The fruiting bodies of truffles are a microhabitat for the growth of bacteria, yeasts, fungi, and... more The fruiting bodies of truffles are a microhabitat for the growth of bacteria, yeasts, fungi, and viruses, which all together represent its “microbiome”. In this review, the mycological component of filamentous fungi and yeasts is examined, and a checklist of these fungi, defined here as truffle-inhabiting fungi (TIF), is provided. The role of these fungi and their effects on the truffle biology (e.g., production of volatile organic compounds and effects on mycorrhizal synthesis) and ecology are examined. The problem of methodology for their detection is also discussed. The traditional technique of isolation in culture makes it possible to highlight a very small percentage of the biodiversity of TIF, since the qPCR has allowed detection of the simultaneous presence of several species of TIF within the gleba. The approach with mass sequencing methods, apart from some tests with DGGE, has not for the moment been used since this concerns obtaining sequences of fungi inside a fungus (truffle) whose copies of DNA are represented at least one million times more compared to those of TIF.
Abstract To date, the ectomycorrhizae formed by silver fir (Abies alba), an ecologically valuable... more Abstract To date, the ectomycorrhizae formed by silver fir (Abies alba), an ecologically valuable and indigenous tree species in many European mountain forests, have been poorly investigated. We characterized the mycorrhizae formed by three Lactarius species (Lac. subsericatus, Lac. intermedius, Lac. salmonicolor) on silver fir, on the basis of material originating from central Italy. The identification of the fungal symbiont was achieved by means of morphoanatomical observations of mycorrhizae, and by comparison of ITS sequences obtained from mycorrhizae and sporocarps of putative fungal partners. Sequences also were obtained from specimens of the same species but from different geographic origin or from closely related Lactarius species. A maximum likelihood analysis of the data was performed. On the whole, the resultant tree is in good agreement with sporocarp and mycorrhiza morphology. RFLP patterns were calculated from sequence data. A discussion on the main morphoanatomical characters distinguishing the Lactarius ectomycorrhizae reported in this study from those already described belonging to related species, is also included. The accuracy of different methods to identify mycorrhizae formed by closely related Lactarius species on silver fir, are discussed.
Tuber mesentericum is an edible European black truffle, apparently easy to recognize, but showing... more Tuber mesentericum is an edible European black truffle, apparently easy to recognize, but showing a high degree of genetic variability. In this study, we performed an integrative taxonomic assessment of the T. mesentericum complex, combining a multilocus phylogeographic approach with morphological analyses, and including authentic specimens of Vittadini, and Berkeley and Broome. We performed maximum likelihood phylogenetic analyses, based on single and concatenated gene datasets (ITS rDNA, β-tubulin, elongation factor 1-α), and including all available sequences from previous studies. Phylogenetic analyses consistently recovered three reciprocally monophyletic and well-supported clades: clade I, with a wide range across Europe; clade II, specimens collected mainly in the Iberian, Italian, and Balkan peninsulas; and clade III, specimens collected almost exclusively in central Italy. Genetic distance between clades ranged from 10.4% to 13.1% at the ITS region. We also designed new prim...
