542 Chiang Mai J. Sci. 2014; 41(3)

Chiang Mai J. Sci. 2014; 41(3) : 542-556 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

Effects of Different Culture Media, Carbon and Nitrogen Sources and Solid Substrates on Growth of Mushrooms Janjira Wiriya [a], Paiboolya Kavinlertvatana [b] and Saisamorn Lumyong [a]* [a] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, . [b] Thai Orchids Lab Co., Ltd. Bangkok 10230, Thailand. *Author for correspondence; e-mail: [email protected]

Received: 9 November 2012 Accepted: 23 February 2013

ABSTRACT In 2009-2011, five different Termitomyces sporocarps were collected from Chiang Mai, Payao and Petchaboon Provinces in Thailand and pure cultures were isolated. Fungi were identified based on morphological and molecular characteristics. One isolate was identified as T. clypeatus while the others probably represent four unknown species. Growth on different carbon, nitrogen and solid substrates was tested. Of ten culture media, malt extract agar was the best for mycelia growth for all isolates. Sucrose was the best carbon source for Termitomyces sp. CMUTM001 and CMUTM002. Fructose was the best carbon source for T. clypeatus CMUTM003 and CMUTM005, while the best growth of isolate CMUTM005 was on glucose. Peptone was the best nitrogen source for enhancing radial growth. In addition, job’s tear grain medium gave the highest mycelial growth and may be a suitable substrate for Termitomyces mycelial inocula production. This study provides valuable information concerning the cultivation of Termitomyces mushrooms.

Keywords: Termitomyces, edible mushroom, culture studies

1. INTRODUCTION The genus Termitomyces (order Provinces). These symbiotic fungi grow on a , family ) is a that special substrate called the “fungus comb” or has a symbiotic relationship with “fungus garden” prepared and maintained by and is widely distributed throughout termites [3, 4]. Fungus nodules (asexual stage) equatorial Africa and Asia [1, 2]. Sporocarps are also found on the fungus comb surface. of Termitomyces appear during the rainy season Fungal mycelia develop on combs (June-September) and are abundant in teak and produce mushrooms in rainy season [5]. plantations and natural forests of northern There are several benefits of Termitomyces for and central Thailand (Kanchanaburi, their termite hosts including producing the Rajburi, Uthaithani, Nonthaburi, Bangkok, enzyme which degrades lignin and improves Saraburi, Petchaboon, Tak and Nakornnayok the digestibility of cellulose for the termites Chiang Mai J. Sci. 2014; 41(3) 543

[6, 7]. In addition, Termitomyces is an abundant were investigated. source of sugar, protein fiber, lipids, vitamins, minerals and medicine used to 2. MATERIALS AND METHODS lower blood pressure, or to treat rheumatism, 2.1 Fungal Isolate kwashiorkor, obesity and diarrhea [8, 9]. Sporocarps of Termitomyces spp. were To increase mushroom production it is collected from Chiang Mai, Payao and desirable to inoculate Termitomyces in natural Petchaboon Provinces, Thailand from habitats and teak plantations. This requires 2009-2011. Sporocarps were wrapped in the good inocula but, there have been few aluminum foil and kept in plastic specimen reports on inocula preparation of Termitomyces. boxes until transported back to the laboratory The fungal mycelium can be grown in where notes on macromorphological controlled conditions using simple media. features and photographs were taken within Tests on suitable growth conditions will lead 24 h. The mycelia were isolated by aseptically to a better understanding of the symbiotic removing a small piece of mycelium from relationship by elucidating the ecological the inside of fresh sporocarps and transferring and physical requirements of sporocarp it to potato dextrose agar (PDA) with 0.05% production [10]. Three are different physical streptomycin sulfate added. Plates for mycelia factors, such as culture media, temperature, isolation were incubated at 30°C in the dark. pH and nutrient (carbon and nitrogen sources) The mycelia emerging from the tissues were affect the fungal mycelial growth [11, 12]. picked up and transferred to fresh PDA. This study focused on the effect of the Cultures were kept on PDA slants and in both culture media, carbon and nitrogen sources sterile distilled water at 4°C and 20% on mycelial growth of Termitomyces spp. in glycerol at -20°C for long-term preservation. pure culture. In addition, suitable solid Five isolates were used in this study substrates for mycelia inocula production (Table 1). Table 1. Origin, date of collection and GenBank accession number of Termitomyces isolates in this study. Isolate Origin Date of collection GenBank no. CMUTM001 Sansai, Chiang Mai 12 Sep 2009 JX866771 CMUTM002 Chondan, Petchaboon 17 Aug 2011 JX866772 CMUTM003 Bhusang, Payao 1 Sep 2010 JX866773 CMUTM004 Muang, Chiang Mai 31 Aug 2010 JX866774 CMUTM005 Sansai, Chiang Mai 8 Sep 2011 JX866775

