Ecological characteristics of truffles growing in Denizli Province, Turkey

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1 WFL Publisher Science and Technology Meri-Rastilantie 3 B, FI Helsinki, Finland info@world-food.net Journal of Food, Agriculture & Environment Vol.12 (2): Ecological characteristics of truffles growing in Denizli Province, Turkey Kutret Gezer 1, Oğuzhan Kaygusuz 1 *, Ali Çelik 1 and Mustafa Işiloğlu 2 1 Biology Department, Faculty of Science and Arts, Pamukkale University, Denizli 20070, Turkey. 2 Biology Department, Science Faculty, Muğla Sıtkı Koçman University, Muğla 48000, Turkey. * okaygusuz03@gmail.com Received 8 February 2014, accepted 4 April Abstract The ecological characteristics of truffle species that grow naturally in Denizli province were investigated in this study. Field and laboratory studies conducted periodically between 2006 and 2010 resulted in the identification of a total of four truffle species belonging to two families. These species are aestivum Vittad., T. borchii Vittad., T. brumale Vittad. and Terfezia boudieri Chatin. In order to understand the fungus-plant, fungus-climate and fungus-soil relationships, the habitat and the climate characteristics of areas where the fungi grow profusely were examined and soil samples from four different localities were analysed. In addition, the physical and chemical properties of the soil were determined. In conclusion, it was determined that the important factors affecting the growth of truffle species in the region were appropriate host plants and the climate, as well as the physical properties of the soil such as ph, lime, texture, saturation and organic matter and the chemical properties of the soil such as the macro- and micronutrients. Key words:, Terfezia, ecology, Denizli, Turkey. Introduction It has been observed that plants grow intensively in areas that are suitable in terms of ecological, climatic, topographic and geological factors, and in parallel to this, the number and types of macrofungi in these areas are plentiful. Turkey is rich in both macrofungi and plants. Many macrofungal varieties grow in Turkey and among them important genera are P. Micheli ex F.H. Wigg. and Terfezia (Tul. & C. Tul.) Tul. & C. Tul. of the class Pezizomycetes, which have been known since ancient times for their ecological and economic values and are commonly referred to as truffles 1. Truffles form mycorrhizal relationships in a variety of habitats with a broad diversity of host plants including gymnosperm and angiosperm trees and herbs 2. They provide their host plants with trace minerals and protection from pathogens, pests, and drought 3-5, and in return they receive organic carbon from their hosts 6. For spore dispersal, an array of animals, including boar, mice and squirrels, are attracted by the aromatic compounds (volatile aldehyde and sulphuric hydrocarbons) and consume the fruit bodies after digging them up 7, 8. The spores then pass unharmed through the digestion system and are dispersed with the dung in potentially new habitats 9. Truffles are subterranean ascocarps, growing just beneath the surface of the soil, making them a little harder to gather than other varieties of fungus, which grow above ground. While growing, they cause three-way splits on the soil. Truffles flourish throughout the autumn, winter and spring, and can be found buried in the soil among fallen leaves and twigs. Truffle species propagate whenever they meet a host, but they will produce truffles only if the right soil environment is present 10. From the floristic point of view, Denizli is located in West Anatolia (Turkey) and is included in the Mediterranean and Irano-Turanian floristic regions 11. Due to its rugged topography and high mountains, temperature and precipitation changes are extreme. Owing to the climatic and soil characteristics of the Denizli region, the truffles show a wide diffusion there. In view of the need to expand our knowledge of the ecology of truffle species growing in Denizli Province, this study aims to determine the ecological characteristics of truffles and to outline the fungus-soil, fungus-habitat and fungus-climate relationships. Materials and Methods Identification of truffles, the habitat and climate analysis: During field trips between 2006 and 2010, the truffle species were photographed in their natural habitats, their morphological and ecological characteristics were recorded and samples were taken to the laboratory. Spores of the samples were examined using a light microscope. The specimens were identified by examining their macroscopic and microscopic features, using current references by Trappe 12, Dennis 13, Breitenbach and Kränzlin 14, Riousset et al. 15. Dried specimens were numbered and placed in polyethylene bags. All specimens were stored in the fungarium of Pamukkale University Mushroom Research and Application Center. Host plant determinations were made according to Davis 16. Detailed climate data were provided by the Denizli Provincial Directorate of Meteorology ( Soil sampling and analysis: Soil samples from four different localities where truffles grow extensively were analysed in order to establish relations between the fungus and the soil. The soil samples (approximately kg) were taken from a depth of about 0 30 cm below the surface after removal of plant debris. The samples were air-dried in the laboratory at 25 C and screened through a 2 mm sieve. The analysis of soil samples was carried Journal of Food, Agriculture & Environment, Vol.12 (2), April

