Nematophagous Fungi: A Review of Their Phosphorus Solubilization Potential

Nematophagous fungi (NF) are a group of diverse fungal genera that benefit plants. The aim of this review is to increase comprehension about the importance of nematophagous fungi and their role in phosphorus solubilization to favor its uptake in agricultural ecosystems. They use different mechanisms, such as acidification in the medium, organic acids production, and the secretion of enzymes and metabolites that promote the bioavailability of phosphorus for plants. This study summarizes the processes of solubilization, in addition to the mechanisms of action and use of NF on crops, evidencing the need to include innovative alternatives for the implementation of microbial resources in management plans. In addition, it provides information to help understand the effect of NF to make phosphorus available for plants, showing how these biological means promote phosphorus uptake, thus improving productivity and yield.


Introduction
Pathogenic threats and nutritional deficiency are the main problems that affect yield and productivity in plant crops [1], increasing the need to apply new microbial agents to solve both obstacles [2]. Thus, NF have been investigated because of their dual ability as nematode controllers and as plant growth promoters that increase the bioavailabilty of nutrients [3].
Phosphorus (P) is the second most important element for plants after nitrogen. It regulates metabolism and plant development and growth, and an adequate supply is needed to keep these metabolic functions [4]. In addition, it is found in abundance in soil, although its uptake is low, which results in a limitation for the productivity of agricultural crops [5]. P can be solubilized by several soil microorganisms (saprotrophic bacteria and fungi) [6]. Phosphate-solubilizing microorganisms (PSM) play an important role in soil by mineralizing organic P, solubilizing inorganic P minerals, and storing P in biomass [7], thus improving plant growth and yield [8]. PSM can solubilize phosphorus by secreting protons and producing organic anions, such as citrate, oxalate, and gluconate [9]. However, the amount of P solubilization depends on the microbial strain and the relationship between carbon sources, P, and organic acid production [10].
Bacteria can release organic acids that solubilize P or produce acid and alkaline phosphatases that mineralize organic P [11]. There are bacterial genera that are known to be very efficient in these mechanisms, such as Arthrobacter, Bacillus, Burkholderia, Natrinema, Bacteria can release organic acids that solubilize P or produce acid and alkaline pho phatases that mineralize organic P [11]. There are bacterial genera that are known to b very efficient in these mechanisms, such as Arthrobacter, Bacillus, Burkholderia, Natrinem Pseudomonas, Rhizobium, and Serratia [12]. Bacteria are superior to fungi in colonizing plan roots, but are less tolerant to acids [13], so fungi have great potential to solubilize P acidic conditions [14]. Fungi, in fact, have a higher capacity to release P compared wit bacteria [15]. They use different mechanisms such as the secretion of fatty acids, the pro duction of enzymes, and the discharge of metabolic substances (siderophores) that ca rapidly metabolize P [16].
Despite the numerous studies on NF as biocontrol agents, the comprehensive an complete understanding of the mechanisms of action is still incipient [17]. In general, th interactions of NF have been evaluated to reduce the densities of plant pathogenic nem todes [18]. Yet, they present other benefits such as the synthesis of phytohormones, as we as favoring the absorption of P [19]. The inoculation of P solubilizing microbial agents seeds, crops, and soil aims at improving agricultural production without affecting so health [20]. Bioformulations based on fungal strains that increase plant yield and are als effective at solubilizing nutritional elements are needed [21]. Therefore, the objective this review is to broaden the understanding of NF and their potential use in P solubiliz tion, focusing on their mechanisms of solubilization and the benefits provided in the nu trition of agricultural crops.

Mechanisms of P solubilization by NF
Plants can harness various forms of P; nevertheless, roots take up negatively charge forms of orthophosphate ions (HPO4 2− -and HPO4 − ) depending on the soil pH [22]. Al hough agriculture practices introduce considerable amounts of phosphate in soil, only 1 to 30% of P is taken up by plants [23]. It generally binds to iron and aluminum oxides an hydroxides, or to calcium in calcareous soils, becoming unavailable for roots due to chem ical precipitation or physical adsorption [24]. Therefore, the application of P itself does n contribute to the improvement of agricultural production systems ( Figure 1). (a) Nematodes can be parasitized and predated by (in clockwise order) trapping fungi, egg-parasite fungi, special device-producing, or toxin-producing and endoparasitic species. (b) Compounds produced by fungi. (c) Chemical reactions of P in soil, binding with elements such as calcium, iron, and aluminum that can then be mineralized or solubilized by the NF compounds. (d) Roots absorbing negatively charged orthophosphate ions. Several microorganisms have been studied in relation to the solubilization of P. Fungi and bacteria use different mechanisms on soluble phosphates, especially acidification of the medium, chelation, exchange reactions, and the production of solubilizing enzymes and of various organic acids [4,8,20,25]. Therefore, some species are able to solubilize and mobilize P for plants, including, several nematode controlling fungi. An example is Trichoderma harzianum, which can acidify the medium, produce chelating metabolites, and produce a redox activity (capable of reducing Fe and Cu) [26].

