Root-free Compartment Research Articles

Nutrient-dependent cross-kingdom interactions in the hyphosphere of an arbuscular mycorrhizal fungus.

The hyphosphere of arbuscular mycorrhizal (AM) fungi is teeming with microbial life. Yet, the influence of nutrient availability or nutrient forms on the hyphosphere microbiomes is still poorly understood. Here, we examined how the microbial community (prokaryotic, fungal, protistan) was affected by the presence of the AM fungus Rhizophagus irregularis in the rhizosphere and the root-free zone, and how different nitrogen (N) and phosphorus (P) supplements into the root-free compartment influenced the communities. The presence of AM fungus greatly affected microbial communities both in the rhizosphere and the root-free zone, with prokaryotic communities being affected the most. Protists were the only group of microbes whose richness and diversity were significantly reduced by the presence of the AM fungus. Our results showed that the type of nutrients AM fungi encounter in localized patches modulate the structure of hyphosphere microbial communities. In contrast we did not observe any effects of the AM fungus on (non-mycorrhizal) fungal community composition. Compared to the non-mycorrhizal control, the root-free zone with the AM fungus (i.e., the AM fungal hyphosphere) was enriched with Alphaproteobacteria, some micropredatory and copiotroph bacterial taxa (e.g., Xanthomonadaceae and Bacteroidota), and the poorly characterized and not yet cultured Acidobacteriota subgroup GP17, especially when phytate was added. Ammonia-oxidizing Nitrosomonas and nitrite-oxidizing Nitrospira were significantly suppressed in the presence of the AM fungus in the root-free compartment, especially upon addition of inorganic N. Co-occurrence network analyses revealed that microbial communities in the root-free compartment were complex and interconnected with more keystone species when AM fungus was present, especially when the root-free compartment was amended with phytate. Our study showed that the form of nutrients is an important driver of prokaryotic and eukaryotic community assembly in the AM fungal hyphosphere, despite the assumed presence of a stable and specific AM fungal hyphoplane microbiome. Predictable responses of specific microbial taxa will open the possibility of using them as co-inoculants with AM fungi, e.g., to improve crop performance.

Arbuscular Mycorrhiza Mediates Efficient Recycling From Soil to Plants of Nitrogen Bound in Chitin.

Symbiosis between plants and arbuscular mycorrhizal (AM) fungi, involving great majority of extant plant species including most crops, is heavily implicated in plant mineral nutrition, abiotic and biotic stress tolerance, soil aggregate stabilization, as well as shaping soil microbiomes. The latter is particularly important for efficient recycling from soil to plants of nutrients such as phosphorus and nitrogen (N) bound in organic forms. Chitin is one of the most widespread polysaccharides on Earth, and contains substantial amounts of N (>6% by weight). Chitin is present in insect exoskeletons and cell walls of many fungi, and can be degraded by many prokaryotic as well as eukaryotic microbes normally present in soil. However, the AM fungi seem not to have the ability to directly access N bound in chitin molecules, thus relying on microbes in their hyphosphere to gain access to this nutrient-rich resource in the process referred to as organic N mineralization. Here we show, using data from two pot experiments, both including root-free compartments amended with 15N-labeled chitin, that AM fungi can channel substantial proportions (more than 20%) of N supplied as chitin into their plants hosts within as short as 5 weeks. Further, we show that overall N losses (leaching and/or volatilization), sometimes exceeding 50% of the N supplied to the soil as chitin within several weeks, were significantly lower in mycorrhizal as compared to non-mycorrhizal pots. Surprisingly, the rate of chitin mineralization and its N utilization by the AM fungi was at least as fast as that of green manure (clover biomass), based on direct 15N labeling and tracing. This efficient N recycling from soil to plant, observed in mycorrhizal pots, was not strongly affected by the composition of AM fungal communities or environmental context (glasshouse or outdoors, additional mineral N supply to the plants or not). These results indicate that AM fungi in general can be regarded as a critical and robust soil resource with respect to complex soil processes such as organic N mineralization and recycling. More specific research is warranted into the exact molecular mechanisms and microbial players behind the observed patterns.

