Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri.

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 µM of acetosyringone, attaining a bacterial optical density of 0.3 OD600; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.

Edible ectomycorrhizal fungi and Cistaceae. A study on compatibility and fungal ecological strategies.

Wild edible mycorrhizal mushrooms are among the most appreciated and prized mushrooms in the world. Despite the cultivation of ectomycorrhizal (ECM) mushrooms has been a growing subject of study worldwide, it has been hampered by the mutualistic lifestyle of the fungi. Although not being obligate symbionts, most of the species of ECM mushrooms only produce fruit bodies in association with trees or shrubs. In the present study, we aimed at understanding certain aspects of the ecology of four different edible ECM fungi: Lactarius deliciosus, Tricholoma equestre, T. portentosum and Boletus fragrans. Despite having a broad distribution worldwide, these fungi inhabit also Mediterranean habitats with understories typically dominated by rockroses (Cistaceae). Studying the ecology of these mutualistic fungi as well as the interaction with these species of shrubs is not only scientifically relevant but also pivotal for the discovery of profitable cultivation protocols. We evaluated the compatibility of these ECM species with five species within Cistaceae family - Cistus ladanifer, C. psilosepalus, C. salviifolius, Halimium halimifolium and Tuberaria lignosa. Each species of fungi proved to be able to establish mycorrhizas with at least 2 different plants species but varied in their host range of the tested Cistaceae. The dissimilarity in terms of host specificity between some fungal species seemed to be connected with the phylogenetic distances of the fungi. A correlation between the colonization percentage of the root systems and the mycelial growth rates in pure culture was found. The connection of these traits might be an important key to understanding the ecological competitor-colonizer tradeoffs of these ECM fungal species. Altogether, our study reports unknown plant-fungi combinations with economical relevance and also adds new insights about the ecology of these species of ECM fungi.

Purification and characterization of Terfezia claveryi TcCAT-1, a desert truffle catalase upregulated in mycorrhizal symbiosis.

Terfezia claveryi Chatin is a mycorrhizal fungus that forms ectendomycorrhizal associations with plants of Helianthemum genus. Its appreciated edibility and drought resistance make this fungus a potential alternative crop in arid and semiarid areas of the Mediterranean region. In order to increase the knowledge about the biology of this fungus in terms of mycorrhiza formation and response to drought stress, a catalase from T. claveryi (TcCAT-1) has been purified to apparent homogeneity and biochemically characterized; in addition, the expression pattern of this enzyme during different stages of T. claveryi biological cycle and under drought stress conditions are reported. The results obtained, together with the phylogenetic analysis and homology modeling, indicate that TcCAT-1 is a homotetramer large subunit size monofunctional-heme catalase belonging to Clade 2. The highest expression of this enzyme occurs in mature mycorrhiza, revealing a possible role in mycorrhiza colonization, but it is not upregulated under drought stress. However, the H2O2 content of mycorrhizal plants submitted to drought stress is lower than in well watered treatments, suggesting that mycorrhization improves the plant's oxidative stress response, although not via TcCAT-1 upregulation.

Mycelium of Terfezia claveryi as inoculum source to produce desert truffle mycorrhizal plants.

Terfezia claveryi Chatin was the first desert truffle species to be cultivated, the mycorrhizal plants being successfully produced by using both desert truffle spores and mycelia. However, it is more advisable to use mycelium than spores whenever possible and profitable. Given the low yields of mycelia obtained using traditional culture methods of this truffle, the medium composition was modified in an attempt to determine its nutritional requirements. For this, an assay involving response surface methodology was performed using Box-Behnken design to find the optimal parameters for the high production of mycelial biomass. The best results were obtained with glucose as carbon source, buffering the pH at 5 during culture, adding a pool of vitamins, and adjusting the optimal concentrations of carbon and nitrogen sources of the MMN medium. Biomass production increased from 0.3 to 3 g L-1 dry weight and productivity increased from 10.7 to 95.8 mg L-1 day-1 dry weight. The produced mycelium was able to colonize Helianthemum roots efficiently, providing more than 50% ectomycorrhizal colonization.

