Desert Truffles Research Articles

Hypogeous fungi in Mediterranean maquis, arid and semi-arid forests

Hypogeous fungi are common in arid and semi-arid areas of the Mediterranean basin and, in particular, they are found in Italy, Spain, northern African countries (especially in Morocco, Algeria, and Tunisia), and in the Middle East. These fungi and, in particular, some species belonging to the genus Tuber and the desert truffles (Terfezia spp. and Tirmania spp.) form ascomata of considerable economic value. In this review, a panorama of hypogeous fungal diversity and information on their ecology in Mediterranean arid and semi-arid ecosystems have been reported; their economical importance and cultivation potential are also underlined.

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.

Desert truffles of the North Algerian Sahara: Diversity and bioecology

This study reports on the bio-ecology of desert truffles collected from the Northern Algerian Sahara. It aims focused on (i) the identification of desert truffle species with a morphometric characterization, (ii) the determination of their geographical distribution, and (iii) the description of the edaphic, climatic and geomorphological characteristics of their natural habitat. The harvest of 78 truffle fruiting bodies from seven different locations resulted in the identification of three species of the family Pezizaceae: Terfezia arenaria (Moris) Trappe, Terfezia claveryi Chatin and Tirmania nivea (Desf.) Trappe. These hypogeous ascomycetes live in mycorrhizal association with Helianthemum lippii (Cistaceae). Desert truffles grow in heterogeneous soils of sandy texture, moderately calcareous (10.19 ± 1.37%), slightly alkaline (7.87 ± 0.22), with low organic matter (0.86 ± 0.1%) and slight phosphorus contents. The development of desert truffles is closely linked with high rainfall occurring during fall and/or winter. The truffles colonize desert depressions Dayas and beds of Wadis, since these geomorphological zones accumulate rainwater, which promotes the growth of both truffles and its host–plant.

Kalaharituber pfeilii and associated bacterial interactions

Truffles are generally known to form a mycorrhizal relationship with plants. Kalaharituber pfeilii (Hennings) Trappe & Kagan-Zur is a species of desert truffle that is found in the southern part of Africa. The life cycle of this truffle has not been fully investigated as there are many unconfirmed plant species that have been suggested as potential hosts. Many mycorrhizal associations often involve other role players such as associated bacteria that may influence the establishment of the mycorrhizal formation and function. As part of an effort to understand the life cycle of K. pfeilii, laboratory experiments were conducted to investigate the role of ascocarp associated bacteria. Bacterial isolates obtained from the truffle ascocarps were subjected to microbiological and biochemical tests to determine their potentials as mycorrhizal helper bacteria. Tests conducted included stimulation of mycelial growth in vitro, indole acetic acid (IAA) production and phosphate solubilising. A total of 17 bacterial strains belonging to the Proteobacteria, Firmicutes and Actinobacteria were isolated from the truffle ascocarps and identified with sequence homology and phylogenetic methods. Three of these isolates showed potential to be helper bacteria in at least one of the media tested through the stimulation of mycelial growth. Furthermore, four isolates produced IAA and one was able to solubilise CaHPO3 in vitro. One isolate, identified as a relative of Paenibacillus sp. stimulated mycelial growth on all the media tested. Other bacterial isolates that showed potential stimulation of mycelial growth were identified molecularly as a Bacillus sp. and two strains of Rhizobium sp. This study has contributed to the existing knowledge on the biotic interactions with K. pfeilii which may be useful in further symbiont and re-synthesis investigations.

Effect of pH and temperature on rheological and calorimetric behavior of desert truffles (Terfezia claveryi)

The effects of pH (4, 7 and 10) and temperature (30, 45, 60, 75 and 90°C) on the oscillatory rheology of desert truffle purees were investigated. Truffle puree exhibited predominant solid-like property (G′>G″), and behaved as a true viscoelastic fluid. Thermal transitions of puree sample exhibited presence of multiple peaks during thermo-rheological scanning of control sample from 30 to 95°C at a heating rate of 2.5°C/min. These peaks are believed to be attributed to the denaturation of various proteins. Change of pH values significantly affects the thermal transitions of truffle puree and gel strengths. Differential scanning calorimetric measurement indicated the presence of multiple peaks exclusively one at higher temperature range (119–125°C). The empirical principle of time–temperature superposition (TTS) was tested to bring experimental oscillatory rheological data at selected temperatures together into a single master curve at a reference temperature of 60°C. The shift factors varied widely with temperature for samples. The elastic modulus superposition (G′−ω) was found to fail in the temperature regime except for the sample at pH value of 4. The failure of TTS was believed to have occurred due to complex nature of protein, its thermal transition and the presence of other constituents in the puree, and temperature response of individual components.

