Traceability of the mycorrhizal symbiosis in the controlled production of edible mushrooms = Traçabilitat de la simbiosi micorízica en la producció controlada de fongs comestibles

[cat] La disponibilitat de la majoria dels fongs micorízics comestibles depèn de la seva fructificació natural. La formació de bolets està relacionada amb les característiques de l'hàbitat i les condicions climàtiques, però aquestes dades no expliquen ni la dinàmica de la fructificació ni del miceli d’aquests fongs. L’establiment de plantacions a partir de plantes micorizades pot ser una alternativa per a l'obtenció de fongs comestibles ectomicorízics. El progrés de les noves tecnologies en la biologia molecular han permès el desenvolupament, implementació i ús de tècniques per a la traçabilitat dels fongs ectomicorízics comestibles. A la tesi s'ha demostrat que la PCR en temps real, amb el disseny d'oligonucleòtids específics per a la detecció i quantificació de miceli extraradical, ens permet determinar la concentració de miceli present al sòl tant a boscos com a plantacions. La micorizació de plantes amb espècies del complex Boletus edulis s'ha aconseguit en condicions de cultiu pur. S’han dissenyat encebadors i sondes específics per l’amplificació de B. edulis i Tuber melanosporum per PCR en temps real. S’ha estudiat la dinàmica estacional del miceli extraradical, les ectomicorizes i la producció de B. edulis i Lactarius deliciosus, a dos boscos de Sòria. També s’ha quantificat el miceli extraradical de Rhizopogon roseolus (a dues plantacions) i de T. melanosporum (a una plantació), des del seu establiment. Per altra banda s’ha quantificat el miceli extraradical de T. melanosporum en una tofonera natural i en set plantacions (d’uns 20 anys). Finalment, s’ha analitzat l'estructura genètica d’una població de T. melanosporum a una plantació productiva, determinat la distribució dels tipus de compatibilitat al sòl, i els genotips de micorizes i ascocarps fent us de marcadors microsatèl•lits. No s'ha trobat relació directa entre les quantitats de miceli del sòl i la producció per cap de les espècies estudiades, però s’ha correlacionat positivament les estructures vegetatives de B. edulis, R. roseolus i T. melanosporum. Els resultats obren la possibilitat d'utilitzar la quantificació de miceli del sòl per PCR en temps real com un bon indicador de la colonització de les arrels en condicions de camp. Les quantitats de miceli extraradical de B. edulis, L. deliciosus i R. roseolus i les micorizes de T. melanosporum s’han correlacionat amb diferents paràmetres climàtics. Finalment, ha estat possible determinar la distribució dels diferents genotips de T. melanosporum a una plantació, on es va trobar una estructura genètica espacial amb un patró de distribució no aleatori, resultant en zones de camp colonitzat per genets que compartien els mateixos tipus de compatibilitat.[eng] The availability of most edible ectomycorrhizal mushrooms depends on their natural fructification. Therefore, mycorrhizal plant production could be an alternative for obtaining ectomycorrhizal edible fungi. The first step to establish a plantation for a certain species of mushrooms is the production of plants, inoculated with the fungus, in order to be outplanted to field. The mycorrhization of plants with species of the Boletus edulis complex has been achieved under pure culture synthesis conditions. The production of B. edulis mycorrhizal plants under nursery conditions could be achieved by acclimation of in vitro-produced mycorrhizal plants, but needs still more research to determine an appropriate procedure. Sporocarp formation of these fungi is linked to habitat characteristics and climate conditions, but these data alone do not explain all the trends of fungal fruiting and dynamics. It could be hypothesized that the amount of soil mycelia could also be related to the production of carpophores. Progress of new technologies in molecular biology and the availability of fungal genomes have led to the development, implementation and use of techniques for the traceability of edible ectomycorrhizal fungi. Through the thesis it has been demonstrated that the real- time PCR technique, with the design of specific oligonucleotides for the detection and quantification of extraradical mycelium in soil, allows us to determine the concentration of soil mycelium in forests and plantations, increasing detection limits over conventional PCR. Specific primers and probes have been designed for B. edulis and Tuber melanosporum for real-time PCR amplification, allowing the traceability of these fungal species in the controlled production of edible mycorrhizal mushrooms. The annual belowground dynamics of extraradical soil mycelium, ectomycorrhizal root tips and sporocarp production of two ectomycorrhizal fungi, B. edulis and Lactarius deliciosus, have been studied in two different pine forests (Pinar Grande and Pinares Llanos, respectively) in Soria in permanent plots. Quantification of extraradical mycelium of Rhizopogon roseolus x Pinus pinea plantations (two in Cabils) and of T. melanosporum x Quercus ilex plantation (one in Cerc), in Catalunya has been carried out from its establishment until 24 or 48 months. T. melanosporum extraradical mycelium has also been quantified by real-time PCR in a natural truffle ground and in seven truffle orchards (around 20 years old) established in Tierra Estella and Valdorba sites (Navarra). Finally, the spatial genetic structure of T. melanosporum population was analyzed in a productive orchard (France). The distribution of the two T. melanosporum mating types was monitored in the soil. Ectomycorrhizas and ascocarps were mapped and genotyped using simple sequence repeat markers and the mating type locus and their genetic profiles were compared. No direct relationships were found between soil mycelium amounts and sporocarp production for any of the studied fungal species, but it was possible to obtain positive correlations between vegetative structures (ectomycorrhiza and extraradical mycelium) for B. edulis, R. roseolus and T. melanosporum. The results obtained open the possibility of using quantification of soil mycelium by real-time PCR as a good indicator for root colonization in field conditions (in natural areas or in manmade orchards), especially when a nondestructive sampling or less time consuming analysis were required. The extraradical mycelial amounts of B. edulis, L. deliciosus and R. roseolus and T. melanosporum mycorrhizas were correlated with climate parameters as temperature, rainfall, solar radiation, relative humidity and evapotranspiration. Moreover, it has been possible to map the distribution of different T. melanosporum genotypes trough the traceability of ascocarps, ectomycorrhizas and soil samples. A pronounced spatial genetic structure was found. A nonrandom distribution pattern of the T. melanosporum was observed, resulting in field patches colonized by genets that shared the same mating types

