Arbuscular Mycorrhiza-Associated Rhizobacteria and Biocontrol of Soilborne Phytopathogens

*Meenakshi Singh, Manjari Mishra, Devendra Kumar Srivastava and Pradeep Kumar Singh*

### **Abstract**

The mutualistic symbiosis of most land plants with arbuscular mycorrhizal (AM) fungi has been shown to favor mineral and water nutrition and to increase resistance to abiotic and biotic stresses. The main mechanisms involved in the control of the disease symptoms and intraradical proliferation of soilborne phytopathogens are due to root colonization with AM fungi. The role of the rhizobacteria is shown to be specifically associated with extraradical network of the AM and mycorrhizosphere. The mycorrhizosphere can form a favorable environment for microorganisms which have potentiality to act antagonistic to pathogen abundance. It makes an additional advantage in identifying rhizobacteria from AM fungi structures or mycorrhizosphere, which often lead to the isolation of organisms having strong properties of antagonism on various soilborne pathogens. The ability of AM fungi to control soilborne diseases is mainly related to their capacity to stimulate the establishment of rhizobacteria against the favorable environment of pathogen within the mycorrhizosphere prior to the root infection. Recent advancement in scientific research has provided more clear picture in understanding the mechanisms involved in AM fungi/rhizobacteria interactions. Herein, this chapter includes the mechanisms of the AM fungi-mediated biocontrol, interactions between AM-associated bacteria and AM fungus extraradical network, AM-associated bacteria and biocontrol activities and unfavorable zone to pathogen development: the mycorrhizosphere.

**Keywords:** AM-associated bacteria (AMB), arbuscular mycorrhizal fungi, biocontrol, mycorrhizosphere, soilborne pathogens

### **1. Introduction**

A majority of land plants in nature are growing symbiotically in relationship with AM fungi. This relationship is well established with the roots of these plants. Soil exploration by the external mycelium of AM fungi increases the nutrient absorptive root surface area and thus favors the host plant in access to nutrients and water [1, 2]. Moreover, as the largest component of the soil microbial biomass [3, 4], AM fungi form widespread mycelial networks within the soil atmosphere, and hyphae harbour important sites for interactions with other soilborne microorganisms. The constricted zone adjacent to soil-living roots is called the

rhizosphere [5]. It is characterized by increased microbial activity and by a specific microbial community structure [6, 7]. Along with root-AM fungi associations, factors influencing the community structure and the biomass of soil microorganisms lead to the establishment of a zone called mycorrhizosphere [8–12]. The zone of soil influenced by only AM fungi is called mycosphere. In the mycorrhizosphere, AM fungi structures and various rhizobacteria (AM fungi-associated rhizobacteria or AMB, e.g. *Paenibacilli*, *Bacilli* and *Pseudomonas* spp.) are generally identified by classical culture-dependent methods [13, 14]. It includes phospholipid fatty acid analysis (PLFA) [15] and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) [13, 16, 17] which reinforce the hypothesis that AM fungi structures constitute important nutrient-rich niches for soilborne microorganisms. *Glomeribacter gigasporarum* (a new taxon of *Burkholderiaceae*) was even described as a Gram-non-cultivable (obligatory) bacterial endosymbiont of spore vacuoles, mycelium and intraradical hyphae of *Gigaspora margarita* [18]. *Glomeribacter gigasporarum* described in detail shows to be widespread within *Gigasporaceae*; it transmitted vertically and contains nitrogen fixation genes [19–21], while in *Gigaspora margarita*, it has been suggested and observed that this AM fungus might fix nitrogen and then deliver it to the symbiotic plant through the associated bacterial population [22]. The effects of this on host plant physiology can be recognized in mycorrhizal root colonization because of the consequence of the activity of specifically AM fungi-associated rhizobacteria.

The beneficial effects of AM fungi on the host plant physiology, in the decrease of intraradical and mycorrhizosphere population and in the decrease of disease symptoms of soilborne pathogens were reported in many biological systems, probably due to synergistic mechanisms [23–25]. The use of chemical pesticides are now avoided and not advocated in fields due to its risks to human health and the environment, and thus the implementation of sustainable agriculture has become essential in crop industry. The perception of the mechanisms involved in the AM fungi-mediated biocontrol will allow to maximize the performance of management of such sustainable agroecosystems and thus authorize the use of AM fungi and its benefits [26]. The main mechanisms involved in the biological control of diseases induced by soilborne phytopathogens start after root colonization with AM fungi especially due to its association with rhizobacteria which constitutes major element for this biocontrol.

### **2. Mechanisms of the AM fungi-mediated biocontrol**

Reduction in the detrimental effects of soilborne pathogens after root colonization with AM fungi was described a long time ago [27, 28] and has been observed on various fungi, stramenopiles, nematodes and bacteria [12, 29]. Carlsen et al. [30] reported the total check of infectivity caused by *Pythium ultimum* on clover plants cv. Sonja by using *Glomus mosseae* as a symbiotic relation partner. For the biological control of pathogen, AM fungus or AM fungi/plant taxa association, conditions of culture, level of root colonization, time of AM fungus or pathogen inoculation and harvest, the mechanisms hypothesized, etc. should be involved [12, 23, 24, 29, 31–35]. The disease symptoms induced by pathogens can systemically be reduced in non-mycorrhizal roots of plants grown in AM fungi-inoculated split-root systems [36]. Various hypotheses have been suggested in an endeavor to elucidate the AM fungi-mediated biocontrol of soilborne phytopathogens. The fact that pathogen-induced symptoms are systemically regulated by AM fungi colonization is related to the establishment of induced systemic resistance (ISR) [37]. ISR is a resistance mechanism induced or acquired in plants which were already undergone

**103**

mycorrhizosphere.

*Arbuscular Mycorrhiza-Associated Rhizobacteria and Biocontrol of Soilborne Phytopathogens*

for pretreatment with a variety of organisms and compounds [e.g. superoxide dismutases (SOD) and peroxidases, pathogenesis-related type 1 proteins (PR-1

Further, higher concentrations of phenolic acids could be detectable in plants which are colonized with AM fungi species subjected for biocontrol activities. Accumulation of jasmonic acid involved in the rhizobacteria-mediated ISR in mycorrhizal roots could be related to the systemic pathogen biocontrol [38, 39]. Cordier et al. [40] identified local cell wall modifications (callose accumulation around arbuscule-containing cortical cells of tomato roots). The synthesis of constitutive and additional isoforms of defense-related enzymes (e.g. chininases, chitosanases, β-1,3-glucanases, peroxidases and SOD) has also been locally detected in mycorrhizal roots [41–43]. The level of production of these enzymes or flavonoids was reported to be unrelated to the capacity of biocontrol of the AM fungi species [30, 44]. The transcript profiling and real-time quantitative PCR used to explore the transcriptional changes triggered by AM fungus colonization revealed a complex pattern of local and systemic changes in gene expression in roots of *Medicago truncatula* [45], and transcripts for defense-related proteins were reported to expressed locally. Furthermore, increase in concentrations of defense-related compounds such as rosmarinic acid, caffeic acids, phenolics and essential oils has not been recorded in colonization with *Glomus mosseae* which was reported for its role in protecting basil plants against *Fusarium oxysporum* f. sp. *basilica.* It highlights and indicates the role of other possible mechanisms in the AM fungus-mediated biocontrol activity which differs to stimulation of systemic and localized plant

The most commonly documented response to AM fungi colonization is an increase in phosphorus nutrition to the host plants which subsequently imparts more dynamic and more resistant properties against pathogen invasion. However, AM fungi-mediated biocontrol is unrelated to the soil phosphorus (P) availability and to the phosphorus status in plant tissues, thus possibly more dependent on

Arbuscular mycorrhizal fungi normally compete for space and nutrients with soilborne pathogens within the zone of mycorrhizosphere and the host roots. Larsen and Bodker [50], using signature fatty acid profiles, demonstrated the decrease in biomass and energy reserves of both *Glomus mosseae* and *Aphanomyces euteiches* co-occupying pea roots; however *Phytophthora nicotianae* and *Glomus mosseae* never reported to occupy simultaneously in the same tomato root tissues [40]. A reduction in the extent of mycorrhizal colonization by different plant pathogens has been reported [51–54] indicating the possible occurrence of competitive interactions. The AM fungus is often inoculated before the attack of pathogen in order to favor biocontrol efficiency [54]. However, *Fusarium solani* f. sp. *phaseoli* genomic DNA quantified using quantitative real-time PCR was significantly reduced not only in the mycorrhizosphere and mycosphere but also in the bulk soil of a compartmentalized soil-root system which was inoculated with *Glomus intraradices*, whereas the AM fungus genomic DNA was not significantly modified by the pathogens in the soil [55]. Reduction in *Fusarium solani* f. sp. *phaseoli* growth as well as decrease in root rot symptoms as a result of colonization with *Glomus intraradices* could not be attributed to the competition for resources and habitat between the two fungi but mostly to the biotic or abiotic characteristic factors of the established

The extraradical network formed by *Glomus intraradices* around the roots of the plants has been reported to show a decrease in the growth of nematodes (e.g. *Radopholus similis* and *Pratylenchus coffeae*) and conidial formation of *Fusarium oxysporum* f. sp. *chrysanthemi*. In vitro aseptic conditions and the above-stated

*DOI: http://dx.doi.org/10.5772/intechopen.89266*

proteins)].

defense mechanisms [46].

other mechanisms [46–49].

*Arbuscular Mycorrhiza-Associated Rhizobacteria and Biocontrol of Soilborne Phytopathogens DOI: http://dx.doi.org/10.5772/intechopen.89266*

for pretreatment with a variety of organisms and compounds [e.g. superoxide dismutases (SOD) and peroxidases, pathogenesis-related type 1 proteins (PR-1 proteins)].

Further, higher concentrations of phenolic acids could be detectable in plants which are colonized with AM fungi species subjected for biocontrol activities. Accumulation of jasmonic acid involved in the rhizobacteria-mediated ISR in mycorrhizal roots could be related to the systemic pathogen biocontrol [38, 39]. Cordier et al. [40] identified local cell wall modifications (callose accumulation around arbuscule-containing cortical cells of tomato roots). The synthesis of constitutive and additional isoforms of defense-related enzymes (e.g. chininases, chitosanases, β-1,3-glucanases, peroxidases and SOD) has also been locally detected in mycorrhizal roots [41–43]. The level of production of these enzymes or flavonoids was reported to be unrelated to the capacity of biocontrol of the AM fungi species [30, 44]. The transcript profiling and real-time quantitative PCR used to explore the transcriptional changes triggered by AM fungus colonization revealed a complex pattern of local and systemic changes in gene expression in roots of *Medicago truncatula* [45], and transcripts for defense-related proteins were reported to expressed locally. Furthermore, increase in concentrations of defense-related compounds such as rosmarinic acid, caffeic acids, phenolics and essential oils has not been recorded in colonization with *Glomus mosseae* which was reported for its role in protecting basil plants against *Fusarium oxysporum* f. sp. *basilica.* It highlights and indicates the role of other possible mechanisms in the AM fungus-mediated biocontrol activity which differs to stimulation of systemic and localized plant defense mechanisms [46].

The most commonly documented response to AM fungi colonization is an increase in phosphorus nutrition to the host plants which subsequently imparts more dynamic and more resistant properties against pathogen invasion. However, AM fungi-mediated biocontrol is unrelated to the soil phosphorus (P) availability and to the phosphorus status in plant tissues, thus possibly more dependent on other mechanisms [46–49].

Arbuscular mycorrhizal fungi normally compete for space and nutrients with soilborne pathogens within the zone of mycorrhizosphere and the host roots. Larsen and Bodker [50], using signature fatty acid profiles, demonstrated the decrease in biomass and energy reserves of both *Glomus mosseae* and *Aphanomyces euteiches* co-occupying pea roots; however *Phytophthora nicotianae* and *Glomus mosseae* never reported to occupy simultaneously in the same tomato root tissues [40]. A reduction in the extent of mycorrhizal colonization by different plant pathogens has been reported [51–54] indicating the possible occurrence of competitive interactions. The AM fungus is often inoculated before the attack of pathogen in order to favor biocontrol efficiency [54]. However, *Fusarium solani* f. sp. *phaseoli* genomic DNA quantified using quantitative real-time PCR was significantly reduced not only in the mycorrhizosphere and mycosphere but also in the bulk soil of a compartmentalized soil-root system which was inoculated with *Glomus intraradices*, whereas the AM fungus genomic DNA was not significantly modified by the pathogens in the soil [55]. Reduction in *Fusarium solani* f. sp. *phaseoli* growth as well as decrease in root rot symptoms as a result of colonization with *Glomus intraradices* could not be attributed to the competition for resources and habitat between the two fungi but mostly to the biotic or abiotic characteristic factors of the established mycorrhizosphere.

The extraradical network formed by *Glomus intraradices* around the roots of the plants has been reported to show a decrease in the growth of nematodes (e.g. *Radopholus similis* and *Pratylenchus coffeae*) and conidial formation of *Fusarium oxysporum* f. sp. *chrysanthemi*. In vitro aseptic conditions and the above-stated

*Biostimulants in Plant Science*

rhizosphere [5]. It is characterized by increased microbial activity and by a specific microbial community structure [6, 7]. Along with root-AM fungi associations, factors influencing the community structure and the biomass of soil microorganisms lead to the establishment of a zone called mycorrhizosphere [8–12]. The zone of soil influenced by only AM fungi is called mycosphere. In the mycorrhizosphere, AM fungi structures and various rhizobacteria (AM fungi-associated rhizobacteria or AMB, e.g. *Paenibacilli*, *Bacilli* and *Pseudomonas* spp.) are generally identified by classical culture-dependent methods [13, 14]. It includes phospholipid fatty acid analysis (PLFA) [15] and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) [13, 16, 17] which reinforce the hypothesis that AM fungi structures constitute important nutrient-rich niches for soilborne microorganisms. *Glomeribacter gigasporarum* (a new taxon of *Burkholderiaceae*) was even described as a Gram-non-cultivable (obligatory) bacterial endosymbiont of spore vacuoles, mycelium and intraradical hyphae of *Gigaspora margarita* [18]. *Glomeribacter gigasporarum* described in detail shows to be widespread within *Gigasporaceae*; it transmitted vertically and contains nitrogen fixation genes [19–21], while in *Gigaspora margarita*, it has been suggested and observed that this AM fungus might fix nitrogen and then deliver it to the symbiotic plant through the associated bacterial population [22]. The effects of this on host plant physiology can be recognized in mycorrhizal root colonization because of the consequence

of the activity of specifically AM fungi-associated rhizobacteria.

**2. Mechanisms of the AM fungi-mediated biocontrol**

The beneficial effects of AM fungi on the host plant physiology, in the decrease of intraradical and mycorrhizosphere population and in the decrease of disease symptoms of soilborne pathogens were reported in many biological systems, probably due to synergistic mechanisms [23–25]. The use of chemical pesticides are now avoided and not advocated in fields due to its risks to human health and the environment, and thus the implementation of sustainable agriculture has become essential in crop industry. The perception of the mechanisms involved in the AM fungi-mediated biocontrol will allow to maximize the performance of management of such sustainable agroecosystems and thus authorize the use of AM fungi and its benefits [26]. The main mechanisms involved in the biological control of diseases induced by soilborne phytopathogens start after root colonization with AM fungi especially due to its association with rhizobacteria which constitutes major element

Reduction in the detrimental effects of soilborne pathogens after root colonization with AM fungi was described a long time ago [27, 28] and has been observed on various fungi, stramenopiles, nematodes and bacteria [12, 29]. Carlsen et al. [30] reported the total check of infectivity caused by *Pythium ultimum* on clover plants cv. Sonja by using *Glomus mosseae* as a symbiotic relation partner. For the biological control of pathogen, AM fungus or AM fungi/plant taxa association, conditions of culture, level of root colonization, time of AM fungus or pathogen inoculation and harvest, the mechanisms hypothesized, etc. should be involved [12, 23, 24, 29, 31–35]. The disease symptoms induced by pathogens can systemically be reduced in non-mycorrhizal roots of plants grown in AM fungi-inoculated split-root systems [36]. Various hypotheses have been suggested in an endeavor to elucidate the AM fungi-mediated biocontrol of soilborne phytopathogens. The fact that pathogen-induced symptoms are systemically regulated by AM fungi colonization is related to the establishment of induced systemic resistance (ISR) [37]. ISR is a resistance mechanism induced or acquired in plants which were already undergone

**102**

for this biocontrol.

negative impacts are not important to affect the developmental stages of all nematodes, and it is also unrelated to the mycelial or spore densities of AM fungus [56–58]. Additionally, in the presence of the AM fungi, significant increase in spore germination and hyphal growth by *Fusarium oxysporum* f. sp. *chrysanthemi* was also reported, and thus, direct inhibition of pathogen by AM fungi structures could not properly be explained for biocontrol [56].

