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.     

Molecular phylogeny of the mycorrhizal desert truffles (Terfezia and Tirmania), host specificity and edaphic tolerance

Terfezia and Tirmania, so called desert truffles, are mycorrhizal fungi mostly endemic to arid and semi-arid areas of the Mediterranean Region, where they are associated with Helianthemum species. The aim of this work was to study the phylogenetic relationships in these pezizalean hypogeous fungi. The restriction fragment length polymorphism (RFLP) and DNA sequences of internal transcribed spacers (ITS) of the nuclear rDNA were studied for several morphological species, Terfezia arenaria, T. boudieri, T. claveryi, T. leptoderma, T. terfezioides (=Mattirolomyces terfezioides), Tirmania nivea and T. pinoyi. The sequences were analyzed with distance and parsimony methods. Phylogenetic analyses indicated a close genetic relationship between Tirmania and Terfezia. They may have arisen from a single evolutionary lineage of pezizalean fungi that developed the hypogeous habit as an adaptation to heat and drought in Mediterranean ecosystems. This analysis also supports the re-establishment of the genus Mattirolomyces. The genera Tirmania and Terfezia were monophyletic, and morphological species corresponded to phylogenetic species. The Tirmania clade comprises desert truffles with smooth spores and amyloid asci, which were found in deserts. The Terfezia clade grouped species found in semi-arid habitats having ornamented and spherical spores. These species are adapted to exploit different types of soil (either acid or basic soils) in association with specific hosts (either basophilous or acidophilous species). Although other factors might also play a role, host specialization and edaphic tolerances (fungus and/or host tolerances) might be the key in the species diversity of these genera.     

Anatomical study on the interaction between the root endophytic fungus <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis> and Chinese cabbage

Chinese cabbage roots colonized by the dematiaceous fungal taxon Heteroconium chaetospira were previously found to become highly resistant to clubroot and Verticillium yellows. The dematiaceous fungus possesses an endophytic nature, but no detailed anatomical studies on endophyte–host plant interactions have so far been provided. Light and electron microscopy revealed that hyphae of H. chaetospira were abundant on and inside the root epidermal cells by 3 weeks following inoculation. The penetration pegs easily breached into epidermal cells, and the infection hyphae penetrated into cortical cells. Some appressorium-like swollen structures formed from intracellular hyphae, but no visible degradation of the host cell walls was evident where the hyphae contacted. No visible signs of host reactions and no invagination of the host plasma membrane around the hyphae were seen in the host cells. By 8 weeks following inoculation, masses of closely packed fungal cells had been formed in some cells of the epidermis and cortical layers, but further hyphal ingress was halted, mostly in the inner cortical cell layer. Thus, root vascular cylinders remained intact.     

Root endophyte symbiosis in vitro between the ectomycorrhizal basidiomycete Tricholoma matsutake and the arbuscular mycorrhizal plant Prunus speciosa

We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or “shiro”. We questioned whether this phenomenon could have occurred because of plant–microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant–fungal symbiosis.     

Molecular dialogues between Trichoderma and roots: Role of the fungal secretome

Trichoderma species are opportunistic fungi residing primarily in soil, tree bark and on wild mushrooms. Trichoderma is capable of killing other fungi and penetrating plant roots, and is commonly used as both a biofungicide and inducer of plant defence against pathogens. These fungi also exert other beneficial effects on plants including growth promotion and tolerance to abiotic stresses, primarily mediated by their intimate interactions with roots. In root–microbe interactions (both beneficial and harmful), fungal secreted proteins play a crucial role in establishing contact with the roots, fungal attachment, root penetration and triggering of plant responses. In Trichoderma–root interactions, the sucrose present in root exudates has been demonstrated to be important in fungal attraction. Attachment to roots is mediated by hydrophobin-like proteins, and secreted swollenins and plant cell wall degrading enzymes facilitate internalization of the fungal hyphae. During the early stage of penetration, suppression of plant defence is vital to successful initial root colonisation; this is mediated by small soluble cysteine-rich secreted proteins (effector-like proteins). Up to this stage, Trichoderma's behaviour is similar to that of a plant pathogen invading root structures. However, subsequent events like oxidative bursts, the synthesis of salicylic acid by the plants, and secretion of elicitor-like proteins by Trichoderma spp. differentiate this fungus from pathogens. These processes induce immunity in plants that help counter subsequent invasion by plant pathogens and insects. In this review, we present an inventory of soluble secreted proteins from Trichoderma that might play an active role in beneficial Trichoderma–plant interactions, and review the function of such proteins where known.     