The roles of mycorrhiza and Penicillium inoculants in phosphorus uptake by biochar-amended wheat

Biochar (BC) application to soil can potentially replace mineral P fertilizers and its effectiveness as fertilizer can be improved by plant inoculation with the phosphate-solubilizing microorganism Penicillium aculeatum (Pa). Arbuscular mycorrhiza (AM) fungi are important in plant P nutrition and may possibly act synergistically with Pa to improve the uptake of BC-P. Responses in wheat to inoculation with Pa and the AM fungus Rhizophagus irregularis were studied in a pot experiment at two levels of BC fertilization. Pots contained a mesh-enclosed, root free compartment with 33P-labelled soil for assessment of the AM contribution to P uptake and for studying if Pa would affect P uptake by the AM fungal hyphae. AM application suppressed wheat growth, albeit AM pathway had a major role in total P uptake at the two lowest P levels (nil or 20 mg BC-P kg−1 soil). Moreover, AM contribution had similar magnitudes in the presence and absence of Pa. Rhizosphere and bulk soil were actively colonized by Pa, both in the presence and absence of AM. The application of Pa or BC at a low rate increased AM-colonized root lengths. Although this was not translated to increased P uptake by wheat, the results suggest that AM and Pa can be combined without showing antagonistic interactions. However, more work is needed to understand how AM and Pa can be combined to increase plant growth.

Phosphorus and carbon availability regulate structural composition and complexity of AM fungal mycelium

The regulation of the structural composition and complexity of the mycelium of arbuscular mycorrhizal (AM) fungi is not well understood due to their obligate biotrophic nature. The aim of this study was to investigate the structure of extraradical mycelium at high and low availability of carbon (C) to the roots and phosphorus (P) to the fungus. We used monoxenic cultures of the AM fungus Rhizophagus irregularis (formerly Glomus intraradices) with transformed carrot roots as the host in a cultivation system including a root-free compartment into which the extraradical mycelium could grow. We found that high C availability increased hyphal length and spore production and anastomosis formation within individual mycelia. High P availability increased the formation of branched absorbing structures and reduced spore production and the overall length of runner hyphae. The complexity of the mycelium, as indicated by its fractal dimensions, increased with both high C and P availability. The results indicate that low P availability induces a growth pattern that reflects foraging for both P and C. Low C availability to AM roots could still support the explorative development of the mycelium when P availability was low. These findings help us to better understand the development of AM fungi in ecosystems with high P input and/or when plants are subjected to shading, grazing or any management practice that reduces the photosynthetic ability of the plant.

Dual plant host effects on two arbuscular mycorrhizal fungi

Abstract Mycorrhizal fungi may simultaneously associate with multiple plant hosts, and the implications of this for the fungi involved are not well understood. To address this question, two arbuscular mycorrhizal fungi (AMF), Glomus clairoideum (a treatment referred to as “Glo”) and Scutellospora fulgida (a treatment referred to as “Scut”), were grown separately in pots that each consisted of two plant compartments separated by a root-free-compartment (RFC). Fungi within each two-plant-compartment pot were exposed to either two individuals of indiangrass (Sorghastrum nutans), two individuals of big bluestem (Andropogon gerardii), or one of each. A non-inoculated treatment (“Non”) was included to help gauge the potential influence of greenhouse contaminant fungi, cross-contamination, or any misidentification of non-AMF hyphae. The two host species had additive effects on the growth of AM hyphae in plant compartments of Scut, Glo, and Non pots, and in the RFCs of Scut pots. In Glo RFCs, however, they were antagonistic in their effects. Synergism between hosts in Non RFCs suggested that any potential contaminants or misidentification could not explain this result. Underyielding was not seen in shoot weight, root weight, or root length in dual host pots, and also therefore could not explain the result. Hyphal growth in the Scut treatment was evenly distributed between the RFC and plant compartments (or marginally skewed toward the RFC), while hyphal growth in the Glo treatment was skewed toward plant compartments (nearer roots). However, hyphal lengths were more highly correlated across plant compartments within a common pot in the Glo treatment, suggesting that this AMF bridged the RFC to experience the entire two-host pot as a single environment to a greater extent than Scut did. These AMF differed in how they responded to both the species composition of the two-host environment and its spatial structure; potential implications for mycorrhizal community dynamics are discussed.

