**1. Introduction**

[17] Leite RG. Cañas C, Forsberg B, Barthem R, Goulding M. Larvas dos Grandes Bagres Migradores. Instituto Nacional de Pesquisas da Amazônia (INPA) - Asociación para

[18] Lewis WM. Saunders III JF. Two new integrating samplers for zooplankton, and wa‐

[19] hlstrom EH, Moser HG. Eggs and larvae of fishes and their role in systematic investi‐ gations and in fisheries. Revue des Travaux de l'Institut des Pêches Maritimes 1976;

[20] Snyder DE. Terminologies of intervals for larval fish development. In: J. Boreman (Ed.) Great Lakes Fish Eggs and Larval Identification. U.S. Fisheries and Wildlife

[21] Araujo-Lima CARM. Larval development and reproductive strategies of Amazonian

[22] Torrente-Vilara G. Heterogeneidade ambiental e diversidade ictiofaunística do tre‐ cho de corredeiras do rio Madeira, Rondônia, Brasil. PhD Thesis. Manaus, Instituto Nacional de Pesquisas da Amazônia (INPA)/Fundação Universitária do Amazonas

[23] Bialetzki A, Sanches PV, Cavicchioli, M, Baumgartner, G, Ribeiro, RP, Nakatani K.. Drift of ichthyoplankton in two channels of the Paraná River, between Paraná and Mato Grosso do Sul states, Brazil. Brazilian Archives of Biology and Technology 1999. http://www.scielo.br/pdf/babt/v42n1/v42n1a08.pdf (accessed 18 September

[24] Marcano D, Cardillo E, Rodriguez C., Poleo G, Gago N, Guerrero H.Y. Seasonal re‐ productive biology of two species of freshwater catfish from the Venezuelan flood‐

[25] Carvalho, N. de O., Filizola Jr., N. P., Santos, P. M. C. dos, and Lima, J. E. F. W., 2000

plains. General and Comparative Endocrinology 2007; 153(1-3): 371-377.


fishes. PhD Thesis. Oban, Argyll, Scotland, University of Sterling; 1990.

la Conservación de la Cuenca Amazónica (ACCA). Lima, Peru; 2007.

ter chemistry. Archiv für Hydrobiologie 1979; 85(2):244-249.

40(3-4) 379-398

158 Biodiversity - The Dynamic Balance of the Planet

1976: 41-58.

(FUA); 2009

2013).

Arbuscular Mycorrhizal Fungi (AMF) are a group of obligate biotrophs, to the extent that they must develop a close symbiotic association with the roots of a living host plant in order to grow and complete their life cycle [1]. The term "mycorrhiza" literally derives from the Greek *mykes* and *rhiza*, meaning fungus and root, respectively. AMF can symbiotically interact with almost all the plants that live on the Earth. They are found in the roots of about 80-90% of plant species (mainly grasses, agricultural crops and herbs) and exchange benefits with their partners, as is typical of all mutual symbiotic relationships [2]. They represent an interface between plants and soil, growing their mycelia both inside and outside the plant roots. AMF provide the plant with water, soil mineral nutrients (mainly phosphorus and nitrogen) and pathogen protection. In exchange, photosynthetic compounds are transferred to the fungus [3].

Taxonomically, all AMF have been affiliated to a monophyletic group of fungi, i.e. the Glomeromycota phylum [4]. They are considered to be living fossils since there is evidence that their presence on our planet dates back to the Ordovician Period, over 460 million years ago [5]. Investigations on AMF taxonomy began in the nineteenth century with the first description of two species belonging to the genus *Glomus* [6]. Since that date, many Glomer‐ omycotan species, genus and families have been discovered and characterized by means of traditional approaches based on the phenotypic characteristics (mainly spore morphology). Molecular DNA sequencing-based analyses have recently contributed to a great extent by shedding light on a previously unseen and profound diversity within this phylum [7].

© 2014 The Author(s). Licensee InTech. 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.

