Competition between Tuber melanosporum and other ectomycorrhizal fungi under two irrigation regimes

A glasshouse experiment was conducted to simulate the competition between artificially introduced Tuber melanosporum (Vitt.) and other symbionts, occurring on outplanted truffle-producing trees. Hazel (Corylus avellana L.) seedlings, previously inoculated with Tuber melanosporum, were rapidly infected with the competing truffle Tuber brumale (Vitt.), added to the soil as spores. Coexistence of both species on the root system was observed. Tuber melanosporum survived and continued to spread. Tuber brumale, which is naturally present in many truffle-orchard soils, protects the roots from other ectomycorrhizal symbionts. Although high water content is unfavourable for the development of T. melanosporum in the absence of other Tube species, Tuber brumale stimulates the development of T. melanosporum under these conditions.     

Suitability of hydroxyapatite and iron phosphate as P sources for Lupinus albus grown in nutrient solution

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.     

Calcareous amendments to soils to eradicate Tuber brumale from T. melanosporum cultivations: a multivariate statistical approach

Calcareous amendments are being used in Tuber melanosporum truffle plantations in attempts to eradicate Tuber brumale. However, there are no studies available which provide soil analysis and statistical data on this topic. We studied 77 soil samples to compare the values for carbonates, pH and total organic carbon in T. brumale truffières with the values for T. melanosporum truffières on contaminated farms and in natural areas. Statistical analyses indicate that the concentrations of active carbonate and total carbonate in the soil are significantly higher in T. brumale truffières than in T. melanosporum truffières, but that there are no significant differences in pH and total organic carbon. We conclude that liming would not suppress T. brumale ectomycorrhizas in contaminated T. melanosporum farms, and calcareous amendments do not therefore seem be a means of eradicating T. brumale in these farms.     

Synthesis and establishment of <Emphasis Type="Italic">Tuber melanosporum</Emphasis> Vitt. ectomycorrhizae on two <Emphasis Type="Italic">Nothofagus</Emphasis> species in Chile

Axenically germinated seedlings of two species of Southern beech (Nothofagus obliqua, N. glauca) from Chile were inoculated with spores of the Périgord black truffle (Tuber melanosporum). Ectomycorrhizal development was monitored for 6 months in the greenhouse and compared to the performance of the natural host species Quercus ilex and Quercus robur. Seedling survival and mycorrhization showed major differences in both Nothofagus species: T. melanosporum readily formed ectomycorrhizae with seedlings of N. obliqua, although at a lower rate than with Q. ilex but at a proportion very similar to Q. robur; survival and colonization rates were high, and seedling growth was not visibly affected by the high soil pH required by T. melanosporum. In contrast, more than 50% of N. glauca seedlings died after inoculation, and mycorrhiza formation was very sparse. In both species, no colonization by adventive ectomycorrhizal fungi could be observed, whereas both species of Quercus showed minor colonization by another fungus, probably Inocybe or Hebeloma. Our results show that it is possible to infect N. obliqua with the Périgord black truffle under greenhouse conditions, which opens up the possibility of cultivating this truffle as a secondary crop during reforestation with N. obliqua in Chile.     

Ascoma genotyping and mating type analyses of mycorrhizas and soil mycelia of Tuber borchii in a truffle orchard established by mycelial inoculated plants

Tuber borchii (the Bianchetto truffle) is a heterothallic Ascomycete living in symbiotic association with trees and shrubs. Maternal and paternal genotype dynamics have already been studied for the black truffles Tuber melanosporum and Tuber aestivum but not yet for T. borchii. In this study, we analysed maternal and paternal genotypes in the first truffle orchard realized with plants inoculated with five different T. borchii mycelia. Our aims were to test the persistence of the inoculated mycelia, if maternal and/or paternal genotypes correspond to inoculated mycelia and to assess the hermaphroditism of T. borchii. The mating type of each isolate as well as those of mycorrhizas, ascomata and extraradical soil mycelia was determined. Moreover, simple sequence repeat (SSR) profiles of maternal and paternal genotypes were assessed in 18 fruiting bodies to investigate the sexual behaviour of this truffle. The maternal genotypes of the fruiting bodies corresponded to those of the inoculated mycelia with only two exceptions. This confirmed that the inoculated mycelia persisted 9 years after plantation. As regards paternal partner, only two had the same genotype as those of the inoculated mycelia, suggesting hermaphroditism. Most of the new paternal genotypes originated from a recombination of those of inoculated mycelia.     

