**2. The impact of humans on AMF biodiversity**

**1. Reference 2. Year 3. Method 4. Target**

166 Biodiversity - The Dynamic Balance of the Planet

**region**

[70] 2007 Clon-seq ITS Plant root

[72] 2005 Clon-seq SSU Plant root

[78] 2003 Clon-seq ITS Plant root

[79] 2003 Clon-seq SSU Plant root

[80] 2002 Clon-seq SSU Plant root

[36] 1999 Clon-seq SSU Plant root

sampling times.

[82] <sup>2002</sup> Clon-seq SSU Plant root Tropical forest

[83] <sup>2002</sup> Clon-seq SSU Plant root Tropical forest

**5. Studied compartment**

[68] 2007 Clon-seq LSU Soil and plant root Arable field (France) 246 12 [69] 2007 Clon-seq SSU Plant root Grassland (Sweden) 185 19

[71] 2006 Clon-seq SSU Plant root Polluted soils (Italy) 115 12

[73] 2004 Clon-seq SSU Plant root Wetland (Germany) 546 35 [74] 2004 Clon-seq LSU Plant root Grassland (Denmark) 158 11 [75] 2004 Clon-seq ITS Plant root Pasture (UK) 30 10 [76] 2004 Clon-seq SSU Plant root Grassland (Japan) 200 8 [77] 2004 Clon-seq SSU Plant root Grassland (UK) 606 9

[81] 2002 Clon-seq SSU Plant root Woodland (UK) 232 13

[84] 2001 Clon-seq SSU Plant root Arable field (UK) 303 8

[85] 1998 Clon-seq SSU Plant root Woodland (UK) 253 6/10b

a-taxa obtained with different primer sets; b: taxa obtained at different study sites; c-taxa obtained from forest ecosystems of different ages and management intensities; d-taxa obtained from roots of different plant species; e-taxa obtained at different

**Table 1.** The table shows an overview of DNA-based studies on the diversity of Arbuscular Micorrhizal (AM) fungal communities. For each study, the following are reported in sequence: 1. Reference, 2. Year of publication, 3. Used method (Clon-seq=cloning and sequencing; NGS=next generation sequencing), 4. Studied DNA region (SSU=Small Subunit; LSU=Large Subunit, ITS=Internal Transcribed Spacer), 5. Compartment from which the DNA was analyzed, 6.

**6.Ecosystem**

Volcanic desert (Japan)

Warm-temperate deciduous forest (Japan)

Afromontane forests (Ethiopia)

> Boreal forest (Estonia)

Seminatural grassland (UK)

(Republic of Panama)

(Republic of Panama)

Seminatural woodland (UK)

**7. AMF sequences**

205 11

394 5

92 20

16 6

88 24

1536 18/23d

558 18

141 6/8e

**8. OTUs**

Most human activities have an arguable impact on the physical and biological aspects of soil. As mentioned before, AMF are among the most widespread soil microorganisms, and each human activity that has an impact on soil, such as agricultural practices, therefore has a side effect on them. These practices, alone or in combination, exert an enormous selective pressure on AMF that shapes their community structure and evolution by modifying several of their biological features, such as sporulation strategy, resource allocation and spatial distribution [86]. As in natural ecosystems, AMF are also present and active in agricultural ecosystems, where they colonize several major arable crops (sorghum, maize, wheat and rice). Many studies have indicated that AMF diversity, effectiveness, abundance and biodiversity decline in agroecosystems subjected to high input practices [41,42]. Modern intensive farming practices that implement deep and frequent tillage, high input inorganic fertilization and pesticide use are evidently a particular threat to AMF. This is surely a drawback for agriculture, since the more AMF biodiversity losses, the fewer AMF functional traits the host plant can benefit from. On the other hand, the activity and diversity of AMF, following conversion from conventional to organic farming, have not yet been investigated thoroughly. However, the available data seem to indicate that AMF respond positively to the transition to organic farming through a progressive enhancement of their activity [87]. Even though it is difficult to dis‐ criminate between the effects that different agricultural treatments exert on AMF communities, they are here considered separately, and their role in shaping AMF communities will be analyzed.