In vitro results of impact studies of the exudates of extraradical AM fungi network or by the mycorrhizal roots on pathogens are in contradiction. Crude extracts from the extraradical network of *Glomus intraradices* is clearly reported for the reduced germination of conidia of *Fusarium oxysporum* f. sp. *chrysanthemi* [59]. Similarly, inhibition in sporulation of pathogen *Phytophthora fragariae* is reported with exudates of strawberry roots which were colonized by *Glomus etunicatum* and *Glomus monosporum* [60]. During the harvest, compared to the exudates of non-AM-inoculated tomato roots, the exudates from in vitro grown AM (*Glomus intraradices*)-inoculated roots were reported either repulsive or more attractive for the zoospores of *Phytophthora nicotianae* [61].

Another example can be seen in the exudates of tomato roots which are reported to double the microconidia germination of *Fusarium oxysporum* f. sp. *lycopersici* in the presence of AM fungi *Glomus mosseae* compared to the exudates from non-mycorrhizal roots [54, 62]. The direct impact of exudates from mycorrhizal plants in the AM fungus-mediated biocontrol activity can directly be measured in soil conditions by quantification of the capacity of root infection by the pathogen [63]. Application of root exudates of tomato plants which are colonized with *Glomus intraradices* or *Glomus mosseae* has not been reported for any positive impact on another tomato plant for the control of pathogen *Phytophthora nicotianae,* while direct inoculation of these AM fungi (i.e. *Glomus intraradices* or *Glomus mosseae*) significantly reduced or controlled the growth of pathogen *Phytophthora nicotianae* in these other tomato plants. Thus, it suggests that exudates from one's mycorrhizal plant will not directly or indirectly inhibit the capacity of pathogen intraradical proliferation on other plants.

From the above it is evident that none of the cited mechanisms is involved in the AM fungus-mediated biocontrol, but it has been shown to happen in every plantfungi system. These mechanisms might act in synergistic way with each other, with one mechanism becoming preponderant depending on the environmental conditions and the plant cultivar-pathogen/AM fungus strain. However, the mechanism related to the capacity of interaction of AM fungi with other soil microorganisms can significantly be attributed as one of the main reasons involved in the control of soilborne diseases.

### **3. Interactions between AM-associated bacteria and AM fungus extraradical network**

The bacterial communities associated with various AM fungal inoculum or spores have been reported to differ from one another based on their association as one found in mycorrhizal isolate and others largely encountered in the mycosphere [15]. The species assemblages of cultivable bacteria from surface-disinfected spores of *Glomus mosseae* and *Glomus intraradices* were influenced both by fungal and plant species where 'spore type' is the important factor. This specificity of interaction in AM fungal species is usually hypothesized to be related to spore size and surface roughness. Under sterile conditions the bacterial adherence to spores or hyphae of AM fungi was demonstrated to be species-specific or depends on bacterial isolate and the fungal vitality [64]. The association competence of rhizobacteria to AM fungal surfaces could be dependent on their ability to form biofilms [65].

**105**

*Arbuscular Mycorrhiza-Associated Rhizobacteria and Biocontrol of Soilborne Phytopathogens*

The roots colonized with *Gigaspora margarita* and its extraradical hyphae demonstrate that extracellular polysaccharides are involved in the in vitro association of *Pseudomonas fluorescens* CHAO to these biological surfaces [66]. *Pseudomonas fluorescens* CHAO have the abilities to form light spots, while two mucoid mutants of this strain by increased production of acidic extracellular polysaccharides formed a large number of clusters on non-mycorrhizal carrot roots, and mutants of *Azospirillum brasilense* and *Rhizobium leguminosarum* affected in extracellular polysaccharide production were strongly impaired in the capacity to attach to mycorrhizal root [67]. Strains of *Burkholderia* on *Gigaspora decipiens* were able to colonize the interior of the spores, and it demonstrates that AM fungal colonization does not occur on AM surfaces only through the biofilm formation [68]. Saprophytic activity of the bacteria was also observed by scanning electron microscopy (SEM) observations of *Glomus geosporum* spores [69]. The growth of *Pseudomonas chlororaphis* was also stimulated in presence of crude extracts, containing AM fungus exudates and mycelial compounds of AM fungi from the extraradical network of in vitro grown

Arbuscular mycorrhizal fungi can stimulate the growth of rhizobacteria by providing nutritional resource through the release of exudates. Exudates collected from tomato roots which were colonized by *Glomus fasciculatum* were reported to attract *Azotobacter chroococcum* and *Pseudomonas fluorescens* more strongly than those collected from non-colonized roots [70]. According to Toljander et al. [71], a bacterial community extracted from soil was significantly affected after 48 h when inoculated with exudates produced by AM fungus mycelia in comparison to a

The reduction in exudation through defoliation of pea plants did not change the PCR-DGGE profile of rhizosphere bacteria, while missing and supplementary bands were observed from the rhizosphere of plants which were pre-colonized with *Glomus intraradices* [72]. PCR-DGGE analysis reported to show no effect on the bacterial community structure of tomato rhizosphere which was treated with pre-colonized (with *Glomus intraradices* or *G. mosseae*) root exudates however direct colonization of root with these AM fungi-induced significant changes [24]. The rhizobacterial community structure modification by AM fungal colonization is usually related poorly to exudate liberation by mycorrhizal roots or by the AM fungal mycelium, and importantly it may be dependant on their physical presence or on direct speciesspecific interactions [24]. It has been noticed that the impact of AM fungus colonization on other soil microorganisms is negative. The overall decrease of microbial activity described after root colonization with AM fungi has been proposed to be due to competition for substrates [73]. In association with cucumber, *Glomus intraradices* possess negative effect on the population of *Pseudomonas fluorescens* DF57. This nega-

tive effect was reported in both rhizosphere and in mycosphere [74].

Most of AM-associated bacteria (AMB) described so far in detail showed antagonistic characteristics towards soilborne pathogens or behaved as mycorrhization helper [16]. Similar studies have been performed by various researchers in aiming to identify AMB with biocontrol activities. A bacterial strain of *Paenibacillus* sp. B2 has been isolated from the mycorrhizosphere of *Glomus mosseae* and identified by phylogeny of its 16S rRNA gene sequence and analytical profile index (API) system. It has been found that it acts antagonistic to various soilborne pathogens under in vitr*o* conditions and reduces necrosis in tomato roots (necrosis caused by *Phytophthora nicotianae*) [75]. This isolate (i.e. bacteria) displayed

**4. AM-associated bacteria and biocontrol activities**

*DOI: http://dx.doi.org/10.5772/intechopen.89266*

*Glomus intraradices* [59].

control composed of culture medium.

*Arbuscular Mycorrhiza-Associated Rhizobacteria and Biocontrol of Soilborne Phytopathogens DOI: http://dx.doi.org/10.5772/intechopen.89266*

The roots colonized with *Gigaspora margarita* and its extraradical hyphae demonstrate that extracellular polysaccharides are involved in the in vitro association of *Pseudomonas fluorescens* CHAO to these biological surfaces [66]. *Pseudomonas fluorescens* CHAO have the abilities to form light spots, while two mucoid mutants of this strain by increased production of acidic extracellular polysaccharides formed a large number of clusters on non-mycorrhizal carrot roots, and mutants of *Azospirillum brasilense* and *Rhizobium leguminosarum* affected in extracellular polysaccharide production were strongly impaired in the capacity to attach to mycorrhizal root [67]. Strains of *Burkholderia* on *Gigaspora decipiens* were able to colonize the interior of the spores, and it demonstrates that AM fungal colonization does not occur on AM surfaces only through the biofilm formation [68]. Saprophytic activity of the bacteria was also observed by scanning electron microscopy (SEM) observations of *Glomus geosporum* spores [69]. The growth of *Pseudomonas chlororaphis* was also stimulated in presence of crude extracts, containing AM fungus exudates and mycelial compounds of AM fungi from the extraradical network of in vitro grown *Glomus intraradices* [59].

Arbuscular mycorrhizal fungi can stimulate the growth of rhizobacteria by providing nutritional resource through the release of exudates. Exudates collected from tomato roots which were colonized by *Glomus fasciculatum* were reported to attract *Azotobacter chroococcum* and *Pseudomonas fluorescens* more strongly than those collected from non-colonized roots [70]. According to Toljander et al. [71], a bacterial community extracted from soil was significantly affected after 48 h when inoculated with exudates produced by AM fungus mycelia in comparison to a control composed of culture medium.

The reduction in exudation through defoliation of pea plants did not change the PCR-DGGE profile of rhizosphere bacteria, while missing and supplementary bands were observed from the rhizosphere of plants which were pre-colonized with *Glomus intraradices* [72]. PCR-DGGE analysis reported to show no effect on the bacterial community structure of tomato rhizosphere which was treated with pre-colonized (with *Glomus intraradices* or *G. mosseae*) root exudates however direct colonization of root with these AM fungi-induced significant changes [24]. The rhizobacterial community structure modification by AM fungal colonization is usually related poorly to exudate liberation by mycorrhizal roots or by the AM fungal mycelium, and importantly it may be dependant on their physical presence or on direct speciesspecific interactions [24]. It has been noticed that the impact of AM fungus colonization on other soil microorganisms is negative. The overall decrease of microbial activity described after root colonization with AM fungi has been proposed to be due to competition for substrates [73]. In association with cucumber, *Glomus intraradices* possess negative effect on the population of *Pseudomonas fluorescens* DF57. This negative effect was reported in both rhizosphere and in mycosphere [74].

### **4. AM-associated bacteria and biocontrol activities**

Most of AM-associated bacteria (AMB) described so far in detail showed antagonistic characteristics towards soilborne pathogens or behaved as mycorrhization helper [16]. Similar studies have been performed by various researchers in aiming to identify AMB with biocontrol activities. A bacterial strain of *Paenibacillus* sp. B2 has been isolated from the mycorrhizosphere of *Glomus mosseae* and identified by phylogeny of its 16S rRNA gene sequence and analytical profile index (API) system. It has been found that it acts antagonistic to various soilborne pathogens under in vitr*o* conditions and reduces necrosis in tomato roots (necrosis caused by *Phytophthora nicotianae*) [75]. This isolate (i.e. bacteria) displayed

*Biostimulants in Plant Science*

properly be explained for biocontrol [56].

the zoospores of *Phytophthora nicotianae* [61].

negative impacts are not important to affect the developmental stages of all nematodes, and it is also unrelated to the mycelial or spore densities of AM fungus [56–58]. Additionally, in the presence of the AM fungi, significant increase in spore germination and hyphal growth by *Fusarium oxysporum* f. sp. *chrysanthemi* was also reported, and thus, direct inhibition of pathogen by AM fungi structures could not

In vitro results of impact studies of the exudates of extraradical AM fungi network or by the mycorrhizal roots on pathogens are in contradiction. Crude extracts from the extraradical network of *Glomus intraradices* is clearly reported for the reduced germination of conidia of *Fusarium oxysporum* f. sp. *chrysanthemi* [59]. Similarly, inhibition in sporulation of pathogen *Phytophthora fragariae* is reported with exudates of strawberry roots which were colonized by *Glomus etunicatum* and *Glomus monosporum* [60]. During the harvest, compared to the exudates of non-AM-inoculated tomato roots, the exudates from in vitro grown AM (*Glomus intraradices*)-inoculated roots were reported either repulsive or more attractive for

Another example can be seen in the exudates of tomato roots which are reported to double the microconidia germination of *Fusarium oxysporum* f. sp. *lycopersici* in the presence of AM fungi *Glomus mosseae* compared to the exudates from non-mycorrhizal roots [54, 62]. The direct impact of exudates from mycorrhizal plants in the AM fungus-mediated biocontrol activity can directly be measured in soil conditions by quantification of the capacity of root infection by the pathogen [63]. Application of root exudates of tomato plants which are colonized with *Glomus intraradices* or *Glomus mosseae* has not been reported for any positive impact on another tomato plant for the control of pathogen *Phytophthora nicotianae,* while direct inoculation of these AM fungi (i.e. *Glomus intraradices* or *Glomus mosseae*) significantly reduced or controlled the growth of pathogen *Phytophthora nicotianae* in these other tomato plants. Thus, it suggests that exudates from one's mycorrhizal plant will not directly or indirectly inhibit the capacity of pathogen intraradical proliferation on other plants. From the above it is evident that none of the cited mechanisms is involved in the AM fungus-mediated biocontrol, but it has been shown to happen in every plantfungi system. These mechanisms might act in synergistic way with each other, with one mechanism becoming preponderant depending on the environmental conditions and the plant cultivar-pathogen/AM fungus strain. However, the mechanism related to the capacity of interaction of AM fungi with other soil microorganisms can significantly be attributed as one of the main reasons involved in the control of

**3. Interactions between AM-associated bacteria and AM fungus** 

The bacterial communities associated with various AM fungal inoculum or spores have been reported to differ from one another based on their association as one found in mycorrhizal isolate and others largely encountered in the mycosphere [15]. The species assemblages of cultivable bacteria from surface-disinfected spores of *Glomus mosseae* and *Glomus intraradices* were influenced both by fungal and plant species where 'spore type' is the important factor. This specificity of interaction in AM fungal species is usually hypothesized to be related to spore size and surface roughness. Under sterile conditions the bacterial adherence to spores or hyphae of AM fungi was demonstrated to be species-specific or depends on bacterial isolate and the fungal vitality [64]. The association competence of rhizobacteria to AM fungal surfaces could be dependent on their ability to form biofilms [65].

**104**

soilborne diseases.

**extraradical network**

cellulolytic, proteolytic, chitinolytic and pectinolytic activities and was reported for antibiotic polymyxin B1 and two other polymyxin-like compounds [76–78]. Moreover, its presence resulted in disorganization of cell walls and/or cell contents of *Phytophthora nicotianae* and *Fusarium oxysporum* as observed in electron microscope. It also increases the root and shoot fresh weights of mycorrhized tomato plants and stimulated *Glomus mosseae* to colonize tomato roots [75].

Under compartmentalized growth system, Mansfeld-Giese et al. [78] identified *Paenibacillus polymyxa* and *P. macerans* from the three different regions, namely mycorrhizosphere, hyphosphere (root-free soil and sand compartments) and from a root-free sand compartment. It was found to be closely associated with *Glomus intraradices*. All *Paenibacilli* strains tested from these AM fungi influenced soil zones and helped in preventing pre-emergence damping-off (caused by *Pythium aphanidermatum*) [79]. Out of 18 cultivable isolates from surface-disinfected spores of *Glomus mosseae*, 14 isolates were identified. These identified isolates were mainly composed of *Bacillus simplex*, *B. niacini*, *B. drententis*, *Paenibacillus* spp. and *Methylobacterium* sp. which were reported to show antagonism to various soilborne pathogens (e.g. *Phytophthora nicotianae*, *Fusarium solani*, *Fusarium oxysporum*, etc.) [80]. Bacteria isolated from surface-decontaminated spores of *Glomus intraradices* and *Glomus mosseae* which were extracted from rhizospheres of *Festuca ovina* and *Leucanthemum vulgare* were classified within two phylogenetic clusters: one corresponding to *Proteobacteria* and the other corresponding to *Actinobacteria* and *Firmicutes* [14]. Under dual culture in vitro assays, bacteria from both clusters were reported antagonistic to *Rhizoctonia solani*. Further, selected bacteria, two isolates of *Stenotrophomonas maltophilia*, three isolates of *Pseudomonas* spp., one isolate each of *Bacillus subtilis* and *Arthro bacterilicis*, were reported to act as antagonistic to *Erwinia carotovora* var. *carotovora*, *Verticillium dahliae*, *Phytophthora infestans* and *Rhizoctonia solani*. In vitro studies revealed that these isolates are responsible for producing siderophores and proteases and thus decrease the weight of rotten potato tissues [81]. The ability of AM fungi to specifically harbor and then to stimulate rhizobacteria with biocontrol properties suggests that these bacteria can directly reduce pathogen development within the mycorrhizosphere and they can strongly contribute to the biocontrol of soilborne diseases.

