http://orcid.org/0000-0001-9647-5307
Abstract
The aim of this study was to investigate the pattern of distribution of mating type (MAT) genes of Tuber indicum in ectomycorhizosphere soils from natural T. indicum-producing areas and cultivated truffle orchards and ascocarp samples from different regions. Quantitative real-time PCR and multiplex PCR were used to weight the copy numbers of MAT1-1-1 and MAT1-2-1 in natural truffle soils and cultivated orchard soils. The effect of limestone on the pattern of truffle MAT genes and the correlation between soil properties and the proportion of MAT genes were also assessed. These results indicated that an uneven and nonrandom distribution of MAT genes was common in truffle-producing areas, cultivated truffle orchards, and ascocarps gleba. The competition between the two mating type genes and the expansion of unbalanced distribution was found to be closely related to truffle fructification. Limestone treatments failed to alter the proportion of the two mating type genes in the soil. The content of available phosphorus in soil was significantly correlated with the value of MAT1-1-1/MAT1-2-1 in cultivated and natural ectomycorhizosphere soils. The application of real-time quantitative PCR can provide reference for monitoring the dynamic changes of mating type genes in soil. This study investigates the distributional pattern of T. indicum MAT genes in the ectomycorhizosphere soil and ascocarp gleba from different regions, which may provide a foundation for the cultivation of T. indicum.
1. Introduction
The truffle Tuber spp. establishes ectomycorrhizal (ECM) symbioses with plant roots (e.g., poplar, oak, willow, and shrubs) [Citation1–3]. The fruit bodies, referred to as truffles, are well-known for their intense aroma, nutritional attributes, and biological activity worldwide [Citation4–6]. The Asian black truffle T. indicum is the most important commercial black truffle in China for the rich flavors of the completely mature ascocarp.
The sequencing of the European black truffle T. melanosporum genome and subsequent studies have revealed that T. melanosporum is heterothallic [Citation7–11]. Thereafter, it has been proven that T. indicum is heterothallic as well [Citation12]. A single heterothallic ascomycete presents one of two alternative mating type (MAT) genes, MAT1-1-1 or MAT1-2-1, and the life cycle can only be completed when different strains with different mating type genes become fused. This process is a precondition for truffle fructification; in other words, the distribution patterns of MATs in truffle populations are the key determinants of successful fruit-body initiation [Citation13,Citation14]. However, it seems contradictory that the distribution patterns of T. melanosporum strains in their hosts are likely the result of competition between different MATs. Murat et al. [Citation15] found each single ECM manifested only one of the two MAT genes, and all of the T. melanosporum ECMs collected from the same host plant shared the same MAT genes. Rubini et al. [Citation16] investigated the distribution of the two MATs in ECMs from a natural T. melanosporum orchard and a nursery-inoculated glasshouse; this study revealed that the ECMs from individual hosts or from sites close to each other (in the range of 3–30 m), as well as the sampling soils from the glasshouse, shared identical MAT profiles. Linde and Selmes [Citation17] found that only approximately half of host trees had both MATs of T. melanosporum, with MAT1-1-1 predominating in established trees in Australia, suggesting a competitive advantage for MAT1-1-1 strains. It is widely believed that ascomycete hyphae need partners exhibiting different mating type genes to complete their life cycle. Thus, it is unreasonable that the distribution patterns of T. melanosporum strains are uneven beneath the same ground. To our knowledge, no research has been performed to investigate if this uneven MAT gene distribution occurs in T. indicum soils.
Meanwhile, the importance of soil physical and chemical properties associated with T. melanosporum, such as pH and carbonates, has been reported. Several statistical studies have indicated that a high concentration of active carbonate in the soil and neutral or slightly acidic pH favors T. melanosporum fruit-body production [Citation18,Citation19]. However, soil properties relating to T. indicum were not adequately investigated. In addition, the correlations between mating type genes and soil properties have not yet been assessed, so it is not yet clear whether it is possible to change the MAT gene distribution by manipulating specific soil properties.
