Impact of microbial inoculation on biomass accumulation by Sulla carnosa provenances, and in regulating nutrition, physiological and antioxidant activities of this species under non-saline and saline conditions.

Bacteria (Pseudomonas sp. and Bacillus sp.) and/or the arbuscular mycorrhizal (AM) fungus Rhizophagus intraradices were able to improve growth, physiological and biochemical characteristics of four Sulla carnosa Desf. provenances (Sidi khlif, Thelja, Kalbia and Kerker) from Tunisia under both saline and non-saline conditions. S. carnosa is a salt-tolerant legume plant, native from North Africa. The intrinsic bacterial characteristics evidenced the fitness of these bacteria to support salt stress and to stimulate plant growth. Bacillus sp. produced more indol acetic acid (IAA) than Pseudomonas sp. and showed a great surviving capacity under salt conditions supporting its capacity to improve plant growth under stress conditions. The microorganisms applied also have a different potential to increase the nutritional and related plant growth parameters. It is noticeable that some provenances reached the highest level of growth when inoculated with Bacillus sp. in Sidi khlif or by Bacillus plus AMF in Kalbia, which increased shoot by 318% and root by 774%. In contrast, in Thelja and Kerker the impact of the test microorganisms was mainly evidenced at increasing nutritional and physiological functions. Salinity reduced some growth and physiological variables as stomatal conductance, photosynthetic pigments and photosynthetic efficiency and increased electrolyte leakage. However, the microbial inoculants compensated these detrimental effects in a degree depending on the S. carnosa provenance. These microorganisms also orchestrate antioxidant activities involved in adaptative responses in S. carnosa provenances. The intrinsic ability of inoculants allow us to select the provenance/microorganism combination which maximizes S. carnosa growth, nutrition and physiological/biochemical responses under salt and non-salt conditions. The results obtained support that the target microbial inocula are beneficial for the ecological stability if this Mediterranean legume.

Divergent composition but similar function of soil food webs of individual plants: plant species and community effects.

Soils are extremely rich in biodiversity, and soil organisms play pivotal roles in supporting terrestrial life, but the role that individual plants and plant communities play in influencing the diversity and functioning of soil food webs remains highly debated. Plants, as primary producers and providers of resources to the soil food web, are of vital importance for the composition, structure, and functioning of soil communities. However, whether natural soil food webs that are completely open to immigration and emigration differ underneath individual plants remains unknown. In a biodiversity restoration experiment we first compared the soil nematode communities of 228 individual plants belonging to eight herbaceous species. We included grass, leguminous, and non-leguminous species. Each individual plant grew intermingled with other species, but all plant species had a different nematode community. Moreover, nematode communities were more similar when plant individuals were growing in the same as compared to different plant communities, and these effects were most apparent for the groups of bacterivorous, carnivorous, and omnivorous nematodes. Subsequently, we analyzed the composition, structure, and functioning of the complete soil food webs of 58 individual plants, belonging to two of the plant species, Lotus corniculatus (Fabaceae) and Plantago lanceolata (Plantaginaceae). We isolated and identified more than 150 taxa/groups of soil organisms. The soil community composition and structure of the entire food webs were influenced both by the species identity of the plant individual and the surrounding plant community. Unexpectedly, plant identity had the strongest effects on decomposing soil organisms, widely believed to be generalist feeders. In contrast, quantitative food web modeling showed that the composition of the plant community influenced nitrogen mineralization under individual plants, but that plant species identity did not affect nitrogen or carbon mineralization or food web stability. Hence, the composition and structure of entire soil food webs vary at the scale of individual plants and are strongly influenced by the species identity of the plant. However, the ecosystem functions these food webs provide are determined by the identity of the entire plant community.

Drought tolerance and antioxidant activities in lavender plants colonized by native drought-tolerant or drought-sensitive Glomus Species.

