Root colonisation and induction of plant defence-associated signalling pathways in Arabidopsis thaliana by Serratia marcescens, Streptomyces galilaeus, and Trichoderma viride

Root colonisation and induction of plant defence-associated signalling pathways in Arabidopsis thaliana by Serratia marcescens, Streptomyces galilaeus, and Trichoderma viride | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Root colonisation and induction of plant defence-associated signalling pathways in Arabidopsis thaliana by Serratia marcescens, Streptomyces galilaeus, and Trichoderma viride Luis Enrique Luna-Hernández, Graciela Huerta-Palacios, Francisco Holguín-Meléndez, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7125135/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Some microorganisms that are antagonistic to phytopathogens can activate induced systemic resistance (ISR) in plants. We sought to determine whether Streptomyces galilaeus CFFSUR-B12, Serratia marcescens CFFSUR-B2, and Trichoderma viride CFFSUR-A21 – strains recognised for their antagonistic capacity – could colonise the roots of and induce resistance in Arabidopsis thaliana . The antagonistic activity of Colletotrichum spp. was determined in dual-culture assays. Strains were inoculated separately in the roots of A. thaliana to study root colonisation and activation of ISR in leaves. PR1 and PDF1.2 expression was monitored by RT-qPCR in leaves. Serratia marcescens CFFSUR-B2 colonised the rhizoplane and endorhizosphere, whereas Str. galilaeus CFFSUR-B12 and T. viride CFFSUR-A21 only colonised the rhizoplane. Serratia marcescens and T. viride induced co-expression of PR1 and PDF1.2 , while Str. galilaeus induced only PDF1.2 expression. These findings reveal new avenues for research into plant disease management in the humid tropics. ISR Serratia Trichoderma Streptomyces PR1 PDF1.2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Worldwide, major agricultural crops are estimated to suffer losses of 20–30% from pests and diseases (Savary et al., 2019 ). These are primarily caused by fungal pathogens (83%) and result in US $ 220 billion in economic losses annually (Agrios 2005 ). This has led to a rise in the use of fungicides which, however, has resulted in an increasing number of fungi to develop resistance, including fungi of medicinal importance (Ishii, 2023 ). Therefore, new technologies and techniques aimed at reducing fungicide dependence without compromising agricultural production efficiency remain the subject of ongoing development. Biocontrol techniques involve strategies for the control of phytopathogenic fungi that use living organisms (including microorganisms) or their metabolites, and strive to minimize the impact of these phytopathogenic fungi and the use of agrochemicals (Galli et al., 2024 ). The action mechanism of microbial biological control agents (MBCAs) can be direct (antibiosis, enzymatic lysis, parasitism, and competition) (Bonaterra et al., 2022 ; Köhl et al., 2019 ) or indirect (induced resistance (IR) in plants) (Maciag et al., 2023 ). IR has been studied extensively and is generally categorized into Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR). Both types are involved in the activation of plant defence mechanisms and differ primarily based on the characteristics of the inducer and the site of induction (Vlot et al., 2021 ). SAR is stimulated by phytopathogenic microorganisms or exogenous application of compounds such as salicylic acid (SA) (Ryals et al., 1994 ). A defence response is activated in the area of interaction between microorganism and plant (foliage or roots) (Vlot et al., 2021 ). The SA route, which is generally assumed to be the main signalling pathway, primarily acts against biotrophic and hemibiotrophic phytopathogens (Vlot et al., 2021 ). In Arabidopsis thaliana , the role of the PR gene family ( PR1 , PR2, PR5 ) in the SA pathway has been recognised as important (Ryals et al., 1996 ). PR1 plays a central role in the activation of plant defences and is used as a marker for the expression of the SA pathway (Pečenková et al., 2022 ; Pieterse et al., 2014 ). The PR gene family has a range of antimicrobial, antifungal, insecticidal, nematocidal, and cell wall degrading functions, and therefore is associated with reducing the impact of pathogens on plants (Alexander et al., 1993 ; Zhang et al., 2018 ). In ISR, the plant defence mechanism is triggered by interaction with non-phytopathogenic microorganisms in the roots, and responses protects aerial tissues (Vlot et al., 2021 ). The ethylene (Et) and jasmonic acid (JA) pathways are the main signalling pathways and act mainly against necrotrophic plant pathogens (Vlot et al., 2021 ). In A. thaliana , the PDF1.2 gene is associated with and used as a marker for the JA/Et signalling pathway (Penninckx et al., 1998 , Kunkel and Brooks, 2002 ). It should be emphasized that the SA and JA/Et pathways are not exclusive to SAR and ISR, and also depend on the characteristics of the microorganisms that interact with the plant, various biotic and abiotic factors, and could happen the cross-talk between these mechanisms (Aerts et al., 2021 ; Dimopoulou et al., 2019 ; Nguyen et al., 2020 ; Pieterse et al., 2012 ). In ISR, non-pathogenic bacteria are initially recognised by plants as potential invaders and trigger a defensive response (Arkhipov et al., 2024 ) by activating pattern recognition receptors (PRRs) – transmembrane proteins capable of recognising microbe-associated molecular patterns (MAMPs), such as quorum-sensing (QS) proteins, siderophores, volatile organic compounds (Monaghan and Zipfel, 2012 ; Zhu et al., 2022 ), or flagellar peptides (Meziane et al., 2005 ). In this case, activation of the ISR resistance mechanism is not necessarily complete. It induces a response known as priming, a process by which a plant prepares its defences against the possible future arrival of phytopathogens (Zhu et al., 2022 ). IR accelerates the response time of a plant to a wide range of pathogens. Moreover, its effects can be long-lasting if the plant is colonised by beneficial microorganisms (Flors et al., 2024 ). Plant-microorganism interactions form a complex system, where various abiotic and biotic factors that determine the efficacy of MBCAs interact (He et al., 2021 ). Successful MBCAs should therefore be able to colonise, compete, and remain in the target habitat to directly or indirectly trigger antagonistic interactions (Bonaterra et al., 2022 ; Ghorbanpour et al., 2018 ; Saeed et al., 2021 ). Evidence suggests that Trichoderma species such as Trichoderma atroviride , Trichoderma harzianum , and Trichoderma viride employ direct as well as indirect mechanisms of actions to mitigate the impact of plant pathogens (Ferreira and Musumeci, 2021 ; Nawrocka and Małolepsza, 2013 ; Sharma and Gothalwal, 2017 ). On the other hand, indirect antagonism that occurs through ISR in other genera, such as Streptomyces and Serratia – both known to inhibit a number of fungal phytopathogens – has remained relatively understudied (Castillo et al., 2016 ; Dow et al., 2023 ; Gutiérrez-Román et al., 2012 , 2015 ; Soenens and Imperial, 2020 ). Several ISR research articles on Streptomyces have been published, although these studies mostly focus on the reduction of phytopathogen-caused symptoms. Their effect on gene expression in the ISR pathway and establishment in roots has remained relatively underexplored (Conn et al., 2008 ; Ebrahimi-Zarandi et al., 2022 ; L. Liu et al., 1995 ; Salla et al., 2014 ). We here present our observations on Streptomyces galilaeus CFFSUR-B12, which previously had been isolated as mycoparasite from an anthracnose-causing fungus on cacao pods. This strain showed promising results as biological control agent (BCA), as it produced secondary metabolites and lysins. Its efficiency, moreover, was evaluated in the laboratory as well as in the field (Castillo, 2016 ). We also present the findings from our study of Serratia marcescens CFFSUR-B2, which had been isolated from pangola-grass compost, and which exhibited enzymatic lysis and prodigiosin production as action mechanisms (Gutiérrez-Román et al., 2015 ). Both strains showed different antagonistic activity to different phytopathogens of agricultural importance, such as Mycosphaerella fijiensis Morelet and Colletotrichum gloeosporioides (Castillo, 2016 ; Castillo et al., 2016 ; Gutiérrez-Román et al., 2012 and 2015 ). Trichoderma viride CFFSUR-A21 was included in the study as fast-growing mycoparasite. This strain was isolated from mango leaf litter and showed direct antagonistic activity against M . fijiensis and C . gloeosporioides . MAMPs have been reported in Serratia as well as Trichoderma , which might indicate that the strains we included in our study can activate ISR (Babenko et al., 2022 ; Contreras-Cornejo et al., 2014 ; Ryu et al., 2013 ; Schenk et al., 2014 ). Also, these strains are of tropical origin and maintain their direct antagonistic activity at temperatures higher than 28 ºC. If their ability to induce systemic resistance can be demonstrated, it would be possible to investigate whether they can induce ISR in crop plants, such as banana, coffee, and cacao. These crops are strongly affected by phytopathogens but have received limited attention in this field of study. The objective of our study was to determine whether Str. galilaeus CFFSUR-B12, Ser. marcescens CFFSUR-B2, and T. viride CFFSUR-A21 – strains that are characterised by their broad antagonistic capacity against phytopathogens – could induce changes in the expression of ISR-associated genes in A. thaliana . Because these strains employ different mechanisms of direct antagonistic action against phytopathogenic fungi, we would expect that if they are capable of activating ISR, they would activate it in different ways. Therefore, the antagonistic activity of the evaluated strains was first tested against two Colletotrichum spp. strains that had been isolated from mango and cacao fruit ( Colletotrichum is a plant pathogen of agricultural importance in Latin America; Zakaria, 2021 ). We then determined their ability to establish in roots and induced expression of PR1 and PDF1.2 (associated with SAR and ISR, respectively) using A. thaliana Col-0 as plant model. Methods Plant culture conditions Arabidopsis thaliana (Col-0) was used as a study model. Seeds were disinfected by immersion in ethanol (70%) for 1 min and then in NaClO (20%, commercial product) for 8 min, followed by rinsing (four washes with sterile water) to remove remaining NaClO. Disinfected seeds were kept in 500 µL of sterile water at 4°C for 48 h and then germinated in Petri dishes containing MS medium (Murashige & Skoog, 1962 ). Fourteen-day-old seedlings with lateral roots and an approximate height of 0.5 cm were transplanted into glass jars containing MS medium and placed in a growth chamber (25 ± 1 ºC, 12 h light-dark photoperiod, light intensity of 8500 lux (ViparSpectra P1000 grow light)). Microorganisms The bacterial strains Ser. marcescens CFFSUR-B2 and Str. galilaeus CFFSUR-B12 were cultured on nutrient agar (NA, pH 7). Trichoderma viride CFFSUR- A21 and the phytopathogenic Colletotrichum spp. strains M07-C2 and M10-F2 (isolated from cacao and mango crops, respectively) were cultured on potato dextrose agar (PDA, pH 7). All microorganisms were incubated at 25ºC. Preparation of inocula Bacterial cell density was determined and adjusted to 1.5x10 8 cells/ml (0.5 McFarland scale) using a McFarland spectrophotometric procedure (λ = 625 nm; Leber, 2016 ; McFarland, 1907 ). Results were verified in culture medium by counting of the colony-forming units (CFU). For fungal inocula, spores were collected from T. viride (7d of growth) and the Colletotrichum strains (at 14 d), and the number of required spores was determined and adjusted using a Neubauer chamber. In vitro antagonism assay The antagonistic activity of the Colletotrichum strains was determined by a dual-culture assay in Petri dishes (90 mm diameter), with 7 biological replicates per treatment (Chen et al., 2017 ; Raymaekers et al., 2020 ; Sari et al., 2021 ). Controls consisted of a mycelial disc (5 mm diameter) of Colletotrichum spp placed 2 cm away from the edge of a Petri dish. The bacterial phytopathogen-antagonist assay was performed on PDA medium supplemented with 10 g/L casein peptone. Bacterial strains (CFFSUR-B2 or CFFSUR-B12) were inoculated along a straight line 2 cm from the edge of the plate. Then, 24 h later, a mycelial disc (5mm diameter) of a phytopathogenic fungus ( Colletotrichum sp M07-C2 or M10-F2) was placed 4 cm away from the bacterial streak (Wonglom et al., 2019 ). In the Trichoderma-Colletotrichum assay, a mycelial disc (5 mm diameter) of Colletotrichum spp was placed 2 cm away from the edge of a plate with PDA medium. After 72 h, a 5 mm diameter mycelial disc of T. viride CFFSUR-A21 was placed at 4 cm of the phytopathogen. The inhibitory effect was estimated by measuring every 24 h the growth radius of the Colletotrichum spp strains towards the antagonist, until the control mycelium completely covered the plate surface. The percentage inhibition of radial growth was calculated using the following formula: % inhibition = (R1-R2) / R1x100, in which R1 represents the radial growth in the control, and R2 radial growth of the phytopathogen against the antagonist (Wonglom et al., 2019 ). The interaction between T. viride and Colletotrichum spp. was assessed using the Bell scale (Bell et al., 1982 ): 1) the antagonist outcompetes the pathogen and entirely covers the surface of the culture medium; 2) the antagonist covers 2/3 of the culture medium; 3) the antagonist covers 1/2 of the culture medium, the remainder being covered by the pathogen; 4) the pathogen covers 3/4 of the culture medium, while the antagonist covers only 1/4; 5) the pathogen entirely covers the culture medium and the antagonist. Data from the bacteria- Colletotrichum antagonism assay were analysed by two-way ANOVA with a subsequent post hoc Tukey HSD test, while data from the Trichoderma - Colletotrichum assay were analysed by two-sample Student's t-test. Analyses were performed using the R statistical software package (v4. 