Ana Rincón

Inoculation of containerized Pinus pinea L. seedlings with seven ectomycorrhizal fungi

Abstract. Containerized Pinus pinea L. seedlings are commonly used for reforestation in the Mediterranean area. While there is an increasing knowledge of the potential ectomycorrhizal fungi associated with Pinus pinea, few studies exist of inoculation techniques with selected ectomycorrhizal fungi. We tested seven ectomycorrhizal fungi for their effectiveness with containerized Pinus pinea seedlings. Hebelomacrustuliniforme, Laccarialaccata and Pisolithustinctorius were applied as vegetative inocula while Melanogasterambiguus, Pisolithus tinctorius, Rhizopogonluteolus, Rhizopogonroseolus and Sclerodermaverrucosum were tested as spore inocula. The inoculum of each fungus was tested at several application rates. Among the fungi tested as vegetative inocula, the highest percentages of ectomycorrhizas were obtained with H. crustuliniforme at all rates tested. The ectomycorrhizas formed by L. laccata varied from 11% to 40% depending on the inoculum rate applied. Vegetative inoculum of Pisolithus tinctorius was only effective at the highest inoculum rates and gave mycorrhization percentages around 60%. Pisolithus tinctorius applied as a spore inoculum formed ectomycorrhizas at a frequency of about 50% at the effective inoculum rates. The rest of the fungi applied as spore inocula produced more than 50% of ectomycorrhizas at the effective spore concentrations. These included the highest percentages of ectomycorrhizas (>80%) obtained with both Rhizopogon species. Differences in growth due to inoculation with the different fungi were not detected and in some cases inoculation even reduced the total biomass accumulated by seedlings. All seedlings reached a size suitable for transplantation.

Field performance in northern Spain of Douglas-fir seedlings Inoculated with ectomycorrhizal fungi

Abstract Experimental plantations were established in northern Spain to determine the effects of different ectomycorrhizal fungi on growth and survival of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) under field conditions. Douglas-fir seedlings were inoculated with Laccariabicolor S238 mycelia in two bareroot nurseries in central France or with spore suspensions of three hypogeous ectomycorrhizal species: Melanogaster ambiguus, Rhizopogon colossus and R. subareolatus, in a Spanish containerised nursery. The effects of ectomycorrhizal inoculation on plant survival after outplanting were limited, being only significant at the Guipuzkoan (Spain) site, when plants inoculated with L.bicolor S238 were compared to non-inoculated plants grown in non-fumigated soil. L. bicolor S238 had a significant effect on plant growth during the phase of bareroot nursery growth and this difference was maintained after field outplanting. Nursery inoculations with M. ambiguus, R. colossus and R. subareolatus improved plant growth during the first 2 and 3 years after field outplanting. The positive effects of the inoculation treatment on seedling height, root collar diameter and stem volume persisted after 5 years of field growth. Inoculation with these ectomycorrhizal fungi may improve the field performance of Douglas-fir seedlings in northern Spain.

Flavodoxin overexpression reduces cadmium-induced damage in alfalfa root nodules.

Flavodoxins are electron carrier flavoproteins that are involved in the response to oxidative stress in bacteria and cyanobacteria. Recently, we obtained Sinorhizobium meliloti bacteria that overexpressed a flavodoxin from the cyanobacterium Anabaena variabilis [Redondo et al. (2009) Plant Physiology 149:1166–1178]. In the present work, tolerance to cadmium was evaluated in free-living transformed S. meliloti and in alfalfa plants nodulated by the flavodoxin-overexpressing rhizobia, in comparison with plants nodulated by wild-type bacteria. Overexpression of flavodoxin protected free-living S. meliloti from cadmium toxicity and had a positive effect on nitrogen fixation of alfalfa plants subjected to cadmium stress. Flavodoxin notably reduced cadmium-induced structural and ultrastructural alterations in alfalfa nodules. Putative protection mechanisms in flavodoxin-overexpressing nodules are discussed. Flavodoxin could have applications as a biotechnological tool to improve the symbiotic performance of alfalfa and other legumes in cadmium polluted soils.

Diversity of Rhizobial Bacteria Isolated from Nodules of the Gypsophyte Ononis tridentata L. Growing in Spanish Soils

The aim of this work is to describe the diversity and phylogeny of rhizobial bacteria associated to nodules of Ononis tridentata L. in different geographical regions of Spain. Twenty-two bacterial isolates were characterized using several molecular techniques (16S amplified ribosomal deoxyribonucleic acid restriction analysis, fingerprinting, and sequencing) and phylogenies were inferred from their 16S and nodC gene sequences. Phylogenetically, the isolates grouped with the genera Rhizobium, Mesorhizobium, Phylobacterium, and Bosea. The nodC gene, essential for nodulation, was detected for the first time in isolates close to the genera Bosea and Phyllobacterium. The bacteria isolated showed a high diversity at the genus, species, and strain level regardless of the geographical origin of the host plant. This is the first report describing bacteria associated to nodules of O. tridentata. This shrub legume is highly prized for the revegetation of gypsum soils in semiarid Mediterranean areas. Our molecular description of bacteria associated to this legume improves the current understanding of the ecology of this plant species. Our findings have implications for formulating suitable bacterial inocula to recover gypsum ecosystems.