This study compared the effectiveness of four arbuscular mycorrhizal (AM) fungal isolates (two autochthonous presumably drought-tolerant Glomus sp and two allochthonous presumably drought-sensitive strains) on a drought-adapted plant (Lavandula spica) growing under drought conditions. The autochthonous AM fungal strains produced a higher lavender biomass, specially root biomass, and a more efficient N and K absorption than with the inoculation of similar allochthonous strains under drought conditions. The autochthonous strains of Glomus intraradices and Glomus mosseae increased root growth by 35% and 100%, respectively, when compared to similar allochthonous strains. These effects were concomitant with an increase in water content and a decline in antioxidant compounds: 25% glutathione, 7% ascorbate and 15% H(2)O(2) by G. intraradices, and 108% glutathione, 26% ascorbate and 43% H(2)O(2) by G. mosseae. Glutathione and ascorbate have an important role in plant protection and metabolic function under water deficit; the low cell accumulation of these compounds in plants colonized by autochthonous AM fungal strains is an indication of high drought tolerance. Non-significant differences between antioxidant activities such as glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) in colonized plants were found. Thus, these results do not allow the generalization that GR, CAT and SOD were correlated with the symbiotic efficiency of these AM fungi on lavender drought tolerance. Plants colonized by allochthonous G. mosseae (the less efficient strain under drought conditions) had less N and K content than those colonized by similar autochthonous strain. These ions play a key role in osmoregulation. The AM symbiosis by autochthonous adapted strains also produced the highest intraradical and arbuscular development and extraradical mycelial having the greatest fungal SDH and ALP-ase activities in the root systems. Inoculation of autochthonous drought tolerant fungal strains is an important strategy that assured the greatest tolerance water stress contributing to the best lavender growth under drought.

An indigenous drought-tolerant strain of Glomus intraradices associated with a native bacterium improves water transport and root development in Retama sphaerocarpa.

The effects of interactions between Bacillus thuringiensis, a drought-adapted bacterium, and two isolates of Glomus intraradices, an arbuscular mycorrhizal (AM) fungus, on Retama sphaerocarpa, a drought-adapted legume, were investigated. The fungal isolates were an indigenous drought-tolerant and a nonindigenous drought-sensitive isolate. Shoot length and root growth, symbiotic parameters, water transport (in terms of percent relative plant water uptake), and volumetric soil moisture and soil enzymatic activities in response to microbial inoculations were evaluated. Retama plants colonized by G. intraradices plus Bacillus possessed similar shoot length after 30 days from sowing compared with noninoculated Retama plants after 150 days. Inoculation with drought-adapted bacterium increased root growth by 201%, but maximum root development was obtained by co-inoculation of B. thuringiensis and the indigenous G. intraradices. Nodules were formed only in plants colonized by autochthonous AM fungi. Relative water uptake was higher in inoculated than in noninoculated Retama plants, and these inoculants depleted soil water content concomitantly. G. intraradices-colonized Retama reached similar shoot length irrespective of the fungal origin, but there were strong differences in relative water uptake by plants colonized by each one of the fungi. Indigenous G. intraradices-colonized roots (evaluated as functional alkaline phosphatase staining) showed the highest intensity and arbuscule richness when associated with B. thuringiensis. The interactive microbial effects on Retama plants were more relevant when indigenous microorganisms were involved. Co-inoculation of autochthonous microorganisms reduced by 42% the water required to produce 1 mg of shoot biomass. This is the first evidence of the effectiveness of rhizosphere bacterium, singly or associated with AM fungus, in increasing plant water uptake, which represents a positive microbial effect on plants grown under drought environments.

Effectiveness of autochthonous bacterium and mycorrhizal fungus on Trifolium growth, symbiotic development and soil enzymatic activities in Zn contaminated soil.

AIMS: This study investigates how autochthonous micro-organisms [bacterium and/or arbuscular mycorrhizal (AM) fungi] affected plant tolerance to Zn contamination. METHODS AND RESULTS: Zinc-adapted and -nonadapted Glomus mosseae strains protected the host plant against the detrimental effect of Zn (600 microg g(-1)). Zn-adapted bacteria increased root growth and N, P nutrition in plants colonized by adapted G. mosseae and decreased the specific absorption rate (SAR) of Cd, Cu, Mo or Fe in plants colonized by Zn-nonadapted G. mosseae. Symbiotic structures (nodule number and extraradical mycelium) were best developed in plants colonized by those Zn-adapted isolates that were the most effective in increasing plant Zn tolerance. The bacterium also increased the quantity and quality (metabolic characteristics) of mycorrhizal colonization, with the highest improvement for arbuscular vitality and activity. Inocula also enhanced soil enzymatic activities (dehydrogenase, beta-glucosidase and phosphatase) and indol acetic acid (IAA) accumulation, particularly in the rhizosphere of plants inoculated with Zn-adapted isolates. CONCLUSIONS: Glomus mosseae strains have a different inherent potential for improving plant growth and nutrition in Zn-contaminated soil. The bacterium increased the potential of mycorrhizal mycelium as inoculum. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycorrhizal performance, particularly that of the autochthonous strain, was increased by the bacterium and both contributed to better plant growth and establishment in Zn-contaminated soils.

