Pål Axel Olsson

The use of phospholipid and neutral lipid fatty acids to estimate biomass of arbuscular mycorrhizal fungi in soil

Cucumber seedlings associated with the arbuscular mycorrhizal (AM) fungi Glomus WUM10 or G. caledanium (BEG 15) were grown in PVC tubes with a lateral root-free compartment and an identical compartment containing both hyphae and roots. The amounts of specific fatty acids, in the neutral lipid and phospholipid fractions, were measured in both compartments and compared with controls without mycorrhiza. The phospholipid fatty acids (PLFAs) 16:1 omega 5, 18:1 omega 7c, 20:4 and 20:5 were present in higher amounts in soil with mycorrhizal hyphae than in soil without mycorrhizal hyphae. The largest relative difference was found in 20:5, but a good correlation existed between 16:1 omega 5 and 20:5 in soil with hyphae. Amounts of these fatty acids were correlated both with length of mycorrhizal hyphae and with amounts of ATP in soil. Conversion factors to calculate hyphal length and AM fungal biomass carbon using the phospholipid fatty acids could thus be estimated; 38 nmol PLFA 16:1 omega 5 mg(-1) AM fungal biomass C (Glomus WUM10) and 22 nmol PLFA 20:5 mg(-1) biomass C. The fatty acid 16:1 omega 5 from the neutral lipid fraction, containing triglycerides, dominated in soils with mycorrhizal hyphae. The amount of 16:1 omega 5 in the neutral lipid fraction decreased during storage of soils, indicating a decrease in storage lipids, while the proportion of 16:1 omega 5 in the phospholipid fraction was almost unaffected. (Less)

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.

Lipid and fatty acid composition of hyphae and spores of arbuscular mycorrhizal fungi at different growth stages

The lipid and fatty acid compositions of Glomus intraradices and G. claroideum mycelia, extracted from quartz sand in a compartmentalized growth system, were analysed. The fungi were grown in association with Cucumis sativus and Trifolium subterraneum, respectively. For both fungi, the fatty acids 16:1 omega 5 and 16:0 dominated in the neutral lipid fraction, and 18:1 omega 7 made up a significant part of the phospholipids. The fatty acids were used as estimators of the amount of neutral lipids and phospholipids of AM fungi as well as to calculate the biomass of different parts of their mycelium. The phospholipid content was higher in hyphae than in spores, whereas the opposite was observed for neutral lipids. In 3-mo-old G. intraradices mycelium, spores accounted for 90% of the external biomass, and calculations indicated that about 20% of the spore biomass consisted of neutral lipids. In both fungi the fatty acid compositions of hyphae and spores were similar regardless of the age of the mycelium. Using the signature fatty acid 16:1 omega 5 to calculate the distribution of AM biomass for a 2-mo-old mycelium of G. claroideum, we found that the fungal biomass was equally distributed between the external mycelium and the internal mycelium in the host root. (Less)

Structure and activity of the bacterial community in the rhizosphere of different plant species and the effect of arbuscular mycorrhizal colonisation

The aim of this study was to determine if arbuscular mycorrhizal (AM) colonisation influences rhizosphere bacteria differently depending on plant species. Thus, the effect of AM colonisation (Glomus intraradices) on rhizosphere bacteria of subterranean clover, cucumber, leek and maize was studied. The bacterial activity was measured as thymidine or leucine incorporation and bacterial numbers as colony forming units and acridine orange direct counts. The phospholipid fatty acid (PLFA) pattern was used to characterise the bacterial community structure and was compared with the community substrate utilisation pattern using Biolog. The bacterial activity and bacterial densities differed between the rhizospheres of the plant species. AM colonisation had a low impact on bacterial activity, but affected bacterial numbers differently depending on the plant species. Only small effects of AM colonisation were detected with the PLFA technique, and no effects were seen with Biolog, while similar effects of the plant species were found for these techniques. Thus, the plant species had greater effects on the bacterial community in the rhizosphere than AM colonisation and the effect of AM differed between plant species.

Fungal Lipid Accumulation and Development of Mycelial Structures by Two Arbuscular Mycorrhizal Fungi

ABSTRACT We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi Scutellospora calospora and Glomus intraradices when colonizing Plantago lanceolata. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1ω5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of G. intraradices increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus. S. calospora does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for G. intraradices. G. intraradices allocated more of its lipids to storage than did S. calospora. Thus, within a species, the fatty acid 16:1ω5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1ω5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.

Carbon dynamics in mycorrhizal symbioses is linked to carbon costs and phosphorus benefits.

The nutrient and carbon (C) allocation dynamics in mycorrhizal hyphal networks cause variation in costs and benefits for individual plants and fungi and influence the productivity, diversity and C cycling in ecosystems. We manipulated light and phosphorus (P) availability in a pot experiment with Trifolium subterraneum colonised by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. Stable (13)C-labelling was used to trace assimilated CO(2) to the mycorrhizal fungus in roots and soil using compound-specific isotope ratio mass spectrometry. We used the neutral lipid fatty acid 16:1omega5 as a signature for AM fungal storage lipids. Both P and shading reduced the AM fungal lipid accumulation in the intraradical mycelium, while only P reduced the amount of lipids in the extraradical mycelium. Recently assimilated plant C was only allocated to the mycorrhizal fungus to a small extent when plant mycorrhizal benefit was reduced by P fertilization, while increasing the plant C cost by shading did not reduce the C flow to the fungus. These results are of importance for our conception of mycorrhizal dynamics during periods of shade in nature.

