Acta Physiologiae Plantarum Histochemistry and Gene Expression Using Glycerol in the Pathosystem Musa X Mycosphaerella Fijiensis

Acta Physiologiae Plantarum Histochemistry and Gene Expression Using Glycerol in the Pathosystem Musa X Mycosphaerella Fijiensis | 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 Acta Physiologiae Plantarum Histochemistry and Gene Expression Using Glycerol in the Pathosystem Musa X Mycosphaerella Fijiensis Ana Paula da Silva Novaes, Andresa Priscila de souza Ramos, Fernanda dos Santos Nascimento, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4311892/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Black Sigatoka (SN) is a disease that causes leaf lesions that impede the photosynthesis process, culminating in fruits of poor quality banana. Hence, environmentally correct measures, which can present sustainable control of the disease in the field, are extremely important. Therefore, the objective of this study was to verify glycerol-mediated resistance responses in banana plants of the Grande Naine cultivar susceptible to BS. The experiment was conducted in randomized blocks with 5 treatments, namely: control, 0, 3, 5 and 8% glycerol and three replicates. The severity of the disease after the first symptoms was assessed every seven days. For gene expression analysis, the signal transduction genes (STRANS) and the recognition and signaling genes (KINLRR) and the reference genes, L2MU and 25S, were used. Histochemistry was performed to detect calosis with the aid of relative dyes of lugol and aniline blue. By means of leaf tissue clearing, it was possible to visualize pathogen structures from 72 HAI. In the analysis of calosis, the production of the compound was identified in the first evaluations in all glycerol treatments. The target resistance genes, STRANS and KINLRR, had higher expression in the treatments with 5-8% glycerol. From the results presented, glycerol appears to be a good resistance inducer for banana plants when used at a dose of 5%, but further studies are needed to verify the best time to apply treatments, inoculation, and doses to be used. Grand Nain resistance KINLRR STRANS. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION The first report of Sigatoka Negra (SN) in Brazil was in 1998, in production regions in the state of Amazonas (Cordeiro 2015). This disease is among the main fungal diseases that attack the banana crop. The symptoms manifest themselves in the leaves of plants through dark streaks, causing their leaf area to be impaired and preventing the photosynthetic process from being conducted. The fruits produced do not have market quality due to premature ripening, which makes them unsuitable for the market, implying financial losses for producers (Gasparotto and Pereira 2010). SN has been advancing in the national territory, recently arriving in the State of Bahia in 2018. In addition to Bahia, SN is located in the states of Acre, Amapá, Amazonas, Espírito Santo, Goiás, Maranhão, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Pará, Paraná, Rio de Janeiro, Rio Grande do Sul, Rondônia, Roraima, Santa Catarina, São Paulo and Tocantins (MAPA 2021). The main means of controlling the disease is through systematic spraying, which damages both man and animals, as well as the environment, with the planting of resistant cultivars being the most environmentally correct measure. However, resistant varieties are not the main ones planted in Brazil and in the world, requiring other control measures that are more feasible and applicable in short term (Garruti et al. 2012). The cultivar Grande Naine (AAA) belongs to the species Musa acuminata , Cavendish subgroup, grown commercially in tropical and subtropical regions (Elbagoury et al. 2022). Grande Naine suffers losses during its cycle, which is affected by severe diseases that limit its development, such as SN, considered one of the main fungal diseases that affect the banana crop. SN caused by the hemibiotrophic fungus in its asexual stage M. fijiensis (ascospores) and in its axesuous form P. fijiensis (conidia) is a polycyclic disease that infects the banana tree several times within a single development cycle (Guzmán et al. 2019). According to Pereira and Gasparotto (2010), 26 applications of systemic fungicides or up to 52 applications of protective fungicides are necessary for effective control of the disease in banana plantations. Unfortunately, the small producer, who is the most representative in the country, most of the time, does not have the conditions to pay the costs of the applications and ends up having losses with the disease. In addition, using the fungicide may be feasible for large producers who have resources, but it can harm the health of humans who consume the fruit and the quality of the environment. Thus, developing sustainable, easily applicable and low-cost practices to control NS are paramount. Glycerol is an economically viable and sustainable alternative that has shown promising results when used in plants for disease control. When applied exogenously to plants, their metabolism produces the compound glycerol-3-phosphate (G3P). This molecule consequently leads to decreased production of oleic acid (18:1- monosaturated fatty acid-omega), causing the induction of resistance in plants against pathogens through the production of salicylic acid (AS) (Zhang et al. 2015; Gao et al. 2011). The responses of hormones, salicylic acid and jasmonate, are most often antagonistic in plants. In the case of glycerol degradation, the decrease in oleium acid production (18:1) simultaneously increases responses mediated by (AS), inhibiting Jasmonate-induced defense responses and consequently increasing plant resistance against biotrophic fungi (Gao et al. 2011). As M. fijiensis is a hemibiotrophic fungus (Churchill et al. 2010) with a biotrophic phase, it is considered that the application of Glycerol can successfully produce compounds that act to increase the resistance of bananas to SN. Therefore, the present study aims to evaluate the efficiency of Glycerol in controlling the fungus M. fijensis in the cultivar Grande Naine de bananeira, susceptible to NS. MATERIAL AND METHODS Plant material The experiment was installed at the Embrapa Cassava and Fruit Farming Unit, located in Cruz das Almas (12◦40′19 ′ ′S, 39◦06′22 ′ ′W ', altitude 220 m above sea level), Bahia, Brazil. The climate is classified as tropical hot, humid, Aw an Am, according to the Köppen classification, with an average annual temperature of 24.5 ◦C, relative humidity of 80%, and average annual rainfall of 1250 mm (AGRITEMPO, 2018). The experiment was conducted in a greenhouse, with 90 banana plants of the Cavendish subgroup, cultivar Grande Naine. The experimental design was completely randomized, with 15 plots, 3 replicates, 6 plants per plot, and 5 treatments: control, water + inoculation, 3, 5, and 8% glycerol. The glycerol solution was sprayed onto the leaves until drained. This application was made every 2 days with an interval of 2 days, for 6 days. Leaves were collected before treatment and before inoculation (time zero) and at times 72 hours after inoculation (HAI), 5, 15, 30, and 60 days after inoculation (DPI). Samples for molecular analysis were stored in aluminum foil envelopes, frozen with liquid nitrogen in a Styrofoam box until placed in the ultrafreezer -80°C. The assembled experiment is shown in Figure 1: A and B. Fig. 1 a) Experiment mounted on DIC in a greenhouse; b) Method of applying the glycerol solution via spray until the leaves run off; c) Banana leaf cv Grande Naine – red arrow - beginning of M. fijiensis lesion. Photo: Ana Paula da Silva Novaes, 2023 For the histology study, samples were diced and stored in karnovsky fixative and FAA in a 2 mL microtube. After each collection, the samples were placed in the vacuum desiccator equipment for 10 minutes and subsequently dehydrated in increasing series of 30-100% ethanol. 2.2. Inoculum preparation The isolate was prepared in the Laboratory of Phytopathology of Embrapa Cassava and Fruticulture. Initially, peaks were performed in solid medium (BDA), remaining until they were transferred to v8 medium for 15 days. After colony growth, a soft bristle toothbrush was scraped on all plates, adding 10 mL of distilled water and counting conidia in a Neubauer chamber to obtain the suspension 104 of the isolate. After 2 days of the last treatment, the plants were inoculated on the abaxial face of the leaves with the aid of a spray bottle until the runoff. 2.3. Symptom assessment Two plants of each plot were evaluated, and of these two plants, 2 leaves were evaluated, giving scores of 0-5, based on the diagrammatic scale of Ganry, Mayer (1972), and Stover (1989) (Fig. 2). Fig. 2 Ganry, Mayer (1972), and Stover (1989) diagrammatic scale The scores obtained at 07-day intervals were transformed into a disease severity index ID (%), based on the formula provided by Mckinney (1923), represented below: ID (%) = ∑(disease score x number of plants with a certain score of disease) x 100/number of plants evaluated by plot x highest score of the scale) The mean ID was estimated for each treatment based on the evaluations performed every 07 days and the number of leaves evaluated. The area under the disease progression curve (AACPD) was estimated according to the formula proposed by Mandden et al. (2007), where n is the number of evaluations, ID is the disease severity index and ( T1-T2 ), the time interval between two consecutive evaluations, as can be seen in the following formula: The AACPD was standardized by the number of assessments performed every 07 days until the end of the assessments. 2.4 Data analysis In order to classify the treatments as resistant to black Sigatoka, the Scott and Knott (1974) cluster test was used at 5% significance, using the "ExpDess" package of the statistical program R (CORE DEVELOPMENT TEAM, 2018). A disease severity index and the area under the disease progress curve were estimated. For the analysis of the relative quantification of gene expression, Microsoft Excel was used using the 2 -ΔΔCT method proposed by Livak and Schmittgen, (2001). Heatmaps with corresponding dendrograms were based on a complete binding cluster by the Euclidean distance method using the heatmap function. 2 of the "plots" and "dev tools" packages were implemented in R (R CORE DEVELOPMENT TEAM, 2018). 2.5. Histochemistry For histochemical analysis, the 1 cm sections were placed in karnovsky and FAA fixative solution, subjected to vacuum for 10 min, stored in fixative solution for 8 days, dehydrated in increasing series of ethanol (30-100%) 1h each. Subsequently, infiltration was performed using part of ethanol+historesin. Polymerization was performed with Historesin Kit (hydroxyethyl methacrylate; Leica Heidelberg, Germany). The cuts were made in a rotating microtome (8mm), placed on slides and stained with dye according to the objective. 2.6. Clarification and coloring of leaves FAA-fixed samples were soaked in Potassium Hydroxide (KOH) solution (10%) for 72h, and then washed with distilled water and placed in Hydrochloric Acid (HCL) for 30 minutes. Then samples were stained with 0.05% trypan blue in solution (2:1:1, lactic acid: Glycerol:water) for 2h. Immediately after dye disposal, the leaf fragments were immersed in lactoglycerol solution (2:1:1, lactic acid: Glycerol:water) and microphotographed under an objective light microscope with a range of 40x. 2.7. Molecular analysis 2.7.1 Total RNA extraction For total RNA extraction, 'Big Naine' plant leaves were collected before glycerol treatment, time 0 (before inoculation), 72 hours post inoculation (HAI), 5, 15, 30, and 60 days post-inoculation. During collection, the samples were stored in liquid nitrogen and then stored in an utrafreezer (-80 °C) until extraction. In the extraction process, the methodology of Ferreira et al. (2019) was used with modifications, where liquid nitrogen was used in the maceration of the samples. To check RNA integrity and concentration after extraction, a 5 µl aliquot of the samples plus 2 µl of GelRed® were observed via 1% agarose gel electrophoresis in 1x TAE buffer (Tris; glacial acetic acid; Milli-Q® water). The extracted RNA was stored in ultrafreezer -80°C for further use. 2.7.2 DNase Treatment The extracted total RNA was treated with DNase TURBOFREE (Ambion®), according to the manufacturer's recommended protocol: in 15µl of total RNA from each sample was added 1µl of DNAse free water, 1µl of Inhibitor (RNase out), 2µl of Buffer (10x turbo DNAse buffer) and 1µl DNAse (turbo DNase). The reaction was incubated in Veriti thermocycler (Applied Biosystems©) at 37°C for 25 min. Then, 2 µl of DNase Inactivation was added, and the sample was incubated for 2 min. at room temperature. Subsequently centrifuged for 1.5 minutes at 10000 rpm, the supernatant transferred to new tubes was stored in ultrafreezer at -80°C. The quality of the treated RNA was conferred on 1% agarose gel with 1 x TAE (Tris; glacial acetic acid; MQ water) and 3 µl of each sample was used. The genes and primers used in the study are shown in Table 1. Table 1- Primers used to evaluate gene expression in Musa spp. X Mycosphaerella fijiensis . Target genes and their respective functions: Signal transduction (STRANS) - signal transduction; Kinase, LRR domains, protein-protein interaction (KINLRR) - recognition, signaling, and references. 