Two bacterial strains isolated from a Zn-polluted soil enhance plant growth and mycorrhizal efficiency under Zn-toxicity.

In this study we investigated the interactions among plant, rhizosphere microorganisms and Zn pollution. We tested the influence of two bacterial strains isolated from a Zn-polluted soil on plant growth and on the symbiotic efficiency of native arbuscular mycorrhizal fungi (AMF) under Zn toxicity. The two bacterial strains exhibited Zn tolerance when cultivated under increasing Zn levels in the medium. However, strain B-I showed a higher Zn tolerance than strain B-II at the two highest Zn levels in the medium (75 and 100 mg l(-1) Zn). Molecular identification placed the strain B-I within the genus Brevibacillus. Our results showed that bacterial strain B-I consistently enhanced plant growth, N and P accumulation, as well as nodule number and mycorrhizal infection which demonstrated its plant-growth promoting (PGP) activity. This strain B-I has been shown to produce IAA (3.95 microg ml) and to accumulate 5.6% of Zn from the growing medium. The enhanced growth and nutrition of plants dually inoculated with the AMF and bacterium B-I was observed at three Zn levels assayed. This effect can be related to the stimulation of symbiotic structures (nodules and AMF colonization) and a decreased Zn concentration in plant tissues. The amount of Zn acquired per root weight unit was reduced by each one of these bacterial strains or AMF and particularly by the mixed bacterium-AMF inocula. These mechanisms explain the alleviation of Zn toxicity by selected microorganisms and indicate that metal-adapted bacteria and AMF play a key role enhancing plant growth under soil Zn contamination.

Brevibacillus brevis isolated from cadmium- or zinc-contaminated soils improves in vitro spore germination and growth of Glomus mosseae under high Cd or Zn concentrations.

In this study we investigated the saprophyte growth of two arbuscular-mycorrhizal fungi (Glomus mosseae isolate) under increasing Cd or Zn levels and the influence of a selected bacterial strain of Brevibacillus brevis. Microorganisms here assayed were isolated from Cd or Zn polluted soils. B. brevis increased the presymbiotic growth (germination rate growth and mycelial development) of Glomus mosseae. Spore germination and mycelial development of both G. mosseae isolate were reduced as much as the amount of Cd or Zn increased in the growth medium. In medium supplemented with 20 microg Cd mL(-1), the spore germination was only 12% after 20 days of incubation, but the coinoculation with B. brevis increased this value to 40% after only 15 days. The addition of 20 microg Cd mL(-1) to the growth medium drastically inhibited hyphal development, but the presence of the bacterium increased hyphal growth of G. mosseae from 195% (without Cd) until 254% (with 20 microg Cd mL(-1)). The corresponding bacterial effect increasing micelial growth ranged from 125% (without Zn) to 232% (200 microg Zn mL(-1)) in the case of G. mosseae isolated from Zn-polluted soil. Mycelial growth under 5 microg Cd mL(-1) (without bacterium) was similarly reduced from that produced at 15 microg Cd mL(-1) in the presence of the bacteria. As well, 50 microg Zn mL(-1) (without bacterium) reduced hyphal growth as much as 200 microg Zn mL(-1) did in the presence of B. brevis. The bacterial effect on the saprophytic growth of G. mosseae in absence of metal may be due to the involvement of indole acetic acid (IAA) produced by these bacteria. The Cd bioaccumulation ability exhibited (76%) by Cd-adapted B. brevis reduced the Cd damage on G. mosseae in Cd-contaminated medium. These capabilities of B. brevis isolates partially alleviate the inhibitory effects of Cd or Zn on the axenic growth of G. mosseae.

