In vitro and in vivo evaluation of citronella essential oil and its compounds in the control of fusarium wilt in tomato plants

In vitro and in vivo evaluation of citronella essential oil and its compounds in the control of fusarium wilt in tomato plants | 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 In vitro and in vivo evaluation of citronella essential oil and its compounds in the control of fusarium wilt in tomato plants Otávio Pereira Araújo, Gustavo Rodrigues Souza, Aldino Venâncio Neto, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7491381/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici is one of the most significant diseases affecting tomato cultivation, leading to substantial economic losses. In response to the growing demand for sustainable alternatives to chemical pesticides, essential oils have emerged as a promising solution for managing phytopathogens. This study investigated citronella essential oil ( Cymbopogon winterianus ) and its major compounds (citronellal, geraniol, and citronellol) as natural alternatives for controlling F. oxysporum f. sp. lycopersici , the causative agent of Fusarium wilt in tomatoes. Chemical analysis identified citronellal as the primary compound, followed by geraniol and citronellol. In vitro tests demonstrated significant antifungal activity, with geraniol and citronellol achieving IC 50 values of 0.144 µL.mL⁻¹ and 0.207 µL.mL⁻¹, respectively, resulting in 100% inhibition of fungal mycelial growth at certain concentrations. Citronellal showed a fungistatic effect with 46% inhibition at 0.500 µL.mL⁻¹, suggesting a role in controlling fungal proliferation without causing complete fungal death. In vivo assays confirmed that geraniol effectively reduced Fusarium wilt symptoms in Santa Cruz tomato plants, achieving comparable effects to commercial fungicides at a concentration of 0.610 µL.mL⁻¹, and thus indicating its potential as a sustainable alternative for plant disease management. These findings underscore the promise of citronella oil and its major components as eco-friendly antifungal agents in agriculture. fusary wilt geraniol citronellol sustainable pest management natural fungicides Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Tomatoes are one of the world's most common crops and significant for small- and medium-scale commercial farmers. It is a fruit with diverse use forms, from natural salads to industrialized products, such as sauces and extracts (Rodriguez et al. 2019). In 2020, around 3,956,559 tomatoes were produced in Brazil, with 1,851,962 tons resulting from production in the Southeast Region (Bissacotti et al. 2021 ). However, in the field, tomato cultivation is affected by diseases caused by a series of phytopathogens capable of reducing productivity and fruit quality, generating significant economic losses for rural producers (Sathiyabama et al. 2015). Among the main phytopathogens that cause diseases in tomatoes are aerial fungi and soil fungi, which are the most difficult to control because pathogens have coevolved with plants for millions of years and are highly adapted to the underground environment in association with the host. (Kobayashi et al. 2018). Among the soil fungi that affect tomato plants is Fusarium oxysporum f. sp. lycopersici , which causes fusarium wilt. It is one of the most harmful fungi to crops as it is difficult to control. This disease is favored when temperatures range from 21°C to 33°C (Kobayashi et al. 2018). The F. oxysporum f. sp. lycopersici infects the plant at practically all stages of development, penetrating through the roots and colonizing the entire plant using the xylem as its principal conductor. The colonization of the xylem by the fungus results in the inhibition of water flow and, subsequently, in the symptoms of wilt. The F. oxysporum f. sp. lycopersici survives in the soil for many years through resistance structures called chlamydospores, even in the absence of a host (Burketova et al., 2015 ), which makes efficient control of this pathogen difficult. Cultivating resistant tomato varieties has been the most effective strategy for managing fusarium wilt in tomatoes (Santos et al. 2021 ). Another form of management used to control this pathogen is the conventional model, which uses synthetic pesticides to reduce the damage caused by the disease (Coppo et al. 2017 ). However, environmental issues arise due to the problem of using pesticides in tomato production, contaminating food, animals, and water reserves (Russiano 2020 ). The demand from the consumer market for a more excellent supply of pesticide-free foods that respect the principles and precepts of sustainability and the conservation of the environment and human well-being is well-known (Pirovani et al. 2015 ). In this scenario, agroecological management strategies emerge to control plant diseases without pesticides' negative impact (López-Aranda et al. 2016 ). An alternative, aiming for more conscious management, would be the use of essential oils (EO), which, based on several studies, have been considered efficient fungicides, presenting promising results in managing several phytopathogens (Gama et al. 2020 ). Studies carried out show positive results using EOs in experiments evaluating the antifungal, insecticidal, and bactericidal effects on plants (Coppo et al. 2017 ; Fonseca et al. 2015 ; Santos et al. 2021 ; Sharma et al. 2017 ). Brazil is a reference in the production of essential oils due to the diversity and richness of the flora in the national territory (Santos et al. 2021 ). A plant that deserves to be highlighted in OE production is citronella ( Cymbopogon winterianus ), a species known for producing household cleaning agents, cosmetics, and perfumery (Oliveira et al. 2020). Because of the above, the objective of this work was to use citronella essential oil and its main compounds to control F. oxysporum f. sp. lycopersici in tomato cultivation. Materials and methods Obtaining the isolate To carry out the experiments, the isolate identified by the acronym CCF 184 of the fungus F. oxysporum f. sp. lycopersici , provided by the Department of Phytopathology of the Federal University of Viçosa. The fungus was cultured in PDA culture medium (potato, dextrose and agar), and added with an amoxicillin solution at a dosage of 0.005 mg, to avoid contamination by opportunistic bacteria. Subsequently, the fungus was placed to grow in a BOD-type incubator greenhouse at 25°C ± 1 ºC, under a 12-hour light/dark photoperiod, at a relative humidity of 70% ±10%, for seven days (period of most significant activity of the fungus) to standardize the age of the strain (group of descendants with a common ancestor who share morphological or physiological similarities). Obtaining citronella essential oil (Cymbopogon winterianus) and major compounds The citronella EO was obtained commercially from Ferquima (Lot 159) and stored in the Phytochemistry laboratory at IFES-Campus de Alegre, in a freezer at -10°C. The commercial citronella EO was analyzed by gas chromatography to determine the chemical composition. A gas chromatograph with a flame ionization detector (GC-FID) and a gas chromatograph coupled to a mass spectrometer (GC-MS) was used, following the methodology adapted from Dos Santos (2021). In both analyses, the following chromatographic conditions were used: fused silica capillary column with stationary phase SH-Rxi-5HT from SHIMADZU (30 m x 0.25 mm x 025 mm); N2 (in GC-FID analysis) and He (in GC-MS analysis) as carrier gas with a flow rate of 3.0 mL/min; the oven temperature follows a program in which it remains at an initial temperature of 40°C for 3 minutes and then gradually increases three °C/minute until it reaches 240°C, staying at this temperature for 5 minutes; injector temperature was 250°C; detector temperature of 280°C and split ratio of 1:30. GC-MS analyses were carried out in equipment operated by electronic impact with an impact energy of 70 eV; scan speed 1,000; scan range of 0.50 fragments/second and detected fragments of 29 to 400 (m/z). The identifications of the chemical components of citronella EO were carried out by comparing their mass spectra with those available in the database of the Willey7, NIST05, NIST05s spectrometers, with the co-injection of standards (mixture of linear n-alkanes, C7 to C40) and by retention indices, LTPRI (Linear Temperature Programmed Retention Indexes). To calculate the retention indices, Eq. 1 was used. The IR index is a retention index with linear temperature programming and is used when the chromatographic run is carried out with linear temperature programming (Muhlen 2009 ). This index describes the retention behavior of the compound of interest compared to that of a mixture of saturated linear hydrocarbons with different numbers of carbon atoms, providing information about the elution sequence of the compound that varies depending on the stationary phase and temperature. The LTPRI calculated for each compound was compared with literature values (Adams 2007 ). $$\:\text{L}\text{T}\text{P}\text{R}\text{I}=100\text{n}+100\left\{\left[\frac{\left({\text{t}{\prime\:}}_{\text{R}\text{i}}\right)-\left({\text{t}{\prime\:}}_{\text{R}\text{n}}\right)}{\left({\text{t}{\prime\:}}_{\text{R}\text{n}}+1\right)-\left({\text{t}{\prime\:}}_{\text{R}\text{n}}\right)}\right]\right\}\:\:\:\:\:\:\:\:\:\:\:\:\:\:\text{E}\text{q}\text{u}\text{a}\text{ç}\text{ã}\text{o}\:1$$ Where: i: is the compound of interest; n: is the number of carbon atoms of the hydrocarbon with a retention time immediately preceding the retention time of i; t'Ri: is the retention time adjusted for the compound of