So far, few successful truffle orchards have been established in mainland China [Citation20]. A T. indicum cultivated orchard area was built in Yanbian County in Panzhihua in 2009 with the aim of establishing a truffle orchard in China through cooperation between the Sichuan Academy of Agricultural Sciences and the Panzhihua Academy of Agricultural and Forestry Sciences. Although the ectomycorrhiza of Castanea mollissima Blume. or Corylus heterophylla Fisch. infected with T. indicum have been observed to be well colonized, to date no fruiting bodies have been found in this orchard. In this paper, we investigated the distribution pattern of MAT genes in ascocarp gleba from different regions. The distribution of MAT genes in soils of cultivated and natural truffle orchards from similar environments was also investigated using a real-time PCR analysis to determine the relationships between the MAT distribution pattern in soil and truffle yield to determine if the fructification of truffles was relative to the MAT distribution. Also, the physical and chemical properties of soil were detected to analyze the correlations between these properties and the MAT genes. To our knowledge, this is the first report on the pattern of distribution of MAT genes of T. indicum in ectomycorhizosphere soils of natural T. indicum-producing areas and cultivated truffle orchards, as well as in ascocarps from different regions. These results may provide guidance for the cultivation of T. indicum.
2. Materials and methods
2.1. Sampling location
Ectomycorhizosphere soils of natural T. indicum-producing areas and cultivated truffle orchards were collected from Yanbian County, Panzhihua City (N26°05′–26°21′, E101°38′–101°41′, Sichuan Province, China) in May 2015. The mean annual temperature in Panzhihua was 19–21 °C, and the mean annual precipitation was 760–1200 mm. The main host plant of T. indicum in the natural truffle-producing areas is Pinus yunnanensis and it grows in sandy loam soil. The altitude of sampling sites ranged from 1620 to 2429 m. Truffles have been found at these sites since 2005.
The T. indicum orchard was built in Yanbian County, Panzhihua City since 2009, which is located near each other geographically in an ecological environment similar to that of natural T. indicum-producing areas. Although the ectomycorrhizae of C. mollissima Blume and Co. heterophylla Fisch infected with T. indicum in the cultivated orchards have been thoroughly studied, no ascocarps had yet been found during the past years. Some of the host trees had calcareous rocks stacked underneath them to investigate the effect of limestone on the distributional pattern of MAT genes.
Thirty-four ascocarps of T. indicum were collected from Sichuan and Yunnan Provinces in southwestern China, to investigate the pattern of distribution of MAT genes in truffle gleba of ascocarps. Information about the collection sites and number of samples from each site is shown in and .
Figure 1. Ectomycorhizosphere soil and ascocarp sampling sites in southwestern China. CJ: Chengjiang County; HD: Huidong County; HL: Huili County; JY: Jinyang County; WS: Weishan County; XGLL: Xianggelila County; YS: Yongsheng County.
2.2. Sampling method, storage and pretreatment
A total of seven ectomycorhizosphere soil samples from natural T. indicum-producing areas and thirteen soil samples from the cultivated orchard were collected ( and ), namely the Control, Ti-S-1, Ti-S-2, Ti-S-3, Ti-S-4, Ti-S-5, Ti-S-6, Art-Control, Cm-Ti-1, Cm-Ti-2, Cm-Ti-3, Cm-Ti-li-1, Cm-Ti-li-2, Cm-Ti-li-3, Ch-Ti-1, Ch-Ti-2, Ch-Ti-3, Ch-Ti-li-1, Ch-Ti-li-2, and Ch-Ti-li-3. “Cm-” means that samples were collected from C. mollissima Blume, while “Ch-” came from Co. heterophylla Fisch. The term “Control” means that the soil samples were collected from the site at which no ascocarp of T. indicum had ever been found in natural field, and Art-Control means the soil samples were collected from the sites at which no truffles were inoculated but were very close to where they were planted (less than 10 m). Soil samples from natural truffle-producing areas were classified into three groups according to yield. The ascocarp yield at Ti-S-1 and Ti-S-2 were ranged from 10 to 100 g per square meter (g/m2), the yield of Ti-S-3 and Ti-S-4 were ranged from 100 to 200 g/m2, and the yield of Ti-S-5 and Ti-S-6 were more than 200 g/m2. They were grouped into low-producing, middle-producing, and high-producing grounds, respectively (). The ascocarp yield from these areas was calculated based on the average yield of the last three years by a local guide. Three replicates, a total of 200 g, of soil samples were randomly collected from each sample site. The soils were checked to make sure forest debris was discarded and the samples were then kept in sterile sealed bags on ice for less than 24 h until transfer to refrigerator at −20 °C for preservation.
