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Effect of different nitrogen and carbon sourcesand concentrations on the mycelial growth ofectomycorrhizal fungi under in-vitro conditionsZahoor Ahmad Itooa & Zafar Ahmad Reshiaa Department of Botany, Biological Invasions Research Lab, University of Kashmir,Hazratbal, Srinagar 190006, IndiaAccepted author version posted online: 29 Sep 2014.Published online: 08 Oct 2014.

To cite this article: Zahoor Ahmad Itoo & Zafar Ahmad Reshi (2014): Effect of different nitrogen and carbon sources andconcentrations on the mycelial growth of ectomycorrhizal fungi under in-vitro conditions, Scandinavian Journal of ForestResearch, DOI: 10.1080/02827581.2014.964756

To link to this article: http://dx.doi.org/10.1080/02827581.2014.964756

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RESEARCH ARTICLE

Effect of different nitrogen and carbon sources and concentrations on the mycelial growthof ectomycorrhizal fungi under in-vitro conditions

Zahoor Ahmad Itoo* and Zafar Ahmad Reshi

Department of Botany, Biological Invasions Research Lab, University of Kashmir, Hazratbal, Srinagar 190006, India

(Received 23 October 2013; accepted 8 September 2014)

Effect of different nitrogen and carbon sources and their concentrations in liquid media on the mycelial growth of sixdifferent ectomycorrhizal (ECM) fungal species was studied. Differences were found in the utilization of the differentnitrogen and carbon sources between the fungal species. All the species showed better mycelial growth in the mediumcontaining ammonium as nitrogen source. Growth was low in all species in medium in which nitrogen was supplied innitrate form. All the ECM isolates investigated showed reduced growth in the medium containing maleic acid as thecarbon source. The effect of glucose and di-ammonium hydrogen orthophosphate concentrations on mycelia growth ofall the six fungal species was studied with ranges for glucose of 2–40 g/l, and for di-ammonium hydrogenorthophosphate of 2–20 g/l. Cortinarius fulvoconicus and Cortinarius flexipes showed maximal mycelial growth at aglucose concentration greater than 20 g/l. Suillus luteus, Scleroderma citrinum, Laccaria laccata, and Tricholomaaurantium showed maximal growth at a glucose concentration of 20 g/l. All six species showed maximal mycelialgrowth at 5–10 g/l of di-ammonium hydrogen orthophosphate concentration and with increased concentration mycelialgrowth in all species decreased.

Keywords: ectomycorrhizal fungi; nitrogen source; carbon source; growth; glucose; maleic acid

Introduction

Ectomycorrhizas are symbiotic associations formedbetween fine roots of forest trees with certain soil fungi.Ectomycorrhizal (ECM) fungi are particularly essentialto the health and growth of forest trees and benefit themin a number of ways. The most important beneficialeffects of ECM fungi include enhancing soil nutrientuptake, particularly for elements with a low mobility inthe soil, such as phosphorus (P), nitrogen (N) and severalmicronutrients, release of nutrients from mineral parti-cles or rock surfaces via weathering, effects on carboncycling, interactions with mycoheterotrophic plants,mediation of plant responses to stress factors such asdrought, soil acidification, toxic metals, and plantpathogens, rehabilitation and regeneration of degradedforest ecosystems, as well as a range of possible interac-tions with groups of other soil microorganisms (Smith &Read 2008; Kemppainen & Pardo 2010; Itoo & Reshi2013). The host supplies carbohydrates to the fungusthrough the root fungus interface, making the relation-ship a mutualistic association. Since ECM fungi areknown to promote the growth of their host symbionts, itbecomes important to develop techniques to culture suchbeneficial fungi for their use in forestry.

In nature, various ecological and physiologicalfactors affect the growth of ECM fungi and their

mutualistic association (Bowen 1994). Physical charac-teristics and nutrient status of soils, especially nitrogenand carbon sources, greatly influence the establishmentand growth of ECM fungi in the field and also undercontrolled conditions in the laboratory (Lilleskov et al.2002). Various workers have examined the effect ofnitrogen (Finlay et al. 1992; Chalot & Brun 1998; Dickieet al. 1998; Sarjala 1999; Rangel-Castro et al. 2002;Daza et al. 2006), carbon (Hatakeyama & Ohmasa 2004;Daza et al. 2006), phosphorus (Sawyer et al. 2003), tem-perature, pH (Sánchez et al. 2001; Daza et al. 2006), andwater stress (Machado & Bragança 1994; Sánchez et al.2001) on the growth of ECM fungi.

ECM fungi vary in their ability to utilize variousnitrogen sources, both organic and inorganic, under fieldand laboratory conditions (Finlay et al. 1992; Hata-keyama & Ohmasa 2004; Daza et al. 2006). In nature,ECM fungi are most prevalent in ecosystems whereorganic sources of nitrogen predominate (Nygren et al.2008) and they are known to absorb organic forms ofnitrogen (Näsholm et al. 1998). ECM fungi are alsoincreasingly exposed to inorganic (NHþ

4 or NO�3) forms

of nitrogen due to anthropogenic sources and ammo-nium-nitrogen is usually the major ionic chemicalspecies available to the ECM fungi (Keeney 1980)and most of the ECM fungi are known to metabolize

*Corresponding author. Email: zahooritoo151@gmail.com

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NHþ4 because enzymes involved in its assimilation are

present (Cairney 1999). Various pure culture studiescarried out by different workers (Finlay et al. 1992;Sarjala 1999; Rangel-Castro et al. 2002; Hatakeyama &Ohmasa 2004) have also shown that ammonium-nitrogen is the preferred form for growth of manyECM fungi. A majority of the higher BasidiomyceteECM fungi are unable to utilize nitrate-nitrogen (Nygrenet al. 2008; Plett & Martin 2011), an attribute generallyconsistent with observations on growth of ECM fungi.

