Ectomycorrhizal Communities in a Tuber Aestivum Vittad. Orchard in Poland

Open Life Sci. 2016; 11: 348–357

Research Article Open Access

Dorota Hilszczańska*, Hanna Szmidla, Jakub Horak, Aleksandra Rosa-Gruszecka Ectomycorrhizal communities in a Tuber aestivum Vittad. orchard in Poland

DOI 10.1515/biol-2016-0046 Received July 6, 2016; accepted October 27, 2016 1 Introduction

Abstract: Cultivation of the Burgundy truffle, Tuber Burgundy truffle (Tuber aestivum Vittad.) is an aestivum Vittad., has become a new agricultural alternative ectomycorrhizal fungus that forms edible hypogeous in Poland. For rural economies, the concept of landscaping ascocarps of considerable economic value. It is well- is often considerably more beneficial than conventional documented in literature that T. aestivum grows in an agriculture and promotes reforestation, as well as land-use ectomycorrhizal symbiosis with many different trees and stability. Considering examples from France, Italy, Hungary shrubs belonging to genera such as Carpinus, Fagus, Tilia, and Spain, truffle cultivation stimulates economic and Populus, Quercus and Corylus [1-3]. social development of small, rural communities. Because Cultivation of the fungus is starting to become a there is no long tradition of truffle orchards in Poland, promising agroforestry alternative for rural areas in knowledge regarding the environmental factors regulating Poland. For a long time, truffles, especially the species the formation of fruiting bodies of T. aestivum is limited. praised by chefs and gourmets for their scent and taste, Thus, knowledge concerning ectomycorrhizal communities were considered rare in Poland, and the Burgundy truffle of T. aestivum host species is crucial to ensuring successful was recorded only once after the Second World War [4]. In Burgundy truffle production. We investigated the the last decade, new data on the distribution of T. aestivum persistence of T. aestivum ectomycorrhizae on roots of and other species of truffles have been reported from hazel (Corylus avellana L.) and oak (Quercus robur L.) and Poland [5,6], as well as from other countries of Central, checked the host-species influence on community structure Eastern and Northern Europe [7-11]. of ectomycorrhizal fungi. The study was conducted in an The persistence of truffle ectomycorrhizae on experimental plantation located in eastern Poland and inoculated seedlings outplanted in the field is one of the established in 2008. We demonstrated that the number most important factors of truffle cultivation success [12-14]. of fungal taxa was not significantly different between However, in many cases, the targeted truffle ectomycorrhizae oak and hazel. However, the species composition differed is replaced by other competitive ectomycorrhizal species between these two host trees. During the three-year study, [15,16]. Understanding the conditions that promote truffle we observed that species richness did not increase with the fructification is essential for detecting species that have age of the plantation. a high possibility of outcompeting targeted truffles [16]. Knowledge about the diversity of species is crucial for Keywords: Burgundy truffle, ectomycorrhizae, hazel, successful conservation efforts and future plantation oak, truffle orchard management. Comparison of the ectomycorrhizal fungi that are associated with different host species within environments with the same conditions would help clarify *Corresponding authors: Dorota Hilszczańska, Department of the importance of host preference in structuring the Forest Ecology, Forest Research Institute, Braci Leśnej 3 Str., ectomycorrhizal community [17]. Sękocin Stary, 05-090 Raszyn, Poland, The aims of our study were i) to examine whether E-mail: [email protected] and how differently T. aestivum mycorrhizae persist in Hanna Szmidla, Aleksandra Rosa-Gruszecka, Department of Forest Protection, Forest Research Institute, Braci Leśnej 3 Str., Sękocin roots of hazel (Corylus avellana L.) and oak (Quercus Stary 05-090 Raszyn, Poland robur L.), ii) to characterize other (non-Tuber) species Jakub Horak, Department of Forest Protection and Entomology, present in the orchard, and iii) to estimate, based on the Faculty of Forestry and Wood Sciences, Czech University of Life ectomycorrhizal fungi community (ECM), the future T. Sciences, Kamýcká 1176, CZ-165 21, Prague 6 – Suchdol, Czech aestivum fructification in the orchard. Republic

© 2016 Dorota Hilszczańska et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Ectomycorrhizal communities in a Tuber aestivum Vittad. orchard in Poland 349