interest; t'Rn: is the retention time adjusted for the hydrocarbon with retention time immediately preceding the retention time of i; t'Rn + 1: is the adjusted retention time of the hydrocarbon with a retention time immediately after the retention time of i. The relative percentage of each EO compound was calculated through the ratio between the integral area of their respective peaks and the total area of all sample constituents, data obtained from analyses carried out by gas chromatography with a flame ionization detector (GC -FID). Compounds with a relative area greater than 0.5% were considered to define the chemical composition. In vitro test of the antifungal activity of citronella essential oil and its major compounds The in vitro tests were conducted in the Phytochemistry laboratory at the Federal Institute of Education, Science and Technology of Espírito Santo, Campus de Alegre, from October 2021 to February 2022. To enable the application of citronella EO and the isolated major compounds, a stock solution was prepared in the form of an emulsion composed of citronella EO and/or major compound, distilled water and Tween 80® emulsifier, with a concentration of 50 µL.mL − 1 , 500 µL/mL − 1 of citronella EO and/or major compound and 100 µL/mL − 1 of Tween 80® were used to prepare the stock solution. From this stock solution, in vitro tests were carried out using the treatments (citronella EO and the main components: cytonellal, cytonelol and geraniol) at the following concentrations: 1.500, 1.000, 0.750, 0.500, 0.300, 0.150, 0.075 and 0.035 µL /mL − 1 . For each treatment, at each concentration, five replications were used. After solidifying the culture medium containing concentrations of citronella EO and/or the major components in 8 cm Petri dishes, 4 mm discs containing the fungus, already grown previously for seven days, were placed in PDA medium. A solution of BDA culture medium and amoxicillin was prepared as a negative control, while the positive control used was the commercial fungicide Tecto SC®, at a concentration of 10 µL.mL − 1 . After mounting the tests, the plates were stored in a BOD-type oven at 25 ± 1 ºC, under a 12-hour light/dark photoperiod, with a relative humidity of 70 ± 10%, for seven days. Seven days after setting up the experiment, two diametrically opposite measurements were taken, with a digital caliper, for each repetition. The mycelial growth data were used to calculate the area under the mycelial growth curve (AACCM). Mycelial growth inhibition was calculated using Eq. (2) (Moumni et al. 2021 ). $$\:Inibição\:do\:crescimento\:micelial\:\left(\%\right)=\left[\right({d}_{c}-{d}_{t})/{d}_{c})\left]*100\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\:\right(Eq.2)$$ Where: d_c and d_t represent the average diameter of mycelial growth of the control and treated fungal strain, respectively. The concentration that inhibits 50% of the growth of fungal mycelium (IC 50 ) was determined from the linear regression equation of the curve between essential oil concentrations. The statistical design used was entirely random. The data were subjected to descriptive, variance, and regression analyses. The Tukey mean test was also used (p < 0.05). Fungicidal and fungistatic test of citronella essential oil and its major compounds After carrying out the in vitro test, the fungal mycelium from the treatment that inhibited 100% of the growth of the fungus, after seven days of incubation, was transferred to a new plate with culture medium prepared as described in item 2.3 and incubated for seven days and re-evaluated. If the fungus grows, the product has a fungistatic effect. If the fungus does not grow again, the product has a fungicidal effect. In vivo test of the antifungal activity of the major compound geraniol in the control of fuse wilting of tomato The in vivo tests were conducted in a greenhouse at the Federal Institute of Education, Science and Technology of Espírito Santo, Campus de Alegre, from November 2022 to February 2023. For in vivo tests, only geraniol, the main compound of citronella EO, was used. Well, this compound was the only one that showed fungicidal action. In other words, it inhibited the mycelial growth of Fusarium oxysporum f. sp. lycopersici under laboratory conditions, causing the fungus to die. Citronella EO and its other major compounds (citronellol and citronellal) presented fungistatic action, hindering the development of F. oxysporum f. sp. lycopersici . In the experiment in the greenhouse, two tomato cultivars, Cereja and Santa Cruz, were used, and they were popularly recommended by fruit producers as resistant and susceptible, respectively. The seeds were purchased from the local agricultural trade in Alegre-ES. The seeds of the two cultivars were sown in 128-cell polystyrene trays containing organic vegetable substrate from the PROVASO® brand. The F. oxysporum f. sp. lycopersici isolate was grown in Petri dishes containing PDA culture medium to obtain the inoculum. The plates were stored in a BOD-type oven at 25 ± 1ºC, under a 12-hour light/dark photoperiod, with a relative humidity of 70 ± 10%, for seven days. The spore suspension was prepared minutes before inoculation. With a fine brush and approximately 10 mL of autoclaved distilled water, the conidia were removed from the Petri dishes and filtered through sterile gauze to remove the hyphae. After this procedure, the conidia were counted in a Neubauer chamber, and the concentration was adjusted to 1x10 6 conidia/mL. Inoculation followed the methodology suggested by Lazaroto et al. ( 2012 ). When the tomato seedlings were at the transplanting point (20 days after planting), with four pairs of definitive leaves, they were removed from the trays, and their roots were washed to remove the substrate. After washing, the roots were cut, using scissors, approximately 2 cm from the end. After cutting, the seedlings had their roots immersed in the conidia suspension for 3 minutes. Then, the seedlings were transplanted into 3L pots containing substrate. To evaluate the efficiency of the geraniol compound in controlling F. oxysporum f. sp. lycopersici in tomato the following treatments were carried out: 1 – emulsion with geraniol, at the concentration determined in the previous experiment IC 10 (0.034 µL.mL − 1 ), IC 50 (0.144 µL.mL − 1 ) and IC 90 (C3 = 0.610 µL .mL − 1 ); 2 – Tecto SC fungicide at a concentration of 10 µL.mL − 1 (positive control); 3 – spraying with water only (negative control). One application was made and the experiment was set up twice. The severity of the disease was measured 23 days after inoculation (time required for the appearance and development of signs of the disease), using a rating scale suggested by Lazaroto et al. ( 2012 ) which ranged from 1 to 5, where: 1- indicates plant without symptoms; 2 - plant without wilting symptoms, with minor vascular discoloration; 3 - plant with symptoms of wilting and vascular discoloration; 4 - plant with severe wilting associated with the presence of chlorosis and leaf necrosis; and 5 - dead plant. All plants were evaluated and the data were used to calculate the severity (S) of fusarium wilt in which: S = (∑N)/nf Eq. 3 Knowing that, N = score for each plant nf = number of plants evaluated The area below the Disease Progress Curve (AACPD), the latent period and incubation period of the disease and the incidence of the disease were also evaluated. This experiment was conducted in a greenhouse at IFES, Campus de Alegre, in a completely randomized statistical design with seven replications. Results Chemical analysis of citronella EO The chemical analysis of citronella EO revealed the presence of 11 compounds (Table 1 ), with citronellal being the major compound with a relative area of 41.08%, followed by geraniol (25.11%) and citronellol (11.83%). The predominant class of compounds in the essential oil is oxygenated monoterpenes (85.28% relative area), with oxygenated sesquiterpenes being the least represented. Table 1 Determination of the chemical composition of citronella EO ( C. winterianus ) a . RT (minutes) RI C b RI T c Compounds Relative Area (%) d 12.924 1024 1024 Limonene 3.27 18.681 1149 1148 Citronellal 41.08 22.252 1227 1223 Citronellol 11.83 23.500 1255 1249 Geraniol 25.11 27.580 1348 1353 Citronellol acetate 3.59 28.826 1377 1379 Geraniol acetate 3.67 29.305 1388 1389 β-Elemene 2.32 32.884 1475 1484 Germacrene D 1.97 33.853 1500 - NI e 1.57 34.598 1520 1522 δ-Cadinene 2.01 35.508 1544 1548 Elemol 3.6 Hydrogenated monoterpenes 3.27 Oxygenated monoterpenes 85.28 Hydrogenated sesquiterpenes 6.30 Oxygenated sesquiterpenes 3.60 a Compounds were identified by LTPRI index (CG−DIC) and mass spectrometry (GC−MS) using an Rtx ® − 5MS column . b Retention index calculated from data from sampling saturated n−alkanes (C7−C40) . c Tabulated retention index (Adams, 2007 and NIST, 2011) . d Compounds with relative areas >1% were identified . e Unidentified compound . In vitro test of the antifungal activity of citronella essential oil and its major compounds The mycelial growth of F. oxysporum f. sp. lycopersici in PDA medium treated with citronella essential oil showed a significant inhibitory effect at the 5% probability level, according to the analysis of variance, across the eight concentrations tested (0.035, 0.075, 0.150, 0.300, 0.500, 0.750, 1.000, and 1.500 µL.mL⁻¹). All concentrations used in the experiment inhibited the mycelial growth of F. oxysporum f. sp. lycopersici , and Tukey's test identified the formation of five distinct groups (Table 2 ). Table 2 Dose-dependent relationship of citronella EO and its three main constituents against