Truffle Production Research Articles

Diversity of endomycorrhizal and ectomycorrhizal fungi in the rhizosphere of Helianthemum ledifolium in the Bni Guil area (Eastern Morocco)

This work aimed to study the diversity of arbuscular mycorrhizal fungi (AMF) and ectomycorrhizal fungi (ECM) present in the rhizosphere of Helianthemum ledifolium in the Bni Guil area (Figuig Province, Morocco), known for its arid climate and desert truffle production. Soil samples from the rhizosphere of Helianthemum ledifolium were taken for spore extraction, while the roots of this host plant were sampled for root observation. The results of this study showed that the density of spores extracted by wet sieving was 165 spores per 50g of the soil. Identification of these spores indicated the presence of 37 species, belonging to three AMF families (Glomeraceae, Acaulosporaceae, and Claroideoglomeraceae) and one ECM family (Cistaceae). The Glomus and Acaulospora genera are the most dominant, with 17 and 14 species, respectively, compared with the other genera. In addition, the frequency of occurrence of the Glomus genus is higher (50%) than that of the other fungal genera present, especially the Terfezia genus, reflecting the scarcity of desert truffles in recent decades. The results of root observations showed the presence of ectomycorrhization with a typical Hartig network and no fungal mantle, despite the dominance of AMF fungi, indicating some host specificity towards ECM fungi.

A way to mechanically activate a suspension of black truffle spores

True truffles, or fungi of the genus Tuber, are among the most valuable, rare, expensive, and mysterious fungi on the planet. The reproduction of truffle fungi in the wild is a complex process that depends on many factors, for example, the presence of suitable host trees, as well as the optimal parameters of temperature, moisture, soil pH, soil symbionts, and other organisms. Successful production and reproduction of truffles require a large number of conditions to be met, primarily the release of their spores from ascii, germination under favorable conditions, and distribution in the environment. The present study aims to develop a method for mechanically breaking ascii in order to release the spores of black truffle. It is shown that homogenization by means of an immersion blender together with a vibrating chopper is a highly effective way to break ascii. The microscopic examination of the specimens at all stages of the experiment revealed that this method leads to the breakdown of at least 85% of ascii and the release of truffle spores. The use of centrifugation approaches enables the separation of black truffle spores from hyphal debris, fungal symbionts, and various associated organisms. Such spores can form mycelium on growth media. The study results can be used to prepare Tuber sp. inoculum and obtain their axenic cultures. Thus, the introduction of methods for releasing spores from ascii improves the efficiency of black truffle cultivation.

Effect of Irrigation Methods on Black Truffle Production

Spain is one of the main producers of black truffle (Tuber melanosporum Vittad.), a fungus of great economic importance. Black truffles are usually cultivated in Quercus ilex orchards, as water availability is one of the most important factors influencing truffle production. Optimizing watering systems is essential to reduce the amount of water wasted. Nevertheless, up to now, no study has been carried out comparing the efficiency of different irrigation systems in truffle plantations. The aim of this study was to compare the efficiency of two different irrigation systems, namely a drip irrigation system and a micro-sprinkler system, in a Quercus ilex plantation situated in Burgos, Spain. Our data showed that there were no differences between the two irrigation systems in terms of truffle yields, the number of truffles, quality (based on truffle size), or the date of truffle harvesting. However, when other parameters were taken into consideration, such as the economic and environmental impact of installing and running these systems, drip irrigation was deemed the superior irrigation system because it uses less water. This study validates for the first time the use of drip irrigation rather than a micro-sprinkler system (the most commonly used in truffle plantations) because of its greater water use efficiency, which is an increasingly important consideration given future climate change scenarios marked by global water scarcity.

Aromatic Plants and Their Associated Arbuscular Mycorrhizal Fungi Outcompete Tuber melanosporum in Compatibility Assays with Truffle-Oaks.