Table 1. Hosts information, soil treatments and copy numbers of MAT genes in soil of cultivated truffle orchards.
Table 2. Host information, soil treatment, and copy numbers of MAT genes in soil from natural T. indicum-producing areas.
2.3. Soil property analysis
Soil samples from the same site were mixed to analyze their properties after being air-dried and sieved to obtain the <2 mm fraction. The pH in water (1:2.5 soil/water ratio) was measured by dissolving air-dried soil. Organic matter content was estimated by the Tyurin method [Citation21]. Total nitrogen was measured by the Kjeldahl method [Citation22]. The alkali solution diffusion method was used to determine effective nitrogen. The baking soda leaching-molybdenum antimony colorimetric method and ammonium acetate extraction-flame photometry [Citation23] was performed to measure available phosphorus and available potassium, respectively. The exchangeable cations of Ca2+ and Mg2+ were determined described as Andrzej et al. [Citation24].
2.4. DNA extraction and PCR reactions
DNA extraction from 200 mg soil was carried out with an E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) following the manufacturer’s guidelines. The concentration and quality of the extracted DNA were determined using a micronucleic acid measuring instrument K5500 (Kaiao, Beijing, China). 1 to 10 ECMs from one plant root were selected under a Greenough stereo microscope, (model Leica S8 APO, Leica Microsystems, Wetzlar, Germany). Universal primers ITS1/ITS2 were used for ITS region amplification to identify ECMs collected from Panzhihua [Citation25]. The i3/i4 and i5/i6 primers [Citation12] were used for MAT genes of T. indicum real-time PCR amplification in soil samples, and MAT 1-1-1 and MAT1-2-1 genes were cloned into E. coli strain JM109 using standard protocols for absolute quantification real-time PCR. Real-time PCR reactions were determined in 20 µL containing 10 µL SYBR® Premix Ex TaqTM II (Takara, Japan), 4 µM of each primer, 4 ng of DNA, and then distilled water was added to a total volume of 20 µL. The real-time PCR cycling conditions included an initial denaturation at 95 °C for 3 min, followed by 39 cycles of denaturing at 95 °C for 30 s, annealing at 65 °C for 20 s, and extension at 72 °C for 30 s; fluorescence was obtained every 0.5 s. At least three replicates per sample were used. Real-time PCR was conducted in a CFX96TM Real-Time PCR detection system (Bio-Rad, Hercules, CA). The P19/P20 and P1/P2 primers [Citation12] were used for MAT gene amplification in the gleba of ascocarps. PCR reactions were performed in a total volume of 50 µL containing 25 µL of 2 × Taq PCR master mix (Tiangen, China), 25 µLM of each primer, and 10 ng of template DNA, to which distilled water was added until a total volume of 50 µL was reached. The MAT gene PCR amplification program contained an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturing at 94 °C for 30 s, annealing at 65 °C for 30 s, and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. PCR products were detected by electrophoresis through a 1.5% agarose gel in 0.5 × TAE buffer, and visualized under UV light. Gene copy numbers were calculated according to Ct values and standard sample DNA copies.
