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3.1 Introduction

It is well known that arbuscular mycorrhizal (AM) fungi are ubiquitous in agricultural and natural ecosystems (Brundrett 1991, 2002) and that most plant species form symbiotic associations with these fungi (Newman and Reddell 1987). The symbiosis can increase the growth and development of plants by enhancing nutrient uptake, especially phosphorus (Ortas 1996; Smith et al. 2004), into plants and by improving water relations of plants (Auge 2001; Boomsma and Vyn 2008; Jacobson 1997; Kyllo et al. 2003; Mohammad et al. 1995). Plants differ in their growth response to mycorrhizal colonization (Monzon and Azcon 1996; Ortas et al. 2002; Pankova et al. 2008). Explanations for differences in growth responses to mycorrhizal colonisation over the full life cycle of plants are complex (Gazey et al. 1992) and new explanations of mycorrhizal function are emerging (Li et al. 2008). Mycorrhizal dependency (Menge et al. 1978; Gerdemann 1975) varies among plant species (Abbott et al. 1995; Pankova et al. 2008), and may influence the abundance and community structure of AM fungi in soil and their infectivity (Crush 1978; Egerton-Warburton et al. 2007; Püschel et al. 2007; Zangaro et al. 2000). AM fungi can also contribute to improving soil physical fertility by enhancing soil aggregation (e.g. Moreno-Espíndolaa et al. 2007; Rillig and Mummery 2006) and protect crops against plant disease (see Douds and Johnson 2003). The soil environment, particularly factors that control soil fertility, strongly influences mycorrhizal function (Abbott and Robson 1982, 1991b; Cardoso and Kuyper 2006).

The diversity of AM fungi is generally low in agriculture compared with indigenous plant communities (Boerner et al. 1996; Helgason et al. 1998; Sieverding 1991). Crop and rotation history also influence the abundance and diversity of AM fungi in agricultural soil (Johnson and Gehring 2007; Douds and Johnson 2003). Therefore, the capacity of AM fungi to form mycorrhizas that contribute substantially to plant nutrition, plant health or soil fertility depends on soil management. Within this context, calibrated bioassays of infectivity have the potential to enable interpretation of mycorrhizal function. However, due to the complexity, diversity and dynamics of mycorrhizal associations in space and time, measurements of mycorrhizal status of roots at one point in time is unlikely to enable full understanding of the interactions between AM fungi and host plants over their life cycle.

3.2 Assessment of Infectivity of AM Fungi in Soil

The term “soil infectivity” was used to define the ability of soil containing propagules of pathogens to initiate infection of host plants (Hornby 1990). Based on this plant pathology concept, “infectivity” of AM fungi in soil has been used with reference to the ability of AM fungi to colonise roots of a host plant (Abbott and Robson 1981) and explored further within the framework of mycorrhiza bioassays (e.g. Gianinazzi-Pearson et al. 1985; Plenchette et al. 1989). The infectivity of AM fungi can differ among host plants even when grown together in the same soil (Abbott et al. 1995). It is also important to be aware that bioassay measurements of infectivity of AM fungi can vary with soil environment, including differences in soil fertility, soil disturbance, host plant type and architecture, temperature, and water availability (Abbott et al. 1995).

Knowledge of the abundance and distribution of AM fungi in soil is acquired from measurements of the length of mycorrhizal root, the proportion of the root length colonised, the length of hyphae in soil, the number and types of spores and other propagules in soil, and the infectivity of the community as a whole (Abbott and Robson 1991a). Colonisation of roots by AM fungi can be estimated directly using microscopic observation of roots and indirectly using bioassays (Abbott and Robson 1981) or molecular assays (Jacquot et al. 2000; Stukenbrock and Rosendahl 2005).

3.2.1 Direct Assessment of AM Fungi

Direct measurement of mycorrhizal colonisation of roots by length of root colonised and percentage of root length colonised is usually based on staining to distinguish the fungi from root tissues (see Brundrett et al. 1984). Colonisation can also be observed in unstained roots (Arias et al. 1987), but most histological methods have limitations because some fungi are not easily visible even after roots are cleared and the fungi stained (Saito et al. 1993). Most assessments of AM fungi in roots do not take into account changes in the intensity of fungal colonisation within the root, but McGonigle et al. (1990) overcame this limitation to some extent by developing a magnified intersection method for assessing the colonisation of roots. This procedure assesses the proportion of root length containing mycorrhizal fungal structures (arbuscules, vesicles and hyphae). In-depth microscopic analysis of colonisation intensity is necessary for more complete understanding of relationships between root colonisation and mycorrhizal function (Gazey et al. 1992), and this will differ for plants with different root structures (Merryweather and Fitter 1998; van der Heijden et al. 1998).