Anatomical Structures of the VA Mycorrhiza in the Apocynaceae (Gentianales)

Roots of 19 Apocynaceae species were studied anatomically with respect to their symbiosis with VAM-fungi. In plants collected from the field, VAM-fungi were established in the root cortex. Also, inoculations with different Glomus species on the cultured plants are very successful in the infection and colonization of the root cortex. After penetration of the rhizodermis, the special exodermal short cells become colonized by winding hyphae. Then, in the root cortex of many Apocynaceae species, the VAM-fungi produce intercellular running hyphae which leads to extensive colonization of the root. Arbuscules develop on intracellular running hyphae, whereas vesicles develop mainly on intercellular hyphae. Except for some special details, this is the most common type of colonization of VAM fungi in flowering plants. But in Amsonia tabernaemontana, Nerium oleander, and Thevetia peruviana, another type of colonization could be observed. In these species, the colonization of the hyphae within the root cortex is only possible by intracellular growth. Intercellular running hyphae in the root are lacking. Therefore, after penetration the colonization in the cortex is cluster-like and strictly limited. Only by additional penetrations from hyphae in the soil, will roots show heavy infestations. This type of growth of the VAM fungi in the root is well known from the Gentianaceae and was explained as a structural incompatibility. In Catharanthus roseus, Pachypodium lamerei, and Trachelospermum jasminoides, intermediate stages of both types of colonization could be described. The results are discussed in the search for possible stimulants for structural incompatibility.     

Colonisation patterns of root tissues byPhytophthora nicotianae var.parasitica related to reduced disease in mycorrhizal tomato

Tomato plants pre-colonised by the arbuscular mycorrhizal fungusGlomus mosseae showed decreased root damage by the pathogenPhytophthora nicotianae var.parasitica. In analyses of the cellular bases of their bioprotective effect, a prerequisite for cytological investigations of tissue interactions betweenG. mosseae andP. nicotianae v.parasitica was to discriminate between the hyphae of the two fungi within root tissues. We report the use of antibodies as useful tools, in the absence of an appropriate stain for distinguishing hyphae ofP. nicotianae v.parasitica from those ofG. mosseae inside roots, and present observations on the colonisation patterns by the pathogenic fungus alone or during interactions in mycorrhizal roots. Infection intensity of the pathogen, estimated using an immunoenzyme labelling technique on whole root fragments, was lower in mycorrhizal roots. Immunogold labelling ofP. nicotianae v.parasitica on cross-sections of infected tomato roots showed that inter or intracellular hyphae developed mainly in the cortex, and their presence induced necrosis of host cells, the wall and contents of which showed a strong autofluorescence in reaction to the pathogen. In dual fungal infections of tomato root systems, hyphae of the symbiont and the pathogen were in most cases in different root regions, but they could also be observed in the same root tissues. The number ofP. nicotianae v.parasitica hyphae growing in the root cortex was greatly reduced in mycorrhizal root systems, and in mycorrhizal tissues infected by the pathogen, arbuscule-containing cells surrounded by intercellularP. nicotianae v.parasitica hyphae did not necrose and only a weak autofluorescence was associated with the host cells. Results are discussed in relation to possible processes involved in the phenomenon of bioprotection in arbuscular mycorrhizal plants.     

Molecular phylogeny of truffles (Pezizales: Terfeziaceae, Tuberaceae) derived from nuclear rDNA sequence analysis.