### **5. Unfavorable zone to pathogen development: the mycorrhizosphere**

The mycorrhizosphere has been hypothesized to comprise of favorable surroundings for the growth and development of microorganisms which works antagonistic to soilborne pathogens proliferation. Undeniably, co-culture of the non-mycorrhizal species (e.g. *Dianthus caryophyllus*) with the mycorrhizal species (e.g. *Tagetes patula*) in the presence of AM fungi (e.g. *Glomus intraradices*) clearly reduces the disease caused by *F*usarium *oxysporum* f. sp. *dianthi* in the plant *Dianthus caryophyllus*. It occurs in a manner which differs in providing nutrition to plants and thus suggests a decline in the pathogen development within the mycorrhizosphere [82]. Moreover, a reduction in the number of infection loci in tomato roots (pre-colonized with *Glomus mosseae* and also inoculated with *Phytophthora nicotianae* zoospores) infers that the pathogen may be affected prior to root penetration in the mycorrhizosphere [83].

The mycorrhizosphere influenced by the rhizobacteria + AM fungus + root tripartite associations presents specific characteristics, in which individual factor influences the others' growth and health. Remarkably in the presence of glycoproteins such as glomalin, AM fungi favor the formation of aggregates which provide

**107**

assistance.

**6. Conclusion**

biocontrol abilities.

**Acknowledgements**

*Arbuscular Mycorrhiza-Associated Rhizobacteria and Biocontrol of Soilborne Phytopathogens*

stable microsites favorable to root and microbe establishment [84, 85]. The AM fungi extraradical network also constitutes specific microsites which favor the growth of some bacteria. Among different plant growth-promoting rhizobacteria, P-solubilizing and N-fixing bacteria has been reported for more efficient synergistic interaction with AM fungi. Increased P and N availability to the plants promotes its growth and probably favors its capacity to counteract pathogen impact [11, 86–88]. Plant growth-promoting rhizobacteria can also display biocontrol properties and impact pathogen proliferation through direct liberation of toxic compounds or by competing for space and nutrients, reduction of Fe and Mn availability, modification of the plant hormone balance and stimulation of plant defense mechanisms [89, 90]. A synergistic or additive impact by dual inoculation of AM fungi with rhizobacteria in controlling pathogens reflects the dependence of biocontrol properties on the combinations of bacterial and fungal species used, nutritional status in soil

Reduction in gall formation and nematode multiplication (which are usually responsible for causing root rot in chick pea) was significantly reported in the tomato plants when its roots were inoculated together with *Glomus intraradices* and bacteria *Pseudomonas striata* and *Rhizobium* sp. [91]. Similar positive reports have been recorded when dual inoculation of *Glomus mosseae* with *Pseudomonas fluorescens* was done [92]. Jaderlund et al. [93] reported the interactions of two plant growth-promoting rhizobacteria, namely, *Pseudomonas fluorescens* SBW25 and *Paenibacillus brasilensis* PB177, with AM fungi *Glomus mosseae* and *Glomus intraradices,* respectively; he investigated it on winter wheat which was infested with *Microdochium nivale* and concluded that this interactions are species-specific between fungi and bacteria. From the above and several other studies, it is clear that microbial antagonist to pathogens, and fungi-plant growth-promoting rhizobacteria, do not exert any negative effect against AM fungi [87]. Thus, such mycorrhization helper bacteria (MHB) are important in promoting mycorrhizal development

The competence of AM fungi to control disease symptoms and the intraradical and rhizosphere proliferation of soilborne pathogens is multifaceted and influenced by different mechanisms possibly acting in a synergetic way with each other. Among these mechanisms, the capacity of extraradical network of AM fungi to stimulate beneficial microorganisms is possibly a strongly responsible factor involved. Different bacteria with high capacities of antagonistic activities against several soilborne pathogens have been reported within AM fungal extraradical structures and in the mycorrhizosphere of several AM fungi species. The AM fungi-mediated biocontrol activities can not solely be due to the AM fungus function but also related strongly to the capacity of the AM fungi to constitute an environment which favors the establishment of rhizobacteria with potential

PKS, and MS thank the SERB, Department of Science and Technology, Government of India, for awarding Fast Track Young Scientist and for the financial

*DOI: http://dx.doi.org/10.5772/intechopen.89266*

and probably other environmental conditions [87].

and may even increase AM fungi impact on pathogens.

### *Arbuscular Mycorrhiza-Associated Rhizobacteria and Biocontrol of Soilborne Phytopathogens DOI: http://dx.doi.org/10.5772/intechopen.89266*

stable microsites favorable to root and microbe establishment [84, 85]. The AM fungi extraradical network also constitutes specific microsites which favor the growth of some bacteria. Among different plant growth-promoting rhizobacteria, P-solubilizing and N-fixing bacteria has been reported for more efficient synergistic interaction with AM fungi. Increased P and N availability to the plants promotes its growth and probably favors its capacity to counteract pathogen impact [11, 86–88].

Plant growth-promoting rhizobacteria can also display biocontrol properties and impact pathogen proliferation through direct liberation of toxic compounds or by competing for space and nutrients, reduction of Fe and Mn availability, modification of the plant hormone balance and stimulation of plant defense mechanisms [89, 90]. A synergistic or additive impact by dual inoculation of AM fungi with rhizobacteria in controlling pathogens reflects the dependence of biocontrol properties on the combinations of bacterial and fungal species used, nutritional status in soil and probably other environmental conditions [87].

Reduction in gall formation and nematode multiplication (which are usually responsible for causing root rot in chick pea) was significantly reported in the tomato plants when its roots were inoculated together with *Glomus intraradices* and bacteria *Pseudomonas striata* and *Rhizobium* sp. [91]. Similar positive reports have been recorded when dual inoculation of *Glomus mosseae* with *Pseudomonas fluorescens* was done [92]. Jaderlund et al. [93] reported the interactions of two plant growth-promoting rhizobacteria, namely, *Pseudomonas fluorescens* SBW25 and *Paenibacillus brasilensis* PB177, with AM fungi *Glomus mosseae* and *Glomus intraradices,* respectively; he investigated it on winter wheat which was infested with *Microdochium nivale* and concluded that this interactions are species-specific between fungi and bacteria. From the above and several other studies, it is clear that microbial antagonist to pathogens, and fungi-plant growth-promoting rhizobacteria, do not exert any negative effect against AM fungi [87]. Thus, such mycorrhization helper bacteria (MHB) are important in promoting mycorrhizal development and may even increase AM fungi impact on pathogens.

### **6. Conclusion**

*Biostimulants in Plant Science*

cellulolytic, proteolytic, chitinolytic and pectinolytic activities and was reported for antibiotic polymyxin B1 and two other polymyxin-like compounds [76–78]. Moreover, its presence resulted in disorganization of cell walls and/or cell contents of *Phytophthora nicotianae* and *Fusarium oxysporum* as observed in electron microscope. It also increases the root and shoot fresh weights of mycorrhized tomato

Under compartmentalized growth system, Mansfeld-Giese et al. [78] identified *Paenibacillus polymyxa* and *P. macerans* from the three different regions, namely mycorrhizosphere, hyphosphere (root-free soil and sand compartments) and from a root-free sand compartment. It was found to be closely associated with *Glomus intraradices*. All *Paenibacilli* strains tested from these AM fungi influenced soil zones and helped in preventing pre-emergence damping-off (caused by *Pythium aphanidermatum*) [79]. Out of 18 cultivable isolates from surface-disinfected spores of *Glomus mosseae*, 14 isolates were identified. These identified isolates were mainly composed of *Bacillus simplex*, *B. niacini*, *B. drententis*, *Paenibacillus* spp. and *Methylobacterium* sp. which were reported to show antagonism to various soilborne pathogens (e.g. *Phytophthora nicotianae*, *Fusarium solani*, *Fusarium oxysporum*, etc.) [80]. Bacteria isolated from surface-decontaminated spores of *Glomus intraradices* and *Glomus mosseae* which were extracted from rhizospheres of *Festuca ovina* and *Leucanthemum vulgare* were classified within two phylogenetic clusters: one corresponding to *Proteobacteria* and the other corresponding to *Actinobacteria* and *Firmicutes* [14]. Under dual culture in vitro assays, bacteria from both clusters were reported antagonistic to *Rhizoctonia solani*. Further, selected bacteria, two isolates of *Stenotrophomonas maltophilia*, three isolates of *Pseudomonas* spp., one isolate each of *Bacillus subtilis* and *Arthro bacterilicis*, were reported to act as antagonistic to *Erwinia carotovora* var. *carotovora*, *Verticillium dahliae*, *Phytophthora infestans* and *Rhizoctonia solani*. In vitro studies revealed that these isolates are responsible for producing siderophores and proteases and thus decrease the weight of rotten potato tissues [81]. The ability of AM fungi to specifically harbor and then to stimulate rhizobacteria with biocontrol properties suggests that these bacteria can directly reduce pathogen development within the mycorrhizosphere and they can strongly

plants and stimulated *Glomus mosseae* to colonize tomato roots [75].

contribute to the biocontrol of soilborne diseases.

tion in the mycorrhizosphere [83].

**5. Unfavorable zone to pathogen development: the mycorrhizosphere**

The mycorrhizosphere has been hypothesized to comprise of favorable surroundings for the growth and development of microorganisms which works antagonistic to soilborne pathogens proliferation. Undeniably, co-culture of the non-mycorrhizal species (e.g. *Dianthus caryophyllus*) with the mycorrhizal species (e.g. *Tagetes patula*) in the presence of AM fungi (e.g. *Glomus intraradices*) clearly reduces the disease caused by *F*usarium *oxysporum* f. sp. *dianthi* in the plant *Dianthus caryophyllus*. It occurs in a manner which differs in providing nutrition to plants and thus suggests a decline in the pathogen development within the mycorrhizosphere [82]. Moreover, a reduction in the number of infection loci in tomato roots (pre-colonized with *Glomus mosseae* and also inoculated with *Phytophthora nicotianae* zoospores) infers that the pathogen may be affected prior to root penetra-

The mycorrhizosphere influenced by the rhizobacteria + AM fungus + root tripartite associations presents specific characteristics, in which individual factor influences the others' growth and health. Remarkably in the presence of glycoproteins such as glomalin, AM fungi favor the formation of aggregates which provide

**106**

The competence of AM fungi to control disease symptoms and the intraradical and rhizosphere proliferation of soilborne pathogens is multifaceted and influenced by different mechanisms possibly acting in a synergetic way with each other. Among these mechanisms, the capacity of extraradical network of AM fungi to stimulate beneficial microorganisms is possibly a strongly responsible factor involved. Different bacteria with high capacities of antagonistic activities against several soilborne pathogens have been reported within AM fungal extraradical structures and in the mycorrhizosphere of several AM fungi species. The AM fungi-mediated biocontrol activities can not solely be due to the AM fungus function but also related strongly to the capacity of the AM fungi to constitute an environment which favors the establishment of rhizobacteria with potential biocontrol abilities.

### **Acknowledgements**

PKS, and MS thank the SERB, Department of Science and Technology, Government of India, for awarding Fast Track Young Scientist and for the financial assistance.

*Biostimulants in Plant Science*

### **Author details**

Meenakshi Singh1 , Manjari Mishra2 , Devendra Kumar Srivastava<sup>3</sup> and Pradeep Kumar Singh3 \*

1 Department of Environmental Studies, Panjab University, Chandigarh, India

2 School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

3 Department of Botany, Eternal University, Baru Sahib, HP, India

\*Address all correspondence to: singhpk@hotmail.co.in

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**109**

*Arbuscular Mycorrhiza-Associated Rhizobacteria and Biocontrol of Soilborne Phytopathogens*

microflora: The mycorrhizosphere effect. Phytopathology. 1988;**78**:366-371

[9] Marschner P, Crowley DE, Lieberei R. Arbuscular mycorrhizal infection changes the bacterial 16S rDNA community composition in the rhizosphere of maize. Mycorrhiza.

[10] Marschner P, Timonen S. Interactions between plant species and mycorrhizal colonization on the bacterial community composition in the rhizosphere. Applied Soil Ecology.

[11] Lioussanne L, Beauregard MS,

St-Arnaud M. Interactions between arbuscular mycorrhiza and soil microorganisms. In: Khasa D,

[12] Singh PK, Vyas D. Biocontrol of plant diseases and sustainable

Piche Y, Coughlan A, editors. Advances in Mycorrhizal Biotechnology: A Canadian Perspective. Ottawa ON: NRC Press; 2009

agriculture. Proceedings of the National Academy of Sciences, India Section B.

bacterienne et de lexudation racinaire de la tomatepar la symbiose mycorhizi

*Phytophthora nicotianae*. Doctoral thesis. University of Montreal, Montreal [In

[15] Rillig MC, Lutgen ER, Ramsey PW, Klironomos JN, Gannon JE. Microbiota

2001;**11**:297-302

2005;**28**:23-36

Hamel C, Jolicoeur M,

2009;**79**(II):110-128

French]. 2007

[13] Lioussanne L. Roles des modifications de la microflore

ennedans le biocontrole sur le

[14] Bharadwaj DP, Lundquist PO, Persson P, Alstrom S. Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores (multitrophic interactions in the rhizosphere). FEMS Microbiology

Ecology. 2008;**65**:310-322

*DOI: http://dx.doi.org/10.5772/intechopen.89266*

Phosphate uptake zones of mycorrhizal and non-mycorrhizal onions. New Phytologist. 1975;**75**:555-561

[2] Smith SE, Read DJ. Mycorrhizal Symbiosis. San Diego, London:

[3] Kabir Z, Ohalloran IP, Fyles JW, Hamel C. Seasonal changes of

arbuscular mycorrhizal fungi as affected by tillage practices and fertilization: Hyphal density and mycorrhizal root colonization. Plant and Soil.

[4] Olsson PA, Thingstrup I, Jakobsen I, Baath F. Estimation of the biomass of arbuscular mycorrhizal fungi in a linseed field. Soil Biology and Biochemistry. 1999;**31**:1879-1887

[5] Hiltner L. Uber neuereer Fahrungen und probleme auf demgebiet der bodenbakteruiligie und unterbes ondererberuck sichtiguang der grundungung und brache (in German). Arbeiten der Deutschen Landwirtschaftlichen Gesellschaft.

[6] Duineveld BM, Kowalchuk GA, Keijzer A, Veen JAV. Analysis of bacterial communities in the rhizosphere of *Chrysanthemum* via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA. Applied and Environmental Microbiology.

[7] Kim JS, Dungan RS, Kwon SW, Weon HY. The community composition of root-associated bacteria of the tomato plant. World Journal of Microbiology and Biotechnology.

[8] Linderman RG. Mycorrhizal interactions with the rhizosphere

Academic Press; 2008

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*Arbuscular Mycorrhiza-Associated Rhizobacteria and Biocontrol of Soilborne Phytopathogens DOI: http://dx.doi.org/10.5772/intechopen.89266*

### **References**

*Biostimulants in Plant Science*

**108**

**Author details**

Meenakshi Singh1

and Pradeep Kumar Singh3

, Manjari Mishra2

\*

\*Address all correspondence to: singhpk@hotmail.co.in

provided the original work is properly cited.

, Devendra Kumar Srivastava<sup>3</sup>

1 Department of Environmental Studies, Panjab University, Chandigarh, India

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

3 Department of Botany, Eternal University, Baru Sahib, HP, India

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[81] Bharadwaj DP, Lundquist PO, Alstrom S. Arbuscular mycorrhizal fungal spore-associated bacteria affect mycorrhizal colonization, plant growth and potato pathogens. Soil Biology and Biochemistry. 2008;**40**:2494-2501

[82] St-Arnaud M, Vujanovic V. Effect of the arbuscular mycorrhizal

symbiosis on plant diseases and pests. In: Hamel C, Plenchette C, editors. Arbuscular Mycorrhizae in Crop Production. NY: Haworth's Food Products Press; 2007. pp. 67-122

[83] Vigo C, Norman JR, Hooker JE. Biocontrol of the pathogen *Phytophthora parasitica* by arbuscular mycorrhizal fungi is a consequence of effects on infection loci. Plant Pathology.