Cultivation of Mediterranean species of Tuber (Tuberaceae) in British Columbia,Canada

Based on an assessment of soil and climatic conditions in British Columbia (BC), the Truffle Association of British Columbia (TABC) determined that the cultivation of Mediterranean Tuber melanosporum and Tuber aestivum might be possible in the warmer parts of the province. With the cooperation of independent truffle growers, TABC assessed the colonization of host tree roots collected from eight truffle orchards planted 2–7 years earlier using morphological and molecular criteria. Both Tuber species persisted on the roots of inoculated trees in six of the eight truffle orchards studied. The identity of Tuber ectomycorrhizas that had been characterized morphologically as differing from those of T. melanosporum and T. aestivum were determined using DNA sequence analysis to belong to three species of truffles native to the Pacific Northwest. One of those species, Tuber anniae, had been previously reported from BC, but the other two, Tuber menseri nom. prov. and Tuber beyerlei, are reported here from BC for the first time. Recently, production of three Périgord black truffles in one truffle orchard and one Burgundy truffle in another orchard demonstrates that these truffles are able to fruit in BC.     

Current state and perspectives of truffle genetics and sustainable biotechnology

Mycorrhizal fungi belonging to the genus Tuber produce, after the establishment of a productive interaction with a plant host, hypogeous fruitbodies of great economic value known as ‘‘truffles’’. This review summarizes the state of art on life cycle, genetic, and biotechnological investigations of Tuber spp. The ascocarp formation in truffles is a consequence of the activation of the sexual phase of the biological cycle. The formation of a dikaryotic secondary mycelium and the karyogamy in the ascal cell (followed by meiosis with ascospores formation) have been hypothesized by several authors but some doubts yet arise from the Tuber cycle by considering that a series of abnormalities have been pointed out in respect to other Ascomycetes. It is unclear if binucleated hyphal cells are derived from the fusion of mononucleated cells belonging to mycelia from different mating types or from one only. According to the karyotypes of Tuber melanosporum, Tuber magnatum, and Tuber borchii, the numbers of hyphal chromosomes suggest a chromosome number of eight (2n); these values are in the range of those of several Ascomycetes and observed for Tuber aestivum (2n=10). The importance and growth in interest during the last years in the fungi protoplasts isolation and transformation techniques can be related to current developments in Tuber genetics and biotechnology. T. borchii could be transformed through liposome-mediated delivery of genetic material as mycelial protoplasts isolation and fusion with liposomes has already been established. On the other hand, Agrobacterium-mediated transformation has been successfully established for T. borchii.     

Impact of active soil carbonate and burn size on the capacity of the rockrose Cistus laurifolius to produce Tuber melanosporum carpophores in truffle culture

There are very few studies on Tuber melanosporum associated with Cistus populations. In central Spain, we confirm that C. laurifolius shows carpophore production of Tuber melanosporum. This study demonstrates a correlation between T. melanosporum production and the size of the 41 burns, with burn size explaining 26 % of the variability in carpophore production. However, statistical analysis of the results indicates that average production of 21 burns with Cistus laurifolius is 73 % lower than the production of 20 burns associated with Quercus/Corylus without Cistus laurifolius in this zone, respectively. C. laurifolius develops small burns, which has an impact on their carpophore production, and their soils have 92 % less active carbonate than the burns associated with Quercus/Corylus in this zone, respectively. The low levels of active carbonate allow Cistus laurifolius to grow, but impair Tuber melanosporum production. We thus provide a mechanism, based on the inability of Cistus laurifolius to grow in highly carbonated soils, which contradicts the well-known fact that C. laurifolius might act as a transmitter of Tuber melanosporum mycorrhizae. These results indicate that this rockrose can not be considered a useful species for truffle culture. We thus recommend that particular attention should be given to the concentration of active carbonate present in the soil in future studies on the relationship between Cistus species and truffles.     

