In the soil, AMF produce and secrete a super glue, glomalin, which aids in soil aggregation (Chi et al. 2018). AMF hyphae in well- aggregated soil improve the capacity of soil to retain water (Querejeta et al. 2003) and promote better root-soil connect, facilitating better water conductance from soil to root (Augé 2004). Thus, AMF help in maintaining continued transportation of water from soil to the plant (Bitterlich et al. 2018). In addition, drought stress also promotes higher water absorption rate in AMF hyphae (Zhang et al. 2018a). This is facilitated by the modulation of aquaporin AQP gene expression on cell membranes by AMF (He et al. 2019; Cheng et al. 2020). Till date, six aquaporin proteins have been identified in AMF: three each in Glomus intraradices (GintAQP1, GintAQPF1, GintAQPF2) and Rhizophagus clarus (RcAQP1, RcAQP2, RcAQP3) (Aroca et al. 2009; Li et al. 2013; Kikuchi et al. 2016). Drought stress induced the expression of GintAQPF1 and GintAQPF2, while the expression of GintAQP1 remained unaltered. Similarly, AMF also modulate PIP (plasma membrane intrinsic protein) genes' expression in the host plants under water- deficit condition; it may be upregulated (Zézé et al. 2008) or downregulated (Aroca et al. 2007). These findings suggest that different aquaporins may impart drought tolerance in different plants. Another view is that aquaporin gene expression is upregulated to improve water uptake by plants and downregulated to prevent water loss from plants (Ruiz-Lozano and Aroca 2010; Cheng et al. 2020).

Along with improved water absorption, AMF also ensure optimum nutrient availability to host plants by increasing their acquisition. Wu and Zou (2009) reported increase in the concentrations of Ca, Fe, P, and K in leaves and roots of trifoliate orange plants following G. versiforme application under both drought and well-watered conditions. Farias et al. (2014) found similar observations in blueberry in which AMF colonization increased NPK concentrations in leaves. Better nutrient uptake in mycorrhizal plants is due to their access to nutrients present in the micro pores owing to their leaner hyphae as well as the ability to activate genes and enzymes involved in assimilation and transportation of nutrients. For example, AMF improves the activity of ammonium transporter protein and nitrate reductase for efficient N uptake and assimilation (Garg 2013; Balestrini et al. 2019), phosphatase activity for efficient P absorption, and metabolism (Abd-Allah et al. 2015; Egamberdieva et al. 2017). In tomato, AMF induced the expression of phosphate transporter genes such as LePT4 and LePT5 to elevate tolerance against drought (Volpe et al. 2018). Balestrini et al. (2019) reported AMF-induced upregulation of peptide transporter, amino acid transporter, and sulfate transporter genes in plants under drought stress. Besides, AMF also up regulated the activities of H<sup>+</sup> -ATPase and Ca2+-ATPase under drought conditions. This, in turn, acidifies the soil environment, allowing for efficient uptake of nutrients and exchange of signals between AMF and plants to increase tolerance to drought (Xu et al. 2018a).

#### 7.5.3 Better Photosynthetic Capacity

Photosynthesis, a key primary metabolic process essential for the synthesis of energy in plant, is drastically affected by drought stress (Ilyas et al. 2021). Moderate and severe levels of drought-induced decrease in leaf number and leaf area reduce the concentration of photosynthetic pigments like chlorophylls and carotenoids (Zhang et al. 2018a; Mashabela et al. 2023), which ultimately hampers photosynthesis in plants (Spinoso-Castillo et al. 2023; El-Sawah et al. 2023). Up regulated expression of chlorophyll- degrading enzymes and down regulation of chlorophyll biosynthetic enzymes also contribute to a decline in chlorophyll content (Ilyas et al. 2021; Saxena et al. 2022). However, AMF inoculation can increase chlorophyll content in plants growing under drought conditions. For example, under drought stress (50% field capacity), Rhizophagus intraradices-colonized Ricinus communis had a significant amount of chlorophylls a and b and total chlorophyll than the non-colonized plants (Zhang et al. 2018a). Similar observations were reported in Cicer arietinum, Zea mays, Abelmoschus esculentus, and Fagopyrum esculentum on AMF inoculation (Hashem et al. 2019; Hu et al. 2020; Jabborova et al. 2021; Mohammadi et al. 2022). Recently, Miceli et al. (2023) demonstrated that Claroideoglomus etunicatum inoculation increased the level of photosynthetic pigments and chlorophyll a/ chlorophyll b ratio in Physalis peruviana under soil moisture- deficit condition, subsequently improving photosynthesis to enhance plant biomass and fruit production.

Drought stress is also known to reduce efficiency of photosynthesis by injuring PSII, and disrupting stomatal conductance, intercellular CO2 concentration, and transpiration (Chen et al. 2022). However, the inimical effects of drought on photosynthetic machinery can be prevented by AMF inoculation to the host plants. AMF can improve quantum efficiency of PS II, stomatal conductance, transpiration, and intercellular CO2 concentration (Mathur et al. 2019; Qian et al. 2023). Application of a consortium of AMF consisting of Rhizoglomus intraradices, Funneliformis geosporum, and F. mosseae in Triticum aestivum enhanced quantum efficiency of PSII (Mathur et al. 2019). Such findings were also demonstrated in Zea mays, Oryza sativa, and Ceratonia siliqua (Quiroga et al. 2019; Tisarum et al. 2020; Hu et al. 2020; Chareesri et al. 2020; Boutasknit et al. 2020). AM symbiosis promoted transpiration, intercellular CO2 concentration, and stomatal conductance in Ricinus communis, Olea europaea, Phaseolus vulgaris, and Paris polyphylla (Zhang et al. 2018a; Ouledali et al. 2019; Al-Amri 2021; Qian et al. 2023) under different levels of water- deficit conditions. Thus, AM symbiosis facilitates plants to overcome drought stress by synthesizing more photosynthetic pigments and preventing damage to the photosynthetic machinery.

#### 7.5.4 Maintenance of ROS Level

ROS is a term for derivatives of O2 generated as byproducts of metabolic reactions in chloroplasts, mitochondria and peroxisomes, plasma membrane, and cell wall (Halliwell and Gutteridge 2015; Huang et al. 2019; Mansoor et al. 2022). ROS include singlet oxygen (1 O2), superoxide anions (O2 -), hydroxyl radicals (OH•), and hydrogen peroxide (H2O2) and are involved in maintaining biochemical and cellular processes like cell cycle, programmed cell death, growth and development, hormonal signaling, and biotic and abiotic stress reactions (Conrad et al. 2016; Mansoor et al. 2022). However, ROS production is elevated under drought conditions leading to oxidative stress causing damage to nucleic acid and proteins, ultimately hampering plant's growth and development (Gechev et al. 2006; Raja et al. 2017; Laxa et al. 2019; Khatri and Rathore 2022). Therefore, ROS concentration needs to be maintained at its normal level (McLachlan 2020; Pammi et al. 2023). Antioxidant defense system, which includes molecules and enzymes, is responsible for maintaining ROS at the normal level (Noctor et al. 2018). Antioxidant molecules include alkaloids, flavonoids, ascorbic acid, carotenoids, glutathione, phenolics, and non-protein amino acids, while enzymatic antioxidants include catalase (CAT), ascorbate peroxidase (APXs), glutathione reductase (GRs), glutathione peroxidase (GPXs), and superoxide dismutase (SOD) (Laxa et al. 2019; Mansoor et al. 2022). Antioxidant molecules scavenge ROS through several mechanisms such as electron donation, redox homeostasis, protection of light- harvesting complex, and maintenance of thylakoid membrane and act as chelators and inhibitors of ROS (Hasanuzzaman et al. 2019, 2020; Pammi et al. 2023). Enzymatic antioxidants catalyze dismutation of ROS to prevent hydroxyl formation, conversion of H2O2 into H2O molecules, oxidation for production of phenoxyl radical, etc. (Gill et al. 2015; Pammi et al. 2023).

Many studies have demonstrated higher level of antioxidant molecules as well as higher activity of enzymes in AMF-colonized plants than their non-colonized counterparts under drought stress. Qian et al. (2023) reported higher saponin VI accumulation in Paris polyphylla when inoculated with Rhizophagus irregularis in water- deficit condition. Similarly, greater carotenoid content was observed in mycorrhizal Brassica juncea, Lactuca sativa, and Cupressus arizonica (Srivastava et al. 2022; Ouhaddou et al. 2022; Aalipour et al. 2023). In addition, Funneliformis mosseae colonization improved coumarin, phenolic compounds, and flavonoids in Olea europaea, Poncirus trifoliata, and Mentha piperita (Tekaya et al. 2022; Liu et al. 2022; Ostadi et al. 2023). Similarly, elevated activities of leaf APX, CAT, APX, and GR were reported in Ephedra foliata upon colonization by a mixture of Funneliformis mosseae, Claroideoglomus etunicatum, and Rhizophagus intraradices under different levels of water stress (Al-Arjani et al. 2020). Most recently, Liu et al. (2023a) observed that a consortium of Rhizophagus clarus, R. irregularis, Glomus lamellosum, and Funneliformis mosseae significantly upped the activities of CAT, APX, SOD, and POD in Arachis hypogaea leaf under different abiotic stresses like drought, salinity, and cold. Higher enzymatic activities of these antioxidants can be partially attributed to enhanced nutritional status in AMF-colonized plants as these enzymes are metalloenzymes, and their activities are dependent on the availability of micronutrients like Fe, Cu, and Mn. Besides, other factors like species of AMF and plant and level and duration of stress also determine their activities (Evelin et al. 2019).

#### 7.5.5 Better Osmotic Adjustment

Organic osmolytes are low- molecular- weight, water- soluble organic compounds that normally accumulate during abiotic stresses. Commonly accumulated organic osmolytes include proline, glycine betaines, polyamines, and sugars (Evelin et al. 2019; Shankar and Evelin 2021). Accumulation of these organic solutes predominantly takes place in the cytoplasm; however, the synthesis of specific osmolytes depends on plant tissue, species of plant, and type of stress involved (Yang and Guo 2018). Under drought stress, concentration of these solutes increases to maintain an optimum water potential gradient and facilitate the flow of water from soil to plant roots, consequently preventing cellular dehydration (Evelin et al. 2019; Saxena et al. 2022). Thus, these solutes participate in osmotic adjustment and can be used to efficiently overcome the deleterious influence of osmotic stress (Tang et al. 2022). Besides, these osmolytes exhibit functional diversity to ameliorate stress effects in plants as they maintain membrane integrity and stabilize structure of protein, enhance ROS defense system to prevent oxidative damage, provide carbon and energy to recover from stress, protect photosynthetic apparatus, prevent K efflux from cytoplasm, and function as signaling molecules (Lokhande and Suprasanna 2012; Shankar and Evelin 2021).

Organic osmolyte accumulation in plants under stress conditions is further enhanced upon inoculation with AMF (Evelin et al. 2019; Tang et al. 2022; Liang et al. 2022). It was observed that proline content was significantly increased in Eucalyptus camaldulensis, Valeriana officinalis, Citrus aurantium, and Saccharum spp. upon inoculation with Gigaspora albida, Funneliformis mosseae, and a mixture of Glomus hoi, G. mosseae, and G. intraradices, respectively (Klinsukon et al. 2021; Amanifar and Toghranegar 2020; Hadian-Deljou et al. 2020; Spinoso-Castillo et al. 2023). The enhanced proline content in plants following AMF inoculation can be due to enhanced activity of enzyme P5CS (Δ′-pyrroline-5-carboxylate synthase), higher expression of proline biosynthetic gene encoding P5CS, enhanced activity of enzyme, glutamate dehydrogenase, and inactivation of enzyme and proline dehydrogenase (Abo-Doma et al. 2011). Besides osmoregulation, proline also participates in other cellular activities like ROS scavenging and stabilization of membrane, proteins, and DNA. It also acts as a reservoir for excessive NAD+ and NADP+ for photosynthesis and respiration and sources of carbon, energy, and nitrogen to recover from stress (Kaur and Asthir 2015; Evelin et al. 2019; Shankar and Evelin 2021). In contrast, there are a few studies that report lesser proline content in the AMF-colonized plants as compared to the non-AMF plants under abiotic stresses. For example, Glycyrrhiza uralensis, Myrtus communis, and Physalis peruviana when inoculated with Rhizophagus irregularis, Funneliformis mosseae, and Claroideoglomus claroideum, respectively, had lesser leaf proline content than control plants (Xie et al. 2018; Azizi et al. 2021; Geneva et al. 2023). Proline is a stress marker and lesser proline accumulation in the AMF-colonized plants may indicate less stress in them (Evelin et al. 2019). Moreover, decreased activity of P5CS could be attributed to lower proline content in Poncirus trifoliata inoculated with Paraglomus occultum (Zhang et al. 2018b).

Glycine betaine is an amphoteric molecule that acts as an osmoregulator and/or osmoprotectant in plants under drought stress (Saxena et al. 2022). AMF-colonized plants showed more glycine betaine concentration than control plants, resulting in higher osmotic potential than control plants under drought stress (Abd-Elghany et al. 2021). Increase in accumulation of glycine betaine could be attributed to enhanced expression of choline monooxygenase and betaine aldehyde dehydrogenase (Zhang et al. 2009). Higher concentration of glycine betaine is correlated to better cell membrane and photosynthetic apparatus protection, scavenging of ROS, and stabilization of protein and enzyme complexes (Sakamoto and Murata 2000; He et al. 2010; Niu et al. 2021).

Free amino acids provide tolerance to plants against drought stress. AMF colonization can further enhance the level of free amino acids in the plant. AM symbiosis in Helianthus tuberosus and Sesamum indicum increased the level of free amino acids (Gholinezhad and Darvishzadeh 2021; Nacoon et al. 2022). Zea mays had higher accumulation of γ-aminobutyric acid, free amino acids, arginine, and ornithine when inoculated with Rhizophagus irregularis as compared to the non-AMF plants growing in water stress conditions (Hu et al. 2020). Similarly, Hu and Chen (2020) observed that Zea mays also accumulated higher concentration of glutamine and γ-aminobutyric acid and increased activity of glutamine synthetase when inoculated with a mixed consortium of Claroideoglomus etunicatum and Rhizophagus irregularis as compared to the control plants.