3.3; R Core Team 2023). The establishment of micro-organisms in the root Eighteen plants of A. thaliana (28 d old) were arranged over 2 bacterial treatments and a treatment control (sterile water). The rhizosphere of the treatment plants was inoculated with 25 µL of a bacterial suspension of 1.5x10 8 cells/ml ( Ser. marcescens or Str. galilaeus , 6 plants each), while the rhizosphere of the 6 control plants was treated with 25 µL of sterile distilled water. To determine the establishment of micro-organisms in the roots, independent root samples were taken from 3 plants per treatment at 3 and 6 d following inoculation (dpi). To determine the establishment of bacteria in the rhizoplane, 2 cm segments were taken from the apical region of lateral roots. The segments were washed gently with sterile water to remove any remaining medium, and the mass of the roots was determined with an analytical balance. To collect bacteria from the rhizoplane, rootlets were placed in 1.5 ml tubes that contained 2 ml sterile water. The rootlets were then subjected to a sonication cycle (Cole-Parmer UC 200): 30 s of sonication at approximately 24000 Hz, followed by 10 s without sonication, five times. They were then vortexed for 5 s using a Vortex-Genie 2 at level 4. Then, 25 µL of undiluted or serially diluted (10 − 1 , 10 − 2 , and 10 − 3 ) supernatant was spread on the surface of a NA-containing Petri dish using a Drigalski spatula. Next, the Petri dishes were incubated at 25 ºC and CFU was counted 48 h later. Three replicates were performed per treatment/dpi/dilution. To determine the establishment of bacteria in the endorhizosphere, ultrasonicated root fragments were disinfected with 2% NaClO (8 min), followed by rinsing with sterile water (4 washes) to remove NaClO residues. The fragments were then macerated with a pistil in 1 ml sterile water, and 25 µL of undiluted or serially diluted (10 − 1 and 10 − 2 ) supernatant was dispersed on the surface of a NA-containing Petri dish for CFU counting. The establishment of T. viride in the roots of A. thaliana was evaluated using the same procedure as for bacteria: the rhizosphere of six 28-day-old plants was inoculated with 20 µL of 2.6 x 10 5 spores/ml of T. viride , while the control treatment consisted of six plants treated with sterile water. The procedure to determine the establishment of T. viride in roots was also the same as for bacteria, but supernatants were cultured on PDA. Also, roots were stained with trypan blue in lactophenol for microscopic observation (Phillips & Hayman, 1970 ). Samples were observed under different magnifications (40x, 100x, 400x, and 1000x) with a Leica DM750 microscope. Photographic images were taken with a Leica ICC50W installed on the microscope. Expression of the genes PR1 and PDF1.2 Forty-five 22-day-old A. thaliana plants were arranged in a completely randomised block design with five treatments ( Str. galilaeus CFFSUR-B12, Ser. marcescens CFFSUR-B2, T. viride CFFSUR-A21, Colletotrichum M10-F2, and water (control)) and nine replicates. The rhizosphere was inoculated with 25 µl of 1.6 x 10 8 CFU/ml of Str. galilaeus , 25 µl of 2.8 X 10 8 CFU/ml of Ser. marcescens , or 20 µl of 5 x 10 5 T. viride spores/ml. In the Colletotrichum M10-F2 treatment, leaves were inoculated with 15 µl of 2.45 x 10 5 spores/ml. In the control treatment, the rhizosphere was treated with 25 µL of sterile water. Treatment response was evaluated by taking 3 plants and then 3 leaves per plant (randomly and without replacement) at 24, 48, and 72 h post inoculation (hpi). Leaves were immediately immersed in the lysis solution of the extraction kit (PureLink RNA Mini Kit) to exclude expression changes. Total RNA extraction was performed immediately after sampling, and extracted RNA was stored at -80 ºC for a maximum of 2.5 months. cDNA was synthesised from 2 µL of RNA using the Maxima First cDNA Synthesis Kit for RT-qPCR, and stored at -80°C until qPCR analysis. Prior to analysis, qPCR primers (Table S1 ) were validated by examining the melting curves for the presence of a single amplicon per primer set (65 ºC to 95 ºC in 0.5 ºC increments), separation on 2% agarose gel (Fig. 1 S, supplementary material), and sequencing of the amplicon. qPCR reactions (10 µL total volume) were performed with 1 µL of cDNA obtained, 0.2 µL of each primer (0.2 pM), 5 µL of SSOAdvanced Universal SYBR Green Supermix (1x), and 3.6 µL of nuclease-free water (2 technical replicates were run). PCR was performed on a CFX96 Touch (Bio-Rad) instrument using the following conditions: initial denaturation at 98°C for 30 s, 40 cycles of 15 s at 98°C and 30 s at 60°C. The relative expression level of PR1 and PDF1.2 was normalised to that of the reference genes UBQ10 and EF1α (Table S1 ) using the using Bio-Rad CFX Maestro™ 1.1 software (v4.2.2433.1219), which performs a calculation based on Pfaffl's method (ΔΔΔCq) (Pfaffl, 2001 ). Results In vitro antagonism assay The capacity of Str. galilaeus , Ser. marcescens , and T. viride to inhibit mycelial growth of Colletotrichum spp. strains was examined using a dual-culture assay. Their antagonistic activity was confirmed, and significant differences for the levels of inhibition and differential sensitivity were observed between Colletotrichum strains and MBCAs. The percentage of growth inhibition induced by Str. galilaeus (p < 0.001) was significantly higher than the inhibition by Ser. marcescens (Fig. 1 ). Colletotrichum strains showed differential sensitivity towards MBCA action mechanisms. Strain M10-F2 was more sensitive to Str. galilaeus (mean = 75.9%; sd = 5.33; p < 0.001) and T. viride (mean = 70.1%; sd = 0.82; p < 0.001) than M07-C2 (Figs. 1 and 2 ). Trichoderma viride CFFSUR-A21 exhibited relatively rapid and aggressive growth, and after 2 d scored 1 on the Bell scale: it completely covered the Petri dish, and grew and sporulated over Colletotrichum spp. Microscopic examination revealed that the hyphae of T. viride coiled round the hyphae of Colletotrichum spp. (Figs. 2 B-C). Figure 2 B shows the presence of an appressorium-like structure, a feature linked with mycoparasitism, on the hyphal tips of T. viride . Presence of microorganisms in the roots Experiments with the inoculation of Ser. marcescens , Str. galilaeus , and T. viride in the roots of A. thaliana showed that these microorganisms were able to establish in the rhizoplane and root endosphere. Serratia marcescens became established as an endophyte and in the rhizoplane, while Str. galilaeus and T. viride only remained in the rhizoplane. From plants that were inoculated with Ser. marcescens , 2.29 × 10 7 CFU/g and 4.68 × 10 7 CFU/g were recovered from rhizoplane root, at 3 and 6 dpi, respectively; and from the internal part (endophytes) 4.13 × 10 5 CFU/g and 3.74 × 10 5 CFU/g were recovered at 3 and 6 dpi, respectively. The observed increase in CFU/g suggests that this strain is able to colonise the roots of A. thaliana . From plants inoculated with Str. galilaeus , only 6.5 × 10 3 CFU/g and 3.92 × 10 3 CFU/g of root were recovered at 3 and 6 dpi, respectively, which suggests that Str. galilaeus remains in the rhizoplane and does not establish as an endophyte. A similar result was observed in plants treated with T. viride and was confirmed by microscopic examination (Fig. 3 ). The mycelium of T. viride formed a sheath covering the rhizoplane of A. thaliana , which caused a clear decrease in the number of absorbing root hairs (Figs. 3 B and 3 C). No mycelial penetration of the epidermal cells was observed at 1000x magnification (Fig. 3 C). Expression of PR1 and PDF1.2 Inoculation of the rhizosphere with Ser. marcescens , Str. galilaeus , or T. viride , and of leaves with the phytopathogen Colletotrichum M10-F2, induced the differential expression of PR1 and/or PDF1.2 (Fig. 4 ) in a time-varying fashion. For PR1 , the highest level of expression was observed 72 h after inoculation of the roots with T. viride , and was almost 40 times as high as in plants that were treated with only water (controls). At 48 hpi, moreover, Ser. marcescens induced a positive up-regulation of PR1 that was five times greater than that induced by the control, and which at 72 hpi increased by 10.5-fold. These results suggest that Ser. marcescens is more quickly recognised by the plant than T. viride , but that the plant’s response to the fungus is stronger. Although the expression of PDF1.2 exhibited greater variability between biological replicates than PR1 , PDF1.2 expression was highest in T. viride -treated plants at 72 hpi and was 47 times higher than in the control. However, PDF1.2 expression was induced earlier in plants treated with Str. galilaeus , and expression levels were 9 times higher at 24 hpi. PDF1.2 expression was also induced by Ser. marcescens , reaching a 26-fold increase at 48 hpi. However, PDF1.2 expression levels did not change significantly in Colletotrichum sp -treated plants, and expression levels remained similar to those of the control. Our results indicate that T. viride as well as Ser. marcescens induce the co-expression of PDF1.2 and PR1 in the leaves of A. thaliana . However, while Ser. marcescens induced the expression of PR1 from 48–72 hpi, PDF1.2 expression was only observed at 48 hpi. In contrast, co-expression of both genes was induced by T. viride at 48–72 hpi. However, the reverse pattern was observed in Str. galilaeus : at 24 hpi, the expression of PDF1.2 was induced but that of PR1 decreased. Yet, as time progressed, the expression of PDF1.2 decreased while that of PR1 remained similar to that of the control, which suggests a differential antagonistic response between both genes. Colletotrichum M10-F2 only induced the expression of PR1 at 72 hpi. Discussion The success of MBCAs in protecting plants against pathogens depends on their ability to establish in and colonise the plant, and to engage different mechanisms of action that directly or indirectly limit pathogen establishment. Understanding and optimising these factors are key to the development and effective application of MBCAs in agriculture (Alabouvette et al., 2009 ; He et al., 2021 ; Whipps, 2001 ). As such, the present study addresses three fundamental aspects: i) to verify the direct antagonistic activity of 3 MBCAs against a pathogen of agricultural importance; ii) to determine the ability of these MBCAs to colonise the plant roots of A. thaliana , and iii) to assess their potential to engage in indirect antagonism through ISR. According to the literature, the 3 micro-organisms evaluated in the present study manage different strategies to inhibit the growth of Colletotrichum . Our findings corroborated that they indeed exhibit different antagonistic capacities: Str. galilaeus