Background Maize (Zea mays L.) is a globally critical crop that faces numerous challenges, including contamination by mycotoxigenic fungi such as Aspergillus spp., which threaten food safety and marketability. This study evaluates the potential of the fungal endophyte Colletotrichum tofieldiae strain Ct0861 as a bioinoculant to enhance maize productivity and investigates its impact on the maize-associated bacterial and fungal microbiomes. Results Field trials showed that Ct0861 treatment enhances biomass and yield compared to controls, irrespective of the application method assayed (seed or spray application). Comprehensive microbiome profiling across soil, rhizosphere, roots, leaves, and grains revealed that Ct0861 inoculation induced subtle, compartment-specific effects on microbial diversity and composition, with similar effects for the two application methods used. Fungal alpha-diversity in grains was significantly reduced, while beta-diversity analyses showed localized shifts, particularly in soil and grain-associated microbial communities. Despite these changes, the core microbiome assemblages remained stable, indicating minimal alteration to the broader microbiome structure. Remarkably, Ct0861 significantly reduced the prevalence of Aspergillus spp. in maize grains, as confirmed by controlled infection assays. This reduction resulted in lower aflatoxin levels, demonstrating the biocontrol potential of Ct0861. Conclusion These findings underscore Ct0861's dual benefits in enhancing crop yield and safety reducing fungal mycotoxin contamination. Further studies are necessary to elucidate the underlying mechanisms and expand its application across diverse agroecosystems. ### Competing Interest Statement S.S. is inventor of the patent WO2015/092104 assigned to UPM and PlantResponse Biotech, S.L. S.D.-G, C.G.-S, P.M., F.B. and S.S. are inventors of the patent applications ES-2969994 and PCT/US23/77169 assigned to UPM and Plant Response, Inc.

Maize (Zea mays L.) is a globally critical crop that faces numerous challenges, including contamination by mycotoxigenic fungi such as Aspergillus spp. The use of fungal endophytes as bioinoculants offers a sustainable strategy to improve plant resilience against biotic and abiotic stresses. Here, we evaluate the potential of Colletotrichum tofieldiae strain Ct0861 as a bioinoculant and its impact on maize-associated bacterial and fungal microbiomes. Field trials demonstrated that Ct0861 enhanced biomass and yield compared to controls, regardless of the application method (seed or foliar). Microbiome profiling showed that Ct0861 induced subtle, compartment-specific changes in microbial diversity and composition, while preserving the stability of core microbiome assemblages. Both microbiome data and qPCR quantification confirmed a significant reduction in Aspergillus spp. abundance in Ct0861-treated plants. Greenhouse assays corroborated these results: Ct0861 reduced A. flavus biomass by up to 90% and significantly lowered aflatoxin levels in infected grains. Dual-culture assays and the absence of Ct0861 in grain samples suggest an indirect biocontrol mechanism, potentially mediated by plant-induced resistance. This study provides the first evidence that Ct0861 acts as a biocontrol agent against mycotoxigenic Aspergillus spp. in maize. Beyond promoting plant growth, Ct0861 enhances food safety by reducing mycotoxin accumulation without disrupting the native microbiome, supporting its potential as a tool for sustainable crop protection.

Plant-associated microorganisms can help crops to alleviate stress and increase the resilience of agricultural ecosystems to climate change. However, we still lack knowledge on the dynamics of soil and plant microbiomes and their response to changing conditions. This information is essential for the development of microbiome-based solutions to improve crop resilience to stressors associated with climate change. In this work, we explored: (i) the conformation of the bacterial and fungal assemblages of different soil and plant compartments (bulk soil, rhizosphere, roots, leaves and grains) along the crop cycle of maize in an open field trial; and (ii) the effect of water restriction on the maize microbiome, comparing optimal irrigation with a 30% reduction of water supply. Our results show a dynamic compartment-driven recruitment of microorganisms with contrasting patterns for bacteria and fungi that were intensified towards the end of the plant cycle. Roots showed the most differentiated bacterial assemblage while fungi conformed a very distinct community in the leaves, suggesting a relevant contribution of aerial fungal propagules to the microbiome of this plant organ. Regarding the grain, bacterial communities looked closer to those in the leaves, while fungal communities were more like those in the root. Despite the reductions in plant growth and yield, the microbiome of limited-watered plants did not show severe alterations. Still, significant impacts were observed within compartments, being fungi more responsive to limited watering than bacteria, with hallmark fungal ASVs for each compartment and irrigation regime. Network analysis suggests that bacteria and fungi may play different roles in the shifts observed under water limitation. Our study highlights the importance of conducting multikingdom analyses for a holistic understanding of the dynamics and evolution of the microbial assemblages in the whole plant and their roles in plant response to environmental stressors.

