Anatomical and morphological characterization of mycorrhizas of five strains of Tuber borchii Vittad

 Tilia platyphyllos Scop. plantlets were inoculated in vitro with five Tuber borchii Vittad. strains (1BO, 17BO, 43BO, 71BO and 10RA) to test their intraspecific variability. The ability of the strains to form mycorrhizas varied, with the mean degree of ectomycorrhizal infection ranging from 50.6% (for 1BO) to 82.1% (for 10RA). The anatomical/morphological characteristics of the resulting mycorrhizas were determined. Although the morphological features of the mycorrhizas and the characteristics of the cystidia were similar for all strains tested, differences were found in the anatomical features of the mantle. The form of the mantle cells was examined in the surface and inner layers (6 and 12 μm deep, respectively) by both conventional and confocal microscopy. These cells were polygonal in 1BO, and 71BO, epidermoid in 43BO and intermediate in 17BO and 10RA. The structure of the mantle also varied and thus provided little information with which to identify T. borchii mycorrhizas. Accepted: 7 July 2000     

Arbutus unedo L. communities in southern Iberian Peninsula mountains

Arbutus unedo L. communities in the southern part of the Iberian Peninsula (Spain and Portugal) were studied, highlighting the differences regarding their floristic composition, biological diversity and plant dynamics according to the dominant geological substratum. Firstly, this type of formation on the little-known decarbonated calcareous substrata was analysed using classification techniques. Later, they were compared to the remaining phytocoenoses in the southern part of the Iberian Peninsula using an ordination analysis. As a result, a new suballiance is proposed together with two plant associations, which will enable us to characterize the strawberry tree groves growing on carbonated substrata and those already proposed for siliceous substrata are confirmed.     

Effect of local competition on resprouting of Arbutus unedo after clipping

Abstract. To evaluate the effects of local competition on the growth and size variability of sprouts following disturbance in a natural population of Arbutus unedo in Catalonia, plants were cut at the base and a neighbor removal experiment was performed. Removal of neighbors resulted in an increase in the number and biomass of sprouts at 2 and 7.5 months after clipping. Number and biomass of sprouts was also correlated with initial plant size (stump area). Inter-genet competition appeared to be symmetric, and acted to delay the onset of interference among sprouts within a genet. Size variability of sprouts on an individual was positively correlated with their density on the stump, supporting the hypothesis that competition among ramets (sprouts) within a genet is asymmetric. Reduced inter-genet competition from neighbor removal resulted in an increase in the number and biomass of sprouts growing from a stump. This resulted in an increase in the asymmetry of competition among sprouts, and therefore an increase in the size variability of these sprouts.     

Isolation and Characterization of Differentially Expressed Genes in the Mycelium and Fruit Body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-β-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

Cloning adult trees of Arbutus unedo L. through somatic embryogenesis

Arbutus unedo L. is a perennial tree, native in the Mediterranean area, and tolerant to stress conditions. Due to its economic potential, there is an increasing demanding for plants by producers and farmers. In order to offer cloned material with assured quality, several micropropagation protocols have been developed, including somatic embryogenesis induction. However, little is known about this process on strawberry tree and a great deal of work is still necessary to successfully clone in A. unedo through somatic embryogenesis. Thus, the main goals of this work were: (i) to test the effect of the genotype on somatic embryogenesis induction, (ii) to analyse the role of adult and young materials on induction, and (iii) to perform a comparative histological analysis between somatic embryos and their zygotic counterparts. Somatic embryogenesis was induced on apical expanded leaves from in vitro shoots of several genotypes in Andersson medium with 3% sucrose and different concentrations of BAP (2.0 mg L^?1) and NAA (0.5, 1.0, 2.0, 5.0, 10.0, 15.0 and 20.0 mg L^?1). Embryogenesis induction rates ranged from 0 to 94.5%. Higher induction rates were achieved on the medium with 2 mg L^?1 BAP and 2 mg L^?1 NAA, and are genotype dependent. After a 3-month induction period, the highest somatic embryogenesis induction rate was 94.5% on genotype AU4. Embryos at different developmental stages were found, as well as abnormal somatic embryos. SEM images showed different anomalies being the most common embryos displaying more than two cotyledons or fused embryos. Embryo germination was not genotype dependent and the maximum embryo conversion rate achieved was 73.5%. However, only 39.21% of the embryos were able to grow into plantlets which displayed a normal chromosome number (2n?=?26). Histological analysis showed differences in the cell organization between somatic and zygotic embryos, as well as several morphological anomalies. Overall, the developed somatic induction protocol proved to be very efficient, with high induction rates achieved, both from seedling and adult material, but its genotype dependent. However, embryo conversion still needs to be improved, in order to fully seize the potential of this micropropagation technique.

