Differences in the composition of arbuscular mycorrhizal fungal communities promoted by different propagule forms from a Mediterranean shrubland.

As it is well known, arbuscular mycorrhizal (AM) colonization can be initiated from the following three types of fungal propagules: spores, extraradical mycelium (ERM), and mycorrhizal root fragments harboring intraradical fungal structures. It has been shown that biomass allocation of AM fungi (AMF) among these three propagule types varies between fungal taxa, as also differs the ability of the different AMF propagule fractions to initiate new colonizations. In this study, the composition of the AMF community in the roots of rosemary (Rosmarinus officinalis L., a characteristic Mediterranean shrub), inoculated with the three different propagule types, was analyzed. Accordingly, cuttings from this species were inoculated with either AMF spores, ERM, or colonized roots extracted from a natural soil. The AMF diversity within the rosemary roots was characterized using terminal restriction fragment length polymorphism (T-RFLP) of the small subunit (SSU) rDNA region. The AMF community established in the rosemary plants was significantly different according to the type of propagule used as inoculum. AMF taxa differed in their ability to initiate new colonizations from each propagule type. Results suggest different colonization strategies for the different AMF families involved, Glomeraceae and Claroideoglomeraceae colonizing mainly from colonized roots whereas Pacisporaceae and Diversisporaceae from spores and ERM. This supports that AMF taxa show contrasting life-history strategies in terms of their ability to initiate new colonizations from the different propagule types. Further research to fully understand the colonization and dispersal abilities of AMF is essential for their rational use in ecosystem restoration programs.

Crop residue stabilization and application to agricultural and degraded soils: A review.

Agricultural activities produce vast amounts of organic residues including straw, unmarketable or culled fruit and vegetables, post-harvest or post-processing wastes, clippings and residuals from forestry or pruning operations, and animal manure. Improper disposal of these materials may produce undesirable environmental (e.g. odors or insect refuges) and health impacts. On the other hand, agricultural residues are of interest to various industries and sectors of the economy due to their energy content (i.e., for combustion), their potential use as feedstock to produce biofuels and/or fine chemicals, or as a soil amendments for polluted or degraded soils when composted. Our objective is review new biotechnologies that could be used to manage these residues for land application and remediation of contaminated and eroded soils. Bibliographic information is complemented through a comprehensive review of the physico-chemical fundamental mechanisms involved in the transformation and stabilization of organic matter by biotic and abiotic soil components.

The composition of arbuscular mycorrhizal fungal communities differs among the roots, spores and extraradical mycelia associated with five Mediterranean plant species.

Arbuscular mycorrhizal fungi (AMF) are essential constituents of most terrestrial ecosystems. AMF species differ in terms of propagation strategies and the major propagules they form. This study compared the AMF community composition of different propagule fractions - colonized roots, spores and extraradical mycelium (ERM) - associated with five Mediterranean plant species in Sierra de Baza Natural Park (Granada, Spain). AMF were identified using 454 pyrosequencing of the SSU rRNA gene. A total of 96 AMF phylogroups [virtual taxa (VT)] were detected in the study site, including 31 novel VT. After per-sample sequencing depth standardization, 71 VT were recorded from plant roots, and 47 from each of the spore and ERM fractions. AMF communities differed significantly among the propagule fractions, and the root-colonizing fraction differed among host plant species. Indicator VT were detected for the root (13 Glomus VT), spore (Paraglomus VT281, VT336, Pacispora VT284) and ERM (Diversispora VT62) fractions. This study provides detailed evidence from a natural system that AMF taxa are differentially allocated among soil mycelium, soil spores and colonized root propagules. This has important implications for interpreting AMF diversity surveys and designing applications of AMF in vegetation restoration.

Arbuscular mycorrhizal fungi native from a Mediterranean saline area enhance maize tolerance to salinity through improved ion homeostasis.

