Using common mycorrhizal networks for controlled inoculation of Quercus spp. with Tuber melanosporum: the nurse plant method.

The high cost and restricted availability of black truffle spore inoculum for controlled mycorrhiza formation of host trees produced for truffle orchards worldwide encourage the search for more efficient and sustainable inoculation methods that can be applied globally. In this study, we evaluated the potential of the nurse plant method for the controlled inoculation of Quercus cerris and Quercus robur with Tuber melanosporum by mycorrhizal networks in pot cultures. Pine bark compost, adjusted to pH 7.8 by liming, was used as substrate for all assays. Initially, Q. robur seedlings were inoculated with truffle spores and cultured for 12 months. After this period, the plants presenting 74 % mycorrhizal fine roots were transferred to larger containers. Nurse plants were used for two treatments of two different nursling species: five sterilized acorns or five 45-day-old, axenically grown Q. robur or Q. cerris seedlings, planted in containers around the nurse plant. After 6 months, colonized nursling plant root tips showed that mycorrhiza formation by T. melanosporum was higher than 45 % in the seedlings tested, with the most successful nursling combination being Q. cerris seedlings, reaching 81 % colonization. Bulk identification of T. melanosporum mycorrhizae was based on morphological and anatomical features and confirmed by sequencing of the internal transcribed spacer region of the ribosomal DNA of selected root tips. Our results show that the nurse plant method yields attractive rates of mycorrhiza formation by the Périgord black truffle and suggest that establishing and maintaining common mycorrhizal networks in pot cultures enables sustained use of the initial spore inoculum.

Comparative study of mycorrhizal susceptibility and anatomy of four palm species.

A morphological and anatomical study of the root systems of the palm species Brahea armata S. Watson, Chamaerops humilis L., Phoenix canariensis Chabaud and Phoenix dactylifera L. has been carried out to determine possible mycorrhizal colonization sites. Furthermore, the arbuscular mycorrhizal (AM) anatomical types formed by the four palm species in association with Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe have been examined. The presence of a continuous sclerenchymatic ring in the outer cortex and aerenchyma in the inner cortex that are anatomical indicators of mycorrhizal nonsusceptibility in all four palm species is observed. The root systems of B. armata and C. humilis present only one group of third-order roots, while the third-order roots of P. canariensis and P. dactylifera may be divided into five different groups: short thick roots, mycorrhizal thickened roots, fine short roots, fine long roots, and pneumatorhizas. Third-order and some second-order roots of B. armata and C. humilis are susceptible to colonization by AM fungi, while only the mycorrhizal thickened roots form mycorrhizas with arbuscules in the Phoenix species. The root system of the Phoenix species also presents AM colonization in fine roots with only intraradical hyphae and spores, but without arbuscules, and pseudomantles of spores anchored in the pneumatorings of the second-order roots, which are described for the first time. The mycorrhizas formed by the four palm species are of an intermediate type, between the Arum and the Paris types, and are characterized by intercalary arbusculate coils and not only by intracellular but also by intercellular fungal growth. Our study suggests that a different degree of adaptation may exist among palm mycorrhizas toward the slow growth of palms and low spore numbers in the soil where they grow.

Ultrastructural localization of acid phosphatase in arbusculate coils of mycorrhizal Phoenix canariensis roots.

Acid phosphatase (ACP) activity has been detected in roots of mycorrhizal and non-mycorrhizal Phoenix canariensis. This enzyme was ultrastructurally localized in arbusculate coils for the first time. This localization was carried out using a cerium-based method, which minimizes non-specific precipitation. The ACP was localized in inter- and intracellular hyphae, in the fungal cytoplasm as well as at the interface and the fungal cell wall and the periarbuscular membrane limiting it. The novel localization of an ACP in the arbuscular mycorrhizal (AM) interface of arbusculate coils suggests that this enzyme may be involved in the phosphorus efflux from the mycorrhizal fungus to the host. The results presented in this article indicate that the role played by ACP in AM symbiosis may be more important than was previously thought and that arbusculate coils are highly relevant when considering nutrient transfer through AM symbiosis.

Genetic variability of Pisolithus isolates associated with native hosts and exotic eucalyptus in the western Mediterranean region.

• Genetic diversity and host specificity of Pisolithus is reported here in exotic (Eucalyptus) and native hosts in the western Mediterranean region. • Polymorphism in the internal transcribed spacer (ITS) sequences of the nuclear rDNA of Pisolithus was analysed. Sequences for 17 isolates associated with native Mediterranean hosts and Eucalyptus were compared with those in the GenBank DNA database using distance and parsimony methods. • Bootstrap analysis showed clustering of all Pisolithus isolates associated with Mediterranean hosts. The ITS sequences suggest the occurrence of several ecological species adapted to exploit different soil types (basic, acid and clayey slate-derived soils), with specificity for particular indigenous hosts. Isolates from eucalypt plantations in Brazil, Kenya and the Mediterranean grouped together with eucalypt-associated Australian isolates. Transfer to native hosts did not occur; the host specificity range of these exotic strains might prevent out-competition and interbreeding with local species. • Pisolithus spp. in eucalypt plantations in the Mediterranean basin are of Australian origin; the co-introduction of the ectomycorrhizal fungi might explain the success of these exotic forest plantations.