2 out in the soil laboratory of the Horticultural Research Institute of Eğirdir (Isparta). The physical features of the soil were determined by appropriate methods, as follows: distribution of soil texture such as sand, clay and silt by the Soil Survey Staff method 17, saturation and ph by Richards 18, organic matter by the modified Walkley-Black method 19, and lime content by the Scheibler calcimeter method 20. Finally, salinity was measured using a conductivity device. The chemical properties of the soil the content of exchangeable potassium, calcium, magnesium and sodium were determined according to Kaçar 21 ; available phosphorus was measured in accordance with Bingham 22 ; and available iron, zinc, copper and manganese were determined according to the Lindsay and Norvell method 23. Results and Discussion Habitat characteristics: Results of macroscopic and microscopic studies indicated that the four species of truffle belonging to the Pezizomycetes class were distributed in one order and two families. Thus, it was determined that three truffle species belonging to the aceae family and one truffle species belonging to the Terfeziaceae family grow naturally in Denizli Province. Nevertheless, the three species of the genus are not generally known in the region, although T. boudieri is recognized in certain places and is consumed as food. Mushroom-habitat relations were investigated in the study in order to demonstrate the characteristics of the habitats where truffles grow (Table 1). A total of three truffle species from the aceae family formed endomycorrhiza with Pinus brutia and P. nigra of the Pinaceae family, and one species of the Terfeziaceae family formed ectomycorrhiza with Helianthemum salicifolium, which belongs to the Cistaceae family. Otherwise, no competitive plants or fungi were found in those habitats during the field work. Truffle species can grow in different habitats depending on the region s climate, altitude, vegetation and soil structure. In our study, the T. boudieri samples were gathered in May, at an altitude of m and a depth of 5 15 cm, with more than one ascocarp and few splits in the soil. The three species of the genus were collected in March-June, at an altitude of m and a depth of 8 15 cm, with a single ascocarp and the cracks in the soil ranging from evident to very evident. According to recent studies, 26 species of truffles have been recorded in Turkey Considering the distribution of truffles in Turkey, species belonging to the aceae family usually grow in the Denizli region, and species belonging to Terfeziaceae family grow profusely in Uşak, Konya, Karaman, Aksaray, Isparta, Şanlıurfa, Elazığ, Malatya and Gaziantep provinces. The widespread growth of wild truffle species in the research area is thought to be due to the appropriate ecology, habitat, soil type and host plants, and environmental and climatic conditions. Table 1. Habitat characteristics of truffles. Studies have revealed that spp. fungi form mycorrhizal relationships with a large variety of hosts such as pine, oak, birch, and willow, and rarely or never form mycorrhiza with arborvitae, cypress, juniper, ash, maple or most fruit trees Likewise, it has been reported that Terfezia spp. fungi form mycorrhiza in various ways not only with H. salicifolium but also with other members of the Cistaceae family Some researchers have found that the maturation period of T. boudieri, known as Boudier s terfez, is usually March and April; the period for T. aestivum or summer truffle is May to November; the period for T. borchii, known as bianchetto, is January to April and that of T. brumale or winter black truffle is December to March 35, 36. Other reports have described species of the genus as generally growing in habitats at m elevation and 5 30 cm below the soil, and species of the Terfezia genus as growing in habitats at m altitude and 5 20 cm below the soil 15, The results of our study regarding the habitat characteristics of truffle species were similar to the data in the above-mentioned reports. Soil characteristics: An examination was made of the physical and chemical analyses of soil samples taken from four different areas where truffles grow profusely in order to demonstrate mushroom-soil relations. The results are given in Table 2. The soils of areas where the four truffle