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Yeasts are abundant and diverse in rhizosphere soil, but information about their importance in agroecosystems is limited. Here we examined maize plant growth response to inoculation with the native maize rhizosphere yeasts Cryptococcus flavus and Candida railenensis in non-mycorrhizal and mycorrhizal maize without and with mineral P fertilization as KH2PO4. Maize plant growth promotion and suppression was observed after inoculation with a native community of arbuscular mycorrhizal (AM) fungi with and without mineral P fertilization, respectively. Inoculation with Cr. flavus reduced shoot biomass of maize plants irrespective of the mycorrhizal status, but only without P fertilization. AM fungal root colonization was increased by C. railenensis without P fertilization, but was reduced by Cr. flavus in combination with mineral P fertilization. In conclusion, our results show that mineral P fertilization strongly modulates interactions between maize, rhizosphere yeasts and AM fungi, which are important to take into account when integrating such root associated microorganisms as biofertilizers in agroecosystems., This study was supported with the grant 179319 from Consejo Nacional de Ciencia y Tecnología (CONACYT). Marcela Sarabia was supported with the Ph.D. fellowship 239182 also from CONACYT.

This study evaluates the responses of Lavandula dentata under drought conditions to the inoculation with single autochthonous arbuscular mycorrhizal (AM) fungus (five fungal strains) or with their mixture and the effects of these inocula with a native Bacillus thuringiensis (endophytic bacteria). These microorganisms were drought tolerant and in general, increased plant growth and nutrition. Particularly, the AM fungal mixture and B. thuringiensis maximized plant biomass and compensated drought stress as values of antioxidant activities [superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase APX)] shown. The AMF-bacteria interactions highly reduced the plant oxidative damage of lipids [malondialdehyde (MDA)] and increased the mycorrhizal development (mainly arbuscular formation representative of symbiotic functionality). These microbial interactions explain the highest potential of dually inoculated plants to tolerate drought stress. B. thuringiensis “in vitro” under osmotic stress does not reduce its PGPB (plant growth promoting bacteria) abilities as indole acetic acid (IAA) and ACC deaminase production and phosphate solubilization indicating its capacity to improve plant growth under stress conditions. Each one of the autochthonous fungal strains maintained their particular interaction with B. thuringiensis reflecting the diversity, intrinsic abilities and inherent compatibility of these microorganisms. In general, autochthonous AM fungal species and particularly their mixture with B. thuringiensis demonstrated their potential for protecting plants against drought and helping plants to thrive in semiarid ecosystems., E. Armada was financed by Ministry of Science and Innovation (Spain). This work was carried out in the framework of the project reference AGL2009-12530-C02-02.

Selenium (Se) is an important micronutrient for human health especially to fortifying the immune system. Chilean Andisols contain low quantities of Se and we have shown that bacteria can be used to enhance Se content of food plants and to improve the antioxidant system against different abiotic stresses. In this study, we prepared inocula based on previously selected Se tolerant endophytic bacteria (Acinetobacter sp. E6.2 and Bacillus sp. E5), which were supplemented with sodium selenite at 5 mM. We studied the effect of this Se amount on bacterial synthesis of organic compounds and Se toxicity, NanoSe size, together with the role of Se in drought stress mitigation. Our results indicated that Acinetobacter sp. E6.2 produced elevated quantities of seleno-methionine (SeMet) and seleno-methyl-selenocysteine (SeMeSeCys) (10 and 3.77 mg kg−1, respectively). However, highly stable NanoSe (Z potential around −40 mV) was the main Se form found in both inocula. The size of NanoSe in Acinetobacter sp. was greater than in Bacillus sp. over time (i.e. 213 ± 3.4 nm and 169 ± 0.92 nm, respectively at 24 h). Bacteria selenium supplementation did not induce oxidative stress due to similar superoxide dismutase (SOD) and catalase (CAT) activities. Selenium did not affect cell tolerance to water deficit due to similar proline and indole acetic acid (IAA) production under drought stress conditions. The findings of this study suggest that these new endophytic bacteria inocula could be effective for Se biofortification., This study was supported by the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT), FONDECYT Postdoctoral Project No. 3130542 from Chilean Government. The authors also acknowledge the technical support by Scientific and Technological Bioresource Nucleus (BIOREN) from Universidad de la Frontera and Horacio Miranda for his collaboration in date analyses.