Extensive morphological convergence or divergence, a common occurrence in fungi, tends to obscure recognition of phylogenetic relationships among Pezizales, widespread filamentous Ascomycetes with either enclosed underground (hypogeous) or exposed (epigeous) fruit bodies, that often establish mutualistic interactions with arboreous plants. Focusing on hypogeous Pezizales commonly known as truffles, we sequenced the 18S rDNA from nine species belonging to three different families (Tuberaceae, Terfeziaceae, and Balsamiaceae). A data set consisting of 1700 secondary structure-aligned sites, including 24 homologous sequences from the GenBank DNA database and using three reconstruction methods, was employed to infer phylogenies in an interval ranging from the subordinal to the subgeneric level. As revealed by the 18S phylogenetic scheme, Balsamiaceae represent a monophyletic clade, comprising the hypogeous taxa Balsamia and Barssia, nested within Helvellaceae. Similarly, the terfeziacean genera Pachyphloeus and Terfezia constitute together with Cazia a distinct hypogeous clade nested within Pezizaceae. The lack of clustering between Terfezia arenaria and Terfezia terfezioides strongly supports the reassignment of the latter taxon to the original monotypic genus Mattirolomyces. Within Tuberaceae, which are sister to the highly evolved Helvellaceae, the genus Tuber cannot be considered monophyletic if Choiromyces is recognized. The paraphyly of Tuber and other relationships that were not supported by high bootstrap values, nor corroborated by morphological evidence, were supported by a parallel analysis of the faster evolving internal transcribed spacer (ITS) rDNA. Distinct episodes of fruit body morphology shifts are discernable in the 18S rDNA phylogenetic tree. In all cases, the shift from an epigeous to a hypogeous form is the most parsimonious interpretation of character transformation, without any instance of character reversal.     

赤松不定根和愈伤组织与松口蘑外生菌根菌的相互作用

本实验报道了以简单的离体培养方式来诱导赤松不定根和愈伤组织与松口蘑的菌根反应。不定根和愈伤组织均起源于无菌苗的下胚轴,接种2周后,菌丝体开始包围不定根。接种3周后,菌丝体出现在不定根皮层细胞间,哈蒂氏网型的形成也同时被确认。在愈伤组织培养物中,细胞间也能观察到菌丝体及拟-哈蒂氏网结构。这是第一个离体条件下成功地诱导赤松培养组织与松口蘑形成外生菌根的报道。     

黄河三角洲滨海盐碱地11个造林树种细根解剖性状对土壤条件的响应

细根作为植物与土壤连接的重要部位,能够反映植物对生存环境的适应性。以黄河三角洲滨海盐碱地不同立地条件下11个造林树种为对象,基于细根分支等级划分1-4级根序并进行解剖特征测定,分析细根解剖性状对滨海盐碱地不同土壤条件的响应规律。结果表明：（1）不同根序的细根直径存在显著差异,细根直径随根序升高呈增大趋势,而同根序的细根直径在不同树种间表现出显著的种间差异（P < 0.05）。1-2级细根皮层厚度、3-4级细根导管密度在树种间的差异均达显著水平（P < 0.05）。（2）在较为严重盐渍化土壤条件下（立地1）,细根皮层厚度较其他立地显著增大,但细根导管密度较小;在轻度盐碱立地条件下（立地3）,细根导管密度较大;较为严重的盐碱立地具有更为发达的细根直径及维管柱直径。（3）树种1-2级细根解剖结构与土壤环境关系最为密切,其中1级根直径与土壤pH值显著正相关（P < 0.05）,与土壤硝态氮含量呈显著负相关（P < 0.05）。对土壤理化性质与细根解剖性状的冗余分析表明,前两个轴的特征值达0.640和0.196,土壤速效养分含量与轴一（RDA1）呈正相关,低级根解剖性状则与轴二（RDA2）呈显著负相关。低级根解剖结构以及土壤的pH值能解释较多树种的差异性,其中低级根直径与皮层厚度对盐碱环境表现出较强的响应。     

海南岛4个热带阔叶树种前5级细根的形态、解剖结构和组织碳氮含量

细根具有复杂的分支系统, 以根序(root order)为取样单元的细根生理生态学研究正在成为根系生态学研究领域的重要内容。该研究以海南岛尖峰岭4个热带阔叶树种海南蕈树(Altingia obovata)、厚壳桂(Cryptocarya chinensis)、山杜英(Elaeocarpus sylvestris)和黄桐(Endospermum chinense)为研究对象, 测定了1-5级细根的形态、解剖结构和组织碳(C)、氮(N)含量, 旨在探讨这些根系特征之间的联系。研究表明: 4个树种的细根形态差异较大, 但在树种水平上直径、根长和组织密度均随着根序的升高而增加, 比根长则随着根序的升高而降低; 低级根(前2级根或前3级根)具有皮层组织, 是典型的吸收根, 而高级根皮层组织消失, 是典型的运输和储藏根; 影响直径大小最重要的因子是皮层厚度, 它可以解释细根直径变异的97%, 而维管束直径仅能解释细根直径变异的70%; 根组织N和C浓度受维管束-根直径比(维根比)的影响, 随着维根比增加, 组织N浓度显著降低, 组织C浓度显著升高。4个树种细根的C/N比的变异受组织N浓度的影响程度为76%, 而受C浓度的影响程度不足10%。上述结果表明, 细根的形态特征、解剖结构和组织化学含量之间存在着紧密联系, 这为我们理解根系结构与功能变异提供了重要依据。     

