Naringenin confers defence against Phytophthora nicotianae through antimicrobial activity and induction of pathogen resistance in tobacco

Abstract Tobacco black shank caused by Phytophthora nicotianae is a serious disease in tobacco cultivation. We found that naringenin is a key factor that causes different sensitivity to P. nicotianae between resistant and susceptible tobacco. The level of basal flavonoids in resistant tobacco was distinct from that in susceptible tobacco. Of all flavonoids with different content, naringenin showed the best antimicrobial activity against mycelial growth and sporangia production of P. nicotianae in vitro. However, naringenin showed very low or no antimicrobial activity to other plant pathogens. We found that naringenin induced not only the accumulation of reactive oxygen species, but also the expression of salicylic acid biosynthesis‐related genes. Naringenin induced the expression of the basal pathogen resistance gene PR1 and the SAR8.2 gene that contributes to plant resistance to P. nicotianae. We then interfered with the expression of the chalcone synthase (NtCHS) gene, the key gene of the naringenin synthesis pathway, to inhibit naringenin biosynthesis. NtCHS‐RNAi rendered tobacco highly sensitive to P. nicotianae, but there was no change in susceptibility to another plant pathogen, Ralstonia solanacearum. Finally, exogenous application of naringenin on susceptible tobacco enhanced resistance to P. nicotianae and naringenin was very stable in this environment. Our findings revealed that naringenin plays a core role in the defence against P. nicotianae and expanded the possibilities for the application of plant secondary metabolites in the control of P. nicotianae.