the mycelial growth of F. oxysporum f. sp. lycopersici . Concentrations µL.mL − 1 0.035 0.075 0.150 0.300 0.500 0.750 1.000 1.500 % inhibition of fungal mycelium Citronella EO 5.71 ± 4.68 f 9.27 ± 1.60 f 21.08 ± 2.05 e 39.30 ± 3.27 d 57.97 ± 2.54 c 69.81 ± 3.68 bc 76.78 ± 3.93 b 92.53 ± 3.91 a Compounds Citronellal 24.93 ± 3.99 b 25.92 ± 3.79 b 26.55 ± 3.47 b 36.98 ± 3.99 a 46.15 ± 4.77 a 49.05 ± 4.77 a 50.40 ± 3.79 a 52.27 ± 3.99 a Citronellol 11.30 ± 3.99 e 13.86 ± 3.79 e 29.84 ± 4.47 d 51.71 ± 4.77 c 100.00 ± 0.00 a 100.00 ± 0.00 a 100.00 ± 0.00 a 100.00 ± 0.00 a Geraniol 10.46 ± 3.22 d 11.80 ± 4.34 d 35.65 ± 2.88 c 61.82 ± 3.67 b 100.00 ± 0.00 a 100.00 ± 0.00 a 100.00 ± 0.00 a 100.00 ± 0.00 a *Means followed by the same letter in the line do not differ at a 5% probability level using the Tukey test. At 0.500 µL.mL⁻¹, citronella EO inhibited 58% of mycelial growth. In contrast, citronellol and geraniol at the same concentration completely inhibited the growth of F. oxysporum f. sp. lycopersici (100% inhibition), suggesting that citronellol and geraniol have greater biological activity and potential as antifungal agents. On the other hand, citronellal, an aldehyde present in greater quantities in citronella EO, showed about 46% inhibition of mycelial growth at 0.500 µL.mL⁻¹. Figure 1 shows representative photographs of the fungicidal trials conducted with citronella EO and its main isolated compounds (citronellal, citronellol, and geraniol) in controlling the mycelial growth of F. oxysporum f. sp. lycopersici at a concentration of 0.500 µL.mL⁻¹. Citronella EO and its major compound, citronellal, exhibited fungistatic effects on the fungus F. oxysporum f. sp. lycopersici . In contrast, citronellol and geraniol showed fungicidal activity, completely inhibiting fungal growth. In Vivo Test of the Antifungal Activity of Geraniol in the Control of Tomato Fusarium Wilt The in vivo test revealed differences between treatments for the Santa Cruz cultivar, whereas no significant difference was observed for the Cereja cultivar (Fig. 2 ). For the Santa Cruz variety, geraniol concentrations C1 (0.034 µL.mL⁻¹) and C2 (0.144 µL.mL⁻¹) showed symptoms of fusarium wilt from the 7th day after inoculation. Disease intensity and progression were observed over time, with plants treated with C1 and C2 concentrations succumbing by day 23. Higher dosages of geraniol resulted in a smaller area under the disease progress curve (AACPD) (Fig. 3 ), indicating reduced tomato fusarium wilt symptoms in the Santa Cruz variety. Discussion The chemical profile of citronella EO identified in this study, dominated by citronellal, geraniol, and citronellol, is consistent with prior research by Verma et al. ( 2020 ) and Tavares et al. ( 2021 ), highlighting citronellal as a major component. However, variations in the composition of citronella oil, as noted by Rodríguez-González et al. ( 2019 ), underscore the influence of genetic, climatic, and environmental factors (Palazzolo et al. 2013 ; Rebey et al. 2012 ). These variations impact the proportion and type of bioactive compounds present, which directly affect the oil’s efficacy and application in disease management. In vitro findings in this study demonstrated that both citronellol and geraniol exhibit potent antifungal activity, achieving 100% inhibition of F. oxysporum f. sp. lycopersici mycelial growth. This total inhibition effect contrasts with the partial suppression observed with citronellal, which exhibited fungistatic activity by reducing growth without causing complete fungal death. The fungistatic action observed in citronellal is particularly significant for sustainable agricultural practices. Fungistatic agents control fungal growth by limiting pathogen proliferation without exerting lethal pressure, which can help mitigate the development of resistance and support environmental conservation. The fungistatic activity of citronella EO, particularly attributed to citronellal, suggests an alternative pathway for managing Fusarium species. Fungistatic agents like citronellal prevent complete fungal eradication, reducing the likelihood of resistance while minimizing toxicity and environmental impact. This activity is advantageous in integrated disease management systems, as it promotes long-term pathogen control without the harsh ecological consequences often associated with synthetic fungicides. According to Seixa et al. (2011), the synergistic effects of multiple fungistatic compounds in citronella EO can enhance its overall efficacy, with combinations of citronellal, citronellol, and geraniol likely working together to inhibit fungal growth effectively. The IC 50 and IC 90 concentration curves for citronella EO and its main components, illustrated in Fig. 4 and Table 3 further validate the antifungal potency of geraniol and citronellol compared to citronella EO alone. These findings suggest that while the complete oil mixture has value, isolating potent components such as geraniol can amplify fungicidal effects. Table 3 Estimated concentrations of citronella EO concentrations and its major compounds that inhibit 50 and 90% of the mycelial growth of F. oxysporum f. sp. lycopersici . IC 50 (µL.mL − 1 ) IC 90 (µL.mL − 1 ) R 2 Citronella EO 0.370 1.986 0.915 Citronellol 0.207 0.819 0.943 Citronellal 1.071 - 0.9183 Geraniol 0.144 0.610 0.9065 The in vivo effectiveness of geraniol, evidenced by its control of fusarium wilt in tomato plants, supports its potential as an alternative to synthetic fungicides. The comparison between geraniol at 0.610 µL.mL⁻¹ and the commercial fungicide TECTO SC shows comparable efficacy in reducing wilt symptoms in the Santa Cruz variety, while likely offering reduced phytotoxicity and environmental persistence (La Torre et al. 2016 ). Plant secondary metabolites, like geraniol, are known for their safer ecological profiles compared to synthetic agents, supporting their inclusion in integrated pest management strategies. Further supporting this potential, studies by Sanap et al. ( 2020 ) and Gonçalves et al. ( 2021 ) confirmed that various essential oils, including citronella, effectively control fungal diseases in tomato plants without phytotoxic effects. This aligns with the results in Figs. 5 , where the cherry tomato variety exhibited resistance to F. oxysporum f. sp. lycopersici , indicating possible innate resilience in certain cultivars. Moreover, Fig. 6 comparing the C3 treatment (0.610 µL.mL⁻¹ of geraniol) with TECTO SC at 10 µL.mL⁻¹ in the Santa Cruz cultivar underscores geraniol's capacity to control fusarium wilt effectively at lower doses. This property aligns with sustainable agriculture goals, where lower input levels of naturally derived agents yield effective results, reducing reliance on synthetic products. Thus, the fungistatic and fungicidal activities of citronella essential oil and its major compounds, especially geraniol and citronellal, represent promising, eco-friendly alternatives for managing Fusarium infections in tomatoes. This study supports further exploration into essential oils as sustainable tools in agricultural disease management, offering reduced phytotoxicity, lower resistance potential, and a favorable environmental profile compared to conventional fungicides. Conclusions The findings of this study underscore the promising antifungal potential of citronella EO and its main constituents, particularly geraniol, citronellol, and citronellal, for sustainable agricultural applications. Geraniol and citronellol exhibited potent fungicidal effects, completely inhibiting F. oxysporum f. sp. lycopersici growth in vitro , with low IC 50 values indicating high efficacy at relatively low concentrations. The fungistatic activity of citronellal, achieving partial inhibition without total fungal eradication, highlights an alternative approach that could reduce resistance pressure and support environmental safety by limiting excessive fungal proliferation rather than completely eliminating the pathogen. In vivo results further validate geraniol’s potential as an effective alternative to conventional fungicides, demonstrating substantial disease suppression in tomato plants without the adverse environmental impacts associated with synthetic chemicals. This study contributes valuable insights into the use of natural compounds for plant disease management, supporting citronella oil and its compounds as viable, eco-friendly options for integrated disease management programs. Future research should explore field trials and the potential synergistic effects of these compounds with other natural agents, advancing their practical application in sustainable agriculture. Declarations Funding Declaration We want to thank the Coordination for the Improvement of Higher Education Personnel (CAPES) and the Fundação de Amparo à Pesquisa do Espírito Santo (FAPES) for the support offered in the development of the research. Competing Interests The authors declare that they have no competing interests. Authors' contributions O.P.A. and G.R.S. wrote the main manuscript text, A.V.N. and A.A.J. contributed to data analysis, O.A.L., S.P.S. and L.A.A. prepared figures 1–3, M.V.A.A. and G.F.S. assisted with data collection, L.A.P., M.F.C.S. and C.S.B. contributed to the experimental design, J.H.A.M. and L.M. supervised the project. All authors reviewed the manuscript. References Adams RP (2007) Identification of essential oil components by gas chromatography/mass spectrometry. Allured Publishing Corporation, Illinois USA, Carol Stream, 4th ed., 804 p Bissacotti AP, Londero P, Costabeber L (2021) Tomate: botânica, produção, composição nutricional e benefícios à saúde. Cad Cienc Tecnol 38:26643 Burketova L, Trda L, Ott PG, Valentova O (2015) Bio-based resistance inducers for sustainable plant protection against pathogens. 