Design and Validation of qPCR-Specific Primers for Quantification of the Marketed Terfezia claveryi and Terfezia crassiverrucosa in Soil.

Desert truffle crop is a pioneer in southeastern Spain, a region where native edible hypogeous fungi are adapted to the semiarid areas with low annual rainfall. Terfezia claveryi Chatin was the first species of desert truffle to be cultivated, and has been increasing in recent years as an alternative rainfed crop in the Iberian Peninsula. However, its behaviour in the field has yet not been investigated. For this purpose, specific primers were designed for the soil DNA quantification of both T. claveryi and Terfezia crassiverrucosa and a real-time qPCR protocol was developed, using the ITS rDNA region as a target. Moreover, a young desert truffle orchard was sampled for environmental validation. The results showed the highest efficiency for the TerclaF3/TerclaR1 primers pair, 89%, and the minimal fungal biomass that could be reliable detected was set at 4.23 µg mycelium/g soil. The spatial distribution of fungal biomass was heterogeneous, and there was not a direct relationship between the quantity of winter soil mycelium and the location/productivity of desert truffles. This protocol could be applied to tracking these species in soil and understand their mycelial dynamics in plantations and wild areas.

Draft Genome Sequence of Pseudomonas mandelii Strain 29, Isolated from the Desert Truffle Terfezia claveryi.

Here, we report the genome sequence of the mycorrhizal helper bacterium (MHB) Pseudomonas mandelii strain 29. This is the genome of an MHB associated with ascocarps of the desert truffle Terfezia claveryi. The genome is complete and consists of 6,302,122 bp and 5,812 predicted protein-coding sequences.

Desert truffle mycorrhizosphere harbors organic acid releasing plant growth-promoting rhizobacteria, essentially during the truffle fruiting season.

Desert truffle is becoming a new crop in semiarid areas. Climatic parameters and the presence of microorganisms influence the host plant physiology and alter desert truffle production. Desert truffle plants present a typical summer deciduous plant phenology divided into four stages: summer dormancy, autumn bud break, winter photosynthetic activity, and spring fruiting. We hypothesize that the bacterial community associated with desert truffle plants will show a seasonal trend linked to their plant growth-promoting rhizobacteria (PGPR) traits. This information will provide us with a better understanding about its potential role in this symbiosis and possible management implementations. Bacteria were isolated from root-adhering soil at the four described seasons. A total of 417 isolated bacteria were phenotypically and biochemically characterized and gathered by molecular analysis into 68 operational taxonomic units (OTUs). They were further characterized for PGPR traits such as indole acetic acid production, siderophore production, calcium phosphate solubilization, and ACCD (1-amino-cyclopropane-1-carboxilatedeaminase) activity. These PGPR traits were used to infer functional PGPR diversity and cultivable bacterial OTU composition at different phenological moments. The different seasons induced shifts in the OTU composition linked to their PGPR traits. Summer was the phenological stage with the lowest microbial diversity and PGPR functions, whereas spring was the most active one. Among the PGPR traits analyzed, P-solubilizing rhizobacteria were harbored in the mycorrhizosphere during desert truffle fruiting in spring.

Different patterns in root and soil fungal diversity drive plant productivity of the desert truffle Terfezia claveryi in plantation.

The desert truffle Terfezia claveryi is one of the few mycorrhizal fungi currently in cultivation in semiarid and arid areas. Agroclimatic parameters seem to affect its annual yield, but there is no information on the influence of biotic factors. In this study, fungal diversity was analysed by high-throughput sequencing of the ITS2 rDNA region from soil and root samples to compare productive and non-productive mycorrhizal plants in a 4-years old plantation (Murcia, Spain). The fungal metaprofile was dominated by Ascomycota phylum. Desert truffle productivity was driven by different patterns of fungal species composition in soil (species replacement) and root (species richness differences). Moreover, positive associations for ectomycorrhizal and negative for arbuscular mycorrhizal guilds were found in productive roots, and positive associations for fungal parasite-plant pathogen guild in non-productive ones. Soil samples were dominated by pathotroph and saprotroph trophic modes, showing positive associations for Aureobasidium pullulans and Alternaria sp. in productive areas, and positive associations for Fusarium sp. and Mortierella sp. were found in non-productive soils. Finally, some significant OTUs were identified and associated to ascocarp producing patches, which could serve as predictive and location markers of desert truffle production.

