FAVOURABLE CULTURE CONDITIONS FOR MYCELIAL GROWTH OF HYDNUM REPANDUM , A MEDICINAL MUSHROOM

In this study, factors such as pH, temperature, carbon and nitrogen sources that affect mycelial growth of Hydnum repandum, a medicinal mushroom, were investigated. Different inoculum media for vegetative inoculum production were also examined. The best suitable pH for mycelial growth was found to be 5.5. Among constant temperatures, the best mycelial growth was obtained at 20 and 25°C. The mycelial growth drastically decreased at 15°C, and no mycelia were obtained at 30°C. Glucose and mannitol were found to be the most suitable carbon sources. Ca(NO3)2 as a nitrogen source gave the best results for mycelial growth. The poorest mycelial growth was noted in sucrose and xylose as carbon sources and in NH4NO3 and (NH4)2HPO4 as nitrogen sources. Peat and peat: vermiculite mixtures (1:4, 1:6, 1:8 and 1:10, v:v) were the best media to use in producing the vegetative inoculum of H. repandum


Introduction
Hydnum repandum (L.ex Fr.) S.F.Gray is an edible, ectomycorrhizal and medicinal mushroom, which belongs to the class Basidiomycetes, the order Cantharellales and the family Hydnaceae (Phillips, 1994).It is commonly known as the Wood Hedgehog or Hedgehog mushroom.This mushroom is easily recognised because of its spore-bearing structures which are shaped like teeth or spines rather than gills.
H. repandum is an important source of income and also valuable human food for rural population due to its nutritional and medical properties.An extract of the culture mycelia showed 70% inhibition against Sarcoma 180 solid cancer in mice, while extracts from the fruit bodies showed 90% inhibition against both Sarcoma 180 and Ehrlich solid cancer in mice (Ohtsuka et al., 1973).Repandiol isolated from H. repandum and H. repandum var.album displayed potent cytotoxic activity against various tumour cells (Takahashi et al., 1992).H. repandum contains 9.16% crude ash, 27.07%crude protein, 7.60% crude cellulose, 3.16% crude fat, and 53.01%carbohydrates (Ertan and Gulyavuz, 1993).Murcia et al. (2002) stated that H. repandum has antioxidant activity.It is also reported that H. repandum is an important nutrient for diet (Alonso et al., 2003).
H. repandum is widely distributed in North America and Europe, found single or in groups in summer or fall, and forms ectomycorrhizal association with hardwoods or conifers (Wikipedia, 2012).This mushroom is also naturally and widely present in the macro fungi flora of Turkey.It is widely distributed in the pine, hornbeam, beech and oak forests in Adana, Antalya, Artvin, Balikesir, Bolu, Eskisehir, Giresun, Gumushane, Istanbul, Izmir, Manisa, Ordu, Samsun and Trabzon provinces of Turkey (Solak et al., 2007).It is sold in the local markets and has high export potential (Peksen and Karaca, 2000) because it is a good delicious edible mushroom, having a sweet, nutty taste, mild odour and a crunchy texture.
There is limited information on mycelial growth conditions of H. repandum.The fastest mycelium growth was recorded in MEPA and PDA among 8 different media including PDA, ME, BAF, MMN, M40, MEPA, PDYA and MYPA.Mycelium type and form in MEPA and PDA media were radial and radial-floccose, respectively (Kibar and Peksen, 2007).However, the determining of a suitable media requires a detailed investigation to establish the most suitable medium that meets certain basic requirements.To find optimal nutritional and environmental conditions for mycelial growth of H. repandum and to produce its vegetative inoculum are necessary and have a great importance from the point of cultivation of this species.The aim of this study was to determine the best mycelial growth conditions (pH, temperature, carbon and nitrogen sources) of H. repandum, and to realise its vegetative inoculum production.

Materials and Methods
The study was conducted in the mycelial production laboratory of Department of Horticulture, Faculty of Agriculture, Ondokuz Mayis University between 2008 and 2009.

Collection, identification and isolation
The fruiting bodies of H. repandum were collected from the pine forests in Sinop province of Turkey in autumn 2008.Identification of mushroom was done using conventional methods (Phillips, 1994).Pure mycelial cultures of H. repandum were obtained using tissue culture method as described by Jonathan and Fasidi (2003).The mycelial culture was maintained on Potato Dextrose Agar (PDA) at 25°C in complete darkness, and the cultures were stored at 4°C.