Organic Acids
Organic acids or anions range from fatty acids to secondary metabolites [22]. They are by-products derived from bacteria and fungi that are able to mobilize P in their microenvironment. Organic acids improve P release so that it can be absorbed by plants [27]. Filamentous fungi use several mechanisms to solubilize minerals and they produce high amounts of organic acids [28]. Species of the genera Aspergillus, Penicillium, Trichoderma, and Fusarium solubilize P by producing different types of organic acids [14,[29][30][31][32]. Furthermore, it is possible that some filamentous fungi such as Aspergillus niger solubilize P when exposed to high salt contents (4% NaCl) [33]. Even on ferric phosphate substrates, A. niger can secrete large amounts of citric and oxalic acids to release P [34]. Therefore, the potential of filamentous fungi in the agrobiotechnology industry is increased, especially because of their properties, for the production of organic acids [35].

Solubilizing Enzymes
Organic P from animal and plant residues can be mineralized by the action of phosphatases [36], such as phytases that hydrolyze the phospho-monoester bonds present in phytates [37]. Nematode controlling fungi can produce phytases during their development or the production of trapping structures [38]. The nematode-capturing fungus Arthrobotrys oligospora forms phytases while building adhesive networks, significantly increasing the release of P [39]. Among the genera of NF that mainly produce phytases and phosphatases under laboratory conditions are the filamentous fungi Aspergillus, Penicillium, and Trichoderma [40].

Siderophore Production
These are low molecular weight organic iron chelating compounds. They can be produced by bacteria, fungi, and plants [40]. Among the most studied fungi for siderophore production are Aspergillus fumigatus and Aspergillus nidulans, which have 55 similar types of siderophores [41,42]. In addition, Penicillium produces siderophores, capable of solubilizing tricalcium phosphate, even in contaminated soils, opening new opportunities in the field of phytoremediation and agrobiotechnology [43]. Therefore, the use of siderophores is increasing in use as a new alternative for agriculture, to replace pesticides and synthetic fertilizers [44].

Potential of NF in P Solubilization
NF can be used in agriculture through the massive production of infective spores or through formulations that improve growth, viability, and efficacy [45]. They use several mechanisms of recognition, signaling, differentiation, and penetration of the cuticle or egg shell of nematodes through mechanical and enzymatic actions [46]. For the purposes of this study, NF are classified according to their mechanism of attack, including trapping, endoparasitic, egg parasitic, toxin-producing, and special device-producing species [47]. NF interact with the environment, performing essential functions to maintain the stability of food webs and the cycling of nutrients in soil [48].
NF widely used in management plans as biological control agents include Purpureocillium lilacinum, Pochonia chlamydosporia, Trichoderma harzianum, Arthrobotrys spp., Hirsutella spp., etc. [49]. Some of these fungi are widely used in agriculture due to their ability to infect and kill arthropods [50]. They are recognized as biopesticides in the management of destructive pests. Some notable examples include Beauveria bassiana [49], Aspergillus fijiensis [51], Cladospo-rium tenuissimum, and Penicillium citrinum [52]. Among NF, some have been reported as P solubilizers. Trichoderma is one of the most studied genera and is considered as a growth promoter, pathogen controller, and nutritional promoter of plants [53,54]. Furthermore, some NF can solubilize P, even under low temperature conditions [55].

Trapping Nematodes-Predatory Fungi
Fungi that capture nematodes have a double function: controlling the populations of infective nematodes and also acting as biofertilizers when applied in soil, being able to solubilizing and mobilize some important nutrients [56,57]. The adhesive networks building fungus Duddingtonia flagrans have been shown to solubilize P both in laboratory [56] and in greenhouse conditions [2]. Likewise, A. oligospora has been used successfully in the solubilization of phosphate rock in in vitro and in vivo conditions (Figure 2), showing promising results in the uptake of P by plants [58]. endoparasitic, egg parasitic, toxin-producing, and special device-producing species [47]. NF interact with the environment, performing essential functions to maintain the stability of food webs and the cycling of nutrients in soil [48].
NF widely used in management plans as biological control agents include Purpureocillium lilacinum, Pochonia chlamydosporia, Trichoderma harzianum, Arthrobotrys spp., Hirsutella spp., etc. [49]. Some of these fungi are widely used in agriculture due to their ability to infect and kill arthropods [50]. They are recognized as biopesticides in the management of destructive pests. Some notable examples include Beauveria bassiana [49], Aspergillus fijiensis [51], Cladosporium tenuissimum, and Penicillium citrinum [52]. Among NF, some have been reported as P solubilizers. Trichoderma is one of the most studied genera and is considered as a growth promoter, pathogen controller, and nutritional promoter of plants [53,54]. Furthermore, some NF can solubilize P, even under low temperature conditions [55].

