Habitat is more important than climate for structuring soil fungal communities associated in truffle sites.

The ectomycorrhizal fungi Tuber melanosporum Vittad. and Tuber aestivum Vittad. produce highly valuable truffles, but little is known about the soil fungal communities associated with these truffle species in places where they co-occur. Here, we compared soil fungal communities present in wild and planted truffle sites, in which T. melanosporum and T. aestivum coexist, in Mediterranean and temperate regions over three sampling seasons spanning from 2018 to 2019. We showed that soil fungal community composition and ectomycorrhizal species composition are driven by habitat type rather than climate regions. Also, we observed the influence of soil pH, organic matter content and C:N ratio structuring total and ectomycorrhizal fungal assemblages. Soil fungal communities in wild sites revealed more compositional variability than those of plantations. Greater soil fungal diversity was found in temperate compared to Mediterranean sites when considering all fungal guilds. Ectomycorrhizal diversity was significantly higher in wild sites compared to plantations. Greater mould abundance at wild sites than those on plantation was observed while tree species and seasonal effects were not significant predictors in fungal community structure. Our results suggested a strong influence of both ecosystem age and management on the fungal taxa composition in truffle habitats.

Tuber indicum and T. lijiangense colonization differentially regulates plant physiological responses and mycorrhizosphere bacterial community of Castanopsis rockii seedlings.

Black truffles and white truffles are widely studied around the world, but their effects on plant growth and physiological responses, and on the mycorrhizosphere bacterial community of the host plant remain unclear. Here, mycorrhizal colonization of Castanopsis rockii by Tuber indicum (Chinese black truffle) and T. lijiangense (Chinese white truffle), respectively, was induced in a greenhouse study, and their effects on host growth, physiological responses and mycorrhizosphere bacterial communities were compared. The results show that colonization of both Tuber species significantly increased leaf photosynthetic rate, leaf P concentration and mycorrhizosphere acid phosphatase activity, as well as richness of mycorrhizosphere bacterial communities of C. rockii seedlings. However, T. indicum colonization on the one hand significantly decreased tartrate content, bacterial acid phosphatase, phoC gene abundance in the mycorrhizosphere, and peroxidase (POD) activity of ectomycorrhizal root tips, but on the other hand increased mycorrhizosphere pH and superoxide dismutase (SOD) of ectomycorrhizal root tips, compared to T. lijiangense colonization. Moreover, principal coordinate and ß-diversity analyses show significant differences in mycorrhizosphere bacterial community composition between T. indicum and T. lijiangese colonized C. rockii seedlings. Finally, the relative abundance of the bacterium Agromyces cerinus significantly correlated to mycorrhizosphere acid phosphatase activity and leaf P concentration, suggesting that this bacterium might play an important role in P mobilization and acquisition. Overall, these results suggest that T. indicum and T. lijiangense differently regulate their host plant's physiological responses and mycorrhizosphere bacterial community.

Factors influencing successful establishment of exotic Pinus radiata seedlings with co-introduced Lactarius deliciosus or local ectomycorrhizal fungal communities.

An introduction of exotic or non-native trees may fail due to a lack of suitable fungal partners. We planted exotic Pinus radiata in Xifeng, Guizhou Southwest China. Strategies to introduce P. radiata seedlings either colonized with an ectomycorrhizal fungus (EcMF), Lactarius deliciosus, or expect them to form familiar/new associations with local EcMF in a new habitat were studied to know how P. radiata could be successfully established over a period of 2.5 years. Plant height and needle nutrient acquisition, the persistence of the co-introduced L. deliciosus, and fungal community composition in rhizosphere soil and root tips were analyzed. In addition, a greenhouse bioassay experiment of local soil to assess the differences in the EcMF community between exotic and native pine seedlings was also conducted. The current results demonstrated that P. radiata could establish in the Xifeng plantation with or without co-introduced L. deliciosus. The co-introduced L. deliciosus might be naturalized with P. radiata in the new area since it has been fruited for 2 years with high relative abundance in mycorrhizosphere soil. L. deliciosus pre-colonization significantly altered the mycorrhizosphere fungal composition and it had a positive correlation with nitrogen acquisition of P. radiata. Host identity had no effect on fungal composition since exotic P. radiata and native P. massoniana recruited similar local fungal communities in early establishment or in plantation. The cosmopolitan species Suillus placidus, with high relative abundance, formed a familiar association with P. radiata. The greenhouse bioassay experiment further showed that Suillus sp. contributed relatively higher total extracellular enzymes by forming ectomycorrhizas with P. radiata and the same type of ectomycorrhiza of P. radiata and P. massoniana showed different enzymatic functions. Our study indicated that exotic P. radiata could be a suitable tree capable to get established successfully in the Xifeng plantation either by interaction with the co-introduced L. deliciosus or with a local EcMF, but we should be cautious about large-scale planting of P. radiata. L. deliciosus persisted in plantation and more attention should be paid to local EcMF community changes induced by the introduced L. deliciosus.