Studies have shown that some microorganisms autochthonous from stressful environments are beneficial when used with autochthonous plants, but these microorganisms rarely have been tested with allochthonous plants of agronomic interest. This study investigates the effectiveness of drought-adapted autochthonous microorganisms [Bacillus thuringiensis (Bt) and a consortium of arbuscular mycorrhizal (AM) fungi] from a degraded Mediterranean area to improve plant growth and physiology in Zea mays under drought stress. Maize plants were inoculated or not with B. thuringiensis, a consortium of AM fungi or a combination of both microorganisms. Plants were cultivated under well-watered conditions or subjected to drought stress. Several physiological parameters were measured, including among others, plant growth, photosynthetic efficiency, nutrients content, oxidative damage to lipids, accumulation of proline and antioxidant compounds, root hydraulic conductivity and the expression of plant aquaporin genes. Under drought conditions, the inoculation of Bt increased significantly the accumulation of nutrients. The combined inoculation of both microorganisms decreased the oxidative damage to lipids and accumulation of proline induced by drought. Several maize aquaporins able to transport water, CO2 and other compounds were regulated by the microbial inoculants. The impact of these microorganisms on plant drought tolerance was complementary, since Bt increased mainly plant nutrition and AM fungi were more active improving stress tolerance/homeostatic mechanisms, including regulation of plant aquaporins with several putative physiological functions. Thus, the use of autochthonous beneficial microorganisms from a degraded Mediterranean area is useful to protect not only native plants against drought, but also an agronomically important plant such as maize., E. Armada was financed by Ministerio de Economía y Competitividad (Spain) (BES-2010-042736). This work was carried out in the framework of the project reference AGL2012-39057-C02.

Cereal crops grown in southern Chilean Andisol provide suboptimal levels of this metalloid for human diet. Certain rhizosphere microorganisms, such as rhizobacteria and arbuscular mycorrhizal fungi can increase the selenium uptake in plants. The purpose of this study was to evaluate selenium acquisition by wheat plants through the co-inoculation of native selenobacteria strains (Stenotrophomonas sp. B19, Enterobacter sp. B16, Bacillus sp. R12 and Pseudomonas sp. R8), both individually and in mixture, as selenonanosphere source with one arbuscular mycorrhizal fungus (Glomus claroideum). Total selenium content in plant tissues and substrate was analyzed. According to our results, significant higher selenium content was found in inoculated plants in comparison to uninoculated controls (P ≤ 0.05). Independently of fungal presence, selenium content in grain from plants inoculated with Enterobacter sp. B16 (236 mg kg−1) was higher than the rest of the strains (116–164 mg kg−1). However, when plants were co-inoculated with a mixture of selenobacteria strains and G. claroideum, selenium content in grain was 23.5% higher (725 mg kg−1) than non-mycorrhizal plants (587 mg kg−1). The results suggest a synergistic effect between the selenobacteria mixture and G. claroideum associated to major biodiversity and demonstrate a great potential of these rhizosphere microorganisms for biofortification of cereals and its derivates., This study was supported by CONICYT Postdoctoral scholarship 3130542 and FONDECYT 1100625 of the Chilean Government. J.J. Acuña is thankful for the financial support of CONICYT through the Doctoral Fellowship Program. M.A. Jorquera also acknowledges financial support by FONDECYT 1120505.

The aim of this study was to assess the effectiveness of inoculation with a native arbuscular mycorrhizal (AM) fungus, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe, or a filamentous fungus, Penicillium aurantiogriseum Dierckx 1901, on the establishment of Coronilla juncea L. seedlings grown in a polluted, semiarid soil. For that, root and shoot biomass, nutrient uptake, mycorrhizal colonisation and nitrate reductase (NR) and phosphatase activities were analysed. Six months after planting, the shoot biomass of C. juncea was increased only by the inoculation with G. mosseae (by about 62% compared with non-mycorrhizal plants). The shoot NR and root acid phosphatase activities were increased more by inoculation with G. mosseae than with P. aurantiogriseum inoculation. The root NR activity and foliar nutrient contents were increased only by the inoculation with the AM fungus. The root Zn and Cu decreased with the AM fungus. In conclusion, the autochthonous AM fungus was an effective inoculant with regard to stimulating growth and alleviating heavy metal toxicity for plants growing on a soil contaminated by multiple heavy metals. Inoculation with an autochthonous, filamentous fungus does not seem to be a good strategy for phytoremediation of such problematic sites., This research was supported by Plan Nacional (Project AGL2006-09453-CO2-01).

The growth of legume plants is usually enhanced by the dual symbiosis of arbuscular mycorrhizal (AM) fungi and Rhizobium bacteria. However, most reports on this topic have been carried out under optimal water regime conditions. Here, four Phaseolus vulgaris varieties were single or dual inoculated with two different AM fungus and/or two different Rhizobium strains. All plants were grown under moderate drought conditions. Surprisingly, most of the biological treatments involving one fungus and one Rhizobium together caused a deleterious effect on plant growth. However, these negative effects were dependent on the P. vulgaris variety used as well as on the symbionts implicated. The results showed that AM symbiosis inhibited nodule development and N2 fixation, causing diminution of plant growth. Therefore, under moderate drought conditions, the dual symbiosis formed by AM fungi and Rhizobium can be deleterious to P. vulgaris growth depending on the plant variety and the symbionts involved. Thus, under these common stress conditions, selection for the appropriated symbionts to each P. vulgaris variety is needed.

