Repression of autocrine pheromone signaling leads to fusaric acid over-production.

Fusaric acid (FA), a picolinic acid derivative, is a natural substance produced by a wide variety of fungal plant pathogens belonging to the Fusarium genus. As a metabolite, fusaric acid exerts several biological activities including metal chelation, electrolyte leakage, repression of ATP synthesis, and direct toxicity on plants, animals and bacteria. Prior studies on the structure of fusaric acid revealed a co-crystal dimeric adduct between FA and 9,10-dehydrofusaric acid. During an ongoing search for signaling genes differentially regulating FA production in the fungal pathogen Fusarium oxysporum (Fo), we found that mutants lacking pheromone expression have an increased production of FA compared to the wild type strain. Noteworthy, crystallographic analysis of FA extracted from Fo culture supernatants showed that crystals are formed by a dimeric form of two FA molecules (1:1 molar stoichiometry). Overall, our results suggest that pheromone signaling in Fo is required to regulate the synthesis of fusaric acid.

Live-Cell Visualization of Early Stages of Root Colonization by the Vascular Wilt Pathogen Fusarium oxysporum.

Fungal phytopathogens induce a variety of pathogenicity symptoms on their hosts. The soilborne vascular wilt pathogen Fusarium oxysporum infects roots of more than 150 different crop species. Initial colonization stages are asymptomatic, likely representing a biotrophic phase of infection, followed by a necrotrophic switch after vascular colonization which results in blockage of the plant xylem and killing of the host. Live-cell microscopy techniques have been successfully employed to study interaction events during fungal colonization of root tissues. This technique is widely used to track fungal development during disease progression. Here, we describe a well-established protocol for generation and screening of fluorescently tagged F. oxysporum transformants, as well as for live-cell imaging of the early colonization stages of F. oxysporum on tomato (Solanum lycopersicum) seedlings. The presented experimental design and techniques involved are also applicable to other root infecting fungi.

Cytosolic pH Controls Fungal MAPK Signaling and Pathogenicity.

Mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes in eukaryotes. In fungal pathogens, conserved MAPK pathways control key virulence functions such as infection-related development, invasive hyphal growth, or cell wall remodeling. Recent findings suggest that ambient pH acts as a key regulator of MAPK-mediated pathogenicity, but the underlying molecular events are unknown. Here, we found that in the fungal pathogen Fusarium oxysporum, pH controls another infection-related process, hyphal chemotropism. Using the ratiometric pH sensor pHluorin we show that fluctuations in cytosolic pH (pHc) induce rapid reprogramming of the three conserved MAPKs in F. oxysporum, and that this response is conserved in the fungal model organism Saccharomyces cerevisiae. Screening of a subset of S. cerevisiae mutants identified the sphingolipid-regulated AGC kinase Ypk1/2 as a key upstream component of pHc-modulated MAPK responses. We further show that acidification of the cytosol in F. oxysporum leads to an increase of the long-chain base (LCB) sphingolipid dihydrosphingosine (dhSph) and that exogenous addition of dhSph activates Mpk1 phosphorylation and chemotropic growth. Our results reveal a pivotal role of pHc in the regulation of MAPK signaling and suggest new ways to target fungal growth and pathogenicity. IMPORTANCE Fungal phytopathogens cause devastating losses in global agriculture. All plant-infecting fungi use conserved MAPK signaling pathways to successfully locate, enter, and colonize their hosts. In addition, many pathogens also manipulate the pH of the host tissue to increase their virulence. Here, we establish a functional link between cytosolic pH (pHc) and MAPK signaling in the control of pathogenicity in the vascular wilt fungal pathogen Fusarium oxysporum. We demonstrate that fluctuations in pHc cause rapid reprogramming of MAPK phosphorylation, which directly impacts key processes required for infection, such as hyphal chemotropism and invasive growth. Targeting pHc homeostasis and MAPK signaling can thus open new ways to combat fungal infection.

