OUR VISION AND AIM

Fungal endophytes (FEs) are a diverse group of fungi that colonize internal plant tissues without causing disease. Many FEs are beneficial, promoting plant growth and enhancing tolerance to biotic and abiotic stresses, what makes them promising candidates for developing novel sustainable solutions for agriculture. However, the great potential of FEs in agriculture remains limited due to inconsistent field performance. In addition, some FEs are closely related to pathogens and may shift toward pathogenic behaviour depending on environmental cues or specific host–fungal genetic determinants. To ensure the safe and effective use of FEs in agriculture, it is essential to understand the mechanisms that govern their interaction with the plant, the conditions that maximize or reduce their benefit, the plant and fungal genes that determine the outcome of the interaction, or the molecules that modulate it.

Our group aims to:
  1. elucidate the molecular and environmental factors that affect fungal endophytism and their relationship with mutualistic and pathogenic interactions with the plant.
  2. effectively apply FEs and their derived molecules to improve crop productivity and agriculture sustainability.

OUR MODEL SYSTEM AND MAJOR ACHIEVEMENTS
Natural FEs of Arabidopsis thaliana
As model system, we use natural FEs of Arabidopsis thaliana. After several years of surveys in ecologically distinct populations across Central Spain, we have established a collection of more than 700 fungal isolates (Figure 1, García et al., 2013). This pioneering work provided the first description of the fungal endophytic assemblages of this model plant species. Screening these isolates under controlled conditions has yielded valuable model systems for hypothesis generation and testing, taking advantage of the extensive molecular and genetic resources available for Arabidopsis.


Figure 1. FEs collection from natural populations of A. thaliana. A. Schematic protocol for the isolation of FEs from A. thaliana plants. B. Arabidopsis plants at a natural site. C. Intercellular endophytic mycelium in a leaf. D. Emerging mycelium from a silique after incubation in a humid chamber.

 

Colletotrichum tofieldiae (Ct)
A major milestone of our research was the discovery of the mutualistic interaction between Colletotrichum tofieldiae (Ct) and A. thaliana. We demonstrated that Ct isolates of our FE collection enhance seed production in Arabidopsis (Patent WO2015/092104). In collaboration with Paul Schulze-Lefert (Max Planck Institute for Plant Breeding Research, Germany), we showed that Ct colonization promotes Arabidopsis growth under phosphate (Pi) limitation (Figure 2, Hiruma et al., 2016). Comparative genomics with related pathogenic Colletotrichum species revealed that mutualistic behaviour is associated with a reduced repertoire of secreted effector proteins and a limited activation of pathogenicity-related genes in planta (Hacquard et al., 2016).


Figure 2. Ct colonizes and promotes growth of Arabidopsis under phosphate (Pi) deficient conditions. A. Representative image of A. thaliana plants grown in low phosphate (Pi) conditions with and without Ct. B. Shoot fresh weight (g) of Arabidopsis plants incubated with Ct, heat killed Ct (HK) or pathogenic C. incanum (Ci) in high or low Pi conditions. C. GFP labelled Ct hyphae penetrating PIP2A-mCherry-labeled Arabidopsis roots. Hiruma et al. (2016) Cell 165: 464–474.
 

More recently, we have demonstrated the plant growth promoting activity of Ct in crops  such as tomato and maize (Figure 3, Díaz-González et al., 2020), expanding the possibilities of studying this fungal endophyte in very different hosts, and opening the door for its use in agriculture. Moreover, microbiome analyses of field trials revealed a reduced abundance of the mycotoxigenic fungus Aspergillus in grains of Ct-treated maize plants, what was confirmed under controlled conditions (Figure 4, Díaz-González et al. 2025, patent WO2024/086627).

 

Figure 3. Ct promotes growth and increases yield of tomato and maize. Representative image of maize (A) and tomato (B) seedlings from seeds inoculated with Ct (Ct0861), or water for control, and grown in MS medium for six days. C. Maize yield (t/ha) under different Ct (Ct0861) treatments compared to control plots in an open-field trial. Díaz-González et al. (2020) Agronomy 10: 1493.

 

 

Figure 4. Ct reduces infection by Asperillus flavus and aflatoxins in maize grains. A. Representative image showing the mycelial growth of A. flavus at 21 days post inoculation (dpi) in cobs of maize plants treated or not with Ct (Ct0861). B. Quantification of A. flavus biomass in grains of maize plants treated or not with Ct (Ct0861) and inoculated with A. flavus at 5 and 21 dpi. Aflatoxin B1 (C) and B2 (D) concentration (µg/kg) in grains of maize plants inoculated or not with Ct (Ct0861) and inoculated with A. flavus at 21 dpi. Asterisks denote statistically significant differences in Student’s T Test (p < 0.05 *, p < 0.01 **). Díaz-González et al. (2025) Plants 14(21), 3236.