Aspergillus niger-treated dry olive cake (DryOC) can be used as a soil organic amendment and the aim of this work was to study the effectiveness of this amendment and a Cd-adapted arbuscular mycorrhizal (AM) fungus in improving Trifolium repens growth and nutrition in Cd-contaminated soil. In a compartmentalized growth system, consisting of a root compartment (RC) and two hyphal compartments (HCs), we investigated the influence of the amendment on intraradical and extraradical AM fungi development. In addition, we studied the viability and infectivity of the detached extraradical mycelium in plants, designated as receptor plants, grown in the HC after removal of the RC. Both the amendment and the AM fungus increased shoot and root biomass and nodulation in both the non-contaminated and Cd-contaminated soils. The positive interaction between the microbiologically treated DryOC and the AM fungus resulted in the highest plant yield, which can be explained by enhanced nutrient acquisition and arbuscular richness as well as by the immobilisation of Cd in amended soils. However, A. niger-treated DryOC had no effect on the extraradical mycorrhizal mycelium development. Although Cd decreased AM hyphal length density, symbiotic infectivity was similar in receptor plants grown in non-contaminated and contaminated soil, thus confirming the AM fungal inoculum potential. The combination of the AM fungus and A. niger-treated DryOC increased plant tolerance to Cd in terms of plant growth and nutrition and can be regarded as an important strategy for reclaiming Cd-contaminated soils., Financial support for this study was obtained from the Comision Interministerial de Ciencia y Tecnología (CICYT) PN REN 2000-1506 GLO. A. Medina is indebted to the Junta de Andalucia (Spain) and the Carl Trigger foundation (Sweden) for scholarship grants.

A mesocosm experiment was conducted to investigate the effect of the addition of Aspergillus niger-treated sugar beet waste, in the presence of rock phosphate, and inoculation with a native, metal-tolerant bacterium, Bacillus thuringiensis, on the stabilisation of soil aggregates of two mine tailings, with differing pH values, from a semiarid Mediterranean area and on the stimulation of growth of Piptatherum miliaceum. Bacterium combined with organic amendment enhanced structural stability (38% in acidic soil and 106% in neutral soil compared with their corresponding controls). Only the organic amendment increased pH, electrical conductivity, water-soluble C, water-soluble carbohydrates and plant growth, in both soils. While in neutral soil both organic amendment and bacterium increased dehydrogenase activity, only organic amendment had a significant effect in acidic soil. This study demonstrates that the use of P. miliaceum in combination with organic amendment and bacterium is a suitable tool for the stabilisation of the soil structure of degraded mine tailings, although its effectiveness is dependent on soil pH., This research was supported by the Plan Nacional (Project AGL2003-05619-CO2-01).

The plant growth, nutrient acquisition, metal translocation and antioxidant activities [ascorbate peroxidase (APX), glutatione reductase (GR), superoxide dismutase (SOD) and catalase (CAT)] were measured in plants growing in a heavy-metal (HM) multicontaminated soil inoculated with selected autochthonous microorganisms [arbuscular mycorrhizal (AM) fungus and/or plant growth promoting bacteria (PGPB)] and/or amended with an Aspergillus niger-treated agrowaste. The treated agrowaste on its own increased root growth by 296% and shoot growth by 504% compared with non-treated control plants. Both chemical and biological treatments, particularly when combined, enhanced plant shoot and root development. The stimulation effect on plant biomass was concomitant with increased AM colonization, P and K assimilation, and reduced metal translocation from soil to plant shoot. The treated residue, particularly through interactions with AM inoculation, produced the expected bioremediation effect, leading to enhanced plant development and successful rehabilitation of contaminated soil. The enhancement of CAT, APX and GR activities caused by AM inoculation suggests that AM colonization helped plants to limit oxidative damage to biomolecules in response to metal stress. The response of the plant's antioxidant activities to the amendment appears to be related to enhanced plant biomass production. The application of amendments and/or microbial inoculations to enhance plant growth and reduce metal translocation in multicontaminated soil could be a promising strategy for remediating HM pollution., This study was supported by “Plan Nacional I+D” Spain (Project AGL 2006-09453-CO2-02).

