Unveiling the Multifaceted Roles of Root Exudates: Chemical Interactions, Allelopathy, and Agricultural Applications

Abstract

Root exudates, compounds secreted by plant roots, play a crucial role in plant–soil interactions and have significant agricultural implications. These substances influence nutrient availability, plant growth, and the surrounding rhizosphere. This review examines the composition, mechanisms, and importance of root exudates, categorizing them as diffusates, secretions, and excretions, each with specific release methods and functions. It highlights the allelopathic effects of root exudates, showing how plants use them to inhibit competitors through chemical signals and nutrient changes. Case studies on crops such as wheat and rice demonstrate the practical relevance of root exudates in agriculture. This review emphasizes the need to understand root exudates to improve sustainable farming and weed control strategies.

1. Introduction

1.1. Background of Root Exudates

The root system of plants anchors the plant and absorbs nutrients. It also secretes root exudates, which interact with the rhizosphere to modify soil properties, enhance nutrient availability, and support long-term adaptation. Root exudates consist of diffusates, secretions, and excretions, which differ in release mechanisms and composition. Diffusates are low-molecular-weight organic compounds that passively diffuse through the root cell membrane into the rhizosphere [1]. Secretions include compounds involved in signaling, nutrient acquisition, and stress resistance, which are actively controlled by the plant [2,3]. Excretions, such as mucus and lysates, facilitate root penetration. Exudates can also be classified by molecular weight, as follows: low-molecular-weight exudates include amino acids, sugars, organic acids, phenols, fatty acids, sterols, vitamins, and secondary metabolites; while high-molecular-weight exudates are mainly proteins and polysaccharides [2,4]. Root exudates, primarily carbon-based molecules, are a key source of organic carbon in the rhizosphere [1,5]. They influence plant relationships with soil microorganisms and neighboring plant roots, sometimes symbiotic, other times competitive, by inhibiting the growth of other organisms. Each plant species has specific microorganisms that colonize its rhizosphere based on the composition of root exudates, which can attract or repel certain microbes. For instance, Leguminosae secrete flavonoids to attract rhizobia for nodulation [6], while basil (Ocimum basilicum L.) releases rosmarinic acid to inhibit unwanted microorganisms, causing cytoskeletal damage and disrupted septa [2,7]. Zhao et al. [8] studied root exudates from both the slow- and fast-growing stages of Arabidopsis thaliana L. in nutrient-depleted soils and found that exudates from the fast-growing stage promoted greater nutrient mineralization and plant growth. This suggests that plants may adjust exudation patterns during different growth phases to optimize microbial recruitment and meet higher nutrient demands. Root exudates also influence nutrient balances in the rhizosphere, including carbon, nitrogen, phosphorus (P), and potassium. Despite many nutrients being present in sufficient quantities in the soil, they are often unavailable to plants due to immobilization in organic matter or adsorption onto minerals [9].

Root exudates enhance nutrient mobilization in the soil, increasing nutrient bioavailability through various mechanisms, often dependent on the plant species. They help release elements immobilized in soil organic matter or adsorbed onto minerals and are produced in greater quantities during nutritional stress to mobilize soil-adsorbed nutrients and attract beneficial microorganisms [10,11]. Andrade et al. studied 15 eucalyptus species under P-deficient and P-sufficient conditions and found that organic acids were the most abundant exudates, with citric and isocitric acids increasing under P scarcity, suggesting that plants alter exudate composition to enhance P availability [12]. Similarly, sugar beet increases the production of citramalic and salicylic acids in P-deficient soils, solubilizing P and raising soil pH to improve nutrient availability [13]. Furthermore, root interactions can affect exudate composition, as seen in a study with hairy vetch (Vicia villosa Roth.) grown in competition with rye (Secale cereale L.). Vetch grown with one rye plant produced higher levels of kaempferol-Rha-Xyl-Gal, a flavonoid, but exudation decreased with increased competition between the species [14].

1.2. Significance of Allelopathic Activity

Root exudates play a key role in allelopathic interactions by releasing allelochemicals, such as phenolic compounds, terpenes, and other secondary metabolites, which can inhibit or promote the growth of neighboring plants. Allelopathy, where one organism produces biomolecules affecting others’ growth, survival, or reproduction, has been known for decades [15]. The release of allelopathic compounds varies based on genotype, development, environment, and competition. These compounds can be released via volatilization, exudates, leaf leaching, or tissue decomposition [16,17]. The allelopathic effect depends on the quantity of metabolites, their availability in soil, and their specificity for target species. Simple molecules are produced consistently, while complex ones are costly and produced when necessary for survival [18]. These molecules can alter various biochemical processes in neighboring plants, including photosynthesis, respiration, and growth [18,19]. Sorgoleone, a hydrophobic p-benzoquinone, is a key allelochemical produced by Sorghum bicolor (L.) Moench roots, with potent phytotoxic effects on weeds and potential applications in sustainable agriculture. It is synthesized and exuded primarily from the root hair cells, often comprising over 80% of the root exudates [20,21,22]. When released into the rhizosphere as sorgoleone, or its reduced form (dihydrosorgoleone) [23], it inhibits seed germination and seedling growth in various species, including weeds, at concentrations as low as 10 µM [24,25,26]. While its effects are most pronounced in weeds, sorgoleone also affects crops, with selectivity varying among plant types [27,28]. Its phytotoxicity is attributed to interference with photosynthesis, H+-ATPase activity, mitochondrial respiration, and water and nutrient uptake [29,30,31,32,33,34,35]. Sorgoleone inhibits photosystem II, disrupting the electron transport chain and reducing energy production [36,37], and its hydrophobic nature allows interaction with lipid membranes [28,38]. The compound’s action varies based on species and environmental conditions. Factors such as soil composition, moisture, and plant competition influence its production, which increases under drought stress or nutrient deficiency [39,40,41,42,43]. Additionally, genetic variations in sorghum cultivars affect sorgoleone levels and efficacy [44,45,46].

In addition to its allelopathic effects, sorgoleone influences soil microbial communities, shaping bacterial composition and activity, which affects nutrient cycling and plant health [47,48,49]. Some bacteria degrade sorgoleone, potentially reducing its phytotoxic effects and promoting ecosystem balance [21,35,50]. This highlights the ecological implications of sorgoleone in agriculture. As a natural herbicide, sorgoleone offers selective phytotoxicity, suppressing weeds while minimizing crop damage [51,52,53]. Field studies show that integrating sorgoleone-producing sorghum into crop rotations reduces weed populations and improves yields [54,55,56], aligning with eco-friendly agricultural practices that reduce reliance on synthetic herbicides [56,57,58].

Mimosine, an allelochemical produced by Mimosa and Leucaena species, particularly Leucaena leucocephala (Lam.), is another compound with strong allelopathic potential. Mimosine, a non-protein amino acid, is found in all plant tissues, with higher concentrations in the younger parts [59]. It is synthesized from serine via a reaction similar to cysteine biosynthesis, involving serine acetyltransferase and mimosine synthase [60]. Mimosine is thought to function in nitrogen storage, stress defense, and promoting iron absorption, as it binds to transition metal ions like Zn (II), Ni (II), Fe (III), Cu (II), and others [61,62]. Leucaena adopts two known strategies for iron absorption due to mimosine’s ability to bind Fe (III). It is hypothesized to act as a phytosiderophore, facilitating iron translocation from the rhizosphere into plant cells, making Leucaena adaptable in alkaline soils [63]. Mimosine also affects interactions with other organisms, causing infertility, growth retardation, cataracts, and alopecia in animals ingesting Leucaena leaves. It can block the cell cycle at the G1 phase and suppress DNA replication elongation [64,65,66]. Allelopathically, mimosine enhances the invasiveness of Leucaena leucocephala, with plant extracts and residues showing inhibitory effects on species like lettuce, rice, maize, Amaranthus hybridus L., and Bidens pilosa L. [67,68,69]. Leaf mimosine concentrations range from 2.5% to 5.75%, and extracts at varying ratios reduced seedling growth, especially root length, dry weight, and fresh weight. Root inhibition was more pronounced than shoot inhibition in rice and Ischaemum rugosum Salisb. [70]. Ishak et al. tested the allelopathic potential of root exudates by growing Leucaena leucocephala, Ageratum conyzoides, Tridax procumbens, and Emilia sonchifolia in the same agar medium. Root growth was reduced by 98% in T. procumbens, 68% in A. conyzoides, and 77% in E. sonchifolia, with greater inhibition near L. leucocephala roots [71]. Although the exact mechanism of mimosine’s effect on germination and development remains unclear, it likely plays a key role in the allelopathic interactions of L. leucocephala, aiding its survival and offering potential for weed biocontrol.