Application of Aspergillus niger-treated agrowaste residue and Glomus mosseae for improving growth and nutrition of Trifolium repens in a Cd-contaminated soil.

The microbial transformation of sugar beet (SB) agrowaste with or without rock-phosphate (RP) has utility for the improvement of plant growth in a Cd (5 microg g-1) artificially contaminated soil, particularly when the soil is co-inoculated with arbuscular mycorrhizal (AM) fungus Glomus mosseae isolated from a Cd-polluted area. Under such Cd-polluted conditions, the limited growth, mineral nutrition, symbiotic developments (nodulation and AM-colonization) and soil enzymatic activities were stimulated using SB or SB+RP as soil amendments and G. mosseae as inoculant. G. mosseae enhanced plant establishment in a higher extent in amended soil; it is probably due to the interactive effect increasing the potential fertility of such compounds and its ability for decreasing Cd transfer from soil to plant. The amount of Cd transferred from soil solution to biomass of AM-colonized plants ranged from 0.09 microg Cd g-1 (in SB+RP-amended soil) to 0.6 microg Cd g-1 (in non-amended soil). Nodule formation was more sensitive to Cd than AM-colonization, and both symbioses were stimulated in amended soils. Not only AM-colonization but also amendments were critical for plant growth and nutrition in Cd-polluted soil. The high effectiveness of AM inoculum increasing nutrients and decreasing Cd in amended soil indicated the positive interaction of these treatments in increasing plant tolerance to Cd contamination.

Interactive effect of Brevibacillus brevis and Glomus mosseae, both isolated from Cd contaminated soil, on plant growth, physiological mycorrhizal fungal characteristics and soil enzymatic activities in Cd polluted soil.

The interaction between two autochthonous microorganisms (Brevibacillus brevis and Glomus mosseae) isolated from Cd amended soil increased plant growth, arbuscular mycorrhizal (AM) colonization and physiological characteristics of the AM infection (measured as SDH or ALP activities). The enhanced plant Cd tolerance after coinoculation with native microorganisms seemed to be a consequence of increased P and K acquisition and, simultaneously, of decreased concentration of Cd, Cr, Mn, Cu, Mo, Fe and Ni in plant tissue. Autochthonous microbial strains were more efficient for nutrient uptake, to immobilize metals and decrease their translocation to the shoot than reference G. mosseae (with or without bacteria). Indole acetic acid produced by B. brevis may be related to its ability for improving root growth, nodule production and AM fungal intra and extraradical development. Dehydrogenase, phosphatase and beta-glucosidase activities, indicative of microbial metabolism and soil fertility, were maximized by the coinoculation of autochthonous microorganisms in cadmium polluted conditions. As a consequence, the use of native microorganisms may result very efficient in bioremediation.

Influence of bacterial strains isolated from lead-polluted soil and their interactions with arbuscular mycorrhizae on the growth of Trifolium pratense L. under lead toxicity.

We isolated two bacterial strains from an experimentally lead (Pb)-polluted soil in Hungary, 10 years after soil contamination. These strains represented the two most abundant cultivable bacterial groups in such soil, and we tested their influence on Trifolium pratense L. growth and on the functioning of native mycorrhizal fungi under Pb toxicity in a second Pb-spiked soil. Our results showed that bacterial strain A enhanced plant growth, nitrogen and phosphorus accumulations, nodule formation, and mycorrhizal infection, demonstrating its plant-growth-promoting activity. In addition, strain A decreased the amount of Pb absorbed by plants, when expressed on a root weight basis, because of increased root biomass due to the production of indoleacetic acid. The positive effect of strain A was not only evident after a single inoculation but also in dual inoculation with arbuscular mycorrhizal fungi. Strain A also exhibited higher tolerance than strain B when cultivated under increasing Pb levels in the spiked soil. Molecular identification unambiguously placed strain A within the genus Brevibacillus. We showed that it is important to select the most tolerant and efficient bacterial strain for co-inoculation with arbuscular mycorrhizal fungi to promote effective symbiosis and thus stimulate plant growth under adverse environmental conditions, such as heavy-metal contamination.