2.7.3. Synthesis of cDNA The cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit to obtain the final volume of 20 µl according to the protocol: 5 µl of the treated RNA, plus 1 µl of 20x enzyme mix, 10 µl of 2x RT Buffer and 4 µl of Nuclease Free water. Subsequently the reaction was incubated in the Veriti thermocycler (Applied Biosystems ©) for 60 minutes at 37°C + 97°C for 5 min + hold 4°C. The cDNAs were stored at -80°C. To verify the viability of the cDNA, conventional PCR with beta-tubulin primer was performed (PODEVIN et al., 2012). RESULTS .1. Symptom assessment The presence of symptoms and confirmation of the presence of the fungus M. fijiensis in plants occurred at 25 DAI. Initial symptoms were light brown color scores on the abaxial face of the leaves. Symptoms in the form of stretch marks began to appear from 55 DAI in plants treated with water and 8% glycerol. At first, treatments 3, 5, and 8% had statistically higher ID values than the inoculated treatment treated with (Table 2). Table 2- Disease severity index (ID) and area under the disease progress curve (AACPD) of glycerol-treated plants at concentrations of 3, 5, and 8%, water-treated plants, and control. Cruz das Almas, 2023. Regression analysis to assess the disease index (ID) and the area under the disease progression curve (AACPD) indicate that there was a drop in severity at 5% glycerol and greater severity in treatment with 8% glycerol (Fig. 3 a and b). Fig. 3 a) Disease graphs of regression plots (ID) and b) of the area under the disease progress curve (AACPD) of Big Naine banana plants, infected with black Sigatoka Histological Analysis 3.2.1. Clarification and coloring of leaves Fig. 4 (a to p) shows lighttening and staining of fungal structures with trypan blue on Big Naine banana leaves prior to inoculation up to 5 DAI. Prior to inoculation, time zero (Fig. 4: a-e), no fungal structures were found. Fig. 4 (a to p):Lightening and staining of fungal structures in Big Naine banana leaves before inoculation up to 5 DAI. a, f and l- control; b, g, m- plant inoculated without glycerol; c, h and n- plants inoculated and treated with 3% glycerol; d, i and o- plants inoculated and treated with 5% glycerol; e, j and p- plants inoculated and treated with 8% glycerol. DAI= days after inoculation, HAI= hours after inoculation, h- hypha, c= conidium, a = ungerminated ascospore, and= stomata. Bars: 200 µm. Cruz das Almas-BA, 2023 Samples collected at 72 HAI show fungal structures in the treatment with water (Fig. 4 g) with the presence of hypha and in plants inoculated and treated with 8% glycerol (Fig. 4 j), with the presence of conidium, spore of asexual reproduction. Thus, it is assumed that in the first five days after inoculation, the plant was being infected in the parenchyma tissue with penetration through the stomata (Beveraggi et al., 1995). No fungal structures were found at 5 DAI in the control treatments (Fig.4 l) and 3% glycerol (Fig. 4 n). In the treatment with water, hyphae continued to appear (Figure 4 M); conidium colonizing stomata in 5% glycerol (Fig. 4 o) and ascospore, ungerminated sex spore, in 8% glycerol (Fig. 4 p). Ascospores produced since the age of 5 days are considered the main structure and can be disseminated to distant areas by the wind. (Favreto; Model and Tonietto 2007). The lightening and staining of fungal structures in leaves of Grande Naine banana with 15, 30, and 60 DAI are shown in Figure 5 (a to p). At 15 DAI, the disease advanced in plants treated with water (Fig. 5 b), with appearance of ascospore, hyphae around the stomata in the 5% glycerol treatment (Figure 5 d) and conidiophores (Fig. 5 e). Control and tre atment 3% continued without structures. Fig. 5 Lightening and staining of fungal structures in leaves of Grande Naine banana with 15, 30 and 60 DAI. A, F and L- control; b, g, m- plant inoculated without Glycerol; c, h and n- plants inoculated and treated with 3% glycerol; d, i and o- plants inoculated and treated with 5% glycerol; e, j and p- plants inoculated and treated with 8% glycerol. DAI= days after inoculation, h- Hypha, c= conidium, co= conidiophore, a = ascospores, st= stomatopodium, e= stomata. Bars: 200 µm At 30 days structures were found in all treatments except control. Hyphae colonizing stomata in the treatments with water and 5% glycerol (Figures 5 G and I), conidiophores in 3% glycerol (Fig 5 h), and ascospore germinated in 8% glycerol (Fig. 5-j). At 60 DAI, hypha colonizing stomata were visualized in the water treatment (Fig. 5 m), mycelium in 3% glycerol (Fig. 5 n), ungerminated ascospore in 5% (Figure 3 O), and the presence of stomatopodium in 8% treatment (Figure 5 p). Until the end of the evaluations, no fungal structures were found in the control treatment. 3.3. Gene Expression Analysis To verify the interaction of glycerol-treated Big Naine plants in interaction with black Sigatoka in molecular analysis, two genes involved in plant-pathogen interaction with recognition and signaling function (KINLRR, Kinase, LRR domains, protein-protein interaction) and signal transduction (STRANS, Signal transduction) (Velame, 2017) were analyzed via RT-qPCR, according to (Figure 6). Expression was normalized by the Musa spp., 25S, and L2MU reference genes (PODEVIN et al., 2012). According to Figure 6 (a and b), genes were expressed differently between treatments and times evaluated. The highest expression of the genes in the final time (60 days) occurred with 5% glycerol. Fig.6 Relative expression profile of genes in Big Naine samples treated with Glycerol and inoculated with M. fijiensis isolate in gration to uninoculated control at time 0h. (a) STRANS (Transduction signal) and (b) KINLRR (Kinase, LRR domains, protein-protein interaction). Quantifications were normalized using the 25S and L2MU reference genes. DAI: Days after inoculation The overall expression of the two selected genes is shown in the heat map (Fig. 7). The analyses show the hierarchical grouping of the cultivar Big Naine according to the expression profiles of the two genes analyzed. The dendrogram lines represent the collection times, and the columns represent the treatments, demonstrating that the responses were different between the genes in relation to the treatments and the evaluated times. Among the treatments, 3% glycerol was the one that obtained the lowest expression of both genes, while the treatment with 5% glycerol showed the highest expression of both target genes, especially at 60 days. This result corroborates the ID and AACPD analyses (Figure 3). Fig. 7 Heatmaps of the relative expression profiles of STRANS (Signal transduction), KINLRR (Kinase, LRR domains, protein-protein interaction in Large Naine samples, at six collection times after glycerol treatment relative to the uninoculated control at time 0h. Quantifications were normalized using the 25S and L2MU reference genes Histochemical Analysis Figure 8 (a to p) shows images of callose detection via fluorescence after aniline blue + lugol staining from time zero to 5 DAI. Production of this compound was visualized from 72 HAI in inoculated plants. At 5 DAI, callose deposition was detected in the palisadedic and lacunose parenchyma in the 5% glycerol treatment. Fig. 8 Cross sections of Grand Naine banana leaves in the control treatments (a, f, l), inoculation + water (b, g, m), 3% glycerol (c, h, n), 5% glycerol (d, i, o), 8% glycerol (e, j, p). The images refer to T0, 72 HAI and 5 DAI. Histochemical test for detection of callose. Ca: callose, HAI: hours after inoculation, HENCE: days after inoculation. Bar: 200 mm. Cruz das Almas-BA, 2023 The cross sections of leaves of Grande Naine banana from 15 to 60 days are shown in Figure 9 (a top). At 15 DAI, no defense response was visualized in plants not treated with Glycerol. At 30 and 60 DAI, all treatments, including the control, detected a defense response. Although the evaluation ofcallose is qualitative, it is possible to identify greater detection (fluorescent parts) in treatments 5 and 8% of Glycerol at 60 DAI. Fig. 9 Cross sections of Grand Naine banana leaves in the control treatments (a, f, l), inoculation + water (b, g, m), 3% glycerol (c, h, n), 5% glycerol (d, i, o), 8% glycerol (e, j, p). The images refer to the 15, 30 and 60 DAI. Histochemical test for callose detection; bar: 200 mm. Cruz das Almas-BA, 2023 Discussion Evaluation of symptoms and histological analysis The characteristic spots of Black Sigatoka are yellow in color, which, over time and the evolution of the pathogen, become brown, grow, and aglutinate, inducing tissue death. The spots in proportion that increase become necrotic, impairing plant photosynthesis (Borah et al. 2022). Ascospores are produced in necrotic tissues, completing their cycle in the necrotrophic phase (Favreto; Model; Tonietto 2007), however, this statement goes against what was observed in our study. The presence of ascospores in the treatments with water and 8% glycerol occurred in non-necrotic leaves. This type of result could be expected in plants treated with 8% glycerol because the high level of this compound, which consequently has a higher amount of carbon, was acting more as an energy source for the fungus (Chanda et al. 2008). M. fijiensis , as a hemibiotrophic fungus, has a biotrophic cycle that lasts 3-4 weeks without causing death to the plant (AGRIOS 2019), which explains the way the symptoms occurred during the 5 weeks of evaluation, not reaching the necrosis phase. Treatment with 8% glycerol showed ascospores and greater severity of symptoms. This result again suggests Glycerol possibly acting as a carbon source for the fungus, corroborating the results obtained for ID and AACPD analysis. Zhang et al. (2015) verified cell death in plants treated with higher doses of Glycerol in the cocoa crop treated with doses above 300 mM, where chlorosis and cell death were visualized at the tips of the leaves. In banana, cultivar Grande Naine, no significant phenotypic differences were seen in plants treated with 3, 5 and 8% Glycerol compared to untreated plants. From the clarification, it was possible to visualize several stages of development of black Sigatoka, from hyphae to spores of sexual reproduction in germinated and non-germinated forms. It has been scientifically proven that the higher the density of stomata, where the fungus M. fijiensis infects the plant, the greater the infection. Grande Naine, a susceptible genotype, has a high density of stomata, which facilitates the penetration of the fungus (SOARES et al., 2022). The plant, with physical and chemical defense responses, uses mechanisms to try to defend itself from the invader. The cell wall restricts the fungal germ tube and biochemical defense is triggered by ITP and ITE by the production of reactive oxygen species and others. Secondary metabolites also act as molecules in the development of acquired systemic resistance (SAR) (Kaur et al. 2022). Plants treated with 8% Glycerol showed similar results to water-treated plants, with some fungal structures in the first hours of evaluation. In the treatment with 5% glycerol, the first visualization of conidia was at 5 DAI, while in the same period the treatment with 8% glycerol presented ungerminated ascospores. Initially, treatment with 3% glycerol demonstrated a higher level of resistance, developing structures from 15 DAI. Li et al. (2016) identified that the higher the dose of Glycerol (1-4%), the greater the resistance developed by wheat plants against the oidium, but considered the best dose at 3% because 4% glycerol presents cell death at the tips of the leaves. Li et al. (2020) found spore germination in ~90% of water-treated plants, suggesting that Glycerol induced resistance against the wheat fungal pathogen powdery mildew. This may be related to the present study in which structures were visualized in the control treatment from the first days and in the treatment with a higher dose of Glycerol, 8%. For wheat, different times of glycerol application were evaluated, before and after inoculation, and it was proven that the best time to start the treatment is 1-2 days before inoculation. Plants treated with Glycerol were highly resistant, while those treated with water only were susceptible (Li et al. 2016). Gene expression The glycerol-mediated induction of defense in plants is related to the increase in glycerol-3-phosphate (G3P) and the decrease in oleic acid (18:1). Glycerol can be used in plants to induce resistance against pathogens, but has