Characterization of Morphology, Volatile Profiles, and Molecular

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Characterization of Morphology, Volatile Profiles and Molecular Markers in Edible Desert Truffles from the Negev Desert Madhu Kamle, Einat Bar, Dalia Lewinsohn, Elinoar Shavit, Nurit Roth-Bejerano, Varda Kagan-Zur, Ofer Guy, Eli Zaadi, Efraim Lewinsohn, and Yaron Sitrit J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04063 • Publication Date (Web): 17 Dec 2016 Downloaded from http://pubs.acs.org on December 19, 2016

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Journal of Agricultural and Food Chemistry

Characterization of Morphology, Volatile Profiles and Molecular Markers in Edible Desert Truffles from the Negev Desert Madhu Kamle1, Einat Bar2, Dalia Lewinsohn3, Elinoar Shavit4, Nurit Roth-Bejerano5, Varda Kagan-Zur5, Ofer Guy6, Eli Zaadi2, Efraim Lewinsohn2, Yaron Sitrit*,1 1

The Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the

Negev, Beer-Sheva, 84105, Israel. 2

Agricultural Research Organization, Institute of Plant Sciences, the Volcani Center,

Bet-Dagan, 50250, Israel. 3

Dalpitriot, Moran 32, Timrat, Israel.

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North American Mycological Association. [email protected]

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Life Sciences Department, Ben-Gurion University of the Negev, Beer-Sheva, 84105.

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Desert Agro-Research Center, Ramat-Negev R&D, D.N. Halutza 85515 Israel.

Corresponding Author: Yaron Sitrit, The Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. FAX: 972-8-646-1984 PHONE: 972-8-647-2705 EMAIL: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Desert truffles are mycorrhizal, hypogeous fungi considered a

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delicacy. Based on morphological characters, we identified three desert truffles

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species that grow in the same habitat in the Negev desert. These include Picoa

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lefebvrei (Pat.), Tirmania nivea (Desf.) Trappe, and Terfezia boudieri (Chatain), all

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associated with Helianthemum sessiliflorum. Their taxonomy was confirmed by

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PCR-RFLP. The main volatiles of fruit bodies of T. boudieri and T. nivea were 1-

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octen-3-ol and hexanal, however volatiles of the latter species further included

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branched chain amino acid derivatives such as 2-methylbutanal and 3-methylbutanal,

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phenylalanine derivatives such as benzaldehyde and benzenacetaldehyde, and

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methionine derivatives such as methional and dimethyldisulfide. The least aromatic

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truffle, Picoa lefebvrei, contained low levels of 1-octen-3-ol as the main volatile.

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Axenic mycelia cultures of T. boudieri displayed a simpler volatile profile compared

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to its fruit bodies. This work highlights differences in the volatile profiles of desert

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truffles, and could hence be of interest for selecting and cultivating genotypes with

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the most likable aroma.

17 Keywords: Helianthemum sessiliflorum, Picoa lefebvrei, Tirmania nivea, Terfezia 18 boudieri, desert truffles, volatiles

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19 INTRODUCTION 20 Desert truffles are edible hypogeous Ascomycetes, mainly of the genera Tirmania and 21 Terfezia. Desert truffles form mycorrhizal relations with their host plants which inhabit 22 arid and semiarid zones.1 This type of symbiosis contributes to the survival of both 23 partners under the harsh conditions prevailing in these areas. The fungus supplies 24 minerals (mainly P ions) and water to the host plant and in return the host, delivers 25 photoassimilates and other nutrients to the fungus.2 The best known desert truffles are 26 members of the Pezizaceae, which also include Terfezia boudieri, Terfezia claveryi, and 27 Tirmania nivea. These hypogeous, mycorrhizal fungi, colonize the roots of plants of the 28 Cistaceae family, mainly Helianthemum species and form edible fruit bodies.3, 4 29 Records found on Amorite cuneiforms (clay tablets) dating back over 4,000 years 30 indicate that desert truffles have been consumed by indigenous people since ancient 31 times.5 Desert truffles are relatives of the well-known and highly prized European 32 truffles, which belong to the Tuberaceae family. Noted among them are the "black 33 diamond of cuisines" i.e. the Perigord truffle (Tuber melanosporum), and the Alba white 34 truffle (Tuber magnatum). Both the Tuberaceae and Pezizaceae are members of the 35 Pezizales order. Because of their intense aromas, truffles belonging to the Tuber genus 36 are normally served raw similarly to a spice added to a dish that creates a boost of 37 flavor. Conversely, desert truffles, are less intense in terms of flavors are generally 38 consumed cooked. The unique aroma characteristics of the European Tuber truffles are 39 associated with the presence of sulfur compounds, such as dimethyl disulphide and 40 dimethyl sulphide, which impart sulfurous notes. They are also associated with the 41 presence of the fatty acid derived 1-octen-3-ol, which imparts an earthy-mushroom 42 aroma. In addition, 3-ethyl-5-methylphenol, 5-methyl-2-propylphenol, β-phenylethanol, 43 2,3-butanedione and 3-ethylphenol are also prominent in European Tuber truffles.6-8

