Two fungal strains, isolated in previous studies and currently preserved at the culture collection of the Fungal Biodiversity Laboratory (FBL) (Sapienza, University of Rome), C. globosum Kunze FBL 205 and M. polyspora (Hotson) Weresub & P. M. LeClair FBL 503, were studied to assess the ability of their culture filtrate to promote plant growth. Prior to the experiment, the strains were reactivated and maintained on malt extract agar (MEA) at 25°C in the dark. MEA was prepared according to the following composition (g/L in distilled water): malt extract, 20; peptone, 1; dextrose, 20; and bacto agar, 20. All components were purchased from Becton Dickinson (Sparks, MD, USA). Each strain's culture filtrate was prepared by inoculating four 4-mm diameter plugs of mycelium, taken from the actively growing margin of a 10-day-old stock culture using a sterile cork borer, in 150 mL Erlenmeyer flask containing 60 mL of malt extract broth (MEB). MEB was prepared with the same abovementioned composition of MEA excluding bacto agar. Five replicates were set up for each strain, and 5 Erlenmeyer flasks were left uninoculated for the control treatment. The flasks were incubated at 25°C on an orbital shaker (ASAL 711/D) at 100 rpm for 14 days.

At the end of the incubation period, the culture medium was recovered and filtered using sterile syringe filters with a 0.45-μm pore size made of mixed cellulose esters (ClearLine^®, Dominique Dutscher SAS, Brumath, France). Culture filtrates from different biological replicates of the same fungal strain were pooled together, and a sample was recovered to perform ¹H-NMR-based metabolomics analysis to identify the metabolites released by the fungus.

Wild chicory (C. intybus) seeds (Fratelli Ingegnoli Spa, Milano, Italy) were surface sterilized for 20 min in a 20% ethanol solution of 15% sodium hypochlorite, followed by 5 rinses in sterile distilled water. Sterilized seeds were plated on Murashige and Skoog (MS) medium (Duchefa Biochemie, Haarlem, The Netherlands) supplemented with 30 g/L of sucrose (Carlo Erba) and 5 g/L of bacto agar (Becton Dickinson Sparks, MD, USA). Ten chicory seeds were placed in each petri dish. Petri dishes were incubated at 25°C under a photoperiod of 16/8 h (light/dark) to promote seed germination. After 15 days, seedlings were transferred to disposable plastic pots of diameter 13 cm and height 10 cm, containing approximately 265 g of autoclaved universal potting soil (COMPO SANA^® Universal potting soil, COMPO Italia Srl, Cesano Maderno, Italy). Pots were incubated in a walk-in chamber at 18/22°C under a photoperiod of 15/9 h (light/dark) and watered every 3 days.

One month after the seedlings were transferred into the pots, 8 mL of the culture filtrate was directly added to the soil of each pot (concentration 30 mL/kg) in 8 different points positioned at two depths according to the scheme reported in Figure 1. In addition to the two treatments with M. polyspora (503) and with C. globosum (205) culture filtrates, two control groups treated with a corresponding volume of distilled water (control water) or with a corresponding volume of uninoculated MEB (MEB) were set up. On the treatment day, plants were watered in the morning and let draining excess for 5/6 h prior to the culture filtrate inoculation. Ten plants were randomly picked and assigned to each treatment.

Fourteen days after the culture filtrate addition, several parameters were measured to assess the plant growth. Growth parameters of five replicates randomly picked for each treatment were evaluated. Plants were removed from the pots and carefully washed to remove soil assuring to avoid root biomass loss. The root biomass was separated into taproot and secondary roots and then dried at 70°C for 48 h in the oven and weighted. The number of leaves per plant was recorded. The leaves were cut at the base, wrapped in a moist paper towel, and stored in containers overnight at 4°C. To determine the total leaf area per pot, water-saturated leaves were blotting dried and their images were acquired and later analyzed using the software ImageJ (version 1.53c, Wayne Rasband, National Institutes of Health, Bethesda, USA. Available online: https://imagej.nih.gov/ij). Following the image acquisition, shoot biomass was oven-dried at 70°C for 48 h to determine the dry weight. Mean specific leaf area (SLA) per pot (total leaf area divided by total dry weight) and root/shoot ratio were also calculated.