Nevertheless, an open debate on the phylogeny of AMF, and in particular concerning some taxonomical groups, is still puzzling scientists [8–10] (Figure 1). Besides a general disagreement about the number of families and genera (Figure 1), what emerges from reference [8] is that Gigasporales are considered to be a separate order from Diversisporales. This is different from what has been reported in the tree on the right side of Figure 1, which was presented in reference [9], and supported by the recent reference [10].

Functionally, AMF form the so-called arbuscular mycorrhizae with plant roots. The most typical AMF structure, which also gives the name to this group of fungi, is the arbuscule (Figure 2). This structure, whose shape recalls that of a small shrub, forms inside the root cortical cells by branching in several very thin hyphae. In this way, the surface area, where the nutritional exchanges between the plant and fungus take place, is maximized. Fungal hyphae that grow between root cortical cells are able to produce other AMF structures, such as intercellular hyphae and vesicles (Figure 2). All these structures that grow inside the plant roots represent the intraradical phase of the fungus. Hyphae also grow outside the plant roots, and generate a network that extends over long distances and explores the soil beyond the nutrient depletion zone that normally characterizes the area surrounding the roots. At the end of the AMF life cycle, or in response to particular environmental conditions, spores (Figure 2) of variable size (up to 400 µm), depending on the species, are produced in the roots and/or in the soil. These, along with external explorative and running hyphae, represent the extraradical phase of the fungus. The synergic action of the intra-and extraradical phases is responsible for the ecological significance of the AMF, a soil-root-living key group of organisms [3].

#### **1.1. The ecological roles of AMF**

Arbuscular mycorrhizal fungi have a high relevance in many ecosystem processes. Since they can be found in many different plant species, they can provide their favorable services to almost all terrestrial ecosystems, from grasslands to forests, deserts and agroecosystems [11]. AMF can play several roles in such environments. The most agriculturally significant and frequently investigated one, from both the ecological and physiological points of view [12], is their positive effect on plant nutrition and, consequently, on plant fitness. In particular, they play a pivotal role in helping the plant uptake phosphorus from the soil [13]. Without AMF, it is rather difficult for the plant to absorb this macroelement from the soil, since it is mainly available in its insoluble organic or inorganic form. Besides phosphorus, AMF can also translocate water and other mineral nutrients (in particular nitrogen) from the soil to the plant. These nutritional exchanges are bidirectional. As a consequence, particularly efficient symbiotic associations have been demonstrated to stabilize through unknown mechanisms, with the plant selecting the most cooperative fungal partners and vice versa [14]. The AMF-inducible recovery of plant nutritional deficiency can inevitably lead to an improvement in plant growth, with a potential positive impact on productivity. Needless to say, AMF have attracted a great deal of interest from the agricultural world over the years [15].

of antioxidants, the increment of osmolyte production or the improvement of abscisic acid regulation [16,17], and the enhancement of plant tolerance to heavy metals [18]; (b) they help plants become established in harsh/degraded ecosystems, such as desert areas and mine spoils [19]; (c) they increase the power of phytoremediation (the removal of pollutants from the soil by plants) by allowing their host to explore and depollute a larger volume of soil [20,21]. Another crucial ecological role played by AMF is their capacity to directly influence the diversity and composition of the aboveground plant community. Several studies have

sequences (ca 1.8 kb).

**Figure 1.** A schematic representation of two recently published and partly controversial phylogenetic trees of the Glomeromycota phylum (reference [8] for the tree on the left side and [9] for the tree on the right side). The one pub‐ lished in reference [8] was based on molecular (SSU, ITS, partial LSU rDNA, and partial β-tubuline gene) and morpho‐ logical analyses (spore wall structures, structures of the spore bases and subtending hyphae, germination, and germination shield structures). The tree published in reference [9] was based on concatenated SSU rDNA consensus

Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management

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AMF are also responsible for other services that favour the plants they colonize: (a) they positively affect plant tolerance towards both biotic (e.g., pathogens) and abiotic stresses (i.e., drought and soil salinity) by acting on several physiological processes, such as the production

Nevertheless, an open debate on the phylogeny of AMF, and in particular concerning some taxonomical groups, is still puzzling scientists [8–10] (Figure 1). Besides a general disagreement about the number of families and genera (Figure 1), what emerges from reference [8] is that Gigasporales are considered to be a separate order from Diversisporales. This is different from what has been reported in the tree on the right side of Figure 1, which was presented in