Plant-associated microorganisms can help crops to alleviate water stress and increase the resilience of agricultural ecosystems to climate change. However, we still lack knowledge on the dynamics of bacterial and fungal microbial kingdoms within the soil and plant microbiomes and the response of these communities to different conditions such us, for example, water restrictions. This information is essential for the development of microbiome-based solutions to improve crop resilience to stressors associated to climate change. In this work, we explored: i) the conformation of the bacterial and fungal assemblages of different soil and plant compartments (bulk soil, rhizosphere, roots, leaves and grains) along the crop cycle of maize in an open field trial; and ii) the effect of water restriction on the maize microbiome comparing optimal irrigation with a 30% reduction of water supply. Our results show that microbial communities are highly structured along soil and plant compartments, with contrasting patterns for bacteria and fungi that were intensified towards the end of the plant cycle. Root showed the most differentiated bacterial assemblage while fungi conformed a very distinct community in the leaf, suggesting a relevant contribution of aerial fungal propagules to the microbiome of this plant organ. Despite the reductions in plant growth and yield, the microbiome of limited-watered plants did not show severe alterations. Still, significant impacts were observed within compartments, being fungi more responsive to limited watering than bacteria. Network analysis suggest that bacteria and fungi may play different roles in the shifts observed under water stress.### Competing Interest StatementThe authors have declared no competing interest.

Plant cell walls are complex structures mainly made up of carbohydrate and phenolic polymers. In addition to their structural roles, cell walls function as external barriers against pathogens and are also reservoirs of glycan structures that can be perceived by plant receptors, activating Pattern-Triggered Immunity (PTI). Since these PTI-active glycans are usually released upon plant cell wall degradation, they are classified as Damage Associated Molecular Patterns (DAMPs). Identification of DAMPs imply their extraction from plant cell walls by using multistep methodologies and hazardous chemicals. Subcritical water extraction (SWE) has been shown to be an environmentally sustainable alternative and a simplified methodology for the generation of glycan-enriched fractions from different cell wall sources, since it only involves the use of water. Starting from Equisetum arvense cell walls, we have explored two different SWE sequential extractions (isothermal at 160 ºC and using a ramp of temperature from 100 to 160 ºC) to obtain glycans-enriched fractions, and we have compared them with those generated with a standard chemical-based wall extraction. We obtained SWE fractions enriched in pectins that triggered PTI hallmarks in Arabidopsis thaliana such as calcium influxes, reactive oxygen species production, phosphorylation of mitogen activated protein kinases and overexpression of immune-related genes. Notably, application of selected SWE fractions to pepper plants enhanced their disease resistance against the fungal pathogen Sclerotinia sclerotiorum. These data support the potential of SWE technology in extracting PTI-active fractions from plant cell wall biomass containing DAMPs and the use of SWE fractions in sustainable crop production.