2.2 Identification features are based on at least 30 measurements Sporocarps were identified based on of each structure. The specimens were dried both morphological characters [1, 2, 13, 14] at 40-45°C and deposited at the Research and a molecular analysis of the internal Laboratory for Excellence in Sustainable transcribed spacer (ITS) regions of nuclear Development of Biological Resources, rDNA. For microscopic study, sections of Faculty of Science, Chiang Mai University, dried sporocarps, including lamellae, cutis, Thailand. In addition, genomic DNA of and pileal context were mounted in 3% KOH each sporocarp was extracted through a or Melzer’s reagent. Size data of anatomical CTAB method [15]. Fresh sporocarps and 544 Chiang Mai J. Sci. 2014; 41(3)

mycelia (1 g) were frozen in liquid nitrogen Germany Catalog no. 740 609.50) and grounded into a fine powder with a mortar following the manufacturer’s protocol. The and pestle. The powder was placed in a purified PCR products were directly 1.5 ml Eppendorf tube and 500 μl extraction sequenced. Sequencing reactions were buffer (2% cetyltrimethylammonium preformed and the sequences were bromide, 100 mM Tris-HCl, 1.2 M NaCl, automatically determined in the genetic 20 mM ethylenediaminetetraacetic acid, analyzer (1ST Base, ) using PCR pH 8.0) added and mixed with the material. primers mentioned above. Sequences were Samples were incubated at 65°C for 60 min used to query GenBank via BLAST (http:/ in a water bath and occasionally mixed. /blast.ddbj.nig.ac.jp/top-e.html) and a An equal volume (500 μl) of chloroform: multiple sequence alignment was carried isoamyl alcohol (24:1) was added to the out using the subroutines in Clustal X [16]. samples and briefly mixed by vortexing. A phylogenetic tree was constructed using After centrifugation for 15 min at 13,000 rpm, the PUAP beta 10 software version 4.0 [17]. the upper aqueous layer was transferred to a new sterile Eppendorf tube. The extraction 2.4 Fungal Cultivation was repeated until the interface was clear. Twenty-five milliliters of each tested The supernatant was removed to a new culture media were poured into Petri dishes Eppendorf tube, containing 2 volumes (9.0 cm, in diameter) after autoclaving at 121°C of cold 100% ethanol. The DNA was for 15 min. A sterile cellophane disc was precipitated at -20°C overnight and placed on the surface of the media. Mycelial centrifuged for 15 min at 14,000 rpm and inocula (5 mm diameter), prepared from 4°C. Then the pellet was washed with cultures grown on PDA at 30°C in darkness 70% ethanol and dried at room temperature. for three weeks, were then transferred to the It was resuspended in 100 ml of 0.002% various tested media. The inoculated plates RNase (5 mg/ml) in TE buffer and incubated were sealed with Parafilm M. After one for one hour at 37C. The suspension was month of incubation, in darkness at 30°C stored at -20°C pending use for amplification. colony diameters on each media were measured. The covered cellophane discs 2.3 Fungal ITS and LSU Regions were removed, dried at 60°C for 48 h and Sequencing and Phylogenetic Analysis maintained in desiccators for 20 min before The internal transcribed spacer (ITS) weighing. Mycelium dry weights were regions of nuclear rDNA were amplified by calculated. Three replications were made for polymerase chain reaction (PCR) with primers each treatment. ITS4 and ITS5 under the following thermal conditions: 94°C for 2 min, 35 revolution 2.5 Effect of Culture Media on Mycelial cycles of 95°C for 30 s, 50°C for 30 s, 72°C Growth for 1 min, and 72°C for 10 min. The PCR Ten culture media were used in this products were checked on 1% agarose gels experiment: fungal-host agar [18], malt stained with ethidium bromide under UV extract agar, modified Hagem agar [19], light. PCR products were purified by using Murashige and Skoog medium [20], oat meal PCR clean up Gel extraction NucleoSpin® agar, PDA, Sabouraud dextrose agar, soil Extract II purification Kit (Macherey-Nagel, extract agar [21], yeast extract agar and Chiang Mai J. Sci. 2014; 41(3) 545