CFSSUR-B12 produces the chitinases Chi 32 and Chi 20, and secondary metabolites (Castillo et al., 2016 ), while Ser. marcescens CFFSUR-B2 uses a combination of chitinases (ChiA, ChiB, ChiC, Chb, and CBP), the pigment prodigiosin, and other metabolites (Gutiérrez-Román et al., 2015 ). Streptomyces galilaeus CFFSUR-B12 was isolated from anthracnose lesions on cacao pods (Castillo et al., 2016 ), which may help explain the high percentage of inhibition of Colletotrichum . Trichoderma viride CFFSUR-A21, a possible parasite that is able to compete, was reported to produce secondary metabolites that can destroy fungal mycelium (Da Costa et al., 2021 ; Druzhinina et al., 2011 ; Harman et al., 2004 ; Nurbailis et al., 2025 ). Because of the used methods, however, it was not possible to determine the effect of secondary metabolites of T. viride on Colletotrichum , nor whether the antagonism exhibited by Str. galilaeus was mediated by antibiosis and/or lysis. Bioprospection studies of micro-organisms for plant-pathogen control rarely address the need to assess the capacity of MBCAs to establish in and/or colonise the plant (Compant et al., 2019 ). Yet, establishment in the roots is a crucial factor in the assessment of the potential of these micro-organisms as biological control agent in ISR. The successful colonisation of A. thaliana by Ser. marcescens CFFSUR-B2, Str. galilaeus CFFSUR-B12, and T. viride CFFSUR-21 is key in the assessment of their potential as inducers of resistance (Kumar et al., 2020 ). At 3 dpi, Ser. marcescens CFFSUR-B2 was able to establish in the rhizoplane and the endorhizosphere. Gyaneshwar et al., 2001 reported that after inoculation, Ser. marcescens could migrate from the rhizosphere to the stems and leaves of rice plants. Results similar to ours have been reported for Ser. marcescens strain AL2-16, an endophytic bacterium isolated from the medicinal plant Achyranthes aspera L. The population of this strain increased from 16.2 × 10 6 to 11.2 × 10 8 CFU/g within 3–5 days after inoculation of the roots of this plant species (Devi et al. 2016 ). Other authors reported that some Serratia strains can colonise plants other than their normal hosts, such as peanuts and maize (Ludueña et al., 2023 ). Streptomyces galilaeus CFFSUR-B12 and T. viride CFFSUR-A21 only colonised the rhizoplane of A. thaliana , although Str. galilaeus was reported to colonise the rhizoplane and endorhizosphere of stems and roots of rice plants (Tian et al., 2007 ). Other Streptomycete species (such as Streptomyces cyaneus and Streptomyces exfoliatus ) were reported to exhibit similar abilities in A. thaliana and lettuce (Bonaldi et al., 2015 ; Chen et al., 2016 ). Differences in colonisation ability between Streptomycete strains could be influenced by tyrosine-encoding genes (Chewning et al., 2019 ), so it would be important to search our strains for the presence of root colonisation-associated genes. It is also necessary to increase the trial duration to confirm that strain CFFSUR-B12 establishes as an endophyte in the roots of A. thaliana. This strain was originally isolated from the epicarp of cacao, and our study demonstrated its ability to colonise the roots of A. thaliana . To the best of our knowledge, this is the first report to describe the colonisation by Str. galilaeus of the roots of a species different from the host it was originally isolated from. Our results show that T. viride CFFSUR-A21 covered the roots of A. thaliana , but did not engage in an endophytic relationship, and also did not colonise the epidermal cells. Various research groups, on the other hand, demonstrated that other T. viride , T. atroviride , and T. harzianum strains were able to colonise the roots of A. thaliana , peppermint, and canola (Garstecka et al., 2023 ; Guo et al., 2020 ; Salas-Marina et al., 2011 ; Tseng et al., 2020 ). Because of their close interaction with the roots of A. thaliana , we explored the possibility that the strains from our study may stimulate ISR (Olanrewaju & Babalola, 2019 ). Our study shows that inoculation of the roots of A. thaliana with Str. galilaeus , Ser. marcescens , or T. viride induced different expression patterns of the defence marker genes PR1 and/or PDF1.2 , which are associated with, respectively, the SA and JA/Et pathways. Inoculation with Ser. marcescens and T. viride induced the co-expression of the SA and JA/Et pathways and translated by the expression of PR1 and PDF1.2 . Hence, the simultaneous expression of PR1 and PDF1.2 by strains CFFSUR-B2 and CFFSUR-A21 might be favoured by a higher degree of root colonisation in comparison to Str. galilaeus . We have not identified the elicitors that stimulated expression in each of these cases, but the simultaneous expression suggests that different types of elicitors are involved. In beneficial bacteria, molecules in the flagella - lipopolysaccharides, siderophores, and quorum-sensing molecules - can act as MAMPS which are recognised by PRRs and induce the priming of plant immunity (Babenko et al., 2022 ; Zhu et al. 2022 ). Serratia strains are characterised by numerous flagella and very active movement. They also produce N-acyl homoserine lactone (AHL), a compound that facilitates the colonisation of plant tissues, aggregation, and biofilm formation. Also, AHL was shown to act as a MAMP (Babenko et al., 2022 ; Ryu et al., 2013 ). Serratia produce a wide range of hydrolytic enzymes, siderophores, and prodigiosin to efficiently control some pests and diseases. Possibly, these molecules are also responsible for the induction of PR1 and PDF1.2 in strain CFFSUR-B2 (Gutierrez-Roman, 2012 and 2015; Meziane et al., 2005 ; Trinh and Nguyen, 2024 ). In the case of T. viride CFFSUR-A21, the induction of PR1 and PDF1.2 may be a function of their role as non-parasitic, soil-dwelling plant-growth promoting fungi (PGPF) that establish mutualistic relationships with plant roots. Several authors reported that this fungus can induce systemic resistance responses to abiotic stress factors (Ahn et al., 2007 ; Segarra et al, 2009 ) and suppress plant pathogens - directly (mycoparasitism, antibiosis, and enzymatic lysis) as well as indirectly - by strengthening immunity in a wide range of plants (Harman et al., 2004 ; Kloepper et al., 2004 ; Pozo and Azcon-Aguilar, 2007; Van Loon et al., 1998 ). Research reports on plant - PGPF interactions described that Trichoderma spp. and mycorrhizal fungi can also induce ISR in plants in a way similar to plant-growth promoting rhizobacteria (Harman et al. 2004 ; Pozo and Azcon-Aguilar, 2007; Vinale et al. 2008 ). Reportedly, this fungus can produce different types of metabolites that can act as elicitors or inducers of resistance (Bisen et al., 2016 ; Harman et al., 2004 ; Woo et al., 2006 ; Woo and Lorito, 2007 ), such as xylanases (Contreras-Cornejo et al., 2014 ; Lotan and Fluhr, 1990 ), avirulence gene-like compounds (Newman et al. 2013 ), low molecular weight compounds that derive from the enzymatic degradation of plant cell walls or fungi (Harman et al., 2004 ; Woo et al., 2006 ; Woo and Lorito, 2007 ). Streptomyces galilaeus induced the expression of PDF1.2 : at 24 hpi, its expression was a 9-fold higher than the control but decreased over time. The expression of PR1 , on the other hand, remained similar to that of the control. Our observations are in agreement with other published reports. Different actinobacteria, including Streptomyces spp., can induce the expression of pathogen resistance, and there is antagonistic cross-communication between the SA and JA/Et pathways (Conn et al., 2008 ; Ryu et al., 2004 ). However, our observations also differ from published reports, in that our strain did not establish as an endophyte, nor induced PR1 expression. It is currently still unclear whether PDF1.2 expression is independent of SA accumulation and which metabolites function as elicitors. To our knowledge, this study presents the first report on the capacity of Str. galilaeus to induce resistance. Most published research reports have focussed on the identification of its secondary metabolites and their possible use in agriculture and medicine (Blumauerová et al., 1979 ; Castillo et al., 2016 ; Kim et al., 1996 ). Inoculation of Colletotrichum M10-F2 in leaves of A. thaliana induced SA-triggered PR1 expression, which according to several authors inhibits biotrophic and hemibiotrophic phytopathogens (Münch et al., 2008 ; Vlot et al., 2021 ). On the other hand, A. thaliana develops necrosis after inoculation with Colletotrichum higginsianum . This pathogenic fungus induces the expression of PR1 and PDF1.2 , possibly because it induces PR1 during the initial stage of the infection, when it acts as a biotrophic pathogen, and PDF1.2 when it enters the necrotrophic phase (Liu et al., 2007 ). A possible explanation for this discrepancy could be that the strain from our study requires more time to enter the necrotrophic phase. Our findings support the idea that co-expression and early activation of the SA and JA/Et pathways in Ser. marcescens CFFSUR-B12 and T. viride CFSUR-A21 can enhance plant defence against biotrophic as well as necrotrophic pathogens, as was reported for Bacillus cereus AR156 as well (Nie et al., 2017 ; Niu et al., 2011 ). However, the mechanisms employed by beneficial microorganisms to induce priming of one or both of the plant defence associated pathways are not yet clear. Also, the response mechanisms they would use against biotrophic, hemibiotrophic, or necrotrophic plant pathogens remain unknown - perhaps more than one mechanism is activated at a time. Plants in nature are known to be simultaneously or sequentially attacked by multiple enemies that often have different strategies and lifestyles. Various authors reported that SA-JA cross-talk allows the plant to prioritise the expression of one pathway over the other, depending on the sequence and nature of the attackers (Kunkel and Brooks 2002 , Verhage et al. 2010 ). Although it was reported that non-pathogenic microorganisms usually induce a low expression level of IR-associated genes, it remains unclear whether the expression level of PR1 and PDF1.2 at inoculation with our MBCAs was sufficient to induce priming. It is necessary to evaluate the induction of PR1 and PDF1.2 genes for longer periods of time, and confirm whether ISR activation offers protection against different types of pathogens. It is important to highlight that the microorganisms studied in the present work have the potential to be included in integrated management programs for tropical plant diseases, given that they can act through different mechanisms of direct antagonism and trigger ISR-related genes through different pathways. Declarations Acknowledgements We are thankful to Yessica Bautista and Jorge Santamaría of the Centro de Investigación Científica de Yucatán (CICY) for the donation of A. thaliana seeds, and to Verónica García for advice on molecular methods. We extend our gratitude to the Laboratorio de Fitopatología of the El Colegio de la Frontera Sur (ECOSUR) in Tapachula for the fungal and bacterial strains used in our study. LELH acknowledges the support from a grant for Master's studies (829983) awarded by the Consejo Nacional de Humanidades, Ciencias y Tecnología (CONAHCYT). Author Contribution Conceptualization: L.E.L-H; K.G-N. and G.H-P. Formal analysis: L.E.L-H. Funding acquisition: K.G-N. and G.H.P. Investigation: L.E.L-H. Methodology: L.E.L-H. Supervision: K.G-N.; G.H-P. and F.H-M. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by L.E.L-H; G.H.P; K.G-N and F.H.M. 