Fourteen days after the culture filtrate addition, five replicates randomly picked were recovered for untargeted metabolomic analysis by ¹H-NMR for each of the treatments: MEB control (Ctrl), C. globosum (205), and M. polyspora (503). Plants were removed from the pots, washed from the soil, and separated into the root and shoot biomass. Both the fractions were immediately frozen with liquid nitrogen to ensure the metabolic quenching and stored at −80°C. For both leaves and roots, 1.5 g of biomass grounded in liquid nitrogen was extracted according to a modified Bligh-Dyer protocol (Giampaoli et al., 2021) using a mixture of methanol, chloroform, and distilled water (3:3:1.2 mL). After overnight incubation at 4°C, samples were centrifuged at 10,000 g for 25 min at 4°C on an Itettich Zentrifugen centrifuge (Germany), and hydroalcoholic and chloroformic fractions were separated and dried under N₂ flow. The dried phases were stored at −80°C until the NMR analysis. The dried residue of the hydro-alcoholic phase was dissolved in 0.6 mL CD₃OD/D₂O (1:2 v/v ratio) containing 3-(trimethylsilyl)-propionic-2,2,3,3,-d₄ acid sodium salt (TSP, 2 mM) as internal standard (chemical shift reference). The dried residue of the chloroformic phase was dissolved in 0.6 mL CDCl₃ (Cambridge Isotope Laboratories, Inc.) (99.8%) containing 1,1,3,3,5,5-hexamethylcyclo-tri-siloxane (HMS) (Sigma-Aldrich, USA) as internal standard (2 mM). All spectra were recorded at 298 K on a Jeol JNM-ECZ 600R spectrometer operating at the proton frequency of 600 MHz and equipped with a multinuclear z-gradient inverse probe head.

Hydroalcoholic ¹H spectra were acquired employing the presat pulse sequence for solvent suppression with 128 transients, a spectral width of 9013.7 Hz, and 64 k data points for an acquisition time of 7.3 s. The recycle delay was set to 7.7 s in order to achieve complete resonance relaxation between successive scansions.

Chloroform ¹H spectra were acquired employing a single-pulse sequence with 128 transients, a spectral width of 9013.7 Hz, and 64 k data points for an acquisition time of 7.3 s. The recycle delay was set to 7.7 s in order to achieve complete resonance relaxation between successive scansions.

Resonance assignment was carried out on the basis of 2D ¹H-¹H TOCSY and ¹H-¹³C HSQC experiments. ¹H-¹H TOCSY spectra were acquired with 64 scans, a spectral width of 9013.7 Hz in both dimensions, a data matrix of 8 k × 256 data points, a recycle delay of 3 s, and a mixing time of 90 ms; ¹H-¹³C HSQC spectra were acquired with 128 scans, a spectral width of 9013.7 Hz, and 30,000 Hz for hydrogen and carbon dimension, respectively, a data matrix of 8 k × 256 data points, a recycle delay of 3 s, and a direct constant of 145 Hz.

Fungal culture filtrate analysis was carried out by adding to 0.35 mL of filtrate an amount of 0.35 mL of D₂O containing 3-(trimethylsilyl)-propionic-2,2,3,3-d₄ acid sodium salt (TSP, 2 mM) as internal chemical shift and concentration standard, and the sample was analyzed by ¹H-NMR employing the presat pulse sequence.

All statistical analyses on growth parameters were carried out using the statistical software R (version 4.1.0) under the R-studio environment (version 1.4.1106). For all parameters belonging to the morphological dataset, the normality and homoscedasticity of the data were tested using Shapiro-Wilk and, as appropriate, Bartlett or Levene test, respectively (packages lawstat and stats). Hereafter, differences between groups were tested using the Mann-Whitney U test, performed through the “Wilcoxon rank sum exact test” function (package stats).

Multivariate PCA and PLS-DA were performed on the data matrix using the Unscrambler ver. 10.5 software (Camo Software AS, Oslo, Norway). Univariate t-test and Pearson's correlation coefficients were calculated with SigmaPlot 14.0 software (Systat Software Inc., San Jose, CA, USA).

