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

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

Effects of Different Culture Media, Carbon andNitrogen Sources and Solid Substrates on Growthof Termitomyces MushroomsJanjira Wiriya [a], Paiboolya Kavinlertvatana [b] and Saisamorn Lumyong [a]*[a] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.[b] Thai Orchids Lab Co., Ltd. Bangkok 10230, Thailand.*Author for correspondence; e-mail: saisamorn.l@cmu.ac.th

Received: 9 November 2012Accepted: 23 February 2013

ABSTRACTIn 2009-2011, five different Termitomyces sporocarps were collected from Chiang

Mai, Payao and Petchaboon Provinces in Thailand and pure cultures were isolated. Fungi wereidentified based on morphological and molecular characteristics. One isolate was identified asT. clypeatus while the others probably represent four unknown species. Growth on differentcarbon, nitrogen and solid substrates was tested. Of ten culture media, malt extract agarwas the best for mycelia growth for all isolates. Sucrose was the best carbon source forTermitomyces sp. CMUTM001 and CMUTM002. Fructose was the best carbon source forT. clypeatus CMUTM003 and CMUTM005, while the best growth of isolate CMUTM005was 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 forTermitomyces mycelial inocula production. This study provides valuable information concerningthe cultivation of Termitomyces mushrooms.

Keywords: Termitomyces, edible mushroom, culture studies

1. INTRODUCTIONThe genus Termitomyces (order

Lyophyllaceae, family Agaricales) is a fungus thathas a symbiotic relationship with termitesand is widely distributed throughoutequatorial Africa and Asia [1, 2]. Sporocarpsof Termitomyces appear during the rainy season(June-September) and are abundant in teakplantations and natural forests of northernand central Thailand (Kanchanaburi,Rajburi, Uthaithani, Nonthaburi, Bangkok,Saraburi, Petchaboon, Tak and Nakornnayok

Provinces). These symbiotic fungi grow on aspecial substrate called the “fungus comb” or“fungus garden” prepared and maintained bytermites [3, 4]. Fungus nodules (asexual stage)are also found on the fungus comb surface.Fungal mycelia develop on termite combsand produce mushrooms in rainy season [5].There are several benefits of Termitomyces fortheir termite hosts including producing theenzyme which degrades lignin and improvesthe digestibility of cellulose for the termites

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[6, 7]. In addition, Termitomyces is an abundantsource of sugar, protein fiber, lipids,vitamins, minerals and medicine used tolower blood pressure, or to treat rheumatism,kwashiorkor, obesity and diarrhea [8, 9].

To increase mushroom production it isdesirable to inoculate Termitomyces in naturalhabitats and teak plantations. This requiresthe good inocula but, there have been fewreports on inocula preparation of Termitomyces.The fungal mycelium can be grown incontrolled conditions using simple media.Tests on suitable growth conditions will leadto a better understanding of the symbioticrelationship by elucidating the ecologicaland physical requirements of sporocarpproduction [10]. Three are different physicalfactors, such as culture media, temperature,pH and nutrient (carbon and nitrogen sources)affect the fungal mycelial growth [11, 12].This study focused on the effect of theculture media, carbon and nitrogen sourceson mycelial growth of Termitomyces spp. inpure culture. In addition, suitable solidsubstrates for mycelia inocula production

were investigated.

2. MATERIALS AND METHODS2.1 Fungal Isolate

Sporocarps of Termitomyces spp. werecollected from Chiang Mai, Payao andPetchaboon Provinces, Thailand from2009-2011. Sporocarps were wrapped inaluminum foil and kept in plastic specimenboxes until transported back to the laboratorywhere notes on macromorphologicalfeatures and photographs were taken within24 h. The mycelia were isolated by asepticallyremoving a small piece of mycelium fromthe inside of fresh sporocarps and transferringit to potato dextrose agar (PDA) with 0.05%streptomycin sulfate added. Plates for myceliaisolation were incubated at 30°C in the dark.The mycelia emerging from the tissues werepicked up and transferred to fresh PDA.Cultures were kept on PDA slants and in bothsterile distilled water at 4°C and 20%glycerol at -20°C for long-term preservation.Five isolates were used in this study(Table 1).