2 Materials and Methods 2.2 Sampling and morphological analyses

To analyse the ectomycorrhizal morphotypes present in root 2.1 Study area tips, five hazel trees and five oaks were randomly chosen (every year, analyses of mycorrhizae were performed on The study was conducted in a plantation located in the same chosen trees). Three soil cores (6 cm diameter, 20 eastern Poland (latitude 51° 8’ 51’’; longitude 23° 28’ 29’’) cm deep) were obtained 25 cm from the base of each tree. at 200 m a.s.l. where the soil parent material is Triassic These three cores per tree were combined to yield a single Muschelkalk. The climate is continental with a mean composite sample. The samples were collected every year annual rainfall of 550 mm and mean annual temperature of the study (2012-2014), in September. Each composite of 8.0°C. The truffle orchard was established in 2008 by sample was individually soaked overnight in tap water and planting 134 seedlings of Quercus robur and 150 seedlings sieved to separate the root fragments and ectomycorrhizae of Corylus avellana, all of which were inoculated with (ECM) from the soil. The clean roots were placed in Petri spores of native Tuber aestivum [18]. The soil physical and dishes filled with water. Only vital roots (identified as chemical properties are shown in Table 1 and were similar swollen, without root hair or covered by fungal mantles) in all areas of the orchard. were considered ECM-colonized roots. The different types of ectomycorrhizae found in each sample were morphotyped Table 1. Physical and chemical properties of the soil in the using a stereomicroscope and a light microscope, with plantation. the anatomo-morphological characteristics described by Measured parameter Agerer [19,20] and two online databases, EctoMycorrhizal Sand (0.2-0.063 mm) % 32.62 Community DataBase (www.emyco.uniss.it) and DEEMY Clay (0.002 mm) % 35.29 (http://www.deemy.de), as references [21,22]. Silt (0.063-0.002 mm) % 32.09 Morphotypes were identified by colour and shape P 0 g kg-1 2.52 2 5 of the mycorrhiza, characteristics of the mantel surface, Calcium cmol+kg-1 27.96 Magnesium cmol+kg-1 0.32 ramification system, and presence and structure of Potassium cmol+kg-1 0.60 rhizomorphs, emanating hyphae and other elements. The water pH 7.6 morphology and colour of the ECMs were evaluated with a CaCO % 19.56 3 dissecting microscope on freshly isolated ectomycorrhizal Organic matter % 2.3 tips. The ectomycorrhizae formed by T. aestivum were C/N 24.3 identified by its ochreous and chestnut brown colour and by the presence of swollen fine roots with tips embedded The experimental plantation of 0.44 ha was established in radially twisted hyphae. The number of living on land that had been abandoned for 19 years prior to mycorrhizae of each morphotype was recorded separately outplanting of seedlings inoculated with of T. aestivum. for each sample. Roots were cut into 2-cm pieces and a Before outplanting of seedlings, the soil was ploughed total of 100 vital tips were counted. to a depth of 50 cm to promote deep soil aeration, and superficial tilling with several passes was later applied to remove herbaceous vegetation. The site was chosen 2.3 Data analysis because truffle species, such as Tuber rufum and T. excavatum, had been found in the forest surrounding the The diversity of ectomycorrhizae on hazel and oak was area. This forest was mainly composed of Q. robur, Carpinus expressed as the number of identified ectomycorrhizal betulus and Populus tremula, with C. avellana in the species (species richness). The relative abundance of each understorey. Seedlings were arranged with 3 m between morphotype (number of root tips of each morphotype / the rows and 4.5 between individuals within the rows. total number of mycorrhizae) was calculated separately Green woven polypropylene fabrics (99 g m-2) were used for each sample. Ecological indicators, such as species in rows for reducing herbaceous cover in this plantation. richness, Shannon diversity index and Simpson In order to control the rapid growth of weeds, soil tilling dominance index, were calculated using EcoComPaC with cultivator tines set at 15 cm depth and manual weed Version 1 [23] (http://prf.osu.cz/kbe/dokumenty/sw/ control with a hoe were performed every year – namely, ComEcoPaC/ComEcoPaC.xls.) in Excel. in May and in August or September, depending on weed Statistical analyses were performed in EstimateS 8.2. growth. and CANOCO 4.5. We focused on predictors that were 350 D. Hilszczańska, et al. testable only within a limited spatial scale. Unreplicated Morphological observations revealed a total of 14 fungal treatments were the only option for our study [24]. We taxa: 10 in oak and 10 in hazel (Table 2 and Fig. 1 a-n). controlled this problem by using randomized techniques Of these, one was assigned at the order level (Pezizales), for taxa richness data [25], as recommended and used seven at the genus level (Inocybe sp., Lactarius sp. 1, elsewhere [26-28]. Lactarius sp. 2, Tomentella sp., Geopora sp., Peziza sp. For the analysis of species richness and for and Alnicola sp.) and five at the species level (Tuber comparisons between tree species and over time, we used aestivum, Amphinema byssoide, Cenococcum geophilum, sample-based species rarefactions (Mao Tau function) Scleroderma areolatum, Laccaria tortilis and Thelephora with 95% confidence intervals [29]. The number of terrestris). In our study, T. aestivum ectomycorrhizae were randomizations was set at 1,000 in EstimateS. present in all 3 years. Data on species composition were log transformed. The The overall species richness of identified ECM fungal length of the gradient in detrended correspondence analysis taxa was variable and ranged from three to nine for oak (DCA) was lower than 3. Redundancy analysis (RDA), and from three to seven for hazel, depending on the year instead of canonical correspondence analysis (CCA), is often (Table 3). The highest mean species richness per sample considered an appropriate method for shorter gradient was noted in 2012 (5.20 for oak and 4.40 for hazel) and the lengths [30]. However, RDA is inappropriate for abundance lowest was noted in 2013 (2.40 for oak and 1.80 for hazel). data involving null abundances [31]; hence, CCA was used The highest diversity index was noted in 2012 for both oak as the most appropriate method for analyses of species (1.58) and hazel (1.57). The evenness index was highest composition. CCA was computed using a randomized (9,999 in 2013 for oak (0.80) and in 2014 for hazel (0.90). The randomizations) split-plot design in CANOCO. highest species dominance index was observed for oak in 2014 (0.53) and for hazel in 2013 (0.47). 3 Results The number of fungal taxa was not significantly different between oak and hazel samples (Fig. 2 a-d). In all tested oak and hazel samples, mycorrhizal The results showed rich colonization of sapling roots by colonization was nearly 100%. Very small proportions of pioneer fungi. The number of fungal species significantly distorted root tips were omitted in the laboratory analysis. decreased in 2013 and 2014 (Fig. 3).