yeast extract-malt extract agar. The final cut from the periphery of a three week- pH value of tested media were adjusted to old growing colony on malt extract agar 5.6 using 1 N HCl and 1 N NaOH. and incubated at 25°C for two months in darkness, with three replicates of each 2.6 Effect of Carbon and Nitrogen Sources experiment. Linear growth of the mycelium on Mycelium Growth was measured every five days and growth A basal medium contained yeast extract rate was estimated [15].

(0.2 g), CaCl2.H2O (0.005 g), FeSO4.7H2O

(0.01 g), KH2PO4 (0.05 g), MgSO4.7HO 2.8 Statistical Analysis

(0.05 g), (NH4)H2PO4 (1.5 g), agar (18 g), All data were analyzed by one-way distilled water 1000 ml and adjusted to pH 5. analysis of variance (ANOVA) in SPSS Carbon sources used were glucose fructose, program version 17.0. The experiment maltose, manitol, starch, sucrose and xylose. followed a completely randomized design Bovine serum albumin (fraction V), egg (CRD) with three replications. Turkey’s test albumin, peptone, soybean meal, urea, and was used for significant differences between

KNO3 were used to replace (NH4)H2PO4. treatments (P<0.05). The inoculated plates with three week-old inocula were incubated at 30°C in the dark 3. RESULTS AND DISCUSSION for one month. 3.1 Identification The morphology of each Termitomyces sp. 2.7 Effect of Solid Substrates on Mycelium was recorded. Termitomyces sp. CMUTM001, Growth pileus 2.8-5.9 cm diameter, convex shape Twelve solid substrates; brown rice grain with perforatorium, surface smooth, center (Oryza sativa), corn grain (Zea mays), job’s tear dark brown to reddish-brown, margin even, grain (Coix lacryma-job), barley grian (Hordeum striate, tuberculate eroded, white to cream. vulgare), black kidney bean (Phasecolus vulgaris), Lamellae free, incurved, 4-6 mm, crowded, mung bean (Phaseolus aureus), rye grain (Secale white to cream. Stipe 3.84.4 × 6.4-11.9 cm, cereale), sorghum grain (Sorghum bicolor), cylindrical, solid, surface white to cream, soybean (Glycine max), wheat grain (Triticum fibrous (Figure 1A, 1B). Context 0.5-5.0 mm, aestivum), rice bran and sawdust were used white. Spores 5.0-7.5 × 3.5-4.0 μm, broadly in this experiment. Each grain was prepared ellipsoid, thin walled, with a few content by boiling for 10-15 min. The substrates (Figure 1c). Basidia 24.0-26.0 × 5.0-8.0 μm were placed in 18 × 180 mm test tubes to (Figure 1D). Cheilocystidia 16.0-25.0 × approximately 10 cm depth and autoclaved 6.0-8.0 μm, pyriform, thin-walled, with at 121°C for 30 min. After cooling, each few contents (Figure 1E). Pinkish cream tube was inoculated with a mycelial plug spore print. 546 Chiang Mai J. Sci. 2014; 41(3)

Figure 1. Termitomyces sp. CMUTM001. A, B, Sporocarps; C, Basidiospores; D, Basidia and E, Cheilocytidia. Bar: A = 5 cm; B = 1 cm; C = 5 μm; D-E = 10 μm.