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Carbohydr Polym 133:400–407 Sharma A, Rajendran S, Srivastava A, Sharma S, Kundu B (2017) Antifungal activities of selected essential oils against Fusarium oxysporum f. sp. lycopersici 1322, with emphasis on Syzygium aromaticum essential oil. J Biosci Bioeng 123:308–313 Tavares LA, Rezende AA, Santos JL, Estevam CS, Silva AM, Schneider JK, Albuquerque-Júnior RL (2021) Cymbopogon winterianus essential oil attenuates bleomycin-induced pulmonary fibrosis in a murine model. Pharmaceutics 13:679 Verma RS, Verma SK, Tandon S, Padalia RC, Darokar MP (2020) Chemical composition and antimicrobial activity of Java citronella ( Cymbopogon winterianus Jowitt ex Bor) essential oil extracted by different methods. J Essent Oil Res 32:449–455. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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. 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Legend: CN- Negative Control (1% v/v Tween 80® solution), CP- Positive Control (TECTO SC®).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7491381/v1/aaddb31120061267b3aebd79.png"},{"id":92197647,"identity":"685f7fac-707e-44d5-909d-1f9214f20c6b","added_by":"auto","created_at":"2025-09-25 16:13:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101248,"visible":true,"origin":"","legend":"\u003cp\u003eThe severity of fusarium wilt at 23 days after inoculation.Legend: C1 (0.034 µL.mL\u003csup\u003e-1\u003c/sup\u003e) C2 (0.144 µL.mL\u003csup\u003e-1\u003c/sup\u003e) C3 (0.610 µL.mL\u003csup\u003e-1\u003c/sup\u003e) CN (Negative control) CP (Positive control) CSI (Concentration without inoculation).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7491381/v1/49c3a6bd76a30a81b2f3e7f5.png"},{"id":92196515,"identity":"80dcbb0d-41f4-43f2-905e-9065c5d8e027","added_by":"auto","created_at":"2025-09-25 15:57:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51690,"visible":true,"origin":"","legend":"\u003cp\u003eThe severity of fusarium wilt at different doses of geraniol and the relationship with the area under the disease progress curve (AACPD). Legend: T1 (0.034 µL.mL\u003csup\u003e-1\u003c/sup\u003e) T2 (0.144 µL.mL\u003csup\u003e-1\u003c/sup\u003e) T3 (0.610 µL.mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7491381/v1/aabe3686195829a280cef975.png"},{"id":92196519,"identity":"9bdf8651-129b-4783-861d-e1134cc6edb8","added_by":"auto","created_at":"2025-09-25 15:57:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":158435,"visible":true,"origin":"","legend":"\u003cp\u003eCurves were used to estimate inhibitory concentrations of 50 and 90% (IC\u003csub\u003e50 \u003c/sub\u003eand IC\u003csub\u003e90\u003c/sub\u003e) using citronella EO and its major compounds against\u003cem\u003e F. oxysporum f. \u003c/em\u003esp\u003cem\u003e. lycopersici. \u003c/em\u003eLegend: A (Citronella essential oil), B (Citronellol), C (Citronellal), D (Geraniol).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7491381/v1/31a69d887897c66e7b7b2d0a.png"},{"id":92196882,"identity":"adda0452-7f83-438f-8565-712db7451f48","added_by":"auto","created_at":"2025-09-25 16:05:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1121117,"visible":true,"origin":"","legend":"\u003cp\u003eTomato plants of the Santa Cruz variety were treated with different concentrations of the formulation in emulsion form, containing the geraniol compound. Legend: C1 (0.034 µL.mL\u003csup\u003e-1\u003c/sup\u003e), C2 (0.144 µL.mL\u003csup\u003e-1\u003c/sup\u003e), C3 (0.610 µL.mL\u003csup\u003e-1\u003c/sup\u003e), CP (Positive Control with TECTO SC), CN (Negative Control with Water Sterile Distilled).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7491381/v1/42a804bfb67d62af1b9fcd26.png"},{"id":92196522,"identity":"44953b68-bb3b-47be-aec2-f6285f03c7e2","added_by":"auto","created_at":"2025-09-25 15:57:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1149839,"visible":true,"origin":"","legend":"\u003cp\u003eCherry variety tomato plants, treated with different concentrations of the formulation in emulsion form, containing the geraniol compound. Legend: C1 (0.034 µL.mL\u003csup\u003e-1\u003c/sup\u003e), C2 (0.144 µL.mL\u003csup\u003e-1\u003c/sup\u003e), C3 (0.610 µL.mL\u003csup\u003e-1\u003c/sup\u003e), CP (Positive Control with TECTO SC), CN (Negative Control with Water Sterile Distilled).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7491381/v1/e491f2e6baf605ac8fbee1cf.png"},{"id":95224095,"identity":"8291847d-16eb-4369-b620-38075543da18","added_by":"auto","created_at":"2025-11-05 16:23:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4809041,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7491381/v1/c4d28371-11ac-44d8-91d5-0e9834355238.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In vitro and in vivo evaluation of citronella essential oil and its compounds in the control of fusarium wilt in tomato plants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTomatoes are one of the world's most common crops and significant for small- and medium-scale commercial farmers. It is a fruit with diverse use forms, from natural salads to industrialized products, such as sauces and extracts (Rodriguez et al. 2019). In 2020, around 3,956,559 tomatoes were produced in Brazil, with 1,851,962 tons resulting from production in the Southeast Region (Bissacotti et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, in the field, tomato cultivation is affected by diseases caused by a series of phytopathogens capable of reducing productivity and fruit quality, generating significant economic losses for rural producers (Sathiyabama et al. 2015). Among the main phytopathogens that cause diseases in tomatoes are aerial fungi and soil fungi, which are the most difficult to control because pathogens have coevolved with plants for millions of years and are highly adapted to the underground environment in association with the host. (Kobayashi et al. 2018).\u003c/p\u003e\u003cp\u003eAmong the soil fungi that affect tomato plants is \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e, which causes fusarium wilt. It is one of the most harmful fungi to crops as it is difficult to control. This disease is favored when temperatures range from 21\u0026deg;C to 33\u0026deg;C (Kobayashi et al. 2018). The \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e infects the plant at practically all stages of development, penetrating through the roots and colonizing the entire plant using the xylem as its principal conductor. The colonization of the xylem by the fungus results in the inhibition of water flow and, subsequently, in the symptoms of wilt. The \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e survives in the soil for many years through resistance structures called chlamydospores, even in the absence of a host (Burketova et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which makes efficient control of this pathogen difficult.\u003c/p\u003e\u003cp\u003eCultivating resistant tomato varieties has been the most effective strategy for managing fusarium wilt in tomatoes (Santos et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Another form of management used to control this pathogen is the conventional model, which uses synthetic pesticides to reduce the damage caused by the disease (Coppo et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, environmental issues arise due to the problem of using pesticides in tomato production, contaminating food, animals, and water reserves (Russiano \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe demand from the consumer market for a more excellent supply of pesticide-free foods that respect the principles and precepts of sustainability and the conservation of the environment and human well-being is well-known (Pirovani et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this scenario, agroecological management strategies emerge to control plant diseases without pesticides' negative impact (L\u0026oacute;pez-Aranda et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAn alternative, aiming for more conscious management, would be the use of essential oils (EO), which, based on several studies, have been considered efficient fungicides, presenting promising results in managing several phytopathogens (Gama et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies carried out show positive results using EOs in experiments evaluating the antifungal, insecticidal, and bactericidal effects on plants (Coppo et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Fonseca et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Santos et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBrazil is a reference in the production of essential oils due to the diversity and richness of the flora in the national territory (Santos et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A plant that deserves to be highlighted in OE production is citronella (\u003cem\u003eCymbopogon winterianus\u003c/em\u003e), a species known for producing household cleaning agents, cosmetics, and perfumery (Oliveira et al. 2020). Because of the above, the objective of this work was to use citronella essential oil and its main compounds to control \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e in tomato cultivation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eObtaining the isolate\u003c/h2\u003e\u003cp\u003eTo carry out the experiments, the isolate identified by the acronym CCF 184 of the fungus \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e, provided by the Department of Phytopathology of the Federal University of Vi\u0026ccedil;osa. The fungus was cultured in PDA culture medium (potato, dextrose and agar), and added with an amoxicillin solution at a dosage of 0.005 mg, to avoid contamination by opportunistic bacteria. Subsequently, the fungus was placed to grow in a BOD-type incubator greenhouse at 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026ordm;C, under a 12-hour light/dark photoperiod, at a relative humidity of 70% \u0026plusmn;10%, for seven days (period of most significant activity of the fungus) to standardize the age of the strain (group of descendants with a common ancestor who share morphological or physiological similarities).