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. 2023 Apr 24;24(8):932–946. doi: 10.1111/mpp.13340

Plant secondary metabolite citral interferes with Phytophthora capsici virulence by manipulating the expression of effector genes

Wen Song 1, Zhiyuan Yin 1, Xinyu Lu 2, Danyu Shen 1,✉, Daolong Dou 1
PMCID: PMC10346372  PMID: 37092279

Abstract

Phytophthora capsici is a notorious pathogen that infects various economically important plants and causes serious threats to agriculture worldwide. Plants deploy a variety of plant secondary metabolites to fend off pathogen attacks, but the molecular mechanisms are largely unknown. In this study, we screened 11 plant secondary metabolites to evaluate their biofumigation effects against P. capsici, and found that citral, carvacrol, and trans‐2‐decenal exhibited strong antimicrobial effects. Intriguingly, a low concentration of citral was effective in restricting P. capsici infection in Nicotiana benthamiana, but it was unable to inhibit the mycelial growth. A high concentration of citral affected the mycelial growth and morphology, zoospore germination, and cell membrane permeability of P. capsici. Further investigations showed that citral did not induce expression of tested plant immunity‐related genes and reactive oxygen species (ROS) production, suggesting that a low concentration of citral could not trigger plant immunity. Moreover, RNA‐Seq analysis showed that citral treatment regulated the expression of some P. capsici effector genes such as RxLR genes and P. cactorum‐fragaria (PCF)/small cysteine‐rich (SCR)74‐like genes during the infection process, which was also verified by reverse transcription‐quantitative PCR assay. Five candidate effector genes suppressed by citral significantly facilitated P. capsici infection in N. benthamiana or inhibited ROS triggered by flg22, suggesting that they were virulence factors of P. capsici. Together, our results revealed that plant‐derived citral exhibited excellent inhibitory efficacy against P. capsici by suppressing vegetative growth and manipulating expression of effector genes, which provides a promising application of citral for controlling Phytophthora blight.

Keywords: antimicrobial activity, mechanism, Phytophthora blight, plant secondary metabolite, virulence gene

Plant‐derived citral exhibited excellent inhibitory efficacy against Phytophthora capsici by suppressing vegetative growth and manipulating the expression of effector genes.

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1. INTRODUCTION

Phytophthora capsici is a highly destructive oomycete pathogen of vegetable crops and causes great yield losses in agricultural production (Gevens et al., 2008; Kamoun et al., 2015; Lamour et al., 2012). Unlike most Phytophthora species with narrow host ranges, P. capsici attacks a wide variety of agriculturally important vegetable crops, such as pepper, snap bean, cucumber, tomato, and pumpkin (Hausbeck & Lamour, 2004; Meitz et al., 2010). P. capsici preferentially causes disease under wet and warm (25–28°C) conditions, and typical symptoms include leaf and stem blight, damping‐off, fruit rot, stem rot, and wilting (Granke et al., 2009). This hemibiotrophic pathogen moves from a biotrophic phase to a necrotrophic phase within 24–48 h (Kamoun et al., 2015). Once introduced to a crop field, P. capsici spreads rapidly and disease is difficult to control (Gobena et al., 2012), therefore P. capsici causes substantial economic losses worldwide.

Management of Phytophthora blight caused by P. capsici relies on modifications in cultural practices, crop rotation, and judicious use of chemical fungicides (Hausbeck & Lamour, 2004). Among these, the application of chemical fungicides is still the most effective control strategy. Some fungicides, such as metalaxyl, have been used against P. capsici for many years (Wang, Liu, et al., 2020). However, the reckless use of chemical fungicides causes serious problems in food safety and environmental pollution (Siegenthaler & Hansen, 2021). Moreover, P. capsici has developed resistance to many of the commonly used fungicides (Dunn et al., 2010; Parra & Ristaino, 2001). Thus, it is urgently required to develop ecofriendly alternatives such as plant‐derived metabolites to achieve control of Phytophthora blight in the field.

Plants have evolved to synthesize a diverse range of plant secondary metabolites (PSMs) to protect themselves against pathogen attacks in case of infection (Hammerbacher et al., 2019; Piasecka et al., 2015; Radulovic et al., 2013). The formation of PSMs is the result of interaction between plants and pathogens over millions of years. PSMs constitute a large group of structurally diversified compounds and are mainly represented by terpenoids, phenylpropanoids, fatty acid derivatives, and amino acid derivatives (Dudareva et al., 2006), most of which do not cause serious toxicity to the environment (Cadena et al., 2018). PSMs are crucial in plant defence against herbivores and pathogens, and many PSMs have been reported to inhibit or even kill pathogens in vitro (Sanchez‐Vallet et al., 2010; Sellam et al., 2007). For example, (S)‐limonene is a volatile phytoanticipin that plays a key role in suppressing Xanthomonas oryzae growth in rice seedlings (Lee et al., 2016). Allyl isothiocyanate causes rapid inhibition of Fusarium solani via hyphal deformity and electrolyte leakage (Li et al., 2020). 10‐deacetylbacatin III significantly inhibits the vegetative and reproductive growth of P. capsici and has both protective and curative activities on pepper plants (Wang et al., 2018). However, it is not clear whether these PSMs play a role in the interference with pathogen pathogenicity during plant–pathogen interaction. Several studies have provided valuable clues regarding this question. The Arabidopsis defence compound sulforaphane functions primarily by inhibiting Pseudomonas syringae type III secretion system (T3SS) genes, which are essential for pathogenesis (Wang, Yang, et al., 2020). Plant polyphenols can directly target P. syringae RhpRS, which results in bacterial virulence being switched off via phosphorylation‐related crosstalk (Xie et al., 2021). These two examples suggest that PSMs can directly target virulence‐related pathways of pathogenic microbes to inhibit microbial virulence in plants. Thus, it is worth exploring more mechanisms of PSMs targeting virulence genes or pathways of pathogenic microbes.

Citral (3,7‐dimethyl‐2,6‐octadienal), an acyclic monoterpene formed by mixing two isomeric compounds (neral and geranial), can be obtained from the essential oils of herbal plants, such as lemon balm (Melissa officinalis), lemongrass (Cymbopogon citratus), and common vervain (Verbena officinalis) (Capetti et al., 2021; Dudai et al., 2005). In addition to its importance in industrial and medical applications, citral has shown promising biological activities, including antifungal, antibacterial, and antioxidant properties (Li et al., 2015). Citral has broad‐spectrum inhibitory effects against various plant pathogens, such as Alternaria alternata (Wang, Jiang, et al., 2019), Penicillium digitatum (OuYang et al., 2016), and Fusarium moniliforme (Kishore et al., 2007). Citral is known to disrupt the cell membrane permeability and membrane integrity of Penicillium italicum by the release of cell constituents, extracellular pH, and leakage of potassium ions (Tao et al., 2014). Citral disrupts the tricarboxylic acid cycle pathway and damages the mitochondrial membrane permeability of P. digitatum (Zheng et al., 2015). RNA‐Seq analysis has revealed that citral suppresses the expression of genes related to the ergosterol biosynthesis pathway to exhibit its antifungal activity against P. digitatum (OuYang et al., 2016). However, information about the molecular inhibitory mechanism of citral on plant pathogens is still limited and thus requires further study.

In this study, we screened 11 plant‐derived PSMs and found that citral, carvacrol, and trans‐2‐decenal exhibited high antimicrobial activity against P. capsici in vitro. We selected citral to explore its antimicrobial mechanisms further. Citral not only inhibited vegetative growth, but also manipulated P. capsici virulence gene expression to achieve control of P. capsici in plants. Together, these findings contribute to advancing our understanding of antimicrobial activity of citral against Phytophthora pathogens. The use of citral represents an efficient alternative in the management of Phytophthora blight.

2. RESULTS

2.1. Screening 11 plant‐derived PSMs with high antimicrobial activity against P. capsici

Many PSMs have effective antimicrobial activities and have been widely used to control plant diseases (Hammerbacher et al., 2019). To screen PSMs for high inhibitory effect on P. capsici, 11 PSMs with antifungal or antibacterial activities reported previously were used in this study (Table S1). Based on the widely used double Petri dish dual‐culture method, the colony diameters of P. capsici after fumigation with 25 μg/mL of each PSM were measured. In contrast to the control treatment, these PSMs exhibited a wide range of antimicrobial activity (Figure 1). (S)‐Limonene, (Z)‐3‐hexenyl butyrate, and carvone could hardly inhibit the mycelial growth of P. capsici, and linalool and eugenol showed a low inhibition rate of less than 30%. The remaining six PSMs had a higher inhibition rate of more than 60%. Notably, citral, carvacrol, and trans‐2‐decenal could greatly inhibit P. capsici with at least 85% inhibition rate (Figure 1a,b), suggesting that the three PSMs could significantly suppress the vegetative growth of P. capsici in vitro. Therefore, these three PSMs were selected for subsequent analysis.

FIGURE 1.

FIGURE 1

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Comparison of the antimicrobial activities of 11 plant secondary metabolites (PSMs) against Phytophthora capsici in vitro. The 11 PSMs exhibited different inhibitory activities on P. capsici colony morphology (a) and inhibition rate (b). The colony was cultured on V8 medium at 25°C, followed by fumigation with 25 μg/mL of each PSM for 2 days. An equal volume of ethanol was used as a control (CK). Bars represent SE for three independent biological experiments.

To further investigate the minimum inhibitory concentration of citral, carvacrol, and trans‐2‐decenal, different concentrations of PSMs were evaluated after fumigation for 3 days. As indicated in Figure 2 and Table S2, citral, carvacrol, and trans‐2‐decenal showed an inhibitory effect on the mycelial growth of P. capsici with IC50 (half maximal inhibitory concentration) values of 17.33, 5.67, and 8.66 μg/mL, respectively. Moreover, at the low concentration level, carvacrol and trans‐2‐decenal had a low inhibition rate, while citral nearly had no inhibitory effect on P. capsici (Figure 2a–c).

FIGURE 2.

FIGURE 2

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The minimum inhibitory concentrations (MICs) of citral, carvacrol, and trans‐2‐decenal to Phytophthora capsici. Each plant secondary metabolite was dissolved in ethanol to obtain a series of diluted concentrations. P. capsici was exposed to different concentrations of citral (a), carvacrol (b), and trans‐2‐decenal (c) for 3 days, and colony morphology, colony diameter, and inhibition rate were evaluated. An equal volume of ethanol was used as a control (CK). The experiment was repeated three times with similar results.

2.2. Low concentration of citral can inhibit P. capsici infection in planta

PSMs have been widely reported to control plant diseases by inhibiting the vegetative growth of plant pathogens (Sellam et al., 2007; Wang, Liu, et al., 2019). However, it is not clear whether PSMs can also control plant diseases through direct inhibition of the pathogenicity. Thus, we selected the low concentration of each PSM that had little or no effect on the vegetative growth of P. capsici (Figure 2), and then examined whether they could affect the pathogenicity of P. capsici on plants. To this end, detached Nicotiana benthamiana leaves were inoculated with P. capsici, followed by fumigation with low concentrations of citral, trans‐2‐decenal, and carvacrol in an airtight container. As shown in Figure 3a–c, there was no significant difference in the lesion area and relative pathogen biomass between fumigation with 1.6 μg/mL of trans‐2‐decenal and the control treatment. Another PSM, carvacrol, resulted in similar results to trans‐2‐decenal (Figure 3a–c). Notably, the N. benthamiana leaves treated with 1.4 μg/mL citral exhibited significantly smaller lesion areas and lower pathogen biomass when compared with the control (Figure 3a–c), and thus citral was selected for further study.

FIGURE 3.

FIGURE 3

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A low concentration of citral directly inhibited Phytophthora capsici infection in Nicotiana benthamiana. (a) Disease symptoms on N. benthamiana leaves after fumigation with low concentrations of each plant secondary metabolite (PSM). Detached N. benthamiana leaves were inoculated with P. capsici, and a low concentration of each PSM was placed adjacent to the leaves. An equal volume of ethanol was used as a control (CK). After 36 h, the inoculated leaves were photographed under UV light. (b) The lesion area of inoculated leaves after fumigation with low concentration of each PSM for 36 h. The lesion area was calculated from three independent biological replicates with at least eight leaves per replicate. (c) Relative biomass of P. capsici in the inoculated leaves after fumigation with each PSM. Infected leaves were collected at 36 h after inoculation for DNA isolation and quantitative PCR analysis. (d) Methodology to test the inhibition effects of citral on P. capsici‐infected N. benthamiana plants. (e) Representative photographs showed that a low concentration of citral could inhibit P. capsici infection in N. benthamiana. Photographs were taken at 36 h after inoculation with P. capsici zoospores. The black‐brown sites showed the infected area, and the bright red areas showed the healthy parts. This assay included three replicates, and each replicate included five N. benthamiana plants. (f) Relative biomass of P. capsici in the inoculated N. benthamiana plants after fumigation with 1.4 μg/mL citral. Asterisks indicate a significant difference according to Student's t test; **p < 0.01.

Furthermore, we tested the inhibitory effects of citral on P. capsici in vivo. First, a double plastic cup dual‐culture method was set up (Figure 3d). The 3‐ to 4‐week‐old N. benthamiana plant was sprayed with P. capsici zoospores. Then 1.4 μg/mL of citral was placed on the cover of the soil surface, and another plastic cup was set upside down on the first one. Sealing film sealed the junction of the two plastic cups (Figure 3d). After 36 h of control treatment, the whole N. benthamiana plant was seriously diseased, and the majority of leaves were wilted (Figure 3e). However, fumigation with 1.4 μg/mL citral significantly limited the development of lesions and also reduced the relative pathogen biomass (Figure 3e,f). Taken together, in vitro and in vivo plant experiments suggested that a low concentration of citral could suppress P. capsici infection in N. benthamiana plants without affecting its mycelial growth.

2.3. Citral affects mycelial growth and morphology, zoospore germination, and cell membrane permeability of P. capsici

To delve deeper into the impact of citral on P. capsici, a thorough examination of various aspects pertaining to vegetative growth was conducted. First, we explored whether citral could affect mycelial growth and morphology. As indicated in Figure 2a, citral had an obviously adverse effect on the mycelial growth of P. capsici, and its inhibition rate could reach 100% at the high concentration of 45.8 μg/mL. Next, we investigated whether citral could affect mycelial morphology by optical microscope observation. As shown in Figure 4a, the mycelia of P. capsici grown on V8 plates presented typical morphology in the control treatment. After fumigation with 1.4 μg/mL citral, the mycelial morphology was similar to that in the control, suggesting that a low concentration of citral not only had no effect on mycelial growth but also had no effect on mycelial morphology. However, after fumigation with 22.9 μg/mL citral for 3 days, the mycelia developed a large number of shorter offshoots at their tops (Figure 4a). These results show that a higher concentration of citral affected the vegetative growth of P. capsici in both colony diameter and mycelial morphology.

FIGURE 4.

FIGURE 4

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Citral affected the mycelial morphology, zoospore germination, and cell membrane permeability of Phytophthora capsici. (a) Effects of citral on the mycelial morphology of P. capsici. After fumigation with 1.4 and 22.9 μg/mL citral for 3 days, the mycelial morphology was observed and photographed under a light microscope. (b) Effects of citral on zoospore germination of P. capsici. An equal number of zoospores was cultured on V8 medium containing 1.4, 11.5, and 22.9 μg/mL citral. The germination rate of zoospores was recorded at 3, 6, and 12 h. (c) The effects of citral on malondialdehyde (MDA) content of P. capsici. After treatment with citral for 48 h, the mycelia of P. capsici were harvested to detect the MDA content using an MDA detection kit. The assays were performed three times. Asterisks indicate a significant difference according to Student's t test; **p < .01. CK, control (ethanol) treatment.

To explore the effects of citral on the secondary infection of P. capsici, we evaluated zoospore germination in the presence of citral. P. capsici zoospores were treated with different concentrations of citral, and the germination rates of zoospores were observed and calculated at 3, 6, and 12 h after treatment. As shown in Figure 4b, the germination rate of P. capsici in the control group reached 100% at 6 h, but it took 12 h to reach 93% after treatment with 1.4 μg/mL citral. When treated with higher citral concentrations of 11.5 and 22.9 μg/mL, the germination rates stayed low and did not exceed 15% even though treatment time was extended (Figure 4b). Therefore, these data indicated that citral in low concentrations delayed P. capsici zoospore germination, and inhibited zoospore germination in high concentrations.

Some reports have suggested that citral could destroy the integrity of the microbial cell membrane to develop its antagonistic activity (Tao et al., 2014). The impact of citral on the cell membrane integrity of P. capsici was evaluated using two separate methods. Malondialdehyde (MDA) is the main product of membrane lipid peroxidation, which has been widely used as an index of the oxidative injury of the cell membrane (Kong et al., 2012). We therefore evaluated whether citral could affect the cell membrane permeability of P. capsici by measuring MDA content in the presence of different concentrations of citral. The results revealed a significant difference in MDA content between the control and each concentration of citral; MDA content increased as the concentration of citral increased (Figure 4c). Additionally, the effect of citral on the membrane integrity was evaluated through the use of propidium iodide (PI), a red fluorescent dye that can enter the cells and stain DNA when the membrane has been damaged (Wang et al., 2022). As shown in Figure S1, the control showed no red fluorescence, indicating that the cell membrane was intact. However, when treated with 1.4 μg/mL citral, a slight red fluorescence was observed. A substantial amount of red fluorescence was observed when treated with 5.7 μg/mL citral. These results suggested that citral could affect P. capsici by disrupting the integrity of its cell membrane.

2.4. Citral does not activate plant defence responses

The above results show that fumigation using a low concentration (1.4 μg/mL) of citral did not affect the vegetative growth of P. capsici, but it significantly inhibited P. capsici infection in N. benthamiana. This phenomenon could occur for two possible reasons: citral could induce plant immunity or inhibit P. capsici pathogenicity. We tested whether citral could enhance plant immunity by measuring the expression changes of four marker genes for plant defence pathways. These genes were Cyp71D20 for pathogen‐associated molecular pattern (PAMP)‐triggered immunity, Plant Defensin 1.2 (PDF1.2) for the jasmonate/ethylene pathway, and Ethylene insensitive 3 (EIN3) and EIN3‐binding F‐box 2 (EBF2) for the ethylene pathway. The N. benthamiana leaves were exposed to 1.4 μg/mL citral, and the transcriptional difference of each gene was measured between citral and control at 0, 6, and 12 hr. As shown in Figure 5a, there was no obvious expression difference for these four genes after fumigation with citral, suggesting that the low concentration of citral did not induce the expression of these plant immunity‐related genes. Furthermore, another index of plant immunity, reactive oxygen species (ROS) production, was examined. Citral was used as a presumptive elicitor to test whether it could trigger ROS in N. benthamiana. The results showed that citral did not induce detectable ROS, whereas the positive control, bacterial flagellin flg22, induced a large amount of ROS (Figure 5b). These results therefore suggest that citral did not activate plant defence responses.

FIGURE 5.

FIGURE 5

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A low concentration of citral did not contribute to plant immunity. (a) Relative expression of four plant defence‐related genes after treatment with citral at different times. The transcript levels of the genes were measured by reverse transcription‐quantitative PCR, and NbActin was used to normalize relative expression. This experiment was repeated three times with similar results. (b) Production of reactive oxygen species in Nicotiana benthamiana treated with 1 μM flg22 or 1.4 μg/mL citral. This assay was repeated three times with similar results. CK, control (ethanol) treatment.

2.5. Citral manipulates the expression of P. capsici candidate effector genes based on RNA‐Seq analysis

Because a low concentration of citral did not induce plant immunity, we speculated that citral might inhibit the expression of virulence genes to reduce P. capsici infection in N. benthamiana. To find further clues, comparative RNA‐Seq analysis was performed. The N. benthamiana leaves were inoculated with P. capsici, followed by fumigation with or without citral. The total RNAs of two groups of samples after 36 h were extracted for subsequent RNA‐Seq sequencing. A total of 67.1 Gb of clean bases and 223.7 million clean reads were produced for six samples. An average of 40.38% and 36.35% clean reads derived from control and citral fumigation samples were mapped to the P. capsici genome, respectively. After filtration with the criteria of p < 0.05 and fold change >2 or <−2, a total of 115 P. capsici differentially expressed genes (DEGs) were identified between the control group and the citral fumigation group (Table S3). These DEGs included 62 up‐regulated and 53 down‐regulated genes (Figure 6a). Intriguingly, 40 DEGs, accounting for 34.78%, encoded secreted proteins. This proportion was significantly higher than that in the whole genome. Further functional annotation of the DEGs encoding the 40 secreted proteins resulted in many known effector genes, including 15 RxLR genes, three PcF/SCR74‐like genes, two elicitin‐like genes, and one NLP (necrosis‐ and ethylene‐inducing peptide 1‐like protein) gene (Figure 6b). This result suggests that fumigation with a low concentration of citral greatly affects the transcriptional expression of P. capsici effector genes. Among these 40 DEGs, 23 were down‐regulated while the remaining 17 were up‐regulated (Figure 6b), suggesting that fumigation with citral mainly suppresses the expression of P. capsici virulence genes during infection. Thus, we focused on the 23 down‐regulated DEGs in this study. These included eight RxLR genes, three PcF/SCR74‐like genes, two glycoside hydrolase family 43 genes, and others. Given that some members of these gene families have been reported to be related to the pathogenesis of Phytophthora pathogens, they were selected for further investigation.

FIGURE 6.

FIGURE 6

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Citral suppressed the expression of Phytophthora capsici virulence genes based on RNA‐Seq analysis. (a) Statistics of up‐regulated and down‐regulated P. capsici genes between citral treatment and the control. (b) Statistics of differentially expressed genes encoding secreted proteins. On the right‐hand side of the figure, various groups of differentially expressed genes (DEGs) encoding virulence proteins are displayed. (c) Validation of RNA‐Seq data by reverse transcription‐quantitative PCR analysis. The PcActin gene was used as the internal control. This assay was repeated three times. Asterisks indicate a significant difference according to Student's t test; **p < 0.01. CK, control (ethanol) treatment.

To validate the expression of candidate effector genes by RNA‐Seq analysis, five RxLR genes (PcAvh254, PcAvh478, PcAvh624, PcAvh214, and PcAvh252) and three PcF/SCR74‐like genes (PcSCR1, PcSCR2, and PcSCR3), were examined using reverse transcription‐quantitative PCR (RT‐qPCR) analysis. All eight genes were down‐regulated based on RNA‐Seq data (Table S3). The RT‐qPCR analysis showed that seven genes excluding PcAvh252 exhibited the same variation tendency of expression after fumigation with citral, which was consistent with the RNA‐Seq results (Figure 6c). These results highlight the accuracy and reproducibility of RNA‐Seq analysis in this study.

2.6. Effector genes inhibited by citral contribute to P. capsici virulence

Based on RNA‐Seq analysis, the expression of a subset of P. capsici candidate virulence genes was suppressed by a low concentration of citral during the interaction between P. capsici and N. benthamiana. Among these repressed virulence genes, RxLR genes and PcF/SCR74‐like genes were relatively enriched (Figure 6b). The two types of candidate effector genes have been reported to play important roles in modulating plant defence responses (Jiang & Tyler, 2012; Lin et al., 2020), raising the question of whether they could interfere with the plant immune system in this study. To test this hypothesis, five repressed P. capsici genes verified by RT‐qPCR (Figure 6c) were selected for subsequent experimental analysis of their virulence functions. The five genes comprised three RxLR genes (PcAvh214, PcAvh254, and PcAvh478) and two PcF/SCR74‐like genes (PcSCR1 and PcSCR2). We investigated whether these five P. capsici genes could induce cell death, inhibit cell death triggered by a well‐known oomycete PAMP INF1 (a major eliticin from Phytophthora infestans) (Kanneganti et al., 2006), facilitate Phytophthora infection, or inhibit ROS caused by flg22.

First, the five P. capsici proteins were transiently expressed in the leaves of N. benthamiana by agroinfiltration, and then cell death induction or suppression assays were performed. In the cell death induction assay, green fluorescent protein (GFP) was used as a negative control. No cell death phenotype was observed for any of the five proteins after 4 days of infiltration (Figure 7a), suggesting that no protein could trigger plant cell death. After that, proteins inhibiting INF1‐induced cell death were also tested by infiltration of INF1 into the expressed proteins or GFP for 24 h. Cell death symptoms were detected at the injection site on overexpression of INF1 on the third day for all samples. After infiltration of each virulence protein, only the positive control Al106 could inhibit cell death induced by INF1, whereas all the five proteins could not (Figure 7a). Immunoblot analysis showed that all five proteins were normally expressed in N. benthamiana (Figure 7b). Next, we examined whether or not they had effects on the ROS production induced by the flg22 PAMP. A luminol‐based assay was carried out to measure ROS production after flg22 treatment in N. benthamiana leaves transiently expressing each of the five proteins or the control GFP. As shown in Figure 7c, three proteins (PcAvh214, PcAvh254, and PcSCR1) were similar to the control GFP, which showed a strong ROS burst in response to flg22. However, the accumulation of ROS was significantly reduced when PcAvh478 or PcSCR2 was expressed (Figure 7c), suggesting that the two proteins suppressed the ROS burst triggered by flg22. Moreover, to explore whether the five proteins could facilitate Phytophthora infection, they were transiently expressed in N. benthamiana using Agrobacterium‐mediated transient expression and then the leaves were inoculated with P. capsici. Compared to the GFP control, the halves of leaves expressing PcAvh478, PcAvh254, and PcSCR1 exhibited much larger lesion areas and relative pathogen biomass than the halves expressing GFP (Figure 7d–f), indicating that these three proteins could promote P. capsici infection in N. benthamiana. In contrast, PcSCR2 and PcAvh214 did not promote P. capsici infection in N. benthamiana leaves. Taken together, these results suggested that the five P. capsici effector genes whose expression was suppressed by citral contributed to P. capsici virulence via either promoting Phytophthora infection or inhibiting ROS triggered by flg22, rather than inducing cell death or inhibiting INF1‐induced cell death (Table 1).

FIGURE 7.

FIGURE 7

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Candidate genes whose expression was inhibited by citral contributed to Phytophthora capsici virulence. (a) Five proteins neither induced cell death nor inhibited the cell death triggered by INF1. Each of the five proteins was infiltrated first, and then INF1 was infiltrated after 24 h. The leaves were photographed after infiltration for 4 days. The numbers in the figure represent different proteins: 1, GFP; 2, PcAvh254; 3, PcAvh214; 4, PcAvh478; 5, PcSCR2; 6, PcSCR1; 7, Al106 + INF1; 8, GFP + INF1; 9, PcAvh254 + INF1; 10, PcAvh214 + INF1; 11, PcAvh478 + INF1; 12, PcSCR2 + INF1; 13, PcSCR1 + INF1. Al106 is an effector from Apolygus lucorum that can inhibit the cell death caused by INF1 and was used as a positive control. This assay was repeated three times. Asterisks indicate a significant difference according to Student's t test; **p < 0.01, *p < 0.05. (b) Immunoblot analysis of five proteins. The asterisks represent the position of the target protein. The anti‐HA antibody was used to detect the expression of the protein. Western blot showed that all the proteins were expressed normally. Ponceau S staining of the RuBisCO protein was used as the equal loading control. (c) Production of reactive oxygen species (ROS) excited by flg22. ROS accumulation was recorded over 30 min. (d) Transient expression of five candidate proteins in Nicotiana benthamiana promoted plant susceptibility to P. capsici. The infiltrated area expressing green fluorescent protein (GFP) or each protein was inoculated with P. capsici. At 36 h postinoculation (hpi), the lesion area was calculated from one experiment that contained eight leaves. This assay was repeated three times with similar results. (e) The lesion area of inoculated leaves expressing each protein. (f) The relative biomass of P. capsici in the inoculated leaves expressing each protein. Infected leaves were collected at 36 hpi and used for DNA extraction and quantitative PCR analysis.

TABLE 1.

Summary of functions of five Phytophthora capsici virulence proteins.

Function PcAvh214 PcAvh254 PcAvh478 PcSCR1 PcSCR2
Promote P. capsici infection No Yes Yes Yes No
Inhibit reactive oxygen species caused by flg22 No No Yes No Yes
Induce cell death No No No No No
Inhibit cell death caused by INF1 No No No No No
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3. DISCUSSION

Plants have evolved a complex defence system to overcome abiotic and biotic stresses. Plants synthesize a large variety of PSMs that can function to protect themselves against herbivores and microbes (Hammerbacher et al., 2019; Zaynab et al., 2018). Some groups of PSMs, including terpenoids, steroids, flavonoids, and alkaloids, have proven roles in plant defence response to pathogens (Sanchez‐Vallet et al., 2010; Sellam et al., 2007). In contrast to the massive use of chemical fungicides, some PSMs have gradually become alternatives due to their environmentally friendly property and antimicrobial activities (Piasecka et al., 2015). Considering Phytophthora blight is a severe threat to agricultural production with increasing pathogen resistance to traditional chemical fungicides, PSMs are becoming promising alternatives against Phytophthora pathogens. At present, only a few PSMs, such as 10‐deacetyl‐bacatin III, zedoary turmeric oil, and esculetin, have been reported to inhibit Phytophthora pathogens (Wang et al., 2018, 2021; Wang, Liu, et al., 2019). In this study, we screened 11 plant‐derived PSMs with known antifungal or antibacterial activities to evaluate their antimicrobial activity against P. capsici. We found that most of the tested PSMs could inhibit the mycelial growth of P. capsici, suggesting that many PSMs had a broad antimicrobial spectrum. Among these tested PSMs, citral, carvacrol, and trans‐2‐decenal had strong inhibitory effects on the mycelial growth of P. capsici, suggesting that the three PSMs had the potential to become promising fungicides against P. capsici in sustainable agriculture.

Understanding the mode of action of antimicrobial PSMs is important for their potential use in the agricultural field. Currently, most studies focus on the effects of PSMs on microbial vegetative growth. Some PSMs, including 10‐deacetyl‐bacatin III, zedoary turmeric oil, and esculetin, have been reported to inhibit mycelial growth and morphology, spore production, and germination of P. capsici (Wang et al., 2018, 2021; Wang, Liu, et al., 2019). It is widely accepted that the normal morphology of mycelia plays a key role in maintaining microbial viability, and zoospores serve as the major survival structure and secondary inoculum source. We found that citral affected mycelial growth, mycelial morphology, and zoospore germination, suggesting that citral could target both the mycelia and zoospores of P. capsici. Previous studies reported that zedoary turmeric oil and esculetin could damage the cell membrane of P. capsici. Likewise, we also found that citral affected the cell membrane permeability of P. capsici. Disruption of the cell membrane usually led to the cellular leakage of soluble proteins as well as nucleic acids from the mycelia, indicating it is a commonly used mode of action of some PSMs with potential as fungicides (Pei et al., 2020).

In addition to affecting microbial vegetative growth, PSMs should theoretically have other modes of antimicrobial action. During the interaction between pathogens and plants, pathogens usually secrete a large variety of virulence proteins into host cells to promote infection. This phenomenon raised a question of whether or not PSMs could interfere with virulence genes, and there is relatively little knowledge about this hypothesis. Recently, an Arabidopsis secondary metabolite sulforaphane was reported to function primarily by inhibiting P. syringae T3SS genes, which were essential for pathogenesis (Wang, Yang, et al., 2020). Another study demonstrated that plant‐derived polyphenols directly targeted RhpRS, the key regulator of P. syringae T3SS, which resulted in reduced bacterial virulence via phosphorylation‐related crosstalk (Xie et al., 2021). These two bacterial studies have supplied support for this hypothesis. In this study, we designed a series of experiments to further explore this hypothesis in P. capsici. At a low concentration (1.4 μg/mL), citral did not affect the mycelial growth of P. capsici. However, it suppressed P. capsici infection in N. benthamiana based on in vitro and in vivo plant experiments. Further study showed that citral did not activate plant defence responses, leading to the possibility that it might affect the pathogenicity of P. capsici. RNA‐Seq analysis indicated that citral manipulated the expression of some virulence genes, which were confirmed to participate in several pathogenesis processes such as the promotion of P. capsici infection and inhibition of ROS triggered by flg22. To the best of our knowledge, this is the first study regarding PSMs directly affecting the pathogenicity of oomycete pathogens via regulation of virulence genes. How citral inhibits the expression of virulence genes is not clear. We speculate that citral might target some regulatory genes such as transcription factors that bind with promoter regions of virulence genes. The detailed action mechanisms need to be further investigated.

In conclusion, the current study identified three PSMs, citral, carvacrol, and trans‐2‐decenal, that exhibited strong antimicrobial activity against P. capsici. Further investigation of citral suggested that it had two antimicrobial modes: inhibiting vegetative growth and manipulating the expression of effector genes of P. capsici. These results support the potential use of citral as a natural antimicrobial agent for protecting crops against devastating Phytophthora disease.

4. EXPERIMENTAL PROCEDURES

4.1. Plant secondary metabolites, microorganisms, and plant materials

The PSMs used in this study were citral (97%, Aladdin), linalool (98%, Macklin), trans‐2‐hexenal (98%, Macklin), carvacrol (97%, Aladdin), nerolidol (98%, Macklin), S‐limonene (98%, Macklin), Z‐3‐hexenyl butyrate (98%, Macklin), nonanal (96%, Macklin), carvone (97%, Macklin), trans‐2‐decenal (95%, Macklin), and eugenol (98.5%, Hushi) (Table S1). All the PSMs were stored at 4°C in the dark and were dissolved in ethanol to different concentrations when used.

P. capsici LT263 was cultivated on V8 agar (10% V8 juice, 0.02% CaCO3, 1.5% agar) plates at 25°C in the dark. Escherichia coli DH5α was used for plasmid construction and was cultured at 37°C. Agrobacterium tumefaciens GV3101, which was used for plasmid transformation, was cultured at 28 °C.

N. benthamiana plants were grown in the greenhouse at 25°C under a 16:8 h (light: dark) photoperiod for 4–6 weeks with a relative humidity of 60%.

4.2. In vitro inhibitory effects of PSMs on P. capsici

The inhibitory effects of PSMs on P. capsici were evaluated according to a previous method with modifications (Ma et al., 2022). Briefly, a mycelial plug (5 mm diameter) of P. capsici was placed at the centre of a Petri dish containing V8 juice medium, and then PSM was added to the upper part of the sealed Petri dish. To conduct the initial screening, we put 1 μL of each PSM on the lid of the culture dish with a space volume of 35 mL, resulting in a concentration of approximately 25 μg/mL. This equates to 25 μg of PSM per 1 mL of space volume. An equal volume of ethanol was used as the control. To evaluate the inhibitory effects of different concentrations of selected PSMs, we initially dissolved the PSM in ethanol to create a stock solution. Working dilutions of citral were prepared with concentrations of 10, 40, 80, 160, and 320 μl/mL (volume of PSM/volume of ethanol). We then added 10 μL of each concentration of citral dilution to the lid of a 60‐mL culture dish. By taking into consideration factors such as volume, density, purity, and relative molecular mass, this resulted in concentrations of 1.4, 5.7, 11.4, 22.9, and 45.8 μg/mL (the quality of PSM/space volume) for citral. We also prepared working dilutions of carvacrol with concentrations of 10, 20, 40, 80, and 160 μl/mL, resulting in concentrations of 1.6, 3.2, 6.5, 9.7, and 12.9 μg/mL. Additionally, working dilutions of trans‐2‐decenal were prepared with concentrations of 10, 20, 30, 40, 80, 160, and 320 μg/mL, which resulted in concentrations of 1.3, 2.7, 3.9, 5.3, 10.7, 21.3, and 42.6 μg/mL. An equal volume of ethanol was used as the control. The colony diameter of P. capsici was measured after fumigation with each PSM for 3 days. The growth inhibition rate was calculated according to the method of Wang, Zhang, et al. (2019).

To investigate the influence of citral on the mycelial morphology of P. capsici, the mycelia of P. capsici were fumigated by citral as described above for 3 days. The mycelia of P. capsici were cut from the leading edge of an actively growing colony, and then mycelial morphology was observed by using a light microscope (Olympus BX phase contrast microscope).

To determine the effects of citral on zoospore germination of P. capsici, equal number of zoospores were acquired. The method of inducing zoospores of P. capsici was performed according to a previous method (Wang, Liu, et al., 2019). Briefly, five fresh marginal mycelial plugs were cut and transferred into a plate containing V8 liquid medium for 3 days. Then the mycelia were washed with sterile water until the mycelia turned white. The washed mycelia were put in an incubator at 25°C for 2 days, and a large number of sporangia grew inside. The plate was transferred to a 4°C refrigerator for 20 min, and then the plate was placed in an incubator at 25°C to release the zoospores. The zoospores were calculated under a light microscope to amount to 100,000 spores/mL. The zoospores were cultured with an equal volume of V8 juice medium containing different concentrations of citral in an airtight container. The germination rate was recorded at 3, 6, and 12 h under the light microscope. These assays were repeated at least three times.

4.3. Detection of the impact of citral on the cell membrane integrity of P. capsici

To determine the effects of citral on the cell membrane permeability of P. capsici, two separate methods were used. First, the MDA content was detected. Three mycelial plugs from the active margin colonies were transferred to a plate containing 8 mL of V8 juice medium. After 24 h, different concentrations of citral were added to the above cultures. After treatment with citral for 48 h, 0.1 g of mycelia per sample was collected and ground in liquid nitrogen. The powder was dissolved in 1 mL of phosphate buffer (pH 7.8), and the suspension was then centrifuged at 10,000 g for 10 min at 4°C. The supernatant was collected for subsequent analysis. Determination of MDA content was performed using a lipid peroxidation MDA assay kit (Beyotime Biotech). Second, a PI staining assay was used. Five mycelial plugs from active margin colonies were transferred to a plate containing 8 mL of V8 juice medium. After incubating for 24 h at 25°C, different concentrations of citral were added to the cultures. Following a 48‐h incubation period, the mycelia were washed three times with sterile distilled water and stained with 200 μM PI for 20 min. Finally, the stained mycelia were washed again with sterile distilled water and observed under a 20× objective lens using a LSM 710 laser scanning microscope (Carl Zeiss). These assays were repeated three times.

4.4. In vitro and in vivo inhibitory activities of PSMs on P. capsici infection

The inoculation airtight container in vitro was 105 mL. The concentration of citral, carvacrol, trans‐2‐decenal used in the assay were 1.4, 1.6, 1.3 μg/m (the quality of PSM/space volume). The stalks of detached N. benthamiana leaves were wrapped in wet cotton. For mycelia inoculation, 5‐mm disks of 3‐day growth medium of P. capsici were inoculated on leaves. At the same time, different concentrations of PSMs were put on the bottle cap beside the leaves. After that, the sealing film was used to ensure an airtight environment. Inoculated leaves were photographed under UV light at 36 hpi, and the lesion area was measured. The assays were repeated three times, and each replicate contained at least eight leaves.

The inoculation airtight container in vivo was a 200‐mL plastic bottle. A concentration of 1.4 μg/mL (the quality of PSM/space volume) citral was used in the experiment. An 800 μL aliquot of zoospore suspension (100,000 spores/mL) of P. capsici was sprayed on the leaves of 3‐ to 4‐week‐old N. benthamiana plants. Citral was then placed on the cover beside the plant, and another plastic cup was placed upside down on the first one. After that, sealing film was used to ensure an airtight environment (Figure 3d). After 36 h, the entire plants were photographed under UV light. The assay was repeated at least three times, and each replicate contained five individual N. benthamiana plants.

The relative biomass of P. capsica was measured by quantitative PCR. At 36 hpi, 0.1 g of inoculated leaves covering the lesion and the aboveground part of N. benthamiana plants was collected for subsequent DNA extraction. PcActin and NbActin were used as the reference genes. The relative biomass of P. capsici represented the genomic DNA ratio of P. capsici to N. benthamiana.

4.5. RNA‐Seq analysis and RT‐qPCR assay

The N. benthamiana leaves were inoculated with P. capsici, followed by fumigation with citral for 36 h. Citral was replaced with ethanol as the control. Three biological replicates per condition were prepared. The total RNAs of two groups of samples were extracted using an RNA‐simple Total RNA Kit (Zoman Biotech) according to the manufacturer's protocol. The RNA libraries were constructed by the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina), followed by sequencing using the Illumina HiSeq X Ten platform. For the produced paired‐end raw reads, low‐quality reads were removed first. The obtained clean reads were mapped to the P. capsici genome (https://genome.jgi.doe.gov/Phyca11/Phyca11.home.html) using HISAT2 (Kim et al., 2015). Using the StingTie tool, the expression of each P. capsici gene was calculated by normalizing it to the fragment per kilobase of exon per million mapped reads (FPKM) value (Pertea et al., 2015). DESeq2 was then used to identify differentially expressed genes with the criteria of p < 0.05 and fold change >2 or fold change <−2 (Love et al., 2014).

The cDNA of P. capsici and N. benthamiana was synthesized from 900 ng of total RNA using HiScript II Q RT Super Mix (Vazyme Biotech). Real‐time PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme Biotech) on an ABI Prism 7500 Fast Real‐Time PCR system. All the primers used for RT‐qPCR assay are listed in Table S4. These data contained three independent biological replicates.

4.6. Functional analysis of P. capsici virulence proteins in N. benthamiana

The candidate RxLR genes and PcF/SCR74‐like genes were cloned from the P. capsici cDNA library by corresponding primers (Table S4). The amplified genes were cloned into the SmaI site of the binary pBin3HA vector, and then anticipated constructs were sequenced for confirmation. The signal peptide of RxLR proteins and PcF/SCR74‐like proteins were predicted by SignalP v. 3.0, and the sequences of RxLR proteins without signal peptide were used for subsequent experiments.

The constructed plasmids were transformed into A. tumefaciens GV3101 by electroporation. The individual bacterial colony PCR and selective antibiotics were used to screen the positive monoclonal colony. The positive monoclonal colony was then cultured in Luria–Bertani medium with selective antibiotics for 48 h at 28°C and 220 rpm. The harvested cells were washed three times at 1180 g for 4 min, and then resuspended in 10 mM MgCl2, 0.05 mM 2‐(N‐morpholino) ethanesulfonic acid, and 10 mM acetosyringone to an appropriate concentration at OD600 (0.5).

For infiltration, the bacteria cell suspension was infiltrated into plant leaves using a syringe without the needle. To test whether P. capsici proteins could trigger cell death in N. benthamiana, recombinant constructs of bacterial cell suspension were agroinfiltrated into 6‐week‐old N. benthamiana leaves. To test whether P. capsici proteins could inhibit cell death caused by INF1, the proteins were agroinfiltrated to N. benthamiana leaves first. After 24 h, INF1 (OD600 = 0.1) was agroinfiltrated to the same injection site, and symptom development was monitored visually 3 days after agroinfiltration with INF1. The assays were repeated at least three times, and each replicate contained nine detached leaves.

For the infection assay, 5‐mm disks of 3‐day‐old P. capsici growth medium were obtained from the leading edge of an actively growing colony. The inoculated leaves had similar sizes and inoculated positions, and P. capsici mycelial disks were inoculated on the back of N. benthamiana leaves. At the same time, 5 μL of double‐deionized water was added to the inoculated site, and the front side of the mycelia disks was put on the inoculated site. The inoculated leaves were kept in high humidity and placed in the climate chamber. After 24–48 h of infection, inoculated leaves were photographed under UV light. The assays were repeated at least three times, and each replicate contained eight detached leaves.

4.7. Western blotting

After 36 h of infiltration in N. benthamiana, a 7‐mm leaf disc was obtained to extract protein. Seventy microlitres of 1% SDS buffer was added to the sample, and then the sample was homogenized, followed by adding 20 μL of 5× sample loading buffer. The mixture was boiled at 100°C for 8 min, followed by centrifuging for 10 min at room temperature. The supernatant was further used for SDS‐PAGE and western blot with anti‐HA antibody (Sigma‐Aldrich).

4.8. Oxidative burst assay

Luminol/peroxidase‐based assay was used to detect the ROS production. First, A grobacterium strains with the indicated constructs were infiltrated in N. benthamiana leaves for 36 h. Disks (5 mm) were cut from N. benthamiana leaves and were incubated in 200 μL per hole of distilled water in a 96‐well plate overnight. After that, the distilled water was replaced by luminescence detection buffer. The 200 μL of luminescence detection buffer was composed of 1 μM flg22, 100 μM luminol, and 200 μg/mL horseradish peroxidase. The luminescence intensity was recorded with a microplate reader (BioTek) for 30 min.

To test whether citral could trigger ROS production in N. benthamiana, we replaced flg22 with citral in the detection buffer. The luminescence detection buffer was composed of 1.4 μg/mL citral, 100 μM luminol, and 200 μg/mL horseradish peroxidase. The entire operation method was the same as above.

4.9. Statistical analysis

All the experiments were performed with at least three independent replicates, and the data are presented as the mean ± SD. Significant differences between treatments were detected using one‐way analysis of variance combined with Fisher's LSD test with p < 0.05.

Supporting information

Figure S1 Propidium iodide (PI) staining assay to evaluate the effect of citral towards cell membrane permeability. The mycelia were treated with citral at the indicated concentrations and stained with PI, followed by observations with a confocal microscope. BF, bright field; PI, PI staining. Scale bar 50 μm

Click here for additional data file. (1.6MB, tif)

Table S1 Details of the 11 plant secondary metabolites used in this study

Click here for additional data file. (54.9KB, docx)

Table S2 The inhibitory effects of three plant secondary metabolites on Phytophthora capsici

Click here for additional data file. (12.7KB, docx)

Table S3 Phytophthora capsici differentially expressed genes identified between control and citral treatment

Click here for additional data file. (18.2KB, xlsx)

Table S4 Primers used in this study

Click here for additional data file. (10.4KB, xlsx)

ACKNOWLEDGEMENTS

This study was supported by grants from the Jiangsu Agricultural Science and Technology Innovation Fund (CX(21)3085), the Technical System of Chinese Herbal Medicine Industry (CARS‐21), the National Natural Science Foundation of China (32070139), and the Jurong Municipal Science and Technology Project (ZA32122).

Song, W. , Yin, Z. , Lu, X. , Shen, D. & Dou, D. (2023) Plant secondary metabolite citral interferes with Phytophthora capsici virulence by manipulating the expression of effector genes. Molecular Plant Pathology, 24, 932–946. Available from: 10.1111/mpp.13340

Wen Song and Zhiyuan Yin contributed equally to this study.

DATA AVAILABILITY STATEMENT

The genes used in this study are deposited in the GenBank database at www.ncbi.nlm.nih.gov/genbank/ with the following accession numbers: PcAvh214 (OP856841), PcAvh254 (OP856845), PcAvh478 (OP856840), PcSCR1 (OP856842), and PcSCR2 (OP856843). The raw RNA sequencing data reported in this study have been deposited in the NCBI Sequence Read Archive at www.ncbi.nlm.nih.gov/sra/ under the accession number PRJNA904659.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Propidium iodide (PI) staining assay to evaluate the effect of citral towards cell membrane permeability. The mycelia were treated with citral at the indicated concentrations and stained with PI, followed by observations with a confocal microscope. BF, bright field; PI, PI staining. Scale bar 50 μm

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Table S1 Details of the 11 plant secondary metabolites used in this study

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Table S2 The inhibitory effects of three plant secondary metabolites on Phytophthora capsici

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Table S3 Phytophthora capsici differentially expressed genes identified between control and citral treatment

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Table S4 Primers used in this study

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Data Availability Statement

The genes used in this study are deposited in the GenBank database at www.ncbi.nlm.nih.gov/genbank/ with the following accession numbers: PcAvh214 (OP856841), PcAvh254 (OP856845), PcAvh478 (OP856840), PcSCR1 (OP856842), and PcSCR2 (OP856843). The raw RNA sequencing data reported in this study have been deposited in the NCBI Sequence Read Archive at www.ncbi.nlm.nih.gov/sra/ under the accession number PRJNA904659.

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