Ingrid M. van Aarle

Phosphatase activity of extra-radical arbuscular mycorrhizal hyphae: A review.

Phosphatase activity of arbuscular mycorrhizal (AM) fungi has attracted attention in three fairly distinct domains: intracellular enzymes with defined metabolic functions that have been studied in intraradical hyphae, histochemical staining of alkaline phosphatase as an indicator of fungal activity measured both intra- and extraradically, and extracellular activity related to mineralization of organic P (Po) compounds that may enhance mycorrhizal utilization of an important nutrient pool in soil. This review focuses on the latter subjects with emphasis on extraradical mycelium (ERM), while it draws on selected data from the vast material available concerning phosphatases of other organisms. We conclude that histochemical staining of alkaline phosphatase is a sensitive and suitable method for monitoring the effect of adverse conditions encountered by ERM both as a symbiotically functional entity in soil, and in vitro without modifying interference of soil or other solid substrates. Furthermore, the quantitative importance of extracellular enzymes for P nutrition of AM plants is estimated to be insignificant. This is concluded from the low quantitative contribution extracellular hyphae of AM fungi give to the total phosphatase activity in soil, and from estimations of which processes that may be rate limiting in organic P mineralization. Maximum values for the former is in the order of a few percent. As for the latter, solubilization of Po seems to be far more important than Po hydrolysis for utilization of Po by AM fungi and plants, as both endogenous soil phosphatase activity and phosphatases of other soil organisms are ubiquitous and abundant. Our discussion of mycorrhizal phosphatases supports the view that extracellular phosphatases of roots and micro-organisms are to a large extent released incidentally into soil, and that the source has limited benefit from its activity.

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.

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.

13C Incorporation into Signature Fatty Acids as an Assay for Carbon Allocation in Arbuscular Mycorrhiza

ABSTRACT The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1ω5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating 13C enrichment of 16:1ω5 and compared it with 13C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [13C]glucose. The 13C enrichment of neutral lipid fatty acid 16:1ω5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for 13C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1ω5 than for the root specific neutral lipid fatty acid 18:2ω6,9. We labeled plant assimilates by using 13CO2 in whole-plant experiments. The extraradical mycelium often was more enriched for 13C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between 13C enrichment in neutral lipid fatty acid 16:1ω5 and total 13C in extraradical mycelia in different systems (r2 = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the 13C enrichment of 16:1ω5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.

Phosphatase activities of arbuscular mycorrhizal intraradical and extraradical mycelium, and their relation to phosphorus availability

We investigated the influence of changes in external phosphorus (P) concentration on the proportion of phosphatase-active structures of the arbuscular mycorrhizal fungus Gigaspora margarita associated with Allium cepa. The P treatment was started when mycorrhizal colonisation had been established, and plant systems were harvested three times after the start of the P treatment. Higher shoot dry weights and P contents were observed in the high-P treated plants at the last harvest. We did not find any change in the proportion of phosphatase-active extraradical mycelium following P treatment. However, the proportion of alkaline phosphatase-active mycelium was positively correlated for extraradical and intraradical mycelium. Also, the proportion of alkaline phosphatase-active arbuscules seemed to increase with the shoot fresh weight, whereas the proportion of acid phosphatase-active arbuscules decreased with higher shoot P concentration and dry weight. We have shown experimentally that the intraradical mycelium of G. margarita, but not the extraradical mycelium, responds metabolically to plant P concentration, and possibly also to external P availability.

Genomics of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon mobility within Gram-negative bacteria

Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway that converts tryptophan to PRN is composed of four genes, prnA through D, whose diversity, genomic context and spread over bacterial genomes are poorly understood. Therefore, we launched an endeavour aimed at retrieving, by in vitro and in silico means, diverse bacteria carrying the prnABCD biosynthetic loci in their genomes. Analysis of polymorphisms of the prnD gene sequences revealed a high level of conservation between Burkholderia, Pseudomonas and Serratia spp. derived sequences. Whole-operon- and prnD-based phylogeny resulted in tree topologies that are incongruent with the taxonomic status of the evaluated strains as predicted by 16S rRNA gene phylogeny. The genomic composition of c. 20 kb DNA fragments containing the PRN operon varied in different strains. Highly conserved and distinct transposase-encoding genes surrounding the PRN biosynthetic operons of Burkholderia pseudomallei strains were found. A prnABCD-deprived genomic region in B. pseudomallei strain K96243 contained the same gene composition as, and shared high homology with, the flanking regions of the PRN operon in B. pseudomallei strains 668, 1106a and 1710b. Our results strongly suggest that the PRN biosynthetic operon is mobile. The extent, frequency and promiscuity of this mobility remain to be understood.