Termitomyces sp. CMUTM002, pileus 2B). Pseudorhiza 3.5-9.0 cm, fusoid above, 1.2-7.5 cm diameter, convex then plan, with a cartilaginous, dark crust. Context centre brown at the disk, white to cream 1.5-3.0 cm thick, white, inflated. Spores towards margin, glabrous, striate at the 6.0-8.0 × 3.5-5.0 μm, ellipsoid, subhyaline, margin. Lamellae breadth 1.0-4.5 mm, free thin-walled (Figure 2C). Basidia 17.0-24.0 × to adnexed, incurved crowded with white 6.0-8.0 μm, bearing four sterigma to cream lamellae. Stipe 10.0-80 × 9.50 μm, (Figure 2D). Cheilocystidia 14.0-28.0 × 6.0- cylindrical, sometimes slightly bulbous at 10.0 μm, pyriform, thin-walled. Peurocystidia ground level; solid; surface white, hairy 16.0-28.0 × 10.0-21.0 μm, obovoid to scales and striates. Partial veil sometimes pyriform (Figure 2E). Salmon pink spore evident, cortinoid on upper stipe (Figure 2A, print.

Figure 2. Termitomyces sp. CMUTM002. A, B, Sporocarps at natural habitat; C, Basidiospores; D, Basidia and E, Cheilocytidia. Bar: A-B = 5 cm; C = 5 μm; D-E = 10 μm. Chiang Mai J. Sci. 2014; 41(3) 547

Termitomyces sp. CMUTM003, pileus 1.0-4.0 cm thick at perforatorium, white, 2.8-8.0 cm diameter, at first pointed-conical, inflated. Spores 4.0-8.0 × 3.0-5.0 μm, later expanding to convex, dark spiniform ellipsoid, hyaline to stramineous, thin-walled perforatorium; surface grayish brown to (Figure 3C). Basidia 17.0-26.0 × 4.0-8.0 μm, ochraceous brown, color paler towards clavate, bearing four sterigma (Figure 3D). margin, dry, silky smooth. Lamellae 2.5- Cheilocystidia 17.0-25.0 × 4.5-7.0 μm, 4.0 mm, free, whitish, crowded; edge entire. clavate to pyriform, hyaline, thin-walled Stipe 5.0-15.0 × 0.4-1.8 cm, cylindrical or with little content (Figure 1E). Peurocystidia wide below, solid; surface whitish to pale 20.0-30.0×1.0-21.0 μm, similar to cheilocystidia. brown longitudinal fribrillose (Figure 3A, Pinkish cream spore print. 3B). Pseudorhiza 5.0-24.0 cm, long Context

Figure 3. Termitomyces sp. CMUTM003. A, B, Sporocarps; C, Basidiospores; D, Basidia and E, Cheilocytidia. Bar: A = 1 cm; B = 5 cm; C = 5 μm; D-E = 10 μm.

Termitomyces sp. CMUTM004, pileus (Figure 4A). Pseudorhiza 3.0-8.5 mm. 10-16 cm diameter, convex shape, finally Context 6.0-8.0 mm, thickened, white to expanding, margin usually remaining cream. Spores 2.5-5.5 μm, ellipsoid shape incurved, center-plane, slight umbo, white (Figure 4B). Basidia 8.5 × 25.5 μm, clavate, to cream colour. Lamellae 6-8 mm, free, four sterigma (Figure 4C). Cheilocystidia white to cream, crowded to close. Stipe 15.70 × 35.20 μm, pyriform (Figure 4D). 6.0-14.0 × 9.5-16.0 cm, equal, solid, white Cream spore print. to cream color, surface dry with fribillose 548 Chiang Mai J. Sci. 2014; 41(3)

Figure 4. Termitomyces sp. CMUTM004. A, Sporocarp; B, Basidiospores; C, Basidia and D, Cheilocytidia. Bar: A = 5 cm; B = 5 μm; C-D = 10 μm.

