Pseudomonas edaphica sp. nov., isolated from rhizospheric soil of Cistus ladanifer L. in Spain.

During a study on biodiversity of bacteria inhabiting rhizospheric soil of rockrose (Cistus ladanifer L.), we isolated a strain coded RD25T in a soil from Northern Spain. The 16S rRNA gene sequence showed 99.5 % identity with respect to the closest related species Pseudomonas brenneri DSM15294T, and 99.4 % with respect to P. paralactis WS4672T. The following related Pseudomonas species showed 99.3 % or less identity, and therefore RD25T was classified within genus Pseudomonas. The phylogenetic analysis of 16S rRNA and the housekeeping genes rpoB, rpoD and gyrB suggested that this strain could be a novel species. The strain RD25T has several polar-subpolar flagella. It can grow at 36 °C, at 0-6 % NaCl concentration and a range of pH 5-9. Positive for arginine dihydrolase and urease production, and negative for reduction of nitrate. The strain is catalase and oxidase positive. Major fatty acids are C16 : 1 ω7c / C16 : 1 ω6c in summed feature 3, C16 : 0, and C18 : 1 ω7c / C18 : 1 ω6c in summed feature 8. The respiratory ubiquinone is Q9. The DNA G+C content was 59.9 mol%. The digital DNA-DNA hybridisation average values (dDDH) ranged between 30-61.2 % relatedness and the ANIb values ranged between 93.9-80.5 % with respect to the type strains of the closely related species. Therefore, the genotypic, genomic, phenotypic and chemotaxonomic data support the classification of strain RD25 as a novel species of genus Pseudomonas, for which the name P. edaphica sp. nov. is proposed. The type strain is RD25T (=LMG 30152T=CECT 9373T).

Effect of forest fire prevention treatments on bacterial communities associated with productive Boletus edulis sites.

Cistus ladanifer scrublands, traditionally considered as unproductive, have nonetheless been observed to produce large quantities of king bolete (Boletus edulis) fruitbodies. These pyrophytic scrublands are prone to wildfires, which severely affect fungi, hence the need for fire prevention in producing C. ladanifer scrublands. In addition, B. edulis productions have severely decreased in the last years. A deeper understanding of the B. edulis life cycle and of biotic and abiotic factors influencing sporocarp formation is needed to implement management practices that facilitate B. edulis production. For example, some bacteria likely are involved in sporocarp production, representing a key part in the triple symbiosis (plant-fungus-bacteria). In this study, we used soil DNA metabarcoding in C. ladanifer scrublands to (i) assess the effect of site history and fire prevention treatment on bacterial richness and community composition; (ii) test if there was any correlation between various taxonomic groups of bacteria and mycelial biomass and sporocarp production of B. edulis; and to (iii) identify indicator bacteria associated with the most productive B. edulis sites. Our results show that site history drives bacterial richness and community composition, while fire prevention treatments have a weaker, but still detectable effect, particularly in the senescent plots. Sporocarp production correlated positively with genera in Verrucomicrobia. Several genera, e.g. Azospirillum and Gemmatimonas, were identified as indicators of the most productive sites, suggesting a potential biological role in B. edulis fructification. This study provides a better understanding of the triple symbiosis (plant-fungus-bacteria) involved in C. ladanifer-B. edulis systems.

Delftia rhizosphaerae sp. nov. isolated from the rhizosphere of Cistus ladanifer.

A bacterial strain, designated RA6T, was isolated from the rhizosphere of Cistus ladanifer. Phylogenetic analyses based on 16S rRNA gene sequence placed the isolate into the genus Delftia within a cluster encompassing the type strains of Delftia lacustris, Delftia tsuruhatensis, Delftia acidovorans and Delftia litopenaei, which presented greater than 97 % sequence similarity with respect to strain RA6T. DNA-DNA hybridization studies showed average relatedness ranging from of 11 to 18 % between these species of the genus Delftia and strain RA6T. Catalase and oxidase were positive. Casein was hydrolysed but gelatin and starch were not. Ubiquinone 8 was the major respiratory quinone detected in strain RA6T together with low amounts of ubiquinones 7 and 9. The major fatty acids were those from summed feature 3 (C16 : 1ω7c/C16 : 1 ω6c) and C16 : 0. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain RA6T should be considered as a representative of a novel species of genus Delftia, for which the name Delftia rhizosphaerae sp. nov. is proposed. The type strain is RA6T (=LMG 29737T= CECT 9171T).

