Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.     

An AM fungus and a PGPR intensify the adverse effects of salinity on the stability of rhizosphere soil aggregates of Lactuca sativa

A mesocosm experiment was conducted to examine the effect of an arbuscular mycorrhizal (AM) fungus (Glomus mosseae (Nicol & Gerd.) Gerd. & Trappe) and a plant growth-promoting rhizobacterium (PGPR) (Pseudomonas mendocina Palleroni), alone or in combination, on the structural stability of the rhizosphere soil of Lactuca sativa L. grown under two levels of salinity. The plants inoculated with P. mendocina had significantly greater shoot biomass than the control plants at both salinity levels, whereas the mycorrhizal inoculation was only effective in increasing shoot biomass at the moderate salinity level. The aggregate stability of soils inoculated with the PGPR and/or G. mosseae significantly decreased with increasing saline stress (about 29% lower than those of soils under non-saline conditions). Only the inoculated soils showed higher concentrations of sodium (Na) under severe saline stress. The severe salinity stress decreased the glomalin-related soil protein (GRSP) concentration, but the highest values of GRSP were recorded in the inoculated soils. Our findings suggest that the use of AM fungi and/or a PGPR for alleviating salinity stress in lettuce plants could be limited by their detrimental effect on soil structural stability.     

Interactions between a plant growth-promoting rhizobacterium,an AM fungus and a phosphate-solubilising fungus in the rhizosphere of Lactuca sativa

This study evaluated the interactions between the inoculation with an arbuscular mycorrhizal fungus, Glomus intraradices Schenck & Smith, a plant growth-promoting rhizobacterium, Bacillus subtilis, and a filamentous soil fungus, Aspergillus niger, with respect to their effects on growth of lettuce plants and on indicators of biological soil quality (microbial biomass C, water-soluble C and carbohydrates and dehydrogenase, urease, acid phosphatase and benzoyl argininamide hydrolyzing protease activities). Water-soluble carbohydrates and microbial biomass were increased only in the rhizosphere soil of G. intraradices-plants. Rhizosphere soil from all microbial inoculation treatments had significantly higher dehydrogenase activity than the control soil, particularly in the soil inoculated with B. subtilis (about 21% higher than control soil). Inoculation with A. niger or B. subtilis increased significantly the urease, protease and phosphatase activities of the rhizosphere soil of the lettuce plants. The foliar P and K contents increased significantly with the B. subtilis or G. intraradices inoculation, alone or in combination. The most effective co-inoculation was observed in the combined treatment of inoculation with G. intraradices and B. subtilis, which synergistically increased plant growth compared with singly inoculated (about 77% greater with respect to the control plants).     

Effects of elevated CO2, water stress, and inoculation with Glomus intraradices or Pseudomonas mendocina on lettuce dry matter and rhizosphere microbial and functional diversity under growth chamber conditions

Purpose  

In this study, we investigated the effects of elevated atmospheric CO₂, drought, and inoculation with a plant-growth-promoting rhizobacterium (PGPR) or an arbuscular mycorrhizal (AM) fungus on microbial community composition and functional diversity in the rhizosphere of Lactuca sativa L. cv. Tafalla.     

Interactions between phosphorus availability and an AM fungus (Glomus intraradices) and their effects on soil microbial respiration, biomass and enzyme activities in a calcareous soil