Termitomyces sp. CMUTM005, pileus pseudorhiza (Figure 5A). Context 0.5-1.0 cm 0.5-2.5 cm diameter, covex , expanding, thick at centre, white, thin-walled. Spores with a papillate umbo; surface white to 5.0-8.0 × 3.0-5.5 μm, ovoid to ellipsoid, cream colour, margin silky striate, smooth. subhyaline, slightly thickened wall (Figure Lamellae 0.5-1.0 mm wide, adnexed to free, 1B). Basidia 18.0-26.0 × 6.0-8.0 μm, white then pinkish, crowded. Stipe 3.0-4.0 × clavate, bearing four sterigma (Figure 5C). 0.3-0.4 cm, slender, cylindrical, solid; surface Cheilocystidia 24.0-62.0 × 13.0-22.0 μm white, smooth, fibrilloso-striate, with very (Figure 5D). Pleurocystidia similar to shot rooting base and lacking a true cheilocystidia but rare. Cream spore print.

Figure 5. Termitomyces sp. CMUTM005. A, Sporocarp; B, Basidiospores; C, Basidia and D, Cheilocytidia. Bar: A = 1 cm; B = 5 μm; C-D = 10 μm. Chiang Mai J. Sci. 2014; 41(3) 549

The morphological study indicated T. clypeatus AB073501. The maximum- that all isolates differed. The morphological parsimony (MP) analysis of the ITS gene characteristics of Termitomyces sp. CMUTM001 of 29 sequences resulted in 788 characters, were similar to T. eurhizus (syn. T. albiceps) of which 316 characters were constant, but differ in basidia and spore size [2, 13]. 127 variable characters were parsimony Termitomyces sp. CMUTM002 was similar to uninformative, and 345 characters were T. cylindricus (syn. T. aurantiacus) but CMU002 parsimony informative. Heuristic searches has a larger stipe than T. cylindricus [2, 13]. resulted in a tree length = 1,094 steps, Termitomyces sp. CMUTM003 was similar to CI = 0.6984, RI = 0.7597, RC = 0.5305 T. clypeatus according to previous studies and HI = 0.3015. One of the maximum- [1, 2, 13]. The morphological characteristics parsimony trees is shown in Figure 6. of Termitomyces sp. CMUTM004 were similar A phylogenetic dendrogram showed that to T. unkowann and T. robustus but differed Termitomyces sp. CMUTM001, CMUTM002, from both species in spore size [1, 14]. and CMUTM005 were separated from Termitomyces sp. CMUTM005 was similar to T. eurhizus, T. cylindricus and T. microcarpus. T. microcarpus but had a different spore print, A strongly supported node separated the later species has a pinkish spore print Termitomyces sp. CMUTM001, CMUTM004 [1, 2]. However, subsequent molecular and CMUTM005. Termitomyces sp. analysis was used to confirm them as the CMUTM003 was closely related to T. clypeatus same species. The sequence of ITS rDNA AB073501 with 100% bootstrap support and gene of five isolates were amplified, from herein is referred to as T. clypeatus. sequenced and deposited in GenBank with Termitomyces sp. CMUTM002 was separated accession numbers JX866771-JX866775 from Termitomyces sp. AB202123 with 100% (Table 1). A BLAST similarity search bootstrap support. Both the data from confirmed that they belong to the genus morphology and molecular characteristics Temitomyces (Table 1). The sequences of showed only one isolate CMUTM003 was Termitomyces sp. CMUTM001, CMUTM004 T. clypeatus. This result is similar to previous and CMUTM005 had 96, 99 and 93% studies that Termitomyces taxonomy similarity with Termitomyces sp. AB073501, classification based on identification keys respectively. Termitomyces sp. CMUTM002 is not sufficient to identify species and showed 91% similarity with Termitomyces molecular techniques need to be employed sp. AB202123. The remaining isolate, [14, 22, 23]. CMUTM003 showed 99% similarity with 550 Chiang Mai J. Sci. 2014; 41(3)