Monophenolase activity of latent Terfezia claveryi tyrosinase: Characterization and histochemical localization.

The monophenolase activity of Terfezia claveryi tyrosinase (EC 1.14.18.1) is described for the first time. This enzyme is fully latent and can only be detected if SDS is present in the reaction medium. Monophenolase activity was localized within the ascocarp using histochemical techniques. A detailed kinetic study of the parameters affecting this activity has been carried out. Both the characteristic lag period and the steady-state rate are affected by pH and the enzyme and substrate concentrations. The presence of catalytic concentrations of o-diphenols affected the lag period but not the steady-state rate. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme activation constant, Kact, which showed a value of 7.2 &mgr;M. The experimental results are compatible with the mechanism previously described for tyrosinases from other sources.

Antioxidant activity of edible fungi (truffles and mushrooms): losses during industrial processing.

The antioxidant properties of two raw truffles (Terfezia claveryi Chatin and Picoa juniperi Vittadini) and five raw mushrooms (Lepista nuda, Lentinus edodes, Agrocybe cylindracea, Cantharellus lutescens, and Hydnum repandum) were tested by subjecting these truffles and mushrooms to different industrial processes (freezing and canning) and comparing them with common food antioxidants (alpha-tocopherol [E-307], BHA [E-320], BHT [E-321], and propyl gallate [E-310]) with regard to their ability to inhibit lipid oxidation. All of the truffles and mushrooms analyzed exhibited higher percentages of oxidation inhibition than did the food antioxidants according to assays based on lipid peroxidation (LOO*), deoxyribose (OH*), and peroxidase (H2O2). Frozen samples exhibited a small reduction in free radical scavenger activity, but the results did not show a significant difference (P < 0.05) with respect to the raw samples, while canned truffles and mushrooms lost some antioxidant activity as a consequence of industrial processing. All of the raw and frozen truffles and mushrooms except frozen Cantharellus improved the stability of oil against oxidation (100 degrees C Rancimat), while canned samples accelerated oil degradation. Antioxidant activity during 30 days of storage was measured by the linoleic acid assay, and all of the samples except canned Terfezia, Picoa, and Hydnum showed high or medium antioxidant activity. The Trolox equivalent antioxidant capacity assay was used to provide a ranking order of antioxidant activity as measured against that of Trolox (a standard solution used to evaluate equivalent antioxidant capacity). The order of raw samples with regard to antioxidant capacity was as follows (in decreasing order): Cantharellus, Agrocybe, Lentinus, Terfezia, Picoa, Lepista, and Hydnum. Losses of antioxidant activity were detected in the processed samples of these truffles and mushrooms.

Autofluorescence detection of arbuscular mycorrhizal fungal structures in palm roots: an underestimated experimental method.

The aim of this study was to reassess the use of autofluorescence for evaluating AM colonization in mycorrhizal roots in the light of criticisms of this method that affirmed that only metabolically inactive arbuscules autofluoresce. It was also investigated whether other mycorrhizal structures, such as hyphae, vesicles and spores, could be detected by autofluorescence, and whether the autofluorescence pattern of AM fungal structures could be exploited methodologically, for example, in the detection and sorting of spores by flow cytometry. Mycorrhizal roots of the palm species Brahea armata, Chamaerops humilis, Phoenix canariensis and Phoenix dactylifera were sectioned and observed by means of fluorescence microscopy. In addition, fungal structures isolated from mycorrhizal roots of P. dactylifera were examined. The same root sections and isolated fungal structures were subjected to vital staining with nitro blue tetrazolium to determine their metabolic state (active or inactive). Moreover, spores of Glomus intraradices, and Glomus clarum were studied by epifluorescence and flow cytometry. Mycorrhizal whole roots of Medicago sativa were also assessed by autofluorescence detection. In contrast to previous reports, the results presented in this paper clearly demonstrate that all fungal structures, both intra- and extraradical, autofluoresced under blue light excitation, regardless of their state (dead or alive). Some arbuscules isolated from roots and mature spores showed further autofluorescence under green light excitation. The source of the autofluorescence was localized in the fungal cell wall. It was shown that AM spores can be detected by flow cytometry. The results support the use of autofluorescence for the evaluation of AM colonization, at least in palm species, and refute previous criticisms of the method.