The arbuscular mycorrhizal fungus Glomus hoi can capture and transfer nitrogen from organic patches to its associated host plant at low temperature

Arbuscular mycorrhizal fungi have been suggested to be of potential benefit in achieving sustainable agriculture systems. However, there is conflicting information on the degree to which arbuscular mycorrhizal fungi (AMF) can grow and function at soil temperatures typical of temperate regions. To resolve this conflict we grew Plantago lanceolata L. inoculated with Glomus hoi (UY 110) in microcosm units maintained at 12/10 °C (day/night). The microcosms had two compartments, one planted and one not. The root-free compartment contained either an organic ( 15N: 13C labelled milled shoot material) or a sand patch. When permitted access, G. hoi proliferated hyphae extensively in the organic patch material. Plant 15N content was a simple function of length density of extra-radical mycelium (ERM) in the patch and c. 6% of host plant N was derived from the patch. These results indicate that G. hoi not only grew at these realistic soil temperatures, but also conferred a nutritional benefit to its host.

Isolation and identification of soil bacteria growing at the expense of arbuscular mycorrhizal fungi

Soil-microorganism symbioses are of fundamental importance for plant adaptation to the environment. Research in microbial ecology has revealed that some soil bacteria are associated with arbuscular mycorrhizal fungi (AMF). However, these interactions may be much more complex than originally thought. To assess the type of bacteria associated with AMF, we initially isolated spores of Glomus irregulare from an Agrostis stolonifera rhizosphere. The spores were washed with sterile water and plated onto G. irregulare mycelium growing in vitro in a root-free compartment of bicompartmented Petri dishes. We hypothesized that this system should select for bacteria closely associated with the fungus because the only nutrients available to the bacteria were those derived from the hyphae. Twenty-nine bacterial colonies growing on the AMF hyphae were subcultured and identified using 16S rRNA gene sequences. All bacterial isolates showed high sequence identity to Bacillus cereus, Bacillus megaterium, Bacillus simplex, Kocuria rhizophila, Microbacterium ginsengisoli, Sphingomonas sp. and Variovorax paradoxus. We also assessed bacterial diversity on the surface of spores by PCR-denaturating gradient gel electrophoresis. Finally, we used live cellular imaging to show that the bacteria isolated can grow on the surface of hyphae with different growing patterns in contrast to Escherichia coli as a control.

Low Concentration of Copper Inhibits Colonization of Soil by the Arbuscular Mycorrhizal Fungus Glomus intraradices and Changes the Microbial Community Structure

Common agricultural practices result in accumulation of copper in agricultural soils worldwide. The effect of bioavailable copper ([Cu](bio)) on colonization of soil by the AM fungus Glomus intraradices and other soil microorganisms was investigated in microcosms containing copper-amended soil. To avoid indirect effects through the plant, copper was only added to root-free microcosm compartments. [Cu](bio) was measured using a Pseudomonas fluorescens biosensor strain. In the range of 0-1.5 μg g(-1) [Cu](bio), a log-log linear relationship between added copper and [Cu](bio) was found. Microbial colonization of the root-free compartment was evaluated by whole-cell fatty acid analysis (WCFA) and amplified rDNA restriction analysis (ARDRA). The WCFA analysis showed that the AM fungus soil colonization was severely inhibited by Cu with a 50% reduction of mycorrhizal growth at 0.26 μg g(-1) [Cu](bio). The growth of other main microbial groups was not significantly affected by copper. However, ARDRA analysis showed a very strong effect of copper on the bacterial community composition probably caused by an increased proportion of Cu-resistant bacteria. Our results suggest that problems with plant yield may arise when converting slightly copper-contaminated soils to land uses such as low-input and sustainable agriculture that are dependent on AM fungal symbiosis.

Suppression of other soil microorganisms by mycelium of arbuscular mycorrhizal fungi in root-free soil

The influence of mycelium of two arbuscular mycorrhizal (AM) fungi, Glomus intraradices and Glomus mosseae, on other soil microorganisms, was examined in root-free soil with and without organic substrate amendment in terms of cellulose. The AM fungi were grown in symbiosis with cucumber in a compartmented growth system, which allowed AM fungal external mycelium to grow into root-free compartments. The fungicide Benomyl was applied to the root-free compartments to create an alternative non-mycorrhizal control treatment. Whole cell biomarker fatty acids were employed to quantify different groups of soil microorganisms including the two AM fungi. Abundance of most microbial groups were reduced by external mycelium of both AM fungi, though differential effects on the microbial community composition were observed between the two AM fungi as revealed from principal component analysis. Inhibition of other soil microorganisms was more pronounced in root-free soil with mycelium of G. mosseae than with mycelium of G. intraradices. In general, cellulose increased the amount of biomarker fatty acids of most groups of soil microorganisms, but cellulose did not affect the influence of AM fungi on other soil microorganisms. Benomyl suppressed growth of the external mycelium of the two AM fungi and had limited non-target effects on other microbial groups. In conclusion, our results show differential effects of external mycelium of AM fungi on other soil microbial communities, though both AM fungi included in the study overall inhibited most microbial groups as examined using whole cell biomarker fatty acids.