Abies nebrodensis (Lojac.) Mattei is a Critically Endangered forest tree included in Appendix I o... more Abies nebrodensis (Lojac.) Mattei is a Critically Endangered forest tree included in Appendix I of the Bern Convention and as a priority species in Annexes II and IV of the Habitats Directive [1]. In situ and ex situ conservation strategies [2] and, more recently, a LIFE Natura project [3] allowed a marked improvement of health conditions of trees and of seedlings renewal. A first attempt to characterize the ectomycorrhizas of A. nebrodensis was carried out by Venturella & Rambelli [4]. In the frame of the activities of the II National Workshop of Ectomycorrhyza (Palermo, 2015), this paper reports the preliminary investigation carried out on the ectomycorrhyzal community of A. nebrodensis in comparison with that of Fagus sylvatica L. Root samples were collected from 12 different trees of the native population of A. nebrodensis and from 12 plants of F. sylvatica growing close to the firs. For each root samples we selected 100-200 mycorrhizas, divided in morphotypes on the basis of their morpho-anatomic features. Each morphotype was also genetically characterized through amplification and direct sequencing of ITS region of rDNA. The preliminary results show a higher richness of ectomycorrhizal species in F. sylvatica compared to A. nebrodensis. Cenococcum geophilum Fr. is the dominant species on F. sylvatica and A. nebrodensis. The higher number of species in F. sylvatica and A. nebrodensis is from genus Cortinarius (Pers.) Gray while Tomentella Pers. ex Pat. and Sebacina Tul & C. Tul. are underepresented
Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, 2017
Abstract The Tuber ascomata (truffles) are a microhabitat for bacteria, viruses, and fungi (yeast... more Abstract The Tuber ascomata (truffles) are a microhabitat for bacteria, viruses, and fungi (yeasts and filamentous fungi). In this survey, we tried to develop a method that would make it possible to define the mycobiome of the truffle-inhabiting filamentous fungi using culture independent molecular methods. The nested quantitative Real-Time PCR allowed us to demonstrate that each truffle is home to multiple species of filamentous fungi and that their DNA is present within the healthy ascoma at the ratio of 10−6 compared to that of the truffle. Probably due to their insignificant presence, Denaturing Gradient Gel Electrophoresis of the amplification of ITS amplicons showed only those of the host. Based on the results, the possibilities of being able to detect the fungicolous fungi present in very small amounts within a fungal host are discussed.
The ectomycorrhizal fungus Tuber magnatum produces the white truffle appreciated worldwide for it... more The ectomycorrhizal fungus Tuber magnatum produces the white truffle appreciated worldwide for its unique aroma. With respect to other Tuber spp. of economic interest, T. magnatum presents a narrower geographical range. This species has, in fact, long been considered endemic to Italy. However, over the last few decades several reports have documented the presence of white truffles in different Mediterranean countries and in particular in various areas of south-east Europe. In this study, samples from several Pannonian and Balkan countries such as Hungary, Serbia, Romania, Bulgaria and Greece have been collected and genotyped with microsatellite markers and the data merged with those available for Italian populations. Our objectives were to test whether Italian and south-east European populations are differentiated and to evaluate the genetic diversity of T. magnatum all over its distributional range. We show the genetic structure of T. magnatum populations with the differentiation o...
Arbutus unedo (the strawberry tree) is a Mediterranean shrub which forms arbutoid mycorrhizae wit... more Arbutus unedo (the strawberry tree) is a Mediterranean shrub which forms arbutoid mycorrhizae with a variety of Asco- and Basidiomycetes. After the discovery of the mycorrhizal symbiosis between A. unedo and Tuber borchii, in this study, arbutoid mycorrhizae were synthetized in greenhouse with Tuber aestivum and Tuber melanosporum. Six months after inoculation, both species colonized the roots of all inoculated A. unedo seedlings, but mature mycorrhizae were only observed after 12 months. Ultrastructure analysis of Tuber arbutoid mycorrhizae was described for the first time, showing, as observed in typical endosymbiosis, a rearrangement of host cells and the creation of an interface compartment with both truffle species. Immunolabelling experiments suggested that pectins are not present in the interface matrix surrounding the intracellular hyphae. Thus, the ability to establish symbiosis with A. unedo seems to be a common feature in the genus Tuber, opening up the possibility to use...
Aflatoxins are mycotoxins produced by some Aspergillus species. They are remarkably toxic and inc... more Aflatoxins are mycotoxins produced by some Aspergillus species. They are remarkably toxic and included among the most known carcinogenic substances. Article 139 of the Italian presidential decree of 30 June 1965, no. 1204 and subsequent amendments, includes hepatocellular carcinoma due to exposure to aflatoxin B1 the list of diseases for which reporting is mandatory. Aflatoxins could contaminate food of plant and animal origin, therefore the areas of work most at risk are the agro-food and, in general, all the activities carried out in humid and poorly ventilated areas, subject to fungal contamination. Although the procedures for controlling aflatoxins in food are now fairly well established, there is still no full knowledge of the risk of workplace exposure. A review of the risks relating to exposure to aflatoxin of workers in the agri-food sector is presented here based on bibliographic data and surveys conducted in some Italian regions by local health authorities and by the Natio...