Alkaline pH, Low Iron Availability, Poor Nitrogen Sources and CWI MAPK Signaling Are Associated with Increased Fusaric Acid Production in Fusarium oxysporum.

Fusaric acid (FA) is one of the first secondary metabolites isolated from phytopathogenic fungi belonging to the genus Fusarium. This molecule exerts a toxic effect on plants, rhizobacteria, fungi and animals, and it plays a crucial role in both plant and animal pathogenesis. In plants, metal chelation by FA is considered one of the possible mechanisms of action. Here, we evaluated the effect of different nitrogen sources, iron content, extracellular pH and cellular signalling pathways on the production of FA siderophores by the pathogen Fusarium oxysporum (Fol). Our results show that the nitrogen source affects iron chelating activity and FA production. Moreover, alkaline pH and iron limitation boost FA production, while acidic pH and iron sufficiency repress it independent of the nitrogen source. FA production is also positively regulated by the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway and inhibited by the iron homeostasis transcriptional regulator HapX. Collectively, this study demonstrates that factors promoting virulence (i.e., alkaline pH, low iron availability, poor nitrogen sources and CWI MAPK signalling) are also associated with increased FA production in Fol. The obtained new insights on FA biosynthesis regulation can be used to prevent both Fol infection potential and toxin contamination.

The Gal4-Type Transcription Factor Pro1 Integrates Inputs from Two Different MAPK Cascades to Regulate Development in the Fungal Pathogen Fusarium oxysporum.

Mitogen-activated protein kinase (MAPK) signaling pathways control fundamental aspects of growth and development in fungi. In the soil-inhabiting ascomycete Fusarium oxysporum, which causes vascular wilt disease in more than a hundred crops, the MAPKs Fmk1 and Mpk1 regulate an array of developmental and virulence-related processes. The downstream components mediating these disparate functions are largely unknown. Here we find that the GATA-type transcription factor Pro1 integrates signals from both MAPK pathways to control a subset of functions, including quorum sensing, hyphal fusion and chemotropism. By contrast, Pro1 is dispensable for other downstream processes such as invasive hyphal growth and virulence, or response to cell wall stress. We further show that regulation of Pro1 activity by these upstream pathways occurs at least in part at the level of transcription. Besides the MAPK pathways, upstream regulators of Pro1 transcription also include the Velvet regulatory complex, the signaling protein Soft (Fso1) and the transcription factor Ste12 which was previously shown to act downstream of Fmk1. Collectively, our results reveal a role of Pro1 in integrating the outputs from different signaling pathways of F. oxysporum thereby mediating key developmental decisions in this important fungal pathogen.

Competitiveness during Dual-Species Biofilm Formation of Fusarium oxysporum and Candida albicans and a Novel Treatment Strategy.

During an infection, a single or multispecies biofilm can develop. Infections caused by non-dermatophyte molds, such as Fusarium spp. and yeasts, such as Candida spp., are particularly difficult to treat due to the formation of a mixed biofilm of the two species. Fusarium oxysporum is responsible for approximately 20% of human fusariosis, while Candida albicans is responsible for superficial mucosal and dermal infections and for disseminated bloodstream infections with a mortality rate above 40%. This study aims to investigate the interactions between C. albicans and F. oxysporum dual-species biofilm, considering variable formation conditions. Further, the ability of the WMR peptide, a modified version of myxinidin, to eradicate the mixed biofilm when used alone or in combination with fluconazole (FLC) was tested, and the efficacy of the combination of WMR and FLC at low doses was assessed, as well as its effect on the expression of some biofilm-related adhesin and hyphal regulatory genes. Finally, in order to confirm our findings in vivo and explore the synergistic effect of the two drugs, we utilized the Galleria mellonella infection model. We concluded that C. albicans negatively affects F. oxysporum growth in mixed biofilms. Combinatorial treatment by WMR and FLC significantly reduced the biomass and viability of both species in mature mixed biofilms, and these effects coincided with the reduced expression of biofilm-related genes in both fungi. Our results were confirmed in vivo since the synergistic antifungal activity of WMR and FLC increased the survival of infected larvae and reduced tissue invasion. These findings highlight the importance of drug combinations as an alternative treatment for C. albicans and F. oxysporum mixed biofilms.