Fermentation of sugar beet waste by Aspergillus niger facilitates growth and P uptake of external mycelium of mixed populations of arbuscular mycorrhizal fungi

Sugar beet waste has potential value as a soil amendment and this work studied whether fermentation of the waste by Aspergillus niger would influence the growth and P uptake of arbuscular mycorrhizal (AM) fungi. Plants were grown in compartmentalised growth units, each with a root compartment (RC) and two lateral root-free compartments (RFC). One RFC contained untreated soil while the other RFC contained soil, which was uniformly mixed with sugar beet waste, either untreated (SB) or degraded by A. niger (ASB) in a rock phosphate (RP)-supplied medium. The soil in each pair of RFC was labelled with 33P and 32P in order to measure P uptake by the AM fungal mycelium, of which length density was also measured. Whole cell fatty acid (WCFA) signatures were used as biomarkers of the AM fungal mycelium and other soil microorganisms. The amount of biomarkers of saprotrophic fungi and both Gram-positive and Gram-negative bacteria was higher in SB than in ASB treatments. Whilst ASB increased growth and activity of AM mycelium, SB had the opposite effect. Moreover, shoot P content was increased by the addition of ASB, and by inoculation with AM fungi. Modification of soil microbial structure and production of exudates by A. niger, as a consequence of fermentation process of sugar beet waste, could possibly explain the increase of AM growth in ASB treatments. On the other hand, the highest P uptake was a result of the solubilisation of rock phosphate by A. niger during the fermentation.

Extraradical mycelium of the arbuscular mycorrhizal fungus Glomus lamellosum can take up, accumulate and translocate radiocaesium under root-organ culture conditions.

Radiocaesium enters the food chain when plants absorb it from soil, in a process that is strongly dependent on soil properties and plant and microbial species. Among the microbial species, arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the root cortex of many plants and develop an extraradical mycelial (ERM) network that ramifies in the soil. Despite the well-known involvement of this ERM network in mineral nutrition and uptake of some heavy metals, only limited data are available on its role in radiocaesium transport in plants. We used root-organ culture to demonstrate that the ERM of the AM fungus Glomus lamellosum can take up, possibly accumulate and unambiguously translocate radiocaesium from a 137Cs-labelled synthetic root-free compartment to a root compartment and within the roots. The accumulation of 137Cs by hyphae in the root-free compartment may be explained by sequestration in the hyphae or by a bottleneck effect resulting from a limited number of hyphae crossing the partition between the two compartments. Uptake and translocation resulted from the incorporation of 137Cs into the fungal hyphae, as no 137Cs was detected in mycorrhizal roots treated with formaldehyde. The importance of the translocation process was indicated by the correlation between 137Cs measured in the roots and the total hyphal length connecting the roots with the labelled compartment. 137Cs may be translocated via a tubular vacuolar system or by cytoplasmic streaming per se.

Bacterial populations associated with mycelium of the arbuscular mycorrhizal fungus Glomus intraradices

The influences of the arbuscular mycorrhizal fungus Glomus intraradices on the culturable aerobic–heterotrophic bacterial communities in the rhizosphere and hyphosphere of cucumber plants ( Cucumis satvius) were investigated. Mycorrhizal and non-mycorrhizal plants were grown in compartmentalised growth units, each with a root compartment and two lateral root-free compartments. Samples representing rhizosphere, root-free soil, root-free sand and washed sand extract were collected 52 days after sowing from treatments both with and without mycorrhiza. No significant difference in total bacterial number was observed between the mycorrhizal and non-mycorrhizal treatment. Fourteen hundred bacterial colonies were isolated and identified by fatty acid methyl ester analysis using the Sherlock system (MIDI Inc.), 87 species within 48 genera were identified with a similarity index >0.30. Pseudomonas, Arthrobacter and Burkholderia were the genera most frequently encountered. Large differences in bacterial community structure were observed between rhizosphere soil, root-free soil/sand and washed sand extract, whereas major differences between mycorrhizal and non-mycorrhizal treatments were observed for a few bacterial species only. Isolates identified as Paenibacillus spp. were more frequently found in the mycorrhizal treatment and especially in the washed sand extract with mycelium of G. intraradices, indicating that bacteria within this genus may live in close association with mycelium of these fungi.