The paper presents a review on health hazards involving operators that cultivate and handle mushr... more The paper presents a review on health hazards involving operators that cultivate and handle mushrooms. Intensive production of edible and medicinal fungi is a common agricultural management all over the world and involves a number of operators whose health must be safeguarded due the number of risks. Growth techniques and environmental conditions for producing and handling mushrooms expose workers to several types of allergens that possibly cause occupational diseases. Namely, mushroom workers suffer from allergic pulmonary diseases and, more rarely, from forms of contact dermatitis. The cause of these clinical manifestations may be found in the exposure to several factors, such as the peculiar production conditions leading to the presence of many allergens (bacteria, moulds, mycotoxins, endotoxins) and the direct contact of workers with some fungal species, which are themselves allergens.
A survey of the truffle Tuber melanosporum genome has shown the presence of 67 programmed cell de... more A survey of the truffle Tuber melanosporum genome has shown the presence of 67 programmed cell death (PCD)-related genes. The 67 genes are all expressed during fruit body (FB) development of T. melanosporum development; their expression has been detected by DNA microarrays and qPCR. A set of 14 PCD-related genes have been chosen, those with the highest identities to the homologs of other species, for a deeper investigation. That PCD occurs during T. melanosporum development has been demonstrated by the TUNEL reaction and transmission electron microscopy. The findings of this work, in addition to the discovery of PCD-related genes in the T. melanosporum genome and their expression during the differentiation and development of the FB, would suggest that one of the PCD subroutines, maybe autophagy, is involved in the FB ripening, i.e., sporogenesis.
The fruiting bodies of truffles are a microhabitat for the growth of bacteria, yeasts, fungi, and... more The fruiting bodies of truffles are a microhabitat for the growth of bacteria, yeasts, fungi, and viruses, which all together represent its “microbiome”. In this review, the mycological component of filamentous fungi and yeasts is examined, and a checklist of these fungi, defined here as truffle-inhabiting fungi (TIF), is provided. The role of these fungi and their effects on the truffle biology (e.g., production of volatile organic compounds and effects on mycorrhizal synthesis) and ecology are examined. The problem of methodology for their detection is also discussed. The traditional technique of isolation in culture makes it possible to highlight a very small percentage of the biodiversity of TIF, since the qPCR has allowed detection of the simultaneous presence of several species of TIF within the gleba. The approach with mass sequencing methods, apart from some tests with DGGE, has not for the moment been used since this concerns obtaining sequences of fungi inside a fungus (truffle) whose copies of DNA are represented at least one million times more compared to those of TIF.
Abstract To date, the ectomycorrhizae formed by silver fir (Abies alba), an ecologically valuable... more Abstract To date, the ectomycorrhizae formed by silver fir (Abies alba), an ecologically valuable and indigenous tree species in many European mountain forests, have been poorly investigated. We characterized the mycorrhizae formed by three Lactarius species (Lac. subsericatus, Lac. intermedius, Lac. salmonicolor) on silver fir, on the basis of material originating from central Italy. The identification of the fungal symbiont was achieved by means of morphoanatomical observations of mycorrhizae, and by comparison of ITS sequences obtained from mycorrhizae and sporocarps of putative fungal partners. Sequences also were obtained from specimens of the same species but from different geographic origin or from closely related Lactarius species. A maximum likelihood analysis of the data was performed. On the whole, the resultant tree is in good agreement with sporocarp and mycorrhiza morphology. RFLP patterns were calculated from sequence data. A discussion on the main morphoanatomical characters distinguishing the Lactarius ectomycorrhizae reported in this study from those already described belonging to related species, is also included. The accuracy of different methods to identify mycorrhizae formed by closely related Lactarius species on silver fir, are discussed.
