Distribution and phylogeography of the genus Mattirolomyces with a focus on the Asian M. terfezioides haplotypes.

Mattirolomyces is an edible commercial sequestrate genus that is globally distributed. From the five described taxa of this genus, Mattirolomyces terfezioides is the most common species in Asia. Our recent attempts to locate M. terfezioides outside its current distribution area in China documented its first records in areas of poplar trees with the lowest known temperature and precipitation averages ever recorded for this species. This peculiar ecology was not reflected on the species-morphological features nor on its phylogenetic position in the genus. The first attempt to apply the phylogenetic network approach to Mattirolomyces revealed its geographic origin in the Asian-Pacific areas prior to frequent long-distance migration events. Based on data from recent study areas, we found that the collections from Inner Mongolia and the Shanxi province were similar to European collections. Asian haplotypes were less distant from the outgroup comparing to collections from Europe, supporting the hypothesis that M. terfezioides was originated from this Chinese area and was subsequently transported to Europe. Exploring M. terfezioides ecology and its mycorrhiza potential to grow in association with poplars would be of great importance for planning cultivation projects of this valuable desert truffle species in Central and Eastern China, a currently underexploited economic sector that deserves further ecological and M. terfezioides mycorrhizal synthesis investigations.

Statically controlled mycogenic-synthesis of novel biologically active silver-nanoparticles using Hafr Al-Batin desert truffles and its antimicrobial efficacy against pathogens.

In our search for new unconventional green-reducing agent, can be applied for biosynthesis of biologically active silver-nanoparticles, fruiting bodies (Ascocarps) of Truffle Terminia sp. were collected from the sandy desert of Hafr Al-Batin, Eastern Region, Saudi Arabia. The desert truffle showed the ability to reduce AgNO3 to Ag0 depending on their high content of proteins (1.74 mg/ml) in the aqueous extract of 30 mg/ml (w/v). The response surface methodology (RSM) with 13 experiments of 2-Factors-5-Levels central composite design was applied for controlling all possible combinations of AgNO3 concentrations and pH values of reaction mixture, which directly affect the particles morphology, size and biological activity. The antimicrobial effectiveness of all synthesized nanoparticles was evaluated against the pathogenic strains by agar diffusion method. The pathogenic Gram-positive Staphylococcus aureus, Bacillus subtilis, Lactobacillus cereus, and Gram-negative Escherichia coli, Salmonella enterica, yeast strain Candida albicans and the fungus Aspergillus niger were evaluated. The biologically active Truffle-AgNPs were characterized by UV-visible spectrophotometry, transmission electron microscopy (TEM), spectrum and dynamic light scatter (DLS), and Fourier Transformed Infrared (FTIR). Results obtained indicated that, the statistically controlled Truffle-AgNPs have great inhibitory role affecting different pathogenic strains, which gained much attention towards application of Hafr Al-Batin-Truffle as reducing and stabilizing biomaterial for green nano-drugs biosynthesis, to resist harmful pathogens threaten human health.

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.

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.

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.

Characterization of natural habitats and diversity of Libyan desert truffles.

Desert truffles have traditionally been used as food in Libya. Desert truffle grows and gives fruit sporadically when adequate and properly distributed rainfall occurs with existence of suitable soil and mycorrhizal host plant. The present study aimed to identify and characterize two kinds of wild desert truffles from ecological and nutritional points that were collected from the studied area. The truffle samples were identified as Terfezia (known as red or black truffle) and Tirmania (known as white truffle). The nutritional values (protein, lipid and carbohydrate) of both Libyan wild truffle (Terfezia and Tirmania) were determined on a dry weight basis and result showed that Tirmania and Terfezia contained 16.3 and 18.5% protein, 6.2 and 5.9% lipid, 67.2 and 65% carbohydrate, respectively, in ascocarp biomass. The soil pH of the upper and lower regions of the Hamada Al-Hamra ranged between 8.2 and 8.5 giving suitable conditions for fructification. The plants, Helianthemum kahiricum and Helianthemum lippii were the dominant plants in Hamada Al-Hamra region found to form a mycorrhiza with desert truffles. The phylogenetic analysis of the genomic rDNA ITS region showed that, out of five collections three represented Tirmania pinoyi (Maire) Malencon, one Tirmania nivea (Desf.) Trappe, and one Terfezia boudieri Chatin.

Molecular and morphological data confirmed the presence of the rare species Mattirolomyces terfezioides in China.

Although the species Mattirolomyces terfezioides (≡ Terfezia terfezioides) has been recorded from China several times but it is really rare taxon with important ecological and economic value, the conspecificity with European material has never been tested by molecular data. We re-examined three specimens labelled as T. terfezioides, one as T. leonis and one as Terfezia sp. in the herbarium HMAS and obtained five ITS and three LSU sequences. Our morphological observation and DNA sequences show that one specimen (HMAS 83766) labelled as M. terfezioides turns out to be Choiromyces sp. and the other four are M. terfezioides. The ITS and (or) LSU sequences of the Chinese samples are identical with or with 99% similarity to those from the European samples, which fully confirms the presence of M. terfezioides in China. The species is currently known from northern China (Hebei Province, Beijing and Shanxi Province). This study shows that M. terfezioides has a Euroasia distribution other than European endemism and such distribution might be explained by the co-occurrence with the potential host tree Robinia pseudoacacia.

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.

The role of pre-symbiotic auxin signaling in ectendomycorrhiza formation between the desert truffle Terfezia boudieri and Helianthemum sessiliflorum.

The ectendomycorrhizal fungus Terfezia boudieri is known to secrete auxin. While some of the effects of fungal auxin on the plant root system have been described, a comprehensive understanding is still lacking. A dual culture system to study pre mycorrhizal signal exchange revealed previously unrecognized root-fungus interaction mediated by the fungal auxin. The secreted fungal auxin induced negative taproot gravitropism, attenuated taproot growth rate, and inhibited initial host development. Auxin also induced expression of Arabidopsis carriers AUX1 and PIN1, both of which are involved in the gravitropic response. Exogenous application of auxin led to a root phenotype, which fully mimicked that induced by ectomycorrhizal fungi. Co-cultivation of Arabidopsis auxin receptor mutants tir1-1, tir1-1 afb2-3, tir1-1 afb1-3 afb2-3, and tir1-1 afb2-3 afb3-4 with Terfezia confirmed that auxin induces the observed root phenotype. The finding that auxin both induces taproot deviation from the gravity axis and coordinates growth rate is new. We propose a model in which the fungal auxin induces horizontal root development, as well as the coordination of growth rates between partners, along with the known auxin effect on lateral root induction that increases the availability of accessible sites for colonization at the soil plane of fungal spore abundance. Thus, the newly observed responses described here of the root to Terfezia contribute to a successful encounter between symbionts.

Mycelium growth stimulation of the desert truffle Terfezia claveryi chatin by ß-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-, ß-, and γ) and two modified (hydroxypropil-ß and methyl-ß) CDs were assayed. The best results were obtained with ß-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.