Induced defense responses in cacao against Phytophthora palmivora (Butler) by Pseudomonas chlororaphis CP07.

Induced defense responses in cacao against Phytophthora palmivora (Butler) by Pseudomonas chlororaphis CP07. | 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 Induced defense responses in cacao against Phytophthora palmivora (Butler) by Pseudomonas chlororaphis CP07. Yulien Miguelez-Sierra, Pierre Bertin, Annia Hernández-Rodríguez This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2987328/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2024 Read the published version in European Journal of Plant Pathology → Version 1 posted 6 You are reading this latest preprint version Abstract The objective of this study was to evaluate the effect of Pseudomonas chlororaphis CP07, isolated from the rhizosphere of cacao, on the induction of defense responses in Theobroma cacao L. against Phytophthora palmivora (Butler) , the causal agent of black rot of the fruit (black pod rot). The in planta greenhouse trial was carried out to determine the reduction of disease symptoms in plants micrografted with three traditional Cuban cacao genotypes of the Trinitario type on UF 677 hybrid rootstocks. The levels of phenylalanine ammonia-lyase (PAL) were determined in micrografts of genotype EICB-371. In genotypes EICB-371 and EICB-385 disease severity was significantly reduced in plants pretreated with the bacteria compared to control plants. In contrast, genotype EICB-384 showed no symptom reduction in plants pretreated with the bacterium. PAL enzyme activity was significantly increased in leaves of plants pretreated with CP07 compared to control plants on days 3 and 4 post-infection with the pathogen. The results suggested that, depending on the genotype, strain CP07 had potential in the protection of T. cacao against P. palmivora in soil substrate and that the interaction of this bacterium with the benefited plant activated defense responses related to the increase of PAL activity in leaves. Theobroma cacao black pod rot fluorescent Pseudomonas induced systemic resistance phenylalanine ammonia-lyase Figures Figure 1 Figure 2 Figure 3 Introduction Black pod rot has been considered the most devastating of the pests affecting Theobroma cacao L. with overall yield losses of 20–30% although in some areas such as Central and West Africa was reported up to 60% of losses (Ploetz, 2016 ). The disease could be caused by several species of the genus Phytophthora which predominate depending on the geographic region. Phytophthora palmivora (Butl.) was reported in all geographic areas of cacao cultivation and was the most common species causing black pod rot (Ali et al. 2017). Management of the disease caused by Phytophthora in T. cacao has included cultural, chemical, biological and genetic methods. Phytosanitary control measures in plantations such as pruning, removal of diseased fruit or branches, water drainage management and shade regulation were part of the cultural methods applied to reduce the incidence of Phytophthora . These practices were not always carried out with the required systematicity which contributed to pathogen inocula persisting in crop areas (Acebo-Guerrero et al. 2012 ; Peter and Chandramohanan, 2014 ). Only a few clones with resistance levels were identified but remained not available for distribution to different growing areas. Breeders have been looking for sources of resistance in cacao trees growing wild in the species' areas of origin in order to create hybrids that can be evaluated locally in their own countries (Thevenin et al. 2012 ). Chemical products, despite their effectiveness and being the main method of disease control, have constituted a polluting element for the cacao agroecosystem, with harmful effects for humans and the rest of the living components found in these areas. In addition, these products resulted costly for producers and therefore had an unfavorable cost-benefit ratio (Falcäo et al. 2014 ). Microbial inoculants, as ecological products, do not generate residues to the environment. Thus, they resulted advantageous and could be incorporated into the integrated management of diseases affecting cacao (Akrofi et al. 2017 ). Research conducted for the selection and use of microbial antagonists of Phytophthora was focused, fundamentally, on in vitro evaluation to determine the antagonistic capacity against the pathogen. However, the evaluation of plant-Phytophthora-microbial antagonist interaction in plant trials, could provide information about the mechanisms involved in antagonist-mediated bioprotection. Rhizobacteria protected their hosts with the activation of defensive mechanisms against pathogens through the phenomenon known as induced systemic resistance (ISR) (De Vleesschauwer y Höfte, 2009; Pieterse et al. 2014 ). In ISR, the production of biochemical compounds involved in the plant defense response was stimulated resulting in enhanced defensive capacity of the entire plant against a broad spectrum of pathogens (Yu et al. 2022 ; Zhu et al. 2022 ). These bacteria, stimulated the massive accumulation of phytoalexins and phenolic compounds, increased the activity of resistance proteins and defense enzymes, and increased cell lignification (Meena, 2014 ). Fluorescent pseudomonads have been considered one of the most effective rhizobacteria for biological control of foliar and soil diseases (De Vleesschauwer y Höfte, 2009; Meena, 2014 ). They have been mostly studied in crop protection against soil-borne plant pathogens such as pathogenic oomycetes (particularly Pythium spp.) and the fungi Fusarium oxysporum and Rhizoctonia solani (Registeri et al. 2012 ). Disease suppression by these rhizobacteria was usually given by pathogen inhibition through competition and/or antagonism, but, in addition, Pseudomonas strains provided plant protection through ISR (Patkowska et al. 2017 ). The enzyme phenylalanine ammonia-lyase (PAL) was used as a biochemical marker in the study of systemic defense responses mediated or not by microorganisms during plant-pathogen interactions. That was due to its involvement in phenylpropanoid biosynthesis, an important point in the regulation of primary and secondary metabolism (Wang et al. 2019 ). The phenylpropanoid pathway was responsible for the production of numerous compounds involved in plant protection against biotic and abiotic stresses (Zhang and Liu, 2015 ). Increased PAL activity was detected in some plant species as a mechanism for the induction of defense responses by antagonistic bacteria. For example, in tomato for Pseudomonas fluorescens Pf1 against Pythium aphanidermatum (Ramamoorthy et al. 2002 ), in rice for Bacillus subtilis GBO3 against Xanthomonas oryzae pv. oryzae (Chithrashreea et al. 2011), and in tomato for Bacillus amyloliquefaciens strains CM-2 and T-5 against Ralstonia solanacearum (Tan et al. 2013 ). In T. cacao - Phytophthora interaction, the activation of defense via PAL has been investigated in relation to resistance of genotypes to the pathogen (Okey et al. 1997 ). However, other enzymes and metabolites of phenylpropanoid pathway have been more studied respect to genetic resistance (Nyadanu et al. 2013 ; Ondobo et al. 2013 ; Rêgo et al. 2023 ). A recent study on defense gene expression in immature leaves of three cacao genotypes (ICS1/CCN51/Pound7) 24 h after infection by P. palmivora showed that a candidate phenylalanine ammonia-lyase gene was induced in all three cacao genotypes (Baruah et al. 2022 ) what evidenced the role of the enzyme in plant defensive capacity against the pathogen. Nevertheless, limited information is available about PAL activation in induced defense responses by microbial antagonists against Phytophthora . Pseudomonas chlororaphis CP07, isolated from the rhizosphere of cacao plants in eastern Cuba was selected for its antagonism to Phytophthora palmivora (Acebo-Guerrero et al. 2015 ). The bacterial strain showed several characteristics for the induction of defense responses against pathogens but the activation of ISR mechanisms in inoculated cacao plants was not investigated through in planta assay. However, inoculation of this bacterial strain into plant roots under controlled conditions significantly reduced disease symptoms caused by inoculation of the pathogen into leaves, with a genotype-dependent effect (Miguelez-Sierra et al. 2019 ). The information provided by these results suggested that the strain could induce systemic defense responses in cacao plants with a potential effect for crop protection against black pod rot. The present work aimed to evaluate the effects of P. chlororaphis CP07 in inducing defense responses in T. cacao against P. palmivora by determining the disease severity reduction under greenhouse conditions and PAL activity in plants previously treated with the bacterial inoculum. Materials and methods Plant material, seedlings preparation and culture conditions The plant material was taken from the collection of the Instituto de Investigaciones Agroforestales (INAF) UCTB Baracoa, Guantánamo, Cuba. The hybrid line UF 677 (International Cacao Germplasm Database [ICGD] 2021) and genotypes EICB-371, EICB-384 and EICB-385 of traditional Cuban cacao identified as