#### **2.1. Tillage: A conventional practice detrimental to AMF**

One of the most ancient and representative agricultural techniques is tillage. Tillage has played a crucial role in the evolution and technological development of agriculture, particularly for food production. The benefits produced by tillage include a better conservation of water and soil fertility, the abatement of weeds and the preparation of a suitable seedbed. To fulfill these tasks, the undisturbed soil is mechanically manipulated in an effort to modify the physical characteristic of the soil and eliminate weeds. The physical, chemical and biological effects of tillage on the soil can be both beneficial and negative, depending on the methods that are used. The inappropriate use of tillage techniques can therefore have a dramatic impact on the soil structure and on soil microorganism community assemblage. It is possible to identify different tilling levels, ranging from a very low impact, "No-tillage", to a high impact, "conventional tillage". A continuum of intermediate conditions lies in between these two extreme situations, e.g. varying frequency and intensity of the plowing.

The mechanical soil disturbance experienced by AMF in tilled agricultural soils has no equivalent in natural ecosystems. This is why tillage has been widely recognized to be one of the principal causes of the modification of the AMF communities that colonize plant roots in agricultural fields [88]. Mycorrhizal diversity, at a family level [88], and the timing of root colonization [89] can be affected negatively. As a consequence, the effectiveness of AMF [90] is likely to be reduced. Periodically repeated mechanical soil disturbance destroys the extra‐ radical mycelial network formed by AMF. This very complex underground structure can reach lengths of up to some tens of meters in one gram of soil [91], and represents a soil "highway" for nutrient transport. For this reason, it is often claimed to be closely correlated to biodiversity, biomass production and the functioning of plant communities [22,25,92].

microbes, favoring more aggressive, antagonistic microbial genotypes [106–108]. This change in competitive balance can alter the evolution of the functional traits of AMF by reprogram‐ ming AMF to reduce their allocation to structures devoted to nutrient exchange (arbuscules and coils), and increase their allocation to internal storage and growth structures (vesicles and intraradical hyphae) [103,109,110]. This is likely to result in an incremented presence of highly competitive AMF which, on the other hand, will be less beneficial to the host crop [111].

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Particular AMF taxa have been found to be more sensitive than others to specific fertilization conditions [42,50,53,65,93,112]. This is probably due to the different taxon-related ability of the AMF taxa to manage nutrient absorption. For instance, *Acaulospora* species have been dem‐ onstrated to be very effective in P uptake, and in the transfer to the host plant, compared to Glomeraceae species [113]. In line with these findings, Acaulosporaceae species have been considered to decrease to a great extent under high input P fertilization [50]. The same thing has been observed for Gigasporaceae in N-enriched soils [50,103]. On the other hand, Glom‐ eraceae species, such as *Rhizophagus intraradices*, are able to cope well with nutrient rich

The choice of crop and rotation made by the farmer has a crucial impact on AMF communities. Even though AMF are commonly recognized as generalist symbionts that show the ability to interact with different plant species, some plant-fungus combinations can perform better than others. The choice of the partner is not univocal, but is believed to be driven by a reciprocal reward mechanism between the two symbionts involved [14]. This means that both the plant and the AMF communities can exert an important role in modifying the community compo‐ sition of the partner [22,23]. Thus, different cultivation practices that involve a variation in plant diversity, such as monoculture, fallow and crop rotation, could show different and

Monoculture can be highly deleterious for AMF communities, and result in a significant reduction in mycorrhizal root colonization [114] and mycorrhizal diversity [115,116]. The effect of continuous monocropping, especially when crops that are not highly dependent on AMFmediated nutrition (e.g. wheat) are used, favors the selection and proliferation of less cooper‐ ative and more aggressive fungal symbionts. These are likely to enact similar behavior to parasitism [102,106]. In addition, intensive tillage treatments, which are necessary in the case of monoculture practices, can overly disperse fungal propagules, thus allowing fewer AMF isolates to dominate the community profile. The dominion of AMF species with a poor mutualistic attitude could be toned down by alternating the cultivation of plant species that are less dependent on AMF with 'break crops', such as *Brassica* [117] or legumes [118]. The former is a non-mycorrhizal crop that can therefore act as an inhibitor of the dominant AMF species proliferation. The latter represent the opposite approach, since legumes are AMFdependent crops that favor the overall propagation of AMF communities. This is the funda‐ mental principle of crop rotation, a practice that can exert a control function that prevents particular AMF from dominating the soil matrix. Hence, crop rotation has the potential of driving AMF communities to be less parasitic [86]. It has been experimentally demonstrated

environments [50,53].