Allelopathy also occurs within the same species, leading to autotoxicity, where plants absorb allelopathic molecules released by their own roots. This is a concern in continuous cropping, where the accumulation of allelochemicals like cinnamic acid and vanillin in eggplants can reduce growth and yield [72]. In rice, momilactones secreted by the roots inhibit neighboring plant growth and affect the plant’s own growth [73]. Autotoxicity can reduce intraspecific competition, benefiting a plant population’s fitness. Phytotoxic tree species are less affected by autotoxicity due to their deep root systems, which prevent phytotoxic molecules from accumulating in the surface soil layers [74]. In many species, autotoxicity affects subsequent generations rather than those producing the phytotoxic molecules. For example, Cunninghamia lanceolata (Lamb.) Hook. produces a cyclic dipeptide that, when accumulated in the soil, inhibits seedling growth, making it difficult to cultivate the species in plantations [75,76]. Other species, such as coffee (Coffea arabica L.), barley (Hordeum vulgare L.), and alfalfa (Medicago sativa L.), also experience autotoxicity [74,77,78,79]. In alfalfa, autotoxicity results from allelopathic molecules released through decomposing residues and root exudates, damaging cell membranes and causing cell death due to ROS accumulation and lipid peroxidation [80]. Managing autotoxicity in alfalfa is challenging in multi-cycle cropping systems. It is important to study secretions, as the concentration of molecules in plant tissues does not directly correspond to the amount exuded [81].

2. Classes of Metabolites in Root Exudates

2.1. Organic Compounds

The rhizosphere, the soil surrounding a plant’s roots, is rich in low-molecular-weight molecules that affect soil physicochemical properties. These molecules, secreted by the plant’s root system, result from specialized metabolism and influence interactions with other organisms and nearby plants. This review focuses on phenolic compounds, organic acids, terpenoids, alkaloids, and benzoxazinoids.

2.1.1. Phenolic Compounds

Phenolic compounds, a significant part of root exudates, are produced via the shikimic acid and phenylpropanoid pathways. They are studied for their antioxidant and defensive properties, as well as their roles in legume–rhizobia symbiosis, auxin transport, pollen production, and tissue pigmentation [82]. Phenolics also influence nutrient availability in the soil and serve as precursors to humic substances upon plant decomposition. These compounds, characterized by a hydroxyl (-OH) group attached to an aromatic benzene ring [83], exhibit diverse structures and phytotoxic actions, which are influenced by the substituents on the ring. Polyphenols, formed by multiple aromatic rings, include compounds like stilbenes, flavonoids, and related derivatives. Flavonoids are classified by oxidation patterns and glycosylation. Quinones, including benzoquinones, naphthoquinones, and anthraquinones, are another group, known for being natural colorants. Lastly, tannins, including hydrolyzable, condensed phlorotannins, are also important phenolic compounds [84].

Flavonoids are abundant compounds in plants, found in seeds, leaves, flowers, stems, and roots. A study by Narasiman et al. [85] on Arabidopsis thaliana (Landsberg erecta accession) using metabolomics revealed that flavonoids made up 37% of secreted secondary metabolites, with quercetin being the most prevalent. Over 4000 flavonoids have been identified in vascular plants, with about 30 known to induce nod genes in nine legume genera under axenic conditions [86]. Flavonoids are classified into two groups: those with an open C3 bridge (e.g., chalcones) and those with a closed C3 bridge forming a third heterocyclic ring. The latter group includes widely produced and secreted compounds such as catechins, epicatechins, anthocyanidins, procyanidins, anthocyanins, flavones, flavonols, dihydroflavonols, isoflavonoids, and neoflavonoids [87,88].

The allelopathic potential of flavonoids is particularly notable in various crop species like rice and canola. Rice root exudates enhance allelopathy against barnyard grass by increasing flavonoid and phenolic acid concentrations in the rhizosphere, crucial for competition in nutrient-poor environments [89]. Canola similarly releases flavonoids that inhibit weed germination and growth [90,91]. The composition of flavonoids in root exudates varies with environmental factors such as soil type, moisture, and plant interactions, modulating their allelopathic effects [92,93,94]. Additionally, flavonoids influence soil microbial communities, affecting nutrient cycling and soil health [95,96], while also disrupting seed germination, hormonal balance, and root development [97,98]. Kato-Noguchi et al. identified specific flavonoids in rice exudates that inhibit weed germination, shedding light on allelopathic biochemical pathways [97]. Research into their precise mechanisms is ongoing.

Beyond allelopathy, flavonoids also attract beneficial soil microbes, enhancing nutrient uptake and plant health [56,99]. More broadly, phenolic compounds, including phenolic acids (e.g., gallic, salicylic, and cinnamic acids), influence soil microbiota. Li [100] demonstrated that phenolic acid accumulation in soil alters fungal communities, increasing pathogenic fungi in consecutive ginseng monocultures, potentially explaining yield declines.

Flavonoids play a key role in plant–microbe interactions, acting as chemoattractants for rhizobial bacteria and inducing nodulation gene expression, essential for Nod factor biosynthesis and root symbiotic infection [101,102,103,104]. Their specificity varies by plant species and influences bacterial targets. Despite extensive research, many aspects of flavonoid–rhizobia interactions remain unexplored. Finally, flavonoids regulate auxin transport in plants. Brown et al. observed that Arabidopsis transparent testa mutants, defective in flavonoid production, exhibited compromised apical dominance and increased auxin transport [105]. Moreover, quercetin-derived flavonoids influence ABA signaling, which is closely linked to stomatal opening [106].

Many plant species secrete phenolic compounds through their roots, though not all exhibit allelopathic activity. These compounds can damage plants via various mechanisms [107], affecting multiple physiological processes. For instance, excessive light exposure increases reactive oxygen species (ROS) production, including triplet chlorophyll, singlet oxygen, and hydroxyl radicals [108]. These radicals damage membranes, enzymes, pigments, and proteins, ultimately harming the entire plant [109,110]. To counteract light stress, plants produce phenols to limit ROS accumulation. However, at high concentrations, phenolics impair photosynthesis, instead of acting as protective molecules, by inhibiting antioxidant enzymes, further increasing harmful ROS levels [101,111]. This phenomenon underlies allelopathic interactions, as shown in a study by Ye et al., where treating cucumber (Cucumis sativus L.) roots with cinnamic acid, an allelopathic root exudate [112], led to reduced leaf area and elevated ROS levels, causing oxidative stress [113].

Phenols and other allelochemicals in the rhizosphere also disrupt plant water balance. They alter root cell membranes, sometimes causing depolarization and ionic imbalances [114,115], leading to water stress and impaired CO₂ uptake [116]. Specifically, phenolics reduce root hydraulic conductivity, leaf water potential, stomatal conductance, and cellular turgor pressure [117,118], ultimately decreasing photosynthetic efficiency and plant growth.

Phenolics can also damage the photosynthetic apparatus. Yang et al. [118] demonstrated that administering o-hydroxyphenylacetic acid, ferulic acid, and p-coumaric acid proportionally decreased chlorophyll and porphyrin content. Excessive ROS presence intensifies oxidative stress, particularly in the presence of O₂ and transition metals like copper (Cu) and iron (Fe), which facilitate phenolic redox cycling. Furthermore, phenols inhibit antioxidant enzymes, exacerbating oxidative damage [101]. Additionally, phenolic compounds influence respiration and transpiration—key processes regulating water exchange, temperature, nutrient uptake, and energy metabolism. Their impact extends beyond photosynthesis, affecting overall plant health and productivity.

When discussing the allelopathic potential of a compound, it is important to note that its effects vary between laboratory and field conditions. Controlled environments are less realistic, as interactions with soil and microbiota can alter the mobility and persistence of compounds. Allelochemicals typically undergo adsorption and desorption onto soil solids, are transported by water, and are biotransformed by soil microorganisms. Microbial retention and degradation mechanisms influence the concentration, bioavailability, and effectiveness of allelochemicals on target plants. Soil microorganisms produce enzymes that catalyze the oxidation and polymerization reactions of phenolic acids [119], and, in carbon-deficient conditions, they mineralize these compounds [120,121]. Abiotic changes in phenolic acids, particularly their high polarity, lead to strong absorption by soil matrices, especially in the presence of organic matter, making them less mobile and bioavailable. However, mixtures of these acids are more mobile than individual compounds [122,123,124].

2.1.2. Organic Acids

Carboxylic acids are common organic acids produced by plants, often found in root secretions. They consist of a carbon skeleton with one or more carboxyl groups (-COOH), which determine their acidity when dissociated in water. These acids are classified as aliphatic or aromatic based on the structure attached to the carboxyl group. Aliphatic acids have a straight or branched carbon chain, while aromatic acids have a benzene ring bonded to the carboxyl group. The most common carboxylic acids secreted by roots include oxalic, citric, malic, succinic, fumaric, and tartaric acids, with varying ratios depending on the species [125]. Some are intermediates in the tricarboxylic acid cycle, involved in fatty acid biosynthesis and amino acid metabolism, and play roles in light-independent photosynthetic processes in C3, C4, and CAM plants [126]. They also help plants to tolerate heavy metals like aluminum and zinc, due to their chelating properties. For example, citric and malic acids were secreted 2–3 times more under phosphorus-deficient conditions in Brassica cultivars grown hydroponically [127]. Their levels also increase in response to stress, providing protection [128]. Additionally, organic acids influence interactions with fungi and bacteria by serving as a carbon source, affecting fungal infections, microbiota development, and degradation. Microorganisms often prefer organic acids over sugars as carbon sources [129]. Plant compounds can be released through active or passive pathways, with passive release depending on the osmotic potential of the surrounding soil and the microorganisms, which aid diffusion. Alternatively, compounds are released due to cell lysis, resulting in the loss of cellular content [130]. The microbial processing of plant-derived organic acids can alter soil pH by decarboxylating anions in these compounds, which plays a key role in mineral absorption [128,131]. For instance, oxalic acid has a dual role during a Sclerotinia sp. fungal infection, as follows: at high concentrations, it induces programmed cell death, whereas, at low concentrations, it enhances resistance [132]. These acids also exhibit allelopathic effects on other plants and are often analyzed spectroscopically to identify and clarify their structure. A notable example is the research by Freitas et al., where ten molecules were identified from root extracts of Banisteria anisandra using spectroscopic methods. These included compounds like 2,8-dihydroxy-6-methoxy-7-methyl-9,10-dihydrophenanthrene-1-carbaldehyde, lupeol, quercetin-3-O-α-rhamnopyranoside, and friedelin. The latter two compounds exhibited strong allelopathic effects on Lactuca sativa shoots and roots at 100 mM, completely inhibiting seedling growth, with partial stimulatory effects at lower concentrations, further confirming the allelopathic potential of Banisteria root extracts [133].