Functionally, AMF form the so-called arbuscular mycorrhizae with plant roots. The most typical AMF structure, which also gives the name to this group of fungi, is the arbuscule (Figure 2). This structure, whose shape recalls that of a small shrub, forms inside the root cortical cells by branching in several very thin hyphae. In this way, the surface area, where the nutritional exchanges between the plant and fungus take place, is maximized. Fungal hyphae that grow between root cortical cells are able to produce other AMF structures, such as intercellular hyphae and vesicles (Figure 2). All these structures that grow inside the plant roots represent the intraradical phase of the fungus. Hyphae also grow outside the plant roots, and generate a network that extends over long distances and explores the soil beyond the nutrient depletion zone that normally characterizes the area surrounding the roots. At the end of the AMF life cycle, or in response to particular environmental conditions, spores (Figure 2) of variable size (up to 400 µm), depending on the species, are produced in the roots and/or in the soil. These, along with external explorative and running hyphae, represent the extraradical phase of the fungus. The synergic action of the intra-and extraradical phases is responsible for the ecological

Arbuscular mycorrhizal fungi have a high relevance in many ecosystem processes. Since they can be found in many different plant species, they can provide their favorable services to almost all terrestrial ecosystems, from grasslands to forests, deserts and agroecosystems [11]. AMF can play several roles in such environments. The most agriculturally significant and frequently investigated one, from both the ecological and physiological points of view [12], is their positive effect on plant nutrition and, consequently, on plant fitness. In particular, they play a pivotal role in helping the plant uptake phosphorus from the soil [13]. Without AMF, it is rather difficult for the plant to absorb this macroelement from the soil, since it is mainly available in its insoluble organic or inorganic form. Besides phosphorus, AMF can also translocate water and other mineral nutrients (in particular nitrogen) from the soil to the plant. These nutritional exchanges are bidirectional. As a consequence, particularly efficient symbiotic associations have been demonstrated to stabilize through unknown mechanisms, with the plant selecting the most cooperative fungal partners and vice versa [14]. The AMF-inducible recovery of plant nutritional deficiency can inevitably lead to an improvement in plant growth, with a potential positive impact on productivity. Needless to say, AMF have attracted a great deal of interest

AMF are also responsible for other services that favour the plants they colonize: (a) they positively affect plant tolerance towards both biotic (e.g., pathogens) and abiotic stresses (i.e., drought and soil salinity) by acting on several physiological processes, such as the production

reference [9], and supported by the recent reference [10].

significance of the AMF, a soil-root-living key group of organisms [3].

**1.1. The ecological roles of AMF**

160 Biodiversity - The Dynamic Balance of the Planet

from the agricultural world over the years [15].

**Figure 1.** A schematic representation of two recently published and partly controversial phylogenetic trees of the Glomeromycota phylum (reference [8] for the tree on the left side and [9] for the tree on the right side). The one pub‐ lished in reference [8] was based on molecular (SSU, ITS, partial LSU rDNA, and partial β-tubuline gene) and morpho‐ logical analyses (spore wall structures, structures of the spore bases and subtending hyphae, germination, and germination shield structures). The tree published in reference [9] was based on concatenated SSU rDNA consensus sequences (ca 1.8 kb).

of antioxidants, the increment of osmolyte production or the improvement of abscisic acid regulation [16,17], and the enhancement of plant tolerance to heavy metals [18]; (b) they help plants become established in harsh/degraded ecosystems, such as desert areas and mine spoils [19]; (c) they increase the power of phytoremediation (the removal of pollutants from the soil by plants) by allowing their host to explore and depollute a larger volume of soil [20,21]. Another crucial ecological role played by AMF is their capacity to directly influence the diversity and composition of the aboveground plant community. Several studies have

process. This aspect has led to the recognition of the importance of this group of organisms in