The interactions between soil P availability and mycorrhizal fungi could potentially impact the activity of soil microorganisms and enzymes involved in nutrient turnover and cycling, and subsequent plant growth. However, much remains to be known of the possible interactions among phosphorus availability and mycorrhizal fungi in the rhizosphere of berseem clover (Trifolium alexandrinum L.) grown in calcareous soils deficient in available P. The primary purpose of this study was to look at the interaction between P availability and an arbuscular mycorrhizal (AM) fungus (Glomus intraradices) on the growth of berseem clover and on soil microbial activity associated with plant growth. Berseem clover was grown in P unfertilized soil (−P) and P fertilized soil (+P), inoculated (+M) and non-inoculated (−M) with the mycorrhizal fungus for 70 days under greenhouse conditions. We found an increased biomass production of shoot and root for AM fungus-inoculated berseem relative to uninoculated berseem grown at low P levels. AM fungus inoculation led to an improvement of P and N uptake. Soil respiration (SR) responded positively to P addition, but negatively to AM fungus inoculation, suggesting that P limitation may be responsible for stimulating effects on microbial activity by P fertilization. Results showed decreases in microbial respiration and biomass C in mycorrhizal treatments, implying that reduced availability of C may account for the suppressive effects of AM fungus inoculation on microbial activity. However, both AM fungus inoculation and P fertilization affected neither substrate-induced respiration (SIR) nor microbial metabolic quotients (qCO₂). So, both P and C availability may concurrently limit the microbial activity in these calcareous P-fixing soils. On the contrary, the activities of alkaline phosphatase (ALP) and acid phosphatase (ACP) enzymes responded negatively to P addition, but positively to AM fungus inoculation, indicating that AM fungus may only contribute to plant P nutrition without a significant contribution from the total microbial activity in the rhizosphere. Therefore, the contrasting effects of P and AM fungus on the soil microbial activity and biomass C and enzymes may have a positive or negative feedback to C dynamics and decomposition, and subsequently to nutrient cycling in these calcareous soils. In conclusion, soil microbial activity depended on the addition of P and/or the presence of AM fungus, which could affect either P or C availability.     

The mycorrhizal fungus Glomus intraradices and rock phosphate amendment influence plant growth and microbial activity in the rhizosphere of Acacia holosericea

Plants inoculated with arbuscular mycorrhizal (AM) fungi utilize more soluble phosphorus from soil mineral phosphate than non-inoculated plants. However, there is no information on the response of soil microflora to mineral phosphate weathering by AM fungi and, in particular, on the catabolic diversity of soil microbial communities.The AM fungus, Glomus intraradices was examined for (i) its effect on the growth of Acacia holosericea, (ii) plant-available phosphate and (iii) soil microbial activity with and without added rock phosphate.After 4-months culture, AM fungal inoculation significantly increased the plant biomasses (by 1.78× and 2.23× for shoot and root biomasses, respectively), while mineral phosphate amendment had no effect in a sterilized soil. After 12-months culture, the biomasses of A. holosericea plants growing in a non-sterilized soil amended with mineral phosphate were significantly higher than those recorded in the control treatment (by 2.5× and 5× for shoot and root biomasses, respectively). The fungal inoculation also significantly stimulated plant growth, which was significantly higher than that measured in the mineral phosphate treatment. When G. intraradices and mineral phosphate were added together to the soil, shoot growth were significantly stimulated over the single treatments (inoculation or amendment) (1.45×). The P leaf mineral content was also higher in the G. intraradices+mineral phosphate treatment than in G. intraradices or rock phosphate amendment. Moreover, the number of fluorescent pseudomonads has been significantly increased when G. intraradices and/or mineral phosphate were added to the soil. By using a specific type of multivariate analysis (co-inertia analysis), it has been shown that plant growth was positively correlated to the metabolization of ketoglutaric acid, and negatively linked to the metabolisation of phenylalanine and other substrates, which shows that microbial activity is also affected.G. intraradices inoculation is highly beneficial to the growth of A. holosericea plants in controlled conditions. This AM symbiosis optimises the P solubilization from the mineral phosphate and affects microbial activity in the hyphosphere of A. holosericea plants.     