Syed et al. [134] demonstrated that aqueous leaf extracts of Tamarindus indica L. are rich in carboxylic acids and can inhibit the growth of lettuce seedlings. When treated with the crude extract (IC₅₀), the seedlings appeared weak and necrotic, particularly at the leaf tips. The primary compounds responsible for these effects were oxalic acid, starting at a concentration of 40 mg L⁻¹, and tartaric acid at 50 mg L⁻¹. In root secretions, many carboxylic acids are also classified as phenolic acids because they contain at least one aromatic ring with a hydroxyl (-OH) group. Examples include p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, caffeic acid, and cinnamic acid. Sorghum roots, for instance, secrete protocatechuic acid and p-coumaric acid, which are believed to contribute to the well-documented allelopathic activity of sorghum against species such as rice, maize [135], and lettuce [136,137].

To better understand the secretion of phenolic acids in wheat, Wu et al. [81] analyzed root exudates from 58 wheat accessions, focusing on the levels of seven phenolic acids, including trans-coumaric, p-hydroxybenzoic, cis-ferulic, and trans-ferulic acids. The study revealed variations in the concentrations of these compounds among the accessions, suggesting that their secretion is an active mechanism rather than a passive diffusion process. This hypothesis is supported by the fact that the abundance of these molecules in shoots and roots did not directly correlate with their exudation levels. However, it is important to note that wheat exerts its main allelopathic effects through sorgoleone [138].

Some researchers have also hypothesized that certain carboxylic acids, such as abietic acid in conifers, are secreted in higher amounts by non-allelopathic species. They suggest that these plants may use these acids as a defense strategy against the toxic effects on germination and growth caused by competing species [139].

2.1.3. Terpenoids

The class of compounds known as terpenoids is primarily divided into two categories, with one deriving from the other. Terpenes are simple molecules composed of a carbon and hydrogen skeleton, where single isoprene units are repeatedly linked without any functional groups. Due to their simple structure, terpenes are nonpolar molecules, making them highly volatile and easily detectable in a gaseous form. They are key components of essential oils of plant origin and are responsible for their characteristic scent.

Terpenoids (or isoprenoids), on the other hand, result from the addition of substituent groups such as aldehydes, ketones, and other oxidative functional groups to the terpene skeleton. These modifications increase their polarity and water solubility [140]. Terpenoids represent one of the most diverse and abundant classes of compounds worldwide and are synthesized by all plant species, as well as by fungi, algae, Archaea, bacteria, and even some invertebrates [141].

In plants, terpenes and terpenoids originate mainly from two distinct biosynthetic pathways, as follows: the mevalonic acid (MVA) pathway, which occurs in the cytosol; and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, which takes place in plastids. Both pathways lead to the production of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate. However, the MVA pathway synthesizes these compounds from mevalonic acid, while the MEP pathway derives them from glyceraldehyde phosphate and pyruvate [142,143].

Terpenoids in plants serve various functions, primarily as defenses against biotic and abiotic threats. They contribute to photosynthetic apparatus maintenance, stabilize cellular lipid membranes, assist in carbon fixation by facilitating energy transfer and disposal, and influence interactions with insects and animals by attracting pollinators or repelling herbivores and parasitoids. Carotenoids, for example, play a crucial role in heat dissipation, preventing oxidative stress in leaves, while also enhancing flower and fruit coloration to aid reproduction. Additionally, terpenoid-based hormones include gibberellins, cytokinins, auxins, brassinosteroids, and strigolactones [144].

Terpenoids are often emitted as volatile organic compounds (VOCs), regulating plant–environment interactions [145]. When not released, they accumulate in vacuoles or specialized structures like glands. Some plants, such as Thymus vulgaris L. and Citrus × limon (L.) Osbeck, synthesize terpenoids in glandular trichomes on leaf surfaces. Isoprene (2-methyl-1,3-butadiene), emitted by 20% of plant species, varies in production among angiosperms, gymnosperms, mosses, and ferns [146]. With an evaporation temperature of 34 °C, isoprene is primarily released in hot periods. Despite its metabolic cost, it protects plants from abiotic stresses, particularly heat and ROS accumulation. Its release increases with temperature and is light-dependent, as it is synthesized using carbon from the Calvin cycle [147]. Isoprene is released more abundantly (up to four times) from the top leaves of crops, suggesting that it helps these leaves to manage heat stress and avoid heat flecks [148]. It is synthesized in the chloroplast stroma through the methylerythritol 4-phosphate pathway, starting from dimethylallyl diphosphate and converted into isoprene by isoprene synthase [149]. While the role of isoprene in mitigating abiotic stress is well documented, its precise physiological role and mechanisms remain unclear. It is thought that isoprene, due to its lipophilic nature, stabilizes thylakoid membranes under high temperatures, reducing ROS production even during water stress. However, recent studies suggest that isoprene acts as a signaling molecule, altering the expression of stress-related genes [150,151]. Xu et al. [52], studying water stress in Populus alba L., found that isoprene emission regulates gene expression, leading to tissue-specific differences in sensitivity to abscisic acid (ABA). They proposed that isoprene enhances the sensitivity of stomatal cells to ABA, improving water-use efficiency. Differences between isoprene-producing and non-producing plants (gene-silencing mutants) are evident at the transcriptomic, proteomic, and metabolomic levels. Non-producing plants exhibit lower expression of genes involved in photosynthesis, ROS defense, light reactions, redox processes, and metabolism [152,153]. A disadvantage of isoprene production is the emission of ozone into the atmosphere in a one-to-one ratio with isoprene molecules, but only in the presence of high NOx concentrations [154].

Plants produce both volatile, low-molecular-weight terpenoids like isoprene and heavier compounds released into the soil, regulating plant–microorganism interactions [155,156]. The most exuded root terpenoids include monoterpenoids, diterpenoids, triterpenoids, and sesquiterpenoids. These can be passively released due to root damage, attracting or repelling insects and herbivores, or actively secreted in response to threats. For example, maize roots release the sesquiterpenoid (E)-β-caryophyllene when attacked by Diabrotica virgifera larvae to attract entomopathogenic nematodes [157]. Heterorhabditis megidis is most responsive to this compound, but Heterorhabditis bacteriophora is more effective in controlling Diabrotica larvae [158]. The terpene synthase 23 (TPS23) enzyme converts farnesyl diphosphate into (E)-β-caryophyllene, and, when maize roots are damaged, the TPS23 gene is upregulated as an indirect defense strategy [159].

Root terpenoids also act as phytoalexins with antioxidant, antimicrobial, and antifungal properties. Arabidopsis thaliana produces various root terpenoids that mediate plant–microbe communication. Mutants lacking rhizatalenes and leucoplastidic diterpenes show increased susceptibility to Bradysia spp. fungus gnat larvae, suffering greater tissue damage [160]. Other terpenoids, such as astellatene, thalianol, marneral, and arabidiol, help to shape the microbial communities on root surfaces. While many root-exuded terpenoids have been identified, their exact role in rhizospheric signaling remains unclear. However, the terpenoid concentration in soil decreases with depth and correlates positively with the C/N ratio. These compounds are hypothesized to influence the nitrogen cycle by inhibiting nitrification or promoting ammoniacal nitrogen immobilization, impacting both plant growth and soil microbiota dynamics [161,162].

2.1.4. Alkaloids

Alkaloids are specialized metabolites found in plants, animals, insects, marine invertebrates, and microorganisms [163]. Over 5500 alkaloids are known [164], many of which have proven medical applications, including analgesic, antibacterial, anti-inflammatory, antidepressant, antimalarial, and anticancer properties [165]. Structurally, they contain a nitrogen atom in a heterocyclic ring, which, along with atomic arrangement, determines their activity. Their potency depends on factors such as dosage, exposure mode and duration, and the sensitivity of the affected organism.

Alkaloids are classified by the precursor origin, chemical structure, or plant genus. Based on precursors, they fall into the following three subclasses: true alkaloids, protoalkaloids, and pseudoalkaloids. True alkaloids and protoalkaloids derive from amino acids like L-phenylalanine, L-tyrosine, L-ornithine, L-histidine, L-lysine, and L-tryptophan, whereas pseudoalkaloids originate from non-amino-acid molecules but remain linked to them through amination or transamination reactions. True alkaloids and pseudoalkaloids feature a nitrogen-containing heterocyclic ring, while protoalkaloids have nitrogen bound in amino groups [166]. Chemically, alkaloids are classified as heterocyclic or non-heterocyclic, depending on whether nitrogen is part of a heterocyclic ring or bound in an aliphatic chain.