Arbuscular Mycorrhizal Fungi and their Value for Ecosystem Management

http://dx.doi.org/10.5772/58231

163

All the services offered by arbuscular mycorrhizal fungi confirm the need to study and describe all their features, including their biology, ecology, taxonomy, phylogeny and biodiversity. Over the years, several techniques have been developed to reach this goal: a brief history is

This group of organisms has a constraining characteristic that makes their study very complex: as obligate symbionts, they cannot be cultivated in vitro, away from their host plant. The development of an artificial system that is capable of going beyond this barrier dates back to the 1980s, when in vitro transformed carrot roots were successfully colonized by AMF species [30]. Thanks to this method, the study of arbuscular mycorrhizae became easier and many researches on both physiology and genetics became possible [31,32]. Over the last two decades, many molecular and physiological mechanisms involved in the symbiotic process between plants and AMF have been discovered and described, thanks to the increasing innovations and opportunities offered by molecular biology. For example, it is now known how the infectious process of AMF arises, and many of the involved genes have been identified

Molecular biology has also revolutionized the analysis of the biodiversity of AMF, making it easier and more accurate to characterize the AMF community composition of large quantities of samples from many different ecosystems, from prairies to savannas, and from grasslands to forests (Table 1). The first studies on the diversity and distribution of AMF were mainly focused on the identification of the species that colonize the roots of a given plant in a given environment [34]. This was mainly due to the deficiency in the available investigation techniques, as they were primarily based on spore surveys and intraradical fungal structure morphological identification by means of microscopy. Such morphological identification surveys were time consuming and often lacked accuracy, since many species were easily confused with others. The situation changed radically when the use of DNA-based techniques became common, and the extraction of DNA from plant tissue was reduced to a few relatively easy steps that could be reproduced in any laboratory [35,36]. The load bearing principle is simple: by sequencing a specific DNA region, it is possible to univocally identify the corre‐ sponding AMF [37]. So far, the most used DNA target regions for AMF identification are located on the ribosomal genes (Small and Large ribosomal Subunits – SSU and LSU, respec‐ tively – and the Internal Transcribed Spacers – ITS1 and ITS2), as they show a rate of variability that is sufficient to discriminate between AMF species/isolates [9]. All this has led to the current era of molecular identification of AMF species [10]. Next-Generation Sequencing (NGS) tools represent a further step forward for biodiversity surveys of all organisms [38], including AMF. Over the last few years, the number of NGS-based AMF biodiversity studies has increased, while the spectrum of the target environments has broadened [39]. Furthermore, new primer pair sets for the specific amplification of AMF DNA sequences, capable of providing higher accuracy and a comprehensive coverage of the whole Glomeromycota phylum, have been

processes related to climate change mitigation [29].

**1.2. Methods used in the study of AMF**

reported in section 1.2.

successfully [33].

**Figure 2.** Extraradical and intraradical phases of AMF growth. The spore (Figure E, a *Scutellospora sp.* spore stained with Melzer's reagent and squeezed) germinates in the bulk soil and approaches the root of a host plant. The fungus penetrates through a hyphopodium (Figure A, stained with 0.1% cotton blue encountered in *Camellia japonica* L. roots) and develops intracellular coils, extracellular vesicles and intracellular arbuscules (Figures B, C, D) within cortical parenchyma, without entering the central cylinder where the vessels are.

confirmed that plant species richness can be altered not only by climatic and edaphic factors, but also by soil microbial assemblages [22–24]. The underlying mechanism is not completely understood, but could be related to the promotion of seedling establishment of secondary plant species [25]. Nevertheless, on some occasions, AMF can also negatively affect the diversity and growth of plants, which is particularly significant for the management of weeds [26]. Last but not least, AMF play a critical role in soil aggregation, thanks to their thick extraradical hyphal network, which envelops and keeps the soil particles compact. It has been suggested that glycoproteins (glomalin and glomalin related proteins) secreted by AMF into the soil could exert a key role in this process [27,28]. These proteins are exuded in great quantities into the soil, and could have implications on carbon sequestration. This potential capability of AMF is likely to contribute to a great extent to the soil ecosystem carbon dioxide (CO2) sequestration process. This aspect has led to the recognition of the importance of this group of organisms in processes related to climate change mitigation [29].