Bacillus terrae sp. nov. isolated from Cistus ladanifer rhizosphere soil.

A bacterial strain designated RA9T was isolated from a root of Cistus ladanifer in Spain. Phylogenetic analyses based on 16S rRNA gene sequences placed the isolate into the genus Bacillus with its closest relatives being Bacillus fortis R-6514T and Bacillus fordii R-7190T with 98.2 % similarity in both cases. DNA-DNA hybridization studies showed mean relatedness values of 29 and 30 %, respectively, between strain RA9T and the type strains of B. fortis and B. fordii. Cells of the isolate were Gram-stain-positive, motile, sporulating rods. Catalase and oxidase were positive. Gelatin, starch and casein were not hydrolysed. Menaquinone MK-7 was the only menaquinone detected and iso-C15 : 0 and anteiso-C15 : 0 were the major fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid, one unidentifed glycolipid and one unidentified lipid. meso-Diaminopimelic acid was detected in the peptidoglycan. The DNA G+C content was 43.1 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain RA9T should be considered as representing a novel species of the genus Bacillus, for which the name Bacillus terrae sp. nov. is proposed. The type strain is RA9T (=LMG 29736T=CECT 9170T).

Mycorrhization between Cistus ladanifer L. and Boletus edulis Bull is enhanced by the mycorrhiza helper bacteria Pseudomonas fluorescens Migula.

Boletus edulis Bull. is one of the most economically and gastronomically valuable fungi worldwide. Sporocarp production normally occurs when symbiotically associated with a number of tree species in stands over 40 years old, but it has also been reported in 3-year-old Cistus ladanifer L. shrubs. Efforts toward the domestication of B. edulis have thus focused on successfully generating C. ladanifer seedlings associated with B. edulis under controlled conditions. Microorganisms have an important role mediating mycorrhizal symbiosis, such as some bacteria species which enhance mycorrhiza formation (mycorrhiza helper bacteria). Thus, in this study, we explored the effect that mycorrhiza helper bacteria have on the efficiency and intensity of the ectomycorrhizal symbiosis between C. ladanifer and B. edulis. The aim of this work was to optimize an in vitro protocol for the mycorrhizal synthesis of B. edulis with C. ladanifer by testing the effects of fungal culture time and coinoculation with the helper bacteria Pseudomonas fluorescens Migula. The results confirmed successful mycorrhizal synthesis between C. ladanifer and B. edulis. Coinoculation of B. edulis with P. fluorescens doubled within-plant mycorrhization levels although it did not result in an increased number of seedlings colonized with B. edulis mycorrhizae. B. edulis mycelium culture time also increased mycorrhization levels but not the presence of mycorrhizae. These findings bring us closer to controlled B. edulis sporocarp production in plantations.

Whose truffle is this? Distribution patterns of ectomycorrhizal fungal diversity in Tuber melanosporum brûlés developed in multi-host Mediterranean plant communities.

In the Mediterranean region, patches of vegetation recovering from disturbance and transiently dominated by shrubs produce one of the world's most prized fungi, the black truffle (Tuber melanosporum). In these successional plant communities, we have fragmentary knowledge of the distribution of T. melanosporum in space among ectomycorrhizal (ECM) host species and in time. Molecular identification of hosts (Restriction Fragment Length Polymorphism) and fungi (Internal Transcribed Spacer sequencing) and quantification of T. melanosporum mycelium (quantitative Polymerase Chain Reaction) were employed to evaluate the presence of T. melanosporum on four dominant ECM host species (Quercus ilex, Quercus  coccifera, Arbutus unedo, Cistus albidus) and the extent to which their respective ECM communities shared fungal diversity, over the course of development of truffle grounds, from recent unproductive brûlés to senescent ones where production has stopped. We found that truffle grounds host rich communities in which multi-host fungal species dominate in frequency. When considering both ECM tips and soil mycelia, we documented a dynamic and spatially heterogeneous pattern of T. melanosporum distribution in soils and a presence of ECM tips restricted to Q. ilex roots. This study advances our knowledge of the ecology of T. melanosporum, and provides insight into the extent of ECM fungal sharing among plant species that dominate Mediterranean landscapes.