Figure 6. Maximum-parsimonious trees inferred from a heuristic search of the internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 sequence alignments of 29 isolates. ionides and C. fallax were used to root the tree. Branches with bootstrap values ≥ 50% are shown at each branch and bar represents 10 substitutions per nucleotide position.

3.2 Effect of Different Culture Media on CMUTM004 showed the smallest colony Mycelia Growth diameter on Hagem agar (26.00±1.00 mm) The effect of ten culture media on and fungal-host agar (25.67±0.58 mm), mycelial growth of five isolates is shown in respectively. The lowest biomass yield of Figure 7. After one month of incubation, Termitomyces sp. CMUTM001 (14.57±3.78 mg/ all fungus isolates grew well on malt extract plate), CMUTM002 (8.43± 0.83 mg/plate), agar, followed by soil extract agar, yeast-malt CMUTM004 (3.03±0.61 mg/plate) and extract agar and PDA. All isolates produced T. clypeatus CMUTM003 (3.20±0.62 mg/plate) white mycelia on culture media. Significantly, were observed on fungal-host agar. The the largest colony diameter and highest results are similar to several other studies biomass yield of all isolates were observed that found growth of mushrooms in pure on malt extract agar. Termitomyces sp. culture was greatly influenced by changes to CMUTM002 showed the largest colony the culture media [11, 12, 24]. Generally, diameter (46.0±1.00 mm) and biomass yield Termitomyces spp. grow well on a variety (60.66±6.93 mg/plate) when compared with culture media such as PDA and malt-yeast other isolates. The smallest colony diameter extract agar [22, 25, 26]. However, this of both Termitomyces sp. CMUTM001 (20.67± research showed that malt extract agar is 0.58 mm) and CMUTM002 (9.00±2.00 mm) the best media for growth of Termitomyces were found on oatmeal agar. Whereas, cultures. Recently, Tibuhwa [27] studied the T. clypeatus CMUTM003 and Termitomyces sp. effect of three culture media on the pure Chiang Mai J. Sci. 2014; 41(3) 551

culture growth of T. aurantiacus and T. salts) and Ghosh media were better than umkowaani and reported that modified Hagem Modess medium. malt extract medium (adding some organic

Figure 7. Colony diameter (A) and biomass yield (B) of five Termitomyces isolates on different culture media. Data means and error bars at each point indicate ± SD. Different letters above each graph indicate the significant difference (P<0.05). PDA = potato dextrose agar, SDA = Sabouraud dextrose agar, MEA = malt extract agar, YMA = yeast extract-malt extract agar, YA = yeast extract agar, MSA = Murashige and Skoog medium, FH = fungal-host agar, OMA = oat meal agar, HA = modified Hagem agar and SEA = soil extract agar.

3.3 Effect of Different Carbon Sources diameter (44.67±1.53 mm) and the highest on Mycelia Growth biomass yield (69.10±15.85 mg/plate) of The growth response of five Termitomyces T. clypeatus CMUTM003 were obtained on isolates to various carbon sources in basal fructose. Termitomyces sp. CMUTM005 showed medium was tested (Figure 8A, 8B). It was the largest colony diameter (36.33±3.06 mm) found that all isolates could grow on all and the highest biomass yield (33.33±15.08 selected carbon sources. All Termitomyces mg/plate) on glucose. This study showed isolates grew well on glucose, fructose, that Termitomyces was able to use various maltose and sucrose. However, sucrose was carbon sources. This result is similar to Kaur the best carbon source for Termitomyces sp. [25] who reported that T. striatus could use CMUTM001 and CMUTM002. The highest lactose, glucose, maltose, raffinose, sucrose colony diameter (43.33±1.15 mm) and the and xylose, and found that the best growth highest biomass yield (45.53±21.70 mg/plate) was obtained from glucose. In addition, of Termitomyces sp. CMUTM004 were found this result agreed that different carbon on fructose. Termitomyces clypeatus CMUTM003 sources elicited different fungal growth. and Termitomyces sp. CMUTM005 grew on For example, the best growth of Psathyrella fructose and glucose which had significant atroumbonata [28] Lentinus subnudus [12] differences. Whereas, the largest colony and Pleurotus ostreatus [29] were found on 552 Chiang Mai J. Sci. 2014; 41(3)