Conserved secreted effectors contribute to endophytic growth and multihost plant compatibility in a vascular wilt fungus.

Fungal interactions with plant roots, either beneficial or detrimental, have a crucial impact on agriculture and ecosystems. The cosmopolitan plant pathogen Fusarium oxysporum (Fo) provokes vascular wilts in more than a hundred different crops. Isolates of this fungus exhibit host-specific pathogenicity, which is conferred by lineage-specific Secreted In Xylem (SIX) effectors encoded on accessory genomic regions. However, such isolates also can colonize the roots of other plants asymptomatically as endophytes or even protect them against pathogenic strains. The molecular determinants of endophytic multihost compatibility are largely unknown. Here, we characterized a set of Fo candidate effectors from tomato (Solanum lycopersicum) root apoplastic fluid; these early root colonization (ERC) effectors are secreted during early biotrophic growth on main and alternative plant hosts. In contrast to SIX effectors, ERCs have homologs across the entire Fo species complex as well as in other plant-interacting fungi, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence and rapid activation of plant immune responses, while ERC deletion in a nonpathogenic isolate led to impaired root colonization and biocontrol ability. Strikingly, some ERCs contribute to Fo infection on the nonvascular land plant Marchantia polymorpha, revealing an evolutionarily conserved mechanism for multihost colonization by root infecting fungi.

Selection of Endophytic Beauveria bassiana as a Dual Biocontrol Agent of Tomato Pathogens and Pests.

Endophytic fungi (EF) can enhance both plant growth and defense barriers against pests and pathogens, contributing to the reduction of chemical pesticides and fertilizers use in agriculture. Beauveria bassiana is an entomopathogenic fungus showing endophytism in several crops, often associated with a good capacity to limit the development of pests and disease agents. However, the diversity of the protective efficacy and plant response to different strains can be remarkable and needs to be carefully assessed for the successful and predictable use of these beneficial microorganisms. This study aims to select B. bassiana strains able to colonize tomato plants as endophytes as well as to control two important disease agents, Botrytis cinerea and Alternaria alternata, and the pest aphid, Macrosiphum euphorbiae. Nine wild-type isolates and one commercial strain were screened for endophytism, then further characterized for plant-growth promotion plus inhibition of disease development and pest infestation. Four isolates proved to have a good control activity against the biotic stressors tested, but only Bb716 was also able to promote plant growth. This work provides a simple workflow for the selection of beneficial EF, paving the way towards more effective use of B. bassiana in Integrate Pest Management (IPM) of tomato.

Chemotropic Assay for Testing Fungal Response to Strigolactones and Strigolactone-Like Compounds.

Current knowledge on the mechanism of strigolactones (SLs) as signaling molecules during specific interactions in the rhizosphere is mainly related to the control of germination of parasitic weed seeds and hyphal branching of arbuscular mycorrhizal fungi. Thus, the role of plant secreted SLs in regulating the growth and development of root-colonizing fungi still remains controversial. Fusarium oxysporum can sense and respond to extracellular signals through oriented germ tube emergence and redirectioning of hyphal growth toward gradients of nutrients, sex pheromones, or plant root exudates. However, chemoattractant activity of SLs against microorganisms living in the soil has not been tested so far. Here we propose a quantitative chemotropic assay to understand if and how soil fungi could sense gradients of SLs and SLs-like sources. In the example case of F. oxysporum, hyphae of fungal representative mutants preferentially grow toward the synthetic SL analog GR24; and this chemotropic response requires conserved elements of the fungal invasive growth mitogen-activated protein kinase (MAPK) cascade.

Chemotropism Assays for Plant Symbiosis and Mycoparasitism Related Compound Screening in Trichoderma atroviride.

