1. Introduction
Cucumber,
Cucumis sativus L. is among the most economically important vegetable crops. China is the dominant producer of cucumber in the world accounting for 70-80% of total world production annually in the last decade (
https://faostat.fao.org/). Many diseases affect cucumber production, the soil-borne fungal Fusarium wilt (FW) is probably the most troublesome and difficult to control, which is particularly true in continuous cropping system and in protected environments. In China, under the continuous cropping system, FW incidences may range from 30 to 90% leading to significant yield loss (Zhou and Wu, 2012). The causal agent is Fusarium
oxysporum f. sp.
cucumerinum (
Foc) (Owen, 1955). Typical symptoms of FW include yellowing, stunting, and death of seedlings, and yellowing and stunting of older plants. Infected plants wilt readily, lower leaves yellow and dry, the xylem tissues turn brown, and the plant may die. The symptoms are worsened when the plants are under stress or during fruiting. This pathogen can survive in plant debris and in soil for many years as chlamydospores (i.e. overwintering spores) and for shorter periods on greenhouse structures between crops as conidia. The large-scale, intensive cucumber production in protected environments such as greenhouses or high tunnels with infected soils increases FW severity and frequency and makes it more difficult to control this disease (Yu. et.al, 2000, Shen. et.al, 2008, Chen. et.al, 2012, Li. et.al, 2016).
Many integrated pest management (IPM) strategies such as deployment of resistant varieties, grafting, fumigation, crop rotation, and biological controls have been proposed to control FW in cucumber (Yu. et.al, 2000, Li. et.al, 2016, Tang. et.al, 2021, Nishioka. et.al, 2022) . Many of the proposed practices are not readily implementable for large scale commercial production. The development of resistant varieties is probably the most economical and environmentally sound measure for IPM of FW in cucumber production. In addition to reduce the disease incidence and yield loss, resistant varieties can also improve the rhizosphere microbial community and soil quality (Yao and Wu, 2010). In China, several studies have evaluated FW resistance in cucumber collections (Mao.et.al,2008, Li. et.al, 2015, Li. et.al, 2018). In a few cases, the FW resistance in different resistance sources has been characterized including the US cucumber inbred lines Wis248, WI2757, WisSMR-18 (Mao. et.al, 2008, D. Netzer, 1976, Vakalounakis,1993, Mao.et.al, 2008, Vakalounakis and Lamprou, 2018), the cucumber germplasm line 9110Gt (Zhang. et.al, 2014), as well as the North China type cucumber lines Rijiecheng (Dong. et.al, 2019) and ‘3461’ (Bartholomew. et.al, 2022). Interestingly, the FW resistance in all these lines seems to be controlled by a single dominant gene. The resistance gene CsChi23 from ‘3461’ has been cloned, which encodes a cucumber class I chitinase with antifungal properties (Bartholomew. et.al, 2022). Interestingly, molecular mapping studies suggest that the single domain resistance gene in all other cucumber lines seems to be located in the same region on cucumber chromosome 2 that harbors a cluster of NB-LRR resistance gene homologs (Zhang. et.al, 2014). However, the identity and exact functions of this gene are unknown.
The molecular mechanisms of resistance gene mediated defense responses have been extensively studied and reviewed (Jones and Dangl,2006, Spoel and Dong,2012, Cui.et.al,2015, Yuan. et.al, 2021, Aerts. et.al, 2022). Briefly, the establishment, penetration into the host cell wall and colonization of host plant by the pathogens are facilitated by various enzymes like pectinases, proteases, polygalacturonases and cellulases that are secreted by the pathogens. To counteract pathogen attacks, upon infection, plant immune receptors recognize diverse pathogen molecules, leading to elicitor triggered immunity (ETI). ETI involves activation of different biochemical pathways for biosynthesis of pathogenesis-related (PR) proteins, callose formation, accumulation of phytoalexins and cell wall modification that comprises lignification (Iqbal. et.al ,2021). Of particular importance is the synthesis of various secondary metabolites such as flavonoids, catecholamines, phenolic acids, phenols, and lignins, which play important roles in disease defense responses (Dixon and Barros, 2019, Campos. et.al, 2021).
