Antifungal activity and mechanism of limonene against Fusarium oxysporum, a pathogen of potato dry rot

Antifungal activity and mechanism of limonene against Fusarium oxysporum, a pathogen of potato dry rot | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Antifungal activity and mechanism of limonene against Fusarium oxysporum , a pathogen of potato dry rot Qianhao Xia , Pan Dong doi: https://doi.org/10.1101/2025.10.06.675995 Qianhao Xia 2 Chongqing Nankai Secondary School , 400030, Chongqing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pan Dong 1 Key Laboratory of Plant Hormones and Development Regulation of Chongqing, School of Life Sciences, Chongqing University , 401331, Chongqing, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: dongpan{at}cqu.edu.cn Abstract Full Text Info/History Metrics Preview PDF Abstract Potato is a pivotal food crop throughout the world. However, potato dry rot, a major potato disease caused by Fusarium species, seriously threatens potato production and quality worldwide. Chemical pesticides are widely used in agricultural practices for disease prevention and control, but their long-term use is harmful to human physical health and the environment. Therefore, there is an urgent need to develop environmentally friendly, antifungal, bio-based fungicides. Limonene, as a main component of plant essential oils, has strong antibacterial activity and is harmless to the human body. Limonene is easily degraded in the natural environment, making it a highly promising bio-based fungicide. However, the use of limonene to control potato dry rot has not yet been reported. This study determines the inhibitory mechanism of limonene on potato dry rot caused by Fusarium oxysporum . Limonene effectively suppressed colony growth of Fusarium oxysporum (half maximal inhibitory concentration (IC 50 ) = 8.32 μL/mL), changed its hyphal morphology, reduced its pathogenicity, decreased its spore germination rate, viability, toxicity, cell membrane integrity, and stability, and caused abnormalities during transcription and translation processes and an imbalance in the plant-–fungus interaction and its intracellular ion homeostasis of transition metals. In addition, limonene affected its stress resistance. The combined antibacterial activity of limonene and chemical fungicides showed that limonene and mancozeb had an additive effect, whereas its combination with hymexazol had a synergistic effect. At the phenotypic, cytological, and transcriptomic levels, this study elucidated the mechanism underlying the antifungal activity of limonene against Fusarium oxysporum . By integrating mechanistic insights with application prospects, this study offers new avenues for developing bio-based, environmentally friendly pesticides and a theoretical foundation for potato dry rot management. 1. Introduction Potato dry rot, a fungal disease caused by multiple Fusarium species, occurs widely worldwide, severely threatening global potato yields. Fusarium oxysporum , a widely distributed pathogenic species, causes wilting in potato plants throughout the growing season and dry rot in potato tubers during storage. Fusarium oxysporum is one of the most prevalent and virulent pathogens causing potato dry rot. It belongs to the taxonomic classification Imperfect fungi, Moniliales, Tuberculariaceae. It was first discovered and isolated from potato stem vascular tissue by Weiss in 1924 [ 1 ]. Fusarium oxysporum employs two widely accepted infection mechanisms. First, hyphae penetrate through plant wounds or root tips, causing vascular blockage within the plant, obstructing internal water and nutrient transport, which leads to plant death. Second, Fusarium oxysporum produces toxins, such as fusaric acid and plant cell wall-degrading enzymes, to destroy plant tissues, clog vascular bundles, and cause plant death. The use of biocontrol agents is a key strategy for suppressing the growth of Fusarium oxysporum . The screening of biocontrol agents remains a vital aspect of disease management. Similar to bioactive substances, microbial agents have promising application prospects. According to Sun et al. [ 5 ], fungi such as JK2015, Bacillus mucilaginosus , JK02, and JK01 inhibit Fusarium oxysporum growth, with JK2015 having the most significant effect. Extensive research has been conducted on the inhibitory effects of chemical pesticides against Fusarium oxysporum . However, the identification and study of the inhibitory activities of bioactive substances with fungistatic properties require further exploration. Bioactive substances with proven efficacy against Fusarium oxysporum , including thymol [ 2 ], chitosan, chito-oligosaccharides [ 3 ], perillaldehyde [ 4 ], L-menthone, and S-carvone [ 46 ], have been investigated. Limonene, a bioactive compound, is a monoterpene widely distributed in plants and a key component of plant essential oils that is characterized by its lemon-like aroma. Limonene is non-toxic and harmless to humans and livestock, readily biodegradable in natural environments, and widely utilized in the food and pharmaceutical industries. In food products, it serves as a natural flavoring agent and taste enhancer and functions as a preservative and antioxidant. In medicine, limonene has demonstrated potent anti-inflammatory, antibacterial, and anticancer potential [ 6 – 9 ]. Additionally, it has been used in insecticide, acaricide, herbicide, and fungicide formulations. For insect control, limonene exhibits varying degrees of lethality, repellency, and fumigant effects against pests, such as Heterodera schachtii [ 10 ], Pseudococcus [ 11 ], Drosophila melanogaster [ 12 ], Tribolium castaneum, Liposcelis bostrychophila , and Sitophilus zeamais [ 13 ]. The efficacy of limonene is positively correlated with the treatment concentration and duration [ 14 ]. Limonene exhibits fumigant effects against mites, such as Panonychus cirri [ 15 ], Tetranychus urticae [ 16 ], and Acarapis tarsonemidae [ 17 ], primarily exerting its activity by acting on the acetylcholinesterase in the nervous system [ 18 ]. Limonene inhibits weed growth by affecting seed germination and seedling development [ 19 ], affecting weeds such as Amaranthus retroflexus L., Convolvulus arvensis L., Rumex crispus L. [ 20 ], Sinapis arvensis L., Lolium rigidum , and Raphanus raphanistrum [ 21 ]. Limonene has demonstrated significant fungicidal activity. Specifically, Sedeek et al. [ 22 ] found that limonene inhibited the growth of four pathogenic fungi— Sclerotium rolfsii, Fusarium semtectium, Botrytis cinerea , and Rhizoctonia solani —by 70–100%, positioning it as a highly promising bio-based reagent with potent antifungal activity. Several studies have demonstrated that limonene inhibits Fusarium species [ 22 , 23 ]. Specifically, lemon essential oil (48.3% limonene content) at concentrations of 0.5–2.0% completely inhibited the growth of Fusarium graminearum [ 24 ]. Syrian rue essential oil (4.19% limonene content) exhibits inhibitory activity against Fusarium species, completely inhibiting the growth of Fusarium culmorum, Fusarium pseudograminearum, Fusarium proliferatum , and Fusarium graminearum at 5, 10, 15, and 20 μL/mL, respectively [ 25 ]. In addition to growth inhibition, studies have shown that limonene effectively suppresses toxin synthesis in pathogenic fungi. Specifically, 0.075 μL/mL limonene significantly inhibited the synthesis of Fumonisin B 1 (FB1) in Fusarium verticillioides [ 26 ], and 0.20 μL/mL limonene significantly suppressed deoxynivalenol (DON) toxin production in Fusarium graminearum and the expression of toxin synthesis-related genes TRI1, TRI4, TRI5, TRI6, TRI6, TRI11, and TRI101 [ 27 ]. Additionally, 3.9 mg/g ylang-ylang essential oil (6.40% limonene content) significantly inhibited DON