Trinitario (Bidot et al. 2015 ) were used. Seedlings and micrografted plants were obtained using the protocol described by Miguelez-Sierra et al. ( 2017 ) for the in vitro micrografting of T. cacao using side graft with axillary buds from young plants. The genotypes EICB-371, EICB-384 and EICB-385 were used as scions and the clone UF 677 was the rootstock. The micrograftings were maintained in a growth room at 25 ± 1 ºC, 16 h/8 h (light/darkness photoperiod), and 23 µmol m − 2 s − 1 of photosynthetic photon flux (PPF). Microbial cultures and inocula The bacterial inocula was obtained from 48 hours' cultures of P. chlororaphis CP07 in King B (KB) Agar. Plated on Ø 90 mm dishes with 10 mL of KB Agar, they were incubated at 28°C. After 24 hours, the plates were rinsed twice with 5 ml of sterile distilled water. Then was extracted with a sterile pipette and placed into a sterile tube. The bacterial suspension was adjusted with sterile distilled water at OD 600 0.6 (10 8 cfu mL − 1 ). P. palmivora Mab 1 cultures were made on Ø 9 mm dishes, with V8 Agar 10 mL and incubated in the dark for 7 days at 24 ˚C. The inoculum consisted of a zoospore suspension prepared for mass zoospore production by 'wet-plate' method as described by Pistininzi et al. ( 2014 ). For that, mycelial plugs were scraped on the bottom of Petri dishes to promote colony growth in V8 broth, and then, the nutrients were drained and dishes washed with sterile distilled water (SDW) to create a stressing environment for sporangial production. The colonies formed at the bottom of dishes were maintained humid without free-flowing water at 22˚C for a week. After that, dishes were flooded with SDW and then placed at 28˚C to trigger zoospores release. Zoospores concentration was adjusted to 10 5 zoospores mL − 1 . The final inoculum consisted of a 1:1 mixture of the zoospore suspension and a low-melting-point agarose solution (2% in sterile distilled water w/v) maintained at 37°C. In planta assay under greenhouse conditions Eight-week-old in vitro micrografted plants were used. The micrografted plants consisted of traditional Cuban cacao genotypes (EICB-371, EICB-384 and EICB-385) grafted on UF 677 rootstocks. Plants were removed from the culture vessels and planted in organic supplemented substrate (Substrat D'argile n° 9 ELEVE DCM®) in plastic pots of 2 L capacity. They were maintained for two weeks under controlled conditions for acclimatization with temperature 25 ± 1°C, relative humidity 88–92%, illumination of 119.85 µmol m − 2 s − 1 (photosynthetic photon flux) and photoperiod 16 h/8 h (light/dark). Irrigation was applied three times a week on alternate days, at a rate of 100 mL of water per plant. After two weeks, plants were transferred to a greenhouse with temperature 25 ± 1°C, relative humidity 70% and 50% natural illumination. Plants were inoculated with 5 mL of P. chlororaphis CP07 inoculum or 5 mL of distilled water was applied to the roots. After 10 days, Mab 1 (10 5 zoospores-mL − 1 ) was inoculated on approximately two-month-old leaves. For this purpose, the zoospore suspension was inoculated on the abaxial side of two leaves per plant with a sterile brush as described by Widmer ( 2009 ), in this case a small paint brush of 0.7 cm in width was used. A total of 100 µL per leaf was inoculated. Inoculated plants were transferred to a 100% humidity chamber for seven days to promote pathogen infection. After seven days, the appearance of symptoms was evaluated on the basis of a leaf symptom scale for Phytophthora spp. infection according to Nyassé et al. ( 1995 ) modified by Acebo-Guerrero et al. ( 2015 ). The scale comprised six values: 0 = no symptoms, 1 = penetration point (very small necrotic spot), 2 = net of points (larger number of necrotic spots), 3 = reticulate patch (spider web-like), 4 = mottled necrotic patch (marbled appearance), and 5 = true necrosis (large brown lesions). Disease severity was calculated as described Yang et al. ( 2009 ): Disease severity = [∑(The number of infected leaves corresponding to the scale value x scale value)/(Total plants x highest scale value)]x 100. Treatments consisted on plants inoculated with P. chlororaphis CP07, plants treated with sterile distilled water and infected with P. palmivora Mab 1 and plants inoculated with strain CP07 and infected with Mab1. Control treatment consisted on plants treated with sterile distilled water. Five plants per treatment were used and the experiment was repeated three times. Determination of PAL activity in leaves and roots. Leaf and root samples were collected on plants maintained under same conditions as the in planta assay in greenhouse which were reserved to this experiment. Plants of EICB-371 micrografts with rootstock UF 677 were used. Samples were taken on days 0, 1, 2, 3 and 4 of infection with P. palmivora Mab 1 from plants of each previously established treatment as described in the in planta assay. Seven plants from each treatment were used in the experiment. Approximately two-month-old leaves and all roots from each plant were used. PAL activity was determined also in roots to investigate the regulation of this mechanism in roots as part of the mutualistic interaction, between genotype EICB-371 and the bacterial strain, that enables the induction of the protective effect against P. palmivora . Samples were macerated independently with liquid nitrogen and kept at -80°C for preservation. The determination of PAL activity was performed according to Olsen et al. ( 2008 ) using L-phenylalanine as substrate. Extraction was performed with 50 mg of leaf or root sample placed in a centrifuge tube with 2 mL of extraction solution (Tris-HCl, 100 mM, pH 8.8; containing 84 µL of β -mercaptoethanol (12 mM) in 100 mL of extraction solution). The tubes were kept on ice and vortexed for 1 min. Samples were centrifuged at 16000 x g, at 4°C for 10 min (Eppendorf Centrifuge 5702®, Germany). The supernatant was stored at -20 ºC until use. The protein concentration in the samples was determined by the method of Bradford ( 1976 ). The absorbance reading was performed at 595 nm in a spectrophotometer (Spectra Max M2, SPE002) using a standard curve of bovine serum albumin (BSA) with six concentrations between 0-500 mg L − 1 . For quantification of PAL activity, 500 mL of enzyme extract, 450 mL of Tris-HCl (pH 8.8) (100 mM) and 50 mL of L-phenylalanine (100 mM) were placed in an Eppendorf tube. The assay was performed in triplicate. The samples were incubated at 37°C for 1 h and the reaction was stopped by the addition of 50 mL of HCl (5 M). They were then centrifuged at 16 000 x g for 15 min (Eppendorf Centrifuge 5702®, Germany). The absorbance reading at 290 nm in spectrophotometer (Spectra Max M2, SPE002) was performed against blanks prepared in the same way as the samples, but only HCl (5 M) was added before L-phenylalanine. The enzymatic activity was obtained from the absorbance readings and the production of cinnamic acid expressed in nmol of product formed per mg of sample per hour (nmol of cinnamic acid mg − 1 h − 1 ) was determined using a standard curve of cinnamic acid with concentrations of 2, 4, 6, 8 and 10 µg L − 1 . Data processing The assays were performed in a completely randomized design. Data which did not conform to normality and homogeneity of variance were analyzed with Kruskal Wallis' ANOVA at p < 0.05. Data which conformed to normality and homogeneity of variance were analyzed with ANOVA and Tukey' Test at p < 0.05. All experiments were repeated three times. Data of one experiment representative of the three replicates were used for figures and standard error is presented as vertical bars in the figures. The program Statistica 8.0 was used for data processing. Results In planta assay under greenhouse conditions Disease severity assessed seven days after infection with the pathogen was reduced in plants treated with P. chlororaphis CP07 of genotypes EICB-371 and EICB-385 (Fig. 1 ) indicating a protective effect of this strain in these genotypes. Genotype EICB-371 with a high susceptibility to Mab 1 showed a drastic reduction of symptoms severity. Genotype EICB-385 showed lower disease severity than the other genotypes in plants inoculated only with the pathogen indicating less susceptibility and disease severity was reduced in plants treated with the bacterium. In EICB-384 there was no significant difference in disease severity between treated and untreated plants. Determination of PAL activity in leaves and roots PAL activity levels in leaves on day 0 post-infection with P. palmivora Mab 1 were significantly higher in leaves of plants pre-treated only with P. chlororaphis CP07 compared to untreated control plants and plants infected with the pathogen (Fig. 2 ). On day 1 post-infection the CP07 treatment differed only from the untreated control. Enzyme activity in leaves on day 2 of pathogen infection in treatments Mab 1 and CP07 + Mab 1 had significant differences with the untreated control. On days 3 and 4 post-infection, the leaves of plants treated with the bacterial strain and infected with the pathogen showed significantly higher levels of PAL activity than the rest of the treatments. In the Mab 1 treatment, the PAL levels on days 3 and 4 were