Receptivity of cloned hazels to artificial ectomycorrhizal infection by Tuber melanosporum and symbiotic competitors

 The cultivation of the black truffle, Tuber melanosporum (Vitt.), is based upon plantation of seedlings artificially infected with Tuber. At the present time, neither the host-plant nor the fungal material used for planting truffle orchards are genetically well defined. Cloning the host-plant was performed as a first step in improvement. A clone of hazel (Corylus avellana L.) was artificially infected with T. melanosporum. The heterogeneity of the root volume was not completely suppressed by cloning but very extensive growth unfavorable to Tuber colonization rarely occurred. The root system was highly receptive to ectomycorrhizal fungi. The percentage of roots infected by T. melanosporum reached a higher level on cloned hazels than uncloned seedlings. The relation between Tuber and other symbionts appeared to depend on the morphology of the root system. T. melanosporum spread more easily on medium-sized root systems whereas the other symbionts (Scleroderma sp., Cenococcum sp., Pulvinula globifera) developed in parallel to the root volume. The practical potential of this system is discussed. Accepted: 4 June 1995     

How the truffle got its mate: insights from genetic structure in spontaneous and planted Mediterranean populations of Tuber melanosporum

The life cycles and dispersal of edible fungi are still poorly known, thus limiting our understanding of their evolution and domestication. The prized Tuber melanosporum produces fruitbodies (fleshy organs where meiospores mature) gathered in natural, spontaneously inoculated forests or harvested in plantations of nursery‐inoculated trees. Yet, how fruitbodies are formed remains unclear, thus limiting yields, and how current domestication attempts affect population genetic structure is overlooked. Fruitbodies result from mating between two haploid individuals: the maternal parent forms the flesh and the meiospores, while the paternal parent only contributes to the meiospores. We analyzed the genetic diversity of T. melanosporum comparatively in spontaneous forests vs. plantations, using SSR polymorphism of 950 samples from South‐East France. All populations displayed strong genetic isolation by distance at the metric scale, possibly due to animal dispersal, meiospore persistence in soil, and/or exclusion of unrelated individuals by vegetative incompatibility. High inbreeding was consistently found, suggesting that parents often develop from meiospores produced by the same fruitbody. Unlike maternal genotypes, paternal mycelia contributed to few fruitbodies each, did not persist over years, and were undetectable on tree mycorrhizae. Thus, we postulate that germlings from the soil spore bank act as paternal partners. Paternal genetic diversity and outbreeding were higher in plantations than in spontaneous truffle‐grounds, perhaps because truffle growers disperse fruitbodies to maintain inoculation in plantations. However, planted and spontaneous populations were not genetically isolated, so that T. melanosporum illustrates an early step of domestication where genetic structure remains little affected.     

Cultivation of Tuber melanosporum in firebreaks: Short-term persistence of the fungus and effect of seedling age and soil treatment

Wildfires are a major threat to Mediterranean forests. Firebreaks are built as a prevention measure, but require a periodic and expensive maintenance. Cultivating the ectomycorrhizal mushroom Tuber melanosporum Vitt. in firebreaks could reduce costs and improve their sustainability. But firebreaks are built on forest soil, considered nonoptimum for T. melanosporum cultivation. A pot experiment was used to study the persistence of T. melanosporum in firebreak soils in the short term, as a first step to assess the viability of these plantations. The influence of seedlings, soil heating, and liming was also tested. During the 2 y after plantation, T. melanosporum mycorrhizas increased their number, showing its ability to proliferate. Percent root colonisation by native fungi importantly increased from month 12 to 22; although T. melanosporum remained dominant, with a colonisation level similar to those in standard truffle plantations. The age of seedlings at the time of planting influenced T. melanosporum proliferation, supporting a key role for nursery seedling quality in the performance of young plantations. Heating the soil before planting reduced the richness of native fungi, suggesting that this could increase plantation success. The results tend to support the viability of T. melanosporum cultivation in firebreaks, and encourage experimental field plantations.     