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Seneviratne (Eds.), Agro-Environmental Sustainability (pp. 113–125). Springer International Publishing. https://doi.org/10.1007/978-3-319-49724-2_6 Soenens A, Imperial J (2020) Biocontrol capabilities of the genus Serratia . Phytochem Rev 19(3):577–587. https://doi.org/10.1007/s11101-019-09657-5 Tian X, Cao L, Tan H, Han W, Chen M, Liu Y, Zhou S (2007) Diversity of cultivated and uncultivated actinobacterial endophytes in the stems and roots of rice. Microb Ecol 53(4):700–707. https://doi.org/10.1007/s00248-006-9163-4 Trinh LL, Nguyen HH (2024) Role of plant-associated microbes in plant health and development: The case of the Serratia genus. Technol Agron 4(1):0–0. https://doi.org/10.48130/tia-0024-0025 Tseng Y-H, Rouina H, Groten K, Rajani P, Furch ACU, Reichelt M, Baldwin IT, Nataraja KN, Shaanker U, R., Oelmüller R (2020) An endophytic Trichoderma strain promotes growth of its hosts and defends against pathogen attack. Front Plant Sci 11:573670. https://doi.org/10.3389/fpls.2020.573670 Van Loon LC, Bakker PAHM, Pieterse CMJ (1998) Systemic resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36(1):453–483. https://doi.org/10.1146/annurev.phyto.36.1.453 Verhage A, Van Wees SCM, Pieterse CMJ (2010) Plant immunity: It’s the hormones talking, but what do they say? Plant Physiol 154(2):536–540. https://doi.org/10.1104/pp.110.161570 Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Woo SL, Lorito M (2008) Trichoderma–plant–pathogen interactions. Soil Biol Biochem 40(1):1–10. https://doi.org/10.1016/j.soilbio.2007.07.002 Vlot AC, Sales JH, Lenk M, Bauer K, Brambilla A, Sommer A, Chen Y, Wenig M, Nayem S (2021) Systemic propagation of immunity in plants. New Phytol 229(3):1234–1250. https://doi.org/10.1111/nph.16953 Whipps JM (2001) Microbial interactions and biocontrol in the rhizosphere. J Exp Bot 52(suppl 1):487–511. https://doi.org/10.1093/jexbot/52.suppl_1.487 Wonglom P, Suwannarach N, Lumyong S, Ito S, Matsui K, Sunpapao A (2019) Streptomyces angustmyceticus NR8-2 as a potential microorganism for the biological control of leaf spots of Brassica rapa subsp. Pekinensis caused by Colletotrichum sp. And Curvularia lunata . Biol Control 138:104046. https://doi.org/10.1016/j.biocontrol.2019.104046 Woo SL, Lorito M (2007) Exploiting the interactions between fungal antagonists, pathogens and the plant for biocontrol. En M. Vurro & J. Gressel (Eds.), Novel Biotechnologies for Biocontrol Agent Enhancement and Management (pp. 107–130). Springer Netherlands. https://doi.org/10.1007/978-1-4020-5799-1_6 Woo SL, Scala F, Ruocco M, Lorito M (2006) The molecular biology of the interactions between Trichoderma spp., phytopathogenic fungi, and plants. Phytopathology® 96(2):181–185. https://doi.org/10.1094/PHYTO-96-0181 Zakaria L (2021) Diversity of Colletotrichum species associated with anthracnose disease in tropical fruit crops—A review. Agriculture 11(4):297. https://doi.org/10.3390/agriculture11040297 Zhang J, Wang F, Liang F, Zhang Y, Ma L, Wang H, Liu D (2018) Functional analysis of a pathogenesis-related thaumatin-like protein gene TaLr35PR5 from wheat induced by leaf rust fungus. BMC Plant Biol 18(1):76. https://doi.org/10.1186/s12870-018-1297-2 Zhu L, Huang J, Lu X, Zhou C (2022) Development of plant systemic resistance by beneficial rhizobacteria: Recognition, initiation, elicitation and regulation. Front Plant Sci 13:952397. https://doi.org/10.3389/fpls.2022.952397 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Supplementary Table 1: Sequences of the primers used in the gene expression assay. Supplementary Figure 1: Validation of primers PR1 (A), PDF1.2 (B), UBQ10 (C), and EF1α (D). Left panel of each section: images of electrophoresis gels (2% agarose) of RT-qPCR amplicons. Right panel: RT-qPCR melting curve, with corresponding no template control (NTC) and threshold. In all cases a single amplicon is observed. Supplementary Figure 2: Mycelial growth of Colletotrichum strains M07-C2 and M10-F2 in interaction with antagonistic microorganisms: (A) Serratia marcescens CFFSUR-B2 and Streptomyces galilaeus CFFSUR-B12; (B) Trichoderma viride CFFSUR-A21. Presented values are the means of seven replicates ± sd. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7125135","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486781698,"identity":"135f3d59-502d-49cd-8487-9318802c7c66","order_by":0,"name":"Luis Enrique Luna-Hernández","email":"","orcid":"","institution":"Grupo Académico de Biotecnología Ambiental. El Colegio de la Frontera Sur (ECOSUR), Unidad Tapachula","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"Enrique","lastName":"Luna-Hernández","suffix":""},{"id":486781699,"identity":"113c7cc8-94d1-461e-ac11-7c8e759ae195","order_by":1,"name":"Graciela Huerta-Palacios","email":"","orcid":"","institution":"Grupo Académico de Biotecnología Ambiental. El Colegio de la Frontera Sur (ECOSUR), Unidad Tapachula","correspondingAuthor":false,"prefix":"","firstName":"Graciela","middleName":"","lastName":"Huerta-Palacios","suffix":""},{"id":486781700,"identity":"ce94c24f-4fa4-45e7-bb75-61951016b58e","order_by":2,"name":"Francisco Holguín-Meléndez","email":"","orcid":"","institution":"El Colegio de la Frontera Sur","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Holguín-Meléndez","suffix":""},{"id":486781701,"identity":"69f93aab-1c64-4a48-9625-5592a401fcfe","order_by":3,"name":"Karina Guillén-Navarro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACCSA+8KCAgYGfNC0JBgwMkg2kaGEAaTE4QKwWyf41hkBbbPKMjzc/fPiz7R6D7owE/FqkJd4YALWkFZudOWZszNtWzGB2hoB9chJnQFoOJ267kcMmzdiWwGB2vIEoLf8TN89/wyb5E6TlMAG/SPP3gLQcSNwgwcMmwUuMLZIz2AqAWpITZ5xJMzbmOZfAQ9AvEucPb/7wocIusb/98MOHP8oS5MxuJBBwmQSaAh4C6oGAn4AzRsEoGAWjYBQwAAAj5EaDlZjhxgAAAABJRU5ErkJggg==","orcid":"","institution":"Grupo Académico de Biotecnología Ambiental. El Colegio de la Frontera Sur (ECOSUR), Unidad Tapachula","correspondingAuthor":true,"prefix":"","firstName":"Karina","middleName":"","lastName":"Guillén-Navarro","suffix":""}],"badges":[],"createdAt":"2025-07-15 01:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7125135/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7125135/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87062163,"identity":"1645df9b-66d1-4ef1-b93d-6a9da3e60033","added_by":"auto","created_at":"2025-07-18 17:22:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75861,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of mycelial growth of two \u003cem\u003eColletotrichum\u003c/em\u003e strains (M07-C2, M10-F2) at 13 d of confrontation with \u003cem\u003eStreptomyces galilaeus\u003c/em\u003e CFFSUR and \u003cem\u003eSerratia marcescens\u003c/em\u003e in a dual-culture assay (PDA + peptone). Bars represent mean percentage inhibition ± standard deviation (sd). Different lowercase letters indicate significant differences according to Tukey HSD's multiple comparison test of means (p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7125135/v1/f1ab0c3143b258d9132a6afd.png"},{"id":87063186,"identity":"a9f12b8c-4ae6-4e94-9f83-2e9181d05acd","added_by":"auto","created_at":"2025-07-18 17:38:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139802,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth inhibition and mycoparasitism of \u003cem\u003eTrichoderma viride \u003c/em\u003eCFFSUR-A21 on \u003cem\u003eColletotrichum \u003c/em\u003espp. strains M07-C2 and M10-F2. \u003cstrong\u003eA)\u003c/strong\u003e Inhibition of mycelial growth of \u003cem\u003eColletotrichum \u003c/em\u003espp. by \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-A21 at 14 days in dual culture (PDA); bars represent mean percentage inhibition ± sd. \u003cstrong\u003eB)\u003c/strong\u003e Hyphae of \u003cem\u003eT. viride\u003c/em\u003eCFFSUR-A21 interacting with \u003cem\u003eColletotrichum\u003c/em\u003e M07-C2; hyphae of \u003cem\u003eT. viride\u003c/em\u003e (red arrow) coiling around hyphae of M10-C2 (black arrow). Also, an appressorium-like structure (blue arrow) in CFFSUR-A21. \u003cstrong\u003eC)\u003c/strong\u003e Mycelium of \u003cem\u003eT. viride\u003c/em\u003e coiling around and covering the mycelium of \u003cem\u003eColletotrichum \u003c/em\u003esp.\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7125135/v1/2ba2e2231f293622b9b82e8d.png"},{"id":87062166,"identity":"467562c3-b3c6-45f2-a7d4-d8ed49201d7f","added_by":"auto","created_at":"2025-07-18 17:22:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":287628,"visible":true,"origin":"","legend":"\u003cp\u003eLateral roots of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e Col-0 stained with trypan blue in lactophenol. \u003cstrong\u003eA)\u003c/strong\u003e Roots and abundant absorbing root hairs (red arrow) of untreated plants. \u003cstrong\u003eB)\u003c/strong\u003e Roots of \u003cem\u003eA. thaliana \u003c/em\u003ecolonised and covered by a mycelial sheath of \u003cem\u003eT. viride\u003c/em\u003eCFFSUR-A21, with reduced number of absorbing root hairs. \u003cstrong\u003eC)\u003c/strong\u003e Root epidermal cells without apparent penetration of \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-A21 hyphae.\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7125135/v1/4f30881cf91fb6440752a3f2.png"},{"id":87061877,"identity":"dd32d725-11ec-48bd-a055-3d734e60e394","added_by":"auto","created_at":"2025-07-18 17:14:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22714,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression pattern of \u003cem\u003ePR1\u003c/em\u003e(A) and \u003cem\u003ePDF1.2\u003c/em\u003e (B) in leaves of \u003cem\u003eA. thaliana\u003c/em\u003e Col-0 induced by microorganism inoculation 24, 48, and 72 hpi. Expression was normalised to reference genes (\u003cem\u003eUBQ10\u003c/em\u003e and \u003cem\u003eEF1α\u003c/em\u003e) and is plotted in comparison to the 24 h control.\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7125135/v1/1fc8f043910654636a95f9ba.png"},{"id":90055998,"identity":"90075e28-8503-4850-b560-732eab74cc65","added_by":"auto","created_at":"2025-08-28 01:16:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1534960,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7125135/v1/fd2cc1b6-cc21-4270-884b-171e84ded38f.pdf"},{"id":87061883,"identity":"bbd79b4f-5627-47db-bbca-41a6c777194b","added_by":"auto","created_at":"2025-07-18 17:14:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":472285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e: Sequences of the primers used in the gene expression assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e: Validation of primers \u003cem\u003ePR1\u003c/em\u003e (A), \u003cem\u003ePDF1.2\u003c/em\u003e(B), \u003cem\u003eUBQ10\u003c/em\u003e (C), and \u003cem\u003eEF1α\u003c/em\u003e (D). Left panel of each section: images of electrophoresis gels (2% agarose) of RT-qPCR amplicons. Right panel: RT-qPCR melting curve, with corresponding no template control (NTC) and threshold. In all cases a single amplicon is observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2\u003c/strong\u003e: Mycelial growth of \u003cem\u003eColletotrichum\u003c/em\u003e strains M07-C2 and M10-F2 in interaction with antagonistic microorganisms: \u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003eSerratia marcescens\u003c/em\u003e CFFSUR-B2 and \u003cem\u003eStreptomyces galilaeus\u003c/em\u003e CFFSUR-B12; \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eTrichoderma viride\u003c/em\u003e CFFSUR-A21. Presented values are the means of seven replicates ± sd.\u003c/p\u003e","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7125135/v1/cd3b7e2b144e39f021c4c2ed.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Root colonisation and induction of plant defence-associated signalling pathways in Arabidopsis thaliana by Serratia marcescens, Streptomyces galilaeus, and Trichoderma viride","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWorldwide, major agricultural crops are estimated to suffer losses of 20–30% from pests and diseases (Savary et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These are primarily caused by fungal pathogens (83%) and result in US\u003cspan\u003e$\u003c/span\u003e 220\u0026nbsp;billion in economic losses annually (Agrios \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This has led to a rise in the use of fungicides which, however, has resulted in an increasing number of fungi to develop resistance, including fungi of medicinal importance (Ishii, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, new technologies and techniques aimed at reducing fungicide dependence without compromising agricultural production efficiency remain the subject of ongoing development.