Changes in biological activity of a degraded Mediterranean soil after using microbially-treated dry olive cake as a biosolid amendment and arbuscular mycorrhizal fungi

A field experiment was carried out to assess the effect of a combined treatment involving addition of Aspergillus niger-treated dry olive cake (DryOC) in the presence of rock phosphate, plus pre-transplant inoculation of seedlings with the arbuscular mycorrhizal (AM) fungi Glomus intraradices, Glomus deserticola or Glomus mosseae, on the establishment of Dorycnium pentaphyllum L., in a degraded semiarid Mediterranean area. Associated changes in soil labile C fractions, enzyme activities and aggregate stability were also observed. One year after planting, the combined treatment of fermented DryOC addition and inoculation with AM fungi, particularly with G. mosseae (on average 328% greater than control plants), had the strongest effect on the shoot biomass of D. pentaphyllum. Only the fermented DryOC addition increased assimilable P, total N and aggregate stability, the greatest increase being in the soil available P content (about four-fold higher than in the non-amended soil). Both the addition of fermented DryOC and the mycorrhizal inoculation treatments significantly increased enzyme activities of rhizosphere soil (dehydrogenase, protease-BAA, acid phosphatase and β-glucosidase). The microbially-treated DryOC proved to be an effective amendment for improving the soil quality which, in turn, enhanced the success of revegetation with mycorrhizal D. pentaphyllum seedlings.     

Elevation of atmospheric CO2 and N-nutritional status modify nodulation, nodule-carbon supply, and root exudation of Phaseolus vulgaris L.

Increased root exudation and a related stimulation of rhizosphere-microbial growth have been hypothesised as possible explanations for a lower nitrogen- (N-) nutritional status of plants grown under elevated atmospheric CO₂ concentrations, due to enhanced plant-microbial N competition in the rhizosphere. Leguminous plants may be able to counterbalance the enhanced N requirement by increased symbiotic N₂ fixation. Only limited information is available about the factors determining the stimulation of symbiotic N₂ fixation in response to elevated CO₂.In this study, short-term effects of elevated CO₂ on quality and quantity of root exudation, and on carbon supply to the nodules were assessed in Phaseolus vulgaris, grown in soil culture with limited (30 mg N kg⁻¹ soil) and sufficient N supply (200 mg N kg⁻¹ soil), at ambient (400 μmol mol⁻¹) and elevated (800 μmol mol⁻¹) atmospheric CO₂ concentrations.Elevated CO₂ reduced N tissue concentrations in both N treatments, accelerated the expression of N deficiency symptoms in the N-limited variant, but did not affect plant biomass production. ¹⁴CO₂ pulse-chase labelling revealed no indication for a general increase in root exudation with subsequent stimulation of rhizosphere microbial growth, resulting in increased N-competition in the rhizosphere at elevated CO₂. However, a CO₂-induced stimulation in root exudation of sugars and malate as a chemo-attractant for rhizobia was detected in 0.5-1.5 cm apical root zones as potential infection sites. Particularly in nodules, elevated CO₂ increased the accumulation of malate as a major carbon source for the microsymbiont and of malonate with essential functions for nodule development. Nodule number, biomass and the proportion of leghaemoglobin-producing nodules were also enhanced. The release of nod-gene-inducing flavonoids (genistein, daidzein and coumestrol) was stimulated under elevated CO₂, independent of the N supply, and was already detectable at early stages of seedling development at 6 days after sowing.     

The bacterial community of tomato rhizosphere is modified by inoculation with arbuscular mycorrhizal fungi but unaffected by soil enrichment with mycorrhizal root exudates or inoculation with Phytophthora nicotianae