significantly lower than the CP07 + Mab 1 treatment. In the roots of micrografted plants, where the rootstock was UF 677 and the bud EICB-371, similar levels of activity were obtained in treated and untreated plants with P. chlororaphis CP07 on all post-infection days (Fig. 3 ). Thus, in these tissues the enzyme activity did not undergo significant variations due to bacterial inoculation. This result indicated that the presence of the bacterial strain in the roots did not stimulate PAL activity above basal levels in this zone. Discussion The results of the greenhouse in planta assay showed the bioprotection effect mediated by P. chlororaphis CP07 on cacao plants potted in substrate with non-sterile soil where there is a community of microorganisms that could interact with the bacterial strain. The reduction of symptom severity caused by P. palmivora in T. cacao plants treated with the bacterium was previously reported by Miguelez-Sierra et al. ( 2019 ) in sterile perlite substrate under controlled conditions with a genotype-dependent effect similar to the present results. The plant protection effect observed in soil conditions could be related to specific traits of the strain which confer competences not only for its activities in the rhizosphere but also for the interaction with cacao plants. Rhizobacteria have shown capacity for rapid establishment in roots and competition for nutrients, which are characteristics of great importance for biocontrol (Dos Santos et al. 2018 ). One of the aspects involved in nutrient competition is the availability of iron, which fluorescent pseudomonads make available to themselves via siderophores (Santoyo et al. 2019 ). In P. chlororaphis CP07, Acebo-Guerrero et al. ( 2015 ) reported the production of pyoverdin and genes for pyochelin and acromobactin. The strain presented high motility, pili and biofilm formation, characteristics that favor the establishment of rhizobacterial colonies on the root surface and contribute to their biocontrol activity (Sivakumar et al. 2019 ). P. chlororaphis CP07 also produced antifungal metabolites like the lytic enzymes proteases and lipases, phenacins that inhibit electron transport, and hydrogen cyanide (HCN) which is a potent inhibitor of metalloenzymes according to Santoyo et al. ( 2019 ). These features have been related to biocontrol interaction of the species Pseudomonas chlororaphis with host plants (Arrebola et al. 2019 ). Similar characteristics benefited antagonism against Fusarium oxysporum f.sp. radicis lycopersici in P. chlororaphis PCL1391, an efficient colonizer of tomato roots (De Weert and Bloemberg, 2007 ). In P. chlororaphis MCC2693, phenazine production was related to biocontrol activity effective against Phytophthora sp. and Fusarium sp. (Jayaprakashvel et al. 2019 ). Also, this characteristic could stimulate plant innate immunity against pathogens according to Ma et al. ( 2016 ). Viscosine-type cyclic lipopeptides present in P. chlororaphis CP07 could have antifungal action and participation in the induction of systemic resistance in plants (Ma et al. 2016 ; Jayaprakashvel et al. 2019 ). For example, masetolide A (viscosin type) produced by Pseudomonas fluorescens SS101 increased resistance to Phytophthora infestans in tomato plants (Tran et al. 2007 ). Additionally, genes involved in the perception of intercellular signal molecules ( quorum sensing ) were found in the genome of P. chlororaphis CP07 (Acebo-Guerrero et al. 2015 ). In this particular, Khan et al. ( 2019 ) and Shrestha et al. ( 2020 ) reported that plants sensitized with quorum sensing molecules activated systemic defense mechanisms against pathogenic bacteria and fungi which may be an alternative for resistance enhancement. Therefore, these characteristics of the strain could favor its permanence in the rhizosphere and protect the plant against P. palmivora Mab 1 and other pathogens. The PAL activity determined as biochemical marker of ISR in cacao plants of genotype EICB-371, was higher in plants infected with P. palmivora Mab 1 compared to control plants not inoculated with the pathogen in day 2 post-infection, which could be a response of plant innate defense against the development of the infection. It is known that activation of enzymes of the phenylpropanoid pathway, including PAL, is an important defense mechanism included in pathogen-induced responses (Zhang and Liu, 2015 ). On the other hand, Iwaro et al. ( 1997 ) proposed that the resistance response of cacao plants to P. palmivora can be manifested both during penetration and in the post-penetration stage of the pathogen. At this stage, the interaction between the plant and the pathogen can trigger defense mechanisms based on pathogen-associated molecular patterns- triggered immunity (PTI), from the recognition of the effectors released by the oomycetes during colonization of host tissues (Stassen and Van den Acherveken, 2011). On days 3 and 4 post infection, PAL activity in leaves of plants untreated with the bacterial strain and infected with the pathogen was similar to control treatment. This could be a consequence of PAL defense response suppression by the pathogen's infective mechanisms. Pathogenic oomycetes can suppress PTI and effector triggered immunity (ETI) via apoplastic effectors and haustorium-secreted effectors within host cells. In this way, they create effector-induced susceptibility thereby promoting disease (Stassen and Van den Acherveken, 2011; Heliwell et al. 2016). At the same time, this response may be a manifestation of the susceptibility of genotype EICB-371 to infection by P. palmivora Mab 1, which was observed in the greenhouse assay. Similarly, Manga et al. ( 2016 ) found significantly lower PAL activity levels in several T. cacao genotypes susceptible to P. megakarya compared to tolerant genotypes. In contrast, plants treated with P. chlororaphis CP07 and inoculated with the pathogen maintained higher PAL levels than untreated plants until the fourth day post-infection. This indicated that PAL defense response induced by the bacterium in leaves remained active and effective during the development of the pathogen attack. Similarly, increased PAL activity as expression of resistance to P. palmivora infection in T. cacao was reported by Okey et al. ( 1997 ). Activation of PAL has been considered one of the elements participating in the interaction of T. cacao with Phytophthora (Rêgo et al. 2023 ). Defense based on increasing the activity of this enzyme and others compounds of phenylpropanoids pathway upon P. palmivora invasion was associated to the accumulation of polyphenols and lignin in stem tissues of tolerant genotypes (Okey et al. 1997 ). Nyadanu et al. ( 2013 ) and Ondobo et al. ( 2013 ) observed that less susceptible genotypes of cacao accumulated more polyphenols and lignin in leaves and pods indicating that the inhibitory effect on pathogen growth depended on the levels of these metabolites in host tissues. In this way, the metabolism of aromatic fungitoxic compounds was promoted with specific effects on pathogen infective mechanisms and lignin deposition acted as barrier to penetration and propagation of the pathogen (Zhang and Liu 2015 ). In the present study, the effects of increased PAL activity could ultimately be related to the significant reduction of disease severity observed in plants of genotype EICB-371 treated with the bacterial strain compared to no-treated plants in the in planta assay. The ISR mediated by beneficial bacteria in T. cacao has not been widely studied, in particular, there are no previous reports on the occurrence of this phenomenon in the interaction of Pseudomonas-Cacao- P. palmivora . Moreover, PAL activation as a mechanism of induction of systemic defense responses by antagonistic bacteria has been described in other plant species (Ramamoorthy et al. 2002 ; Chithrashree et al. 2011; Tan et al. 2013 ; Dias et al. 2017 ). Meena ( 2014 ) pointed out that a fundamental element in the increase of resistance due to rhizobacteria-induced ISR is the induction of compounds of the phenylpropanoid pathway in which PAL was a key enzyme. The presence of the bacterial strain in the roots did not stimulate PAL activity above basal levels in this zone. Pieterse et al. ( 2014 ) described that ISR elicitor microorganisms can suppress the root immune response upon colonization protecting themselves from antimicrobial compounds produced by the recognition of molecular patterns associated with the microorganism and allowing to establish the mutualistic relationship with the host. This effect was detected in several beneficial ISR-inducing microorganisms such as Trichoderma , Bacillus subtilis FB17 and P. fluorescens WCS417r (Pieterse et al. 2014 )d putida RRF3, a rice rhizosphere isolate (Kandaswamy et al. 2019 ). In the case of P. chlororaphis CP07, regulation of the defense response via PAL in roots could be one of the mechanisms favoring its interaction with the host plant. The results showed that, in the protective effect of P. chlororaphis CP07 against P. palmivora Mab 1 in genotype EICB-371, PAL activity was stimulated in leaves what could be used as marker of defense responses against the pathogen mediated by the bacterial strain. This information would allow the selection of genotypes with the greatest potential for the induction of defenses by the strain. The study of these aspects can generate new perspectives for the use of P. chlororaphis CP07 in the protection of T. cacao against pathogens that affect the crop, particularly in the integrated management of black pod rot. Declarations Acknowledgments This work was supported by the Project 'Design and strengthening of an agroecological cacao production system in Cuba' of ARES (Académie de Recherche et d'Enseignement supérieur, Belgium) and the Project PN223LH010-009 'Contributions to knowledge for the agroecological management of black pod rot in Theobroma cacao L.' of the National Program of Basic Sciences of the Ministry of Science, Technology and Environment (CITMA) of Cuba. Our sincere gratitude to Dr. Stanley Lutts from Université catholique de Louvain, Belgium, for their support to the research. The authors thank to the Instituto de Investigaciones Agroforestales (INAF) UCTB Baracoa gene Banks for providing the plant material. The authors have no relevant financial or non-financial interests to disclose. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. 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S., Jayashree, S., Lozano, G. L., Miles, J. (2019). Evaluation of InSeq to Identify Genes Essential for Pseudomonas aeruginosa PGPR2 Corn Root Colonization. G3: Genes Genomes Genetics Early Online . http://doi.org/10.1534/g3.118.200928 . Stassen, J. H. M., & Van den Ackerveken, G. (2011). How do oomycete effectors interfere with plant life? Current Opinion in Plant Biology , 14 , 407–414. https://doi.org/10.1016/j.pbi.2011.05.002 . Tan, S., Dong, Y., Liao, H., Huang, J., Song, S., Xu, Y., & Shen, Q. (2013). Antagonistic bacterium Bacillus amyloliquefaciens induces resistance and controls the bacterial wilt of tomato. Pest Management Science , 69 (11), 1245–1252. https://doi.org/10.1002/ps.3491 . Thevenin, J. M., Rossi, V., Ducamp, M., Doare, F., Condina, V., & Lachenaud, P. (2012). Numerous Clones Resistant to Phytophthora palmivora in the ‘‘Guiana’’ Genetic Group of Theobroma cacao L. Plos One , 7 (7), e40915. https://doi.org/10.1371/journal.pone.0040915 . Tran, H., Ficke, A., Asiimwe, T., Höfte, M., & Raaijmakers, J. M. (2007). Role of the cyclic lipopeptide massetolide A in biological control of Phytophthora infestans and in colonization of tomato plants by Pseudomonas fluorescens . New Phytologist , 175 , 731–742. https://doi.org/10.1111/j.1469-8137.2007.02138.x . Wang, R., Wang, G. L., & Ning, Y. (2019). PALs: Emerging Key Players in Broad-Spectrum Disease Resistance. Trends in Plant Science , 24 (9), 785–787. https://doi.org/10.1016/j.tplants.2019.06.012 . Widmer, T. L. (2009). Infective potential of sporangia and zoo-spores of Phytophthora ramorum . Plant Disease , 93 , 30–35. https://doi.org/10.1094/PDIS-93-1-0030 . Yu, Y., Gui, Y., Li, Z., Jiang, C., Guo, J., & Niu, D. (2022). Induced Systemic Resistance for Improving Plant Immunity by Beneficial Microbes. Plants , 11 , 386. https://doi.org/10.3390/plants11030386 . Yang, D., Wang, B., Wang, J., Chen, Y., & Zhou, M. (2009). 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Cite Share Download PDF Status: Published Journal Publication published 25 Nov, 2024 Read the published version in European Journal of Plant Pathology → Version 1 posted Reviewers agreed at journal 26 Jan, 2024 Reviewers invited by journal 11 Jan, 2024 Editor invited by journal 03 Jan, 2024 Editor assigned by journal 17 Nov, 2023 First submitted to journal 16 Nov, 2023 Editorial decision: Revision 11 Jun, 2023 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-2987328","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266776612,"identity":"f85f1d53-929e-4114-8505-44f33ca262a0","order_by":0,"name":"Yulien Miguelez-Sierra","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYFAC5oYDIMr+MDOIlpAhQgsjRAvDcbYEkBYeorRA6PM8BiCKsBaDG4mNBz7u2GbP2Mzz+dWNGgseBvbDRzcQ0NJwcOaZ24nNzLzbrHOOAR3Gk5Z2g5CWw7xttxPYgFqMc9iAWiR4zAhr+dt2256HmeeZcc4/YrUwtt1mnMHMw/w4t40ILZJnHjYc7G27nbiBmc2MObdPgoeNkF/4jicf/vAT6DAD/sOPP+d8q5PjZz98DK8WhQMINpsEmMSnHATkGxBs5g+EVI+CUTAKRsHIBAAb2E2vtzjWyQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-5438-4412","institution":"Universidad de Guantanamo","correspondingAuthor":true,"prefix":"","firstName":"Yulien","middleName":"","lastName":"Miguelez-Sierra","suffix":""},{"id":266776613,"identity":"4dfdf3e8-417d-4c29-ac95-5cdc689d7fb7","order_by":1,"name":"Pierre Bertin","email":"","orcid":"","institution":"Université catholique de Louvain: Universite Catholique de Louvain","correspondingAuthor":false,"prefix":"","firstName":"Pierre","middleName":"","lastName":"Bertin","suffix":""},{"id":266776614,"identity":"b7fd767a-a757-411a-b297-315b2e68b48d","order_by":2,"name":"Annia Hernández-Rodríguez","email":"","orcid":"","institution":"University of Havana: Universidad de la Habana","correspondingAuthor":false,"prefix":"","firstName":"Annia","middleName":"","lastName":"Hernández-Rodríguez","suffix":""}],"badges":[],"createdAt":"2023-05-26 23:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2987328/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2987328/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10658-024-02982-2","type":"published","date":"2024-11-25T15:58:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49889333,"identity":"05aeffce-66a7-4813-9b5b-98049d8fef24","added_by":"auto","created_at":"2024-01-19 19:45:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16830,"visible":true,"origin":"","legend":"\u003cp\u003eDisease severity in micrografted plants of different genotypes (EICB-371, EICB-384 and EICB-385 grafted on UF 677 rootstocks) seven days after inoculation with \u003cem\u003eP. palmivora \u003c/em\u003eMab 1. Plants treated and untreated with \u003cem\u003eP. chlororaphis \u003c/em\u003eCP07 grown in greenhouses were used. Means with common letters do not differ significantly according to the Kruskal Wallis Multiple Comparison Test (p\u0026lt;0.05). Vertical bars represent the standard error of the mean.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2987328/v1/fbb9c8432507f86231115e17.png"},{"id":49889335,"identity":"59a1849f-bea0-4289-8cb6-592c7e02694b","added_by":"auto","created_at":"2024-01-19 19:45:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":21462,"visible":true,"origin":"","legend":"\u003cp\u003ePAL activity detected in leaves of micrografted plants (scion EICB-371 and rootstock UF 677) treated and untreated with \u003cem\u003eP. chlororaphis \u003c/em\u003eCP07. Control were plants treated with sterile distilled water. Inoculation with \u003cem\u003eP. palmivora \u003c/em\u003eMab 1 was performed 10 days after treatment with the bacterium. Data analysis was performed with ANOVA and Tukey's Test (p\u0026lt;0.05). Vertical bars represent means ± standard error.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2987328/v1/5fac048ffe43c0437a35b4a6.png"},{"id":49889677,"identity":"bdf5b330-3ca1-461e-ba17-b8a133d6c161","added_by":"auto","created_at":"2024-01-19 19:53:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22644,"visible":true,"origin":"","legend":"\u003cp\u003ePAL activity detected in roots of micrografted \u003cem\u003eT. cacao \u003c/em\u003eplants (bud EICB-371 and rootstock UF 677) treated and untreated with \u003cem\u003eP. chlororaphis \u003c/em\u003eCP07. Control were plants treated with sterile distilled water. Inoculation with \u003cem\u003eP. palmivora \u003c/em\u003eMab 1 was performed on leaves 10 days after treatment with the bacterium. Data analysis was performed with ANOVA (p\u0026lt;0.05). Vertical bars represent means ± standard error.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2987328/v1/66523af5312ecf02a78a198c.png"},{"id":70391182,"identity":"5572bd0f-b371-4e31-8e33-0d5e71f8ce7d","added_by":"auto","created_at":"2024-12-02 17:30:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":532193,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2987328/v1/8cee3430-2a02-4404-b2d8-214917c2e09a.pdf"}],"financialInterests":"","formattedTitle":"Induced defense responses in cacao against Phytophthora palmivora (Butler) by Pseudomonas chlororaphis CP07.