\u003c/p\u003e\u003cp\u003eBiocontrol techniques involve strategies for the control of phytopathogenic fungi that use living organisms (including microorganisms) or their metabolites, and strive to minimize the impact of these phytopathogenic fungi and the use of agrochemicals (Galli et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe action mechanism of microbial biological control agents (MBCAs) can be direct (antibiosis, enzymatic lysis, parasitism, and competition) (Bonaterra et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Köhl et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or indirect (induced resistance (IR) in plants) (Maciag et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). IR has been studied extensively and is generally categorized into Systemic Acquired Resistance (SAR) and Induced Systemic Resistance (ISR). Both types are involved in the activation of plant defence mechanisms and differ primarily based on the characteristics of the inducer and the site of induction (Vlot et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). SAR is stimulated by phytopathogenic microorganisms or exogenous application of compounds such as salicylic acid (SA) (Ryals et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). A defence response is activated in the area of interaction between microorganism and plant (foliage or roots) (Vlot et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The SA route, which is generally assumed to be the main signalling pathway, primarily acts against biotrophic and hemibiotrophic phytopathogens (Vlot et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the role of the PR gene family (\u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePR2, PR5\u003c/em\u003e) in the SA pathway has been recognised as important (Ryals et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). \u003cem\u003ePR1\u003c/em\u003e plays a central role in the activation of plant defences and is used as a marker for the expression of the SA pathway (Pečenková et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pieterse et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The PR gene family has a range of antimicrobial, antifungal, insecticidal, nematocidal, and cell wall degrading functions, and therefore is associated with reducing the impact of pathogens on plants (Alexander et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn ISR, the plant defence mechanism is triggered by interaction with non-phytopathogenic microorganisms in the roots, and responses protects aerial tissues (Vlot et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The ethylene (Et) and jasmonic acid (JA) pathways are the main signalling pathways and act mainly against necrotrophic plant pathogens (Vlot et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In \u003cem\u003eA. thaliana\u003c/em\u003e, the \u003cem\u003ePDF1.2\u003c/em\u003e gene is associated with and used as a marker for the JA/Et signalling pathway (Penninckx et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Kunkel and Brooks, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). It should be emphasized that the SA and JA/Et pathways are not exclusive to SAR and ISR, and also depend on the characteristics of the microorganisms that interact with the plant, various biotic and abiotic factors, and could happen the cross-talk between these mechanisms (Aerts et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dimopoulou et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nguyen et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pieterse et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn ISR, non-pathogenic bacteria are initially recognised by plants as potential invaders and trigger a defensive response (Arkhipov et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) by activating pattern recognition receptors (PRRs) – transmembrane proteins capable of recognising microbe-associated molecular patterns (MAMPs), such as quorum-sensing (QS) proteins, siderophores, volatile organic compounds (Monaghan and Zipfel, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), or flagellar peptides (Meziane et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In this case, activation of the ISR resistance mechanism is not necessarily complete. It induces a response known as priming, a process by which a plant prepares its defences against the possible future arrival of phytopathogens (Zhu et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIR accelerates the response time of a plant to a wide range of pathogens. Moreover, its effects can be long-lasting if the plant is colonised by beneficial microorganisms (Flors et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Plant-microorganism interactions form a complex system, where various abiotic and biotic factors that determine the efficacy of MBCAs interact (He et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Successful MBCAs should therefore be able to colonise, compete, and remain in the target habitat to directly or indirectly trigger antagonistic interactions (Bonaterra et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ghorbanpour et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Saeed et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEvidence suggests that \u003cem\u003eTrichoderma\u003c/em\u003e species such as \u003cem\u003eTrichoderma atroviride\u003c/em\u003e, \u003cem\u003eTrichoderma harzianum\u003c/em\u003e, and \u003cem\u003eTrichoderma viride\u003c/em\u003e employ direct as well as indirect mechanisms of actions to mitigate the impact of plant pathogens (Ferreira and Musumeci, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nawrocka and Małolepsza, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sharma and Gothalwal, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). On the other hand, indirect antagonism that occurs through ISR in other genera, such as \u003cem\u003eStreptomyces\u003c/em\u003e and \u003cem\u003eSerratia\u003c/em\u003e – both known to inhibit a number of fungal phytopathogens – has remained relatively understudied (Castillo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Dow et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gutiérrez-Román et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Soenens and Imperial, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several ISR research articles on \u003cem\u003eStreptomyces\u003c/em\u003e have been published, although these studies mostly focus on the reduction of phytopathogen-caused symptoms. Their effect on gene expression in the ISR pathway and establishment in roots has remained relatively underexplored (Conn et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ebrahimi-Zarandi et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; L. Liu et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Salla et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe here present our observations on \u003cem\u003eStreptomyces galilaeus\u003c/em\u003e CFFSUR-B12, which previously had been isolated as mycoparasite from an anthracnose-causing fungus on cacao pods. This strain showed promising results as biological control agent (BCA), as it produced secondary metabolites and lysins. Its efficiency, moreover, was evaluated in the laboratory as well as in the field (Castillo, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We also present the findings from our study of \u003cem\u003eSerratia marcescens\u003c/em\u003e CFFSUR-B2, which had been isolated from pangola-grass compost, and which exhibited enzymatic lysis and prodigiosin production as action mechanisms (Gutiérrez-Román et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Both strains showed different antagonistic activity to different phytopathogens of agricultural importance, such as \u003cem\u003eMycosphaerella fijiensis\u003c/em\u003e Morelet and \u003cem\u003eColletotrichum gloeosporioides\u003c/em\u003e (Castillo, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Castillo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gutiérrez-Román et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e and \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eTrichoderma viride\u003c/em\u003e CFFSUR-A21 was included in the study as fast-growing mycoparasite. This strain was isolated from mango leaf litter and showed direct antagonistic activity against \u003cem\u003eM\u003c/em\u003e. \u003cem\u003efijiensis\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e. \u003cem\u003egloeosporioides\u003c/em\u003e. MAMPs have been reported in \u003cem\u003eSerratia\u003c/em\u003e as well as \u003cem\u003eTrichoderma\u003c/em\u003e, which might indicate that the strains we included in our study can activate ISR (Babenko et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Contreras-Cornejo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ryu et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Schenk et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Also, these strains are of tropical origin and maintain their direct antagonistic activity at temperatures higher than 28 ºC. If their ability to induce systemic resistance can be demonstrated, it would be possible to investigate whether they can induce ISR in crop plants, such as banana, coffee, and cacao. These crops are strongly affected by phytopathogens but have received limited attention in this field of study.\u003c/p\u003e\u003cp\u003eThe objective of our study was to determine whether \u003cem\u003eStr. galilaeus\u003c/em\u003e CFFSUR-B12, \u003cem\u003eSer. marcescens\u003c/em\u003e CFFSUR-B2, and \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-A21 – strains that are characterised by their broad antagonistic capacity against phytopathogens – could induce changes in the expression of ISR-associated genes in \u003cem\u003eA. thaliana\u003c/em\u003e. Because these strains employ different mechanisms of direct antagonistic action against phytopathogenic fungi, we would expect that if they are capable of activating ISR, they would activate it in different ways. Therefore, the antagonistic activity of the evaluated strains was first tested against two \u003cem\u003eColletotrichum\u003c/em\u003e spp. strains that had been isolated from mango and cacao fruit (\u003cem\u003eColletotrichum\u003c/em\u003e is a plant pathogen of agricultural importance in Latin America; Zakaria, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We then determined their ability to establish in roots and induced expression of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e (associated with SAR and ISR, respectively) using \u003cem\u003eA. thaliana\u003c/em\u003e Col-0 as plant model.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003ePlant culture conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Col-0) was used as a study model. Seeds were disinfected by immersion in ethanol (70%) for 1 min and then in NaClO (20%, commercial product) for 8 min, followed by rinsing (four washes with sterile water) to remove remaining NaClO. Disinfected seeds were kept in 500 µL of sterile water at 4°C for 48 h and then germinated in Petri dishes containing MS medium (Murashige \u0026amp; Skoog, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). Fourteen-day-old seedlings with lateral roots and an approximate height of 0.5 cm were transplanted into glass jars containing MS medium and placed in a growth chamber (25 ± 1 ºC, 12 h light-dark photoperiod, light intensity of 8500 lux (ViparSpectra P1000 grow light)).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicroorganisms\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe bacterial strains \u003cem\u003eSer. marcescens\u003c/em\u003e CFFSUR-B2 and \u003cem\u003eStr. galilaeus\u003c/em\u003e CFFSUR-B12 were cultured on nutrient agar (NA, pH 7). \u003cem\u003eTrichoderma viride\u003c/em\u003e CFFSUR- A21 and the phytopathogenic \u003cem\u003eColletotrichum\u003c/em\u003e spp. strains M07-C2 and M10-F2 (isolated from cacao and mango crops, respectively) were cultured on potato dextrose agar (PDA, pH 7). All microorganisms were incubated at 25ºC.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of inocula\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBacterial cell density was determined and adjusted to 1.5x10\u003csup\u003e8\u003c/sup\u003e cells/ml (0.5 McFarland scale) using a McFarland spectrophotometric procedure (λ = 625 nm; Leber, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; McFarland, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1907\u003c/span\u003e). Results were verified in culture medium by counting of the colony-forming units (CFU).\u003c/p\u003e\u003cp\u003eFor fungal inocula, spores were collected from \u003cem\u003eT. viride\u003c/em\u003e (7d of growth) and the \u003cem\u003eColletotrichum\u003c/em\u003e strains (at 14 d), and the number of required spores was determined and adjusted using a Neubauer chamber.