Pseudoexon exclusion by antisense therapy in methylmalonic aciduria (MMAuria)

Development of pseudoexon exclusion therapies by antisense modification of pre‐mRNA splicing represents a type of personalized genetic medicine. Here we present the cellular antisense therapy and the cell‐based splicing assays to investigate the effect of two novel deep intronic changes c.1957–898A>G and c.1957–920C>A identified in the methylmalonyl–coenzyme A (CoA) mutase (MUT) gene. The results show that the nucleotide change c.1957–898A>G is a pathological mutation activating pseudoexon insertion and that antisense morpholino oligonucleotide (AMO) treatment in patient fibroblasts leads to recovery of MUT activity to levels 25 to 100% of control range. On the contrary, the change c.1957–920C>A, identified in two fibroblasts cell lines in cis with c.1885A>G (p.R629G) or c.458T>A (p.D153V), appears to be a rare variant of uncertain clinical significance. The functional analysis of c.1885A>G and c.458T>A indicate that they are the disease‐causing mutations in these two patients. The results presented here highlight the necessity of scanning the described intronic region for mutations in MUT‐affected patients, followed by functional analyses to demonstrate the pathogenicity of the identified changes, and extend previous work of the applicability of the antisense approach in methylmalonic aciduria (MMAuria) for a novel intronic mutation. Hum Mutat 30:1–7, 2009.

Influence of the fertilisation method in controlled ectomycorrhizal inoculation of two Mediterranean pines

The influence of the fertilisation method: soluble (SF) vs. slow-release fertiliser (SRF) and of inoculation with Laccaria laccata (Scop.) Fr., Pisolithus tinctorius (Pers.) Coker & Couch and Melanogaster ambiguus (Vittad.) Tul & C. Tul. on ectomycorrhizal colonization and growth of Pinus pinea L. and Pinus pinaster Ait. was evaluated. For both pines, mycorrhization with L. laccata was not affected by the fertilisation method. Percentages of ectomycorrhizas (ECM) formed by P. tinctorius were dependent on the fertilisation method, the inoculum type (vegetative or spores) and the pine species involved. ECM formed by M. ambiguus were increased with fertilisation in both pines. Inoculation significantly improved P. pinea biomass when seedlings were fertilised with SRF whereas no effect was found in non-fertilised ones. For non-fertilised P. pinaster, inoculation with L. laccata and both inocula of P. tinctorius increased seedling biomass whereas fertilisation neutralised the fungal effect. Fertilisation increased P. pinea and P. pinaster biomass, independently of the inoculation treatment.RésuméL’impact sur le degré de mycorhization et la croissance de jeunes plants de Pinus pinea L. et de Pinus pinaster Ait., de deux méthodes de fertilisation (fertilisant soluble (FS) et fertilisant à libération lente) et d’une inoculation contrôlée avec Laccaria laccata (Sco.) Fr., Pisolithus tinctorius (Pers.) Coker et Couch et Melanogaster ambiguus (Vittad.) Tul et C. Tul. Pour les deux pins, la mycorhization avec Laccaria laccata n’a pas été modifiée par la méthode de fertilisation. Le pourcentage d’ectomycorrhizes (ECM) formé by P. tinctorius dépendait de la méthode de fertilisation, su type d’inoculum (spores ou inoculum végétatif) et de l’espèce de pin. La fertilisation a augmenté les ECM produites par Melanogaster ambiguus chez les deux pins. L’inoculation a augmenté significativement la biomasse des semis de Pinus pinea lorsqu’ils ont été fertilisés avec SRF tandis qu’aucun effet n’a été trouvé pour les traitements non fertilisés. Pour les semis non fertilisés de Pinus pinaster, l’inoculation avec Laccaria laccata et avec les deux inoculums de Pisolithus tinctorius a augmenté la biomasse des semis tandis que la fertilisation a neutralisé l’effet de l’inoculation. La fertilisation a augmenté la biomasse de Pinus pinaster et de Pinus pinea indépendamment du traitement d’inoculation utilisé.

Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain

Abstract Although Pinus pinea L. is an important forest species in the Mediterranean region, few reports exist on its ectomycorrhizal associates. Sixty isolates, obtained from fungal sporocarps collected in mixed forests of P.pinea in Catalonia (northeastern Spain), were tested for ectomycorrhiza formation on containerized P. pinea seedlings when applied as mycelial inoculum produced in peat-vermiculite. A total of 17 isolates, in 8 genera (Amanita, Hebeloma, Laccaria, Lactarius, Pisolithus, Rhizopogon, Scleroderma and Suillus), formed ectomycorrhizas and the percentages of mycorrhizal short roots varied among isolates and species from 13% to 89%. Some of these fungi are cited for the first time in association with P. pinea. The results indicate further fungal candidates for controlled inoculation of P. pinea seedlings in the nursery.

Structure and phylogenetic diversity of post-fire ectomycorrhizal communities of maritime pine

Environmental disturbances define the diversity and assemblage of species, affecting the functioning of ecosystems. Fire is a major disturbance of Mediterranean pine forests. Pines are highly dependent on the ectomycorrhizal (EM) fungal symbiosis, which is critical for tree recruitment under primary succession. To determine the effects of time since fire on the structure and recovery of EM fungal communities, we surveyed the young Pinus pinaster regenerate in three sites differing in the elapsed time after the last fire event. Pine roots were collected, and EM fungi characterized by sequencing the internal transcribed spacer (ITS) and the large subunit (LSU) regions of the nuclear ribosomal (nr)-DNA. The effects of the elapsed time after fire on the EM community structure (richness, presence/absence of fungi, phylogenetic diversity) and on soil properties were analysed.Fungal richness decreased with the elapsed time since the fire; although, the phylogenetic diversity of the EM community increased. Soil properties were different depending on the elapsed time after fire and particularly, the organic matter, carbon-to-nitrogen (C/N) ratio, nitrogen and iron significantly correlated with the assemblage of fungal species. Ascomycetes, particularly Tuberaceae and Pezizales, were significantly over-represented on saplings in the burned site. On seedlings, a significant over-representation of Rhizopogonaceae and Atheliaceae was observed in the most recently burned site, while other fungi (i.e. Cortinariaceae) were significantly under-represented. Our results are consistent with the hypothesis that fire can act as a selective agent by printing a phylogenetic signal on the EM fungal communities associated with naturally regenerated pines, pointing out to some groups as potential fire-adapted fungi.

Effects of salt stress and rhizobial inoculation on growth and nitrogen fixation of three peanut cultivars

Increasing soil salinity represents a major constraint for agriculture in arid and semi-arid lands, where mineral nitrogen (N) deficiency is also a frequent characteristic of soils. Biological N fixation by legumes may constitute a sustainable alternative to chemical fertilisation in salinity-affected areas, provided that adapted cultivars and inoculants are available. Here, the performance of three peanut cultivars nodulated with two different rhizobial strains that differ in their salt tolerance was evaluated under moderately saline water irrigation and compared with that of N-fertilised plants. Shoot weight was used as an indicator of yield. Under non-saline conditions, higher yields were obtained using N fertilisation rather than inoculation for all the varieties tested. However, under salt stress, the yield of inoculated plants became comparable to that of N-fertilised plants, with minor differences depending on the peanut cultivar and rhizobial strain. Our results indicate that N fixation might represent an economical, competitive and environmentally friendly choice with respect to mineral N fertilisation for peanut cultivation under moderate saline conditions.

Compartmentalized and contrasted response of ectomycorrhizal and soil fungal communities of Scots pine forests along elevation gradients in France and Spain

Fungi are principal actors of forest soils implied in many ecosystem services and the mediation of trees responses. Forecasting fungal responses to environmental changes is necessary for maintaining forest productivity, although our partial understanding of how abiotic and biotic factors affect fungal communities is restricting the predictions. We examined fungal communities of Pinus sylvestris along elevation gradients to check potential responses to climate change-associated factors. Fungi of roots and soils were analysed at a regional scale, by using a high-throughput sequencing approach. Overall soil fungal richness increased with pH, whereas it did not vary with climate. However, when representative sub-assemblages, i.e. Ascomycetes/Basidiomycetes, and families were analysed, they differentially answered to climatic and edaphic variables. This response was dependent on where they settled, i.e. soil versus roots, and/or on their lifestyle, i.e. mycorrhizal or not, suggesting different potential functional weights within the community. Our results revealed a highly compartmentalized and contrasted response of fungal communities in forest soils. The different response of fungal sub-assemblages indicated a range of possible selective direct and indirect (i.e. via host) impacts of climatic variations on these communities, of unknown functional consequences, that helps in understanding potential fungal responses under future global change scenarios.