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBlack pod rot has been considered the most devastating of the pests affecting \u003cem\u003eTheobroma cacao\u003c/em\u003e L. with overall yield losses of 20\u0026ndash;30% although in some areas such as Central and West Africa was reported up to 60% of losses (Ploetz, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The disease could be caused by several species of the genus \u003cem\u003ePhytophthora\u003c/em\u003e which predominate depending on the geographic region. \u003cem\u003ePhytophthora palmivora\u003c/em\u003e (Butl.) was reported in all geographic areas of cacao cultivation and was the most common species causing black pod rot (Ali et al. 2017). Management of the disease caused by \u003cem\u003ePhytophthora\u003c/em\u003e in \u003cem\u003eT. cacao\u003c/em\u003e has included cultural, chemical, biological and genetic methods. Phytosanitary control measures in plantations such as pruning, removal of diseased fruit or branches, water drainage management and shade regulation were part of the cultural methods applied to reduce the incidence of \u003cem\u003ePhytophthora\u003c/em\u003e. These practices were not always carried out with the required systematicity which contributed to pathogen inocula persisting in crop areas (Acebo-Guerrero et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Peter and Chandramohanan, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Only a few clones with resistance levels were identified but remained not available for distribution to different growing areas. Breeders have been looking for sources of resistance in cacao trees growing wild in the species' areas of origin in order to create hybrids that can be evaluated locally in their own countries (Thevenin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Chemical products, despite their effectiveness and being the main method of disease control, have constituted a polluting element for the cacao agroecosystem, with harmful effects for humans and the rest of the living components found in these areas. In addition, these products resulted costly for producers and therefore had an unfavorable cost-benefit ratio (Falc\u0026auml;o et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Microbial inoculants, as ecological products, do not generate residues to the environment. Thus, they resulted advantageous and could be incorporated into the integrated management of diseases affecting cacao (Akrofi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Research conducted for the selection and use of microbial antagonists of \u003cem\u003ePhytophthora\u003c/em\u003e was focused, fundamentally, on \u003cem\u003ein vitro\u003c/em\u003e evaluation to determine the antagonistic capacity against the pathogen. However, the evaluation of plant-Phytophthora-microbial antagonist interaction in plant trials, could provide information about the mechanisms involved in antagonist-mediated bioprotection. Rhizobacteria protected their hosts with the activation of defensive mechanisms against pathogens through the phenomenon known as induced systemic resistance (ISR) (De Vleesschauwer y H\u0026ouml;fte, 2009; Pieterse et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In ISR, the production of biochemical compounds involved in the plant defense response was stimulated resulting in enhanced defensive capacity of the entire plant against a broad spectrum of pathogens (Yu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These bacteria, stimulated the massive accumulation of phytoalexins and phenolic compounds, increased the activity of resistance proteins and defense enzymes, and increased cell lignification (Meena, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Fluorescent pseudomonads have been considered one of the most effective rhizobacteria for biological control of foliar and soil diseases (De Vleesschauwer y H\u0026ouml;fte, 2009; Meena, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). They have been mostly studied in crop protection against soil-borne plant pathogens such as pathogenic oomycetes (particularly \u003cem\u003ePythium\u003c/em\u003e spp.) and the fungi \u003cem\u003eFusarium oxysporum\u003c/em\u003e and \u003cem\u003eRhizoctonia solani\u003c/em\u003e (Registeri et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Disease suppression by these rhizobacteria was usually given by pathogen inhibition through competition and/or antagonism, but, in addition, \u003cem\u003ePseudomonas\u003c/em\u003e strains provided plant protection through ISR (Patkowska et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe enzyme phenylalanine ammonia-lyase (PAL) was used as a biochemical marker in the study of systemic defense responses mediated or not by microorganisms during plant-pathogen interactions. That was due to its involvement in phenylpropanoid biosynthesis, an important point in the regulation of primary and secondary metabolism (Wang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The phenylpropanoid pathway was responsible for the production of numerous compounds involved in plant protection against biotic and abiotic stresses (Zhang and Liu, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Increased PAL activity was detected in some plant species as a mechanism for the induction of defense responses by antagonistic bacteria. For example, in tomato for \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e Pf1 against \u003cem\u003ePythium aphanidermatum\u003c/em\u003e (Ramamoorthy et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), in rice for \u003cem\u003eBacillus subtilis\u003c/em\u003e GBO3 against \u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv. oryzae (Chithrashreea et al. 2011), and in tomato for \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e strains CM-2 and T-5 against \u003cem\u003eRalstonia solanacearum\u003c/em\u003e (Tan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In \u003cem\u003eT. cacao\u003c/em\u003e-\u003cem\u003ePhytophthora\u003c/em\u003e interaction, the activation of defense via PAL has been investigated in relation to resistance of genotypes to the pathogen (Okey et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). However, other enzymes and metabolites of phenylpropanoid pathway have been more studied respect to genetic resistance (Nyadanu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ondobo et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; R\u0026ecirc;go et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A recent study on defense gene expression in immature leaves of three cacao genotypes (ICS1/CCN51/Pound7) 24 h after infection by \u003cem\u003eP. palmivora\u003c/em\u003e showed that a candidate phenylalanine ammonia-lyase gene was induced in all three cacao genotypes (Baruah et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) what evidenced the role of the enzyme in plant defensive capacity against the pathogen. Nevertheless, limited information is available about PAL activation in induced defense responses by microbial antagonists against \u003cem\u003ePhytophthora\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e CP07, isolated from the rhizosphere of cacao plants in eastern Cuba was selected for its antagonism to \u003cem\u003ePhytophthora palmivora\u003c/em\u003e (Acebo-Guerrero et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The bacterial strain showed several characteristics for the induction of defense responses against pathogens but the activation of ISR mechanisms in inoculated cacao plants was not investigated through \u003cem\u003ein planta\u003c/em\u003e assay. However, inoculation of this bacterial strain into plant roots under controlled conditions significantly reduced disease symptoms caused by inoculation of the pathogen into leaves, with a genotype-dependent effect (Miguelez-Sierra et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The information provided by these results suggested that the strain could induce systemic defense responses in cacao plants with a potential effect for crop protection against black pod rot.\u003c/p\u003e \u003cp\u003eThe present work aimed to evaluate the effects of \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 in inducing defense responses in \u003cem\u003eT. cacao\u003c/em\u003e against \u003cem\u003eP. palmivora\u003c/em\u003e by determining the disease severity reduction under greenhouse conditions and PAL activity in plants previously treated with the bacterial inoculum.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePlant material, seedlings preparation and culture conditions\u003c/p\u003e \u003cp\u003eThe plant material was taken from the collection of the Instituto de Investigaciones Agroforestales (INAF) UCTB Baracoa, Guant\u0026aacute;namo, Cuba. The hybrid line UF 677 (International Cacao Germplasm Database [ICGD] 2021) and genotypes EICB-371, EICB-384 and EICB-385 of traditional Cuban cacao identified as Trinitario (Bidot et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) were used.\u003c/p\u003e \u003cp\u003eSeedlings and micrografted plants were obtained using the protocol described by Miguelez-Sierra et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) for the \u003cem\u003ein vitro\u003c/em\u003e micrografting of \u003cem\u003eT. cacao\u003c/em\u003e using side graft with axillary buds from young plants. The genotypes EICB-371, EICB-384 and EICB-385 were used as scions and the clone UF 677 was the rootstock. The micrograftings were maintained in a growth room at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026ordm;C, 16 h/8 h (light/darkness photoperiod), and 23 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of photosynthetic photon flux (PPF).