Identification and In Situ Distribution of a Fungal Gene Marker: The Mating Type Genes of the Black Truffle

Truffles are ectomycorrhizal fungi harvested mainly in human managed agroforestry ecosystems. Truffle production in truffle orchards faces two important bottlenecks or challenges: the initiation of the sexual reproduction and the growth of the ascocarps during several months. The black Périgord truffle, Tuber melanosporum, is a heterothallic species and the mating type genes (MAT1-1 and M1T1-2) have been characterized. In this context, the unraveling of the T. melanosporum mating type strains distribution in truffle orchards is a critical starting point to provide new insights into its sexual reproduction. The aim of this chapter is to present the protocol used to characterize the T. melanosporum mating type present in a truffle orchard from ascocarps, hazel mycorrhizal root tips, and/or soil samples, by polymerase chain reactions using specific primers for those genes, but it can be adapted for other fungal species

Dynamique de la reproduction sexuée.

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Five years investigation of female and male genotypes in périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction strategies

The Périgord black truffle (Tuber melanosporum Vittad.) is a heterothallic ascomycete that establishes ectomycorrhizal symbiosis with trees and shrubs. Small-scale genetic structures of female genotypes in truffle orchards are known, but it has not yet been studied in male genotypes. In this study, our aim was to characterize the small-scale genetic structure of both male and female genotypes over five years in an orchard to better understand the T. melanosporum sexual reproduction strategy, male genotype dynamics, and origins. Two-hundred forty-one ascocarps, 475 ectomycorrhizas, and 20 soil cores were harvested and genotyped using microsatellites and mating type genes. Isolation by distance analysis revealed pronounced small-scale genetic structures for both female and male genotypes. The genotypic diversity was higher for male than female genotypes with numerous small size genotypes suggesting an important turnover due to ascospore recruitment. Larger and perennial female and male genotypes were also detected. Only three genotypes (1.5%) were found as both female and male genotypes (hermaphrodites) while most were detected only as female or male genotype (dioecy). Our results suggest that germinating ascospores act as male genotypes, but we also proposed that soil mycelium could be a reservoir of male genotypes

Comparative genomics and population genetics provide new insights on the life cycle of the black truffle of Périgord (Tuber melanosporum Vittad.)