[84] Rillig MC, Mummey DL. Mycorrhizas and soil structure. New

Phytologist. 2006;**171**:41-53

[85] Singh PK, Singh M, Tripathi BN. Glomalin: an arbuscular mycorrhizal fungal soil protein. Protoplasma.

[86] Johansson JF, Paul LR, Finlay RD.

mycorrhizosphere and their significance

Microbial interactions in the

2000;**49**:509-514

2013;**250**:663-669

[74] Ravnskov S, Nybroe O, Jakobsen I. Influence of an arbuscular mycorrhizal fungus on *Pseudomonas fluorescens* DF57 in rhizosphere and hyphosphere soil. New Phytologist. 1999;**142**:113-122

Martinotti G, Gianinazzi S. Isolation

[76] Budi SW, van Tuinen D, Arnould C, Dumas-Gaudot E, Gianinazzi-Pearson V,

Gianinazzi S. Hydrolytic enzyme activity of *Paenibacillus* sp. strain B2 and effects of the antagonistic bacterium on cell integrity of two soil-borne pathogenic fungi. Applied Soil Ecology.

[77] Selim S, Negrel J, Govaerts C, Gianinazzi S, van Tuinen D. Isolation and partial characterization of antagonistic peptides produced by *Paenibacillus* sp. strain B2 isolated from the sorghum mycorrhizosphere. Applied and Environmental Microbiology.

[78] Mansfeld-Giese K, Larsen J, Bodker L. Bacterial populations

mycorrhizosphere of a bacterium compatible with arbuscular mycorrhiza development and antagonistic towards soilborne fungal pathogens. Applied and Environmental Microbiology.

[75] Budi SW, van Tuinen D,

from the *Sorghum bicolour*

1999;**65**:5148-5150

2000;**15**:191-199

2005;**71**:6501-6507

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[87] Barea JM, Pozo MJ, Azcon R, Azcon-Aguilar C. Microbial co-operation in the rhizosphere. Journal of Experimental Botany. 2005;**6**:1761-1778

[88] Artursson V, Finlay RD, Jansson JK. Interactions between arbuscular mycorrhizal fungi and bacteria and their potential for stimulating plant growth. Environmental Microbiology. 2006;**8**:1-10

[89] Nehl DB, Allen SJ, Brown JF. Deleterious rhizosphere bacteria: An integrating perspective. Applied Soil Ecology. 1997;**5**:1-20

[90] Bowen GD, Rovira AD. The rhizosphere and its management to improve plant growth. Advances in Agronomy. 1999;**66**:1-102

[91] Akhtar MS, Siddiqui ZA. Biocontrol of a root rot disease complex of chickpea by *Glomus intraradices*, *Rhizobium* sp. and *Pseudomonas striata*. Crop Protection. 2008;**27**:410-417

[92] Siddiqui ZA, Mahmood I. Effect of a plant growth promoting bacterium, an AM fungus and soil types on the morphometrics and reproduction of *Meloidogyne javanica* on tomato. Applied Soil Ecology. 1998;**8**:77-84

[93] Jaderlund L, Arthurson V, Granhall U, Jansson JK. Specific interactions between arbuscular mycorrhizal fungi and plant growth-promoting bacteria: as revealed by different combinations. FEMS Microbiology Letters. 2008;**287**:174-180

**117**

**Chapter 8**

**Abstract**

biotic or abiotic stresses is analyzed.

nature of intracellular hyphal development [4, 5].

**1. Introduction**

Ectomycorrhizal Fungi as

*José Alfonso Domínguez-Núñez and Ada S. Albanesi*

Ectomycorrhizal (ECM) fungi play a fundamental role in the nutrient cycle in terrestrial ecosystems, especially in forest systems. In this chapter, the value of ECM fungi is reviewed from a global framework, not only to increase the production of edible fruit bodies and biomass of plants but also for the regular practices of reforestation and restoration of ecosystems, with implicit applications in biofertilization, bioremediation, and control of soil pathogens. Ecological functions of the ECM fungi are briefly reviewed. The direct implications of the ECM fungi in forestry are described. To do so, its role as a biotechnological tool in forest nursery production is briefly analyzed, as well as the role of mycorrhizal helper bacteria (MHB). Subsequently, the direct role as biofertilizers of the ECM fungi in forest management is discussed: reforestation, plantation management, and ecosystem restoration. The importance of ECM fungi to increase the tolerance of plants against

**Keywords:** forestry, ectomycorrhiza, restoration, sustainable development, nutrients

It was Albert Bernhard Frank (1885), a forest pathologist, who for the first time introduced the term mycorrhiza. In the Greek language, "mykes" refers to fungus and "rhiza" refers to root. Since Frank's description of mycorrhizal association in the 1880s [1], a lot of work has been generated by different investigators as a consequence of which it is estimated that 86% of terrestrial plant species are benefited as they acquire their mineral nutrients via mycorrhizal roots [2]. These groups of fungi establish a symbiotic relationship with the roots of plants, called mycorrhizas. Frank established two large subdivisions of mycorrhizas, ecto- and endomycorrhizas. Ectomycorrhizal fungi form mantle and Hartig network of intercellular hyphae in the roots of forest species. Endomycorrhizas are classified as arbuscular mycorrhizas, ericoid mycorrhizas, arbutoid mycorrhizas, monotropoid mycorrhizas, ectendomycorrhizas, or orchid mycorrhizas [3]. The Arbuscular Mycorrhizal fungi (AM) form arbuscules and vesicles, they are more variable than ECM fungi since they form symbiosis with trees and herbaceous plants. Each of these categories is characterized by the invasion of plant root cells by fungal hyphae but differs in the

Ectomycorrhizal fungi are predominantly *Basidiomycetes*, some *Ascomycetes*, and a very few *Zygomycetes*. In these symbiotic structures, the Hartig network is the interface for the metabolic exchange between the fungus and the root. The mycorrhizal mantle is connected to the filaments of fungi that extend into the soil

Biofertilizers in Forestry

### **Chapter 8**

### Ectomycorrhizal Fungi as Biofertilizers in Forestry

*José Alfonso Domínguez-Núñez and Ada S. Albanesi*

### **Abstract**

Ectomycorrhizal (ECM) fungi play a fundamental role in the nutrient cycle in terrestrial ecosystems, especially in forest systems. In this chapter, the value of ECM fungi is reviewed from a global framework, not only to increase the production of edible fruit bodies and biomass of plants but also for the regular practices of reforestation and restoration of ecosystems, with implicit applications in biofertilization, bioremediation, and control of soil pathogens. Ecological functions of the ECM fungi are briefly reviewed. The direct implications of the ECM fungi in forestry are described. To do so, its role as a biotechnological tool in forest nursery production is briefly analyzed, as well as the role of mycorrhizal helper bacteria (MHB). Subsequently, the direct role as biofertilizers of the ECM fungi in forest management is discussed: reforestation, plantation management, and ecosystem restoration. The importance of ECM fungi to increase the tolerance of plants against biotic or abiotic stresses is analyzed.

**Keywords:** forestry, ectomycorrhiza, restoration, sustainable development, nutrients

### **1. Introduction**

It was Albert Bernhard Frank (1885), a forest pathologist, who for the first time introduced the term mycorrhiza. In the Greek language, "mykes" refers to fungus and "rhiza" refers to root. Since Frank's description of mycorrhizal association in the 1880s [1], a lot of work has been generated by different investigators as a consequence of which it is estimated that 86% of terrestrial plant species are benefited as they acquire their mineral nutrients via mycorrhizal roots [2]. These groups of fungi establish a symbiotic relationship with the roots of plants, called mycorrhizas. Frank established two large subdivisions of mycorrhizas, ecto- and endomycorrhizas. Ectomycorrhizal fungi form mantle and Hartig network of intercellular hyphae in the roots of forest species. Endomycorrhizas are classified as arbuscular mycorrhizas, ericoid mycorrhizas, arbutoid mycorrhizas, monotropoid mycorrhizas, ectendomycorrhizas, or orchid mycorrhizas [3]. The Arbuscular Mycorrhizal fungi (AM) form arbuscules and vesicles, they are more variable than ECM fungi since they form symbiosis with trees and herbaceous plants. Each of these categories is characterized by the invasion of plant root cells by fungal hyphae but differs in the nature of intracellular hyphal development [4, 5].

Ectomycorrhizal fungi are predominantly *Basidiomycetes*, some *Ascomycetes*, and a very few *Zygomycetes*. In these symbiotic structures, the Hartig network is the interface for the metabolic exchange between the fungus and the root. The mycorrhizal mantle is connected to the filaments of fungi that extend into the soil (extraradical mycelium), directly involved in the mobilization, absorption, and translocation of soil nutrients and water to the roots. Molecular clock analysis on the reconciled tree suggested that ECM fungi evolved far later than the appearance of the last common ancestor of brown and white rot fungi about 300 mya [6]. These results supported the long-standing hypothesis that ECM fungi evolved polyphyletically from multiple saprophytic species. More than 7000 species of fungi form ectomycorrhizas [7], many of them with important commercial trees such as poplar, birch, oak, pine, and spruce [8]. The reproductive structures (fruiting bodies) of the macromycetes are known as mushrooms when they grow in the soil and, like truffles, when they grow underground.

The community of mycorrhizal fungi can be determinant in the structure of the plant community [9]. Therefore, the identification of the mycobiont partner and its functional structure [10] are fundamental to understand the ecological importance of this symbiotic relationship. ECM fungal diversity studies were initially based on studies of fruiting bodies and, more recently, on the direct identification of ectomycorrhizal morphoanatomical characters [11]. Despite recent advances in the use of molecular techniques, there are still many advantages associated with classical methods for studying ECM fungal diversity. For the recognition of fungal relationship and type of mycorrhizal association is advantageous over molecular method [7]. Sometimes morphoanatomical-based taxonomy is not well supported by molecular taxonomy. To overcome such discrepancy, the combined approach of morphoanatomical and molecular characterization of ectomycorrhizas in combination with phylogeny was applied [12].

Most of the cultivated species of edible fungi are saprophytes, and only some of them are ECM fungi [13]. The tickets (*Boletus edulis*), the chanterelles (*Cantharellus* spp.), the matsutake mushroom (*Tricholoma matsutake*), and the truffle (many species of the *Tuber* genus) are some ECM fungi for which the crop has been studied [14–16]. The black truffle or Périgord, *Tuber melanosporum*, is widely grown, while other species of ECM mushrooms have not yet been cultivated, including fungi porcini (*Boletus edulis* S.) and the high-priced Italian fungus, white truffles (*Tuber magnatum*).

### **2. Ecological functions of ECM fungi**

In different forest ecosystems, ECM fungi have been reported to play an important role in seedling survival, establishment, and growth [3, 17, 18]. Researches have confirmed that ECM fungi play a key role in terrestrial ecosystems as drivers of global carbon and nutrient cycles [19].

Some of these traditionally known functions of the ECM fungi on the ecosystem are:

ECM fungi increase the water and nutrient supply plant, extending the volume of land accessible to the plants.

Different fungal species (drought-sensitive hydrophilic or drought-tolerant hydrophobic) can have different effects on hydraulic redistribution patterns [20]. The mechanisms to enhance the acquisition of P by tree mycorrhizal roots are the extension of extramatrical mycorrhizal hyphae, the increase of inorganic P transfer, the increase of inorganic P transporters in the fungus/soil interface, the mobilization of organic P (labile) by emission of phosphatases, and the mobilization of mineral insoluble P by the emission of organic acids (LMWOAs) [21, 22].

The mechanisms of improvement in nitrogen (N) absorption would be the intervention in the mineral N cycle (NH4 + , NO3 <sup>−</sup>) and the assimilation of organic N (by emitting proteases, chitinases, and others) [23, 24].

**119**

*Ectomycorrhizal Fungi as Biofertilizers in Forestry DOI: http://dx.doi.org/10.5772/intechopen.88585*

and mineral or organic forms of N, such as NH4

in signaling in ectomycorrhizal symbioses [26].

abiotic) environmental stresses.

**3. Applications: ECM fungi to forestry**

+

aradical mycelium. NH3/NH4

Currently, recent advances in the knowledge of nutrient translocation processes in the fungus-plant and fungus-soil interaction are especially interesting, in particular the priority role of transporters of P, N, and C [25]. The inorganic P

absorbed by transporters specialized located in the fungal membrane in the extr-

from the symbiotic interface to the cells of the plant through selective transporters. Transporters of hexoses import carbon of plant origin into the fungus. The nutritional strategies seem to be different between symbiotic and pathogenic fungi, for example, in the translocation of C. Even different transport strategies have been found between ECM symbionts *Ascomycota* and *Basidiomycota*. The understanding of the different systems of transporters or nutrient channels involved both at the level of the extraradical mycelium and at the level of the symbiotic interface will clarify in the future the processes of nutrition in the plant-fungus and fungus-soil interaction. Also, looking at the fungal factors in the establishment of the symbiotic relationship, chitin-related molecules seem to be shared by pathogenic and arbuscular mycorrhizal fungi, opening the question of whether they could also function

On the other hand, the ECM colonization of the root can provide protection against soil pathogens [27]. Also, the non-nutritive benefits to plants due to changes in water relations, the level of phytohormones, the assimilation of carbon, etc. have already been verified [3]. The carbon is transferred through the ECM fungal mycelium that connects different species of plants. This can reduce competition among plants and contribute to the stability and diversity of ecosystems [28]. The extraradical mycelium of the ECM fungi provides a direct pathway for the translocation of photosynthesized carbon to microsites in the soil and a large surface area for interaction with other microorganisms [29, 30]. Recently, Hupperts et al. [31] proposed two competing models to explain carbon mobilization by ectomycorrhizal fungi. "Saprotrophy model", where decreased allocation of carbon may induce saprotrophic behaviour in ectomycorrhizal fungi, resulting in the decomposition of organic matter to mobilize carbon and second, "nutrient acquisition model", where decomposition may instead be driven by the acquisition of nutrients locked within soil organic matter compounds. Moreover, epigeous and hypogeal sporocarps of ECM fungi are important food sources for placental and marsupial mammals [32]. The ectomycorrhizal roots, the mycelium, and the fruiting bodies of the fungi are important as food sources and habitats for invertebrates [33]. The hyphal networks produced by ECM fungi significantly alter and improve the structure of the soil [34]. In a global way, the ECM fungi improve the plant tolerance to (biotic and

Much of our understanding of the functions of ECM fungi has come from research directed toward practical application in forestry. Some of the most common criteria considered for the selection of a most valued species or strain of ECM fungi (some of them implicit in others) are the abiotic criteria: climatic conditions such as temperature, insolation, and humidity; improvement of soil properties, such as texture and permeability; abiotic soil stress mitigation; soil contamination mitigation; soil metal mobilization; or nutrient cycling. There may also be criteria regarding the host, such as the plant/fungus specificity, the improvement of plant health, or the increase in the biomass of the plant. Finally, there are criteria regarding the fungus, such as abundance, effectiveness, propagules competitiveness,

+ , NO3

and inorganic P (from polyphosphates) are imported

<sup>−</sup>, and amino acids (AA), are

*Ectomycorrhizal Fungi as Biofertilizers in Forestry DOI: http://dx.doi.org/10.5772/intechopen.88585*

*Biostimulants in Plant Science*

truffles, when they grow underground.

tion with phylogeny was applied [12].

**2. Ecological functions of ECM fungi**

global carbon and nutrient cycles [19].

intervention in the mineral N cycle (NH4

of land accessible to the plants.