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44 Unlike the European Tuber truffles, in which the aroma constituents have been 45 identified and thoroughly described, the volatiles that impart the unique aromas of 46 desert truffles have received very little attention.9, 10 47 At least three different truffle species are known to share hosts and habitats in some 48 areas of the Negev desert, but the extent of overlap is unknown. Moreover, desert truffle 49 taxonomy is often difficult to define since many of the truffles' macro- and micro50 morphological characteristics are very similar, which may lead to misidentifications. 51 The lack of consistent phenotypic and molecular markers are major obstacles for 52 studying the distribution, typing and fructification of hypogeous fungi.11-13 We 53 previously described a molecular marker-based system, which was developed to 54 distinguish between three T. boudieri genotypes found in Israel.12 It was therefore of 55 interest to us to determine if a similar system would be adequate for the differentiation 56 of other desert truffle species. 57 The interest in developing desert truffles as alternative crops for arid zones such as the 58 Negev desert has increased in recent years, due to global warming and desertification 59 processes. The desert truffle T. clavieri, a close relative of T. boudieri, is already 60 cultivated in Spain and in the Middle East.14, 15 Nevertheless, the domestication of other 61 desert truffle species has not been successful yet. 62 In this work we describe some of the morphological characteristics of different desert 63 truffle species in the Negev desert in Israel and test the suitability of the ITS-based 64 PCR-RFLP method to distinguish among them. We analyzed the volatiles of the truffles 65 fruit bodies and of axenically grown T. boudieri cultures were also evaluated. This is the 66 first work that highlights differences in the volatile profiles of desert truffles, and could 67 hence be of interest for selecting and eventually cultivating types with superior aroma 68 properties.

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69 MATERIALS AND METHODS 70 Chemicals 71 All the standards used for volatile identifications and other reagents for tissue extraction 72 were purchased from Sigma-Aldrich (WGK, Germany). The SPME fiber used for 73 volatile extraction and the alkane solution (C7–C40), which was employed to calculate 74 the linear retention index of each analyte were purchased from Supelco (PA, USA). 75 Collection of Fruit Bodies from the Wild and at Experimental Plots. Fruit bodies 76 were collected from the wild in the area near Kibbutz Ze'elim, in the north-western part 77 of the Negev (34° 32′ N 31° 12′ E). The area consists of sandy dunes and calcareous 78 hills dominated by sporadic patches of Helianthemum spp. and Artemisia monosperma. 79 The average precipitation in the area is 120 mm/year, and temperatures may reach 260C 80 in spring during the fruiting season (Jan-Apr). Samples were collected during the spring 81 of 2015. An experimental plot was established in the Ramat Negev experimental station, 82 about 30 km south of Beer-Sheva, Israel. H. sessiliflorum plants were inoculated with T. 83 boudieri spores and the plants were grown in an open field (sandy dune) with drip 84 irrigation of fresh water (1.5 L/plant twice a week from May to November). Desert 85 truffle fruit body samples were collected from January to April 2015. 86 Axenic Cultures. T. boudieri spores were collected by grinding fruit bodies and 87 sterilized by sequential immersion in a 1% (v/v) hypochlorite and a 70% (v/v) ethanol 88 solutions for 5 min each. Mycelia of germinated spores were propagated on solid 89 Fontana medium in the dark at 250C as described before.

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One isolate termed 3-27

90 was used for volatile analyses. For that, mycelium (in three replicates) was grown on a 91 cellophane sheet that was placed onto the plates. Three weeks thereafter, mycelia was 92 carefully scraped from the cellophane and immediately extracted (see below).

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93 Morphological Analyses. Fresh fruit bodies were ground with a mortar and pestle in 94 sterile water, and the asci and ascospores were observed under a light microscope. 95 Species were identified by spore shape, specific ornamentation and size. 96 DNA Isolation. Three fresh fruit bodies of the three desert truffle species were 97 extracted as replicates. For total genomic DNA extraction, 100 mg of fresh tissues were 98 pulverized in liquid nitrogen using a pestle and mortar. The tissue was then processed 99 for DNA extraction using DNeasy plant mini kit (Qiagen) according to the 100 manufactures' protocols. 101 Molecular Characterization. The ITS region was amplified by PCR with the 102 ITS1/ITS4 pair of primers.

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To determine the ITS types the method employed by

103 Aviram et al., 2004 was followed. PCR amplification was performed and the cycling 104 parameters included an initial denaturation at 950C for 3 min, followed by 35 cycles at 105 950C for 90 s, 500C for 45 s, 720C for 90s and a final extension at 720C for 5 min. The 106 PCR reaction of 50 µl contained 0.1 µg of gDNA, 50 pmol of each primer, 0.2 mM 107 dNTPs, 0.5 U Taq- Polymerase (Fermentas) and Buffer (10 mM Tris-HCl, 1.5 mM 108 MgCl2, 0.1 mg/ml BSA). Negative controls (without DNA) were included in each 109 experiment. The amplified products were separated on 1.2 % (w/v) agarose gel run in 110 TAE buffer. 111 PCR-RFLP and Sequencing. One-fifth of the amplified PCR reaction was digested for 112 2 to 3 hrs at 370C with four units of the restriction enzyme Hinf I. The restriction 113 digestion was run in a 2.5% (w/v) agarose gel and stained with ethidium bromide. The 114 PCR amplicons were purified through PCR purification kit (Fermentas) and sequenced 115 using ITS1/4 as primers at the center for genomic technologies at the Hebrew University 116 of Jerusalem. The obtained nucleotide sequences were aligned with nBLAST at NCBI 117 site.