Desert truffle genomes reveal their reproductive modes and new insights into plant-fungal interaction and ectendomycorrhizal lifestyle.

Desert truffles are edible hypogeous fungi forming ectendomycorrhizal symbiosis with plants of Cistaceae family. Knowledge about the reproductive modes of these fungi and the molecular mechanisms driving the ectendomycorrhizal interaction is lacking. Genomes of the highly appreciated edible desert truffles Terfezia claveryi Chatin and Tirmania nivea Trappe have been sequenced and compared with other Pezizomycetes. Transcriptomes of T. claveryi × Helianthemum almeriense mycorrhiza from well-watered and drought-stressed plants, when intracellular colonizations is promoted, were investigated. We have identified the fungal genes related to sexual reproduction in desert truffles and desert-truffles-specific genomic and secretomic features with respect to other Pezizomycetes, such as the expansion of a large set of gene families with unknown Pfam domains and a number of species or desert-truffle-specific small secreted proteins differentially regulated in symbiosis. A core set of plant genes, including carbohydrate, lipid-metabolism, and defence-related genes, differentially expressed in mycorrhiza under both conditions was found. Our results highlight the singularities of desert truffles with respect to other mycorrhizal fungi while providing a first glimpse on plant and fungal determinants involved in ecto to endo symbiotic switch that occurs in desert truffle under dry conditions.

Elevated atmospheric CO2 modifies responses to water-stress and flowering of Mediterranean desert truffle mycorrhizal shrubs.

Predicted increases in atmospheric concentration of carbon dioxide (CO2 ) coupled with increased temperatures and drought are expected to strongly influence the development of most of the plant species in the world, especially in areas with high risk of desertification like the Mediterranean basin. Helianthemum almeriense is an ecologically important Mediterranean shrub with an added interest because it serves as the host for the Terfezia claveryi mycorrhizal fungus, which is a desert truffle with increasingly commercial interest. Although both plant and fungi are known to be well adapted to dry conditions, it is still uncertain how the increase in atmospheric CO2 will influence them. In this article we have addressed the physiological responses of H. almeriense × T. claveryi mycorrhizal plants to increases in atmospheric CO2 coupled with drought and high vapor pressure deficit. This work reports one of the few estimations of mesophyll conductance in a drought deciduous Mediterranean shrub and evaluates its role in photosynthesis limitation. High atmospheric CO2 concentrations help desert truffle mycorrhizal plants to cope with the adverse effects of progressive drought during Mediterranean springs by improving carbon net assimilation, intrinsic water use efficiency and dispersal of the species through increased flowering events.

Spring stomatal response to vapor pressure deficit as a marker for desert truffle fruiting.

The cultivation of desert truffle Terfezia claveryi using Helianthemum almeriense as a host plant has recently become a solid alternative crop in the Mediterranean region due to its adaptation to arid and semiarid ecosystems, which are expected to increase during the following years because of climate change. However, management models are still being developed in order to improve and stabilize the production, which varies greatly from one year to another. According to gatherers and farmers, one of the key factors for desert truffle production is the plant phenology in spring, which, in turn, depends on environmental conditions. In this manuscript, we have characterized the physiological, morphological, and molecular responses of the mycorrhizal plants in spring, coinciding with the fructification period of the plant and fungal species. Thanks to this characterization, a sigmoidal relationship between stomatal conductance and vapor pressure deficit (VPD) was found, which can be used as a marker of plant phenological switch. In order to confirm that this phenology status is related to desert truffle fructification, this marker has been successfully correlated to total truffle production. The results of this manuscript suppose a big step forward that will help to develop management models for the desert truffle crop.