     

Cloning and characterization of PKC-homologous genes in the truffle species Tuber borchii and Tuber magnatum

The protein kinases C (PKCs) define a growing family of ubiquitous signal transducting serine/threonine kinases that control ion conductance channels, release of hormones and cell growth and proliferation. Degenerated oligonucleotides were used as primers for polymerase chain reactions to amplify PKC-related sequences from the white truffle species Tuber magnatum and Tuber borchii. The deduced amino acid sequences of cloned sequences reveal domains homologous to the regulatory and kinase domains of PKC-related proteins, but lack typical Ca(2+)-binding domain and therefore should be classified as nPKCs. Both contain a large extended N-terminus which is found exclusively in fungi PKCs. Phylogenetic analysis of the kinase domain demonstrates high homology with known filamentous fungi isoenzymes.     

Characterization and complementation of a Fus3/Kss1 type MAPK from Tuber borchii, TBMK

Mitogen-activated protein kinases (MAPK) are used by organisms to transduce extra cellular signals from the environment in cellular events such as proliferation and differentiation. In the present study, we have characterized the first MAPK from the ectomycorrhizal fungus Tuber borchii (TBMK) which belongs to the YERK1 (yeast extra cellular regulated kinase) subfamily. TBMK is present as a single copy in the genome and the codified protein was phosphorylated during the interaction with the host plant, Tilia americana. Complementation studies showed that TBMK restores pheromone signaling in Saccharomyces cerevisiae and partially restores invasive growth of Fusarium oxysporum that lack the fmk1 gene. This suggests a protein kinase activity and its involvement in the infection processes. Hence, TBMK could play an important role during the pre-symbiotic phase of T. borchii with its host plant in the modulation of genes necessary for the establishment of symbiosis leading to the synthesis of functional ectomycorrhizae.     

Structural analysis of the rDNA intergenic spacer of Tuber borchii

The sequence and characterisation of the entire nuclear rDNA intergenic spacer (IGS) for the genus Tuber are presented. Sequence analyses showed that the organisation of the Tuber borchii rDNA IGS is typical of rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. Direct repeats, symmetry elements, tandem repeats and possible areas of recombination were found. The putative ends of the 25S and 17S rDNA were identified. The presence of 5S rDNA in the IGS region was excluded.     

Phylogenetic Characterization and In Situ Detection of a Cytophaga-Flexibacter-Bacteroides Phylogroup Bacterium in Tuber borchii Vittad. Ectomycorrhizal Mycelium

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.     

Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T. borchii mycelial growth

Isolation and physiological and molecular characterisation of culturable bacterial strains belonging to actinomycetes, pseudomonads and aerobic spore-forming bacteria were carried out on mycorrhizal root tips of Quercus robur var. peduncolata infected by Tuber borchii. Cellular density of the three bacterial groups in ectomycorrhizal root tips was estimated to be 1.3+/-0.11 x 10(6) cfu g(-1) dry weight for total heterotrophic bacteria and 1.08+/-0.6 x 10(5) (mean+/-S.E.), 1.3+/-0.3 x 10(5) and 1.4+/-0.2 x 10(5) cfu g(-1) dry weight for pseudomonads, actinomycetes and spore-forming bacteria respectively. Identification of pseudomonads by the Biolog system indicated, besides the most represented species Pseudomonas fluorescens (biotypes B, F and G), the occurrence of strains belonging to Pseudomonas corrugata. Amplified ribosomal DNA restriction analysis of actinomycetes and spore formers revealed at least three and six different groups of patterns, respectively. Many bacterial isolates were able to induce variations in growth rates of T. borchii mycelium; among these, 101 strains showed antifungal activity, whereas 17 isolates, belonging to spore formers, were able to increase mycelial growth up to 78% when compared to uninoculated mycelial growth. The potential role of these populations in the development and establishment of mycorrhizas is discussed.     