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro antagonism assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antagonistic activity of the \u003cem\u003eColletotrichum\u003c/em\u003e strains was determined by a dual-culture assay in Petri dishes (90 mm diameter), with 7 biological replicates per treatment (Chen et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Raymaekers et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sari et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Controls consisted of a mycelial disc (5 mm diameter) of \u003cem\u003eColletotrichum\u003c/em\u003e spp placed 2 cm away from the edge of a Petri dish.\u003c/p\u003e\u003cp\u003eThe bacterial phytopathogen-antagonist assay was performed on PDA medium supplemented with 10 g/L casein peptone. Bacterial strains (CFFSUR-B2 or CFFSUR-B12) were inoculated along a straight line 2 cm from the edge of the plate. Then, 24 h later, a mycelial disc (5mm diameter) of a phytopathogenic fungus (\u003cem\u003eColletotrichum\u003c/em\u003e sp M07-C2 or M10-F2) was placed 4 cm away from the bacterial streak (Wonglom et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the \u003cem\u003eTrichoderma-Colletotrichum\u003c/em\u003e assay, a mycelial disc (5 mm diameter) of \u003cem\u003eColletotrichum\u003c/em\u003e spp was placed 2 cm away from the edge of a plate with PDA medium. After 72 h, a 5 mm diameter mycelial disc of \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-A21 was placed at 4 cm of the phytopathogen. The inhibitory effect was estimated by measuring every 24 h the growth radius of the \u003cem\u003eColletotrichum\u003c/em\u003e spp strains towards the antagonist, until the control mycelium completely covered the plate surface. The percentage inhibition of radial growth was calculated using the following formula: % inhibition = (R1-R2) / R1x100, in which R1 represents the radial growth in the control, and R2 radial growth of the phytopathogen against the antagonist (Wonglom et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe interaction between \u003cem\u003eT. viride\u003c/em\u003e and \u003cem\u003eColletotrichum\u003c/em\u003e spp. was assessed using the Bell scale (Bell et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1982\u003c/span\u003e): 1) the antagonist outcompetes the pathogen and entirely covers the surface of the culture medium; 2) the antagonist covers 2/3 of the culture medium; 3) the antagonist covers 1/2 of the culture medium, the remainder being covered by the pathogen; 4) the pathogen covers 3/4 of the culture medium, while the antagonist covers only 1/4; 5) the pathogen entirely covers the culture medium and the antagonist.\u003c/p\u003e\u003cp\u003eData from the bacteria-\u003cem\u003eColletotrichum\u003c/em\u003e antagonism assay were analysed by two-way ANOVA with a subsequent post hoc Tukey HSD test, while data from the \u003cem\u003eTrichoderma\u003c/em\u003e-\u003cem\u003eColletotrichum\u003c/em\u003e assay were analysed by two-sample Student's t-test. Analyses were performed using the R statistical software package (v4. 3.3; R Core Team 2023).\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe establishment of micro-organisms in the root\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEighteen plants of \u003cem\u003eA. thaliana\u003c/em\u003e (28 d old) were arranged over 2 bacterial treatments and a treatment control (sterile water). The rhizosphere of the treatment plants was inoculated with 25 µL of a bacterial suspension of 1.5x10\u003csup\u003e8\u003c/sup\u003e cells/ml (\u003cem\u003eSer. marcescens\u003c/em\u003e or \u003cem\u003eStr. galilaeus\u003c/em\u003e, 6 plants each), while the rhizosphere of the 6 control plants was treated with 25 µL of sterile distilled water.\u003c/p\u003e\u003cp\u003eTo determine the establishment of micro-organisms in the roots, independent root samples were taken from 3 plants per treatment at 3 and 6 d following inoculation (dpi). To determine the establishment of bacteria in the rhizoplane, 2 cm segments were taken from the apical region of lateral roots. The segments were washed gently with sterile water to remove any remaining medium, and the mass of the roots was determined with an analytical balance. To collect bacteria from the rhizoplane, rootlets were placed in 1.5 ml tubes that contained 2 ml sterile water. The rootlets were then subjected to a sonication cycle (Cole-Parmer UC 200): 30 s of sonication at approximately 24000 Hz, followed by 10 s without sonication, five times. They were then vortexed for 5 s using a Vortex-Genie 2 at level 4. Then, 25 µL of undiluted or serially diluted (10\u003csup\u003e− 1\u003c/sup\u003e, 10\u003csup\u003e− 2\u003c/sup\u003e, and 10\u003csup\u003e− 3\u003c/sup\u003e) supernatant was spread on the surface of a NA-containing Petri dish using a Drigalski spatula. Next, the Petri dishes were incubated at 25 ºC and CFU was counted 48 h later. Three replicates were performed per treatment/dpi/dilution. To determine the establishment of bacteria in the endorhizosphere, ultrasonicated root fragments were disinfected with 2% NaClO (8 min), followed by rinsing with sterile water (4 washes) to remove NaClO residues. The fragments were then macerated with a pistil in 1 ml sterile water, and 25 µL of undiluted or serially diluted (10\u003csup\u003e− 1\u003c/sup\u003e and 10\u003csup\u003e− 2\u003c/sup\u003e) supernatant was dispersed on the surface of a NA-containing Petri dish for CFU counting.\u003c/p\u003e\u003cp\u003eThe establishment of \u003cem\u003eT. viride\u003c/em\u003e in the roots of \u003cem\u003eA. thaliana\u003c/em\u003e was evaluated using the same procedure as for bacteria: the rhizosphere of six 28-day-old plants was inoculated with 20 µL of 2.6 x 10\u003csup\u003e5\u003c/sup\u003e spores/ml of \u003cem\u003eT. viride\u003c/em\u003e, while the control treatment consisted of six plants treated with sterile water. The procedure to determine the establishment of \u003cem\u003eT. viride\u003c/em\u003e in roots was also the same as for bacteria, but supernatants were cultured on PDA. Also, roots were stained with trypan blue in lactophenol for microscopic observation (Phillips \u0026amp; Hayman, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). Samples were observed under different magnifications (40x, 100x, 400x, and 1000x) with a Leica DM750 microscope. Photographic images were taken with a Leica ICC50W installed on the microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of the genes\u003c/b\u003e \u003cb\u003ePR1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePDF1.2\u003c/b\u003e\u003c/p\u003e\u003cp\u003eForty-five 22-day-old \u003cem\u003eA. thaliana\u003c/em\u003e plants were arranged in a completely randomised block design with five treatments (\u003cem\u003eStr. galilaeus\u003c/em\u003e CFFSUR-B12, \u003cem\u003eSer. marcescens\u003c/em\u003e CFFSUR-B2, \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-A21, \u003cem\u003eColletotrichum\u003c/em\u003e M10-F2, and water (control)) and nine replicates. The rhizosphere was inoculated with 25 µl of 1.6 x 10\u003csup\u003e8\u003c/sup\u003e CFU/ml of \u003cem\u003eStr. galilaeus\u003c/em\u003e, 25 µl of 2.8 X 10\u003csup\u003e8\u003c/sup\u003e CFU/ml of \u003cem\u003eSer. marcescens\u003c/em\u003e, or 20 µl of 5 x 10\u003csup\u003e5\u003c/sup\u003e \u003cem\u003eT. viride\u003c/em\u003e spores/ml. In the \u003cem\u003eColletotrichum\u003c/em\u003e M10-F2 treatment, leaves were inoculated with 15 µl of 2.45 x 10\u003csup\u003e5\u003c/sup\u003e spores/ml. In the control treatment, the rhizosphere was treated with 25 µL of sterile water. Treatment response was evaluated by taking 3 plants and then 3 leaves per plant (randomly and without replacement) at 24, 48, and 72 h post inoculation (hpi). Leaves were immediately immersed in the lysis solution of the extraction kit (PureLink RNA Mini Kit) to exclude expression changes. Total RNA extraction was performed immediately after sampling, and extracted RNA was stored at -80 ºC for a maximum of 2.5 months.\u003c/p\u003e\u003cp\u003ecDNA was synthesised from 2 µL of RNA using the Maxima First cDNA Synthesis Kit for RT-qPCR, and stored at -80°C until qPCR analysis. Prior to analysis, qPCR primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were validated by examining the melting curves for the presence of a single amplicon per primer set (65 ºC to 95 ºC in 0.5 ºC increments), separation on 2% agarose gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS, supplementary material), and sequencing of the amplicon. qPCR reactions (10 µL total volume) were performed with 1 µL of cDNA obtained, 0.2 µL of each primer (0.2 pM), 5 µL of SSOAdvanced Universal SYBR Green Supermix (1x), and 3.6 µL of nuclease-free water (2 technical replicates were run). PCR was performed on a CFX96 Touch (Bio-Rad) instrument using the following conditions: initial denaturation at 98°C for 30 s, 40 cycles of 15 s at 98°C and 30 s at 60°C. The relative expression level of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e was normalised to that of the reference genes \u003cem\u003eUBQ10\u003c/em\u003e and \u003cem\u003eEF1α\u003c/em\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) using the using Bio-Rad CFX Maestro™ 1.1 software (v4.2.2433.1219), which performs a calculation based on Pfaffl's method (ΔΔΔCq) (Pfaffl, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIn vitro antagonism assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe capacity of \u003cem\u003eStr. galilaeus\u003c/em\u003e, \u003cem\u003eSer. marcescens\u003c/em\u003e, and \u003cem\u003eT. viride\u003c/em\u003e to inhibit mycelial growth of \u003cem\u003eColletotrichum\u003c/em\u003e spp. strains was examined using a dual-culture assay. Their antagonistic activity was confirmed, and significant differences for the levels of inhibition and differential sensitivity were observed between \u003cem\u003eColletotrichum\u003c/em\u003e strains and MBCAs. The percentage of growth inhibition induced by \u003cem\u003eStr. galilaeus\u003c/em\u003e (p \u0026lt; 0.001) was significantly higher than the inhibition by \u003cem\u003eSer. marcescens\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eColletotrichum\u003c/em\u003e strains showed differential sensitivity towards MBCA action mechanisms. Strain M10-F2 was more sensitive to \u003cem\u003eStr. galilaeus\u003c/em\u003e (mean = 75.9%; sd = 5.33; p \u0026lt; 0.001) and \u003cem\u003eT. viride\u003c/em\u003e (mean = 70.1%; sd = 0.82; p \u0026lt; 0.001) than M07-C2 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eTrichoderma viride\u003c/em\u003e CFFSUR-A21 exhibited relatively rapid and aggressive growth, and after 2 d scored 1 on the Bell scale: it completely covered the Petri dish, and grew and sporulated over \u003cem\u003eColletotrichum\u003c/em\u003e spp.\u003c/p\u003e\u003cp\u003eMicroscopic examination revealed that the hyphae of \u003cem\u003eT. viride\u003c/em\u003e coiled round the hyphae of \u003cem\u003eColletotrichum\u003c/em\u003e spp. (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows the presence of an appressorium-like structure, a feature linked with mycoparasitism, on the hyphal tips of \u003cem\u003eT. viride\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePresence of microorganisms in the roots\u003c/b\u003e\u003c/p\u003e\u003cp\u003eExperiments with the inoculation of \u003cem\u003eSer. marcescens\u003c/em\u003e, \u003cem\u003eStr. galilaeus\u003c/em\u003e, and \u003cem\u003eT. viride\u003c/em\u003e in the roots of \u003cem\u003eA. thaliana\u003c/em\u003e showed that these microorganisms were able to establish in the rhizoplane and root endosphere. \u003cem\u003eSerratia marcescens\u003c/em\u003e became established as an endophyte and in the rhizoplane, while \u003cem\u003eStr. galilaeus\u003c/em\u003e and \u003cem\u003eT. viride\u003c/em\u003e only remained in the rhizoplane.