The values of growth parameters, evaluated 14 days after culture filtrate application, are reported in Table 1. Plant growth promotion effect, in terms of increase of biomass production, has been observed only in plants treated with M. polyspora culture filtrate. In fact, a statically significant difference (p < 0.05) in the total dry weight of the plant has been observed solely by comparing M. polyspora treatment with both water and MEB control, and the same significant result has been observed considering the dry weight of the shoot. Regarding the dry weights of the total root system and of the lateral roots, the only significant difference (p < 0.05) observed is between M. polyspora treatment and the MEB control, while no statistically significant differences among all treatments have been recorded in the taproot dry weight.

Focusing on shoot parameters, while no statistically significant differences have been observed in the numbers of leaves, a statically significant difference (p < 0.05) in leaf area has been observed comparing M. polyspora treatment with both MEB control and C. globosum treatment. It is interesting to note that the leaf area values of the plants treated with C. globosum culture filtrate resulted to be lower than controls, although it did not result to be statistically significant. Finally, no significant differences were observed among the treatments concerning the indexes of root/shoot ratio and SLA.

The filtrate composition is reported in Supplementary Table S1. ¹H-NMR analysis of C. globosum culture filtrate showed a decrease in all amino acids including leucine, isoleucine, valine, threonine, lysine, tyrosine, phenylalanine, glutamate, and acetate that resulted to be consumed by the fungus and an increase in peptone, ethanol, and fumarate that were released. A slight decrease in glucose, maltose, and fructose was also observed. Conversely, in M. polyspora culture filtrate, a high increase in glucose associated with a decrease in maltose was observed. No variation in amino acids except for alanine and lysine was observed. A production of adenosine and guanosine phosphates nucleotides (AXP and GXP, respectively) was also detected.

The inspection of the 600 MHz ¹H-NMR spectra obtained from hydroalcoholic and chloroformic extracts of chicory leaves and roots revealed the presence of 49 molecules univocally identified. A total of 46 metabolites including amino acids, organic acids, sugars, organic compounds, fatty acids, secondary metabolites, and other compounds were integrated. Only the molecules univocally identified were considered for the study, and their quantification was performed by integration of their NMR signals. Due to the overcrowding of ¹H-NMR spectra, only those signals that did not overlap with other resonances were considered for integration. Comparing the spectra obtained from leaves and roots, it was possible to observe both qualitative and quantitative differences, while the spectral comparison among the control group and the treatments showed only quantitative differences. Examples of ¹H-NMR spectra are reported in Supplementary Figures S1–S4, and the table of resonance assignment is reported in Supplementary Table S2. Quantitative analysis of the phytochemical composition of C. intybus roots and leaves are reported in Supplementary Tables S3, S4, respectively.

To examine an overview of the whole NMR data set, a preliminary unsupervised PCA was performed in leaves and roots separately. In leaves, PC1 and PC2 components explained 42% of the total variation, and PCA score plot revealed a clustering of the samples according to the treatment with the fungal filtrate (Supplementary Figure S5). In roots, 41% of the total variation was explained by the two main components, with the first and second contributing 24 and 17%, respectively (Supplementary Figure S6).

With the aim of refining the sample grouping observed in the unsupervised PCA model, PLS-DA discriminant analyses were performed to identify the most important metabolites that discriminated the treatments with C. globosum and M. polyspora separately in leaves and roots.

In leaves treated with C. globosum filtrate, PLS-DA analysis provided a model (R2 =0.96; Q2 =0.62) with three discriminant components explaining 34, 76, and 15% of the variance (Figure 2). The corresponding PLS-DA score plot revealed a clear separation of the leaves treated with C. globosum filtrate (205) from control samples.

The levels of campesterol, chlorophyll a and b, β-sitosterol, valine, oleic acid, and isoleucine decreased in C. globosum group compared with controls (Figure 3).

In roots treated with C. globosum filtrate, the PLS-DA model showed a clear separation of samples with good descriptive parameters (R2 =0.97; Q2 =0.75) (Figure 4). A significant decrease in β-sitosterol, linolenic acid (ω-3), and phospholipids and a significant increase in phenylalanine, chicoric acid, triterpenes, 3-OH-butyrate, monoacylglycerols, and alanine were observed in C. globosum compared with control samples (Figure 3).