Higher plants contain the highest alkaloid concentrations, storing them in leaf vacuoles or the apoplast, where they remain inactive and non-toxic to the cell [167]. While not essential for plant survival, alkaloids regulate growth and store nitrogen [168,169]. They serve a defensive role, affecting herbivore central nervous systems when ingested and sometimes acting as DNA intercalants, causing mutations and genotoxicity [166]. Alkaloid production is widespread across various plant families, including Berberidaceae, Amaryllidaceae, Liliaceae, Leguminosae, Papaveraceae, Ranunculaceae, Solanaceae, Boraginaceae, Apocynaceae, Asclepiadaceae, Rutaceae, and Malvaceae [166]. Species within the same genus typically produce similar molecules, allowing alkaloids to be categorized. For instance, opiates are characteristic of the genus Papaver, particularly Papaver somniferum L., and include codeine, noscapine, thebaine, and papaverine [170,171]. The Solanaceae family also contains genera that produce tropane, pyrrolidine-pyridine, and steroid alkaloids (and their glycosylated forms), such as solanine, solanidine, solasodine, tomatidine, tomatine, canine, scopolamine, atropine, nicotine, and cocaine [172]. Some Ephedra species (Ephedraceae family) produce specialized metabolites from phenylalanine, like ephedrine, pseudoephedrine, phenylpropanolamine, and cathinone [173]. Alkaloids are stored by plants for normal physiological functions and to defend against threats like predators and parasites and are also used to reduce competition from other plants. These compounds can be released both above and below ground, depending on the plant’s needs and external stimuli. In plant–microbe interactions, some alkaloids inhibit fungal and bacterial growth. For example, tomatidine is effective against Staphylococcus aureus [174], lysergol against Escherichia coli [175], and quinoline alkaloids from Waltheria indica L. against the fungus Candida albicans [176].

There is limited information on alkaloid-mediated allelopathic plant–plant interactions, particularly when alkaloids are secreted by roots. However, studies on seeds and seedlings of model species have assessed the effects of alkaloids on germination and growth. Allelopathic activity was observed with berberine, sanguinarine, and gramine on lettuce seeds, where germination and seedling growth were partially inhibited [170]. Tests have also been conducted on other species, despite lettuce being commonly used as a model organism.

In 2018, Ogunsusi et al. [177] investigated the allelopathic potential of a metabolic extract (leaves and stems) from Crotalaria retusa L. They administered a raw alkaloidal fraction (0.5–100 µg/mL) to brown bean (Phaseolus vulgaris L.) seedlings, using strychnine as a reference alkaloid. The treatment reduced germination rates and altered metabolic parameters in the seedlings. Specifically, proline, reduced glutathione, and ascorbic acid levels increased in the stems and leaves, while sugar and protein levels declined, indicating stress. The mechanism of action involved oxidative stress induction, leading to increased reactive oxygen species (ROS) production and metabolic imbalances, ultimately reducing seedling growth.

Lei et al. [178] studied the allelopathic effects of alkaloids from Sophora alopecuroides L., including aloperine, matrine, oxymatrine, oxysophocarpine, sophocarpine, and sophoridine. These compounds, both individually and as a mixture, were tested in distilled water and soil on two dicotyledons, amaranth (Amaranthus retroflexus L.) and alfalfa, and two monocotyledons, rye and green foxtail (Setaria viridis (L.) Beauv.), grown in pots and Petri dishes. The results showed that sophocarpine and the alkaloid mixture most strongly inhibited root growth, though the effect was weaker in the pots due to the complexity of the growth medium. Hormonal assays on alfalfa confirmed the phytotoxicity of these alkaloids, revealing altered levels of indole-3-acetic acid, abscisic acid (ABA), and cytokinins. Additionally, increased malondialdehyde content and enhanced peroxidase activity suggested photooxidative damage caused by ROS accumulation. These findings indicate that S. alopecuroides alkaloids may provide a competitive advantage in natural conditions.

2.1.5. Benzoxazinoids

Benzoxazinoids (BXZs), also known as hydroxamic acids, are specialized metabolites mainly produced by Graminaceae species like maize, wheat, and rye (Secale cereale L.), as well as some dicotyledonous families, including Plantaginaceae, Acanthaceae, Lamiaceae, Calceolariaceae, and Ranunculaceae [179]. These compounds have an oxazinone ring attached to a benzene ring, with variable substituents affecting their properties. Benzoxazinoids are classified into two groups: benzoxazinones and benzoxazolinones. Benzoxazinones can be further split into lactams, hydroxamic acids, and methyl derivatives, depending on the substituent at the N position. Lactams contain an N-hydro group, hydroxamic acids an N-hydroxy group, and methyl derivatives an N-methoxy group. The N substituent influences the molecule’s activity [180,181]. The most common benzoxazolinones are found in Scoparia dulcis L., with N-hydroxy and N-methoxy derivatives, though N-hydroxy compounds are more prevalent in plants [182]. During detoxification, glucose may attach to these compounds, reducing toxicity and allowing storage in vacuoles [183].

Active benzoxazinoids like 2,4-Dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), along with byproducts such as benzoxazolin-2-one (BOA) and 6-methoxy-benzoxazolin-2-one (MBOA), are significant allelochemicals in wheat [184]. In maize, their biosynthesis involves the enzyme BX1 converting indole-3-glycerolphosphate (IGP) into indole, followed by enzymes BX2 – BX5 oxidizing the molecule to produce HBOA and DIBOA, with BX5 being specifically involved in N-hydroxylation [185,186]. DIMBOA is formed from DIBOA via methylation, which alters its properties. These compounds are toxic, requiring glucosylation by enzymes BX6 and BX7 for safe storage in vacuoles [187,188]. Glucosylated forms are released as defensive aglycones after enzymatic hydrolysis. These aglycones, once released into the soil, undergo degradation, often in microbial communities, and become more stable forms like benzoxazolinones, which possess higher allelopathic potential than their glycosylated counterparts [189,190,191,192].

This class of molecules is important for acting as antifeedants, attractants, or repellents against insects, influencing symbiosis development, exhibiting antimicrobial and allelopathic activities, and playing a role in plant defense regulation [193,194]. For example, Maag et al. studied BX synthesis dynamics in maize after exposure to the herbivore Spodoptera littoralis Boisduval. They found that BX1 gene transcripts, which catalyze the first step in BX biosynthesis, were upregulated after insect infestation, with peak expression occurring at the feeding site. This suggests BXs’ involvement in plant defense against abiotic stress and that their synthesis can be induced by herbivores [195].

The release of benzoxazinoids into the soil occurs through root hair exudation, though the exact mechanism remains unknown (passive diffusion has been proposed but not confirmed). Root exudation levels vary between species and even within the same cultivar.

Benzoxazinoids are known for inhibiting germination and seedling growth, either through root secretion or the decomposition of dead tissues. The primary mechanism behind their allelopathic action involves oxidative stress and ROS generation, which cause membrane damage, lipid peroxidation, and protein oxidation. These disruptions impair essential physiological processes like protein synthesis, lipid metabolism, photosynthesis, and auxin-mediated hormonal balance (critical for lateral root growth), ultimately leading to plant death [196,197,198].

Studies on the phytotoxicity of benzoxazinoids have demonstrated their allelopathic activity against various weeds and non-weed species. Rye, a major producer of these compounds, was the first species in which benzoxazinoids were identified [199]. The highest concentrations are found in young rye tissues, and the compounds are used as a natural weed suppressant when rye is planted, as a cover crop. These allelochemicals inhibit weed germination and early-stage growth [200]. Their allelopathic effect has also been observed on crops like white mustard (Sinapis alba L.), wild oat (Avena fatua L.), and ryegrass [201].

Building on previous successful weed control results, Mejías et al. [201] synthesized eight compounds inspired by benzoxazinones to test on ryegrass, purslane (Portulaca oleracea L.), and barnyard grass (Echinochloa crus-galli (L.) P. Beauv). The synthetic molecules showed strong inhibition of root growth in dicot weeds, with effects comparable to an herbicide used as a positive control. The use of BXZ-producing plants for weed control in organic farming (through mulching or competition) offers potential. Additionally, identifying QTLs for allelopathic traits could enhance crop allelopathic activity through marker-assisted selection.

3. Allelopathic Mechanisms

3.1. Chemical Signaling

Plants, as sessile organisms, have developed chemical communication mechanisms to cope with adversity and interact with other living beings. They use chemical signals to detect and identify nearby plants, pathogens, and herbivorous animals. In response to competitors, plants regulate the production of potentially phytotoxic substances, which are exuded in varying amounts depending on the surrounding plant types and quantities [202]. This applies to allelopathic molecules, which are produced differently in the presence of potentially harmful plant species [203,204]. These substances are primarily released as volatile organic compounds (VOCs) in a gaseous form or exuded through roots into the rhizosphere. VOCs serve as signal molecules between plants, with the message depending on the specific molecule and the emitter’s health status. This communication often warns nearby plants of potential threats like herbivores or pathogens, prompting them to increase the production of defensive metabolites. VOCs also help plants to recognize neighboring species, distinguishing related plants from others [19,205,206]. Common VOC classes include terpenoids, isoprenoids, alcohols, esters, and hormones [207].