Ethnomycological study on wild mushrooms in Pu'er Prefecture, Southwest Yunnan, China.

BACKGROUND: Yunnan is rich in fungal diversity and cultural diversity, but there are few researches on ethnomycology. In addition, extensive utilization of wild edible fungi (WEF), especially the ectomycorrhizal fungi, threatens the fungal diversity. Hence, this study aims to contribute to the ethnomycological knowledge in Pu'er Prefecture, Yunnan, China, including information on the fungal taxa presented in markets and natural habitats, with emphasis in ectomycorrhizal fungi (EMF). METHODS: Semi-structured interviews with mushroom vendors in markets and with mushroom collectors in natural habitats were conducted. Information related to local names, habitat, fruiting time, species identification, price, cooking methods and preservation methods of wild edible mushrooms were recorded. Wild edible fungi were collected from forests, and morphological and molecular techniques were used to identify fungal species. RESULTS: A total of 11 markets were visited during this study. The 101 species collected in the markets belonged to 22 families and 39 genera, and about 76% of them were EMF. A wealth of ethnomycological knowledge was recorded, and we found that participants in the 45-65 age group were able to judge mushroom species more accurately. Additionally, men usually had a deepest mushroom knowledge than women. A total of 283 species, varieties and undescribed species were collected from natural habitats, and about 70% of them were EMF. Mushroom species and recorded amounts showed correspondence between markets and the natural habitats on different months. CONCLUSION: The present study shows that Pu'er Prefecture is rich in local mycological knowledge and fungal diversity. However, it is necessary to continue the research of ethnomycological studies and to design and conduct dissemination of local knowledge in order to preserve it, since it currently remains mainly among the elderly population.

New qPCR protocol to detect Diplodia corticola shows phoretic association with the oak pinhole borer Platypus cylindrus.

BACKGROUND: Botryosphaeria canker (causal agent: Diplodia corticola) is considered one of the most important diseases of cork oak (Quercus suber) stands since it necrotizes the subero-phellodermic layer preventing cork regeneration after harvesting. One of the most intriguing etiological issues of this disease is its plausible spreading through trunk borer insects. In this study, we highlight the phoretic relationship between D. corticola and the oak pinhole borer Platypus cylindrus (Coleoptera, Curculionidae) which massively colonizes debarked cork oaks in southern Europe. We extracted DNA from 154 adults of P. cylindrus collected in six cork oak stands in north-eastern Spain during the summer of 2021. We developed a new nested quantitative PCR-based protocol for quick detection of D. corticola carried by insects. RESULTS: The use of real time amplification of a highly specific mitochondrial marker allowed us to detect spore loads down to a single conidium within the first 29 cycles of qPCR. The 29.62% of insect pools (corresponding to 31.16% of studied insects) resulted in an estimated spore load higher than one conidium/insect, with a moderate value of mean spore load for the whole dataset (~33 conidia/insect). Estimated spore load was significantly higher in May and August, regardless of insect abundance in the field. CONCLUSION: This study provides new tools for diagnosis of this emergent pathogen that would be useful for developing monitoring strategies and epidemiological studies. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Lack of Phylogenetic Differences in Ectomycorrhizal Fungi among Distinct Mediterranean Pine Forest Habitats.