Summary Pattern‐triggered immunity (PTI) is activated in plants upon recognition by pattern recognition receptors (PRRs) of damage‐ and microbe‐associated molecular patterns (DAMPs and MAMPs) derived from plants or microorganisms, respectively. To understand better the plant mechanisms involved in the perception of carbohydrate‐based structures recognized as DAMPs/MAMPs, we have studied the ability of mixed‐linked β‐1,3/1,4‐glucans (MLGs), present in some plant and microbial cell walls, to trigger immune responses and disease resistance in plants. A range of MLG structures were tested for their capacity to induce PTI hallmarks, such as cytoplasmic Ca 2+ elevations, reactive oxygen species production, phosphorylation of mitogen‐activated protein kinases and gene transcriptional reprogramming. These analyses revealed that MLG oligosaccharides are perceived by Arabidopsis thaliana and identified a trisaccharide, β‐ d ‐cellobiosyl‐(1,3)‐β‐ d ‐glucose (MLG43), as the smallest MLG structure triggering strong PTI responses. These MLG43‐mediated PTI responses are partially dependent on LysM PRRs CERK1, LYK4 and LYK5, as they were weaker in cerk1 and lyk4 lyk5 mutants than in wild‐type plants. Cross‐elicitation experiments between MLG43 and the carbohydrate MAMP chitohexaose [β‐1,4‐ d ‐(GlcNAc) 6 ], which is also perceived by these LysM PRRs, indicated that the mechanism of MLG43 recognition could differ from that of chitohexaose, which is fully impaired in cerk1 and lyk4 lyk5 plants. MLG43 treatment confers enhanced disease resistance in A. thaliana to the oomycete Hyaloperonospora arabidopsidis and in tomato and pepper to different bacterial and fungal pathogens. Our data support the classification of MLGs as a group of carbohydrate‐based molecular patterns that are perceived by plants and trigger immune responses and disease resistance.

Immune responses in plants can be triggered by damage/microbe-associated molecular patterns (DAMPs/MAMPs) upon recognition by plant pattern recognition receptors (PRRs). DAMPs are signaling molecules synthesized by plants or released from host cellular structures (e.g., plant cell walls) upon pathogen infection or wounding. Despite the hypothesized important role of plant cell wall-derived DAMPs in plant-pathogen interactions, a very limited number of these DAMPs are well characterized. Recent work demonstrated that pectin-enriched cell wall fractions extracted from the cell wall mutant impaired in Arabidopsis Response Regulator 6 (arr6), that showed altered disease resistance to several pathogens, triggered more intense immune responses than those activated by similar cell wall fractions from wild-type plants. It was hypothesized that arr6 cell wall fractions could be differentially enriched in DAMPs. In this work, we describe the characterization of the previous immune-active fractions of arr6 showing the highest triggering capacities upon further fractionation by chromatographic means. These analyses pointed to a role of pentose-based oligosaccharides triggering plant immune responses. The characterization of several pentose-based oligosaccharide structures revealed that β-1,4-xylooligosaccharides of specific degrees of polymerization and carrying arabinose decorations are sensed as DAMPs by plants. Moreover, the pentasaccharide 33-α-L-arabinofuranosyl-xylotetraose (XA3XX) was found as a highly active DAMP structure triggering strong immune responses in Arabidopsis thaliana and enhancing crop disease resistance.

Facing rising global food demand in a sustainable way is a great challenge of modern agriculture. Thus, the increase of crop productivity and resilience in an adverse climate scenario is urgently needed. Fungal endophytes have been described as potential biological tools to improve plant yield and tolerance to biotic and abiotic stresses; however, their application in agriculture needs further research. The fungal endophyte Colletotrichum tofieldiae strain Ct0861 establishes a mutualistic interaction with Arabidopsis thaliana, promoting plant growth and silique production at low phosphate conditions. Until now, its ability to colonize and confer benefits to other plant species remained unexplored. Here, we show that Ct0861 colonizes and promotes growth in vitro of maize (Zea mays L.) and tomato (Solanum lycopersicum L.) seedlings, resulting in significantly larger shoot length and weight. Greenhouse and field experiments in optimal nutritional conditions showed an increase between 12% and 22% of yield in both tomato and maize. The inoculated plants were not suffering from phosphate starvation, which points at different modes of action not elucidated yet. These results indicate that the beneficial effect of Ct0861 may extend to other plant species of economic importance, making Ct0861 a potentially valuable inoculant.