Volatile substances are not only active signaling molecules above the soil, but are also released by roots, serving similar functions [208]. Root exudates, which include molecules like (-)-loliolide, salicylic acid, jasmonic acid, and luteolin, enable communication between neighboring plants. These substances influence the production of metabolites that affect competitor growth and can impact microbial communities and nutrient availability in the rhizosphere.

An experiment by Wang et al. [209] involving soft wheat (Triticum aestivum L. cv Jing-411) and 60 other plant species revealed that root exudates from different plants caused wheat to produce allelopathic molecules that inhibited the growth of nearby plants. These allelochemicals caused a shift in the root orientation of neighboring plants, making them less susceptible to the toxic molecules [210]. The presence and abundance of nearby species seem to influence the production of allelochemicals, with wheat roots showing greater DIMBOA production when exposed to exudates from different species rather than conspecifics. The number of competing plants also correlates with the amount of DIMBOA secreted by wheat, indicating a competitive response.

Several plant species, including sorghum, corn, rice, barley, buckwheat (Fagopyrum esculentum Moench), and alfalfa, produce allelopathic molecules. This suggests allelopathy is both a defense mechanism against unwanted neighbors and a form of interspecies communication.

3.2. Effects on Plant Growth

Allelopathy is an evolutionary strategy that plants use to compete with other species, affecting processes like seed germination, root growth, and photosynthesis, which are crucial for survival (Table 1). It can be species-specific or affect surrounding plants, sometimes causing auto-toxicity [211]. The effectiveness of allelochemicals depends on their persistence in the environment, influenced by the soil, climate, microbial communities, and molecular structure [212]. Typically, allelochemicals have a shorter half-life in the soil than synthetic herbicides, due to their more biodegradable structures [19]. These factors make studying allelopathic mechanisms complex, as in vitro conditions are challenging to replicate. Allelochemicals are often tested using crop residues, aqueous/ethanolic extracts, or exudates. Crop residues, particularly from Brassicaceae species like Brassica, Raphanus, Sinapis, and Eruca, are highly active in allelopathy, releasing phenolic compounds and glucosinolates that break down into harmful isothiocyanates in the presence of water and myrosinase enzymes. Isothiocyanates reduce seed numbers and the germination of weeds like Digitaria sanguinalis, Portulaca oleracea, and Taraxacum officinale [213,214]. Extracts from plants like sorghum, sunflower, and mulberry are also widely studied, using aqueous or less polar matrices depending on the molecules being extracted. Crop residues and extracts are easier to access than exudates, which must be collected directly from the plant, especially root exudates.

Over the years, tests conducted have shown how allelochemicals can cause the following:

• Inhibition of germination;
• Inhibition of root development and growth;
• Inhibition of/uncontrolled increase in cell division, with consequences on seedling development;
• Morphological alterations of the root system and shoots.

Carvalho and colleagues observed phytotoxic symptoms in lettuce seeds and seedlings [215]. They tested five ethanol extracts from species of Amaranthus (A. spinosum, A. viridis, A. deflexus, A. hybridus, and A. retroflexus), known for their biologically active metabolites. Phytotoxicity tests showed a dose-dependent decrease in lettuce seed germination and root and shoot growth. Microscopically, the extracts caused mitotic changes, including sticky chromosomes. Further screening of root exudates from five weed species (Cyperus rotundus, Marselia quadrifolia, Ludwigia hyssopifolia, Pistia stratiotes, and Colocasia esculenta) on mung bean (Vigna radiata) and cowpea (Vigna unguiculata) showed a 10–50% reduction in germination and shorter radicles and hypocotyls [216]. Akter et al. [217] tested exudates from 15 allelopathic species on wheat, finding the greatest inhibition in germination from Commelina benghalensis (from 86.33% to 64.0%) and the greatest reduction in shoot length from Mikania micrantha. These treatments also led to reduced chlorophyll a and b, lower antioxidant enzyme activity (catalase and superoxide dismutase), and increased malondialdehyde due to oxidative stress.

Table 1. Root exudates tested for their allelopathic potential with their respective effects on treated plants.

Tested Exudates	Species Tested with Exudate	Methodology	Phytotoxic Effects Observed	References
Ageratum conyzoides Centella asiatica Commelina benghalensis Cynodon dactylon Heliotropium indicum Leucas aspera Marsilea quadrifolia Mikania micrantha Phyllanthus niruri Physalis heterophylla Polygonum hydropiper Rotala indica Sida acuta Solanum nigrum Spilanthes acmella	Triticum aestivum	In vitro germination inhibition test in Petri dishes with filter paper. Germination and growth inhibition tests in pots with garden soil.	The most inhibitory exudate for T. aestivum germination was from C. benghalensis (22%). A total of 10.41% of inhibition in the shoot growth by M. micrantha exudate. Presence of byproducts resulting from oxidative stress.	[217]
Colocasia esculenta Cyperus rotundus Ludwigia hyssopifolia Marselia quadrifolia Colocasia esculenta	Vigna radiata Vigna unguiculata	In vitro germination inhibition test in Petri dishes with filter paper.	Inhibition of germination up to 30% for V. radiata and up to 50% for V. unguiculata. Reduction in root growth dependent on the species’ sensitivity to different exudates.	[216]
Triticum aestivum (cv. Adesso, Element, Maurizio, and NS 40S)	Lolium rigidum Portulaca oleracea	In vitro tests with equal-compartment-agar method employing pregerminated seeds.	T. aestivum cv. Maurizio caused, in both treated species, an inhibition in germination. Reduction in total weight, shoot length, and root length (56%, 55%, and 94%, respectivley) on L. rigidum, and reduction in total weight and root length (84% and 86%, respectivley) in P. oleracea.	[218]
Ageratum conyzoides	Lactuca sativa	Germination and growth inhibition test in pots with infested and not-infested forest soil.	Inhibition of germination of 6.67% in a dose-dependent manner. Significant mitotic inhibition in the root cells.	[219]
Bidens pilosa	Pteris multifida	Test in in vitro conditions using applications of undecane and palmitic acid, main compounds in B. Pilosa root exudate.	Down-regulation of alpha−linolenic acid, starch, and sucrose metabolism by the undecane. Reduction in flavonoid biosynthesis, arginine biosynthesis, pentose, and glucuronate interconversions, and the proteins related to spliceosome pathway production by the palmitic acid.	[220]
Bidens pilosa	Lactuca sativa, Phaseolus vulgaris, Zea mays, and Sorghum bicolor	Root exudates recirculating system.	Inhibition of germination and growth ranging from 30 to 50%, monocots are more sensitive than dicots.	[221]
Oryza sativa	Echinochloa crus-galli	Co-cultivation with rice seedlings in a bioassay medium.	Inhibition of roots and stem growth.	[222]
Triticum turgidum durum cv. Khapli	Lolium rigidum	In vitro growth inhibition tests in beakers with agar medium.	Inhibition of root and shoot growth with a maximum effect after 6–8 days of treatment.	[223]
Triticum aestivum cv. Pishgam	Amaranthus retroflexus	Growth tests in pots with sterilized soil and peat moss. Intercropping with ratios (wheat:amaranth) 100:0, 75:25, 50:50, and 25:75.	As wheat increased (particularly at 75:25), there was a reduction in the fresh weight of the roots and chlorophyll content. Additionally, there was a decrease in shoot protein content (−42%) but an increase in roots (+285%). Induction of oxidative stress.	[92]
Amaranthus retroflexus	Triticum aestivum cv. Pishgam	Growth tests in pots with sterilized soil and peat moss. Intercropping with ratios (amaranth:wheat) 0:100, 25:75, 50:50, and 75:25.	As amaranth increased (particularly at 75:25), there was a decrease in the fresh weight of the roots, but an increase in protein content (74%) at the same. Induction of oxidative stress.	[92]
Hordeum vulgare	Bromus diandrus Lolium rigidum Hordeum vulgare	Growth and germination tests in in vitro conditions with filter paper. “Seed-to-seed” protocol.	Greater inhibitory effect on weeds, affecting their root and coleoptile growth. In B. diandrus, inhibition ranged from 65 to 74% for radicle growth and 42% for coleoptile growth (25 barley seeds). In L. rigidum, inhibition ranged from 55 to 65% for radicle growth and 18% for coleoptile growth (25 barley seeds).	[224]
Heracleum mantegazzianum Heracleum sphondylium Dactylis glomerata Plantago lanceolata	Centaurea jacea Dactylis glomerata Plantago lanceolata	Tests in pots with sterilized garden soil and sand (ratio 2:3). Soil microbiota treatments (added vs. sterilized) and activated carbon treatments (20 mL of powder vs. without powder).	D. glomerata exudates suppressed C. jacea biomass in soil without carbon, while P. lanceolata exudates also suppressed biomass, but within the same species. The addition of activated carbon in the soil reversed the negative effects of all exudates on the tested plants.	[225]
Solanum rostratum	Triticum aestivum Brassica campestris	Growth and germination in vitro tests in Petri dishes with agar medium (exudate concentrations: 0.1 g fw/mL, 0.2 g fw/mL, and 0.4 g fw/mL).	The germination rate decreased in both wheat (by approximately 18.5%) and cabbage (by approximately 23%) with increasing concentrations of exudates. The wheat shoot growth decreased starting from the concentration of 0.1 g fw/mL. Conversely, the cabbage shoot growth increased, although not significantly.	[226]
Oryza sativa	Oryza sativa Cyperus difformis Echinochloa crus-galli Eclipta prostrata Leptochloa chinensis	Tests with window rhizoboxes and root segregation methods.	Rice interfered with weeds by altering root placement patterns and root interactions, except for wild rice.	[210]
Sorghum bicolor	Triticum aestivum Triticum durum Hordeum spontaneum Avena fatua Phalaris minor	Growth and chlorophyll content tests in a greenhouse with a modified stair step tool.	Phalaris minor was the most sensitive plant to sorghum root exudates. Compared to other species, its dry weight, and its length (41.68 cm, 294.87 mg) were strongly inhibited, along with its chlorophyll content.	[227]
Sorghum bicolor Solidago canadensis	Bromus sterilis Veronica persica Youngia japonica Rumex acetosa	Germination and growth tests in pots using coco peat and sand (1:1) in a glasshouse.	The invasive species exhibited variable growth responses, while the native species showed greater sensitivity to the exudates across all evaluated parameters in both shoot and root growth.	[228]
Tithonia diversifolia	Amaranthus dubius Solanum melongena	Germination, growth, and chlorophyll tests were conducted in in vitro conditions (Petri dishes with filter paper) and subsequently in pots filled with humus soil.	Reduction in germination rates was observed for both species (from 91% to 72.5% in A. dubius, and from 53.3% to 12.5% in S. melongena). Shoot length and leaf area of A. dubius were significantly inhibited.	[229]
Ageratina adenophora	Osbeckia stellata Elsholtzia blanda	Growth tests in a greenhouse were conducted using pots filled with soil from uninvaded areas and root leachate (10 g root/100 mL distilled water).	Adenophora root leachate reduced the shoot length of E. blanda and the chlorophyll content in both tested weeds.	[230]