Table 2. Relative abundance (%) of ectomycorrhizal taxa associated with roots of oak (Quercus robur L.) and hazel (Corylus avellana L.) in Tuber aestivum plantations in Chełm.

Relative abundance of mycorrhizal fungal taxa

Taxa of mycorrhizal fungi 2012 2013 2014

Oak Hazel Oak Hazel Oak Hazel

Tuber aestivum 35.81 17.07 32.60 48.41 33.96 43.18 Inocybe sp. 37.08 0.00 49.80 0.00 64.69 0.00 Lactarius sp. 1 0.00 27.99 0.00 0.00 0.00 0.00 Lactarius sp. 2 7.64 0.00 0.00 0.00 0.00 0.00 Amphinema byssoides 1.47 0.00 0.00 0.00 0.00 0.00 Cenococcum geophilum 6.61 0.00 0.00 0.00 1.35 31.59 Scleroderma areolatum 3.20 1.40 0.00 0.00 0.00 0.00 Geopora sp. 2.97 0.00 0.00 0.00 0.00 0.00 Tomentella sp. 2.94 3.36 13.80 0.00 0.00 13.18 Laccaria tortilis 2.27 0.00 0.00 0.00 0.00 0.00 Peziza sp. 0.00 5.74 3.80 0.00 0.00 0.00 Pezizales sp. 0.00 36.32 0.00 0.00 0.00 0.00 Thelephora terrestris 0.00 8.12 0.00 48.86 0.00 12.05 Alnicola sp. 0.00 0.00 0.00 2.73 0.00 0.00 Ectomycorrhizal communities in a Tuber aestivum Vittad. orchard in Poland 351

a b

c d

e f

g h

i j

Figure 1. Plan views of mycorrhizae observed on hazel (Corylus avellana L.) and oak (Quercus robur L.) from Tuber aestivum plantations in Chełm; Tuber aestivum (a), Inocybe sp. (b), Lactarius sp. 1 (c), Lactarius sp. 2 (d), Amphinema byssoides (e), Cenococcum geophilum (f), Scleroderma areolatum (g), Geopora sp. (h), Tomentella sp. (i), Laccaria tortilis (j), Peziza sp. (k), Pezizales sp. (l), Thelephora terrestris (m), and Alnicola sp. (n). 352 D. Hilszczańska, et al.