\u003c/p\u003e \u003cp\u003eMicrobial cultures and inocula\u003c/p\u003e \u003cp\u003eThe bacterial inocula was obtained from 48 hours' cultures of \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 in King B (KB) Agar. Plated on \u0026Oslash; 90 mm dishes with 10 mL of KB Agar, they were incubated at 28\u0026deg;C. After 24 hours, the plates were rinsed twice with 5 ml of sterile distilled water. Then was extracted with a sterile pipette and placed into a sterile tube. The bacterial suspension was adjusted with sterile distilled water at OD\u003csub\u003e600\u003c/sub\u003e 0.6 (10\u003csup\u003e8\u003c/sup\u003e cfu mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). \u003cem\u003eP. palmivora\u003c/em\u003e Mab 1 cultures were made on \u0026Oslash; 9 mm dishes, with V8 Agar 10 mL and incubated in the dark for 7 days at 24 ˚C. The inoculum consisted of a zoospore suspension prepared for mass zoospore production by 'wet-plate' method as described by Pistininzi et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For that, mycelial plugs were scraped on the bottom of Petri dishes to promote colony growth in V8 broth, and then, the nutrients were drained and dishes washed with sterile distilled water (SDW) to create a stressing environment for sporangial production. The colonies formed at the bottom of dishes were maintained humid without free-flowing water at 22˚C for a week. After that, dishes were flooded with SDW and then placed at 28˚C to trigger zoospores release. Zoospores concentration was adjusted to 10\u003csup\u003e5\u003c/sup\u003e zoospores mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The final inoculum consisted of a 1:1 mixture of the zoospore suspension and a low-melting-point agarose solution (2% in sterile distilled water w/v) maintained at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn planta\u003c/em\u003e assay under greenhouse conditions\u003c/p\u003e \u003cp\u003eEight-week-old \u003cem\u003ein vitro\u003c/em\u003e micrografted plants were used. The micrografted plants consisted of traditional Cuban cacao genotypes (EICB-371, EICB-384 and EICB-385) grafted on UF 677 rootstocks. Plants were removed from the culture vessels and planted in organic supplemented substrate (Substrat D'argile n\u0026deg; 9 ELEVE DCM\u0026reg;) in plastic pots of 2 L capacity. They were maintained for two weeks under controlled conditions for acclimatization with temperature 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity 88\u0026ndash;92%, illumination of 119.85 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (photosynthetic photon flux) and photoperiod 16 h/8 h (light/dark). Irrigation was applied three times a week on alternate days, at a rate of 100 mL of water per plant.\u003c/p\u003e \u003cp\u003eAfter two weeks, plants were transferred to a greenhouse with temperature 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity 70% and 50% natural illumination. Plants were inoculated with 5 mL of \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 inoculum or 5 mL of distilled water was applied to the roots. After 10 days, Mab 1 (10\u003csup\u003e5\u003c/sup\u003e zoospores-mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was inoculated on approximately two-month-old leaves. For this purpose, the zoospore suspension was inoculated on the abaxial side of two leaves per plant with a sterile brush as described by Widmer (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), in this case a small paint brush of 0.7 cm in width was used. A total of 100 \u0026micro;L per leaf was inoculated. Inoculated plants were transferred to a 100% humidity chamber for seven days to promote pathogen infection. After seven days, the appearance of symptoms was evaluated on the basis of a leaf symptom scale for \u003cem\u003ePhytophthora\u003c/em\u003e spp. infection according to Nyass\u0026eacute; et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) modified by Acebo-Guerrero et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The scale comprised six values: 0\u0026thinsp;=\u0026thinsp;no symptoms, 1\u0026thinsp;=\u0026thinsp;penetration point (very small necrotic spot), 2\u0026thinsp;=\u0026thinsp;net of points (larger number of necrotic spots), 3\u0026thinsp;=\u0026thinsp;reticulate patch (spider web-like), 4\u0026thinsp;=\u0026thinsp;mottled necrotic patch (marbled appearance), and 5\u0026thinsp;=\u0026thinsp;true necrosis (large brown lesions). Disease severity was calculated as described Yang et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e): Disease severity = [\u0026sum;(The number of infected leaves corresponding to the scale value x scale value)/(Total plants x highest scale value)]x 100.\u003c/p\u003e \u003cp\u003eTreatments consisted on plants inoculated with \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07, plants treated with sterile distilled water and infected with \u003cem\u003eP. palmivora\u003c/em\u003e Mab 1 and plants inoculated with strain CP07 and infected with Mab1. Control treatment consisted on plants treated with sterile distilled water. Five plants per treatment were used and the experiment was repeated three times.\u003c/p\u003e \u003cp\u003eDetermination of PAL activity in leaves and roots.\u003c/p\u003e \u003cp\u003eLeaf and root samples were collected on plants maintained under same conditions as the \u003cem\u003ein planta\u003c/em\u003e assay in greenhouse which were reserved to this experiment. Plants of EICB-371 micrografts with rootstock UF 677 were used. Samples were taken on days 0, 1, 2, 3 and 4 of infection with \u003cem\u003eP. palmivora\u003c/em\u003e Mab 1 from plants of each previously established treatment as described in the \u003cem\u003ein planta\u003c/em\u003e assay. Seven plants from each treatment were used in the experiment. Approximately two-month-old leaves and all roots from each plant were used. PAL activity was determined also in roots to investigate the regulation of this mechanism in roots as part of the mutualistic interaction, between genotype EICB-371 and the bacterial strain, that enables the induction of the protective effect against \u003cem\u003eP. palmivora\u003c/em\u003e. Samples were macerated independently with liquid nitrogen and kept at -80\u0026deg;C for preservation.\u003c/p\u003e \u003cp\u003eThe determination of PAL activity was performed according to Olsen et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) using L-phenylalanine as substrate. Extraction was performed with 50 mg of leaf or root sample placed in a centrifuge tube with 2 mL of extraction solution (Tris-HCl, 100 mM, pH 8.8; containing 84 \u0026micro;L of β -mercaptoethanol (12 mM) in 100 mL of extraction solution). The tubes were kept on ice and vortexed for 1 min. Samples were centrifuged at 16000 x g, at 4\u0026deg;C for 10 min (Eppendorf Centrifuge 5702\u0026reg;, Germany). The supernatant was stored at -20 \u0026ordm;C until use. The protein concentration in the samples was determined by the method of Bradford (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). The absorbance reading was performed at 595 nm in a spectrophotometer (Spectra Max M2, SPE002) using a standard curve of bovine serum albumin (BSA) with six concentrations between 0-500 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor quantification of PAL activity, 500 mL of enzyme extract, 450 mL of Tris-HCl (pH 8.8) (100 mM) and 50 mL of L-phenylalanine (100 mM) were placed in an Eppendorf tube. The assay was performed in triplicate. The samples were incubated at 37\u0026deg;C for 1 h and the reaction was stopped by the addition of 50 mL of HCl (5 M). They were then centrifuged at 16 000 x g for 15 min (Eppendorf Centrifuge 5702\u0026reg;, Germany). The absorbance reading at 290 nm in spectrophotometer (Spectra Max M2, SPE002) was performed against blanks prepared in the same way as the samples, but only HCl (5 M) was added before L-phenylalanine.\u003c/p\u003e \u003cp\u003eThe enzymatic activity was obtained from the absorbance readings and the production of cinnamic acid expressed in nmol of product formed per mg of sample per hour (nmol of cinnamic acid mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was determined using a standard curve of cinnamic acid with concentrations of 2, 4, 6, 8 and 10 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData processing\u003c/h2\u003e \u003cp\u003eThe assays were performed in a completely randomized design. Data which did not conform to normality and homogeneity of variance were analyzed with Kruskal Wallis' ANOVA at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data which conformed to normality and homogeneity of variance were analyzed with ANOVA and Tukey' Test at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All experiments were repeated three times. Data of one experiment representative of the three replicates were used for figures and standard error is presented as vertical bars in the figures. The program Statistica 8.0 was used for data processing.