This study investigated how two different N sources used as fertilizer (NO3− or NH4+) interact with an inoculated arbuscular mycorrhizal (AM) fungus (Glomus etunicatum) in an Andisol from southern Chile. The effect of NO3− or NH4+ on mycorrhizal and non-mycorrhizal wheat plants was measured on key root–soil interface activities: pH, acid phosphatase (P-ase) activity and P availability. Root AM colonization, extraradical mycelium length and spore number were also examined at three stages of AM symbiosis development (120, 150 and 240 days after sowing, DAS). The effect of N-source on AM propagule formation was used as an index of the quality and vigor of AM colonization. Mycorrhizal root length was greater with NO3− than with NH4+ at all times. The NO3− source also improved extraradical mycelium density, which reached its maximum at 150 DAS. At each harvest the spore number in the rhizosphere soil was also greater with NO3− fertilization. This NO3− effect on spore formation ranged from 20% at a 120 DAS to 287% at a 240 DAS increase, compared with NH4+. Extraradical mycelium and AM efficiency for P acquisition appeared to be related. The particular fungus/plant metabolism as affected by N sources (NO3− or NH4+) applied did not result in differential plant growth or in changes in N plant acquisition, but affected AM development and activity. Differences in soil pH, available P or P-ase activity in soil seems not to be responsible for the improved physiological status of mycorrhizal development in NO3− fed plants. Mycorrhizal propagule formation in this soil and the high persistence of extraradical mycelium are important factors which may have a strong influence on the next crop, and thus, this aspects should be considered when a cropping system is designed. The influence of N sources on AM performance is of ecological and practical interest in volcanic soils when conventional management is used., This work was supported by Fondecyt, grant 1990756, from Comision Nacional Cientifica y Tecnologica, Chile. The authors also acknowledge financial support given to R. Azcon for travel and expenses while staying in Chile through Andes Foundation project C-13755-28.

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., A Marie Curie Fellowship of the European Commission for Almudena Medina supported this work.

The growth of clover (Trifolium repens ) and its uptake of N, P and Ni were studied following inoculation of soil with Rhizobium trifolii, and combinations of two Ni-adapted indigenous bacterial isolates (one of them was Brevibacillus brevis) and an arbuscular mycorrhizal (AM) fungus (Glomus mosseae). Plant growth was measured in a pot experiment containing soil spiked with 30 (Ni I), 90 (Ni II) or 270 (Ni III) mg kg−1 Ni-sulphate (corresponding to 11.7, 27.6 and 65.8 mg kg−1 available Ni on a dry soil basis). Single inoculation with the most Ni-tolerant bacterial isolate (Brevibacillus brevis) was particularly effective in increasing shoot and root biomass at the three levels of Ni contamination in comparison with the other indigenous bacterial inoculated or control plants. Single colonisation of G. mosseae enhanced by 3 fold (Ni I), by 2.4 fold (Ni II) and by 2.2 fold (Ni III) T. repens dry weight and P-content of the shoots increased by 9.8 fold (Ni I), by 9.9 fold (Ni II) and by 5.1 fold (Ni III) concomitantly with a reduction in Ni concentration in the shoot compared with non-treated plants. Coinoculation of G. mosseae and the Ni-tolerant bacterial strain (B. brevis) achieved the highest plant dry biomass (shoot and root) and N and P content and the lowest Ni shoot concentration. Dual inoculation with the most Ni-tolerant autochthonous microorganisms (B. brevis and G. mosseae) increased shoot and root plant biomass and subtantially reduced the specific absorption rate (defined as the amount of metal absorbed per unit of root biomass) for nickel in comparison with plants grown in soil inoculated only with G. mosseae. B. brevis increased nodule number that was highly depressed in Ni I added soil or supressed in Ni II and Ni III supplemented soil. These results suggest that selected bacterial inoculation improved the mycorrhizal benefit in nutrients uptake and in decreasing Ni toxicity. Inoculation of adapted beneficial microorganisms (as autochthonous B. brevis and G. mosseae) may be used as a tool to enhance plant performance in soil contaminated with Ni., A. Vivas wants to thank the Fundación Gran Mariscal de Ayacucho (Venezuela) for the scholarship. The project was further supported by the CSIC-HAS bilateral agreement, the Hung. National Research Fund (OTKA T046610) and the COST Action 8.38 and the EU-fp 5 QLK (Mycorem) program, which are highly acknowledged.

9 pages, 2 figures, 4 tables., The effects of mycorrhizal inoculation, with an arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck and Smith) or with a mixture of three AM fungi (G. intraradices, G. deserticola (Trappe, Bloss. and Menge) and G. mosseae (Nicol and Gerd.) Gerd. and Trappe), and addition of composted sewage sludge or Aspergillus niger-treated dry olive cake (DOC) residue on aggregate stabilisation of the rhizosphere soil of Juniperus oxycedrus were studied. The influence of such structural improvements on the establishment of J. oxycedrus was evaluated. Six months after planting, the inoculation with the mixture of three AM fungi and the combination of G. intraradices with both organic amendments had significantly enhanced the structural stability. Water soluble C and carbohydrates values were increased only with the addition of composted sewage sludge or fermented DOC. The addition of both organic amendments, particularly fermented DOC, decreased significantly the dehydrogenase, urease, protease and β-glucosidase activities. Rhizosphere soil from the inoculation treatments had higher dehydrogenase and β-glucosidase activities than control soil. Both the organic amendments addition and the mycorrhizal inoculation treatments increased significantly shoot biomass in J. oxycedrus. AM inoculation treatments were more effective with respect to increasing shoot biomass than the addition of organic amendments alone, and there were no significant differences between the two mycorrhization treatments (on average, about 53% higher with respect to control plants and about 18% higher with respect to plants grown in the amended soil)., This research was supported by the Seneca Foundation (Project PI-69/00815/FS/01) and by CICYT (Project AGL2003-05619-CO2-01).