even been considered toxic in plants when used in high concentrations (Zhang et al. 2015). The study of gene expression in banana plants infected by the M. fijiensis isolate contributes to the identification of the beginning of plant defense, since visually this was not possible due to the initial phase being biotrophic and not presenting symptoms initially (Rodríguez et al. 2016). In the study performed here, it was possible to identify that the signaling gene was expressed early on in the infection. In a study with resistant and susceptible varieties, Rodríguez et al. (2016) were able to identify several defense genes in the resistant genotype, Calcutta 4, and different induction in the susceptible one, Williams. In Calcutta 4, induction was early, as in the present study; in susceptible plants, there was gene induction at the beginning of infection. A transduction signal developed by plants after glycerol treatment in response to the pathogen may be salicylic acid, an important hormone for acquired systemic resistance (Xá et al. 2001) and oleic acid (Mendel et al.2012); its response is dose-dependent (Zhang et al. 2015). This explains the induction of the STRANS and KINLRR genes in banana plants, the higher the glycerol dose, the higher the expression of these genes. Signal transduction is activated when the plant recognizes the presence of the pathogen. When recognizing, it releases Ca2 +, considered the main mediator against plant stress (Ding et al. 2022). The KINLRR gene is a leucine-rich kinase gene. This gene acts as a receptor that detects abnormalities created by biotic and abiotic factors in plants (Senseverino et al. 2010; Antolín-Loovera, Binder and parniske 2012). Leucine-rich proteins are effector-triggered immunity (ETI). The ETI is activated after the pathogen manages to overcome the host's first line of defense, acting as a second line of defense (Noman, Aqeel, Lou 2019). Signal transduction in plants involves several lines of plant defense to prevent the development of the pathogen, where the responses are the creation of reactive oxygen species (ROS), pathogenesis-related genes, callose deposition, salicylic acid, and mitogen-activated protein kinase (MAPK) signaling, among others (Liu and Lam 2019). Hormones are molecules that act on the plant in various physiological processes, including plant defense. Among the main ones, jasmonic acid, salicylic acid, ethylene, abscisic acid, auxin, gibberellin stand out (Shigenaga and Argueso 2016). Treatment with 5% glycerol had long-term positive responses for the two genes tested. STRANS gene expression in this treatment remained high at 60 DAI, while for KINLRR, it decreased compared to 30 DAI. Comparing treatments 5 and 8% glycerol, the treatment that had the most gene expression, 5% glycerol, maintained this result until the end of the evaluations. The way Glycerol acts in banana plants still needs several studies to prove, but the increase in G3P and the decrease in oleic acid occurs through the acylation between the two in plasmids, the elevation of G3P contributes to signaling through salicylic acid (AS) and jasmonic acid (AJ) (Kachroo et al. 2004). Ethylene is also part of signaling, and each hormone activates a pathway individually, together, or antagonistically, but each response depends on the plant x pathogen interaction (Chandra-shekara et al. 2007). Related to plant defense against pathogens, induction against necrotrophic microorganisms occurs through JA, the hormone responsible for defense against necrotrophic pathogens (Matric et al. 2016). LRR is an effector recognition gene that causes virulence proteins to become avirulent, it has a second line of defense known as ETI, which was previously called gene-to-gene or race-specific resistance (Jones and Dangl 2006). LRR induces resistance in plants attacked by biotrophic or hemibiotrophic microorganisms, that is, organisms that allow the plant to remain alive for at least one period (Glazebrook, 2005), such as the case of the fungus M. fijiensis , which is a hemibiotrophic fungus. The KINLRR gene was expressed during the first hour of evaluation during the interaction of Musa spp. x M. fijiensis , demonstrating that the expression of this gene was rapid in the detection of stress, recognizing the presence of the pathogen in only 1 HAI and induced resistance in most of the evaluated times (Oliveira 2020). Signal transduction acts on the plant when pathogens invade it. The signals cooperate for resistance with the activation of molecules such as salicylic acid, jasmonate, and ethylene, which are considered precursors of acquired systemic resistance (Yang, Shah, and Klessig 1997; Kachroo et al. 2004). With signal transduction, plant defense mechanisms are activated and the pathogen's development is impaired (Dangue and Jones 2001). Mandel et al. (2012) point out that these signals, such as nitric oxide, are involved in the oleic acid pathway (18:1). Not only are hormones part of the signal transduction pathway, but also reactive species and pending calcium signaling, and these can act in interaction (Tiwari 2018). In wheat culture, Glycerol induced plant hormone signal transduction through auxin, PP2C and Jasmonate Zim Domain (JAZ). The positive induction of JA by Glycerol could contribute to wheat resistance to the fungus of Oide (Li et al. 2020). For the target gene KINLRR, the positive expression with the dose of 5% glycerol was decreasing in the final moments of the evaluations, which can be deduced that more applications of this compound are necessary during the period of infection for the plant to maintain a level of resistance without so many oscillations in the expressions of these resistance genes. In the present study, the regression result, together with the gene expression data, demonstrated a tendency of the cultivar Big Naine to respond more resistantly to M. fijiensis isolate at a concentration of 5% glycerol. This result differs from responses with other plants in which the 3% dose was the most expressed resistance to plants (LI et al. 2016; LI et al. 2020; Gazolla et al. 2019). Histochemistry This is the first study on the Musa spp. x M. fijiensis interaction in response to Glycerol, as well as the first to use histochemistry to visualize defense mechanisms for the defined treatments. The detection of callose in the first days after inoculation indicates that the application of Glycerol led to a tendency to condition the plant to a state of resistance to fungal attack. Soares et al. (2022) did not identify the presence of callose in banana plants in susceptible genotypes Grande Naine and Akondro Mainty infected with Black Sigatoka, evaluated up to 21 Dai. Callose is a beta-1,3-glucan polysaccharide that is present in the cell wall of several plants, aiding in development and contributing as a defense response against biotic and abiotic factors (Chen and Kim, 2009). As a structural mechanism, it provides cell wall resistance as a form of containment to prevent pathogen penetration, thus preventing associations with each other (Stangarlin et al. 2011). As it is a pioneering work, it is believed that the use of Glycerol to control SN in bananas of the cultivar Grande Naine seems promising. However, further studies involving more applications of Glycerol prior to inoculation, as well as the use of a greater number of genes in the gene expression study, will be able to respond to the responses more robustly. The possibility of using this compound in banana plantations of cultivar GN will bring great benefits to the production chain of the crop, with great impact on the environment and human health. Conclusions Based on the studies presented in the present work, Glycerol seems to be a good inducer of resistance for banana plants when used first, at a dose of 5%, but more studies need to be conducted to verify the best time of application of the treatments, inoculation, and doses to be used. Declarations Competitive interest We declare that this work has no competitive interest. Author contribution Writing, preparation and original draft: APSN, genetic material, EPA, methodology, APSN, ARSR, FSN, ASR, LCSJ, RSSS, EPA, LSR and CFF. Data analysis: APSN, RMFS and conceptualization: TAOM. Acknowledgments Embrapa Mandioca e Fruticultura for the technical and laboratory support for developing the research; the coordination of the Improvement of Higher Education Personnel and research (CAPES and CNPq) and the Bahia State Research Support Foundation for granting the scholarships (FAPESB). Funding This research was funded by IITA/The Bill and Melinda Gates Foundation-Accelerated Breeding of Better Bananas. ID OPP1093845. Data Availability Data will be made available upon request. References Agrios. Disponivel em: https://agrio.app/library/Sigatoka-negra. Acesso em: 10/12/2023. AGRITEMPO. Agritempo: sistema de monitoramento agrometeorológico. http: //www.agritempo.gov. br/agroclima/sumario, acesso em 22/10/2021 Antolín-loovera MK, Binder A, Parniske M. (2012) Receptor kinase singnaling pathways in plant-microbe interactions. Annual Review of Phytopathology, Lawrence 50:451-473. Beveraggi A, Mourichon X, Sallé G (1995) Étude comparée des premiéres étapes de l´infection chez des bananiers sensibles et résistants infectés par Cerscospora fijiensi (Mycosphaerella fijiensis) agent responsable de la maladies des raies noires. Canadian Journal of Botany 3:1328-1337. https:// doi.org/10.1139/b95-144 Borah S, Saikia B, Bora D, Bhorali P (2022) Expression analysis of defense response genes upon Sigatoka infection in Musa paradisiaca cultivar Kachkal. Indian Journal of Genetics 217-223. https:doi.org/ 10.31742/IJGPB.82.2.11 Chanda B, Venugopal SC, Kulshrestha S, Navarre D A, Downie B, Vaillancourt L, Kachroo A, Kachroo P (2008) Glycerol-3- phosphate levels are associated with basal resistance to the hemibiotrophic fungus Colletotrichum higginsianum in Arabidopsis. Plant Physiol 147:2017-2029. https:doi.org/ 10.1104/pp.108.121335 Chandra-shekara AC, Venugopal SC, Barman SR, Kachroo A, Kachroo P (2007) Plastidial fatty acid levels regulate resistancegene-dependent defense signaling inArabidopsis. PNAS, p. 7277- 7282 DOI: 10.1073pnas.0609259104. 79 Chen XY, Kim JY (2009) Callose synthesis in higher plants. Plant signaling e Behavior. P. 489-492. https:doi.org//10.4161/psb.4.6.8359 Churchill ACL (2010) Mycosphaerella fijiensis, the black leaf streak pathogen of banana: progress towards understanding pathogen biology and detection, disease development, and the challenges of control. Molecular Plant Pathology 307-328. https:doi.org/1111/j.1364-3703.2010.00672.x Cordeiro ZJM (2015) Pragas Quarentenárias e melhoramento preventivo. Embrapa. https://www.embrapa.br/busca-de-publicacoes/-/publicacao/1074071/pragas-quarentenarias-e-melhoramento-preventivo . Acesssdo em 22 de junho de 2023 Elbagoury MM, Turoop P, Runo S, Sila DN, Madivoli ES (2022) Postharvest treatments of banana (Musa acuminata cv.’Grand Nain’, AAA) during cold and rupening temperatures with chitosan nanoparticles to alleviate chilling injury and maintain antioxindant activity. Horticulture, Enviroment, and Biotechnology. 63:677-699 Dangue JL, Jones JDG (2001) Plant pathogens and integrated defence responses to infection. Nature 826–833 Ding LN, yue-tao L, yuan-zhen W, Li T Geng R, Cao J, Wei Z, Tan XL (2022) Plant disease reaistance related signaling pathways: recent progress and Future prospects. Int. J. Mol. Sci. 23:20. https:doi.org/10.3390/ijms232416200 Favreto R, Model NS, Tonietto A (2007) Sigatoka negra, fatores de ambiente e sistemas agroflorestais em bananis do Rio Grande do Sul. Pesquisa Agropecuária Gaucha 95-104. Garruti DS, Matias ML, Facundo HVV, Silva EO, Costa JN, SILVA M AAP (2012) .Acception of banana cultivars rsistant to black Sigatoka by the consumer market of northeast brazilian region. Ciência rural 42 https:doi.org/10.1590/s0103-84782012000500030. Gasparotto L, Pereira JCR (2010) Sigatoka Negra: desafio para a bananicultura brasileira. https://ainfo.cnptia.embrapa.br/digital/bitstream/item/71420/1/sigatoka-negra-desafio-para-a-bananicultura.pdf. Acessado em: 02 de fevereiro de 2023 Gazolla LCOF, Almeida DP, Castro ISL, Castro AG, Arruda LVS, Vieira NM, Mantovani HC, Zambolim L, Caixeta ET, Mendes TAO (2019) Transcriptional regulation of glycerol-3- phosphate metabolism induces resistance to coffee rust 5. Guzmán M, Pérez-Vicente L, Carlier J, Abadie C, de Lapeyre de Belaire, L., Carreel, F.(2019) Doenças fúngicas da folhagem. http://www.biologico.sp.gov.br/publicacoes/comunicados-documentos-tecnicos/comunicados-tecnicos/doencas-fungicas-das-plantas-medicinais-aromaticas-e-condimentares-%E2%80%93-parte-aerea#:~:text=Entre%20as%20ocorr%C3%AAncias%20de%20doen%C3%A7as,se%20os%20o%C3%ADdios%20e%20m%C3%ADldios. Access 23 de janeiro de 2024 Glazebrook J (2005) Mecanismos contrastantes de defesa contra patógenos biotróficos e necrotróficos. Anu. Rev. Fitopatol. 