Based on a multi-locus phylogeny of a combined dataset of ITS, LSU, tef1-α, and rpb2 and comprehensive morphological analyses, we describe three new species from the Melanosporum group of genus Tuber and synonymize T. pseudobrumale and T. melanoexcavatum. Phylogenetically, the three newly described species, T. yunnanense, T. melanoumbilicatum and T. microexcavatum, differ significantly in genetic distance from any previously known species. Morphologically, T. yunnanense is distinctly different from its closest phylogenetically related species, T. longispinosum, due to its long shuttle-shape spores (average the ratio of spore length to spore width for all spores (Qm) = 1.74). Tuber melanoumbilicatum differs from the other species in having a cavity and long shuttle-shaped spores (Qm = 1.65). Although T. microexcavatum sampled ascomata have relatively low maturity, they can be distinguished from its closely related species T. pseudobrumale by the ascomata size, surface warts, and spore number per asci; additionally, phylogenetic analysis supports it as a new species. In addition, molecular analysis from 22 newly collected specimens and Genebank data indicate that T. pseudobrumale and T. melanoexcavatum are clustered into a single well-supported clade (Bootstrap (BS) = 100, posterior probabilities (PP) = 1.0); and morphological characteristics do not differ. Therefore, based on the above evidence and publication dates, we conclude that T. melanoexcavatum is a synonym of T. pseudobrumale. By taking into account current knowledge and combining the molecular, multigene phylogenetic clade arrangement and morphological data, we propose that the Melanosporum group should be divided into four subgroups. Diagnostic morphological features and an identification key of all known species in the Melanosporum group are also included. Finally, we also provide some additions to the knowledge of the characterization of T. pseudobrumale, T. variabilisporum, and T. pseudohimalayense included in subgroup 1 of the Melanosporum group.

Ectomycorrhizal symbiosis is a cornerstone of ecosystem health, facilitating nutrient uptake, stress tolerance, and biodiversity maintenance in trees. Optimizing Pinus armandii–Tuber indicum mycorrhizal synthesis enhances the ecological stability of coniferous forests while supporting high-value truffle cultivation. This study conducted a pot experiment to compare the effects of three root nutrient regulations—Aolu 318S (containing N-P₂O₅-K₂O in a ratio of 15-9-11 (w/w%)), Aolu 328S (11-11-18), and Youguduo (19-19-19)—on the mycorrhizal synthesis of P. armandii–T. indicum. The results showed that root nutrient supplementation significantly improved the seedling crown, plant height, ground diameter, biomass dry weight, and mycorrhizal infection rate of both the control and mycorrhizal seedlings, with the slow-release fertilizers Aolu 318S and 328S outperforming the quick-release fertilizer Youguduo. The suitable substrate composition in this experiment was as follows: pH 6.53–6.86, organic matter content 43.25–43.49 g/kg, alkali-hydrolyzable nitrogen 89.25–90.3 mg/kg, available phosphorus 83.69–87.32 mg/kg, available potassium 361.5–364.65 mg/kg, exchangeable magnesium 1.17–1.57 mg/kg, and available iron 33.06–37.3 mg/kg. It is recommended to mix the Aolu 318S and 328S solid fertilizers evenly into the substrate, with a recommended dosage of 2 g per plant. These results shed light on the pivotal role of a precise N-P-K ratio regulation in fostering sustainable ectomycorrhizal symbiosis, offering a novel paradigm for integrating nutrient management with mycorrhizal biotechnology to enhance forest restoration efficiency in arid ecosystems.

Hydnobolites is an ectomycorrhizal fungal genus with hypogeous ascomata in the family Pezizaceae (Pezizales). Molecular analyses of Hydnobolites using both single (ITS) and concatenated gene datasets (ITS-nLSU) showed a total of 223 sequences, including 92 newly gained sequences from Chinese specimens. Phylogenetic results based on these two datasets revealed seven distinct phylogenetic clades. Among them, the ITS phylogenetic tree confirmed the presence of at least 42 phylogenetic species in Hydnobolites. Combined the morphological observations with molecular analyses, five new species of Hydnobolites translucidus sp. nov., H. subrufus sp. nov., H. lini sp. nov., H. sichuanensis sp. nov. and H. tenuiperidius sp. nov., and one new record species of H. cerebriformis Tul., were illustrated from Southwest China. Macro- and micro-morphological analyses of ascomata revealed a few, but diagnostic differences between the H. cerebriformis complex, while the similarities of the ITS sequences ranged from 94.4 to 97.2% resulting in well-supported clades.

Tuber is a rare edible and medicinal fungus with a global distribution. The Macrosporum lineage within Tuber is a relatively small and infrequent clade, which has been receiving increasing attention due to the presence of an endangered species, T. gigantosporum. However, the species diversity, phylogenetic relationships, geographic distribution, and ecological characteristics of the Macrosporum group remain poorly understood. Through extensive sampling and combining the accurate and available global specimen and mycorrhizal data, 19 Tuber specimens from Southwest China were studied based on morphology, molecular systematics, and ecology. The specimens were identified as T. calosporum, T. glabrum, and T. sinomacrosporum within the Macrosporum group, with detailed descriptions provided, particularly supplementing the taxonomic features of T. glabrum. A classification key for the Macrosporum group species is also provided. The geographic distribution of specimens and diverse hosts suggest that the Macrosporum clade displays wider ecological adaptability. This study is important to better understand the diversity and conservation measures of Tuber resources throughout the world.