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118 Analysis of Fruit Bodies and Mycelia Volatiles by SPME Extraction and GC-MS. 119 SPME extraction. Fresh samples (4 individual fruit bodies for each species) were 120 frozen in liquid nitrogen and immediately ground using an IKA® A11 electric mill 121 (IKA Werke GMBH&Co.KG, Staufen, Germany). The frozen powder (120 mg of 122 idividual fruit bodies) or 0.05 to 0.1 mg axenically grown mycelia were homogenized 123 with 1 ml of a 20% (w/v) NaCl solution. One g of solid NaCl was added to the samples 124 (to inhibit enzymatic reactions) and poured into a 10 ml glass vial containing 0.1 µg of 125 2-heptanone as an internal standard. The vials were sealed and kept at 4°C until 126 analysis. As controls vials containing 20% NaCl only were used. The volatiles were 127 adsorbed for 30 min by an autosampler device (HS-SPME MPS2, Gerstel, Mülheim, 128 Germany) at 50°C using a 65 µm PDMS/DVB/CAR fiber (polydimethylsiloxane / 129 divinylbenzene/Carboxene), Supelco (PA, USA). The fiber was inserted into the 130 injection port of the GC-MS for 5 min for desorption of the volatiles in a splitless mode. 131 GC-MS Conditions 132 The SPME fiber was injected into an Agilent GCMS 6890 system coupled to a 133 quadrupole mass spectrometer detector 5973N (CA, USA). The instrument was 134 equipped with a Restek Rxi-5sil MS column (30 m length × 0.25 mm i.d., 0.25 µm film 135 thickness, stationary phase 95% dimethyl- 5% diphenyl polysiloxane). He (9.65 psi) 136 was used as a carrier gas with splitless injection. The injector temperature was 250 °C 137 and the detector temperature was 280 °C. The following conditions were used for 138 analyses: initial temperature 50 °C for 1 min, followed by a ramp of 5 °C/min to 200 139 °C, and 10 °C/min up to 300 °C (5 min). A quadrupole mass detector with electron 140 ionization at 70 eV was used to acquire the MS data in the range of 41 to 350 m/z. A 141 mixture of straight-chain alkanes (C7-C23) was injected into the column under the 142 above-mentioned conditions for determination of retention indices. The identification of 7



143 the volatiles was performed by comparison of their retention indices (KI) with those 144 found in literature and by comparison of spectral data with authentic standards or with 145 the W10N14 and QuadLib 2205 GC-MS libraries as indicated. 146 RESULTS AND DISCUSSION 147 Desert Truffles Collection and Initial Characterization. Previous surveys carried out 148 during the years 2009 and 2010 indicated that a mixed, multi-species desert truffle 149 population growing in association with H. sessiliflorum plants is present in the Negev 150 desert area (Figure 1a). Even so, during the consecutive drought years (about 80 151 mm/year) of 2011, 2012 and 2013 we were largely unsuccessful in finding any fruit 152 bodies of desert truffles in the same area, with the exception of a few fruit bodies of T. 153 boudieri. This apparent lack of fruit bodies, which was further corroborated by 154 information obtained from the local population significantly increased the market price 155 of the truffles during these years. It is noteworthy that in 2015 and 2016, which were 156 relatively rainy years (~130 mm), all three truffle species described here were amply 157 represented in the surveyed area. These collections yielded a mixed population of desert 158 truffles. The most abundant truffle collected consisted of rough, uneven, multi-lobbed, 159 brown fruit bodies on average 1 to 5 cm in diameter, which is characteristic of T. 160 boudieri. Smooth, white fruit bodies measuring 5 to 15 cm in diameter, typical of T. 161 nivea, were also very prominent. Less prominent were smaller (1 to 3 cm in diameter) 162 brown, multi-lobbed fruit bodies, typical of P. lefebvrei. Remarkably, all species were 163 collected in association with the roots of H. sessiliflorum and were located by the typical 164 cracks that form in the sand's biocrust surface above the developing fruit body. All 165 truffles were generally found at a depth of 5 to 20 cm. Still, the fruit bodies of T. nivea 166 were often present at shallower depths, and from time to time were found completely 167 exposed, and above ground. While it is well established that different species of desert 8


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168 truffles may share hosts and habitats, it is nevertheless noteworthy that these three 169 truffle species were found in association with H. sessiliflorum, and only in rare cases 170 under other species of Helianthemum such as H. salicifolium (L.) Mill, H. lippii, H. 171 vesicarium Boiss., and H. stipulatum. H. sessiliflorum usually grows in relatively 172 homogenous patches of its own. However, in the past few years the area has 173 experienced consecutive periods of drought and the populations of H. sessiliflorum have 174 receded. The vegetation in this area has been replaced by drought tolerant species such 175 as Artemisia monosperma Delile (Figure 1a), partially explaining the decrease in truffle 176 yields. In 2015, which was a relatively rainy year (130 mm), the wet conditions 177 apparently promoted the development of fruit bodies in all three species despite the 178 diminution of the host areas. In contrast to these observations, the desert truffles in the 179 well irrigated experimental plots adjacent to the non-irrigated wild areas, produced fruit 180 bodies each year (Figure 1b). It is also noteworthy that between the two edible truffle 181 species, T. nivea almost disappeared in the wild during the drought years, whereas small 182 numbers of T. boudieri fruit bodies, often negligible, were still collected in these years. 183 This may suggest that either T. boudieri or its fruiting process may be more drought 184 tolerant than T. nivea. This observation is currently being investigated and further 185 clarified since it may imply that T. boudieri may be sturdier and more appropriate for 186 development as a new crop for arid zones. The fruit bodies collected from the irrigated 187 experimental plot (Figure 1b) included T. boudieri as expected, but the occurrence of P. 188 lefebvrei fruit bodies was very prominent, although the plants were not originally 189 inoculated with this desert truffle. It is possible that a natural inoculum was present in 190 the soil before the establishment of the experimental plot, thus contributing to the 191 formation of symbiotic associations between H. sessiliflorum and P. lefebvrei.