43:205–227. Jones JDG, Dangl JL (2006) The plant immune system. Nature 323-329 Li Y, Qiu L, Liu X, Zang Q, Zhaunsun X, Fahima T, Krugman T, Sun Q, Xie C (2020) Glycerol-Induced Powdery Mildew Resistance in Wheat by Regulating Plant Fatty Acid Metabolism, Plant Hormones Cross-Talk, and Pathogenesis-Related Genes. International Journal of Molecular Sciences 21:1-19. https:doi.org/10.3390/ijms21020673 Li Y, Song N, Zhao C, Li F, Geng M, Wang Y, Liu W, Xie C, Sun Q (2016) Application of glycerol for induced Powdery Mildew resistance in Triticum aestivum L. Frontiers in Physiology 7:1-13. https:doi.org/10.3389/fphys.2016.00413. Livak KJ, Schmittgen TD (2001) Analysis of Relative Gene Expression Data Using RealTime Quantitative PCR and the 2 -ΔΔCt Method. Methods 25:402-408 Kachroo A, Venugopal SC, Lapchyk L, Falcone D, Hildebrand D, Kachroo P (2004) Oleic acid levels regulated by glycerolipid metabolism modulate defense gene expression in Arabidopsis. PNAS. V. 101. Kaur S, Samota, MK, Choudhary M, Pandey AK, Sharma A, Thakur J (2022) How do plantas defend themselves against pathogens-biochemical mecanismos and genética interventions. Physiology and Molecular Biologia of plants 28:485-504. https:doi.org/10.1007/s1298-022-01146-y Mandal MK, Chandra-Shekara AC, Jeong RD, Yu K, Zhu S, Chanda B, Navarre D, Kachroo A, Kachroo P (2012) Oleic acid dependent modulation of nitric oxide associated1 protein levels regulates nitric oxide mediated defense signaling in Arabidopsis. The Plant Cell 24:1654–1674. https:doi.org/10.1105/tpc.112.096768. Ministério da Agricultura e Pecuária. https://www.gov.br/agricultura/pt-br. Acesso em 23 de novembro de 2021. Noman A, Aqeel M, Lou Y (2019) PRRs and NB-LRRs: From sinal perception tô activation of plant innate immunity. Internacional Jornal of Molecular Sciences. 20:1-21 https:doi.org/10.3390/ijms20081882. Oliveira WDS (2020) Interação genótipo x microrganismos benéficos e indução de resistência a Fusarium oxysporum f. sp. cubense em bananeira. Dissertação, Universidade Federal do Recôncavo da Bahia. Podevin N, Krauss A, Henry I, Swwennen R, Remy S (2012) Selection and validation of reference genes for quantitative RT-PCR expression. Studies of the non-model crop Musa. Mol Breeding 30:1237-1252. https:doi.org/10.1007/s11032-012-9711-1. 81 R CORE TEAM. (2018) R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing Rodríguez HA, Rodriguez-Arango E, Morales J, Kema G, Rango RE (2016) Defense gene expression. Associated with biotrophic pgase of Mycosphaerella fijiensis M. Morelaet infection in banana. The American phytopathological Society 100:1170-1175. https:doi.org/10.1094/PDIS-08-15- 0950-RE. Senseverino W, Roma G, Simone M, Faino L, Melito S, Stupka E, Frusciante L, Ercolano M. (2010) R. PRGdb: a bioinformatics platform for plant resistance gene analysis. Nucleic Acids Research 38:814–821 https:doi.org/10.1093/nar/gkp978. SHIGENAGA, A. M.; ARGUESO, C. T. (2016) No hormone to rule them all: interactions of plant hormones during the responses of plants to pathogens. Seminars in cell e developmental biology. v. 56, p. 174-189. https:doi.org/10.1016/j.semcdb.2016.06.005. Soares JMS, Rocha AJ, Nascimento FS, Amorim VBO, Ramos A, Ferreira CF, Haddad F, Amorim EP (2022) Gene expression., histology and histochemistry inthe interaction between Musa sp. And Peseudocercospora fijiensis. Plants. V. 11. https:doi.org/10.3390/plants1151953. Stangarlin JR, Kuhn OJ, Toledo MV, Portz RL, KRF, Pascholati SF (2011) A defesa vegetal contra fitopatógenos. Scientia Agraria Paranaensis 10:1-29 https:doi.org/10.18188/sap.v10i1.5268. Stover RH (1972) Banana, plantain and abaca disease. London: Commonwealth Agricultural Bureaux 316 Tiwari S, Lata C (2018) Heavy Metal Stress, Signaling, and Tolerance Due to Plant-Associated Microbes: An Overview. Frontiers 6:9:452. https:doi.org/10.3389/fpls.2018.00452. Velame KVC (2017) validação diferencialmente expressos e estimativa da agressividade de isolados de Fusarium oxysporum f. sp. Cubense. Tese, Universidade Estadual de Feira de Santana. Xá J, Kachroo P, Nandi A, Klessig DF (2001) Uma mutação recessiva no O gene SSI2 de Arabidopsis confere expressão independente de SA e NPR1 de genes PR e resistência contra patógenos bacterianos e oomicetos. Planta 25:563–574 Yang Y, Shah J, Klesig DF (1997) Signal perception and transduction in plant defense responses. Genes Development. 11:1621-1639. https:doi.org/10.1101/gad.11.13.1621. Zhang Y, Th PLS, Imova SENM, Inan GL (2015) Application of glycerol as a foliar spray activates the defence response and enhances disease resistance of Theobroma cacao. Molecular Plant Pathology. 16:27-37. https:doi.org/10.1111/mpp.12158 Tables Tables 1 to 2 are available in the Supplementary Files section Supplementary Files Table1.docx Table2.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 11 Jun, 2024 Reviewers invited by journal 11 Jun, 2024 Editor assigned by journal 25 Apr, 2024 First submitted to journal 23 Apr, 2024 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4311892","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312962364,"identity":"5d439e3a-eda8-410e-b199-bb0a45cdfa91","order_by":0,"name":"Ana Paula da Silva Novaes","email":"","orcid":"","institution":"UFRB: Universidade Federal do Reconcavo da Bahia","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Paula da Silva","lastName":"Novaes","suffix":""},{"id":312962365,"identity":"3dad831c-be11-4afc-9387-e5e2fb33c5ea","order_by":1,"name":"Andresa Priscila de souza 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12:09:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4311892/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4311892/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59115839,"identity":"f5e002ba-2541-4a5a-b486-8de4addfa1b8","added_by":"auto","created_at":"2024-06-26 14:01:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":897826,"visible":true,"origin":"","legend":"\u003cp\u003ea) Experiment mounted on DIC in a greenhouse; b) Method of applying the glycerol solution via spray until the leaves run off; c) Banana leaf cv Grande Naine – red arrow - beginning of \u003cem\u003eM. fijiensis \u003c/em\u003elesion. Photo: Ana Paula da Silva Novaes, 2023\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/af263440f3d338aa42e7a416.png"},{"id":59116605,"identity":"bcd9e07d-5a57-49b4-a54b-a8cd48469abe","added_by":"auto","created_at":"2024-06-26 14:09:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":132121,"visible":true,"origin":"","legend":"\u003cp\u003eGanry, Mayer (1972), and Stover (1989) diagrammatic scale\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/7a01170758120d2e037ad7b4.png"},{"id":59115837,"identity":"977f104e-f032-443d-bb22-6554d71adfad","added_by":"auto","created_at":"2024-06-26 14:01:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":870819,"visible":true,"origin":"","legend":"\u003cp\u003ea) Disease graphs of regression plots (ID) and b) of the area under the disease progress curve (AACPD) of Big Naine banana plants, infected with black Sigatoka\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/0914351532c6701348ab5c55.png"},{"id":59116606,"identity":"210ce8dd-f82d-4dba-9a8f-b2ee4f79d4d1","added_by":"auto","created_at":"2024-06-26 14:09:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11006576,"visible":true,"origin":"","legend":"\u003cp\u003e(a to p)shows lighttening and staining of fungal structures with trypan blue on Big Naine banana leaves prior to inoculation up to 5 DAI. Prior to inoculation, time zero (Fig. 4: a-e), no fungal structures were found.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 4\u003c/strong\u003e(a to p):\u003cstrong\u003e \u003c/strong\u003eLightening and staining of fungal structures in Big Naine banana leaves before inoculation up to 5 DAI. a, f and l- control; b, g, m- plant inoculated without glycerol; c, h and n- plants inoculated and treated with 3% glycerol; d, i and o- plants inoculated and treated with 5% glycerol; e, j and p- plants inoculated and treated with 8% glycerol. DAI= days after inoculation, HAI= hours after inoculation, h- hypha, c= conidium, a = ungerminated ascospore, and= stomata. Bars: 200 µm. Cruz das Almas-BA, 2023\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/fe90baf28523effad7d83e60.png"},{"id":59116607,"identity":"e3778e6f-36bf-4eff-b064-61ecefb661c2","added_by":"auto","created_at":"2024-06-26 14:09:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8455604,"visible":true,"origin":"","legend":"\u003cp\u003eLightening and staining of fungal structures in leaves of Grande Naine banana with 15, 30 and 60 DAI. A, F and L- control; b, g, m- plant inoculated without Glycerol; c, h and n- plants inoculated and treated with 3% glycerol; d, i and o- plants inoculated and treated with 5% glycerol; e, j and p- plants inoculated and treated with 8% glycerol. DAI= days after inoculation, h- Hypha, c= conidium, co= conidiophore, a = ascospores, st= stomatopodium, e= stomata. Bars: 200 µm\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/ca48e1425a47e1e4d51a7dc2.png"},{"id":59115842,"identity":"a7ec2d14-53b5-4bab-b815-e2fe2a364b16","added_by":"auto","created_at":"2024-06-26 14:01:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":590959,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression profile of genes in Big Naine samples treated with Glycerol and inoculated with \u003cem\u003eM. fijiensis isolate in\u003c/em\u003egration to uninoculated control at time 0h. (a) STRANS (Transduction signal) and (b) KINLRR (Kinase, LRR domains, protein-protein interaction). Quantifications were normalized using the 25S and L2MU reference genes. DAI: Days after inoculation\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/aa33883c272afe6f8a6dde14.png"},{"id":59115841,"identity":"dfe41f53-5e7c-42d1-a018-09672c65ca2c","added_by":"auto","created_at":"2024-06-26 14:01:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":128369,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmaps of the relative expression profiles of STRANS (Signal transduction), KINLRR (Kinase, LRR domains, protein-protein interaction in Large Naine samples, at six collection times after glycerol treatment relative to the uninoculated control at time 0h. Quantifications were normalized using the 25S and L2MU reference genes\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/4a39780ae4b6cd9c45e079e9.png"},{"id":59115844,"identity":"9dd9af29-bc72-4a7d-ab77-bdec0c545279","added_by":"auto","created_at":"2024-06-26 14:01:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":9001066,"visible":true,"origin":"","legend":"\u003cp\u003eCross sections of Grand Naine banana leaves in the control treatments (a, f, l), inoculation + water (b, g, m), 3% glycerol (c, h, n), 5% glycerol (d, i, o), 8% glycerol (e, j, p). The images refer to T0, 72 HAI and 5 DAI. Histochemical test for detection of callose. Ca: callose, HAI: hours after inoculation, HENCE: days after inoculation. Bar: 200 mm. Cruz das Almas-BA, 2023\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/dde75336a744fd09817575e7.png"},{"id":59115847,"identity":"d348c25f-e1dc-4f04-8766-4e9b558ef8a4","added_by":"auto","created_at":"2024-06-26 14:01:32","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":8947481,"visible":true,"origin":"","legend":"\u003cp\u003eCross sections of Grand Naine banana leaves in the control treatments (a, f, l), inoculation + water (b, g, m), 3% glycerol (c, h, n), 5% glycerol (d, i, o), 8% glycerol (e, j, p). The images refer to the 15, 30 and 60 DAI. Histochemical test for callose detection; bar: 200 mm. Cruz das Almas-BA, 2023\u003c/p\u003e","description":"","filename":"figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/52702888e30cb6e0070920f1.png"},{"id":59117484,"identity":"7f31c7b6-6d39-4fc4-ac07-c82d2256500a","added_by":"auto","created_at":"2024-06-26 14:18:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":83744660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/c0bc5b21-bb8d-4643-bc3d-0f6c07a474c9.pdf"},{"id":59116604,"identity":"623071b1-e73d-42a3-9b40-cd90216c7e2c","added_by":"auto","created_at":"2024-06-26 14:09:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14047,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/74ea070d08d89f6bbc73fc9d.docx"},{"id":59115836,"identity":"8bb1a65a-7d58-43a7-8b02-daabc15734e3","added_by":"auto","created_at":"2024-06-26 14:01:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15214,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4311892/v1/4322a9e4d8910be74fd740de.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eActa Physiologiae Plantarum Histochemistry and Gene Expression Using Glycerol in the Pathosystem Musa X Mycosphaerella Fijiensis\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe first report of Sigatoka Negra (SN) in Brazil was in 1998, in production regions in the state of Amazonas (Cordeiro 2015). This disease is among the main fungal diseases that attack the banana crop. The symptoms manifest themselves in the leaves of plants through dark streaks, causing their leaf area to be impaired and preventing the photosynthetic process from being conducted. The fruits produced do not have market quality due to premature ripening, which makes them unsuitable for the market, implying financial losses for producers (Gasparotto and Pereira 2010).