2.2 IdentificationSporocarps were identified based on

both morphological characters [1, 2, 13, 14]and a molecular analysis of the internaltranscribed spacer (ITS) regions of nuclearrDNA. For microscopic study, sections ofdried sporocarps, including lamellae, cutis,and pileal context were mounted in 3% KOHor Melzer’s reagent. Size data of anatomical

features are based on at least 30 measurementsof each structure. The specimens were driedat 40-45°C and deposited at the ResearchLaboratory for Excellence in SustainableDevelopment of Biological Resources,Faculty of Science, Chiang Mai University,Thailand. In addition, genomic DNA ofeach sporocarp was extracted through aCTAB method [15]. Fresh sporocarps and

Table 1. Origin, date of collection and GenBank accession number of Termitomycesisolates in this study.

Isolate Origin Date of collection GenBank no.CMUTM001 Sansai, Chiang Mai 12 Sep 2009 JX866771CMUTM002 Chondan, Petchaboon 17 Aug 2011 JX866772CMUTM003 Bhusang, Payao 1 Sep 2010 JX866773CMUTM004 Muang, Chiang Mai 31 Aug 2010 JX866774CMUTM005 Sansai, Chiang Mai 8 Sep 2011 JX866775

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mycelia (1 g) were frozen in liquid nitrogenand grounded into a fine powder with a mortarand pestle. The powder was placed in a1.5 ml Eppendorf tube and 500 μl extractionbuffer (2% cetyltrimethylammoniumbromide, 100 mM Tris-HCl, 1.2 M NaCl,20 mM ethylenediaminetetraacetic acid,pH 8.0) added and mixed with the material.Samples were incubated at 65°C for 60 minin a water bath and occasionally mixed.An equal volume (500 μl) of chloroform:isoamyl alcohol (24:1) was added to thesamples and briefly mixed by vortexing.After centrifugation for 15 min at 13,000 rpm,the upper aqueous layer was transferred to anew sterile Eppendorf tube. The extractionwas repeated until the interface was clear.The supernatant was removed to a newEppendorf tube, containing 2 volumesof cold 100% ethanol. The DNA wasprecipitated at -20°C overnight andcentrifuged for 15 min at 14,000 rpm and4°C. Then the pellet was washed with70% ethanol and dried at room temperature.It was resuspended in 100 ml of 0.002%RNase (5 mg/ml) in TE buffer and incubatedfor one hour at 37C. The suspension wasstored at -20°C pending use for amplification.

2.3 Fungal ITS and LSU RegionsSequencing and Phylogenetic Analysis

The internal transcribed spacer (ITS)regions of nuclear rDNA were amplified bypolymerase chain reaction (PCR) with primersITS4 and ITS5 under the following thermalconditions: 94°C for 2 min, 35 revolutioncycles of 95°C for 30 s, 50°C for 30 s, 72°Cfor 1 min, and 72°C for 10 min. The PCRproducts were checked on 1% agarose gelsstained with ethidium bromide under UVlight. PCR products were purified by usingPCR clean up Gel extraction NucleoSpin®

Extract II purification Kit (Macherey-Nagel,

Germany Catalog no. 740 609.50)following the manufacturer’s protocol. Thepurified PCR products were directlysequenced. Sequencing reactions werepreformed and the sequences wereautomatically determined in the geneticanalyzer (1ST Base, Malaysia) using PCRprimers mentioned above. Sequences wereused to query GenBank via BLAST (http://blast.ddbj.nig.ac.jp/top-e.html) and amultiple sequence alignment was carriedout using the subroutines in Clustal X [16].A phylogenetic tree was constructed usingthe PUAP beta 10 software version 4.0 [17].