The PLS-DA analysis performed to highlight metabolic differences related to M. polyspora treatment in C. intybus leaves showed an excellent sample separation (R2 = 0.99; Q2 = 0.62) (Figure 5). Similar to C. globosum treatment, a significant decrease in chlorophyll a and b, campesterol, acetate, and β-sitosterol and a significant increase in 4-OH-benzoate were observed in leaves of plants treated with M. polyspora filtrate compared with control samples (Figure 6). In the PLS-DA model (R2 = 0.77; Q2 = 0.52) of root samples (Figure 7), discriminant variables such as triterpenes, phospholipids, linolenic acid (ω-3), campesterol, xantophylls, and tryptophan were significantly decreased, while 4-OH-benzoate, 3-OH-butyrate, and monoacylglycerol were significantly increased in roots treated with M. polyspora filtrate compared with controls (Figure 6).

Fungal culture filtrates have consistently been reported as effective biostimulants. In the environment, fungi due to a complex extracellular metabolism exert their influence on plants also through the release of diffusible metabolites in soils. Therefore, learning from nature, the metabolites released in the culture medium during growth in controlled conditions may be used to simulate these interactions and effectively stimulate plant growth.

In this study, we showed that soil application of M. polyspora culture filtrate promotes biomass production, both in shoot and root, and leaf area extension in C. intybus plants (Table 1). Considering that no statistically significant differences in the number of leaves were observed, the increase in biomass production seems to be related to the significant increase in leaf area observed. The significant increase in root biomass more specifically occurred in the lateral roots biomass since the taproot biomass did not show differences among the treatments. An increase in lateral roots may have resulted in a more effective exploration of the soil and therefore an increased efficiency in nutrient uptake leading to a higher growth.

In this study, M. polyspora is reported for the first time to have a direct plant growth-promoting effect. This effect is in line with what is reported in previous studies on the fungal culture filtrate biostimulant effect. For example, the culture filtrate of Serendipita indica, which, like M. polyspora, is an anamorphic species belonging to the Basidiomycota phylum, has been widely reported to increase the biomass production and other growth parameters including plant height, leaves length and width, root length and number, and fruit and seeds production in Helianthus annus (common sunflower), C. intybus (chicory), Zea mays (maize), Bacopa monniera (water hyssop), and Nicotiana tabacum (tobacco) plants (Varma et al., 1999; Bagde et al., 2011, 2013; Rashnoo et al., 2020).

Several other fungal strains, including Alternaria alternata, Aspergillus fumigatus, Byssochlamys spectabilis, C. globosum, Cladosporium sp., Fusarium tricinctum, Fusarium proliferatum, Gibberella spp., Penicillium melinii, Penicillium citrinum, Penicillium oxalicum, Penicillium sp., Phoma herbarum, Sclerotium rolfsii, Shimizuomyces paradoxus, Trichoderma virens, Trichoderma pseudokoningii, and Trichoderma harzianum, have been reported for their culture filtrates biostimulant effect determining an increase in growth parameters in several plant species including rice, cucumber, chickpeas, wheat, canola, tobacco, and pearl millet (Singh et al., 2003; Khan et al., 2008, 2015; Hamayun et al., 2009, 2010; Hwang et al., 2011; Sung et al., 2011; Rahman et al., 2012; López-Bucio et al., 2015; Murali and Amruthesh, 2015; Bilal et al., 2018; Haruma et al., 2018; Zhai et al., 2018; Zhou et al., 2018; Jahagirdar et al., 2019; Naziya et al., 2019; Baron et al., 2020; Ozimek and Hanaka, 2020; Khalmuratova et al., 2021; Tarroum et al., 2021).

