Isabel Brito

Techniques for Arbuscular Mycorrhiza Inoculum Reduction

Given the ubiquous presence of AM fungi, a major constraint to the evaluation of the activity of AM colonization has been the need to account for the indigenous soil native inoculum. This has to be controlled (i.e., reduced or eliminated) if we are to obtain a true control treatment for analysis of arbuscular mycorrhizas in natural substrates. There are various procedures possible for achieving such an objective, and the purpose of this chapter is to provide details of a number of techniques and present some evaluation of their advantages and disadvantages.

New Tools to Investigate Biological Diversity and Functional Consequences

To exploit the opportunities offered by our increased understanding of arbuscular mycorrhiza (AM) diversity and the potential to manage it requires greater knowledge of the indigenous AM fungi (AMF) involved in the symbiosis with target plants. Our ability to fully describe AMF diversity is still at early stage in terms of the taxonomic units present, despite recent developments in DNA sequencing capacity. The information required is the number of species or taxonomic groups present, the number of individuals that can be identified as belonging to the same group, and the level of variability there is within such a group in terms of their capability to enhance growth. With the techniques available it is possible to be precise over the molecular makeup of individuals but it is more difficult to be certain as to which species they may belong, particularly if that species has been mainly been identified through its morphological characteristics, especially those of its spores, rather than from DNA sequencing. Consequently it is operational taxonomic units (OTUs) or virtual taxa that are used to describe taxonomic groups, which are identified solely through molecular techniques. Nevertheless variability exists in both species and OTUs. Effectiveness of a mycorrhizal symbiosis depends on environmental conditions, but is also influenced by the community of higher plants as well as the local soil biota. Establishing linkages between genetic and the functional diversity under field conditions with biotic and abiotic stresses is more challenging. Successful assemblages of AMF combine the ability to acquire P and to protect host plants against pathogens or abiotic stresses, consistent with greater diversity being associated with greater ecosystem functioning through enhanced trait richness. To capitalize on the potential benefits from managing AMF diversity, appropriate solutions are needed for different agroecosystems and applicable on a site-by-site basis. Identifying genetic and molecular markers that allows quantitative assessment of the potential to exploit indigenous AMF is crucial. Within AMF hyphae two markers have been identified in the ribosomal DNA that can be used to identify OTUs. Similarly mitochondrial large ribosomal subunit sequences have also been used. Short-term considerations suggest that more effort is needed to identify key markers in major crops for AMF assemblages that function effectively to offset major biotic and abiotic stresses.To exploit the opportunities offered by our increased understanding of arbuscular mycorrhiza (AM) diversity and the potential to manage it requires greater knowledge of the indigenous AM fungi (AMF) involved in the symbiosis with target plants. Our ability to fully describe AMF diversity is still at early stage in terms of the taxonomic units present, despite recent developments in DNA sequencing capacity. The information required is the number of species or taxonomic groups present, the number of individuals that can be identified as belonging to the same group, and the level of variability there is within such a group in terms of their capability to enhance growth. With the techniques available it is possible to be precise over the molecular makeup of individuals but it is more difficult to be certain as to which species they may belong, particularly if that species has been mainly been identified through its morphological characteristics, especially those of its spores, rather than from DNA sequencing. Consequently it is operational taxonomic units (OTUs) or virtual taxa that are used to describe taxonomic groups, which are identified solely through molecular techniques. Nevertheless variability exists in both species and OTUs. Effectiveness of a mycorrhizal symbiosis depends on environmental conditions, but is also influenced by the community of higher plants as well as the local soil biota. Establishing linkages between genetic and the functional diversity under field conditions with biotic and abiotic stresses is more challenging. Successful assemblages of AMF combine the ability to acquire P and to protect host plants against pathogens or abiotic stresses, consistent with greater diversity being associated with greater ecosystem functioning through enhanced trait richness. To capitalize on the potential benefits from managing AMF diversity, appropriate solutions are needed for different agroecosystems and applicable on a site-by-site basis. Identifying genetic and molecular markers that allows quantitative assessment of the potential to exploit indigenous AMF is crucial. Within AMF hyphae two markers have been identified in the ribosomal DNA that can be used to identify OTUs. Similarly mitochondrial large ribosomal subunit sequences have also been used. Short-term considerations suggest that more effort is needed to identify key markers in major crops for AMF assemblages that function effectively to offset major biotic and abiotic stresses.