*magnatum*).

tem are:

(extraradical mycelium), directly involved in the mobilization, absorption, and translocation of soil nutrients and water to the roots. Molecular clock analysis on the reconciled tree suggested that ECM fungi evolved far later than the appearance of the last common ancestor of brown and white rot fungi about 300 mya [6]. These results supported the long-standing hypothesis that ECM fungi evolved polyphyletically from multiple saprophytic species. More than 7000 species of fungi form ectomycorrhizas [7], many of them with important commercial trees such as poplar, birch, oak, pine, and spruce [8]. The reproductive structures (fruiting bodies) of the macromycetes are known as mushrooms when they grow in the soil and, like

The community of mycorrhizal fungi can be determinant in the structure of the plant community [9]. Therefore, the identification of the mycobiont partner and its functional structure [10] are fundamental to understand the ecological importance of this symbiotic relationship. ECM fungal diversity studies were initially based on studies of fruiting bodies and, more recently, on the direct identification of ectomycorrhizal morphoanatomical characters [11]. Despite recent advances in the use of molecular techniques, there are still many advantages associated with classical methods for studying ECM fungal diversity. For the recognition of fungal relationship and type of mycorrhizal association is advantageous over molecular method [7]. Sometimes morphoanatomical-based taxonomy is not well supported by molecular taxonomy. To overcome such discrepancy, the combined approach of morphoanatomical and molecular characterization of ectomycorrhizas in combina-

Most of the cultivated species of edible fungi are saprophytes, and only some of them are ECM fungi [13]. The tickets (*Boletus edulis*), the chanterelles (*Cantharellus* spp.), the matsutake mushroom (*Tricholoma matsutake*), and the truffle (many species of the *Tuber* genus) are some ECM fungi for which the crop has been studied [14–16]. The black truffle or Périgord, *Tuber melanosporum*, is widely grown, while other species of ECM mushrooms have not yet been cultivated, including fungi porcini (*Boletus edulis* S.) and the high-priced Italian fungus, white truffles (*Tuber* 

In different forest ecosystems, ECM fungi have been reported to play an important role in seedling survival, establishment, and growth [3, 17, 18]. Researches have confirmed that ECM fungi play a key role in terrestrial ecosystems as drivers of

Some of these traditionally known functions of the ECM fungi on the ecosys-

ECM fungi increase the water and nutrient supply plant, extending the volume

Different fungal species (drought-sensitive hydrophilic or drought-tolerant hydrophobic) can have different effects on hydraulic redistribution patterns [20]. The mechanisms to enhance the acquisition of P by tree mycorrhizal roots are the extension of extramatrical mycorrhizal hyphae, the increase of inorganic P transfer, the increase of inorganic P transporters in the fungus/soil interface, the mobilization of organic P (labile) by emission of phosphatases, and the mobilization of mineral

The mechanisms of improvement in nitrogen (N) absorption would be the

+ , NO3

<sup>−</sup>) and the assimilation of organic N

insoluble P by the emission of organic acids (LMWOAs) [21, 22].

(by emitting proteases, chitinases, and others) [23, 24].

**118**

Currently, recent advances in the knowledge of nutrient translocation processes in the fungus-plant and fungus-soil interaction are especially interesting, in particular the priority role of transporters of P, N, and C [25]. The inorganic P and mineral or organic forms of N, such as NH4 + , NO3 <sup>−</sup>, and amino acids (AA), are absorbed by transporters specialized located in the fungal membrane in the extraradical mycelium. NH3/NH4 + and inorganic P (from polyphosphates) are imported from the symbiotic interface to the cells of the plant through selective transporters. Transporters of hexoses import carbon of plant origin into the fungus. The nutritional strategies seem to be different between symbiotic and pathogenic fungi, for example, in the translocation of C. Even different transport strategies have been found between ECM symbionts *Ascomycota* and *Basidiomycota*. The understanding of the different systems of transporters or nutrient channels involved both at the level of the extraradical mycelium and at the level of the symbiotic interface will clarify in the future the processes of nutrition in the plant-fungus and fungus-soil interaction. Also, looking at the fungal factors in the establishment of the symbiotic relationship, chitin-related molecules seem to be shared by pathogenic and arbuscular mycorrhizal fungi, opening the question of whether they could also function in signaling in ectomycorrhizal symbioses [26].

On the other hand, the ECM colonization of the root can provide protection against soil pathogens [27]. Also, the non-nutritive benefits to plants due to changes in water relations, the level of phytohormones, the assimilation of carbon, etc. have already been verified [3]. The carbon is transferred through the ECM fungal mycelium that connects different species of plants. This can reduce competition among plants and contribute to the stability and diversity of ecosystems [28]. The extraradical mycelium of the ECM fungi provides a direct pathway for the translocation of photosynthesized carbon to microsites in the soil and a large surface area for interaction with other microorganisms [29, 30]. Recently, Hupperts et al. [31] proposed two competing models to explain carbon mobilization by ectomycorrhizal fungi. "Saprotrophy model", where decreased allocation of carbon may induce saprotrophic behaviour in ectomycorrhizal fungi, resulting in the decomposition of organic matter to mobilize carbon and second, "nutrient acquisition model", where decomposition may instead be driven by the acquisition of nutrients locked within soil organic matter compounds. Moreover, epigeous and hypogeal sporocarps of ECM fungi are important food sources for placental and marsupial mammals [32]. The ectomycorrhizal roots, the mycelium, and the fruiting bodies of the fungi are important as food sources and habitats for invertebrates [33]. The hyphal networks produced by ECM fungi significantly alter and improve the structure of the soil [34]. In a global way, the ECM fungi improve the plant tolerance to (biotic and abiotic) environmental stresses.

### **3. Applications: ECM fungi to forestry**

Much of our understanding of the functions of ECM fungi has come from research directed toward practical application in forestry. Some of the most common criteria considered for the selection of a most valued species or strain of ECM fungi (some of them implicit in others) are the abiotic criteria: climatic conditions such as temperature, insolation, and humidity; improvement of soil properties, such as texture and permeability; abiotic soil stress mitigation; soil contamination mitigation; soil metal mobilization; or nutrient cycling. There may also be criteria regarding the host, such as the plant/fungus specificity, the improvement of plant health, or the increase in the biomass of the plant. Finally, there are criteria regarding the fungus, such as abundance, effectiveness, propagules competitiveness,

fungus growth rate, or edibility. Other criteria may be the conservation of native biodiversity, the functioning of the ecosystem, human health, food, nutraceutical value, etc. [30, 35].

### **3.1 ECM fungi in forest nurseries**

Since the late 1950s, mycorrhizal fungi were utilized as biofertilizers to promote plant growth, because of their ability to increase the plant uptake of P, N, mineral nutrients, and water [36–38]. The idea of inoculating ECM fungi on seedlings in plant nurseries was developed by Fortin [39]. Vozzo and Hacskaylo [40] while working on ECM in the United States experimentally demonstrated that field survival and growth of tree seedlings with specific potential ECM enhance the performance of seedlings and contribute to the proper functioning of forest ecosystems.

Although successful inoculation of tree seedlings (already planted) in the field has been known, nursery inoculation is more common. Seedlings inoculated in the nursery can establish a healthy ECM system before planting. The challenge in the controlled synthesis of the ectomycorrhizal symbiosis is to produce a quality mycorrhizal plant, only colonized by the desired fungus. Accurate identification of the inoculum used and avoiding contamination during the growth of the inoculated plants are essential parts of the production process to avoid the introduction of unwanted species and to avoid the mixing of their genetic material with indigenous species [41]. The appropriate selection of suitable plant-host species is essential for the success of mycorrhization [42]. Relatively fast-growing fungi are generally preferred for inoculation because of their short incubation period. Unfortunately, many otherwise desirable ECM fungi grow slowly. According to Marx [43], fresh cultures are preferred to cultures repeatedly transferred and stored for several years. He further suggests passing important fungus cultures through a host inoculation and mycorrhiza formation followed by re-isolation, every few years to maintain mycorrhiza-forming capacity. Moreover, fungi, which produce large hyphal stands of rhizomorphs in the culture of the soil, may be superior in soil exploration and mineral uptake to those which lack rhizomorphic growth. On the other hand, the fruiting of the ECM fungi species is not based solely on the mycorrhizal state of the seedlings. After planting, in addition to the presence of indigenous competitors, the biotic and physicochemical characteristics of the soil also influence the persistence and spread of the cultivated fungus [44]. The type of ECM material used for inoculation can affect the success of a mycorrhizal inoculation program. In addition to remaining viable during storage and transport, the inoculant must also maintain its infectivity for several months after its introduction [45].

There are three main sources of fungal inoculum: soil, spores, and mycelium.

Initially, the soil or humus collected from the mycorrhizal plantation area was frequently used. Its main disadvantage is the lack of control of ECM species in the soil or of microorganisms and harmful germs. Another problem with this type of inoculant is that large amounts of soil are required to inoculate nursery plants. This method is widely used in developing countries, although it is currently discarded in mycorrhization programs. Also, planting mycorrhizal "nurse" seedlings or incorporating chopped roots of ECM hosts into nursery beds as a source of fungi for neighboring young seedlings has been successful [46].

Other sources of inoculum are the spores of fruit bodies collected in the field. The main advantages are that the spores do not require the extension of the aseptic culture and that the spore inoculum is not heavy [47]. Most of the recent research has been with *Pisolithus tinctorius*. Inoculation with spores of *Rhizopogon* species also appears promising. Abundant *Rhizopogon* mycorrhizas formed on seedlings produced from the coated seed of *Pinus radiata* D. Don with

**121**

different origin.

*Ectomycorrhizal Fungi as Biofertilizers in Forestry DOI: http://dx.doi.org/10.5772/intechopen.88585*

loid chips [47].

multimicrobial inoculant.

*3.1.1 Ectomycorrhizal helper bacteria*

basidiospores of *Rhizopogon luteolus* [5]. However, it has three main drawbacks: (a) significant quantities of fruiting bodies are required and may not be available each year, (b) the success of the inoculation is highly dependent on the viability of the spores, and (c) the lack of genetic definition. Freeze-drying and storage at a low temperature in the dark is helpful to maintain its viability. The spores can be mixed with physical supports before the soil inoculation; suspended in water and soaked in the soil; sprinkled, sprayed or pelleted, and emitted to the ground; and encapsulated or coated on the seeds, and they can be embedded in hydrocol-

The most appropriate inoculum is the use of hyphae in a solid or liquid medium or substrate. Hyphae are cultivated mainly from sterile parts of fruiting bodies, less frequently from mycorrhiza due to their low (approx. 5–20%) success rate [48] and rarely from sclerotia [49] or sexual spores [50]. It is considered the most appropriate method since it allows the selection of particular strains of a fungus previously tested for its ability to promote the growth of plants [43]. Many species do grow well in culture, e.g., most species of *Suillus*, *Hebeloma*, *Laccaria*, *Amanita*, *Rhizopogon*, and *Pisolithus* genus. Liquid substrates have the advantage over solids because they are easily mixed and produce more uniform conditions for crop growth, but the risk of bacterial contamination and costs are higher [45]. On the other hand, the main advantages of the solid medium [51] are the reduction of bacterial contamination due to the lower water content, the low costs of the equipment, and the simplified design of the bioreactors. The main drawbacks of the use of mycelial inocula are that several species of ECM fungi are difficult to grow under laboratory conditions, or growth is very slow (due to the absence of their symbiont), and it is not always easy to produce large amounts of inoculum viable for large-scale nursery inoculation programs. Some advances have been made using mycelium encapsulated in "beads" of calcium alginate (e.g., [52]), but they have to be refrigerated. Inoculant beads can remain viable for several months under refrigeration, although the results vary between fungal species. For several species, the mycelial inoculum has been tested with trees of economic interest. This technique has great potential for the inoculation of seedlings in reforestation programs. For example, Rossi et al. [45] designed a bioreactor with the capacity to produce inoculum for 300 000 seedlings, enough to reforest 200 hectares. Based on a global demand of 3.0 billion cubic meters of wood, an estimated 4.3 tons of mycelium would be needed to inoculate 12 billion seedlings (5 g of dry mycelium per plant [45]). An advantage of alginate gel is the possibility of preparing a

The concept of "mycorrhiza helper bacteria" (MHB) was introduced in a "Tansley Review": Helper Bacteria—a new dimension of mycorrhizal symbiosis [53], which has led to new research in the plant-fungus model system, as for the meaning of these bacteria that promote the formation of mycorrhizas and cause many physiological effects of mutualistic interaction. In general, the ability of some microorganisms to influence the formation and functioning of the symbiosis is known, through activities of various kinds such as the activation of infective propagules of the fungus in presymbiotic stages [54], facilitating the formation of entry points in the root [55] and increase of the growth rate [56]. The MHB improve mycorrhiza formation, although the same MHB can benefit mycorrhization for certain fungi and be negative for others [57]. The above reflects the fungal specificity by isolate, which exemplifies the genetic distance between isolates of

*Ectomycorrhizal Fungi as Biofertilizers in Forestry DOI: http://dx.doi.org/10.5772/intechopen.88585*

*Biostimulants in Plant Science*

**3.1 ECM fungi in forest nurseries**

value, etc. [30, 35].

fungus growth rate, or edibility. Other criteria may be the conservation of native biodiversity, the functioning of the ecosystem, human health, food, nutraceutical

Since the late 1950s, mycorrhizal fungi were utilized as biofertilizers to promote plant growth, because of their ability to increase the plant uptake of P, N, mineral nutrients, and water [36–38]. The idea of inoculating ECM fungi on seedlings in plant nurseries was developed by Fortin [39]. Vozzo and Hacskaylo [40] while working on ECM in the United States experimentally demonstrated that field survival and growth of tree seedlings with specific potential ECM enhance the performance of seedlings and contribute to the proper functioning of forest ecosystems. Although successful inoculation of tree seedlings (already planted) in the field has been known, nursery inoculation is more common. Seedlings inoculated in the nursery can establish a healthy ECM system before planting. The challenge in the controlled synthesis of the ectomycorrhizal symbiosis is to produce a quality mycorrhizal plant, only colonized by the desired fungus. Accurate identification of the inoculum used and avoiding contamination during the growth of the inoculated plants are essential parts of the production process to avoid the introduction of unwanted species and to avoid the mixing of their genetic material with indigenous species [41]. The appropriate selection of suitable plant-host species is essential for the success of mycorrhization [42]. Relatively fast-growing fungi are generally preferred for inoculation because of their short incubation period. Unfortunately, many otherwise desirable ECM fungi grow slowly. According to Marx [43], fresh cultures are preferred to cultures repeatedly transferred and stored for several years. He further suggests passing important fungus cultures through a host inoculation and mycorrhiza formation followed by re-isolation, every few years to maintain mycorrhiza-forming capacity. Moreover, fungi, which produce large hyphal stands of rhizomorphs in the culture of the soil, may be superior in soil exploration and mineral uptake to those which lack rhizomorphic growth. On the other hand, the fruiting of the ECM fungi species is not based solely on the mycorrhizal state of the seedlings. After planting, in addition to the presence of indigenous competitors, the biotic and physicochemical characteristics of the soil also influence the persistence and spread of the cultivated fungus [44]. The type of ECM material used for inoculation can affect the success of a mycorrhizal inoculation program. In addition to remaining viable during storage and transport, the inoculant must also maintain its infectivity for several months after its

There are three main sources of fungal inoculum: soil, spores, and mycelium. Initially, the soil or humus collected from the mycorrhizal plantation area was frequently used. Its main disadvantage is the lack of control of ECM species in the soil or of microorganisms and harmful germs. Another problem with this type of inoculant is that large amounts of soil are required to inoculate nursery plants. This method is widely used in developing countries, although it is currently discarded in mycorrhization programs. Also, planting mycorrhizal "nurse" seedlings or incorporating chopped roots of ECM hosts into nursery beds as a source of fungi for

Other sources of inoculum are the spores of fruit bodies collected in the field. The main advantages are that the spores do not require the extension of the aseptic culture and that the spore inoculum is not heavy [47]. Most of the recent research has been with *Pisolithus tinctorius*. Inoculation with spores of *Rhizopogon* species also appears promising. Abundant *Rhizopogon* mycorrhizas formed on seedlings produced from the coated seed of *Pinus radiata* D. Don with

neighboring young seedlings has been successful [46].

**120**

introduction [45].

basidiospores of *Rhizopogon luteolus* [5]. However, it has three main drawbacks: (a) significant quantities of fruiting bodies are required and may not be available each year, (b) the success of the inoculation is highly dependent on the viability of the spores, and (c) the lack of genetic definition. Freeze-drying and storage at a low temperature in the dark is helpful to maintain its viability. The spores can be mixed with physical supports before the soil inoculation; suspended in water and soaked in the soil; sprinkled, sprayed or pelleted, and emitted to the ground; and encapsulated or coated on the seeds, and they can be embedded in hydrocolloid chips [47].