2.5. Statistical analysis
Data are presented as means ± standard deviation (SD) of three biological replicates for each treatment. A least significant difference test was performed using the one-way ANOVA, and Spearman’s rank-order correlation was used to determine correlations between the MAT1-1-1/MAT1-2-1 and soil properties.
3. Results
3.1. Distribution of mating type genes in orchard and natural-truffle-producing soils
For real-time PCR, standard curves were generated; eight serial dilutions containing 108–101 positive sense standard sample DNA copies were prepared (15–20 ng/µL). The standard curve covered a dynamic range of six or seven log units of concentration and showed a strong linear relationship, with high coefficient of determination (R2) of 0.999 and a high amplification efficiency (87.7%–94.0%).
Both MAT1-1-1 and MAT1-2-1 were successfully detected using absolute quantification real-time PCR in ectomycorhizosphere soil from the cultivated truffle orchard (). No MAT1-1-1 gene amplification was obtained in Ti-S-3, Ti-S-4, or Ti-S-6. MAT1-2-1 gene amplification was successfully obtained in all soil samples from natural truffle-producing areas (), indicating the unbalanced distribution of two kinds of mating type genes in those areas. In the cultivated truffle orchard, where truffle production was 0, MAT1-2-1 gene was more abundant than the MAT1-1-1 in most samples. Their ratio of MAT1-1/MAT1-2-1 ranged from 0.16 to 1.12. Compared to MAT genes in the cultivated truffle orchard, the two MAT genes in soils from natural truffle-producing areas were highly uneven. In low-producing areas, the proportion of MAT1-1-1 gene to MAT1-2-1 was as high as 12.08 and 148.33, and MAT1-1-1 gene occupies a dominant position relative to the MAT1-2-1 gene. In soil in which truffle yield was more than 100 g/m2, MAT1-2-1 gene occupied the dominant position, and even in some samples, only MAT1-2-1 gene could be detected. These results indicated that ascocarps production is closely related to the competition between two mating type genes and the expansion of the unbalanced distribution of two kinds of mating type genes.
MAT1-1-1/MAT1-2-1 values were also calculated to assess the distribution situation of two MAT genes in cultivated orchard soils of different host species (). The value of MAT1-1-1/MAT1-2-1 was greater in C. mollissima–T. indicum ground soil than in Co. heterophylla–T. indicum ground soils, indicating that the distribution of mating type genes might be associated with the hosts. Limestone plays an important role in the success of truffle fructification in orchards. No significant correlations were found between limestone treatment and the distribution of MAT genes.
3.2. Mating types of T. indicum gleba
The patterns of distribution of MAT genes in ascocarps gleba from different truffle-producing areas were investigated in this paper. Mating type genes of all 34 ascocarps gleba were amplified using changes in PCR reaction cycles and the concentration of DNA template (). Among 34 ascocarps, twenty-one of their gleba presented MAT1-2-1 mating type, thirteen of their gleba presented MAT1-1-1 mating types. Samples from each site showed nonrandom distribution of two mating types. For example, samples from Huidong and Weishan presented only one mating types, MAT1-2-1 were obviously greater than MAT1-1-1 in samples from Jinyang and Huili ().
Table 3. Information of sample sources, total number of samples, and two different gleba mating types.
3.3. Characteristics of T. indicum-producing soils in natural and cultivated orchard soils
The pH of the ectomycorhizosphere soil samples collected from the natural truffle-producing areas and cultivated orchards were neutral or acidic (pH: 6.10–7.19) (). In total, most of both natural and cultivated soil properties were significantly different from their matched controls. Sand content value in all natural samples was significantly higher than that of the natural-control. The value of total nitrogen, available calcium (ACa), and silt was lower than that of the natural-control. Clay values in natural samples were significantly higher than that of the natural-control in most samples with Ti-S-1 as an exception. For the cultivated orchards soils, available calcium in soils of truffle cultivated orchards was increased by calcareous rock stacked under hosts (). However, there was no significant correlation between available calcium and the value of |(MAT1-1-1/MAT1-2-1)| in cultivated and natural samples (). Available phosphorus was found to be significantly closely correlated with the MAT1-1-1/MAT1-2-1 in ectomycorhizosphere soil.