and zearalenone (ZEA) toxin production in Fusarium graminearum [ 28 ]. Holy basil essential oil (3.73% limonene content) suppressed ZEA toxin synthesis in Fusarium graminearum by reducing the expression of toxin synthesis-related genes PKS4 and PKS13 [ 29 ]. Limonene inhibits the growth of some Fusarium species by disrupting fungal cell membrane and cell wall integrity and reducing its ATPase activity [ 30 ]. For instance, Zhang et al. [ 31 ] found that lemongrass essential oil (1.77% limonene content) acts on the cell membrane of Fusarium graminearum , disrupting its integrity and causing protein leakage. This oil also inhibits the activity of its pectin methyl-galacturonase, suppressing its growth and reducing its pathogenicity. A 5% limonene solution can disrupt the cell membrane, cell wall, and vacuole structures of Fusarium graminearum , interfering with its energy metabolism pathways and inhibiting its growth [ 27 ]. Although studies have confirmed the inhibitory effects of limonene on Fusarium oxysporum , its specific antibacterial mechanism against this pathogen remains unclear. Therefore, this study investigated the inhibitory effects of limonene on Fusarium oxysporum in potato considering the phenotype, cytology, application potential, and transcriptomics. This study aimed to reveal the antibacterial activity and mechanism of limonene against Fusarium oxysporum , thereby providing new insights for the development of environmentally friendly, bio-based pesticides to control potato dry rot and offering potential solutions for managing this disease. 2. Results 2.1 Limonene Inhibits Fusarium Oxysporum Growth In this experiment, dimethyl sulfoxide (DMSO) was used as the organic solvent. After culturing for 5 days in Petri dishes, comparative analysis revealed that the higher the limonene concentration within the range of 2.5–10 μL/mL, the smaller the Fusarium oxysporum colony diameter, the more pronounced the mycelial growth inhibition ( Figure 1A,I ), and the higher the inhibition rate ( Figure 1J ). This indicates a significant dose-dependent inhibitory effect of limonene on Fusarium oxysporum . Regression analysis using GraphPad Prism 10 software showed that the half maximal inhibitory concentration (IC 50 ) value of limonene was 8.32 μL/mL. Download figure Open in new tab Figure 1. Phenotypic analysis. (A) Fusarium oxysporum growth after 8 days of treatment with different limonene concentrations. (B) Untreated hyphae with limonene, 10 μm electron microscopy image. (C) Mycelia treated with limonene at the half maximal inhibitory concentration (IC50) concentration, photographed under a 10 μm electron microscope. (D) Untreated membrane surface with limonene, 50 μm electron microscopy image. (E) Surface of bacterial membrane treated with limonene at the IC 50 concentration, 50 μm electron microscopy image. (F) Spore germination in the limonene-treated group after 6 h of cultivation, observed under a 40-x microscope. (G) Spore germination in the control (CK) group after 6 h of cultivation, observed under a 40-x microscope. (H) Incidence of Fusarium oxysporum on potato slices treated with different limonene concentrations after 5 days of growth. (I) Diameter of Fusarium oxysporum colonies cultured on media containing different limonene concentrations for 5 days. (J) Inhibition rate of different limonene concentrations on Fusarium oxysporum after 5 days. (K) Pathogenic area of Fusarium oxysporum under different limonene concentrations after 5 days. (L) Inhibition rate of CK and the treatment group after 6 h of cultivation. Each process was repeated at least three times, and the data are the average ± standard deviation. The asterisk ( * ) indicates significant differences according to the t-test ( ** P < 0.01; *** P < 0.001; **** P < 0.0001). 2.2 Limonene Affects Fusarium Oxysporum Hyphal Morphology As shown in Figure 1B , compared to the control group, limonene-treated Fusarium oxysporum colonies exhibited denser hyphal growth and thicker hyphal layers. Scanning electron microscopy (SEM) observations revealed that hyphae in the control group exhibited a sturdier and fuller morphology, with more regular growth and tighter arrangement ( Figure 1B, D ). In contrast, hyphae in the treated group appeared thinner and shriveled, showing wrinkles on their surfaces. Their growth patterns were more chaotic, with increased twisting and entanglement ( Figure 1C, E ). The results indicate that limonene treatment significantly affected the morphology of Fusarium oxysporum mycelium at both the macroscopic and microscopic levels. 2.3 Effect of Limonene on the Pathogenicity of Fusarium Oxysporum When limonene-treated Fusarium oxysporum infected potato tubers, observations after 5 days revealed a sharp decrease in the disease area at limonene concentrations ranging from 0 to 10 μL/mL, indicating significantly reduced fungal pathogenicity. The effect of limonene on Fusarium oxysporum f. sp. solani pathogenicity exhibited a pronounced dosedependent relationship ( Figure 1H, K ). At limonene concentrations between 10 and 40 μL/mL, the potato lesion area remained unchanged, stabilizing around 10 mm. A slight rebound in lesion area occurred at 20 μL/mL due to: (1) highconcentration limonene undergoing oxidation on exposed potato cells, producing high-viscosity byproducts that formed a physical barrier obstructing cellular respiration and accelerating potato cell death, which caused the lesion area to expand. (2) Fusarium oxysporum leaves minor white starch residues after growth, which mix with mycelium, visually enlarging the white lesion area and thus affecting measurement data. 2.4 Limonene Reduces the Spore Germination Rate Microscopy observations revealed that Fusarium oxysporum predominantly produced ovoid and reniform minor conidia, with major conidia being extremely rare. Observation under a 40× microscope after 6 h revealed a significantly reduced spore germination rate in the limonene-treated group (at the IC 50 concentration) compared to the control ( Figure 1F, G ). Spore germination in limonene-containing medium was substantially lower than that in the control group ( Figure 1L ), indicating that limonene significantly inhibits Fusarium oxysporum spore germination. 2.5 Limonene Reduces Spore Activity The red dye propidium iodide (PI) stains dead cells that have lost cell wall integrity, and the green dye fluorescein diacetate (FDA) stains live cells. In this experiment, the staining rates of both dyes were calculated by counting the number of red and green fluorescent cells in the treatment and control groups, reflecting cell viability. In the control (CK) group, most cells were stained with FDA, and few were stained with PI ( Figure 2A –C). Live cells accounted for 82%, whereas dead cells constituted less than 10% ( Figure 2I, J ). The limonene-treated group exhibited predominantly PI-stained cells, and few were stained with FDA ( Figure 2D–F ). Live cells constituted 36%, whereas dead cells accounted for 60% ( Figure 2I, J ). These results indicate that limonene significantly reduces spore viability. Not all spores were successfully stained during the experiment; thus, the sum of staining percentages within the same group did not reach 100%. Increasing the dye concentration or prolonging the staining duration could enhance the dye staining efficiency. Download figure Open in new tab Figure 2. Cytological analysis. (A) Spores in the negative control (CK) group. (B) Spores stained with FDA in the CK group. (C) Spores stained with PI in the CK group. (D) Spores treated with limonene at the IC 50 . (E) Spores stained with FDA after treatment with limonene at the IC 50 . (F) Spores stained with PI after treatment with limonene at the IC 50 . (G) Microscopic images of mycelium fluorescence staining in the CK group. (H) Microscopic images of mycelium fluorescence staining in the limonene treatment group. (I) Spore staining rate of FDA fluorescent dyes. (J) Spore staining rate of PI fluorescent dye. Each process was repeated at least three times, and the data are the average ± standard deviation. The asterisk ( * ) indicates a significant difference according to a t-test ( **** P < 0.0001). 