The most appropriate inoculum is the use of hyphae in a solid or liquid medium or substrate. Hyphae are cultivated mainly from sterile parts of fruiting bodies, less frequently from mycorrhiza due to their low (approx. 5–20%) success rate [48] and rarely from sclerotia [49] or sexual spores [50]. It is considered the most appropriate method since it allows the selection of particular strains of a fungus previously tested for its ability to promote the growth of plants [43]. Many species do grow well in culture, e.g., most species of *Suillus*, *Hebeloma*, *Laccaria*, *Amanita*, *Rhizopogon*, and *Pisolithus* genus. Liquid substrates have the advantage over solids because they are easily mixed and produce more uniform conditions for crop growth, but the risk of bacterial contamination and costs are higher [45]. On the other hand, the main advantages of the solid medium [51] are the reduction of bacterial contamination due to the lower water content, the low costs of the equipment, and the simplified design of the bioreactors. The main drawbacks of the use of mycelial inocula are that several species of ECM fungi are difficult to grow under laboratory conditions, or growth is very slow (due to the absence of their symbiont), and it is not always easy to produce large amounts of inoculum viable for large-scale nursery inoculation programs. Some advances have been made using mycelium encapsulated in "beads" of calcium alginate (e.g., [52]), but they have to be refrigerated. Inoculant beads can remain viable for several months under refrigeration, although the results vary between fungal species. For several species, the mycelial inoculum has been tested with trees of economic interest. This technique has great potential for the inoculation of seedlings in reforestation programs. For example, Rossi et al. [45] designed a bioreactor with the capacity to produce inoculum for 300 000 seedlings, enough to reforest 200 hectares. Based on a global demand of 3.0 billion cubic meters of wood, an estimated 4.3 tons of mycelium would be needed to inoculate 12 billion seedlings (5 g of dry mycelium per plant [45]). An advantage of alginate gel is the possibility of preparing a multimicrobial inoculant.

### *3.1.1 Ectomycorrhizal helper bacteria*

The concept of "mycorrhiza helper bacteria" (MHB) was introduced in a "Tansley Review": Helper Bacteria—a new dimension of mycorrhizal symbiosis [53], which has led to new research in the plant-fungus model system, as for the meaning of these bacteria that promote the formation of mycorrhizas and cause many physiological effects of mutualistic interaction. In general, the ability of some microorganisms to influence the formation and functioning of the symbiosis is known, through activities of various kinds such as the activation of infective propagules of the fungus in presymbiotic stages [54], facilitating the formation of entry points in the root [55] and increase of the growth rate [56]. The MHB improve mycorrhiza formation, although the same MHB can benefit mycorrhization for certain fungi and be negative for others [57]. The above reflects the fungal specificity by isolate, which exemplifies the genetic distance between isolates of different origin.

Among the mechanisms presented by the MHB are:


MHB belong to a wide range of genera (*Burkholderia*, *Paenibacillus* [67]; *Pseudomonas*, *Bacillus* [68]; *Streptomyces* [69]). However, the molecular mechanisms by which MHB induce the growth of ECM fungi are not well described. Recently, changes in expression of genes involved in the development of certain ECM fungi have been studied at the molecular level in confrontations with MHB [70–73].

Research in mycorrhizas should, therefore, strive toward an improved understanding of the functional and molecular mechanisms involved in interactions in the mycorrhizosphere, in order to develop ad hoc biotechnology that allows the application of optimized combinations of microorganisms as effective inoculators within sustainable systems of plant production [74].

### *3.1.2 Polymicrobial formulations*

A polymicrobial formulation containing a diverse mixture of beneficial rhizosphere microorganisms with multiple functionalities is attractive because combining different classes of soil organisms can take advantage of multiple plant growthpromoting mechanisms and could be applied to multiple crops [75–79]. A key concept in constructing effective polymicrobial multifunctional formulations is the selection and use of a right combination of rhizosphere bacteria and fungi that are mutually compatible, have complementary functionalities, effectively colonize the rhizosphere of the crop(s) of interest, and bring about a synergistic promotion of growth and yield of crop(s) [75, 80–82]. It is to be expected that well-designed multifunctional formulations such as the one described would be a welcome addition to the fastgrowing inoculant enterprises worldwide. Such an inoculant is also expected to be eco-friendly and suitable for organic farming and other integrated production systems, where synthetic fertilizer inputs are not allowed or restricted by law. However, construction of such complex formulations is technically demanding [83].

**123**

*Ectomycorrhizal Fungi as Biofertilizers in Forestry DOI: http://dx.doi.org/10.5772/intechopen.88585*

were present [85, 86].

strategies of forest management [91].

obtaining productions from 6 to 7 years of implantation.

Ectomycorrhizal fungi exhibit synergistic interactions with other plantbeneficial organisms such as symbiotic N2-fixers. For example, ectomycorrhizal symbiosis enhanced the efficiency of inoculation of two *Bradyrhizobium* strains on the growth of legumes [84]. It is also of interest that similar synergies were seen when AM fungus (*Glomus mosseae*), ECM fungus (*Pisolithus tinctorius*), and *Bradyrhizobium* sp*.* were used together to inoculate *Acacia nilotica*; enhancement of N2 fixation, growth, and dry biomass were observed when all three organisms

Also, using plant growth-promoting microorganism (PGPM) strains that form stable and effective biofilms could be a strategy for producing commercially viable inoculant formulations [78, 87]. A majority of plant-associated bacteria found on roots and in the soil are found to form biofilms [88]. Bacterial, fungal, and bacteria/ fungal biofilms were suggested as possible inoculants. This is a novel and interesting idea, but to what extent this approach would be practiced remains to be seen [83].

**3.2 Application of ECM fungi in forest management: restoration of ecosystems**

The inoculation of ECM fungi can be done with the objective of producing edible carpophores but also because of its considerable value in forest management; in particular, they have had great importance in reforestation programs where it was expected that the quality and economic productivity of the plantations would increase [89]. The success of the plantations with mycorrhizal seedlings from the nursery depends on their ability to quickly access the nutrients and water available within the soil matrix [90]. The relationships between the various native edible ECM fungi have been, until relatively recently, insufficiently considered in the

In ectomycorrhizal plantations (productive or conservation reforestations), a consequence of the recognition of the advantages of fungal diversity in ecosystems will be an increase in the refusal to introduce potentially dominant species in mixed communities. On the other hand, unfortunately, it seems that many of those fungi selected for optimal colonization in the nursery have been poor competitors in the field, especially when the planting sites contained indigenous populations of mycorrhizal fungi. There are several possible explanations for the inoculation failure (from the nursery) to produce beneficial effects in the planting sites. Probably, among the most important of these is the inability of inoculum introduced to persist in the roots of the plant after the transfer of the nursery to the field. The soil conditions experienced in the nursery and with the plant growing in a container are very different from those of most of the planting sites; in addition, the raising, storage, and transport of seedlings can reduce the vigor of fine roots and their fungal associates. Species such as *Pisolithus tinctorius* (15 sub spp.), in circumstances such as degraded environments, with absence or scarcity of autochthonous mycorrhizal populations, have achieved the greatest success in inoculation programs [92]. In the case of edible ECM fungi, such as *Tuber melanosporum* (black truffle), the establishment of mycorrhizal plantations has always aimed at the production of carpophores, leaving aside the contribution of ecological functions of the symbiosis (in the plant, in the soil, and, in general, in the ecosystem) [93]. The example of mycorrhizal plantations for truffle production has been generally successful [94],

In the restoration of ecosystems, the biofertilization, the bioremediation, and the biocontrol of soil pathogens are prominent roles of the ectomycorrhizal fungi. Degraded ecosystems are the result of a wide range of characteristics and factors related to unfavorable land management or industrial activities. Environmental degradation of the soil is increasing worldwide at an alarming rate due to erosion,

*Ectomycorrhizal Fungi as Biofertilizers in Forestry DOI: http://dx.doi.org/10.5772/intechopen.88585*

*Biostimulants in Plant Science*

Among the mechanisms presented by the MHB are:

production of growth factors [58].

a.Promotion of the establishment of the symbiosis by stimulation of the mycelial extension. The germination of spores and mycelial growth are improved by the

b.Increased contact and colonization root-fungus: increase in the number of lateral roots, mediated by the production of phytohormones [59] and the improve-

ment of radical colonization by induction of flavonoid production [60].

them with available nitrogen derived from atmospheric nitrogen (N2).

MHB belong to a wide range of genera (*Burkholderia*, *Paenibacillus* [67]; *Pseudomonas*, *Bacillus* [68]; *Streptomyces* [69]). However, the molecular mechanisms by which MHB induce the growth of ECM fungi are not well described. Recently, changes in expression of genes involved in the development of certain ECM fungi have been studied at the molecular level in confrontations with MHB [70–73]. Research in mycorrhizas should, therefore, strive toward an improved understanding of the functional and molecular mechanisms involved in interactions in the mycorrhizosphere, in order to develop ad hoc biotechnology that allows the application of optimized combinations of microorganisms as effective inoculators

A polymicrobial formulation containing a diverse mixture of beneficial rhizosphere microorganisms with multiple functionalities is attractive because combining different classes of soil organisms can take advantage of multiple plant growthpromoting mechanisms and could be applied to multiple crops [75–79]. A key concept in constructing effective polymicrobial multifunctional formulations is the selection and use of a right combination of rhizosphere bacteria and fungi that are mutually compatible, have complementary functionalities, effectively colonize the rhizosphere of the crop(s) of interest, and bring about a synergistic promotion of growth and yield of crop(s) [75, 80–82]. It is to be expected that well-designed multifunctional formulations such as the one described would be a welcome addition to the fastgrowing inoculant enterprises worldwide. Such an inoculant is also expected to be eco-friendly and suitable for organic farming and other integrated production systems, where synthetic fertilizer inputs are not allowed or restricted by law. However,

construction of such complex formulations is technically demanding [83].

within sustainable systems of plant production [74].

*3.1.2 Polymicrobial formulations*

c.Reduction of the impact of adverse environmental factors on the mycelium of the mycorrhizal fungus. Bacteria can detoxify soils, restoring their conductivity, similarly freeing them from contamination generated by heavy metals [61], and reducing the concentrations of phenolic antagonist compounds produced by the same mycorrhizal fungi [62]. The rhizospheric microorganisms also have an effect on the growth of the plants, reaching a synergistic effect, where the presence of the microfungus and the other microorganism produces an increase in the growth, vigor, and protection of the plant [63]. These effects are based on activities such as the acquisition of nutrients, inhibition of the growth of pathogenic fungi [64], and improvement of the root ramification [65]. In recent years, a potential capacity of bacteria associated with ectomycorrhizas to fix atmospheric nitrogen has been suggested [66]. Several studies suggest a real possibility that the bacteria present in mycorrhizal tissues contribute to the nutritional needs of both the fungus (ascocarp development) and consequently the plants, by providing

**122**

Ectomycorrhizal fungi exhibit synergistic interactions with other plantbeneficial organisms such as symbiotic N2-fixers. For example, ectomycorrhizal symbiosis enhanced the efficiency of inoculation of two *Bradyrhizobium* strains on the growth of legumes [84]. It is also of interest that similar synergies were seen when AM fungus (*Glomus mosseae*), ECM fungus (*Pisolithus tinctorius*), and *Bradyrhizobium* sp*.* were used together to inoculate *Acacia nilotica*; enhancement of N2 fixation, growth, and dry biomass were observed when all three organisms were present [85, 86].

Also, using plant growth-promoting microorganism (PGPM) strains that form stable and effective biofilms could be a strategy for producing commercially viable inoculant formulations [78, 87]. A majority of plant-associated bacteria found on roots and in the soil are found to form biofilms [88]. Bacterial, fungal, and bacteria/ fungal biofilms were suggested as possible inoculants. This is a novel and interesting idea, but to what extent this approach would be practiced remains to be seen [83].

### **3.2 Application of ECM fungi in forest management: restoration of ecosystems**

The inoculation of ECM fungi can be done with the objective of producing edible carpophores but also because of its considerable value in forest management; in particular, they have had great importance in reforestation programs where it was expected that the quality and economic productivity of the plantations would increase [89]. The success of the plantations with mycorrhizal seedlings from the nursery depends on their ability to quickly access the nutrients and water available within the soil matrix [90]. The relationships between the various native edible ECM fungi have been, until relatively recently, insufficiently considered in the strategies of forest management [91].

In ectomycorrhizal plantations (productive or conservation reforestations), a consequence of the recognition of the advantages of fungal diversity in ecosystems will be an increase in the refusal to introduce potentially dominant species in mixed communities. On the other hand, unfortunately, it seems that many of those fungi selected for optimal colonization in the nursery have been poor competitors in the field, especially when the planting sites contained indigenous populations of mycorrhizal fungi. There are several possible explanations for the inoculation failure (from the nursery) to produce beneficial effects in the planting sites. Probably, among the most important of these is the inability of inoculum introduced to persist in the roots of the plant after the transfer of the nursery to the field. The soil conditions experienced in the nursery and with the plant growing in a container are very different from those of most of the planting sites; in addition, the raising, storage, and transport of seedlings can reduce the vigor of fine roots and their fungal associates. Species such as *Pisolithus tinctorius* (15 sub spp.), in circumstances such as degraded environments, with absence or scarcity of autochthonous mycorrhizal populations, have achieved the greatest success in inoculation programs [92]. In the case of edible ECM fungi, such as *Tuber melanosporum* (black truffle), the establishment of mycorrhizal plantations has always aimed at the production of carpophores, leaving aside the contribution of ecological functions of the symbiosis (in the plant, in the soil, and, in general, in the ecosystem) [93]. The example of mycorrhizal plantations for truffle production has been generally successful [94], obtaining productions from 6 to 7 years of implantation.

In the restoration of ecosystems, the biofertilization, the bioremediation, and the biocontrol of soil pathogens are prominent roles of the ectomycorrhizal fungi. Degraded ecosystems are the result of a wide range of characteristics and factors related to unfavorable land management or industrial activities. Environmental degradation of the soil is increasing worldwide at an alarming rate due to erosion,

acidity, salinization, compaction, depletion of organic matter, and water scarcity. On the contrary, in a healthy ecosystem, there is a balanced microbiota of the soil, in such a way that the potential of pathogenic and mycorrhizal fungi coexists in apparent harmony. Ectomycorrhizal fungi can survive in extreme habitats with high or low temperature [95, 96], salt and metal concentration [97, 98], drought [99], and other circumstances related to the degradation of the ecosystem. The importance of ECM fungi in the balance of the ecosystem can be enormous, since they can be used to increase the tolerance of plants against biotic or abiotic stresses, especially their capacity to fix heavy metals or to degrade a wide variety of persistent organic compounds; to interact with soil bacteria; to attack fungi, bacteria, and pathogenic nematodes; and to improve the vegetative growth and the nutritional status of its symbiont plant.

It has been documented by several authors that mycorrhizal fungi improve the disease resistance of their host plant primarily by direct competition, enhanced or altered plant growth, nutrition and morphology, induced resistance, and development of antagonist microbiota. Direct competition or inhibition is reported to be due to the production and release of antibiotics and physical sheathing by the mantle of ECM [27, 100–102]. For example, ECM fungi have been shown to protect trees from *Phytophthora cinnamomi* infection along with supporting their survival and growth in comparison to non-mycorrhizal seedlings [35, 101, 102]. Thus, ECM fungi can also be used as a fungicide in nursery plantations for better growth, survival, and establishment of seedlings.

Under drought stress, ectomycorrhizal symbiosis has been documented to possess a remarkable capacity to the uptake of water and alter hydraulic properties of plant roots by altering both apoplastic and symplastic pathways and by their impact on plant aquaporins (AQPs) [103–106]. A symbiosis between plants and ECM fungi has been documented to help plants to cope with salt stress [97, 107–109]. Li et al. [110] reported that there is ECM fungus-mediated remodeling of ion flux which helps to maintain K+/Na+ homeostasis by increasing the release of Ca2+. Also, ECM fungi have been reported to change the plant phytohormone balance during salt stress [111, 112]. Research efforts are still in progress to select new pioneer symbiotic couples for land reforestation [113].