glucose, fructose and manitol, respectively. carbon compounds tested, mannose enhanced Gbolagade [30] reported that among seventeen the best biomass yield of Lepiota procera.

Figure 8. Growth of five Termitomyces isolates on basal medium with different carbon and nitrogen sources. A, C, colony diameter on different carbon and nitrogen sources, respectively. B, D, biomass yield on different carbon and nitrogen sources, respectively. Data means and error bars at each point indicate ± SD. Different letters above each graph indicate the significant difference (P<0.05). BSA = bovine serum albumin, EG = egg albumin and SB = soy bean meal.

3.4 Effect of Different Nitrogen Sources peptone.The highest biomass yield of on Mycelia Growth Termitomyces sp. CMUTM001 (51.00 ± 2.38 Significant variation in fungal growth mg/plate), CMUTM002 (45.63 ± 1.97 mg/ among the two inorganic (KNO3 and plate) and CMUTM005 (48.60 ± 3.40 mg/

(NH4)H2PO4) and five organic nitrogen plate) were obtained on KNO3. Small colony (bovine serum albumin, egg albumin, diameter and biomass yield of all isolates peptone, soybean meal and urea) sources was found on urea. The result is similar to were observed (Figure 8C, 8D). Peptone was previous studies that report the best in vitro the best nitrogen source to promote fungal growth of mushroom on nitrogen sources growth in terms of radial growth. In addition, depended on fungal species or isolate and

KNO3 and egg albumin supported fairly good nitrogen sources. For example, optimum radial growth. The highest biomass yield of mycelia growth of Pleurotus ostreatus was T. clypeatus CMUTM003 (64.73 ± 2.62 mg/ achieved when urea was used as a nitrogen plate) and Termitomyces sp. CMUTM004 source [29]. Gbolagade et al. [12] found (53.20±1.83 mg/plate) was obtained on that yeast extract enhanced the greatest Chiang Mai J. Sci. 2014; 41(3) 553

mycelial growth of Lentinus ubnudus and barley grain, job’s tear grain and sorghum peptone was the best nitrogen source for grain had the thickest mycelial growth in the growth of L. procera [30]. This present comparison with other substrates based on study showed that all Termitomyces a visual assessment. All fungus isolates grew isolates could use varied nitrogen sources significantly faster on job’s tear grain and for growth and peptone was the best T. clypeatus CMUTM003 showed the organic nitrogen source for fungal growth, highest growth rate (1.25±0.05 mm/day) which is similar to the findings of Kaur when compared with other isolates in this [25] who reported that T. striatus could use study. The lowest mycelial growth various nitrogen sources and the highest rate of all strains was obtained on rice bran. growth was obtained from peptone. In This present study shows that job’s tear grain addition, inorganic nitrogen sources such can be used as a suitable substrate for inocula as CH3COONH4, (NH4)H2PO4, KNO3, production of Termitomyces spp. This result and NaNO3 supported fairly good growth was different from the solid substrates used of T. striatus. for inocula production of other cultivated mushrooms. For example, sorghum grain is 3.5 Effect of Solid Substrates on Mycelia the most popular solid substrate for inocula Growth production of Agaricus spp., Coprinus spp., The mycelium growth rates of Pleurotus spp. and Volvariella spp. in Termitomyces spp. in test tube culture on Thailand [31-33]. Furthermore, wheat grain various substrates were investigated and are is used in A. blazei inocula production in shown in Figure 9. The results indicated Brazil [34] and P. ostreatus inocula that mycelial growth rate varied for production in Ghana uses sorghum and different solid substrates and fungal millet grains [35]. isolates. After two months of incubation,