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192 ITS-PCR RFLP Markers Developed to Discriminate Among Species. Molecular 193 typing using species-specific multiplex-PCR is a useful tool in modern taxonomy and 194 has been utilized for the identification of several Tuber species.18 Kagan-Zur et al., 1999 195

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reported on the usefulness of ITS-based identification of T. boudieri collected in the

196 Negev desert. Further work by Ferdman et al., 2009 13 demonstrated that the fruit bodies 197 of T. boudieri, which were collected in one location and were comprised of three cryptic 198 species and their phylogenetic diversity, could be resolved by ITS-PCR analysis. We 199 tested the possibility that similar molecular tools could be utilized to distinguish 200 between the different desert truffle species growing in the Negev dessert. To this end an 201 ITS-PCR system coupled with RFLP markers, using HinfI restriction enzyme was 202 developed revealing a distinct pattern for each of the three desert truffle species (Figure 203 2). The resulting amplicons were as follows: T. nivea 640 bp, T. boudieri 600 bp and P. 204 lefebvrei 600 bp. The DNA restriction fragment pattern of T. nivea amplicon gave four 205 distinct fragments of 200, 180, 160 and 100 bp (Table 1, Figure 2). The restriction 206 analysis of P. lefebvrei amplified ITS revealed three distinct fragments of 200, 160 and 207 100 bp, thus enabling an easy differentiation between these two truffles (Figure 2). For 208 T. boudieri, the restriction with HinfI also clearly discriminated the three genotypes I, II 209 and III, as was previously described.12, 13 Type I, the most abundant genotype, displayed 210 a pattern of two fragments 300 and 240 bp. Type II displayed two 310 and 250 bp 211 fragments, while type III displayed only one fragment size of 305 bp. To corroborate the 212 above findings the amplified ITS fragments of each species were sequenced. Multiple 213 sequence alignment confirmed that all three fungi belong to the same Pezizales clade 214 (data not shown). We conclude that the restriction banding patterns of the ITS-PCR and 215 RFLP can be used to unambiguously differentiate between these three desert truffle 216 species and also between the genotypes of T. boudieri, as previously noted.13 The

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217 molecular markers developed in this work enable the convenient classification of 218 unidentified truffle fruit bodies collected in natural habitats. Zitouni-Haouar et al., 2015 219

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described the morphology of several original collections of P. juniperi and P.

220 lefebvrei from Europe, North Africa and the Middle East and created a phylogenetic tree 221 of this broad collection based on molecular data of ITS sequencing. They concluded that 222 P. lefebvrei accessions collected in the Middle East and North Africa form a separate 223 clade from the European accessions. Our BLAST analyses indicate that P. lefebvrei 224 collected in the Negev desert belongs to this Middle Eastern-North African clade and 225 showed the highest homology to the Algerian originated accession BMBD33. This 226 accession BMBD33 belongs to the lineage VI, which produces spores that are verrucous 227 and smooth as we found in samples of the Negev area (Figure 3 j, k). 228 Morphological Characterization of Dessert Truffles 229 To visually distinguish between the three desert truffle species we compared the 230 morphology of the fruit bodies, asci and spores. T. nivea is characterized by sub-globose 231 to ovoid, white fruit bodies, with a pedicel which often breaks the soil biocrust and 232 appears above the ground surface, unlike the other species (Figure 3a). Its spores 233 (Figure 3b) are white-brown, oval to ellipsoidal, smooth with a guttle filled with lipid 234 (Alsheikh & Trappe, 1983a). Unripe spores of T. nivea contain an eye-ball-like structure 235 which disappears upon reaching maturity (Figure 3c). Its asci contain 6 to 8, club236 shaped spores, which display a pedicel structure (Figure 3d). 237 T. boudieri forms hypogeous fruit bodies that are usually irregular, mostly lobbed. The 238 fruit body is brown and is carried on a basal stem cord attached to the roots of 239 Helianthemum plants (Figure 3e). The spore is ornamented, pale yellow-brown, with a 240 large guttule 21 (Figure 3f, 3g). Its ornamentation is 1 to 3 µm thick and may appear like

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241 spines, but this may vary depending on the degree of maturity (Figure 3g). The asci, 242 which display short pedicels, are globose and contain 5 to 8 spores each (Figure 3h). 243 Picoa lefebvrei forms hypogeous fruit bodies that develop in close association with the 244 roots of Helianthemum (Figure 3i). Its fruit body is sub-globose, dark brown, fragile and 245 tends to break into many lobs. Ascospores are globose to oval, with a large lipid 246 guttule22 (Figure 3j, 2l). At maturity the spores are ornamented and warty (Figure 3k). 247 Unripe spores may contain an eyeball-like structure which disappears upon maturity. Its 248 asci are club-shaped, with long pedicles containing 6 to 8 spores per ascus (Figure 3l). 249 Our descriptions, which are in accordance with earlier similar descriptions were used as 250 the morphological basis for our taxonomical classification.21-26 251 Volatile Composition. Desert truffles have a particularly long history of use. However, 252 while desert truffles have been well known to indigenous peoples of the Middle East 253 since biblical times5, the potential of these species for modern intensive cultivation has 254 only recently begun to be explored. As a crop, desert truffles may have a remarkable 255 agronomical potential that has not yet been fully appreciated. Sensory evaluations of 256 desert truffle aromas by gatherers and consumers indicate that the fruit bodies of T. 257 nivea are more aromatic than those of T. boudieri, and this observation is reflected by 258 their market price.