| INTRODUC TI ON
Plant-pathogenic oomycetes pose a serious threat to agriculture, horticulture, forestry, aquaculture and natural ecosystems (Wang, Tyler, et al., 2019). They encompass over 100 species that cause devastating diseases in both plants and animals. The Phytophthora species are the most serious pathogens. They infect a broad group of hosts and cause diseases such as root rot, late blight, downy blight, and downy mildew (Derevnina et al., 2016;Kamoun et al., 2015;Panabieres et al., 2016). Tobacco black shank (TBS) disease, caused by Phytophthora nicotianae, threatens production in tobaccoproducing areas across the world (Gallup et al., 2018). Infections mainly occur in the adult stage of tobacco, resulting in significant yield loss and quality reduction. Roots and stems are the main parts infected, leading to progressive decay of the diseased tissues, such as root and stem necrosis, wilting, and chlorosis, and finally death Csinos & Minton, 1983;Gallup et al., 2006).
Mycelia, oospores and chlamydospores from P. nicotianae are found in soil and plant tissues in compost through the winter and can survive for more than 3 years. Soil is therefore the main source of infection, while manure and irrigation promote the spread of pathogens (Antonopoulos et al., 2010;Gallup et al., 2018).
TBS is a very difficult disease to control due to the diversity and high adaptability of P. nicotianae (Gallup et al., 2018;Ji et al., 2014).
Its management relies on the integration of different approaches, including planting resistant varieties, fungicide applications, crop rotation, management of water and fertilizer (Shew & Lucas, 1991).
So far, four physiological races (0, 1, 2 and 3) of P. nicotianae have been reported. The undomesticated Nicotiana species are an important source of resistant varieties. Tobacco cultivars resistant to race 0 of P. nicotianae conferred by the genes Php or Phl have been widely used across the world; however, cultivars have become susceptible to P. nicotianae as there has been a gradual shift of pathogen populations from race 0 to race 1, making the strategy of TBS-resistant cultivars ineffective in disease control (Johnson et al., 2002;Li et al., 2006;Sullivan et al., 2005). In addition, P. nicotianae has a large arsenal of secreted proteins, termed effectors, that act as weapons to overcome the host resistance and promote infection (Hou et al., 2019;Lee et al., 2018). These are the main reasons that crop rotation is highly recommended to avoid disease outbreaks and propagation of P. nicotianae in the field (Yong et al., 2010). Pesticide application is another important approach to managing TBS. Several fungicides such as mefenoxam and potassium phosphite have been applied for many years but resistance in Phytophthora has been reported for both compounds (Hao et al., 2019;Ji et al., 2014). Some new fungicides against oomycetes, such as fluopicolide, ethaboxam, oxathiapiprolin and mandipropamid, with different modes of action have been developed and they have shown higher efficiency in treatment of Phytophthora (Hao et al., 2019;Ji et al., 2014;Qu et al., 2016). These new fungicides provide another highly effective approach against pathogens in addition to rotation and proper use of all existing fungicides. However, more attention should be paid to the development of resistance to these new fungicides among the populations of P. nicotianae (Panabieres et al., 2016;Qu et al., 2016).
Biocontrol has been shown to be an alternative way to manage TBS efficiently, mainly using biocontrol strains and new compounds that have been isolated from natural organisms. Among biocontrol strains, Pseudomonas fluorescens and Bacillus spp. have been widely studied and used due to the antimicrobial components they release, such as peptides and enzymes (Choudhary & Johri, 2009;Fira et al., 2018;Rajaofera et al., 2018). These antimicrobial components can improve plant growth, as well as induce a plant immune response to abiotic stress (Choudhary & Johri, 2009;Sun et al., 2017). In addition, some biocontrol strains, such as the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens Ba168, secrete proteins and peptides that target pathogens directly, for example, by damaging the cell wall and membrane of P. nicotianae .
In addition, various natural agents produced from plants are another source of antimicrobial components. Some secondary metabolites are not involved in growth, development or reproduction of plants directly. Rather, they perform special functions under a given set of conditions such as pathogen attack, water deficit or extreme temperature (Bartwal et al., 2012). The essential oil eugenol and diallyl disulphides have been confirmed to be effective against TBS by destroying mycelial cell membrane integrity, causing an increase in cell membrane permeability and leading to cell death (Jing et al., 2017;. Phytoalexins with antiseptic, anti-inflammatory, antioxidant or antimicrobial activities accumulate soon after pathogen infection (Chripkova et al., 2016;Kumar et al., 2006).
Over 9000 categories of flavonoids have been found in various plants, making them one of the largest families of secondary metabolites (Wang et al., 2011). They are essential factors that are involved in aspects of plant development and defence, and flower and fruit quality (Treutter, 2005;Wang et al., 2011). Flavonoids often accumulate in specialized cells and there are only a few studies on their function against pathogens (Kariu et al., 2017;Martínez-Castillo et al., 2018;Paczkowski et al., 2017;Tattini et al., 2004). Naringenin is one of the major flavonoids and is mainly found in citrus fruits, including tangerine, lemon, orange and grapefruit (Manchope et al., 2017).
We found that naringenin conferred defence against P. nicotianae by inhibition of mycelial growth and sporangia production. Through metabolomics analysis, we found that basal flavanones and flavonols in resistant tobacco varieties were distinct from those in susceptible tobacco varieties. Of all flavanones and flavonols, we discovered that naringenin inhibited P. nicotianae most effectively. However, naringenin showed no or very low inhibition activity towards other plant pathogens. We also found that naringenin induced plant resistance to P. nicotianae by inducing the accumulation of O 2 − and H 2 O 2 , the expression of genes regulating salicylic acid (SA) biosynthesis and SA signalling, and the expression of genes regulating plant basal defence and resistance to P. nicotianae. Knockdown of chalcone synthase (CHS), a gene encoding a rate-limiting enzyme of the naringenin sythesis pathway, rendered tobacco highly susceptible to P.
nicotianae, but it kept the same susceptibility to other plant pathogens. Interestingly, exogenous application of naringenin to tobacco enhanced resistance to TBS without affecting the agronomic traits.
Our findings reveal that naringenin showed antimicrobial activity towards P. nicotianae and is therefore a potential botanical pesticide in tobacco disease control for the future.