Table 4. Physical and chemical properties of natural T. indicum-producing soils and cultivated plantation soils.
Table 5. Spearman correlation coefficient between soil properties and mating type gene distribution in cultivated plantation soils.
4. Discussion
Several studies have reported that active carbonate favors the growth of T. melanosporum because active carbonate constitutes an important reserve of exchangeable calcium ions (Ca2+) [Citation18,Citation26]. Riousset et al. [Citation27] reported that T. indicum developed in soils free from calcium carbonate, with a moderate pH, and rich in organic matter. However, other authors indicated that T. indicum inhabits calcareous soils. García found that T. indicum ectomycorrhizae develop well in calcareous substrates that are rich in active carbonate [Citation28]. Granetti et al. [Citation29] indicated that T. indicum inhabits calcareous substrates with a pH that varies from 5.5 (due to organic matter) to 8.5. Fourré et al. [Citation30] reported that T. indicum occurs on calcareous plateaus at 2000–3000 m. In our study, T. indicum inhabited neutral or acidic soils (pH 6.10–7.18). It is quite interesting to see in our study that active calcium in natural samples was significantly (p < .05) lower than that of the control, indicating that T. indicum might adapt to a wide range of calcium conditions in soil. Most of natural and cultivated soil properties were found significantly different from their corresponding controls, which indicated that the colonization of truffle mycelia could affect the properties of ectomycorhizosphere soil. Compared to the controls, the natural truffle producing soils have higher sand and clay content, which may be beneficial to oxygen acquisition for truffle production and discharge of excess water. Soils treated with stacking calcareous rock had a relatively higher exchangeable Ca2+ compared to samples without limestone treatment, and correlation analysis showed that exchangeable Ca2+ in soils showed no significant correlation with the ratio of MAT1-1-1/MAT1-2-1 in soils. However, available phosphorus was found significantly correlated with the MAT1-1-1/MAT1-2-1 in ectomycorhizosphere soil. The result provides a potential way to affect the proportion of the mating type genes in ectomycorhizosphere soil. In the cultivated orchards, T. indicum are associated with some indigenous host trees, such as pines, C. mollissima and Asian Quercus [Citation31]. In our study, C. mollissima and Co. heterophylla were considered due to their relative high colonization rate with T. indicum. No significant difference was found for soil properties between the soils of the two host trees. However, the value of MAT1-1-1/MAT1-2-1 in C. mollissima–T. indicum ground soils was greater than in Co. heterophylla–T. indicum ground soils, indicating that the distribution of mating type genes might be associated with the hosts. Further investigation is needed to study the relationships between MAT gene distribution of T. indicum and its hosts.
The Asian black truffle T. indicum was proven to be heterothallic by Belfiori in 2013 [Citation12]. The distribution pattern of European black truffle T. melanosporum strains on their hosts was found to share an identical MAT profile with the underground soil, which is not conducive to the formation of its ascocarps. The presence of two MAT idiomorphs (MAT1-2 and MAT1-1) in the T. melanosporum orchard was determined by Osting and Tedersoo [Citation14], and MAT-gene determination was successful in T. melanosporum root tip samples in their study. They reported that each DNA sample of T. melanosporum analyzed displayed the presence of either the MAT1-2 or the MAT1-1 idiomorph. Previous studies indicated that although black truffle spores of both mating types coexisted on young seedlings, one of the mating types was progressively supplanted over time [Citation32]. These findings are consistent with the results of this study, which showed that in natural-truffle-producing-areas, the MAT1-2-1 gene could be detected in only three of the collected soil samples (Ti-S-3, Ti-S-4, and Ti-S-6), suggesting that the MAT1-1-1 gene may be excluded over time. Soil samples were also collected to investigate the distribution of T. melanosporum mycelia with different MATs by Murat et al. [Citation15], in which at least one MAT gene was amplified in 45 of the 48 soil DNA samples studied. In our study, we found that ascocarp production was closely related to the competition between two mating type genes and the expansion of the unbalanced distribution of two kinds of mating type genes, which supported previous studies on T. melanosporum soils. Furthermore, ascocarps gleba from each site showed a nonrandom distribution of the two mating types. For example, samples from Huidong and Weishan contained only one mating type, MAT1-2-1, which was more plentiful than MAT1-1-1 in samples from Jinyang and Huili. Absolute quantitative real-time PCR has been reported to be sensitive and accurate and can be used to detect genes in environmental samples with low copy numbers [Citation33]. The application of absolute quantification real-time PCR technology in this study has helped us calculate the distribution of MAT genes in ectomycorhizosphere soil and the ratio of the two mating types. This suggests that monitoring the dynamic changes in MAT genes in the soil and ECMs may have good prospects for use.