\u003c/p\u003e\u003cp\u003eFrom plants that were inoculated with \u003cem\u003eSer. marcescens\u003c/em\u003e, 2.29 × 10\u003csup\u003e7\u003c/sup\u003e CFU/g and 4.68 × 10\u003csup\u003e7\u003c/sup\u003e CFU/g were recovered from rhizoplane root, at 3 and 6 dpi, respectively; and from the internal part (endophytes) 4.13 × 10\u003csup\u003e5\u003c/sup\u003e CFU/g and 3.74 × 10\u003csup\u003e5\u003c/sup\u003e CFU/g were recovered at 3 and 6 dpi, respectively. The observed increase in CFU/g suggests that this strain is able to colonise the roots of \u003cem\u003eA. thaliana\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eFrom plants inoculated with \u003cem\u003eStr. galilaeus\u003c/em\u003e, only 6.5 × 10\u003csup\u003e3\u003c/sup\u003e CFU/g and 3.92 × 10\u003csup\u003e3\u003c/sup\u003e CFU/g of root were recovered at 3 and 6 dpi, respectively, which suggests that \u003cem\u003eStr. galilaeus\u003c/em\u003e remains in the rhizoplane and does not establish as an endophyte. A similar result was observed in plants treated with \u003cem\u003eT. viride\u003c/em\u003e and was confirmed by microscopic examination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The mycelium of \u003cem\u003eT. viride\u003c/em\u003e formed a sheath covering the rhizoplane of \u003cem\u003eA. thaliana\u003c/em\u003e, which caused a clear decrease in the number of absorbing root hairs (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). No mycelial penetration of the epidermal cells was observed at 1000x magnification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of\u003c/b\u003e \u003cb\u003ePR1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePDF1.2\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInoculation of the rhizosphere with \u003cem\u003eSer. marcescens\u003c/em\u003e, \u003cem\u003eStr. galilaeus\u003c/em\u003e, or \u003cem\u003eT. viride\u003c/em\u003e, and of leaves with the phytopathogen \u003cem\u003eColletotrichum\u003c/em\u003e M10-F2, induced the differential expression of \u003cem\u003ePR1\u003c/em\u003e and/or \u003cem\u003ePDF1.2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) in a time-varying fashion.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003ePR1\u003c/em\u003e, the highest level of expression was observed 72 h after inoculation of the roots with \u003cem\u003eT. viride\u003c/em\u003e, and was almost 40 times as high as in plants that were treated with only water (controls). At 48 hpi, moreover, \u003cem\u003eSer. marcescens\u003c/em\u003e induced a positive up-regulation of \u003cem\u003ePR1\u003c/em\u003e that was five times greater than that induced by the control, and which at 72 hpi increased by 10.5-fold. These results suggest that \u003cem\u003eSer. marcescens\u003c/em\u003e is more quickly recognised by the plant than \u003cem\u003eT. viride\u003c/em\u003e, but that the plant’s response to the fungus is stronger.\u003c/p\u003e\u003cp\u003eAlthough the expression of \u003cem\u003ePDF1.2\u003c/em\u003e exhibited greater variability between biological replicates than \u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePDF1.2\u003c/em\u003e expression was highest in \u003cem\u003eT. viride\u003c/em\u003e-treated plants at 72 hpi and was 47 times higher than in the control. However, \u003cem\u003ePDF1.2\u003c/em\u003e expression was induced earlier in plants treated with \u003cem\u003eStr. galilaeus\u003c/em\u003e, and expression levels were 9 times higher at 24 hpi. \u003cem\u003ePDF1.2\u003c/em\u003e expression was also induced by \u003cem\u003eSer. marcescens\u003c/em\u003e, reaching a 26-fold increase at 48 hpi. However, \u003cem\u003ePDF1.2\u003c/em\u003e expression levels did not change significantly in \u003cem\u003eColletotrichum sp\u003c/em\u003e-treated plants, and expression levels remained similar to those of the control.\u003c/p\u003e\u003cp\u003eOur results indicate that \u003cem\u003eT. viride\u003c/em\u003e as well as \u003cem\u003eSer. marcescens\u003c/em\u003e induce the co-expression of \u003cem\u003ePDF1.2\u003c/em\u003e and \u003cem\u003ePR1\u003c/em\u003e in the leaves of \u003cem\u003eA. thaliana\u003c/em\u003e. However, while \u003cem\u003eSer. marcescens\u003c/em\u003e induced the expression of \u003cem\u003ePR1\u003c/em\u003e from 48–72 hpi, \u003cem\u003ePDF1.2\u003c/em\u003e expression was only observed at 48 hpi. In contrast, co-expression of both genes was induced by \u003cem\u003eT. viride\u003c/em\u003e at 48–72 hpi. However, the reverse pattern was observed in \u003cem\u003eStr. galilaeus\u003c/em\u003e: at 24 hpi, the expression of \u003cem\u003ePDF1.2\u003c/em\u003e was induced but that of \u003cem\u003ePR1\u003c/em\u003e decreased. Yet, as time progressed, the expression of \u003cem\u003ePDF1.2\u003c/em\u003e decreased while that of \u003cem\u003ePR1\u003c/em\u003e remained similar to that of the control, which suggests a differential antagonistic response between both genes. \u003cem\u003eColletotrichum\u003c/em\u003e M10-F2 only induced the expression of \u003cem\u003ePR1\u003c/em\u003e at 72 hpi.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe success of MBCAs in protecting plants against pathogens depends on their ability to establish in and colonise the plant, and to engage different mechanisms of action that directly or indirectly limit pathogen establishment. Understanding and optimising these factors are key to the development and effective application of MBCAs in agriculture (Alabouvette et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; He et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Whipps, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). As such, the present study addresses three fundamental aspects: i) to verify the direct antagonistic activity of 3 MBCAs against a pathogen of agricultural importance; ii) to determine the ability of these MBCAs to colonise the plant roots of \u003cem\u003eA. thaliana\u003c/em\u003e, and iii) to assess their potential to engage in indirect antagonism through ISR.\u003c/p\u003e\u003cp\u003eAccording to the literature, the 3 micro-organisms evaluated in the present study manage different strategies to inhibit the growth of \u003cem\u003eColletotrichum\u003c/em\u003e. Our findings corroborated that they indeed exhibit different antagonistic capacities: \u003cem\u003eStr. galilaeus\u003c/em\u003e CFSSUR-B12 produces the chitinases Chi 32 and Chi 20, and secondary metabolites (Castillo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), while \u003cem\u003eSer. marcescens\u003c/em\u003e CFFSUR-B2 uses a combination of chitinases (ChiA, ChiB, ChiC, Chb, and CBP), the pigment prodigiosin, and other metabolites (Gutiérrez-Román et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eStreptomyces galilaeus\u003c/em\u003e CFFSUR-B12 was isolated from anthracnose lesions on cacao pods (Castillo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which may help explain the high percentage of inhibition of \u003cem\u003eColletotrichum\u003c/em\u003e. \u003cem\u003eTrichoderma viride\u003c/em\u003e CFFSUR-A21, a possible parasite that is able to compete, was reported to produce secondary metabolites that can destroy fungal mycelium (Da Costa et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Druzhinina et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Harman et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Nurbailis et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Because of the used methods, however, it was not possible to determine the effect of secondary metabolites of \u003cem\u003eT. viride\u003c/em\u003e on \u003cem\u003eColletotrichum\u003c/em\u003e, nor whether the antagonism exhibited by \u003cem\u003eStr. galilaeus\u003c/em\u003e was mediated by antibiosis and/or lysis.\u003c/p\u003e\u003cp\u003eBioprospection studies of micro-organisms for plant-pathogen control rarely address the need to assess the capacity of MBCAs to establish in and/or colonise the plant (Compant et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Yet, establishment in the roots is a crucial factor in the assessment of the potential of these micro-organisms as biological control agent in ISR.\u003c/p\u003e\u003cp\u003eThe successful colonisation of \u003cem\u003eA. thaliana\u003c/em\u003e by \u003cem\u003eSer. marcescens\u003c/em\u003e CFFSUR-B2, \u003cem\u003eStr. galilaeus\u003c/em\u003e CFFSUR-B12, and \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-21 is key in the assessment of their potential as inducers of resistance (Kumar et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). At 3 dpi, \u003cem\u003eSer. marcescens\u003c/em\u003e CFFSUR-B2 was able to establish in the rhizoplane and the endorhizosphere. Gyaneshwar et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2001\u003c/span\u003e reported that after inoculation, \u003cem\u003eSer. marcescens\u003c/em\u003e could migrate from the rhizosphere to the stems and leaves of rice plants. Results similar to ours have been reported for \u003cem\u003eSer. marcescens\u003c/em\u003e strain AL2-16, an endophytic bacterium isolated from the medicinal plant \u003cem\u003eAchyranthes aspera\u003c/em\u003e L. The population of this strain increased from 16.2 × 10\u003csup\u003e6\u003c/sup\u003e to 11.2 × 10\u003csup\u003e8\u003c/sup\u003e CFU/g within 3–5 days after inoculation of the roots of this plant species (Devi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Other authors reported that some \u003cem\u003eSerratia\u003c/em\u003e strains can colonise plants other than their normal hosts, such as peanuts and maize (Ludueña et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eStreptomyces galilaeus\u003c/em\u003e CFFSUR-B12 and \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-A21 only colonised the rhizoplane of \u003cem\u003eA. thaliana\u003c/em\u003e, although \u003cem\u003eStr. galilaeus\u003c/em\u003e was reported to colonise the rhizoplane and endorhizosphere of stems and roots of rice plants (Tian et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Other Streptomycete species (such as \u003cem\u003eStreptomyces cyaneus\u003c/em\u003e and \u003cem\u003eStreptomyces exfoliatus\u003c/em\u003e) were reported to exhibit similar abilities in \u003cem\u003eA. thaliana\u003c/em\u003e and lettuce (Bonaldi et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Differences in colonisation ability between Streptomycete strains could be influenced by tyrosine-encoding genes (Chewning et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), so it would be important to search our strains for the presence of root colonisation-associated genes. It is also necessary to increase the trial duration to confirm that strain CFFSUR-B12 establishes as an endophyte in the roots of \u003cem\u003eA. thaliana.\u003c/em\u003e This strain was originally isolated from the epicarp of cacao, and our study demonstrated its ability to colonise the roots of \u003cem\u003eA. thaliana\u003c/em\u003e. To the best of our knowledge, this is the first report to describe the colonisation by \u003cem\u003eStr. galilaeus\u003c/em\u003e of the roots of a species different from the host it was originally isolated from.\u003c/p\u003e\u003cp\u003eOur results show that \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-A21 covered the roots of \u003cem\u003eA. thaliana\u003c/em\u003e, but did not engage in an endophytic relationship, and also did not colonise the epidermal cells. Various research groups, on the other hand, demonstrated that other \u003cem\u003eT. viride\u003c/em\u003e, \u003cem\u003eT. atroviride\u003c/em\u003e, and \u003cem\u003eT. harzianum\u003c/em\u003e strains were able to colonise the roots of \u003cem\u003eA. thaliana\u003c/em\u003e, peppermint, and canola (Garstecka et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Salas-Marina et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Tseng et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Because of their close interaction with the roots of \u003cem\u003eA. thaliana\u003c/em\u003e, we explored the possibility that the strains from our study may stimulate ISR (Olanrewaju \u0026amp; Babalola, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur study shows that inoculation of the roots of \u003cem\u003eA. thaliana\u003c/em\u003e with \u003cem\u003eStr. galilaeus\u003c/em\u003e, \u003cem\u003eSer. marcescens\u003c/em\u003e, or \u003cem\u003eT. viride\u003c/em\u003e induced different expression patterns of the defence marker genes \u003cem\u003ePR1\u003c/em\u003e and/or \u003cem\u003ePDF1.2\u003c/em\u003e, which are associated with, respectively, the SA and JA/Et pathways. Inoculation with \u003cem\u003eSer. marcescens\u003c/em\u003e and \u003cem\u003eT. viride\u003c/em\u003e induced the co-expression of the SA and JA/Et pathways and translated by the expression of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e. Hence, the simultaneous expression of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e by strains CFFSUR-B2 and CFFSUR-A21 might be favoured by a higher degree of root colonisation in comparison to \u003cem\u003eStr. galilaeus\u003c/em\u003e. We have not identified the elicitors that stimulated