None of the measurements mentioned above provide information about the activity of the fungi present inside roots or in soil, so histochemical procedures are required to determine whether the fungi are living or dead. Hyphae and vesicles can be observed inside roots, even in dead roots, and enzyme assays can be used to determine whether the fungi are alive (e.g. MacDonald and Lewis 1978; Saito 1995; Saito et al. 1993).

As root systems develop, changes occur in the abundance of fungi in the root system either as a whole or in terms of the relative abundance of different AM fungi; sequential sampling may be necessary. As suggested above, the architecture of the root system also needs to be considered when interpreting colonisation data. For example, calculation of the proportion of root length colonised for the whole of a woody root system is of little significance compared with a measurement that is restricted to the fine (most active) roots of the same plant. Similarly, calculations of the proportion of root length colonised on root systems of plants with highly differentiated root systems might be of little value without quantifying the relative abundance of different types and ages of roots. Mycorrhizal colonisation of plants that shed roots extensively may be difficult to determine accurately.

Morphological structures of AM fungi inside roots can be distinctive within some communities of fungi (Abbott 1982; Merryweather and Fitter 1998) but overall the fungi are structurally diverse (Dickson et al. 2007; Dodd et al. 2000; Smith and Smith 1997). The recognition of morphological similarities among AM fungi occurring in a soil community requires extensive experience based on observations of mycorrhizas formed in pure pot cultures of fungi isolated from one location and studied on a standard host plant (Abbott 1982). The morphology of a fungus within roots of different host plants can differ (Lackie et al. 1987; Smith and Smith 1997), so, while morphological characteristics may provide a tool for understanding the dynamics of colonisation of some root systems by distinct morphotypes (e.g. Abbott and Robson 1982; Merryweather and Fitter 1998; Scheltema et al. 1987), molecular characterisation is required to distinguish among fungi within morphotypes (e.g. Jacquot et al. 2000; Stukenbrock and Rosendahl 2005).

Combined with methods for determining physiological activity within hyphae (e.g. MacDonald and Lewis 1978; Saito 1995), direct morphological and molecular features of AM fungi can be used to determine the abundance, dynamics, diversity and activity of AM fungi inside roots (Solaiman and Abbott 2004; Wubet et al. 2004).

3.2.2 Indirect Assessment: Mycorrhiza Bioassay

Mycorrhiza bioassays involve use of bait plants grown in a soil sample; roots are examined for the presence of AM fungi after a set time. Glasshouse bioassays have been used to determine the infectivity of AM fungi in a range of situations (Table 3.1).

Table 3.1 Examples of studies using bioassays to assess the infectivity of AM fungi (after Djuuna 2006)
Full size table

Generally, bioassay plants are grown in defined conditions using soil collected from the field, and the extent of mycorrhizal colonisation is assessed after a predetermined period. Bioassays of this type have been used to show differences among soils in the infectivity of AM fungi present (e.g. Abbott and Robson 1982; Gianinazzi-Pearson et al. 1985). Few studies have evaluated the relationship between the infectivity assessed in a bioassay and the development of mycorrhizas in situ at the site from which the bioassay soil was collected (see Abbott and Robson 1991a). Steps in each mycorrhiza bioassay (soil sampling, bioassay conditions, calibration and testing field predictions) all need to be selected with the purpose of the analysis well defined in advance (Abbott et al. 1995).

Soil sampling: Soil samples can be collected as bulk soil (disturbed soil samples) or as soil cores (undisturbed soil). Sampling needs to be representative of each sampling point. Soil samples should be used quickly or stored at low temperature until used. Consideration should be given to whether or not to allow the soil to dry, and this will depend on the biology of the AM fungi expected to be present. Prior understanding of the fungi present is therefore advantageous.

Bait plants: Host plants such as grass or legume species are good bait plants for bioassays, but other species are suitable if they are highly mycorrhizal. Plants with fast-growing roots in the soil and environmental conditions used in the bioassay, and plants with roots which are easy to clear and stain for AM fungi are most useful as bait plants (Brundrett et al. 1996).

Duration of bioassays: Brundrett and Abbott (1995) noted that the duration of bioassays should not exceed the growth stage required for an adequate level of mycorrhizas to form. If a longer time is chosen before the bait plants are assessed, the plateau level of colonisation may mask differences in rate of colonisation (Abbott and Robson 1981; Sanders et al. 1977). While it is necessary to stop the bioassay before colonisation reaches the plateau, this time might depend on the communities of AM fungi, soil conditions, growing conditions and species of bait plant, highlighting the need for customised bioassays.