'Prepackaged symbioses': propagules on roots of the myco-heterotrophic plant Arachnitis uniflora

Arachnitis uniflora, a myco-heterotrophic plant species, has fleshy tuberous roots colonized by the arbuscular mycorrhizal fungal genus Glomus (Phylum Glomeromycota). These roots produce apical and lateral propagules, both reported here for the first time. The objective of the study was to characterize the ontogeny and structure of the propagules, and to determine their function. Scanning electron microscopy, laser scanning confocal microscopy and light microscopy were used to study the ontogeny and structure of the propagules. Propagules developed either from cortical parenchyma cells or from cells immediately beneath the root cap; they developed a shoot meristem and cells in the basal region which were colonized by various fungal structures including hyphae and vesicles. These propagules may detach from the roots, establishing new plants.     

40种常见药用草本植物根系解剖特征研究

植物的根系具有水分吸收和运输的功能,部分还有重要的药用价值。该研究观察了40种药用草本植物粗根的解剖特征,比较了不同功能类群的差异性并分析了性状的相关性,同时结合系统发育和自然分布区气候因子解释了根系性状的种间变异性。结果表明,草本植物根系解剖特征种间差异显著;与双子叶植物相比,单子叶植物的根皮层占比更高,而中柱占比更小,揭示了两者根系吸收和运输策略的差异性。性状网络分析表明,木质部面积占比具有高的度和紧密度,是其中的中心性状,木质部面积占比与韧皮部面积占比正相关但与皮层占比负相关。除导管频度和皮层占比外,其他特征均未显示出显著的系统发育信号,说明根系性状受系统发育的影响较少。在单子叶草本植物中,年平均降水量与皮层占比呈正相关;而在双子叶草本中,年平均气温与导管直径正相关。该研究进一步揭示了药用草本植物根系结构的多样性和适应性。     

Land-use change impact on mycorrhizal symbiosis in female and male plants of wild <Emphasis Type="Italic">Carica papaya</Emphasis> (Caricaceae)

It has been acknowledged that land-use change has negative effects on genetic diversity and sex ratio in dioecious species, but less attention has been paid on the influence that land-use change has on the biotic interactions, especially between dioecious species and arbuscular mycorrhizal (AM) fungi. AM mutualism involves reciprocal transfer of carbohydrates and mineral nutrients between the host plant’s roots and these fungi. Here, we report spatial and temporal variation in AM colonization in dioecious wild Carica papaya plants growing in sites with different land use intensity. We tagged, recorded the basal stem circumference and collected roots of reproductive female and male Carica papaya plants in three wild sites during dry and rainy seasons of western Mexico. We also collected soil samples in each site to conduct soil chemical analyses. The sexes of C. papaya did not show significant differences in the frequency (percentage of root colonized by intraradical fungal structures) and abundance (length of intraradical hyphae) of AM fungi but the higher AM colonization was observed during the dry season, and in the site with the lowest disturbance. There was no relationship between soil chemistry and AM colonization. Overall, our findings suggest that land-use intensity has a negative effect on AM colonization and we discuss the consequences of habitat loss for the reproductive female and male plants, the implications of decreasing AM colonization for wild Carica papaya plants an important species that provides a source of genetic variation for the C. papaya varieties.     

Extraradical mycelium of the arbuscular mycorrhizal fungus Glomus lamellosum can take up,accumulate and translocate radiocaesium under root-organ culture conditions

Radiocaesium enters the food chain when plants absorb it from soil, in a process that is strongly dependent on soil properties and plant and microbial species. Among the microbial species, arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the root cortex of many plants and develop an extraradical mycelial (ERM) network that ramifies in the soil. Despite the well-known involvement of this ERM network in mineral nutrition and uptake of some heavy metals, only limited data are available on its role in radiocaesium transport in plants. We used root-organ culture to demonstrate that the ERM of the AM fungus Glomus lamellosum can take up, possibly accumulate and unambiguously translocate radiocaesium from a 137Cs-labelled synthetic root-free compartment to a root compartment and within the roots. The accumulation of 137Cs by hyphae in the root-free compartment may be explained by sequestration in the hyphae or by a bottleneck effect resulting from a limited number of hyphae crossing the partition between the two compartments. Uptake and translocation resulted from the incorporation of 137Cs into the fungal hyphae, as no 137Cs was detected in mycorrhizal roots treated with formaldehyde. The importance of the translocation process was indicated by the correlation between 137Cs measured in the roots and the total hyphal length connecting the roots with the labelled compartment. 137Cs may be translocated via a tubular vacuolar system or by cytoplasmic streaming per se.     

Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots

A very large number of plant species are capable of forming symbiotic associations with arbuscular mycorrhizal (AM) fungi. The roots of these plants are potentially capable of absorbing P from the soil solution both directly through root epidermis and root hairs, and via the AM fungal pathway that delivers P to the root cortex. A large number of phosphate (P) transporters have been identified in plants; tissue expression patterns and kinetic information supports the roles of some of these in the direct root uptake pathways. Recent work has identified additional P transporters in several unrelated species that are strongly induced, sometimes specifically, in AM roots. The primary aim of the work described in this paper was to determine how mycorrhizal colonisation by different species of AM fungi influenced the expression of members of the Pht1 gene families in the cereals Hordeum vulgare (barley), Triticum aestivum (wheat) and Zea mays (maize). RT-PCR and in-situ hybridisation, showed that the transporters HORvu;Pht1;8 (AY187023), TRIae;Pht1;myc (AJ830009) and ZEAma;Pht1;6 (AJ830010), had increased expression in roots colonised by the AM fungi Glomus intraradices,Glomus sp. WFVAM23 and Scutellospora calospora. These findings add to the increasing body of evidence indicating that plants that form AM associations with members of the Glomeromycota have evolved phosphate transporters that are either specifically or preferentially involved in scavenging phosphate from the apoplast between intracellular AM structures and root cortical cells. Operation of mycorrhiza-inducible P transporters in the AM P uptake pathway appears, at least partially, to replace uptake via different P transporters located in root epidermis and root hairs. Electronic Supplementary Material Supplementary material is available for this article at     

Homokaryotic and heterokaryotic hyphae in Terfezia

Mycelia of Terfezia pfeilii (Ascomycetes) were obtained by two methods, i.e., from the sterile hyphae of fresh fruit bodies or by germinating ascospores. Nuclear staining revealed the existence of multinucleate cells in all mycelia. Paired nuclei were observed only in mycelia obtained from sterile hyphae proliferation, while single nuclei were found in mycelia originating from singly germinated spores. Co-cultivation of mycelia from two different ascospores apparently facilitated plasmogamy, resulting in mycelia with paired nuclei. Terfezia boudieri cultures originating from sterile hyphae also exhibit paired nuclei, indicating the possible existence of a long-term heterokaryon. The timing of plasmogamy and karyogamy in Terfezia is discussed.     

Nitrate uptake by bean (Phaseolus vulgaris L.) roots under phosphate deficiency

Bean (Phaseolus vulgaris L.) plants were cultured for 19 d on complete or on phosphate deficient culture media. Low inorganic phosphate concentration in the roots decreased ATP level and nitrate uptake rate. The mechanisms which may control nitrate uptake rate during phosphate deficiency were examined. Plasma membrane enriched fractions from phosphate sufficient and phosphate deficient plants were isolated and compared. The decrease in total phospholipid content was observed in plasma membranes from phosphate deficient roots, but phospholipid composition was similar. No changes in ATPase and proton pumping activities measured in isolated plasma membrane of phosphate sufficient and phosphate deficient bean roots were noted. The electron microscope observations carried out on cortical meristematic cells of the roots showed that active ATPases were found in plasma membrane of both phosphate sufficient and phosphate deficient plants. The decrease in inorganic phosphate concentration in roots led to increased nitrate accumulation in roots, accompanied by a corresponding alterations in NO₃ distribution between shoots and roots. Nitrate reductase activity in roots of phosphate deficient plants estimated in vivo and in vitro was reduced to 50–60% of the control. The increased NO₃ concentration in root tissue may be explained by decreased NR activity and lower transport of nitrate from roots to shoots. Therefore, the reduction of nitrate uptake during phosphate starvation is mainly a consequence of nitrate accumulation in the roots.     

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.