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eObtaining citronella essential oil (Cymbopogon winterianus) and major compounds\u003c/h3\u003e\n\u003cp\u003eThe citronella EO was obtained commercially from Ferquima (Lot 159) and stored in the Phytochemistry laboratory at IFES-Campus de Alegre, in a freezer at -10\u0026deg;C. The commercial citronella EO was analyzed by gas chromatography to determine the chemical composition. A gas chromatograph with a flame ionization detector (GC-FID) and a gas chromatograph coupled to a mass spectrometer (GC-MS) was used, following the methodology adapted from Dos Santos (2021). In both analyses, the following chromatographic conditions were used: fused silica capillary column with stationary phase SH-Rxi-5HT from SHIMADZU (30 m x 0.25 mm x 025 mm); N2 (in GC-FID analysis) and He (in GC-MS analysis) as carrier gas with a flow rate of 3.0 mL/min; the oven temperature follows a program in which it remains at an initial temperature of 40\u0026deg;C for 3 minutes and then gradually increases three \u0026deg;C/minute until it reaches 240\u0026deg;C, staying at this temperature for 5 minutes; injector temperature was 250\u0026deg;C; detector temperature of 280\u0026deg;C and split ratio of 1:30.\u003c/p\u003e\u003cp\u003eGC-MS analyses were carried out in equipment operated by electronic impact with an impact energy of 70 eV; scan speed 1,000; scan range of 0.50 fragments/second and detected fragments of 29 to 400 (m/z).\u003c/p\u003e\u003cp\u003eThe identifications of the chemical components of citronella EO were carried out by comparing their mass spectra with those available in the database of the Willey7, NIST05, NIST05s spectrometers, with the co-injection of standards (mixture of linear n-alkanes, C7 to C40) and by retention indices, LTPRI (Linear Temperature Programmed Retention Indexes). To calculate the retention indices, Eq.\u0026nbsp;1 was used. The IR index is a retention index with linear temperature programming and is used when the chromatographic run is carried out with linear temperature programming (Muhlen \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This index describes the retention behavior of the compound of interest compared to that of a mixture of saturated linear hydrocarbons with different numbers of carbon atoms, providing information about the elution sequence of the compound that varies depending on the stationary phase and temperature. The LTPRI calculated for each compound was compared with literature values (Adams \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{L}\\text{T}\\text{P}\\text{R}\\text{I}=100\\text{n}+100\\left\\{\\left[\\frac{\\left({\\text{t}{\\prime\\:}}_{\\text{R}\\text{i}}\\right)-\\left({\\text{t}{\\prime\\:}}_{\\text{R}\\text{n}}\\right)}{\\left({\\text{t}{\\prime\\:}}_{\\text{R}\\text{n}}+1\\right)-\\left({\\text{t}{\\prime\\:}}_{\\text{R}\\text{n}}\\right)}\\right]\\right\\}\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\text{E}\\text{q}\\text{u}\\text{a}\\text{\u0026ccedil;}\\text{\u0026atilde;}\\text{o}\\:1$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere: i: is the compound of interest; n: is the number of carbon atoms of the hydrocarbon with a retention time immediately preceding the retention time of i; t'Ri: is the retention time adjusted for the compound of interest; t'Rn: is the retention time adjusted for the hydrocarbon with retention time immediately preceding the retention time of i; t'Rn\u0026thinsp;+\u0026thinsp;1: is the adjusted retention time of the hydrocarbon with a retention time immediately after the retention time of i.\u003c/p\u003e\u003cp\u003eThe relative percentage of each EO compound was calculated through the ratio between the integral area of their respective peaks and the total area of all sample constituents, data obtained from analyses carried out by gas chromatography with a flame ionization detector (GC -FID). Compounds with a relative area greater than 0.5% were considered to define the chemical composition.\u003c/p\u003e\n\u003ch3\u003eIn vitro test of the antifungal activity of citronella essential oil and its major compounds\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e tests were conducted in the Phytochemistry laboratory at the Federal Institute of Education, Science and Technology of Esp\u0026iacute;rito Santo, Campus de Alegre, from October 2021 to February 2022.\u003c/p\u003e\u003cp\u003eTo enable the application of citronella EO and the isolated major compounds, a stock solution was prepared in the form of an emulsion composed of citronella EO and/or major compound, distilled water and Tween 80\u0026reg; emulsifier, with a concentration of 50 \u0026micro;L.mL \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 500 \u0026micro;L/mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of citronella EO and/or major compound and 100 \u0026micro;L/mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Tween 80\u0026reg; were used to prepare the stock solution. From this stock solution, in vitro tests were carried out using the treatments (citronella EO and the main components: cytonellal, cytonelol and geraniol) at the following concentrations: 1.500, 1.000, 0.750, 0.500, 0.300, 0.150, 0.075 and 0.035 \u0026micro;L /mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. For each treatment, at each concentration, five replications were used. After solidifying the culture medium containing concentrations of citronella EO and/or the major components in 8 cm Petri dishes, 4 mm discs containing the fungus, already grown previously for seven days, were placed in PDA medium. A solution of BDA culture medium and amoxicillin was prepared as a negative control, while the positive control used was the commercial fungicide Tecto SC\u0026reg;, at a concentration of 10 \u0026micro;L.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After mounting the tests, the plates were stored in a BOD-type oven at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026ordm;C, under a 12-hour light/dark photoperiod, with a relative humidity of 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, for seven days.\u003c/p\u003e\u003cp\u003eSeven days after setting up the experiment, two diametrically opposite measurements were taken, with a digital caliper, for each repetition. The mycelial growth data were used to calculate the area under the mycelial growth curve (AACCM). Mycelial growth inhibition was calculated using Eq.\u0026nbsp;(2) (Moumni et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Inibi\u0026ccedil;\u0026atilde;o\\:do\\:crescimento\\:micelial\\:\\left(\\%\\right)=\\left[\\right({d}_{c}-{d}_{t})/{d}_{c})\\left]*100\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\:\\right(Eq.2)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere: d_c and d_t represent the average diameter of mycelial growth of the control and treated fungal strain, respectively.\u003c/p\u003e\u003cp\u003eThe concentration that inhibits 50% of the growth of fungal mycelium (IC\u003csub\u003e50\u003c/sub\u003e) was determined from the linear regression equation of the curve between essential oil concentrations. The statistical design used was entirely random. The data were subjected to descriptive, variance, and regression analyses. The Tukey mean test was also used (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003ch3\u003eFungicidal and fungistatic test of citronella essential oil and its major compounds\u003c/h3\u003e\n\u003cp\u003eAfter carrying out the in vitro test, the fungal mycelium from the treatment that inhibited 100% of the growth of the fungus, after seven days of incubation, was transferred to a new plate with culture medium prepared as described in item 2.3 and incubated for seven days and re-evaluated. If the fungus grows, the product has a fungistatic effect. If the fungus does not grow again, the product has a fungicidal effect.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo test of the antifungal activity of the major compound geraniol in the control of fuse wilting of tomato\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e tests were conducted in a greenhouse at the Federal Institute of Education, Science and Technology of Esp\u0026iacute;rito Santo, Campus de Alegre, from November 2022 to February 2023.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e tests, only geraniol, the main compound of citronella EO, was used. Well, this compound was the only one that showed fungicidal action. In other words, it inhibited the mycelial growth of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e under laboratory conditions, causing the fungus to die. Citronella EO and its other major compounds (citronellol and citronellal) presented fungistatic action, hindering the development of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn the experiment in the greenhouse, two tomato cultivars, Cereja and Santa Cruz, were used, and they were popularly recommended by fruit producers as resistant and susceptible, respectively. The seeds were purchased from the local agricultural trade in Alegre-ES. The seeds of the two cultivars were sown in 128-cell polystyrene trays containing organic vegetable substrate from the PROVASO\u0026reg; brand.