Quasi-targeted metabolomics revealed isoliquiritigenin and lauric acid associated with resistance to tobacco black shank

ABSTRACT

Tobacco black shank (TBS), caused by Phytophthora nicotianae, is a severe disease. Plant root exudates play a crucial role in mediating plant-pathogen interactions in the rhizosphere. However, the specific interaction between key secondary metabolites present in root exudates and the mechanisms of disease resistance remains poorly understood. This study conducted a comprehensive comparison via quasi-targeted metabolomic analysis on the root exudate metabolites from the tobacco cultivar Yunyan87 and K326, both before and after inoculation with P. nicotianae. The results showed that the root exudate metabolites changed after P. nicotianae inoculation, and the root exudate metabolites of different tobacco cultivar was significantly different. Furthermore, homovanillic acid, lauric acid, and isoliquiritigenin were identified as potential key compounds for TBS resistance based on their impact on the mycelium growth of the pathogens. The pot experiment showed that isoliquiritigenin reduced the incidence by 55.2%, while lauric acid reduced it by 45.8%. This suggests that isoliquiritigenin and lauric acid have potential applications in the management of TBS. In summary, this study revealed the possible resistance mechanisms of differential metabolites in resistance of commercial tobacco cultivar, and for the first time discovered the inhibitory effects of isoliquiritigenin and homovanillic acid on P. nictianae, and attempt to use plants secondary metabolites of for plant protection.

KEYWORDS:

• Tobacco
• Phytophthora nicotianae
• quasi-targeted metabolomics
• root exudates
• isoliquiritigenin
• lauric acid

Introduction

Tobacco black shank (TBS), caused by Phytophthora nictianae, is a serious soil-borne plant pathogen that infects over 255 plant species spanning 90 families.^1,2 The dormant forms of P. nictianae exhibit high resistance to stress, while currently commercially cultivated tobacco varieties have low disease resistance,^3,4 resulting in significant economic losses in China.^5,6 Synthetic chemicals have traditionally been employed in agricultural production to control the spread of crop diseases.⁷ However, people have gradually become aware of the numerous negative impacts of these chemicals, including the increasingly serious “3 R” (Resistance, Resurgence and Residue).⁸ To address these concerns, biocontrol has emerged as a promising alternative to chemical treatments.⁹

Currently, four physiological races (0, 1, 2, and 3) of P. nicotianae have been reported. Race 0 is widely distributed, while Race 1 is less adaptable. Both races 0 and 1 of P. nicotianae have been observed in China.^2,10,11 Tobacco cultivars resistant to TBS can be categorized into multiple types: those with resistance conferred by multiple quantitative trait loci (QTLs) primarily derived from “Florida 301” and “Beinhart 1000”, those with resistance mediated by single homologous genes (such as the phl gene from N. longiflora and the php gene from N. plumbaginifolia), and those with resistance governed by a single dominant resistance locus Wz from N. rustica.^2,10 It is worth noting that the cultivar K326 resistance was sourced from the “Florida 301”. Grafting, where highly resistant plants serve as rootstocks and high-yield crops as scions, can avoid the above problems to some extent¹² and improve fruit quality.¹³ For instance, grafting using the resistant cultivar “Gexin3” as a rootstock enhanced the resistance of the susceptible cultivar “Xiaohuangjing1025” to TBS.⁴ However, when there is a shift from race 0 to 1, the original resistant cultivar may lose its effectiveness.¹⁴ Therefore, developing new disease-resistant cultivars still requires significant time and effort.

Natural products (NPs) are chemical substances derived from microorganisms, plants, or animals and are extensively utilized in the food industry, agriculture, and pharmaceuticals.¹⁵ However, the isolation of natural products from microorganisms has reached a bottleneck,¹⁶ so the effort to isolate natural products from plants should be strengthened in the future. Plant secondary metabolites are associated with plant resilience to stress and responses to changes in the environment.¹⁵ The secondary metabolites mainly include terpenoids, phenolics, and nitrogen-containing compounds.¹⁷ Among these, terpenoids, exemplified by citronellal, demonstrate the ability to impede the formation of biofilms by inhibiting Streptococcus albicans.^18,19 Similarly, phenolic such as flavonoid naringenin impedes the growth of P. nicotianae by inducing the expression of genes related to salicylic acid biosynthesis.¹¹ Furthermore, nitrogen-containing compounds, represented by berberine, exert their growth-inhibitory effects on Ralstonia solanacearum through modulation of cell permeability.²⁰ Due to the rapid accumulation of antioxidant and antibacterial compounds via metabolic pathways following pathogenic microbial infections in plants,²¹ the accumulation of these secondary metabolites is a key feature of the host-pathogen interaction.²² The plant metabolome is considered the link between the genotype and the phenotype, and the detection of multiple differentially accumulated metabolites (DAMs) in plants can be achieved through metabolomic approaches.^23,24