Baroja-Fernández et al. (2021) recently reported the efficacy of T. harzianum, A. alternata, and Penicillium aurantiogriseum culture filtrates in promoting plant growth of Capsicum annuum (peppers). Moreover, the culture filtrates of the three fungal species were found to contain considerable concentrations of glucose and fructose, and a complex mixtures of amino acids, some of which were previously reported to be involved in signaling mechanisms for environmental changes. Similarly, in M. polyspora culture filtrate, we detected essential amino acids (e.g., alanine, valine, lysine, and leucine), adenosine and guanosine phosphates nucleotides (AXP and GXP), and glucose at higher concentrations than in MEB (Supplementary Table S1). The higher concentrations of these metabolites may explain the significant variations of growth parameters in treated chicory plants compared with both the controls. Specifically, in M. polyspora filtrate, alanine and lysine were higher than the control, while other amino acids (leucine, valine, threonine, glutamate, tyrosine, phenylalanine) occurred in quite similar concentrations. Indeed, fungi can synthesize lysine de novo through the so-called α-aminoadipate pathway with α-ketoglutarate as the precursor, while alanine can be formed by transamination from glutamate and pyruvate by glutamate-pyruvate aminotransferase (Smith and Berry, 1976; Zabriskie and Jackson, 2000). Amino acids along with other growth factors present in culture filtrates of some Fusarium species have been reported to promote the growth of Oryza sativa's (rice) roots (Ram, 1959). Moreover, the presence of essential amino acids in culture filtrates can confer stress resistance to plants, such as alanine in hypoxic conditions in plants (Podlešáková et al., 2019; Baroja-Fernández et al., 2021). Lysine has been reported to be involved in plant growth and stress resistance, as well. In fact, roots can take up several amino acids, including lysine, and directly incorporate them into new cell biomass and utilize them for respiration (Owen and Jones, 2001). Moreover, foliar application of iron-conjugated lysine on Brassica napus (rapeseed) has been reported to increase plant growth, biomass production, chlorophyll content, and essential micronutrients uptake and to reduce oxidative stress enhancing antioxidant enzyme activities in response to chromium stress condition (Zaheer et al., 2020).

Furthermore, the presence of glucose and adenosine and guanosine phosphates nucleotides, as energy sources, can exert a plant growth promotion activity in chicory. In particular, this higher level of glucose compared with MEB may be explained by the extracellular hydrolysis of maltose, leading to the formation of two molecules of glucose, as reported in Aspergillus niger through the maltase enzyme (Yuan et al., 2008; Hamad et al., 2015). Indeed, maltose in M. polyspora filtrate was almost completely depleted, and it is reasonable to think that this was determined by the release of hydrolytic enzymes, as this cellulolytic strain in different development stages can metabolize polysaccharides, hexoses, and oligosaccharides (Pinzari et al., 2014). Conversely, C. globosum culture filtrate in our study did not exert any effect in increasing the biomass production and the other morphological parameter's values. Nevertheless, C. globosum and its metabolites have been previously reported as an effective plant growth promoter of C. annuum (pepper), Brassica juncea (mustard), Solanum lycopersicum (tomato), Pennisetum americanum (pearl millet), Z. mays (maize), and N. tabacum (tobacco). In all the abovementioned species, an increase in biomass production was recorded, and, as appropriate, C. globosum caused an increase also in various morphological and physiological parameters of growth and development such as shoot growth, plant height, root length, leaf area, chlorophyll content, stomatal conductance, transpiration rate, seed germination, and nutrient uptake (Tarafdar and Gharu, 2006; Abou Alhamed and Shebany, 2012; Khan et al., 2012; Kumar et al., 2021; Singh et al., 2021; Tarroum et al., 2021). Unfortunately, no other studies addressing the effect of C. globosum on C. intybus are available in the literature, and therefore, it is not possible to determine whether its inefficiency is related to the test conditions in this study or to the specific plant-fungal strain interaction. In fact, a PGPF that results effective in promoting the growth of a given plant species may be less effective or even not present the same beneficial effect at all upon another plant species. Moreover, environmental conditions may also affect the beneficial effect of a PGPF (Hossain et al., 2017).

Beneficial microorganisms are known to release diffusible substances that promote plant growth. Consistently, soil application of fungal culture filtrates can affect plant metabolism, growth, and yield (Baroja-Fernández et al., 2021).

However, how this treatment acts in plants is largely unknown. In this work, we characterized the responses of chicory (C. intybus) plants cultured under greenhouse conditions to soil application of culture filtrates obtained from C. globosum (205) and M. polyspora (503).

¹H-NMR-based metabolomics analysis of C. intybus roots after treatment with C. globosum and M. polyspora culture filtrates revealed common metabolic threads involving the increase of 3-OH-butyrate and monoacylglycerols associated with the decrease of unsaturated fatty acids (UFAs) such as linolenic acid, sterols including campesterol and β-sitosterol, and phospholipids. We outlined the metabolic network involving 3-OH-butyrate, phospholipids, sterols, and fatty acids occurring in C. intybus roots after treatment with C. globosum (205) and M. polyspora (503) (Figure 8).