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e isolate was grown in Petri dishes containing PDA culture medium to obtain the inoculum. The plates were stored in a BOD-type oven at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026ordm;C, under a 12-hour light/dark photoperiod, with a relative humidity of 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, for seven days. The spore suspension was prepared minutes before inoculation. With a fine brush and approximately 10 mL of autoclaved distilled water, the conidia were removed from the Petri dishes and filtered through sterile gauze to remove the hyphae. After this procedure, the conidia were counted in a Neubauer chamber, and the concentration was adjusted to 1x10\u003csup\u003e6\u003c/sup\u003e conidia/mL.\u003c/p\u003e\u003cp\u003eInoculation followed the methodology suggested by Lazaroto et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). When the tomato seedlings were at the transplanting point (20 days after planting), with four pairs of definitive leaves, they were removed from the trays, and their roots were washed to remove the substrate. After washing, the roots were cut, using scissors, approximately 2 cm from the end. After cutting, the seedlings had their roots immersed in the conidia suspension for 3 minutes. Then, the seedlings were transplanted into 3L pots containing substrate.\u003c/p\u003e\u003cp\u003eTo evaluate the efficiency of the geraniol compound in controlling \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e in tomato the following treatments were carried out: 1 \u0026ndash; emulsion with geraniol, at the concentration determined in the previous experiment IC\u003csub\u003e10\u003c/sub\u003e (0.034 \u0026micro;L.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), IC\u003csub\u003e50\u003c/sub\u003e (0.144 \u0026micro;L.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and IC \u003csub\u003e90\u003c/sub\u003e (C3\u0026thinsp;=\u0026thinsp;0.610 \u0026micro;L .mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); 2 \u0026ndash; Tecto SC fungicide at a concentration of 10 \u0026micro;L.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (positive control); 3 \u0026ndash; spraying with water only (negative control). One application was made and the experiment was set up twice.\u003c/p\u003e\u003cp\u003eThe severity of the disease was measured 23 days after inoculation (time required for the appearance and development of signs of the disease), using a rating scale suggested by Lazaroto et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) which ranged from 1 to 5, where: 1- indicates plant without symptoms; 2 - plant without wilting symptoms, with minor vascular discoloration; 3 - plant with symptoms of wilting and vascular discoloration; 4 - plant with severe wilting associated with the presence of chlorosis and leaf necrosis; and 5 - dead plant.\u003c/p\u003e\u003cp\u003eAll plants were evaluated and the data were used to calculate the severity (S) of fusarium wilt in which:\u003c/p\u003e\u003cp\u003eS = (\u0026sum;N)/nf Eq.\u0026nbsp;3\u003c/p\u003e\u003cp\u003eKnowing that,\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;score for each plant\u003c/p\u003e\u003cp\u003enf\u0026thinsp;=\u0026thinsp;number of plants evaluated\u003c/p\u003e\u003cp\u003eThe area below the Disease Progress Curve (AACPD), the latent period and incubation period of the disease and the incidence of the disease were also evaluated.\u003c/p\u003e\u003cp\u003eThis experiment was conducted in a greenhouse at IFES, Campus de Alegre, in a completely randomized statistical design with seven replications.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eChemical analysis of citronella EO\u003c/h2\u003e\u003cp\u003eThe chemical analysis of citronella EO revealed the presence of 11 compounds (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with citronellal being the major compound with a relative area of 41.08%, followed by geraniol (25.11%) and citronellol (11.83%). The predominant class of compounds in the essential oil is oxygenated monoterpenes (85.28% relative area), with oxygenated sesquiterpenes being the least represented.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDetermination of the chemical composition of citronella EO (\u003cem\u003eC. winterianus\u003c/em\u003e)\u003csup\u003ea\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRT (minutes)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRI\u003csub\u003eC\u003c/sub\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRI\u003csub\u003eT\u003c/sub\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCompounds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRelative Area (%)\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12.924\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLimonene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e18.681\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCitronellal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e41.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e22.252\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1227\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1223\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCitronellol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.83\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1255\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1249\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGeraniol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e27.580\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1348\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1353\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCitronellol acetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e28.826\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1377\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1379\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGeraniol acetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e29.305\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1388\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1389\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eβ-Elemene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e32.884\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1475\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1484\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGermacrene D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e33.853\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNI\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e34.598\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1520\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1522\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eδ-Cadinene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e35.508\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1544\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1548\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eElemol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eHydrogenated monoterpenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eOxygenated monoterpenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e85.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eHydrogenated sesquiterpenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eOxygenated sesquiterpenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea Compounds were identified by LTPRI index (CG\u0026minus;DIC) and mass spectrometry (GC\u0026minus;MS) using an Rtx\u003c/sup\u003e \u0026reg;\u003csup\u003e\u0026minus;\u0026thinsp;5MS column\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003csup\u003eb Retention index calculated from data from sampling saturated n\u0026minus;alkanes (C7\u0026minus;C40)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003csup\u003ec Tabulated retention index (Adams, 2007 and NIST, 2011)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003csup\u003ed Compounds with relative areas \u0026gt;1% were identified\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003csup\u003ee Unidentified compound\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003etest of the antifungal activity of citronella essential oil and its major compounds\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mycelial growth of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e in PDA medium treated with citronella essential oil showed a significant inhibitory effect at the 5% probability level, according to the analysis of variance, across the eight concentrations tested (0.035, 0.075, 0.150, 0.300, 0.500, 0.750, 1.000, and 1.500 \u0026micro;L.mL⁻\u0026sup1;). All concentrations used in the experiment inhibited the mycelial growth of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e, and Tukey's test identified the formation of five distinct groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDose-dependent relationship of citronella EO and its three main constituents against the mycelial growth of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u003cp\u003eConcentrations \u0026micro;L.