Beneficial native bacteria improve survival and mycorrhization of desert truffle mycorrhizal plants in nursery conditions.

Sixty-four native bacterial colonies were isolated from mycorrhizal roots of Helianthemum almeriense colonized by Terfezia claveryi, mycorrhizosphere soil, and peridium of T. claveryi to evaluate their effect on mycorrhizal plant production. Based on the phylogenetic analysis of the 16S rDNA partial sequence, 45 different strains from 17 genera were gathered. The largest genera were Pseudomonas (40.8 % of the isolated strains), Bacillus (12.2 % of isolated strains), and Varivorax (8.2 % of isolated strains). All the bacteria were characterized phenotypically and by their plant growth-promoting rhizobacteria (PGPR) traits (auxin and siderophore production, phosphate solubilization, and ACC deaminase activity). Only bacterial combinations with several PGPR traits or Pseudomonas sp. strain 5, which presents three different PGPR traits, had a positive effect on plant survival and growth. Particularly relevant were the bacterial treatments involving auxin release, which significantly increased the root-shoot ratio and mycorrhizal colonization. Moreover, Pseudomonas mandelii strain 29 was able to considerably increase mycorrhizal colonization but not plant growth, and could be considered as mycorrhiza-helper bacteria. Therefore, the mycorrhizal roots, mycorrhizosphere soil, and peridium of desert truffles are environments enriched in bacteria which may be used to increase the survival and mycorrhization in the desert truffle plant production system at a semi-industrial scale.

Morphological characterization of mycorrhizae formed between three Terfezia species (desert truffles) and several Cistaceae and Aleppo pine.

Six Cistaceae species, Helianthemum ledifolium, Helianthemum lippii, Fumana procumbens, Cistus albidus, Cistus incanus, Cistus salvifolius, and Pinus halepensis (Aleppo pine) were inoculated with three mycorrhizal desert truffles, Terfezia leptoderma, Terfezia boudieri, and Terfezia claveryi under greenhouse conditions, on soil originating from desert truffle natural habitat in Algeria. The syntheses have led to the formation of typical endomycorrhizae in annual Cistaceae (H. ledifolium) and perennial ones (H. lippii and F. procumbens) and an ectomycorrhiza with a less developed sheath in Cistus species and Aleppo pine. These results demonstrate the plasticity of Terfezia species to form different mycorrhizal types. The formation of an endomycorrhiza with H. ledifolium and F. procumbens and a sheathing ectomycorrhiza with P. halepensis inoculated by T. leptoderma in in vivo culture conditions was obtained for the first time.

Species-specific ITS primers for the identification of Picoa juniperi and Picoa lefebvrei and using nested-PCR for detection of P. juniperi in planta.

Desert truffles, hypogeous Pezizales (Ascomycota), are difficult to identify due to evolutionary convergence of morphological characters among taxa that share a similar habitat and mode of spore dispersal. Also, during their symbiotic phase, these are barely distinguishable morphologically, and molecular probes are needed for their identification. We have developed a PCR-based method for the identification of Picoa juniperi and Picoa lefebvrei based on internal transcribed spacers of rDNA. Two PCR primers specific for P. lefebvrei (FLE/RLE) and two specific for P. juniperi (FJU/RJU) were designed. A collection of samples from different geographical areas representing diversity of these species were examined for unique regions of internal transcribed spacers 1, 2 and 5.8S gene of rDNA (ITS) compared to other closely related species. Annealing temperatures and extension times were optimized for each set of primers for maximum specificity and efficiency. They proved to be efficient to specifically detect the presence of P. juniperi and P. lefebvrei by PCR and neither set amplified purified DNA from other truffle species as well as some ascomycetous fungi. The partial small subunit of ribosomal DNA genes of P. juniperi were amplified with the genomic DNA extracted from Helianthemum ledifolium var. ledifolium roots by nested polymerase chain reaction (PCR) using the universal fungal primer pair ITS1/ITS4 and specific primer pair FTC/RTC, which was designed based on internal transcribed spacer 1, 2 and 5.8S gene of rDNA sequences of P juniperi. The nested-PCR was sensitive enough to re-amplify the direct-PCR product, resulting in a DNA fragment of 426 bp. The efficacy of nested-PCR showed that it could re-amplify the direct-PCR product and detect 200 fg genomic DNA.