Table 1. Root exudates tested for their allelopathic potential with their respective effects on treated plants.

Tested Exudates	Species Tested with Exudate	Methodology	Phytotoxic Effects Observed	References
Ageratum conyzoides Centella asiatica Commelina benghalensis Cynodon dactylon Heliotropium indicum Leucas aspera Marsilea quadrifolia Mikania micrantha Phyllanthus niruri Physalis heterophylla Polygonum hydropiper Rotala indica Sida acuta Solanum nigrum Spilanthes acmella	Triticum aestivum	In vitro germination inhibition test in Petri dishes with filter paper. Germination and growth inhibition tests in pots with garden soil.	The most inhibitory exudate for T. aestivum germination was from C. benghalensis (22%). A total of 10.41% of inhibition in the shoot growth by M. micrantha exudate. Presence of byproducts resulting from oxidative stress.	[217]
Colocasia esculenta Cyperus rotundus Ludwigia hyssopifolia Marselia quadrifolia Colocasia esculenta	Vigna radiata Vigna unguiculata	In vitro germination inhibition test in Petri dishes with filter paper.	Inhibition of germination up to 30% for V. radiata and up to 50% for V. unguiculata. Reduction in root growth dependent on the species’ sensitivity to different exudates.	[216]
Triticum aestivum (cv. Adesso, Element, Maurizio, and NS 40S)	Lolium rigidum Portulaca oleracea	In vitro tests with equal-compartment-agar method employing pregerminated seeds.	T. aestivum cv. Maurizio caused, in both treated species, an inhibition in germination. Reduction in total weight, shoot length, and root length (56%, 55%, and 94%, respectivley) on L. rigidum, and reduction in total weight and root length (84% and 86%, respectivley) in P. oleracea.	[218]
Ageratum conyzoides	Lactuca sativa	Germination and growth inhibition test in pots with infested and not-infested forest soil.	Inhibition of germination of 6.67% in a dose-dependent manner. Significant mitotic inhibition in the root cells.	[219]
Bidens pilosa	Pteris multifida	Test in in vitro conditions using applications of undecane and palmitic acid, main compounds in B. Pilosa root exudate.	Down-regulation of alpha−linolenic acid, starch, and sucrose metabolism by the undecane. Reduction in flavonoid biosynthesis, arginine biosynthesis, pentose, and glucuronate interconversions, and the proteins related to spliceosome pathway production by the palmitic acid.	[220]
Bidens pilosa	Lactuca sativa, Phaseolus vulgaris, Zea mays, and Sorghum bicolor	Root exudates recirculating system.	Inhibition of germination and growth ranging from 30 to 50%, monocots are more sensitive than dicots.	[221]
Oryza sativa	Echinochloa crus-galli	Co-cultivation with rice seedlings in a bioassay medium.	Inhibition of roots and stem growth.	[222]
Triticum turgidum durum cv. Khapli	Lolium rigidum	In vitro growth inhibition tests in beakers with agar medium.	Inhibition of root and shoot growth with a maximum effect after 6–8 days of treatment.	[223]
Triticum aestivum cv. Pishgam	Amaranthus retroflexus	Growth tests in pots with sterilized soil and peat moss. Intercropping with ratios (wheat:amaranth) 100:0, 75:25, 50:50, and 25:75.	As wheat increased (particularly at 75:25), there was a reduction in the fresh weight of the roots and chlorophyll content. Additionally, there was a decrease in shoot protein content (−42%) but an increase in roots (+285%). Induction of oxidative stress.	[92]
Amaranthus retroflexus	Triticum aestivum cv. Pishgam	Growth tests in pots with sterilized soil and peat moss. Intercropping with ratios (amaranth:wheat) 0:100, 25:75, 50:50, and 75:25.	As amaranth increased (particularly at 75:25), there was a decrease in the fresh weight of the roots, but an increase in protein content (74%) at the same. Induction of oxidative stress.	[92]
Hordeum vulgare	Bromus diandrus Lolium rigidum Hordeum vulgare	Growth and germination tests in in vitro conditions with filter paper. “Seed-to-seed” protocol.	Greater inhibitory effect on weeds, affecting their root and coleoptile growth. In B. diandrus, inhibition ranged from 65 to 74% for radicle growth and 42% for coleoptile growth (25 barley seeds). In L. rigidum, inhibition ranged from 55 to 65% for radicle growth and 18% for coleoptile growth (25 barley seeds).	[224]
Heracleum mantegazzianum Heracleum sphondylium Dactylis glomerata Plantago lanceolata	Centaurea jacea Dactylis glomerata Plantago lanceolata	Tests in pots with sterilized garden soil and sand (ratio 2:3). Soil microbiota treatments (added vs. sterilized) and activated carbon treatments (20 mL of powder vs. without powder).	D. glomerata exudates suppressed C. jacea biomass in soil without carbon, while P. lanceolata exudates also suppressed biomass, but within the same species. The addition of activated carbon in the soil reversed the negative effects of all exudates on the tested plants.	[225]
Solanum rostratum	Triticum aestivum Brassica campestris	Growth and germination in vitro tests in Petri dishes with agar medium (exudate concentrations: 0.1 g fw/mL, 0.2 g fw/mL, and 0.4 g fw/mL).	The germination rate decreased in both wheat (by approximately 18.5%) and cabbage (by approximately 23%) with increasing concentrations of exudates. The wheat shoot growth decreased starting from the concentration of 0.1 g fw/mL. Conversely, the cabbage shoot growth increased, although not significantly.	[226]
Oryza sativa	Oryza sativa Cyperus difformis Echinochloa crus-galli Eclipta prostrata Leptochloa chinensis	Tests with window rhizoboxes and root segregation methods.	Rice interfered with weeds by altering root placement patterns and root interactions, except for wild rice.	[210]
Sorghum bicolor	Triticum aestivum Triticum durum Hordeum spontaneum Avena fatua Phalaris minor	Growth and chlorophyll content tests in a greenhouse with a modified stair step tool.	Phalaris minor was the most sensitive plant to sorghum root exudates. Compared to other species, its dry weight, and its length (41.68 cm, 294.87 mg) were strongly inhibited, along with its chlorophyll content.	[227]
Sorghum bicolor Solidago canadensis	Bromus sterilis Veronica persica Youngia japonica Rumex acetosa	Germination and growth tests in pots using coco peat and sand (1:1) in a glasshouse.	The invasive species exhibited variable growth responses, while the native species showed greater sensitivity to the exudates across all evaluated parameters in both shoot and root growth.	[228]
Tithonia diversifolia	Amaranthus dubius Solanum melongena	Germination, growth, and chlorophyll tests were conducted in in vitro conditions (Petri dishes with filter paper) and subsequently in pots filled with humus soil.	Reduction in germination rates was observed for both species (from 91% to 72.5% in A. dubius, and from 53.3% to 12.5% in S. melongena). Shoot length and leaf area of A. dubius were significantly inhibited.	[229]
Ageratina adenophora	Osbeckia stellata Elsholtzia blanda	Growth tests in a greenhouse were conducted using pots filled with soil from uninvaded areas and root leachate (10 g root/100 mL distilled water).	Adenophora root leachate reduced the shoot length of E. blanda and the chlorophyll content in both tested weeds.	[230]

4. Collection Methods for Root Exudates

The composition of root exudates plays a crucial role in the allelopathic activity of plants. However, collecting representative samples of these exudates is challenging, as smaller molecules can be absorbed by soil or transformed by microorganisms, affecting the analysis results [231]. After collection, exudates undergo purification and concentration to remove solids, root cells, and microorganisms before analysis [232,233]. The main difficulty lies in faithfully collecting the exudates from the roots and growth medium.