Combined effects of clay immobilized Azospirillum brasilense and Pantoea dispersa and organic olive residue on plant performance and soil properties in the revegetation of a semiarid area.

The reestablishment of autochthonous shrubs species is an essential strategy for recovering degraded soils under semiarid Mediterranean areas. A field experiment was carried out to assess the effectiveness of an immobilized microbial inoculant (Azospirillum brasilense and Pantoea dispersa) and the addition of organic olive residue (alperujo), for plant growth promotion of Cistus albidus L. and enhancement of soil properties. Sixteen months after planting, the microbial inoculant and organic residue combined treatment was the most effective for stimulating the root dry weight of C. albidus (by 133% with respect to control plants) and microbial inoculant was the most effective treatment for increasing the shoot dry weight of plants (by 106% with respect to control plants). Available phosphorus and potassium content in the amended soils was about 100 and 70% respectively higher than the non-amended soil. Total C, total organic C and microbial biomass C content and enzyme activities (dehydrogenase, urease and protease) of the rhizosphere of C. albidus were increased by microbial inoculant and organic residue combined, but not by the microbial inoculation and organic residue applied independently. The combined treatment, involving microbial inoculant and the addition of the organic residue, had an additive effect improving the biochemical and microbiological quality of the soil.

Hebeloma species associated with Cistus.

The genus Hebeloma has a number of species highly specific to Cistus and others that occur with several host genera. This paper discusses the species of Hebeloma that appear to be ectomycorrhizal with Cistus, judging from their occurrence when Cistus is the only available host. The previously unknown species H. plesiocistum spec. nov. is described. We also provide a key to the known Hebeloma associates of Cistus. Molecular analyses based on ITS sequence data further illustrate the distinctness of the newly described species and difficulties in the species delimitation with view to H. erumpens. Specific associations with Cistus may have evolved more than once within the genus Hebeloma.

Mycorrhizal synthesis between Boletus edulis species complex and rockroses (Cistus sp.).

Ectomycorrhizas of Boletus aereus, Boletus edulis, and Boletus reticulatus were synthesized with Cistus sp. under laboratory conditions using synthesis tubes filled with a mixture of sterilized peat-vermiculite and nutrient solution. The fungal strains isolated from sporocarps were identified by molecular techniques. The inoculated seedlings were grown for 4-5 months. The ectomycorrhizas formed were described based on standard morphological and anatomical characters. The three ectomycorrhizas described were very similar, with white monopodial-pinnate morphology, a three-layered plectenchymatous mantle on plan view and boletoid rhizomorphs.

Impact of active soil carbonate and burn size on the capacity of the rockrose Cistus laurifolius to produce Tuber melanosporum carpophores in truffle culture.

There are very few studies on Tuber melanosporum associated with Cistus populations. In central Spain, we confirm that C. laurifolius shows carpophore production of Tuber melanosporum. This study demonstrates a correlation between T. melanosporum production and the size of the 41 burns, with burn size explaining 26% of the variability in carpophore production. However, statistical analysis of the results indicates that average production of 21 burns with Cistus laurifolius is 73% lower than the production of 20 burns associated with Quercus/Corylus without Cistus laurifolius in this zone, respectively. C. laurifolius develops small burns, which has an impact on their carpophore production, and their soils have 92% less active carbonate than the burns associated with Quercus/Corylus in this zone, respectively. The low levels of active carbonate allow Cistus laurifolius to grow, but impair Tuber melanosporum production. We thus provide a mechanism, based on the inability of Cistus laurifolius to grow in highly carbonated soils, which contradicts the well-known fact that C. laurifolius might act as a transmitter of Tuber melanosporum mycorrhizae. These results indicate that this rockrose can not be considered a useful species for truffle culture. We thus recommend that particular attention should be given to the concentration of active carbonate present in the soil in future studies on the relationship between Cistus species and truffles.

