Stay tuned!
Subscribe to the newsletter to stay informed about our new training courses, our new products and our latest tips for growing your truffle farm!
Référence: Gómez-Molina, E.; Marco, P.; Garcia-Barreda, S.; González, V.; Sánchez, S. Effect of Selected Truffle-Associated Bacteria and Fungi on the Mycorrhization of Quercus ilex Seedlings with Tuber melanosporum. BioTech 2025, 14, 69. https://doi.org/10.3390/biotech14030069
Mycorrhizal symbiosis is now recognized as being much more than a simple bilateral relationship between fungi and plant roots. Soil bacteria and fungi play an essential role in the complex biological processes of nutrient exchange and signaling between soil fungi and plant roots. Many microorganisms identified from truffle mycorrhizae, truffle ascocarps, or in the soil are believed to possess certain plant growth-promoting (PGP) activities. Plant Growth-Promoting) or mycorrhization aid (MH – Mycorrhizal Helper). Bacteria such as Bradyrhizobium, Pseudomonas, Rhizobium, Variovorax, and Ensifer often dominate the bacterial community of truffle ascocarps and are known for their active role in mycorrhization and host plant development.
There is growing interest in the use of specific bacteria to improve mycorrhizal colonization of truffle-inoculated seedlings in nurseries. Several studies have already evaluated the effect of selected bacteria on root colonization by T. melanosporum in various host species such as Corylus avellana, Pinus halepensis, Pinus nigra, Quercus faginea and Quercus ilex. However, these studies reported contrasting effects on truffle mycorrhization levels and mainly used commercial strains rather than strains isolated directly from the mycorrhizosphere or ascocarps of T. melanosporum, with the exception of Mamoun and Olivier (1992 Plant and Soil). In addition, information on Quercus ilex, although it is the main host of T. melanosporum in Spain and widely used in France and Italy, remains limited.
In this context, Eva Gomez-Molina and colleagues, in a study published in 2025 in the journal BioTech, evaluated the ability of twelve endophytic bacterial and fungal strains, isolated from the environment of T. melanosporum (gleba, mycorrhizae, and burnt material), to improve the establishment and formation of black truffle mycorrhizae on the roots of Q. ilex seedlings. in nurseries. The study also examined their effect on plant growth. The researchers hypothesized that (i) microbial species living in association with truffles could play a positive role in the establishment of mycorrhizae of T. melanosporum, and (ii) co-inoculation of truffles with these microorganisms could positively influence the growth of Q. ilex plants.. The mycorrhizal status and vegetative quality of the plants are indeed relevant factors for the overall quality of seedlings inoculated by T. melanosporum.
The experiment involved thirteen microbial strains, twelve of which were isolated from the black truffle environment and one, Bradyrhizobium japonicum. (BJ, DSM accession number: 30131), was purchased from the Spanish Type Culture Collection. Among these strains, eleven were bacterial and two were fungal ( Tulasnella tubericola – TT and Trichoderma harzianum – TH). The ten strains isolated from truffle gleba came from ascocarps harvested in a plantation in eastern Spain. Strain TT was isolated from a mycorrhiza of T. melanosporum, and strain TH was isolated from the soil inside a T. melanosporum burn site.. After identification using sequencing-based methods, the strains were prepared as inoculum, cultured in liquid medium, and then encapsulated in alginate beads prior to inoculation.
Mature ascocarps of T. melanosporum used as inoculum were harvested from truffle fields in the province of Huesca (Spain), cleaned, surface sterilized, then dehydrated and reduced to a fine powder. The acorns of Q. ilex, originating from the Spanish region of Sistema Ibérico, were surface sterilized and germinated. In June 2018, seedlings that had developed 6 to 8 leaves and lateral roots were transplanted into pots and simultaneously inoculated with the selected microbial strains and T. melanosporum. Microbial inoculation was carried out by mixing 2.5 mL of alginate beads per pot into the potting substrate (white peat and black peat with a pH adjusted to 7.5). Truffle inoculation was carried out by root dusting with a talc carrier, at a rate of 1.5 g of fresh truffle per plant. Twelve replicates were prepared per treatment, and two control treatments were included: a procedural control (P-Con) with alginate beads without microbial content, and an absolute control (A-Con) without added beads. Additional plants without truffle inoculum were also grown to ensure that the truffle mycorrhizae came solely from the inoculation provided.
The plants were kept in a greenhouse for ten months. In April 2019, the stem length and collar diameter of the seedlings were measured. After careful cleaning, the root systems were analyzed to assess colonization by ectomycorrhizal fungi, using the INIA-Aragón method. The roots were divided into three depth segments, and at least 100 root tips per segment were counted and classified as non-mycorrhizal, mycorrhizal by T. melanosporum or by contaminating morphotypes, the only contaminant found being Sphaerosporella brunnea. Four plants were excluded due to poor growth, bringing the final sample size to 176.