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSN has been advancing in the national territory, recently arriving in the State of Bahia in 2018. In addition to Bahia, SN is located in the states of Acre, Amapá, Amazonas, Espírito Santo, Goiás, Maranhão, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Pará, Paraná, Rio de Janeiro, Rio Grande do Sul, Rondônia, Roraima, Santa Catarina, São Paulo and Tocantins (MAPA 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe main means of controlling the disease is through systematic spraying, which damages both man and animals, as well as the environment, with the planting of resistant cultivars being the most environmentally correct measure. However, resistant varieties are not the main ones planted in Brazil and in the world, requiring other control measures that are more feasible and applicable in \u0026nbsp; short term (Garruti et al. 2012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cultivar Grande Naine (AAA) belongs to the species \u003cem\u003eMusa acuminata\u003c/em\u003e, Cavendish subgroup, grown commercially in tropical and subtropical regions (Elbagoury et al. 2022). Grande Naine suffers losses during its cycle, which is affected by severe diseases that limit its development, such as SN, considered one of the main fungal diseases that affect the banana crop.\u003c/p\u003e\n\u003cp\u003eSN caused by the hemibiotrophic fungus in its asexual stage \u003cem\u003eM. fijiensis\u003c/em\u003e (ascospores) and in its axesuous form \u003cem\u003eP. fijiensis\u003c/em\u003e (conidia) is a polycyclic disease that infects the banana tree several times within a single development cycle (Guzmán et al. 2019).\u003c/p\u003e\n\u003cp\u003eAccording to Pereira and Gasparotto (2010), 26 applications of systemic fungicides or up to 52 applications of protective fungicides are necessary for effective control of the disease in banana plantations. Unfortunately, the small producer, who is the most representative in the country, most of the time, does not have the conditions to pay the costs of the applications and ends up having losses with the disease. In addition, using the fungicide may be feasible for large producers who have resources, but it can harm the health of humans who consume the fruit and the quality of the environment. Thus, developing sustainable, easily applicable and low-cost practices to control NS are paramount.\u003c/p\u003e\n\u003cp\u003eGlycerol is an economically viable and sustainable alternative that has shown promising results when used in plants for disease control. When applied exogenously to plants, their metabolism produces the compound glycerol-3-phosphate (G3P). This molecule consequently leads to decreased production of oleic acid (18:1- monosaturated fatty acid-omega), causing the induction of resistance in plants against pathogens through the production of salicylic acid (AS) (Zhang et al. 2015; Gao et al. 2011).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe responses of hormones, salicylic acid and jasmonate, are most often antagonistic in plants. In the case of glycerol degradation, the decrease in oleium acid production (18:1) simultaneously increases responses mediated by (AS), inhibiting Jasmonate-induced defense responses and consequently increasing plant resistance against biotrophic fungi (Gao et al. 2011). As \u003cem\u003eM. fijiensis\u003c/em\u003e is a hemibiotrophic fungus (Churchill et al. 2010) with a biotrophic phase, it is considered that the application of Glycerol can successfully produce compounds that act to increase the resistance of bananas to SN.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore, the present study aims to evaluate the efficiency of Glycerol in controlling the fungus \u003cem\u003eM. fijensis in\u003c/em\u003e the cultivar Grande Naine de bananeira, susceptible to NS.\u0026nbsp;\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003ePlant material\u003c/p\u003e\n\u003cp\u003eThe experiment was installed at the Embrapa Cassava and Fruit Farming Unit, located in Cruz das Almas (12◦40\u0026prime;19 \u0026prime; \u0026prime;S, 39◦06\u0026prime;22 \u0026prime; \u0026prime;W \u0026apos;, altitude 220 m above sea level), Bahia, Brazil. The climate is classified as tropical hot, humid, Aw an Am, according to the K\u0026ouml;ppen classification, with an average annual temperature of 24.5 ◦C, relative humidity of 80%, and average annual rainfall of 1250 mm (AGRITEMPO, 2018).\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted in a greenhouse, with 90 banana plants of the Cavendish subgroup, cultivar Grande Naine. The experimental design was completely randomized, with 15 plots, 3 replicates, 6 plants per plot, and 5 treatments: control, water + inoculation, 3, 5, and 8% glycerol. The glycerol solution was sprayed onto the leaves until drained. This application was made every 2 days with an interval of 2 days, for 6 days. Leaves were collected before treatment and before inoculation (time zero) and at times 72 hours after inoculation (HAI), 5, 15, 30, and 60 days after inoculation (DPI). Samples for molecular analysis were stored in aluminum foil envelopes, frozen with liquid nitrogen in a Styrofoam box until placed in the ultrafreezer -80\u0026deg;C. The assembled experiment is shown in Figure 1: A and B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 1\u003c/strong\u003e a) Experiment mounted on DIC in a greenhouse; b) Method of applying the glycerol solution via spray until the leaves run off; c) Banana leaf cv Grande Naine \u0026ndash; red arrow - beginning of \u003cem\u003eM. fijiensis\u0026nbsp;\u003c/em\u003elesion. Photo: Ana Paula da Silva Novaes, 2023\u003c/p\u003e\n\u003cp\u003eFor the histology study, samples were diced and stored in karnovsky fixative and FAA in a 2 mL microtube. After each collection, the samples were placed in the vacuum desiccator equipment for 10 minutes and subsequently dehydrated in increasing series of 30-100% ethanol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.2. Inoculum preparation\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe isolate was prepared in the Laboratory of Phytopathology of Embrapa Cassava and Fruticulture. Initially, peaks were performed in solid medium (BDA), remaining until they were transferred to v8 medium for 15 days. After colony growth, a soft bristle toothbrush was scraped on all plates, adding 10 mL of distilled water and counting conidia in a Neubauer chamber to obtain the suspension\u003csup\u003e104\u003c/sup\u003e of the isolate. After 2 days of the last treatment, the plants were inoculated on the abaxial face of the leaves with the aid of a spray bottle until the runoff.\u003c/p\u003e\n\u003cp\u003e2.3. Symptom assessment\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwo plants of each plot were evaluated, and of these two plants, 2 leaves were evaluated, giving scores of 0-5, based on the diagrammatic scale of Ganry, Mayer (1972), and Stover (1989) (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2\u003c/strong\u003e Ganry, Mayer (1972), and Stover (1989) diagrammatic scale\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The scores obtained at 07-day intervals were transformed into a disease severity index ID (%), based on the formula provided by Mckinney (1923), represented below:\u003c/p\u003e\n\u003cp\u003eID (%) = \u0026sum;(disease score x number of plants with a certain score of disease) x 100/number of plants evaluated by plot x highest score of the scale)\u003c/p\u003e\n\u003cp\u003eThe mean ID was estimated for each treatment based on the evaluations performed every 07 days and the number of leaves evaluated.\u003c/p\u003e\n\u003cp\u003eThe area under the disease progression curve (AACPD) was estimated according to the formula proposed by Mandden et al. (2007), where \u003cem\u003en\u003c/em\u003e is the number of evaluations, \u003cem\u003eID\u003c/em\u003e is the disease severity index and (\u003cem\u003eT1-T2\u003c/em\u003e), the time interval between two consecutive evaluations, as can be seen in the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe AACPD was standardized by the number of assessments performed every 07 days until the end of the assessments.\u003c/p\u003e\n\u003cp\u003e2.4 Data analysis\u003c/p\u003e\n\u003cp\u003eIn order to classify the treatments as resistant to black Sigatoka, the Scott and Knott (1974) cluster test was used at 5% significance, using the \u0026quot;ExpDess\u0026quot; package of the statistical program R (CORE DEVELOPMENT TEAM, 2018). A disease severity index and the area under the disease progress curve were estimated.\u003c/p\u003e\n\u003cp\u003eFor the analysis of the relative quantification of gene expression, Microsoft Excel was used using the 2 -\u0026Delta;\u0026Delta;CT method proposed by Livak and Schmittgen, (2001). Heatmaps with corresponding dendrograms were based on a complete binding cluster by the Euclidean distance method using the heatmap function. 2 of the \u0026quot;plots\u0026quot; and \u0026quot;dev tools\u0026quot; packages were implemented in R (R CORE DEVELOPMENT TEAM, 2018).\u003c/p\u003e\n\u003cp\u003e2.5. Histochemistry\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor histochemical analysis, the 1 cm sections were placed in karnovsky and FAA fixative solution, subjected to vacuum for 10 min, stored in fixative solution for 8 days, dehydrated in increasing series of ethanol (30-100%) 1h each. Subsequently, infiltration was performed using part of ethanol+historesin. Polymerization was performed with Historesin Kit (hydroxyethyl methacrylate; Leica Heidelberg, Germany). The cuts were made in a rotating microtome (8mm), placed on slides and stained with dye according to the objective.\u003c/p\u003e\n\u003cp\u003e2.6. Clarification and coloring of leaves\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFAA-fixed samples were soaked in Potassium Hydroxide (KOH) solution (10%) for 72h, and then washed with distilled water and placed in Hydrochloric Acid (HCL) for 30 minutes. Then samples were stained with 0.05% trypan blue in solution (2:1:1, lactic acid: Glycerol:water) for 2h. Immediately after dye disposal, the leaf fragments were immersed in lactoglycerol solution (2:1:1, lactic acid: Glycerol:water) and microphotographed under an objective light microscope with a range of 40x.\u003c/p\u003e\n\u003cp\u003e2.7. Molecular analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.7.1 Total RNA extraction\u003c/p\u003e\n\u003cp\u003eFor total RNA extraction, \u0026apos;Big Naine\u0026apos; plant leaves were collected before glycerol treatment, time 0 (before inoculation), 72 hours post inoculation (HAI), 5, 15, 30, and 60 days post-inoculation. During collection, the samples were stored in liquid nitrogen and then stored in an utrafreezer (-80 \u0026deg;C) until extraction.\u003c/p\u003e\n\u003cp\u003eIn the extraction process, the methodology of Ferreira et al. (2019) was used with modifications, where liquid nitrogen was used in the maceration of the samples. To check RNA integrity and concentration after extraction, a 5 \u0026micro;l aliquot of the samples plus 2 \u0026micro;l of GelRed\u0026reg; were observed via 1% agarose gel electrophoresis in 1x TAE buffer (Tris; glacial acetic acid; Milli-Q\u0026reg; water). The extracted RNA was stored in ultrafreezer -80\u0026deg;C \u0026nbsp; \u0026nbsp; \u0026nbsp;for further use.\u003c/p\u003e\n\u003cp\u003e2.7.2 DNase Treatment\u003c/p\u003e\n\u003cp\u003eThe extracted total RNA was treated with DNase TURBOFREE (Ambion\u0026reg;), according to the manufacturer\u0026apos;s recommended protocol: in 15\u0026micro;l of total RNA from each sample was added 1\u0026micro;l of DNAse free water, 1\u0026micro;l of Inhibitor (RNase out), 2\u0026micro;l of Buffer (10x turbo DNAse buffer) and 1\u0026micro;l DNAse (turbo DNase). The reaction was incubated in Veriti thermocycler (Applied Biosystems\u0026copy;) at 37\u0026deg;C for 25 min. Then, 2 \u0026micro;l of DNase Inactivation was added, and the sample was incubated for 2 min. at room temperature. Subsequently centrifuged for 1.5 minutes at 10000 rpm, the supernatant transferred to new tubes was stored in ultrafreezer at -80\u0026deg;C. The quality of the treated RNA was conferred on 1% agarose gel with 1 x TAE (Tris; glacial acetic acid; MQ water) and 3 \u0026micro;l of each sample was used. The genes and primers used in the study are shown in Table 1.