| Metabolite profiling of basal flavanone and flavonol in resistant tobacco varieties is distinct from that in susceptible tobacco varieties
Typical resistant and susceptible cultivated tobacco varieties to TBS were used in this study. The typical cultivated tobacco varieties in China are Beinhart 1000-1 (BH-1) and Xiaohuangjin 1025 (XHJ), which show resistance and susceptibility to P. nicotianae, respectively ( Figure 1a). The disease index of BH-1 is significantly lower than that of XHJ after inoculation with P. nicotianae (Figure 1b), and the colonization of P. nicotianae in XHJ is higher than that in BH-1 ( Figure 1c). To test whether there is a difference in the basal level of metabolites between the resistant and susceptible tobacco varieties in uninfected roots, the flavonoids of BH-1 and XHJ were analysed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to evaluate the metabolite profiles.
As shown in Table S1, 166 metabolites were identified and characterized by its distinct retention time and mass-to-charge ratio (m/z). Through metabolite analysis, 16 flavanones were characterized. The content of nine flavanones, including liquiritigenin and naringenin, was slightly higher and the content of three flavanones, including isosakuranetin and hesperetin, was significantly higher in BH-1 compared to XHJ (Table S3). We also characterized 44 flavonols, of which the content of seven was significantly higher in BH-1 than XHJ, such as kaempferol and quercetin (Table S4). These results indicate that there is a significant difference in the basal level of flavonoids between resistant and susceptible tobacco varieties, and this difference might be related to resistance to P. nicotianae in the plants.  (Table S5).

| Naringenin showed antimicrobial activity on P. nicotianae in vitro
In contrast, none of the flavonols, such as kaempferol, quercetin or reutinum, showed any inhibitory activity on the mycelial growth of even at concentrations up to 200 mg/L ( Figure S1a,b and Table S5).
Naringenin also inhibited growth of other oomycetes, such as Phytophthora capsici, with an EC 50 value of 50.11 mg/L, but showed low inhibition activity on Pythium aphanidermatum ( Figure S2 and Table S6). However, naringenin showed very low or no inhibitory activity on other plant pathogens such as Sclerotinia sclerotiorum, Botrytis cinerea, and Fusarium graminearum ( Figure S3). We next tested whether naringenin affected the reproduction of P. nicotianae by microscopic observation. As shown in Figure 2c,d and Table S6, the EC 50 and EC 90 values of naringenin for inhibition of P. nicotianae sporangia production were 2.01 mg/L and 6.62 mg/L, respectively. These values are significantly lower than the EC 50 for mycelial growth. Furthermore, we used reverse transcription-quantitative PCR (RT-qPCR) to confirm that cell growth and reproduction-related genes were down-regulated when P. nicotianae was treated with naringenin ( Figure S4). These results indicate that naringenin inhibits not only the mycelial growth but also the reproduction of P. nicotianae.

| Naringenin induces accumulation of reactive oxygen species
Normally, plants are resistant to the invasion of most pathogens through a burst of reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ) and the superoxide anion (O 2 − ) (Bray, 2000). The increased H 2 O 2 content promotes the up-regulation of genes that are associated with the plant defence response (Bray, 2000). Flavonoids have been shown to be antioxidant agents by clearing away ROS (Turek & Stintzing, 2013

| Naringenin induced expression of salicylic acid biosynthesis-related genes and signalling in tobacco
The hormone salicylic acid (SA) is required for the activation of immune responses to biotrophic pathogens in plants (van Butselaar & F I G U R E 1 Metabolite profiling of resistant (BH-1) and susceptible (XHJ) tobacco lines in response to Phytophthora nicotianae. Disease symptoms observed following root inoculation with a spore suspension of P. nicotianae at 5 days postinoculation (dpi). (a) Phenotypes of BH-1 and XHJ in response to P. nicotianae. (b) Disease index of P. nicotianae on BH-1 and XHJ (n = 3, error bars, SD). XHJ is significantly different from BH-1 according to a Mann-Whitney test (**p < 0.01). (c) Biomass of P. nicotianae in BH-1 and XHJ (n = 3, error bars, SD). XHJ is significantly different from BH-1 by a Mann-Whitney test (**p < 0.01). (d) A volcano plot of metabolites of BH-1 compared to XHJ. (e) The proportion of the number with a higher content of metabolites in each class of metabolites. The red numbers indicate the number of metabolites with significantly higher content, and the blue numbers indicate the total number of the metabolites detected in the metabolome. (f) A heatmap of 15 metabolites with higher content in BH-1 than XHJ. The left three columns are from roots of XHJ and the right three columns are from roots of BH-1; each column represents a biological replicate.