The growth of external AM fungal mycelium in sand dunes and in experimental systems

We estimated the biomass and growth of arbuscular mycorrhizal (AM) mycelium in sand dunes using signature fatty acids. Mesh bags and tubes, containing initially mycelium-free sand, were buried in the field near the roots of the dune grass Ammophila arenaria L. AM fungal mycelia were detected at a distance of about 8.5 cm from the roots after 68 days of growth by use of neutral lipid fatty acid (NLFA) 16:1ω5. The average rate of mycelium extension during September and October was estimated as 1.2 mm day−1. The lipid and fatty acid compositions of AM fungal mycelia of isolates and from sand dunes were analysed and showed all to be of a similar composition. Phospholipid fatty acids (PLFAs) can be used as indicators of microbial biomass. The mycelium of G. intraradices growing in glass beads contained 8.3 nmol PLFAs per mg dry biomass, and about 15% of the PLFAs in G. intraradices, G. claroideum and AM fungal mycelium extracted from sand dunes, consisted of the signature PLFA 16:1ω5. We thus suggest a conversion factor of 1.2 nmol PLFA 16:1ω5 per mg dry biomass. Calculations using this conversion factor indicated up to 34 μg dry AM fungal biomass per g sand in the sand dunes, which was less than one tenth of that found in an experimental system with Glomus spp. growing with cucumber as plant associate in agricultural soil. The PLFA results from different systems indicated that the biomass of the AM fungi constitutes a considerable part of the total soil microbial biomass. Calculations based on ATP of AM fungi in an experimental growth system indicated that the biomass of the AM fungi constituted approximately 30% of the total microbial biomass.

Phosphorus Effects on Metabolic Processes in Monoxenic Arbuscular Mycorrhiza Cultures

The influence of external phosphorus (P) on carbon (C) allocation and metabolism as well as processes related to P metabolism was studied in monoxenic arbuscular mycorrhiza cultures of carrot (Daucus carota). Fungal hyphae of Glomus intraradices proliferated from the solid minimal medium containing the colonized roots into C-free liquid minimal medium with different P treatments. The fungus formed around three times higher biomass in P-free liquid medium than in medium with 2.5 mminorganic P (high-P). Mycelium in the second experiment was harvested at an earlier growth stage to study metabolic processes when the mycelium was actively growing. P treatment influenced the root P content and [13C]glucose administered to the roots 7 d before harvest gave a negative correlation between root P content and13C enrichment in arbuscular mycorrhiza fungal storage lipids in the extraradical hyphae. Eighteen percent of the enriched13C in extraradical hyphae was recovered in the fatty acid 16:1ω5 from neutral lipids. Polyphosphate accumulated in hyphae even in P-free medium. No influence of P treatment on fungal acid phosphatase activity was observed, whereas the proportion of alkaline-phosphatase-active hyphae was highest in high-P medium. We demonstrated the presence of a motile tubular vacuolar system inG. intraradices. This system was rarely seen in hyphae subjected to the highest P treatment. We concluded that the direct responses of the extraradical hyphae to the P concentration in the medium are limited. The effects found in hyphae seemed instead to be related to increased availability of P to the host root.

Soil bacteria respond to presence of roots but not to mycelium of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) cucumber seedlings and uncolonized controls were grown in growth chambers which allowed separation of compartments with roots from compartments with the extraradical mycelium alone. Two fungi, Glomus invermaium Hall and G. caledonium (Nicol. and Gerd.) Trappe and Gerdemann, were used. Bacterial numbers (direct and viable count) and activities (thymidine incorporation) were highest in the root compartment, but were not affected by the AM mycelium after 30 days of plant growth. The soil was stored after harvest for 16 d at 13 degrees C to study the effect of disconnected mycorrhizal hyphae on bacterial activity. This treatment increased bacterial activity in mycorrhizal treatments compared to non-mycorrhizal control soils. The highest increase was found in the root compartment. The bacterial community structure was studied by analyzing the phospholipid fatty acid (PLEA) pattern. The bacteria specific PLFAs cy17:0 and cy19:0 increased in both experiments in the root compartments. The PLFAs 15:0 and 17:0, which are usually considered to be bacteria specific, also increased due to the presence of roots, but it was shown that these fatty acids were present in aseptically grown cucumber roots, and thus not bacteria specific. No bacterial PLFAs were affected by the presence of mycorrhiza. Copyright (C) 1996 Elsevier Science Ltd (Less)

Tit for tat? A mycorrhizal fungus accumulates phosphorus under low plant carbon availability

The exchange of carbohydrates and mineral nutrients in the arbuscular mycorrhizal (AM) symbiosis must be controlled by both partners in order to sustain an evolutionarily stable mutualism. Plants downregulate their carbon (C) flow to the fungus when nutrient levels are sufficient, while the mechanism controlling fungal nutrient transfer is unknown. Here, we show that the fungus accumulates nutrients when connected to a host that is of less benefit to the fungus, indicating a potential of the fungus to control the transfer of nutrients. We used a monoxenic in vitro model of root organ cultures associated with Glomus intraradices, in which we manipulated the C availability to the plant. We found that G. intraradices accumulated up to seven times more nutrients in its spores, and up to nine times more in its hyphae, when the C pool available to the associated roots was halved. The strongest effect was found for phosphorus (P), considered to be the most important nutrient in the AM symbiosis. Other elements such as potassium and chorine were also accumulated, but to a lesser extent, while no accumulation of iron or manganese was found. Our results suggest a functional linkage between C and P exchange.