In this study, we have determined whether the arbuscular mycorrhizal (AM) symbiosis is able to alter the pattern of Δ1-pyrroline-5-carboxylate synthetase (p5cs) gene expression under drought stress and whether such possible alteration functions in the protection of the host plants against drought. To achieve this, we cloned a P5CS-encoding gene from Glycine max (gmp5cs) and another from Lactuca sativa (lsp5cs) and analyzed their contribution to the response against drought in control and AM soybean and lettuce plants. The analysis of gmp5cs and lsp5cs gene expression showed that these genes were up-regulated by drought stress. The highest gene expression was found in non-inoculated plants subjected to drought. A contrasting result was obtained in soybean plants singly inoculated with Bradyrhizobium japonicum, where the gmp5cs gene showed little up-regulation in roots under drought stressed conditions. Moreover, both soybean and lettuce AM plants showed lower p5cs transcript accumulation under drought stress than non-inoculated plants. The present results indicate that the induction of p5cs gene is not a mechanism by which the AM symbiosis protects their host plant against drought., This work and R. Porcel were financed by CICYT-FEDER (Project AGL2002-03952).

12 pages, 1 figure, 7 tables., A field experiment was undertaken to evaluate the effectiveness of mycorrhizal inoculation with Glomus intraradices (Schenck & Smith) or Glomus deserticola (Trappe, Bloss. & Menge) and the addition of sugar beet (SB), rock phosphate and Aspergillus niger with respect to the establishment of Dorycnium pentaphyllum L. seedlings, in a semiarid Mediterranean area, and the improvement of the physical-chemical, biochemical and biological properties of the soil. Eighteen months after planting, amended soil had higher available phosphorus and aggregate stability levels than control soil (165 and 157%, respectively). All assayed treatments had higher soil total carbohydrates and water soluble C values and enzyme activities (dehydrogenase, urease, acid phosphatase and β-glucosidase), and lower soil pH and bulk density than control soil. Biomass C and dehydrogenase, urease and β-glucosidase activities of the rhizosphere of G. deserticola-inoculated plants were higher than those of the rhizosphere of G. intraradices-inoculated plants. Mycorrhizal inoculation treatments stimulated significantly the production of shoot biomass, to a higher extent than the addition of the amendment alone to soil or the combined treatment. The highest levels of mycorrhizal colonisation and of foliar N and K were recorded in the seedlings inoculated with G. deserticola. The shoot biomass of G. deserticola-inoculated D. pentaphyllum plants was greater (about 219% with respect to control plants) than that of G. intraradices (about 116% with respect to control plants)., This research was supported by the EC + CICYT co-financed FEDER programme (REN 2000-1724- CO3-01).

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., Financial support for this study was obtained from Comision Interministerial de Ciencia y Tecnologı́a (CICYT) PN REN 2000-1506 GLO. One of us (A. Medina) is indebted to Junta de Andalucia for scholarship grants.

In the present study, we have investigated whether the ubiquitous rhizosphere soil organism Sinorhizobium meliloti has a plant growth promoting (PGP) effect on non-leguminous plant species. Such PGP activity was investigated for both a wild type strain and its genetically modified (GM) derivative, which had an enhanced biofertilizer capability. The PGP effect of these rhizobial strains was tested in interaction with two arbuscular-mycorrhizal (AM) fungi: G. mosseae or G. intraradices on lettuce (Lactuca sativa L.) plants. Both rhizobial strains were efficient in increasing lettuce biomass and also induced modifications on root morphology, particularly in mycorrhizal plants; thus these strains behave as plant growth promoting rhizobacteria. In non-mycorrhizal plants, both strains exhibited a similar growth promoting effect on lettuce. However, both rhizobial strains differed in mycorrhizal plants with regard to (i) biomass production, (ii) the length of axis and lateral roots, and (iii) the number of lateral roots formed; effects which were, in turn, affected by the AM fungus involved. Microbial treatments were more effective on root growth and morphology at earlier developmental stages (20 days of plant growth) but, in a later stage (after 40 days), the microbial effects were more relevant at increasing plant biomass. The interaction between the GM rhizobial strain and G. mosseae produced the highest growth promoting effect (476% over control), in spite of the fact that G. intraradices showed a quicker and higher colonization ability than G. mosseae. Microbial interactions inducing PGP effects did not benefit AM colonization nor the succinate dehydrogenase activity in the AM fungal mycelium. Irrespective of the underlying mechanisms, which are being now investigated, the interactions between rhizobial strains, as free-living saprophs, and AM fungi are noteworthy, and depend on the microbial genotype involved., Two of the authors (C.G. and C.A.) are grateful to AECI, Spain, for financial support during their stay at the EEZ-CSIC. This research was supported by the European Union, TMR Network ERB FMR XCT 960039.