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Conversely, the fruit bodies of P. lefebvrei have little culinary

259 value to humans due to their small size, and are often consumed by birds and rodents. 28 260 The aroma properties of foods is determined by blends of volatile organic compounds. 261 Accordingly, the total levels of volatiles were ~2-fold higher in the fruit bodies of T. 262 nivea than in the fruit bodies of T. boudieri, which in turn, were more than 10-fold 263 higher than in those of P. lefebvrei (Table 2). 264 The main volatile constituent found in all the analyzed fruit bodies was 1-octen-3-ol 265 (Table 2), which possesses a typical earthy-mushroomy odor and is accumulated and

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266 emitted by many edible mushroom species. 1-Octen-3-ol is largely used in the food 267 industry to confer mushroom notes to food products and is also an insect attractant.28 1268 Octen-3-ol is derived from the catabolism of lipids, such as linoleic acid.29 Other lipid269 derived volatiles, such as hexanal and pentanal are also prominent in all desert truffle 270 fruit body samples (Table 2). Different fruit bodies differ in the levels of other short 271 chain aldehyde constituents. Lipid-derived short chain alcohols and aldehyde volatiles 272 are important constituents of many fruits and vegetables often imparting fresh notes and 273 grassy aromas.30 274 In addition to the lipid derived volatiles, the fruit bodies of T. nivea also accumulated 275 moderate levels of 3-methylbutanal and 2-methylbutanal, both derived from the 276 catabolism of branched-chain amino acids.31 3-Methylbutanal is biosynthetically 277 derived from leucine and confers unique flavors to many foods including chocolate, 278 beer and peaches. 2-Methylbutanal is derived from isoleucine and imparts nutty notes to 279 many food products. In addition, T. nivea's volatiles include substantial levels of 280 benzaldehyde (almonds) and benzeneacetaldehyde (flowery notes). Both aromatic 281 derivatives are known to originate from the catabolism of phenylalanine.30,

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282 addition, low levels of the potent odorant methional were detected in T. nivea fruit 283 bodies (Table 2). Methional is a sulfur-containing metabolite derived from the 284 catabolism of methionine31 contributing to the aroma of potato chips. Methional is 285 readily degraded to methanethiol which, in turn forms dimethyl sulfide (also present in 286 the fruit bodies of T. nivea, Table 2) and other important sulfur metabolites. Sulfur 287 volatiles are key flavor components of the European Tuber truffles. 6, 32 The fruit bodies 288 of T. nivea also contain such compounds albeit in much lower levels than in their 289 European counterparts.

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290 Interestingly, amino acid derived volatiles were seemingly absent in the fruit bodies of 291 both T. boudieri and P. lefebvrei, which provides a possible explanation for the 292 differences emerging from the sensory evaluations. The volatile profile results were 293 similar both in fruit bodies collected from the wild, and in those that were grown under 294 agricultural experimental conditions (Sitrit et al. unpublished data). 295 It is of interest to assess the potential of producing fungal volatiles in vitro for use in the 296 food industry. Attempts to axenically culture T. nivea and P. lefebvrei have so far been 297 unsuccessful in our hands. However, the conditions needed to culture axenic T. boudieri 298 mycelia were successfully developed. The axenically grown mycelia of T. boudieri 299 displayed volatiles at much lower levels (Table 2) and only the major components found 300 in the fruit bodies, including 1-octen-3-ol and hexanal, were discernible (Table 2). Other 301 minor differences were detected in the volatile components. These include fatty acid 302 derived volatiles that were found in the axenically grown mycelia at much lower levels 303 than in the fruit bodies of T. boudieri (Table 2). Also, volatiles derived from amino 304 acids such as benzothiazole, which is known to be a potent antimicrobial agent33 and is 305 present at relatively high levels in the fruit bodies of T. nivea and T. boudieri, were 306 present at lower levels in the axenically grown mycelia (Table 2). It is well documented 307 that some beneficial bacteria are associated with the truffle fruit bodies and contribute to 308 the Tuber truffle aroma34 and it is therefore, possible that benzothiazole protects the 309 fruit bodies from spoilage bacteria. Scents play a key role in mediating the 310 communication between organisms. Fruit body volatiles may attract spore disperser 311 organisms such as rodents, other mammals and birds that consume fruit bodies. Other 312 odorants may protect fruit bodies from predation or repel pathogenic fungi and 313 bacteria.35

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314 Finally, this study and earlier works clearly indicate that desert truffles could be a 315 promising new crop for agricultural arid and semi-arid zones in the world. However, 316 more work is needed to study the factors that control their organoleptic quality which is 317 essential for the future success of these crops. 318 Acknowledgments 319 The author (MK) gratefully acknowledges the PBC Post-doc Fellowship by The 320 Council of Higher Education, Israel. 321 AUTHOR INFORMATION 322 Corresponding Author 323 * Tel: 972-8-6472705. Fax: 972-6472984.Email: [email protected]. 324 Funding 325 This work was supported by the Israel Ministry of Science and Technology, grant 3326 4766, to Katif Research Center for Coastal Desert Development and the Chief Scientist 327 of the Israel Ministry of Agriculture grants 857-0626-11 and 16-13-0008. 328 Notes 329 The authors declare no competing financial interest. 330