The high value of black truffle recompenses the slow growth of the fungus when established in the field. Adding a secondary crop, such as medicinal and aromatic plants (MAPs), could further enhance the sustainability of truffle production agro-forest systems. The dual cultures of ectomycorrhizal truffle-oak seedlings and MAPs (lavender, thyme, and sage) previously inoculated and non-inoculated with native arbuscular mycorrhizal fungi (AMF), were established to evaluate plant-fungi relationships. After 12 months in a shadehouse, plants' growth, mycorrhizal colonization, and extraradical soil mycelium (both of Tuber melanosporum and AMF) were measured. Overall, truffle-oaks' growth was negatively affected by the presence of MAPs, especially when inoculated with AMF. In turn, the presence of truffle-oaks barely affected the co-cultured MAPs, and only lavenders showed a significant growth reduction. All AMF-inoculated MAPs showed higher shoot and root biomass than non-inoculated ones. Compared to truffle-oaks growing alone, the presence of co-cultured MAPs, especially when they were AMF-inoculated, significantly decreased both the ectomycorrhizas and soil mycelium of T. melanosporum. These results reveal the strong competition between AMF and T. melanosporum and warn about the need for the protection of intercropping plants and their associated symbiotic fungi to avoid reciprocal counterproductive effects in mixed truffle-oak-AMF-MAP plantations.

Whitish truffles found in Finland: soil characteristics, and identification based on morphological and molecular properties.

The growing demand for truffles has led to a global pursuit for their occurrence in the wild, and studies concerning their cultivation. Although European countries such as Italy, France and Spain have been known for truffle production, truffle hunting is new to Finland. The present study reports for the first time, the findings of Tuber maculatum in Finland on the basis of morphological and molecular analysis. The chemical characteristics of soil samples collected from the truffle sites have also been discussed. The species of the Tuber samples were identified primarily using morphological analysis. Molecular analysis was carried out for the confirmation of the identity of the species. Two phylogenetic trees were constructed based on internal transcribed spacer (ITS) sequences produced in this study and including representative sequences of whitish truffles available in GenBank. The truffles were identified as T. maculatum and T. anniae. This study could be considered as a basis for encouraging research on findings and identification of truffles in Finland.

Evaluation of Pecan [Carya illinoinensis (Wangenh.) K. Koch] Cultivars for Possible Cultivation for Both Fruit and Truffle Production in the Puglia Region, Southeastern Italy

Yield and different nut parameters were measured for two growing seasons on mature (28–29 years) trees of 11 pecan cultivars grown in an experimental orchard located in the Puglia Region, Southeastern Italy. ‘Shoshoni’ and ‘Shawnee’ pecan seedlings were inoculated with three truffle species (Tuber borchii—known as the ‘whitish truffle’, T. aestivum—called the ‘summer truffle’, and T. melanosporum—the common ‘Black truffle’) and investigated for six months. The level of ectomycorrhizal colonization was assessed 6 and 12 months after inoculation. Results indicated that ‘Wichita’, ‘Shoshoni’, and ‘Pawnee’ performed well in the pedoclimatic conditions of the area with a yield higher than 20 kg/tree and a kernel dry weight of ≅3 g. These preliminary yield results suggested that some pecan cultivars could deserve consideration for cultivation in the Puglia Region, whereas others with low yield and a stronger alternate bearing should not be considered. Plant height, number of leaves, chlorophyll content (expressed as a SPAD unit) and stem diameter partially indicated the increase in ecological fitness in truffles-inoculated plants. Successful mycorrhization indicated ‘Shoshoni’ and ‘Shawnee’ as suitable to establish ectomycorrhizal symbiosis with T. aestivum, T. borchii, and T. melanosporum cultivation under Puglia climatic conditions. The results also showed that the applied protocol was adequate to obtain healthy mycorrhized seedlings appropriate for commercialization and plantation for truffles production on pecan in the future.

First molecular identification of Suillia gigantea (Meigen, 1830) (Diptera: Heleomyzidae)

In 2017, the presence of the fly Suillia gigantea (Meigen, 1830) was noted in Poland, after many years of research related to the ecology of insects associated with the fruiting bodies of hypogeous fungi. Finally, in 2020, after further studies, the distribution of the truffle fly in Poland was confirmed. Six adults were reared from larvae inhabiting the fruiting bodies of Burgundy truffle (Tuber aestivum Vittad. (1831)). The fungi were harvested in southern Poland. Morphological and genetic analyses of the insect specimens were performed. For the first time, the DNA sequence of this fly species was isolated. This is the first record of S. gigantea in Poland, although it has already been reported from neighbouring countries. The development of S. gigantea, also known as the truffle fly, is associated with hypogeous fungi, mainly belonging to the genus Tuber P. Micheli ex F.H. Wigg. (1780). The diptera larvae develop inside the fruiting bodies. This type of damage causes great losses in truffle production. Containment of these losses is of great interest to the truffle industry. Knowledge of truffle-inhabiting insects is crucial for the development of effective methods to protect truffle crops in Europe.