2.4 Fungal CultivationTwenty-five milliliters of each tested

culture media were poured into Petri dishes(9.0 cm, in diameter) after autoclaving at 121°Cfor 15 min. A sterile cellophane disc wasplaced on the surface of the media. Mycelialinocula (5 mm diameter), prepared fromcultures grown on PDA at 30°C in darknessfor three weeks, were then transferred to thevarious tested media. The inoculated plateswere sealed with Parafilm M. After onemonth of incubation, in darkness at 30°Ccolony diameters on each media weremeasured. The covered cellophane discswere removed, dried at 60°C for 48 h andmaintained in desiccators for 20 min beforeweighing. Mycelium dry weights werecalculated. Three replications were made foreach treatment.

2.5 Effect of Culture Media on MycelialGrowth

Ten culture media were used in thisexperiment: fungal-host agar [18], maltextract agar, modified Hagem agar [19],Murashige and Skoog medium [20], oat mealagar, PDA, Sabouraud dextrose agar, soilextract agar [21], yeast extract agar and

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yeast extract-malt extract agar. The finalpH value of tested media were adjusted to5.6 using 1 N HCl and 1 N NaOH.

2.6 Effect of Carbon and Nitrogen Sourceson Mycelium Growth

A basal medium contained yeast extract(0.2 g), CaCl2.H2O (0.005 g), FeSO4.7H2O(0.01 g), KH2PO4 (0.05 g), MgSO4.7HO(0.05 g), (NH4)H2PO4 (1.5 g), agar (18 g),distilled water 1000 ml and adjusted to pH 5.Carbon sources used were glucose fructose,maltose, manitol, starch, sucrose and xylose.Bovine serum albumin (fraction V), eggalbumin, peptone, soybean meal, urea, andKNO3 were used to replace (NH4)H2PO4.The inoculated plates with three week-oldinocula were incubated at 30°C in the darkfor one month.

2.7 Effect of Solid Substrates on MyceliumGrowth

Twelve solid substrates; brown rice grain(Oryza sativa), corn grain (Zea mays), job’s teargrain (Coix lacryma-job), barley grian (Hordeumvulgare), black kidney bean (Phasecolus vulgaris),mung bean (Phaseolus aureus), rye grain (Secalecereale), sorghum grain (Sorghum bicolor),soybean (Glycine max), wheat grain (Triticumaestivum), rice bran and sawdust were usedin this experiment. Each grain was preparedby boiling for 10-15 min. The substrateswere placed in 18 × 180 mm test tubes toapproximately 10 cm depth and autoclavedat 121°C for 30 min. After cooling, eachtube was inoculated with a mycelial plug

cut from the periphery of a three week-old growing colony on malt extract agarand incubated at 25°C for two months indarkness, with three replicates of eachexperiment. Linear growth of the myceliumwas measured every five days and growthrate was estimated [15].

2.8 Statistical AnalysisAll data were analyzed by one-way

analysis of variance (ANOVA) in SPSSprogram version 17.0. The experimentfollowed a completely randomized design(CRD) with three replications. Turkey’s testwas used for significant differences betweentreatments (P<0.05).

3. RESULTS AND DISCUSSION3.1 Identification

The morphology of each Termitomyces sp.was recorded. Termitomyces sp. CMUTM001,pileus 2.8-5.9 cm diameter, convex shapewith perforatorium, surface smooth, centerdark brown to reddish-brown, margin even,striate, tuberculate eroded, white to cream.Lamellae free, incurved, 4-6 mm, crowded,white to cream. Stipe 3.84.4 × 6.4-11.9 cm,cylindrical, solid, surface white to cream,fibrous (Figure 1A, 1B). Context 0.5-5.0 mm,white. Spores 5.0-7.5 × 3.5-4.0 μm, broadlyellipsoid, thin walled, with a few content(Figure 1c). Basidia 24.0-26.0 × 5.0-8.0 μm(Figure 1D). Cheilocystidia 16.0-25.0 ×6.0-8.0 μm, pyriform, thin-walled, withfew contents (Figure 1E). Pinkish creamspore print.

