlorophyll a-b binding protein 8, photosystem I reaction center subunit N, sedoheptulose-1,7-bisphosphatase, and transketolase; while severe drought down-regulated LHC II proteins, ferredoxin-NADP reductase, ATP synthase subunits ε and ß, and carbonic anhydrase isoform X2. The glutamate-glyoxylate aminotransferase 2 and glycine dehydrogenase were down-regulated upon moderate drought, while catalase 2 and glycine cleavage system H protein 3 were up-regulated. Severe drought up-regulated glycolate oxidase, glycine cleavage system H protein 3, and aminomethyl transferase, but most of photorespiration-related proteins were only found in control plants. Down-regulation of chaperones and antioxidant enzymes and increased lipid peroxidation in stressed plants were observed upon both stress severities. Therefore, the decreases in Pn and failure in preventing oxidative damages through adjustments in NPQ and photorespiration- and antioxidant-related proteins accounted for drought sensitivity in açaí.

Adaptation and tolerance mechanisms developed by mycorrhizal Bipinnula fimbriata plantlets (Orchidaceae) in a heavy metal-polluted ecosystem.

The adaptation and performance of orchid mycorrhizae in heavy metal-polluted soils have been poorly explored. In the present study, proteomic and metabolic approaches were used to detect physiological changes in orchid roots established in a heavy metal-polluted soil and to ascertain whether mycorrhizal fungi affect the metabolic responses of roots. Young Bipinnula fimbriata plantlets were established in control and heavy metal-polluted soils in a greenhouse. After 14 months, exudation of root organic acids, phenolics, percentage of mycorrhization, mineral content, and differential protein accumulation were measured. More root biomass, higher root colonization, and higher exudation rates of citrate, succinate, and malate were detected in roots growing in heavy metal-polluted soils. Higher accumulation of phosphorus and heavy metals was found inside mycorrhizal roots under metal stress. Under non-contaminated conditions, non-mycorrhizal root segments showed enhanced accumulation of proteins related to carbon metabolism and stress, whereas mycorrhizal root segments stimulated protein synthesis related to pathogen control, cytoskeleton modification, and sucrose metabolism. Under heavy metal stress, the proteome profile of non-mycorrhizal root segments indicates a lower induction of defense mechanisms, which, together with the stimulation of enzymes related to carotenoid biosynthesis and cell wall organization, may positively influence mycorrhizal fungi colonization. The results point to different metabolic strategies in mycorrhizal and non-mycorrhizal root segments that are exposed to heavy metal stress. The results indicate that root colonization by mycorrhizal fungi is stimulated to alleviate the negative effects of heavy metals in the orchids.

Differential accumulation of proteins in oil palms affected by fatal yellowing disease.

There is still no consensus on the true origin of fatal yellowing, one of the most important diseases affecting oil palm (Elaeis guineensis Jacq.) plantations. This study involved two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-UPLC-MSE) analyses to identify changes in protein profiles of oil palms affected by FY disease. Oil palm roots were sampled from two growing areas. Differential accumulation of proteins was assessed by comparing plants with and without symptoms and between plants at different stages of FY development. Most of the proteins identified with differential accumulation were those related to stress response and energy metabolism. The latter proteins include the enzymes alcohol dehydrogenase and aldehyde dehydrogenase, related to alcohol fermentation, which were identified in plants with and without symptoms. The presence of these enzymes suggests an anaerobic condition before or during FY. Transketolase, isoflavone reductase, cinnamyl alcohol dehydrogenase, caffeic acid 3-O-methyltransferase, S-adenosylmethionine synthase, aldehyde dehydrogenase and ferritin, among others, were identified as potential marker proteins and could be used to guide selection of FY-tolerant oil palm genotypes or to understand the source of this anomaly. When comparing different stages of FY, we observed high accumulation of alcohol dehydrogenase and other abiotic stress related-proteins at all disease stages. On the other hand, biological stress-related proteins were more accumulated at later stages of the disease. These results suggest that changes in abiotic factors can trigger FY development, creating conditions for the establishment of opportunistic pathogens.

Mycorrhizal compatibility and symbiotic seed germination of orchids from the Coastal Range and Andes in south central Chile.

Little is known about Orchidaceae plants in Chile and their mycorrhizal associations, a key issue for designing protective actions for endangered species. We investigated root fungi from seven terrestrial orchid species to identify potential mycorrhizal fungi. The main characteristics of Rhizoctonia-like fungi were observed under light microscopy, and isolates were identified through PCR-ITS sequencing. Molecular identification of fungal sequences showed a high diversity of fungi colonizing roots. Fungal ability to germinate seeds of different orchids was determined in symbiotic germination tests; 24 fungal groups were isolated, belonging to the genera Tulasnella, Ceratobasidium, and Thanatephorus. Furthermore, dark septate and other endophytic fungi were identified. The high number of Rhizoctonia-like fungi obtained from adult orchids from the Coastal mountain range suggests that, after germination, these orchids may complement their nutritional demands through mycoheterotrophy. Nonetheless, beneficial associations with other endophytic fungi may also co-exist. In this study, isolated mycorrhizal fungi had the ability to induce seed germination at different efficiencies and with low specificity. Germin ation rates were low, but protocorms continued to develop for 60 days. A Tulasnella sp. isolated from Chloraea gavilu was most effective to induce seed germination of different species. The dark septate endophytic (DSE) fungi did not show any effect on seed development; however, their widespread occurrence in some orchids suggests a putative role in plant establishment.