Sugars are major osmoprotectants that contribute to nearly 50% of a plant's osmotic potential by providing carbon to recover from stress and aiding in osmotic adjustment (Santander et al. 2017). Under stress conditions, total soluble sugar increased in Malus hupehensis, Cinnamomum migao, and Fagopyrum esculentum when inoculated with Rhizophagus irregularis, Glomus lamellosum, and G. etunicatum and a consortium of Funneliformis mosseae, Rhizophagus irregularis, and R. fasciculatus, respectively, as compared to the non-AMF plants (Huang et al. 2020; Liao et al. 2021; Mohammadi et al. 2022). Tekaya et al. (2022) reported higher accumulation of mannitol in Olea europaea when colonized by Rhizophagus irregularis as compared to the non-mycorrhizal plants. Under osmotic stress, AM symbiosis also improved glucose content in Oryza sativa and Ephedra foliata (Tisarum et al. 2020; Al-Arjani et al. 2020). The increase in these sugars can be attributed to increased photosynthesis and carbohydrate metabolism in the AMF-colonized plants under abiotic stress conditions (Santander et al. 2017; Tarnabi et al. 2020).

Polyamines are low- molecular-weight polycationic nitrogen-containing compounds of aliphatic nature. They are found in all living organisms. During stress, it takes part in several physiological responses like osmoregulation, stabilization of membranes, ROS scavenging, cellular pH modulation, and photosynthesis (Pang et al. 2007; Chen et al. 2019; Liang et al. 2022). AMF colonization modulated polyamine levels in host plants under drought stress. Putrescine content was enhanced by Rhizophagus irregularis in Zea mays under drought stress (Hu et al. 2020). Interestingly, Hu and Chen et al. (2020) observed a decrease in putrescine content in Zea mays when inoculated with Rhizophagus irregularis.

Drought stress also induces dynamism in accumulation of other osmolytes, organic acids. Organic acids prevent the buildup of toxic chloride ions, while providing cellular ion homeostasis, pH regulation, and osmotic adjustment (Guo et al. 2010). AM symbiosis can modulate the accumulation of organic acid in plants. Zea mays inoculated with Rhizophagus irregularis improved concentration of malic acid and total organic acids under water- deficit conditions (Hu et al. 2020; Hu and Chen 2020). Besides malic acid, Hu and Chen et al. (2020) observed increased activity of GABA transaminase and malic enzymes in AMF-colonized maize plants. AMF could provide protection to enzymes of organic acid biosynthesis as well as initiate closure of stomata (Sheng et al. 2011; Hu and Chen 2020; Hu et al. 2020). Increase in organic acids could positively regulate the Calvin cycle to promote synthesis of sugar for plant's growth and development and confer stress tolerance (Guo et al. 2010). The exact mechanisms behind the alteration in organic acids in the AMF-colonized plants are yet to be established and require further intensive research.

#### 7.5.6 Maintaining Higher Unsaturated Fatty Acid: Saturated Fatty Acid Ratio

Drought tolerance in plants is also associated with a greater unsaturated fatty acid/ saturated fatty acid ratio (Mahnaz et al. 2020). AM symbiosis has been found to regulate metabolism of fatty acids in conferring drought tolerance to the host plants. In inoculation with AMF, F. mosseae induced a higher UFA (C18:1, C18:2, and C18:3N3)/SFA (C18:0) ratio in roots of trifoliate orange plants as compared to the non-mycorrhizal plants (Wu et al. 2019; Hu et al. 2020). In sesame plants, inoculation with F. mosseae and Rhizophagus intraradices imparted drought tolerance by maintaining higher UFA/SFA ratio and better antioxidant defense (Gholinezhad and Darvishzadeh 2021). Higher UFA in mycorrhizal plants may be attributed to the induction of FA desaturase 2 and FA desaturase 6 genes involved in desaturation of fatty acids (Wu et al. 2019). AMF-induced maintenance of higher UFA levels prevents the host plants from drought-induced oxidative damage by maintaining membrane fluidity. Alternatively, AMF also induce sporulation and their growth indirectly by increasing the level of C14:0 levels in the host plants (Sugiura et al. 2020; Meng et al. 2021a).

#### 7.5.7 Modulation of Phytohormones

Phytohormones, also known as plant growth regulators, play a crucial role in growth and development. Drought stress alters endogenous phytohormone levels in plants. However, AMF colonization has been shown to modulate levels of phytohormones in imparting tolerance against drought stress in the host plants (Table 7.1). Abscisic acid (ABA) is an important phytohormone that acts as a key signal molecule in roots for AM colonization. AM symbiosis upregulated biosynthesis of ABA resulting in higher ABA content in Solanum lypersicum which further induced closing of stomata, thereby preventing transpiration and water loss (Chitarra et al. 2016). Higher level of ABA is essential for maintaining structural fluidity and efficient regulation of transport ions in plants (Yang et al. 2014). Under drought stress conditions, a mixed consortium of Funneliformis mosseae, Claroideoglomus etunicatum, and Rhizophagus intraradices increased the content of ABA in Ephedra foliata (Al-Arjani et al. 2020). More recently, Qian et al. (2023) demonstrated that colonization with Rhizophagus irregularis increased ABA content in Paris polyphylla var. yunnanensis under drought stress. Increase in ABA can be attributed to key genes such as 14-3-3 genes involved in ABA signaling. AM symbiosis induced the expressions of PcGRF10 and PcGRF11 in Populus cathayana as their expressions were correlated with improved osmoregulation and antioxidant defense system (Han et al. 2022). However, it has also been shown that ABA content in the AMF-colonized plants could reduce under stress conditions, indicating that ABA regulation in the AMF-colonized host plants could vary (Cheng et al. 2021). Reduction of ABA content in Cichorium intybus was observed under drought stress upon Rhizophagus irregularis inoculation (Langeroodi et al. 2020). Chen et al. (2020) also reported reduced ABA content in Catalpa bungei upon Rhizophagus intraradices inoculation under drought stress. Similarly, ABA content was lesser in R. irregularis- colonized Olea europaea than in control plants (Tekaya et al. 2022). The decrease in ABA content in the mycorrhizal plants can be due to the role of ABA in developing fungal mycelium in host plants (Kandowangko et al. 2009; Goicoechea et al. 2010).

AM symbiosis imparts a positive influence on salicylic acid (SA) in improving carbohydrate metabolism and ion homeostasis and formation of fungal structures like arbuscules and vesicles, resulting in increased AM colonization (Liu et al. 2018). Moreover, salicylic acid also regulates root aquaporins to modulate water conductivity and control root water transport (Quiroga et al. 2018). Higher levels of SA were observed in mycorrhizal Olea europaea than in control plants under drought stress (Tekaya et al. 2022).

Phytohormone jasmonic acid (JA) also takes part in improving plant's tolerance against abiotic and biotic stresses. Levels of jasmonic acid increased in stress conditions (Gul et al. 2022). Liu et al. (2016) and Zhang et al. (2019) reported that AM symbiosis enhanced the level of methyl jasmonate in trifoliate orange grown under drought conditions. As JA promotes accumulation of osmolytes and enhance

the antioxidant defense system (Qiu et al. 2014a, b), AM-induced enhancement of JA protects host plants from drought stress.

Strigolactones are a class of plant hormone that participate in the detection of fungal structures and establishment of AM symbiosis in the host plants (Chauhan et al. 2022). They are also involved in regulatory roles against abiotic stress. Under osmotic stress, production of strigolactones was down regulated; however, with the application of AM fungi, levels of strigolactones were improved along with ABA, consequently promoting AM symbiosis and related benefits to counter deleterious effects of osmotic stress (López-Ráez 2016). Higher levels of strigolactones in Sesbania cannabina could be as a result of high ABA in the mycorrhizal plants, as ABA induces production of strigolactones that improve the ability of the plant to tolerate abiotic stress (Ren et al. 2018).

There are also other phytohormones whose levels were modulated upon AM symbiosis in abiotic stress conditions. Indole-3-acetic acid (IAA), an auxin, dramatically improved in Ephedra foliata and Camellia sinensis in the presence of mixture AMF (Funneliformis mosseae, Claroideoglomus etunicatum, and Rhizophagus intraradices) and C. etunicatum, respectively, under osmotic stress (Al-Arjani et al. 2020; Liu et al. 2023b). Induction of auxin synthesis is key to enhanced growth of root hairs that will improve plant nutrition and help plant to resist the negative effects of osmotic stress (Liu et al. 2018). Chen et al. (2020) reported an increase in IAA, along with gibberellins, IAA/ABA, and GA3/ABA in Catalpa bungei in the presence of Rhizophagus intraradices under drought stress and a decrease in ABA and zeatin. The role of hormones in improving tolerance against osmotic stress under AM symbiosis is not fully understood; therefore, more research should be directed towards studies involving phytohormones and AM fungi under stress conditions.

#### 7.5.8 Influence on LEA Proteins, Dehydrins, and ATPase

LEA proteins are rich in glycine, alanine, and serine and consist of a diverse family of minute hydrophilic polypeptides (10–30 kDa) (Karpinska et al. 2022). They are housed in subcellular compartments like cytosol, mitochondria, and chloroplast and are regulated by phytohormones like ABA (Huang et al. 2016; Artur et al. 2019). Some LEA proteins function as molecular chaperones under abiotic stress and help in the restoration of proteins into their natural conformations, thus enhancing stress tolerance (Kovacs et al. 2008). Expression of genes LEA proteins could contribute to drought tolerance. Gh\_A08G0694, a LEA3 family gene when overexpressed in Gossypium hirsutum, in collaboration with other key stress- tolerant genes such as glyceraldehyde-3-phosphate dehydrogenase and a voltage-dependent anion channel 1 and other regulatory networks could promote drought tolerance (Shiraku et al. 2022). Wang et al. (2023b) reported that ZmNHL1 protein encoded by a LEA-2 protein family could improve activities of POD and SOD and lower electrolyte leakage and promote drought tolerance in Zea mays.

Dehydrins or group II LEA are the LEA proteins which are most widely described. They are low molecular weight, thermostable, and hydrophilic and generally assemble in the later phase of embryogenesis in response to abiotic stress and widely scattered in bryophytes, angiosperms, and gymnosperms (Yu et al. 2018). Response of dehydrins varies with levels of drought stress. For instance, expressions of ShDHN and SiDhn2 reached the maximum level (12-fold change) when subjected to 6 and 3 h of drought conditions, respectively (Qiu et al. 2014a, b; Liu et al. 2015). Under drought stress, MtCAS31 responds positively to drought by promoting autophagy of MtPIP2;7 proteins and reducing hydraulic conductivity of roots, consequently minimizing water loss (Li et al. 2020). Overexpression of CaDHN3 , a dehydrin gene of Capsicum annum enhanced tolerance against waterdeficit condition in Arabidopsis thaliana by promoting phenotypic characteristics and enzymatic antioxidant defense system (Meng et al. 2021b).

The contribution of AM symbiosis in the light of LEA proteins and dehydrins under drought is limited. However, Porcel et al. (2005) observed that AMF can alter the accumulation dehydrin pattern in Glycine max and Lactuca sativa under drought stress. Expression of LEA genes such as Islea 1, gmlea 8, and gmlea 10 was lesser in the mycorrhizal plants than in the non-AMF plants under drought conditions, thus indicating that AM symbiosis does not involve LEA proteins as a drought tolerance mechanism. In contrast, AMF contributed to drought tolerance by increased accumulation of dehydrins (Ruiz-Lozano et al. 2008).

H+ -ATPase is a membrane protein that generates the H+ electrochemical gradient which regulates solute and nutrient transport through the membrane (Garry et al. 2007). The activity of plasma membrane H+ -ATPase under drought is inhibited causing net H+ influx which results in depolarization of membrane potential. Therefore, higher activity of H+ -ATPase is crucial to prevent cellular H+ influx and K<sup>+</sup> influx in order to maintain K+ homoeostasis (Zhang et al. 2018c). Dong et al. (2013) observed that over expression of MdVHA-A, a H<sup>+</sup> -ATPase subunit A, improved the activity of vacuole H+ -ATPase to confer drought tolerance in Malus domestica, thus making it a potential gene candidate for drought tolerance. It was also shown that the activity of H<sup>+</sup> -ATPase in plasma membranes could be promoted by drought- induced augmentation of polyamines like spermine and spermidine in Triticum aestivum (Du et al. 2015). Transcriptional study of four H+ -ATPase genes in Solanum lypersicum namely, SlHA1, SlHA4, SlHA2, and SlHA8, was investigated and it was reported that in the presence of Rhizophagus irregularis, SlHA2 and SlHA8 in leaf and root were upregulated, while SlHA1 and SlHA4 remained unaffected; thus, AM regulation of HA genes depends on HA homologous genes and the host plants (Liu et al. 2016). Further, expression of SlHA8 was linked to arbuscule development and nitrogen uptake (Liu et al. 2020). Under drought stress, Funneliformis mosseae distinctly improved the activity of H+ -ATPase in roots and leaf by 22.61% and 26.06%, respectively, than in control Poncirus trifoliata plants. Transcriptome study also revealed that expression of PtAHA2 in both leaf and roots was significantly improved by 9.50- fold and 20.92-fold, respectively, in the mycorrhizal plants (Cheng et al. 2021).

Drought stress triggers the level of calcium (Ca2+ ), an important intracellular second messenger, resulting in the aggregation of nucleic acids and proteins, membrane lipid disintegration, and phosphatase precipitation, consequently leading to death of cells (Knight and Knight 2001; Case et al. 2007). The enhanced accumulation of cytosolic Ca2+ ions is effluxed out to restore Ca2+ homeostasis by antiporters and a high- affinity, low- capacity transporter known as Ca2+ ATPases (Bose et al. 2011). Besides, Ca2+ ATPases also play a remarkable role in stomatal regulation, pollen development, floral inflorescence architecture, and gibberellin signaling (Huda et al. 2013). However, there is a lack of studies that provide direct data on the role of Ca2+ ATPases in stress tolerance. Drought stress triggered overexpression of OsACA6, a Ca2+ ATPases gene in transgenic Oryza sativa, and this modulated scavenging machinery of ROS and synthesis of proline. In addition, overexpressing transgenic lines showed higher content of chlorophyll, photosynthesis, stability of membrane, and reduced loss of water, thus ensuring drought tolerance (Huda et al. 2013). Under drought condition, the activity of Ca2+ ATPases along with H+ -ATPase was induced in extraradical hyphae of R. irregularis in symbiosis with Daucus carota. This caused acidification of rhizosphere soil which promoted plant nutrition and exchange of signals between the plant and the AMF (Xu et al. 2018a). More recently Sheteiwy et al. (2021) also noted improved activitie s of Ca2+ ATPases along with H+ -ATPase in soybean plant in the presence of AMF (Acaulospora laevis, Septoglomus deserticola, and R. irregularis) to confer drought tolerance. However, the studies concerning induction of ATPase activities, especially Ca2+ ATPases under drought conditions in the presence of AM fungi, are limited. Therefore, extensive research should be directed towards understanding the regulation of ATPase in different plants by different AMF at different levels of drought stress.