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

Termitomyces sp. CMUTM002, pileus1.2-7.5 cm diameter, convex then plan,centre brown at the disk, white to creamtowards margin, glabrous, striate at themargin. Lamellae breadth 1.0-4.5 mm, freeto adnexed, incurved crowded with whiteto cream lamellae. Stipe 10.0-80 × 9.50 μm,cylindrical, sometimes slightly bulbous atground level; solid; surface white, hairyscales and striates. Partial veil sometimesevident, cortinoid on upper stipe (Figure 2A,

2B). Pseudorhiza 3.5-9.0 cm, fusoid above,with a cartilaginous, dark crust. Context1.5-3.0 cm thick, white, inflated. Spores6.0-8.0 × 3.5-5.0 μm, ellipsoid, subhyaline,thin-walled (Figure 2C). Basidia 17.0-24.0 ×6.0-8.0 μm, bearing four sterigma(Figure 2D). Cheilocystidia 14.0-28.0 × 6.0-10.0 μm, pyriform, thin-walled. Peurocystidia16.0-28.0 × 10.0-21.0 μm, obovoid topyriform (Figure 2E). Salmon pink sporeprint.

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.

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Termitomyces sp. CMUTM003, pileus2.8-8.0 cm diameter, at first pointed-conical,later expanding to convex, dark spiniformperforatorium; surface grayish brown toochraceous brown, color paler towardsmargin, dry, silky smooth. Lamellae 2.5-4.0 mm, free, whitish, crowded; edge entire.Stipe 5.0-15.0 × 0.4-1.8 cm, cylindrical orwide below, solid; surface whitish to palebrown longitudinal fribrillose (Figure 3A,3B). Pseudorhiza 5.0-24.0 cm, long Context

1.0-4.0 cm thick at perforatorium, white,inflated. Spores 4.0-8.0 × 3.0-5.0 μm,ellipsoid, hyaline to stramineous, thin-walled(Figure 3C). Basidia 17.0-26.0 × 4.0-8.0 μm,clavate, bearing four sterigma (Figure 3D).Cheilocystidia 17.0-25.0 × 4.5-7.0 μm,clavate to pyriform, hyaline, thin-walledwith little content (Figure 1E). Peurocystidia20.0-30.0×1.0-21.0 μm, similar to cheilocystidia.Pinkish cream spore print.

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

Termitomyces sp. CMUTM004, pileus10-16 cm diameter, convex shape, finallyexpanding, margin usually remainingincurved, center-plane, slight umbo, whiteto cream colour. Lamellae 6-8 mm, free,white to cream, crowded to close. Stipe6.0-14.0 × 9.5-16.0 cm, equal, solid, whiteto cream color, surface dry with fribillose

(Figure 4A). Pseudorhiza 3.0-8.5 mm.Context 6.0-8.0 mm, thickened, white tocream. Spores 2.5-5.5 μm, ellipsoid shape(Figure 4B). Basidia 8.5 × 25.5 μm, clavate,four sterigma (Figure 4C). Cheilocystidia15.70 × 35.20 μm, pyriform (Figure 4D).Cream spore print.

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

Termitomyces sp. CMUTM005, pileus0.5-2.5 cm diameter, covex , expanding,with a papillate umbo; surface white tocream colour, margin silky striate, smooth.Lamellae 0.5-1.0 mm wide, adnexed to free,white then pinkish, crowded. Stipe 3.0-4.0 ×0.3-0.4 cm, slender, cylindrical, solid; surfacewhite, smooth, fibrilloso-striate, with veryshot rooting base and lacking a true

pseudorhiza (Figure 5A). Context 0.5-1.0 cmthick at centre, white, thin-walled. Spores5.0-8.0 × 3.0-5.5 μm, ovoid to ellipsoid,subhyaline, slightly thickened wall (Figure1B). Basidia 18.0-26.0 × 6.0-8.0 μm,clavate, bearing four sterigma (Figure 5C).Cheilocystidia 24.0-62.0 × 13.0-22.0 μm(Figure 5D). Pleurocystidia similar tocheilocystidia but rare. Cream spore print.