Mycelium growth stimulation of the desert truffleTerfezia claveryichatin by β‐cyclodextrin

The commercial value of Terfezia claveryi, an edible desert truffle with important gastronomic, nutritional, and antioxidant properties, has led to growing interest in its cultivation. The erratic and slow growth of T. claveryi mycelium in vitro represents an impairment to obtain mycorrhizal plants, and it makes necessary to find a new culture medium able to overcome these drawbacks. In this work, we analyze the effect of cyclodextrins (CDs) on the growth of T. claveryi mycelium. Different parameters, including colony diameter, growth rate, and colony fresh weight, were evaluated, both in the presence and absence of these encapsulant agents. The results obtained confirm the ability of CDs to stimulate the growth of T. claveryi mycelium when present in the culture medium. A similar effect was observed when CDs were added to the culture medium of Tuber melanosporum. Three natural (α-, β-, and γ) and two modified (hydroxypropil-β and methyl-β) CDs were assayed. The best results were obtained with β-cyclodextrin, but no improvement was observed with its chemically modified derivatives. CDs complex the different compounds present in the culture medium which impair mycelial growth.

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.

Immobilization of a Polyphenol Oxidase Extract from Terfezia leonis Tul. Desert Truffle in Multilayered Silica Films for Dopamine Biosensing

An optical biosensor based on the immobilization of a crude extract from Terfezia leonis Tul. Desert truffle in silica films was developed for dopamine detection. The polyphenol oxidase activity of the extract was first compared with commercial tyrosinase, showing better stability in slightly acidic conditions and under mild heating conditions. The sol-gel encapsulation process was found favourable for the stability and preservation of the enzymatic conversion of dopamine into dopachrome. The range of linear detection of dopamine was 0.1 mM–1.5 mM with a two minutes response time. These results demonstrate the benefits of associating a cost-effective enzymatically-active extract with water-based robust silica hosts for biosensor design.

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.

First extraction of polyphenol oxidase from edible desert truffle (Terfezia leonis Tul.) and its thermal behavior

Polyphenol oxidase (PPO) is a common enzyme with large applications in food processing and analysis, especially based on their monophenolase activity. In this context, extraction and surfactant-mediated activation of PPO from desert truffle Terfezia leonis Tul. were successfully achieved. In the presence of l-tyrosine, the cresolase activity was optimal in the pH 5–6 domain and in the 35–45 °C temperature range. In the presence of pyrocatechol, the catecholase activity was optimal at neutral pH and 30 °C. Kinetics studies revealed higher affinity of PPO for l-tyrosine than for pyrocatechol. Both enzyme–substrate complexes were structurally robust, and their thermosensitivity was mainly related to entropy changes. These properties may reflect the adaptation to desert conditions where T. leonis grows and should be useful for the development of enzymatic catalysts and sensors.

Nutraceutical potential, antioxidant and antibacterial activities of Terfezia boudieri Chatin, a wild edible desert truffle from Tunisia arid zone

Nutritional composition, antioxidant and antibacterial properties of Terfezia boudieri Chatin, a desert truffle largely distributed in Southern Tunisia were evaluated. Carbohydrates were the most abundant macronutrients (62.03g/100g) followed by proteins (26.12g/100g) in T. boudieri truffle (dry mass). The ash content was 4.49g/100g dry mass and potassium, phosphorus, iron, and calcium were found to be particularly abundant in T. boudieri. The fat content of the truffle was 8g/100g dry mass, characterized by its higher content of the essential linoleic acid (n-6) (54.18%). Eight volatile compounds were identified by GC and GC/MS and they were dominated by fatty acids. The results of ascorbic acid, total carotenoids and total anthocyanins expressed on a truffle dry mass were 12.20mg/100g, 1.43mg/100g and 35.40mg/100g, respectively. The methanolic extract, containing the highest amounts of phenolics and flavonoids, displayed the highest DPPH radical-scavenging activity (IC50=0.20mg/ml) and the lipid peroxidation inhibitory activity (IC50=0.36mg/ml). Furthermore, the truffle extracts were tested for their antibacterial activity against seven species of bacteria. The methanolic extract also exhibited remarkable inhibitory activity on the tested strains, which minimum inhibitory concentration values ranged from 0.25 to 1.3mg/ml.