Various sampling methods have been developed, including hydroponic, semi-hydroponic, and soil-based cultures, as well as hybrid approaches (Figure 1) [234,235]. However, hydroponic cultures are not fully representative of field conditions, and semi-hydroponic systems may lack adequate air for roots. Soil-based methods, such as pot cultivation in rhizotrons or rhizoboxes [236], are more natural but require root washing, which can stress plants and alter exudate composition. Recent hybrid methods attempt to improve root development and exudate collection, but they still face the root washing issue. An experiment tested whether a recovery period after washing could improve the representativeness of collected exudates [237]. In this experiment, seedlings of three European grassland species (Holcus lanatus, Rumex acetosa, and Trifolium repens) were grown in soil, and their root exudates were collected using a hybrid method after three recovery times (0, 3, and 7 days). The plants in the 3- and 7-day recovery groups were kept in aerated hydroponic conditions. The exudate profiles were analyzed using gas chromatography and compared to leachates from plants with deliberately damaged roots. The study concluded that a recovery period of at least 3 days is necessary for obtaining representative exudate samples, and the hybrid method effectively distinguished between the species.

In 2024, Döll et al. proposed a sampling method using pots with tomato plants. The effects of various parameters (collection medium, recovery period, and exudation duration) were evaluated. The LC-MS analysis revealed that the most important factor for quality exudate samples was the recovery period, with plants that had no recovery showing almost double the concentration of primary metabolites. A 24-hour recovery period was recommended to prevent solute loss. The ideal collection medium was ultrapure water, and a 4-hour exudation period was found to be optimal for representative sampling. Glass beads added complexity without benefits [238]. While a fully efficient protocol without root washing is still being explored, using a permeable membrane in rhizoboxes could potentially eliminate the need for washing and avoid root damage [239,240].

5. Species with Noteworthy Allelopathic Root Exudates

Over time, many plant species have been recognized as allelopathic in agriculture. Below are two examples of crops and the allelopathic effects of their root exudates on competing plants, including weeds. This chapter also discusses two weed species that release phytotoxic root exudates, making it difficult to cultivate target species.

5.1. Crops

5.1.1. Wheat

In 2023, global wheat production reached 783 million tons, with about 95% coming from hexaploid Triticum aestivum (bread wheat) and the remaining 5% from tetraploid Triticum turgidum (durum wheat) [241]. Wheat has notable allelopathic potential due to the presence of various specialized metabolites found in different parts of the plant. The highest release of these allelopathic compounds typically occurs during straw degradation, when the plant is used as mulch, and through root exudation [242]. The main allelochemicals identified in wheat include hydroxamic acids, phenolic acids such as chlorogenic, caffeic, and ferulic acids, short-chain fatty acids (like palmitic and stearic), and benzoxazinoids (BXZs) (Table 2) [81,115,223].

Research has shown that wheat’s allelopathic activity, particularly when competing with other plants, can cause physical and metabolic changes in the plant, triggering the production of more allelopathic metabolites [243]. A study conducted on wheat root exudates, when cultivated with common purslane and annual ryegrass, showed that the presence of these weeds significantly increased the production of vanillic, ferulic, syringic, and p-coumaric acids, as well as BXZs. This increase in allelochemical secretions led to a 29–62% reduction in ryegrass growth, along with a notable decrease in photosynthetic pigment production, demonstrating wheat’s ability to inhibit weed growth through its root exudates [189].

Additionally, another study focusing on bread wheat (cv. Pishgam) identified various allelopathic molecules, including terpenoids, phenolic compounds, fatty alcohols, steroids, fatty acids, and alkanes, in wheat root exudates. These compounds influenced the activation of the antioxidant system in both wheat and the weed Amaranthus retroflexus [92]. Interestingly, this study also showed that increasing the density of wheat sowing further reduced A. retroflexus growth, highlighting the potential for wheat to exert allelopathic effects in a density-dependent manner.

While durum wheat is mainly used for products like pasta, semolina, and couscous, allelopathic research on this species is less extensive. However, studies suggest that the leaves of durum wheat are particularly rich in allelopathic compounds, making their extracts more biologically active than those derived from other parts of the plant [244].

Genetic studies have also explored the allelopathic potential of wheat. A study involving 453 wheat accessions from 50 countries assessed their ability to inhibit annual ryegrass growth. The results showed a wide range of inhibition (9.7% to 90.9%), indicating the genetic variability in allelopathic potential among different wheat cultivars. Further research has identified two potential quantitative trait loci (QTLs) on chromosomes 1A and 2B, which may play a role in regulating the production of allelopathic compounds in wheat [245,246,247].

Table 2. Principal class of allelochemicals produced by wheat with their respective production site and allelopathic effect.

Chemical Class	Compound	Allelopathic Effect	Production Site	References
Phenolic acids	p-Hydroxybenzoic acid	Oxidative stress, membrane depolarization, hydraulic conductivity reduction, affection of respiration and transpiration, and inhibition of germination and plant growth.	Root, shoot, leaf, seed, and straw.	[81,247,248,249,250]
	Vanillic acid
	Cis-coumaric acid
	Syringic acid
	Trans-coumaric acid
	Trans-ferulic acid
Hydroxamic acids	DIBOA	Physiological, biochemical, and oxidative stress, affection of photosynthesis and respiration, damage in membrane transport, germination, and root and shoot growth.	Root, shoot, and leaf.	[218,223,242,248]
	DIMBOA

Table 2. Principal class of allelochemicals produced by wheat with their respective production site and allelopathic effect.

Chemical Class	Compound	Allelopathic Effect	Production Site	References
Phenolic acids	p-Hydroxybenzoic acid	Oxidative stress, membrane depolarization, hydraulic conductivity reduction, affection of respiration and transpiration, and inhibition of germination and plant growth.	Root, shoot, leaf, seed, and straw.	[81,247,248,249,250]
	Vanillic acid
	Cis-coumaric acid
	Syringic acid
	Trans-coumaric acid
	Trans-ferulic acid
Hydroxamic acids	DIBOA	Physiological, biochemical, and oxidative stress, affection of photosynthesis and respiration, damage in membrane transport, germination, and root and shoot growth.	Root, shoot, and leaf.	[218,223,242,248]
	DIMBOA

5.1.2. Rice

Rice (Oryza sativa L. subsp. indica and japonica) is one of the most cultivated cereal crops globally, with an annual production of approximately 500 million tons [251]. Much like wheat, rice also produces allelochemicals as a strategy to combat competition. The primary organs involved in the production of these allelopathic molecules are the leaves, straws, and roots. These allelochemicals mainly belong to the classes of phenolic acids, terpenes, and terpenoids, with a significant concentration of momilactones, which are particularly important for the allelopathic effects of rice (Table 3) [252,253].

The phenolic acids and terpenes in rice have been discussed earlier. Momilactones are a distinct class of compounds that are synthesized from geranylgeranyl diphosphate through a series of cyclization reactions. At the genetic level, the production of momilactones is regulated by a gene cluster located on chromosome 4 of the rice genome [254,255,256]. The primary momilactones produced by rice are momilactone A (MA) and momilactone B (MB). While both compounds differ in their chemical structures, with MB containing an oxygen bridge at the C3 position of the benzene ring, it is MB that is hypothesized to be the main compound responsible for the allelopathic activity of rice. While the concentration of MA in rice organs is generally higher, MB has been found to exert superior suppressive effects in Arabidopsis thaliana [73,257]. Interestingly, MA is more closely associated with resistance to pathogens in rice, which might explain why it is less secreted from rice roots compared to MB [74]. This family of compounds is known to significantly inhibit the germination and root development of common weed species found in rice fields, highlighting their role in rice’s competitive strategy [252,257].

Previous studies have demonstrated the critical role of momilactones in rice’s allelopathic effects. For example, the simultaneous cultivation of rice mutants unable to produce momilactones with various weed species, such as barnyard grass and lettuce, resulted in significantly higher weed growth compared to cultivation with wild-type rice [94,255,258]. This suggests that the presence of momilactones is central to the allelopathic activity in rice. Supporting this hypothesis, a study by Xu et al. [259] investigated mutants with gene knockouts (cps4 and ksl4), which lack the ability to synthesize momilactones. These mutants were tested against wild-type rice, and the results showed a complete absence of allelopathic activity in the mutants. This finding further strengthened the connection between momilactones and the innate allelopathic properties of rice [73,99].