Free and encapsulated cells of Yarowia lipolytica produced citric acid in repeated batch-shake-flask fermentation on either calcium carbonate or rock phosphate supplemented medium, the latter being simultaneously solubilized by the organic acid.' Agar-encapsulated cells on calcium carbonate supplemented medium produced the highest amount of citric acid productivity of 0.234 g/l per h. Alginate and k-carrageenan appeared to be unsuitable cell-carriers when rock phosphate was supplied to the medium solution instead of calcium carbonate. The soluble phosphorus concentration depended on the mode of biocatalyst application (as freely suspended or encapsulated cells) and the initial concentration of rock phosphate in the cultivation medium. Rock phosphate clearly affected the behaviour of Y. lipolytica and determined the level of acid productivity and corresponding P-solubilization., M. Vassileva and N. Vassilev are grateful to the Spanish CICYT for the contracts. Authors are also grateful to the Joint FAD/IAEA Division, Vienna, United Nations and IMPHOS for the support.

The effect of arbuscular-mycorrhizal (AM) fungus Glomus fasciculatum on growth and N form assimilation was measured on onion (Allium cepa) grown under well-watered (−0.04 MPa) or drought conditions (−0.17 MPa). Two uninoculated control treatments, one provided with phosphate, were also addressed. These three treatments were supplemented with 2.0 mM nitrogen as nitrate and ammonium in a 1/1 ratio. Shoots and root weights, percentage of root colonized and glutamine synthetase (GS) (EC 6.3.1.2.) and nitrate reductase (NR) (EC 1.6.6.1) activity in shoot and root tissue were determined when water was maintained at (−0.04 MPa) or (−0.17 MPa) in the growth medium. The growth of G. fasciculatum-colonized plants was comparable to that of uncolonized P-supplemented plants under well-watered or drought conditions but mycorrhizal plants reached a higher specific and total GS activity in shoots and roots than P-fertilized plants growth at −0.04 MPa. The mycorrhizal effect on GS activity under water stress (−0.17 mPa) was evident only in roots being comparable to that found in P-fertilized plants. The proportion of GS in roots was increased in AM plants under whatever soil water conditions. The most marked increasing effect of AM-colonization on NR activity was in root tissue. Under water limitations the effectiveness of G. fasciculatum increasing NR activity in plant was enhanced. The proportion of nitrate assimilation into root was increased in AM plants particularly under well-watered conditions. Mycorrhizal modifications in the GS and NR distribution into root and shoot compartments may account for some physiological effect from mycorrhizal colonization. These results are further evidence of a direct effect on absorption, translocation and assimilation of both N forms by the endomycorrhizal system. That mycorrhizal plants can utilize nitrate form more efficiently than ammonium under drought conditions is consistent with more recent studies on the AM effect on N uptake from a neutral-alkaline soil. Results here presented suggest that either AM fungi increase the nitrogen forms assimilation in the host plant (regardless of P content) or the AM fungi have such enzymatic activities per se. This last assumption is supported by the relative high increase of NR and GS activities found in the roots of mycorrhizal plants. Nevertheless while NR was maintained increased in mycorrhizal roots under water stress the GS activity was not affected. This suggests the AM ability to provide an active nitrate acquisition in particular in water stressed environment. The different proportion of nitrate and ammonium assimilation into shoot and root compartments may account to modify physiological mycorrhizal responses related to plant sensitivity to drought.

Lavender plants (Lavandula spp.) are small woody shrubs which belong to the natural succession in certain plant communities of semiarid mediterranean ecosystems in the southeast of Spain and they thrive in areas which are threatened by desertification. Lavender plants form arbuscular mycorrhizas and have been described as mycorrhizal-dependent species, but they are also known to display a stimulated root phosphatase activity in conditions of low-nutrient supply. Consequently, a series of microcosm experiments were carried out in order to identify the relative importance of the mycotrophic habit in comparison with phosphatase production as mechanisms which account for plant establishment and nutrient uptake, using four soils typical of the degraded area of study. It was found that lavender plants were obligatorily mycorrhizal species under the prevailing conditions of their natural habitat. The specific activity of root-associated acid phosphatase in lavender was enhanced in P-deficient, non-mycorrhizal, plants, but apparently this stimulated phosphatase activity did not result in an increased ability of the plant to take up P. Low organic phosphate content in the test soils may account for the absence of plant response to acid phosphatase activity. However, it is much more likely that this ineffectiveness is due to the fact that P-deficiency is concomitant, in the degraded test habitats, with deficiencies in other nutrients. Thus plant growth is being limited by an overall lack of nutrient supply. Mycorrhizal inoculation largely improved P, N and K uptake thereby restoring the biochemical cycling of plant nutrients. In conclusion, lavender plants must be mycorrhizal in order to thrive in the degraded soils from desertification threatened areas in typical mediaterranean ecosystems., We thank the CICYT Project AMB 95-0699 for supporting this research.