Till date, most studies have indicated that ECM plants accumulate less metal inside their tissue and grow better than non-mycorrhizal plants when exposed to heavy metal stress [114–118]. Also, Meharg and Cairney [119] revised potential ways in which ectomycorrhizal fungi might support rhizosphere remediation of persistent organic pollutants (POPs). Krupa and Kozdrój [120] documented the importance of mycorrhizal fungi in forming an efficient biological barrier for checking the movement of heavy metals into the host tissues. Recently, the importance of LMW organic acids and metal chelating agents (such as siderophores) from ECM fungi in the fixation of metal ions and their transmission or not to the root of the host plant has been described [121]. The cellular mechanisms involved in detoxification of heavy metals by mycorrhizal fungi include biosorption of metals to fungal cell wall, chelation of metal ion in the cytosol by compounds such as glutathione and metallothioneins, metal exclusion mechanisms in metal-tolerant ECM fungi, and the compartmentation of metals in the vacuole, where metal ions are probably complexed in a chemically inactive form [98, 118, 122, 123].

### **4. Conclusions**

The ectomycorrhizal fungi are predominantly *Basidiomycetes* and *Ascomycetes*, which establish a symbiotic relationship with the roots of forest plants, and these

**125**

**Author details**

José Alfonso Domínguez-Núñez1

Ciudad Universitaria, Madrid, Spain

provided the original work is properly cited.

*Ectomycorrhizal Fungi as Biofertilizers in Forestry DOI: http://dx.doi.org/10.5772/intechopen.88585*

are directly involved in the mobilization, absorption, and translocation of soil nutrients and water to the roots. Most of the known cultivated species of edible fungi are saprophytes, and some of them are ectomycorrhizal fungi, but there is a promising potential in the study and knowledge of new species of ECM fungi as potential wild collected edible mushrooms. ECM fungi play a key role in terrestrial ecosystems as drivers of global carbon and nutrient cycles; in the fungus-plant interface, the role of C and nutrient transporters seems a priority. Research in ectomycorrhizal fungi should focus on better understanding the functional and molecular mechanisms involved in fungus-plant and fungus-soil interactions. For decades, our understanding of the functioning of ectomycorrhizal fungi has allowed us their application in the forest area. In the nursery, the inoculation of ECM fungi is a more common method to produce ectomycorrhizal forest seedlings, and the mycelial inoculation has great potential in reforestation programs. We should aim to find the appropriate technology for the commercial techniques of multiplication and large-scale inoculation of the mycorrhizal inoculum and the application of optimized combinations of plant-microorganisms (e.g., MHB, PGPB) adopted under well-defined environmental and soil conditions. The role of ECM fungi as biofertilizers in bioremediation or biocontrol in plantations, reforestation, and environmental restoration has been fundamental up to now, and its importance in the balance of the ecosystem can be enormous, increasing the tolerance of plants against biotic and abiotic stress. The application of ectomycorrhizal fungi in current environmental problems as the oaks or pines decline, or the phytoremediation of contaminated soils, seems promising. Research is still underway to select new

pioneer symbiotic relationships for land restoration and reforestation.

\* and Ada S. Albanesi<sup>2</sup>

1 E.T.S.I de Montes, Forestal y del Medio Natural, Universidad Politécnica de Madrid,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Departamento de Microbiología, Facultad de Agronomía y Agroindustrias, Universidad Nacional Santiago del Estero, Santiago del Estero, Argentina

\*Address all correspondence to: josealfonso.dominguez@upm.es

*Ectomycorrhizal Fungi as Biofertilizers in Forestry DOI: http://dx.doi.org/10.5772/intechopen.88585*

*Biostimulants in Plant Science*

status of its symbiont plant.

survival, and establishment of seedlings.

otic couples for land reforestation [113].

acidity, salinization, compaction, depletion of organic matter, and water scarcity. On the contrary, in a healthy ecosystem, there is a balanced microbiota of the soil, in such a way that the potential of pathogenic and mycorrhizal fungi coexists in apparent harmony. Ectomycorrhizal fungi can survive in extreme habitats with high or low temperature [95, 96], salt and metal concentration [97, 98], drought [99], and other circumstances related to the degradation of the ecosystem. The importance of ECM fungi in the balance of the ecosystem can be enormous, since they can be used to increase the tolerance of plants against biotic or abiotic stresses, especially their capacity to fix heavy metals or to degrade a wide variety of persistent organic compounds; to interact with soil bacteria; to attack fungi, bacteria, and pathogenic nematodes; and to improve the vegetative growth and the nutritional

It has been documented by several authors that mycorrhizal fungi improve the disease resistance of their host plant primarily by direct competition, enhanced or altered plant growth, nutrition and morphology, induced resistance, and development of antagonist microbiota. Direct competition or inhibition is reported to be due to the production and release of antibiotics and physical sheathing by the mantle of ECM [27, 100–102]. For example, ECM fungi have been shown to protect trees from *Phytophthora cinnamomi* infection along with supporting their survival and growth in comparison to non-mycorrhizal seedlings [35, 101, 102]. Thus, ECM fungi can also be used as a fungicide in nursery plantations for better growth,

Under drought stress, ectomycorrhizal symbiosis has been documented to possess a remarkable capacity to the uptake of water and alter hydraulic properties of plant roots by altering both apoplastic and symplastic pathways and by their impact on plant aquaporins (AQPs) [103–106]. A symbiosis between plants and ECM fungi has been documented to help plants to cope with salt stress [97, 107–109]. Li et al. [110] reported that there is ECM fungus-mediated remodeling of ion flux which helps to maintain K+/Na+ homeostasis by increasing the release of Ca2+. Also, ECM fungi have been reported to change the plant phytohormone balance during salt stress [111, 112]. Research efforts are still in progress to select new pioneer symbi-

Till date, most studies have indicated that ECM plants accumulate less metal inside their tissue and grow better than non-mycorrhizal plants when exposed to heavy metal stress [114–118]. Also, Meharg and Cairney [119] revised potential ways in which ectomycorrhizal fungi might support rhizosphere remediation of persistent organic pollutants (POPs). Krupa and Kozdrój [120] documented the importance of mycorrhizal fungi in forming an efficient biological barrier for checking the movement of heavy metals into the host tissues. Recently, the importance of LMW organic acids and metal chelating agents (such as siderophores) from ECM fungi in the fixation of metal ions and their transmission or not to the root of the host plant has been described [121]. The cellular mechanisms involved in detoxification of heavy metals by mycorrhizal fungi include biosorption of metals to fungal cell wall, chelation of metal ion in the cytosol by compounds such as glutathione and metallothioneins, metal exclusion mechanisms in metal-tolerant ECM fungi, and the compartmentation of metals in the vacuole, where metal ions

are probably complexed in a chemically inactive form [98, 118, 122, 123].

The ectomycorrhizal fungi are predominantly *Basidiomycetes* and *Ascomycetes*, which establish a symbiotic relationship with the roots of forest plants, and these

**124**

**4. Conclusions**

are directly involved in the mobilization, absorption, and translocation of soil nutrients and water to the roots. Most of the known cultivated species of edible fungi are saprophytes, and some of them are ectomycorrhizal fungi, but there is a promising potential in the study and knowledge of new species of ECM fungi as potential wild collected edible mushrooms. ECM fungi play a key role in terrestrial ecosystems as drivers of global carbon and nutrient cycles; in the fungus-plant interface, the role of C and nutrient transporters seems a priority. Research in ectomycorrhizal fungi should focus on better understanding the functional and molecular mechanisms involved in fungus-plant and fungus-soil interactions. For decades, our understanding of the functioning of ectomycorrhizal fungi has allowed us their application in the forest area. In the nursery, the inoculation of ECM fungi is a more common method to produce ectomycorrhizal forest seedlings, and the mycelial inoculation has great potential in reforestation programs. We should aim to find the appropriate technology for the commercial techniques of multiplication and large-scale inoculation of the mycorrhizal inoculum and the application of optimized combinations of plant-microorganisms (e.g., MHB, PGPB) adopted under well-defined environmental and soil conditions. The role of ECM fungi as biofertilizers in bioremediation or biocontrol in plantations, reforestation, and environmental restoration has been fundamental up to now, and its importance in the balance of the ecosystem can be enormous, increasing the tolerance of plants against biotic and abiotic stress. The application of ectomycorrhizal fungi in current environmental problems as the oaks or pines decline, or the phytoremediation of contaminated soils, seems promising. Research is still underway to select new pioneer symbiotic relationships for land restoration and reforestation.

### **Author details**

José Alfonso Domínguez-Núñez1 \* and Ada S. Albanesi<sup>2</sup>

1 E.T.S.I de Montes, Forestal y del Medio Natural, Universidad Politécnica de Madrid, Ciudad Universitaria, Madrid, Spain

2 Departamento de Microbiología, Facultad de Agronomía y Agroindustrias, Universidad Nacional Santiago del Estero, Santiago del Estero, Argentina

\*Address all correspondence to: josealfonso.dominguez@upm.es

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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2007;**45**:277-286

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2008;**180**:260-263

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[54] Azcón-Aguilar C, Barea JM. Arbuscular mycorrhizas and biological control of soil-borne plant pathogens - an overview of the mechanisms involved. Mycorrhiza. 1996;**6**:457-464

[55] Linderman RG. Mycorrhizal interactions with the Rhizosphere Microflora - the rhizosphere effect. Phytopathology. 1988;**78**:366-371

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[76] Gravel V, Antoun H, Tweddell RJ. Growth stimulation and growth yield improvement of greenhouse tomato plants by inoculation with *Pseudomonas putida* and *Trichoderma atroviride*: Possible role of indole acetic acid (IAA). Soil Biology and Biochemistry.

[77] Hayat R, Ali S, Amara U, Khalid R, Ahmed I. Soil beneficial bacteria and their role in plant growth promotion: A review. Annales de Microbiologie.

[78] Malusa E, Sas-Paszt L, Ciesielska J.

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2005;**168**:205-216

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**130**

Microbial inoculation for improving the growth and health of micropropagated strawberry. Applied Soil Ecology. 2004;**27**:243-258

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[81] Barea JM, Azcón R, Azcón-Aguilar C. Interactions between mycorrhizal fungi and bacteria to improve plant nutrient cycling and soil structure. In: Varma A, Buscot F, editors. Microorganisms in Soils: Roles in Genesis and Functions, Vol. 3. Heidelberg: Springer Berlin; 2005. pp. 195-212

[82] Hata S, Kobae Y, Banba M. Interactions between plants and arbuscular mycorrhizal fungi. In: Kwang WJ, editor. International Review of Cell and Molecular Biology, Vol. 281. San Diego, USA: Academic Press; 2010. pp. 1-48

[83] Reddy CA, Saravanan RS. Polymicrobial multi-functional approach for enhancement of crop productivity. In: Sariaslani S, Gadd GM, editors. Advances in Applied Microbiology. Vol. 82. Michigan, USA: Elsevier; 2013. pp. 53-113

[84] Andre S, Galiana A, Le Roux C, Prin Y, Neyra M, Duponnois R. Ectomycorrhizal symbiosis enhanced the efficiency of inoculation with two *Bradyrhizobium* strains and *Acacia holosericea* growth. Mycorrhiza. 2005;**15**:357-364

[85] Saravanan RS, Natarajan K. Effect of *Pisolithus tinctorius* on the nodulation and nitrogen fixing potential of *Acacia nilotica* seedlings. Kavaka. 1996;**24**:41-49

[86] Saravanan RS, Natarajan K. Effect of ecto- and endomycorrhizal fungi along with *Bradyrhizobium sp*. on the growth and nitrogen fixation in

*Acacia nilotica* seedlings in the nursery. Journal of Tropical Forest Science. 2000;**12**:348-356

[87] Seneviratne G, Zavahir J, Bandara W, Weerasekara M. Fungalbacterial biofilms: Their development for novel biotechnological applications. World Journal of Microbiology and Biotechnology. 2008;**24**:739-743

[88] Ude S, Arnold DL, Moon CD, Timms-Wilson T, Spiers AJ. Biofilm formation and cellulose expression among diverse environmental *Pseudomonas* isolates. Environmental Microbiology. 2006;**8**:1997-2011

[89] Garbaye J. Use of mycorrhizas in forestry. In: Strullu DG, editor. Les mycorhizes des Arbres et Plantes Cultivées. Paris, France: Lavoisier; 1990. pp. 197-248

[90] Duñabeitia M, Rodríguez N, Salcedo I, Sarrionandia E. Field mycorrhization and its influence on the establishment and development of the seedlings in a broadleaf plantation in the Basque country. Forest Ecology and Management. 2004;**195**:129-139

[91] Dahlberg A, Genney DR, Heilmann-Clausen J. Developing a comprehensive strategy for fungal conservation in Europe: Current status and future needs. Fungal Ecology. 2010;**3**(2):50-64

[92] McAfee BJ, Fortin JA. Competitive interactions of ectomycorrhizal mycobionts under field conditions. Canadian Journal of Botany. 1986;**64**:848-852

[93] Domínguez JA, Selva J, Rodríguez Barreal JA, de Omeñaca S. The influence of mycorrhization with *Tuber melanosporum* in the afforestation of a Mediterranean site with *Quercus ilex* and *Quercus faginea*. Forest Ecology and Management. 2006;**231**:226-233

[94] Olivier JM, Savignac JC, Sourzat P. Truffe et Trufficulture. Perigueux, France: Ed Fanlac; 1996

[95] Tibbett M, Cairney JWG. The cooler side of mycorrhizas: Their occurrence and functioning at low temperatures. Canadian Journal of Botany. 2007;**85**:51-62

[96] Geml J, Timling I, Robinson CH, Lennon N, Nusbaum HC, Brochmann C, et al. Antarctic community of symbiotic fungi assembled by long-distance dispersers: Phylogenetic diversity of ectomycorrhizal basidiomycetes in Svalbard based on soil and sporocarp DNA. Journal of Biogeography. 2011;**39**:74-88

[97] Guerrero-Galán C, Calvo-Polanco M, Zimmermann SD. Ectomycorrhizal symbiosis helps plants to challenge salt stress conditions. Mycorrhiza. 2019;**29**:291-301. DOI: 10.1007/ s00572-019-00894-2

[98] Colpaert JV, Wevers JHL, Krznaric E, Adriaensen K. How metaltolerant ecotypes of ectomycorrhizal fungi protect plants from heavy metal pollution. Annals of Forest Science. 2011;**68**:17-24

[99] Azul AM, Sousa JP, Agerer R, Martín MP, Freitas H. Land use practices and ectomycorrhizal fungal communities from oak woodlands dominated by *Quercus suber* L. considering drought scenarios. Mycorrhiza. 2010;**20**:73-88

[100] Blom JM, Vannini A, Vettraino AM, Hale MD, Godbold DL. Ectomycorrhizal community structure in a healthy and a *Phytophthora*-infected chestnut (*Castanea sativa* Mill.) stand in central Italy. Mycorrhiza. 2009;**20**:25-38. DOI: 10.1007/s00572-009-0256-z

[101] Branzanti MB, Rocca E, Pisi A. Effect of ectomycorrhizal fungi on chestnut ink disease. Mycorrhiza.

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[102] Corcobado T, Moreno G, Azul AM, Solla A. Seasonal variations of ectomycorrhizal communities in declining *Quercus ilex* forests: Interactions with topography, tree health status and *Phytophthora cinnamomi* infections. Forestry. 2015;**88**:257-266. DOI: 10.1093/forestry/ cpu056

[103] Lehto T, Zwiazek JJ. Ectomycorrhizas and water relations of trees: A review. Mycorrhiza. 2011;**21**(2):71-90. DOI: 10.1007/ s00572-010-0348-9

[104] Maurel C, Plassard C. Aquaporins: For more than water at the plantfungus interface? New Phytologist. 2011;**190**(4):815-817. DOI: 10.1111/j.1469-8137.2011.03731.x

[105] Nehls U, Dietz S. Fungal aquaporins: Cellular functions and ecophysiological perspectives. Fungal Aquaporins: Cellular functions and Ecophysiological perspectives. Applied Microbiology and Biotechnology. 2014;**98**(21):8835-8851. DOI: 10.1007/ s00253-014-6049-0

[106] Xu H, Kemppainen M, El Kayal W, Lee SH, Pardo AG, Cooke JEK, et al. Overexpression of *Laccaria bicolor* aquaporin JQ585595 alters root water transport properties in ectomycorrhizal white spruce (*Picea glauca*) seedlings. New Phytologist. 2015;**205**:757-770. DOI: 10.1111/nph.13098

[107] Ishida TA, Nara K, Ma S, Takano T, Liu S. Ectomycorrhizal fungal community in alkaline-saline soil in northeastern China. Mycorrhiza. 2009;**19**(5):329-335. DOI: 10.1007/ s00572-008-0219-9

[108] Luo ZB, Li K, Gai Y, Gobel C, Wildhagen H, Jiang XN, et al. The ectomycorrhizal fungus (*Paxillus* 

**133**

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*involutus*) modulates leaf physiology of poplar towards improved salt tolerance. Environmental and Experimental Botany. 2011;**72**:304-311. DOI: 10.1016/j. protects pines from zinc stress. New Phytologist. 2004;**161**:549-555. DOI: 10.1046/j.1469-8137.2003.00941.x

Colpaert JV. Copper-adapted *Suillus luteus*, a symbiotic solution for pines colonizing Cu mine spoils. Applied and Environmental Microbiology. 2005;**71**:7279-7284. DOI: 10.1128/ AEM.71.11.7279-7284.2005

[115] Adriaensen K, Vrålstad T, Noben JP, Vangronsveld J,

[116] Jourand P, Ducousso M, Reid R, Majorel C, Richert C, Riss J, et al. Nickel-tolerant ectomycorrhizal *Pisolithus albus* ultramafic ecotype isolated from nickel mines in New Caledonia strongly enhance growth of the host plant *Eucalyptus globulus* at toxic nickel concentrations. Tree Physiology. 2010;**30**:1311-1319. DOI:

10.1093/treephys/tpq070

s00468-013-0973-y

[118] Luo ZB, Wua C, Zhang C, Lic H, Lipkad U, Polle A. The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environmental

[119] Meharg AA, Cairney JWG. Ectomycorrhizas: Extending the capacities of rhizosphere remediation?