Understanding whether the occurrences of ectomycorrhizal species in a given tree host are phylogenetically determined can help in assessing different conservational needs for each fungal species. In this study, we characterized ectomycorrhizal phylogenetic composition and phylogenetic structure in 42 plots with five different Mediterranean pine forests: i.e., pure forests dominated by P. nigra, P. halepensis, and P. sylvestris, and mixed forests of P. nigra-P. halepensis and P. nigra-P. sylvestris, and tested whether the phylogenetic structure of ectomycorrhizal communities differs among these. We found that ectomycorrhizal communities were not different among pine tree hosts neither in phylogenetic composition nor in structure and phylogenetic diversity. Moreover, we detected a weak abiotic filtering effect (4%), with pH being the only significant variable influencing the phylogenetic ectomycorrhizal community, while the phylogenetic structure was slightly influenced by the shared effect of stand structure, soil, and geographic distance. However, the phylogenetic community similarity increased at lower pH values, supporting that fewer, closely related species were found at lower pH values. Also, no phylogenetic signal was detected among exploration types, although short and contact were the most abundant types in these forest ecosystems. Our results demonstrate that pH but not tree host, acts as a strong abiotic filter on ectomycorrhizal phylogenetic communities in Mediterranean pine forests at a local scale. Finally, our study shed light on dominant ectomycorrhizal foraging strategies in drought-prone ecosystems such as Mediterranean forests.

White mulch and irrigation increase black truffle soil mycelium when competing with summer truffle in young truffle orchards.

The black truffle (Tuber melanosporum Vittad.) and the summer truffle (Tuber aestivum Vittad.) are two of the most appreciated edible fungi worldwide. The natural distributions of both species partially overlap. However, the interspecific interactions between these truffles and how irrigation and mulching techniques impact the dynamics between them are still unknown. Here, an experimental truffle plantation with Quercus ilex was established in Maials (Catalonia, Spain), combining three soil mulch treatments (white mulch, black mulch and bare soil as a control) and two irrigation regimes (irrigated and non-irrigated as a control) to investigate truffle mycelial dynamics in soil when both truffle species co-occur. The development of truffle mycelium in two different seasons (spring and autumn) in two consecutive years (2017 and 2018) was quantified using qPCR. Truffle mycelia of both species showed greatest development under white mulch. When mycelia of both truffle species co-occurred in soil, irrigation combined with white mulch resulted in greater quantities of T. melanosporum mycelial biomass, whereas the control irrigation treatment favoured the development of T. aestivum. Mulch treatments were also advantageous for seedling growth, which was expressed as root collar diameter and its increment during the study period. Significant relationships between root collar diameter and root growth and the amount of mycelial biomass in the soil were observed for both truffle species. Our results indicate the potential advantages of using white mulch to support irrigation in truffle plantations located in areas with dry Mediterranean climatic conditions to promote the development of Tuber mycelium.

A new edible Rhizopogon species from Southwest China, and its mycorrhizal synthesis with two native pines.

A new Rhizopogon species associated with Pinus was discovered at local wild mushroom markets and Pinus armandii forests from March to July in Southwest China where it is considered a delicacy. Based on morphological and molecular phylogenetic analyses, the collections were described as Rhizopogon songmaodan sp. nov. belonging to the subgenus Versicolores. The new species described here increases the current number of Rhizopogon species known in China to ten. R. songmaodan establishes ectomycorrhizal associations with P. armandii which was confirmed by comparing rDNA ITS sequences from basidiomata and ectomycorrhizal root tips. Mycorrhizal synthesis via spore inoculation between R. songmaodan and two native pine species, Pinus armandii and P. yunnanensis was successfully carried out in a greenhouse study. The ease of R. songmaodan inoculation onto pine species, and the high market demand of its sporocarps, could make R. songmaodan a good candidate for cultivation in Southwest China.

Time and dose of irrigation impact Tuber melanosporum ectomycorrhiza proliferation and growth of Quercus ilex seedling hosts in young black truffle orchards.