Fluctuations in the number of anaerobic sulphur mineralizers, autotrophic sulphur oxidizers and sulphate-reducing microorganisms in pot cultures of arbuscular mycorrhizal and non-mycorrhizal plants were examined at 0, 15, 30, 45 and 60 days after planting using the most probable number enumeration method. Two arbuscular mycorrhizal (AM) fungi' belonging to different Glomus species were assayed. Populations of S-cycling microorganisms varied along the growth period for both AM fungi colonized and uncolonized plants, with significant quantitative and qualitative changes in the mycorrhizal compared with the non-mycorrhizal root zone. The occurrence of autotrophic sulphur oxidizers in pot cultures colonized with G. fasciculatum was significantly higher than in non-mycorrhizal ones throughout the plant growth period. The population size of this group was similar in non-mycorrhizal and G. mosseae colonized soil. The population of anaerobic sulphur mineralizing microorganisms was significantly decreased by mycorrhizal colonization. In contrast, pot cultures colonized by G. mosseae or G. fasciculatum had a higher quantity of sulphate-reducing bacteria than the control. The fact that the autotrophic S oxidizers and sulphate-reducing root-associated microorganisms were selectively stimulated by each one of the AM fungi assayed is a noticeable aspect. The maximum activity of the microbial processes regarding specific groups was at 15 days for the mineralizing, 45 days for the oxidizing and 0 days for the reducing microorganisms. In the case of autotrophic S oxidizers, the maximum population was achieved by 30 days in G. fasciculatum colonized treatment. From the data on occurrence of physiological grouping of sulphur-cycling microorganisms, we can deduce that a different ecological situation existed in the rhizosphere developed by non-mycorrhizal plants compared with those colonized by each one of the mycorrhizal endophytes., One of us (EAL) is on leave from the Department of Microbiology, Escuela Nacional de Ciencias Biologicas, IPN (Mexico), and she is a scholarship holder of the Banco de Mexico.

Olive mill waste water (OMW), supplemented or not with ammonium sulphate and rock phosphate (RP), was applied as a medium in a shake-flask repeated-batch fermentation with Aspergillus niger immobilized on polyurethane sponge. Compared to other treatments, the results showed higher growth of the immobilized mycelium and significant reduction of the total phenols when the waste material was enriched with RP and ammonium sulphate (N). The immobilized fungus solubilized the RP with a maximum level of soluble P of 0·58 g/litre reached during the fourth batch cycle of the OMW+RP treatment. Depending on the medium composition, three types of treated OMW were produced which could be further used for various purposes.

Aspergillus niger, an acid-producing filamentous fungus, was immobilized on polyurethane foam. Various amounts of foam cubes and spore suspension were tested in order to obtain an efficient immobilization process. The best combination selected for further experiments was 0.2 g polyurethane foam and 3 ml spore suspension. Immobilized cells were reused, with higher levels of acid formation being maintained for longer periods (at least 240 h) than for free cells. The highest titratable acidity, of about 315 mmole/l, was reached with 0.5 cm foam cubes after 3 × 48 h batch cultures in a laboratory shake-flask experiment. Rock phosphate (2.5 g/l and 5.0 g/l) solubilization was carried out in repeated 48 h batch fermentation using A. niger immobilized on polyurethane foam cubes. The greatest accumulation of soluble P, approximately 360 μg/ml, was obtained after the second batch in flasks with 5 g/l rock phosphate, but process efficiency was higher at lower concentration of the rock phosphate. After 10 days of culture a total production of 1.2–1.6 mg/ml soluble P was obtained depending on the concentration of rock phosphate in the medium.

