Search

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.

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.

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.

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.

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.

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 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.

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.

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 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.