Growth and interactions of arbuscular mycorrhizal fungi in soils from limestone and acid rock habitats

Arbuscular mycorrhizal (AM) development in different soil types, and the influence of AM fungal hyphae on their original soil were investigated. Plantago lanceolata, which can grow in soils of a very wide pH range, was grown in two closely related limestone soils and an acid soil from rock habitats. Plants were colonised by the indigenous AM fungal community. The use of compartmented systems allowed LIS to compare soil with and without mycorrhizal hyphae. Root colonisation of P. lanceolata was markedly higher in the limestone soils (30-60%) than in the acid soil (5 -20%), both in the original habitat and in the experimental study. Growth of extraradical AM fungal hyphae was detected in the limestone soils, but not in the acid soil, using the signature fatty acid 16: 1 omega5 as biomass indicator. Analysis of signature fatty acids demonstrated an increased microbial biomass in the presence of AM fungal hyphae as judged for example from ail increased amount of NLFA 16:0 with 30 nmol g(-1) in one of the limestone soils. Bacterial activity, but not soil phosphatase activity, was increased by around 25% in the presence of mycorrhizal hyphae in the first harvest of limestone soils. AM fungal hyphae can thus stimulate microorganisms. However, no effect of AM hyphae was observed on the soil pH or organic matter content in the limestone soils and the available P was not depleted

Elemental composition in vesicles of an arbuscular mycorrhizal fungus, as revealed by PIXE analysis

We investigated element accumulation in vesicles of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, extracted from the roots of inoculated leek plants. The elemental composition (elements heavier than Mg) was quantified using particle-induced X-ray emission (PIXE), in combination with scanning transmission ion microscopy (STIM). In vesicles, P was the most abundant of the elements analysed, followed by Ca, S, Si and K. We analysed 12 vesicles from two root systems and found that the variation between vesicles was particularly high for P and Si. The P content related positively to Si, Zn and K, while its relation to Cl fitted to a negative power function. Vesicle transects showed that P and K were present in central parts, while Ca was present mainly near the vesicle surfaces. The results showed that P is an important part (0.5% of the dry weight) of the vesicle content and that the distribution of some elements, within mycelia, may be strongly correlated.

Fenpropimorph and fenhexamid impact phosphorus translocation by arbuscular mycorrhizal fungi

Fenpropimorph and fenhexamid are sterol biosynthesis inhibitor (SBI) molecules widely used to control diseases in agriculture. Both molecules, at increasing concentrations, have been shown to impact on the non-target arbuscular mycorrhizal (AM) fungi. Root colonization, spore production and mycelium architecture, including the branched absorbing structures which are thought to be involved in phosphorus (P) uptake, were affected. In the present study, we investigated the capacity of Glomus sp. MUCL 43204 to take up, transfer and translocate labelled P to Medicago truncatula in the presence of these SBI molecules. We used a strict in vitro cultivation system associating an autotrophic plant of M. truncatula with the AM fungus. In addition, the effects of both SBI molecules on the proportion of hyphae with alkaline phosphatases (ALP), succinate dehydrogenase (SDH) activity and on the expression of the mycorrhiza-specific plant phosphate transporter MtPT4 gene were examined. We demonstrated that the two SBI molecules impacted the AM fungus. This was particularly evidenced for fenpropimorph. A decrease in P transport and ALP and SDH activities associated with the extraradical mycelium and MtPT4 expression level was noted. These three factors were closely related to the development of the AM fungus, suggesting a direct impact not only on the AM fungal growth but also on the physiology and metabolic activities of the AM fungus. These results further emphasized the interest on the autotrophic in vitro culture system as an alternative to pot experiments to investigate the mechanisms behind the impact of disease control molecules on the non-target AM fungal symbionts.

Plantago lanceolata L. and Rumex acetosella L. differ in their utilisation of soil phosphorus fractions

To establish relationships between soil phosphorus (P) fractions and leaf P, a mycorrhizal species (Plantago lanceolata L.) was compared with a typically non-mycorrhizal species (Rumex acetosella L.) in a glasshouse experiment. The plants were grown in 40 soils from non-fertilised, abandoned pastures or abandoned arable fields and leaf P concentration were found to be related to various soil P fractions after six weeks of growth. The differences in the P fractions in soil can account for a large share of the variation in leaf P concentration in both species, but the two species differed in their utilisation of P fractions. Leaf P concentration of R. acetosella was more related to extractable soil P than that of P. lanceolata. Rumex acetosella showed a higher maximum P concentration. The P fractions accounting for the largest share of the variation in leaf P concentration was the Bray 1 extractable and the weak oxalate (1 mM) extractable P, and for P. lanceolata also the Na2SO4+NaF extractable P fraction. P extracted with these methods accounted for up to 80% of the variation in P concentration in leaves of R. acetosella and 65% of the variation in leaves of P. lanceolata. More P extractable with weak oxalate, Na2SO4+NaF and strong oxalate (50 mM) was released from the soil than was taken up by the plants during the experimental period. The Bray 1 extractable P fraction, however, decreased in both unplanted and planted soils. Phosphatase release was not induced in any of the plants during the experimental period, indicating that they were not mobilising soil organic P. However, some of the methods extracted a large share of the organic P and still explained much of the variation in leaf P concentration. Mycorrhizal colonisation of P. lanceolata was inversely related to the extractable soil P. The consistently fast P uptake of R. acetosella indicates that this species have a high demand for P. The differences in P utilisation between R. acetosella and P. lanceolata could be caused by their different mycorrhizal status.