species grow naturally are slightly alkaline (ph ) and non-salty ( %). Pedological analysis showed a clear separation between the four truffles sites. Texture, saturation, lime and organic matter were Table 2. Mushroom-soil relations. MUSHROOMS FEATURES borchii aestivum brumale Terfezia boudieri Sand (%) Clay (%) Silt (%) Sandy and Sandy and Sandy and Silty and Texture Clayey Silty Clayey Clayey Physical Saturation (%) Features Salinity (%) ph Lime (%) Organic Chemical Features FEATURES MUSHROOMS borchii aestivum brumale Terfezia boudieri Vegetation Pinus brutia and Pinus nigra Pinus nigra Pinus nigra Helianthemum salicifolium Types of mycorrhiza Endomycorrhiza Endomycorrhiza Endomycorrhiza Ectomycorrhiza Altitude (m) Season May May-June March May Type of fruit-bodies Singly Singly Singly Collectively Status of the soil Evident More evident More evident More evident Depth (cm) Recognition Unknown Unknown Unknown Known Matter (%) P (ppm) K (ppm) Ca (ppm) Mg (ppm) Na (ppm) Fe (ppm) Zn (ppm) Cu (ppm) Mn (ppm) Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014

3 found to be the best parameters to describe the different ecological requirements of the four truffle species: T. borchii required sandy and clayey soil with a medium structure (68%), which was moderately calcareous (2.90%) and high in organic matter (4.30%); T. aestivum required sandy and silty soil, with a fine structure (95%), low calcium content (2.20%) and medium organic matter content (4.00%); T. brumale required sandy and clayey soil with a medium structure (72%), moderately calcareous conditions (3.10%) and a high level of organic matter (4.25%); T. boudieri required silty and clayey soil with a low structure (47%), highly calcareous conditions (36.00%) and a low organic matter content (2.20%). Researchers have reported that most Terfezia species occupy dry to desert habitats in various soil types: in Africa and Asia sandy, calcareous, or gravelly gypsiferous, gypsiferous-saline, sandy saline soils, or, in southern Europe, clayey-calcareous to acid soils. Terfezia species are recorded in Turkey as growing in sandy soils 27 and in sandy-clayey-limy soils in impoverished steppe ecosystems within the Anatolian climate region 34. Many spp. grow naturally in sandy-clayey, calcareous humusclayey soils or humus-sandy clayey soils with small calcareous stones 15, 40. Truffles prefer a soil with ph and a high level of calcium: these conditions are very important for the formation of mycorrhiza and the development of mycelium. Low ph has a negative effect on truffles in the wild, as it leads to a reduction in the calcium content of the soil. One of the curious aspects of many truffle soils is their relatively high organic matter levels compared with other alkaline soils 15, 38, 40. However, soil organic matter can decline in alkaline conditions. Organic matter is a source of essential nutrients and energy for macrofungi as it is for all other living things in the soil, and gives the soil a spongy structure by linking its separate particles and improving the physical properties of the soil 41. The desired soil conditions for truffle development are a high ph and well aerated granular soil with a moderate level of organic matter (up to 10%) 42. Our findings were supported by previous findings in the aforementioned studies 40, 42. In terms of the chemical properties of the soil, it was determined that the soils of the four different localities had medium contents of zinc ( ppm), copper ( ppm) and manganese ( ppm), a low content of sodium ( ppm) and a very high content of iron ( ppm). Phosphorus content was observed to be very variable from medium to high (7 20 ppm). While the soils in the areas where the species T. borchii, T. aestivum and T. brumale grew were high in potassium ( ppm), very high in calcium ( ppm) and high in magnesium ( ppm), the soil in the locality where T. boudieri grew had a medium level of potassium and calcium (127 ppm and 3539 ppm, respectively) and was low in magnesium (138 ppm). In Europe, the chemical characteristics of the soils of some truffle orchards have been reported to be rich in iron with high levels of calcium and magnesium, moderate levels of phosphorus, and low levels of sodium 42. Other truffle soils have been indicated to contain extractable calcium ( ppm), phosphorus ( ppm), potassium ( ppm), magnesium ( ppm), sodium ( ppm) and iron ( ppm) 43. Except for some varying values, these data were similar to the data in our study. Concentrations of macro-elements including phosphorus, calcium, potassium, magnesium and sodium may vary markedly among truffle species, their developmental stages, their habitats, and the analytical methods used, so that few generalities are possible. Fungi, especially mycorrhizal species (truffles among them) are particularly efficient in the uptake of phosphorus as compared to plants. Therefore, it is not surprising that phosphorus concentrations tend to