In Mediterranean climate, young truffle-oak orchards are subjected to drought episodes that can compromise the development of Tuber melanosporum. We investigated the responses of T. melanosporum to water supply in three periods: May to July, August to October, and May to October. In each period, five water doses were established: 0, 25, 50, 75, and 100% of the reference evapotranspiration (ETo). Five orchards were planted with Quercus ilex inoculated with T. melanosporum, and in each orchard, we arranged a two-factorial design with irrigation period and irrigation dose as main factors to test their combined effects on the development of both T. melanosporum and Q. ilex after 3 years in the field. Irrigation period significantly interacted with irrigation doses for the absolute presence per seedling of T. melanosporum mycorrhizae. Irrigation in May-July increased significantly T. melanosporum colonization in seedlings irrigated with 50% ETo dose compared to the 0% ETo dose. A similar pattern with smaller differences in means was observed in August-October period, but the irrigation doses did not change T. melanosporum colonization when we watered from May to October. We found ectomycorrhizae different from T. melanosporum in 51% of the seedlings studied, but their presence was marginal. Our results suggest that a moderate irrigation dose promotes seedling growth and number of fine root tips per unit of fine root length, which may be potentially colonized by T. melanosporum.

Can NPK fertilizers enhance seedling growth and mycorrhizal status of Tuber melanosporum-inoculated Quercus ilex seedlings?

Although successful cultivation of the black truffle (Tuber melanosporum) has inspired the establishment of widespread truffle orchards in agricultural lands throughout the world, there are many unknowns involved in proper management of orchards during the 6-10 years prior to truffle production, and there are conflicting results reported for fertilizer treatments. Here, we systematically evaluate the combined effects of nitrogen, phosphorous, and potassium with different doses of each element, applied to either foliage or roots, on plant growth parameters and the mycorrhizal status of outplanted 3-year-old seedlings in five experimental Quercus ilex-T. melanosporum orchards. Fertilization did not significantly improve seedling aboveground growth, but the plants treated with the fertilizer 12-7-7 applied to the roots (HNr) displayed longer field-developed roots. Only the fertilizer with the highest dose of K (10-6-28) applied to the foliage (HKf) increased the probability of fine root tip colonization by T. melanosporum in field-developed roots. However, the plants treated with the same fertilizer applied to the soil (HKr) presented the highest probability for colonization by other competing mycorrhizal soil fungi. Potassium seems to have an important role in mycorrhizal development in these soils. Apart from T. melanosporum, we found 14 ectomycorrhizal morphotypes, from which seven were identified to species level, three to genus, two to family, and two remained unidentified by their morphological characteristics and DNA analyses.

Mycelial abundance and other factors related to truffle productivity in Tuber melanosporum-Quercus ilex orchards.

Relative quantification of DNA from Tuber melanosporum mycelia was performed by conventional and real-time PCR in soil from trees in three truffle orchards of different ages to determine: (1) whether burn appearance is related to the amount of T. melanosporum mycelium in soil, and (2) whether productivity onset and truffle production are related to (a) the amount of T. melanosporum mycelium in soil, (b) tree height and diameter, (c) burn extension and (d) surface rock cover. The burn seems to appear only after a certain amount of mycelium has formed. Precociously productive trees presented higher quantities of mycelium than nonproductive trees in the productivity onset study, while highly productive trees presented less quantities of mycelium than nonproductive trees in the productivity study. Trees with high but not excessive surface rock cover showed greater truffle production. Larger trees tended to display a burn earlier than smaller trees.

Detection of Tuber melanosporum DNA in soil.

Our objectives were (i) to develop a molecular method to detect mycelia of Tuber melanosporum (black truffle) in soil and (ii) to test for mycelial distribution around two truffle-bearing Quercus ilex trees in a truffle orchard. Isolation of total DNA from soil was performed, followed by PCR amplification with T. melanosporum-specific primers and restriction analysis. To address the detection sensitivity level, soil samples were inoculated with known amounts of gleba of T. melanosporum. The detection limit was >/=11.4 mug of hyphae g(-1) of soil. Mycelium was detected primarily within the area defined by the truffle burn and within the top 35 cm of the soil in all directions from the trees.