Arbuscular mycorrhizal (AM) fungi have been shown to induce the biocontrol of soilborne diseases, to change the composition of root exudates and to modify the bacterial community structure of the rhizosphere, leading to the formation of the mycorrhizosphere. Tomato plants were grown in a compartmentalized soil system and were either submitted to direct mycorrhizal colonization or to enrichment of the soil with exudates collected from mycorrhizal tomato plants, with the corresponding negative controls. Three weeks after planting, the plants were inoculated or not with the soilborne pathogen Phytophthora nicotianae growing through a membrane from an adjacent infected compartment. At harvest, a PCR-Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments amplified from the total DNA extracted from each plant rhizosphere was performed. Root colonization with the AM fungi Glomus intraradices or Glomus mosseae induced significant changes in the bacterial community structure of tomato rhizosphere, compared to non-mycorrhizal plants, while enrichment with root exudates collected from mycorrhizal or non-mycorrhizal plants had no effect. Our results support that the effect of AM fungi on rhizosphere bacteria would not be mediated by compounds present in root exudates of mycorrhizal plants but rather by physical or chemical factors associated with the mycelium, volatiles and/or root surface bound substrates. Moreover, infection of mycorrhizal or non-mycorrhizal plants with P. nicotianae did not significantly affect the bacterial community structure suggesting that rhizosphere bacteria would be less sensitive to the pathogen invasion than to mycorrhizal colonization. Of 96 unique sequences detected in the tomato rhizosphere, eight were specific to mycorrhizal fungi, including two Pseudomonas, a Bacillus simplex, an Herbaspirilium and an Acidobacterium. One Verrucomicrobium was common to rhizospheres of mycorrhizal plants and of plants watered with mycorrhizal root exudates.     

Physiological characteristics (SDH and ALP activities) of arbuscular mycorrhizal colonization as affected by Bacillus thuringiensis inoculation under two phosphorus levels

The effect of Bacillus thuringiensis (B.t.) inoculation on plant growth and on the intra- and extraradical mycorrhizal development of lettuce roots colonized by Glomus mosseae or Glomus intraradices was examined in an inert, soil-less substrate. Histochemical determination of succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities which indicate active fungal metabolism was carried out at two phosphorus (P) levels. The presence of B.t. increased extra- and intraradical colonization [measured as frequency (%F), intensity (%I) and percentage of arbuscules (%A)] for both arbuscular mycorrhizal fungi (AMF) rather than plant growth or nutrition regardless P level. Under the lowest level of P fertilization, B.t. enhanced to a similar extent the extra- and intraradical development of both endophytes, but the proportion of fungal tissue showing SDH or ALP was increased in G. intraradices-colonized plants. [SDH: 458% (M) and 512% (A); ALP: 358% (M) and 300% (A)]. P supply decreased G. intraradices colonization to a higher extent than G. mosseae. Nevertheless, the totality of G. intraradices structures developed in P-amended medium showed intraradical o extraradical activity, while in G. mosseae-colonized roots, SDH and ALP activities highly decreased relative to fungal tissue determined by TB staining as affected by P. Our results show that bacterial inoculation compensates the negative effect of P on the intraradical fungal growth and vitality. P amendment reduced in a higher extent G. intraradices infection intensity (non-vital and vital staining) and G. mosseae activity (ALP staining). Thus, big differences in the proportion of SDH-active infection showing ALP activity in mycelium developed by each endophyte were noted at the highest P level. Physiological plant parameters such as photosynthetic activity did not explain specific changes on each arbuscular-mycorrhizal fungus as affected by P or B.t. inoculation. The increased extraradical mycelium development and metabolic fungal activity as a result of B.t. inoculation positively affected N and P plant content and photosynthetic rate in G. intraradices-colonized plants under the lowest P conditions. In general, the increased metabolically active fungal biomass in co-inoculated plants was irrespective of P level and was not related to the P plant uptake from the inert soil-less substrate. These results show the bacterial effect increasing the physiological and metabolic status of AM endophytes, which not only confirms but also extends previous findings on arbuscular mycorrhizae-bacteria interactions. The present study emphasizes the ecological and practical importance of rhizosphere free-living bacteria as mycorrhizae-helper microorganisms.     