Soil salinity restricts plant growth and productivity. Na(+) represents the major ion causing toxicity because it competes with K(+) for binding sites at the plasma membrane. Inoculation with arbuscular mycorrhizal fungi (AMF) can alleviate salt stress in the host plant through several mechanisms. These may include ion selection during the fungal uptake of nutrients from the soil or during transfer to the host plant. AM benefits could be enhanced when native AMF isolates are used. Thus, we investigated whether native AMF isolated from an area with problems of salinity and desertification can help maize plants to overcome the negative effects of salinity stress better than non-AM plants or plants inoculated with non-native AMF. Results showed that plants inoculated with two out the three native AMF had the highest shoot dry biomass at all salinity levels. Plants inoculated with the three native AMF showed significant increase of K(+) and reduced Na(+) accumulation as compared to non-mycorrhizal plants, concomitantly with higher K(+) /Na(+) ratios in their tissues. For the first time, these effects have been correlated with regulation of ZmAKT2, ZmSOS1 and ZmSKOR genes expression in the roots of maize, contributing to K(+) and Na(+) homeostasis in plants colonized by native AMF.

Native arbuscular mycorrhizal fungi isolated from a saline habitat improved maize antioxidant systems and plant tolerance to salinity.

High soil salinity is a serious problem for crop production because most of the cultivated plants are salt sensitive, which is also the case for the globally important crop plant maize. Salinity stress leads to secondary oxidative stress in plants and a correlation between antioxidant capacity and salt tolerance has been demonstrated in several plant species. The plant antioxidant capacity may be enhanced by arbuscular mycorrhizal fungi (AMF) and it has been proposed that AM symbiosis is more effective with native than with collection AMF species. Thus, we investigated whether native AMF isolated from a dry and saline environment can help maize plants to overcome salt stress better than AMF from a culture collection and whether protection against oxidative stress is involved in such an effect. Maize plants inoculated with three native AMF showed higher efficiency of photosystem II and stomatal conductance, which surely decreased photorespiration and ROS production. Indeed, the accumulation of hydrogen peroxide, the oxidative damage to lipids and the membrane electrolyte leakage in these AM plants were significantly lower than in non-mycorrhizal plants or in plants inoculated with the collection AMF. The activation of antioxidant enzymes such as superoxide dismutase or catalase also accounted for these effects.

Septoglomus altomontanum, a new arbuscular mycorrhizal fungus from mountainous and alpine areas in Andalucía (southern Spain).

A new arbuscular mycorrhizal (AM) fungus was found in Sierra Nevada National Park of Andalucía (Southern Spain). It forms intraradical hyphae, vesicles and arbuscles, typical characteristics of Glomeromycetes. The spores are dark reddish brown to dark reddish black, 132-205 µm diam, and are formed on pigmented subtending hyphae whose pores are regularly closed by a thick septum at the spore base but without support of introverted wall thickening. Phylogenetic analyses on concatenate sequences of the partial SSU, ITS region and the partial LSU of the rDNA confirm the new species, described here as Septoglomus altomontanum, in a monophyletic clade next to S. africanum. An identification key to all Septoglomus species described is given. The new fungus can unequivocally be distinguished from all other Septoglomus species by the combination of spore size, colour and spore wall structure, and especially by the shape and colour of the subtending hyphae. Septoglomus altomontanum has so far been found only in soils with pH 5.9-6.7, located in mountainous and alpine altitudes (1800-3100 m asl) of Sierra Nevada which is well known for a high degree of plant endemism. While it is a frequent fungus in this area, it has so far not been found in lower altitudes in Andalucía.

Temporal dynamics of arbuscular mycorrhizal fungi colonizing roots of representative shrub species in a semi-arid Mediterranean ecosystem.

Arbuscular mycorrhizal (AM) symbiosis plays an important role in improving plant fitness and soil quality, particularly in fragile and stressed environments, as those in certain areas of Mediterranean ecosystems. AM fungal communities are usually affected by dynamic factors such as the plant community structure and composition, which in turn are imposed by seasonality. For this reason, a one-year-round time-course trial was performed by sampling the root system of two representative shrubland species (Rosmarinus officinalis and Thymus zygis) within a typical Mediterranean ecosystem from the Southeast of Spain. The 18S rDNA gene, of the AM fungal community in roots, was subjected to PCR-SSCP, sequencing, and phylogenetic analysis. Forty-three different AM fungal sequence types were found which clustered in 16 phylotypes: 14 belonged to the Glomeraceae and two to the Diversisporaceae. Surprisingly, only two of these phylotypes were related with sequences of morphologically defined species: Glomus intraradices and Glomus constrictum. Significant differences were detected for the relative abundance of some phylotypes while no effects were found for the calculated diversity indices. These results may help to design efficient mycorrhizal-based revegetation programs for this type of ecosystems.