Diffuse competition for heterogeneous substrate in soil among six species of wood-decomposing basidiomycetes

Competition among six wood decay fungi was studied using 15×15 mm wood blocks placed in 250×250 mm plastic trays filled with unsterilized sand or clay. The wood blocks were preinoculated with Heterobasidion annosum (Fr.) Bref., Resinicium bicolor (Alb. & Schw. ex Fr.) Parm., Phanerochaete sanguinea (Fr.) Hjortstam, Coniophora sp. DC. ex Me"rat, Armillaria borealis Marxmuller and Korhonen and Hypholoma capnoides (Fr.) Kummer before they were combined in all possible combinations in the trays. Two methods were used, one with all wood blocks inoculated, and one with sterilized non-inoculated wood blocks distributed between the inoculated ones. Wood blocks preinoculated with the six species were also used in a pairwise competition test. Following incubation for 9 months in darkness at 21°C, mycelia were reisolated and identified. R. bicolor was most successful at invading through the soil and replacing other species in the wood blocks. P. sanguinea, Coniophora sp. and H. capnoides also had some success.     

Can NPK fertilizers enhance seedling growth and mycorrhizal status of Tuber melanosporum-inoculated Quercus ilex seedlings?

Although successful cultivation of the black truffle (Tuber melanosporum) has inspired the establishment of widespread truffle orchards in agricultural lands throughout the world, there are many unknowns involved in proper management of orchards during the 6–10 years prior to truffle production, and there are conflicting results reported for fertilizer treatments. Here, we systematically evaluate the combined effects of nitrogen, phosphorous, and potassium with different doses of each element, applied to either foliage or roots, on plant growth parameters and the mycorrhizal status of outplanted 3-year-old seedlings in five experimental Quercus ilex–T. melanosporum orchards. Fertilization did not significantly improve seedling aboveground growth, but the plants treated with the fertilizer 12-7-7 applied to the roots (HNr) displayed longer field-developed roots. Only the fertilizer with the highest dose of K (10-6-28) applied to the foliage (HKf) increased the probability of fine root tip colonization by T. melanosporum in field-developed roots. However, the plants treated with the same fertilizer applied to the soil (HKr) presented the highest probability for colonization by other competing mycorrhizal soil fungi. Potassium seems to have an important role in mycorrhizal development in these soils. Apart from T. melanosporum, we found 14 ectomycorrhizal morphotypes, from which seven were identified to species level, three to genus, two to family, and two remained unidentified by their morphological characteristics and DNA analyses.     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

Integration of Pythium nunn and Trichoderma harzianum isolate T-95 for the biological control of Pythium damping-off of cucumber

Two biological control agents, Pythium nunn and Trichoderma harzianum isolate T-95, were combined to reduce Pythium damping-off of cucumber in greenhouse experiments lasting 3–4 weeks. T. harzianum T-95, a rhizosphere competent mutant, was applied to seeds and P. nunn was applied to pasteurized and raw soils naturally and artificially infested with Pythium ultimum. Some treatments were also amended with bean leaves to enhance the activity of P. nunn. The biological control of Pythium damping-off was evaluated in a Colorado soil (Nunn sandy loam) and an Oregon soil mix, which were replanted twice after 2 and 3 months. Interactions between P. nunn and T-95 were detected in the Colorado but not the Oregon soil. No consistent evidence of antagonism between P. nunn and T. harzianum was seen, and significant interactions were detected in the Colorado, but not the Oregon soil. In the first planting of some treatments, the combination of P. nunn and T. harzianum gave greater control of damping-off than either applied alone. P. nunn was most effective in soils that were pasteurized or amended with bean leaves. T. harzianum controlled Pythium damping-off in the Colorado, but not the Oregon soil. In both soils, disease declined over time in treatments amended with bean leaves but without P. nunn or T. harzianum added. This suppression was greater in the Colorado soil, which contained an indigenous population of P. nunn. This work demonstrates that two compatible biological control agents can be combined to give additional control of a soil-borne plant pathogen.     