Figure 9. Growth rate of five Termitomyces isolates on different solid substrates. Data means and error bars at each point indicate ± SD. Different letters above each graph indicate the significant difference (P<0.05). SG = sorghum grain, BG = barley grian, RG = rye grain, CG = corn grain, SBG = soybean, JB = job’s tear grain, BKG = black kidney bean, MGB = mung bean, WG = wheat grain, GG = GABA rice grain, SD = sawdust and RB = rice bran. 554 Chiang Mai J. Sci. 2014; 41(3)

4. CONCLUSIONS [5] Moriya S., Inoue T., Traprab Y., Among the ten tested culture media Johjima T., Suwanarit P., Sangwanit and twelve solid substrates, malt extract agar U., Vongkaluang N. and Kudo T., and job’s tear grain were the most suitable Fungal community analysis of fungus medium for mycelial growth and solid gardens in termite nests, Microbes Environ., 2005; 20: 243-252. substrate for inocula production. Different carbon and nitrogen sources showed [6] Jojima T., Inoue T., Ohkuma M., different effects on fungal growth. All Noparatnaraporn N. and Kudo T., isolates grew well on glucose, fructose, Chemical analysis of food processing maltose and sucrose, and peptone enhanced by the fungus-growing termite the greatest radial growth. Future studies gilvus, Sociobiol., 2003; 42: 815-824. will attempt the selection of suitable techniques for large scale Termitomyces [7] Taprab Y., Johjima T., Maeda Y., inocula production and inoculation programs, Moriya S., Trakulnaleamsai S., which are important for the management of Noparatnaraporn N., Ohkuma M. teak plantations in Thailand. and Kudo T., Symbiotic fungi produce laccase potentially involved in phenol degradation in fungus combs of ACKNOWLEDGEMENT fungus growing termite Thailand, This project received support from the Appl. Environ. Microbiol., 2005; 71: Thai Orchid Lab Co., Ltd., TRF-Master 7696-7704. research grants (WRG WI525S062), the Graduate School of Chiang Mai University [8] Breene W.M., Nutritional and medicinal value of specialty and Chiang Mai University. We are grateful mushrooms, J. Food Prot., 1990; 53: to Keegan H. Kennedy for improving the 883-894. English. [9] Nabubuya A., Muyonga J.H. and REFERENCES Kabasa J.D., Nutritional and hypocholesterolemic properties of [1] Heim R., Termites et Champignons. Termitomyces microcarpus mushroom, Soci t Nouvelle Des Editions Boub , Afr. J. Food Agric. Nutri. Dev., 2010; Paris, 1977. 10: 2235-2257. [2] Pegler D.N. and Vanhaecke M., [10] Yamada A., Inoue T., Wiwatwitaya Termitomyces of south-east Asia, Kew D., Ohkuma M., Kudo T., Abe T. and Bulletin, 1994; 49: 717-736. Sugimoto A., Carbon mineralization [3] Okane I. and Nakagiri A., Taxonomy by termites in tropical forest with of an anamorphic xylariaceous fungus emphasis on fungus combs, Ecol. Res., from a termite net found together with 2005; 20: 453-460. Xylaria angulosa, Mycosci., 2007; 48: [11] Nasim G., Malik S.H., Bajwa R., 240-249. Afzal M. and Mian S.W., Effect of [4] Froslev T.G., Aanen D.K., Laessoe T. three different culture media on and Rosendahl S., Phylogenetic mycelia growth of oyster and Chinese relationships of Termitomyces and mushroom, J. Biol. Sci., 2001; 1: 1130- related taxa, Mycol. Res., 2003; 107: 1133. 1277-1286. Chiang Mai J. Sci. 2014; 41(3) 555

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