Expression analysis of aquaporins from desert truffle mycorrhizal symbiosis reveals a fine-tuned regulation under drought.

We have performed the isolation, functional characterization, and expression analysis of aquaporins in roots and leaves of Helianthemum almeriense, in order to evaluate their roles in tolerance to water deficit. Five cDNAs, named HaPIP1;1, HaPIP1;2, HaPIP2;1, HaPIP2;2, and HaTIP1;1, were isolated from H. almeriense. A phylogenetic analysis of deduced proteins confirmed that they belong to the water channel proteins family. The HaPIP1;1, HaPIP2;1, and HaTIP1;1 genes encode functional water channel proteins, as indicated by expression assays in Saccharomyces cerevisiae, showing divergent roles in the transport of water, CO2, and NH3. The expression patterns of the genes isolated from H. almeriense and of a previously described gene from Terfezia claveryi (TcAQP1) were analyzed in mycorrhizal and nonmycorrhizal plants cultivated under well-watered or drought-stress conditions. Some of the studied aquaporins were subjected to fine-tuned expression only under drought-stress conditions. A beneficial effect on plant physiological parameters was observed in mycorrhizal plants with respect to nonmycorrhizal ones. Moreover, stress induced a change in the mycorrhizal type formed, which was more intracellular under drought stress. The combination of a high intracellular colonization, together with the fine-tuned expression of aquaporins could result in a morphophysiological adaptation of this symbiosis to drought conditions.

The role of phosphorus in the ectendomycorrhiza continuum of desert truffle mycorrhizal plants.

The influence of inorganic and organic phosphorus (P) and the absence of P in the culture medium on the type of mycorrhizal colonization formed (ecto-, ectendo-, or endomycorrhiza) during Helianthemum almeriense x Terfezia claveryi symbiosis in in vitro conditions was analyzed. This is the first time that the relative proportions of the different mycorrhizal types in mycorrhizal roots of H. almeriense have been quantified and statistically analyzed. The relative proportions of the mycorrhizal types depended on the P source in the medium, suggesting that it is the organic P form that induces the formation of intracellular colonization. The above association should be considered as a continuum between intra- and intercellular colonizations, the most appropriate term for defining it being ectendomycorrhiza. The influence of the endogenous concentration of P on plant growth was also analyzed. P translocation was observed from shoot to roots, especially in mycorrhizal plants because mycorrhizal roots showed higher growth than non-mycorrhizal roots and/or because of an extra P demand from mycelium inside the roots. Soluble and cell wall acid phosphatases activities from H. almeriense roots were kinetically characterized at optimum pH (5.0), using p-nitrophenyl phosphate as substrate, with K (m) values of 3.4 and 1.8 mM, respectively. Moreover, the plant acid phosphatase and fungal alkaline phosphatases activities were histochemically localised in mycorrhizal H. almeriense roots by fluorescence with enzyme-labelled fluorescence substrate.

The aquaporin TcAQP1 of the desert truffle Terfezia claveryi is a membrane pore for water and CO(2) transport.

Terfezia claveryi is a hypogeous mycorrhizal fungus belonging to the so-called "desert truffles," with a good record as an edible fungus and of considerable economic importance. T. claveryi improves the tolerance to water stress of the host plant Helianthemum almeriense, for which, in field conditions, symbiosis with T. claveryi is valuable for its survival. We have characterized cDNAs from T. claveryi and identified a sequence related to the aquaporin gene family. The full-length sequence was obtained by rapid amplification of cDNA ends and was named TcAQP1. This aquaporin gene encoded a functional water-channel protein, as demonstrated by heterologous expression assays in Saccharomyces cerevisiae. The mycorrhizal fungal aquaporin increased both water and CO(2) conductivity in the heterologous expression system. The expression patterns of the TcAQP1 gene in mycelium, under different water potentials, and in mycorrhizal plants are discussed. The high levels of water conductivity of TcAQP1 could be related to the adaptation of this mycorrhizal fungus to semiarid areas. The CO(2) permeability of TcAQP1 could be involved in the regulation of T. claveryi growth during presymbiotic phases, making it a good candidate to be considered a novel molecular signaling channel in mycorrhizal fungi.