Trichoderma atroviride is a mycoparasitic fungus used as biological control agent to protect plants against fungal pathogens. Successful biocontrol is based on the perception of signals derived from both the plant symbiont and the fungal prey. Here, we applied three different chemotropic assays to study the chemosensing capacity of T. atroviride toward compounds known or suspected to play a role in the mycoparasite/plant or host/prey fungal interactions and to cover the complete spectrum of T. atroviride developmental stages. Purified compounds, including nutrients, the fungal secondary metabolite 6-amyl-α-pyrone (6-pentyl-α-pyrone, 6-PP) and the plant oxylipin 13-(s)-HODE, as well as culture supernatants derived from fungal preys, including Rhizoctonia solani, Botrytis cinerea and Fusarium oxysporum, were used to evaluate chemotropic responses of conidial germlings, microcolonies and fully differentiated mycelia. Our results show that germlings respond preferentially to compounds secreted by plant roots and T. atroviride itself than to compounds secreted by prey fungi. With the progression of colony development, host plant cues and self-generated signaling compounds remained the strongest chemoattractants. Nevertheless, mature hyphae responded differentially to certain prey-derived signals. Depending on the fungal prey species, chemotropic responses resulted in either increased or decreased directional colony extension and hyphal density at the colony periphery closest to the test compound source. Together these findings suggest that chemotropic sensing during germling development is focused on plant association and colony network formation, while fungal prey recognition develops later in mature hyphae of fully differentiated mycelium. Furthermore, the morphological alterations of T. atroviride in response to plant host and fungal prey compounds suggest the presence of both positive and negative chemotropism. The presented assays will be useful for screening of candidate compounds, and for evaluating their impact on the developmental spectrum of T. atroviride and other related species alike. Conidial germlings proved particularly useful for simple and rapid compound screening, whereas more elaborate microscopic analysis of microcolonies and fully differentiated mycelia was essential to understand process-specific responses, such as plant symbiosis and biocontrol.

A bacterial endophyte exploits chemotropism of a fungal pathogen for plant colonization.

Soil-inhabiting fungal pathogens use chemical signals released by roots to direct hyphal growth towards the host plant. Whether other soil microorganisms exploit this capacity for their own benefit is currently unknown. Here we show that the endophytic rhizobacterium Rahnella aquatilis locates hyphae of the root-infecting fungal pathogen Fusarium oxysporum through pH-mediated chemotaxis and uses them as highways to efficiently access and colonize plant roots. Secretion of gluconic acid (GlcA) by R. aquatilis in the rhizosphere leads to acidification and counteracts F. oxysporum-induced alkalinisation, a known virulence mechanism, thereby preventing fungal infection. Genetic abrogation or biochemical inhibition of GlcA-mediated acidification abolished biocontrol activity of R. aquatilis and restored fungal infection. These findings reveal a new way by which bacterial endophytes hijack hyphae of a fungal pathogen in the soil to gain preferential access to plant roots, thereby protecting the host from infection.

Structure of Fungal α Mating Pheromone in Membrane Mimetics Suggests a Possible Role for Regulation at the Water-Membrane Interface.

Fusarium oxysporum is a highly destructive plant pathogen and an emerging pathogen of humans. Like other ascomycete fungi, F. oxysporum secretes α-pheromone, a small peptide that functions both as a chemoattractant and as a quorum-sensing signal. Three of the ten amino acid residues of α-pheromone are tryptophan, an amino acid whose sidechain has high affinity for lipid bilayers, suggesting a possible interaction with biological membranes. Here we tested the effect of different lipid environments on α-pheromone structure and function. Using spectroscopic and calorimetric approaches, we show that this peptide interacts with negatively charged model phospholipid vesicles. Fluorescence emission spectroscopy and nuclear magnetic resonance (NMR) measurements revealed a key role of the positively charged groups and Trp residues. Furthermore, NMR-based calculation of the 3D structure in the presence of micelles, formed by lipid surfactants, suggests that α-pheromone can establish an intramolecular disulfide bond between the two cysteine residues during interaction with membranes, but not in the absence of lipid mimetics. Remarkably, this oxidized version of α-pheromone lacks biological activity as a chemoattractant and quorum-sensing molecule. These results suggest the presence of a previously unidentified redox regulated control of α-pheromone activity at the surface of the plasma membrane that could influence the interaction with its cognate G-protein coupled receptor.