\u003c/p\u003e\n\u003cp\u003eTable 1- Primers used to evaluate gene expression in \u003cem\u003eMusa\u0026nbsp;\u003c/em\u003espp. X \u003cem\u003eMycosphaerella fijiensis\u003c/em\u003e. Target genes and their respective functions: Signal transduction (STRANS) - signal transduction; Kinase, LRR domains, protein-protein interaction (KINLRR) - recognition, signaling, and references.\u003c/p\u003e\n\u003cp\u003e2.7.3. Synthesis of cDNA\u003c/p\u003e\n\u003cp\u003eThe cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit to obtain the final volume of 20 \u0026micro;l according to the protocol: 5 \u0026micro;l of the treated RNA, plus 1 \u0026micro;l of 20x enzyme mix, 10 \u0026micro;l of 2x RT Buffer and 4 \u0026micro;l of Nuclease Free water. Subsequently the reaction was incubated in the Veriti thermocycler (Applied Biosystems \u0026copy;) for 60 minutes at 37\u0026deg;C + 97\u0026deg;C for 5 min + hold 4\u0026deg;C. The cDNAs were stored at -80\u0026deg;C. To verify the viability of the cDNA, conventional PCR with beta-tubulin primer was performed (PODEVIN et al., 2012).\u003c/p\u003e"},{"header":"RESULTS ","content":"\u003cp\u003e.1. Symptom assessment\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe presence of symptoms and confirmation of the presence of the fungus \u003cem\u003eM. fijiensis\u0026nbsp;\u003c/em\u003ein plants occurred at 25 DAI. Initial symptoms were light brown color scores on the abaxial face of the leaves. Symptoms in the form of stretch marks began to appear from 55 DAI in plants treated with water and 8% glycerol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt first, treatments 3, 5, and 8% had statistically higher ID values than the inoculated treatment treated with (Table 2).\u003c/p\u003e\n\u003cp\u003eTable 2- Disease severity index (ID) and area under the disease progress curve (AACPD) of glycerol-treated plants at concentrations of 3, 5, and 8%, water-treated plants, and control. Cruz das Almas, 2023.\u003c/p\u003e\n\u003cp\u003eRegression analysis to assess the disease index (ID) and the area under the disease progression curve (AACPD) indicate that there was a drop in severity at 5% glycerol and greater severity in treatment with 8% glycerol (Fig. 3 a and b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 3\u003c/strong\u003e a) Disease graphs of regression plots (ID) and b) of the area under the disease progress curve (AACPD) of Big Naine banana plants, infected with black Sigatoka\u003c/p\u003e\n\u003cp\u003eHistological Analysis\u003c/p\u003e\n\u003cp\u003e3.2.1. Clarification and coloring of leaves\u003c/p\u003e\n\u003cp\u003eFig. 4 (a to p)\u0026nbsp;shows lighttening and staining of fungal structures with trypan blue on Big Naine banana leaves prior to inoculation up to 5 DAI.\u0026nbsp;Prior to inoculation, time zero (Fig. 4: \u0026nbsp;a-e), no fungal structures were found.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u003cstrong\u003eFig. 4\u003c/strong\u003e (a to p):Lightening and staining of fungal structures in Big Naine banana leaves before inoculation up to 5 DAI. a, f and l- control; b, g, m- plant inoculated without glycerol; c, h and n- plants inoculated and treated with 3% glycerol; d, i and o- plants inoculated and treated with 5% glycerol; e, j and p- plants inoculated and treated with 8% glycerol. DAI= days after inoculation, HAI= hours after inoculation, h- hypha, c= conidium, a = ungerminated ascospore, and= stomata. Bars: 200 µm. Cruz das Almas-BA, 2023\u003c/p\u003e\n\u003cp\u003eSamples collected at 72 HAI show fungal structures in the treatment with water (Fig. 4 g) with the presence of hypha and in plants inoculated and treated with 8% glycerol (Fig. 4 j), with the presence of conidium, spore of asexual reproduction. Thus, it is assumed that in the first five days after inoculation, the plant was being infected in the parenchyma tissue with penetration through the stomata (Beveraggi et al., 1995).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo fungal structures were found at 5 DAI in the control treatments (Fig.4 l) and 3% glycerol (Fig. 4 n). In the treatment with water, hyphae continued to appear (Figure 4 M); conidium colonizing stomata in 5% glycerol (Fig. 4 o) and ascospore, ungerminated sex spore, in 8% glycerol (Fig. 4 p). Ascospores produced since the age of 5 days are considered the main structure and can be disseminated to distant areas by the wind. (Favreto; Model and Tonietto 2007).\u003c/p\u003e\n\u003cp\u003eThe lightening and staining of fungal structures in leaves of Grande Naine banana with 15, 30, and 60 DAI are shown in Figure 5 (a to p). At 15 DAI, the disease advanced in plants treated with water (Fig. 5 b), with appearance of ascospore, hyphae around the stomata in the 5% glycerol treatment (Figure 5 d) and conidiophores (Fig. 5 e). Control and tre atment 3% continued without structures.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u003cstrong\u003eFig. 5\u003c/strong\u003e Lightening and staining of fungal structures in leaves of Grande Naine banana with 15, 30 and 60 DAI. A, F and L- control; b, g, m- plant inoculated without Glycerol; c, h and n- plants inoculated and treated with 3% glycerol; d, i and o- plants inoculated and treated with 5% glycerol; e, j and p- plants inoculated and treated with 8% glycerol. DAI= days after inoculation, h- Hypha, c= conidium, co= conidiophore, a = ascospores, st= stomatopodium, e= stomata. Bars: 200 µm\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt 30 days structures were found in all treatments except control. Hyphae colonizing stomata in the treatments with water and 5% glycerol (Figures 5 G and I), conidiophores in 3% glycerol (Fig 5 h), and ascospore germinated in 8% glycerol (Fig. 5-j).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt 60 DAI, hypha colonizing stomata were visualized in the water treatment (Fig. 5 m), mycelium in 3% glycerol (Fig. 5 n), ungerminated ascospore in 5% (Figure 3 O), and the presence of stomatopodium in 8% treatment (Figure 5 p). Until the end of the evaluations, no fungal structures were found in the control treatment.\u003c/p\u003e\n\u003cp\u003e3.3. Gene Expression Analysis\u003c/p\u003e\n\u003cp\u003eTo verify the interaction of glycerol-treated Big Naine plants in interaction with black Sigatoka in molecular analysis, two genes involved in plant-pathogen interaction with recognition and signaling function (KINLRR, Kinase, LRR domains, protein-protein interaction) and signal transduction (STRANS, Signal transduction) (Velame, 2017) were analyzed via RT-qPCR, according to (Figure 6). Expression was normalized by the \u003cem\u003eMusa\u003c/em\u003e spp., 25S, and L2MU reference genes (PODEVIN et al., 2012).\u003c/p\u003e\n\u003cp\u003eAccording to Figure 6 (a and b), genes were expressed differently between treatments and times evaluated. The highest expression of the genes in the final time (60 days) occurred with 5% glycerol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig.6\u003c/strong\u003e Relative expression profile of genes in Big Naine samples treated with Glycerol and inoculated with \u003cem\u003eM. fijiensis isolate in\u003c/em\u003e gration to uninoculated control at time 0h. (a) STRANS (Transduction signal) and (b) KINLRR (Kinase, LRR domains, protein-protein interaction). Quantifications were normalized using the 25S and L2MU reference genes. DAI: Days after inoculation\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The overall expression of the two selected genes is shown in the heat map (Fig. 7). The analyses show the hierarchical grouping of the cultivar Big Naine according to the expression profiles of the two genes analyzed. The dendrogram lines represent the collection times, and the columns represent the treatments, demonstrating that the responses were different between the genes in relation to the treatments and the evaluated times.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the treatments, 3% glycerol was the one that obtained the lowest expression of both genes, while the treatment with 5% glycerol showed the highest expression of both target genes, especially at 60 days. This result corroborates the ID and AACPD analyses (Figure 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 7\u003c/strong\u003e Heatmaps of the relative expression profiles of STRANS (Signal transduction), KINLRR (Kinase, LRR domains, protein-protein interaction in Large Naine samples, at six collection times after glycerol treatment relative to the uninoculated control at time 0h. Quantifications were normalized using the 25S and L2MU reference genes\u003c/p\u003e\n\u003cp\u003eHistochemical Analysis\u003c/p\u003e\n\u003cp\u003eFigure 8 (a to p) shows images of callose detection via fluorescence after aniline blue + lugol staining from time zero to 5 DAI. Production of this compound was visualized from 72 HAI in inoculated plants. At 5 DAI, callose deposition was detected in the palisadedic and lacunose parenchyma in the 5% glycerol treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 8\u0026nbsp;\u003c/strong\u003eCross sections of Grand Naine banana leaves in the control treatments (a, f, l), inoculation + water (b, g, m), 3% glycerol (c, h, n), 5% glycerol (d, i, o), 8% glycerol (e, j, p). The images refer to T0, 72 HAI and 5 DAI. Histochemical test for detection of callose. Ca: callose, HAI: hours after inoculation, HENCE: days after inoculation. Bar: 200 mm. Cruz das Almas-BA, 2023\u003c/p\u003e\n\u003cp\u003eThe cross sections of leaves of Grande Naine banana from 15 to 60 days are shown in Figure 9 (a top).\u0026nbsp;At 15 DAI, no defense response was visualized in plants not treated with Glycerol. At 30 and 60 DAI, all treatments, including the control, detected a defense response. Although the evaluation ofcallose is qualitative, it is possible to identify greater detection (fluorescent parts) in treatments 5 and 8% of Glycerol at 60 DAI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 9\u003c/strong\u003e Cross sections of Grand Naine banana leaves in the control treatments (a, f, l), inoculation + water (b, g, m), 3% glycerol (c, h, n), 5% glycerol (d, i, o), 8% glycerol (e, j, p). The images refer to the 15, 30 and 60 DAI. Histochemical test for callose detection; bar: 200 mm. Cruz das Almas-BA, 2023\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEvaluation of symptoms and histological analysis\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;The characteristic spots of Black Sigatoka are yellow in color, which, over time and the evolution of the pathogen, become brown, grow, and aglutinate, inducing tissue death. The spots in proportion that increase become necrotic, impairing plant photosynthesis (Borah et al. 2022).\u003c/p\u003e\n\u003cp\u003eAscospores are produced in necrotic tissues, completing their cycle in the necrotrophic phase (Favreto; Model; Tonietto 2007), however, this statement goes against what was observed in our study. The presence of ascospores in the treatments with water and 8% glycerol occurred in non-necrotic leaves. This type of result could be expected in plants treated with 8% glycerol because the high level of this compound, which consequently has a higher amount of carbon, was acting more as an energy source for the fungus (Chanda et al. 2008).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eM. fijiensis\u003c/em\u003e, as a hemibiotrophic fungus, has a biotrophic cycle that lasts 3-4 weeks without causing death to the plant (AGRIOS 2019), which explains the way the symptoms occurred during the 5 weeks of evaluation, not reaching the necrosis phase. \u0026nbsp;Treatment with 8% glycerol showed ascospores and greater severity of symptoms. This result again suggests Glycerol possibly acting as a carbon source for the fungus, corroborating the results obtained for ID and AACPD analysis.\u003c/p\u003e\n\u003cp\u003eZhang et al. (2015) verified cell death in plants treated with higher doses of Glycerol in the cocoa crop treated with doses above 300 mM, where chlorosis and cell death were visualized at the tips of the leaves. In banana, cultivar Grande Naine, no significant phenotypic differences were seen in plants treated with 3, 5 and 8% Glycerol compared to untreated plants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom the clarification, it was possible to visualize several stages of development of black Sigatoka, from hyphae to spores of sexual reproduction in germinated and non-germinated forms. It has been scientifically proven that the higher the density of stomata, where the fungus \u003cem\u003eM. fijiensis\u003c/em\u003e infects the plant, the greater the infection. Grande Naine, a susceptible genotype, has a high density of stomata, which facilitates the penetration of the fungus (SOARES et al., 2022). The plant, with physical and chemical defense responses, uses mechanisms to try to defend itself from the invader. The cell wall restricts the fungal germ tube and biochemical defense is triggered by ITP and ITE by the production of reactive oxygen species and others. Secondary metabolites also act as molecules in the development of acquired systemic resistance (SAR) (Kaur et al. 