Symbiotic efficiency of autochthonous arbuscular mycorrhizal fungus (G. mosseae) and Brevibacillus sp. isolated from cadmium polluted soil under increasing cadmium levels.

The effect of inoculation with indigenous naturally occurring microorganisms (an arbuscular mycorrhizal (AM) fungus and rhizosphere bacteria) isolated from a Cd polluted soil was assayed on Trifolium repens growing in soil contaminated with a range of Cd. One of the bacterial isolate showed a marked PGPR effect and was identified as a Brevibacillus sp. Mycorrhizal colonization also enhanced Trifolium growth and N, P, Zn and Ni content and the dually inoculated (AM fungus plus Brevibacillus sp.) plants achieved further growth and nutrition and less Cd concentration, particularly at the highest Cd level. Increasing Cd level in the soil decreased Zn and Pb shoot accumulation. Coinoculation of Brevibacillus sp. and AM fungus increased shoot biomass over single mycorrhizal plants by 18% (at 13.6 mg Cd kg(-1)), 26% (at 33.0 mg Cd kg(-1)) and 35% (at 85.1 mg Cd (kg(1)). In contrast, Cd transfer from soil to plants was substantially reduced and at the highest Cd level Brevibacillus sp. lowered this value by 37.5% in AM plants. Increasing Cd level highly reduced plant mycorrhization and nodulation. Strong positive effect of the bacterium on inocula, are important in plant Cd tolerance and development in Cd polluted soils.

Cloning of cDNAs encoding SODs from lettuce plants which show differential regulation by arbuscular mycorrhizal symbiosis and by drought stress.

In the present study three cDNA fragments were cloned using degenerate primers for Mn-sod genes and PCR: two showed a high degree of identity with Mn-sods from plants and the third with Fe-sod. Arbuscular mycorrhizal (AM) symbiosis down-regulated their expression pattern under well-watered conditions. In contrast, AM symbiosis in combination with drought stress considerably increased the expression of the Mn-sod II gene and this correlated well with plant tolerance to drought. These results would suggest that mycorrhizal protection against oxidative stress caused by drought may be an important mechanism by which AM fungi protect the host plant against drought.

Compatibility of a wild type and its genetically modified Sinorhizobium strain with two mycorrhizal fungi on Medicago species as affected by drought stress.

The effect of double inoculation with two strains of Sinorhizobium meliloti [the wild type (WT) strain GR4 and its genetically modified (GM) derivative GR4(pCK3)], and two species of arbuscular mycorrhizal (AM) fungi (Glomus deserticola and Glomus intraradices) was examined in a microcosm system on three species of Medicago (M. nolana, M. rigidula, M. rotata). Two water regimes (80 and 100% water holding capacity, WHC) were assayed. The efficiency of each AM fungus increasing plant growth, nutrient content, nodulation and water-stress tolerance was related to the Sinorhizobium strains and Medicago species. This indicates selective and specific compatibilities between microsymbionts and the common host plant. Differential effects of the mycorrhizal isolates were not associated with their colonizing ability. Nodulation and mycorrhizal dependency (MD) changed in each plant genotype in accordance with the Sinorhizobium strain and AM fungi involved. Generally, Medicago sp. MD decreased under water-stress conditions even when these conditions did not affect AM colonization (%). Proline accumulation in non-mycorrhizal plant leaves was increased by water stress, except in M. rotata plants. Differences in proline accumulation in AM-colonized plants suggest that both the AM fungus and the Sinorhizobium strain were able to induce different degrees of osmotic adjustment. Mycorrhizal plants nodulated by the WT strain accumulated more proline in M. rigidula and M. rotata under water stress than non-mycorrhizal plants. Conversely, mycorrhizal plants nodulated by the GM strain accumulated less proline in response to both AM colonization and drought. These results indicated changes in the synthesis of this nitrogenous osmoregulator product associated with microbial inoculation and drought tolerance. Mycorrhizal plants nodulated by the GM Sinorhizobium strain seem to suffer less from the detrimental effect of water stress, since under water limitation relative plant growth, percentage of AM colonization, root dry weight and the highest R/S ratio remained the same. The fact that GM nodulated plants are better adapted to drought stress could be of practical interest and the management of GM microorganism inoculation may be crucial for biotechnological approaches to improving crop yield in dry environments.