Soil Biology and Biochemistry.

2004;**20**(4):427-430. DOI:

[120] Krupa P, Kozdrój J. Accumulation of heavy metals by ectomycorrhizal fungi colonizing birch trees growing in an industrial desert soil. World Journal of Microbiology and Biotechnology.

10.1023/B:WIBI.0000033067.64061.f3

and Experimental Botany. 2014;**108**:47-62. DOI: 10.1016/j.

envexpbot.2013.10.018

2000;**32**:1475-1484

[117] Kayama M, Yamanaka T. Growth characteristics of ectomycorrhizal seedlings of *Quercus glauca*, *Quercus salicina*, and *Castanopsis cuspidata* planted on acidic soil. Trees. 2014;**28**:569-583. DOI: 10.1007/

[109] Richard F, Roy M, Shahin O, Sthultz C, Duchemin M, Joffre R, et al. Ectomycorrhizal communities in a Mediterranean forest ecosystem dominated by *Quercus ilex*: Seasonal dynamics and response to drought in the surface organic horizon. Annals of Forest Science. 2011;**68**:57-68. DOI:

envexpbot.2011.04.008

10.1007/s13595-010-0007-5

[110] Li J, Bao S, Zhang Y, Ma X, Mishra-Knyrim M, Sun J, et al. *Paxillus* 

*involutus* strains MAJ and NAU mediate K(+)/Na(+) homeostasis in ectomycorrhizal *Populus x canescens* under sodium chloride stress. Plant Physiology. 2012;**159**:1771-1786. DOI:

[111] Luo ZB, Janz D, Jiang X, Göbel C, Wildhagen H, Tan Y, et al. Upgrading root physiology for stress tolerance by ectomycorrhizas: Insights from metabolite and transcriptional profiling into reprogramming for stress anticipation. Plant Physiology. 2009;**151**:1902-1917. DOI: 10.1104/

[112] Szuba A. Ectomycorrhiza of *Populus*. Forest Ecology and Management. 2015;**347**:156-169. DOI:

10.1016/ j.foreco.2015.03.012

Bissonnette C, Bélanger P,

DOI: 10.1139/cjm-2015-0703

[113] Beaudoin-Nadeau M, Gagné A,

Fortin J, Roy S, et al. Performance of ectomycorrhizal alders exposed to specific Canadian oil sands tailing stressors under in vivo bipartite

symbiotic conditions. Canadian Journal of Microbiology. 2016;**62**(7):543-549.

[114] Adriaensen K, van der Lelie D, Van Laere A, Vangronsveld J, Colpaert JV. A zinc-adapted fungus

10.1104 /pp.112.195370

pp.109.143735

*Ectomycorrhizal Fungi as Biofertilizers in Forestry DOI: http://dx.doi.org/10.5772/intechopen.88585*

*Biostimulants in Plant Science*

[94] Olivier JM, Savignac JC, Sourzat P. Truffe et Trufficulture. Perigueux, France: Ed Fanlac; 1996

Canadian Journal of Botany.

2007;**85**:51-62

2011;**39**:74-88

s00572-019-00894-2

2011;**68**:17-24

[98] Colpaert JV, Wevers JHL,

Krznaric E, Adriaensen K. How metaltolerant ecotypes of ectomycorrhizal fungi protect plants from heavy metal pollution. Annals of Forest Science.

[99] Azul AM, Sousa JP, Agerer R, Martín MP, Freitas H. Land use practices and ectomycorrhizal fungal communities from oak woodlands dominated by *Quercus suber* L. considering drought scenarios. Mycorrhiza. 2010;**20**:73-88

[100] Blom JM, Vannini A, Vettraino AM, Hale MD, Godbold DL. Ectomycorrhizal community structure in a healthy and a *Phytophthora*-infected chestnut (*Castanea sativa* Mill.) stand in central Italy. Mycorrhiza. 2009;**20**:25-38. DOI:

10.1007/s00572-009-0256-z

[101] Branzanti MB, Rocca E, Pisi A. Effect of ectomycorrhizal fungi on chestnut ink disease. Mycorrhiza.

[95] Tibbett M, Cairney JWG. The cooler side of mycorrhizas: Their occurrence and functioning at low temperatures.

1999;**9**:103-109. DOI: 10.1007/

Solla A. Seasonal variations of ectomycorrhizal communities in declining *Quercus ilex* forests: Interactions with topography, tree health status and *Phytophthora cinnamomi* infections. Forestry.

[103] Lehto T, Zwiazek JJ.

2011;**190**(4):815-817. DOI: 10.1111/j.1469-8137.2011.03731.x

[105] Nehls U, Dietz S. Fungal aquaporins: Cellular functions and ecophysiological perspectives. Fungal Aquaporins: Cellular functions and Ecophysiological perspectives. Applied Microbiology and Biotechnology. 2014;**98**(21):8835-8851. DOI: 10.1007/

s00572-010-0348-9

s00253-014-6049-0

DOI: 10.1111/nph.13098

s00572-008-0219-9

[107] Ishida TA, Nara K, Ma S,

[108] Luo ZB, Li K, Gai Y, Gobel C, Wildhagen H, Jiang XN, et al. The ectomycorrhizal fungus (*Paxillus* 

[102] Corcobado T, Moreno G, Azul AM,

2015;**88**:257-266. DOI: 10.1093/forestry/

Ectomycorrhizas and water relations of trees: A review. Mycorrhiza. 2011;**21**(2):71-90. DOI: 10.1007/

[104] Maurel C, Plassard C. Aquaporins: For more than water at the plantfungus interface? New Phytologist.

[106] Xu H, Kemppainen M, El Kayal W, Lee SH, Pardo AG, Cooke JEK, et al. Overexpression of *Laccaria bicolor* aquaporin JQ585595 alters root water transport properties in ectomycorrhizal white spruce (*Picea glauca*) seedlings. New Phytologist. 2015;**205**:757-770.

Takano T, Liu S. Ectomycorrhizal fungal community in alkaline-saline soil in northeastern China. Mycorrhiza. 2009;**19**(5):329-335. DOI: 10.1007/

s005720050007

cpu056

[96] Geml J, Timling I, Robinson CH, Lennon N, Nusbaum HC, Brochmann C, et al. Antarctic community of symbiotic fungi assembled by long-distance dispersers: Phylogenetic diversity of ectomycorrhizal basidiomycetes in Svalbard based on soil and sporocarp DNA. Journal of Biogeography.

[97] Guerrero-Galán C, Calvo-Polanco M, Zimmermann SD. Ectomycorrhizal symbiosis helps plants to challenge salt stress conditions. Mycorrhiza. 2019;**29**:291-301. DOI: 10.1007/

**132**

*involutus*) modulates leaf physiology of poplar towards improved salt tolerance. Environmental and Experimental Botany. 2011;**72**:304-311. DOI: 10.1016/j. envexpbot.2011.04.008

[109] Richard F, Roy M, Shahin O, Sthultz C, Duchemin M, Joffre R, et al. Ectomycorrhizal communities in a Mediterranean forest ecosystem dominated by *Quercus ilex*: Seasonal dynamics and response to drought in the surface organic horizon. Annals of Forest Science. 2011;**68**:57-68. DOI: 10.1007/s13595-010-0007-5

[110] Li J, Bao S, Zhang Y, Ma X, Mishra-Knyrim M, Sun J, et al. *Paxillus involutus* strains MAJ and NAU mediate K(+)/Na(+) homeostasis in ectomycorrhizal *Populus x canescens* under sodium chloride stress. Plant Physiology. 2012;**159**:1771-1786. DOI: 10.1104 /pp.112.195370

[111] Luo ZB, Janz D, Jiang X, Göbel C, Wildhagen H, Tan Y, et al. Upgrading root physiology for stress tolerance by ectomycorrhizas: Insights from metabolite and transcriptional profiling into reprogramming for stress anticipation. Plant Physiology. 2009;**151**:1902-1917. DOI: 10.1104/ pp.109.143735

[112] Szuba A. Ectomycorrhiza of *Populus*. Forest Ecology and Management. 2015;**347**:156-169. DOI: 10.1016/ j.foreco.2015.03.012

[113] Beaudoin-Nadeau M, Gagné A, Bissonnette C, Bélanger P, Fortin J, Roy S, et al. Performance of ectomycorrhizal alders exposed to specific Canadian oil sands tailing stressors under in vivo bipartite symbiotic conditions. Canadian Journal of Microbiology. 2016;**62**(7):543-549. DOI: 10.1139/cjm-2015-0703

[114] Adriaensen K, van der Lelie D, Van Laere A, Vangronsveld J, Colpaert JV. A zinc-adapted fungus

protects pines from zinc stress. New Phytologist. 2004;**161**:549-555. DOI: 10.1046/j.1469-8137.2003.00941.x

[115] Adriaensen K, Vrålstad T, Noben JP, Vangronsveld J, Colpaert JV. Copper-adapted *Suillus luteus*, a symbiotic solution for pines colonizing Cu mine spoils. Applied and Environmental Microbiology. 2005;**71**:7279-7284. DOI: 10.1128/ AEM.71.11.7279-7284.2005

[116] Jourand P, Ducousso M, Reid R, Majorel C, Richert C, Riss J, et al. Nickel-tolerant ectomycorrhizal *Pisolithus albus* ultramafic ecotype isolated from nickel mines in New Caledonia strongly enhance growth of the host plant *Eucalyptus globulus* at toxic nickel concentrations. Tree Physiology. 2010;**30**:1311-1319. DOI: 10.1093/treephys/tpq070

[117] Kayama M, Yamanaka T. Growth characteristics of ectomycorrhizal seedlings of *Quercus glauca*, *Quercus salicina*, and *Castanopsis cuspidata* planted on acidic soil. Trees. 2014;**28**:569-583. DOI: 10.1007/ s00468-013-0973-y

[118] Luo ZB, Wua C, Zhang C, Lic H, Lipkad U, Polle A. The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environmental and Experimental Botany. 2014;**108**:47-62. DOI: 10.1016/j. envexpbot.2013.10.018

[119] Meharg AA, Cairney JWG. Ectomycorrhizas: Extending the capacities of rhizosphere remediation? Soil Biology and Biochemistry. 2000;**32**:1475-1484

[120] Krupa P, Kozdrój J. Accumulation of heavy metals by ectomycorrhizal fungi colonizing birch trees growing in an industrial desert soil. World Journal of Microbiology and Biotechnology. 2004;**20**(4):427-430. DOI: 10.1023/B:WIBI.0000033067.64061.f3

### *Biostimulants in Plant Science*

[121] Machuca A. Metal-chelating agents from ectomycorrhizal fungi and their biotechnological potential. In: Rai M, Varma A, editors. Diversity and Biotechnology of Ectomycorrhizas. Soil Biology. Berlin, Heidelberg: Springer-Verlag; 2011:**25**

[122] Daghino S, Martino E, Perotto S. Model systems to unravel the molecular mechanisms of heavy metal tolerance in the ericoid mycorrhizal symbiosis. Mycorrhiza. 2016;**26**:263-274. DOI: 10.1007/s00572-015-0675-y

[123] Krpata D, Fitz W, Peintner U, Langer I, Schweiger P. Bioconcentration of zinc and cadmium in ectomycorrhizal fungi and associated aspen trees as affected by level of pollution. Environmental Pollution. 2009;**157**:280-286. DOI: 10.1016/j.envpol.2008.06.038

**135**

**Chapter 9**

**Abstract**

**1. Introduction**

Microbes for Iron Chlorosis

Peach [*Prunus persica* (L.) Batsch] suffers from iron chlorosis when grown in calcareous soils due to low iron availability. Traditionally, soil and foliar application of ferrous sulphate, Fe-EDTA, Fe-EDDHA chelates, etc. is used as a corrective measure of chlorosis**.** The latter practice is quite effective. However, variable responses have been reported. Therefore, foliar spray cannot yet be considerd as a reliable method to control lime-induced chlorosis. Bioremediation constitutes innovative approaches for chlorosis correction. Iron fixations in calcareous soil, iron uptake by plants, and advance detection techniques and correction strategies in plants for iron chlorosis have been discussed in this chapter. The microbe-mediated correction

**Keywords:** peach, *Prunus persica*, calcareous soil, iron chlorosis, bioremediation

Peach [*Prunus persica* (L.) Batsch] is one of the most common temperate region fruit crops of the world. China, Italy, the USA, Greece, Spain, Turkey, Iran, Chile, etc. are the major producing countries [1]. This stone fruit crop belongs to the family Rosaceae. Peach [*Prunus persica* var. *vulgaris* Maxim.] with round and fuzzy fruit, the nectarine [*Prunus persica var. nectarina* (Aiton) Maxim.] with round fruit but without pubescence (fuzz), and the flat peach [*Prunus persica* var. *platicarpa* Bailey] with flat-shaped fruit are the three categories [2]. Iron, the fourth most prevalent element preceded by O, Si, and Al in the earth's crust and soils, is classified as an essential micronutrient for plant growth. It is a multifunctional element [3], required for the different physicochemical processes of plants, and plays an important role in chlorophyll activation, chloroplast membrane structure, photosynthesis, respiration, and synthesis of many heme proteins and iron–sulphur (Fe-S) clusters as cofactors of proteins that function in the fundamental life of plants [4–6]. Higher plants use two general mechanisms (strategies I and II) for iron acquisition with low iron availability in soil [7]. Calcareous soil gives lower iron availability abreast with a diminishing uptake efficiency by plant roots specially of a plant that depends on ferric reductase activity, because of higher soil pH and bicarbonate concentration [8, 9]. Out of a total of 13.4 billion ha global land surface, 1.5 billion ha is used in crop production, including arable lands plus lands under permanent crops [10, 11]. 30% of the soils in the world are calcareous in nature. They limit the iron availability for plant growth and development, not due to the

Remediation in Peach

*Saurabh Kumar Singh*

strategies are identified as eco-friendly.

iron status of the soil but due to their solubility [12].

### **Chapter 9**

*Biostimulants in Plant Science*

Verlag; 2011:**25**

[121] Machuca A. Metal-chelating agents from ectomycorrhizal fungi and their biotechnological potential. In: Rai M, Varma A, editors. Diversity and Biotechnology of Ectomycorrhizas. Soil Biology. Berlin, Heidelberg: Springer-

[122] Daghino S, Martino E, Perotto S. Model systems to unravel the molecular mechanisms of heavy metal tolerance in the ericoid mycorrhizal symbiosis. Mycorrhiza. 2016;**26**:263-274. DOI:

10.1007/s00572-015-0675-y

[123] Krpata D, Fitz W, Peintner U, Langer I, Schweiger P. Bioconcentration of zinc and cadmium in ectomycorrhizal fungi and associated aspen trees as affected by level of pollution. Environmental Pollution. 2009;**157**:280-286. DOI: 10.1016/j.envpol.2008.06.038

**134**