Heterologous Expression of PKPI and Pin1 Proteinase Inhibitors Enhances Plant Fitness and Broad-Spectrum Resistance to Biotic Threats.

Kunitz-type (PKPI) and Potato type I (Pin1) protease inhibitors (PIs) are two families of serine proteinase inhibitors often associated to plant storage organs and with well known insecticidal and nematicidal activities. Noteworthy, their ability to limit fungal and bacterial pathogenesis in vivo or to influence plant physiology has not been investigated in detail. To this aim, we generated a set of PVX-based viral constructs to transiently and heterologously express two potato PKPI (PKI1, PKI2) and three potato Pin1 (PPI3A2, PPI3B2, PPI2C4) genes in Nicotiana benthamiana plants, a widely used model for plant-pathogen interaction studies. Interestingly, transgenic plants expressing most of the tested PIs showed to be highly resistant against two economically important necrotrophic fungal pathogens, Botrytis cinerea and Alternaria alternata. Unexpectedly, overexpression of the PKI2 Kunitz-type or of the PPI2C4 and PPI3A2 Potato type I inhibitor genes also lead to a dramatic reduction in the propagation and symptom development produced by the bacterial pathogen Pseudomonas syringae. We further found that localized expression of PPI2C4 and PKI2 in N. benthamiana leaves caused an increase in cell expansion and proliferation which lead to tissue hypertrophy and trichome accumulation. In line with this, the systemic expression of these proteins resulted in plants with enhanced shoot and root biomass. Collectively, our results indicate that PKPI and Pin1 PIs might represent valuable tools to simultaneously increase plant fitness and broad-spectrum resistance toward phytopathogens.

Expansion Microscopy for Cell Biology Analysis in Fungi.

Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.

The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set of lineage-specific chromosomes.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation.

The Role of Volatile Organic Compounds and Rhizosphere Competence in Mode of Action of the Non-pathogenic Fusarium oxysporum FO12 Toward Verticillium Wilt.

Verticillium wilts caused by Verticillium spp. are among the most challenging plant diseases to control and affect numerous hosts worldwide. Due to the lack of effective, conventional control methods, integrated control strategies provide a promising approach to manage these diseases. The non-pathogenic Fusarium oxysporum strain FO12 was reported in previous studies to be an effective biocontrol agent against Verticillium dahliae, however, its mode of action remains to be elucidated. In this study, complementary in vitro and in vivo experiments were conducted in order to explore the implications of inhibitory substances and rhizosphere competence in antagonistic effects of FO12 against V. dahliae and V. longisporum. Volatile organic compounds and soluble substances produced by FO12, which caused significant inhibition of mycelial growth and microsclerotia viability in the two tested Verticillium species, were identified by means of gas and liquid chromatography-mass spectrometry. We showed that the antagonistic effect of F. oxysporum FO12 is partially due to the production of bioactive compounds such as 3-methyl-1-butanol and 2-methyl-1-butanol, among others. Several metabolic pathways of FO12 were altered upon contact with V. dahliae ELV22 volatiles. The reduced production of alpha, alpha-trehalose, a metabolite used in starch and sucrose metabolism, suggests that the biocontrol agent activates its stress response in the presence of the phytopathogen. Microscopic analysis using sGFP-tagged FO12 on oil seed rape as a model plant suggests that the biocontrol strain is an efficient root colonizer, which could compete with V. dahliae in the same ecological niche. The findings obtained in this study provide new insights into the mode of action of this potential biocontrol agent, which are relevant for controlling Verticillium wilt through an ecologically friendly approach.

NADPH oxidase regulates chemotropic growth of the fungal pathogen Fusarium oxysporum towards the host plant.