Trapping Nematodes-Predatory Fungi
Fungi that capture nematodes have a double function: controlling the populations of infective nematodes and also acting as biofertilizers when applied in soil, being able to solubilizing and mobilize some important nutrients [56,57]. The adhesive networks building fungus Duddingtonia flagrans have been shown to solubilize P both in laboratory [56] and in greenhouse conditions [2]. Likewise, A. oligospora has been used successfully in the solubilization of phosphate rock in in vitro and in vivo conditions (Figure 2), showing promising results in the uptake of P by plants [58].

Endoparasitic and Egg-parasitic Fungi
Endoparasitic fungi are obligate pathogens that infect nematodes. Conidia of the obligate endoparasitic fungus Drechmeria coniospora are ingested or adhere to the cuticle of its hosts [59]. The spores produce a germination tube that enters the nematode, producing a mycelium that, by degrading internal tissues, exploits the host nutrients for growth [60]. One of the most studied fungal parasites of nematode eggs is the fungus Pochonia chlamydosporia, able to reduce populations of several plant parasitic nematodes, including species of Globodera, Heterodera, Meloidogyne, Nacobbus, and Rotylenchulus [61]. Pochonia chlamydo-

Endoparasitic and Egg-parasitic Fungi
Endoparasitic fungi are obligate pathogens that infect nematodes. Conidia of the obligate endoparasitic fungus Drechmeria coniospora are ingested or adhere to the cuticle of its hosts [59]. The spores produce a germination tube that enters the nematode, producing a mycelium that, by degrading internal tissues, exploits the host nutrients for growth [60]. One of the most studied fungal parasites of nematode eggs is the fungus Pochonia chlamydosporia, able to reduce populations of several plant parasitic nematodes, including species of Globodera, Heterodera, Meloidogyne, Nacobbus, and Rotylenchulus [61]. Pochonia chlamydosporia benefits the plant, as it is able to control nematode infections, shortening the flowering and fruiting times (up to 5 and 12 days), as well as significantly increasing root growth [62]. This parasitic fungus produces phosphatases, organic acids, and propionic acid, which promote the depolymerization of phosphate compounds [63,64]. Furthermore, in association with the predatory fungus D. flagrans, they can significantly increase nutrient uptake, especially P, by up to 70% in tomato plants [3]. Therefore, research studies suggest the use of bioformulated P. chlamydosporia for the management of phytoparasitic nematodes, with the potential to support plant nutrition [65].
Purpureocillium is a further fungal genus used for the reduction of phytoparasitic nematode eggs and females. Some species attack the gall-forming nematodes Meloidogyne spp. [66], and show a P solubilization capability in laboratory conditions [67], as well as promoting tomato seedling growth in greenhouse conditions [68]. These fungi may secrete hydrolytic enzymes and siderophores that solubilize P and promote plant growth [69].

Toxin Production
Toxin-producing fungi are characterized by the release of compounds that paralyze nematodes and produce a rapid and systemic cell necrosis in multiple tissues throughout their bodies. In the order of the Agaricales, one representative is Pleurotus ostreatus, which is a toxin-producing fungus capable of producing necrosis after the contact of hyphae with the cilia of the sensory system of nematodes [70]. The fungus has P solubilizing properties [71] and is applied as a biofertilizer based on biomasses obtained as mycelial by-products [72].
Piriformospora indica is an endosymbiotic fungus belonging to the order Sebacinales (Basidiomycota), capable of producing metabolites that inhibit nematode populations [73]. This species has shown synergy with phosphate-solubilizing bacteria, sustaining the growth of chickpea plants [74]. Studies have indicated that the transfer of P by P. indica to the host plant has a significant effect on its development [75].

Filamentous Fungi with Nematophagous Activity
In addition to the examples mentioned above, there are a variety of fungi that do not use mechanisms based on capture, parasitism, toxins, or special devices, so it is presumed that hydrolytic enzymes are a key in the nematode infection process [76]. Among the known genera that work as both biocontrol and P solubilizers are Trichoderma, Fusarium, Penicillium, and Aspergillus. Filamentous fungi can secrete hydrolytic enzymes, organic acids, and low molecular weight natural products, which confer several functions, including P solubilization [77], and are able to solubilize calcium and iron phosphates [78]. Thus, they hold great potential for the development of biofertilizers, contributing to soil fertility and promoting plant growth [79], which are essential in sustainable agriculture (Table 1) [80].
Filamentous fungi such as Trichoderma spp. are often dominant in soil microbial communities and have the ability to colonize roots [81]. Trichoderma spp. are marketed all over the world for their potential as biocontrol and biostimulant agents for numerous agricultural crops. The application of Trichoderma spp. and related metabolites allegedly improves crop productivity, nutrient supply, and defense against plant pathogens [54,82], as it combines P solubilization and nematode predation.

