Comparison of bacterial communities in roots of selected trees with and without summer truffle (Tuber aestivum) ectomycorrhiza

Due to its physical properties and chemical composition, the soil is a suitable environment for the development of microorganisms, especially bacteria and fungi. Their biological activity affects, among other things, the fertility of the soil, as well as the availability to plants of nutrients derived from the decomposition of the biomass by microorganisms (Galus-Barchan and Paśmionka 2014; Pociejowska et al. 2014). Depending on environmental factors, the quantitative and qualitative composition of microorganisms may vary over time. Differences in bacterial counts may also result from their populations’ diversity, sometimes even within one site (Clark 1971; Steinauer et al. 2016).

The layer of soil most populated by microorganisms is the rhizosphere. This layer contains several to several hundred times more bacteria than the soil zone outside the rhizosphere. Foster (1988) states that in one gram of dry matter within the rhizosphere, there are 10¹⁰–10¹² bacterial cells, and outside it, no more than 10⁸ cells per gram of soil. In the rhizosphere, there are microorganisms with a different qualitative composition than in soil without plants. The relationships between microbes and plants are complex. Bacteria and fungi living in the rhizosphere are dependent on hosts (plant roots), nutrients, other microbial populations and climatic conditions that stimulate or inhibit their diversity (Bardgett 2011; Lau and Lennon 2011; Eisenhauer et al. 2017).

A diversified microbiome can be observed in the roots of plants. The plant’s condition is strongly influenced by the microbial community, which in turn is influenced by the host (Beckers et al. 2017; Proenca et al. 2017). This microbiome is made up of microorganisms that have a beneficial (plant growth-promoting rhizobacteria, PGPR), neutral (neutral rhizobacteria) or deleterious (deleterious rhizosphere microorganisms, DRMO) effect on plant development (Kurek and Kobus 1990). Symbiosis is one example of the beneficial influence of microorganisms on plant development.

It is well known that through symbiosis, organisms acquire capabilities and properties that they would not otherwise show (Frey-Klett et al. 2011). The Poole study (2001) shows that the rhizosphere layer is dominated by bacterial species belonging primarily to the genera Pseudomonas and Bacillus, which have beneficial influences on the plant, such as increasing its health and reducing potential infections (Badura 2005). These are free-living bacteria that mineralize organic compounds and fix molecular nitrogen. Soil bacteria that symbiotically fix nitrogen are, for example, commonly found in legumes and include Rhizobium and Frankia (Błaszczyk 2010).

For many years, the direct influence of rhizospheric microorganisms on the stimulation of plant growth was associated with their ability to fix nitrogen. Rhizospheric bacteria have received particular attention because of their potential use in stimulating the colonization of mycorrhizal roots and, consequently, the industrial production of fungi, including truffles (Boersma et al. 2009, 2010; Antony-Babu et al. 2014). Free-living bacteria (PGPR) have a beneficial effect on plants by stimulating their growth directly and indirectly. Bacterial communities isolated from root secretions have shown an effect on stimulating the development of mycorrhizal mycelia (Ali and Jackson 1988). Some rhizospheric microorganisms can also induce changes in the quantity and composition of root secretions (Deveau et al. 2007). Direct support consists of stimulating mineral nutrition, for example, by facilitating the uptake of nitrogen, dissolving phosphorus compounds or binding iron by siderophores. This also includes the synthesis of phytohormones that affect plant development, that is, auxins, gibberellins and cytokinins, as well as lowering ethylene levels that affect plant rooting. Indirect support, on the other hand, is associated with bacterial-based biological control of phytopathogens. Bacteria can exhibit various symbiotic effects against fungi and also inhibit pathogen development through antagonism (Tsukamoto et al. 2002; Frey-Klett et al. 2007), improve spore distribution (Citterio et al. 1995; Splivallo et al. 2015), and provide vitamins and growth regulators (Rangel-Castro et al. 2002; Riedlinger et al. 2006).