Ambispora granatensis, a new arbuscular mycorrhizal fungus, associated with Asparagus officinalis in Andalucia (Spain).

A new dimorphic fungal species in the arbuscular mycorrhiza-forming Glomeromycota, Ambispora granatensis, was isolated from an agricultural site in the province of Granada (Andalucía, Spain) growing in the rhizosphere of Asparagus officinalis. It was propagated in pot cultures with Trifolium pratense and Sorghum vulgare. The fungus also colonized Ri T-DNA transformed Daucus carota roots but did not form spores in these root organ cultures. The spores of the acaulosporoid morph are 90-150 µm diam and hyaline to white to pale yellow. They have three walls and a papillae-like rough irregular surface on the outer surface of the outer wall. The irregular surface might become difficult to detect within a few hours in lactic acid-based mountings but are clearly visible in water. The structural central wall layer of the outer wall is only 0.8-1.5 µm thick. The glomoid spores are formed singly or in small, loose spore clusters of 2-10 spores. They are hyaline to pale yellow, (25)40-70 µm diam and have a bilayered spore wall without ornamentation. Nearly full length sequences of the 18S and the ITS regions of the ribosomal gene place the new fungus in a separate clade next to Ambispora fennica and Ambispora gerdemannii. The acaulosporoid spores of the new fungus can be distinguished easily from all other spores in genus Ambispora by the conspicuous thin outer wall.

Entrophospora nevadensis, a new arbuscular mycorrhizal fungus from Sierra Nevada National Park (southeastern Spain).

A new fungal species in the arbuscular mycorrhiza-forming Glomeromycetes, Entrophospora nevadensis, was isolated from soil near the roots of several endemic and endangered plant species (e.g. Plantago nivalis and Alchemilla fontqueri) growing in Sierra Nevada National Park (Granada, Andalucia, Spain). The fungus was propagated in trap cultures on Plantago nivalis and Sorbus hybrida and in pure cultures on Trifolium pratense and Sorghum vulgare. Spores are yellow brown to brown, 90-115 .m diam and form singly in soil, in the neck of adherent sporiferous saccules that form either terminally or intercalary on mycelial hyphae. Spores have two three-layered walls and conspicuous, 6-12 microm long, spiny, thorn-like projections on the outer wall consisting of hyaline to subhyaline, evanescent tips and yellow brown to brown, persistent bases. In aging spores these projections are usually shorter (1-2.8 microm) and dome-shaped or rounded, sometimes with a central pit on top where the evanescent tip has sloughed off. Molecular analysis with partial sequences of the 18S ribosomal gene places the fungus within the Diversisporales. The new fungus was found in soil near plants with different living strategies but growing in high altitude soils with acidic pH, high soil moisture and organic carbon content, and close to streams.

Arbuscular mycorrhizal fungi, Bacillus cereus, and Candida parapsilosis from a multicontaminated soil alleviate metal toxicity in plants.