#### 7.6 Conclusions

The benefits of AMF in ameliorating drought stress are well documented. AMF symbiosis have proved to be a key tool in bestowing drought tolerance and consequently mitigate drought- induced damages in plants. The ameliorative mechanisms under drought stress include AMF-induced adaptive changes in structure, biochemistry, and physiology of the host plants. However, the current study finds that the following research areas need further investigation to gain full understanding of AMF-induced drought tolerance in host plants:

are insufficient to decipher the mechanisms of drought tolerance at molecular level.

Acknowledgment AT is thankful to the Ministry of Tribal Affairs for National Fellowship for ST students.

#### References

oldest plant symbiosis. Proceedings of the National Academy of Sciences 110(50): 20117–20122. https://doi.org/10.1073/pnas.1313452110

## Chapter 8 Arbuscular Mycorrhizal Fungi in Plant Tolerance to Organic Pollutants and Associated Food Safety

Hamideh Bakhshayeshan-Agdam, Mahdieh Houshani, and Seyed Yahya Salehi-Lisar

Abstract Industrialization and the modern world have led to environmental contamination by organic pollutants, many of which are persistent and could engender hazardous disorders in ecosystems and living organisms including humans. Organic pollutants are phytotoxic compounds and affect the plant biochemical and physiological processes. However, plants can uptake these compounds leading to inclusion of pollutants into food chains. Nowadays, several studies have been carried out to explore and develop novel strategies that could lessen or eradicate pollution while posing no deleterious effects on the environment. Bioremediation (including phytoremediation) is green technology for the remediation of contaminated soils and water. The majority of plant species form symbiosis with mycorrhizal fungi which usually leads to improved plant resistance to organic contaminations. Therefore, plant root inoculation with mycorrhizal fungi could be a biological solution for the problem of organic pollutants.

Keywords Organic pollutants · Arbuscular mycorrhizal fungi (AMF) · Bioremediation · Crops

#### 8.1 Introduction

Large amounts of mineral and organic pollutants are released into the environment because of human activities such as agriculture and mining, which causes soil pollution and ultimately the accumulation of these pollutants in agricultural products (Wang et al. 2019). For example, pesticides as contaminations are widely used to secure crops from pathogens and weeds. Besides, other organic contaminations, such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), phthalates (PAEs), and petroleum hydrocarbons, generally have long

H. Bakhshayeshan-Agdam · M. Houshani · S. Y. Salehi-Lisar (✉)

Department of Plant, Cell and Molecular Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, East Azerbaijan, Iran

e-mail<EMAIL_ADDRESS>
<sup>©</sup> The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 G. J. Ahammed, R. Hajiboland (eds.), Arbuscular Mycorrhizal Fungi and Higher Plants, https://doi.org/10.1007/978-981-99-8220-2\_8

persistence in agricultural soils. A lot of these organic pollutants are mutagens, carcinogens, and teratogens (Wang et al. 2019; Casida 2017). Biological mechanisms are commonly used for the elimination of these toxic compounds from the environment (Fecih and Baoune 2019). Symbiotic interactions, mycorrhizal associations, in particular, are valuable tools for the bioremediation of polluted ecosystems (Whitacre 2015). Mycorrhizoremediation, the use of mycorrhizae in bioremediation,

is one of the biological ways that could alleviate the matter of persistent organic pollutants (POPs) that results in removing the pollutants from soils and increases the host plant growth and development in contaminated soils (Fecih and Baoune 2019). In the current chapter, the arbuscular mycorrhizal fungi (AMF)-induced plant response to organic pollutants, the role of AMF in plant-based remediation of organic pollutants, the positive effect of AMF inoculation on host plants grown in contaminated environments, as well as their application for ensuring food safety are discussed.

#### 8.2 Mycorrhiza: Definition and Types

Mycorrhiza is a unique symbiotic relationship between beneficial fungi and host plant roots. This symbiosis can be divided into two types: ectomycorrhiza and endomycorrhiza.

#### 8.2.1 Ectomycorrhiza

Ectomycorrhizae are formed by basidiomycetes and ascomycetes with the roots of plants mainly from cold temperate regions, such as beech, birch, pine, willow, oak, spruce, and fir (Smith and Read 2008; Ganugi et al. 2019). Ectomycorrhizal symbiosis is featured in intercellular fungi hyphae net called as the "Hartig Net." The Hartig Net includes vastly branched hyphae attaching the root epidermal and cortical cells. The fungus creates a covering of hyphae around the root and completely covers the tip of the root, but never enters the cells (Ganugi et al. 2019; Dighton and Allen 1991).

#### 8.2.2 Endomycorrhiza

Endomycorrhizae are found in over 80% of vascular plants. Endomycorrhiza is distinct from ectomycorrhiza in formation and function. The fungus hyphae grow inside the plant roots and even transpire to the root cell walls. They have more invasive relationship than ectomycorrhiza (Weber 2005; Ganugi et al. 2019). Endomycorrhizae are divided into five types including arbuscular mycorrhizae, ericaceous mycorrhizae, arbutoid mycorrhizae, monotropoid, and orchidaceous mycorrhizae (Ganugi et al. 2019). Arbuscular mycorrhizae are the most widespread and important relationship in the plant kingdom.

#### <IP_ADDRESS> Arbuscular Mycorrhizae Fungi (AMFs)

Arbuscular mycorrhiza symbiosis is the largest association between over 100,000 plant species and a few 100 AMF morphotypes (Chen et al. 2018). AMFs are obligate biotrophs and indeed need plant photosynthetic products which are necessary for their metabolism (Begum et al. 2019).

The penetration of the fungus into the root can be done in various ways including (1) appressorium generation from which intracellular hyphae originate, (2) penetration through a radical hair, and (3) entering across the cells of external layers of roots that are often dead (Ganugi et al. 2019).

#### 8.3 Benefits of AMFs for Plants

Based on the obtained results from fossil and molecular data, the symbiosis of mycorrhizal fungi and plants dates back to the time of the first plant appearance on land, which is about 400–450 million years ago (Smith and Read 2008; Selosse et al. 2015). The symbiosis between mycorrhizal fungi and plants has many advantages for plants:

#### 8.3.1 Effects on Plant Growth and Nutrition

Among the symbiotic microorganisms, AMFs are widely distributed and establish symbiotic relationships with most crops (Posta and Hong Duc 2020). In many studied plant species, AMFs improve growth (Bona et al. 2017; Gamalero et al. 2004; Gogoi and Singh 2011; Nakmee et al. 2016; Parihar and Bora 2018) and uptake of mineral elements like nitrogen and phosphorus (Jansa et al. 2019; Song et al. 2020). AMFs enhance the mineral absorption in the plants as a result of an escalation in the absorption network of nutrients around the roots, which makes the roots available to a large amount of soil (Smith and Smith 2011; Begum et al. 2019). Besides, the fungal hyphae are very tiny compared with the roots of plants, so they can enter the tiny pores and subsequently absorb more nutrients. The direct transfer of nutrients to the inner layers of the root tissue has also been proposed as a mechanism to increase the uptake of elements. Molecular data have shown that during the coexistence of plants with AMFs, the expression of transporter genes such as aquaporins, phosphate, ammonium, nitrate, sulfur, and zinc transporters increases (Diagne et al. 2020). Experimental trials have shown that inoculation with AMFs leads to increased plant leaf area and higher contents of essential elements such as phosphorus, nitrogen, potassium, and calcium and finally boosts plant growth (Begum et al. 2019). In fact, AMFs support plants nutritionally, especially in stressful situations.

#### 8.3.2 Alleviation of Stress in Plants

Many studies were carried out to examine the mitigating effects of AM fungi on plants under different stresses. The results revealed that these fungi could improve the tolerance of inoculated host plants to a wide range of stress including biotic and abiotic stress (Diagne et al. 2020).

Drought is a devastating abiotic stress affecting plant growth and development (Salehi-Lisar and Bakhshayeshan-Agdam 2016; Ganugi et al. 2019). Inoculation of plants with AMFs makes them resistant to drought and increases the stability of plants in stressful conditions (Diagne et al. 2020). The increase of water absorption by AMFs is done mainly through the development of more absorption surface by fungal hyphae (Augé 2001), as well as increasing the access to fine soil pores and improving the apoplastic flow of water (Diagne et al. 2020).

Heat and cold stress are also considered as important stress factors affecting plants worldwide (Begum et al. 2019; Hasanuzzaman et al. 2013). Plant inoculation with AMFs increases the resistance of plants to temperature stress by improving the water and mineral element absorption, improving the efficiency and capacity of photosynthesis, alleviation of oxidative stress, and inducing osmotic adaptation (Caradonia et al. 2019; Zhu et al. 2017).

AMF could be a remedy for chemical pesticides in agriculture, leading to higher sustainability and eliminating public health hazards. Indeed, it is well-known that AMF could lessen the soil-borne pathogens that caused damage in plants (Ganugi et al. 2019).

The soil structure is adversely affected by intensive agricultural practices. The hyphae of AMFs are one of the chief binding factors involved in stabilizing soil aggregates (Miller and Jastrow 2000).

#### 8.4 Environmental Pollutants: Types and Problems

The pollution of the environment because of the spreading of industrial and urban wastes produced through anthropogenic acts is one of the significant environmental worries in the world (Jacob and Cherian 2013). Environmental contaminants polluting the water and land resources result in the reduction of food quality and health risks in living organisms (Dean et al. 2001; Houshani et al. 2021). The common pollutants exist abundantly in the environments such as PAHs, pesticides, PCBs, and explosives as organic compounds and inorganic compounds such as metals, metalloids, and radionuclides (Testiati et al. 2013; Vane et al. 2014). Their long-lived toxicity, persistence in the environment, and their large amount require a lot of attention (Aina et al. 2006; Parrish et al. 2005). Organic pollutants can disrupt different plant segments and activities like membranes, metabolism, water and nutrient uptake, and other disorders. In the same manner, these organic contaminations have the ability to cause toxicity and have xenobiotic and carcinogenic properties (Zeb et al. 2022). Human and wildlife exhibition to such steady and toxic compounds leads to their accumulation and movement into plants and also natural and agricultural food chains (Yan et al. 2004).

#### 8.4.1 Persistent Organic Pollutants (POPs)

Organic pollutants are toxic and can harmfully affect living organisms. There are different sources for releasing these pollutants including accidental release, industrial functions (such as chemical and petrochemical plants), agriculture (such as pesticides), and military activities (such as explosives, chemical weapons) (Ensley and Raskin 1999; Ashraf 2017). Further, organic contaminations include various types of compounds like solvents (such as trichloroethylene), explosives (such as trinitrotoluene (TNT) and cyclotrimethylenetrinitramine), polycyclic aromatic hydrocarbons (PAHs), petroleum products (such as benzene, toluene, ethylbenzene, and xylene: BTEX), polychlorinated biphenyles (PCBs), and pesticides (such as atrazine, chlorpyrifos, 2, 4-D) (Singh and Jain 2003; Zeb et al. 2022). As a consequence of standing against biological, chemical, and photolytic transformation processes, they could accumulate in the environment and could be moved by wind and water and thus affect living organisms far from where they are released (Cheng et al. 2017).

#### 8.5 Organic Pollutant Effects on Plants

Many organic pollutants are plant toxic compounds and influence numerous biochemical and physiological activities in plants. These organic pollutants can make acute and chronic damages to plants based on the concentrations and duration of exposure (Kmentova 2003; Cheng et al. 2017). The long-lived effect of toxic organic pollutants can impact the sustainability and performance of ecosystems, reduce biomass production and oxygen release through photosynthesis, and finally lead to the destruction of the environment (Vanova 2009). In addition, the severity of the effects of organic pollutants on plants depends on the ontogenetic phase of growth, the physiological condition of the organisms, the morphological and size features of the leaf surface, cuticle enterability, transpiration rate and leaf length, and the enzymes involved in organic pollutants metabolism such as peroxidases, hydrolases, and cytochrome P450 causing the reactivity of plants to these pollutants (Greipsson 2011). These pollutants cause morphological, cytological, genetic, and metabolic disorganization; for example, through the induction of oxidative stress, they inhibit and reduce seed germination and disrupt the functioning of the photosynthetic apparatus (Reynoso-Cuevas et al. 2008; Tomar and Jajoo 2014).

#### 8.5.1 Seed Germinations

Seed germination and root system formation are vital stages of plant growth influenced by various factors as external factors. Accordingly, as one of the outside factors affecting plants, pollutants can influence all phases of plant life cycle from germination to reproduction (Maila and Cloete 2002; Reynoso-Cuevas et al. 2008; Houshani et al. 2021). Investigators have demonstrated that the reactivity of maize, barley, millet, and lettuce seed germination to PAHs as an organic pollutant is more dependent on the length of the root (Kmentova 2003; Kummerova et al. 2012, Houshani et al. 2019). Salehi-Lisar and Deljoo (2015) reported that organic pollutants had a damaging effect on the germination speed of alfalfa, sunflower, and wheat and caused a decrease in their growth.