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.

Identification of an Alternative rRNA Post-transcriptional Maturation of 26S rRNA in the Kingdom Fungi.

Despite of the integrity of their RNA, some desert truffles present a non-canonical profile of rRNA where 3.3 kb is absent, 1.8 kb is clear and a band of 1.6 kb is observed. A similar rRNA profile was identified in organisms belonging to different life kingdoms, with the exception of the Kingdom Fungi, as a result of a split LSU rRNA called hidden gap. rRNA profiles of desert truffles were analyzed to verify the presence of the non-canonical profile. The RNA of desert truffles and yeast were blotted and hybridized with probes complementary to LSU extremes. RACE of LSU rRNA was carried out to determine the LSU rRNA breakage point. LSU rRNA of desert truffles presents a post-transcriptional cleavage of five nucleotides that generates a hidden gap located in domain D7. LSU splits into two molecules of 1.6 and 1.8 kb. Similar to other organisms, a UAAU tract, downstream of the breakage point, was identified. Phylogenetic comparison suggests that during fungi evolution mutations were introduced in the hypervariable D7 domain, resulting in a sequence that is specifically post-transcriptionally cleaved in some desert truffles.

Significance of oxygen transport through aquaporins.

Aquaporins are membrane integral proteins responsible for the transmembrane transport of water and other small neutral molecules. Despite their well-acknowledged importance in water transport, their significance in gas transport processes remains unclear. Growing evidence points to the involvement of plant aquaporins in CO2 delivery for photosynthesis. The role of these channel proteins in the transport of O2 and other gases may also be more important than previously envisioned. In this study, we examined O2 permeability of various human, plant, and fungal aquaporins by co-expressing heterologous aquaporin and myoglobin in yeast. Two of the most promising O2-transporters (Homo sapiens AQP1 and Nicotiana tabacum PIP1;3) were confirmed to facilitate O2 transport in the spectrophotometric assay using yeast protoplasts. The over-expression of NtPIP1;3 in yeasts significantly increased their O2 uptake rates in suspension culture. In N. tabacum roots subjected to hypoxic hydroponic conditions, the transcript levels of the O2-transporting aquaporin NtPIP1;3 significantly increased after the seven-day hypoxia treatment, which was accompanied by the increase of ATP levels in the apical root segments. Our results suggest that the functional significance of aquaporin-mediated O2 transport and the possibility of controlling the rate of transmembrane O2 transport should be further explored.

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.

Transcript profiling of aquaporins during basidiocarp development in Laccaria bicolor ectomycorrhizal with Picea glauca.

Sporocarp formation is part of the reproductive stage in the life cycle of many mycorrhizal macrofungi. Sporocarp formation is accompanied by a transcriptomic switch and profound changes in regulation of the gene families that play crucial roles in the sporocarp initiation and maturation. Since sporocarp growth requires efficient water delivery, in the present study, we investigated changes in transcript abundance of six fungal aquaporin genes that could be cloned from the ectomycorrhizal fungus Laccaria bicolor strain UAMH8232, during the initiation and development of its basidiocarp. Aquaporins are intrinsic membrane proteins facilitating the transmembrane transport of water and other small neutral molecules. In controlled-environment experiments, we induced basidiocarp formation in L. bicolor, which formed ectomycorrhizal associations with white spruce (Picea glauca) seedlings. We profiled transcript abundance corresponding to six fungal aquaporin genes at six different developmental stages of basidiocarp growth and development. We also compared physiological parameters of non-inoculated to mycorrhizal seedlings with and without the presence of basidiocarps. Two L. bicolor aquaporins--JQ585592, a functional channel for CO2, NO and H2O2, and JQ585595, a functional water channel--showed the greatest degree of upregulation during development of the basidiocarp. Our findings point to the importance of aquaporin-mediated transmembrane water and CO2 transport during distinct stages of basidiocarp development.