profound effects on AMF community assemblages.

**2.3. Crop rotation**

An ecological shift in AMF communities is particularly noticeable when frequently and infrequently tilled agroecosystems are compared [42,63,88,93]. This is probably due to the different tolerance to hyphal disruption among the different AMF species [94,95]. Although AMF species can colonize plants from spores, this process often requires a certain amount of time. Faster root colonization can be reached in the presence of a viable and well-structured underground mycelial network that facilitates AMF proliferation and speeds up plant root penetration [96]. On the other hand, AMF species differ greatly in their capacity to restart colonization from fragmented mycelium or root fragments [97]. Intense tillage could be a factor that favors those AMF species that are more able to proliferate from fragmented hyphae or root fragment [98], and could therefore determine a shift in AMF community assemblages. A clear example of this is the large presence of Glomeraceae species found in tilled soil all over the world [99]. AMF species belonging to this group are able to randomly connect hyphae in close proximity after disruption, a condition that can easily be found in disturbed soil. This allows these species to proliferate more easily and to rapidly become dominant over slowgrowing AMF. The members of the Gigasporaceae family, for example, use spores as the main source of root colonization, but do not regrow from hyphal fragments [97].

#### **2.2. Fertilization**

Another agricultural practice that has major ecological fall-outs is chemical fertilization. This practice is often claimed to be fundamental in improving the growth performance of plants, but it is sometimes abused. In addition to the environmental drift and the possible pollution of underground water reservoirs, the presence in the soil of high levels of fertilizer dramatically alters the interaction between plants and microbial communities. The central role of arbuscular mycorrhizae in plant nutrition makes them very susceptible to changes in soil nutrient availability. Generally, in a nutrient-rich environment, a plant can directly uptake enough nutrient from the soil, without the "catering" service provided by the AMF partners. As a result, the dependency of plants on their AMF partners gradually diminishes, and AMF community richness and diversity decline [42,53,100,101]. It is thought that fertilization can alter the performance of this symbiosis, making microbial partners costly, and even parasitic [102]. It has been hypothesized that the enrichment of soil resources, due to high input fertilization, could lead to a reduction in plant allocation to roots and mycorrhizas [103], and an accumulation of nutrient resources in epigeous plant sinks [104]. A reduction in host plant resource allocation to the fungal partners can therefore result in a decrease in AMF root colonization [105], and an increase in fungal competition for limited C resources. Moreover, this reduction in host nutrient availability is thought to shift the competitive balance between microbes, favoring more aggressive, antagonistic microbial genotypes [106–108]. This change in competitive balance can alter the evolution of the functional traits of AMF by reprogram‐ ming AMF to reduce their allocation to structures devoted to nutrient exchange (arbuscules and coils), and increase their allocation to internal storage and growth structures (vesicles and intraradical hyphae) [103,109,110]. This is likely to result in an incremented presence of highly competitive AMF which, on the other hand, will be less beneficial to the host crop [111].

Particular AMF taxa have been found to be more sensitive than others to specific fertilization conditions [42,50,53,65,93,112]. This is probably due to the different taxon-related ability of the AMF taxa to manage nutrient absorption. For instance, *Acaulospora* species have been dem‐ onstrated to be very effective in P uptake, and in the transfer to the host plant, compared to Glomeraceae species [113]. In line with these findings, Acaulosporaceae species have been considered to decrease to a great extent under high input P fertilization [50]. The same thing has been observed for Gigasporaceae in N-enriched soils [50,103]. On the other hand, Glom‐ eraceae species, such as *Rhizophagus intraradices*, are able to cope well with nutrient rich environments [50,53].