Efficiency of the traditional practice of traps to stimulate black truffle production, and its ecological mechanisms

The black truffle Tuber melanosporum was disseminated all over the world, propelled by the development of a wide variety of empirical practices. A widespread practice, called ‘truffle trap’, consists of placing pieces of truffles into excavations dug under host trees, and of collecting truffle in these traps in the next years. This research aims at (1) evaluating the effect of this practice on fruitbody production based on the analysis of 9924 truffle traps installed in 11 orchards across T. melanosporum native area in France and (2) exploring the mechanisms involved in fruitbody emergence using traps where the genotypes of introduced truffles were compared with those of fruitbodies collected in the same traps. We confirmed that truffle traps provide a major and highly variable part of truffle ground production, representing up to 89% of the collected fruitbodies. We evidenced a genetic link between introduced spores and collected fruitbodies, and then demonstrated that truffle growers provide paternal partners for mating with local maternal mycelia. We also highlighted that soil disturbance stimulate the vegetative development of established maternal mycelia. This research supports that a widely used traditional practice enhances fruitbody production by shaping favorable conditions and providing sexual partners required for fruiting.

Presence and production of Tuber aestivum in relation with the soil environment of a semi-natural experimental truffle orchard in Spoleto, Central Italy

Truffles produced by Tuber species (Ascomycota) represent an additional income in economically marginal areas, especially if cultivating mycorrhized seedlings in dedicated truffle orchards. However, despite the great progress made in cultivation, truffle production has not increased substantially. One of the reasons may be the lack of knowledge on soil as a key component of truffle species habitats. This is the case for Tuber aestivum, whose soil environment has been studied less than those of T. magnatum and T. melanosporum due to the lower economic value of its ascocarps. We did the present study in an experimental truffle orchard submitted to a light tillage before our investigation and characterized by the presence of different host tree species and soil variation in space. We assessed whether truffle production and T. aestivum mycelial DNA in soil varied depending on symbiont tree species, soil type or both by comparing sub-areas characterized by the presence/absence of: i) two landforms; ii) four host tree species; and iii) the truffle brûlé, i.e., the area lacking herbaceous cover when root colonization by T. aestivum occurs.Results show that combined variations in chemical and physical properties determined by soil forming processes can make an area suitable or not for T. aestivum. The establishment of brûlé and higher ascocarp production occurred in the more alkaline and less compact soil type, where the resistance to ascocarp volume enlargement is lower and aeration of the soil surface layer is higher. The tillage carried out in the truffle orchard presumably mimicked the effect of the brûlé on the soil structure, allowing the enlargement of the productive area outside the brûlé itself and showing how the management of a truffle plantation, even if sub-optimal for T. aestivum colonization, can influence its productive capacity. Our results further suggest that even in the best environmental conditions not all host trees will enter in production, while in less suitable soil conditions little or no production should be expected.

Soil Moisture and Black Truffle Production Variability in the Iberian Peninsula

The relationship between modelled root zone soil moisture (SM) and black truffle production in the Iberian Peninsula was studied. Previous works have investigated the influence that precipitation exerts on truffle yield highlighting the importance of water for the growth of black truffle. However, SM had not been used until now due to the lack of suitable databases. The SM series from the LISFLOOD hydrological rainfall–runoff model was used in this study. Annual black truffle yield series from 175 locations in Spain was correlated with SM for the period 1991–2012. For this, different approaches were applied considering daily, weekly and monthly temporal scales. The same analysis was carried out using precipitation data to compare the behaviors of both variables related to truffle production variability. The results obtained show critical periods in terms of soil water content in summer (June–September) and during October–November months. Moreover, a clear delay between precipitation and SM influence on black truffle was observed. The results obtained in this study highlight the importance of SM for black truffle production, since this variable truly expresses the available water for this fungus, which completes its entire life cycle living below ground.

Bacterial Communities in the Fruiting Bodies and Background Soils of the White Truffle Tuber magnatum.