All the services offered by arbuscular mycorrhizal fungi confirm the need to study and describe all their features, including their biology, ecology, taxonomy, phylogeny and biodiversity. Over the years, several techniques have been developed to reach this goal: a brief history is reported in section 1.2.

#### **1.2. Methods used in the study of AMF**

confirmed that plant species richness can be altered not only by climatic and edaphic factors, but also by soil microbial assemblages [22–24]. The underlying mechanism is not completely understood, but could be related to the promotion of seedling establishment of secondary plant species [25]. Nevertheless, on some occasions, AMF can also negatively affect the diversity and growth of plants, which is particularly significant for the management of weeds [26]. Last but not least, AMF play a critical role in soil aggregation, thanks to their thick extraradical hyphal network, which envelops and keeps the soil particles compact. It has been suggested that glycoproteins (glomalin and glomalin related proteins) secreted by AMF into the soil could exert a key role in this process [27,28]. These proteins are exuded in great quantities into the soil, and could have implications on carbon sequestration. This potential capability of AMF is likely to contribute to a great extent to the soil ecosystem carbon dioxide (CO2) sequestration

parenchyma, without entering the central cylinder where the vessels are.

162 Biodiversity - The Dynamic Balance of the Planet

**Figure 2.** Extraradical and intraradical phases of AMF growth. The spore (Figure E, a *Scutellospora sp.* spore stained with Melzer's reagent and squeezed) germinates in the bulk soil and approaches the root of a host plant. The fungus penetrates through a hyphopodium (Figure A, stained with 0.1% cotton blue encountered in *Camellia japonica* L. roots) and develops intracellular coils, extracellular vesicles and intracellular arbuscules (Figures B, C, D) within cortical This group of organisms has a constraining characteristic that makes their study very complex: as obligate symbionts, they cannot be cultivated in vitro, away from their host plant. The development of an artificial system that is capable of going beyond this barrier dates back to the 1980s, when in vitro transformed carrot roots were successfully colonized by AMF species [30]. Thanks to this method, the study of arbuscular mycorrhizae became easier and many researches on both physiology and genetics became possible [31,32]. Over the last two decades, many molecular and physiological mechanisms involved in the symbiotic process between plants and AMF have been discovered and described, thanks to the increasing innovations and opportunities offered by molecular biology. For example, it is now known how the infectious process of AMF arises, and many of the involved genes have been identified successfully [33].

Molecular biology has also revolutionized the analysis of the biodiversity of AMF, making it easier and more accurate to characterize the AMF community composition of large quantities of samples from many different ecosystems, from prairies to savannas, and from grasslands to forests (Table 1). The first studies on the diversity and distribution of AMF were mainly focused on the identification of the species that colonize the roots of a given plant in a given environment [34]. This was mainly due to the deficiency in the available investigation techniques, as they were primarily based on spore surveys and intraradical fungal structure morphological identification by means of microscopy. Such morphological identification surveys were time consuming and often lacked accuracy, since many species were easily confused with others. The situation changed radically when the use of DNA-based techniques became common, and the extraction of DNA from plant tissue was reduced to a few relatively easy steps that could be reproduced in any laboratory [35,36]. The load bearing principle is simple: by sequencing a specific DNA region, it is possible to univocally identify the corre‐ sponding AMF [37]. So far, the most used DNA target regions for AMF identification are located on the ribosomal genes (Small and Large ribosomal Subunits – SSU and LSU, respec‐ tively – and the Internal Transcribed Spacers – ITS1 and ITS2), as they show a rate of variability that is sufficient to discriminate between AMF species/isolates [9]. All this has led to the current era of molecular identification of AMF species [10]. Next-Generation Sequencing (NGS) tools represent a further step forward for biodiversity surveys of all organisms [38], including AMF. Over the last few years, the number of NGS-based AMF biodiversity studies has increased, while the spectrum of the target environments has broadened [39]. Furthermore, new primer pair sets for the specific amplification of AMF DNA sequences, capable of providing higher accuracy and a comprehensive coverage of the whole Glomeromycota phylum, have been