How the FW resistance genes regulate defense responses against the Foc pathogen infection in cucumber is largely unknown. Chitinases are pathogenesis-related (PR) proteins that have been shown to play an important role in FW resistance (Bartholomew. et.al, 2022, Bartholomew. et.al, 2019, Xu. et.al, 2021). Zhang et al. (2016) (Zhang. et.al, 2016) conducted comparative proteomic analysis of roots between two resistant and a susceptible cucumber lines and identified 15 over accumulated proteins that were involved in defense and stress responses, oxidation-reduction, metabolism, transport and other processes, and jasmonic acid and redox signaling components. Xu et al. (2021) (Xu. et.al, 2021) also compared the proteomes of the FW resistant Rijiecheng and susceptible Superina and identified 210 and 243 differentially regulated proteins in response to Foc infection with 32 predominantly expressed in Superina and significantly up-regulated after Foc inoculation. Dong et al. (2020) (Dong. et.al, 2020) conducted transcriptome analysis in cucumber and suggested that ethylene-mediated defense responses play an important role against Foc infection in cucumber.
Integrated omics (genome, transcriptome, metabolomes and proteomes) approaches have provided powerful tools for understanding the molecular mechanisms of R-gene mediate defense responses in different crop plants (Kumar. et.al, 2016, Chen. et.al, 2019, Szymanski. et.al, 2020, Chai. et.al, 2021, Duan. et.al, 2022, Hussain. et.al, 2023). From our previous work, we identified a highly inbred cucumber line (NR) with high resistance to FW. Preliminary observations found that FW resistance in NR is controlled a single domain gene A spontaneous susceptible mutant plant (NS) was isolated from the resistant line (NR). The objective of this study was to investigate the transcriptomes and metabolomes of the two near isogenic lines (NILs) to understand resistance gene mediated defense responses. We evaluated the FW resistance of the NILs. We further conducted RNA-Seq and widely targeted metabolomic analyses using the two NILs. Comparative and integrated analyses of the transcriptomes and metabolomes identified key genes, phenolic acids and flavonoid secondary metabolites that may play important roles in FW resistance in NR.
3. Discussion
3.1. Accumulation of Phenolic Compounds is Positively Correlated with FW Resistance in NR
FW is a serious soilborne disease which is very difficult to control especially under continuous cropping in protected environments, which is popular in cucumber production in China (He. et.al, 2022). Development of host resistance is a critical component in IPM of diseases in crop production. Although several resistance sources to FW have been identified (see introduction), most cucumber varieties in commercial production in China are FW susceptible. The only FW resistance gene cloned is a single dominant gene
Foc from the north China inbred line ‘3461’ which encodes a class I chitinase (Bartholomew. et.al, 2022). Overall, our understanding of the molecular mechanisms of FW resistance in cucumber is very limited. In this study, we developed NILs, NR and NS for FW resistance, which belong to the South China cucumber type ecotype. In multiple screening tests in both plastic greenhouses (natural infection) and the laboratory screening (artificial infection), the NR exhibited consistent and stable FW resistance (
Figure 1 and
supplemental Figure S1B). Based on results from the present study, this resistance gene is probably different from the
Foc gene reported in 3461 (Bartholomew. et.al, 2022).
To understand the gene regulatory network in resistance gene mediated defense responses to
Foc infection in NR NIL, we conducted both metabolome and transcriptome profiling of the two NILs before (0 dpi) and after (4 dpi)
Foc inoculation using the same set of four samples (NR0d, NS0d, NR4d and NS4d) with three biological replications. Many differentially accumulated metabolites (DMAs) and differentially expressed genes (DEGs) were identified in comparison between the resistant and susceptible NILs (
Figure 2 and
Figure 3;
Tables S1-8). Further analysis of the metabolomic data found significantly more accumulation of phenolic compounds (phenolic acids and flavonoids) in NR than in NS in response to
Foc infection (
Figure 2). In consistent with this, DEGs response for biosynthesis or regulation of secondary metabolite biosynthesis in the flavonoid biosynthesis pathway were highly enriched (
Figure 3;
Table S6-7). These data strongly suggest that accumulation of phenolic compounds from the phenylpropane and flavonoid biosynthesis pathways are contributing to the FW resistance in NR.