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331 REFERENCES 332 (1) Moreno, R.; Alvarado, P.; Majon, J. L. Hypogeous desert fungi. In Desert Truffles, 333 Kagan-Zur, V., Roth-Bejerano, N.; Sitrit, Y.; Morte, A., Eds.; Book Series on Soil 334 Biology, Springer-Verlag, Berlin, Heidelberg, Germany, 2014; pp 217-241. 335 (2) Smith, S.E.; Read, D. J. Mycorrhizal symbiosis. 3rd ed.; Academic Pr, London, UK. 336 (3) Kovacs, G. M.; Trappe, J. M. Nomenclatural history and genealogies of desert 337 truffles. In Desert Truffles, Kagan-Zur, V.; Roth-Bejerano, N.; Sitrit, Y.; Morte, A., 338 Eds.; Book Series on Soil Biology, Springer-Verlag, Berlin, Heidelberg, Germany, 339 2014; pp 21-37. 340 (4) Shavit, E. The history of desert truffle use. In Desert Truffles, Kagan-Zur, V., Roth341 Bejerano, N.; Sitrit, Y.; Morte, A., Eds.; Book Series on Soil Biology, Springer-Verlag, 342 Berlin, Heidelberg, Germany, 2014; pp 217-241. 343 (5) Shavit, E. Truffles Roasting in the Evening Fires. Fungi Maga. 2008, 1, 18-23. 344 (6)Cullere, L.; Ferreira, V.; Chevret, B.; Venturini, M. E.; Sanchez-Gimeno, A. C.; 345 Blanco, D. Characterisation of aroma active compounds in black truffles (Tuber 346 melanosporum) and summer truffles (Tuber aestivum) by gas chromatography347 olfactometry. Food Chem. 2010, 122, 300-306. 348 (7) Diaz, P.; Ibáñez, E.; Señoráns, F. J.; Reglero, G. Truffle aroma characterization by 349 headspace solid-phase microextraction. J. Chrom. A. 2003, 1017, 207–214. 350 (8) Splivallo, R.; Ottonello, S.; Mello, A.; Karlovsky, P. Truffle volatiles: from 351 chemical ecology to aroma biosynthesis. New Phytol. 2011, 189, 688–699. 352 (9) Omer, E. A.; Smith, D. L.; Wood, K. V.; El-Menshawi, B. S. The volatiles of desert 353 truffle: Tirmania nivea. Plant Food Hum. Nutr. 1994, 45, 247-249. 354 (10) Martinez-Tome, M.; Maggi, L.; Jimenez-Monreal, A.M.; Murcia, M.A.; Mari 355 J.A.T. Nutrition and antioxidant properties of Terfezia and Picoa. In Desert Truffles,

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356 Kagan-Zur, V.; Roth-Bejerano, N.; Sitrit, Y.; Morte, A., Eds.; Book Series on Soil 357 Biology, Springer-Verlag, Berlin, Heidelberg, Germany, 2014; pp 3-20. 358 (11) Bordallo, J. J.; Rodriguez, A. Cryptic and new species. In Desert Truffles, Kagan359 Zur, V.; Roth-Bejerano, N.; Sitrit, Y.; Morte, A., Eds. Book Series on Soil Biology, 360 Springer-Verlag, Berlin, Heidelberg, Germany, 2014; pp 39-53. 361 (12) Aviram, S.; Roth-Bejarano, N.; Kagan-Zur, V. Two ITS forms co-inhabiting a 362 single genet of an isolate of Terfezia boudieri (Ascomycotina), a desert truffle. Anton. 363 Leeuw. Int. J. 2004, 85, 169-174. 364 (13) Ferdman, Y.; Sitrit, Y.; Li, Y.-F.; Roth Bejarano, N.; Kagan-Zur, V. Cryptic 365 species in the Terfezia boudieri complex. Anton. Leeuw. Int. J. 2009, 95, 351-362. 366 (14) Morte, A.; Honrubia, M.; Gutierez, A. Biotechnology and cultivation of desert 367 truffles. In Mycorrhiza 3rd ed.; Varma, A., Ed.; Springer, Heidelberg, Berlin, Germany, 368 2008; pp 467-483. 369 (15) Morte, A.; Andrino, A. Domestication: Preparation of mycorrhizal seedlings. In 370 Desert Truffles, Kagan-Zur, V.; Roth-Bejerano, N.; Sitrit, Y.; Morte, A., Eds.; Book 371 Series on Soil Biology, Springer-Verlag, Berlin, Heidelberg, Germany, 2014; pp 343372 365. 373 (16) Roth-Bejerano, N.; Livne, D.; Kagan-Zur, V. Helianthemum-Terfezia relations in 374 different growth media. New Phytol. 1990 114, 235-238. 375 (17) White, T. J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of 376 fungal ribosomal RNA genes for phylogenetic. In PCR Protocols: A guide to methods 377 and applications, Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J Eds.; Academic 378 Press, New York, US, 1990; pp. 315-322.

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379 (18) Paolocci F.; Rubini, A.; Granetti, B.; Arcioni, S. Rapid molecular approach for a 380 reliable identification of Tuber spp. ectomycorrhizae. FEMS. Microbiol. Ecol. 1999, 28, 381 23-30. 382 (19) Kagan-Zur, V.; Kuang, J.; Tabak, S.; Taylor, F.; Roth-Bejerano, N. Potential 383 verification of a host plant for the desert truffle Terfezia pfeilii by molecular methods. 384 Mycol. Res. 1999 103, 1270–1274. 385 (20) Zitouni-Haouar, FE-H.; Alvarado, P.; Sbissi, I.; Boudabous, A.; Fortas, 386 Z.; Moreno, G.; Manjón, J. L.; Gtari, M. Contrasted Genetic Diversity, Relevance of 387 Climate and Host Plants, and Comments on the Taxonomic Problems of the Genus 388 Picoa (Pyronemataceae, Pezizales). PLoS ONE, 2015, 10, e0138513. 389 (21) Moreno, G.; Diez, J.; Manjon, J. L. Phaeagium lefebvrei and Tirmania nivea, Two 390 Rare hypogeous fungi from Spain. Mycol. Res. 2000, 104, 378-381. 391 (22) Alsheikh, M.; Trappe, J. M. Taxonomy of Phaeangium lefebvrei: A desert Truffle 392 eaten by Birds. Can. J. Bot. 1983, 61, 1919-1925. 393 (23) Akyuz, M.; Kırbag, S.; Bircan, B.; Gurhan, Y. Diversity and distribution of arid394 semi arid truffle (Terfezia and Picoa) in Elazığ-Malatya region of Turkey. Mycosphere, 395 2015, 6, 766–783. 396 (24) Alsheikh, M.; Trappe, J. M. Desert truffles: The Genus Tirmania. Trans. Brit. 397 Mycol. Soc., 1983, 81, 83-90. 398 (25) Sbissi, I.; Neffati, M.; Murat, C.; Gtari, M. Phylogeneticn affiliation of the Desert 399 Truffles Picoa juniper and Picoa lefebvrei. Anton. Leeuw. Int. J. 2010, 98, 429-436. 400 (26) Jamali, S.; Banihashemi, Z. Hosts and Distribution of Desert Truffles in Iran, based 401 on morphological and molecular criteria. J. Agr. Sci. Tech. 2012, 14, 1379-1396.