Plant metabolites exuded from roots have emerged as pivotal mediators in plant-microbe interactions, governing the structure and diversity of soil microbial communities. Recent research has unveiled variations in root exudate metabolites among cultivars following infection by pathogenic microorganisms.³ In this study, a quasi-targeted metabolomics was used to thoroughly compare the root exudate metabolites from two tobacco cultivars, Yunyan87 and K326, under both inoculated and non-inoculated conditions with P. nicotianae. The relationship between the root exudates of tobacco cultivars, which possess disease resistance traits mediated by QTLs, and their ability to resist diseases was analyzed. Furthermore, certain compounds with potential for application in disease control were assessed. The study uncovered the potential of isoliquiritigenin and lauric acid in controlling TBS. These findings lay a robust foundation for utilizing root exudates as a means to control diseases.

Material and methods

Materials

The tested tobacco cultivars Yunyan87 and K326, the main cultivars planted in China, were provided by Baise Tobacco Company and Hezhou Tobacco Company in Guangxi. The P. nicotianae LY-1 (GenBank accessions: OR037749 for ITS) with virulent pathogenicity was isolated from tobacco at Leye County, Guangxi, China.

Preparation of zoospores of Phytophthora nicotianae

P. nicotianae was inoculated onto oat meal agar (OMA) medium and cultured at 26°C for 14 days in darkness. Following this, 15 mL of a 0.1% KNO₃ solution was added to each petri dish, which was then returned to the 26°C to continue the dark incubation, thereby promoting the production of sporangia. After 3 days, the dishes were removed and subjected to a chilling process at 4°C for 30 minutes to facilitate the release of zoospores. The resulting zoospores were filtered and washed using gauze to obtain P. nicotianae zoospore suspensions. Finally, spore suspensions with a concentration of 1 × 10⁵ spore/mL was prepared by counting under a microscope using hemocytometer.

Disease resistance assay of tobacco cultivar Yunyan87 and K326

The resistance of the Yunyan87 and K326 cultivars to TBS was assessed through the artificial induction of the disease in a greenhouse nursery. Sterilized soil mix was used to sow the K326 and Yunyan87 tobacco seeds. When the tobacco plants reached the 4-leaf stage, tobacco seedlings of similar size were selected and transplanted to a seedling tray with 32 compartments (54 cm × 28 cm). All plants were placed in a greenhouse at 26 ± 3°C and 60–70% relative humidity, with a photoperiod of 12 h and cultured until the 6-leaf stage for inoculation with P. nicotianae. After the roots of the tobacco seedlings were carefully cut, 15 mL of the prepared P. nicotianae spore suspensions (10⁵ spore/mL) were introduced to the roots of both Yunyan87 and K326 through irrigation, and irrigated with 15 mL of sterile water as a control. The incidence of TBS was investigated 4 days and 8 days post-inoculation, respectively. Each treatment repeated three times, and every replicate had 32 seedlings for each cultivar.

Collection and treatment of root exudates

Root exudates were collected by root soaking.⁴ The treatments consisted of non-inoculated root exudates of Yunyan87 (Y), non-inoculated root exudates of K326 (K), P. nicotianae inoculated root exudates of Yunyan87 (Yi), and P. nicotianae inoculated root exudates of K326 (Ki). Prior to inoculation and 4 days after inoculation, three seedlings from each cultivar were carefully selected for the collection of root exudates, and repeated 3 times. The two cultivars of tobacco were taken out of the seedling tray, and their roots were thoroughly rinsed with deionized water to remove any soil particles attached to the root surface. Following this preparatory step, the roots were placed in a 100 mL conical flask containing 75 mL of sterile water. Under conditions of aeration and darkness, the seedlings were allowed to secrete their exudates for a duration of 12 hours. Root exudates were filtered with 0.22 μm millipore filter and then placed in a lyophilizer for vacuum freeze-drying. Afterwards, the root exudates were stored at −80°C for further analysis.

Effect of root exudates on mycelium growth of P. nicotianae

The growth rate method was used to determine the effect of root exudates on the mycelial growth of P. nicotianae. The root exudate was diluted to the final concentration of 10 mg/mL. Subsequently, it was added to OMA that had been cooled to 45°C in a 1:1 ratio, and then poured it into petri dish (Φ = 9 cm). To prevent bacterial contamination, chloramphenicol was added at a concentration of 30 µg per milliliter of medium. A P. nicotianae mycelium cake (0.80 cm in diameter) was inoculated at the center of each OMA plate and cultured at 26°C for 4 days. The mycelium diameter was measured using the cross method to determine the inhibition rate of root exudates, and OMA plate containing the same content of chloramphenicol as the control, each treatment had three replicates.

HPLC-MS/MS analysis of root exudates

Freeze-dried sample (100 mg) was resuspended and homogenized by gently agitating it with prechilled 80% methanol using a vortex mixer. The samples were then incubated on ice for 5 minutes and centrifuged at 15,000 g at 4°C for 20 minutes. A portion of the supernatant was taken and diluted with LC-MS grade water, resulting in a final concentration comprising 53% methanol. The diluted samples were transferred to suitable tubes and centrifuged again at 15,000 g at 4°C for 20 minutes. The recovered supernatant was then used as the material for subsequent analysis. To ensure the reproducibility and reliability of the LC-MS system, quality control (QC) samples were created by mixing equal volumes of samples from each experimental treatment. For analysis, each treated sample of 2 µL was placed in the HPLC-MS/MS system,²⁵ with each treatment repeated three times.

The LC-MS/MS analyses were conducted using an ExionLC™ AD system (SCIEX) coupled with a QTRAP® 6,500+ mass spectrometer (SCIEX) in Novogene Co., Ltd. (Beijing, China). Samples were injected onto a Xselect HSS T3 (2.1 × 150 mm, 2.5 μm) using a 20-min linear gradient at a flow rate of 0.4 mL/min for the positive/negative polarity mode. The eluents were eluent A (0.1% Formic acid-water) and eluent B (0.1% Formic acid-acetonitrile).²⁶ The solvent gradient was set as follows: 2% B for 2 minutes, 2–100% B over 15.0 minutes, 100% B for 17.0 minutes, 100–2% B over 17.1 minutes, and 2% B for 20 minutes. Mass spectrometry was acquired by using electrospray ionization (ESI) in both negative and positive ionization modes. The conditions for ion detection mass spectrometry included a curtain gas pressure of 35 psi, a collision gas set to medium, an ionspray voltage of 5,500 V for positive ionization modes, and −2,500 V for negative ionization modes. The temperature was maintained at 550°C, while the ion source gas 1 and ion source gas 2 were set at 60.

Identification and quantification of metabolites and data analysis

The experimental samples were analyzed using Multiple Reaction Monitoring (MRM) based on the in-house database provided by Novogene. Metabolite quantification was carried out using Q3, while the identification of metabolites was determined using Q1, Q3, retention time (RT), declustering potential (DP), and collision energy (CE). The data files generated from the HPLC-MS/MS analysis were processed using SCIEX OS Version 1.4 to integrate and correct the peaks. The key parameters for peak processing were set with a minimum peak height of 500, a signal-to-noise ratio of 5, and a Gaussian smooth width of 1. The area under each peak represented the relative content of the corresponding substance.

Principal components analysis (PCA) and Partial least squares discriminant analysis(PLS-DA) were performed using metaX (a flexible and comprehensive software for processing metabolomics data).²⁷ Univariate analysis (t-test) were used to calculate the statistical significance (P-value). Metabolites with VIP > 1 and P-value <0.05, as well as fold change > 1.5 or FC < 0.667²⁸ were considered to be differential metabolites.

The effect of compounds on mycelium growth of P. nicotianae

The growth rate method was used to determine the effect of compounds on the mycelial growth of P. nicotianae. The 6 tested different compounds including fatty acid (lauric acid, oleic acid), organic acids (homovanillic acid, homogentisic acid), alkaloid (lycorine), flavonoids (isoliquiritigenin). The tested compound was dissolved in DMSO and subsequently diluted to the desired concentration. It was then filtered using a 0.22 μm millipore filter. The resulting solution was added to OMA, which had been cooled to 45°C, to ensure final concentrations of 50 µg/mL, 100 µg/mL, and 500 µg/mL, respectively. A P. nicotianae mycelium cake (0.80 cm in diameter) was inoculated at the center of each OMA plate and cultured at 26°C for 4 days. The mycelium diameter was measured using the cross method to determine the inhibition rate of compounds and OMA plate containing the same content of DMSO as the control, each treatment had three replicates.

Based on the results, lauric acid, homovanillic acid, and isoliquiritigenin were identified as compounds with potent inhibitory properties. To further investigate their effects on the mycelial growth of P. nicotianae, a series of concentration gradients were established. The mycelial growth was measured, and the data were used to determine the toxic regression equation and 50% of maximal effect (EC₅₀) were computed.

Evaluation of pot control effect of compounds against TBS

To assess the control potential of compounds against TBS, the control effect of two potent monomers, namely lauric acid and isoliquiritigenin, against TBS in potted plants was determined using the methodology outlined in Section 2.3. The method of inoculating P. nicotianae cake with wounded at the base of the stem was used to determine the effectiveness of potted control. When the tobacco Yunyan87 plants reached the 6-leaf stage, tobacco seedlings of similar size were selected and transferred to a seedling tray with 32 compartments (54 cm × 28 cm). After the seedlings had passed the recover period, the base of their stems was gently scratched using tweezer. Subsequently, a P. nicotianae mycelium cake with a diameter of 0.80 cm was applied, and the cake was covered with soil to maintain moisture. A solution of 0.2 mg/mL isoliquiritigenin and a solution of 0.5 mg/mL lauric acid were prepared. These solutions were sprayed onto the stem base of the tobacco plants 24 hours post-inoculation, at a volume of 5 mL per plant. Additionally, a 64% oxadixyl·mancozeb wettable powder, a commonly used pesticide for controlling TBS in production, was used as a comparative treatment at the recommended concentration of 2.37 mg/mL (effective concentration). A control group was sprayed with sterile water containing equal amounts of DMSO. Each treatment was replicated three times, with each replicate consisting of 32 seedlings.

Data statistics

Excel 2010 and DPS 7.05 software were used for data analysis. Differences between groups were tested using one-way ANOVA, followed by duncan’s multiple range test. p < 0.05 indicates that the differences are statistically significant.

Results

Tobacco cultivar K326 is resistant to TBS

The resistance of the Yunyan87 and K326 cultivars to TBS was assayed by inoculating of P. nicotianae. Figure 1(a) portrays the discernible disparity in the incidence of TBS between Yunyan87 and K326 on both the 4th and 8th days. Notably, Yunyan87 exhibited an incidence rate of approximately 59.4% four days after inoculation, while the incidence rate of K326 is only 6.3%. As time progressed, this discrepancy became more apparent. After 8 days, Yunyan87 showcased an incidence rate of 83.3%, whereas K326 displayed a significantly lower incidence rate of 20.7% (Figure 1b). These findings reveal that K326 is resistant to TBS when compared to Yunyan87.

Figure 1. Resistance identification of Yunyan87 and K326 against TBS. (a) The incidence rate of Yunyan87 and K326 inoculated with P. nicotianae for 4 and 8 days. (b) TBS symptoms of tobacco plants 8 days with P. nicotianae (***, p < 0.001; ****, p < 0.0001; t test; the error bars represent the standard deviation of three repetitions).

Root exudates derived from K326 exhibit an inhibitory effect

The impact of different root exudates on the growth of P. nicotianae was quantified using the growth rate method. As shown in Figure 2(a), there was a significant difference in the impact of root exudates on mycelial growth of P. nicotianae between cultivar Yunyan87 and K326. The mycelial growth inhibition of root exudates from Yunyan87 without pathogen infection was 2.7%, while the inhibition of K326 root exudates was about 15%. The root exudates derived from K326 exhibit strong inhibitory effect.

Figure 2. Effects of root exudates from Yunyan87 and K326 on the mycelial growth of P. nicotianae. (a) The colony diameter of P. nicotianae treated with root exudates of Yunyan87 and K326 for 4 days. (b) Mycelial growth of P. nicotianae after 4 days treatment with root exudates of Yunyan87 and K326. Yi and Y represent root extract from pathogen inoculation and non-inoculation of Yunyan87, respectively; Ki and K represent root extract from pathogen inoculation and non-inoculation of K326, respectively; different lowercase letters marked on the column indicate significant differences between groups at p < 0.05; the error bars represent the standard deviation of three repetitions.

The inhibitory efficacy of diverse resistant root exudates against P. nicotianae varies, and the presence of pathogen infection can likewise influence the inhibitory potential of root exudates on P. nicotianae. Notably, the inhibitory activity of K326 root exudates on mycelial growth was intensified following P. nicotianae infection, culminating in an inhibition rate of 55.2%, significantly higher than that observed in the non-inoculated K326 group. Similarly, the introduction of P. nicotianae amplified the inhibition of root exudates derived from Yunyan87, resulting in an inhibition rate of 8.8% (Figure 2b). It is evident that the infection of P. nicotianae augment the inhibitory efficacy of root exudates from both cultivars against mycelial growth.

Root exudate metabolites are more affected by the inoculation of P. nicotianae

To elucidate the relationship between disease resistance and metabolites in cultivars, we employed HPLC-MS/MS to analyze the root exudates of both inoculated and non-inoculated Yunyan87 and K326 cultivars. In order to evaluate the reliability of the metabolite detection data, correlation analysis was conducted on the TIC plots of different QC samples. As shown in Fig. S1, the response intensity and retention time of each chromatographic peak basically overlapped, indicating that the QC sample has good repeatability and the stability of the experimental instrument. The data in this study has good repeatability and reliability.

The HPLC-MS/MS analysis results showed that each sample of root exudates encompassed an impressive compendium of 1,165 metabolites, mainly including esters, phenols, organic acids, fatty acids, terpenoids, alcohols, flavonoids, amino acids, and alkaloids, etc. In order to elucidate the dissimilarities within the metabolome of each sample group, principal component analysis (PCA) and hierarchical clustering analysis (HCA) were used to analyze 1,165 metabolites. PCA (Figure 3a) and HCA (Figure 3b) can clearly distinguish the inoculated and non-inoculated tobacco cultivars, the farthest distance between these two categories of tobacco cultivars serves as testament to the marked dissimilarity in metabolites. Evidently, these findings suggest that the root exudate metabolites are more affected by the inoculation of P. nicotianae than by genotypes.

Figure 3. Principal component analysis (a) and hierarchical clustering analysis (b) of metabolites of different tobacco cultivar non-inoculation and inoculation. Yi and Y represent root extract from pathogen inoculation and non-inoculation of Yunyan87, respectively; Ki and K represent root extract from pathogen inoculation and non-inoculation of K326, respectively.

Differential compounds analysis

Yi and Y represent root extract from pathogen inoculation and non-inoculation of Yunyan87, respectively; Ki and K represent root extract from pathogen inoculation and non-inoculation of K326, respectively. Comparing the root exudate of plants treated with Y and K is represented as Y vs. K, and the others are consistent. In Y vs. K, Yi vs. Ki, Yi vs. Y, and Ki vs. K, a tally of 59, 206, 198, and 38 differential compounds, totaling 398, were successfully identified. Figure 4(a) depicts the distribution of down-regulated and up-regulated compounds (Table S1). Notably, the root exudates of K326 exhibited a higher abundance of organic acids, fatty acids, phenols, coumarin, terpenoids, and flavonoids, while Yunyan87 displayed a greater richness of amino acids, phenylpropanoids, and alkaloids.

Figure 4. The numbers of differential compounds before and after inoculation of P. nicotianae in both tobacco cultivars. (a) Histogram. (b) Venn diagram. Yi and Y represent root extract from pathogen inoculation and non-inoculation of Yunyan87, respectively; Ki and K represent root extract from pathogen inoculation and non-inoculation of K326, respectively.

In combination with possible disease resistance association from references, inhibitory effects of root exudate and differential compounds Venn diagram (Figure 4b), 6 important differential compounds including fatty acids, organic acids, alkaloids, and flavonoids were selected from the 398 differential compounds for further verification (Table 1). In the absence of pathogen infection, the concentration of isoliquiritigenin in K326 root exudates surpassed that observed in Yunyan87 root exudates. In the presence of pathogenic infection, there was a notable augmentation the accumulation of lauric acid, oleic acid, homogenic acid, homovanillic acid, and lycoline in K326 root exudates. A similar pattern was observed in Yunyan87 root exudates, where the accumulation of homovanillic acid and isoliquiritigenin was significantly enhanced.

Table 1. Potential key differential metabolites in tobacco root exudates of tobacco Yunyan87 and K326.

Compound	Iron mode	Log2 Y/K			Log2 Yi/Ki			Log2 Yi/Y			Log2 Ki/K
		FC	Change	Pvalue	FC	Change	Pvalue	FC	Change	Pvalue	FC	Change	Pvalue
Lauric acid	ESI-	1.68	-	0.3985	0.81	-	0.3125	2.80	-	0.0684	5.83	up	0.0000
Oleic acid	ESI+	1.14	-	0.8569	0.53	down	0.0402	1.66	-	0.2089	3.59	up	0.0233
Homogentisic acid	ESI-	0.87	-	0.5727	0.69	-	0.0388	3.09	-	0.0708	3.91	up	0.0040
Homovanillic acid	ESI-	1.035	-	0.8174	0.94	-	0.8213	3.46	up	0.0226	3.82	up	0.0128
Lycorine	ESI+	5.31	-	0.1313	0.67	-	0.3393	1.84	-	0.2694	14.61	up	0.0021
Isoliquiritigenin	ESI-	0.61	down	0.0396	1.14	-	0.7290	1.40	up	0.0459	1.41	-	0.2522

Isoliquiritigenin has the best inhibitory effect

The impact of selected compounds on the growth of P. nicotianae was quantified using the growth rate method. As shown in Table 2, the impacts of the 6 compounds tested on the mycelial growth of P. nicotianae exhibit variability. With the increase of concentration, most compounds display enhanced inhibitory effects on the mycelial growth of P. nicotianae. Notably, isoliquiritigenin have the best inhibitory effect, followed by homovanillic acid and lauric acid (Figure 5). At a low concentration of 50 µg/mL, the inhibition rates of isoliquiritigenin exceed 75%, while the remaining compounds yield feeble inhibitory outcomes. As concentrations increase, the inhibition rates of homovanillic acid and lauric acid surpass 65%, while homogentisic acid attains inhibition rates of more than 35%.

Figure 5. Mycelial growth of P. nicotianae after 4 days treatment with isoliquiritigenin, homovanillic acid and lauric acid with strong inhibitory activity.

Table 2. Effects of compounds on the growth of mycelium of P. nicotianae.

Compound	Mycelial growth inhibition (%)
	Treatment with 50 µg/mL	Treatment with 100 µg/mL	Treatment with 500 µg/mL
Isoliquiritigenin	79.0 ± 0.66 a	87.3 ± 0.76 a	94.1 ± 0.66 a
Lauric Acid	8.1 ± 1.37 b	18.4 ± 0.66 b	65.1 ± 0.66 c
Lycorine	1.1 ± 0.38 c	14.7 ± 1.37 c	27.9 ± 1.01 e
Homovanillic acid	2.0 ± 0.66 c	15.4 ± 0.76 c	68.6 ± 0.38 b
Homogentisic Acid	1.1 ± 0.38 c	2.0 ± 0.66 d	37.3 ± 0.76 d
Oleic Acid	0.7 ± 1.14 c	0.7 ± 0.66 d	14.5 ± 2.37 f

The data in the table are the means and standard deviation of three replicates, and different lower case letters represent significant level at P < 0.05.

The evaluation of the impact of isoliquiritigenin, homovanillic acid, and lauric acid on the growth of P. nicotianae was conducted by using the growth rate method at varying concentrations of 12.5 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, and 200 µg/mL for isoliquiritigenin, and 50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL, and 600 µg/mL for homovanillic acid and lauric acid. The toxic regression equations and EC₅₀ of these three compounds were acquired and presented in Table 3. Among them, isoliquiritigenin demonstrated the most potent anti-fungal activity, with EC₅₀ of 14.5 µg/mL, while homovanillic acid and lauric acid exhibited EC₅₀ of 239.2 µg/mL and 413.3 µg/mL, respectively.

Table 3. Inhibitory activity of four compounds on mycelium growth of P. nicotianae.

Compound	Toxic regression equation	Correlation coefficient	EC50 (ug/mL）	95% Confidence interval
Isoliquiritigenin	y = 1.3659×+7.5111	0.9658	14.5	−2.6429~-1.0339
Homovanillic acid	y = 2.6019×+6.6162	0.9867	239.2	−0.8636~-0.3787
Lauric Acid	y = 1.643×+5.6305	0.9101	413.3	−0.8253 ~ 0.0578

The data in the table are the means and standard deviation of three replicates, and different lower case letters represent significant level at P < 0.05.

Isoliquiritigenin and lauric acid as effective agents for managing of TBS

Based on the initial investigation, isoliquiritigenin showed the most significant inhibitory effect on mycelial growth of P. nicotianae, while previous studies have indicated the potential of lauric acid in controlling TBS. Consequently, for the subsequent potted control experiments, isoliquiritigenin and lauric acid were chosen as the focal compounds.

To assess the efficacy of two compounds, a solution of 0.2 mg/mL isoliquiritigenin and a solution of 0.5 mg/mL lauric acid were prepared. Additionally, a 64% oxadixyl·mancozeb wettable powder was employed as a comparative treatment. These solutions were sprayed onto the stem base of tobacco plants 24 hours post- P. nicotianae inoculation at a volume of 5 mL per plant.

As depicted in Figure 6(a), 24.9% the tobacco seedlings were symptomatic 4 days after inoculation with P. nicotianae, where the stem base was wounded.The incidence rate of TBS decreased significantly by 9.4% when treated with oxadixyl·mancozeb. Figure 6(a,b) show that the control had a near-total demise of tobacco seedlings 11 days post-inoculation. However, both isoliquiritigenin and lauric acid, as well as oxadixyl·mancozeb, demonstrated a significant reduction in incidence. Oxadixyl·mancozeb showed the most effective prevention efficacy, resulting in a substantial 61.5% reduction in incidence compared to the control. Moreover, isoliquiritigenin and lauric acid showed a notable decrease in incidence, with reductions of 55.2% and 45.8% respectively, compared to the control. These findings highlight the potential of isoliquiritigenin and lauric acid as effective agents for managing of TBS.

Figure 6. Pot control effect of isoliquiritigenin, lauric acid and oxadixyl·mancozeb against TBS. (a) The TBS incidence of Yunyan87 inoculated with P. nicotianae for 4 and 11 days after treatment with two compounds and oxadixyl·mancozeb. (b) TBS symptoms in tobacco plants 11 days post-inoculation with P. nicotianae after treatment with three compounds and oxadixyl·mancozeb. Different lowercase letters marked on the column indicate significant differences between groups at p < 0.05; the error bars represent the standard deviation of three repetitions.

Discussion

Root exudates play a crucial role in the intricate interplay between plants, soil, and pathogens, constituting a crucial determinant for the occurrence of soil-borne diseases.²⁹ It has been observed that root exudates derived from disease-resistant and susceptible cultivars exert contrasting effects on pathogens. Specifically, while root exudates from susceptible cultivars can stimulate pathogen growth, those emanating from disease-resistant cultivars demonstrate the ability to impede pathogen proliferation.^30,31 Root exudates directly or indirectly participate in the recognition of host plants and pathogens, induction of plant disease resistance signal transduction and plant defense response.³² Previous studies have reported that root exudates from disease-resistant cultivar can effectively inhibit the growth of P. nicotianae, while those derived from susceptible cultivars can stimulate the growth of P. nicotianae.³ This study measured the effect of different root exudates on the mycelial growth of P. nicotianae using the growth rate method. Consistent with previous studies, our results show that the root exudates of disease-resistant cultivar K326 exhibited a more potent inhibitory effect on the growth of P. nicotianae compared to tobacco cultivar Yunyan87. Furthermore, the infection of P. nicotianae augmented the inhibitory ability of the root exudates from both cultivars on mycelial growth. These results suggest that K326 contains a higher abundance of disease-resistant compounds, and the pathogen’s infection serves to stimulate the accumulation of such compounds.

This study is based on LC-MS for quasi-targeted metabolomics analysis of root exudates of the main tobacco cultivar Yunyan87 and K326 in China, both inoculated and non-inoculated with pathogen. Quasi-Targeted Metabolomics is a new type of metabolomics detection technology based on LC-MS/MS, which combines the high-throughput advantages of untargeted metabolomics with the precision and sensitivity of targeted metabolomics. The compound identification results are consistent with previously reported categories of tobacco, arabidopsis, and wheat root exudates.^3,33,34 Tobacco root exudates include esters, phenols, organic acids, fatty acids, terpenoids, alcohols, flavonoids, amino acids, alkaloids, and among others. The results of PCA and HCA reveal a closer clustering pattern among the root exudates from different cultivars subjected to the same treatment, indicating that the influence of pathogenic infection on the root exudates metabolomics surpasses that of plant genotypes. In contrast to the present study, metabolomics analysis from Zhang showed that the root exudate metabolites were more profoundly influenced by plant genotypes rather than pathogenic infection.³

The main differential metabolites identified for disease resistance include fatty acids, organic acids, alkaloids, terpenoids, and flavonoids, among them, fatty acids, organic acids, and isoflavones exhibit superior inhibitory effects. Fatty acid compounds are known for their ability to resist pathogen infection,³⁵ among which lauric acid is related to plant disease resistance.³⁶ Previous studies have shown that an increase in fatty acid content in eggplants can enhance their resistance to verticillium wilt.³⁷ In this study, tobacco was subjected to P. nicotianae infection, wherein the levels of lauric acid in K326 exhibited a significant elevation compared to the non-inoculated counterparts. It is postulated that the presence of lauric acid may be intricately linked to the augmented suppression of P. nicotianae by the root extract derived from the infected K326.

Organic acids in plant root exudates play a crucial role in regulating plants and microorganisms, it can promote plant roots to absorb soil nutrients, improve plant growth environment, and promote plant growth.^38,39 Homovanillic acid has traditionally been used as an indicator of human health, but its specific role in plants remains elusive. In this study, the levels of homovanillic acid in both cultivars were significantly upregulated subsequent to P. nicotianae infection, suggesting a plausible correlation with the enhanced inhibitory effect of root exudates from both inoculated cultivars on P. nicotianae.

Flavonoids, a class of polyphenolic compounds, have garnered considerable attention owing to their remarkable capacity to inhibit various pathogen.⁴⁰ It has been reported that isoliquiritigenin has various pharmacological properties such as anti-inflammatory, anti-microbial, antioxidative, anti-cancer as well as immunoregulatory effects.^41–44 Furthermore, investigations have revealed the commendable antibacterial activity of isoliquiritigenin against Escherichia coli, Bacillus cereus, Staphylococcus aureus, Erwinia carotovora and Bacillus subtilis.⁴⁵ This study observed that the levels of isoliquiritigenin in K326 were significantly higher than those in Yunyan87, potentially accounting for the heightened inhibitory prowess of root exudates derived from K326 against P. nicotianae.

The effects of six selected main differentially metabolites on the mycelial growth of P. nicotianae were assayed. The results showed that isoliquiritigenin, lauric acid, and homovanillic acid exhibited robust inhibitory effects, substantiating that tobacco root exudates contain a substantial quantity of anti-fungal substances, which can directly inhibit the growth of pathogens and enable plants to resist pathogen infection. With the exception of lauric acid, which has been previously reported to inhibit the P. nicotianae,³ and there has been no literature on the anti-fungal activity of the other two compounds as agents against P. nicotianae. To further validate the efficacy of isoliquiritigenin and lauric acid in controlling TBS, pot experiments were conducted. Consistent with a previous study,³ a significant reduction in the disease index of TBS when the concentration of lauric acid was 0.5 mg/mL. Moreover, 0.2 mg/mL isoliquiritigenin demonstrated favorable control effects on TBS. Compared to pesticides, the utilization of these compounds is less likely to induce pathogen resistance, facilitates natural degradation, and exerts a lesser impact on the ecological environment. The experimental outcomes suggest that root exudates may be the cause of tobacco resistance, with isoliquiritigenin and lauric acid exhibiting conspicuous control effects on TBS. However, it is imperative to conduct further investigations to explore the impact of numerous other compounds present in root exudates on tobacco resistance.

Conclusions

The root exudates of disease-resistant cultivar K326 exhibited a more pronounced inhibitory effect on P. nicotianae compared to tobacco cultivar Yunyan 87. The root exudates of K326 were found to be rich in organic acids, fatty acids, phenols, coumarins, terpenoids, and flavonoids, whereas Yunyan 87 exhibited a higher abundance of amino acids, phenylpropanoids, and alkaloids. Isoliquiritigenin, homovanillic acid and lauric acid hold promise in the control of tobacco black shank. The relationship between root exudates and the mechanisms underlying plant disease resistance is exceedingly intricate. This study merely provides a preliminary glimpse into the association between tobacco disease resistance and root exudates, necessitating further experimentation to delve deeper into the disease resistance mechanisms governed by these root exudates.

Author contributions

G.Y, and D.Z. conceived and designed the experiments. F.S, and P.S. conduced the experiments. F.S., Y.L, and D.F. analyzed the data. P.S, and F.S. wrote the manuscript. All authors contributed to the manuscript and approved the submitted version.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All the data relevant to this study are included in the article or uploaded as Supplementary Materials.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592324.2024.2332019

Additional information

Funding

This work was funded by the Science and Technology Planning Project of Tobacco Company of Guangxi Zhuang Autonomous Region [No.202045000020084].