| Naringenin enhanced plant resistance to P. nicotianae
Flavonoids have been shown to be critical to plant defence on pathogenic bacteria and fungi through the induction of PR genes (Mierziak et al., 2014). Quercetin and its derivatives are capable of inducing pathogen resistance to both bacteria and fungi (Jia et al., 2010;Parvez et al., 2004;Yang et al., 2016). To examine whether naringenin induces basal pathogen resistance, we analysed the expression of PR1 and SAR8.2 genes in tobacco after naringenin treatment using RT-qPCR. The SAR8.2 gene is a gene that controls plant resistance to P. nicotianae (Shi et al., 2022). As expected, the expression levels of PR1 and SAR8.2 with naringenin treatment were approximately 1.4-fold and 3.5-fold higher than those of the control, respectively (Figure 3e,f). These results suggest that naringenin induces basal pathogen resistance.

| Interference of naringenin biosynthesis led to more susceptibility to P. nicotianae in tobacco
The flavonoid biosynthesis pathway has been well studied and most intermediate enzyme steps have been characterized (see Figure 4a).
The pathway starts with the conversion of phenylalanine to cinnamic acid by phenylalanine ammonia-lyase (PAL  (Kreuzaler & Hahlbrock, 1972;Pandey et al., 2015). We first tested the expression of NtCHS, NtPAL, NtC4H and Nt4CL in BH-1 and XHJ without and with inoculation of P. nicotianae. We found that, compared to XHJ, NtCHS was more highly expressed in BH-1 even when infected with P. nicotianae, indicating that NtCHS might R. solanacearum even at concentrations up to 400 mg/L (Table S7).
Furthermore, the biomass of P. nicotianae in NtCHS-RNAi mutants was up to six times more than that in WT by DNA quantification of the oomycete ( Figure S7). These results show that interference in naringenin synthesis in tobacco induced more susceptibility to P. nicotianae and naringenin might be a specific antimicrobial agent to P. nicotianae because it showed very low or no activity towards other plant pathogens.

| Exogenous application of naringenin enhanced resistance to P. nicotianae
To explore the possibility of applying naringenin as a natural antimicrobial agent in the management of TBS in the field, we treated susceptible tobacco HD seedlings with naringenin before they were inoculated with P. nicotianae. HD seedlings inoculated with P. nicotianae but without naringenin treatment were used as the control. After 10 days, we found that HD seedlings treated with naringenin were significantly more resistant to P. nicotianae compared to the control (Figure 5a,b). Accordingly, the disease index in HD treated with naringenin was dramatically lower than in the control (Figure 5c). More importantly, the application of naringenin did not negatively affect the agronomic traits of tobacco, such as plant height, leaf length, leaf width, knot spacing and stem girth ( Figure S8). In addition, to test the impact of naringenin stability on infection by pathogens, we checked the susceptibility of HD tobacco to P. nicotianae 3, 7, 15 and 30 days after treatment with naringenin. The results showed that HD treated with naringenin for 30 days still had the same susceptibility to P. nicotianae as HD treated with naringenin for 3 days, and both were significantly more resistant to P. nicotianae compared to HD without naringenin treatment. These results show that exogenous application of naringenin improved resistance to P. nicotianae significantly and showed good stability of antimicrobial activity in the environment ( Figure S9). Therefore, naringenin was shown to be a potential antimicrobial agent for the management of TBS in the field.