Tuber mesentericum is an edible European black truffle, apparently easy to recognize, but showing... more Tuber mesentericum is an edible European black truffle, apparently easy to recognize, but showing a high degree of genetic variability. In this study, we performed an integrative taxonomic assessment of the T. mesentericum complex, combining a multilocus phylogeographic approach with morphological analyses, and including authentic specimens of Vittadini, and Berkeley and Broome. We performed maximum likelihood phylogenetic analyses, based on single and concatenated gene datasets (ITS rDNA, β-tubulin, elongation factor 1-α), and including all available sequences from previous studies. Phylogenetic analyses consistently recovered three reciprocally monophyletic and well-supported clades: clade I, with a wide range across Europe; clade II, specimens collected mainly in the Iberian, Italian, and Balkan peninsulas; and clade III, specimens collected almost exclusively in central Italy. Genetic distance between clades ranged from 10.4% to 13.1% at the ITS region. We also designed new prim...
Abies nebrodensis (Lojac.) Mattei is a Critically Endangered forest tree included in Appendix I o... more Abies nebrodensis (Lojac.) Mattei is a Critically Endangered forest tree included in Appendix I of the Bern Convention and as a priority species in Annexes II and IV of the Habitats Directive [1]. In situ and ex situ conservation strategies [2] and, more recently, a LIFE Natura project [3] allowed a marked improvement of health conditions of trees and of seedlings renewal. A first attempt to characterize the ectomycorrhizas of A. nebrodensis was carried out by Venturella & Rambelli [4]. In the frame of the activities of the II National Workshop of Ectomycorrhyza (Palermo, 2015), this paper reports the preliminary investigation carried out on the ectomycorrhyzal community of A. nebrodensis in comparison with that of Fagus sylvatica L. Root samples were collected from 12 different trees of the native population of A. nebrodensis and from 12 plants of F. sylvatica growing close to the firs. For each root samples we selected 100-200 mycorrhizas, divided in morphotypes on the basis of their morpho-anatomic features. Each morphotype was also genetically characterized through amplification and direct sequencing of ITS region of rDNA. The preliminary results show a higher richness of ectomycorrhizal species in F. sylvatica compared to A. nebrodensis. Cenococcum geophilum Fr. is the dominant species on F. sylvatica and A. nebrodensis. The higher number of species in F. sylvatica and A. nebrodensis is from genus Cortinarius (Pers.) Gray while Tomentella Pers. ex Pat. and Sebacina Tul & C. Tul. are underepresented
Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, 2017
Abstract The Tuber ascomata (truffles) are a microhabitat for bacteria, viruses, and fungi (yeast... more Abstract The Tuber ascomata (truffles) are a microhabitat for bacteria, viruses, and fungi (yeasts and filamentous fungi). In this survey, we tried to develop a method that would make it possible to define the mycobiome of the truffle-inhabiting filamentous fungi using culture independent molecular methods. The nested quantitative Real-Time PCR allowed us to demonstrate that each truffle is home to multiple species of filamentous fungi and that their DNA is present within the healthy ascoma at the ratio of 10−6 compared to that of the truffle. Probably due to their insignificant presence, Denaturing Gradient Gel Electrophoresis of the amplification of ITS amplicons showed only those of the host. Based on the results, the possibilities of being able to detect the fungicolous fungi present in very small amounts within a fungal host are discussed.
The ectomycorrhizal fungus Tuber magnatum produces the white truffle appreciated worldwide for it... more The ectomycorrhizal fungus Tuber magnatum produces the white truffle appreciated worldwide for its unique aroma. With respect to other Tuber spp. of economic interest, T. magnatum presents a narrower geographical range. This species has, in fact, long been considered endemic to Italy. However, over the last few decades several reports have documented the presence of white truffles in different Mediterranean countries and in particular in various areas of south-east Europe. In this study, samples from several Pannonian and Balkan countries such as Hungary, Serbia, Romania, Bulgaria and Greece have been collected and genotyped with microsatellite markers and the data merged with those available for Italian populations. Our objectives were to test whether Italian and south-east European populations are differentiated and to evaluate the genetic diversity of T. magnatum all over its distributional range. We show the genetic structure of T. magnatum populations with the differentiation o...