Influence of temperature and rainfall on fruit and seed production of Arbutus unedo L.

Correlations were found between certain climatic parameters and fresh weight, dry weight and seed number of the fruit of Arbutus unedo L. In five stations in central Italy. Temperature and rainfall were found to affect fruit production. For the parameters fresh weight and number of seeds, the first months of development were important and fresh weight was also influenced by rainfall in the last monthS. Dry weight did not show statistically significant correlation with any one of the considered parameterS. However, the pattern of temperature and rainfall over the 12-month period of fruit ripening determined the characteristics of the fruit.     

Effects of nursery preconditioning through mycorrhizal inoculation and drought in Arbutus unedo L. plants

The influence of a water deficit treatment and mycorrhizal inoculation with Pisolithus tinctorius (Pers.) Coker and Couch on the water relations, gas exchange, and plant growth in Arbutus unedo L. plants was studied in order to evaluate the hardening process during the nursery period. The ability to withstand the adverse conditions after transplantation was also studied. Mycorrhizal and non-mycorrhizal seedlings of A. unedo were pot-grown for 4 months in a greenhouse (nursery period), during which time two irrigation treatments, well watered (100% water holding capacity, leaching 20% of the applied water) and deficit irrigation (50% of the well watered), were applied. Subsequently, the plants were transplanted to the field and well irrigated (transplanting period), after which and until the end of the experiment they received no water (establishment period). At the end of the nursery period, both water deficit and mycorrhizae were seen to have altered the plant morphology. Mycorrhizal plants had lower leaf area and improved leaf color parameters, while the water deficit increased root dry weight and the root/shoot ratio. Mycorrhizal plants had higher leaf water potential values than non-inoculated plants. Mycorrhizae increased stomatal conductance and photosynthesis values, especially in stressed plants. Drought led to an osmotic adjustment and a decrease in the leaf water potential values at turgor loss point in the mycorrhizal plants. Cell wall rigidity, measured as increased bulk modulus of elasticity, was decreased by the mycorrhizae effect. After transplanting, no differences were found in the water relations or gas exchange values between treatments. During the establishment period, the plants that had been exposed to both drought and mycorrhizae showed a better water status (higher leaf water and turgor potential values) and higher gas exchange values. In conclusion, water deficit and mycorrhizal inoculation of A. unedo plants in nursery produced changes in tissue water relations, gas exchange, and growth, related with the acclimation process in the seedlings, which could provide better resistance to drought and stress conditions following planting.     

A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii

An ammonium transporter cDNA, named TbAMT1, was isolated from the ectomycorrhizal ascomycetous truffle Tuber borchii. The polypeptide encoded by TbAMT1 (52 kDa) functionally complements ammonium uptake-defective yeast mutants and shares sequence similarity with previously characterized ammonium transporters from Saccharomyces (Mep) and Arabidopsis (AtAMT1). Structural characteristics common to the Mep/Amt family and peculiar features of the Tuber transporter have been evidenced by a detailed topological model of the TbAMT1 protein, which predicts 11 transmembrane helices with an N terminus(OUT)/C terminus(IN) orientation. As revealed by uptake/competition experiments conducted in yeast, TbAMT1 is a high-affinity transporter with an apparent K(m) for ammonium of 2 microM. The TbAMT1 mRNA was very slowly, yet specifically upregulated in nitrogen-deprived T. borchii mycelia. Instead, a much faster return to basal expression levels was observed upon resupplementation of either ammonium or nitrate, which thus appear to be utilized as equally effective nitrogen sources by Tuber mycelia.     