Phenolic compounds are secondary metabolites in plants which may include phenolic acids (e.g., chlorogenic acid, caffeic acid, p-hydroxybenzoic acid, ferulic acid, 4-coumaric acid and gallic acid), flavonoids (e.g., flavanones, flavonols and proanthocyanidins), tannins, stilbenes, and lignans which are mainly synthesized by the shikimic acid, chorismate and phenylalanine metabolic pathways (Dixon and Barros, 2019, Palacio. et.al, 2012) . Phenolic compounds have diverse functions in plant growth and development, reproduction, and defense, which may as antioxidants, structural polymers (lignin), attractants (flavonoids and carotenoids), UV screens (flavonoids), signal compounds (salicylic acid and flavonoids) and defense response chemicals (tannins and phytoalexins) (Dixon and Barros, 2019, Dicko. et.al, 2005, Visioli. et.al, 2011, Lima. et.al, 2019, Weisshaar and Jenkins, 1998). The roles of phenolic compounds in plant disease resistances have been extensively documented (Dixon and Barros, 2019, Kneusel. et.al, 1988, Jun Tsuji. et.al, 1992). For example, phenolic acids are key components of plant resistance to different pathogens (bacteria, fungi and viruses) (Santi M. Mandal, 2010, Boiteux. et.al, 2014, Zhang. et.al, 2022). Upon pathogen infection, plant may accumulate large amount phenolic acids (Mikulic-Petkovsek. et.al, 2013). Phenolic acids are key signaling molecules can be released rapidly from new roots during seed germination and seedling growth which may contribute to soil-borne pathogens (Santi M. Mandal, 2010, Ndakidemi and Dakora, 2003). Microbial changes influenced by signals from phenolic acids may have ecological effects on plant-microbial interactions (Shaw. et.al, 2006). Accumulation of flavonoids is part of the general defense responses in plants (Ndakidemi and Dakora, 2003, Li.et.al, 2011, Roy.et.al, 2018, Theunis. et.al, 2004). The antifungal effects of flavonoids were mainly manifested in inhibiting the growth of fungal colonies, spore germination and bud tube length (Xu.et.al, 2022). Therefore, data from present study were consistent with previous findings and support a critical role of phenolic acids in defense against Foc infection.
3.2. Key phenolic Acids and Flavonoids and Biosynthetic or Regulatory Genes Associated with FW Resistance
Joint analysis of the metabolomic and transcriptomic data using Pearson’s correlation coefficients identified important associations between 13 DAMs (phenolic acids/flavonoids) and 20 DEGs (
Figure 4;
Figure S3;
Tables S11 and S12). Among the four phenolic acids, mudanoside A is one of the end products of phenolic acid compounds with gallic acid as the precursor of its synthesis which is catalyzed a hydrolase that seems to be associated with the function of
Csa1G043010 (
Figure 4), which is involved in the biosynthesis of tulitin, a defensive chemical with antibacterial activity against a variety of bacterial and fungal strains (Lima. et.al, 2019, Milinčić. et.al, 2021, Zhu. et.al, 2022). While gallic acid can synthesize and induce rice rhizobia resistance to rhizobia (Sarma and Singh, 2003). Hydrolase coding gene (
Csa1G043010) hydrolyzes ferulic acid to produce phenolic compounds 3,4'-Dihydroxy-3',5'-dimethoxypropiophenone, which can hydrolyze caffeic shikimic acid into caffeic acid and shikimic acid. Ferulic acid not only inhibits the growth of anthrax, but also induces resistance of rice rhizobia to rhizobia. It can also regulate plant root growth (Santi M. Mandal,2010, Sarma and Singh, 2003, Zhijun.et.al, 2022). It is speculated that the high expression of these genes in the regulation pathway of phenolic acid synthesis may lead to the accumulation of these phenolic acid compounds, thus improving the resistance of plants to FW.