Differently from animals, in which 3-OH-butyrate is an intermediate metabolite of fatty acids, the physiological importance and the metabolism of 3-OH-butyrate in plants are not fully understood and characterized. Mierziak et al. (2020) demonstrated that 3-OH-butyrate occurs naturally in flax and could act as a regulatory molecule that most likely influences the expression of genes involved in DNA methylation, thereby altering DNA methylation levels. Recent studies showed that plants contain enzymes such as β-ketothiolase (EC 2.3.1.9) and acetoacetyl-CoA-reductase (EC 1.1.1.36), which are involved in the synthesis of 3-OH-butyrate as in bacteria and animals (Xu et al., 1997; Beaudoin et al., 2009; Jin et al., 2012; Tsuda et al., 2016; Mierziak et al., 2021). In plants, biosynthesis of fatty acids and biosynthesis of sterols, which takes place via mevalonate pathway in plastids and endoplasmic reticulum, occur from acetyl-CoA and acetoacetyl-CoA through NADPH/NADP⁺ recycling and reductase/synthase enzyme activities. The treatment with C. globosum (205) and M. polyspora (503) culture filtrates stimulates a common response in C. intybus roots involving the synthesis of 3-OH-butyrate through the decrease of the synthesis of fatty acids and sterols, as a mechanism balancing the NADPH/NADP⁺ ratio.

In most plants, the predominant unsaturated fatty acids (UFAs) are three 18-carbon (C18) species, i.e., 18:1 (oleate), 18:2 (linoleate), and 18:3 (α-linolenate) (He et al., 2020). UFAs compounds play multiple crucial roles and are deeply associated with both abiotic and biotic stresses. Besides being membrane ingredients and modulators in glycerolipids, as well as carbon and energy reserve in triacylglycerols (TAGs), C18 UFAs serve as intrinsic antioxidants, precursors of various bioactive molecules [typically the stress hormone jasmonic acid (JA)], and stocks of extracellular barrier constituents such as cutin and suberin (He et al., 2020). The predominant sterols in plants, such as β-sitosterol, campesterol, and stigmasterol, are precursors of a group of plant hormones the brassinosteroids, such as gibberellins and abscisic acid, which regulate plant growth and development (Valitova et al., 2016). Plant lipids, released from the roots into the rhizosphere, facilitate signaling and actively shape the microbiome inhabiting the rhizosphere and the subsequent colonization of their root tissues. Lipids play essential roles as the “chemical language” that facilitates the exchange of resources and modulates the cell responses by inhibiting pathogen attack or enhancing microbial symbiosis. The recruitment of the rhizobiome into the plant vicinity is mediated by rhizodeposits. The release of rhizodeposits comes with a wide variety of substances, such as sugars, amino acids, organic acids, enzymes, growth factors and vitamins, flavanones and purines/nucleotides, and miscellaneous substances. In our study, we observed a decrease in root phospholipids, fatty acids, and sterols that are probably released from roots into the rhizosphere as chemical signals and/or chemotactic attractors to facilitate the recruitment, nutrition, shaping, and tuning of the microbial communities. The perception of these compounds could lead to the stimulation of regulatory or signaling cascades that cause various responses in the microbes. We also observed an increase in monoacylglycerols (MAG) levels. MAG comprise the bulk of oil storage in plant tissues and are involved in many regulatory processes such as cell signaling and intracellular trafficking (Macabuhay et al., 2022).

¹H-NMR-based metabolomics analysis of C. intybus leaves after treatment with C. globosum and M. polyspora culture filtrates showed the reduction in chlorophylls a and b content not associated with leaf greening decrease during the plant development and/or treatment with fungal culture filtrates. Dynamic control of chlorophyll levels determined by the relative rates of chlorophyll anabolism and catabolism processes, that largely occur in chloroplasts, ensures optimal photosynthesis and plant fitness. The accumulation of adequate amounts of chlorophyll is therefore vital for plants to establish photosynthetically active chloroplasts during leaf greening. Furthermore, optimized chlorophyll degradation is not only essential for the detoxification of free chlorophyll released but also indispensable for the remobilization of nutrients during leaf development and senescence. Thus, efficient photosynthesis, plant fitness, and yield are critically dependent on the dynamic regulation of chlorophyll levels in response to various developmental and environmental cues (Wang et al., 2020).