Bacterial populations associated with mycelium of the arbuscular mycorrhizal fungus Glomus intraradices

Abstract The influences of the arbuscular mycorrhizal fungus Glomus intraradices on the culturable aerobic-heterotrophic bacterial communities in the rhizosphere and hyphosphere of cucumber plants (Cucumis satvius) were investigated. Mycorrhizal and non-mycorrhizal plants were grown in compartmentalised growth units, each with a root compartment and two lateral root-free compartments. Samples representing rhizosphere, root-free soil, root-free sand and washed sand extract were collected 52 days after sowing from treatments both with and without mycorrhiza. No significant difference in total bacterial number was observed between the mycorrhizal and non-mycorrhizal treatment. Fourteen hundred bacterial colonies were isolated and identified by fatty acid methyl ester analysis using the Sherlock system (MIDI Inc.), 87 species within 48 genera were identified with a similarity index >0.30. Pseudomonas, Arthrobacter and Burkholderia were the genera most frequently encountered. Large differences in bacterial community structure were observed between rhizosphere soil, root-free soil/sand and washed sand extract, whereas major differences between mycorrhizal and non-mycorrhizal treatments were observed for a few bacterial species only. Isolates identified as Paenibacillus spp. were more frequently found in the mycorrhizal treatment and especially in the washed sand extract with mycelium of G. intraradices, indicating that bacteria within this genus may live in close association with mycelium of these fungi.

Changes in the extractability of cations (Ca, Mg and K) in the rhizosphere soil of Norway spruce ( Picea abies ) roots

Nutrient concentrations in the rhizosphere soil can be higher, lower or remain unchanged compared to the bulk soil, but relatively little is known about such changes for basic cations in the rhizosphere of tree roots. A modified root container technique of studying rhizosphere processes was employed. Plexiglas cylinders were horizontally split by a membrane with 30 μM mesh size into an upper compartment for root growth and a root-free lower compartment, each with an inner diameter of 5 cm and a height of 10 cm. One 2-year-old Norway spruce (Picea abies) seedling was transplanted from a nursery into each cylinder. Plants were not specifically inoculated, but roots were colonised by a mix of ectomycorrhizal fungi originating from the nursery. The nutrient poor mineral soil used in the experiment was taken from a forest site in Bayerischer Wald, southern Germany. The soil was either supplied with a mix of Ca, Mg and K, or not supplied with these cations. Plants were harvested 30 weeks after transplanting. The nylon membrane between the root compartments restricted root growth to the upper compartment, so that by the end of the experiment a root mat was formed at the top side of the membrane. In the lower compartment, soil nearest to the root mat was regarded as rhizosphere soil while soil in a distance from the root mat was regarded as bulk soil. In the upper compartment, rhizosphere soil was obtained at the end of the experiment by gently shaking the roots. The soils were analysed for Ca, Mg and K contents following two different soil extraction methods. In the fertilised treatment, H2O-extractable Ca and Mg were accumulated in the rhizosphere. In contrast, K (NH4Cl-extraction) was depleted in the rhizosphere. In the bottom tube, the depletion of K (NH4Cl-extraction) was restricted to 1 cm distance from the root mat. In unfertilised soil, Ca, Mg and K concentrations did not differ clearly between rhizosphere and bulk soils. The results indicated that the occurrence of cation gradients in the rhizosphere depended on the level of soil nutrient supply. Distinct rhizosphere effects were measured by conventional soil extraction methods only when the soil was freshly fertilised with mineral elements prior to the experiment. In this case, K depletion in the rhizosphere reflected higher K uptake by the fertilised Norway spruce plants. For low-nutrient soils, novel techniques are required to follow subtle changes in the rhizosphere.