Tuber magnatum Picco is a greatly appreciated truffle species mainly distributed in Italy. Its price and characteristics mostly depend on its geographical origin. Truffles represent a fundamental step of the life cycle of Tuber species promoting spore dissemination. They consist of two main parts, gleba, the inner part, and peridium, which is in direct contact with ground soil. Within the truffle and around in the growing soil, both the occurrence and abundance of different microbial species seem to play an essential role in truffle production. The development of the next-generation sequencing (NGS) based technology has greatly improved to deepen the role of the composition of microbial communities, thus improving the knowledge of the existing relationships between microbial taxa in a specific condition. Here, we applied a metabarcoding approach to assess the differences in T. magnatum samples collected from three areas in Tuscany (Italy). Peridium and gleba were analyzed separately with the aim to distinguish them based on their microbial composition. Also, soil samples were collected and analyzed to compare productive and unproductive truffle grounds to confirm the presence of specific patterns linked to truffle production. Results indicate that differences occurred between truffle compartments (gleba and peridium) as well as between analyzed soils (productive and unproductive), with distinctive taxa associated. Furthermore, findings also demonstrated specific characteristics associated with truffle collection areas, thus indicating a degree of microbial selection related to different environments.

Black Truffles Affect Quercus aliena Physiology and Root-Associated nirK- and nirS-Type Denitrifying Bacterial Communities in the Initial Stage of Inoculation.

Truffles (Tuber spp.) are edible ectomycorrhizal fungi with high economic value. Bacteria in ectomycorrhizosphere soils are considered to be associated with the nutrient uptake of truffles and hosts. Whether Tuber spp. inoculation can affect the growth of Quercus aliena, the ectomycorrhizosphere soil, and the rhizosphere nirK and nirS-denitrifier communities at the ectomycorrhizae formation stage is still unclear. Therefore, we inoculated Q. aliena with the black truffles Tuber melanosporum and Tuber indicum, determined the physiological activity and morphological indices of Q. aliena seedlings, analyzed the physicochemical properties of ectomycorrhizosphere soils, and applied DNA sequencing to assess the nirK and nirS- denitrifier community structure in ectomycorrhizosphere soils. Peroxidase activity was higher in the seedlings inoculated with T. melanosporum than in the T. indicum inoculation and uninoculated control treatments. The available phosphorus contents were lower and nitrate contents were higher in those with truffle inoculation, and T. melanosporum treatment differed more from the control than the T. indicum treatment. The richness of the nirK-community was highest in the T. indicum treatment and lowest in the uninoculated treatment. The differences in nirK-community composition across treatments were not statistically significant, but the nirS communities were different. The nirS-type bacteria correlated with three environmental factors (pH, available phosphorus, and nitrate contents), whereas the nirK-type bacteria were only associated with the nitrate contents. Generally, this work revealed that inoculation with Tuber spp. would change a few nutrient contents and richness of nirK-type bacteria and had little effects on growth of Q. aliena seedlings in the initial stage of inoculation. The results of this study may provide in-depth insights into the relationships between Tuber spp. and hosts, which should be taken into account when developing truffle production methods.

The hidden value of non-timber forest products: income contribution of the Basilicata wild truffle

The Basilicata region (South of Italy) is land of truffles where the gastronomic, economic and cultural awareness has developed for this non-timber forest product only in the past decade. Little is known about truffle production and its social, economic and environmental implications. In this article we investigate the Basilicata truffle sector by devoting particular attention to the truffle hunters who gather the truffles from the forests. The data for the analysis were collected through a survey with the aim of describing the gathering activity (people involved, specie and quantities collected, etc.) and assessing its significance as a source of income. Results show that truffles can provide local communities with earning opportunities. However, the truffle sector needs to be protected and enhanced.

Microbial communities of ascocarps and soils in a natural habitat of Tuber indicum.