To be successful plant pathogens, microbes use "effector proteins" to manipulate host functions to their benefit. Identifying host targets of effector proteins and characterizing their role in the infection process allow us to better understand plant-pathogen interactions and the plant immune system. Yeast two-hybrid analysis and coimmunoprecipitation were used to demonstrate that the Phytophthora infestans effector AVIRULENCE 2 (PiAVR2) interacts with all three BRI1-SUPPRESSOR1-like (BSL) family members from potato (Solanum tuberosum). Transient expression of BSL1, BSL2, and BSL3 enhanced P. infestans leaf infection. BSL1 and BSL3 suppressed INFESTIN 1 elicitin-triggered cell death, showing that they negatively regulate immunity. Virus-induced gene silencing studies revealed that BSL2 and BSL3 are required for BSL1 stability and show that basal levels of immunity are increased in BSL-silenced plants. Immune suppression by BSL family members is dependent on the brassinosteroid-responsive host transcription factor CIB1/HBI1-like 1. The P. infestans effector PiAVR2 targets all three BSL family members in the crop plant S. tuberosum. These phosphatases, known for their role in growth-promoting brassinosteroid signaling, all support P. infestans virulence and thus can be regarded as susceptibility factors in late blight infection.

Pathogens secrete effector proteins to interfere with plant innate immunity, in which Ca2+/calmodulin (CaM) signalling plays key roles. Thus far, few effectors have been identified that directly interact with CaM for defence suppression. Here, we report that SFI5, an RXLR effector from Phytophthora infestans, suppresses microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) by interacting with host CaMs. We predicted the CaM-binding site in SFI5 using in silico analysis. The interaction between SFI5 and CaM was tested by both invitro and invivo assays. MTI suppression by SFI5 and truncated variants were performed in a tomato protoplast system. We found that both the predicted CaM-binding site and the full-length SFI5 protein interact with CaM in the presence of Ca2+. MTI responses, such as FRK1 upregulation, reactive oxygen species accumulation, and mitogen-activated protein kinase activation were suppressed by truncated SFI5 proteins containing the C-terminal CaM-binding site but not by those without it. The plasma membrane localization of SFI5 and its ability to enhance infection were also perturbed by loss of the CaM-binding site. We conclude that CaM-binding is required for localization and activity of SFI5. We propose that SFI5 suppresses plant immunity by interfering with immune signalling components after activation by CaMs.

Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato. X. euvesicatoria bacteria interfere with plant cellular processes by injecting effector proteins into host cells through the type III secretion (T3S) system. About 35 T3S effectors have been identified in X. euvesicatoria 85-10, and a few of them were implicated in suppression of pattern-triggered immunity (PTI). We used anArabidopsis thaliana pathogen-free protoplast based assay to identify X. euvesicatoria 85-10 effectors that interfere with PTI signaling induced by the bacterial peptide flg22. Of 33 tested effectors, 17 inhibited activation of a PTI-inducible promoter. Among them, nine effectors also interfered with activation of an abscisic acid inducible promoter. However, effectors that inhibited flg22-induced signaling did not affect phosphorylation of mitogen-activated protein (MAP) kinases acting downstream of flg22 perception. Further investigation of selected effectors revealed that XopAJ, XopE2, and XopF2 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. The effectors XopF2, XopE2, XopAP, XopAE, XopH, and XopAJ inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria. Finally, selected effectors were found to localize to various plant subcellular compartments. These results indicate that X. euvesicatoria bacteria utilize multiple T3S effectors to suppress flg22-induced signaling acting downstream or in parallel to MAP kinase cascades and suggest they act through different molecular mechanisms.