Differential expression of fungal genes at preinfection and mycorrhiza establishment between Terfezia boudieri isolates and Cistus incanus hairy root clones.

Changes in gene expression by isolates of Terfezia boudieri during mycorrhization with Cistus incanus hairy roots were followed. Four fungus-hairy root clone combinations were cultivated under two sets of conditions, in which the root and the fungus were separated by a cellophane sheet or were allowed physical contact. One of the combinations produced endomycorrhizas, the other three solely ectomycorrhizas. Fragments isolated by cDNA-AFLP analysis from cellophane-separated cultures (preinfection) were used to identify differentially expressed genes by reverse Northern analysis. Genes showing no homology to known sequences constituted the largest group under both growth conditions. Some fungal genes were expressed transiently, while others exhibited altered expression patterns as conditions changed from individually growing through the preinfection stage to mycorrhizas. Genes expressed exclusively under combinations allowing either ectomycorrhiza or endomycorrhiza under a particular condition were detected. Our results point, for the first time, to some of the genes that might be involved in determining the type of association that will be formed: ecto- or endomycorrhiza.

An overview of Cistus ectomycorrhizal fungi.

The genus Cistus comprises a group of about 20 shrub species found in wide areas throughout the whole Mediterranean region to the Caucasus. Being one of the main constituents of the Mediterranean-type maquis, this plant genus is peculiar in that it has developed a range of specific adaptations to resist summer drought and frequent disturbance events, such as fire and grazing. In addition, it can form both ectomycorrhizas and arbuscular mycorrhizas. In this paper, we review the information available on the ectomycorrhizal fungi of Cistus across its entire geographic range, as gathered and critically sifted from both published literature sources and personal observations. Although the resulting data matrix was based primarily on accounts of sporocarp inventories in the field, existing knowledge on the features of Cistus natural and synthesized ectomycorrhizas was also included and discussed. In total, more than 200 fungal species belonging to 40 genera have been reported so far to be associated with Cistus. An analysis of the pattern of ectomycorrhizal diversity and host specificity revealed that members of the Cortinariaceae and Russulaceae make the most of both Cistus-aspecific and Cistus-specific mycobionts. Further studies are needed to expand our preliminary knowledge of the mycorrhizal ecology and biology of Cistus and its fungal associates, focusing on topics such as mycobiont diversity, host specificity, fungal succession, mycorrhizal influence on stress tolerance, and impact of disturbances, while comparing the findings with those from other ecosystems.

Characterization and identification of field ectomycorrhizae of Boletus edulis and Cistus ladanifer.

Field ectomycorrhizae sampled under Boletus edulis and Cistus ladanifer have been characterized and described in detail based on standard morphological and anatomical characters. The described ectomycorrhiza has traits typical of Boletales: whitish with three differentiated plectenchymatous layers in the mantle in plan view forming ring-like structures and rhizomorphs with highly differentiated hyphae. The inflated, smooth cystidia-like clavate end cells on the surface of the rhizomorphs and their slightly twisted external hyphae are additional characterizing features. The Hartig net occupies 1 1/2 rows of cortical cells, partly reaching the endodermis. Not all hyphae have clamps. The identification of the fungal symbiont as B. edulis was confirmed by ITS rDNA sequence comparison between mycorrhizas and sporocarps. The singularity of this symbiotic association, as well as its ecological and practical implications, are discussed.

Analysis of mycorrhizal associations formed by Cistus incanus transformed root clones with Terfezia boudieri isolates.

One clone (M-2), out of several Agrobacterium rhizogenes transformed root clones of Cistus incanus, formed ecto- or endomycorrhiza in vitro with two isolates of Terfezia boudieri collected in Israel. All other clone-fungal isolate combinations formed ectomycorrhiza. The endomycorrhiza-forming isolate secreted smaller amounts of auxin than an ectomycorrhiza-forming isolate. Addition of 2,4-dichlorophenoxyacetic acid (2,4-D) led to ectomycorrhiza formation by the M-2 clone on low P medium. Endomycorrhizas were formed by both M-2 and a control clone with the same T. boudieri isolates on high P medium with 2,4-D. The M-2 clone of C. incanus exhibited greater sensitivity to exogenous auxins (IAA and 2,4-D) than other clones, and clonal sensitivity to auxin was increased tenfold under low P conditions. Results are discussed in relation to phosphate and auxin influence on T. boudieri-C. incanus interaction.