Statistical analyses were performed to test the effects of microorganisms on mycorrhization, plant growth, and distribution of colonization on the root system.
Ten months after inoculation, the results revealed average root mycorrhization by T. melanosporum. of 22.8% (standard deviation: 11.5). All seedlings showed mycorrhizae of T. melanosporum, with the exception of a few in certain treatments (two in the treatment with T. harzianum, one in the treatment with B. japonicum, and three in the treatment with Ensifer adhaerens – EA2). Statistical analysis showed that at least one of the co-inoculated microorganisms had a significant effect on root colonization by T. melanosporum in Q. ilex seedlings (F = 6553, p-value = 0.003, n = 176).
Post hoc analysis indicated that AT2 treatment (Agrobacterium tumefaciens strain 2) was the only one to show significantly higher levels of mycorrhization than the procedural control (P-Con). This is a key finding of the study, highlighting the potential role of this strain as an auxiliary bacterium for mycorrhization. Strain AT1, another strain of Agrobacterium tumefaciensalso showed a marginally significant positive effect on mycorrhization (p = 0.055). In addition, the strains Variovorax sp. (Vsp) and the commercial strain Bradyrhizobium japonicum (BJ) showed marginally significant positive effects on mycorrhization by T. melanosporum (p = 0.069 and p = 0.068, respectively).
Conversely, treatment with the TH strain (Trichoderma harzianum)showed significantly lower mycorrhization values compared to most other co-inoculation treatments. This inhibitory effect contradicts a previous study that reported a significant improvement in truffle mycorrhization by T. harzianum.
The remaining strains did not show any significant effects on seedling mycorrhization. This includes two native strains of Pseudomonas (Psp1 and Psp2), as well as the two strains of Ensifer adhaerens (EA1, EA2) and the two strains of Kocuria rhizophila (KR1, KR2).
The only contaminating ectomycorrhizal fungus detected in the samples was S. brunnea.. This fungus appeared in 3.4% of the plants inoculated with truffles, with an average root colonization value of 0.2%. Statistical analysis showed that the frequency of occurrence of S. brunnea was not significantly affected by the co-inoculated microorganisms (z-value = 0.002, p = 0.99, n = 176). The additional plants not inoculated with truffles also showed mycorrhizae of S. brunnea, but no mycorrhizae of T. melanosporum or other ectomycorrhizal fungi. These observations confirm that differences in colonization by T. melanosporum can be attributed to the treatments applied.
Seedlings inoculated with T. melanosporum had an average stem length of 11.7 cm (standard deviation: 3.1) and an average collar diameter of 4.3 mm (standard deviation: 0.8). Stem length was not significantly affected by the co-inoculated microorganisms (F = 1.72, p = 0.056, n = 176). Collar diameter showed significant differences between some of the co-inoculated microorganisms (F = 3.07, p < 0.001, n = 176), but none of the co-inoculation treatments showed significant differences compared to the control treatments. This means that the microorganisms tested had no noticeable impact on the vegetative growth of the plants. Q. ilex under the conditions of the study.
Statistical analysis revealed no significant interaction between co-inoculated microorganisms and root depth segments (F = 1.16, p = 0.27, n = 528). However, the percentage of root colonization by T. melanosporum was significantly affected by depth (F = 214.84, p < 0.001, n = 528), with colonization decreasing with depth for all co-inoculation treatments. This suggests that the microorganisms did not alter the vertical distribution of truffle mycorrhizae, which naturally tend to be more abundant in the upper layers of the soil.
The results of this study make important contributions to understanding microbial interactions in the truffle mycorrhizosphere and improving the production of truffle seedlings in nurseries.
The most significant finding is that co-inoculation of T. melanosporum with strain AT2 (Agrobacterium tumefaciens) resulted in significantly higher colonization levels compared to the control. Strain AT1 also showed a marginally significant positive effect. Agrobacterium tumefaciens is a widespread soil bacterium commonly associated with roots, tubers, or underground stems. Although recent studies have reclassified most species of the genus Agrobacterium under the genus Rhizobium, this AT2 strain has demonstrated mycorrhization-promoting (MH) capabilities. The Rhizobium and other members of the Rhizobiaceae family are recognized for their plant growth-promoting properties (PGP) and their role as mycorrhizal auxiliary bacteria. Although DNA analysis has not allowed the AT2 strain to be classified accurately due to its similarity to several strains of Agrobacterium and Rhizobium, phylogenetic reconstruction supported its classification as A. tumefaciens.. It is possible that this strain belongs to one of the complexes. A. tumefaciens, which includes both pathogenic and non-pathogenic microorganisms (such as Rhizobium radiobacter). The MH capabilities of the AT2 strain, similar to those found in other Rhizobium such as Rhizobium pusense, make it a promising candidate for improving the quality of seedlings mycorrhized by black truffles in nurseries. The precise mechanisms by which the AT2 strain contributes to this process remain to be elucidated, but MH bacteria can act in various ways: by mobilizing soil nutrients, fixing atmospheric nitrogen, producing growth factors, and protecting the root-fungus system against pathogens. These activities can induce spore germination, improve hyphal growth, promote root branching and root-fungus contact, and mitigate chemical or biological stresses.