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.075\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.750\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003e% inhibition of fungal mycelium\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitronella EO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.68\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e39.30\u0026thinsp;\u0026plusmn;\u0026thinsp;3.27\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e57.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e69.81\u0026thinsp;\u0026plusmn;\u0026thinsp;3.68\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e76.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e92.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompounds\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitronellal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.47\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e46.15\u0026thinsp;\u0026plusmn;\u0026thinsp;4.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e49.05\u0026thinsp;\u0026plusmn;\u0026thinsp;4.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e50.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e52.27\u0026thinsp;\u0026plusmn;\u0026thinsp;3.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitronellol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.30\u0026thinsp;\u0026plusmn;\u0026thinsp;3.99\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29.84\u0026thinsp;\u0026plusmn;\u0026thinsp;4.47\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e51.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.77\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeraniol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.46\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.34\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.88\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e61.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e*Means followed by the same letter in the line do not differ at a 5% probability level using the Tukey test.\u003c/p\u003e\u003cp\u003eAt 0.500 \u0026micro;L.mL⁻\u0026sup1;, citronella EO inhibited 58% of mycelial growth. In contrast, citronellol and geraniol at the same concentration completely inhibited the growth of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e (100% inhibition), suggesting that citronellol and geraniol have greater biological activity and potential as antifungal agents. On the other hand, citronellal, an aldehyde present in greater quantities in citronella EO, showed about 46% inhibition of mycelial growth at 0.500 \u0026micro;L.mL⁻\u0026sup1;. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows representative photographs of the fungicidal trials conducted with citronella EO and its main isolated compounds (citronellal, citronellol, and geraniol) in controlling the mycelial growth of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e at a concentration of 0.500 \u0026micro;L.mL⁻\u0026sup1;. Citronella EO and its major compound, citronellal, exhibited fungistatic effects on the fungus \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e. In contrast, citronellol and geraniol showed fungicidal activity, completely inhibiting fungal growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vivo Test of the Antifungal Activity of Geraniol in the Control of Tomato Fusarium Wilt\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e test revealed differences between treatments for the Santa Cruz cultivar, whereas no significant difference was observed for the Cereja cultivar (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For the Santa Cruz variety, geraniol concentrations C1 (0.034 \u0026micro;L.mL⁻\u0026sup1;) and C2 (0.144 \u0026micro;L.mL⁻\u0026sup1;) showed symptoms of fusarium wilt from the 7th day after inoculation. Disease intensity and progression were observed over time, with plants treated with C1 and C2 concentrations succumbing by day 23. Higher dosages of geraniol resulted in a smaller area under the disease progress curve (AACPD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating reduced tomato fusarium wilt symptoms in the Santa Cruz variety.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe chemical profile of citronella EO identified in this study, dominated by citronellal, geraniol, and citronellol, is consistent with prior research by Verma et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and Tavares et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), highlighting citronellal as a major component. However, variations in the composition of citronella oil, as noted by Rodr\u0026iacute;guez-Gonz\u0026aacute;lez et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), underscore the influence of genetic, climatic, and environmental factors (Palazzolo et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rebey et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These variations impact the proportion and type of bioactive compounds present, which directly affect the oil\u0026rsquo;s efficacy and application in disease management.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e findings in this study demonstrated that both citronellol and geraniol exhibit potent antifungal activity, achieving 100% inhibition of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e mycelial growth. This total inhibition effect contrasts with the partial suppression observed with citronellal, which exhibited fungistatic activity by reducing growth without causing complete fungal death. The fungistatic action observed in citronellal is particularly significant for sustainable agricultural practices. Fungistatic agents control fungal growth by limiting pathogen proliferation without exerting lethal pressure, which can help mitigate the development of resistance and support environmental conservation.\u003c/p\u003e\u003cp\u003eThe fungistatic activity of citronella EO, particularly attributed to citronellal, suggests an alternative pathway for managing \u003cem\u003eFusarium\u003c/em\u003e species. Fungistatic agents like citronellal prevent complete fungal eradication, reducing the likelihood of resistance while minimizing toxicity and environmental impact. This activity is advantageous in integrated disease management systems, as it promotes long-term pathogen control without the harsh ecological consequences often associated with synthetic fungicides. According to Seixa et al. (2011), the synergistic effects of multiple fungistatic compounds in citronella EO can enhance its overall efficacy, with combinations of citronellal, citronellol, and geraniol likely working together to inhibit fungal growth effectively.\u003c/p\u003e\u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e and IC\u003csub\u003e90\u003c/sub\u003e concentration curves for citronella EO and its main components, illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e further validate the antifungal potency of geraniol and citronellol compared to citronella EO alone. These findings suggest that while the complete oil mixture has value, isolating potent components such as geraniol can amplify fungicidal effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEstimated concentrations of citronella EO concentrations and its major compounds that inhibit 50 and 90% of the mycelial growth of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;L.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIC\u003csub\u003e90\u003c/sub\u003e (\u0026micro;L.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitronella EO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.370\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.986\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.915\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitronellol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.207\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.819\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.943\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitronellal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.071\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.9183\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGeraniol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.144\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.610\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.9065\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e effectiveness of geraniol, evidenced by its control of fusarium wilt in tomato plants, supports its potential as an alternative to synthetic fungicides. The comparison between geraniol at 0.610 \u0026micro;L.mL⁻\u0026sup1; and the commercial fungicide TECTO SC shows comparable efficacy in reducing wilt symptoms in the Santa Cruz variety, while likely offering reduced phytotoxicity and environmental persistence (La Torre et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Plant secondary metabolites, like geraniol, are known for their safer ecological profiles compared to synthetic agents, supporting their inclusion in integrated pest management strategies.\u003c/p\u003e\u003cp\u003eFurther supporting this potential, studies by Sanap et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and Gon\u0026ccedil;alves et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) confirmed that various essential oils, including citronella, effectively control fungal diseases in tomato plants without phytotoxic effects. This aligns with the results in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, where the cherry tomato variety exhibited resistance to \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e, indicating possible innate resilience in certain cultivars.