Stability of desiccated rhizosphere soil aggregates of mycorrhizal Juniperus oxycedrus grown in a desertified soil amended with a composted organic residue

Adequate soil structural stability favours the establishment and viability of a stable plant cover, protecting the soil against water erosion in desertified Mediterranean environments. We studied the effect of soil drying-rewetting, inoculation with a mixture of three exotic arbuscular mycorrhizal (AM) fungi (Glomus intraradices Schenck & Smith, Glomus deserticola (Trappe, Bloss. & Menge) and Glomus mosseae (Nicol & Gerd.) Gerd. & Trappe) and addition of a composted organic residue on aggregate stabilisation of the rhizosphere soil of Juniperus oxycedrus. The AM fungi and composted residue produced similar increases in plant growth, independently of the water conditions. Under well-watered conditions, the highest percentages of stable aggregates were recorded in the amended soil, followed by the soil inoculated with AM fungi. Excepting microbial biomass C, the soil drying increased labile C fractions (water soluble C, water soluble and total carbohydrates), whereas the rewetting decreased significantly such C fractions. Desiccation caused a significant increase in aggregate stability of the rhizosphere soil of all plants, particularly in the amended and inoculated plants. In all treatments, the aggregates formed after soil drying were unstable, since, in the rewetting, they disappear, reaching the initial levels before soil drying. Our results suggest that the aggregation mechanisms developed by rhizosphere microbial community of the amended and inoculated plants under water stress can be particularly relevant in desertified soils exposed to long desiccation periods.     

Differential effects of soil disturbance and plant residue retention on function of arbuscular mycorrhizal (AM) symbiosis are not reflected in colonization of roots or hyphal development in soil

The effects of soil disturbance and residue retention on the functionality of the symbiosis between medic (Medicago truncatula L.) and arbuscular mycorrhizal fungi (AMF) were assessed in a two-stage experiment simulating a crop rotation of wheat (Triticum aestivum L.) followed by medic. Plants were inoculated or not with the AMF, Glomus intraradices and Gigaspora margarita, separately or together. The contribution of the arbuscular mycorrhizal (AM) pathway for P uptake was determined using ³²P-labeled soil in a small hyphal compartment accessible only to hyphae of AMF. In general AM colonization was not affected by soil disturbance or residue application and disturbance did not affect hyphal length densities (HLDs) in soil. At 4 weeks disturbance had a negative effect on growth and phosphorus (P) uptake of plants inoculated with G. margarita, but not G. intraradices. By 7 weeks disturbance reduced growth of plants inoculated with G. margarita or AMF mix and total P uptake in all inoculated plants. With the exception of plants inoculated with G. margarita in disturbed soil at 4 weeks, the AM pathway made a significant contribution to P uptake in all AM plants at both harvests. Inoculation with both AMF together eliminated the negative effects of disturbance on AM P uptake and growth, showing that a fungus insensitive to disturbance can compensate for loss of contribution of a sensitive one. Application of residue increased growth and total P uptake of plants but decreased ³²P in plants inoculated with the AMF mix in disturbed soil, compared with plants receiving no residue. The AMF responded differently to disturbance and G. intraradices, which was insensitive to disturbance, compensated for lack of contribution by the sensitive G. margarita when they were inoculated together. Colonization of roots and HLDs in soil were not good predictors of the outcomes of AM symbioses on plant growth, P uptake or P delivery via the AM pathway.     

Changes in microbial communities and carbohydrate profiles induced by the mycorrhizal fungus (Glomus intraradices) in rhizosphere of olive trees (Olea europaea L.)

The influence of inoculation of olive trees with arbuscular mycorrhizal (AM) fungi, Glomus (G) intraradices, on microbial communities and sugar concentrations, were examined in rhizosphere of olive trees (Olea europaea L.). Analyses of phospholipid and neutral lipid fatty acids (PLFA and NLFA, respectively) were then used to detect changes in microbial community structure in response to inoculation of plantlets with G. intraradices.Microscopic observations studies revealed that the extraradical mycelium of the fungus showed formation of branched absorbing structures (BAS) in rhizosphere of olive tree. Root colonization with the AM fungi G. intraradices induced significant changes in the bacterial community structure of olive tree rhizosphere compared to non-mycorrhizal plants. The largest proportional increase was found for the fatty acid 10Me18:0, which indicated an increase in the number of actinomycetes in mycorrhizal rhizosphere soil, whereas the PLFAs i15:0, a15:0, i16:0, 16:1ω7 and cy17:0 which were used as indicators of bacteria decreased in mycorrhizal treatment compared to non-mycorrhizal control treatment. A highest concentration of glucose and trehalose and a lowest concentration of fructose, galactose, sucrose, raffinose and mannitol were detected in mycorrhizal rhizosphere soil. This mycorrhizal effect on rhizosphere communities may be a consequence of changes in characteristics in the environment close to mycorrhizal roots.     