#### 8.5.2 Metabolism and Photosynthesis

Recent research has shown that organic pollutants have a profound effect on plant metabolism and mainly have a negative and destructive effect on photosynthesis and synthesis of lipids, amino acids, proteins, nucleic acids, and hormones. For instance, PCBs and herbicides as an organic pollutants have been found to be detrimental to cell metabolism (Brain et al. 2010; Cheng et al. 2017). In parallel, it was found that these pollutants can inhibit photosynthesis in plants through the prevention of photosynthetic pigment biosynthesis, making green leaves and stems white and translucent (Liu et al. 2010; Kaspary et al. 2014; Cheng et al. 2017). Organic contamination can restrain the photosynthesis process. In addition, these pollutants can perforate chloroplasts and affect the initial ways of photosynthesis (Zobiole et al. 2009; Amaia et al. 2013). Correspondingly, the stability and activity of the photosynthetic structure can be affected by organic pollutant contamination and also they can interrupt the electron transport chain by producing quinone compounds and binding to plastoquinones. In addition, these contaminants can inhibit photosystem II (PSII) on both sides (Kummerova et al. 2006; Houshani & Salehi-Lisar 2020).

#### 8.5.3 Oxidative Stress

Plants have enzymatic and non-enzymatic defense techniques to mitigate oxidative stress induced by organic pollutants (Yoshiyama et al. 2013). The oxidative stress induced by these pollutants and their connection with morphological disorders has been previously stated in different plant species such as Arabidopsis thaliana (Alkio et al. 2005). Tarigholizadeh et al. (2021) reported that PAHs could cause oxidative stress in Panicum miliaceum. Treated plants have shown a remarkable increase in antioxidant enzyme activity along with a substantial increase in non-enzymatic antioxidant levels potentially in order to detoxify reactive oxygen species.

#### 8.6 Plant Resistance to POPs: General Mechanisms

Plants have the potential to uptake contaminations from the environment by the root system or aerial parts. Also, they can remove or reduce pollutant concentration in the environment by different strategies like rhizoremediation, phytoextraction, phytostabilization, phytovolatilization, phytodegradation, and rhizodegradation. These strategies form the basis of phytoremediation technology (Etim 2012). Accordingly, phytoremediation is named rhizodegradation when degradation occurs in the rhizosphere, and it is also named phytostimulation when it includes microbes. In some samples, the pollutants are deactivated in the root zone called phytostabilization. The blended processes of the rhizosphere within the root promote phytoremediation called rhizoremediation. Phytoextraction refers to assembling contaminations in collective plant tissues—uniquely shoot tissues. This approach is notably utilized for phytoremediation of inorganic pollutants. In some cases, plants can break down contaminations inside their tissues; this process is named phytodegradation and is mainly appropriate for organic contaminations. Some pollutants can also be volatilized by plants and this process is called phytovolatilization (Etim 2012; Kafle et al. 2022). The organic pollutants are chiefly degraded as plant responses to organic pollutants by two mechanisms: phytodegradation or phytotransformation and rhizoremediation (Arthur et al. 2005).

#### 8.6.1 Transformation of POPs

The transformation of organic contaminations in plants is done by enzymes (Vanova 2009). It is a characteristic of a process that is species-specified and may be specific to that plant and occur only in a particular tissue or organ or even at a particular growth step (Kmentova 2003; Vanova 2009). The transformation of pollutants in plants is divided into three stages, which include conversion, conjugation, and elimination reactions (Fig. 8.1).

In the primary stage, oxidation, reduction, and hydrolysis reactions are created by enzymes such as cytochrome P450 or carboxylesterases. The reduction reactions are of little importance in transformation, but oxidation is the most involved reaction in detoxification of the organic pollutants. The important reaction in the stage of oxidation is monooxygenation (hydroxylation) where the addition of oxygen to

Fig. 8.1 Various stages of metabolism of organic pollutants in plant cell

toxicant molecule occurs (Korte et al. 2000). It is mentioned that the top-said stage is placed within the endoplasmatic reticulum or mitochondria and these reactions may adjust combinations and able to conjugate with amines, alcohols, and acids in the next stage of detoxification (Korte et al. 2000; Syed et al. 2010).

In the subsequent stage (conjugation), the conjugated forms of pollutants are created by adding compounds like sugars, amino acids, organic acids, and peptides. Reactions are carried out by enzymes such as transferases. For example, glucosyltransferase and glutathione transferase enzymes add sugars and glutathione to pollutants, respectively. Then, malonyltransferase enzyme adds malonic acid after adding glucose (Knejzlik et al. 2000; Collins 2011).

The third stage is called detoxification. The conjugated compounds constructed in the former stage are transported to the vacuole by ABC transporters. On the other hand, the metabolites accumulated in the vacuole are catalyzed by hydrolytic reactions inside the vacuole and then again transferred to the cytoplasm for other catabolism (Brazier-Hicks et al. 2008). Also, plants can store soluble conjugated compounds in the vacuole and transport insoluble conjugated compounds to the cell wall for further catabolism (Trapp and Mcfarlane 1995). Moreover, numerous organic contaminations that possess carboxyl, sulfhydryl, and hydroxyl or amine groups are located into lignin or to other cell wall components like hemicellulose, cellulose, or protein (Vanova 2009; Kang et al. 2010).

#### 8.6.2 Phytodegradation

This concept is considered a process using plants to remove or degrade or reduce the toxicity of environmental pollutants (Singh et al. 2017; Kafle et al. 2022). Plant degradation includes the direct destruction of organic pollutants, and this action occurs through the discharge of enzymes from the roots or through metabolic activities in plant tissues. In phytodegradation, organic pollutants are absorbed by roots and metabolized into smaller toxic substances in plant tissues. Some studies have reported that different leguminous and grass plant species have the ability to break down organic contaminants (Adam and Duncan 1999). Some tropical plants significantly have the potential to degrade organic pollutants, and this potential is due to the inherent characteristics of the plant itself including deep fibrous root systems and high hydrocarbon tolerance (Dzantor et al. 2000). Plant intercropping has the ability to increase the speed of breakdown of organic contaminations in comparison with the cultivation of plant individually in soil (Chouychai et al. 2009; Cheema et al. 2010). For example, plants such as alfalfa (Medicago sativa), corn (Zea mays), rice (Oryza sativa), rapeseed (Brassica napus), and Festuca arundinacea can increase the capability of degradation in polluted environments (Cheema et al. 2008; Du et al. 2011).

#### 8.7 AMFs and Plant Resistance to POPs

AMFs could modify soil structure and plant health and decline the contaminant accumulation in the plants as well. AMFs improve the plant resistance to organic pollutants and enhance plant growth, resulting in higher biomass production in contaminated soils. AMFs reduce organic pollutant residues in the plant shoots and increase the accumulation of pollutants (POPs in particular) in plant roots. AMFs could improve the resistance of plants to POPs in various ways including (1) biomass increase in plants via improved mineral nutrition and water availability, (2) enhanced activities of contaminant-degrading enzymes, (3) alleviation of the harmful effects of oxidative stress induced by pollutants, (4) accumulation of pollutants by AMF structures, (5) reduction of pesticide and herbicide use in agricultural fields, and (6) stimulation of growth of microorganisms involved in the degradation of contaminants in the soil (Wang et al. 2019; Fecih and Baoune 2019). It was observed that AM causes significant degradation of PAHs as one type of organic pollutant. The demotion of pollutants has been observed in plants with mycorrhizal treatments much more and faster (Joner and Leyval 2003). Similarly, Gao et al. (2011) reported greater degradation of pyrene and phenanthrene as PAHs in plant soils with AM cultivation than in soils without AM cultivation. Ectomycorrhizal fungi in pure culture are able to secrete oxidative enzymes such as polyphenol oxidases, laccases, tyrosinases, or peroxidases and these enzymes can degrade PAHs in spike soils under controlled environmental conditions (Gramss and Rudeschko 1998; Genney et al. 2004).

#### 8.8 Use of AMFs for Management of Polluted Environment and Agriculture

One of the most successful bioremediation techniques is the AMF-assisted remediation. In this technique, improvement of soil construction and enhancement of nutrient uptake by plants and the efficiency of POP bioremediation increase considerably. In order to improve the bioremediation process, the adapted strains of AMFs must be selected and used for plant inoculation in polluted ecosystems (Fecih and Baoune 2019). Gao et al. (2010) reported that AMF hyphae have a great potential to uptake PAHs. Combined use of AMFs and rhizobia can be effective in enhancing the phytoremediation efficiency of PAHs such as phenanthrene and pyrene by plants (Ren et al. 2017). Teng et al. (2010) suggested that synergistic interactions between AMFs and rhizobium may have great potential to enhance the phytoremediation of agricultural soil contaminated with weathered PCBs.

#### 8.9 AMFs and Food Safety

Food supply and safety is one of the most important issues for a growing global population. Application of excess chemicals (such as fertilizers, herbicides, pesticides, etc.) in agriculture is not a sustainable approach for the future because it causes severe environmental challenges such as negative impacts on soils, human and ecosystem health, accumulation of biocide residues in the environments, and finally affecting plant productivity and product safety. Therefore, chemicalized agriculture is an important challenge for food safety in the future (George and Ray 2023). In recent years, enough and safe food production to feed the world population has become a serious problem worldwide. Recently there have been many reasons for the extensive production of AMF inocula. Favorable effects on health, plant growth and development, ecosystem restoration, disease management, and phytoremediation improvement are the most important reasons among them (Siddiqui et al. 2008). Organic farming is developing day by day and its general principles are the elimination of most synthetic materials, soil management through the addition of organic materials, and the use of crop rotation (Gosling et al. 2006). Indeed, AMFs are capable not only of relieving lower fertilizer inputs (especially phosphorus) in organic systems but also of helping to phytoremediation of POPs (Fecih and Baoune 2019). Figure 8.2 briefly illustrates the role of AMF in improving food safety.

Fig. 8.2 Sources of POP entry into plants and roles of AMF in reducing food chain contamination and improving food safety

#### 8.10 Conclusions

Environmental pollution influences soil characteristics and reduces plant growth and yield along with its impact on food safety and human health. Production of sufficient and safe food for the growing population of the globe is one of the most important concerns in many countries. In many regions farmers cultivate crops in polluted soils or use polluted water for irrigation, leading to contamination of foods directly or indirectly. So, the management of contaminated lands for crop cultivation is an urgent need. Many strategies can be used to improve the cultivation of crops and to decrease the risk of food contamination in polluted soils. Remediation of the environment by different techniques is the most effective strategy for preventing the entry of contaminants into the food chain. Mycorrhizal fungi (especially AMFs) have great potential in the degradation of some organic pollutants. Therefore, colonization with AM fungi helps to protect plants and decrease contaminant entry into crop plants and the food chain. More biochemical, physiological, and molecular studies are needed to determine the role of AMF in removing organic pollutants and the involved mechanisms. These researches could help to progress in the usage of AMF as candidates to assist phytoremediation and restoration of the environments polluted with organic contaminants.

#### References

Dighton J, Allen MF (1991) The ecology of Mycorrhizae. J Ecol 79(3):867

## Chapter 9 Arbuscular Mycorrhizal Fungi (AMF)-Mediated Control of Foliar Fungal Diseases

Priyakshi Kashyap, Indrani Sharma, Sampurna Kashyap, and Niraj Agarwala

Abstract Plants witness a variety of disease incidences throughout their life, ultimately resulting in reduced plant growth and productivity. Climate change or human interventions have aggravated the incidences of various plant diseases, among which foliar fungal diseases are serious threats. Arbuscular mycorrhizal fungi (AMF) are a mutualistic group of organisms that play a significant role in enhancing plant growth and resilience under varied environmental circumstances. Moreover, it is well established that AMF confers tolerance against several foliar fungal diseases. This chapter highlights how fungal foliar diseases affect plant health and the various roles of AMF in providing resistance to different crop plants. In addition, AMF-mediated alterations in the root system architecture (RSA), modulation of reactive oxygen species (ROS), and reinforcement of the physical barrier that prevents pathogen invasion and establishment have been discussed in detail. Furthermore, the intricate cross talk between AMF and phytohormones or plant metabolites has also been explored. Overall, harnessing the potential of AMF in imparting tolerance against foliar fungal diseases might reduce the reliance on chemical fungicides, thereby introducing an environment-friendly approach for plant protection.

Keywords Arbuscular mycorrhizal fungi · Foliar fungal diseases · Plant growthpromoting rhizobacteria · Defense responses · Root exudation · Rhizosphere niche

P. Kashyap

Chaiduar College, Gohpur, Gohpur, Assam, India

I. Sharma · S. Kashyap · N. Agarwala (✉)

Department of Botany, Gauhati University, Guwahati, Assam, India e-mail<EMAIL_ADDRESS>
<sup>©</sup> The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 G. J. Ahammed, R. Hajiboland (eds.), Arbuscular Mycorrhizal Fungi and Higher Plants, https://doi.org/10.1007/978-981-99-8220-2\_9

#### 9.1 Introduction

The outbreak of plant pathogens in newer areas and their extension to multiple plant hosts along with the emergence of new pathogenic strains has caused a major threat to global food security and sustainable crop production. Alterations in weather patterns of a geographical location might create an environment more conducive for insects, pests, or pathogens (Pawlowski and Hartman 2020), which directly affect plant life and ultimately cause a threat to mankind. Among various plant diseases, fungal diseases have caused major crop loss around the world. For example, in the 1840s, potato blight disease caused by the fungus Phytophthora infestans, had created havoc in Ireland, where millions of people lost their lives and livelihoods (Badri and Vivanco 2009). Foliar fungal diseases are one of the key constraints affecting the productivity of crop plants. Blight, wilt, powdery mildew, anthracnose, leaf spot, etc. are some of the well-known foliar fungal diseases, which infect majority of the crop plants causing a heavy loss in crop yield. With the rise in plant disease incidences, agronomists have searched for several alternative ways to achieve disease-free sustainable agriculture. Among various plant disease management practices, the use of AMF, a group of obligate fungal symbionts, in the agricultural sector has gained momentum in recent times. AMF belonging to the group Glomeromycotina are the most comprehensive plant root symbionts, which supply soil nutrients like phosphorous (P) and nitrogen (N) to the plant roots via extra- and intra-radical mycorrhizal hyphae in exchange for lipid and sugar molecules (Marro et al. 2022).