2.6 Limonene Does Not Affect Chitin Distribution in Fusarium Oxysporum Cell Walls Fluorescent brightener 28 (FB 28) stains chitin in fungal cell walls. In this experiment, after staining cultured fungi for 5 min and observing them under a fluorescence microscope, it was evident that Fusarium oxysporum hyphae were uniformly stained with blue fluorescence, and the septal structures on the hyphae were stained bright blue in both the CK ( Figure 2G ) and treated groups ( Figure 2H ). These results indicate that the chitin distribution in Fusarium oxysporum cell walls remained uniform before and after treatment, with no apparent structural defects induced by limonene. This suggests that limonene did not significantly affect the chitin distribution in Fusarium oxysporum cell walls. 2.7 Effect of Limonene on the Partial Stress Resistance of Fusarium Oxysporum The following five external growth environments were evaluated: high temperature, low temperature, high salinity, oxidative stress, and ultraviolet (UV) radiation. The colony growth status after 5 days of cultivation is shown in Figure 3A . GraphPad Prism 10 software was used to calculate the colony diameter and inhibition rates across treatment groups ( Figure 3B, C ). High and low temperatures, high salinity, and oxidative stress had significant inhibitory effects on the growth of Fusarium oxysporum , with high temperature having the strongest effect and UV irradiation having the weakest effect. When limonene was combined with chemical pesticides, the growth inhibition rates of Fusarium oxysporum under low and high temperatures, high salinity, and oxidative stress significantly increased compared to the limonene-only inhibition group and the groups subjected to stress alone. These results indicate that limonene significantly influences the stress resistance of Fusarium oxysporum , increasing its environmental sensitivity and reducing its pathogenicity. The combined treatment of high-temperature stress and limonene had pronounced effects on Fusarium oxysporum . Download figure Open in new tab Figure 3. Application potential analysis. (A) Growth of Fusarium oxysporum colonies under different stressors. (B) Bar chart of the Fusarium oxysporum colony diameter under different stress groups. (C) Bar chart of the growth inhibition rates of Fusarium oxysporum colonies under different stress groups. (D) Growth of Fusarium oxysporum colonies under different treatments. (E) Bar chart of the Fusarium oxysporum colony diameter under different treatment groups. (F) Bar chart of the growth inhibition rate of Fusarium oxysporum under different treatment groups. Each process was repeated at least three times, and the data are the average ± standard deviation. The asterisk ( * ) indicates a significant difference according to a t-test ( * P < 0.1; ** P < 0.01; *** P < 0.001). 2.8 Synergistic or Additive Effects of Limonene Combined with Chemical Fungicides Two commonly used fungicides—hymexazol and mancozeb—were selected to investigate the combined fungicidal efficacy of limonene and chemical pesticides. Through a literature review and cross-verification, the IC 50 of hymexazol and mancozeb against Fusarium oxysporum was determined to be 49.401 and 109.53 μg/mL, respectively [ 3 , 43 – 45 ]. When chemical pesticides were mixed with limonene and added to potato dextrose agar (PDA) medium for Fusarium oxysporum cultivation, the combined pesticide–limonene treatment group exhibited significantly enhanced inhibition compared to pesticide use alone ( Figure 3D, E ). Specifically, the inhibition rate was 22% in the medium containing only limonene and 27% in the medium containing only mancozeb; however, when combined, it reached 46%. In the medium containing only hymexazol, the inhibition rate was 44%; however, under the combined action of limonene and hymexazol, it reached 65% ( Figure 3F ). Calculations using the Jin formula [ 48 ] (see Section 4.8 ) revealed a Q-value of 1.068 (0.85 < Q 1.15) for synergistic inhibition by limonene and hymexazol. Thus, the combined use of mancozeb and limonene had an additive effect against Fusarium oxysporum , and the combination of hymexazol and limonene had a synergistic effect. This experiment demonstrates that the combined use of limonene and certain pesticides in agriculture can effectively reduce the reliance on chemical fungicides (pesticides) and enhance sterilization efficacy. 2.9 Analysis of Fusarium Oxysporum Transcriptome Sequencing Results Following Limonene Treatment 2.9.1 Overall Analysis of Transcriptome Sequencing Data Compared to the DMSO negative control group, 1884 differentially expressed genes (DEGs) were detected in limonene-treated Fusarium oxysporum , including 1027 downregulated genes and 857 upregulated genes ( Figure 4A ). Boxplots of the gene expression levels revealed that the overall gene expression was reduced in the limonene-treated samples compared to the control group ( Figure 4B ). Gene Ontology (GO) enrichment analysis revealed the top five terms: RNA polymerase II DNA-binding transcription factor activity, transmembrane transport, RNA polymerase II cis-regulatory sequence-specific DNA binding, DNA template transcription, and specific sequence DNA binding ( Figure 4C ). Furthermore, among the 20 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, 17 were related to metabolism, primarily involving carbohydrate metabolism and amino acid metabolism pathways ( Figure 4D ). Furthermore, analysis of protein–protein interaction networks (PPINs) revealed a general downregulation trend in most networks, with genes in the ribosome assembly-related protein interaction network showing significant overall downregulation ( Figure 4E ). Download figure Open in new tab Figure 4. Transcriptome analysis. (A) Significant difference gene histogram. The vertical axis represents the corresponding number of upregulated and downregulated genes. Red represents the upregulated genes, and blue represents the downregulated genes. (B) Gene expression box plot, showing the distribution of expression levels in each sample. The horizontal axis represents the sample, and the vertical axis represents log (FPKM+1). The box plot for each region includes five statistical measures (maximum, upper quartile, median, lower quartile, and minimum), with the upper and lower quartiles containing 50% of the data. Box plots are designed for continuous variables. The dashed line is used to visually distinguish the differences in gene expression levels between the treatment group and the negative control group. (C) GO enrichment analysis bubble chart: the horizontal axis represents the proportion of genes in the corresponding entry to all genes in the entry, and the vertical axis represents different gene function entries. The size of the circle represents the number of genes enriched in the corresponding entry, and the larger the circle, the more genes enriched in the pathway. Color represents enrichment significance; circles indicate that the gene function is associated with both upregulated and downregulated genes; the upper triangle indicates only upregulated genes; and the lower triangle indicates only down-regulated genes. (D) KEGG enrichment analysis bar chart. The horizontal axis represents pathway names, and the vertical axis represents the number of enriched genes. The enriched pathways and the degree of enrichment are shown in the bar chart. (E) Protein–protein interaction network (PPIN) diagram showing the differential gene protein interaction network, with circles representing genes, red circles representing upregulated genes, and blue circles representing downregulated genes. The line represents the interaction between genes, and the larger the circle, the more genes are connected to it. (F) Gene set enrichment analysis (GSEA) curve of genes related to steroid synthesis. (G) GSEA curve of ribosome synthesis-related genes. 2.9.2 Transcriptional Dysfunction in Fusarium Oxysporum Induced by Limonene Transcription factors serve as critical interfaces between genetic regulatory information and the transcription system in cells, governing gene expression levels [ 33 ]. Impairment of overall transcription factor activity may lead to reduced or abnormal gene expression, disrupting key biological processes, such as intracellular signaling, metabolic pathways, and cell cycle regulation. In this study, GO enrichment analysis revealed 167 genes associated with RNA polymerase II-specific DNA-binding transcription factor activity, of which 143 were downregulated. Among the downregulated genes, 106 were associated with RNA polymerase II cis-regulatory region sequence-specific DNA-binding transcription factor-related genes ( Table 1 ), suggesting that limonene induces widespread abnormal transcription factor gene expression, reducing the overall efficiency and accuracy of gene transcription in Fusarium oxysporum cells. This leads to cellular dysfunction, including slowed growth rates, metabolic abnormalities, and diminished responsiveness to external stimuli, inhibiting the growth of Fusarium oxysporum . View this table: View inline View popup Download powerpoint Table 1. Downregulated genes associated with specific DNA-binding transcription factor activity of polymerase II (top 20). 2.9.3 Limone-Induced Disruption of Plant– Fusarium oxysporum Interactions The lysine motif (LysM) is a widely distributed domain across prokaryotes and eukaryotes that recognizes and binds to polysaccharides, such as chitin in fungal cell walls, playing a crucial role in plant–fungal interactions. In plants, LysM functions as a pattern recognition receptor that identifies chitin on fungal surfaces, triggering chitinase release to defend against fungal invasion. To facilitate infection, fungi secrete effector molecules containing LysM (similar to those on plant cell surfaces) onto their cell walls to disrupt chitin-triggered plant immune activation [ 34 ]. In this study, GO and KEGG enrichment analyses and raw data screening identified 32 downregulated genes among 47 genes associated with amino acid transmembrane transporter activity pathways. Of these, 16 downregulated genes were related to lysine transmembrane transporter activity ( Table 2 ), demonstrating that limonene reduces lysine transmembrane transport efficiency, which indicates that limonene impedes extracellular lysine acquisition in Fusarium oxysporum , decreasing the intracellular lysine content. Of the 15 lysine synthesis genes screened, 14 were downregulated ( Table 3 ), demonstrating limonene’s inhibitory effect on intracellular lysine synthesis in Fusarium oxysporum , which reduces intracellular lysine levels. As lysine is a key precursor for LysM synthesis, inhibition of intracellular lysine production leads to a gradual decrease in the LysM content on the cell wall. This reduces the infection efficiency when confronted with plant immune activation, making the fungal cell wall more susceptible to degradation by plant-secreted chitinases and inducing fungal cell apoptosis. Thus, by inhibiting the intracellular lysine content through two pathways, limonene suppresses Fusarium oxysporum growth on plants. View this table: View inline View popup Download powerpoint Table 2. Genes related to lysine transmembrane transporter activity. View this table: View inline View popup Download powerpoint Table 3. Genes related to intracellular lysine synthesis. 2.9.4 Reduction in Fusarium oxysporum Toxicity Induced by Limonene DON is a highly toxic secondary metabolite produced by Fusarium species, inducing wilting and yield reduction in crops and triggering dizziness and vomiting in humans [ 35 ]. According to Ma [ 36 ], putrescine, as a representative polyamine, significantly induces the expression of FgTRI genes related to DON toxin synthesis, enhancing the toxicity of Fusarium oxysporum . In this study, GO enrichment analysis and raw data screening identified 37 putrescine transportrelated genes, of which 30 were downregulated ( Table 4 ). The differential expression of these genes likely reduced intracellular putrescine levels, indirectly decreasing DON synthesis efficiency in Fusarium oxysporum . This weakened its virulence and thus its growth. View this table: View inline View popup Download powerpoint Table 4. Genes related to putrescine transport (top 20). 2.9.5 Limonene-Induced Disruption of Intracellular Transition Metal Ion Homeostasis in Fusarium Oxysporum Fungi require transition metals, such as copper, iron, manganese, and zinc, to influence cellular biological events and serve as structural components of proteins or catalytic elements in enzymatic reactions, regulating crucial intracellular biochemical processes [ 37 ]. Copper ions are essential for fungi, serving as enzyme cofactors in biochemical processes and playing critical roles in aerobic respiration, detoxification, signal transduction, and iron transport [ 38 ]. Copper ion uptake begins with transporters on the cell membrane that facilitate its non-directional transport across the membrane [ 39 ]. Therefore, the activity of membrane copper transporters directly influences intracellular copper levels. Excessive free copper within cells can catalyze Fenton-type reactions, inducing oxidative stress and impairing cellular functions [ 40 ]. In this study, GO enrichment analysis identified 26 DEGs directly associated with copper transporter activity, of which 17 were downregulated ( Table 5 ). Limonene directly influenced the intracellular copper ion content of Fusarium oxysporum by suppressing the expression of copper transporters, disrupting intracellular copper ion homeostasis, and inhibiting fungal activity. Furthermore, raw data screening revealed that 72.5% of the 40 DEGs associated with intracellular iron ion homeostasis were downregulated ( Table 6 ) and that 54% of the 11 DEGs associated with intracellular manganese ion homeostasis regulation were downregulated ( Table 7 ). These results indicate that limonene reduces the activity and growth of Fusarium oxysporum by inducing an imbalance in intracellular transition metal homeostasis. View this table: View inline View popup Download powerpoint Table 5. Downregulated genes related to copper ion transmembrane transporter View this table: View inline View popup Download powerpoint Table 6. Downregulated genes related to maintaining intracellular iron ion homeostasis (top 20). View this table: View inline View popup Download powerpoint Table 7. Downregulated genes related to maintaining intracellular manganese ion homeostasis 2.9.6 Limonene-Induced Decline in Cell Membrane Integrity and Stability of Fusarium Oxysporum Ergosterol is a type of steroid. As a key component of many microbial cell membranes and the characteristic sterol of fungi, ergosterol is an essential constituent of fungal cell membranes. Its stable structure and high specificity play a crucial role in ensuring cell viability, membrane fluidity, and membrane integrity [ 41 , 42 ]. When ergosterol production decreases in fungal cells, its content on the fungal cell membrane decreases, which reduces the fluidity and integrity of the fungal cell membrane, thereby affecting cellular life activities. Gene set enrichment analysis (GSEA) of steroid synthesis-related genes revealed a significant downregulation trend with backward enrichment ( Figure 4F ). KEGG enrichment analysis indicated that 18 of 28 steroid synthesis-related genes were downregulated. Further validation with raw data confirmed that 11 of these downregulated genes were involved in ergosterol synthesis ( Table 8 ). This indicates that limonene disrupts membrane fluidity and integrity in Fusarium oxysporum cells by inhibiting intracellular ergosterol synthesis, thereby suppressing its growth. View this table: View inline View popup Download powerpoint Table 8. Downregulated genes related to ergosterol synthesis. 2.9.7 Limonene-Induced Abnormalities in Fusarium oxysporum Translation Function During translation, the small ribosomal subunit (SSU) first binds to mRNA and then associates with the large ribosomal subunit (LSU) to initiate translation. When LSU levels decrease, the cellular translation efficiency declines because the SSU cannot properly pair with the LSU. Reduced translation efficiency diminishes the protein synthesis capacity of fungal cells, slowing fungal growth, weakening metabolic activity, and altering cellular morphology. In this experiment, GSEA revealed that genes associated with intracellular ribosome synthesis exhibited a downward trend and were enriched in the downstream region ( Figure 4G ). KEGG enrichment analysis and PPIN ( Figure 4E ) showed that 59 of the 63 genes related to ribosome biosynthesis were downregulated. Among the downregulated genes, 26 were associated with LSU synthesis ( Table 9 ). These results indicate that limonene inhibits the overall translation efficiency of Fusarium oxysporum cells by suppressing LSU synthesis, impairing protein production and inhibiting fungal growth. View this table: View inline View popup Download powerpoint Table 9. Downregulated LSU synthesis-related genes (top 20). 3. Discussion In recent years, studies have demonstrated that bioactive compounds extracted from plants or animals exhibit excellent antibacterial effects against Fusarium oxysporum , such as thymol [ 24 ], chitosan, chito-oligosaccharides [ 23 ], perillaldehyde [ 4 ], L-menthone, and S-carvone [ 46 ]. This study found that limonene similarly has a pronounced inhibitory effect on the growth of Fusarium oxysporum , significantly suppressing colony development, altering the mycelial structure, reducing the pathogenicity, spore activity, and stress resistance, disrupting cell membrane integrity and stability, and causing abnormalities in biochemical processes, including fungal cellular transcription, translation, fungus–plant interactions, toxin synthesis, and ion homeostasis. Transcriptome sequencing is widely used to analyze molecular mechanisms. This study employed a multi-faceted approach combining analyses to validate the identified inhibitory pathways. Through integrated analysis and verification using GO, KEGG, PPIN, GSEA, and raw data screening, six highly probable inhibitory pathways were identified. Of these, the DON toxin inhibition and ergosterol biosynthesis pathways showed high consistency with findings from other relevant studies. For example, Dambolena et al. [ 26 ] demonstrated that limonene significantly inhibits FB1 synthesis in Fusarium verticillioides . Jian et al. [ 27 ] reported that limonene suppresses the expression of DON toxin synthesisrelated genes in Fusarium graminearum . These findings align with our study’s conclusions, supporting that limonene inhibits DON synthesis. Additionally, studies have shown that plant essential oils containing limonene disrupt Fusarium cell membranes. For instance, Zhang et al. [ 31 ] demonstrated that lemongrass essential oil acts on the cell membranes of Fusarium graminearum , compromising their integrity. This aligns with our study’s conclusions, demonstrating that limonene likely disrupts ergosterol synthesis, damaging the fungal cell membrane structure. The other four inhibitory pathways were similarly supported by results from at least three analytical methods. Potato dry rot typically occurs during transportation and storage. Consequently, environmental conditions significantly influence its development. Stress analysis revealed that under limonene treatment, the pathogen Fusarium oxysporum f. sp. patatorum had reduced tolerance to high temperatures (37°C). Although high temperatures and limonene effectively inhibit the growth of Fusarium oxysporum , they pose significant hazards to potato storage. According to Rastovski et al. [ 47 ], the optimal storage temperature for edible potatoes is 2–4°C, which is far below the 37°C used in high-temperature stress studies, indicating that combined high-temperature stress and limonene treatment lacks practical value. In addition to high-temperature stress, the combined application of low-temperature stress (4°C), high-salinity stress, and limonene exhibited significant inhibitory effects on the growth of Fusarium oxysporum . Notably, low-temperature stress (4°C) falls within the optimal potato storage range. Under these conditions, limonene treatment provides the most favorable conditions for controlling Fusarium oxysporum during storage. Chemical pesticides are extensively used in modern agricultural production. Contemporary agriculture is exploring the combined use of bioactive substances with chemical pesticides to reduce chemical pesticide use and enhance antimicrobial efficacy, thus mitigating environmental issues arising from excessive pesticide application. This study revealed that the combination of limonene with mancozeb had an additive effect against Fusarium oxysporum , and its combination with hymexazol exhibited a synergistic effect. The concentration of hymexazol used was 49.401 μg/mL, which is significantly lower than the mancozeb concentration of 109.53 μg/mL, indicating that the combination of limonene and hymexazol has promising application prospects. hymexazol has long been used in agriculture to control various plant fungal diseases, such as Fusarium wilt, damping-off, and yellow wilt. When synergized with limonene, it can reduce the reliance on this chemical pesticide and enhance its antimicrobial efficacy. 4. Materials and Methods 4.2 Effect of Limonene on Fusarium Oxysporum Hyphal Growth To prepare the PDA medium, 200 g of potato, 20 g of glucose, and 15 g of agar were combined. Peeled and diced potatoes were simmered in deionized water until they became soft and mushy. After straining, glucose and agar powder were added to the filtrate and simmered while stirring constantly until the agar dissolved. The solution was poured into glass containers and autoclaved at 121°C for 1.5 h. On a sterile bench, limonene was emulsified in 0.3 mL DMSO and then added to PDA at 60°C. The final volume was adjusted to 30 mL to achieve limonene concentrations of 40.0, 20.0, 10.0, 5.0, 2.5, and 0 μL/mL. In a Petri dish, 10 mL was added. A 6-mm-diameter circular agar plug with mycelium, harvested from the periphery of a 5-day-old Fusarium oxysporum PDA plate, was placed at the center of a plate containing limonene-diluted PDA medium. The 0 μL/mL limonene + DMSO group was considered as CK. Each group (limonenetreated and CK) included three biological replicates. The prepared PDA plates were incubated at 28°C for 5 days and then observed. Colony diameters were measured using the cross-measurement method. The inhibition rate (%) was calculated using the following formula: Inhibition rate (%) = [(Control colony diameter (C) − Treated colony diameter (T)] / (Control colony diameter (C) − Disc diameter (D)) × 100%. GraphPad Prism 10 software was used to plot curves and calculate the IC 50 . 