Estimation of the biomass of arbuscular mycorrhizal fungi in a linseed field

Abstract

Linseed was grown in field plots included in a long-term P fertilisation experiment (0, 15 or 30 kg P ha⁻¹ yr⁻¹ for 20 yr). Two months before sowing, half of each plot was applied with dazomet to prevent the formation of arbuscular mycorrhiza (AM). The biomass of different groups of micro-organisms was estimated 28, 51 and 72 d after sowing based on amounts of certain fatty acids extracted from the soil. Dazomet application strongly suppressed colonisation of the linseed roots by AM fungi throughout the experiment. In plots with no dazomet application, root colonisation by the AM fungi increased from harvests 1 to 3 as judged both from microscopical estimates and from quantitative analysis of the AM fungal indicative fatty acid 16:1ω5. These methods also revealed that AM formation was reduced in P-fertilised plots. The phospholipid fatty acid (PLFA) 16:1ω5 decreased in dazomet-treated soil, and it was assumed that the PLFA 16:1ω5 remaining in treated soil originated from bacteria. The biomass of the extraradical AM mycelium could then be estimated by multiplying the difference in PLFA 16:1ω5 between dazomet treated and nontreated soils by a conversion factor. This calculation indicated that the biomass of the extraradical mycelium of AM fungi was about 10 times as high as the biomass of intraradical mycelium and that the extraradical mycelium constituted the largest fraction of the soil microbial biomass. Dazomet application also decreased the biomass of saprophytic fungi in the soil as indicated by the amount of PLFA 18:2ω6,9, while analyses of bacteria-specific fatty acids indicated that the bacterial biomass in the soil was not affected by either dazomet or P application.

Introduction

Arbuscular mycorrhizas (AM) are known for efficiently taking up phosphorus and increase the inflow of phosphorus to plants (Sanders and Tinker, 1971, Jakobsen, 1986).

The AM fungal mycelium exists both within the host root (intraradical mycelium) and outside of it (extraradical mycelium). Quantification of the fungal colonisation of the root is fairly straightforward using biochemical (Hepper, 1977, Bethlenfalvay and Ames, 1987, Olsson et al., 1997) or microscopical (Phillips and Hayman, 1970) techniques, and the latter can be combined with morphometric methods to increase the resolution of the measurements (Toth and Toth, 1982, Toth et al., 1991). Quantification of extraradical mycelium using microscopy is difficult, especially in field studies, since it is necessary to differentiate AM fungal hyphae from those of saprophytic fungi. AM hyphal-length values obtained in attempts at such quantification in field soil vary between 2 and 85 m g⁻¹ soil (Trent et al., 1994, Miller et al., 1995), whereas in artificial growth systems (like pot cultures), values are typically within the range of 2–20 m g⁻¹ soil (e.g. Gazey et al., 1992, Pearson and Jakobsen, 1993). Conversion factors can be used to calculate biomass values based on these hyphal lengths. However, no data are available on the AM fungal biomass in field soil. Data are also lacking on the partitioning of the AM fungal biomass between intraradical and extraradical components in the field. Pot culture studies suggest that more than 80% of the biomass can be found outside the root (Bethlenfalvay et al., 1982, Olsson et al., 1995). Furthermore, there are no field data comparing the biomass of AM fungi with the biomasses of other groups of soil microorganisms.

The analysis of phospholipid fatty acids (PLFAs) extracted from soil has been used to determine how microorganisms are affected by management practices and pollution in forest (Bååth et al., 1992, Frostegård et al., 1993a) and agricultural soils (Haack et al., 1994, Cavigelli et al., 1995). Some PLFAs have been used to estimate the biomass of specific groups of organisms (Sundh et al., 1995, Frostegård and Bååth, 1996), although most PLFAs are common to many soil organisms and are therefore not useful as signature compounds. The PLFA 16:1ω5 has proved to be suitable for estimating the biomass of AM fungi in roots since this PLFA is not commonly found in plants and since the amount of this PLFA correlated well with microscopical estimates of root colonisation (Peng et al., 1993, Olsson et al., 1997). The PLFA 16:1ω5 has also been used to estimate the extraradical biomass in pot cultures, where the amount of this PLFA was well correlated with the AM hyphal length (Olsson et al., 1997). However, since PLFA 16:1ω5 is not unique to AM fungi, being present in other organisms such as bacteria (Walker, 1969, Nichols et al., 1986, Olsson et al., 1995), a nonmycorrhizal control is required to quantify the amount of PLFA 16:1ω5 present in organisms other than AM fungi. This background, originating primarily from bacteria, does not pose much of a problem in controlled pot experiments where nonmycorrhizal controls can be used (Olsson et al., 1995, Olsson et al., 1997). However, the establishment of nonmycorrhizal control plots in field experiments requires that anti-fungal soil fumigants be applied in order to exclude the AM fungi (Carey et al., 1992, Merryweather and Fitter, 1996).

Our study was performed in a linseed field where dazomet application had been employed to prevent AM root colonisation in control plots (Thingstrup et al., 1998). Linseed is usually strongly colonised by AM fungi (Dugassa et al., 1996) and depends on them as a source of P at low to intermediate levels of P availability (Thompson, 1996, Thingstrup et al., 1998). The dazomet treatment was used in combination with quantitative analyses of specific fatty acids in order to estimate the AM fungal biomass in soil and in linseed roots in the field. The experiment was conducted in a field included in a long-term P fertilisation experiment, thus allowing the effect of soil P content to be studied. Besides affecting the biomass of AM fungi, P might also affect the abundance of energy storage in the AM fungal mycelium (Olsson et al., 1997). Neutral lipids, such as triglycerides, are abundant in AM fungi (Cooper and Lösel, 1978), and the neutral lipid fatty acids (NLFAs) of AM fungi are largely restricted to storage structures, like spores, in the soil and to vesicles in the root (Olsson et al., 1997). The partitioning of the AM fungal biomass in soil between hyphae and storage structures can therefore be studied by comparing the amounts of NLFA and PLFA 16:1ω5.

Our objectives were to estimate the AM fungal biomass in soil and roots in a field situation and to compare it with the biomasses of bacteria and saprophytic fungi based on a quantitative analysis of signature fatty acids.