#### **2.3. Crop rotation**

agricultural fields [88]. Mycorrhizal diversity, at a family level [88], and the timing of root colonization [89] can be affected negatively. As a consequence, the effectiveness of AMF [90] is likely to be reduced. Periodically repeated mechanical soil disturbance destroys the extra‐ radical mycelial network formed by AMF. This very complex underground structure can reach lengths of up to some tens of meters in one gram of soil [91], and represents a soil "highway" for nutrient transport. For this reason, it is often claimed to be closely correlated to biodiversity,

An ecological shift in AMF communities is particularly noticeable when frequently and infrequently tilled agroecosystems are compared [42,63,88,93]. This is probably due to the different tolerance to hyphal disruption among the different AMF species [94,95]. Although AMF species can colonize plants from spores, this process often requires a certain amount of time. Faster root colonization can be reached in the presence of a viable and well-structured underground mycelial network that facilitates AMF proliferation and speeds up plant root penetration [96]. On the other hand, AMF species differ greatly in their capacity to restart colonization from fragmented mycelium or root fragments [97]. Intense tillage could be a factor that favors those AMF species that are more able to proliferate from fragmented hyphae or root fragment [98], and could therefore determine a shift in AMF community assemblages. A clear example of this is the large presence of Glomeraceae species found in tilled soil all over the world [99]. AMF species belonging to this group are able to randomly connect hyphae in close proximity after disruption, a condition that can easily be found in disturbed soil. This allows these species to proliferate more easily and to rapidly become dominant over slowgrowing AMF. The members of the Gigasporaceae family, for example, use spores as the main

biomass production and the functioning of plant communities [22,25,92].

source of root colonization, but do not regrow from hyphal fragments [97].

Another agricultural practice that has major ecological fall-outs is chemical fertilization. This practice is often claimed to be fundamental in improving the growth performance of plants, but it is sometimes abused. In addition to the environmental drift and the possible pollution of underground water reservoirs, the presence in the soil of high levels of fertilizer dramatically alters the interaction between plants and microbial communities. The central role of arbuscular mycorrhizae in plant nutrition makes them very susceptible to changes in soil nutrient availability. Generally, in a nutrient-rich environment, a plant can directly uptake enough nutrient from the soil, without the "catering" service provided by the AMF partners. As a result, the dependency of plants on their AMF partners gradually diminishes, and AMF community richness and diversity decline [42,53,100,101]. It is thought that fertilization can alter the performance of this symbiosis, making microbial partners costly, and even parasitic [102]. It has been hypothesized that the enrichment of soil resources, due to high input fertilization, could lead to a reduction in plant allocation to roots and mycorrhizas [103], and an accumulation of nutrient resources in epigeous plant sinks [104]. A reduction in host plant resource allocation to the fungal partners can therefore result in a decrease in AMF root colonization [105], and an increase in fungal competition for limited C resources. Moreover, this reduction in host nutrient availability is thought to shift the competitive balance between

**2.2. Fertilization**

168 Biodiversity - The Dynamic Balance of the Planet

The choice of crop and rotation made by the farmer has a crucial impact on AMF communities. Even though AMF are commonly recognized as generalist symbionts that show the ability to interact with different plant species, some plant-fungus combinations can perform better than others. The choice of the partner is not univocal, but is believed to be driven by a reciprocal reward mechanism between the two symbionts involved [14]. This means that both the plant and the AMF communities can exert an important role in modifying the community compo‐ sition of the partner [22,23]. Thus, different cultivation practices that involve a variation in plant diversity, such as monoculture, fallow and crop rotation, could show different and profound effects on AMF community assemblages.