Soil-inhabiting fungal pathogens use chemical signals to locate and colonise the host plant. In the vascular wilt fungus Fusarium oxysporum, hyphal chemotropism towards tomato roots is triggered by secreted plant peroxidases (Prx), which catalyse the reductive cleavage of reactive oxygen species (ROS). Here we show that this chemotropic response requires the regulated synthesis of ROS by the conserved fungal NADPH oxidase B (NoxB) complex, and their transformation into hydrogen peroxide (H2 O2 ) by superoxide dismutase (SOD). Deletion of NoxB or the regulatory subunit NoxR, or pharmacological inhibition of SOD, specifically abolished chemotropism of F. oxysporum towards Prx gradients. Addition of isotropic concentrations of H2 O2 rescued chemotropic growth in the noxBΔ and noxRΔ mutants, but not in a mutant lacking the G protein-coupled receptor Ste2. Prx-triggered rapid Nox- and Ste2-dependent phosphorylation of the cell wall integrity mitogen-activated protein kinase (CWI MAPK) Mpk1, an essential component of the chemotropic response. These results suggest that Ste2 and the CWI MAPK cascade function downstream of NoxB in Prx chemosensing. Our findings reveal a new role for Nox enzymes in directed hyphal growth of a filamentous pathogen towards its host and might be of broad interest for chemotropic interactions between plants and root-colonising fungi.

Autocrine pheromone signalling regulates community behaviour in the fungal pathogen Fusarium oxysporum.

Autocrine self-signalling via secreted peptides and cognate receptors regulates cell development in eukaryotes and is conserved from protozoans to mammals1,2. In contrast, secreted peptides from higher fungi have been traditionally associated with paracrine non-self-signalling during sexual reproduction3. For example, cells of the model fungus Saccharomyces cerevisiae fall into two distinct mating types (MAT), which produce either a- or α-pheromone and the cognate receptors Ste2 or Ste3, respectively4. Inappropriate autocrine pheromone signalling (APS) during mating is prevented by downregulation of the self-pheromone receptor4,5 and by a-type cell-specific cleavage of α-pheromone through the protease Bar1 (refs. 6-8). While APS can be artificially induced by manipulation of the pheromone secrete-and-sense circuit7,9-11, its natural occurrence in ascomycete fungi has not been described. Here, we show that Fusarium oxysporum-a devastating plant pathogen that lacks a known sexual cycle12-co-expresses both pheromone-receptor pairs, resulting in autocrine regulation of developmental programmes other than mating. We found that unisexual populations of MAT1-1 cells (α-type idiomorphs13) secrete and sense both a- and α-pheromone, and that their perception requires the cognate receptors and conserved elements of the cell wall integrity mitogen-activated protein kinase cascade. We further show that F. oxysporum uses APS to regulate spore germination in a cell-density-dependent manner, whereby the α-Ste2 interaction leads to repression of conidial germination while the a-Ste3 interaction relieves repression. Our results reveal the existence of a regulatory function for peptide pheromones in the quorum-sensing-mediated control of fungal development.

Root Exudates of Stressed Plants Stimulate and Attract Trichoderma Soil Fungi.

Plant roots release complex mixtures of bioactive molecules, including compounds that affect the activity and modify the composition of the rhizosphere microbiome. In this work, we investigated the initial phase of the interaction between tomato and an effective biocontrol strain of Trichoderma harzianum (T22). We found that root exudates (RE), obtained from plants grown in a split-root system and exposed to various biotic and abiotic stress factors (wounding, salt, pathogen attack), were able to stimulate the growth and act as chemoattractants of the biocontrol fungus. On the other hand, some of the treatments did not result in an enhanced chemotropism on Fusarium oxysporum f. sp. lycopersici, indicating a mechanism that may be selective for nonpathogenic microbes. The involvement of peroxidases and oxylipins, both known to be released by roots in response to stress, was demonstrated by using RE fractions containing these molecules or their commercial purified analogs, testing the effect of an inhibitor, and characterizing the complex pattern of these metabolites released by tomato roots both locally and systemically.