Instrumental monitoring of the birth and development of truffles in a Tuber melanosporum orchard.

Mycorrhizal symbiotic plants, soil suitability, temperature, and humidity are, by general consensus, considered decisive factors in truffle production. However, experimental approaches to define the environmental conditions that stimulate formation of truffle primordia and promote their growth to maturity have been lacking. By analysis of data of many atmospheric and soil parameters collected since 2009 within a Tuber melanosporum orchard, the trends of metabolic activity, detected as CO2 production in the soil, have been identified as the most reliable parameter to indicate the 'birth' of the truffle primordia. They seem to be produced when mycelial activity is intense and undergoes water stress, after which it resumes. About 6-18 days after recovery of metabolic activity, we could collect primordia of T. melanosporum. Many die or develop too early and consequently rot or are eaten by insect larvae. These events occur several times during summer and autumn, those that 'sprout' in late summer or later grow steadily and reach maturity. Using a particular ground-penetrating radar (GPR) setup to discriminate truffles, we could identify individual truffles in the soil after they have enlarged to at least 6 mm in diameter and follow their growth in volume and diameter over time. These two instrumental methods (CO2 sensor and GPR), although yet to be improved, open new important perspectives to better understand truffle biology and manage truffle orchards to support the newly acquired demonstration of the fundamental role of host plants for the nutrient transfer to the ectomycorrhiza-mycelium-fruiting body complex of T. melanosporum.

Assessment of ectomycorrhizal fungal communities in the natural habitats of Tuber magnatum (Ascomycota, Pezizales).

The ectomycorrhizal (ECM) fungal communities of four natural Tuber magnatum truffle grounds, located in different Italian regions (Abruzzo, Emilia-Romagna, Molise, and Tuscany), were studied. The main objective of this study was to characterize and compare the ECM fungal communities in the different regions and in productive (where T. magnatum ascomata were found) and nonproductive points. More than 8,000 (8,100) colonized root tips were counted in 73 soil cores, and 129 operational taxonomic units were identified using morphological and molecular methods. Although the composition of the ECM fungal communities studied varied, we were able to highlight some common characteristics. The most plentiful ECM fungal taxa belong to the Thelephoraceae and Sebacinaceae families followed by Inocybaceae and Russulaceae. Although several ectomycorrhizas belonging to Tuber genus were identified, no T. magnatum ectomycorrhizas were found. The putative ecological significance of some species is discussed.

Biochemical, electrophoretic and immunohistochemical aspects of malate dehydrogenase in truffles (Ascomycotina).

The malate dehydrogenase (MDH; EC 1.1.1.37; L-malate-NAD(+)-oxidoreductase) activities of truffles of the genus Tuber (Tuber melanosporum Vittad., Tuber brumale Vittad., Tuber aestivum Vittad., Tuber magnatum Pico, Tuber rufum Pico) have been characterized with regard to the K(m) and V(max) values in the direct and reverse reactions. The isoelectrofocusing has revealed bands showing pI values ranging from pH 5.85 to 7.8. The MDH of T. melanosporum has been partially purified by hydroxyapatite treatment, DEAE-cellulose and Sephadex G-75 columns. With the partially purified T. melanosporum MDH activity polyclonal anti-T. melanosporum MDH antibodies have been prepared and used to localize MDH in the mycorrhizae and ascocarps of T. melanosporum. These antibodies inhibit T. melanosporum MDH activity as well as that of T. magnatum but not that of rabbit liver; this supports the specificity of the MDH antibodies used to localize MDH in truffle tissues.

Effect of tyrosinase inhibitors on Tuber borchii mycelium growth in vitro.

This paper reports the effect of the tyrosinase (monophenol o-diphenol:oxygen oxidoreductase; EC 1.14.18.1) inhibitors diethyldithiocarbamate (DETC), L-tropolone, kojic acid, phenylthiourea (PTU) and L-mimosine on the in vitro growth of Tuber borchii (a white truffle) mycelium. A significant inhibitory effect on mycelium growth was observed for DETC, PTU and L-tropolone (0% growth compared to control at 100 microg ml(-1) DETC, PTU and L-tropolone and at 10 microg ml(-1) DETC and L-tropolone). As a comparison the action of the same inhibitors was also tested on the growth and pigmentation of the mould Cladosporium sphaerospermum. In the presence of CuSO(4) 10(-6) M T. borchii mycelium acquired pigmentation (as rounded aggregates compared to control revealed by SEM microscopy). Tyrosinase activity in the extract from T. borchii mycelium (18-day culture) was detected spectrophotometrically.

Melanogenesis, tyrosinase expression, and reproductive differentiation in black and white truffles (Ascomycotina).

White and black truffles of the genus Tuber are Ascomycotina as well as Neurospora crassa, which expresses tyrosinase dependently on the reproductive cycle. Tyrosinase expression dependent on reproductive differentiation has been also described in black truffles. We present novel and comparative work on melanogenic activities in black and white truffles that both express true tyrosinases. L-tyrosine 3-monooxygenase and L-DOPA oxidase activities colocalize as histochemically detected and are similarly located in white and black truffles, from the hypothecium through the sporogenic hyphae to asci and spores. Sulfur components of truffle flavours reversibly inhibit tyrosinase. The respiratory phenotype of truffle mitochondria is discussed in relation to reproductive differentiation and melanogenesis.

Truffle melanogenesis: correlation with reproductive differentiation and ascocarp ripening.

The present work uses histochemical techniques to investigate the correlation between reproductive differentiation and age of truffles (Tuber aestivum and Tuber melanosporum) with melanin synthesis. The dopa oxidase and tyrosine hydroxylase activities of tyrosinase have been localized within the ascocarp and melanin localization was performed by the Schmorl's reaction. A true tyrosinase is present in truffles, able to oxidize both l-tyrosine and l-dopa. The tyrosinase activity is on in the young ascocarps (in the peridium, hypothecium, and fertile veins) and off in the ripe ones, thus it appears correlated with the age and differentiation of the sporogenic hyphae that arise from the hypothecium; a similar correlation has been previously described in Neurospora crassa, which is an ascomycete as well as the truffles.