Determination of antimicrobial effect, antioxidant activity and phenolic contents of desert truffle in Turkey

Terfezia boudieri Chatin (Scop.) Pers., is a famous macrofungus in the world as well as in Turkey for its pleasant aroma and flavour. People believe that this mushroom has some medicinal properties. Therefore, it is consumed as food and for medicinal purposes. Chloroform, acetone and methanol extracts of T. boudieri were tested to reveal its antimicrobial activity against four Gram-positive and five Gram-negative bacteria, and one yeast using a micro dilution method. In this study, the highest minimum inhibitory concentration (MIC) value was observed with the acetone extract (MIC, 4.8 µg/mL) against Candida albicans. Maximum antimicrobial effect was also determined with the acetone extract (MIC, 39-78 µg/mL). The scavenging effect of T. boudieri on 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals was measured as 0.031 mg/mL at 5 mg/mL concentration, and its reducing power was 0.214 mg/mL at 0.4 mg/mL. In addition, the phenolic contents were determined as follows: the catechin was 20 mg/g, the ferulic acid was 15 mg/g, the p-coumaric acid was 10 mg/g, and the cinnamic acid was 6 mg/g. The results showed that T. boudieri has antimicrobial activity on the gram negative and positive bacteria as well as yeast, and it also has a high antioxidant capacity. Therefore, T. boudieri can be recommended as an important natural food source.

A Reward for Patience and Suffering: Ethnomycology and Commodification of Desert Truffles among Sahrawi Refugees and Nomads of Western Sahara

A Reward for Patience and Suffering: Ethnomycology and Commodification of Desert Truffles among Sahrawi Refugees and Nomads of Western Sahara. This paper reflects on the role of ethnobiological knowledge and practices for refugees’ agency by focusing on the use and commodification of desert truffles among the Sahrawi refugees of Western Sahara. Historically, desert truffles of the genera Terfezia and Tirmania have been an important food and medicinal resource for Saharan nomads. Today, after becoming refugees following war and forced displacement, the Sahrawi still harvest truffles for their use values, but most are sold in the Algerian town of Tindouf. This paper addresses Sahrawi food, medicinal, and veterinary uses of desert truffles, and the on–going process of commodification sustained by a high international demand and the need for cash income. This process of commodification has both helped refugees to generate income and triggered a recovery of traditional knowledge around desert truffles. However, it has also led to increasing harvesting pressure and competition among truffle collectors, thus giving rise to the risk of unsustainable harvest levels.

Tirmania pinoyi: Chemical composition, in vitro antioxidant and antibacterial activities and in situ control of Staphylococcus aureus in chicken soup

Desert truffle, Tirmania pinoyi (Maire) Malençon from Libya was chemically characterized in nutritional value, primary and secondary metabolites. Antioxidant (scavenging activity, reducing power and inhibition of lipid peroxidation) and antibacterial activity of its methanolic extract alone and in combination with potassium metabisulfite (E224) were evaluated under in vitro conditions. Furthermore, the effect of the extract on the control of Staphylococcus aureus was examined in a contaminated soup under in situ conditions. T. pinoyi was shown to be rich in carbohydrates (82.60g/100g dw) and proteins (8.06g/100g dw), being identified four free sugars (rhamnose, fructose, trehalose and mannitol) and three tocopherol isoforms (β-, δ- and γ-tocopherols). Oleic (32.29%), linoleic (29.72%) and palmitic (27.97%) acids were the main fatty acids. p-Hydroxybenzoic (300.65μg/100g dw) and cinnamic (8.30μg/100g dw) acids were quantified, as also four organic acids: quinic (0.83g/100g dw), malic (0.35g/100g dw), fumaric (0.05g/100g dw) and citric (1.26g/100g dw) acids. The methanolic extract of the mushroom showed in vitro antioxidant and antimicrobial activities. Sterilized distillated water containing 0.02% Tween 80 proved to be the most convenient solvent for in situ antimicrobial activity; the extract successfully inhibited the growth of S. aureus in chicken soup in a dose dependent manner.

A new record of the desert truffle Picoa lefebvrei in Saudi Arabia

A new record of Picoa lefebvrei from Saudi Arabia is reported accompanied by notes on its taxonomy, ecology, and distribution.

Diversity and antimicrobial properties of lactic acid bacteria isolated from rhizosphere of olive trees and desert truffles of Tunisia.