4.2 Effect of Limonene on Fusarium Oxysporum Hyphal Morphology To prepare potato dextrose broth (PDB), 200 g of potato was mixed with 20 g of glucose. Peeled and diced potatoes were simmered in deionized water until soft and mushy. After filtering, glucose was added to the filtrate and simmered until the glucose dissolved. The solution was transferred to a glass container and autoclaved at 121°C for 1.5 h. On a sterile bench, limonene was emulsified in 0.2 mL DMSO and added to PDB at 60°C to a final volume of 20 mL. After transfer to a 50-mL centrifuge tube, the limonene concentration was adjusted to the IC 50 value. Spores were washed from 5-day-old Fusarium oxysporum cultures on PDA with sterile water, and the spores in the suspension were counted using a hemocytometer. The spore concentration was diluted to 10 6 spores/mL with water and then added to PDB. The centrifuge tube was sealed with plastic wrap. CK was 20 mL PDB containing only 0.2 mL DMSO. There were three biological replicates per group. After 5 days of incubation, mycelium was removed, mounted on slides, and observed under a microscope. PDA containing limonene at the IC 50 concentration was prepared using the method described in Section 4.1 , with the DMSO group considered as CK. There were three biological replicates for each group. A 6-mm-diameter fungal plug from 5-day-old Fusarium oxysporum cultures on PDA plates was used to inoculate the center of the medium and was incubated at 28°C for 3 days before removal. Using forceps, an appropriate amount of biofilm was removed from the medium and observed under a field emission scanning electron microscope (SU8600, Hitachi, Tokyo, Japan). 4.3 Effect of Limonene on the Pathogenicity of Fusarium Oxysporum Potatoes were peeled and cut into rectangular slices measuring 4 cm × 3 cm × 0.7 cm. Fusarium oxysporum colonies were grown on PDA plates supplemented with different limonene concentrations (0, 2.5, 5, 10, 20, and 40 μL/mL) for 5 days. A 6-mm-diameter plug was cut from the outer edge of each colony, following the method described in Section 2.2 . The resulting fungal plug was placed at the center of a potato slice, which was then placed on filter paper and moistened with sterile water on a tray. The tray was covered with plastic wrap and sealed with another tray. After incubating at room temperature (25°C) for 5 days, the lesion diameters were measured using the cross-section method, and inhibition curves were plotted using GraphPad Prism 10 software. 4.4 Effect of Limonene on the Germination Rate of Fusarium Oxysporum Spores were washed from 5-day-old Fusarium oxysporum cultures on PDA with sterile water, and the spores in the suspension were counted using a hemocytometer. The spore concentration was diluted to 10 6 spores per milliliter with water. Following the method described in Section 4.1 , PDA medium containing limonene at the IC 50 concentration and the CK medium were prepared. Using a glass rod, the spore suspension was spread evenly on the PDA medium and incubated in a constant-temperature incubator at 28°C in the dark for 6 h. Spore germination was observed under an inverted fluorescence phase contrast microscope (Leica, Wetzlar, Germany) at 40× magnification. Germination was considered when the germ tube length reached half the spore length. Photographs were taken using a five-point sampling method. The total number of spores and the number of germinated spores under the lens were counted. The germination rate was calculated using the following formula: Germination rate = (Number of germinated spores/Total number of spores) × 100%. The germination rates of the treatment and control groups were compared. There were three biological replicates for each group. 4.5 Effect of Limonene on the Spore Activity of Fusarium Oxysporum Diluted spore suspensions and PDB medium were prepared as described in Section 4.2 , and the limonene concentration in PDB was adjusted to the IC 50 value. CK was PDB without limonene addition. There were three biological replicates per group. The prepared PDB was incubated in a constant-temperature shaking incubator at 28°C for 10 h. The supernatant was removed by centrifugation at 6000 r/min for 5 min, and the spores were resuspended in sterile water. FDA and PI solutions were added to the resuspended spore suspension at concentrations of 10 and 5 μg/mL, respectively, and incubated in the dark for 15–20 and 5 min, respectively, for staining. They were then observed under the same microscope described in Section 4.4 . FDA stains viable cells green at an excitation wavelength of 488–530 nm, and PI stains dead cells red at an excitation wavelength of 493–636 nm. The staining rates for FDA and PI were calculated using the five-point method with the following formula: Staining rate for fluorescent dye A = (Number of successfully stained spores/Total number of spores) × 100%. 4.6 Effect of Limonene on Cell Wall Integrity in Fusarium Oxysporum Diluted spore suspensions and PDB medium were prepared as described in Section 4.2 . Spore suspensions were added to PDB and incubated at 28°C in the dark. When the spore germination rate reached 85% (germination was defined as the length of the germ tube reaching half the length of the spore), limonene was added at the IC 50 concentration. Culture was continued in the dark at 28°C for 24 h, with an established CK group. After incubation, mycelia were stained with FB 28 for 5 min and observed under a fluorescence microscope to compare the fluorescence distribution differences between treated and CK groups. There were three biological replicates for each group. 4.7 Effect of Limonene on the Stress Sensitivity of Fusarium Oxysporum Growth conditions significantly influence the development status of Fusarium oxysporum , and altering the external environments is a common physical method for inhibiting fungal growth. In this experiment, five growth conditions were established to determine the effect of limonene on the stress resistance of Fusarium oxysporum : high temperature (37°C), low temperature (4°C), UV irradiation, high salinity (NaCl), and oxidative stress (H 2 O 2 ). Four experimental groups were established under each growth condition: CK, limonene (at IC 50 concentration), stress, and stress + limonene. There were three biological replicates for each group. For high-temperature stress, Fusarium oxysporum was first incubated at 37°C for 24 h before being transferred to normal conditions (28°C in the dark). For low-temperature stress, the fungus was incubated at 4°C for 24 h before being transferred to normal conditions. For UV irradiation stress, Fusarium oxysporum was exposed to UV light for 30 min before being placed under normal conditions for cultivation. For high-salt stress, a 0.3 M NaCl solution was used, and the strain was directly placed under normal conditions for cultivation. For oxidative stress, a 0.5 mM H 2 O 2 solution was used, and the strain was directly placed under normal conditions for cultivation. The culture media used in the experiment were prepared according to the methods described in Section 4.1 . After 5 days of incubation, the colony diameters were measured using the cross-section method, and inhibition rates were calculated using the formula given in Section 4.1 . Figures were plotted using GraphPad Prism 10 software. 