Soil is inhabited not only by bacteria. It is also the natural habitat for fungi, including the genus Tuber from the division Ascomycota. Truffles (genus Tuber) are found, apart from North America and Asia, in many European countries including France, Italy, Spain and Poland as a natural component of forest ecosystems (Blom et al. 2011; Hilszczańska 2016). Truffles are colonized in all stages of their life cycle by various micro-organisms including bacteria (Vahdatzadeh et al. 2015), Cryptococcus yeast (Zacchi et al. 2003), and moulds and viruses (Splivallo et al. 2015). Microorganisms accompany fungi/truffles in the symbiotic stage when they remain in an ectomycorrhizal relationship with the plant, as well as in the sexual stage (ascomata) and the saprotrophic stage (free-living mycelium). The development of truffle mycorrhiza, as well as the growth of bacteria, is regulated by temperature, humidity, structure and soil chemistry. Some microbes called mycorrhiza helper bacteria (MHB) stimulate mycorrhizal colonization of host roots and consequently affect the development of truffle ascomata (Gryndler et al. 2013). To date, the microbial communities best recognized are those that occur in the ascomata of the four truffle species appreciated in the kitchen: the white truffle (Tuber magnatum), the whitish truffle (T. borchii), the black truffle (T. melanosporum) and the summer truffle (T. aestivum) (Hilszczańska 2016; Siebyła and Hilszczańska 2020).

Research in many countries shows that the role of bacteria and fungi in the functioning of forest soils is not yet fully understood. This is especially true for the evaluation of bacterial diversity and their relationship with fungi (Baldrian et al. 2012), particularly regarding the summer truffle, in which communities of the coexisting bacteria were usually identified by the 454 method, PhyloChip (Gryndler and Hršelová 2012; Mello et al. 2013; Deveau et al. 2016). Nowadays, owing to the use of modern molecular methods such as next-generation sequencing (NGS), it is possible to get a thorough understanding of the composition of microbial communities that inhabit the roots, including those that are conducive to truffle growth (Gryndler et al. 2013).

This study aimed to elucidate the bacterial communities associated with the ascomata of the summer truffle T. aestivum found in s elected locations in Poland. Two different methods were applied in this study: a) the classical method, that is, the plate culture method, which involves the identification of bacteria in samples from the roots at the sites where summer truffle ascomata are observed and in the samples where no summer truffle ascomata are observed, and b) the molecular method consisting of the isolation of DNA of both the Sanger and NGS methods. Sanger sequencing was used to identify the isolated bacteria based on DNA isolated from pure strains, and the NGS method was used to analyse the DNA isolated from roots.

It has been assumed that the ‘truffle’ sites will exhibit a specific composition of the bacterial microbiome, which may help monitor soils conducive to truffle development and yield.

The involvement, abundance, metabolic activity, and role of bacteria in shaping the forest soil environment are not yet fully understood. Bacteria in forest soils can serve many functions, including helping plants adapt to adverse conditions, that is, lack of water or excessive salinity, assisting in the formation of mycorrhizal systems, increasing the resistance of trees to stressors, and inhibiting the development of pathogens (Tsukamoto et al. 2002; Frey-Klett et al. 2007; Tarkka and Frey-Klett 2008). Soil bacteria show various endophytic and symbiotic effects on fungi, including enhancing the distribution of their spores (Citterio et al. 1995; Splivallo et al. 2015) or providing vitamins and growth regulators (Rangel-Castro et al. 2002; Riedlinger et al. 2006). Bacteria are found not only in soil and green plant tissue, but also in wood and roots (Kubiak et al., 2018). The abundance and composition of soil bacterial communities depends on the type of host plant, on the activity and nutrients of plant roots and their secretions, on fungi coexisting in the substrate, but also on climatic conditions (Bardgett 2011, Lau and Lennon 2011; Eisenhauer et al. 2017). Ambient temperature influences the number of bacteria, the ratio of bacteria to fungi, but also favourable interactions between species.

Studies by Deveau et al. (2016) in relation to T. melanosporum showed significant differences in t he percentage of bacterial classes and subclass Proteobacteria in the soil on ectomycorrhizal roots during the winter months (November, December and January). Comparison of the number of OTU-level bacteria in soil and ectomycorrhizal roots and their interactions showed significant differences over time (Deveau et al. 2016). The number of bacteria belonging to Acidobacteria, Bacteroidetes and Betaproteobacteria showed different variability depending on the time of sampling and community location – soil bacteria showed seasonally higher numbers than ectomycorrhizal root bacteria, whose communities were more stable. In the studies of Deveau et al. (2016), the number (in %) of bacteria belonging to the class Betaproteobacteria was highest in September, and bacteria from the cluster Bacteroidetes (Microscilla) in November, compared to other dates, while the proportion of Acidobacteria (Candidatus Koribacter) increased from September to January. It was also shown that bacteria belonging to the genera Thermoleophilum and Pseudonocardia colonised ectomycorrhizal roots more frequently than soil in November. The dominant bacterial communities inhabiting ectomycorrhizal roots were bacteria belonging to the classes Alphaproteobacteria and Actinobacteria (genus Thermoleophilum).

