Saprophytic Growth of Arbuscular Mycorrhizal Fungi

C. Azc6N-AGUILAR, B. BAGO and J. M. BAREA!

1 Introduction

1.1 General Concepts

Strictly speaking the title of this chapter is a contradiction. If "saprophytic

growth" is growth exhibited by an organism in a free-living status, it is obvious
that this term cannot apply to arbuscular mycorrhizal fungi (AMF) as none
of the 130 species of AMF have yet been successfully cultured axenically.
These fungi have a low, or negligible, saprophytic ability and can apparently
produce viable propagules only upon the biotrophic colonization of a suscep-
tible host root. They are thus considered physiologically obligate symbionts
and the related literature reflects the failure to grow them on synthetic media
(Azc6n-Aguilar and Barea 1992). There are descriptions, however, of limited
saprophytic development of AMF which takes place either in soil, prior to any
contact with the host root, or even "in vitro" (Azc6n-Aguilar and Barea 1985;
Koske and Gemma 1992; Giovannetti et al. 1996). It is this saprophytic growth
which will be discussed in this chapter.

1.2 Terminology

The terms "saprophytic growth" or "host-free growth" of AMF are used to

refer to plant-independent mycelial development. Additionally, the literature
on the topic contains several other terms related to the culture of AMF.
Possible misconceptions, however, make it advisable to give accurate defini-
tions for such terms. These have been provided by Williams (1992). In sum-
mary, the term "axenic" must be used to refer to the growth of a single species
(for example an isolate of an AMF) in the absence of whole, live organisms
or living cells of any other species. "Monoxenic" describes a culture containing
organisms or cells of two species (for example an AMF and a root organ
culture, or an AMF and a soil bacterium growing together on an agar plate).

1 Estaci6n Experimental del Zaidin, CSIC, Profesor Albareda 1, 18008 Granada,

Spain

A. Varma, B. Hock (Eds.), Mycorrhiza, 2nd Ed.

© Springer-Verlag Berlin Heidelberg 1999
392 c. Azc6n-Aguilar et al.

"Dixenic" must be applied to a culture containing organisms or cells of

three species, etc. As stated by Williams (1992) the terms "aseptic", "sterile",
"artificial", "pure", "bacteria-free" etc. are ambiguous or imprecise.

1.3 Metabolic Capabilities of AMF

Nowadays it is accepted that spores of AMF possess the suitable genetic

information and biosynthetic abilities to germinate once the appropriate,
simple, physicochemical conditions of moisture, temperature, and pH (Barea
1986) have been provided. Over 90% germination can be obtained axenically
on water agar in the absence of any mineral or organic supply (Azc6n-Aguilar
et al. 1986a). The spores readily absorb soluble substances and this has allowed
a determination of the metabolic abilities of non-symbiotic AMF. A number
of factors and conditions can affect the germination rate and stimulate a host-
free mycelial development, as will be discussed later (Sect. 3).
It was thought that the AMF failed to grow on nutrient media because
they had a biochemical lesion or a metabolic block. Thus, the addition of
metabolic inhibitors and labelled metabolites to germinating spores, together
with assays for the presence of several enzymes, have been key tools to
study the biosynthetic ability of AMF. The final aims of these assay were to
define the reasons for the inability of these fungi to grow saprophytically
under axenic conditions. The experimental approaches used and the con-
clusions reached have been reviewed by Hepper (1984), Siqueira et al. (1985),
Siqueira (1987) and Azc6n-Aguilar et al. (1991) who discussed the published
work on this topic. In summary, the synthesis of cytoplasmic proteins, some
forms of RNA, and mitochondrial DNA takes place during germination and
subsequent hyphal growth from the spores. Protein synthesis during germina-
tion was preprogrammed in stored messenger RNA, whereas new RNA was
required for hyphal growth. The fungus, however, is able to accomplish a
limited amount of hyphal growth. It thus appears that AMF do not have
defects which would prevent their host-free growth due to limitation of
protein or nucleic acid synthesis. In this sense, AMF resemble saprophytic
fungi more than obligate biotrophic fungi. The germinating spores of AMF
have been shown to possess glutamate dehydrogenase (DH) activity, succinate
DH glyceraldehyde-3-phosphate DH, and glucose 6-phospate DH. 13C-NMR
spectroscopy revealed their ability in absorbing and metabolizing glucose,
fructose, acetate and COz (Pfeffer et al. 1998). They are also capable of
reoxidizing the reduced cofactors NADH and NADPH generated during
spore germination. The synthesis of RNA, proteins, neutral carbohydrates,
amina acids, ATP, and organic acids can be detected a few minutes after
spore imbibition. The triglyceride reserve in AMF spores is broken down into
free fatty acids which are then transformed into different lipid classes. It seems
that lipid reserves are not exhausted during germination and subsequent germ
tube growth.