Phosphate transport by hyphae of field communities of arbuscular mycorrhizal fungi at two levels of P fertilization

This study was conducted to elucidate the effect of P fertilisation on the function of field communities of arbuscular mycorrhizal fungi (AMF) measured as P transport to flax. Two methods were applied to soil from a long-term field experiment with NaHCO3-extractable soil P levels of 24 and 50 mg kg-1in an experiment under controlled conditions: i) Measurement of plant growth and P uptake in the presence or absence of the fungicide benomyl and ii) measurement of hyphal P transport from a root-free compartment labelled with 32P. Benomyl successfully prevented mycorrhizal function. The absolute contribution of AMF to plant P uptake was of the same magnitude with or without P fertilisation at 27 days after sowing. Therefore, even though plants grown at the higher soil P level had greater P uptake, the relative contribution of AMF to P uptake was greater at the lower P level than at the higher P level (77 and 49% of total P uptake, respectively). The AMF in P-fertilized soil transported less P32 from the root-free compartment to the plant after 23 days than the AMF in unfertilized soil, but this difference disappeared in plants harvested after 27 and 32 days. The production of hyphae was largely similar in both fertilization treatments, indicating that the capacity for P uptake and transport by hyphae of the two AMF communities was similar.

Uptake of P from apatite by Pinus sylvestris seedlings colonised by different ectomycorrhizal fungi

The role of ectomycorrhizal (EM) fungi in increasing apatite dissolution was tested in a pot system with Pinus sylvestris (L.) seedlings growing in a sand/peat mixture. Non-mycorrhizal seedlings and seedlings inoculated with one of three different isolates of EM fungi were grown for 210 days in pots divided into a root-containing and a root-free compartment. The EM mycelium was allowed to colonise the root-free compartment, to which apatite had been added as a P source in half of the pots. All mineral nutrients except P were supplied in the form of a balanced nutrient solution. Seedlings grown with apatite as their P source grew significantly better and had higher P concentrations (1.1-1.5 mg/g) compared with seedlings growing without any P source (0.6-1.0 mg/g), indicating that they were able to use apatite-P. A weathering budget based on P uptake indicated that 6.7-18.9 mg apatite was weathered per pot which corresponds to 0.3-0.9% of the added apatite. A similar budget based on Sr uptake indicated that the apatite weathering rate was lower (0.13-0.3%). One Suillus variegatus isolate and an unidentified fungus had a significant positive influence on the dissolution of apatite, while another S. variegatus isolate had colonised roots poorly and did not influence weathering significantly. Oxalic acid was detected in root-free soil and was especially abundant in compartments colonised by S. variegatus. The concentration of oxalic acid was correlated to the concentration of phosphate in the soil solution of root-free soil, indicating that oxalic acid influences apatite dissolution.

Effects of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal fungi in symbiosis with cucumber

The effect of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal (AM) fungi in symbiosis with cucumber plants was studied in compartmentalised growth systems. Hyphae of Glomus intraradices Schenck & Smith (BEG87) or G. caledonium (Nicol. & Gerd.) Trappe & Gerdeman (BEG15) grew into lateral root-free compartments. Non-mycorrhizal plants served as control. The soil in half of the growth units of each mycorrhizal treatment was inoculated with P. fluorescens DF57. P. fluorescens DF57 enhanced hyphal length density of one of the AM fungi, G. caledonium, but this was not reflected in a higher hyphal transport of P from the root-free soil to the plant. The total P content was higher in plants grown in symbiosis with G. intraradices than in plants in the other treatments. G. caledonium and P. fluorescens DF57 had a synergistic effect in that total P content in plants inoculated with G. caledonium was higher in the presence than in the absence of P. fluorescens DF57.

Combined effects of soil disturbance and fallowing on plant and fungal components of mycorrhizal corn ( Zea mays L.)