Agronomic Opportunities to Modify Cropping Systems and Soil Conditions Considered Supportive of an Abundant, Diverse AMF Population

There is a great functional diversity within and between different species of arbuscular mycorrhizal fungi (AMF) in terms of the benefits they may confer to host plants, such as the acquisition of nutrients or protection from biotic and abiotic stresses. It is critical to understand how the various practices available for use within production systems, particularly those compatible with the sustainable intensification of agriculture, impact AMF, and their diversity. In commercial crop production farmers need to prepare the land for seeding, protect the developing and maturing plants from pests and diseases, provide a suitable supply of nutrients and water, and ensure a timely harvest. Tillage systems vary greatly in the extent of their disturbance of soil in terms of the depth and fragmentation, affecting AMF abundance and diversity. In contrast to inversion tillage, the AMF colonization rate of crops under no-till starts earlier and develops faster due to the presence in the soil of an intact extraradical mycelium, which enhances the role of AMF in the uptake of nutrients and the protection against stresses. Rational use of applied nutrients, supplied either through the application of organic amendments or inorganic fertilizers, which is essential to maintain soil productivity, is compatible with maintaining an abundant and diverse AMF population, especially in association with no-till systems. Crop rotation has been the traditional approach to ensure that neither pests nor diseases of a particular crop build up to epidemic proportions in the soil or field environment. There is a relationship between the diversity of plant material above ground and the AMF present below the soil surface. Reducing soil disturbance by tillage, adding organic amendments, keeping harvest residues and use of cover crops all help to increase soil organic matter, which in turn plays an important role in reducing application of mineral fertilizer and the need for herbicides. By employing cropping practices that achieve these goals, it might be possible to improve AMF diversity to levels identical to that in natural ecosystems. There is an urgent need to use the new generation of molecular tools for the evaluation of effects of cropping systems on biodiversity of AMF associated with field crops, especially for AMF from different functional groups.

Chapter 5 - Impacts on Host Plants of Interactions Between AMF and Other Soil Organisms in the Rhizosphere*

There is a considerable evidence of both cooperation and synergism between groups of organisms concentrated around mycorrhiza rather than the rhizosphere of plants being inhabited by a very diverse population of competing organisms. A huge increase has taken place in the detailed understanding of the microbial environment surrounding plant root systems and of the processes involved in the establishment of the mycorrhiza symbiosis. It seems that if the interaction between microbes and plants is of particular interest to the development of a sustainable agriculture, the relationship is carefully choreographed through complex signaling systems. This development has also allowed a more holistic approach to the investigation of mycorrhiza and the possibility for optimizing the beneficial aspects of the symbiosis. Much of our detailed knowledge of the interaction between arbuscular mycorrhizal fungi (AMF), bacteria, and plants comes from legumes, members of the Fabaceae, which form symbiotic relationships with both AMF and nitrogen fixing “rhizobia.” The three groups of organisms establish a tripartite interaction that may also involve additional endophytic partners. There is considerable similarity in the development of the symbiosis between the contrasting microbial symbionts – fungi and bacteria – and the host legume. Although there is no competition for infection sites between AMF and rhizobia, there can be resources from the host plant. The benefits from mycorrhiza in the tripartite interaction are enhanced when the host plant is colonized early, especially from an intact extraradical mycelium (ERM). Such AMF colonization can also stimulate more rapid formation of root nodules by rhizobia. An increased rate of photosynthesis or greater green leaf area can be triggered in the host plant in response to AMF colonization, which enhances the available carbon resources within the tripartite symbiosis. The interactions between AMF and other bacteria is less well understood but some of the species that are found in close association with AMF have been shown to enhance the formation of mycorrhiza on receptive hosts and most of the “mycorrhiza helper bacteria” have some beneficial effects on the development of the mycorrhizal host plant. The interactions with soil fauna, particularly grazing arthropods, do not suggest that AMF or their host plants are adversely affected by these activities.

Management of Biological and Functional Diversity in Arbuscular Mycorrhizal Fungi Within Cropping Systems

The potential benefits of arbuscular mycorrhizal fungi (AMF) are crucial aspects for the sustainable intensification of agriculture. However, in industrialized regions injudicious use of both manure and excessive application of fertilizers and pesticides are imposing unacceptable environmental impacts and in regions where there is already an urgent need to improve land productivity, the resources required to enhance productivity are scarce. The intentional use of AMF within agricultural cropping systems has received little attention, especially for large-scale production. Three key aspects prevent the full exploitation of AMF in agricultural ecosystems: the benefits of arbuscular mycorrhiza symbiosis (AM) are dependent on the biological diversity of both partners; the time required to achieve an effective colonization may restrict the benefits when protection against stresses are a major role of AM; the large cost of commercial inoculum coupled with its lack of biological diversity. These concerns clearly establish that the need to develop strategies for the management of indigenous AMF within different cropping system, specifically focusing on how to control the AMF biological diversity in the roots of the crops and to enhance early colonization. The answer to the latter is well-established in the literature and is the maintenance in the soil of an intact extraradical mycelium (ERM) to act as preferential AMF propagule. Under these conditions, colonization starts earlier and develops faster, enhancing the role of AMF in bioprotection of host plants. The management of AMF diversity present in the roots of a crop is still the major difficulty within the cropping systems. However, in a plant succession it appears possible to manage the AMF biological diversity present in the roots of the second plant if intact ERM developed by the first is the preferential initiator of colonization. This approach significantly improves the role of AMF in the protection of plants against biotic and abiotic stresses. Therefore the right choice of crop rotation, including cover crops or even the weeds that germinate before the seeding of the crop, and the adoption of appropriate tillage techniques that maintain the integrity of ERM, provide the potential to overcome the two major limitations to the intentional use of indigenous AMF within cropping systems. Criteria to select the plants to be included in a succession must be developed considering their different functional groups, which can be identified at least at the family level. For example, the Fabaceae and Poacea, harbor distinct AMF communities. These two plant families include many cultivated plants and, depending on the prevailing stresses, they can certainly provide useful candidates for this strategy. Numerous research requirements are identified but modern genomics technology brings the possibility closer that compatible assemblages of AMF and helper bacteria will be discovered.