Although phylogenetically unrelated, filamentous oomycetes and fungi establish similar structures to colonize plants and they represent economically the most important microbial threat to crop production. In mutualistic interactions established by root-colonizing fungi, clear differences to pathogens can be seen, but there is mounting evidence that their infection strategies and molecular interactions have certain common features. To infect the host, fungi and oomycetes employ similar strategies to circumvent plant innate immunity. This process involves the suppression of basal defence responses which are triggered by the perception of conserved molecular patterns. To establish biotrophy, effector proteins are secreted from mutualistic and pathogenic microbes to the host tissue, where they play central roles in the modulation of host immunity and metabolic reprogramming of colonized host tissues. This review article discusses key effector mechanisms of filamentous pathogens and mutualists, how they modulate their host targets and the fundamental differences or parallels between these different interactions. The orchestration of effector actions during plant infection and the importance of their localization within host tissues are also discussed.

The necrotrophic white mold fungus Sclerotinia sclerotiorum (S. sclerotiorum) is pathogenic to a broad range of plant species, including the Brassicaceae model plant Arabidopsis thaliana (Boland and Hall, 1994; Bolton et al., 2006). In Arabidopsis thaliana (A. thaliana), the semi-purified proteinaceous S. sclerotinorum elicitor SCFE1 (Sclerotinia culture filtrate elicitor 1) is sensed at the plasma membrane by the receptor-like protein RLP30 and triggers strong immune responses (Zhang et al., 2013), similar to the bacterial elicitor flagellin (Felix et al., 1999). Elicitation of plant defenses with SCFE1 is a tool to dissect the signaling pathway involving RLP30 and to study immunity to necrotrophic fungi. Here, we describe a simple protocol to axenically grow S. sclerotiorum. Further, we present a two-step liquid chromatography-based method for the partial purification of SCFE1 from culture filtrate (Figure 1A-B). Measurement by gas chromatography of the emission of the plant stress hormone ethylene is proposed as a bioassay to monitor elicitor activity in the fractions throughout the purification procedure (Figure 1C).Figure 1. Two-step chromatographic fractionation of S. sclerotiorum culture filtrate to obtain semi-purified SCFE1. A. Purification scheme of SCFE1. Crude filtrate (CF) is loaded onto a Sepharose SP cation exchange chromatography column. The eluate (S1) is diluted 10-fold and loaded onto a Source 15S cation exchange chromatography column. Elution is performed with a linear gradient of 0 to 0.3 M KCl and elution fractions of 0.5 ml (F1 - F100) are collected. FT = flow-through. B. Chromatogram of the SCFE1-containing fractions eluted from a Source 15S cation exchange chromatography column. The black line represents the protein elution profile monitored with OD280 nm. The grey line shows the increasing conductivity of the elution buffer. ma.u. = milli-arbitrary units. mS = milli-Siemens. C. Ethylene response in Arabidopsis Col-0 to SCFE1-containing fractions obtained by two-step cation exchange chromatography. Arabidopsis Col-0 leaf pieces are treated with 15 µl CF, undiluted and 10-fold diluted S1 as well as the Source 15S elution fractions (Fx). Treatment with 0.5 µM flg22 is used as a positive control for ethylene production. No treatment and treatment with 15 µl buffers A and B are used as negative controls. In this representative purification, SCFE1 is contained in fractions F24 to F52. Bars represent average values (n=2) ± S.D.

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta . The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter ( pFRK1-Luc ) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana , a non-host plant species of P. infestans . From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans .

Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

Transient expression in plant mesophyll protoplasts allows rapid characterisation of gene functions in vivo in a simplified and synchronized manner without bias due to the use of bacteria-based gene or protein delivery systems. It offers the possibility to test whether microbial effectors can subvert early events of plant immune signaling that are activated upon recognition of Microbe-Associated Molecular Patterns (MAMPs), the so-called MAMP-triggered immunity (MTI). Here, we describe the isolation and transfection with effector genes of Arabidopsis thaliana and Solanum lycopersicum mesophyll protoplasts, the use of a non-invasive luciferase reporter assay and a simple method to detect activated Mitogen-Activated Protein Kinases (MAPKs) to identify and study, in a medium-throughput manner, new effectors suppressing early signal transduction events of MTI.

Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites.