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Desert truffles are edible hypogeous (forming fruit bodies below ground) fungi that grow in semi-arid and arid areas. They are highly valued for both their culinary and medicinal properties in the Mediterranean basin, the Middle East and the Gulf areas. Desert truffles form mycorrhizae mostly with plants belonging to the Cistaceae family, mainly with Helianthemum species. These truffles are still, usually, collected from the wild, but loss of habitats due to urbanization, desertification, intensive agriculture and global warming, along with an urgent need to develop new crops adapted to arid conditions, are currently hastening efforts towards their domestication. Here, we sum up the successful research leading to cultivation of this crop, based on plots that were established in sandy to silt soils with high pH values and low mineral contents. We report suitable methods for production of mycorrhized seedlings and preferred planting methods. We found that under natural conditions yields are affected by water availability, so irrigation regimes to ensure good yields were sought. Although good yields were indeed obtained in some years, fluctuations in yields over the years were significant; the reasons for this are not entirely clear and are currently under study. This crop is particularly well suited to relatively marginal conditions but prospects for establishment of desert truffles as a niche crop for arid and semi-arid areas depend on further improvements in yields.

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 μM of acetosyringone, attaining a bacterial optical density of 0.3 OD600; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.

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.

Desert truffles are mycorrhizal, hypogeous fungi considered a delicacy. On the basis of morphological characters, we identified three desert truffle species that grow in the same habitat in the Negev desert. These include Picoa lefebvrei (Pat.), Tirmania nivea (Desf.) Trappe, and Terfezia boudieri (Chatain), all associated with Helianthemum sessiliflorum. Their taxonomy was confirmed by PCR-RFLP. The main volatiles of fruit bodies of T. boudieri and T. nivea were 1-octen-3-ol and hexanal; however, volatiles of the latter species further included branched-chain amino acid derivatives such as 2-methylbutanal and 3-methylbutanal, phenylalanine derivatives such as benzaldehyde and benzenacetaldehyde, and methionine derivatives such as methional and dimethyl disulfide. The least aromatic truffle, P. lefebvrei, contained low levels of 1-octen-3-ol as the main volatile. Axenic mycelia cultures of T. boudieri displayed a simpler volatile profile compared to its fruit bodies. This work highlights differences in the volatile profiles of desert truffles and could hence be of interest for selecting and cultivating genotypes with the most likable aroma.

A Tuber melanosporum plantation established in 1994/1995 on Kibbutz Bar’am (in the Upper Galilee, Israel) gradually lost its T. melanosporum mycorrhiza. In 1999, T. aestivum inoculated seedlings were inadvertently introduced into the plantation to fill the gaps between trees. A single T. melanosporum fruit body was found in 1999. Although no truffles were found after 1999 and until 2009, in that year and in 2010, truffles were collected. Morphological and molecular analyses proved these to be T. aestivum. Thus, the intentionally introduced T. melanosporum mycorrhiza was replaced by that of another introduced mycorrhizal fungus, T. aestivum. Local oak species produced higher yields compared to introduced host species known to be good T. melanosporum plant symbionts. The yield was comparable to that reported for young commercial orchards, but the fruiting season was earlier than in Europe.

The Israeli Negev is the habitat of at least two brown desert truffles, the most abundant of which is the complex known as Terfezia boudieri. Using molecular phylogenetic approaches, we were able to demonstrate the existence within this complex of three cryptic species exhibiting physiological, but not morphological, discriminating characters. A culture harboring two distinct internal transcribed spacer (ITS) types led us to the discovery of long-term heterokaryons in the glebal outgrowth of these truffles (Ascomycetes). Desert truffles form endomycorrhizas or Terfezia-type ectomycorrhizas. By following the formation of mycorrhizas in a laboratory system, we were able to unravel some of the underlying conditions responsible for the establishment of each type of association. Using a similar system, we also isolated genes involved in determining mycorrhizal type. Some of these genes were further analyzed, leading to identification of a peptide of unknown function that is involved in the determination process.

Motivated by the increasing interest in the black Perigord truffle (Tuber melanosporum), we have been seeking to develop a controlled cultivation method for this celebrated fungus. Our strategy is based on establishing optimal conditions for mycorrhizal development in detached media by mimicking natural productive truffle beds. Cistus incanus, a Mediterranean shrub, was used as host. The components of the growth medium—polystyrene foam flakes, Perlite, peat, and dolomite powder—provided the coarse and fine porosity required for adequate drainage, appropriate balance between aeration and moisture, the desired pH (7.5-8.2), and support of mycorrhizal development. Optimum rhizosphere temperatures for inoculation and continued mycorrhizal development were 20-25 °C. Inoculation with mycorrhized root fragments proceeded more rapidly than traditional inoculation with a carpophore suspension, particularly at suboptimal growth medium temperatures. A keystone of our system is to determine the optimum root volume re...

Progress was made towards a reliable in vitro system for mycorrhizing Cistus incanus seedlings with Tuber melanosporum. A rich growth medium favored extensive growth of mycorrhized Ri-transformed roots (MTR) but inhibited mycelial outgrowth into the medium. A minimal medium, on the other hand, inhibited MTR growth but supported considerable mycelial outgrowth into the medium. While the presence of a C.␣incanus propagule clearly enhanced mycelial growth into the minimal medium, a highly significant factor appeared to be the use of MTR inoculant, which supported mycorrhizal development to the Hartig net stage. The advantages of MTR for in vitro mycorrhization of host plant seedlings are discussed.

A Tuber melanosporum plantation established in 1994/5 on Kibbutz Bar’am (in the Upper Galilee, Israel, Fig. 1) gradually lost its T. melanosporum mycorrhiza. When checked in 1998, only about 70% of the trees maintained their original mycorrhiza (Kagan-Zur et al. 2001) and was deteriorating constantly. A number of trees perished. Several different tree species were initially introduced. These included both ACTA MYCOLOGICA Vol. 47 (2): 169–171 2012

The effect of Ca2+ on mycelial growth in Terfezia boudieri was studied. Terfezia boudieri Chatin (Ascomycotinae) occurs in mycorrhizal association with Helianthemum shrubs in deserts with calcareous soils. External Ca2+ stimulated mycelial growth in both liquid media and solidified substrates. The response to Ca2+ was very faint in well-aerated culture but pronounced in mycelia immersed in the medium, indicating dependence on mycelial aeration. 2,4-Dinitrophenol (DNP), an inhibitor of oxidative phosphorylation, and succinate, a potential cytoplasm acidifier, inhibited mycelial growth but enhanced the stimulatory effect of Ca2+. This effect was reduced by the Ca2+ channel blocker verapamil.

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.

Two fruit-bodies of Terfezia boudieri Chatin, each exhibiting a mixture of two ITS -RFLP profiles, were found in the Negev desert of Israel. A mycelial culture obtained from glebal out-growth maintained the double profile, as did proliferating cultures established using single hyphae isolated from the original cultures. The main difference between the two ITS variants lies in a 21 bp deletion in the smaller variant. The question whether both variants are contained within a single nucleus or occupy different nuclei sharing the same cytoplasm is discussed.

Seedlings of Vangueria infausta, an indigenous Kalahari tree, were used as plant symbionts in a pot experiment designed to measure the effect of three local vesicular–arbuscular mycorrhizal (VAM) fungal communities on mineral acquisition and concentrations in host shoots. VAM significantly affected seedlings size. The highest level of VAM mycorrhizae occurred in soil with intermediate P concentration. Two VAM communities had the highest mycorrhizal response in their soils of origin. The presence of VAM caused a significant depletion of P from soils. VAM communities differed in their ability to promote P, Ca and N uptake. A significant interaction between VAM community and soil type—in terms of Ca and N concentration in shoots—indicates a local adaptation of the VAM species.

We set out to establish root cultures of a host plant with the aim of obtaining dual cultures of Tuber melanosporum mycorrhiza on transformed roots. Seedlings of Cistus incanus germinated under sterile conditions from seeds collected in the wild were treated with Agrobacterium rhizogenes. Nine hairy roots collected from different seedlings were cultured individually by repeated subculturing. The hairy root clones differed in growth rates and in morphology (branching frequency and distance between side roots). Root growth in a liquid medium exhibited a lag phase of about 2 weeks and an exponential phase lasting about 12 days before the start of the stationary phase. Hairy roots could be kept alive on medium M, a special solid minimal medium (low in Fe2+, BO43–, Ca2+, Cu2+ and Zn2+, very low in PO43– and lacking MoO42–, NH4+ and Co2+), for more than 7 months. T. melanosporum could be grown on the same medium for long periods only by subculturing the fungus with the roots. A mycorrhizal association developed between the roots and the T. melanosporum mycelium within 3 months. The association consisted of elongated roots with a mantle and a Hartig net surrounding two to three layers of cortical cells. Swollen, club-like root tips were discernible 5 months after inoculation. The mycorrhized roots could be subcultured and propagated on medium M and maintain the mycorrhizal association.

In the framework of efforts to introduce Tuber melanosporum as a cultivated crop to Israel, spores of the truffle, obtained from fruit-bodies procured in Italy and France, were used to inoculate oak seedlings and hazel suckers. Typical T. melanosporum mycorrhizas were observed 3 months after inoculation on roots of both plant species. One- to two-year-old mycorrhizal seedlings were outplanted at a number of experimental sites and irrigated regularly. Two sites characterized by alkaline soil but differing in soil composition and climatic conditions were chosen for the present study. DNA of ascocarps used for inoculation, DNA of re-isolated cultures and fungal DNA taken from tree mycorrhizas 4 years after outplanting were compared with T. melanosporum reference cultures by molecular methods. All T. melanosporum profiles proved to be identical except for one belonging to a reference culture, which exhibited an unusual HinfI ITS-RFLP pattern. A single base substitution, responsible for the different HinfI restriction site, distinguished the ITS region of this culture from a published T. melanosporum ITS sequence. ITS restriction polymorphism analyses determined that roots of all potted plants tested and many 4-year-old trees from the two experimental plots (irrespective of soil and climatic differences) were colonized by T. melanosporum.

The mating system and ploidy level of truffles, both mycorrhizal and sterile fruit-body hyphae are the subject of conflicting hypotheses. The current dogma concerning Ascomycetes, supported by recent studies on truffles, maintains that the mycorrhizing mycelium is homokaryotic and that the short-lived ascogenous heterokaryotic hyphae resulting from the fertilization process occur only in truffle primordia. Therefore the gleba and sterile veins are also homokaryotic. Other studies show heterokaryons both in gleba and mantle hyphae of some truffle species, leading to the conclusion that long- term heterokaryons do exist in truffles. The matter is discussed. Some fundamental aspects concerning the life cycle of truffles (Pezizales) (1) have yet to be fully elucidated. Among these are the mating system and the ploidy level of both mycorrhizal and sterile fruit-body hyphae. Both are the subject of conflicting hypotheses. Tuber and Terfezia hyphal cells, like those of other As- comycetes, are multinucleate (e.g. (2)). Ascertaining whether a mycelial culture is a homo or heterokaryon is, therefore, not straightforward. Nowadays, however, the prevailing per- ception, which is based on in-depth research, casts doubt on the long-term subsistence of natural vegetative heterokary- ons in most filamentous Ascomycetes (e.g. (3)). The hypothesis that tends to be accepted today - a hy- pothesis that has been thoroughly investigated - maintains that the vegetative mycelium of such fungi is homokaryotic and that plasmogamy occurs only in the primordia of the fruit bodies. Plasmogamy in the developing fruit body leads to the formation of heterokaryotic ascogenous hyphae, from which, after karyogamy and meiosis, haploid ascospores develop (e.g. (2)). As a result, the heterokaryotic phase is brief and is restricted to the developing ascocarps. Yet, it had long been known that long-term heterokary- ons exist in Neurospora crassa, Pezizomycotina (4). This was later shown to be dependent on the existence of identical het alleles in the individual strains forming the heterokaryon (5). Wild type Neurospora tetrasperma isolates are invaria- bly heterokaryons, mostly selfing, but maintain some level of outcrossing in nature, as well as in the laboratory (6). This phenomenon is also dependent on het allele identity in both nuclei forming the long-term heterokaryion (ibid). Thus con- ditions under which long term ascomyceteous heterokaryons may be maintained - do exist. No het genes were, so far, identified or demonstrated in truffles. But Terfezia boudieri and Kalaharituber pfeilii het- erokaryon mycelial cultures originating from glebal out- growth seem to be the rule rather than exception: In glebal K. pfeilii and T. boudieri cultures, the hyphal cells invariably

The 930,000 km2 Kalahari covers nearly all of Botswana, the eastern third of Namibia, and Northern Cape Province of South Africa. It is a basin-like, arid and semiarid plain 900 m or more in elevation with a long history of use of truffles by its native people. Three genera and species of desert truffles have been recorded from the Kalahari: Kalaharituber pfeilii is a monotypic endemic. Eremiomyces has also been reported from Spain and Mattirolomyces is a cosmopolitan genus, at least in terms of xeric habitats, but their Kalahari species, E. echinulatus and M. austroafricanus, are endemic. Kalaharituber pfeilii has been prized as a food by the San back into their prehistory. It is harvested for personal use but also for sale to distributors for both domestic use and European markets. Depending on rain in a region where rain is undependable, its production understandably varies strongly from year to year.

It has long been known that under suboptimal conditions mycorrhized plants fare better than their non-mycorrhized counterparts. This applies to desert truffles as well: when conditions are less than favorable, plants that enter into mycorrhizal associations with desert truffles exhibit higher levels of transpiration, stomatal conductance, and photosynthesis. Furthermore, studies of the gene expression of a fungal aquaporin in the mycorrhizal state revealed negative correlations with plant physiological parameters, suggesting that there is some form of communication between the symbionts. In particular, fine-tuning of plant aquaporin gene expression is responsive to water availability. Consistent with results reported for other mycorrhizal fungi, mineral acquisition was higher in plants mycorrhized by desert truffles, as was photosynthesis. Girdling led to noticeable reduction in carbon assimilation in non-mycorrhized plants but only to a slight reduction in mycorrhized ones. In addition, not only was general chlorophyll content higher in plants forming associations with desert truffles, but the ratio of chlorophyll b to chlorophyll a was higher too, leading to improved light harvesting under the lower irradiance conditions typical of mornings and afternoons. Correspondingly, the calculated activation energy for onset of photosynthesis was lower for desert truffle-mycorrhized plants.

Each of the two members of the mycorrhizal couple, the plant and the fungus, has developed mechanisms for signaling the other prior to the actual formation of the mycorrhizal association. The symbiotic pair Terfezia boudieri (Pezizaceae, a desert truffle) and Helianthemum sessiliflorum (Cistaceae) was studied with a view to determining how fungal secretion of auxin—a plant hormone—promotes establishment of mycorrhizas, on the one hand, and how plant chemoattractants affect fungal growth, on the other. In view of the known effects of auxin—inhibition of tap root growth, enhancement of secondary root development, bending of the direction of root growth towards the horizontal (negative chemotropism)—it seems that auxin secretion by the fungus is designed to keep the root tips (which are the fungal penetration sites) within its reach. Indeed, fungi grow about ten times more slowly than developing roots. Inhibition of root hair formation, another effect of fungal auxin, makes the plant dependent on the fungus for its nutrients. It is known that fungal oriented growth towards plant roots is very pronounced under limiting sugar availability and much weaker under high sugar concentrations. Our research has recently focused on isolation of potential plant chemoattractants; so far, one as yet unidentified compound with a molecular weight of about 535 has been found to possess chemotropic activity.

The effects of extended heat stress on polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) gene expression at mRNA, protein and activity levels in ripening tomato fruits were investigated. Steady state levels of PG mRNA declined at temperatures of 27 degrees C and above, and a marked reduction in PG protein and activity was observed at temperatures of 32 degrees C and above. Exogenous ethylene treatment did not reverse heat stress-induced inhibition of PG gene expression. Transfer of heat-stressed fruits to 20 degrees C partly restored PG mRNA accumulation, but the rate of PG mRNA accumulation declined exponentially with duration of heat stress. Heat stress-induced inhibition of PME mRNA accumulation was recoverable even after 14 days of heat stress. In fruits held at 34 degrees C, both PG and PME protein and activity continued to accumulate for about 4 days, but thereafter PG protein and activity declined while little change was observed in PME protein and activity. In spite of increases in mRNA levels of both PG and PME during the recovery of heat-stressed fruit at 20 degrees C, levels of PG protein and activity declined in fruits heat-stressed for four or more days while PME protein and activity levels remained unchanged. Collectively, these data suggest that PG gene expression is being gradually and irreversibly shut off during heat stress, while PME gene expression is much less sensitive to heat stress.

Ascocarps of the Kalahari desert truffle, T. pfeilii, were discovered attached by a ‘stalk’ consisting of sand particles and a mesh of mycelial tissue. Rhizomorphs were found in the vicinity of this ‘stalk’. The ascocarps occurred mainly on the southern, shady, side of shrubs and trees, the roots of some of which appeared to have developed mycorrhizal associations. Local lore maintains that truffles are found under grasses. Perennial Gramineae were found in close proximity to all the T. pfeilii ascocarps, some of which were mycorrhizal. However, roots of bushes and trees were also invariably found at a shallow depth beneath each truffle. At one site, over 20 fruiting bodies were found in a 20 m2 watered lawn of Cynodon dactylon under the shade of an Acacia erioloba. Soils at T. pfeilii sites had a lower pH and lower calcium content than those reported from sites where northern African members of this genus occur.

summary Activated charcoal added to artificial growth medium enhances initial association between the mycelium of the desert truffle Terfezia leonis Tul. and the roots of Helianthemum sessiliforum (Defs). Pers. in vitro. This is due, at least in part, to adsorption of iron.

The host plant Helianthemum sessiliflorum was inoculated with the mycorrhizal desert truffle Terfezia boudieri Chatin, and the subsequent effects of the ectomycorrhizal relationship on host physiology were determined. Diurnal measurements revealed that mycorrhizal (M) plants had higher rates of photosynthesis (35%), transpiration (18%), and night respiration (49%) than non-mycorrhizal (NM) plants. Consequently, M plants exhibited higher biomass accumulation, higher shoot-to-root ratios, and improved water use efficiency compared to NM plants. Total chlorophyll content was higher in M plants, and the ratio between chlorophyll a to chlorophyll b was altered in M plants. The increase in chlorophyll b content was significantly higher than the increase in chlorophyll a content (2.58- and 1.52-fold, respectively) compared to control. Calculation of the photosynthetic activation energy indicated lower energy requirements for CO(2) assimilation in M plants than in NM plants (48.62 and 61.56 kJ mol(-1), respectively). Continuous measurements of CO(2) exchange and transpiration in M plants versus NM plants provided a complete picture of the daily physiological differences brought on by the ectomycorrhizal relationships. The enhanced competence of M plants to withstand the harsh environmental conditions of the desert is discussed in view of the mycorrhizal-derived alterations in host physiology.

Selfed and auxin-treated fruits of tomato (Lycopersicon esculentum Mill.) were obtained from tetraploid and diploid plants and compared to determine why selfed tetraploid fruits are smaller. Application of auxin to ovaries produced tetraploid fruits as large as normally set (selfed) diploid fruits but led to reduced fruit size in diploid fruits, reflecting a further potential for tetraploid fruit development. The selfed tetraploid fruits were found to be smaller because they had fewer cells per fruit. Cell size (diameter) at the end of fruit development was similar for all fruit types. DNA per cell did not closely match cell size during development, especially during the cell division stage. Protein per cell also failed to follow cell size. Protein per DNA showed one peak at around day 10 of fruit development and possibly a second one at day 30.

A spontaneous tomato (Lycopersicon esculentum Mill.) triploid was studied with a view to its commercialization. Fruits induced by auxin contained 50% more DNA and 30% more protein than their diploid counterpart. The fruits were 50% larger than those of the diploid counterpart and were juicy but seedless. All fruit quality characteristics checked (polygalacturonase activity, reducing sugars content, electrical conductivity, pH, titratable acidity, pigment content, and shelf life) were comparable to the diploid except for ethylene evolution rate, which was lower than that of the diploid counterpart, and flavor, which was superior. The line seems suitable for agricultural cultivation. Triploid tomato plants are not readily obtainable through crosses between tetraploid and diploid tomato cultivars (Cooper and Brink, 1945; Jorgensen, 1928; Nilsson, 1950). However, they do arise occasionally in tomato plots and are readily identified by their vigor and fruitlessness (Rick, 1945). In a study aimed at estimating the commercial potential of fruits from triploid tomatoes, we searched for triploids among the commercial greenhouses of the Negev (southern area of Israel). A triploid plant discovered in a greenhouse of the tomato hybrid cultivar FC121 was verified by chromosome count, and was found to descend from a haploid female and a diploid male parent (Kagan-Zur et al., 1991). Triploid tomato plants often do not bear fruits in nature (Jorgensen, 1928; Nilsson, 1950; Rick, 1945). If induced by artificial means, fruits have virtually no seeds due to chromosomal imbalance of the gametes (Strickberger, 1976). Tetraploid tomato plants bear smaller fruits that contain about one-tenth as many seeds as isogenic diploid lines (Kagan-Zur and Mizrahi, 1987; Nilsson, 1950). Fruit size has been correlated with the number of seeds developing in the fruit (Varga and Bruinsma, 1976) and seed number correlated with auxin level (Mapelli et al., 1978). In diploid plants, it is possible to meet the auxin demands of the ovary and produce seedless parthenocarpic fruit by exogenous application of an auxin to emasculated flowers (BungerKibler and Bangerth, 1982/3; Mapelli et al., 1978). We assumed that the same treatment would induce fruit set and development on triploid plants. As polyploidy usually causes gigas appearance of plant organs (Jorgensen, 1928; Rick and Butler, 1956; Saimbhi and Brar, 1978; Tarn and Hawkes, 1986), and the smaller fruit size of tetraploids is correlated with lack of auxin, we surmised that auxin treatment would cause triploid fruits to surpass normal diploid fruits in size. We undertook this study to assess the possibility of obtaining triploid fruits, and to study their physiology and potential as a crop. Materials and Methods Plant material. A triploid of the tomato hybrid cultivar FC121 was identified in a commercial greenhouse in the Negev area for publication 18 Dec. 1989. We thank the Israel Endowment Fund the Isreael Ministry of Agriculture for partial financial support of The cost of publishing this paper was defrayed in part by the payment arges. Under postal regulations, this paper therefore must be hereby vertisement solely to indicate this fact. he Dept. of Biology. and propagated through cuttings together with the diploid hybrid obtained from the same source. Parent lines-of ‘FC121’ were grown from seeds (kindly provided by its breeder, N. Kedar). The parent plants were initially grown from germinated seeds, then propagated from cuttings as needed. Culture. Seeds were sown in vermiculite in a greenhouse, maintained under natural light conditions, and irrigated with Hoagland’s solution (Hoagland and Arnon, 1950). Triploids as well as ‘FC121’ diploids were propagated through cuttings, which were allowed to root in half-strength Hoagland’s solution. Threeweek-old seedlings and rooted cuttings were transferred to dark, opaque 10-liter buckets containing 1 vermiculite : 1 perlite : 1 C1 Finnish peat (by volume). Plants were irrigated with halfstrength Hoagland’s solution every other day. Ten plants of each cultivar were cultivated and trained to a single stem. Only the first four fruits on each inflorescence were allowed to develop. Flowers were either manually pollinated, tagged, and allowed to develop, or emasculated and treated with auxin. A commercial diluted solution of auxin [“NO Seed”, Bruinsma Ltd. Holland, consisting of 0.1 g of b -naphthoxy-acetic acid/liter in water and a drop of the surfactant Tween 20] was applied manually to emasculated diploid flowers and to nonemasculated triploid flowers at full anthesis. Plants were grown in four consecutive seasons—winter, spring, summer, and fall. Cell size. Thin slices of fresh pericarp sectioned with a razor blade were observed under a microscope with a calibrated ocular at 400 × magnification. The same cell layer was measured in each sample. The length and width were taken to the largest and smallest diameter of a cell, respectively. Cell volume was approximated as: (width) × (length) = volume. DNA content. Pericarp tissue (0.1 g) was frozen in liquid N and ground with methanol. After centrifugation for 10 min at 1350 × g in a bench centrifuge (Runne Heidelberg Mod 100-2; Heidelberg, Germany) the supernatant was discarded; this was repeated until a white pellet was obtained. The pellet was resuspended in 2.0 ml of 0.5 N perchloric acid and heated to 70C for 45 min. Following centrifugation as above, the supematant served for the DNA assay. DNA was assayed according to Richards (1974) using diphenylamine in glacial acetic acid and acetaldehyde. Protein content. For protein extraction, 2.0 ml of 1 M NaOH was added to ground pericarp after extraction of pigments (see below). The samples were then boiled for 20 rein, centrifuged at 1350 × g, and the supernatant set aside. This procedure was repeated twice at room temperature. All supernatants of a sample were combined and 0.4 ml was taken for protein determination. Protein content was determined using the method of Lowry et al. (1951), Na2C O3 and CuSO4 being added to the samples followed by Folin–Ciocalteus reagent. J. Amer. Soc. Hort. Sci. 116(2):228-231. 1991. Table 1. Effect of ploidy level and mode of fruit-set on fruit size and time from set to first color in hybrid tomatoes and their parent lines. zNOA = 0.1 g β -naphthoxy-acetic acid/liter. The averages include > 30 fruits per parental line and at least 60 fruits per hybrid line. yMean separation by Duncan’s multiple range test. Ethylene evolution. Ten fruits of each line were picked at 80% development (100% development being the time from anthesis to first red color in fruits) (Lyons and Pratt, 1964) and individually placed in the flowing air system described by Kopeliovitch et al. (1980). Ethylene evolution rate was measured daily by withdrawing a gas sample with a syringe and analyzing its contents by gas chromatography (Kopeliovitch et al., 1980). Each fruit was frozen 10 days after the onset of ethylene evolution, so that fruits all were at a comparable physiological stage when thawed for chemical analysis. Polygalacturonase activity. Polygalacturonase (PG) (EC 3.2. 1.15.) activity was measured as described by Mizrahi et al. (1976). Frozen tissue (40 g) was diced and homogenized in 1.0 M NaCl, then filtered and centrifuged (600 × g). The supernatant was, dialyzed against cold water. This served as a crude extract of the pectolytic enzyme. Ten milliliters of 4% sodium polypectate was added to 5 ml of crude extract. Substrate and extract were mixed in a viscosimeter (Cannon 200, United States), based on the principle that viscosity can be determined by recording the time needed for the solution to pass through a narrow calibrated glass column in the viscosimeter. Measurements were taken every 2 rein, and the activity was expressed as the time needed for a 50% decrease in the initial viscosity. Pigment analysis. Pigments were analyzed by weighing pericarp discs 11 mm in diameter and extracting each disk with 5 ml of 4 acetone : 5 hexane (v/v). After centrifugation, the optical density of the supernatant was read at several wavelengths with a Kontron UNIKON 810 spectrophotometer (Hager and Meyer-Bertenrath, 1967). Chemical analyses. Fruit tissue (10 g) was homogenized with 5 ml of water in a Virtis homogenizer, the homogenate centrifuged in a Sorvall RC2-B centrifuge (12000 × g, 10 rein), and the supernatant solution analyzed. Both pH and electrical conductivity were recorded using El-Hama Instruments PBS 710 and TH 250 (Tel Aviv), respectively. The amount of reducing sugars was measured according to the method of Sumner (1921) with dinitro-salycilic acid reagent. Acidity was estimated by titration with NaOH standardized against a KOH volumetric solution (BDH lg104 3u, Dorset, England). The analyses were performed on individual fruits from the ethylene evolution studies 10 days after the last of the tomatoes had entered the stage of ethylene evolution. The experiment was performed on three occasions several months apart, with essentially the same results, and all data were pooled. Sensory tests. Flavor was estimated using fresh fruits under red light (to avoid effect of color on taste estimation). Several fruits of each line were cut so that each slice contained pericarp and jelly and the slices were mixed together. Evaluations were requested to rate the samples as: 1 (worst), 2 (medium), or 3 (4 in one test) (best) (Kopeliovitch et al., 1982). Average flavor scores were calculated.

A spontaneous tomato (Lycopersicon esculentum Mill.) triploid hybrid was analyzed by isozyme and restriction fragment length polymorphism profiles. The double chromosome complement donor was shown to be the male parent, contrary to the prevailing hypothesis.

Phylogenetic analyses have corroborated the discovery of three internal transcribed spacer (ITS) Types in Terfezia boudieri isolates in the course of earlier studies and have emphasized the divergence of Type 2 from Types 1 and 3. The application of molecular and physiological tools described below, revealed the existence of cryptic species within T. boudieri. The markers used include sequences taken from the 5' end of the ribosomal large subunit gene, a chitin synthase partial sequence, beta-tubulin partial sequence and amplified fragment length polymorphism (AFLP)-based markers. Following initial sequencing of a single PCR amplified sample for each Type, mass analysis of specimens relied on RFLP differences between the Types. Over 100 fruit bodies, 30 or more specimens for each ITS Type, were tested with each of the markers. The markers analysis divided the isolates into three groups, each correlated to a specific ITS Type. Two of the physiological traits examined: mycelial proliferation and mycorrhiza formation, consistently showed responses paralleling the ITS Types; the data presented suggest that T. boudieri is comprised of three cryptic species.

SUMMARY The Israeli desert truffle, Terfezia leonis Tul., failed to enter into association with its plant symbiont, Helianthemum sessiliflorum (Defs.) Pers., when cultivated together on an agar medium consisting of inorganic salts. Addition of activated charcoal to the growth medium brought about rapid and extensive association. Activated charcoal did not enhance fungal growth in the absence of the plant seedling. The stimulating effect of the charcoal is probably related to its adsorption characteristics and may not be due to contact or abrasion between seedling roots and charcoal particles.

Changes in gene expression by isolates of Terfezia boudieri during mycorrhization with Cistus incanus hairy roots were followed. Four fungus-hairy root clone combinations were cultivated under two sets of conditions, in which the root and the fungus were separated by a cellophane sheet or were allowed physical contact. One of the combinations produced endomycorrhizas, the other three solely ectomycorrhizas. Fragments isolated by cDNA-AFLP analysis from cellophane-separated cultures (preinfection) were used to identify differentially expressed genes by reverse Northern analysis. Genes showing no homology to known sequences constituted the largest group under both growth conditions. Some fungal genes were expressed transiently, while others exhibited altered expression patterns as conditions changed from individually growing through the preinfection stage to mycorrhizas. Genes expressed exclusively under combinations allowing either ectomycorrhiza or endomycorrhiza under a particular condition were detected. Our results point, for the first time, to some of the genes that might be involved in determining the type of association that will be formed: ecto- or endomycorrhiza.

The ITS region including the 5.8S rRNA gene as well as the 5′ end of the 28S rRNA gene of hypogeous Pezizaceae and Tuberaceae were studied to clarify the generic placement of two southern African desert truffles, Terfezia pfeilii and Choiromyces echinulatus. The results show that neither species belongs in the genus to which it has been assigned on the basis of morphological characters. As expected, two Choiromyces spp. grouped close to the representative of the Tuberaceae (Tuber melanosporum). However, C. echinulatus diverged from the other Choiromyces species and emerged near members of the genus Terfezia, being even closer to that genus than T. pfeilii. Two new genera and new species combinations, Kalaharituber gen. nov. with K. pfeilii (syn. T. pfeilii) comb. nov. and Eremiomyces gen. nov. with E. echinulatus (syn. C. echinulatus) comb. nov. are therefore introduced to accomodate these taxa. Both genera are closely related to Terfezia, and thus are placed in the Pezizaceae.

Independent spontaneous triploid tomato plants (Lycopersicon esculentum Mill.) were collected among diploid hybrids growing in commercial greenhouses. Ploidy levels were verified by counting chromosomes, and the donor of the double genome dose was determined by restriction fragment length polymorphism (RFLP) analysis. The TG101 probe, which is tightly linked to the Tm-2 (a) locus, revealed different restriction patterns between TMV-resistant and TMV-susceptible parent lines. The parent donor which provided two genomes to the triploid was identified by comparing the relative intensity of alleles in the triploid with that in the diploid. The results indicate that both parents can serve as a double genome donor.

The Terfeziaceae family of fungi belongs to the Tuberales, Ascomycetes, and includes several genera of hypogeaus mushrooms, of which the better known and culinary appreciated are the genera of the Terfezias and Tirmanias, on which this presentation focuses. Members of these genera are mycorrhizal, usually occupy the same habitat, and often share the same plant hosts. Fruit bodies may range from about 30 to 300g per truffle, the main body being about 100g. They are subglobous, and many times lobed (Figures 1 and 2).

SummaryIsogenic diploid and tetraploid tomato plants, cv Moneymaker, were grown in nutrient solution in a greenhouse. Fruits were detached at known physiological stages and analysed for various ripening parameters. The rate of ethylene evolution by tetraploid tomatoes reached its peak four days after its detectable onset, in contrast to six days for the diploid tomato. The rates of evolution at the peak were similar for both types of tomato. At the same physiological age, in respect of the onset of ethylene evolution, the pH, titratable acidity, electrical conductivity and sugar content of the juice of ripe tetraploid tomato fruits were no different from those of diploid fruits. The pigment content was lower and the activity of pectolytic enzymes (polygalacturonase and polymethylgalacturonase) was higher in tetraploid fruits. Their taste was inferior to that of the diploids. The relevance to the ripening process of the ethylene level and of the time interval between the onset of the upsurge in ethylene ev...

The question of whether bacteriophage Mu prefers replication forks for lysogenic integration into Escherichia coli chromosomes was tested by using two different systems. In the first, inactivation of genes was scored in synchronized cultures infected by Mu at various times. No increase in the mutation frequency of a gene was found after infection at the time of its replication. In the second, the composition of colonies formed by bacteria lysogenized by Mu was determined; the newly formed lysogens should give rise to mixed colonies (containing lysogenized as well as nonlysogenized bacteria), uniform colonies, or both, depending on the mode of integration. Both types of colonies were found, and the fraction of uniform colonies was proportional to the relative length of the unreplicated segment of an average chromosome in the culture. The results in both systems clearly preclude the possibility that a lysogenizing Mu integrates with high preference at the chromosome replication forks.

Homogenates of dark-pretreated leaves yield two particulate fractions in density gradient centrifugation: one contains chlorophyll (chloroplasts) while a second fraction contains ribulose-1, 5-bisphophate carboxylase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and catalase. Addition of a microbody-rich pellet to chloroplasts isolated from dark-pretreated plants largely enhances both oxygen evolution and CO2-fixation into organic compounds. The pathway of CO2 reduction may be part of a membrane system which, under suitable conditions, may separate from the chloroplast as a distinct cytoplasmic entity, having physical properties similar to those of microbodies.

The complete Escherichia coli ftsQ coding sequence, together with part of the ftsA coding sequence, has been cloned upstream of the lacZ open reading frame in a lambda-vector (lambda JFL100). Cells which are lysogenic for lambda JFL100 transcribe the cloned lacZ from promoter(s) within the ftsQ and ftsA sequences. The level of beta-galactosidase produced is dependent on growth rate (and/or cell size) and is derepressed in an ftsA-deficient mutant. Transcription during the cell cycle is restricted to the time of cell division.

Glutaraldehyde fixation in 0.33 M sorbitol without any buffer reveals changes in the staining properties of the envelopes of chloroplasts of pea plants kept in the light or in the dark prior to fixation. After dark pretreatment the outer double membrane of the chloroplast does not adsorb heavy metals, resulting in a “white” unstained rim instead of the usual membrane. All other membranes of the cell, including chloroplast grana, are not affected and stain normally. Light pretreatment of the plants allows the usual staining of the outer membrane of the chloroplats. Fixation carried out in the medium usually used to isolate intact CO2 fixing chloroplasts (sorbitol+buffer+ions) reverses the above process and results in unstained envelopes of chloroplasts from preilluminated leaves, while the envelopes of chloroplasts from leaves kept in the dark stain normally. Glutaraldehyde-fixed chloroplats isolated from preilluminated leaves show a very basic isoelectric point during electrofocusing, while fixed chloroplasts from predarkened tissue exhibit an isoelectric point at about pH 7.

The capacity of a microbody-rich fraction (MBL) to stimulate photosynthesis of isolated chloroplasts was previously reported (1). Leaf extracts from plants kept in darkness for 24 h prior to homogenization yielded, after density gradient centrifugation, two peaks of RuBPCase: one in the chlorophyll band and the second with catalase (2). Consequently we studied interactions between isolated chloroplasts and the (MBL) and the effect of the latter on the photosynthetic rates of isolated chloroplasts. MBL enhanced photosynthesis of chloroplasts isolated from pea seedlings, kept in darkness for 24 h before homogenization, by 300% (1,2). Chloroplasts isolated from pea seedlings kept in the light could be enhanced by MBL to a lesser extent (50%). Marked differences were observed in the staining properties of the outer membrane of chloroplasts in leaf sections from plants kept in the light or in darkness (3). These differences of chloroplast envelopes may reflect changes in membrane composition and permeability due to light conditions. Gigot et al (4), observed portions of the stroma envolved by a membrane leaving the chloroplasts during the process of protoplast isolation. Vaughn et al (5) observed association of vesicles containing nitrate reductase, with chloroplasts, suggesting a possible fusion with the chloroplast envelopes.