| DISCUSS ION
Plants have evolved diverse biosynthetic routes to produce a range of small organic molecules referred to as secondary metabolites. The disease index with or without the treatment of naringenin after HD was inoculated with P. nicotianae (n = 3, error bars = SD). HD is the main cultivated fluecured tobacco variety but is susceptible to tobacco black shank. Significant difference compared to control by the Mann-Whitney test (**p < 0.01).
such as P. capsici. Naringenin showed no or very low inhibitory activity towards plant pathogens such as S. sclerotiorum and B. cinerea.
Interestingly, there was dramatic inhibition of sporangia production when P. nicotianae was treated with a very low concentration of naringenin. The reason for the inhibition of sporangia production by naringenin might be that genes regulating the reproductive process in P. nicotianae are directly suppressed by naringenin. We used RT-qPCR to confirm that genes regulating cell growth and reproduction were down-regulated after P. nicotianae was treated with naringenin.
Naringenin is thus the first flavonoid to be identified as a antimicrobial agent against P. nicotianae; it might affect the expression of genes regulating the growth and reproduction of P. nicotianae directly.
Flavonoids are well known as ROS scavengers and powerful antioxidants (Pannala et al., 2001;Rice-Evans, 2001). Flavonoids decrease the ROS level through inhibition of pro-oxidant enzymes, including cyclo-oxygenase and lipoxygenase (Eghbaliferiz & Iranshahi, 2016). Phenolics and carotenoids are chemical groups that prevent oxidative damage as a result of their ability to decrease the ROS level, and they also exhibit pro-oxidant activities in vitro in the presence of metal ions (Eghbaliferiz & Iranshahi, 2016).
Like phenolics and carotenoids, flavonoids act as pro-oxidants at physiological pH. In this study, we showed that naringenin induced the accumulation of ROS, which suggests that naringenin might act as a pro-oxidant. An et al. also showed that naringenin induced an ROS burst in plants as a defence against P. syringae (An et al., 2021). SA has been shown to induce pathogen resistance through ROS accumulation and increased expression of PR genes.
We also found that genes regulating SA biosynthesis, SA signalling and basal pathogen resistance were enhanced at the transcriptional level by treatment with naringenin . Furthermore, SAR8.2, a gene in tobacco that regulates plant resistance to P. nicotianae (Shi et al., 2022), was substantially up-regulated by treatment with naringenin. These results indicate that naringenin induces plant-pathogen resistance. Naringenin accumulates soon after plants are infected with biotrophic pathogens such as Rhizobium leguminosarum bv. viceae, Pseudomonas syringae pv. pisi and Plasmodiophora brassicae (Makarova et al., 2016). However, we did not detect significant accumulation of naringenin in tobacco after infection with P. nicotianae by metabolite analysis. Interestingly, naringenin has been shown to induce pathogen resistance against P. syringae through the activation of NPR1 in Arabidopsis 2021, but did not show any antimicrobial activity against P. syringae even at high concentrations in vitro (An et al., 2021). Our results demonstrate that naringenin confers plant defence against P. nicotianae through induction of plant-pathogen resistance in addition to its antimicrobial activity.
The key genes in the metabolic pathway of naringenin synthesis include PAL, 4CL, C4H, and CHS (Kreuzaler & Hahlbrock, 1972;Pandey et al., 2015). Our results showed that the expression level of NtPAL, NtC4H and Nt4CL did not show a significant change except in the later period (5 days) after BH-1 had been infected by P. nicotianae ( Figure S5). Remarkably, only NtCHS had a higher expression level in BH-1 than that in XHJ, even after BH-1 was treated with P. nicotianae. The accummulation of naringenin is positively correlated with the expression of CHS (Pandith et al., 2019).
In this study, without infection of P. nicotianae, the basal expression level of NtCHS in resistant tobacco variety BH-1 was higher than that in susceptible tobacco variety XHJ. Correspondingly, the content of naringenin in BH-1 was higher than that in XHJ. CHS syringae, naringenin could also be expected to induce resistance to R. solanacearum. CHS regulates flower colour, fertility and gas substances, and we found that CHS was also involved in resistance to P. nicotianae. We also found that TBS-susceptible tobacco variety HD became resistant to P. nicotianae after treatment with naringenin. Naringenin showed high activity with a good stability in the environment ( Figure S9). This leads to the potential application of naringenin as a natural antimicrobial agent in the management of TBS in the field in the future.
In conclusion, for first time we found that naringenin might be an antimicrobial secondary metabolite against P. nicotianae.
Naringenin inhibited P. nicotianae through not only its antimicrobial activity, but also its induction of defences against plant pathogens ( Figure S10). Our discoveries will enrich the means of prevention and control for TBS. Future work needs to address the question of whether naringenin targets proteins or genes in P. nicotianae directly, and whether naringenin interferes in the interaction between P. nicotianae and tobacco. Notably, the development of naringenin as a plant-derived fungicide against P. nicotianae is important work for the future. score)] × 100. Three-month-old tobacco plants were infected with P. nicotianae according to a described method (Zhang et al., 2018).