Mycorrhizal association between the desert truffle Terfezia boudieri and Helianthemum sessiliflorum alters plant physiology and fitness to arid conditions.

The host plant Helianthemum sessiliflorum was inoculated with the mycorrhizal desert truffle Terfezia boudieri Chatin, and the subsequent effects of the ectomycorrhizal relationship on host physiology were determined. Diurnal measurements revealed that mycorrhizal (M) plants had higher rates of photosynthesis (35%), transpiration (18%), and night respiration (49%) than non-mycorrhizal (NM) plants. Consequently, M plants exhibited higher biomass accumulation, higher shoot-to-root ratios, and improved water use efficiency compared to NM plants. Total chlorophyll content was higher in M plants, and the ratio between chlorophyll a to chlorophyll b was altered in M plants. The increase in chlorophyll b content was significantly higher than the increase in chlorophyll a content (2.58- and 1.52-fold, respectively) compared to control. Calculation of the photosynthetic activation energy indicated lower energy requirements for CO(2) assimilation in M plants than in NM plants (48.62 and 61.56 kJ mol(-1), respectively). Continuous measurements of CO(2) exchange and transpiration in M plants versus NM plants provided a complete picture of the daily physiological differences brought on by the ectomycorrhizal relationships. The enhanced competence of M plants to withstand the harsh environmental conditions of the desert is discussed in view of the mycorrhizal-derived alterations in host physiology.

Phylogenetic affiliation of the desert truffles Picoa juniperi and Picoa lefebvrei.

The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation.

Assessing the diversity of AM fungi in arid gypsophilous plant communities.

In the present study, we used PCR-Single-Stranded Conformation Polymorphism (SSCP) techniques to analyse arbuscular mycorrhizal fungi (AMF) communities in four sites within a 10 km(2) gypsum area in Southern Spain. Four common plant species from these ecosystems were selected. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, SSCP analysis, sequencing and phylogenetic analyses. A total of 1443 SSU rRNA sequences were analysed, for 21 AM fungal types: 19 belonged to the genus Glomus, 1 to the genus Diversispora and 1 to the Scutellospora. Four sequence groups were identified, which showed high similarity to sequences of known glomalean species or isolates: Glo G18 to Glomus constrictum, Glo G1 to Glomus intraradices, Glo G16 to Glomus clarum, Scut to Scutellospora dipurpurescens and Div to one new genus in the family Diversisporaceae identified recently as Otospora bareai. There were three sequence groups that received strong support in the phylogenetic analysis, and did not seem to be related to any sequences of AM fungi in culture or previously found in the database; thus, they could be novel taxa within the genus Glomus: Glo G4, Glo G2 and Glo G14. We have detected the presence of both generalist and potential specialist AMF in gypsum ecosystems. The AMF communities were different in the plant studied suggesting some degree of preference in the interactions between these symbionts.

Alternaria jesenskae sp. nov., a new species from Slovakia on Fumana procumbens (Cistaceae).

Alternaria jesenskae sp. nov. recovered from seeds of a shrubby perennial plant Fumana procumbens (Cistaceae) in Slovakia is described and illustrated. The new taxon can be clearly separated from the other related large-spored and filament-beaked Alternaria species based on sequences of the ITS1, 5.8S and ITS2 region as well as by its distinctive morphology. Even though the molecular data have shown close relatedness with A. multirostrata, the new species is morphologically most similar to A. tomatophila distinguished primarily by the pronounced colony pigmentation, conidial septation and beak branching.

Two ITS forms co-inhabiting a single genet of an isolate of Terfezia boudieri (Ascomycotina), a desert truffle.