In conclusion, the phenomenon of uneven and nonrandom distribution of MAT genes in truffle-producing areas or ascocarps gleba was here found to be shared across different soil groups. The competition between the two mating type genes and the subsequent expansion of the unbalanced distribution is considered to be closely related to truffle fructification. Limestone treatments failed to alter the proportion of the two mating type genes in the soil. The use of real-time quantitative PCR can provide a reference for monitoring the dynamic changes of mating type genes in soil. Further studies are needed to investigate the competition, fusion, and uneven distribution of mating type genes and the associated environmental factors that may promote fructification in truffles.
Acknowledgements
The authors would also like to thank LetPub for its linguistic assistance during the preparation of this manuscript.
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References
- Tang YJ, Liu RS, Li HM. Current progress on truffle submerged fermentation: a promising alternative to its fruiting bodies. Appl Microbiol Biotechnol. 2015;99:2041–2053.
- Arai H, Tamai Y, Yajima T, et al. Ectomycorrhizal fungal communities associated with Quercus dentata in a coastal broadleaf forest. Mycosphere. 2017;8:561–567.
- Barseghyan GS, Wasser SP. Species diversity of hypogeous ascomycetes in Israel. Mycobiology. 2010;38:15965.
- Fillet M, Heugen JCV, Servais AC, et al. Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatographyelectrospray ionization tandem mass spectrometry. J Chromatogr A. 2002;949:225–233.
- Gao JM, Zhang AL, Chen H, et al. Molecular species of ceramides from the ascomycete truffle Tuber indicum. Chem Phys Lipids. 2004;131:205–213.
- Islam MT, Mohamedali A, Garg G, et al. Unlocking the puzzling biology of the black Perigord truffle Tuber melanosporum. J Proteome Res. 2013;12:5349–5356.
- Martin F, Kohler A, Murat C, et al. Perigord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis. Nature. 2010;464:1033–1038.
- Kues U, Martin F. On the road to understanding truffles in the underground. Fungal Genet Biol. 2011;48:555–560.
- Murat C, Riccioni C, Belfiori B, et al. Distribution and localization of microsatellites in the Perigord black truffle genome and identification of new molecular markers. Fungal Genet Biol. 2011;48:592–601.
- De la Varga H, Le Tacon F, Lagoguet M, et al. Five years investigation of female and male genotypes in périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction strategies. Environ Microbiol. 2017;19:2604–2615.
- Rubini A, Belfiori B, Riccioni C, et al. Tuber melanosporum: mating type distribution in a natural plantation and dynamics of strains of different mating types on the roots of nursery-inoculated host plants. New Phytol. 2011;189:723–735.
- Belfiori B, Riccioni C, Paolocci F, et al. Mating type locus of Chinese black truffles reveals heterothallism and the presence of cryptic species within the T. indicum species complex. PLoS One. 2013;8:e82353.
- Rubini A, Riccioni C, Belfiori B, et al. Impact of the competition between mating types on the cultivation of Tuber melanosporum: Romeo and Juliet and the matter of space and time. Mycorrhiza. 2014;24(Suppl 1):S1927.