k l

m n

Figure 2. Number of fungal taxa found on roots of oak and hazel in the orchard studied here. Number of taxa found in (a) 2012, (b) 2013, (c) 2014 and (d) all years together. Black squares represent oak samples; grey circles represent hazel; whiskers show 95% confidence intervals; 1,000 randomizations were used. Ectomycorrhizal communities in a Tuber aestivum Vittad. orchard in Poland 353

Table 3: Species richness, Shannon diversity, evenness and Simpson dominance indices of ectomycorrhizal communities associated with oak (Quercus robur L.) and hazel (Corylus avellana L.) in Tuber aestivum plantations in Chełm.

2012 2013 2014

Oak Hazel Oak Hazel Oak Hazel

Number of samples 5.00 5.00 5.00 5.00 5.00 5.00 Total species richness 9.00 7.00 4.00 3.00 3.00 4.00 Average species richness 5.20 4.40 2.40 1.80 2.00 3.00 Shannon diversity index (H’) 1.58 1.57 1.11 0.80 0.71 1.25 Evenness (J’) 0.72 0.81 0.80 0.73 0.64 0.90 Simpson dominance index (λ) 0.28 0.25 0.37 0.47 0.53 0.32

Similarity among samples according to species composition is indicated in Fig. 4 a-d, and the first axis of the CCA explained 42.19% of the variance in the data in 2012, 37.21% in 2013, 55.16% in 2014 and 43.94% in all years together. The second axis explained 16.51% of the variance in 2012, 21.59% in 2013, 27.84% in 2014 and 18.88% in all years together. Similarities among samples from oak and hazel indicated that there was a clear difference between samples from these two species in the first and third year, with only one diverging sample from hazel in the second year. The same result as for the first and third year was observed for all years analysed together. In total, samples from oak appeared to be more similar than those from hazel. Figure 3. Total number of ectomycorrhizal taxa associated with oak Ectomycorrhizal species composition on roots of and hazel in the three study years. Sample-based rarefactions (solid hazel and oak differed significantly in all studied years, lines) and 95% confidence intervals (dashed lines) are shown. The number of species in 2012 (green), 2013 (orange) and 2014 (blue) is based on canonical correspondence analyses, in 2012 (F shown = 5.46; P < 0.001), 2013 (F = 3.62; P < 0.001) and 2014 (F = 7.58; P < 0.001). The total explained variance was highest aestivum was examined. Ectomycorrhizal communities in 2013 (85.97%), followed by 2014 (48.70%) and 2012 on root tips in natural and cultivated truffle plantations (40.54%). Composition of the entire fungal communities in the Mediterranean region have been amply investigated during three seasons explained 43.20% of the variance (F [16,32-41]. = 6.09; P < 0.001). Ectomycorrhizae, such as Inocybe sp., From our data, we determined that ectomycorrhizae Lactarius sp. 2, Geopora sp., Laccaria tortilis, Scleroderma of Peziza sp., Lactarius sp., T. terrestris, Pezizales sp. and areolatum and Amphinema byssoides, colonized the Alnicola sp. were more frequent on the roots of hazel largest number of tips of oak roots. Peziza sp., Lactarius than on those of oak. Benucci et al. [16] investigated sp. 1, Thelephora terrestris, Pezizales sp. and Alnicola sp. ectomycorrhizae of hornbeam and hazel in a T. aestivum were more abundant on roots of hazel. The preference of orchard and found fungi belonging to Thelephoraceae T. aestivum, Cenococcum geophilum and Tomentella sp. and Peziza michelli exclusively on the roots of hazel trees. was rather indifferent for oak and hazel (Fig. 5). Taxon Host effects may be important factors that determine richness did not increase with the age of the plantation. the structure of ectomycorrhizal diversity. A preference for a certain host taxon over other taxa can affect the 4 Discussion ectomycorrhizal fungi community directly, and this preference is stronger for host species occurring later To the best of our knowledge, this report describes in plant community succession [42]. Examples of a the first study in Poland in which the fungal below- preference for a particular host plant have also been ground community of oak and hazel inoculated with T. found. For example, Iotti et al. [39] found that, in the case 354 D. Hilszczańska, et al.