Moreover, root secretions in rice play a significant role in kin recognition, which has been linked to allelopathic interactions. Xu and colleagues [260] examined whether rice cultivars could recognize their kin and how this might influence other factors such as weed abundance, plant morphology, and soil microbial communities. In their experiment, rice cultivars were bred for kin recognition abilities and were grown alongside the following four common weed species: Echinochloa crus-galli, Leptochloa chinensis, Cyperus difformis, and Eclipta prostrata. The study revealed that there were no significant differences in microbial communities between kin and non-kin groups. However, the root morphology of the rice plants grown in related groups exhibited notable changes. The rice in the kin group developed more intrusive roots directed towards the weeds, while the weeds exhibited avoidance behaviors by growing roots away from the rice roots. Furthermore, genetic relatedness resulted in a reduction in allelochemical production in rice, while still maintaining a significant inhibition of weed growth. These findings suggest that kin recognition in rice may regulate root morphology, competitiveness, and chemical defense, influencing the overall growth dynamics between rice and surrounding weeds [261].

Table 3. Principal class of allelochemicals produced by rice with their respective production site and allelopathic effect.

Chemical Class	Compound	Allelopathic Effect	Production Site	References
Phenolic acids	Salicylic acid	Affection of photosynthesis and metabolism. Inhibition of germination, roots growth, and plant growth.	Roots and leaves.	[262,263,264]
	Ferulic acid
	p-Hydroxybenzoic acid
	Vanillic acid
	p-Coumaric acid
	2,4-Dimethoxybenzoic acid
Flavonoids	Tricin	Inhibitory activity on weeds and pathogens.	Roots	[91,263,265]
Momilactones	Momilactone A	Inhibition of germination and seedling growth.	Roots, leaves, husks, and seeds.	[73,260,266]
	Momilactone B

Table 3. Principal class of allelochemicals produced by rice with their respective production site and allelopathic effect.

Chemical Class	Compound	Allelopathic Effect	Production Site	References
Phenolic acids	Salicylic acid	Affection of photosynthesis and metabolism. Inhibition of germination, roots growth, and plant growth.	Roots and leaves.	[262,263,264]
	Ferulic acid
	p-Hydroxybenzoic acid
	Vanillic acid
	p-Coumaric acid
	2,4-Dimethoxybenzoic acid
Flavonoids	Tricin	Inhibitory activity on weeds and pathogens.	Roots	[91,263,265]
Momilactones	Momilactone A	Inhibition of germination and seedling growth.	Roots, leaves, husks, and seeds.	[73,260,266]
	Momilactone B

5.2. Weeds

5.2.1. Bidens pilosa L.

Bidens pilosa L., commonly known as “blackjack”, is an annual or biennial weed in the Asteraceae family. Native to South America, it has spread to tropical and subtropical regions, including Colombia, Brazil, Peru, Uganda, Kenya, China, Australia, and Hawaii [267]. It is recognized as a medicinal plant, a food source, and as a weed. B. pilosa can significantly reduce yields in crops such as Brassica chinensis L., Abutilon theophrasti Medic., and Echinochloa crusgalli L. It also inhibits germination and growth in species like Cynodon dactylon (L.) Pers. and Digitaria sanguinalis (L.) Scop [268].

Approximately 201 metabolites have been described in this plant, mainly flavonoids (centaureidin, centaurein, luteolin, and cypotiloin), terpenoids, phenylpropanoids, aromatic compounds, and aliphatic compounds. These molecules contribute to its medicinal properties, including anti-inflammatory, antioxidant, anticancer, antiallergic, antidiabetic, and antimicrobial effects [269,270]. Allelochemicals have been found in the leaves, stems, roots, extracts, and root exudates of B. pilosa [271,272,273]. Some of these include well-known allelochemicals such as p-coumaric acid, caffeic acid, ferulic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid, quercetin, undecane, and palmitic acid, although the allelopathic activity of some is still to be verified [220,274].

In a study using a recirculation system that prevented direct contact between donor and acceptor plants, B. pilosa root exudates were continuously administered to various plant species (lettuce, beans, corn, and sorghum). This resulted in a 30–50% inhibition of germination and growth, with monocots being more sensitive than dicots [221]. In a more recent experiment [220], the native fern Pteris multifida Poir. was exposed to two key root exudates of blackjack, undecane and palmitic acid, for 10 days. Gametophytes treated with undecane exhibited morphological anomalies, chlorosis, and a significant reduction in protein production (about 270 types of proteins) compared to the palmitic acid treatment (35 types). Further analysis revealed that undecane negatively regulated fatty acid biosynthesis, damaging the cell and chloroplast membranes, ultimately leading to cell death. These results encourage further research to identify the allelochemicals responsible for the potential allelopathic effects of B. pilosa root exudates.

5.2.2. Ageratum conyzoides L.

Ageratum conyzoides L., also commonly known as “goat weed”, is an annual plant species belonging to the Asteraceae family, typical of tropical and subtropical regions in West Africa, Asia, and South America. It is not an edible plant, but has been used for medicinal purposes as a purgative, febrifuge, ophthalmia treatment, and for colic, ulcers, and wound dressing [275]. The toxic properties of this plant species are known and are hypothesized to be due to the alkaloid pyrrolizidine found in the plant and its hydroalcoholic extract (LD50 in mice = 600 mg/kg body weight). In addition to pyrrolizidine, other interesting molecules have been found, such as phenols, phenolic esters, coumarins, terpenoids, steroids, flavonoids, and chromenes [276]. Thanks to many of these allelochemicals, A. conyzoides can suppress the growth of fungal genera such as Aspergillus, Alternaria, Candida, Fusarium, Phytophthora, and Pythium [277,278].

Given the richness and variability of compounds produced by goat weed, research has been conducted on the allelopathic potential of this species. To test the activity of A. conyzoides root exudates, soil from a healthy forest and soil from an area colonized by the weed were used, both being placed in pots for microcosm investigation. Lettuce was sown in pots, and root parameters such as germination percentage, potential, speed, and root length were evaluated [219]. These parameters decreased for the lettuce seedlings sown in the invaded soil, showing minimal stimulating activity and suggesting that these root exudates may have both allelopathic and stimulatory activity. Unfortunately, information specifically regarding root exudates is scarce in the literature; however, numerous screening activities and studies exist on using A. conyzoides extracts. Aqueous extracts from different parts of goat weed have been tested on parthenium weed (Parthenium hysterophorus L.) at various concentrations (from 0 to 10%). The extract with the strongest herbicidal action was from the leaves, concentrated at 10%, inhibiting seed germination [279]. Inflorescence and root extracts also showed significant herbicidal effects, with inhibition rates of 28–89% and 31–95%, respectively. Previously, Kato-Noguchi [280] observed the allelopathic power of these extracts and tested them on lettuce, Amaranthus caudatus L., and Digitaria sanguinalis L. Scop., obtaining similar results. In 2020, Lalrindiki et al. [281] tested a leaf extract of A. conyzoides (in methanol), recording the presence of phenols (8.6 ± 0.446 mg/g), flavonoids (27.8 ± 0.685 mg/g), alkaloids (2.06 ± 0.53 mg/g), quinones, terpenoids, and tannins. Subsequently, the extract was tested on Zea mays seeds in Petri dishes, confirming the allelopathic activity of this extract with increasing concentration. Leaf extracts of goat weed obtained using different solvents were tested on Amaranthus spinosus L. to identify the most active extracted fraction and characterize it. Specifically, n-hexane, ethyl acetate, and methanol were used sequentially, while distilled water and 2,4-D (2,4-dichlorophenoxyacetic acid) were used as controls. The ethyl acetate extract was the most active of the three, producing effects comparable to the control with 2,4-D only 21 days after administration (at 20% concentration) on A. spinosus. In order of activity, the second-best extract was the n-hexane extract, and the methanol extract was the last [282]. Considering this, A. conyzoides could be an excellent source of allelochemical compounds, although it is still unclear how this plant exerts its allelopathic potential through root exudates.

6. Conclusions

Root exudates represent a fundamental aspect of plant–soil interactions, encompassing various compounds that significantly influence the rhizosphere’s chemical and biological properties. This review has highlighted the complex nature of root exudates, including their classification, mechanisms of release, and their vital roles in allelopathy. By producing diffusates, secretions, and excretions, plants can modify soil properties, attract beneficial microorganisms, deter pathogens, and inhibit competing vegetation.

The review has also underscored the importance of specific classes of organic compounds in root exudates—such as phenolic compounds, organic acids, terpenoids, alkaloids, and benzoxazinoids—and their allelopathic potential.

Case studies on crops like wheat and rice demonstrate the practical applications of understanding root exudates in enhancing agricultural productivity and managing weed growth. These insights pave the way for more sustainable farming practices, leveraging the natural allelopathic properties of plants to reduce reliance on synthetic herbicides.

Understanding and harnessing the power of root exudates can lead to innovative and sustainable agricultural practices. By appreciating these compounds’ diverse functions and impacts, we can better manage soil health, promote beneficial microbial interactions, and develop natural strategies for weed control. This knowledge contributes to more productive and resilient agricultural systems and supports environmental sustainability by reducing reliance on synthetic herbicides and fertilizers. Embracing the complexity of root exudates and their ecological roles can pave the way for advancements in agroecology and integrated pest management, ultimately fostering a more sustainable and harmonious relationship between agriculture and the environment.