The synthesis of flavonoids in plants is a complex metabolic process, which is controlled by a series of enzymes and varies according to species and tissues (Mikulic-Petkovsek. et.al, 2013, Sade. et.al, 2015). In the study, we identified four flavonoid DAMs that were significantly more accumulated in NR including 1 intermediate product tricin-4'-O-glucoside and 2 end-products 6-hydroxykaempfero-7-O-glucoside and gallocatechin 3-O-gallate (
Table S11). Three differentially expressed genes were strongly correlated with them, namely, carboxylesterase gene (
Csa6G401340), UDP glycosyltransferase gene (
Csa4G620550) and plant disease resistance protein gene (
Csa6G084580). Compared with NS4d, the chalcone synthase gene (CHS) (
Csa6G401340) was up-regulated 2.13 times in NR4d, which is the first key gene in phenylpropane biosynthesis to flavonoid biosynthesis. It regulates the formation of Tricin-4'-O-glucoside, which is an antioxidant flavonoid in plants and enhances the disease resistance of plants by improving the antioxidant activity of plants (Zhong. et.al, 2022). Flavanols, flavonols and flavones are important subclasses of flavonoid compounds (Chen. et.al, 2021). FLS (
Csa4G620550) flavonol synthase coding gene was significantly upregulated in NR4d, 2.16 times of NS4d. This gene encodes UDP glycosyltransferase and catalyzes the formation of flavonol glucosides (Li. et.al, 2021). LAR (
Csa6G084580) was 2.12 times more prevalent in NR4d than in NS4d, and it was associated with the synthesis of plant disease resistance response protein. In flax, three key genes of flavonoids, CHS, CHI and DFR, were synthesized by transgenic method, resulting in a significant increase in the contents of flavanones, flavonoids and flavanols. Increased flax resistance to Fusarium acarium (Lorenc-Kukuła. et.al, 2007).
In addition to biosynthetic genes, many DEGs for transcription factors (TFs) were also identified from RNA-Seq (
Table S9). TFs, especially MYB and bZip TFs have been shown to play important roles in regulating phenolic compound accumulation for disease resistance (Bartholomew. et.al, 2022, Nuruzzaman. et.al, 2013, Shim. et.al, 2013, Liu. et.al, 2016, Jin. et.al, 2017, Tang. et.al, 2022). For example, MYB TFs plays an important role in plant defense response to biological stress that triggers a wide range of plant defenses. SpMYB (Solanum pimpinelifolium L3708) was significantly expressed in tobacco after infection with fusarium oxysporum. Overexpression of SpMYB in tobacco increased resistance to Fusarium oxysporum, and peroxidase, superoxide dismutase and phenylalanine increased the activity of ammonia lyase in transgenic plants (Wang. et.al, 2015, Xie. et.al, 2022). The DEGs associated with DAMs included four genes for MYB family transcription factors (
Csa1G071840, Csa2G403160, Csa4G638510, Csa1G586860) and one for a bZIP transcription factor (
Csa2G403160). The MYB transcription factor coding gene (
Csa4G638510) activated the transcription of the auxin response gene IAA19 in response to auxin. Shikimic acid produces phenylalanine under the action of the R2R3MYB transcription factor coding gene (
Csa1G071840), the transcription factor encoding gene can promote shimoic acid pathway to regulate volatile benzene and phenylpropane-activated EPSPS, ADTI, CFTA, CCoAOMT1 genes (Van Moerkercke. et.al, 2011, Shaipulah. et.al, 2016). Sallic Acid (SA) is an important phenolic acid compound. In recent years, PBS3 gene has been identified as the most critical enzyme encoding gene in the SA biosynthetic pathway (Rekhter. et.al, 2019). SA is an important plant disease resistance mediator. In our pathway, it is synthesized by the bZIP transcription factor encoding gene (
Csa2G403160). This gene can mediate auxin and salicylic acid induced transcription in cauliflower Mosaic virus, and can interact with NPR1 to induce systemic acquired resistance in plants (Després. et.al, 2003). At the same time, SA can be reached by Salicylic Acid 5-Hydroxylase and participate in pathogen sensitivity. It is widely expressed from seedling to adult stage (Rekhter. et.al, 2019, Zhang. et.al, 2017, Torrens-Spence. et.al, 2019). SA is hydroxylated by the MYB transcription factor coding gene (
Csa1G586860) to form Diisooctyl phthalate and Bis (2-ethylhexyl) phthalate, this gene activates SA-mediated defense and resistance to pathogens (Shim. et.al, 2013). In summary, we speculate that these transcription factor coding genes may be related to FW resistance genes.