We investigated if the limited development of Trifolium repens growing in a heavy metal (HM) multicontaminated soil was increased by selected native microorganisms, bacteria (Bacillus cereus (Bc)), yeast (Candida parapsilosis (Cp)), or arbuscular mycorrhizal fungi (AMF), used either as single or dual inoculants. These microbial inoculants were assayed to ascertain whether the selection of HM-tolerant microorganisms can benefit plant growth and nutrient uptake and depress HM acquisition. The inoculated microorganisms, particularly in dual associations, increased plant biomass by 148% (Bc), 162%, (Cp), and 204% (AMF), concomitantly producing the highest symbiotic (AMF colonisation and nodulation) rates. The lack of AMF colonisation and nodulation in plants growing in this natural, polluted soil was compensated by adapted microbial inoculants. The metal bioaccumulation abilities of the inoculated microorganisms and particularly the microbial effect on decreasing metal concentrations in shoot biomass seem to be involved in such effects. Regarding microbial HM tolerance, the activities of antioxidant enzymes known to play an important role in cell protection by alleviating cellular oxidative damage, such as superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase, were here considered as an index of microbial metal tolerance. Enzymatic mechanisms slightly changed in the HM-adapted B. cereus or C. parapsilosis in the presence of metals. Antioxidants seem to be directly involved in the adaptative microbial response and survival in HM-polluted sites. Microbial inoculations showed a bioremediation potential and helped plants to develop in the multicontaminated soil. Thus, they could be used as a biotechnological tool to improve plant development in HM-contaminated environments.

Microbial co-operation in the rhizosphere.

Soil microbial populations are immersed in a framework of interactions known to affect plant fitness and soil quality. They are involved in fundamental activities that ensure the stability and productivity of both agricultural systems and natural ecosystems. Strategic and applied research has demonstrated that certain co-operative microbial activities can be exploited, as a low-input biotechnology, to help sustainable, environmentally-friendly, agro-technological practices. Much research is addressed at improving understanding of the diversity, dynamics, and significance of rhizosphere microbial populations and their co-operative activities. An analysis of the co-operative microbial activities known to affect plant development is the general aim of this review. In particular, this article summarizes and discusses significant aspects of this general topic, including (i) the analysis of the key activities carried out by the diverse trophic and functional groups of micro-organisms involved in co-operative rhizosphere interactions; (ii) a critical discussion of the direct microbe-microbe interactions which results in processes benefiting sustainable agro-ecosystem development; and (iii) beneficial microbial interactions involving arbuscular mycorrhiza, the omnipresent fungus-plant beneficial symbiosis. The trends of this thematic area will be outlined, from molecular biology and ecophysiological issues to the biotechnological developments for integrated management, to indicate where research is needed in the future.

Temporal temperature gradient gel electrophoresis (TTGE) as a tool for the characterization of arbuscular mycorrhizal fungi.

The aim of this study was to assess the feasibility of using temporal temperature gradient electrophoresis (TTGE) of PCR-amplified 18S rDNA fragments of different Glomus species for their detection and characterization. Screening of Glomus clarum, Glomus constrictum, Glomus coronatum, Glomus intraradices, Glomus mosseae and Glomus viscosum by PCR-TGGE revealed that the NS31-AM1 region of the 18S rRNA gene contained insufficient variation to discriminate between them. In contrast, TTGE analysis of the NS31-Glo1 region, which was obtained by nested PCR of the NS31-AM1 amplicon, showed that each species was characterized by a specific TTGE fingerprint. However, isolates of the same species could not be distinguished. The nested PCR-TTGE approach developed allowed identification of the Glomus species colonising the roots of different plant species.

Influence of a Bacillus sp. on physiological activities of two arbuscular mycorrhizal fungi and on plant responses to PEG-induced drought stress.

The effects of bacterial inoculation (Bacillus sp.) on the development and physiology of the symbiosis between lettuce and the arbuscular mycorrhizal (AM) fungi Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe and Glomus intraradices (Schenck and Smith) were investigated. Plant growth, mineral nutrition and gas-exchange values in response to bacterial inoculation after PEG-induced drought stress were also evaluated. In AM plants, inoculation with Bacillus sp. enhanced fungal development and metabolism, measured as succinate dehydrogenase (SDH) and alkaline phosphatase (ALP) activities, more than plant growth. Under non-stressed conditions, G. intraradices colonization increased all plant physiological values to a higher extent when in dual inoculation with the bacterium. Under stress conditions, the bacterium had an important stimulatory effect on G. intraradices development. Under such conditions, the effects of the bacterium on photosynthetic rate, water use efficiency (WUE) and stomatal conductance of lettuce plants differed with the fungus species. Plant-gas exchange was enhanced in G. intraradices- and reduced in G. mosseae-colonized plants when co-inoculated with Bacillus sp. Thus, the effects of each fungus on plant physiology were modulated by the bacterium. Stress was detrimental, particularly in G. intraradices-colonized plants without the bacterium, reducing intra and extraradical mycelium growth and vitality (SDH), as well as plant-gas exchange. Nevertheless, Bacillus sp. inoculation improved all these plant and fungal parameters to the same level as in non-stressed plants. The highest amount of alive and active AM mycelium for both fungi was obtained after co-inoculation with Bacillus sp. These results suggest that selected free-living bacteria and AM fungi should be co-inoculated to optimize the formation and functioning of the AM symbiosis in both normal and adverse environments.