Figure 4. Similarity of samples from oak (black) and hazel (grey) roots according to ectomycorrhizal species composition. First and second axis of the detrended correspondence analyses for (a) 2012, (b) 2013, (c) 2014 and (d) all years together. of T. borchii, the ectomycorrhizae on oak roots differed significantly from those on pine roots. Ectomycorrhizal species belonging to Inocybe and Lactarius are fairly common in various plant communities [43] and in environments in which truffles are produced [44,45]. Thelephoraceae species are common morphotypes on root tips in boreal forests [45] and in environments of truffle production [16,34-36,39,41,47]. The genus Tomentella is widespread in orchards with Tuber spp. [16,34,36,37,39,41,47]. Additionally, the species Cenococcum geophilum is a well-known cosmopolitan ectomycorrhizal fungus living in a wide range of environmental conditions [48,49] and is very abundant in truffle plantations [37]. The common presence of this fungus is related to its ubiquity on tree roots of the surrounding forest areas [50]. According to several authors [37,51], Cenococcum has only been detected in mature plantations. In our plantation, however, morphotypes of this fungus were present on roots of 4-year-old oaks and, in the case of Figure 5. Similarity between oak and hazel according to composition hazel, they appeared on roots of 6-year-old saplings. of ectomycorrhizal taxa. First and second axis of canonical corres- Perhaps the difference is due to the nearby old deciduous pondence analysis. Ectomycorrhizal communities in a Tuber aestivum Vittad. orchard in Poland 355 stand. The presence of forests near a plantation, up to 75 [57], their lack of presence in our orchard bodes well for m [13], may be a factor that clearly influences the fungal the future of T. aestivum fructification. Another factor diversity in truffle orchards. that justifies this conclusion is the presence of a T. rufum In our study, T. aestivum ectomycorrhizae were present fruiting body that was discovered in August 2014 very close in all 3 years and we observed that species richness did to the stem of a hazel tree. According to Olivier et al. [14], not increase with the age of the plantation. This result its appearance is an indicator of future good production is surprising since usually the number of fungal species of T. melanosporum. T. rufum Pico grows in many truffle in plantations increases with time [43,52]. This may be orchards and has been repeatedly noted in French and due to the plantation management. Findings of Sanchez Spanish plantations and, to a lesser extent, in Italy and [52] showed that tillage can decrease the presence of the USA [57]. Scleroderma sp. and other ectomycorrhizae belonging to the long-distance exploration type, as rhizomorphs are Acknowledgments: This research was funded by the broken in the process. In 2013, the tillage was performed State Forest National Forest Holding (Project No OR twice, in spring and autumn, to limit rapid growth of 271.3.6.2015), the Polish Ministry of Science and Higher weeds and increase the soil aeration, which possibly Education (Project No 240 318) and the Forest Research favoured the development of T. aestivum ectomycorrhizae. Institute (Project No 260102). However, tillage practices (that had been done in the orchards, such as mulching, weed control, plougthing) Authors’ contributions: Participation of authors: DH could cause a detriment of ectomycorrhizal diversity as designed the study, interpreted the data and wrote the well. Most ECM roots are located in the top layer of soil paper; HS carried out mycorrhizae analysis and helped to (down to 20 cm depth), due to the richness in organic draft the manuscript; JH performed the statistical analysis matter and the high concentration of nutrients in this and interpreted the data; and ARG helped to draft the layer [53-55]. This soil layer was clearly affected by soil manuscript. tillage, and hence recolonization of broken roots by fast-growing fungal species might be the main cause of Conflict of interest: The authors declare nothing to reduction in ECM fungal diversity. Such an explanation is disclose. supported by results obtained in two T. magnatum truffle orchards, where after tillage, the number of fungal species decreased from 45 to 2 in one and from 22 to 18 in the other References [56]. 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