Changes in rhizosphere microbial activity mediated by native or allochthonous AM fungi in the reafforestation of a Mediterranean degraded environment

This study was carried out in a semiarid degraded area to assess the effectiveness of mycorrhizal inoculation with a mixture of native arbuscular mycorrhizal (AM) fungi or an allochthonous AM fungus (Glomus claroideum), on the establishment of Olea europaea subsp. sylvestris L. and Retama sphaerocarpa (L.) Boissier in this area. Associated changes in the soil microbiological properties and aggregate stability related to these AM inocula were also recorded. Eighteen months after planting, G. claroideum had increased available P in the rhizosphere of both shrub species. In general, both inoculation treatments increased water-soluble C and water-soluble and total carbohydrates, G. claroideum being the most effective inoculum, particularly in R. sphaerocarpa. The mixture of native AM fungi was the most effective treatment for increasing the aggregate stability of R. sphaerocarpa soil, while that of O. europaea was increased only by G. claroideum. Increased (dehydrogenase, urease, protease-BAA, acid phosphatase and -glucosidase) enzyme activities, in particular of dehydrogenase and acid phosphatase, were recorded in the rhizosphere of both mycorrhizal shrub species. The mixture of native AM fungi was the most effective treatment for stimulating the growth of O. europaea and R. sphaerocarpa (11.6-fold and 3.3-fold, respectively, greater than control plants). The establishment of mycorrhizal shrub species favoured the reactivation of soil microbial activity, which was linked to an increase in aggregate stability.     

Fraxinus-Glomus-Pisolithus symbiosis: Plant growth and soil aggregation effects

It has been established that arbuscular mycorrhizal (AM) fungi are involved in the conservation of soil structure. However, the effect of ectomycorrhizal (EM) fungi alone or in interaction with AM fungi in soil structure has been much less studied. This experiment evaluated EM and AM fungi effects on soil aggregation and plant growth. Ash plants (Fraxinus uhdei) were grown in pots, and were inoculated with Glomus intraradices and Pisolithus tinctorius separately but also in combination. Our results showed that F. uhdei established a symbiotic association with EM and AM fungi, and that these organisms, when interacting, showed synergistic and additive effects on plant growth compared to singly inoculated treatments. EM and AM fungi prompted changes in root morphology and increased water-stable aggregates. AM fungi affect mainly small-sized macroaggregates, while EM and EM-AM fungi interaction mainly affected aggregates bigger than 0.5 mm diameter. These results suggest that ectomyccorrhizal as well as arbuscular mycorrhizal fungi should be considered in restoration programs with Fraxinus plants.     

Short and long-term effects of elevated CO2 on Lolium perenne rhizodeposition and its consequences on soil organic matter turnover and plant N yield

It is still unclear whether elevated CO₂ increases plant root exudation and consequently affects the soil microbial biomass. The effects of elevated CO₂ on the fate of the C and nitrogen (N) contained in old soil organic matter pools is also unclear. In this study the short and long-term effects of elevated CO₂ on C and N pools and fluxes were assessed by growing isolated plants of ryegrass (Lolium perenne) in glasshouses at elevated and ambient atmospheric CO₂ and using soil from the New Zealand FACE site that had >4 years exposure to CO₂ enrichment. Using ¹⁴CO₂ pulse labelling, the effects of elevated CO₂ on C allocation within the plant-soil system were studied. Under elevated CO₂ more root derived C was found in the soil and in the microbial biomass 48 h after labelling. The increased availability of substrate significantly stimulated soil microbial growth and acted as priming effect, enhancing native soil organic matter decomposition regardless of the mineral N supply. Despite indications of faster N cycling in soil under elevated CO₂, N availability to plants stayed unchanged. Soil previously exposed to elevated CO₂ exhibited a higher N cycling rate but again there was no effect on plant N uptake. With respect to the difficulties of extrapolating glasshouse experiment results to the field, we concluded that the accumulation of coarse organic matter observed in the field under elevated CO₂ was probably not created by an imbalance between C and N but was likely to be due to more complex phenomena involving soil mesofauna and/or other nutrients limitations.     