This study set out to determine the effect of drought stress on nitrate reductase (NR, EC 1.6.6.1.) activity in mycorrhizal plants, and to see if the maintenance of this enzymatic activity under stress conditions is a factor involved in the drought tolerance of mycorrhizal plants. Lactuca sativa L. plants were inoculated with three arbuscular mycorrhizal (AM) fungi, Glomus deserticola (Trappe. Bloss. and Menge), G. fasciculatum (Thax. and Gerd.) Gerd. and Trappe or G. mosseae (Nicol. and Gerd.) Gerd and Trappe or remained uninoculated (plus or less P fertilization). The plants were grwn under controlled conditions at constant soil water potential (close to −0.04 MPa) or at −0.17 MPa during the last six weeks of plant growth. Results obtained showed that mycorrhizal plants had higher NR activity (NRA) than the uninoculated treatments, particularly under water stress conditions. Control plants had 57% less NRA than G. deserticola-colonized ones under well watered conditions, with a reduction in NRA of 79% when the plants were subjected to drought stress. Under well-watered conditions the P-fertilized plants showed similar or higher growth and P content than the G. mosseae and G. fasciculatum mycorrhizal ones, the NRA being lower in P-fertilized than in AM plants. These results suggest that either the AM fungi increase the NRA in the host plant (regardless of the P content) or the AM fungi have such enzymatic activity per se. Besides, under the experimental conditions, plants colonized by different AM fungi showed different NR activities. It was concluded that drought stress decreased NRA, but much less in mycorrhizal than in uninoculated plants. This effect may be a factor in the drought tolerance of mycorrhizal plants., This study was supported by CICYT-Spain (Project AGR 91-0605-C02-O1).

This study reports the effects of three selected arbuscular mycorrhizal- (AM-) forming species (Glomus mosseae, Glomus fasciculatum or Glomus caledonium) in comparison with autoctone endophytes by determining changes in plant growth, nutritional and symbiotic parameters in four species of Medicago: M. trunculata, M. rigidula, M. polimorpha and M. rotata. The relative susceptibility to and dependence upon AM fungi of Medicago species was also evaluated. Results showed a high functional compatibility between Medicago species and autoctone endophytes, and a specific plant response to individual Glomus species. Differences among the Medicago species in their reaction to Glomus species ranged from negative to highly positive. A combination of host and AM fungal species indicates that a specific compatibility exists among symbionts. Nutrient uptake as a result of AM colonization also indicated a wide degree of responsiveness in each species according to the associated fungal symbiont to the plant. A positive effect on N, P and K, and negative on Ca and Mg nutrition was found in all mycorrhizal treatments, especially with the autoctone endophytes. Mycorrhizal infectivity could not be related to endophyte effectiveness, with generally fewer and larger Rhizobium nodules found on the most effective mycorrhizal treatments. Results support the role of indigenous AM endophytes in the efficacy of mycorrhizal symbiosis and the importance of host-endophyte selection to maximize growth and nutrition of Medicago. This study shows that AM symbiotic efficiency attributed to Medicago is dependent on endophyte association and plant species. The importance of selecting suitable AM fungi is of practical interest for improving the effectiveness of the tripartite symbiosis., The work was supported by CICYT, Project AGR 91-0605-C02-01.

The purpose of this experiment was to investigate the effect of components from a plant growth-promoting rhizobacterium (PGPR) or phytohormones on the growth and nutrient assimilation of a legume with N 2 -fixing Rhizobium . Half of these plants were inoculated with the vesiscular-arbuscular mycorrhizal (VAM) fungi Glomus mosseae . Hedysarum coronarium VAM and non-VAM plants at either 1 day, 15 days or both (1 plus 15) times were treated with components from the PGPR culture: (1) washed cells; (2) culture filtrate; (3) complete bacterial culture; or (4) a mixture of phytohormones. Results show that components from the PGPR culture behaved similarly in increasing growth in non-mycorrhizal plants irrespective of time of treatment. In mycorrhizal plants, the single addition of washed cells or bacterial culture at 15 days was significantly less effective than repeated applications. However, phytohormones were better when supplied on young mycorrhizal plants at 15 days after sowing. However, maximum growth and nutrient uptake was reached in mycorrhizal plants supplied with PGPR culture filtrate in repeated treatments. A specific interaction between the tripartite plant- Rhizobium VAM symbiosis and the PGPR components can be inferred depending on time of application of bacterial fraction.

We have conducted some experiments aimed to help the introduction in new habitats of Hedysarum coronarium L., a legume of high potential for animal nutrition. A survey was first undertaken to examine whether H. coronarium is nodulated and mycorrhizal in its natural growing area. Pot experiments were then set up to assess the feasibility of field inoculation in soils (new habitats) previously studied for some of their physical, chemical and biological (microbial) characteristics. Finally, a field experiment was carried out in a selected soil. Rhizobium and the VA mycorrhizal fungus were both successfully established in the rhizosphere of H. coronarium growing in the field. They stimulated the yield of plant material and the nutrient uptake by the plants beyond that achieved by adding a standard dose of a compound N-P-K fertilizer., This study was supported by a grant of Caja General de Ahorros de Granada (Spain).

Germination and growth of chlamydospores of the vesicular-arbuscular (VA) mycorrhizal fungus Glomus mosseae on water-agar medium under axenic conditions were compared after treatment with washed cells, cell-free supernatants and complete bacterial cultures. Spore germination was not affected by bacterial treatments, but the additions of complete bacterial cultures and cell-free supernatants significantly (P ⩽ 0.05) stimulated hyphal growth and the number of new vegetative vesicles formed per germinated resting spore.