In our study, the treatment of C. intybus plants with C. globosum culture filtrate triggered the phenylpropanoid pathway through an increase of phenylalanine and chicoric acid in roots. The biosynthetic pathway of chicoric acid in plants is putative and still not well known, although it is generally understood to form via shikimic acid/phenylpropanoid pathway as other phenolics, analogous to the conjugation of caffeic acid derivatives of rosmarinic acid or chlorogenic acid (Legrand et al., 2016; Peng et al., 2019). Putative metabolic pathway involved in chicoric acid biosynthesis in C. intybus roots is shown in Figure 9.

The entry point is the aromatic amino acid phenylalanine (Phe) arising from the shikimate pathway. Deamination of Phe by Phe ammonia lyase (PAL) leads to cinnamic acid. Cinnamate-4-hydroxylase and 4-coumarate coenzyme A (CoA) ligase (4CL) generate p-coumaroyl-CoA from cinnamic acid. Thereafter, hydroxycinnamoyl transferases (HCTs) convert the CoA-thioester to coumaroyl quinate or coumaroyl shikimate which is subsequently hydroxylated by p-coumarate-30-hydroxylase to form the caffeoyl derivatives. A recent study carried out on purple coneflower highlighted two types of acyltransferases distributed in distinct subcellular compartments and involved in the biosynthesis of chicoric acid. In the cytosol, the BAHD acyltransferase family including a tartaric acid hydroxycinnamoyl transferases (HTT) and a quinate hydroxycinnamoyl transferases (HQT) use caffeoyl CoA from phenylpropanoid metabolism as an acyl donor to synthesize caftaric acid and chlorogenic acid, respectively (D'Auria, 2006). Both products are then transported into the vacuole where CAS, a specialized serine carboxypeptidase-like (SCPL) acyltransferase, uses chlorogenic acid as its acyl donor and transfers the caffeoyl group to caftaric acid to generate chicoric acid (Fu et al., 2021).

For millennia, humans have used plant specialized metabolites as herbal medicines. Chicoric acid is a very promising natural compound, which occurs in a variety of plant species such as C. intybus, Echinacea purpurea L. (purple coneflower), Ocimum basilicum L. (basil), Lactuca sativa L. (lettuce), Taraxacum officinale (dandelion), Cucurbita pepo L. (squash), and Borago officinalis L. (borage) (Lee and Scagel, 2013). According to the literature data, chicoric acid plays an important role in plant defense against different diseases caused by viruses, bacteria, fungi, nematodes, and insects (Nishimura and Satoh, 2006; Cheynier et al., 2013).

As reported above, the treatment of C. intybus plants with M. polyspora culture filtrate determined significant variations of growth parameters in chicory plants including the dry weight of the total plant, shoot, total roots, lateral roots, and leaf area that increased compared with control plants. A relationship between growth parameters and metabolic changes was observed. In particular, M. polyspora culture filtrate triggered the phenylpropanoid pathway through an increase of 4-OH-benzoate, which is generated from aromatic amino acids produced via the shikimate pathway (Figure 10).

The shikimic acid is converted into L-phenylalanine through a chorismic acid intermediate. Thus, the L-phenylalanine is converted into p-coumaric, salicylic, and p-hydroxybenzoic acids, which serve as precursors for other derivatives of phenolic acids. It is thought that hydroxybenzoic acids can be produced from structurally analogous hydroxycinnamic acids in coenzyme A (CoA)-dependant (β-oxidative) or CoA-independent (non-β-oxidative) pathways or the combination of both of them, which have been determined as occurring in peroxisomes and mitochondria (Widhalm and Dudareva, 2015). Benzoic acids serve as precursors for a wide variety of essential compounds and natural products playing crucial roles in plant fitness and in defense response activation. In this context, 4-OH-benzoate showed in vitro antifungal effects on Eutypa lata growth (Amborabé et al., 2002).