Laccaria bicolor aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides).

The development of ectomycorrhizal associations is crucial for growth of many forest trees. However, the signals that are exchanged between the fungus and the host plant during the colonization process are still poorly understood. In this study, we have identified the relationship between expression patterns of Laccaria bicolor aquaporin LbAQP1 and the development of ectomycorrhizal structures in trembling aspen (Populus tremuloides) seedlings. The peak expression of LbAQP1 was 700-fold higher in the hyphae within the root than in the free-living mycelium after 24 h of direct interaction with the roots. Moreover, in LbAQP1 knock-down strains, a non-mycorrhizal phenotype was developed without the Hartig net and the expression of the mycorrhizal effector protein MiSSP7 quickly declined after an initial peak on day 5 of interaction of the fungal hyphae with the roots. The increase in the expression of LbAQP1 required a direct contact of the fungus with the root and it modulated the expression of MiSSP7. We have also determined that LbAQP1 facilitated NO, H2 O2 and CO2 transport when heterologously expressed in yeast. The report demonstrates that the L. bicolor aquaporin LbAQP1 acts as a molecular signalling channel, which is fundamental for the development of Hartig net in root tips of P. tremuloides.

Hydraulic adjustments in aspen (Populus tremuloides) seedlings following defoliation involve root and leaf aquaporins.

MAIN CONCLUSION: Changes in root and leaf hydraulic properties and stimulation of transpiration rates that were initially triggered by defoliation were accompanied by corresponding changes in leaf and root aquaporin expression. Aspen (Populus tremuloides) seedlings were subjected to defoliation treatments by removing 50, 75 % or all of the leaves. Root hydraulic conductivity (Lpr) was sharply reduced in plants defoliated for 1 day and 1 week. The decrease in L pr could not be prevented by stem girdling and it was accompanied in one-day-defoliated plants by a large decrease in the root expression of PIP1,2 aquaporin and an over twofold decrease in hydraulic conductivity of root cortical cells (L pc). Contrary to L pr and L pc, 50 and 75 % defoliation treatments profoundly increased leaf lamina conductance (K lam) after 1 day and this increase was similar in magnitude for both defoliation treatments. Transpiration rates (E) rapidly declined after the removal of 75 % of leaves. However, E increased by over twofold in defoliated plants after 1 day and the increases in E and K lam were accompanied by five- and tenfold increases in the leaf expression of PIP2;4 in 50 and 75 % defoliation treatments, respectively. Defoliation treatments also stimulated net photosynthesis after 1 day and 3 weeks, although the increase was not as high as E. Leaf water potentials remained relatively stable following defoliation with the exception of a small decrease 1 day after defoliation which suggests that root water transport did not initially keep pace with the increased transpirational water loss. The results demonstrate the importance of root and leaf hydraulic properties in plant responses to defoliation and point to the involvement of PIP aquaporins in the early events following the loss of leaves.

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.

Effect of water stress on in vitro mycelium cultures of two mycorrhizal desert truffles.

The ability of two species of desert truffle, Terfezia claveryi strain TcS2 and Picoa lefebvrei strain OL2, to tolerate water stress in pure culture has been investigated. Both T. claveryi and P. lefebvrei strains exhibited a mycelium growth pattern characteristic of drought tolerant species. However, they were only tolerant to moderate water stress, below -1.07 MPa, with the P. lefebvrei isolate being slightly more drought tolerant than the T. claveryi isolate. The increased alkaline phosphatase (ALP) activity observed in both fungi at moderate water stress with respect to the control indicated the functional adaptation of these mycelia to these drought conditions. ALP activity can be used as an indicator of the metabolic activity of these fungi. Slight water stress (-0.45 MPa) could improve mycelial inoculum production of these desert truffles. Moreover, P. lefebvrei could be a good candidate for further desert truffle mycorrhizal plant cultivation programmes in semiarid Mediterranean areas.