Challenges to Agriculture Systems

Food production has to be greatly increased simply to feed a population growing from 7 billion to in excess of 9 billion over the next 35 years and we still have more than a billion undernourished people. To increase global food production is an unprecedented challenge in the history of agriculture, particularly if we consider that the solutions adopted in the past are much less of an option. Previous solutions have been to increase the area made available for agriculture and to enhance land productivity by an increase in crop yields, with the latter being particularly important. Only limited areas of new land are available for adoption by agriculture but soil degradation and urbanization are removing considerable areas from the existing productive land bank. In consequence, intensification of production is going to be essential. At the same time there is an urgent need to reduce the environmental impacts of food production. It will be crucial to close the gap in yield between the climatic potential and what farmers achieve across the different regions of the world, particularly those areas where the difference is greatest due to environmental, economic, and social conditions. The world is not in a position to ignore the possible contribution from any technological solution on ideological grounds and the concept of sustainable intensification of agriculture has to be on the agenda. Among the possible solutions the intentional use of beneficial soil microbes in agricultural systems is only in its early days. There is a much greater need than ever to find ways of exploiting the benefits from the microbes in our soils and to develop tools that will help farmers implement strategies related to sustainable soil use and management. Our focus is on arbuscular mycorrhizal fungi (AMF) that can impact several soil processes, including the cycling of phosphorus and nitrogen, their acquisition by plants and reducing losses of nitrogen by leaching or volatilization, as well as play other crucial roles within the agricultural ecosystem. AMF can protect their host plants from both biotic and abiotic stresses, including root pathogens, toxic metals, and water shortage. Managing the soil microbiota, particularly AMF, has the potential not only to increase production, while decreasing the incorporation of inputs, with the potential to be applied to productive and marginal soils and used in regions of the world where the resources required by farmers are scarce.

The Significance of an Intact Extraradical Mycelium and Early Root Colonization in Managing Arbuscular Mycorrhizal Fungi

Early root colonization is crucial if the potential benefits from arbuscular mycorrhiza (AM) are to be optimized, especially when protection against biotic or abiotic stresses is involved, so that the AM is well-established as the host plant encounters the stress. Of the different arbuscular mycorrhizal fungi (AMF) propagules capable of infecting roots of a host plant, colonization from an intact extraradical mycelium (ERM) occurs earlier and develops faster than from spores, colonized root segments, or fragmented hypha. There is also evidence that the biochemical recognition dialogue between AMF and the host plant leading to a functional mycorrhiza is different for spores and intact ERM.