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlants treated with 8% Glycerol showed similar results to water-treated plants, with some fungal structures in the first hours of evaluation. In the treatment with 5% glycerol, the first visualization of conidia was at 5 DAI, while in the same period the treatment with 8% glycerol presented ungerminated ascospores. Initially, treatment with 3% glycerol demonstrated a higher level of resistance, developing structures from 15 DAI. Li et al. (2016) identified that the higher the dose of Glycerol (1-4%), the greater the resistance developed by wheat plants against the oidium, but considered the best dose at 3% because 4% glycerol presents cell death at the tips of the leaves.\u003c/p\u003e\n\u003cp\u003eLi et al. (2020) found spore germination in ~90% of water-treated plants, suggesting that Glycerol induced resistance against the wheat fungal pathogen powdery mildew. This may be related to the present study in which structures were visualized in the control treatment from the first days and in the treatment with a higher dose of Glycerol, 8%.\u003c/p\u003e\n\u003cp\u003eFor wheat, different times of glycerol application were evaluated, before and after inoculation, and it was proven that the best time to start the treatment is 1-2 days before inoculation. Plants treated with Glycerol were highly resistant, while those treated with water only were susceptible (Li et al. 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGene expression\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The glycerol-mediated induction of defense in plants is related to the increase in glycerol-3-phosphate (G3P) and the decrease in oleic acid (18:1). Glycerol can be used in plants to induce resistance against pathogens, but has even been considered toxic in plants when used in high concentrations (Zhang et al. 2015). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study of gene expression in banana plants infected by the \u003cem\u003eM. fijiensis\u003c/em\u003e isolate contributes to the identification of the beginning of plant defense, since visually this was not possible due to the initial phase being biotrophic and not presenting symptoms initially (Rodríguez et al. 2016). In the study performed here, it was possible to identify that the signaling gene was expressed early on in the infection.\u003c/p\u003e\n\u003cp\u003eIn a study with resistant and susceptible varieties, Rodríguez et al. (2016) were able to identify several defense genes in the resistant genotype, Calcutta 4, and different induction in the susceptible one, Williams. In Calcutta 4, induction was early, as in the present study; in susceptible plants, there was gene induction at the beginning of infection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; A transduction signal developed by plants after glycerol treatment in response to the pathogen may be salicylic acid, an important hormone for acquired systemic resistance (Xá et al. 2001) and oleic acid \u0026nbsp;(Mendel et al.2012); its response is dose-dependent (Zhang et al. 2015). This explains the induction of the STRANS and KINLRR genes in banana plants, the higher the glycerol dose, the higher the expression of these genes. Signal transduction is activated when the plant recognizes the presence of the pathogen. When recognizing, it releases\u003csup\u003eCa2\u003c/sup\u003e+, considered the main mediator against plant stress (Ding et al. 2022).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The KINLRR gene is a leucine-rich kinase gene. This gene acts as a receptor that detects abnormalities created by biotic and abiotic factors in plants (Senseverino et al. 2010; Antolín-Loovera, Binder and parniske 2012). Leucine-rich proteins are effector-triggered immunity (ETI). The ETI is activated after the pathogen manages to overcome the host's first line of defense, acting as a second line of defense (Noman, Aqeel, Lou 2019).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Signal transduction in plants involves several lines of plant defense to prevent the development of the pathogen, where the responses are the creation of reactive oxygen species (ROS), pathogenesis-related genes, callose deposition, salicylic acid, and mitogen-activated protein kinase (MAPK) signaling, among others (Liu and Lam 2019). Hormones are molecules that act on the plant in various physiological processes, including plant defense. Among the main ones, jasmonic acid, salicylic acid, ethylene, abscisic acid, auxin, gibberellin stand out (Shigenaga and Argueso 2016).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Treatment with 5% glycerol had long-term positive responses for the two genes tested. STRANS gene expression in this treatment remained high at 60 DAI, while for KINLRR, it decreased compared to 30 DAI. Comparing treatments 5 and 8% glycerol, the treatment that had the most gene expression, 5% glycerol, maintained this result until the end of the evaluations. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe way Glycerol acts in banana plants still needs several studies to prove, but the increase in G3P and the decrease in oleic acid occurs through the acylation between the two in plasmids, the elevation of G3P contributes to signaling through salicylic acid (AS) and jasmonic acid (AJ) (Kachroo et al. 2004). Ethylene is also part of signaling, and each hormone activates a pathway individually, together, or antagonistically, but each response depends on the plant x pathogen interaction (Chandra-shekara et al. 2007). Related to plant defense against pathogens, induction against necrotrophic microorganisms occurs through JA, the hormone responsible for defense against necrotrophic pathogens (Matric et al. 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLRR is an effector recognition gene that causes virulence proteins to become avirulent, it has a second line of defense known as ETI, which was previously called gene-to-gene or race-specific resistance (Jones and Dangl 2006). LRR induces resistance in plants attacked by biotrophic or hemibiotrophic microorganisms, that is, organisms that allow the plant to remain alive for at least one period (Glazebrook, 2005), such as the case of the fungus \u003cem\u003eM. fijiensis\u003c/em\u003e, which is a hemibiotrophic fungus.\u003c/p\u003e\n\u003cp\u003eThe KINLRR gene was expressed during the first hour of evaluation during the interaction of \u003cem\u003eMusa\u003c/em\u003e spp. x \u003cem\u003eM. fijiensis\u003c/em\u003e, demonstrating that the expression of this gene was rapid in the detection of stress, recognizing the presence of the pathogen in only 1 HAI and induced resistance in most of the evaluated times (Oliveira 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSignal transduction acts on the plant when pathogens invade it. The signals cooperate for resistance with the activation of molecules such as salicylic acid, jasmonate, and ethylene, which are considered precursors of acquired systemic resistance (Yang, Shah, and Klessig 1997; Kachroo et al. 2004). With signal transduction, plant defense mechanisms are activated and the pathogen's development is impaired (Dangue and Jones 2001). Mandel et al. (2012) point out that these signals, such as nitric oxide, are involved in the oleic acid pathway (18:1). Not only are hormones part of the signal transduction pathway, but also reactive species and pending calcium signaling, and these can act in interaction (Tiwari 2018).\u003c/p\u003e\n\u003cp\u003eIn wheat culture, Glycerol induced plant hormone signal transduction through auxin, PP2C and Jasmonate Zim Domain (JAZ). The positive induction of JA by Glycerol could contribute to wheat resistance to the fungus of Oide (Li et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;For the target gene KINLRR, the positive expression with the dose of 5% glycerol was decreasing in the final moments of the evaluations, which can be deduced that more applications of this compound are necessary during the period of infection for the plant to maintain a level of resistance without so many oscillations in the expressions of these resistance genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, the regression result, together with the gene expression data, demonstrated a tendency of the cultivar Big Naine to respond more resistantly to \u003cem\u003eM. fijiensis\u003c/em\u003e isolate at a concentration of 5% glycerol. This result differs from responses with other plants in which the 3% dose was the most expressed resistance to plants (LI et al. 2016; LI et al. 2020; Gazolla et al. 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistochemistry\u003c/p\u003e\n\u003cp\u003eThis is the first study on the \u003cem\u003eMusa\u0026nbsp;\u003c/em\u003espp. \u003cem\u003ex M. fijiensis\u003c/em\u003e interaction in response to Glycerol, as well as the first to use histochemistry to visualize defense mechanisms for the defined treatments. The detection of callose in the first days after inoculation indicates that the application of Glycerol led to a tendency to condition the plant to a state of resistance to fungal attack. Soares et al. (2022) did not identify the presence of callose in banana plants in susceptible genotypes Grande Naine and Akondro Mainty infected with Black Sigatoka, evaluated up to 21 Dai.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCallose is a beta-1,3-glucan polysaccharide that is present in the cell wall of several plants, aiding in development and contributing as a defense response against biotic and abiotic factors (Chen and Kim, 2009). As a structural mechanism, it provides cell wall resistance as a form of containment to prevent pathogen penetration, thus preventing associations with each other (Stangarlin et al. 2011).\u003c/p\u003e\n\u003cp\u003eAs it is a pioneering work, it is believed that the use of Glycerol to control SN in bananas of the cultivar Grande Naine seems promising. However, further studies involving more applications of Glycerol prior to inoculation, as well as the use of a greater number of genes in the gene expression study, will be able to respond to the responses more robustly. The possibility of using this compound in banana plantations of cultivar GN will bring great benefits to the production chain of the crop, with great impact on the environment and human health.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u0026nbsp;Based on the studies presented in the present work, Glycerol seems to be a good inducer of resistance for banana plants when used first, at a dose of 5%, but more studies need to be conducted to verify the best time of application of the treatments, inoculation, and doses to be used.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompetitive interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that this work has no competitive interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWriting, preparation and original draft: APSN, genetic material, EPA, methodology, APSN, ARSR, FSN, ASR, LCSJ, RSSS, EPA, LSR and CFF. Data analysis: APSN, RMFS and conceptualization: TAOM. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEmbrapa Mandioca e Fruticultura for the technical and laboratory support for developing the research; the coordination of the Improvement of Higher Education Personnel and research (CAPES and CNPq) and the Bahia State Research Support Foundation for granting the scholarships (FAPESB).\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by IITA/The Bill and Melinda Gates Foundation-Accelerated Breeding of Better Bananas. ID OPP1093845.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Data will be made available upon request.\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n\u003cli\u003eAgrios. Disponivel em: https://agrio.app/library/Sigatoka-negra. Acesso em: 10/12/2023. AGRITEMPO. Agritempo: sistema de monitoramento agrometeorol\u0026oacute;gico. http: //www.agritempo.gov. br/agroclima/sumario, acesso em 22/10/2021\u003c/li\u003e\n\u003cli\u003eAntol\u0026iacute;n-loovera MK, Binder A, Parniske M. (2012) Receptor kinase singnaling pathways in plant-microbe interactions. Annual Review of Phytopathology, Lawrence 50:451-473. \u003c/li\u003e\n\u003cli\u003eBeveraggi A, Mourichon X, Sall\u0026eacute; G (1995) \u0026Eacute;tude compar\u0026eacute;e des premi\u0026eacute;res \u0026eacute;tapes de l\u0026acute;infection chez des bananiers sensibles et r\u0026eacute;sistants infect\u0026eacute;s par Cerscospora fijiensi (Mycosphaerella fijiensis) agent responsable de la maladies des raies noires. Canadian Journal of Botany 3:1328-1337. https:// doi.org/10.1139/b95-144\u003c/li\u003e\n\u003cli\u003eBorah S, Saikia B, Bora D, Bhorali P (2022) Expression analysis of defense response genes upon Sigatoka infection in Musa paradisiaca cultivar Kachkal. Indian Journal of Genetics 217-223. https:doi.org/ 10.31742/IJGPB.82.2.11\u003c/li\u003e\n\u003cli\u003eChanda B, Venugopal SC, Kulshrestha S, Navarre D A, Downie B, Vaillancourt L, Kachroo A, Kachroo P (2008) Glycerol-3- phosphate levels are associated with basal resistance to the hemibiotrophic fungus Colletotrichum higginsianum in Arabidopsis. Plant Physiol 147:2017-2029. https:doi.org/ 10.1104/pp.108.121335\u003c/li\u003e\n\u003cli\u003eChandra-shekara AC, Venugopal SC, Barman SR, Kachroo A, Kachroo P (2007) Plastidial fatty acid levels regulate resistancegene-dependent defense signaling inArabidopsis. PNAS, p. 7277- 7282 DOI: 10.1073pnas.0609259104. 