Management of indigenous plant-microbe symbioses aids restoration of desertified ecosystems.

Disturbance of natural plant communities is the first visible indication of a desertification process, but damage to physical, chemical, and biological soil properties is known to occur simultaneously. Such soil degradation limits reestablishment of the natural plant cover. In particular, desertification causes disturbance of plant-microbe symbioses which are a critical ecological factor in helping further plant growth in degraded ecosystems. Here we demonstrate, in two long-term experiments in a desertified Mediterranean ecosystem, that inoculation with indigenous arbuscular mycorrhizal fungi and with rhizobial nitrogen-fixing bacteria not only enhanced the establishment of key plant species but also increased soil fertility and quality. The dual symbiosis increased the soil nitrogen (N) content, organic matter, and hydrostable soil aggregates and enhanced N transfer from N-fixing to nonfixing species associated within the natural succession. We conclude that the introduction of target indigenous species of plants associated with a managed community of microbial symbionts is a successful biotechnological tool to aid the recovery of desertified ecosystems.

The plasma membrane H(+)-ATPase gene family in the arbuscular mycorrhizal fungus Glomus mosseae.

To identify genes that encode plasma membrane H(+)-ATPases in the arbuscular mycorrhizal fungus Glomus mosseae two sets of degenerate primers matching highly conserved motifs present in all plant and fungal ATPases were designed. Nested PCR-amplification of G. mosseae genomic DNA using the designed degenerate primers was carried out. Sequence analysis of the cloned PCR products identified five different clones (GmHA1, GmHA2, GmHA3, GmHA4 and GmHA5) encoding putative plasma membrane H(+)-ATPases. Comparison of the deduced amino-acid sequences of GmHA1-GmHA5 indicate that GmHA1, GmHA3 and GmHA4 are highly identical, while GmHA2 and GmHA5 are more divergent. The evolutionary and functional significance of the divergence found among the different members of the H(+)-ATPase gene family in G. mosseae is discussed.

Diversity of arbuscular mycorrhizal fungus populations in heavy-metal-contaminated soils.

High concentrations of heavy metals have been shown to adversely affect the size, diversity, and activity of microbial populations in soil. The aim of this work was to determine how the diversity of arbuscular mycorrhizal (AM) fungi is affected by the addition of sewage-amended sludge containing heavy metals in a long-term experiment. Due to the reduced number of indigenous AM fungal (AMF) propagules in the experimental soils, several host plants with different life cycles were used to multiply indigenous fungi. Six AMF ecotypes were found in the experimental soils, showing consistent differences with regard to their tolerance to the presence of heavy metals. AMF ecotypes ranged from very sensitive to the presence of metals to relatively tolerant to high rates of heavy metals in soil. Total AMF spore numbers decreased with increasing amounts of heavy metals in the soil. However, species richness and diversity as measured by the Shannon-Wiener index increased in soils receiving intermediate rates of sludge contamination but decreased in soils receiving the highest rate of heavy-metal-contaminated sludge. Relative densities of most AMF species were also significantly influenced by soil treatments. Host plant species exerted a selective influence on AMF population size and diversity. We conclude based on the results of this study that size and diversity of AMF populations were modified in metal-polluted soils, even in those with metal concentrations that were below the upper limits accepted by the European Union for agricultural soils.

Application of an encapsulated filamentous fungus in solubilization of inorganic phosphate.

Spores of Aspergillus niger were encapsulated in agar, calcium alginate and k-carrageenan and further applied in citric acid production during six repeated batch cultivations. Rock phosphate (RP) at concentrations of 3 g l-1 and 7 g l-1 was supplemented to the culture medium to test encapsulated-fungus solubilizing capability. The highest average citric acid productivity of 0.15 g l-1 h-1 was reached with alginate-bead-encapsulated A. niger on RP-free culture medium while agar seemed to be the most suitable carrier on RP-supplemented medium. Accordingly, the highest average soluble P concentration of 0.20 g l-1 batch-1 was obtained with agar-cell beads as compared with other encapsulated systems.