Truffles are the fruiting bodies of hypogeous fungi in the genus Tuber. Some truffle species usually grow in an area devoid of vegetation, called brûlé, but limited knowledge is available on the microbial composition and structure of them. Here, we investigated the bacterial and fungal communities of Tuber indicum ascocarps and soils inside and outside a characteristic brûlé from a poplar plantation with no truffle production history in northeastern China using a high-throughput sequencing approach. A predominance of members of the bacterial phylum Proteobacteria was observed in all samples. Members of Bacillus were the main genera in the ascocarps, while members of Lysobacter and unidentified Acidobacteria were more abundant in the soil. In addition, members of Gibberella, Fusarium, and Absidia were the dominant fungi in the ascocarps, while members of Tuber were enriched in the ascocarps and soils inside the brûlé. Some mycorrhization helper bacteria (Rhizobium) and ectomycorrhiza-associated bacteria (Lysobacter) were detected, indicating their potential roles in the complex development of underground fruiting bodies and brûlé formation. These findings may contribute to the protection and cultivation of truffles.

Risk and reward of the global truffle sector under predicted climate change

Climate change has been described as the main threat for the cultivation and growth of truffles, but hydroclimate variability and model uncertainty challenge regional projections and adaptation strategies of the emerging sector. Here, we conduct a literature review to define the main Périgord truffle growing regions around the world and use 20 global climate models to assess the impact of future trends and extremes in temperature, precipitation and soil moisture on truffle production rates and price levels in all cultivation regions in the Americas, Europe, South Africa, and Australasia. Climate model simulations project 2.3 million km2 of suitable land for truffle growth will experience 50% faster aridification than the rests of the global land surface, with significantly more heat waves between 2070 and 2099 CE. Overall, truffle production rates will decrease by ∼15%, while associated price levels will increase by ∼36%. At the same time, a predicted increase in summer precipitation and less intense warming over Australasia will likely alleviate water scarcity and support higher yields of more affordable truffles. Our findings are relevant for truffle farmers and businesses to adapt their irrigation systems and management strategies to future climate change.

Desert truffle mycorrhizosphere harbors organic acid releasing plant growth\u2013promoting rhizobacteria, essentially during the truffle fruiting season

Desert truffle is becoming a new crop in semiarid areas. Climatic parameters and the presence of microorganisms influence the host plant physiology and alter desert truffle production. Desert truffle plants present a typical summer deciduous plant phenology divided into four stages: summer dormancy, autumn bud break, winter photosynthetic activity, and spring fruiting. We hypothesize that the bacterial community associated with desert truffle plants will show a seasonal trend linked to their plant growth–promoting rhizobacteria (PGPR) traits. This information will provide us with a better understanding about its potential role in this symbiosis and possible management implementations. Bacteria were isolated from root-adhering soil at the four described seasons. A total of 417 isolated bacteria were phenotypically and biochemically characterized and gathered by molecular analysis into 68 operational taxonomic units (OTUs). They were further characterized for PGPR traits such as indole acetic acid production, siderophore production, calcium phosphate solubilization, and ACCD (1-amino-cyclopropane-1-carboxilatedeaminase) activity. These PGPR traits were used to infer functional PGPR diversity and cultivable bacterial OTU composition at different phenological moments. The different seasons induced shifts in the OTU composition linked to their PGPR traits. Summer was the phenological stage with the lowest microbial diversity and PGPR functions, whereas spring was the most active one. Among the PGPR traits analyzed, P-solubilizing rhizobacteria were harbored in the mycorrhizosphere during desert truffle fruiting in spring.

Soil fungal community and mating type development of Tuber melanosporum in a 20-year chronosequence of black truffle plantations

Black truffle plantations are established on the basis that Tuber melanosporum Vittad. spreads from artificially inoculated trees. Although truffle cultivation has progressed tremendously over the past 30 years, the ecological processes underlying T. melanosporum mycelium expansion and its interactions with the rest of the fungal community over time are not completely understood. Controversy exists on how mating type distribution evolves with time and its incidence on truffle production. We studied the soil fungal community and mating type distribution in plantations before truffle production (3, 5 and 7 years and after plantation establishment) and during the production period (10, 14 and 20 years) at three distances from the tree stem: 40, 100 and 200 cm. We found that T. melanosporum developed steadily over the years as extraradical mycelium, first at the nearest part of the tree and later up to 200 cm. T. melanosporum development was not correlated with changes in other ectomycorrhizal fungi and was negatively correlated with lower relative abundance of arbuscular mycorrhizal fungi and of non-root associated guilds such as moulds, yeasts and plant pathogens. Mating type frequency did not change across years. Twenty years after establishment no signs of replacement of T. melanosporum by other fungi nor biases in mating type abundance were observed in soil mycelia, indicating that T. melanosporum can colonize and dominate the surrounding soil in mature Quercus ilex L. plantations.