expression in each of these cases, but the simultaneous expression suggests that different types of elicitors are involved. In beneficial bacteria, molecules in the flagella - lipopolysaccharides, siderophores, and quorum-sensing molecules - can act as MAMPS which are recognised by PRRs and induce the priming of plant immunity (Babenko et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eSerratia\u003c/em\u003e strains are characterised by numerous flagella and very active movement. They also produce N-acyl homoserine lactone (AHL), a compound that facilitates the colonisation of plant tissues, aggregation, and biofilm formation. Also, AHL was shown to act as a MAMP (Babenko et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ryu et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). \u003cem\u003eSerratia\u003c/em\u003e produce a wide range of hydrolytic enzymes, siderophores, and prodigiosin to efficiently control some pests and diseases. Possibly, these molecules are also responsible for the induction of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e in strain CFFSUR-B2 (Gutierrez-Roman, 2012 and 2015; Meziane et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Trinh and Nguyen, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the case of \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-A21, the induction of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e may be a function of their role as non-parasitic, soil-dwelling plant-growth promoting fungi (PGPF) that establish mutualistic relationships with plant roots. Several authors reported that this fungus can induce systemic resistance responses to abiotic stress factors (Ahn et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Segarra et al, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and suppress plant pathogens - directly (mycoparasitism, antibiosis, and enzymatic lysis) as well as indirectly - by strengthening immunity in a wide range of plants (Harman et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kloepper et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Pozo and Azcon-Aguilar, 2007; Van Loon et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Research reports on plant - PGPF interactions described that \u003cem\u003eTrichoderma\u003c/em\u003e spp. and mycorrhizal fungi can also induce ISR in plants in a way similar to plant-growth promoting rhizobacteria (Harman et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Pozo and Azcon-Aguilar, 2007; Vinale et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Reportedly, this fungus can produce different types of metabolites that can act as elicitors or inducers of resistance (Bisen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Harman et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Woo et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Woo and Lorito, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), such as xylanases (Contreras-Cornejo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lotan and Fluhr, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), avirulence gene-like compounds (Newman et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), low molecular weight compounds that derive from the enzymatic degradation of plant cell walls or fungi (Harman et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Woo et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Woo and Lorito, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eStreptomyces galilaeus\u003c/em\u003e induced the expression of \u003cem\u003ePDF1.2\u003c/em\u003e: at 24 hpi, its expression was a 9-fold higher than the control but decreased over time. The expression of \u003cem\u003ePR1\u003c/em\u003e, on the other hand, remained similar to that of the control. Our observations are in agreement with other published reports. Different actinobacteria, including \u003cem\u003eStreptomyces\u003c/em\u003e spp., can induce the expression of pathogen resistance, and there is antagonistic cross-communication between the SA and JA/Et pathways (Conn et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ryu et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). However, our observations also differ from published reports, in that our strain did not establish as an endophyte, nor induced \u003cem\u003ePR1\u003c/em\u003e expression. It is currently still unclear whether \u003cem\u003ePDF1.2\u003c/em\u003e expression is independent of SA accumulation and which metabolites function as elicitors. To our knowledge, this study presents the first report on the capacity of \u003cem\u003eStr. galilaeus\u003c/em\u003e to induce resistance. Most published research reports have focussed on the identification of its secondary metabolites and their possible use in agriculture and medicine (Blumauerová et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Castillo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInoculation of \u003cem\u003eColletotrichum\u003c/em\u003e M10-F2 in leaves of \u003cem\u003eA. thaliana\u003c/em\u003e induced SA-triggered \u003cem\u003ePR1\u003c/em\u003e expression, which according to several authors inhibits biotrophic and hemibiotrophic phytopathogens (Münch et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Vlot et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). On the other hand, \u003cem\u003eA. thaliana\u003c/em\u003e develops necrosis after inoculation with \u003cem\u003eColletotrichum higginsianum\u003c/em\u003e. This pathogenic fungus induces the expression of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e, possibly because it induces \u003cem\u003ePR1\u003c/em\u003e during the initial stage of the infection, when it acts as a biotrophic pathogen, and \u003cem\u003ePDF1.2\u003c/em\u003e when it enters the necrotrophic phase (Liu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). A possible explanation for this discrepancy could be that the strain from our study requires more time to enter the necrotrophic phase.\u003c/p\u003e\u003cp\u003eOur findings support the idea that co-expression and early activation of the SA and JA/Et pathways in \u003cem\u003eSer. marcescens\u003c/em\u003e CFFSUR-B12 and \u003cem\u003eT. viride\u003c/em\u003e CFSUR-A21 can enhance plant defence against biotrophic as well as necrotrophic pathogens, as was reported for \u003cem\u003eBacillus cereus\u003c/em\u003e AR156 as well (Nie et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Niu et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, the mechanisms employed by beneficial microorganisms to induce priming of one or both of the plant defence associated pathways are not yet clear. Also, the response mechanisms they would use against biotrophic, hemibiotrophic, or necrotrophic plant pathogens remain unknown - perhaps more than one mechanism is activated at a time.\u003c/p\u003e\u003cp\u003ePlants in nature are known to be simultaneously or sequentially attacked by multiple enemies that often have different strategies and lifestyles. Various authors reported that SA-JA cross-talk allows the plant to prioritise the expression of one pathway over the other, depending on the sequence and nature of the attackers (Kunkel and Brooks \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Verhage et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough it was reported that non-pathogenic microorganisms usually induce a low expression level of IR-associated genes, it remains unclear whether the expression level of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e at inoculation with our MBCAs was sufficient to induce priming. It is necessary to evaluate the induction of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e genes for longer periods of time, and confirm whether ISR activation offers protection against different types of pathogens. It is important to highlight that the microorganisms studied in the present work have the potential to be included in integrated management programs for tropical plant diseases, given that they can act through different mechanisms of direct antagonism and trigger ISR-related genes through different pathways.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eWe are thankful to Yessica Bautista and Jorge Santamaría of the \u003cem\u003eCentro de Investigación Científica de Yucatán\u003c/em\u003e (CICY) for the donation of \u003cem\u003eA. thaliana\u003c/em\u003e seeds, and to Verónica García for advice on molecular methods. We extend our gratitude to the \u003cem\u003eLaboratorio de Fitopatología\u003c/em\u003e of the \u003cem\u003eEl Colegio de la Frontera Sur\u003c/em\u003e (ECOSUR) in Tapachula for the fungal and bacterial strains used in our study. LELH acknowledges the support from a grant for Master's studies (829983) awarded by the \u003cem\u003eConsejo Nacional de Humanidades, Ciencias y Tecnología\u003c/em\u003e (CONAHCYT).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: L.E.L-H; K.G-N. and G.H-P. Formal analysis: L.E.L-H. Funding acquisition: K.G-N. and G.H.P. Investigation: L.E.L-H. Methodology: L.E.L-H. Supervision: K.G-N.; G.H-P. and F.H-M. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by L.E.L-H; G.H.P; K.G-N and F.H.M. The first draft of the manuscript was written by L.E.L-H and K.G-N and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAerts N, Pereira Mendes M, Van Wees SCM (2021) Multiple levels of crosstalk in hormone networks regulating plant defense. Plant J 105(2):489\u0026ndash;504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/tpj.15124\u003c/span\u003e\u003cspan address=\"10.1111/tpj.15124\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAgrios GN (2005) \u003cem\u003ePlant pathology\u003c/em\u003e (Fifth edition). 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Front Plant Sci 13:952397. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2022.952397\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2022.952397\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ISR, Serratia, Trichoderma, Streptomyces, PR1, PDF1.2","lastPublishedDoi":"10.21203/rs.3.rs-7125135/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7125135/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSome microorganisms that are antagonistic to phytopathogens can activate induced systemic resistance (ISR) in plants. We sought to determine whether \u003cem\u003eStreptomyces galilaeus\u003c/em\u003e CFFSUR-B12, \u003cem\u003eSerratia marcescens\u003c/em\u003e CFFSUR-B2, and \u003cem\u003eTrichoderma viride\u003c/em\u003e CFFSUR-A21 \u0026ndash; strains recognised for their antagonistic capacity \u0026ndash; could colonise the roots of and induce resistance in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. The antagonistic activity of \u003cem\u003eColletotrichum\u003c/em\u003e spp. was determined in dual-culture assays. Strains were inoculated separately in the roots of \u003cem\u003eA. thaliana\u003c/em\u003e to study root colonisation and activation of ISR in leaves. \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e expression was monitored by RT-qPCR in leaves. \u003cem\u003eSerratia marcescens\u003c/em\u003e CFFSUR-B2 colonised the rhizoplane and endorhizosphere, whereas \u003cem\u003eStr. galilaeus\u003c/em\u003e CFFSUR-B12 and \u003cem\u003eT. viride\u003c/em\u003e CFFSUR-A21 only colonised the rhizoplane. \u003cem\u003eSerratia marcescens\u003c/em\u003e and \u003cem\u003eT. viride\u003c/em\u003e induced co-expression of \u003cem\u003ePR1\u003c/em\u003e and \u003cem\u003ePDF1.2\u003c/em\u003e, while \u003cem\u003eStr. galilaeus\u003c/em\u003e induced only \u003cem\u003ePDF1.2\u003c/em\u003e expression. These findings reveal new avenues for research into plant disease management in the humid tropics.\u003c/p\u003e","manuscriptTitle":"Root colonisation and induction of plant defence-associated signalling pathways in Arabidopsis thaliana by Serratia marcescens, Streptomyces galilaeus, and Trichoderma viride","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 17:14:18","doi":"10.21203/rs.3.rs-7125135/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94944f4d-0e2b-4267-a545-edf8489a6c7d","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-28T01:08:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-18 17:14:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7125135","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7125135","identity":"rs-7125135","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}