3.3. Antimicrobial Effect of Phthalate Derivatives
This is example 1 of an equation: Phthalates are widely known as polymer materials in plasticizer. However, Phthalate compounds can be discovered in secondary metabolites of plants, animals and microorganisms since 1967 (Zhang. et.al, 2018, Roy, 2020). Eichhornia crassipes can produce mono-(2-ethyl hexyl) phthalate which shows bioactivity against Chl. Vulgaris (Sultan. et.al, 2009). Traditional medicinal plants produce an abundance of phthalate compounds with a variety of activities. These compounds isolated from the hairy vetch buds of Viciavillosa Roth, it showed inhibitory effects against phytopathogenic strains such as Rhizobium Cheonan 493 and Bacillus subtilis (Islam. et.al, 2013). Isolated from Sysimbrium officinale showed broad-spectrum antimicrobial activity against gram-positive and pathogenic fungi at a concentration of 0.5 mg/mL (Blažević. et.al, 2010). Phthalates were detected in the soil of tomatoes grown after biosolids application and radishes grown after compost (Mo. et.al, 2008, Sablayrolles. et.al, 2013). From the fruits of Acanthopanax sessiliflorus (Araliaceae), 13 different phthalates were isolated (D. T. Asilbekova.e t.al, 1985). In 2020, N. Kumari et al. published the isolation of dibutyl phthalate (5) as secondary metabolites of an actinomycetes strain grown on actinomycete isolation agar. However, in the same study tert-butylcalix arene, clearly, a synthetic product, was also found as a purported secondary metabolite of the actinomycetes strain (Kumari. et.al, 2020). Phthalic acid has been found in a number of plant extracts, such as in the ethyl acetate extract of Bridelia ovata and ethanolic extracts of licorice (Glycyrrhiza glabra) leaves, sometimes in concert with phthalates (Mohan and Anand, 2019, Poofery. et.al, 2020).
In this study, metabolic analysis identified two phenolic acid compounds, diisooctyl phthalate and bis(2-ethylhexyl) phthalate that were significantly more accumulated in NR than in NS at 4 dpi (Tables S1-2). In vivo assays suggested that they had inhibitory effect on
Foc growth (
Figure 4). The root exudates of Barnyard grass (
Echinochloa crusgalli) contain diisooctyl phthalate, which reduces the germination and growth of monocotyledonous plants, lettuce and rice (Xuan. et.al, 2006). Diisooctyl phthalate is secreted from the water hyacinth (
Eichhornia crassipes), and it possess strong inhibitory effects on Chlorella vulgaris (Liang. et.al, 2008). Bis (2-ethyl hexyl) phthalate can be produced by the strain of the fungus Cladosporium sp. F14 (Qi. et.al, 2009). Bis(2-ethylhexyl) phthalate isolated from the flower of Procera gigantea was found to be active against the grampositive bacteria staphylococcus aureus, bacillus subtilis, btreptococcus equosemens and sarcina lutea and against the gram-negative bacteria closteridium perfringens, pseudomonas aeruginosa and shigella dysenteriae (Habib and Karim, 2009, El-Sayed, 2013).
Author Contributions
Conceptualization, H.C. and Z.L.; methodology, K.Y. and G.Z.; software, K.Y. and C.C. F.Z.; validation, K.Y., Z.G. and C.C.; formal analysis, H.C., Z.L. and L.W.; investigation, K.Y.; resources, H.C., C.C. and X.L.; data curation, K.Y., X.L., F.Z. and L.W.; writing—original draft preparation, K.Y., G.Z., C.C. and F.Z.; writing—review and editing, H.C .;visualization, H.C.; supervision, H.C. and Z.L.; project administration, H.C., K.Y., G.Z., C.C.; funding acquisition, H.C., C.C. and X.L. All authors have read and agreed to the published version of the manuscript.