AMF are protective symbionts that help the host plants to survive well in any diseased condition by providing the plants with defensive benefits and thereby shaping balanced ecological dynamics (Eck et al. 2022). About 80% of terrestrial plants live in direct association with different groups of AM fungi, which determines the overall health and fitness of the host plant (van der Heijden et al. 2015; Weng et al. 2022). Mutualistic interactions among the host plants and mycorrhizal partner determine the belowground ecosystem as well as regulate nutrient cycling (Sharma et al. 2023). However, being ubiquitous in nature, the potential of AMF in governing the fate of a host plant undergoing pathogen infection under natural conditions is not yet fully understood.

The distribution, abundance, and diversity of plant species in a natural ecosystem are influenced by the negative effect of any biotic or abiotic stress. Biotic stress-like disease conditions lead to the alteration in the normal physio-biochemical process of the plants, ultimately hampering growth and development. AMF provides nutritional and defensive support to the associated plant and also improves plant primary and secondary metabolism and the mobilization of less mobile elements, thus enhancing plant protection from pathogens (Delavaux et al. 2017; Kaur and Suseela 2020; Dey and Ghosh 2022). AMF association provides the host plant with an augmented state of defense, called defense priming, where they regulate the expression of host defense genes, hence enhancing the efficiency of plant defense mechanisms (Goddard et al. 2021; Eck et al. 2022). Besides, enhancing the plant survivability under any pathogen attack, AMF also help the plants to tolerate abiotic stresses like drought, high temperature, salinity, metal toxicity, etc. (Begum et al. 2019). The rational use of AMF with other microorganisms maximizes plant tolerance to diseases by competing with harmful pathogens (Devi et al. 2022). Studies have also revealed that the combined application of AMF and other beneficial plant microorganisms like plant growth-promoting rhizobacteria (PGPRs) impacts plant root exudation, thereby altering the rhizosphere niche (Ji et al. 2022). Furthermore, deciphering the far-reaching changes in plant metabolome, microbiome, and molecular reprogramming under the influence of AMF in diseased and non-diseased conditions will provide better outcomes in AMF-mediated plant disease management.

This chapter highlights the role of AMF in controlling foliar fungal diseases and the mechanisms associated with AMF-mediated disease resistance. The chapter also aims to describe the benefits of AMF in plant adaptation and provides a glimpse of how AMF along with other plant-beneficial microbes enhances plant overall health.

#### 9.2 The Impact of Foliar Diseases on Plants

The changes in climatic conditions have a significant effect on the occurrence and progression of crop plant diseases as well as the distribution of disease-causing pathogens. Additionally, the changes in agricultural practices and the use of highyielding crop plants may have a profound role in shifting both the pathogen diversity and the threat they possess (Launay et al. 2014). For instance, survey data on the severity index of Phytophthora cinnamomi, a fungal pathogen affecting oak trees in relation to winter temperature (Marĉais et al. 1996), reveals winter survival of P. cinnamomi plays a crucial role in limiting canker disease in oaks (Bergot et al. 2004).

Plant-microbe interactions undoubtedly brought various changes in plant adaptation to challenging environmental circumstances. These interactions might negatively impact the plants, causing several diseases. Foliar diseases have significant effects on plant morphology and physiology where the leaf architecture, color, and the process of photosynthesis get negatively afflicted. They often manifest in visible signs such as leaf spots, wilting, and discoloration, which help in identifying specific foliar diseases like late blight of potato, Fusarium or Verticillium wilt, anthracnose, and rust in wheat (Paliwal and Joshi 2022). Fungal pathogens are the primary cause of the majority of foliar diseases, with around 8000 fungal species known to cause plant diseases (Agrios 2009). Wilt diseases, observed in crops like eggplant (Bletsos et al. 2003), melon (Ros et al. 2005), cotton (Cai et al. 2009), tomato (Bidellaoui et al. 2019), and others, are primarily caused by the fungi belonging to Fusarium and Verticillium spp. Similarly, rice plants are susceptible to blast disease caused by Magnaporthe oryzae, which leads to the formation of elliptical reddish spots on the leaf sheath (Younas et al. 2023).

Fungal infection in the plant tissues begins with spore germination followed by germ tube elongation and mycelial penetration inside the host tissues. The mycelial growth phase is considered as the determining phase in triggering fungal crop disease epidemics (Magarey et al. 2007); it is also the phase that is most sensitive to variations in climatic conditions. Subsequent epidemics take place in suitable weather conditions where temperature and water availability play an important role in determining the intensity of infections (Launay et al. 2014). Plant pathogenic fungi utilize the natural openings, injured cells, and tissues to enter the host plants using varied mechanical and chemical methods (Knogge 1998). Based on these strategies, fungal pathogens are classified as biotrophs, hemibiotrophs, and necrotrophs, where the biotrophs directly feed on the living host tissues using a specialized structure known as haustoria; necrotrophs on the other hand, rely on the dead tissues and infected areas of the host plant. On the contrary, hemibiotrophic pathogens can eventually switch from a biotrophic to a necrotrophic mode (Stotz et al. 2014). For example, Puccinia graminis, which causes rust on wheat, is a biotrophic fungus, while Botrytis cinerea, causing mold disease in various plant species, is a necrotrophic fungus (De Silva et al. 2017), and P. infestans, causing potato late blight, is an example of hemibiotrophic fungus (Stotz et al. 2014).

Meanwhile, plants are found to evolve a broad range of direct and indirect resistance mechanisms against varied foliar pathogens. Infection by pathogen triggers several signaling pathways like extracellular-signal-regulated kinase (ERK) and mitogen-activated protein kinases (MAPKs) which leads to hypersensitive response (HR) and prevents the spreading of disease (Hammond-Kosack and Rudd 2008). Plants utilize effector-triggered defense (ETD) where receptor-like proteins (RLPs) engage receptor-like kinase SOBIR1 against the response of pathogenic fungi. The host cell death is stimulated typically by ETD after a period of endophytic pathogen growth (Stotz et al. 2014). On the contrary, plant response toward pathogenic fungi also leads to several changes in the metabolic profile of the host plants. Xiao et al. (2022) reported the enhancement of calcium signaling pathway-related genes such as CMLs, CNGCs, CPKs, and RBOHDs in strawberry resistant cultivar against B. cinerea. Furthermore, a network involving six different phytohormones, especially auxin (IAA) and jasmonic acid (JA) and genes associated with amino acid and phenylpropanoid biosynthesis, contributed to the strawberry flower's resistance to B. cenerea. Similarly, ascorbic acid, citric acid, glucose, galactose, total phenolics, and flavonoids were found to be positively correlated with B. cinerea resistance (Zhao et al. 2022). Furthermore, plant-released chemical signaling molecules play a pivotal role in the recruitment of microbial symbionts which promotes plant growth and development under stressed circumstances (Sharma et al. 2023). AMF make close associations with most of the terrestrial plants which facilitates the bidirectional exchange of nutrients, i.e., carbon-based photosynthates and soil minerals, between plants and AMF. These symbiotic associations play an important role in plant ecosystem functioning (Sharma et al. 2017).

#### 9.3 Benefits of AMF to Plants

Symbiotic relationships between AMF and the plants depend upon varied environmental factors where the soil characteristics and the host plant defense also play an important role (Poveda et al. 2022). Plant roots colonized by AMF are known to influence host plant morphology, physiology, and expression patterns of several host genes associated with the terpenoid biosynthesis pathways. AMF-induced changes in the level of secondary metabolites, phytohormones, proteins, and external hyphal networks have a profound effect on plant defense strategy (Sharma et al. 2017; Weng et al. 2022). AMF-colonized plants show an increase in shoot and root length and also the biomass of the host plant (Han et al. 2023). Furthermore, soil chemical and physical properties along with rhizospheric microbial communities are greatly influenced by the roots colonized by AMF, and such changes play a significant role in alleviating numerous plant abiotic and biotic stresses (Miozzi et al. 2019; Weng et al. 2022). AM fungi show diverse functional activities ranging from the mobilization of soil nutrients, fixation of atmospheric nitrogen, and production of phytohormones, siderophores, and antibiotics. AMF-colonized durum wheat cultivars, Saragolla and Odisseo, showed an enhancement in the abundance of Bacteroidetes and Actinobacteria in the host plants (Agnolucci et al. 2019); particularly, AM fungi Funneliformis mosseae synergistically regulated the abundance of Rhodococcus sp. in Saragolla cultivar, while an increase in Microbacterium and Streptomyces spp. was observed in both the durum wheat cultivars (Ujvári et al. 2021). Similarly, Han et al. (2023) reported a shift in endophytic microbial diversity in AMF-colonized host plant (Lettuce) where Sphingomonadales, Betaproteobacteria, and Burkholderiales showed a positive correlation with AMF colonization.

Besides nutrition, mycorrhizal association provides several other benefits to the plants. AMF is reported to act as a biofertilizer which aids in the uptake of nutrients and thus increases plant growth. AMF association alters the physical and chemical environment of the plant rhizosphere, thereby modulating the rhizospheric microbiota (Dowarah et al. 2022; Sharma et al. 2023). Changes in rhizospheric microflora might synergistically affect the plant-beneficial microorganisms, thus inhibiting the outbreak of pathogenic strains (Xavier and Boyetchko 2004). Maize plants inoculated with AM fungus Claroideoglomus etunicatum altered the bacterial and fungal communities of rhizospheric soil (Hao et al. 2022). The symbiosis between AMF and Artemisia annua roots modulates the use of various carbon sources by rhizomicrobiota affecting the survivability and functionality of rhizospheric microbes, which in return improves nutrient acquisition by A. annua (Ma et al. 2021). Furthermore, AMF-induced changes in root exudation directly impact the rhizospheric microorganisms. The enzymatic activities of rhizospheric soil as well as the abundance of the bacterial community were found to be increased in the maize rhizosphere when the plants were in association with AMF. However, the AMF association negatively regulated the rhizospheric bacterial diversity, but a reduction in soil-borne diseases of maize was observed (Ma et al. 2022). Hence, AMF-mediated alteration in the rhizospheric microbial communities and root exudation profiles of host plant confers tolerance to various bacterial and fungal diseases.

Genes for phosphate transporter (LePT3, LePT4, LePT5) in tomato plants were found to be upregulated when the roots were colonized by AM fungi (Nagy et al. 2005). AM fungi form a network of fungal hyphae which helps in enhancing the water-holding capacity (Songachan 2023). Mycorrhizal plants can combat water stress conditions as the water-absorbing surface is increased by the fungal hyphal network which helps in increased absorption of water even from the soil with low water potential (Augé et al. 2001). AMF in the soil increase the production of glomalin or glomalin-related soil protein (GRSP), which helps in soil binding and thus aids in overcoming erosion of soil and water infiltration (Sharma et al. 2017). AMF can reduce heavy metal toxicity in the host plant and the soil by increasing the expression of metal transporters (Ahammed et al. 2023). These transporters help AM fungi to take up heavy metals from the soil and thus lead to environmental cleanup (Göhre and Paszkowski 2006). A Zn transporter, GintZnT1, is reported to be upregulated in Glomus intraradices when exposed to high Zn concentration (González-Guerrero et al. 2005). As per evidence put forward by Lazcano et al. (2014), it is also found that tomato plants with mycorrhizal association have the potential to reduce N2O emissions during nitrogen fixation, which is a potent greenhouse gas. Further research involving the use of AMF and selected rhizospheric microbes along with other non-microbial substrates in field conditions might improve plant resistance to various diseases.

AMF-associated plants also acquire additional benefits in developing resilience capacity towards abiotic and biotic stresses (Delavaux et al. 2017). Meanwhile, AMF associations and their benefits vary among species, taxonomic groups, and types of stresses encountered by the host plant. For example, a global meta-analysis report on the effects of AMF species and taxonomic groups on stressed and non-stressed plants reveals positive effect sizes of F. mosseae, Rhizophagus intraradices, and Diversispora versiformis under drought-stressed conditions; Septoglomus deserticola, etc. under heavy metal stress; D. versiformis, R. fasciculatus, etc. under salinity stress; and Gigaspora margarita and R. fasciculatus under biotic stress conditions of the host plant species (Marro et al. 2022). In a study conducted by Begum et al. (2021), inoculation of AMF was reported to significantly increase the tolerance of tobacco plants against drought stress. It also leads to the increased production of antioxidants which protects the plant from oxidative damage during drought stress. Similarly, Orine et al. (2022), through an experimental study, revealed the importance of plant metabolites such as rutin and alpha-tomatine, accumulated in AMF-associated tomato plant leaves. The accumulated metabolites play a key role in the host plant contributing toward more plasticity and greater phenotypical space in terms of growth and defense against drought-stressed conditions. Under salt stress, AMF association induces increased expression of the chloroplast gene which as a result increases the photosynthesis efficiency in the host plant (Chandrasekaran et al. 2019). Simultaneously, mycorrhizal fungi associated with the host plants act as a protective shield, preventing the host from damage caused by plant pathogens. Considerably, endless studies reported the beneficial role of AMF association in reducing plant diseases caused by pathogenic bacteria, nematodes, and fungi under varied environmental circumstances (Weng et al. 2022). For example, AMF, G. mossae inoculation in the soybean plant results in the suppression of Pseudomonas syringae infection (Miransari 2011). Mycorrhizal fungi reduce varied nematocidal diseases of soybeans, cucumbers, cotton, alfalfa, peach, tomatoes, kidney beans, and citrus to various extents by parasitizing the cysts of nematodes (de Sá and Campos 2020; Rodrigues e Silva et al. 2021). Whether symbiotic or pathogenic interactions, various fungi have developed diverse strategies to infect the host plants, where pathogen infection leads to plant death in most cases. Phytopathogenic fungi are the most dominant group of organisms causing ecological and economic threats to the crop plants (Doehlemann et al. 2017). For example, Ceylon (Sri Lanka) was one of the great coffee producers until the 1870s. Coffee rust caused by Hemileia vastatrix in Ceylon during 1875 declined its production by 95% by completely destroying the coffee plantations (Avelino et al. 2015). Thus, fungal diseases pose a major risk to world crop production, and formulation of immediate effective measures seeks concentration from the scientific community. Here, in this chapter, we are going to discuss various strategies employed by AMF in controlling foliar diseases caused by pathogenic fungi which might cause serious threats to crop plantations.