Figure 5. Termitomyces sp. CMUTM005. A, Sporocarp; B, Basidiospores; C, Basidiaand D, Cheilocytidia. Bar: A = 1 cm; B = 5 μm; C-D = 10 μm.

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The morphological study indicatedthat all isolates differed. The morphologicalcharacteristics of Termitomyces sp. CMUTM001were similar to T. eurhizus (syn. T. albiceps)but differ in basidia and spore size [2, 13].Termitomyces sp. CMUTM002 was similar toT. cylindricus (syn. T. aurantiacus) but CMU002has a larger stipe than T. cylindricus [2, 13].Termitomyces sp. CMUTM003 was similar toT. clypeatus according to previous studies[1, 2, 13]. The morphological characteristicsof Termitomyces sp. CMUTM004 were similarto T. unkowann and T. robustus but differedfrom both species in spore size [1, 14].Termitomyces sp. CMUTM005 was similar toT. microcarpus but had a different spore print,the later species has a pinkish spore print[1, 2]. However, subsequent molecularanalysis was used to confirm them as thesame species. The sequence of ITS rDNAgene of five isolates were amplified,sequenced and deposited in GenBank withaccession numbers JX866771-JX866775(Table 1). A BLAST similarity searchconfirmed that they belong to the genusTemitomyces (Table 1). The sequences ofTermitomyces sp. CMUTM001, CMUTM004and CMUTM005 had 96, 99 and 93%similarity with Termitomyces sp. AB073501,respectively. Termitomyces sp. CMUTM002showed 91% similarity with Termitomycessp. AB202123. The remaining isolate,CMUTM003 showed 99% similarity with

T. clypeatus AB073501. The maximum-parsimony (MP) analysis of the ITS geneof 29 sequences resulted in 788 characters,of which 316 characters were constant,127 variable characters were parsimonyuninformative, and 345 characters wereparsimony informative. Heuristic searchesresulted in a tree length = 1,094 steps,CI = 0.6984, RI = 0.7597, RC = 0.5305and HI = 0.3015. One of the maximum-parsimony trees is shown in Figure 6.A phylogenetic dendrogram showed thatTermitomyces sp. CMUTM001, CMUTM002,and CMUTM005 were separated fromT. eurhizus, T. cylindricus and T. microcarpus.A strongly supported node separatedTermitomyces sp. CMUTM001, CMUTM004and CMUTM005. Termitomyces sp.CMUTM003 was closely related to T. clypeatusAB073501 with 100% bootstrap support andfrom herein is referred to as T. clypeatus.Termitomyces sp. CMUTM002 was separatedfrom Termitomyces sp. AB202123 with 100%bootstrap support. Both the data frommorphology and molecular characteristicsshowed only one isolate CMUTM003 wasT. clypeatus. This result is similar to previousstudies that Termitomyces taxonomyclassification based on identification keysis not sufficient to identify species andmolecular techniques need to be employed[14, 22, 23].

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

3.2 Effect of Different Culture Media onMycelia Growth

The effect of ten culture media onmycelial growth of five isolates is shown inFigure 7. After one month of incubation,all fungus isolates grew well on malt extractagar, followed by soil extract agar, yeast-maltextract agar and PDA. All isolates producedwhite mycelia on culture media. Significantly,the largest colony diameter and highestbiomass yield of all isolates were observedon malt extract agar. Termitomyces sp.CMUTM002 showed the largest colonydiameter (46.0±1.00 mm) and biomass yield(60.66±6.93 mg/plate) when compared withother isolates. The smallest colony diameterof both Termitomyces sp. CMUTM001 (20.67±0.58 mm) and CMUTM002 (9.00±2.00 mm)were found on oatmeal agar. Whereas,T. clypeatus CMUTM003 and Termitomyces sp.