| Plant materials and P. nicotianae inoculation
The biomass of P. nicotianae in infected tobacco plants was quantified using a slight modification of a previously described method (Park et al., 2012). Quantitative PCR was performed using a Light Cycler 96 Real-Time PCR detection system (Roche, http://techn ical-suppo rt.roche.com). The biomass of P. nicotianae was calculated using the threshold cycle value (C t ) of P. nicotianae WS21 DNA against the C t of tobacco genomic actin DNA.

| Inoculation of tobacco with R. solanacearum
After 12 at 28°C for 14 days. The inoculum method was used as described (Zhang et al., 2018). Then 0.1% KNO 3 was added to the PDA plate containing P. nicotianae and kept at 4°C for 25 min, then kept in the light for 30 min at 25°C. The concentration of the spore suspension was determined by a cellometer Auto T4 (Nexcelom Cellometer) and adjusted to 10 4 spores/ml.

| Antimicrobial activity test for flavonoids
All flavonoid standards used in this study were purchased from the Solarbio Company. First, 20 mg of standard flavonoid was dissolved in

| Metabolite estimation and analysis
Eight-week-old seedlings of BH-1 and XHJ grown in Hoagland hy-  Three independent biological replicates were used. The sequences of all primers are shown in Table S8.

| The production of NtCHS-RNAi lines
The construction of NtCHS-RNAi and generation of transgenic tobacco lines have been described previously (Chen et al., 2019).
PCR identification was used to amplify the transformed seedling DNA with RNAi-F and RNAi-R primers to determine whether the RNAi vector was successfully introduced. For the transformed lines, the silencing efficiency of NtCHS was identified by RT-qPCR.

| Exogenous application of naringenin
Under simulated field conditions, Nicotiana tabacum 'Honghuadajinyuan' (HD) was cultured for 3 months in pots. Then, the stem base of the HD seedlings was treated with 100 ml of solution including 0.04 g of naringenin 1 week prior their inoculation with P. nicotianae. Naringenin was added only once. HD seedlings with inoculation by P. nicotianae but without prior treatment of naringenin were used as a negative control. After 10 days, the phenotype of TBS was evaluated using an empirical six-point scale (YC/T39-1996, China), where 0 represents a highly resistant response and 9 represents a highly susceptible response. Disease index scores based on disease severity were used for assessment and calculated using the following formula: disease index (%) = [Σ(disease evaluation scale score × number of plants with each scale score)/(total number of plants observed × the highest disease evaluation scale score)] × 100. Shuai for providing seeds of NtCHS-RNAi lines of tobacco plants.

CO N FLI C T O F I NTE R E S T
No conflict of interest declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.