#### 9.4 Role of AMF in Controlling Fungal Foliar Diseases

AMF forms hyphal interactions with the roots of most agriculturally important crop plants. This interaction promotes great effectiveness in the uptake of soil nutrients confining low mobility like P, resulting in an enhancement in the crop nutrient availability and growth. Due to their ability in promoting health benefits to the associated crop plants, they possess potential economic concerns in agricultural fields (Abarca et al. 2023). AMF-associated plants show significant resistance toward biotic and abiotic stress factors. This protective nature of AMF toward environmental factors might be due to varied mechanisms associated with an increment in nutrient acquisition (Mustafa et al. 2016) and induction of systemic acquired resistance (SAR) (Jung et al. 2012) in the host plant. Foliar fungal diseases develop a wide range of symptoms that differ to a great extent depending on the pathogen type and the host plant. Some of the major fungal foliar diseases and their control measures using AMF are discussed below and enlisted in Table 9.1.

#### 9.4.1 Blight

The symptomatic occurrence of yellowish to brownish spots ranging in diameter from 2 to 30 mm on the leaves results in blight, which later on spreads over a large

portion of the foliage (Klinsukon et al. 2021). A study carried out by Zhengjia and Xiangdong (1991), reveal the importance of vesicular arbuscular mycorrhizal (VAM) fungi G. mosseae against Fusarium blight caused by F. vasinfectum. Cotton plants inoculated with G. mosseae result in a significant decrease in the severity of disease symptoms like vascular discoloration caused by F. vasinfectum. Hu-zhe et al. (2005) reported that microbial infection can reduce the incidence of blight disease in pepper plants caused by P. capsici. Pepper plants infected by P. capsici along with the mycorrhizal fungus G. intraradices show reduced mortality and infection in comparison to those plants which were infected by the pathogen alone. Similarly, AM fungi G. fasciculatum, G. etunicatum, G. mosseae, and G. margarita showed enhancement in the growth and development as well as reduction in the blight of pepper plant caused by P. capsici (Ozgonen and Erkilic 2007). Tomato plants infected by the fungus Alternaria solani cause early blight, which results in damping off of seedlings, collar rot, and destruction of leaves and fruits (Rotem 1994). G. intraradices colonization significantly reduces the symptoms caused by A. solani in tomato plants when compared with non-mycorrhizal plants (Fritz et al. 2006). Potato plants inoculated with Glomus sp. induce two pathogenesis-related genes, PR1 and PR2 in the leaves of the host plant. The expression of PR genes ultimately shows systemic resistance against the pathogenic fungus P. infestans causing potato late blight disease (Gallou et al. 2011). In a similar study conducted by Song et al. (2011b) on the effect of AMF, G. versiforme upon tomato plants infected by A. solani, it was revealed that G. versiforme inoculation showed an enhancement in the expression of three defense-related genes, namely, PR1, PR2, and PR3, in the leaves of the host plant. Mosquera-Espinosa et al. (2013), in their study, depicted the role of mycorrhizal fungi Ceratobasidium spp., which were isolated from orchid and evaluated for their ability to control the disease severity caused by Rhizoctonia solani in rice plants. A significant reduction in the symptoms of sheath blight disease was observed in pre-inoculated host plants compared to the control. AM fungus G. margarita KKU-BH-01, in association with the roots of eucalyptus plants, was reported to reduce leaf blight disease caused by Cylindrocladium quinqueseptatum. Moreover, plants pre-inoculated with AMF, prior to pathogen infection, showed 33.4% reduction in the disease (Klinsukon et al. 2021). Moarrefzadeh et al. (2022) successfully inoculated two varieties of chickpea, Saral and Bivanij, with five strains of AM fungi R. irregularis, G. versiform, F. mosseae, G. fasciculatum, and G. margarita and observed their disease control ability against Ascochyta blight caused by Ascochyta rabiei. All four strains of AMF except G. fasciculatum in the Saral variety significantly reduced the disease extremity. Furthermore, R. irregularis and G. versiform showed the highest disease suppression capacity, i.e., 46.15% and 42.30%, respectively, on the variety Saral and G. fasciculatum by 40% on the variety Bivanij. In another recent experiment with tomato plants, AM fungi G. clarum was identified as having an important role in reducing the incidence of early blight disease caused by A. solani (Joseph and Olumide 2022).

#### 9.4.2 Wilt

Wilt is caused by soil-borne pathogenic fungi that infect the host plant via roots, ultimately clogging the vascular system and resulting in yellowing, wilting, and death of the infected plants (Noman et al. 2023). F. oxysporum and V. dahliae are found to cause the majority of wilt diseases in a variety of plant species (Usharani et al. 2022). AM fungus G. etunicatum BEG168 inoculation on cucumber seeds at the germination stage showed resistance toward F. oxysporum f. sp. cucumerinum. Mycorrhizal colonization increases the production of plant secondary metabolites (Hao et al. 2005), which might result in plant tolerance toward Fusarium wilt disease. AMF-associated cyclamen plants exhibited higher antioxidant contents and enhanced plant defense against Fusarium wilt, thereby reducing disease incidences (Maya and Matsubara 2013). Ren et al. (2015), in an experimental study, revealed the importance of p-coumaric acid and malic acid secreted in the rhizospheric region of the host plant watermelon (Citrullus lanatus), co-inoculated with AMF G. mosseae and pathogenic fungus F. oxysporum f. sp. niveum (FON). The alteration in the composition of the exudate resulted in the stimulation of defense signaling molecules in the host plant ultimately enhancing plant disease tolerance capacity. A similar study was conducted in in vitro conditions, using tomato as a host plant and AM fungus Glomus sp. as an antagonist against F. oxysporum. The study depicted the potential role of Glomus sp. to control the pathogen. Furthermore, the secretion of various exudates around the roots has resulted in a decrease in disease incidences, thereby improving the plant growth. (Kumari and Prabina 2019).

#### 9.4.3 Powdery Mildew

Symptoms of powdery mildew are easily recognized by the presence of a white powder-like substance on the leaf surface of the host plants. Host plants may undergo leaf chlorosis and an overall reduction in plant growth and development due to powdery mildew. Lowe et al. (2012) have reported that AM fungi (G. caledonium, G. fasiculatum, and G. mossae) in combination with chitosan spray significantly reduces the effect of powdery mildew, resulting in higher fruit yield and runner production in strawberry plants. Barley roots colonized with Piriformospora indica, a basidiomycetous fungus, upregulate the expression of heat-shock proteins (Hsp70) and defense genes PR5, PR2, and PR1 against Blumeria graminis f. sp. hordei, a leaf pathogen causing powdery mildew. The pathogen results in the induction of a distinct set of genes, LRR receptor kinases, and WRKY transcription factors after 12 h of postinoculation (hpi) and vesicle-localized gene products after 24 hpi, which were altered by the root-associated fungus P. indica. P. indica results shift in the metabolic profile of the host plant, making it unsuitable for pathogen colonization (Molitor et al. 2011). Wheat plants inoculated with AM fungi R. irregularis singly and in combination with G. mosseae showed resistance of 25% and 43%, respectively, toward a fungal pathogen B. graminis (Mustafa et al. 2013). In a similar study conducted by Mustafa et al. (2014), AM fungus F. mosseae was reported to decrease powdery mildew infection by 73% in wheat plants caused by B. graminis f. sp. tritici. Reduction in the disease symptoms results in the accumulation of defense enzymes such as lipoxygenase and peroxidase and induces plant systemic acquired resistance (SAR). Additionally, on testing three different AM fungi, viz., F. mosseae, and Solrize®, two commercially available mycorrhizal inocula and R. irregularis, a laboratory sample, against the powdery mildew pathogen B. graminis f. sp. tritici in wheat plants, it was found that all the three inocula exhibit biocontrol activity against B. graminis with different extents, i.e., 74%, 58%, and 34%, respectively. Hence, from the study, it can be concluded that the extent of protection against B. graminis is determined by the type of AM fungi involved in the mycorrhizal association (Mustafa et al. 2016). In an experiment carried out by Yousefi et al. (2018), apple seedlings treated with powdery mildew-causing fungal pathogen Podosphaera leucotricha were given different treatments with two fungicides (Flint and Stroby) and AM fungus Glomus sp. After 6 weeks, seedlings in association with AM fungi are reported to show higher resistance to the pathogen in comparison to the fungicide treatments.

#### 9.4.4 Other Fungal Foliar Diseases

Besides the aforementioned fungal diseases, mycorrhizal fungi associations with the host plants can also effectively control several other fungal foliar diseases. Anthracnose disease caused by Colletotrichum gloeosporioides reduces the growth and biomass production of the host plant. Additionally, C. gloeosporioides infection shows a significant reduction in the plant antioxidant contents like ascorbic acid, ascorbate peroxidase (APX), superoxide dismutase (SOD), and polyphenols. AM fungus G. fasciculatum, inoculation induces drastic changes in growth parameters and in the activity of antioxidants in cyclamen, infected with C. gloeosporioides (Maya and Matsubara 2013). A similar study conducted by Chen et al. (2023b) reported an increase in the activity of antioxidants {(SOD, catalase (CAT) and peroxidase (POD)} in tea plants inoculated with AM fungi R. intraradices BGC JX04B. Tea plants inoculated with AM fungi significantly reduce the anthracnose disease severity caused by Colletotrichum camelliae. AM fungi alone or in a combined form with PGPR play a significant role in controlling plant fungal diseases. For example, rust disease in wheat caused by Puccinia graminis Pers. f. sp. tritici can be reduced by mycorrhizal treatment alone or in combination with Trichoderma harzianum HL1 and T. viride HL5 (El-Sharkawy et al. 2018).

F AM fungi with grass fungal endophyte (Epichloe) suppressed the leaf spot disease incidence of perennial ryegrass caused by Bipolaris sorokiniana. AM alone or in association with (Epichloe) can increase the lignin contents of the host plant infected with the fungal pathogen B. sorokiniana (Guo et al. 2019). Similarly, AMF with two PGPR, viz., P. fluorescens (Pf) and T. viride (Tv), results in highest fungal disease control efficiency of 65.60% (AMF + Tv) and 48.40% (AMF + Pf + Tv) in rice plant against B. oryzae, a fungal pathogen causing brown spot disease (Saleh and El-Akshar 2020). Black sigatoka disease caused by Mycosphaerella fijiensis,a fungal pathogen in banana, was found to be controlled by AM fungus, R. irregularis MUCL 41833. Four banana cultivars (Batard, C292, CRBP39, and F568) inoculated with R. irregularis were grown in a greenhouse and were observed for the effects of the pathogen on the plantlets. After 35 days of growth, it was observed that AM fungi inoculated CRBP39 and F568 showed more resistance than Batard and C292 cultivars toward M. fijiensis. The overall study signifies that AMF inoculation resists the development of host plant infectious symptoms by M. fijiensis at an early age of infection (Anda et al. 2020). Symbiotic association of AM fungi R. intraradices, with alfalfa (Medicago sativa), decreases the incidence of leaf spot disease caused by the fungus Phoma medicaginis by 39.48% and also reduces its disease index by 56.18% (Li et al. 2021). A soil-borne fungus Physoderma maydis causing brown spot disease in Zea mays was effectively controlled by AM fungi (Glomus sp. and Gigaspora sp.). Two different varieties of maize, CMS9015 and CMS8704, were inoculated with AM fungi in combination and were observed for their potential to control P. maydis and found that the incidence and severity of the disease is reduced by 33.7% and 35%, respectively (Adamou et al. 2023).

#### 9.5 AMF-Mediated Mechanisms Associated with Plant Disease Management

The use of biocontrol agents in ameliorating the havoc of plant diseases has gained momentum in recent years. With the advances in the use of biological control agents, the application of AMF as a plant disease management tool has also got the attention of the scientific community. AMF association induces resistance in the infected plants through various mechanisms involving structural and chemical changes, improving nutrient uptake by the plants, changing the rhizospheric niche, competing with the pathogenic microbes, and activating the host plant defense system (Fig. 9.1) (Weng et al. 2022).

#### 9.5.1 AMF-Induced Structural Changes in Plants

AMF association induces structural/morphological changes in the roots of the host plants ultimately creating a barrier for the entry of pathogens. Plant roots undergo changes like thickening, lignification, excessive branching, increase in the cell layers, etc. when they are symbiotically associated with the AMF, thus slowing

Fig. 9.1 Diagrammatic representation of various AMF-induced mechanisms in host plants associated with improved plant disease tolerance. Created with BioRender.com

down the pathogen from entering the plant tissue (Gao et al. 2018; Basyal and Emery 2021). Moreover, increased root growth and branching enhance the mineral nutrition of the plants ultimately strengthening the plant defense system (Matsubara et al. 1995). Similarly, the deposition of lignin at various plant parts like leaves, roots, and xylem tissue provides mechanical strength to the plants, thus inhibiting pathogen invasion (Goicoechea et al. 2010; Sanmartín et al. 2020). Tomato plants in association with AMF (G. mosseae and G. intraradices) were found to have altered root anatomy, eventually hindering Phytophthora parasitica infection kinetics (Pozo et al. 2002). Yanan et al. (2015) in their study demonstrated that strawberry plants treated with AMF resulted in a reduction in wilt disease caused by F. oxysporum. Plants in association with AMF had shown an increase in hydroxyproline-rich glycoprotein (HRGP) and lignin contents in the root tissue of strawberry plants leading to cell wall thickening and contributing to host defense. Similarly, wheat plants inoculated with AM fungus F. mosseae showed biocontrol activity against the foliar pathogen B. graminis f. sp. tritici. Inoculation of F. mosseae induced systemic resistance in the leaves by forming papillae at the B. graminis penetration site, thus enhancing the wheat plant's resistance to powdery mildew (Mustafa et al. 2017). Furthermore, HPRGs are sugar proteins that protect the cell wall from degradation by various enzymes secreted by pathogens like proteases, cellulases, etc. Medicago sativa in symbiosis with AM fungi was found to produce more amount of HPRGs, which act as a defense molecule against Phoma medicaginis (Gao et al. 2018).

