biotic and abiotic stresses, and ecosystem biodiversity [2, 4]. Indeed,
inoculation with AM fungi can reduce the need for fertilizers, while
increasing crop resilience against diverse stresses under lab and field
conditions [5]. Because its benefits for the host plant, AM symbiosis has an immediate potential for application in modern and sustainable agriculture as biofertilizers and bioprotection agents.
However, to take advantage of all these benefits, a good and functional symbiosis is required. Therefore, reliable quantification of
colonization levels is key, not only for research but also from the
agronomic point of view.
AM symbiosis is a very dynamic system, which establishment
and functioning depends on a high degree of coordination between
the two partners. The success of the association relies on a continuous molecular dialogue that orchestrates the complex symbiotic
programs that are regulated by certain apocarotenoids, such as
strigolactones, ABA, and C 13 α-ionol glycosides (blumenols) and
C 14 mycorradicin derivatives [6, 7]. Apocarotenoids are a class of
compounds that play important roles in nature, derived from carotenoids by oxidative cleavage. In AM symbiosis, strigolactones are
important during the presymbiotic stage by enhancing spore germination and hyphal development [8, 9]. Blumenols and mycorradicin (known as the yellow pigments) seems to be more important
at later stages of the symbiosis. Indeed, their accumulation correlates with arbuscule occurrence within the roots, being associated
with a well-established and functional symbiosis [7, 10]. Therefore,
these compounds constitute a reliable and quantitative metabolic
marker for a functional AM symbiosis. In the present work, we
present two different methods for the quantification of mycorrhization levels. The first one is a traditional method based on the
histochemical analysis of the fungal structures within the roots, by
means of ink staining. The second one consists in the analytical
quantification by liquid chromatography associated to mass spectrometry of the apocarotenoids C 13 α-ionol glycosides (blumenols)
and C 14 mycorradicin derivatives (Fig. 1).
2 Materials
2.1 Histochemical
Quantification
This method, known as the gridline intersect method [11], is the
most frequently used to quickly evaluate the presence of mycorrhiza and quantify the percentage of colonized roots within a
microscope field (Fig. 2). It is a reliable, simple, and nonexpensive
method. Here, we show a slightly modified protocol described by
Vierheilig et al. [12] , which avoids the use of the toxic compound
trypan blue [12]. All solutions should be prepared using deionized
water and analytical grade reagents., and they should be stored at
room temperature.
294
Juan M. Garcı ´a et al.
inoculation with AM fungi can reduce the need for fertilizers, while
increasing crop resilience against diverse stresses under lab and field
conditions [5]. Because its benefits for the host plant, AM symbiosis has an immediate potential for application in modern and sustainable agriculture as biofertilizers and bioprotection agents.
However, to take advantage of all these benefits, a good and functional symbiosis is required. Therefore, reliable quantification of
colonization levels is key, not only for research but also from the
agronomic point of view.
AM symbiosis is a very dynamic system, which establishment
and functioning depends on a high degree of coordination between
the two partners. The success of the association relies on a continuous molecular dialogue that orchestrates the complex symbiotic
programs that are regulated by certain apocarotenoids, such as
strigolactones, ABA, and C 13 α-ionol glycosides (blumenols) and
C 14 mycorradicin derivatives [6, 7]. Apocarotenoids are a class of
compounds that play important roles in nature, derived from carotenoids by oxidative cleavage. In AM symbiosis, strigolactones are
important during the presymbiotic stage by enhancing spore germination and hyphal development [8, 9]. Blumenols and mycorradicin (known as the yellow pigments) seems to be more important
at later stages of the symbiosis. Indeed, their accumulation correlates with arbuscule occurrence within the roots, being associated
with a well-established and functional symbiosis [7, 10]. Therefore,
these compounds constitute a reliable and quantitative metabolic
marker for a functional AM symbiosis. In the present work, we
present two different methods for the quantification of mycorrhization levels. The first one is a traditional method based on the
histochemical analysis of the fungal structures within the roots, by
means of ink staining. The second one consists in the analytical
quantification by liquid chromatography associated to mass spectrometry of the apocarotenoids C 13 α-ionol glycosides (blumenols)
and C 14 mycorradicin derivatives (Fig. 1).
2 Materials
2.1 Histochemical
Quantification
This method, known as the gridline intersect method [11], is the
most frequently used to quickly evaluate the presence of mycorrhiza and quantify the percentage of colonized roots within a
microscope field (Fig. 2). It is a reliable, simple, and nonexpensive
method. Here, we show a slightly modified protocol described by
Vierheilig et al. [12] , which avoids the use of the toxic compound
trypan blue [12]. All solutions should be prepared using deionized
water and analytical grade reagents., and they should be stored at
room temperature.
294
Juan M. Garcı ´a et al.