Tuber borchii fruit body: 2-dimensional profile and protein identification

The formation of the fruit body represents the final phase of the ectomycorrhizal fungus T. borchii life cycle. Very little is known concerning the molecular and biochemical processes involved in the fructification phase. 2-DE maps of unripe and ripe ascocarps revealed different protein expression levels and the comparison of the electropherograms led to the identification of specific proteins for each developmental phase. Associating micropreparative 2-DE to microchemical approaches, such as N-terminal sequencing and 2-D gel-electrophoresis mass-spectrometry, proteins playing pivotal roles in truffle physiology were identified.     

Phospholipase A2 up-regulation during mycorrhiza formation in Tuber borchii

TbSP1 is a secreted and surface-associated phospholipase A(2) previously found to be up-regulated in C- or N-deprived free-living mycelia from the ectomycorrhizal ascomycete Tuber borchii. As nutrient limitation is considered an important environmental factor favouring the transition to symbiotic status, TbSP1 was suggested to be involved in the formation of mycorrhizas. An in vitro symbiosis system between Cistus incanus and T. borchii was set up: TbSP1 mRNA levels in free-living mycelia and in mycorrhizas sampled in different districts of the plant-fungus interaction were examined. In the same samples, TbSP1 protein expression was analysed by immunoelectron microscopy. A substantially enhanced TbSP1 mRNA expression, compared with nutrient-limited but free-living mycelia, was detected in the presence of the plant and reached maximal levels in fully developed mycorrhizas. A similar expression trend was revealed by immunolocalization experiments. We have shown that TbSP1 appears to respond to two partially overlapping yet distinct stimuli: nutrient starvation and mycorrhiza formation.     

Molecular markers for the identification of the ectomycorrhizal fungus Tuber borchii

Five species of white truffle were classified using PCR-based techniques. RAPD (random amplified polymorphic DNA) fingerprints and specific pairs of primers were used. A RAPD fragment was constant in Tuber borchii Vittad. isolates and polymorphic among the other species. Two molecular markers specific for T. borchii were developed from the sequence of the non-polymorphic RAPD fragment and from regions flanking the 5'-3' ends of a truffle gene. These markers were applied in the identification of T. borchii fruit bodies, mycelia and mycorrhizas, allowing us to monitor the development of this fungus during its entire life cycle.     

The volatile organic compounds from the mycelium of Tuber borchii Vitt

The mycelium of T. borchii (characterized by DNA analysis) grown in sterile liquid medium produced some VOCs. The VOCs were retained on carbographs by passing a flow of helium, isolated and characterized in a GC-MS equipment after a thermal desorption. The compounds present in the VOCs from the mycelium cultures, but not in the VOCs from the control cultures, contained 29 compounds. The main compounds were 1,3-ditertbutylbenzene (16.1 ng/l), 3-methylheptane (9.2 ng/l), butan-2-one (8.8 ng/l), ethynylbenzene (5.6 ng/l), and octan-3-one (4.9 ng/l).     

Contribution of diffusional and non-diffusional limitations to midday depression of photosynthesis in Arbutus unedo L.

It is still unknown whether the midday depression of photosynthesis under severe water stress, frequently observed in plants growing in a Mediterranean-type climate, is primarily a consequence of diffusional or non-diffusional limitations. We carried out combined measurements of gas exchanges and chlorophyll fluorescence in field-grown Arbutus unedo L. trees during late spring and mid summer, and a quantitative limitation analysis was performed to distinguish between the different limitations to photosynthesis, i.e., diffusional [D _L = stomatal (S _L) + mesophyll (MC_L)] and non-diffusional (carboxylation capacity and electron transport, B _L) limitations. Light-saturated assimilation at ambient CO₂ (A _max), stomatal conductance to water vapour (g _sw) and maximum carboxylation rate (V _cmax C _i) showed a marked midday depression during both periods. The total limitations tended to increase during the day and were remarkably similar in June and July (50 and 48%, respectively); on a daily basis, D _L was similar to B _L (about 23%) in June; whereas, in July the former was predominant (38 and 4%, respectively). We concluded that the midday depression in photosynthesis was largely caused by diffusional limitations, with non-diffusional limitations playing a smaller role. Although stomatal closure was the main diffusional limitation, the decline in mesophyll conductance was not negligible during the hottest and driest period.