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402 (27) Mandeel, Q. A.; Al-Laith, A. A. Ethnomycological aspects of the desert truffle 403 among native Bahraini and non-Bahraini people of the Kingdom of Bahrain. J. 404 Ethnopharmacol. 2007, 110,118–129. 405 (28) Hall D. R.; Beevor P. S.; Cork A.; Nesbitt B. F.; Vale G. A. 1-Octen-3-ol. A potent 406 olfactory stimulant and attractant for tsetse isolated from cattle odours. Int. J. Trop. 407 Insect Sc. 1984, 5, 335-339. 408 (29) Wurzenberger, M.; Grosch, W. The formation of 1-octen-3-ol from the 10409 hydroperoxide isomer of linoleic acid by a hydroperoxide lyase in mushrooms 410 (Psalliota bispora). Biochim. Biophys. Acta 1984, 794, 25-30. 411 (30) Schwab, W.; Davidovich-Rikanati, R.; Lewinsohn, E. Biosynthesis of plant412 derived flavor compounds. Plant J. 2008, 54, 712-732. 413 (31) Gonda, I.; Bar, E.; Portnoy, V.; Lev, S.; Burger, J.; Schaffer, A. A.; Tadmor, Y.; 414 Gepstein, S.; Giovannoni, J .J.; Katzir, N.; Lewinsohn, E. Branched-chain and aromatic 415 amino acid catabolism into aroma volatiles in Cucumis melo L. fruit. J. Exp. Bot. 2010, 416 61, 1111-1123. 417 (32) Vita, F.; Taiti, C.; Pompeiano, A.; Bazihizina, N.; Lucarotti, V.; Mancuso, S.; Alpi, 418 A. Volatile organic compounds in truffle (Tuber magnatum Pico): comparison of 419 samples from different regions of Italy and from different seasons. Sci. Rep. 2015, 420 5:12629 DOII: 10.1038/srep12629. 421 (33) Keri, R. S.; Patil, M. R.; Patil, S. A.; Budagumpi, S. A comprehensive review in 422 current developments of benzothiazole-based molecules in medicinal chemistry. Eur. J. 423 Med. Chem. 2015, 89, 207-251. 424 (34) Splivallo, R.; Deveau, A.; Valdez,N.; Kirchhoff,N.; Frey-Klett, P.; Karlovsky, P. 425 Bacteria associated with truffle-fruiting bodies contribute to truffle aroma. Environ. 426 Microbiol. 2014, 17, 2647-60.

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427 (35) Morath, S. U.; Hung, R.; Bennett, J. W. Fungal volatile organic compounds: A 428 review with emphasis on their biotechnological potential. Fungal Biol. Rev. 2012, 26, 429 73-83. 430 FIGURE CAPTIONS 431 Figure 1. A natural landscape of Helianthemum spp. habitat and experimental plot. (A) 432 Growth area in the dunes of the Negev desert. Note Artemisia monosperma (dashed 433 arrow) invading the patches of Helianthemum sessiliflorum (solid arrow). (B) Plot of H. 434 sessiliflorum inoculated with Terfezia boudieri on a dune at the Ramat Negev 435 experimental station. 436 Figure 2. PCR-RFLP band patterns of desert truffles DNA digested with HinfI. Terfezia 437 boudieri Type I, Type II, Type III corresponding to lanes TI, TII, TIII. Tirmania nivea 438 Lane T.n. comprising of 200, 180, 160 and 100 bp bands. Picoa lefebvrei lane P.l, bands 439 sizes 200, 160 and 100 bp. DNA Ladder lane L (100 bp). 440 Figure 3. Morphological characteristics of the desert truffles. Tirmania nivea fruiting 441 body growing above ground (a) mature spore (b) unripe spore (c) asci with ascospores 442 with lipid guttules (d) Terfezia boudieri fruiting body carried on "leg" (e) mature-spore 443 (f) spore with warty ornamentation (g) asci containing ascospores, (h) Picoa lefebvrei 444 fruiting body growing between the hosts' roots. The color of the truffle is dark brown 445 outside and white inside. The white spots seen in the photograph are due to wounding 446 and exposure of inner tissues. (i) mature spore (j) spore with warty ornamentation (k) 447 asci containing ascospores (l) Spores and asci were visualized under light microscope. 448 Bars, 10 µm. 449

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TABLES Table 1. DNA-fragment sizes of the three desert truffle species analyzed by PCR-RFLP. DNA was extracted from fruit bodies and PCR amplified employing IT1, IT4 primers and then digested with HinfI. The restriction products were separated on a 2.5% agarose gel.

T. boudieri Type I (bp) 300

T. boudieri Type II (bp) 310

240

250

T. boudieri Type III (bp) 305

Picoa lefebvrei

Tirmania nivea

(bp) 200

(bp) 200 180

160 100

160 100

Table 2. Volatile composition of the desert truffle fruit bodies and T. boudieri axenically grown mycelia. The data presented are averages of four replicates ± S.E. Biosynthetic pathway

T. nivea

T. boudieri

(µg/g fw)

(µg/g fw)

(µg/g fw)

T. boudieri axenic mycelia (µg/g fw)

3-methylbutan-1-ol

0.04 ± 0.03

b.d.