Truffle Market Evolution: An Application of the Delphi Method

Background: The objective of this study was to analyze the current situation of the truffle sector in the main producing countries of the Mediterranean area. Additionally, we identified the challenges for the future and the priority actions to develop the truffle sector in the region. Methods: We used a Delphi process approach, and we selected a total of 17 expert panelists in different positions within the supply chain of the target countries (Spain, France, Italy, Croatia, and Greece). Results: The results obtained allowed us to have a complete description of the current truffle supply chain. We confirmed an evolution of the sector due to the cultivation success of several Tuber species. The maturity of the sector has produced shifts in the roles that form the traditional truffle supply chain operators. We confirmed the trend of a decrease of collectors that hunt truffles in the wild and sell to small travelling buyers, whilst truffle hunters that collect for farmers and specialty wholesalers are emerging. However, a trend of truffle price decrease in the last few years has alerted the sector. Conclusions: As production increases due to truffle cultivation, it will be necessary to promote truffle consumption. We identified actions to develop the truffle sector: (a) strengthen the link between truffles, tourism, and gastronomy; (b) increase the effort at European level for the recognition of truffle production, helping to develop truffle culture and marketing; (c) increase the awareness and consumption of truffles among consumers; and (d) develop tourism workshops for truffle farmers.

Different patterns in root and soil fungal diversity drive plant productivity of the desert truffle Terfezia claveryi in plantation.

The desert truffle Terfezia claveryi is one of the few mycorrhizal fungi currently in cultivation in semiarid and arid areas. Agroclimatic parameters seem to affect its annual yield, but there is no information on the influence of biotic factors. In this study, fungal diversity was analysed by high-throughput sequencing of the ITS2 rDNA region from soil and root samples to compare productive and non-productive mycorrhizal plants in a 4-years old plantation (Murcia, Spain). The fungal metaprofile was dominated by Ascomycota phylum. Desert truffle productivity was driven by different patterns of fungal species composition in soil (species replacement) and root (species richness differences). Moreover, positive associations for ectomycorrhizal and negative for arbuscular mycorrhizal guilds were found in productive roots, and positive associations for fungal parasite-plant pathogen guild in non-productive ones. Soil samples were dominated by pathotroph and saprotroph trophic modes, showing positive associations for Aureobasidium pullulans and Alternaria sp. in productive areas, and positive associations for Fusarium sp. and Mortierella sp. were found in non-productive soils. Finally, some significant OTUs were identified and associated to ascocarp producing patches, which could serve as predictive and location markers of desert truffle production.

Mycorrhization of pecans with European truffles (Tuber spp., Tuberaceae) under southern subtropical conditions

Pecan trees are known to form ectomycorrhizae with a wide range of fungi, including commercial truffles that can be cultivated in agroforestry systems. Here, we investigated whether pecans can be successfully mycorrhized by Tuber spp. and whether the mycorrhization affects growth parameters and nutrient content of two productive pecan cultivars under southern subtropical conditions. We also sought to define procedures of mycorrhization adapted to the subtropics. The seedlings of two pecan cultivars (Barton and Importada) were inoculated with both Tuber aestivum and T. brumale and grown in a nursery for 12 months. The levels of ectomycorrhizal colonization and the morphological and chemical parameters of the seedlings were analyzed. The proposed mycorrhization protocol was successful, as all truffle-inoculated seedlings exhibited a high percentage of mycorrhized fine roots, on average 70% with T. aestivum and 88% with T. brumale. In both pecan cultivars, mycorrhization by the Tuber species reached levels at which all seedlings would qualify as suitable for commercialization. Notably, the cultivar Barton inoculated with T. brumale exhibited higher total biomass accumulation and growth of roots and shoots. Shoot and root parameters did not show nutrient deprivation. The nutrient status after one year of cultivation in a nursery also supported that the applied protocol was adequate to maintain healthy mycorrhized seedlings with suitable nutritional status for out-planting of seedlings in a commercial plantation. To our knowledge, this is the first study on the mycorrhization of pecan seedlings with European truffles under southern subtropical conditions. Our results suggested that the tested cultivars have a high potential for truffle production considering the necessary substrate, site, and management adaptations for selected commercial truffle species and the requirements for pecan orchards.