The differences in root colonization by bacteria in the years 2017–2018 may have been due to meteorological conditions. The total precipitation in the vegetative season was ~ 1.5 times higher in 2017 compared to 2018. The difference between the averages of the two measuring stations was higher by ~ 1.00 in 2017 than in 2016 and by ~ 2.18 in 2017 than in 2018. From the results obtained by Siebyła and Hilszczańska (2020) on the identification of soil bacterial communities, it can be concluded that the weather conditions (here: in the autumn season of 2017) resulted in an increase in the amount of bacterial population. Similar relationships are described by Przemieniecki et al. (2021), who point to the significant influence of weather conditions at different times of the year on the activity of the bacterial microbiome in Armillaria ostoyae rhizomorphs inhabiting the roots of Scots pine trees.

The Sielianinov hydrothermal coefficient in presented research was above 2 at that time, which indicates high water saturation of the soil after the dry summer months in 2015 and 2016. In 2017, the Sielianinov hydrothermal coefficient (average of results in both stations) was ~2.19, twice as high as in 2018 when it was ~1.1.

Research by Deveau et al. (2016) concerning the analysis of ectomycorrhizal communities associated with Tuber melanosporum showed the dominance of Proteobacteria clusters, including Alphaproteobacteria (9%), Betaproteobacteria (3%), Deltaproteobacteria (4%), Gammaproteobacteria (1%), Actinobacteria (20%), Bacteroidetes (9%), Firmicutes (1%), Verrucomicrobia (1%), Acidobacteria (0.2%) and Planctomycetes (0.4%). Similar results to Deveau et al. (2016) were obtained by Antony-Babu et al. (2014), however, sampling was carried out in a different month and year. In this study, Proteobacteria (20.2–65.1%) and Firmicutes (3.4–11.2%) were the dominant groups, as was observed through the classical method (Sanger method). The use of NGS sequencing allowed us to distinguish five phyla: Proteobacteria (49%), Actinobacteria (26%), Bacteroidetes (11.4%), Acidobacteria (6%), Firmicutes (4.8%) and others (3.3%). A higher percentage of OTU in this study may be due to sampling of different trees, in contrast to the studies carried out by Deveau et al. (2016), where root samples were taken from C. avellana only. In this study, the alpha biodiversity index using the Shannon’s index was 6.34–7.78, similar to the result obtained by Deveau et al. (2016) of 6.44–7.00. In the case of studies by Antony-Babu et al. (2014), Shannon’s index was the highest, equal to 7.25. A similar difference was observed for the Chao index. In our studies, the value of the Chao index ranged from 120 (sample G2) to 362 (W4), while in the studies of Antony-Babu et al. (2014) it was as high as 10,948.

Studies conducted by López-Mondéjar et al. (2015) and Deveau et al. (2016) indicate that the bacterial community in soil changes over time. Changes in the composition of the microbiome depending on the season may therefore affect the growth of Tuber fungi. However, the research presented here indicates that the bacterial community is affected not only by time but also by meteorological conditions, location, tree species and host plant. The results obtained by the Sanger method showed the dominance of bacteria of the genera Pseudomonas, Erwinia, Serratia, Brevibacterium and Bradyrhizobium (only in 2018), and Acinetobacter (only in 2018) in tree roots on truffle sites. The NGS sequencing gave a much more detailed picture of the bacterial communities than the Sanger sequencing for the Pseudomonas and Bradyrhizobium genera, which were only detected in the control variant. This confirms the hypothesis regarding the possibilities of practical use of the NGS method to determine the potential for truffle occurrence and, therefore, its yield.

The NGS sequencing of root samples showed differences in the proportion of Agromyces bacteria, depending on the variant. These studies confirm previous studies by Siebyła et al. (2020), which concerned soil analysis using Sanger sequencing. Interestingly, in the case of Lysobacter bacteria, the results of NGS sequencing of soil samples contradict the results obtained by the same method (NGS sequencing) for root samples. Studies by Siebyła et al. (unpubl.) concerning the NGS sequencing of soil samples show that there is an important positive relationship between the occurrence of Lysobacter species in s oil and t he presence of T. aestivum summer truffle ascomata. The NGS sequencing of both root and soil samples showed a negative correlation between the presence of Bradyrhizobium bacteria in the soil and the occurrence of summer truffle ascomata.