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e comparing the C3 treatment (0.610 \u0026micro;L.mL⁻\u0026sup1; of geraniol) with TECTO SC at 10 \u0026micro;L.mL⁻\u0026sup1; in the Santa Cruz cultivar underscores geraniol's capacity to control fusarium wilt effectively at lower doses. This property aligns with sustainable agriculture goals, where lower input levels of naturally derived agents yield effective results, reducing reliance on synthetic products. Thus, the fungistatic and fungicidal activities of citronella essential oil and its major compounds, especially geraniol and citronellal, represent promising, eco-friendly alternatives for managing \u003cem\u003eFusarium\u003c/em\u003e infections in tomatoes. This study supports further exploration into essential oils as sustainable tools in agricultural disease management, offering reduced phytotoxicity, lower resistance potential, and a favorable environmental profile compared to conventional fungicides.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe findings of this study underscore the promising antifungal potential of citronella EO and its main constituents, particularly geraniol, citronellol, and citronellal, for sustainable agricultural applications. Geraniol and citronellol exhibited potent fungicidal effects, completely inhibiting \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e growth \u003cem\u003ein vitro\u003c/em\u003e, with low IC\u003csub\u003e50\u003c/sub\u003e values indicating high efficacy at relatively low concentrations. The fungistatic activity of citronellal, achieving partial inhibition without total fungal eradication, highlights an alternative approach that could reduce resistance pressure and support environmental safety by limiting excessive fungal proliferation rather than completely eliminating the pathogen. \u003cem\u003eIn vivo\u003c/em\u003e results further validate geraniol’s potential as an effective alternative to conventional fungicides, demonstrating substantial disease suppression in tomato plants without the adverse environmental impacts associated with synthetic chemicals. This study contributes valuable insights into the use of natural compounds for plant disease management, supporting citronella oil and its compounds as viable, eco-friendly options for integrated disease management programs. Future research should explore field trials and the potential synergistic effects of these compounds with other natural agents, advancing their practical application in sustainable agriculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to thank the Coordination for the Improvement of Higher Education Personnel (CAPES) and the Fundação de Amparo à Pesquisa do Espírito Santo (FAPES) for the support offered in the development of the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eO.P.A. and G.R.S. wrote the main manuscript text, A.V.N. and A.A.J. contributed to data analysis, O.A.L., S.P.S. and L.A.A. prepared figures 1–3, M.V.A.A. and G.F.S. assisted with data collection, L.A.P., M.F.C.S. and C.S.B. contributed to the experimental design, J.H.A.M. and L.M. supervised the project. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdams RP (2007) Identification of essential oil components by gas chromatography/mass spectrometry. Allured Publishing Corporation, Illinois USA, Carol Stream, 4th ed., 804 p\u003c/li\u003e\n\u003cli\u003eBissacotti AP, Londero P, Costabeber L (2021) Tomate: bot\u0026acirc;nica, produ\u0026ccedil;\u0026atilde;o, composi\u0026ccedil;\u0026atilde;o nutricional e benef\u0026iacute;cios \u0026agrave; sa\u0026uacute;de. Cad Cienc Tecnol 38:26643\u003c/li\u003e\n\u003cli\u003eBurketova L, Trda L, Ott PG, Valentova O (2015) Bio-based resistance inducers for sustainable plant protection against pathogens. Biotechnol Adv 33:994\u0026ndash;1004\u003c/li\u003e\n\u003cli\u003eCoppo JC, Stangarlin JR, Mioranza TM, Coltro-Roncato S, Kuhn OJ, Schwan-Estrada KRF (2017) Rev Cienc Agroambientais 15:92\u0026ndash;99\u003c/li\u003e\n\u003cli\u003eCruz TPD, Alves FR, Mendon\u0026ccedil;a RF, Costa AV, Junior WJ, Pinheiro PF, Marins AK (2015) Atividade fungicida do \u0026oacute;leo essencial de \u003cem\u003eCymbopogon winterianus\u003c/em\u003e Jowit (Citronela) contra \u003cem\u003eFusarium solani\u003c/em\u003e. Biosci \u003cem\u003eJ\u003c/em\u003e 31:1\u0026ndash;8\u003c/li\u003e\n\u003cli\u003eDevi KP, Nisha SA, Sakthivel R, Pandian SK (2010) Eugenol (an essential oil of clove) acts as an antibacterial agent against \u003cem\u003eSalmonella typhi\u003c/em\u003e by disrupting the cellular membrane. J Ethnopharmacol 130:107\u0026ndash;115\u003c/li\u003e\n\u003cli\u003edos Santos ATB, Junior JSZ, Parreira LA, de Abreu KMP, de Oliveira Bernardes C, de Carvalho JR, Menini L (2021) Chemical identification and insecticidal effect of \u003cem\u003eTephrosia vogelii\u003c/em\u003e essential oil against \u003cem\u003eCerosipha forbesi\u003c/em\u003e in strawberry crop. Crop Prot 139:1\u0026ndash;6\u003c/li\u003e\n\u003cli\u003eFonseca MCM, Lehner MDS, Gon\u0026ccedil;alves MG, Paula J\u0026uacute;nior TJ, Silva AF, Bonfim FPG, Prado AL (2015) Potencial de \u0026oacute;leos essenciais de plantas medicinais no controle de fitopat\u0026oacute;genos. Rev Bras Plantas Med 17:45\u0026ndash;50\u003c/li\u003e\n\u003cli\u003eGama BF, Paiva GA, Silva LS, Medeiros TR, Rodrigues APS, Gibbert LF, Campos OR (2020) Use of essential oils in the control of the fungus \u003cem\u003eFusarium oxysporum\u003c/em\u003e isolated in northern MT. 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Ind Crops Prod 36:238\u0026ndash;245\u003c/li\u003e\n\u003cli\u003eRodr\u0026iacute;guez-Gonz\u0026aacute;lez \u0026Aacute;, \u0026Aacute;lvarez-Garc\u0026iacute;a S, Gonz\u0026aacute;lez-L\u0026oacute;pez \u0026Oacute;, da Silva F, Casquero PA (2019) Insecticidal properties of \u003cem\u003eOcimum basilicum\u003c/em\u003e and \u003cem\u003eCymbopogon winterianus\u003c/em\u003e against \u003cem\u003eAcanthoscelides obtectus\u003c/em\u003e, insect pest of the common bean (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.). Insects 10:151\u003c/li\u003e\n\u003cli\u003eRussiano CGS (2020) Volatility of essential oils in the control of \u003cem\u003eFusarium oxysporum\u003c/em\u003e in cherry tomato seeds. 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J Biosci Bioeng 123:308\u0026ndash;313\u003c/li\u003e\n\u003cli\u003eTavares LA, Rezende AA, Santos JL, Estevam CS, Silva AM, Schneider JK, Albuquerque-J\u0026uacute;nior RL (2021) \u003cem\u003eCymbopogon winterianus\u003c/em\u003e essential oil attenuates bleomycin-induced pulmonary fibrosis in a murine model. Pharmaceutics 13:679\u003c/li\u003e\n\u003cli\u003eVerma RS, Verma SK, Tandon S, Padalia RC, Darokar MP (2020) Chemical composition and antimicrobial activity of Java citronella (\u003cem\u003eCymbopogon winterianus\u003c/em\u003e Jowitt ex Bor) essential oil extracted by different methods. J Essent Oil Res 32:449\u0026ndash;455.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"fusary wilt, geraniol, citronellol, sustainable pest management, natural fungicides","lastPublishedDoi":"10.21203/rs.3.rs-7491381/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7491381/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFusarium wilt caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e is one of the most significant diseases affecting tomato cultivation, leading to substantial economic losses. In response to the growing demand for sustainable alternatives to chemical pesticides, essential oils have emerged as a promising solution for managing phytopathogens. This study investigated citronella essential oil (\u003cem\u003eCymbopogon winterianus\u003c/em\u003e) and its major compounds (citronellal, geraniol, and citronellol) as natural alternatives for controlling \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e, the causative agent of Fusarium wilt in tomatoes. Chemical analysis identified citronellal as the primary compound, followed by geraniol and citronellol. \u003cem\u003eIn vitro\u003c/em\u003e tests demonstrated significant antifungal activity, with geraniol and citronellol achieving IC\u003csub\u003e50\u003c/sub\u003e values of 0.144 \u0026micro;L.mL⁻\u0026sup1; and 0.207 \u0026micro;L.mL⁻\u0026sup1;, respectively, resulting in 100% inhibition of fungal mycelial growth at certain concentrations. Citronellal showed a fungistatic effect with 46% inhibition at 0.500 \u0026micro;L.mL⁻\u0026sup1;, suggesting a role in controlling fungal proliferation without causing complete fungal death. \u003cem\u003eIn vivo\u003c/em\u003e assays confirmed that geraniol effectively reduced Fusarium wilt symptoms in Santa Cruz tomato plants, achieving comparable effects to commercial fungicides at a concentration of 0.610 \u0026micro;L.mL⁻\u0026sup1;, and thus indicating its potential as a sustainable alternative for plant disease management. These findings underscore the promise of citronella oil and its major components as eco-friendly antifungal agents in agriculture.\u003c/p\u003e","manuscriptTitle":"In vitro and in vivo evaluation of citronella essential oil and its compounds in the control of fusarium wilt in tomato plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 15:56:55","doi":"10.21203/rs.3.rs-7491381/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7778e32d-cc6b-483f-bd10-e261fa2c0d9b","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-04T08:23:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 15:56:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7491381","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7491381","identity":"rs-7491381","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}