CMUTM004 showed the smallest colonydiameter on Hagem agar (26.00±1.00 mm)and fungal-host agar (25.67±0.58 mm),respectively. The lowest biomass yield ofTermitomyces sp. CMUTM001 (14.57±3.78 mg/plate), CMUTM002 (8.43± 0.83 mg/plate),CMUTM004 (3.03±0.61 mg/plate) andT. clypeatus CMUTM003 (3.20±0.62 mg/plate)were observed on fungal-host agar. Theresults are similar to several other studiesthat found growth of mushrooms in pureculture was greatly influenced by changes tothe culture media [11, 12, 24]. Generally,Termitomyces spp. grow well on a varietyculture media such as PDA and malt-yeastextract agar [22, 25, 26]. However, thisresearch showed that malt extract agar isthe best media for growth of Termitomycescultures. Recently, Tibuhwa [27] studied theeffect of three culture media on the pure

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culture growth of T. aurantiacus and T.umkowaani and reported that modifiedmalt extract medium (adding some organic

salts) and Ghosh media were better thanHagem Modess medium.

Figure 7. Colony diameter (A) and biomass yield (B) of five Termitomyces isolates on differentculture media. Data means and error bars at each point indicate ± SD. Different lettersabove 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 extractagar, YA = yeast extract agar, MSA = Murashige and Skoog medium, FH = fungal-hostagar, OMA = oat meal agar, HA = modified Hagem agar and SEA = soil extract agar.

3.3 Effect of Different Carbon Sourceson Mycelia Growth

The growth response of five Termitomycesisolates to various carbon sources in basalmedium was tested (Figure 8A, 8B). It wasfound that all isolates could grow on allselected carbon sources. All Termitomycesisolates grew well on glucose, fructose,maltose and sucrose. However, sucrose wasthe best carbon source for Termitomyces sp.CMUTM001 and CMUTM002. The highestcolony diameter (43.33±1.15 mm) and thehighest biomass yield (45.53±21.70 mg/plate)of Termitomyces sp. CMUTM004 were foundon fructose. Termitomyces clypeatus CMUTM003and Termitomyces sp. CMUTM005 grew onfructose and glucose which had significantdifferences. Whereas, the largest colony

diameter (44.67±1.53 mm) and the highestbiomass yield (69.10±15.85 mg/plate) ofT. clypeatus CMUTM003 were obtained onfructose. Termitomyces sp. CMUTM005 showedthe largest colony diameter (36.33±3.06 mm)and the highest biomass yield (33.33±15.08mg/plate) on glucose. This study showedthat Termitomyces was able to use variouscarbon sources. This result is similar to Kaur[25] who reported that T. striatus could uselactose, glucose, maltose, raffinose, sucroseand xylose, and found that the best growthwas obtained from glucose. In addition,this result agreed that different carbonsources elicited different fungal growth.For example, the best growth of Psathyrellaatroumbonata [28] Lentinus subnudus [12]and Pleurotus ostreatus [29] were found on

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glucose, fructose and manitol, respectively.Gbolagade [30] reported that among seventeen

carbon compounds tested, mannose enhancedthe best biomass yield of Lepiota procera.