The functionality of plant roots gets altered during symbiosis with AMF (de Vries et al. 2021). Analysis of mycorrhizal and non-mycorrhizal roots of Camellia sinensis L. revealed that AMF (R. intraradices) association amends the RSA and increased the sugar content of tea roots (Chen et al. 2021), indicating that change in RSA and root sugar content might modulate the rhizomicrobiome of C. sinensis, which may directly or indirectly hamper the pathogen infection in plants. Thus, AMF-mediated structural changes in root and leaf morphology or anatomy protect the host plant from any type of pathogen interventions and minimize disease incidences.

#### 9.5.2 AMF-Induced Enhancement in Plant Nutrition

Plants exchange their photosynthetic outcomes with AMF and in return acquire nutrients and water from the mycorrhizal partner (Smith and Read 2008). AMF helps the plants in uptaking nutrients like N, P, and other essential micronutrients by inducing a series of chemical signaling between the AMF and host plant (Dowarah et al. 2022), which assists plant growth and development. The mycelia formed by AMF develop into a huge branching system that helps in better absorption of water and minerals from the soil. Being an inevitable element for plant functions, P is required by plants during various developmental stages. The utilization of inorganic P (Pi) from the soil is not an easy task for many plant species due to its low mobility and solubility in the soil (Wang et al. 2023). Since plants alone cannot absorb sufficient Pi for their utilization, they seek help from the mycorrhizal fungal partner and fulfill their need (Ferrol et al. 2019). Likewise, AMF also helps in the uptake of both organic and inorganic N by the plants, but the mechanisms of action involved in AMF-induced N acquisition are not yet fully understood (Savolainen and Kytöviita 2022). AMF colonization enhances plant nutrient uptake as their extraradical hyphae explore beneath the soil by expanding the host plant rhizosphere (Schaefer et al. 2021) through enzymatic or physical actions. Furthermore, mycorrhizal hyphae can penetrate deep into the soil due to their thinner appearance, thus improving the efficiency of nutrient acquisition by the host plant (Ebbisa 2022).

Cucumber plants symbiotically associated with AMF had shown increased uptake of inorganic nutrients such as N, P, and potassium (K) from the soil which eventually enhanced the defense system of cucumber plants aiding in resisting Fusarium wilt disease (Wang et al. 2012). A tripartite interaction involving AMF, leguminous plants, and Rhizobium sp. helps in N uptake by the host plant. In an intercropping system consisting of soybean and maize plants, when inoculated with AM fungus, G. mosseae and Rhizobium strain SH212 exhibited an enhanced N fixation rate in soybean plants. Furthermore, improved N fixation efficacy stimulated the transfer of N from soybean to maize plants, thus improving the productivity of both plants (Meng et al. 2015). AM fungi F. mosseae BGC HEB02 and R. intraradices BGC HEB07D in interaction with wheat plants increased the levels of P and zinc (Zn); especially wheat plants in association with R. intraradices significantly increased the Zn concentration 1.13–2.76 times than that of non-inoculated plants (Ma et al. 2019). Similarly, in tomato plants, a maximum uptake of N, P, and K was observed when inoculated with AMF (Kumari and Prabina 2019). High-throughput phenotyping of various sorghum genotypes unveiled that colonization of sorghum roots by the AM fungus R. irregularis boosts P, iron (Fe), and Zn concentration in the maize grains (Watts-Williams et al. 2022). However, Artemisia ordosica plants inoculated with F. mosseae, an AM fungus, lowered the availability of leaf P, indicating that AMF association may not always enhance plant nutrient levels. Moreover, a lowering in leaf P content due to the symbiosis between A. ordosica and F. mosseae negatively regulated the population of a foliar herbivore Chrysolina aeruginosa, emphasizing that AMF-mediated reduction in P concentration of A. ordosica leaves might have a positive role in limiting the damages caused by C. aeruginosa (Wang et al. 2023). In addition to the enhancement in plant nutrient uptake, AMF association also contributes to the sequestration of heavy metals (HMs) present in HM-polluted soil (Khan et al. 2022), suggesting that AMF interaction may have the potential to alleviate the toxic effect of HM stress. The further implication of AMF with other microbial or nonmicrobial substances might help the plants to survive under challenging environmental conditions.

#### 9.5.3 AMF-Induced Changes in Plant Metabolites

About 4–20% of the photosynthetic output of the host plant either in sugar or lipid form is utilized by the AMF for their proper nutrition (Kaur and Suseela 2020). Acquisition of photosynthetically fixed carbon by the mycorrhizal partner in return generates a carbon sink in host plant roots and activates the photosynthetic machinery (Bago et al. 2000; Kaschuk et al. 2009), thereby forming a positive feedback cycle. Symbiosis by AM fungi stimulates the production of various types of plant metabolites, which play a significant role in plant defense against foliar fungal pathogens. AMF-induced modulation in the root metabolite profile synergistically alters the metabolites of aboveground plant parts (Schweiger and Müller 2015; Wang et al. 2018), thus helping the plant from various harmful phyllospheric and rhizospheric microbes. Moreover, AMF association initiates the shikimic acid pathway in the host plant, which produces several plant secondary metabolites like phytoalexins, phenolics, antimicrobial compounds, and various other enzymes. Several studies have also revealed that mycorrhizal plants' primary and secondary metabolite profiles differ from those of non-mycorrhizal plants (Kaur and Suseela 2020).

Pepper plants pre-inoculated with AM fungus G. mosseae have enhanced tolerance against the pepper blight pathogen P. capsica in both greenhouse and field conditions. Furthermore, the study also revealed that inoculation of G. mosseae enhanced the production of plant secondary metabolites, phytoalexin, and capsidiol which can have a role in defense against P. capsica (Ozgonen and Erkilic 2007). Corn variety Gaoyou-115, susceptible to R. solani, a fungus responsible for sheath blight disease, when inoculated with the AM fungus G. mosseae, a significant reduction in disease outbreak, was observed. Furthermore, the interaction of G. mosseae upregulated the production of 2,4-dihydroxy-7-methoxy-2 H-1,4-benzoxazin-3(4H)-one (DIMBOA) in the leaves and roots of corns, helping the plants to cope with sheath blight disease (Song et al. 2011a). Previous studies involving the role of DIMBOA in controlling sheath blight disease also reflected that an increase in DIMBOA concentration in the host plant inhibits the hyphal growth of R. solani (Song et al. 2011a). Similarly, cereal crops in association with AMF had shown an increase in the concentration of secondary metabolites like flavones, phenolic acid, and phytic acid (Ryan and Angus 2003; Pepe et al. 2018), which are well known for their key regulatory role in plant stress alleviation. Additionally, the quantity of plant secondary metabolites like alkaloids, flavonoids, organic acids, and terpenoids was also found to be increased in plants interacting with various AM fungi (Card et al. 2016; Latz et al. 2018; Kaddes et al. 2019). AMF-induced changes in plant primary and secondary metabolic pathways and changes in hormonal cross-talk can induce the photosynthetic machinery of the host plants, thereby increasing the photosynthetic output (Khan et al. 2022).

n AMF symbiosis also increases antioxidant enzyme synthesis which reduces oxidative damage caused by various fungal pathogens to the plants. Tomato plants infected with vascular wilt disease, when inoculated with a consortium of AMF comprising Claroideoglomus etunicatum, F. mosseae, and R. intraradices, a induction in plant systemic defense, were observed involving the production of fusaric acid, which helped in reducing wilting symptoms in tomato plants. Moreover, inoculation of the AMF consortium also enhanced the concentration of antioxidative enzymes involving CAT, glutathione reductase (GR), etc. and reduced the toxic effect of ROS generated due to F. oxysporum f. sp. lycopersici (Hashem et al. 2021). Similarly, evaluating the effect of AMF in tomato plants infected with V. dahliae revealed that AMF interaction enhanced the intensity of antioxidants like CAT, SOD, and polyphenol oxidase (PPO) in tomato roots and leaves and finally improved tomato tolerance against V. dahliae (Ait Rahou et al. 2021). In addition to that, AM fungus G. viscosum showed effective biocontrol ability against verticillium wilt disease in artichoke plants by modulating the activity of antioxidant enzymes like SOD, APX, and monodehydroascorbate reductase (MDHAR). Further, the association of G. viscosum also increased the concentration of glutathione (GSH) and ascorbate (ASC) in artichoke plants, while a reduction in hydrogen peroxidase (H2O2) and lipid peroxidation levels was observed (Villani et al. 2021). Deciphering the effect of AMF on tea anthracnose incidences unraveled that mycorrhizal inoculation significantly increases the content of CAT, POD, and superoxide ions in tea plants and inhibited the proliferation of C. camelliae, thus protecting the tea plants from damages caused by anthracnose disease (Chen et al. 2023b). AMF-induced changes in plant metabolic profile directly or indirectly influence plant tolerance to any pathogenic fungal challenges. Furthermore, AMF also modulates the expression of defense signaling molecules in plants and also alters the composition of plantassociated microbiota, ultimately improving plant disease resistance.

#### 9.5.4 AMF-Induced Hormonal Signaling

Plant response to environmental fluctuations or pathogen attack is also regulated by the modulation in plant defense hormonal signaling. Plant hormones are required in trace amounts and play an important role in plant development. Various plant hormones like IAA, abscisic acid (ABA), brassinosteroid (BR), cytokinin (CK), ethylene (ET), gibberellic acid (GA), JA, and salicylic acid (SA) play a crucial role in the establishment of AMF-mediated plant defense (Schmitz and Harrison 2014). From various studies, it has been established that SA resists the infection of biotrophic pathogens and induces SAR in plants, while JA and ET inhibit saprotrophic pathogens and activate induced systemic resistance (ISR) in plants (Hause et al. 2007). The combined application of G. mosseae and JA/SA elicitors on Fusarium wilt undergoing tomato plants resulted in a significant reduction in disease incidences. Furthermore, the level of phytohormones like GA, IAA, zeatin, and zeatin-riboside was found to be significantly enhanced in diseased tomato plants after the application of AMF and JA/SA inducers, indicating that changes in hormonal levels might have a positive correlation with the disease resistance (El-Khallal 2007). Likewise, in tomato plants infected with B. cinerea when inoculated with the mycorrhizal fungus G. mosseae, an upregulation in Pin II (a JA marker gene) was observed. Mycorrhiza-induced enhancement in the Pin II expression conferred ISR in infected tomato plants suggesting that modulation in JA concentration might have primed the plants against B. cinerea (Jung et al. 2009).

The real-time qPCR analysis on the effect of Glomus sp. MUCL41833 on lateblight infected potato plants unveiled that AMF induction enhanced the expression of defense genes associated with JA, SA, and JA/ET pathways (Gallou et al. 2011). Analyzing the cross-talk of G. mosseae, G. intraradices, and the beneficial non-mycorrhizal fungus T. harzianum on melon plants showed induction in 1-aminocyclopropane-1-carboxylic acid (ACC), IAA, ABA, JA, SA, and zeatin levels. In addition to the modified hormonal profile, the treated melon plants also exhibited an enhanced tolerance against the Fusarium wilt disease (Martínez-Medina et al. 2011). In a recent study, it was found that Salvia miltiorrhiza plants when pre-inoculated with AM fungus G. versiforme, a significant reduction in the wilt incidence was observed. Further investigation also revealed that the inoculation of mycorrhizal fungus also upregulated the JA and SA signaling pathways, thereby enhancing the host tolerance to Fusarium wilt (Pu et al. 2022). Further studies involving the modulation in phytohormone levels due to AMF association and tolerance to fungal foliar disease need attention from the scientific community.

#### 9.6 Association of AMF with Other Microbes for Enhancing the Efficiency of Plant Stress Tolerance

The stress tolerance potential of AMF can be amplified by using various strategies. AMF in combination with other microorganisms shows enhanced efficiency in controlling various plant diseases and other abiotic stresses. To minimize the use of chemical fertilizers, the combined application of AMF and PGPR in crop fields has gained momentum in recent years. Tomato plants inoculated with AM fungi G. mosseae and G. versiforme along with PGPR, and Bacillus polymyxa provided control against Meloidogyne incognita. Furthermore, the study revealed that the AMF association increased the population of PGPR in the rhizospheric region, whereas PGPR enhanced the colonization of AMF in tomato roots (Liu et al. 2012). The study indicates that inoculation of plants with compatible AMF and PGPR enhances plant tolerance against any biotic stress.

A consortium of AMF and two PGP bacteria B. subtilis and P. fluorescens resulted in a significant reduction in the infection of Sclerotium rolfsi on common bean plants growing in P-deficient soil. Moreover, the synergistic effect of AMF and PGP bacteria enhanced P and Fe uptake by the diseased plants, which ultimately improved plant development under biotic stress (Mohamed et al. 2019). Triticum durum L. and Vicia faba L. are the two most important economic crops of the Mediterranean semiarid region. Research involving the inoculation of a consortium of AMF-PGPR-rhizobia revealed that combined inoculation of all three enhances soil fertility and wheat/faba bean productivity. Furthermore, inoculation of AMF-PGPR-rhizobia improved the content of nutrients like N, P, calcium (Ca), potassium (K), and sodium (Na) in the shoot tissue of both crops along with an increase in shoot protein and sugar content (Raklami et al. 2019). Similarly, tomato plants inoculated with AM fungus F. mosseae and PGPR strain Paraburkholderia graminis C4D1M and enhanced tomato seedling growth and the efficacy of PSII by reducing the leakage of electrolyte (Caradonia et al. 2019).

Summer squash inoculated with AMF (G. etunicatu, G. harzianum, G. intraradices, and G. monosporum) and PGPR strains B. subtilis and T. harzianum resulted in an elevated level of free amino acids, total proteins, free phenolics, chlorophyll, and carotenoid contents in the host plant. Furthermore, the application of AMF with PGPRs lowered the disease severity index of Fusarium wilt in the summer squash plants (El-Sharkawy and Abdelrazik 2022). Evaluating the effect of the combined application of AMF and PGPR revealed that the mixture of both microbial substrates enhanced rice plants' nutritional content and yield. In addition to that, the consortium of AMF and PGPR significantly enhanced the levels of inorganic nutrients like boron (B), N, P, and K in rice field soils, thus providing the plants with sufficient nutrients for proper growth and yield (Chen et al. 2023a). Similarly, inoculation of AMF and PGPR consortium on date palms undergoing Fusarium wilt decreased the disease incidences. Further investigation also revealed that the reduction in disease progression was due to the activation of the phenylpropanoid pathway in date palms, induced by the synergistic effect of AMF and PGPR (Ziane et al. 2023). Hence, the combined application of AMF and other plant-beneficial microorganisms plays crucial functions in improving plant-soil characteristics and also ameliorates the ability of host plants to cope with any environmental fluctuations. Further, the large-scale application of AMF and their consortia in field conditions will help in achieving climate-smart crops for a sustainable future.