Changes in soil aggregation and glomalin-related soil protein content as affected by the arbuscular mycorrhizal fungal species Glomus mosseae and Glomus intraradices

Arbuscular mycorrhizal (AM) fungi are key organisms of the soil/plant system, influencing soil fertility and plant nutrition, and contributing to soil aggregation and soil structure stability by the combined action of extraradical hyphae and of an insoluble, hydrophobic proteinaceous substance named glomalin-related soil protein (GRSP). Since the GRSP extraction procedures have recently revealed problems related to co-extracting substances, the relationship between GRSP and AM fungi still remains to be verified. In this work the hypothesis that GRSP concentration is positively correlated with the occurrence of AM fungi was tested by using Medicago sativa plants inoculated with different isolates of Glomus mosseae and Glomus intraradices in a microcosm experiment. Our results show that (i) mycorrhizal establishment produced an increase in GRSP concentration - compared to initial values - in contrast with non-mycorrhizal plants, which did not produce any change; (ii) aggregate stability, evaluated as mean weight diameter (MWD) of macroaggregates of 1-2 mm diameter, was significantly higher in mycorrhizal soils compared to non-mycorrhizal soil; (iii) GRSP concentration and soil aggregate stability were positively correlated with mycorrhizal root volume and weakly correlated with total root volume; (iv) MWD values of soil aggregates were positively correlated with values of total hyphal length and hyphal density of the AM fungi utilized.The different ability of AM fungal isolates to affect GRSP concentration and to form extensive and dense mycelial networks, which may directly affect soil aggregates stability by hyphal enmeshment of soil particles, suggests the possibility of selecting the most efficient isolates to be utilized for soil quality improvement and land restoration programs.     

Can a mixed stand of N2-fixing and non-fixing plants restrict N2O emissions with increasing CO2 concentration?

Initial effects of elevated atmospheric CO₂ concentration on N₂O fluxes and biomass production of timothy/red clover were studied in the laboratory. The experimental design consisted of two levels of atmospheric CO₂ (ca. 360 and 720 μmol CO₂ mol⁻¹) and two N fertilisation levels (5 and 10 g N m⁻²). There was a total of 36 mesocosms comprising sandy loam soil, which were equally distributed in four thermo-controlled greenhouses. In two of the greenhouses, the CO₂ concentration was kept at ambient concentration and in the other two at doubled concentration. Forage was harvested and the plants fertilised three times during the basic experiment, followed by harvest, a fertilisation with the double amount of nitrogen and rise of water level. Under elevated CO₂, harvestable and total aboveground dry biomass production of a mixed Trifolium/Phleum stand was increased at both N treatments compared to ambient CO₂. The N₂O flux rates under ambient CO₂ were significantly higher at both N treatments during the early growth of mixed Phleum/Trifolium mesocosms compared to the N₂O flux rate under elevated CO₂. However, when the conditions were favourable for denitrification at the end of the experiment, i.e. N availability and soil moisture were high enough, the elevated CO₂ concentration enhanced the N₂O efflux.     