Comparative effects of four mycorrhizal fungi on loblolly pine seedlings growing in a greenhouse in a Piedmont soil

Summary A greenhouse study was conducted to evaluate the effect of ectomycorrhizae on loblolly pine (Pinus taeda L.) growing in a Piedmont soil. Pine seedlings were inoculated with one of four species of fungi (Scleroderma aurantium, Pisolithus tinctorius, Thelophora terrestris, andRhizopogon roseolus). The seedlings were grown in pots containing a Cecil sandy clay loam amended to create a gradient of extractable P ranging from 5.9 to 52.5 g/g. After ten months, all colonized seedlings were significantly larger than control seedlings. However, of the four fungi,Scleroderma aurantium mediated a far superior shoot growth response to increasing levels of soil P; the seedlings were significantly larger than those colonized by any other fungus and also had the largest root systems and greatest degree of mycorrhizal colonization.     

Gas exchange and photosynthesis of Eucalyptus camaldulensis seedlings inoculated with different ectomycorrhizal symbionts

Eucalyptus camaldulensis Dehnh. seedlings inoculated with Pisolithus tinctorius (Pers.) Coker & Couch and Thelephora terrestris Ehrl. per Fr. were grown in well watered soil (_s –0.03 MPa) or subjected to a long-term soil water stress of up to –1.0 MPa over 13-week period in a glasshouse. After 13 weeks, all seedling containers were watered to field capacity and then water was withheld from the E. camaldulensis seedlings to induce a short-term drought. Diurnal measurements of seedling photosynthesis rate (A), leaf stomatal conductance (g) and leaf water potential (_p) were completed before, during, and after the short term drought. Although they were growing in an equal soil volume, photosynthesis rate (A), leaf stomatal conductance and leaf water potential (_p) of larger seedlings with P. tinctorius ectomycorrhizae were similar to those of smaller seedlings colonized with T. terrestris during the short-term drought period. Seedlings inoculated with Pisolithus tinctorius maintained higher photosynthesis rates over the course of the short-term drought. Thus, P. tinctorius ectomycorrhizae appear to be more efficient than those of T. terrestris in assisting seedlings to maintain gas exchange and photosynthesis under limited soil moisture conditions.     

Availability of cadmium and zinc accumulated in the leaves of Thlaspi caerulescens incorporated into soil

When grown on contaminated soil, hyperaccumulator plants contain high concentrations of metals which may return to the soil after senescence. This work was undertaken to assess the availability of Cd and Zn associated to the leaves of the hyperaccumulator Thlaspi caerulescens after incorporation into an uncontaminated soil. A Zn- and Cd- accumulator population of T. caerulescens was grown on a Cd- and Zn- contaminated soil previously labelled with ¹⁰⁹Cd. Leaves (TCL) were harvested, dried, ground and incorporated into the soil at a rate of 2.07 mg Cd kg⁻¹ and 51.9 mg Zn kg⁻¹. Then a pot experiment was conducted for 3 months with rye grass (Lolium perenne) and T. caerulescens. Rye grass was harvested monthly and T. caerulescens at the end of the experiment. Plant biomass was measured, along with the concentration of Cd, Zn and ¹⁰⁹Cd. Results showed that water-extractable metals in TCL were 69% for Zn and 33% for Cd. Addition of TCL to soil, depleted growth of rye grass, and improved that of T. caerulescens. At harvest, concentrations of both metals were increased in plants by TCL. Concentrations of Cd in rye grass increased with the cut number, while that of Zn decreased slightly. Rye grass extracted 1.6% of the total Cd and 0.9% of the total Zn, and T. caerulescens extracted up to 22.4% of the Cd and 7% of the Zn. About 94% of the Cd in rye grass and 86% in T. caerulescens was derived from TCL. In conclusion, metals associated with leaves of the hyperaccumulator T. caerulescens were very mobile after incorporation into the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of soil liming and vesicular-arbuscular-mycorrhizal inoculation on the growth and micronutrient content of the teff plant

In a greenhouse experiment involving an acid soil teff [Eragrostis tef (Zucc.) Trotter] plants failed to grow unless the soil was limed or inoculated with either of two vesicular-arbuscular-mycorrhizal (VAM) fungi,Glomus mosseae orGlomus macrocarpum. Plant growth increased by liming and to a lesser extent by VAM fungal inoculation. Liming also enhanced root colonization by VAM fungi. Shoot micronutrient content generally increased as a result of inoculation, and decreased by increased lime applications.