A total of 119 lactic acid bacteria (LAB) were isolated, by culture-dependant method, from rhizosphere samples of olive trees and desert truffles and evaluated for different biotechnological properties. Using the variability of the intergenic spacer 16S-23S and 16S rRNA gene sequences, the isolates were identified as the genera Lactococcus, Pediococcus, Lactobacillus, Weissella, and Enterococcus. All the strains showed proteolytic activity with variable rates 42% were EPS producers, while only 10% showed the ability to grow in 9% NaCl. In addition, a low rate of antibiotic resistance was detected among rhizospheric enterococci. Furthermore, a strong antibacterial activity against plant and/or pathogenic bacteria of Stenotrophomonas maltophilia, Pantoea agglomerans, Pseudomonas savastanoi, the food-borne Staphylococcus aureus, and Listeria monocytogenes was recorded. Antifungal activity evaluation showed that Botrytis cinerea was the most inhibited fungus followed by Penicillium expansum, Verticillium dahliae, and Aspergillus niger. Most of the active strains belonged to the genera Enterococcus and Weissella. This study led to suggest that environmental-derived LAB strains could be selected for technological application to control pathogenic bacteria and to protect food safety from postharvest deleterious microbiota.

Antioxidant and Antiradical Properties of Methanolic Extracts from Algerian Wild Edible Desert Truffles (Terfezia and Tirmania, Ascomycetes)

Wild edible truffles (namely, Terfezia leonis, Tirmania pinoyi, and T. nivea) were commercially obtained from Southern Algeria and methanolic extracts were prepared from these truffles. Their antioxidant and antiradical properties were studied by using five analytical methods: scavenging capacity on 1,1-diphenyl-2-picrylhydrazyl (DPPH·), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS·+), superoxide anion (O2·-) radicals, ferric reducing antioxidant power (FRAP), and ferricyanide/Prussian blue assay. Chemical constituents contributing to these activities were also investigated. T. leonis had the highest total phenolics, total carotenoids, and anthocyanin contents. At 2.6 mg/mL, scavenging effects on the DPPH· radical were 92.47%, 53.06%, and 41.34% for T. leonis, T. pinoyi, and T. nivea, respectively. T. leonis showed the most potent radical scavenging activities on DPPH·, ABTS·+, and O2·- radicals, with EC50 values of 1.08, 1.35, and 7.27 mg/mL, respectively. On the other hand, T. leonis exhibited the highest reductive capabilities. On the basis of the EC50 values, T. leonis had good antioxidant and antiradical properties. These results showed that methanolic extracts from these three truffles species had effective antioxidant and antiradical properties. Therefore, wild edible desert truffles could serve as an easily accessible item of food rich in natural antioxidants, as a possible food supplement, or even as a pharmaceutical agent.

The Aquaporin TcAQP1 of the Desert Truffle Terfezia claveryi Is a Membrane Pore for Water and CO2 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.

English

The antibacterial activity of various fractions of ethyl acetate extract isolated from edible fungi, Tirmania pinoyi (Maire) Malençon, growing in Algeria, was investigated. Extraction was done by the Soxhlet and the fractions obtained were purified with silica-gel column. Two fractions of ethyl acetate extract were tested against the bacteria Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633,Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 14028 andEnterococcus sp. ATCC 29212 using the agar diffusion technique. These fractions were preliminary analyzed by gas chromatography-mass spectrometry (GC-MS). The results show inhibition zones, with diameters between10 and 22 mm, againstB. subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538. The fractions were not effective against the other investigated bacteria. The GC-MS showed the presence of several products, some of them have antibacterial activity in the literature.   Key words: Edible fungi, desert truffle, Tirmania pinoyi, antibacterial activity, gas chromatography-mass spectrometry (GC-MS), ascocarps.

The diversity of Terfezia desert truffles: new species and a highly variable species complex with intrasporocarpic nrDNA ITS heterogeneity

Desert truffles belonging to Terfezia are well known mycorrhizal members of the mycota of the Mediterranean region and the Middle East. We aimed to test (i) whether the morphological criteria of Terfezia species regularly collected in Spain enable their separation and (ii) whether the previously hypothesized edaphic/biotic specificity of one group could be confirmed by study of a larger number of specimens. The species T. arenaria and T. claveryi can be identified unambiguously by morphological characters. We consider T. leptoderma as a distinct species while several lineages of similar spiny spored Terfezia truffles with cellular peridium were detected that have no obvious anatomical differences. Several species treated generally as synonyms of T. olbiensis have been described in this group, and because they cannot be unambiguously assigned to separate lineages we propose to consider the group as the T. olbiensis species complex. A high level of intrasporocarpic variation of the nrDNA ITS was detected in the T. olbiensis species complex, especially in one of its lineages. We detected no exclusive specificity to either plant associates or soil, except in T. leptoderma, which was associated with Quercus spp. and cistaceous plants on acidic soils. Nevertheless the clades showed a tendency either to associate with Quercus/Helianthemum/Cistus or Pinus hosts. Specimens having distinct anatomical features, reticulate spores and cellular peridium formed a separate group in the molecular phylogenetic analyses of nrDNA ITS and LSU regions; for these specimens we propose a new species, Terfezia alsheikhii sp. nov.