 

Host-dependent transcriptomic responses of Ct

We have compared the transcriptomes of Ct interactions with maize or Arabidopsis, identifying differentially expressed fungal genes potentially involved in host colonization. Most of them showed host-specific expression, indicating a specialized fungal response (Figure 5). The differential response is specially observed in transporters and genes associated with secondary metabolism.

 


Figure 5. Ct transcriptional response during interactions with maize and Arabidopsis. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in the Ct transcriptome during interactions with (A) maize at 7 days post-inoculation (dpi) and (B) Arabidopsis at 6 dpi, compared to axenic culture. GO terms related to transporters and secondary metabolism are highlighted in red and blue, respectively. (C) Venn diagrams showing Ct transporter genes (left) and secondary metabolism genes (right) that are upregulated or downregulated, and either shared between or specific to interactions with maize (4 and 7 dpi) and Arabidopsis.

 

Role of benzoxacinoids (Bx) in Ct–maize interaction

Our RNA-seq analysis of maize seedlings at early stages of the interaction with Ct have revealed a prominent role of maize secondary metabolite pathways related to defense (Figure 6). Strikingly, the BX biosynthetic pathway is specifically repressed in roots of maize colonized by Ct. Our experiments show that BXs inhibit maize colonization by Ct and its beneficial effect on the plant.


Figure 6. Benzoxazinoids (BXs) are repressed in Ct-maize interaction (A,B) and inhibit maize colonization by Ct (C, D) and Ct beneficial effect on the plant (E). A. Heat map showing maize gene expression in mock and Ct treated seedlings 4 or 7-days post inoculation (dpi). Asterisks indicate significant differences in Ct treated vs mock (LRT test with a Benjamini-Hochberg correction, Padj< 0.05). B. Relative expression levels of the initial genes of BXs biosynthetic pathway ZmBX1 and ZmBX2 quantified by qRtPCR using specific primers. ZmACTIN gene was used as reference. C. Ct biomass measured by fungal DNA quantification using Ct specific primers normalized to maize β-tubulin expression in roots and shoots of maize seedlings at 4 or 7 dpi. D. Ct biomass measured as in (C) in roots of W22 wild type and BXs deficient bx1 mutant maize plants at 7dpi. E. Root and shoot weight of W22 and bx1 mutant plants mock or Ct treated at 7 dpi. The percentage change in Ct-treated vs mock plants is shown.



KEY QUESTIONS WE ARE WORKING ON

How do FEs benefit plants, and what conditions enhance their beneficial effects?
We investigate how FEs enhance plant growth, stress tolerance, and pathogen resistance. To uncover EFs modes of action, we test their effects under different environmental conditions using an integrative approach that combines phenotypic, physiological, metabolic, and transcriptomic data.

What enables FEs to colonize diverse plant hosts without causing disease?
We analyse plant and fungal transcriptomes to identify common and host-specific genes essential for colonization. Functional analyses using plant and fungal mutants help us determine the roles of these key genes.

What do FEs obtain from their plant partners?
In collaboration with Marcel Bucher (CEPLAS, University of Cologne, Germany), we track labelled elements (P, N, C) to study the bidirectional nutrient exchange between FEs and different hosts. We are especially interested in the transporters and molecules that are key to sustain balanced and stable mutualistic interactions.

How can FEs be effectively applied in agriculture?
We collaborate with industry partners  to screen our FE collections for beneficial interactions and apply our knowledge to develop new solutions for sustainable agriculture.

 

Díaz González, Sandra - Postdoctoral Fellow

Gómez Vílchez, Lucía - PhD Student

González Sanz, Carlos - PhD Student

Martínez Arias, Clara - Assistant Lecturer

Monteoliva García, Gonzalo - PhD Student

Pérez Moreno, Lucía - TFG Student

Sacristán Benayas, María Soledad - Associate Professor

Szygut, Sylwia - Technician

Wang, Bo - PhD Student

  1. PID2024-161830OB-I00. New insights into the molecular mechanisms behind the beneficial interaction between the fungal endophyte Colletotrichum tofieldiae and the plant. 2025-2028. Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación (MICIU/AEI), Spain. PI: Soledad Sacristán.