Arbutus unedo (the strawberry tree) is a Mediterranean shrub which forms arbutoid mycorrhizae wit... more Arbutus unedo (the strawberry tree) is a Mediterranean shrub which forms arbutoid mycorrhizae with a variety of Asco- and Basidiomycetes. After the discovery of the mycorrhizal symbiosis between A. unedo and Tuber borchii, in this study, arbutoid mycorrhizae were synthetized in greenhouse with Tuber aestivum and Tuber melanosporum. Six months after inoculation, both species colonized the roots of all inoculated A. unedo seedlings, but mature mycorrhizae were only observed after 12 months. Ultrastructure analysis of Tuber arbutoid mycorrhizae was described for the first time, showing, as observed in typical endosymbiosis, a rearrangement of host cells and the creation of an interface compartment with both truffle species. Immunolabelling experiments suggested that pectins are not present in the interface matrix surrounding the intracellular hyphae. Thus, the ability to establish symbiosis with A. unedo seems to be a common feature in the genus Tuber, opening up the possibility to use...
Aflatoxins are mycotoxins produced by some Aspergillus species. They are remarkably toxic and inc... more Aflatoxins are mycotoxins produced by some Aspergillus species. They are remarkably toxic and included among the most known carcinogenic substances. Article 139 of the Italian presidential decree of 30 June 1965, no. 1204 and subsequent amendments, includes hepatocellular carcinoma due to exposure to aflatoxin B1 the list of diseases for which reporting is mandatory. Aflatoxins could contaminate food of plant and animal origin, therefore the areas of work most at risk are the agro-food and, in general, all the activities carried out in humid and poorly ventilated areas, subject to fungal contamination. Although the procedures for controlling aflatoxins in food are now fairly well established, there is still no full knowledge of the risk of workplace exposure. A review of the risks relating to exposure to aflatoxin of workers in the agri-food sector is presented here based on bibliographic data and surveys conducted in some Italian regions by local health authorities and by the Natio...
The paper presents a review on health hazards involving operators that cultivate and handle mushr... more The paper presents a review on health hazards involving operators that cultivate and handle mushrooms. Intensive production of edible and medicinal fungi is a common agricultural management all over the world and involves a number of operators whose health must be safeguarded due the number of risks. Growth techniques and environmental conditions for producing and handling mushrooms expose workers to several types of allergens that possibly cause occupational diseases. Namely, mushroom workers suffer from allergic pulmonary diseases and, more rarely, from forms of contact dermatitis. The cause of these clinical manifestations may be found in the exposure to several factors, such as the peculiar production conditions leading to the presence of many allergens (bacteria, moulds, mycotoxins, endotoxins) and the direct contact of workers with some fungal species, which are themselves allergens.
A survey of the truffle Tuber melanosporum genome has shown the presence of 67 programmed cell de... more A survey of the truffle Tuber melanosporum genome has shown the presence of 67 programmed cell death (PCD)-related genes. The 67 genes are all expressed during fruit body (FB) development of T. melanosporum development; their expression has been detected by DNA microarrays and qPCR. A set of 14 PCD-related genes have been chosen, those with the highest identities to the homologs of other species, for a deeper investigation. That PCD occurs during T. melanosporum development has been demonstrated by the TUNEL reaction and transmission electron microscopy. The findings of this work, in addition to the discovery of PCD-related genes in the T. melanosporum genome and their expression during the differentiation and development of the FB, would suggest that one of the PCD subroutines, maybe autophagy, is involved in the FB ripening, i.e., sporogenesis.
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