Interactions of arbuscular-mycorrhizal fungi and Bacillus strains and their effects on plant growth,microbial rhizosphere activity (thymidine and leucine incorporation) and fungal biomass (ergosterol and chitin)

The effects of two Bacillus strains (Bacillus pumillus and B. licheniformis) on Medicago sativa plants were determined in single or dual inoculation with three arbuscular-mycorrhizal (AM) fungi and compared to P-fertilization. Shoot and root plant biomass, values of thymidine and leucine incorporation as well as ergosterol and chitin in rhizosphere soil were evaluated to estimate metabolic activity and fungal biomass, respectively, according to inoculation treatments. For most of the plant parameters determined, the effectiveness of AM fungal species was influenced by the bacterial strain associated. Dual inoculation of Bacillus spp. and AM fungi did not always significantly increase shoot biomass compared to single AM-colonized plants. The most efficient treatment in terms of dry matter production was the dual Glomus deserticola plus B. pumillus inoculation, which produced similar shoot biomass and longer roots than P-fertilization and a 715% (shoot) and 190% (root length) increase over uninoculated control. The mycorrhizas were more important for N use-efficiency than for P use-efficiency, which suggests a direct mycorrhizal effect on N nutrition not mediated by P uptake. Both chemical and biological treatments affected thymidine and leucine incorporation in the rhizosphere soil differently. Thymidine was greater in inoculated than in control rhizospheres and B. licheniformis was more effective than B. pumillus in increasing thymidine. Non-inoculated rhizospheres showed the lowest thymidine and leucine values, which shows that indigenous rhizosphere bacteria increased with introduced inocula. The highest thymidine and leucine values found in P-fertilized soils indicate that AM plants are better adapted to compete with saprophytic soil bacteria for nutrients than P-amended plants. Chitin was only increased by coinoculation of B. licheniformis and G. intraradices. B. pumillus increased ergosterol (indicative of active saprophyte fungal populations) in the rhizosphere of AM plants and particularly when colonized by G. mosseae. The different AM fungi have different effects on bacterial and/or fungal saprophytic populations and for each AM fungus, this effect was specifically stimulated or reduced by the same bacterium. This is an indication of ecological compatibilities between microorganisms. Particular Glomus–bacterium interactions (in terms of effect on plant growth responses or rhizosphere population) do not seem to be related to the percentage of AM colonization. The effect on plant growth and stimulation of rhizosphere populations, as a consequence of selected microbial groups, may be decisive for the plant establishment under limiting soil conditions.     

Interactions between the arbuscular mycorrhizal fungus Glomus intraradices and the plant growth promoting rhizobacteria Paenibacillus polymyxa and P. macerans in the mycorrhizosphere of Cucumis sativus

Interactions between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and bacteria from the genus Paenibacillus (P. macerans and P. polymyxa) were examined in a greenhouse pot experiment with Cucumis sativus with and without organic matter amendment (wheat bran). P. polymyxa markedly suppressed AM fungus root colonization irrespective of wheat bran amendment, whereas P. macerans only suppressed AM fungus root colonization in combination with wheat bran amendment. Dual inoculation with P. macerans and G. intraradices in combination with wheat bran amendment also caused severe plant growth suppression. Inoculation with G. intraradices was associated with increased levels of dehydrogenase activity and available P in the growth substrate suggesting that mycorrhiza formation accelerated the decomposition of organic matter resulting in mobilization of phosphorus. Inoculation with both Paenibacillus species increased all measured microbial fatty acid biomarkers in the cucumber rhizosphere, except for the AM fungus biomarker 16:1ω5, which was reduced, though not significantly. Similarly, inoculation with G. intraradices increased all measured microbial fatty acid biomarkers in the cucumber rhizosphere, except for the Gram-positive bacteria biomarker 15:0 anteiso, which was overall decreased by G. intraradices inoculation. In combination with wheat bran amendment G. intraradices inoculation caused a 39% reduction in the amount of 15:0 anteiso in the treatment with P. polymyxa, suggesting that G. intraradices suppressed P. polymyxa in this treatment. In conclusion, plant growth promoting species of Paenibacillus may have suppressive effects of AM fungi and plant growth, especially in combination with organic matter amendment. The use of an inert plant growth media in the present study allowed us to study rhizosphere microbial interactions in a relative simple substrate with limited interference from other soil biota. However, the results obtained in the present work mainly show potential interactions and should not be directly extrapolated to a soil situation.