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eIn planta\u003c/em\u003e assay under greenhouse conditions\u003c/p\u003e \u003cp\u003eDisease severity assessed seven days after infection with the pathogen was reduced in plants treated with \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 of genotypes EICB-371 and EICB-385 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) indicating a protective effect of this strain in these genotypes. Genotype EICB-371 with a high susceptibility to Mab 1 showed a drastic reduction of symptoms severity. Genotype EICB-385 showed lower disease severity than the other genotypes in plants inoculated only with the pathogen indicating less susceptibility and disease severity was reduced in plants treated with the bacterium. In EICB-384 there was no significant difference in disease severity between treated and untreated plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDetermination of PAL activity in leaves and roots\u003c/p\u003e \u003cp\u003ePAL activity levels in leaves on day 0 post-infection with \u003cem\u003eP. palmivora\u003c/em\u003e Mab 1 were significantly higher in leaves of plants pre-treated only with \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 compared to untreated control plants and plants infected with the pathogen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On day 1 post-infection the CP07 treatment differed only from the untreated control. Enzyme activity in leaves on day 2 of pathogen infection in treatments Mab 1 and CP07\u0026thinsp;+\u0026thinsp;Mab 1 had significant differences with the untreated control. On days 3 and 4 post-infection, the leaves of plants treated with the bacterial strain and infected with the pathogen showed significantly higher levels of PAL activity than the rest of the treatments. In the Mab 1 treatment, the PAL levels on days 3 and 4 were significantly lower than the CP07\u0026thinsp;+\u0026thinsp;Mab 1 treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the roots of micrografted plants, where the rootstock was UF 677 and the bud EICB-371, similar levels of activity were obtained in treated and untreated plants with \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 on all post-infection days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Thus, in these tissues the enzyme activity did not undergo significant variations due to bacterial inoculation. This result indicated that the presence of the bacterial strain in the roots did not stimulate PAL activity above basal levels in this zone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of the greenhouse \u003cem\u003ein planta\u003c/em\u003e assay showed the bioprotection effect mediated by \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 on cacao plants potted in substrate with non-sterile soil where there is a community of microorganisms that could interact with the bacterial strain. The reduction of symptom severity caused by \u003cem\u003eP. palmivora\u003c/em\u003e in \u003cem\u003eT. cacao\u003c/em\u003e plants treated with the bacterium was previously reported by Miguelez-Sierra et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) in sterile perlite substrate under controlled conditions with a genotype-dependent effect similar to the present results. The plant protection effect observed in soil conditions could be related to specific traits of the strain which confer competences not only for its activities in the rhizosphere but also for the interaction with cacao plants. Rhizobacteria have shown capacity for rapid establishment in roots and competition for nutrients, which are characteristics of great importance for biocontrol (Dos Santos et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). One of the aspects involved in nutrient competition is the availability of iron, which fluorescent pseudomonads make available to themselves via siderophores (Santoyo et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07, Acebo-Guerrero et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported the production of pyoverdin and genes for pyochelin and acromobactin. The strain presented high motility, pili and biofilm formation, characteristics that favor the establishment of rhizobacterial colonies on the root surface and contribute to their biocontrol activity (Sivakumar et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e\u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 also produced antifungal metabolites like the lytic enzymes proteases and lipases, phenacins that inhibit electron transport, and hydrogen cyanide (HCN) which is a potent inhibitor of metalloenzymes according to Santoyo et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These features have been related to biocontrol interaction of the species \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e with host plants (Arrebola et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similar characteristics benefited antagonism against \u003cem\u003eFusarium oxysporum f.sp. radicis lycopersici\u003c/em\u003e in \u003cem\u003eP. chlororaphis\u003c/em\u003e PCL1391, an efficient colonizer of tomato roots (De Weert and Bloemberg, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In \u003cem\u003eP. chlororaphis\u003c/em\u003e MCC2693, phenazine production was related to biocontrol activity effective against \u003cem\u003ePhytophthora\u003c/em\u003e sp. and \u003cem\u003eFusarium\u003c/em\u003e sp. (Jayaprakashvel et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Also, this characteristic could stimulate plant innate immunity against pathogens according to Ma et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eViscosine-type cyclic lipopeptides present in \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 could have antifungal action and participation in the induction of systemic resistance in plants (Ma et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jayaprakashvel et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, masetolide A (viscosin type) produced by \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e SS101 increased resistance to \u003cem\u003ePhytophthora infestans\u003c/em\u003e in tomato plants (Tran et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Additionally, genes involved in the perception of intercellular signal molecules (\u003cem\u003equorum sensing\u003c/em\u003e) were found in the genome of \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 (Acebo-Guerrero et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this particular, Khan et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Shrestha et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that plants sensitized with \u003cem\u003equorum sensing\u003c/em\u003e molecules activated systemic defense mechanisms against pathogenic bacteria and fungi which may be an alternative for resistance enhancement. Therefore, these characteristics of the strain could favor its permanence in the rhizosphere and protect the plant against \u003cem\u003eP. palmivora\u003c/em\u003e Mab 1 and other pathogens.\u003c/p\u003e \u003cp\u003eThe PAL activity determined as biochemical marker of ISR in cacao plants of genotype EICB-371, was higher in plants infected with \u003cem\u003eP. palmivora\u003c/em\u003e Mab 1 compared to control plants not inoculated with the pathogen in day 2 post-infection, which could be a response of plant innate defense against the development of the infection. It is known that activation of enzymes of the phenylpropanoid pathway, including PAL, is an important defense mechanism included in pathogen-induced responses (Zhang and Liu, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). On the other hand, Iwaro et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) proposed that the resistance response of cacao plants to \u003cem\u003eP. palmivora\u003c/em\u003e can be manifested both during penetration and in the post-penetration stage of the pathogen. At this stage, the interaction between the plant and the pathogen can trigger defense mechanisms based on pathogen-associated molecular patterns- triggered immunity (PTI), from the recognition of the effectors released by the oomycetes during colonization of host tissues (Stassen and Van den Acherveken, 2011).\u003c/p\u003e \u003cp\u003eOn days 3 and 4 post infection, PAL activity in leaves of plants untreated with the bacterial strain and infected with the pathogen was similar to control treatment. This could be a consequence of PAL defense response suppression by the pathogen's infective mechanisms. Pathogenic oomycetes can suppress PTI and effector triggered immunity (ETI) via apoplastic effectors and haustorium-secreted effectors within host cells. In this way, they create effector-induced susceptibility thereby promoting disease (Stassen and Van den Acherveken, 2011; Heliwell et al. 2016). At the same time, this response may be a manifestation of the susceptibility of genotype EICB-371 to infection by \u003cem\u003eP. palmivora\u003c/em\u003e Mab 1, which was observed in the greenhouse assay. Similarly, Manga et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found significantly lower PAL activity levels in several \u003cem\u003eT. cacao\u003c/em\u003e genotypes susceptible to \u003cem\u003eP. megakarya\u003c/em\u003e compared to tolerant genotypes.