International audienceTruffles are ectomycorrhizal fungi living in symbiosis with many trees and shrubs. Inoculated seedlings with truffles are available since forty years and the interest in truffle cultivation is increasing worldwide. However, novel management guidelines for sustainable truffle orchards, in which the production is less unpredictable even in the climate change context, are needed. A better understanding of the biological and ecological mechanisms driving the truffle life cycle is one of the approaches to reach these goals. The genomic resources available for Tuber melanosporum allowed us to unravel its sexual reproduction mode by identifying mating type genes and to characterize new polymorphic markers allowing population genetics analyses. The study of genet distribution in truffle orchards showed a non-random spatial distribution of ectomycorrhizas formed by both T. melanosporum mating types, as well as a rapid yearly turnover of these genets. This analysis revealed a striking competition between genets based on their mating-type genes to colonize plant root system and questioned the localization of both parents in the initiation of the sexual cycle. To gain additional information on T. melanosporum genetic diversity, we re-sequenced the nuclear genome of six geographic accessions. A total of 442,326 single nucleotide polymorphisms (SNPs) corresponding to 3,540 SNPs/Mbps were identified. Based on their SNP density, samples clustered according to their geographic origin and the putative role of the last glaciation in T. melanosporum phylogeography is confirmed. Additionally, genomic regions and genes potentially subjected to positive or purifying selection were identified. Finally, a large-scale analysis of T. melanosporum populations at European scale using highly polymorphic simple sequence repeats (SSR) is also in progress

Comparative genomics and population genetics provide new insights on the life cycle of the black truffle of Périgord (Tuber melanosporum Vittad.)

International audienceTruffles are ectomycorrhizal fungi living in symbiosis with many trees and shrubs. Inoculated seedlings with truffles are available since forty years and the interest in truffle cultivation is increasing worldwide. However, novel management guidelines for sustainable truffle orchards, in which the production is less unpredictable even in the climate change context, are needed. A better understanding of the biological and ecological mechanisms driving the truffle life cycle is one of the approaches to reach these goals. The genomic resources available for Tuber melanosporum allowed us to unravel its sexual reproduction mode by identifying mating type genes and to characterize new polymorphic markers allowing population genetics analyses. The study of genet distribution in truffle orchards showed a non-random spatial distribution of ectomycorrhizas formed by both T. melanosporum mating types, as well as a rapid yearly turnover of these genets. This analysis revealed a striking competition between genets based on their mating-type genes to colonize plant root system and questioned the localization of both parents in the initiation of the sexual cycle. To gain additional information on T. melanosporum genetic diversity, we re-sequenced the nuclear genome of six geographic accessions. A total of 442,326 single nucleotide polymorphisms (SNPs) corresponding to 3,540 SNPs/Mbps were identified. Based on their SNP density, samples clustered according to their geographic origin and the putative role of the last glaciation in T. melanosporum phylogeography is confirmed. Additionally, genomic regions and genes potentially subjected to positive or purifying selection were identified. Finally, a large-scale analysis of T. melanosporum populations at European scale using highly polymorphic simple sequence repeats (SSR) is also in progress

Fine-scale spatial genetic structure of the black truffle (Tuber melanosporum) investigated with neutral microsatellites and functional mating type genes

The genetic structure of ectomycorrhizal (ECM) fungal populations results from both vegetative and sexual propagation. In this study, we have analysed the spatial genetic structure of Tuber melanosporum populations, a heterothallic ascomycete that produces edible fruit bodies. Ectomycorrhizas from oaks and hazels from two orchards were mapped and genotyped using simple sequence repeat markers and the mating type locus. The distribution of the two T. melanosporum mating types was also monitored in the soil. In one orchard, the genetic profiles of the ascocarps were compared with those of the underlying mycorrhizas. A pronounced spatial genetic structure was found. The maximum genet sizes were 2.35 and 4.70 m in the two orchards, with most manifesting a size < 1 m. Few genets persisted throughout two seasons. A nonrandom distribution pattern of the T. melanosporum was observed, resulting in field patches colonized by genets that shared the same mating types. Our findings suggest that competition occurs between genets and provide basic information on T. melanosporum propagation patterns that are relevant for the management of productive truffle orchards