Agricultural Solutions: Application of NF in P Solubilization
Although P is added to the soil of agricultural crops, chemical fertilizer synthesis is an energy-consuming process with long-term impacts on the environment in terms of eutrophication, fertility decrease, and carbon footprint [97]. Nematophagous species have been used for plant growth promotion and to sustain yields, especially members of the genera Trichoderma, Purpureocillium, Pochonia, Fusarium, Arthrobotrys, and Verticillium [98].
Several NF are beneficial because of the production of phytohormones, antibiotics, or siderophores, which benefit long-cycle and short-cycle crops [99] (Table 2), as shown by the P solubilization activity in alkaline soils, which increase the yields of corn and wheat [100,101]. These NF potentially shorten the maturity period of crops, improve fruit quality, increase the availability of soluble P, and improve soil biodiversity [102]. Thus, there is a positive link between the application of these fungal strains and the P content in soil and plants [103].
Currently, several investigations suggest the use of fungi as bioinoculants to increase the yield in agricultural crops [104], as well as ornamentals. P solubilization has been in fact also reported in shrubs within ornamental greenhouses, as demonstrated by the application of Mortierella sp. on seedlings of Leucaena leucocephala [105].

Pleurotus ostreatus Maize
Increase in root and shoot lengths, fresh and dry root weights, fresh and dry shoot weights, chlorophyll content, and nutrient uptake [71] Trichoderma, Purpureocillium Banana Active production of indole-3-acetic acid IAA and solubilize insoluble phosphate [99] Trichoderma Soy bean Plant growth promotion increase of P uptake efficiency [82,87] Aspergillus niger, A. fumigatus, Penicillium pinophilum Wheat and faba bean Yield of wheat grains and faba bean seed production [108] Penicillium oxalicum Wheat and maize Replace chemical fertilizer in alkaline soils. Improved crop production [101] Penicillium sp. Penicillium oxalicum Maize Increased uptake of P by plants and increased availability of P in soil [100]

Future Research Perspectives
The main P source in the world, phosphate rock, is fundamental for food production. It is strongly threatened as it is a finite resource. P, for the most part, is limited to a few countries in the world, particularly Morocco, which holds 75% of the world reserve [113]. Therefore, in recent years, the challenge of ensuring sustainable global P management to achieve world food security has been evidenced [114]. Given the current agricultural practices dependent on the continuous supply of commercial P-based fertilizers [115], the need to search for new sustainable strategies to manage the P availability in agricultural fields has increased [116]. Therefore, biological activators must be used to accelerate the bioavailability of P for plants, making NF important not only for the biological control of nematodes, but also for P solubilization. Their applications are also in accordance with sustainable agricultural practices for developing countries [117].
Currently, microbial-based fertilizers are not only considered for productivity and economic benefits, but also for their use as environmental-friendly products [18]. The aim is to guarantee less damage to water quality, increase nutrients recycling, reduce the consumption of resources, improve soil health, and increase the biodiversity of beneficial microorganisms [118]. Therefore, there is a demand to join efforts towards the search and discovery of microfungi, especially from little-explored natural regions [119], with the potential to sustain crop nutrition [120]. In fact, the excessive use of fertilizers affects soil health, adding to the presence of pathogens and pests [121].
Filamentous fungi with a dual activity (nematicide and P solubilizers) have versatile capacities to synthesize biocompounds such as enzymes, organic acids, and metabolites [122]. Especially, for P bioavailability, they can biologically produce high concentrations of organic acids, offering new knowledge through the detection of genes related to the mechanisms of phosphate solubilization [24,120,123]. Biotechnology applications may offer sustainable solutions based on microorganisms that are tolerant to new environmental conditions, including the microbiota that favors plant nutrition [124].

Conclusions
Several NF have the ability to suppress nematode parasites and improve the uptake of nutritional elements in order to promote plant growth and development. They have been successfully applied as biofertilizers and biocontrol agents to establish beneficial ecological relationships within their environment. For P solubilization, they rely on different mechanisms, such as a decrease in pH and the production of siderophors, organic acids, and enzymes. The wide variety of mechanisms for P solubilization in NF can be harnessed to reduce the dependence on P-based fertilizers in agriculture. The availability of new enzymes such as phytases promotes the search for beneficial microorganisms to develop environmentally-friendly and sustainable management plans.