\u003c/p\u003e \u003cp\u003eIn contrast, plants treated with \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 and inoculated with the pathogen maintained higher PAL levels than untreated plants until the fourth day post-infection. This indicated that PAL defense response induced by the bacterium in leaves remained active and effective during the development of the pathogen attack. Similarly, increased PAL activity as expression of resistance to \u003cem\u003eP. palmivora\u003c/em\u003e infection in \u003cem\u003eT. cacao\u003c/em\u003e was reported by Okey et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Activation of PAL has been considered one of the elements participating in the interaction of \u003cem\u003eT. cacao\u003c/em\u003e with Phytophthora (R\u0026ecirc;go et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Defense based on increasing the activity of this enzyme and others compounds of phenylpropanoids pathway upon \u003cem\u003eP. palmivora\u003c/em\u003e invasion was associated to the accumulation of polyphenols and lignin in stem tissues of tolerant genotypes (Okey et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Nyadanu et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and Ondobo et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) observed that less susceptible genotypes of cacao accumulated more polyphenols and lignin in leaves and pods indicating that the inhibitory effect on pathogen growth depended on the levels of these metabolites in host tissues. In this way, the metabolism of aromatic fungitoxic compounds was promoted with specific effects on pathogen infective mechanisms and lignin deposition acted as barrier to penetration and propagation of the pathogen (Zhang and Liu \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the present study, the effects of increased PAL activity could ultimately be related to the significant reduction of disease severity observed in plants of genotype EICB-371 treated with the bacterial strain compared to no-treated plants in the \u003cem\u003ein planta\u003c/em\u003e assay. The ISR mediated by beneficial bacteria in \u003cem\u003eT. cacao\u003c/em\u003e has not been widely studied, in particular, there are no previous reports on the occurrence of this phenomenon in the interaction of Pseudomonas-Cacao-\u003cem\u003eP. palmivora\u003c/em\u003e. Moreover, PAL activation as a mechanism of induction of systemic defense responses by antagonistic bacteria has been described in other plant species (Ramamoorthy et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Chithrashree et al. 2011; Tan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Dias et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Meena (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) pointed out that a fundamental element in the increase of resistance due to rhizobacteria-induced ISR is the induction of compounds of the phenylpropanoid pathway in which PAL was a key enzyme.\u003c/p\u003e \u003cp\u003eThe presence of the bacterial strain in the roots did not stimulate PAL activity above basal levels in this zone. Pieterse et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) described that ISR elicitor microorganisms can suppress the root immune response upon colonization protecting themselves from antimicrobial compounds produced by the recognition of molecular patterns associated with the microorganism and allowing to establish the mutualistic relationship with the host. This effect was detected in several beneficial ISR-inducing microorganisms such as \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e FB17 and \u003cem\u003eP. fluorescens\u003c/em\u003e WCS417r (Pieterse et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)d \u003cem\u003eputida\u003c/em\u003e RRF3, a rice rhizosphere isolate (Kandaswamy et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the case of \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07, regulation of the defense response via PAL in roots could be one of the mechanisms favoring its interaction with the host plant.\u003c/p\u003e \u003cp\u003eThe results showed that, in the protective effect of \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 against \u003cem\u003eP. palmivora\u003c/em\u003e Mab 1 in genotype EICB-371, PAL activity was stimulated in leaves what could be used as marker of defense responses against the pathogen mediated by the bacterial strain. This information would allow the selection of genotypes with the greatest potential for the induction of defenses by the strain. The study of these aspects can generate new perspectives for the use of \u003cem\u003eP. chlororaphis\u003c/em\u003e CP07 in the protection of \u003cem\u003eT. cacao\u003c/em\u003e against pathogens that affect the crop, particularly in the integrated management of black pod rot.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Project \u0026apos;Design and strengthening of an agroecological cacao production system in Cuba\u0026apos; of ARES (Acad\u0026eacute;mie de Recherche et d\u0026apos;Enseignement sup\u0026eacute;rieur, Belgium) and the Project PN223LH010-009 \u0026apos;Contributions to knowledge for the agroecological management of black pod rot in \u003cem\u003eTheobroma cacao\u003c/em\u003e L.\u0026apos; of the National Program of Basic Sciences of the Ministry of Science, Technology and Environment (CITMA) of Cuba. Our sincere gratitude to Dr. Stanley Lutts from Universit\u0026eacute; catholique de Louvain, Belgium, for their support to the research. The authors thank to the Instituto de Investigaciones Agroforestales (INAF) UCTB Baracoa gene Banks for providing the plant material. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman and animals rights\u0026nbsp;\u003c/strong\u003eNo human and/or animal participants were involved in this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003eAll authors consent to this submission.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAcebo-Guerrero, Y., Hern\u0026aacute;ndez-Rodr\u0026iacute;guez, A., Heydrich-P\u0026eacute;rez, M., Jaziri, E., M., \u0026amp; Hern\u0026aacute;ndez-Lauzardo, A. 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Development of plant systemic resistance by beneficial rhizobacteria: Recognition, initiation, elicitation and regulation. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, 952397. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.3389/fpls.2022.952397\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Theobroma cacao, black pod rot, fluorescent Pseudomonas, induced systemic resistance, phenylalanine ammonia-lyase","lastPublishedDoi":"10.21203/rs.3.rs-2987328/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2987328/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study was to evaluate the effect of \u003cem\u003ePseudomonas chlororaphis \u003c/em\u003eCP07, isolated from the rhizosphere of cacao, on the induction of defense responses in \u003cem\u003eTheobroma cacao \u003c/em\u003eL. against \u003cem\u003ePhytophthora palmivora \u003c/em\u003e(Butler)\u003cem\u003e, \u003c/em\u003ethe\u003cem\u003e \u003c/em\u003ecausal agent of black rot of the fruit (black pod rot). The \u003cem\u003ein planta \u003c/em\u003egreenhouse trial was carried out to determine the reduction of disease symptoms in plants micrografted with three traditional Cuban cacao genotypes of the Trinitario type on UF 677 hybrid rootstocks. The levels of phenylalanine ammonia-lyase (PAL) were determined in micrografts of genotype EICB-371. In genotypes EICB-371 and EICB-385 disease severity was significantly reduced in plants pretreated with the bacteria compared to control plants. In contrast, genotype EICB-384 showed no symptom reduction in plants pretreated with the bacterium. PAL enzyme activity was significantly increased in leaves of plants pretreated with CP07 compared to control plants on days 3 and 4 post-infection with the pathogen. The results suggested that, depending on the genotype, strain CP07 had potential in the protection of \u003cem\u003eT. cacao \u003c/em\u003eagainst \u003cem\u003eP. palmivora \u003c/em\u003ein soil substrate and that the interaction of this bacterium with the benefited plant activated defense responses related to the increase of PAL activity in leaves.\u003c/p\u003e","manuscriptTitle":"Induced defense responses in cacao against Phytophthora palmivora (Butler) by Pseudomonas chlororaphis CP07.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-19 19:45:12","doi":"10.21203/rs.3.rs-2987328/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-01-26T13:28:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-11T20:36:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"European Journal of Plant Pathology","date":"2024-01-04T02:55:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-17T13:28:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2023-11-16T19:44:10+00:00","index":"","fulltext":""},{"type":"decision","content":"Revision","date":"2023-06-11T16:07:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"850c1311-834a-44e3-b8fd-10e1d8a826ad","owner":[],"postedDate":"January 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-02T17:27:56+00:00","versionOfRecord":{"articleIdentity":"rs-2987328","link":"https://doi.org/10.1007/s10658-024-02982-2","journal":{"identity":"european-journal-of-plant-pathology","isVorOnly":false,"title":"European Journal of Plant Pathology"},"publishedOn":"2024-11-25 15:58:30","publishedOnDateReadable":"November 25th, 2024"},"versionCreatedAt":"2024-01-19 19:45:12","video":"","vorDoi":"10.1007/s10658-024-02982-2","vorDoiUrl":"https://doi.org/10.1007/s10658-024-02982-2","workflowStages":[]},"version":"v1","identity":"rs-2987328","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2987328","identity":"rs-2987328","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}