Effect of initial pH and temperature on mycelial growth
The effect of initial pH and temperature on mycelial growth of H. repandum was investigated on Potato Dextrose Yeast Agar (PDYA) medium.PDYA medium was composed of potato 200 g, dextrose 20 g, yeast extract 2 g, agar 20 g and 1000 ml of distilled water.pH of PDYA medium was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 by means of pH metre with the addition of 1 N NaOH or HCl prior to autoclaving and then The media were autoclaved at 121 o C for 30 minutes.After cooling, PDYA media were poured into the 9 cm diameter Petri dishes.Mycelial agar discs of 5 mm diameter cut from the margin of an actively growing colony of subcultures were carefully placed in the centre of new Petri plates filled with PDYA.Inoculated Petri plates were sealed with parafilm and placed into separate incubators to incubate at 15, 20, 25 and 30°C under dark conditions.The experimental design was a completely randomised design with 5 replications.

Effect of carbon and nitrogen sources on mycelial growth
To screen the effect of different carbon and nitrogen sources on the mycelial growth of H. repandum, 7 carbon and 6 nitrogen sources were tested.Glucose, lactose, maltose, dextrose, mannitol, xylose and sucrose were used as carbon sources and each of them was added into PDYA medium at the rate of 20 g/l.The medium without carbon served as the control (C-free) (Kadiri and Fasidi, 1994).Malt extract, yeast extract, peptone, (NH4) 2 HPO 4 , NH 4 NO 3 and Ca(NO 3 ) 2 were used as nitrogen sources and each of them was added into PDYA medium at a concentration of 2 g/l.The control (N-free) medium was constituted without nitrogen (Daza et al., 2006).Sterilisation and inoculation were carried out as described for the pH and temperature above.The inoculated plates were incubated at 25 o C under dark conditions.The experiment was carried out in completely randomised design with 9 replications.Duration of complete mycelium running, mycelial growth area and colony diameter was measured as mycelial growth parameters in the experiments.Duration of complete mycelium running was determined as the number of days from the mycelium inoculation to time that petri plate was completely covered by mycelium.Mycelial growth area (cm 2 ) covered by mycelia was marked when the mycelia growth was completed in any of the Petri dishes, and measured with a digital planimeter.Mycelial colony diameter (mm) was determined by daily measurements in four different point of mycelial colony in the plates with a digital calliper.The average values which were calculated from these measurements were multiplied by two.

Vegetative inoculum preparation
To produce vegetative inoculum of H. repandum, 5 different vegetative inoculum media consisting of peat and peat: vermiculite mixtures (1:4, 1:6, 1:8 and 1:10 v:v) were used.Vegetative inoculum media, in 1000 ml glass culture bottles containing 800 ml of peat or different peat: vermiculite mixtures, were autoclaved at 121°C for 1.5 h.After 24 h, vegetative inoculum media was moistened with 200 ml MEPA liquid medium and autoclaved again at 121°C for 30 min.After cooling, the culture bottles were inoculated with 4 mycelial discs of 5 mm diameter.The inoculated bottles were incubated at 20±2 °C under dark conditions.The experiment was carried out in completely randomised design with 4 replications.pH of vegetative inoculum media was determined both after sterilisation and after mycelial growth was completed (Rowell, 1996).Mycelial growth rate (cm day -1 ) was determined by daily measurements from the two different sections of the culture bottles.Mycelial growth (cm) on the 18th day was also determined in the trial.

Statistical analysis
Data obtained from the experiments were subjected to analysis of variance using the SPSS statistical programme, and means showing statistical significance were compared using Duncan's multiple range test.