Soil disturbance may reduce the effect of mycorrhizae on plant growth and nutrient uptake through its effects on the integrity of the extraradical hyphal network. A growth-chamber experiment was conducted to evaluate the survival of extraradical arbuscular mycorrhizal (AM) fungal hyphae when detached from the host root system, and to understand the effects of soil disturbance on the ability of these hyphae to colonize plant roots and to reestablish mycorrhizal associations in previously disturbed soils. The experiment consisted of establishing AM fungi in pots divided into two compartments by a nylon mesh (37 μm), by growing corn ( Zea mays L.) in one of the compartments for 6 weeks in an unsterilized agricultural field soil. The mesh prevented the growth of corn roots from one side of the pot to the other, while allowing the passage of the AM hyphae. After establishment of AM fungi the following treatments were performed: soil in the two compartments was either disturbed by sieving through 2 mm mesh (D) or undisturbed (U) leading to four combined disturbance treatments: (1) both compartments undisturbed (UU); (2) root compartment disturbed and root-free compartment undisturbed (DU); (3) root compartment undisturbed and root-free compartment disturbed (UD); and (4) both compartments disturbed (DD). The effects of fallows of four different durations; 0, 30, 60 and 90 d were also measured in the same experiment giving a total of 16 treatments. Soils were disturbed at the beginning of the experiment in the root compartment, and after each fallow period in the root-free compartment. Immediately after disturbance of the soil in the root-free compartment, corn was planted and grown for 30 d to test the combined effects of fallow and soil disturbance on AM formation and nutrient content. Soil disturbance had no adverse effect on AM efficiency if test plants were planted immediately after disturbing the root-free compartment. However, AM efficiency decreased with increasing length of fallow. Lengths of total and metabolically active extraradical hyphae in the root-free compartments were measured before each fallow. Significantly less hyphal lengths were observed in pots where the soil of the root compartment had been disturbed. Test plant shoot weight was highest in UU and lowest in DD treated pots. Phosphorus content by the test plants was twice as high in UU as in DD. Test plants in undisturbed (UU) pots had greater Zn and Cu contents than in DU, UD or DD pots. Content of P, Zn and Cu in test plants was reduced by about 40%, 63% and 70%, respectively, by 90 d of fallow, compared to 0 d fallow.

Growth of arbuscular mycorrhizal mycelium in calcareous dune sand and its interaction with other soil microorganisms as estimated by measurement of specific fatty acids

Fatty acid analysis was used for determining the extent of the development of the external mycelium of AM fungi (mixed inoculum from a sand dune) growing from roots of Festuca rubra and Plantago lanceolata in calcareous dune sand. The plants were raised in chambers specially designed to permit the growth of AM mycelium in root-free compartments. In two separate experiments, growth of external mycelium in the root-free compartments was detected and the amount of mycelium was estimated using the indicator of AM fungal biomass, phospholipid fatty acid (PLFA) 16:1 omega 5. The results showed that the PLFA 16:1 omega 5 was suitable for estimating the mycelium emerging from the mixed inoculum obtained from the field roots of F: rubra and P lanceolata. The PLFA 16:1 omega 5 showed external mycelium to become established in the root-free compartments within a period of 3 weeks and the amount of mycelium to continue to increase at 6 and 9 weeks. Increases in neutral lipid fatty acid (NLFA) 16:1 omega 5 (indicator of storage lipids) over time were inconsistent between the two experiments, but appeared to follow patterns of sporulation in each experiment. In both experiments, the root-free compartment was colonised by saprophytic fungi to a greater extent in the case of non-mycorrhizal than of AM treatment, as indicated by an increase in PLFA 18:2 omega 6,9 (indicator of saprophytic fungi). The absence of an increase in the case of AM treatment indicates that AM fungal mycelium can negatively affect the growth of saprophytic fungi in this soil type. This result was, however, only weakly supported by measurements of ergosterol content. The analysis of bacteria specific PLFAs showed that bacterial biomass was not affected by the AM mycelium.

Nitrate depletion and pH changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic culture.

The effect of the extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Smith & Schenck on nitrate uptake and on the pH of the medium was studied in a monoxenic culture with tomato (Lycopersicon esculentum Mill. var. Vendor) roots obtained from root organ culture. The symbiosis was established in compartmented Petri dishes containing agar media amended with the pH indicator bromocresol purple. A pattern of pH changes was revealed as the symbiosis progressed in the media of the Petri dish compartments containing the dual, arbuscular-mycorrhizal fungi/root, culture as well as in the media of the hyphae, root-free compartments, in which the extraradical hyphae developed extensively, coming from the compartment containing the symbiosis. The colour changes in the media were measured spectrophotometrically, whilst maintaining the monoxenic conditions. The extraradical hyphae of G. intraradices strongly increased the pH of nutrient-free medium when supplied with nitrate, whereas the pH decreased m the absence of this N source. The hyphae developing from germinated spores and growing in axenic, nitrate-amended media did not induce any increase in pH. Nitrogen analysis revealed that a depletion of nitrate in the media accompanied increased pH. These results point towards an active uptake of nitrate by the extraradical mycelium of G. intraradices, probably coupled to a H+ -symport mechanism. The pH changes induced by AM fungal hyphae and the possible influence of the establishment of a functional symbiosis on these pH changes are discussed.