#### 9.7 Field Application of AMF: constraints and possible measures

Application of AMF in conventional agriculture practices still remains an unaccomplished task due to various hindrances. Excessive use of chemicals, crop rotation using non-mycorrhizal plants, and intensive tillage practices hampers the symbiotic association and efficiency of AMF. Thus, the natural soil is the main reservoir for a diversity of AM fungi, but due to anthropogenic interventions, the population and diversity of AMF are much lower in the agricultural field (Mbuthia et al. 2015). For instance, high doses of P-fertilization inhibit AM symbiosis with the crop plants (Balzergue et al. 2013). Overdose of P in agricultural fields allows the plants to utilize plenty of P from the soil without exchanging the soluble sugars (Garcia-Caparros et al. 2021), thereby limiting the mycorrhizal appressoria formation. Thus, the requirement of P by the host plant and its availability in the agricultural soil determines the extent of mycorrhization. Similar to P, the application of a disproportionate amount of N in the soil negatively impacts AM colonization (Lin et al. 2020). Furthermore, the use of fungicides and pesticides in the crop plants also degrades the AMF association (Channabasava et al. 2015; Kuila and Ghosh 2022), thereby making it more difficult for the AMF to function properly in the field conditions. Hence, minimal use of chemical substances might enhance the field applicability of AMF in conferring plant disease resistance.

One of the wide practices in conventional cultivation is the tillage of agricultural land. It has been estimated from various studies that tillage can crash the AMF spores and cause physical damage to them by disrupting the hyphal networks (Brito et al. 2012). Because of the tillage system, the soil layers get altered in the fields, ultimately making the soil environment suppressive for AMF. In addition to that, no-till activity for a longer duration also reduces soil AMF population (Schlüter et al. 2018). Thus, minimizing the tillage practices in agricultural land may help in the proper proliferation of AM fungi. Moreover, crop rotation is another factor that hampers the proper growth and functioning of AMF. The use of non-mycorrhizal partner plants during crop rotation adversely affects AMF development (Kuila and Ghosh 2022). Therefore, the rational use of various agricultural practices will help in overcoming constraints associated with AMF application. Furthermore, appropriate preparation of mycorrhizal inoculum and their compatibility with the host and soil samples will also determine the effectiveness of AMF in plant disease management.

#### 9.8 Conclusion and Future Prospects

Control of plant disease by means of chemical substances hampers the ecosystem processes and poses a threat to mankind. To avoid/minimize such problems in the agricultural sector, alternative means have been taken into consideration in the recent decade. Due to the widespread availability and eco-friendly nature, the use of AMF in controlling plant diseases has gained popularity nowadays. AMF association enhances plant growth and development and also provides tolerance to various biotic and abiotic stresses. Besides having so many advantages in the application of AMF for agricultural purposes, a few limitations also exist for which the AMF-based solutions are unable to replace/reduce the use of chemicals. Furthermore, the tripartite interaction involving AMF, host plants, and indigenous microbes associated with the host may not always act synergistically to improve plant functionality in field conditions. Thus, depending upon the colonization, survival rate, and potency of plant disease control, a careful selection of the AMF species is very necessary. Furthermore, the interaction between the plant and microbes must also be precisely screened by understanding the mechanism of biocontrol. The application of AMF individually or in consortium with other microbes can provide an eco-friendly approach to plant disease management. However, understanding the in-depth mechanisms and other environmental/edaphic factors involved in AMF-mediated plant disease control will help to devise more effective future strategies.

Acknowledgement The authors are thankful to DST, Govt. of India for providing DST-FIST Support to Department of Botany, Gauhati University and DST-PURSE support to Gauhati University, where this work was carried out.

#### References

## Chapter 10 Applications of Arbuscular Mycorrhizal Fungi in Controlling Root-Knot Nematodes

Maryluce Albuquerque da Silva Campos

Abstract Meloidogyne nematodes cause diseases in economically important plants. These sedentary endoparasites modify plant roots, creating feeding sites and leading to the formation of root galls. Arbuscular mycorrhizal fungi (AMF) form mutualistic associations with many plants, enhancing nutrient uptake and overall plant health. AMF can also provide protection against pathogens, making them valuable for biocontrol. Studies have shown that AMF can reduce the number of Meloidogyne galls and eggs while improving plant growth and nutrient absorption, potentially outperforming chemical pesticides. AMF affect Meloidogyne infection at various stages, such as making roots less attractive to nematodes and reducing giant cell formation in galls. There is an increase in the production of protective molecules, compounds, and defense genes in mycorrhizal plants infected by Meloidogyne, standing out phenolic compounds and defense enzymes like peroxidase and polyphenol oxidase. The activation of defense genes and pathways is suggested to play a role in the tolerance of mycorrhizal plants to Meloidogyne. However, there is still a need for further research to understand the physiological and genetic modifications that occur in plants infected by Meloidogyne and associated with AMF.

Keywords Meloidogyne · AMF · Mycorrhiza · Bioprotection · Sustainability

#### 10.1 Introduction

Among phytonematodes, which are nematodes causing diseases in plants, those of the genus Meloidogyne stand out for causing diseases in many economically important plants, and in some of these, they are quite aggressive and challenging to control. These nematodes are classified as sedentary endoparasites, settling close to the central cylinder of the plant root, modifying the root cells by creating their own feeding site. In the places where these feeding sites are located, the root becomes

M. A. da Silva Campos (✉)

University of Pernambuco (UPE) Campus Petrolina, Petrolina, Pernambuco, Brazil e-mail<EMAIL_ADDRESS>
<sup>©</sup> The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 G. J. Ahammed, R. Hajiboland (eds.), Arbuscular Mycorrhizal Fungi and Higher Plants, https://doi.org/10.1007/978-981-99-8220-2\_10

swollen, forming the so-called galls, a term commonly used to name these nematodes, known as root-knot nematodes (Perry and Wright 1998).

Arbuscular mycorrhizal fungi (AMF), found in the soil, form a mutualistic symbiotic association called mycorrhiza with 80% of plants. They absorb nutrients from the soil, which are provided to the plants in exchange for carbohydrates and lipids. This association is quite ancient, and it is believed to have been essential for the migration of plants from aquatic environments to terrestrial ones. AMF belong to the phylum Glomeromycota (Tedersoo et al. 2018), which comprises approximately 300 species that do not exhibit specificity toward their host; in other words, the same species can associate with any plant species. However, preferences exist, considered compatibility between the fungus and the plant, making the association established between the AMF species and the plant species a promising one, with the robust development of both symbionts and better adaptation to the conditions to which they are exposed (Silva et al. 2004; Püschel et al. 2021; Amanifar et al. 2019; Coninx et al. 2017).

Considering the benefits arising from mycorrhization with AMF, tolerance to biotic stresses, such as pathogens, is well documented in the literature. In this way, these fungi can be used in biocontrol, reducing the number of pathogenic individuals, while allowing better plant development and growth and increased nutrient absorption (Hajra et al. 2015).

Numerous articles address the topic of biocontrol using AMF in plants infected by Meloidogyne. In most articles, with experiments conducted in greenhouses or in the field, the presence of AMF reduces the number of galls and parasite eggs, while the plant develops well and produces flowers and fruits normally, in addition to having a higher content of nutrients such as P, N, and K, so the plant is in a situation of tolerance to the pathogen in the presence of AMF (Campos 2020). This scenario depends on the species and isolate of AMF used, as well as the plant species and cultivar, and the factors that can influence this association, such as the substrate.

Some articles highlight that the use of AMF in controlling Meloidogyne is equal to or greater than the use of chemical pesticides (Saad et al. 2012; Odeyemi et al. 2013; Abo-Korah 2022). Thus, the use of AMF in controlling Meloidogyne would be as efficient as the use of chemical products that are harmful to nature, accumulating in the soil and contaminating the plants, while AMF are natural soil organisms that do not cause any damage to nature, on the contrary, they contribute to the maintenance of ecosystems and cultivation areas.

It is worth noting that the addition of other beneficial microorganisms, as well as natural substrates used in plant production, such as organic fertilizers, plant extracts with nematicidal effects, can positively contribute to the beneficial effect of AMF.

Although there are numerous articles dealing with the use of AMF for Meloidogyne control, most only highlight the best results according to the tests performed, providing specific information about plant growth, production, and the reduction of Meloidogyne quantity. Few articles address possible physiological and genetic modifications, making this topic still in its early stages and in need of elucidation for a better understanding of the action of AMF in infected plants. Therefore, this chapter aims to provide a brief description of the main causes related

Fig. 10.1 Mechanisms provided by arbuscular mycorrhizal fungi in the protection of plants infected by Meloidogyne. Created in BioRender.com

to the bioprotection conferred by AMF on plants infected by Meloidogyne and address the latest developments on this topic, citing some possible strategies (Fig. 10.1).

#### 10.2 Key Known Causes of Mycorrhizal Plant Tolerance to Meloidogyne

The mechanisms involved in reducing the quantity of Meloidogyne in plants associated with AMF range from competition for space and nutrients to the activation of plant defense mechanisms, with an increase in the number of genes involved in this process, as well as possible protective molecules (Sá and Campos 2020; Campos 2020; Ahamad et al. 2023; Malviya et al. 2023). However, studies regarding the biochemical, physiological, and genetic description of protection against Meloidogyne modulated by AMF are scarce; thus, there are still many gaps regarding this topic.

It is important to highlight that plants associated with AMF are more robust, as these fungi enhance nutrient and water absorption, making these plants better nourished and vigorous and thus better equipped to withstand the presence of the pathogen (Kolawole et al. 2018; Campos 2020). Table 10.1 summarizes the effects on AMF, Meloidogyne, and plant parameters in experiments with AMF and Meloidogyne. Furthermore, the presence of AMF associating with plants can positively influence soil microbiota by stimulating other beneficial organisms (Tiwari et al. 2017; Campos 2020).

A highly enlightening aspect related to the tolerance of mycorrhizal plants to Meloidogyne is the negative influence during the preinfection, infection, and postinfection phases exerted by AMF. In the preinfection phase, it was observed that the presence of AMF in plants rendered the roots less attractive for nematode infection. This could be attributed to a reduction in the quantity of root exudates or alteration of these exudates. Another possibility could be the accumulation of phenols, such as phytoalexins, flavonoids, and isoflavonoids, which render the root less suitable for pathogen penetration (Morandi 1996). In addition to an increase in the amount of lignin in the roots (Malviya et al. 2023).

In regard to infection, a lower quantity of giant cells in galls was observed in the presence of FMA (Siddiqui and Mahmood 1998), in addition to the reduced size of these cells (Hajra et al. 2015). Another characteristic observed in roots associated with FMA is smaller nematodes, which take longer to develop into adults (Vos et al. 2012a). These modifications result in lower egg production by Meloidogyne, reducing the reproductive rate, a characteristic associated with the postinfection phase, as observed in most articles related to this topic (Campos 2020).

The causes mentioned above require more detailed studies that can elucidate how each one occurs, which molecules are involved, and which mechanisms could be employed, in addition to the steps involved.

#### 10.3 Protective Molecules, Compounds and Defense Genes in Mycorrhizal Plants Infected by Meloidogyne

The most innovative study regarding the utilization of FMA in the control of Meloidogyne involves the qualification and quantification of protective molecules, defense compounds, as well as defense-related genes. Several molecules and compounds produced by the plant are considered important for plant defense. While there are reports of many different types of defense molecules and compounds, few are known in relation to mycorrhizal plants infected by Meloidogyne. Most articles quantify these defense compounds, most of which belong to the phenols group. When it comes to molecules, which are also quantified and/or tested for activity, defense enzymes stand out in the studies.

Positive correlations of AMF with plant growth, phenolic content, photosynthetic pigments, and defense enzyme activity were observed by Ahamad et al. (2023) in mycorrhizal and Meloidogyne incognita-infected carrot plants (Daucus carota L.). In the same article, the authors observed that the activity of peroxidase and polyphenol oxidase, defense enzymes, increased in the presence of AMF and Meloidogyne.

Table 10.1 (continued)

NO not observed

For various phenolic compounds, negative effects on nematode chemotaxis, motility, or survival have been reported (Wuyts et al. 2006). The quantification of phenolic compounds in the root was higher in the treatment with a mixture of AMF in the presence of M. incognita in Impatiens balsamina L. plants and fertilized soil. The same result was observed regarding the quantification of antioxidants, although in the latter case, the result extended to the aerial part of the plant (Banuelos et al. 2014). In the article, both compounds are considered plant defense compounds. There was a higher concentration of phenolics in cassava (Manihot esculenta Crantz.) plants inoculated with AMF in the presence of Meloidogyne spp., both in treatments where AMF and nematodes were inoculated simultaneously and in treatments where nematodes were inoculated 30 days after AMF inoculation (Sery et al. 2016).

In chamomile plants (Matricaria recutita L.) infected with M. incognita, the inoculation with the AMF Glomus intraradices, as well as the bacteria Bacillus megaterium and the filamentous fungus Trichoderma harzianum, resulted in the best treatment, exhibiting higher content of flavonoids and phenols, along with greater antioxidant capacity (Gupta et al. 2017).

Sharma and Sharma (2017a) in a study exploring the quantification of defense enzymes in tomato plants (Solanum lycopersicum L.) infected with M. incognita and inoculated with the beneficial bacteria Pseudomonas jessenii and the AMF Rhizophagus irregularis, the researchers observed that the treatment containing all microorganisms led to an increase in the quantity of polyphenol oxidase, peroxidase, malondialdehyde, hydrogen peroxide, superoxide dismutase, as well as an increase in the quantity of phenols. In another study by the same authors, they observed higher activity of the enzymes polyphenol oxidase, peroxidase, and superoxide dismutase, while there was a reduction in the content of malondialdehyde and hydrogen peroxide compounds in tomato plants inoculated with R. irregularis and infected by M. incognita (Sharma and Sharma 2017b).