Differential response of delta13C and water use efficiency to arbuscular mycorrhizal infection in two aridland woody plant species.

During a revegetation field experiment in Southeast Spain, we measured foliar carbon isotope ratios (delta13C) and gas exchange parameters in order to evaluate the influence of arbuscular mycorrhizal (AM) infection on the water use efficiency (WUE) of two semiarid woodland species. WUE during drought was significantly enhanced by inoculation with Glomus intraradices in Olea europaea ssp sylvestris, but not in Rhamnus lycioides. While Olea is a long-lived, slow-growing evergreen tree with a conservative water use strategy, Rhamnus is a drought-deciduous shrub with a shorter lifespan; these differences may explain their dissimilar patterns of physiological response to inoculation with the same AM fungus. Differences in delta13C and WUE between Olea and Rhamnus were larger when comparing AM inoculated than non-inoculated seedlings. This result suggests that some of the interspecific variability in delta13C observed for aridland plant communities may be due to different physiological responses to mycorrhization.

Antioxidant activities in mycorrhizal soybean plants under drought stress and their possible relationship to the process of nodule senescence.

• The mechanisms by which the mycorrhizal symbiosis protects soybean ( Glycine max ) plants against premature nodule senescence induced by drought stress is investigated here by evaluating the activity of a set of antioxidant enzymes in relation to nodule senescence. • Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activity was determined in well watered or drought-stressed soybean plants inoculated with Bradyrrhizobium japonicum alone or in combination with Glomus mosseae . • In roots, only GR activity was higher in mycorrhizal than in non-mycorrhizal plants. The other antioxidant activities were similar, or lower (APX), in droughted, mycorrhizal plants than in the corresponding nonmycorrhizal ones. Similarly, in nodules, SOD, CAT and APX activities were lower in droughted, mycorrhizal plants than in nonmycorrhizal plants whereas, again, GR activity was higher in nodules from mycorrhizal plants. • We propose that the consistently higher GR activity in roots and nodules of mycorrhizal plants might have contributed to decreased oxidative damage to biomolecules, which are involved in premature nodule senescence. Additional drought-avoidance mechanisms induced by the AM symbiosis might also contribute to the lower oxidative stress in mycorrhizal plants.

Mycorrhizosphere interactions to improve plant fitness and soil quality.

Arbuscular mycoruhizal fungi are key components of soil microbiota and obviously interact with other microorganisms in the rhizosphere, i.e. the zone of influence of plant roots on microbial populations and other soil constituents. Mycorrhiza formation changes several aspects of plant physiology and some nutritional and physical properties of the rhizospheric soil. These effects modify the colonization patterns of the root or mycovrhizas (mycorrhizosphere) by soil microorganisms. The rhizosphere of mycorrhizal plants, in practice a mycorrhizosphere, harbors a great array of microbial activities responsible for several key ecosystem processes. This paper summarizes the main conceptual principles and accepted statements on the microbial interactions between mycorrhizal fungi and other members of rhizosphere microbiota and discusses current developments and future trends concerning the following topics: (i) effect of soil microorganisms on mycorrhiza formation; (ii) mycorrhizosphere establishment; (iii) interactions involved in nutrient cycling and plant growth; (iv) interactions involved in the biological control of plant pathogens; and (v) interactions to improve soil quality. The main conclusion is that microbial interactions in the rhizosphere of mycorrhizal plants improve plant fitness and soil quality, critical issues for a sustainable agricultural development and ecosystem functioning.