Predicting mycorrhizal colonisation in the field: An additional value of mycorrhiza bioassays is that they may be used to predict mycorrhiza development in agricultural or horticultural plants (Abbott et al. 1995). However, management practices imposed after collection of the soil can influence the subsequent field colonisation (Abbott and Robson 1991b). Therefore, if the intention is to use the bioassay to predict colonisation later in the season, a calibration is needed to take into account any effect of management practices imposed after soil is collected for the bioassay on expected colonisation in the field.

A bioassay for predicting mycorrhiza formation could use soil collected before the start of the season combined with (1) information about the proposed agricultural practice, (2) soil conditions, and (3) knowledge of how the AM fungi in this soil are expected to respond to these conditions and practices. Furthermore, in order to consider using a mycorrhiza bioassay to predict the rate and extent of colonisation of roots by AM fungi, the bioassay needs to be conducted under defined conditions.

Mycorrhiza bioassays have been used to quantify seasonal and spatial variations in inoculum potential of AM fungi (Brundrett and Abbott 1995; Brundrett et al. 1996; Jasper et al. 1991; Scheltema et al. 1987). Bioassay plants grown in undisturbed soil cores are likely to closely represent field conditions, but mixed soil samples can be used as part of a standardized bioassay procedure. However, if field soil is disturbed, infectivity of the fungi present may be altered (Miller et al. 1995), so interpretation of the data could be complicated by the conditions under which the assessment is conducted.

Quantification of the rate of colonisation of bioassay plants by different morphotypes or genetically distinguishable groups of AM fungi in field soil could be used to estimate the potential of the components of the community to develop mycorrhizas (Abbott et al. 1995). Alternatively, it is possible that this may not represent the true potential infectivity of each group of fungi if there were no competing fungi present. Observations can be complicated by competition among the AM fungi during colonisation of roots (Solaiman and Abbott 2003).

3.3 Purpose of Bioassays for Infectivity Assessment of AM Fungi

The majority of ecological studies of AM fungi include a measure of mycorrhizal colonisation. However, colonisation of roots by AM fungi is a complex process and it is difficult to represent such a dynamic process in a single measurement made at one point in time. The complex characteristics of mycorrhizal associations depend on root growth and architecture as well as fungal diversity in the soil and inside roots (Abbott and Robson 1984).

The infectivity of AM fungi changes with spore maturation or dormancy (Tommerup 1983), in response to the presence of roots of different forms and age (e.g. associated with the nature and quantity of root exudates) and as the fungi proceed through stages in their life cycle (Pearson and Schweiger 1993). In the latter case, the infectivity of AM fungi associated with mycorrhizal roots paralleled the pattern of sporulation (and transfer of energy resources), despite the presence of fungi in the roots detected by staining. In natural ecosystems with little physical disturbance of soil, roots may be colonised by hyphae associated with adjacent plant species or by new hyphae from germinated spores. When soil is disturbed (e.g. with farming practices), the infectivity of AM fungi can be altered (Evans and Miller 1990). The extent of change may depend on the abundance of propagules of AM fungi present in the soil (Jasper et al. 1989). Soil disturbance in natural ecosystems can reduce the infectivity of AM fungi, especially where propagule abundance is low (Jasper et al. 1987).

A direct measurement of the presence of AM fungi in roots at a particular time results from colonisation events that have taken place during the life of the plant. In contrast, a measure of infectivity of AM fungi using a bioassay bait plant represents the capacity of the hyphae to colonise roots at a particular point in time. A measure of infectivity of AM fungi assessed in a bioassay can be made at different stages in the plant’s growth cycle and interpreted in terms of the ability of the hyphae in soil or in the roots to colonise new roots.

3.4 Conclusions

Mycorrhiza bioassays measure the infectivity in soil at a point in time, representing the potential of AM fungal propagules present in soil to colonise roots. However, the conditions of the bioassay may not be the same as those under field conditions; therefore, it is possible that fungi are present in soil in an infective state but not able to colonise roots for some reason, perhaps related to the physiological state of the roots or due to competition from other AM fungi. Bioassays of infectivity of AM fungi have potential to assist in predicting how roots might become colonised, but calibrations are necessary that take into account an understanding of the relationships between mycorrhizal colonisation under the expected soil conditions (i.e. that take into account intervening management practices such as fertilizer addition, crop or tillage practice). Therefore, several factors need to be considered before conducting a mycorrhizal bioassay, including the time of soil sampling, choice of bait plant, handling of soil samples, and length of the bioassay, and precautions are necessary when interpreting mycorrhiza bioassay data.