79\u003c/li\u003e\n\u003cli\u003eChen XY, Kim JY (2009) Callose synthesis in higher plants. Plant signaling e Behavior. P. 489-492. https:doi.org//10.4161/psb.4.6.8359\u003c/li\u003e\n\u003cli\u003eChurchill ACL (2010) Mycosphaerella fijiensis, the black leaf streak pathogen of banana: progress towards understanding pathogen biology and detection, disease development, and the challenges of control. Molecular Plant Pathology 307-328. https:doi.org/1111/j.1364-3703.2010.00672.x\u003c/li\u003e\n\u003cli\u003eCordeiro ZJM (2015) Pragas Quarenten\u0026aacute;rias e melhoramento preventivo. Embrapa. https://www.embrapa.br/busca-de-publicacoes/-/publicacao/1074071/pragas-quarentenarias-e-melhoramento-preventivo . Acesssdo em 22 de junho de 2023\u003c/li\u003e\n\u003cli\u003eElbagoury MM, Turoop P, Runo S, Sila DN, Madivoli ES (2022) Postharvest treatments of banana (Musa acuminata cv.\u0026rsquo;Grand Nain\u0026rsquo;, AAA) during cold and rupening temperatures with chitosan nanoparticles to alleviate chilling injury and maintain antioxindant activity. Horticulture, Enviroment, and Biotechnology. 63:677-699\u003c/li\u003e\n\u003cli\u003eDangue JL, Jones JDG (2001) Plant pathogens and integrated defence responses to infection. Nature 826\u0026ndash;833\u003c/li\u003e\n\u003cli\u003eDing LN, yue-tao L, yuan-zhen W, Li T Geng R, Cao J, Wei Z, Tan XL (2022) Plant disease reaistance related signaling pathways: recent progress and Future prospects. Int. J. Mol. Sci. 23:20. https:doi.org/10.3390/ijms232416200 \u003c/li\u003e\n\u003cli\u003eFavreto R, Model NS, Tonietto A (2007) Sigatoka negra, fatores de ambiente e sistemas agroflorestais em bananis do Rio Grande do Sul. Pesquisa Agropecu\u0026aacute;ria Gaucha 95-104.\u003c/li\u003e\n\u003cli\u003eGarruti DS, Matias ML, Facundo HVV, Silva EO, Costa JN, SILVA M AAP (2012) .Acception of banana cultivars rsistant to black Sigatoka by the consumer market of northeast brazilian region. Ci\u0026ecirc;ncia rural 42 https:doi.org/10.1590/s0103-84782012000500030. \u003c/li\u003e\n\u003cli\u003eGasparotto L, Pereira JCR (2010) Sigatoka Negra: desafio para a bananicultura brasileira. https://ainfo.cnptia.embrapa.br/digital/bitstream/item/71420/1/sigatoka-negra-desafio-para-a-bananicultura.pdf. Acessado em: 02 de fevereiro de 2023\u003c/li\u003e\n\u003cli\u003eGazolla LCOF, Almeida DP, Castro ISL, Castro AG, Arruda LVS, Vieira NM, Mantovani HC, Zambolim L, Caixeta ET, Mendes TAO (2019) Transcriptional regulation of glycerol-3- phosphate metabolism induces resistance to coffee rust 5. \u003c/li\u003e\n\u003cli\u003eGuzm\u0026aacute;n M, P\u0026eacute;rez-Vicente L, Carlier J, Abadie C, de Lapeyre de Belaire, L., Carreel, F.(2019) Doen\u0026ccedil;as f\u0026uacute;ngicas da folhagem. http://www.biologico.sp.gov.br/publicacoes/comunicados-documentos-tecnicos/comunicados-tecnicos/doencas-fungicas-das-plantas-medicinais-aromaticas-e-condimentares-%E2%80%93-parte-aerea#:~:text=Entre%20as%20ocorr%C3%AAncias%20de%20doen%C3%A7as,se%20os%20o%C3%ADdios%20e%20m%C3%ADldios. Access 23 de janeiro de 2024\u003c/li\u003e\n\u003cli\u003eGlazebrook J (2005) Mecanismos contrastantes de defesa contra pat\u0026oacute;genos biotr\u0026oacute;ficos e necrotr\u0026oacute;ficos. Anu. Rev. Fitopatol. 43:205\u0026ndash;227.\u003c/li\u003e\n\u003cli\u003eJones JDG, Dangl JL (2006) The plant immune system. Nature 323-329\u003c/li\u003e\n\u003cli\u003eLi Y, Qiu L, Liu X, Zang Q, Zhaunsun X, Fahima T, Krugman T, Sun Q, Xie C (2020) Glycerol-Induced Powdery Mildew Resistance in Wheat by Regulating Plant Fatty Acid Metabolism, Plant Hormones Cross-Talk, and Pathogenesis-Related Genes. International Journal of Molecular Sciences 21:1-19. https:doi.org/10.3390/ijms21020673 \u003c/li\u003e\n\u003cli\u003eLi Y, Song N, Zhao C, Li F, Geng M, Wang Y, Liu W, Xie C, Sun Q (2016) Application of glycerol for induced Powdery Mildew resistance in Triticum aestivum L. Frontiers in Physiology 7:1-13. https:doi.org/10.3389/fphys.2016.00413. \u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD (2001) Analysis of Relative Gene Expression Data Using RealTime Quantitative PCR and the 2 -\u0026Delta;\u0026Delta;Ct Method. Methods 25:402-408\u003c/li\u003e\n\u003cli\u003eKachroo A, Venugopal SC, Lapchyk L, Falcone D, Hildebrand D, Kachroo P (2004) Oleic acid levels regulated by glycerolipid metabolism modulate defense gene expression in Arabidopsis. PNAS. V. 101.\u003c/li\u003e\n\u003cli\u003eKaur S, Samota, MK, Choudhary M, Pandey AK, Sharma A, Thakur J (2022) How do plantas defend themselves against pathogens-biochemical mecanismos and gen\u0026eacute;tica interventions. Physiology and Molecular Biologia of plants 28:485-504. https:doi.org/10.1007/s1298-022-01146-y\u003c/li\u003e\n\u003cli\u003eMandal MK, Chandra-Shekara AC, Jeong RD, Yu K, Zhu S, Chanda B, Navarre D, Kachroo A, Kachroo P (2012) Oleic acid dependent modulation of nitric oxide associated1 protein levels regulates nitric oxide mediated defense signaling in Arabidopsis. The Plant Cell 24:1654\u0026ndash;1674. https:doi.org/10.1105/tpc.112.096768.\u003c/li\u003e\n\u003cli\u003eMinist\u0026eacute;rio da Agricultura e Pecu\u0026aacute;ria. https://www.gov.br/agricultura/pt-br. Acesso em 23 de novembro de 2021. \u003c/li\u003e\n\u003cli\u003eNoman A, Aqeel M, Lou Y (2019) PRRs and NB-LRRs: From sinal perception t\u0026ocirc; activation of plant innate immunity. Internacional Jornal of Molecular Sciences. 20:1-21 https:doi.org/10.3390/ijms20081882. \u003c/li\u003e\n\u003cli\u003eOliveira WDS (2020) Intera\u0026ccedil;\u0026atilde;o gen\u0026oacute;tipo x microrganismos ben\u0026eacute;ficos e indu\u0026ccedil;\u0026atilde;o de resist\u0026ecirc;ncia a Fusarium oxysporum f. sp. cubense em bananeira. Disserta\u0026ccedil;\u0026atilde;o, Universidade Federal do Rec\u0026ocirc;ncavo da Bahia.\u003c/li\u003e\n\u003cli\u003ePodevin N, Krauss A, Henry I, Swwennen R, Remy S (2012) Selection and validation of reference genes for quantitative RT-PCR expression. Studies of the non-model crop Musa. Mol Breeding 30:1237-1252. https:doi.org/10.1007/s11032-012-9711-1. 81 \u003c/li\u003e\n\u003cli\u003eR CORE TEAM. (2018) R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing\u003c/li\u003e\n\u003cli\u003eRodr\u0026iacute;guez HA, Rodriguez-Arango E, Morales J, Kema G, Rango RE (2016) Defense gene expression. Associated with biotrophic pgase of Mycosphaerella fijiensis M. Morelaet infection in banana. The American phytopathological Society 100:1170-1175. https:doi.org/10.1094/PDIS-08-15- 0950-RE. \u003c/li\u003e\n\u003cli\u003eSenseverino W, Roma G, Simone M, Faino L, Melito S, Stupka E, Frusciante L, Ercolano M. (2010) R. PRGdb: a bioinformatics platform for plant resistance gene analysis. Nucleic Acids Research 38:814\u0026ndash;821 https:doi.org/10.1093/nar/gkp978. \u003c/li\u003e\n\u003cli\u003eSHIGENAGA, A. M.; ARGUESO, C. T. (2016) No hormone to rule them all: interactions of plant hormones during the responses of plants to pathogens. Seminars in cell e developmental biology. v. 56, p. 174-189. https:doi.org/10.1016/j.semcdb.2016.06.005. \u003c/li\u003e\n\u003cli\u003eSoares JMS, Rocha AJ, Nascimento FS, Amorim VBO, Ramos A, Ferreira CF, Haddad F, Amorim EP (2022) Gene expression., histology and histochemistry inthe interaction between Musa sp. And Peseudocercospora fijiensis. Plants. V. 11. https:doi.org/10.3390/plants1151953. \u003c/li\u003e\n\u003cli\u003eStangarlin JR, Kuhn OJ, Toledo MV, Portz RL, KRF, Pascholati SF (2011) A defesa vegetal contra fitopat\u0026oacute;genos. Scientia Agraria Paranaensis 10:1-29 https:doi.org/10.18188/sap.v10i1.5268.\u003c/li\u003e\n\u003cli\u003eStover RH (1972) Banana, plantain and abaca disease. London: Commonwealth Agricultural Bureaux 316 \u003c/li\u003e\n\u003cli\u003eTiwari S, Lata C (2018) Heavy Metal Stress, Signaling, and Tolerance Due to Plant-Associated Microbes: An Overview. Frontiers 6:9:452. https:doi.org/10.3389/fpls.2018.00452.\u003c/li\u003e\n\u003cli\u003eVelame KVC (2017) valida\u0026ccedil;\u0026atilde;o diferencialmente expressos e estimativa da agressividade de isolados de Fusarium oxysporum f. sp. Cubense. Tese, Universidade Estadual de Feira de Santana.\u003c/li\u003e\n\u003cli\u003eX\u0026aacute; J, Kachroo P, Nandi A, Klessig DF (2001) Uma muta\u0026ccedil;\u0026atilde;o recessiva no O gene SSI2 de Arabidopsis confere express\u0026atilde;o independente de SA e NPR1 de genes PR e resist\u0026ecirc;ncia contra pat\u0026oacute;genos bacterianos e oomicetos. Planta 25:563\u0026ndash;574\u003c/li\u003e\n\u003cli\u003eYang Y, Shah J, Klesig DF (1997) Signal perception and transduction in plant defense responses. Genes Development. 11:1621-1639. https:doi.org/10.1101/gad.11.13.1621. \u003c/li\u003e\n\u003cli\u003eZhang Y, Th PLS, Imova SENM, Inan GL (2015) Application of glycerol as a foliar spray activates the defence response and enhances disease resistance of Theobroma cacao. Molecular Plant Pathology. 16:27-37. https:doi.org/10.1111/mpp.12158\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-physiologiae-plantarum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acpp","sideBox":"Learn more about [Acta Physiologiae Plantarum](http://link.springer.com/journal/11738)","snPcode":"11738","submissionUrl":"https://www.editorialmanager.com/acpp/default2.aspx","title":"Acta Physiologiae Plantarum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Grand Nain, resistance, KINLRR, STRANS.","lastPublishedDoi":"10.21203/rs.3.rs-4311892/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4311892/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Black Sigatoka (SN) is a disease that causes leaf lesions that impede the photosynthesis process, culminating in fruits of poor quality banana. Hence, environmentally correct measures, which can present sustainable control of the disease in the field, are extremely important. Therefore, the objective of this study was to verify glycerol-mediated resistance responses in banana plants of the Grande Naine cultivar susceptible to BS. The experiment was conducted in randomized blocks with 5 treatments, namely: control, 0, 3, 5 and 8% glycerol and three replicates. The severity of the disease after the first symptoms was assessed every seven days. For gene expression analysis, the signal transduction genes (STRANS) and the recognition and signaling genes (KINLRR) and the reference genes, L2MU and 25S, were used. Histochemistry was performed to detect calosis with the aid of relative dyes of lugol and aniline blue. By means of leaf tissue clearing, it was possible to visualize pathogen structures from 72 HAI. In the analysis of calosis, the production of the compound was identified in the first evaluations in all glycerol treatments. The target resistance genes, STRANS and KINLRR, had higher expression in the treatments with 5-8% glycerol. From the results presented, glycerol appears to be a good resistance inducer for banana plants when used at a dose of 5%, but further studies are needed to verify the best time to apply treatments, inoculation, and doses to be used.","manuscriptTitle":"Acta Physiologiae Plantarum Histochemistry and Gene Expression Using Glycerol in the Pathosystem Musa X Mycosphaerella Fijiensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-26 14:01:26","doi":"10.21203/rs.3.rs-4311892/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-06-11T13:05:11+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-11T06:06:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-25T05:53:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Physiologiae Plantarum","date":"2024-04-23T08:09:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-physiologiae-plantarum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acpp","sideBox":"Learn more about [Acta Physiologiae Plantarum](http://link.springer.com/journal/11738)","snPcode":"11738","submissionUrl":"https://www.editorialmanager.com/acpp/default2.aspx","title":"Acta Physiologiae Plantarum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5d3c6c03-cb5e-46d4-a4fd-be94e7ce3dbc","owner":[],"postedDate":"June 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-04T09:14:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-26 14:01:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4311892","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4311892","identity":"rs-4311892","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}