Monoculture can be highly deleterious for AMF communities, and result in a significant reduction in mycorrhizal root colonization [114] and mycorrhizal diversity [115,116]. The effect of continuous monocropping, especially when crops that are not highly dependent on AMFmediated nutrition (e.g. wheat) are used, favors the selection and proliferation of less cooper‐ ative and more aggressive fungal symbionts. These are likely to enact similar behavior to parasitism [102,106]. In addition, intensive tillage treatments, which are necessary in the case of monoculture practices, can overly disperse fungal propagules, thus allowing fewer AMF isolates to dominate the community profile. The dominion of AMF species with a poor mutualistic attitude could be toned down by alternating the cultivation of plant species that are less dependent on AMF with 'break crops', such as *Brassica* [117] or legumes [118]. The former is a non-mycorrhizal crop that can therefore act as an inhibitor of the dominant AMF species proliferation. The latter represent the opposite approach, since legumes are AMFdependent crops that favor the overall propagation of AMF communities. This is the funda‐ mental principle of crop rotation, a practice that can exert a control function that prevents particular AMF from dominating the soil matrix. Hence, crop rotation has the potential of driving AMF communities to be less parasitic [86]. It has been experimentally demonstrated that crop rotation promotes higher AMF diversity [115,119], and can reshape AMF commun‐ ities derived from agricultural fields to be more diverse and similar to the ones detected in natural ecosystems [87].

tion-related costs. The impossibility of rapidly assessing AMF colonization on the host plant, together with the complexity of AMF species identification, also contribute to the pitfalls of inoculum agricultural usability. Moreover, the management of the high amount of inoculum necessary for extensive use is very challenging. It has been suggested that AMF is more suitable for plant production systems that involve a transplant stage, as inoculation is carried out more easily, and smaller quantities of inoculum are needed. At a first glance, establishing an openfield, large-scale inoculation treatment would seem technically impractical and economically prohibitive. However, once AMF biodiversity has been restored, AMF-friendly practices, such as fall cover cropping [122], can be put in place in order to help the AMF persist. If no detri‐ mental agricultural practices are carried out, the biodiverse mycelial network will remain unaltered and infective in the future. For example, in revegetation schemes, it would be totally impractical to restore an entire degraded land, which often appears as a highly extended surface, through inoculation. A particular approach must be considered when it is necessary to face these situations. First, the ability of specific cover crop mixtures and even target indigenous plant species to elevate the native AMF inoculum has to be taken into account as a potentially successful selective management tool to aid the recovery of desertified ecosystems [123]. However, since ecosystem functioning is supported by a close liaison between the aboveground plant diversity and belowground AMF diversity [22], the excessive loss of AMF propagules in degraded ecosystems could, in some cases, preclude either natural or artificial revegetation. For this reason, an inoculation step may also be needed. Although it would be too laborious and expensive to re-introduce AMF and cover plants into entire lands, a smallerscale approach should be adopted. Taking inspiration from the idea of creating the so-called "fertility islands" [124], only small patches of cover plants could be inoculated with AMF. This could lead, in time, but with reduced costs, to the re-establishment of a mycelial network that would also be able to allow native plant species to quickly recover the nutrient impoverished

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171

Hence, AMF restoration would only represent an initial cost and, if soil AMF persistence is favoured, this cost could be subjected to amortization over the years. This makes the applica‐ tion of AMF particularly attractive since, as already demonstrated [125,126], it could provide considerable savings for growers and for degraded land recovery projects, in comparison to conventional fertilization. It is important that the end-users cultivate a portion of their crop without inoculum in order to assess the cost-effectiveness and the beneficial effects on plant fitness due to AMF inoculation [127]. Growers are starting to understand the significance of sustainable agricultural systems, and of reducing phosphorus inputs using AMF inocula, especially in the case of high value crops, such as potted ornamental plants. These crops can easily be regarded as the result of organic crop farming, and be sold at a premium price to an eco-friendly orientated consumer class. However, the absence of solid inoculation practices still represents a problem, and applied research should therefore be focused on defining the

best inoculum formulation strategies [128] and imparting know-how to the growers.

Since large-scale AMF production is impractical for growers, the significance of AMF has not been ignored by the commercial sector, and many AMF-based inocula are nowadays available for sale. AMF inoculum production began in the 1980s and flourished in the 1990s. Nowadays,

land.