Two fruit-bodies of Terfezia boudieri Chatin, each exhibiting a mixture of two ITS -RFLP profiles, were found in the Negev desert of Israel. A mycelial culture obtained from glebal out-growth maintained the double profile, as did proliferating cultures established using single hyphae isolated from the original cultures. The main difference between the two ITS variants lies in a 21 bp deletion in the smaller variant. The question whether both variants are contained within a single nucleus or occupy different nuclei sharing the same cytoplasm is discussed.

In vitro interaction of the truffle Terfezia terfezioides with Robinia pseudoacacia and Helianthemum ovatum.

The type of the in vitro root interactions of Terfezia terfezioides with the plants Robinia pseudoacacia and Helianthemum ovatum was investigated including detailed anatomical and ultrastructural characterization. No difference in growth was detected at different phosphate concentrations on agar synthetic medium between the inoculated and control plants during a short-time cultivation. The fungal colonization of the roots increased with higher phosphate level in both plant species, but was always lower in R. pseudoacacia roots. Septate hyphae formed frequently intracellular branched coils in dead cortical cells. In H. ovatum intercellular hyphae were observed forming finger-like structures reminiscent of Hartig-net structures in ectomycorrhizae. A loose hyphal envelope covered the root surface of both colonized and noncolonized roots. The features resembled similar structures described earlier during the mycorrhizae of different Terfezia species. Our detailed anatomical and ultrastructural study shows that the in vitro root interactions of the T. terfezioides cannot be considered unambiguously as mycorrhiza.

Morphological characterization of the mycorrhiza formed by Helianthemum almeriense Pau with Terfezia claveryi Chatin and Picoa lefebvrei (Pat.) Maire.

This work presents the first anatomical description of the mycorrhizal systems of Helianthemum almeriense, and of the structure and ultrastructure of the mycorrhizae formed by this plant species with the ascomycetes Terfezia claveryi and Picoa lefebvrei. Four different mycorrhizal systems are described, the club-shaped mycorrhiza being the most abundant. The type of mycorrhiza formed depended on the mycorrhiza culture conditions, but not on the fungal species. For both fungal species, H. almeriense formed an endomycorrhiza in natural field conditions, an ecto- and ectendomycorrhiza without a sheath in pot cultures, and an ectomycorrhiza with a characteristic sheath and Hartig net in in vitro cultures. This is the first report of a typical sheath in Helianthemum-desert truffle mycorrhizal associations. The results support the idea that culture conditions can induce changes in mycorrhiza morphology and that there is no clear barrier between the two main types of mycorrhiza organization in Helianthemum species. The ultrastructural study confirmed the regular presence of T. claveryi intracellular hyphae in direct contact with the host wall, a localization which seems to be a characteristic of the T. claveryi mycorrhiza organization. The P. lefebvrei mycorrhiza organization was characterized by intracellular hyphae with large amounts of electron-dense globules, probably with a lipidic content, and a warty ornamentation on the wall of the root external hyphae.

Studies on the root associations of the truffle Terfezia terfezioides.

The paper contains an overview of the results of the studies made on the truffle Terfezia terfezioides, particularly the investigations related to the associations of this fungus with plants. Twelve plant species originated from a natural habitat of the fungus were supposed to be connected with T. terfezioides based on the anatomy of the endogenous fungal structures in their roots. Aseptic experiments were carried out on modified MMN substrates with different phosphate concentrations to study the interaction of T. terfezioides with Robinia pseudoacacia and Helianthemum ovatum. The colonization of the roots of black locust was always weaker than that of Helianthemum. The main characteristics were the intracellular coiled, branched, frequently septated hyphae in dead root cells. The intercellular hyphae formed Hartig-net with finger like structures only in Helianthemum, the interactions could not be considered unambiguously as mycorrhizae. There was no difference between the RFLP profiles of the nr DNA ITS of nineteen fruit bodies collected at the same time from the habitat and the ITS of three randomly chosen specimens were identical on sequence level, too. These invariability makes to design species specific PCR primers possible to check unambiguously the host plants.