  2. EoI-CSPINT08-MOLEXBPI. Edaphic and genetic effects on molecular exchange and feedback mechanisms underlying beneficial plant-fungal endophyte interaction. 2024-2025. Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación (MICIU/AEI), Spain (CEX2020-000999-S-20-3). Co-PIs: Soledad Sacristán/Marcel Bucher.xxxxxxxx

      

  3. EoI-MCBGP21 PLANTADAPT_05_NUTRI-HEAT. Deciphering novel plant responses and plant-microbiome interactions under heat stress and nutrition scarcity (μNUTRI-HEAT). 2023 - 2025. Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación (MICIU/AEI), Spain (CEX2020-000999-S-20-3). PI: Juan Carlos del Pozo. Co-PIs: Stephan Pollmann/Mar Castellano/Soledad Sacristan/Jaime Huerta-Cepas.

      

  4. PID2021-123697OB-I00. Análisis integrado del modo de acción del hongo endófito Colletotrichum tofieldiae en cultivos. 2022-2026. Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación (MICIU/AEI), Spain.   PI: Soledad Sacristán.



  5. TED2021-130317B-I00. Transición hacia una agricultura sostenible basada en la economía circular y en nuevas soluciones de base biológica. 2022-2024. Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación (MICIU/AEI), Spain and European Union NextGenerationEU/PRTR.   PIs: Antonio Molina/Juan Carlos del Pozo.



  6. EOI-TSP3-03. Toward a Sustainable Agriculture: Understanding Root Responses to Phosphate Starvation to improve plant nutrition and productivity. 2018-2022. Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación (MICIU/AEI), Spain and Severo Ochoa Excellence program (SEV-2016-0672 (2017-2021).  PI: Juan Carlos del Pozo. Co-Pis: Soledad Sacristán/ Mar Castellano.

      

  7. BFU2015-72484-EXP. Sobrecompensación en respuesta a un hongo no patogénico. 2017-2020. Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación (MICIU/AEI), Spain.  PI: Soledad Sacristán.



     

FUNDING FOR HIRING PERSONNEL
  1. PID2024-161830OB-I00 FPI Predoctoral contract. 2026-2029. Funded by MICIU/AEI /10.13039/501100011033 and ESF+



  2. PEJ-2024-TL_BIO-33107 Laboratory Technician contract for Sylwia Szygut. 2025-2027. Funded by Consejería de Educación, Ciencia y Universidades de Comunidad de Madrid and ESF+.



  3. CSC 202309150001 Predoctoral contract for Bo Wang. 2024-2027. Funded by the China Scholarship Council



  4. PRE 2022 105238 FPI Predoctoral contract for Gonzalo Monteoliva García 2024-2027. Funded by MICIU/AEI /10.13039/501100011033 and ESF+



  5. PRE2022-103983 FPI Predoctoral contract for Carlos González Sanz 2024-2027. Funded by MICIU/AEI /10.13039/501100011033 and ESF+



  6. Margarita Salas Grant for junior doctors (RD 289/2021) for Sandra Díaz González 2022-2023. Funded by the Spanish Ministry of Science, Innovation and Universities (MCIU/AEI/https://doi.org/10.13039/501100011033) and the European Union – NextGenerationEU/PRTR



  7. PEJ-2020-AI/BIO-Research Assistant contract for Carlos González Sanz. 2021-2022. Funded by Consejería de Educación, Ciencia y Universidades de Comunidad de Madrid and ESF



  8. PEJ-2018-TL/BIO-11749 Laboratory Technician contract for Rubén Martín Antoranz. 2019-2020. Funded by Consejería de Educación, Ciencia y Universidades de Comunidad de Madrid and ESF



  9. PEJ2018-005339-A. Laboratory Technician contract for Palmira del Prado Polonio. 2019-2020. Funded by MICIU/AEI /10.13039/501100011033



  10. BES-2017-082152. Predoctoral contract for Carlos Conesa Quintana 2018-2022. Funded by MICIU/AEI /10.13039/501100011033 and ESF Investing in your future.