Results and Discussion
Means of mycelial growth characteristics obtained from different temperature and pH applications are presented in Table 1.Statistical analysis of the data on the mycelial growth parameters revealed that significant differences were found among temperatures, pH and temperature × pH interactions with regards to duration of complete mycelium running, mycelial growth area and mycelial colony diameter.The best temperatures for mycelial growth of H. repandum were found to be 20 and 25°C.The mycelial growth drastically decreased at 15°C (Table 1).It may be sourced from the reducing metabolic activities of the fungus that allow the absorption of essential nutrients needed for growth (Garraway  , 1984).In the present study, no mycelial growth was observed at 30°C (Table 1).Inhibition of mycelium growth at 30°C can be attributed to denaturation of important enzymes which catalyse fungal metabolic processes (Jonathan et al., 2004).The findings of this study were in agreement with Kalyoncu et al. (2009) who found that optimal temperatures for six Morchella species were 25 and 20°C.
The most suitable mycelial growth was obtained at pH 5.0-6.5, while the mycelial growth at pH 4.0 and 4.5 evidently decreased (Table 1).These results were in agreement with Stanbury et al. (1995) who stated that ectomycorrhizal fungi grow better at mild acidic conditions.Temperature and pH are accepted to be important environmental factors that control the fungal development (Johansson, 2002).Temperature × pH interactions were significant (P<0.01).The best mycelial growth was determined in pH between 5.0 and 6.5 at 20 and 25°C (Table 1).Kibar and Peksen (2011a) reported that best mycelial growth was determined in pH between 4.5 and 6.0 at 25°C in both Lactarius pyrogalus and L. controversus.
The effect of carbon sources on mycelia growth of H. repandum was significant.Among carbon sources, the best mycelial growth area and mycelial colony diameter were obtained in the media including mannitol and glucose (Table 2).Glucose and mannitol were the most suitable carbon sources for the mycelial growth of A. caesarea (Daza et al., 2006) and L. pyragolus and L. controversus (Kibar and Peksen, 2011a).Guler and Ozkaya (2008) reported that malt extract agar, wheat agar, potato dextrose agar and complete medium yeast extract agar media containing glucose, sucrose, maltose and starch were the best vegetative mycelial growth of Morchella conica.
The lowest mycelial growth area and colony diameter was determined in C-free control medium and media supplemented with sucrose and xylose (Table 2).This result was parallel with the suggestion of Hatakeyama and Ohmasa (2004) who found that the mycelial growth of Suillus and Boletinus was poor in sucrose contained medium.  1 Mycelial growth area and mycelial colony diameter were measured when it was completed in any of Petri plates at the end of the 11th day after mycelium inoculation for the current study; **Means followed by different letters in the same columns are statistically different according to Duncan's multiple range test (P<0.01).
The results of the study carried out to determine nitrogen requirement of H. repandum indicated that Ca(NO 3 ) 2 was the best nitrogen source (P<0.01).This was followed by peptone.Mycelial growth area and mycelial colony diameter of media containing (NH 4 ) 2 HPO 4 and NH 4 NO 3 were lower than that of the control and the other nitrogen sources (Table 3).It is generally asserted that ammonium is the most commonly used nitrogen source for most ECM fungi (Rangel-Castro et al., 2002;Sangtiean and Schmidt, 2002;Daza et al., 2006), but the results from the present study were contrary to the reports of these researchers.Initial pH values of the vegetative inoculum media ranged from 5.47 to 7.30.pH of media at the end of mycelial growth was found between 6.08 and 7.22.In vegetative inoculum media studies, the effects of vegetative inoculum media on mycelial growth were found nonsignificant (Table 4).The best mycelial growth for T. terreum was obtained from peat:vermiculite mixtures in the rate of 1:4 and 1:6 (Kibar and Peksen, 2011b).In another study, peat: vermiculite mixture in the rate of 1:4 was found to be the most suitable vegetative inoculum medium for L. pyrogalus (Kibar and Peksen, 2011a).In many studies, it was reported that the most appropriate media to obtain the vegetative inoculum of ectomycorrhizal mushrooms were peat, vermiculite, sphagnum moss and perlite mixtures moistened with different liquid nutrient media (Santiago-Martínez et al., 2003;Parlade et al., 2004;Flores et al., 2005).ns: non-significant In conclusion, the most suitable mycelial growth of H. repandum was obtained at 20 and 25°C, pH 5.5, by using glucose and mannitol as carbon sources and Ca(NO 3 ) 2 as nitrogen source.The poorest mycelial growth was recorded in sucrose and xylose as carbon sources and NH 4 NO 3 and (NH 4 ) 2 HPO 4 as nitrogen sources.It can also be concluded that the vegetative inoculum of H. repandum can be produced by cultivating the fungus in media which are prepared by peat and peat:vermiculite mixtures.To know the most suitable nutritional and environmental conditions for mycelial growth of H. repandum and to produce its vegetative inoculum would be useful for successful cultivation of this mushroom.

Table 1 :
Effect of temperature and initial pH on the mycelial growth of H. repandum on PDYA 1 Data were arcsine-square root transformed prior to statistical analyses.*Means followed by different letters are statistically different by Duncan's multiple range test (P<0.05),**Means followed by different letters are statistically different according to Duncan's multiple range test (P<0.01).

Table 2 :
Effect of carbon sources on the mycelial growth of H. repandum

Table 3 :
Effect of nitrogen sources on the mycelial growth of H. repandum Mycelial growth area and mycelial colony diameter were measured when it was completed in any of Petri plates at the end of the 11th day after mycelium inoculation for the current study; **Means followed by different letters in the same columns are statistically different according to Duncan's multiple range test (P<0.01).

Table 4 :
pH values of the vegetative inoculum media and their effects on the mycelial growth of H. repandum