4.8 Synergistic Effects of Limonene and Chemical Fungicides Hymexazol and mancozeb are commonly used pesticides for controlling potato dry rot. PDA was prepared according to the method described in Section 4.1 . Pesticides were then added to the prepared PDA to achieve hymexazol and mancozeb concentrations of 10, 20, 40, 80, 160, and 200 mg/L. After incubating the inoculated media at 28°C for 5 days, the colony diameters were measured, and the IC 50 values for hymexazol and mancozeb were calculated using GraphPad Prism 10 software. The IC 50 values of hymexazol and mancozeb against Fusarium oxysporum can also be obtained from literature sources [ 3 , 43 – 45 ]. To determine the combined inhibitory effect of limonene and the two fungicides, this experiment comprised four groups: CK, limonene (at IC 50 concentration), fungicide, and limonene + fungicide. There were three biological replicates for each group. For all treatment groups, the fungicide concentration was set at the IC 50 , and the limonene concentration was set at its IC 50 . After incubating the medium at 28°C for 5 days, the colony diameter was measured, and the inhibition rates were calculated using the formula given in Section 4.1 . Finally, the Jin formula [ 48 ] (Q = E × (A + B) / (EA + EB − EA × EB)) was applied to determine the Q value, identifying the synergistic interaction type between limonene and the two pesticides. Here, E(A+B), EA, and EB denote the inhibition rates of the combination of two drugs, drug A alone, and drug B alone, respectively. Q < 0.85, 0.85 < Q 1.15 indicate antagonistic, additive, and synergistic effects, respectively. 4.9 Transcriptome Sequencing Analysis of Fusarium Oxysporum Following Limonene Treatment To further investigate the mechanism by which limonene affects Fusarium oxysporum , transcriptome sequencing was performed after treatment with limonene at the IC 50 concentration. First, 0.2 mL DMSO and limonene was added to PDB and then diluted to 20 mL in a 50-mL centrifuge tube to achieve a limonene concentration of 8.32 μL/mL (IC 50 ). This process was repeated nine times to prepare nine centrifuge tubes. Second, 0.2 mL DMSO was added to PDB, and the volume was adjusted to 20 mL in a 50-mL centrifuge tube as CK. This step was repeated nine times, preparing nine centrifuge tubes. Third, a Fusarium oxysporum spore suspension was added to the centrifuge tubes to achieve a spore concentration of 10 −5 . The tubes were sealed and incubated in a constant-temperature shaking incubator. Fourth, according to Zhu et al. [ 32 ], Fusarium oxysporum enters the logarithmic growth phase after 3 days of incubation. After 5 days of growth, the tubes were removed from the incubator and centrifuged at a low speed, and the medium was discarded. The cells were washed two or three times with sterile water and centrifuged at 5000 r/min for 5 min. The supernatant was discarded, and the cell pellet was collected. Finally, the experimental group was divided into three subgroups, each containing three centrifuge tubes. The bacterial cells from each subgroup were pooled, transferred to 2-mL screw-cap conical centrifuge tubes, and frozen in liquid nitrogen for 3–4 h. Samples were then stored at −80°C. Transcriptome sequencing was performed by Tsingke Biotechnology Co., Ltd. (Chongqing, China). The sequencing results were quantified to determine the expression level using String Tie v2.2.1. Differential expression analysis was performed using the R v4.4.1 package ‘DESeq2.’ Functional enrichment analyses, including GO enrichment analysis, KEGG pathway enrichment analysis, and GSEA, were conducted using the R v4.4.1 package ‘cluster Profiler.’ In addition, BLAST was employed to compare the target gene against proteins in the STRING database ( https://cn.string-db.org/ ) and to identify homologous proteins, and a PPIN was subsequently constructed based on their interaction relationships. In the research, GO enrichment analysis was performed using the GO database ( http://www.geneontology.org/ ). Pathway enrichment analysis was conducted using the KEGG database ( https://www.kegg.jp/ ). The reference genome of Fusarium oxysporum was downloaded from: http://ftp.ensemblgenomes.org/pub/fungi/release-59/fasta/fungi_ascomycota3_collection/fusarium_oxysporum_gca_900096695/dna/ . The corresponding gene annotation was obtained from: https://ftp.ensemblgenomes.ebi.ac.uk/pub/fungi/release-59/gff3/fungi_ascomycota3_collection/fusarium_oxysporum_gca_900096695/ . 5. Conclusions Limonene, as a widely available and inexpensive bioactive compound, has potential in fungicidal and insecticidal applications. This study showed that limonene significantly inhibited Fusarium oxysporum , the pathogen causing potato dry rot, and had a significant effect on its colony growth, mycelial morphology, pathogenicity, and spore activity. It may inhibit the growth of Fusarium by disrupting cell membrane integrity and stability, leading to abnormalities in biochemical processes, such as fungal transcription, translation, fungus–plant interactions, toxin synthesis, and ion homeostasis. Furthermore, limonene could be combined with abiotic stressors or traditional pesticides to achieve enhanced inhibitory effects, demonstrating significant potential for reducing fungicide use and protecting the environment. References 1. ↵ Weiss F. Survey for potato wilt in Pennsylvania and Southern New York . American Journal of Potato Research , 1924 , 1 ( 11 ): 243 – 244 . OpenUrl 2. ↵ Liu Y , Liu S , Lou X , et al. Antifungal activity and mechanism of thymol against Fusarium oxysporum, a pathogen of potato dry rot, and its potential application . Postharvest Biology and Technology , 2022 , 192 : 112025 . OpenUrl 3. ↵ Ren Jie . Study on the mechanism of chitosan/Chito oligosaccharide in preventing and controlling potato dry rot . Chongqing University , 2021 . doi: 10.27670/d.cnki.gcqdu.2021.003276 . OpenUrl CrossRef 4. ↵ Liu S. Study on the mechanism of perillaldehyde in preventing and controlling potato dry rot . Chongqing University , 2023 . 5. ↵ Sun Q , Li C , Yi X. The antibacterial effect of several biocontrol bacteria on Fusarium oxysporum and the rapid detection of their inclusive growth . Chinese Journal of Tropical Agriculture , 2019 , 39 ( 12 ): 36 – 41 .DOI: CNKI: SUN: RDNK.0.2019-12-008. OpenUrl 6. ↵ Ma D C , Dou D L , Liu F. Potential and application of plant volatile organic compounds in agricultural disease control . Chinese Journal of Pesticide Science , 2022 , 24 ( 4 ): 682 – 691 . OpenUrl 7. Dassanayake M K , Chong C H , Khoo T J , et al. Synergistic field crop pest management properties of plant-derived essential oils in combination with synthetic pesticides and bioactive molecules: a review . Foods , 2021 , 10 ( 9 ): 2016 . OpenUrl PubMed 8. Das S , Ghosh A , Mukherjee A. Nanoencapsulation-based edible coating of essential oils as a novel green strategy against fungal spoilage, mycotoxin contamination, and quality deterioration of stored fruits: an overview . Frontiers in Microbiology , 2021 , 12 : 768414 . OpenUrl PubMed 9. ↵ Ravichandran C , Badgujar P C , Gunde V P , et al. Review of toxicological assessment of D-limonene, a food and cosmetics additive . Food and Chemical Toxicology , 2018 , 120 : 668 – 680 . OpenUrl PubMed 10. ↵ Ibrahim M A , Kainulainen P , Aflatuni A , et al. Insecticidal, repellent, antimicrobial activity and phytotoxicity of essential oils: with special reference to limonene and its suitability for control of insect pests . Agriculture and Food Science in Finland , 2001 , 10 ( 3 ): 243 – 259 . OpenUrl 11. ↵ Hollingsworth R G. Limonene, a citrus extract, for control of mealybugs and scale insects . Journal of Economic Entomology , 2005 , 98 ( 3 ): 772 – 779 . OpenUrl CrossRef PubMed 12. ↵ Zhang R F , Jin Y , Shi G L. Biological activity of D-limonene, the main component of orange peel . Journal of Zhejiang Agricultural Sciences , 2013 ( 1 ): 73 – 74 . 13. ↵ Yang S. Study on fumigation activities of plant essential oil to stored grain pest . Wuhan: Huazhong Agricultural University , 2008 . 14. ↵ Hebeish A , Fouda M M G , Hamdy I A , et al. Preparation of durable insect repellent cotton fabric: limonene as insecticide[J] . Carbohydrate Polymers , 2008 , 74 ( 2 ): 268 – 273 . OpenUrl 15. ↵ Zhang X X. Study on the activity of D-limonene in the control of Panonychus citri McGregor . Guangzhou : South China Agricultural University , 2018 . 