Figure 8. Growth of five Termitomyces isolates on basal medium with different carbonand 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 eachgraph 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 Sourceson Mycelia Growth

Significant variation in fungal growthamong the two inorganic (KNO3 and(NH4)H2PO4) and five organic nitrogen(bovine serum albumin, egg albumin,peptone, soybean meal and urea) sourceswere observed (Figure 8C, 8D). Peptone wasthe best nitrogen source to promote fungalgrowth in terms of radial growth. In addition,KNO3 and egg albumin supported fairly goodradial growth. The highest biomass yield ofT. clypeatus CMUTM003 (64.73 ± 2.62 mg/plate) and Termitomyces sp. CMUTM004(53.20±1.83 mg/plate) was obtained on

peptone.The highest biomass yield ofTermitomyces sp. CMUTM001 (51.00 ± 2.38mg/plate), CMUTM002 (45.63 ± 1.97 mg/plate) and CMUTM005 (48.60 ± 3.40 mg/plate) were obtained on KNO3. Small colonydiameter and biomass yield of all isolateswas found on urea. The result is similar toprevious studies that report the best in vitrogrowth of mushroom on nitrogen sourcesdepended on fungal species or isolate andnitrogen sources. For example, optimummycelia growth of Pleurotus ostreatus wasachieved when urea was used as a nitrogensource [29]. Gbolagade et al. [12] foundthat yeast extract enhanced the greatest

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mycelial growth of Lentinus ubnudus andpeptone was the best nitrogen source forthe growth of L. procera [30]. This presentstudy showed that all Termitomycesisolates could use varied nitrogen sourcesfor growth and peptone was the bestorganic nitrogen source for fungal growth,which is similar to the findings of Kaur[25] who reported that T. striatus could usevarious nitrogen sources and the highestgrowth was obtained from peptone. Inaddition, inorganic nitrogen sources suchas CH3COONH4, (NH4)H2PO4, KNO3,and NaNO3 supported fairly good growthof T. striatus.

3.5 Effect of Solid Substrates on MyceliaGrowth

The mycelium growth rates ofTermitomyces spp. in test tube culture onvarious substrates were investigated and areshown in Figure 9. The results indicatedthat mycelial growth rate varied fordifferent solid substrates and fungalisolates. After two months of incubation,

barley grain, job’s tear grain and sorghumgrain had the thickest mycelial growth incomparison with other substrates based ona visual assessment. All fungus isolates grewsignificantly faster on job’s tear grain andT. clypeatus CMUTM003 showed thehighest growth rate (1.25±0.05 mm/day)when compared with other isolates in thisstudy. The lowest mycelial growthrate of all strains was obtained on rice bran.This present study shows that job’s tear graincan be used as a suitable substrate for inoculaproduction of Termitomyces spp. This resultwas different from the solid substrates usedfor inocula production of other cultivatedmushrooms. For example, sorghum grain isthe most popular solid substrate for inoculaproduction of Agaricus spp., Coprinus spp.,Pleurotus spp. and Volvariella spp. inThailand [31-33]. Furthermore, wheat grainis used in A. blazei inocula production inBrazil [34] and P. ostreatus inoculaproduction in Ghana uses sorghum andmillet grains [35].

Figure 9. Growth rate of five Termitomyces isolates on different solid substrates. Datameans and error bars at each point indicate ± SD. Different letters above each graphindicate 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 = blackkidney bean, MGB = mung bean, WG = wheat grain, GG = GABA rice grain,SD = sawdust and RB = rice bran.

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4. CONCLUSIONSAmong the ten tested culture media

and twelve solid substrates, malt extract agarand job’s tear grain were the most suitablemedium for mycelial growth and solidsubstrate for inocula production. Differentcarbon and nitrogen sources showeddifferent effects on fungal growth. Allisolates grew well on glucose, fructose,maltose and sucrose, and peptone enhancedthe greatest radial growth. Future studieswill attempt the selection of suitabletechniques for large scale Termitomycesinocula production and inoculation programs,which are important for the management ofteak plantations in Thailand.

ACKNOWLEDGEMENTThis project received support from the

Thai Orchid Lab Co., Ltd., TRF-Masterresearch grants (WRG WI525S062), theGraduate School of Chiang Mai Universityand Chiang Mai University. We are gratefulto Keegan H. Kennedy for improving theEnglish.

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