  11. DI-15-07906. Predoctoral contract for Industrial Doctorate for Sandra Díaz González. 2016-2020. Characterization of endophyte microorganisms of plants for the development of commercial formulations that improve the physiology and productivity of crops. Funded by MICIU/AEI /10.13039/501100011033. PI. Frederic Brunner (Plant Response Biotec S.L)

Rodríguez de Lope, M.M., Sánchez-Pajares, I.R., Herranz, E., López-Vázquez, C.M., González-Moro, A., Rivera-Tenorio, A., González-Sanz, C., Sacristán, S., Chicano-Gálvez, E., de la Cuesta, F. 2025. A Compendium of Bona Fide Reference Markers for Genuine Plant Extracellular Vesicles and Their Degree of Phylogenetic Conservation. Journal of Extracellular Vesicles 14, e70147. DOI: 10.1002/jev2.70147

Catarecha, P., King, E., Díaz-González, S., Caro, E., Sacristán, S., Del Pozo Benito, J.C. 2025. Heat Stress and Soil Thermal Gradients Shape Root-Associated Fungal Community Recruitment. Frontiers in Microbiology 16. DOI: 10.3389/fmicb.2025.1334648

Díaz-González, S., González-Bodí, S., González-Sanz, C., Marín, P., Brunner, F., Sacristán, S. 2025. Maize associated bacterial and fungal microbiomes show contrasting conformation patterns dependent on plant compartment and water availability. BMC Plant Biology 25, 448. DOI: 10.1186/s12870-025-06465-2

Díaz-González, S., Andrés, M.F., González-Sanz, C., Sacristán, S., González-Coloma, A. 2025. Nematicidal and antifeedant activity of ethyl acetate extracts from culture filtrates of Arabidopsis thaliana fungal endophytes. Scientific Reports 15, 11332. DOI: 10.1038/s41598-025-94939-6

Montesinos, Á., Sacristán, S., del Prado-Polonio, P., Arnaiz, A., Díaz-González, S., Diaz, I., Santamaria, M.E. 2024. Contrasting plant transcriptome responses between a pierce-sucking and a chewing herbivore go beyond the infestation site. BMC Plant Biology 24, 120. DOI: 10.1186/s12870-024-04806-1

Fiallo-Olivé, E., Palacio-Bielsa, A., Sacristán, S. 2023. Plant Pathogenic Microorganisms: State-of-the-Art Research in Spain. Microorganisms 11, 816. DOI: 10.3390/microorganisms11030816

Poveda, J., Baptista, P., Sacristán, S., Velasco, P. 2022. Editorial: Beneficial effects of fungal endophytes in major agricultural crops. Frontiers in Plant Science 13. DOI: 10.3389/fpls.2022.1061112

Poveda, J., Díaz-González, S., Díaz-Urbano, M., Velasco, P., Sacristán, S. 2022. Fungal endophytes of Brassicaceae: Molecular interactions and crop benefits. Frontiers in Plant Science 13. DOI: 10.3389/fpls.2022.932288

Sacristán, S., Goss, E.M., Eves-van den Akker, S. 2021. How Do Pathogens Evolve Novel Virulence Activities?. Molecular Plant-Microbe Interactions® MPMI-09-20-0258-IA. DOI: 10.1094/MPMI-09-20-0258-IA

Díaz-González, S., Marín, P., Sánchez, R., Arribas, C., Kruse, J., González-Melendi, P., Brunner, F., Sacristán, S. 2020. Mutualistic Fungal Endophyte Colletotrichum tofieldiae Ct0861 Colonizes and Increases Growth and Yield of Maize and Tomato Plants. Agronomy 10, 1493. DOI: 10.3390/agronomy10101493

Muñoz-Barrios, A., Sopeña-Torres, S., Ramos, B., López, G., Del Hierro García, I., Díaz-González, S., González-Melendi, P., Mélida, H., Fernández-Calleja, V., Mixão, V., Martín Dacal, M., Marcet-Houben, M., Gabaldón, T., Sacristan, S., Molina, A. 2020. Differential expression of fungal genes determines the lifestyle of Plectosphaerella strains during Arabidopsis thaliana colonization. Molecular Plant-Microbe Interactions®. DOI: 10.1094/MPMI-03-20-0057-R

Valverde, S., Vidiella, B., Montañez, R., Fraile, A., Sacristán, S., García-Arenal, F. 2020. Coexistence of nestedness and modularity in host–pathogen infection networks. Nature Ecology & Evolution. DOI: 10.1038/s41559-020-1130-9

Conesa, C.M., Saez, A., Navarro-Neila, S., de Lorenzo, L., Hunt, A.G., Sepúlveda, E.B., Baigorri, R., Garcia-Mina, J.M., Zamarreño, A.M., Sacristán, S., del Pozo, J.C. 2020. Alternative Polyadenylation and Salicylic Acid Modulate Root Responses to Low Nitrogen Availability. Plants 9, 251. DOI: 10.3390/plants9020251