Bacterial colonization of the phyllosphere of mediterranean perennial species as influenced by leaf structural and chemical features.

In this study, we assessed various leaf structural and chemical features as possible predictors of the size of the phyllosphere bacterial population in the Mediterranean environment. We examined eight perennial species, naturally occurring and coexisting in the same area, in Halkidiki (northern Greece). They are Arbutus unedo, Quercus coccifera, Pistacia lentiscus, and Myrtus communis (evergreen sclerophyllous species), Lavandula stoechas and Cistus incanus (drought semi-deciduous species), and Calamintha nepeta and Melissa officinalis (non-woody perennial species). M. communis, L. stoechas, C. nepeta, and M. officinalis produce essential oil in substantial quantities. We sampled summer leaves from these species and (1) estimated the size of the bacterial population of their phyllosphere, (2) estimated the concentration of different leaf constituents, and (3) studied leaf morphological and anatomical features and expressed them in a quantitative way. The aromatic plants are on average more highly colonized than the other species, whereas the non-woody perennials are more highly colonized than the woody species. The population size of epiphytic bacteria is positively correlated with glandular and non-glandular trichome densities, and with water and phosphorus contents; it is negatively correlated with total phenolics content and the thickness of the leaf, of the mesophyll, and of the abaxial epidermis. No correlation was found with the density of stomata, the nitrogen, and the soluble sugar contents. By regression tree analysis, we found that the leaf-microbe system can be effectively described by three leaf attributes with leaf water content being the primary explanatory attribute. Leaves with water content >73% are the most highly colonized. For leaves with water content <73%, the phosphorus content, with a critical value of 1.34 mg g(-1) d.w., is the next explanatory leaf attribute, followed by the thickness of the adaxial epidermis. Leaves higher in phosphorus (>1.34 mg g(-1) d.w.) are more colonized, and leaves with the adaxial epidermis thicker than 20.77 microm are the least colonized. Although these critical attributes and values hold true only within the Mediterranean ecosystem studied and the range of observations taken, they are important because they provide a hypothesis to be tested in other Mediterranean ecosystems and other biomes. Such comparative studies may give insight as to the general properties governing the leaf-microbe system.

Establishment of two ectomycorrhizal shrub species in a semiarid site after in situ amendment with sugar beet, rock phosphate, and Aspergillus niger.

A field experiment was carried out to assess the effectiveness of the addition of sugar beet, rock phosphate, and Aspergillus niger directly into the planting hole, and the mycorrhizal inoculation of seedlings with Scleroderma verrucosum, for promotion of plant growth of Cistus albidus L. and Quercus coccifera L. and enhancement of soil physicochemical, biochemical, and biological properties, in a degraded semiarid Mediterranean area. One year after planting, the available phosphorus content in the amended soils of both species was about fourfold higher than in the nonamended soil. Amendment addition increased the aggregate stability of the rhizosphere of C. albidus (by 56% with respect to control soil) while the mycorrhizal inoculation increased only the aggregate stability of the rhizosphere of Q. coccifera (by 13% with respect to control soil). Biomass C content and enzyme activities (dehydrogenase, urease, protease-BAA, acid phosphatase, and beta-glucosidase) of the rhizosphere of C. albidus were increased by amendment addition but not by mycorrhizal inoculation. Both treatments increased enzyme activities of the rhizosphere of Q. coccifera. The mycorrhizal inoculation of the seedlings with S. verrucosum was the most effective treatment for stimulating the growth of C. albidus (by 469% with respect to control plants) and Q. coccifera (by 74% with respect to control plants). The combined treatment, involving mycorrhizal inoculation of seedlings and addition of the amendment directly into soil, had no additive effect on the growth of either shrub species.