Diversity in Arbuscular Mycorrhizal Fungi

Despite the fact that arbuscular mycorrhizal fungi (AMF) are the most abundant symbionts in terrestrial ecosystems, only about 240 species have been described, based on the morphological features of their spores. Modern molecular techniques have identified many more taxonomic groups, indicating that the overall AMF diversity has been significantly underestimated. However, such information does not permit the differentiation between functional and biological variation within AMF on the impacts on plant growth and community composition. The great functional diversity evident in AMF from genetically different isolates, even those within species, may play an important role in determining plant diversity, productivity, and ecosystem variability. Exchange of phosphorus and photosynthate between plants and AMF, colonization rates, growth of extraradical mycelium (ERM), and differential gene expression of the host plant induced by AMF are important factors in functional diversity. However, the extent to which these and other determining functions are distributed within and between different taxa is still unclear. The mutual interaction between hosts and AMF is critical in determining the benefits to the former. Maintenance of a sustainable mixed plant population therefore depends on the preservation of a diverse AMF population, based on the concept that larger AMF communities have greater probability of containing more diversity in phenotypic traits. Employing this strategy may help to ensure availability of the essential number of species required for ecosystems functioning. Evidence of the coexistence of distinct evolutionary lineages resulting from phylogenetic trait conservatism can enhance ecosystem functioning because of functional complementarity. Thus any change in populations or loss of diversity within agroecosystems may result in changes to productivity or loss of resilience to adverse conditions because of alteration in the ability of AMF to sustain multiple functions in above- and belowground ecosystems. Management practices such as crop rotation, tillage, and phosphorus fertilization influence AMF diversity and, consequently, AMF functioning. Agronomic systems that minimize negative effects on AMF diversity will enable the proper functioning of ecosystems and will provide the desired agricultural benefits, however, appropriate practices still need to be further developed. The combination of crop sequence selection and tillage practices that maintain the ERM intact appears to be a possible approach to selecting a consortium of AMF that can be induced to colonize and improve the productivity of more than one crop.Despite the fact that arbuscular mycorrhizal fungi (AMF) are the most abundant symbionts in terrestrial ecosystems, only about 240 species have been described, based on the morphological features of their spores. Modern molecular techniques have identified many more taxonomic groups, indicating that the overall AMF diversity has been significantly underestimated. However, such information does not permit the differentiation between functional and biological variation within AMF on the impacts on plant growth and community composition. The great functional diversity evident in AMF from genetically different isolates, even those within species, may play an important role in determining plant diversity, productivity, and ecosystem variability. Exchange of phosphorus and photosynthate between plants and AMF, colonization rates, growth of extraradical mycelium (ERM), and differential gene expression of the host plant induced by AMF are important factors in functional diversity. However, the extent to which these and other determining functions are distributed within and between different taxa is still unclear. The mutual interaction between hosts and AMF is critical in determining the benefits to the former. Maintenance of a sustainable mixed plant population therefore depends on the preservation of a diverse AMF population, based on the concept that larger AMF communities have greater probability of containing more diversity in phenotypic traits. Employing this strategy may help to ensure availability of the essential number of species required for ecosystems functioning. Evidence of the coexistence of distinct evolutionary lineages resulting from phylogenetic trait conservatism can enhance ecosystem functioning because of functional complementarity. Thus any change in populations or loss of diversity within agroecosystems may result in changes to productivity or loss of resilience to adverse conditions because of alteration in the ability of AMF to sustain multiple functions in above- and belowground ecosystems. Management practices such as crop rotation, tillage, and phosphorus fertilization influence AMF diversity and, consequently, AMF functioning. Agronomic systems that minimize negative effects on AMF diversity will enable the proper functioning of ecosystems and will provide the desired agricultural benefits, however, appropriate practices still need to be further developed. The combination of crop sequence selection and tillage practices that maintain the ERM intact appears to be a possible approach to selecting a consortium of AMF that can be induced to colonize and improve the productivity of more than one crop.

The Roles of Arbuscular Mycorrhiza and Current Constraints to Their Intentional Use in Agriculture

Arbuscular mycorrhiza (AM) is the oldest and most widespread mutualistic symbiosis known. Colonization of host plants with arbuscular mycorrhizal fungi (AMF) provides a wide range of benefits to the host. The most obvious advantage is the ability to explore a larger volume of soil, by means of the extraradical mycelium (ERM), than plant roots alone can reach, allowing a better acquisition of water and nutrients. Although the uptake and transport of P is the most obvious benefit, absorption of other immobile nutrients, e.g., Zn and mobile nutrients, including N and S, are also significant. Several other ecological benefits are associated with AMF colonization plants, including alleviation of biotic, particularly soilborne diseases, and abiotic stresses, such as toxicity of metal ions and salinity. The formation and maintenance of soil structure is another relevant aspect of AMF that enhances soil functions that impact on the above benefits. In addition, communication between plants through common mycorrhizal networks and interactions with other soil microbes, as in the case of the tripartite symbioses between legume plants (Fabaceae), AMF, and nitrogen-fixing rhizobia are also important benefits of this symbiosis. To capitalize on the possible range of benefits an early and fast colonization of the host plant is essential; between possible AMF propagule types, an intact ERM is the most effective for this objective. Despite the apparent lack of specificity between AMF and host plant species, preferential associations exist with greater or lesser efficiency of the AM. Consequently a diverse population of AMF together with different host plant species increases the possibilities for successful combinations. Despite considerable evidence of the wide range of benefits in crop production granted by the AM symbiosis, there are constrains to the intentional use of AMF in agriculture. Major limitations are associated with the cost and efficacy of commercially available inoculum. Therefore the solution needs to be found through exploiting indigenous AMF, by adopting appropriate cropping systems, including diversified crop rotations and conservation tillage. Some preconceived ideas of incompatibility between common management practices and AMF, e.g., the use of P fertilizer rates or breeding programs that do not consider AM traits have been shown to be wrong.