0.52 ± 0.18

b.d.a

730

2-methylbutan-1-ol

0.04 ± 0.03

b.d.

0.12 ± 0.03

b.d.

734

MSb, KIc, ASd MS, KI, AS

1115

MS, KI, AS

Compound name

2-phenylethanol t

2-phenylbutan-1-ol 3-methylbutanal

t

2-methylbutanal Amino acids

3methylsulfanylpropanal benzaldehyde 2-phenylacetaldehyde butane-2,3-dionet

KI

Identification method

b.d.

b.d.

0.19 ± 0.09

b.d.

b.d.

b.d.

0.18 ± 0.13

b.d.

1256

MS

9.39 ± 1.38 20.39 ± 2.79

3.21 ± 0.43

0.26 ± 0.06

b.d.

655

1.83 ± 0.2

0.13 ± 0.04

MS, KI MS, KI, AS

0.73 ± 0.09

b.d.

b.d.

8.55 ± 1

b.d.

0.01 ± 0.01

b.d.

957

MS, KI, AS

4.62 ± 0.78

b.d.

0.04 ± 0.01

b.d.

1041

MS, KI, AS

0.04 ± 0.03

b.d.

597

MS, KI

769

MS, KI, AS

1227

MS, KI

739

MS, KI

0.02 ± 0.02

b.d.

b.d. b.d.

668 907

MS, KI, AS

butane-2,3-diol

b.d.

b.d.

0.24 ± 0.14

b.d.

1,3-benzothiazolet

1.49 ± 0.31

2.25 ± 0.17

0.05 ± 0.01

0.05 ± 0.01

0.16 ± 0.08

b.d.

0.01 ± 0.01

0.36 ± 0.36

b.d.

0.07 ± 0.04

b.d.

595

MS, KI, AS

hexan-1-ol

b.d.

0.04 ± 0.03

0.18 ± 0.12

b.d.

862

MS, KI, AS

heptan-2-ol

0.04 ± 0.01

0.01 ± 0.01

0.26 ± 0.14

b.d.

887

MS, KI, AS

heptanal

0.61 ± 0.13 62.99 ± 13.73

0.39 ± 0.17

0.03 ± 0.02

0.01 ± 0

903

MS, KI, AS

2.58 ± 0.82

980

MS, KI, AS

(methyldisulfanyl) methanet acetic acid

Fatty acids

P. lefebvrei

oct-1-en-3-ol

30.79 ± 6.04

7.85 ± 1.01

21


b.d.


Biosynthetic pathway

T. nivea

T. boudieri

(µg/g fw)

(µg/g fw)

(µg/g fw)

T. boudieri axenic mycelia (µg/g fw)

4.36 ± 0.77

b.d.

0.13 ± 0.05

0.12 ± 0.05

1069

MS, KI, AS

4.53 ± 0.74

0.18 ± 0.18

0.01 ± 0.01

0.02 ± 0

600

MS, KI

3.97 ± 1.33

6.74 ± 2.7

0.09 ± 0.07

0.14 ± 0.02

691

MS, KI

b.d.

b.d.

0.18 ± 0.07

b.d.

755

MS, KI

b.d.

b.d.

744

1.62 ± 0.52

802

MS MS, KI, AS

P. lefebvrei

Compound name

oct-2-en-1-ol butanal

t

pentanal

t

2-methylpentanal

t

2-ethylbutanalt

0.05 ± 0.05 23.03 ± 3.05 0.12 ± 0.09

hexanal (E)-hex-2-enal

Page 22 of 25

0.74 ± 0.17

KI

Identification method

24.29 ± 7.24

0.68 ± 0.5

b.d.

0.01 ± 0.01

b.d.

847

MS, KI, AS

954

MS, KI, AS

(E)-hept-2-enal

2.92 ± 0.46

2.49 ± 1.22

0.13 ± 0.11

0.21 ± 0.1

octanal

0.04 ± 0.04

b.d.

0.02 ± 0.01

0.05 ± 0.01

1005

MS, KI, AS

(E)-oct-2-enal

2.55 ± 0.38

1.43 ± 0.83

0.21 ± 0.1

0.14 ± 0.07

1056

MS, KI, AS

1.04 ± 0.18

0.26 ± 0.22

0.06 ± 0.05

0.03 ± 0.02

1218

0.62 ± 0.31

b.d.

0.02 ± 0.02

0.01 ± 0

1298

1.42 ± 0.59

b.d.

0.17 ± 0.15

0.01 ± 0.01

1321

MS, KI,AS

0.91 ± 0.17

0.11 ± 0.04

0.09 ± 0.06

0.03 ± 0.02

971

MS, KI,AS

0.11 ± 0.08

0.55 ± 0.4

0.04 ± 0.03

0.03 ± 0.01

1044

MS, KI

(2E,4E)-nona-2,4dienalt (2E,4Z)-deca-2,4dienalt (2E,4E)-deca-2,4dienal oct-1-en-3-one (E)-oct-3-en-2-one

t

a: b.d. : below detection limit b: Identified by comparison with W10N14 and QuadLib 2205 GC-MS libraries. c: Identified by comparison of Kovats Index from the literature. d: Identified by comparison of MS and KI with an authentic standard. T=t: tentatively identified

22


MS, KI MS, KI

Page 23 of 25


FIGURES

Figure 1.

Figure 2.

23



Figure 3. 450

24


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TOC Graphic

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Characterization of Morphology, Volatile Profiles, and Molecular

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