16. ↵ Badawy M E I , Abdelgaleil S A M , Mahmoud N F , et al. Preparation and characterization of essential oil and monoterpene nanoemulsions and acaricidal activity against two-spotted spider mite (Tetranychus urticae Koch) . International Journal of Acarology , 2018 , 44 ( 7 ): 330 – 340 . OpenUrl 17. ↵ Ellis M D , Baxendale F P. Toxicity of seven monoterpenoids to tracheal mites (Acari: Tarsonemidae) and their honey bee (Hymenoptera: Apidae) hosts when applied as fumigants . Journal of Economic Entomology , 1997 , 90 ( 5 ): 1087 – 1091 . OpenUrl CrossRef 18. ↵ Abdelgaleil S A M , Mohamed M I E , Badawy M E I , et al. Fumigant and contact toxicities of monoterpenes to Sitophilus oryzae L. and Tribolium castaneum Herbst and their inhibitory effects on acetylcholinesterase activity . Journal of Chemical Ecology , 2009 , 35 ( 5 ): 518 – 525 . OpenUrl CrossRef PubMed 19. ↵ Isman M B , Miresmailli S , Machial C. Commercial opportunities for pesticides based on plant essential oils in agriculture, industry and consumer products . Phytochemistry Reviews , 2011 , 10 ( 2 ): 197 – 204 . OpenUrl 20. ↵ Bozhuyuk A U. Herbicidal activity and chemical composition of two essential oils on seed germinations and seedling growths of three weed species . Journal of Essential Oil-Bearing Plants , 2020 , 23 ( 4 ): 821 – 831 . OpenUrl 21. ↵ Amri I , Gargouri S , Hamrouni L , et al. Chemical composition, phytotoxic and antifungal activities of Pinus pinea essential oil . Journal of Pest Science , 2012 , 85 ( 2 ): 199 – 207 . OpenUrl 22. ↵ Sedeek M S , Al-Mahallawi A M , Hussien R A A , et al. Hexosomal dispersion: a nano-based approach to boost the antifungal potential of Citrus essential oils against plant fungal pathogens . Molecules , 2021 , 26 ( 20 ): 6284 . OpenUrl PubMed 23. ↵ Marei G I K , Rasoul M A A , Abdelgaleil S A M. Comparative antifungal activities and biochemical effects of monoterpenes on plant pathogenic fungi . Pesticide Biochemistry and Physiology , 2012 , 103 ( 1 ): 56 – 61 . OpenUrl CrossRef 24. ↵ Krzyśko-Łupicka T , Walkowiak W , Białoń M. Comparison of the fungistatic activity of selected essential oils relative to Fusarium graminearum isolates . Molecules , 2019 , 24 ( 2 ): 311 . OpenUrl PubMed 25. ↵ Bouajaj S , Romane A , Benyamna A , et al. Essential oil composition, phytotoxic and antifungal activities of Ruta chalepensis L . leaves from High Atlas Mountains (Morocco). Natural Product Research , 2014 , 28 ( 21 ): 1910 – 1914 . OpenUrl PubMed 26. ↵ Dambolena J S , Lopez A G , Canepa M C , et al. Inhibitory effect of cyclic terpenes (limonene, menthol, menthone and thymol) on Fusarium verticillioides MRC 826 growth and fumonisin B1 biosynthesis . Toxicon , 2008 , 51 ( 1 ): 37 – 44 . OpenUrl PubMed 27. ↵ Jian Y Q , Chen X , Ma H Q , et al. Limonene formulation exhibited potential application in the control of mycelial growth and deoxynivalenol production in Fusarium graminearum . Frontiers in Microbiology , 2023 , 14 : 1161244 . OpenUrl PubMed 28. ↵ Kalagatur N K , Mudili V , Kamasani J R , et al. Discrete and combined effects of Ylang-Ylang (Cananga odorata) essential oil and gamma irradiation on growth and mycotoxins production by Fusarium graminearum in maize . Food Control , 2018 , 94 : 276 – 283 . OpenUrl CrossRef 29. ↵ Kalagatur N K , Mudili V , Siddaiah C , et al. Antagonistic activity of Ocimum sanctum L. essential oil on growth and zearalenone production by Fusarium graminearum in maize grains . Frontiers in Microbiology , 2015 , 6 : 892 . OpenUrl PubMed 30. ↵ Hou T Y , Sana S S , Li H Z , et al. Essential oils and its antibacterial, antifungal and anti-oxidant activity applications: a review . Food Bioscience , 2022 , 47 : 101716 . OpenUrl 31. ↵ Zhang Z L , Mo Z L , Zhang X L , et al. The antifungal activity and action mechanism of lemongrass (Cymbopogon flexuosus) essential oil against Fusarium avenaceum . Journal of Essential Oil-Bearing Plants , 2022 , 25 ( 3 ): 536 – 547 . OpenUrl 32. ↵ Zhu Y , Che J , Xiao R , et al. Growth characteristics of Fusarium oxysporum Schl . China Agricultural Bulletin , 2007 , 23 ( 8 ): 373 – 376 . OpenUrl 33. ↵ Kadonaga J T , et al. Regulation of RNA polymerase II transcription by sequence-specific DNA binding . Cell , 2004 , 116 : 247 – 257 . OpenUrl CrossRef PubMed Web of Science 34. ↵ Gao F , Zhang B S , Zhao J H , et al. Deacetylation of chitin oligomers increases virulence in soil-borne fungal pathogens . Nature Plants , 2019 , 5 ( 11 ): 1167 – 1176 . OpenUrl PubMed 35. ↵ Sun J , Zhao C C , Du W , et al. Research advances in enzymatic detoxification of deoxynivalenol . Science and Technology of Cereals, Oils and Foods , 2024 , 32 ( 2 ): 121 – 126 . OpenUrl 36. ↵ Ma T. Study on the mechanism of putrescine regulating the synthesis of DON toxin in Fusarium graminearum infection . Zhejiang University , 2021 : 28 – 31 . 37. ↵ Moraes D , et al. Fungal Stress Mechanisms and Responses . Chapter seven , 2024 , 189 – 229 . 38. ↵ Smith A D , Logeman B L , Thiele D J. Copper acquisition and utilization in fungi . Annual Review of Microbiology , 2017 , 71 : 597 – 623 . OpenUrl CrossRef PubMed 39. ↵ Festa R A , Thiele D J. Copper: An essential metal in biology . Current Biology , 2011 , 21 ( 21 ): R877 – R883 . OpenUrl CrossRef PubMed 40. ↵ Imlay J A , Linn S. DNA damage and oxygen radical toxicity . Science , 1988 , 240 ( 4857 ): 1302 – 1309 . OpenUrl Abstract / FREE Full Text 41. ↵ Li W , Liu J , Shi J. Study on the extraction process and content determination of ergosterol in Cordyceps militaris from different habitats in Shandong . Chinese Journal of Anti-infective pharmacy . 2014 : 302 – 305 . 42. ↵ Li Y , Li M , Li S , et al. Study on extraction and purification of ergosterol from yeast . Military Medical Sciences , 2021 , 45 ( 3 ): 192 – 198 . OpenUrl 43. ↵ Ning N , Liu Q , Xian W , et al. Indoor toxicity determination of five fungicides against potato dry rot fungi . Journal of Qinghai University , 2021 , 39 ( 1 ): 31 – 37 . OpenUrl 44. Zhao D , Wei W , Zhang D , et al. Study on indoor fungicide screening and disease prevention of potato dry rot . Hubei Agricultural Sciences , 2017 , 56 ( 17 ): 3268 - 3271 +3279. OpenUrl 45. ↵ Wang L , Xu T , Li L , et al. Biological characteristics and indoor fungicide screening of potato dry rot pathogen . Journal of Xinjiang Agricultural University , 2016 , 39 ( 3 ): 222 – 226 . OpenUrl 46. ↵ Al-Mughrabi , K.I. , Coleman , W.K. , Vikram , A. , Poirier , R. , Jayasuriya , K.E. , 2013 . Effectiveness of essential oils and their combinations with aluminum starch octenyl succinate on potato storage pathogens . J. Essent. Oil Bear. Plants 16 , 23 – 31 . OpenUrl 47. ↵ Rastovski A , van Es A. Storage of Potatoes: Post-Harvest Behaviour, Store Design, Storage Practice, Handling . Wageningen, The Netherlands: Pudoc , 1987 . 48. ↵ Jin Z , Tang Y , Li X. About the evaluation of drug combination . J Clin Pharmacol . 2004 , 44 ( 7 ): 698 – 705 . OpenUrl View the discussion thread. Back to top Previous Next Posted October 06, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Antifungal activity and mechanism of limonene against Fusarium oxysporum, a pathogen of potato dry rot Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Antifungal activity and mechanism of limonene against Fusarium oxysporum , a pathogen of potato dry rot Qianhao Xia , Pan Dong bioRxiv 2025.10.06.675995; doi: https://doi.org/10.1101/2025.10.06.675995 Share This Article: Copy Citation Tools Antifungal activity and mechanism of limonene against Fusarium oxysporum , a pathogen of potato dry rot Qianhao Xia , Pan Dong bioRxiv 2025.10.06.675995; doi: https://doi.org/10.1101/2025.10.06.675995 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)