be high in fungi. Mineral uptake by different species may differ within a given habitat. Mycorrhizal fungi, which specialize in the uptake of soil minerals, often have a higher mineral content because the soil is generally higher in that mineral. Climate characteristics: Climate characteristics were examined in order to describe fungus-climate relations. Generally, truffles prefer growing in areas with cool winters, damp warm springs and hot summers, with an intermittent annual rainfall of around 28 inches. Basic climatic data presented in Table 3 indicate that the Denizli region of Turkey has warm winters with a mean daily temperature of 6.7 C, and hot summers with a mean daily temperature of 26.4 C. Accumulated degree days (>10 C) are Average annual rainfall in the research area is 568 mm. Annual sunshine hours are 2585, and summer sunshine hours over the growing period are In order to thrive, truffles require suitable hosts and soil types as well as particular climate conditions. The climate data of some truffle producing regions of the world are given in Table 3. The climate of the area examined fits well into the requirement range of truffle species: the annual rainfall of the Denizli region (568 mm) is close to the annual precipitation of Spain (569 mm) and Italy (589 mm). Summer and winter mean daily temperatures are typical for a Mediterranean climate, with hot summer temperatures which are Table 3. Comparison of climatic conditions of important (wild or cultivated) truffle producing countries*. Latitude ( : ) Annual rainfall (mm) Accumulated degree days (>10 C) Mean daily temperature in summer ( C) Mean daily temperature in winter ( C) Annual sunshine hours Summer sunshine hours (April-Sept./ Sept.-April) Turkey (Denizli) 37 : Australia (Warwick) 28 : Croatia (Rijeka) 45 : Denmark (Copenhagen) 55 : France (Lyon) 45 : Italy (Bologna) 44 : New Zealand (Waipukurau) 40 : North America (Washington) 36 : Spain (Madrid) 40 : Sweden (Gotland) 57 : Switzerland (Lugano) 46 : UK (Durham) 54 : * All data, except the data regarding Turkey, were obtained by Hall et al. 35. Journal of Food, Agriculture & Environment, Vol.12 (2), April

4 similar to Italy (24.6 C) and North America (25.5 C), and warm winters, which are similar to Croatia (5.0 C) and New Zealand (7.1 C). Generally, the climatic requirement for the production of truffles appears to range within the common bounds of a wet, cool winter and a cool to warm summer with intermittent rain during the warmer months. Sourzat 44 describes a favourable climate as a warm and wet spring, dry summers with intermittent storms, autumns without early frost and winters that do not have prolonged conditions below -10 C (air temperature). Other climatic factors, which are often considered important in producing truffles, include monthly values of day length, hours of sunshine, humidity, vapor pressure deficits and evaporation. In terms of annual sunshine hours and summer sunshine hours, the research area is close to Spain (Table 3). Thus, more similarities have been observed to exist between European truffle growing regions and the Denizli region than to other areas. Both areas are on the western seaboard of the continent close to the 40 th parallel, and experience similar seasonal variations of temperature and precipitation. Conclusions In this study, we investigated the ecological characteristics of the truffle species growing in Denizli Province in Turkey. The fungussoil, fungus-habitat and fungus-climate relations were determined by examining the ecological requirements of truffle species. The appropriate host plants and the climate, as well as the physical properties of the soil such as ph, lime, texture, saturation and organic matter and the chemical properties of the soil such as the macro and micro-nutrients, were observed to be the important factors affecting the growth of truffle species in the region. Moreover, abiotic factors such as forest fires, drought and severe frost, which affect host plants, along with biotic factors such as logging and wrong grazing, can directly affect truffle growth because the development of truffles continues throughout the year. It is thought that great contributions can be made to the region as well as to Turkey by the establishment of truffle plantations, which could have ecological and economic importance for the Denizli region with its appropriate vegetation, ecology, climate and soil type. Acknowledgements We would like to thank Denizli Provincial Directorate of Meteorology for climatic data. References 1 Hall, I. R., Yun, W. and Amicucci, A Cultivation of edible ectomycorrhizal mushrooms. Trends Biotechnol. 21: Rossi, L., Bartolacci, B., Potenza, L., Bertini, L., Barbieri, E. and Stocchi, V Identification of white truffle species using RAPD markers. Plant Soil 219: Landeweert, R., Hoffland, E., Finlay, R. D., Kuyper, T. W. and Van, B. 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