- Otsing E, Tedersoo L. Temporal dynamics of ectomycorrhizal fungi and persistence of Tuber melanosporum in inoculated Quercus robur seedlings in North Europe. Mycorrhiza. 2015;25:61–66.
- Murat C, Rubini A, Riccioni C, et al. Finescale spatial genetic structure of the black truffle (Tuber melanosporum) investigated with neutral microsatellites and functional mating type genes. New Phytol. 2013;199:176–187.
- Rubini A, Belfiori B, Riccioni C, et al. Isolation and characterization of MAT genes in the symbiotic ascomycete Tuber melanosporum. New Phytol. 2011;189:710–722.
- Linde CC, Selmes H. Genetic diversity and mating type distribution of Tuber melanosporum and their significance to truffle cultivation in artificially planted truffieres in Australia. Appl Environ Microbiol. 2012;78:6534–6539.
- García-Montero LG, Quintana A, Valverde-Asenjo I, et al. Calcareous amendments in truffle culture: a soil nutrition hypothesis. Soil Biol Biochem. 2009;41:1227–1232.
- Alonso PR, Agreda T, Agueda B, Aldea J, MartinezPena F, Modrego MP. Soil physical properties influence “black truffle” fructification in plantations. Mycorrhiza. 2014;24(Suppl 1):S5564.
- Zambonelli A, Bonito GM. Edible ectomycorrhizal mushrooms: current knowledge and future prospects. Vol 34. Berlin: Springer Science & Business Media; 2013.
- Ruma K, Sunil K, Prakash HS. Bioactive potential of endophytic Myrothecium sp. isolate M1CA102, associated with Calophyllum apetalum. Pharm Biol. 2014;52:665–676.
- Bremner JM, Mulvaney CS. Nitrogentotal. In: Methods of soil analysised. Madison (WI): American Society of Agronomy; 1982. p. 595–624.
- Mehlich A. Comprehensive methods in soil testing. Raleigh (NC): Agronomic Division, North Carolina Department of Agriculture; 1982.
- Andrzej B, Leszek B, Anna O, et al. Instrumental analysis of metals profile in poison pax (Paxillus involutus) collected at two sites in Bory Tucholskie. Chem Analityczna. 2009;54:1297–1308.
- White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications. New York: Academic Press; 1990. p. 315–322.
- García-Montero LG, Casermeiro MA, Hernando J, et al. Soil factors that influence the fruiting of Tuber melanosporum (black truffle). Aust J Soil Res. 2006;44:731–738.
- Riousset L, Riousset G, Chevalier G, Bardet MC. Truffes d’Europe et de Chine. Paris: INRA; 2001.
- Garcia-Montero LG, Di Massimo G, Manjon JL, et al. New data on ectomycorrhizae and soils of the Chinese truffles Tuber pseudoexcavatum and Tuber indicum, and their impact on truffle cultivation. Mycorrhiza. 2008;19:7–14.
- Granetti B,D, Angelis A, Materozzi G. Umbria terra di tartufi. Regione Umbria, Assessorato regionale agricoltura, Caccia, Pesca, Terni; 2005. 303 p.
- Fourré G, Riousset L, Riousset G. Ces “truffes de l’Inde” qui nous arrivent de Chine. Bull Féd Assoc Mycol Médit. 1996;9:3–21.
- Geng LY, Wang XH, Yu FQ, et al. Mycorrhizal synthesis of Tuber indicum with two indigenous hosts, Castanea mollissima and Pinus armandii. Mycorrhiza. 2009;19:461–467.
- Selosse M-A, Schneider-Maunoury L, Taschen E, et al. Black Truffle, a hermaphrodite with forced unisexual behaviour. Trends Microbiol. 2017;25:784–787.
- Sebastião FA, Lemos EGM, Pilarski F. Validation of absolute quantitative real-time PCR for the diagnosis of Streptococcus agalactiae in fish. J Microbiol Methods. 2015;119:168–175.