Contrary to some expectations based on previous studies, the study did not demonstrate a significant effect for the two native Pseudomonas strains on the levels of mycorrhization by T. melanosporum.. Previous research on the effect of Pseudomonas on truffle mycorrhization have reported contrasting effects, ranging from temporary negative effects to positive effects for certain strains on Pinus halepensis, but not significant on Pinus nigra and Quercus faginea.. The non-significant results of this study for native strains of Pseudomonas, as well as contradictory results from other studies, suggest that this approach has limited value in directly improving the mycorrhization levels of truffle seedlings in nurseries. However, it is important to note that previous studies have shown that inoculation with certain strains of Pseudomonas fluorescens could improve growth and drought tolerance in other tree species. An assessment of the plant growth-promoting (PGP) capabilities of microorganisms associated with truffles could therefore be relevant in the future, especially under stressful conditions such as nutrient limitation.
The study showed marginally significant positive effects of Variovorax sp. (Vsp) strains and the commercial strain Bradyrhizobium japonicum (BJ) on mycorrhization by T. melanosporum.. These genera are among the most frequently detected in truffle ascocarps and mycorrhizae. However, another study found that co-inoculation of a strain of Bradyrhizobium with T. melanosporum did not significantly improve mycorrhization and reduced fine root density. This discrepancy highlights the need to evaluate more native strains of these genera to confirm their MH capabilities.
The TH strain (Trichoderma harzianum) showed colonization by T. melanosporum. significantly lower than most other microbial treatments. This inhibitory effect is consistent with the recognized role of T. harzianum as a biocontrol agent, characterized by its fungicidal activity. This fungus is known for its production of extracellular chitinases, antibiotics (such as gliotoxin), and other secondary metabolites, as well as for its hyperparasitic behavior and its ability to colonize the rhizosphere and other plant organs on a massive scale. Researchers have also questioned the use of Trichoderma spp. as biocontrol agents in forest nurseries due to their antagonism towards ectomycorrhizal colonization. These results are also consistent with previous experiments in which several strains of T. harzianum native to truffle soils showed inhibitory effects in vitro on the mycelial growth of Armillaria mellea and S. brunnea.
Overall, the results of this study do not demonstrate that the bacteria and fungi associated with T. melanosporum systematically possess mycorrhization-promoting (MP) capabilities. The insignificant effects observed for many taxa could indicate that they do not play a major role in mycorrhiza formation or plant growth promotion under the conditions of the study. It is also possible that they are involved in specific metabolic or physiological processes that are not directly reflected in mycorrhization rates or seedling size, or that the combined action of several taxa is necessary for an effective impact, as suggested by other studies.
The study was conducted under standard nursery conditions, recognizing that the uncontrolled presence of microbial populations in the substrate and greenhouse environment could influence the results. However, the objective was to reflect common nursery practices, where the quality of truffle plants depends on the proportion of roots colonized by ectomycorrhizal fungi. The treatments, including controls, were applied to seedlings from the same batch, using the same substrate and grown under homogeneous conditions. Furthermore, no significant differences in colonization rates by S. brunnea was not found between treatments, reinforcing the conclusion that the differences observed in colonization by T. melanosporum are attributable to specific microbial treatments.
In conclusion, this study successfully identified a strain of Agrobacterium tumefaciens (AT2), isolated from the gleba of T. melanosporum, which significantly improved the mycorrhization of Q. ilex roots by T. melanosporum. under nursery conditions. This result supports the hypothesis that certain microorganisms naturally present in the mycorrhizosphere of truffles may act as auxiliary bacteria in mycorrhization. On the other hand, colonization by T. melanosporum was significantly lower in the presence of the Trichoderma harzianum (TH) strain, and other rhizobacterial strains (of the genera Pseudomonas, Ensifer, or Variovorax) showed no significant effect on either truffle mycorrhization or seedling growth.
Under current nursery practices, which involve highly controlled and aseptic conditions that reduce the diversity of indigenous microorganisms in the mycorrhizosphere, these results highlight the potential of controlled addition of specific microbial strains with mycorrhization-promoting capabilities to improve the quality of inoculated plants and symbiotic efficiency under routine nursery production conditions. However, further research is needed to gain a deeper understanding of the interactions within the mycorrhizosphere that could contribute to improving the quality of nursery plants. Better characterization of the mechanisms of action of these MH bacteria, particularly through complete genome sequencing to confirm strain identity, and studies evaluating their PGP capabilities, could open up new avenues for optimizing black truffle cultivation.
Subscribe to the newsletter to stay informed about our new training courses, our new products and our latest tips for growing your truffle farm!