In respect of carbon, ECM fungi are believed toobtain much of the carbon (C) necessary for growth fromthe host trees. Carbon flows from host trees to ECMfungi through finely branched Hartig net hyphae whichserve as an interface between plant and fungus wherecells are adapted to the exchange of plant-derivedcarbohydrates for fungus-derived nutrients. Carbohy-drates that are taken up by hyphae of the Hartig net aredesignated for transport toward the soil-growingmycelium (Jordy et al. 1998). Sucrose, which is themain source of carbon for ECM fungi, is excreted byplant root cells into the common apoplast of the plant–fungus interface and is hydrolyzed by a plant-derivedinvertase in ECM symbiosis (Nehls 2004). The lack ofan invertase in many ECM fungi (Hatakeyama &Ohmasa 2004; Daza et al. 2006) is a profound differencefrom phytopathogenic (Walters et al. 1996; Voegele et al.2001) and also ericoid mycorrhizal fungi (Hughes &Mitchell 1996). The low cell wall-degrading activitycompared with wood- and litter-degrading and alsoericoid mycorrhizal fungi, in combination with the lackof invertase activity makes ECM fungi dependent on theactivity of the plant partner (Smith & Read 2008), whichmight be essential for the function of this type ofsymbiosis. These ECM fungi are also known for theirstrong relation with forest plants.

Sucrose (the main carbon content of phloem sap) ishydrolyzed to glucose and fructose by plant-derived cellwall bound acid invertases at the root–fungus interface(Nehls & Hampp 2000). The monosaccharides thusproduced at the root–fungus interface are then taken upby both the fungal partner and root cortical cells (Nehls &Hampp 2000). Fungal hexose transporters allow thetransfer of glucose and fructose to the fungus and enableits growth (Nehls et al. 2001a; 2001b; Nehls & Hampp2000). The Hartig net hyphae strongly increase thehexose uptake capacity in ECM fungi. The expressionand activity of proteins involved in trehalose biosyn-thesis that are mainly localized in hyphae of the Hartignet are also increased, indicating an important functionof trehalose in generation of a strong carbon sink byfungal hyphae (Nehls 2008). The Hartig net hyphae atthe root fungus interface also generate a strong gradientas fungal cells continuously convert glucose and fruct-ose into compounds that are not used by the plant hostsuch as mannitol, arabitol, glycerol, and non-reducing

saccharides such as trehalose; thus, sucrose flowscontinuously from host cells to Hartig net hyphae atthe root–fungus interface (Hampp & Schaeffer 1999).These sugars produced are the main soluble carbohy-drates found in ECM fungi (Martin et al. 1998). Thus,sucrose concentration in the apoplast of tree roots is veryhigh (Hayashi & Chino 1986) and shows fluctuationsduring seasonal changes or in response to a change inenvironmental conditions. ECM fungi may be able togrow in relatively high concentrations of sugars whenapoplast sugar concentration is higher.

Mycorrhizal fungi can also obtain C directly fromsoils by decomposing soil organic matter. Simple organiccompounds, such as amino acids, could also represent asignificant C source for mycorrhizal fungi as ECM fungican also assimilate intact amino acids while in associ-ation with a host plant (Taylor et al. 2004). Amino acidtransporters that allow uptake of amino acids from soilsolution have also been identified in several species ofECM fungi (Nehls et al. 1999; Chalot et al. 2002). ECMfungi decompose soil organic C as an alternate C sourcewhen supplies of photosynthates from the host plant arelow or unavailable. Some ECM fungi produce highextracellular enzyme activity during the winter monthswhen photosynthetic rates decline indicating that fungisupply carbon to host plants during these months (Buéeet al. 2007; Mosca et al. 2007). Fungi may also facilitatethe formation of new tissues during spring by supplyingcarbon to their host plants (Courty et al. 2007).

Because the utilization of carbohydrates by fungi hasbeen reported to be dependent on the amount of nitrogensource present (Eaton & Ayres 2002), to elucidate thenutritional characteristics of ECM fungi, growth tests inhigh concentrations of carbon sources in the presence ofvarying amounts of nitrogen are necessary. In the presentstudy, the mycelial growth of six species of fungi wasstudied with a range of nitrogen and carbon sources anddifferent concentrations of glucose (2–40 g/l) andammonium (2–20 g/l) to characterize the nutritionalrequirements of these fungi.

Materials and methods

Fungal species used and isolation of pure cultures

In the present study, six ECM species viz., Sclerodermacitrinum, Suillus luteus, Laccaria laccata, Cortinariusfulvoconicus, Cortinarius flexipes, and Tricholoma aur-antium were used to study the effect of different nitrogenand carbon sources at various concentrations on theirmycelial growth in pure cultures. These species wereassociated with Cedrus deodara and Pinus wallichianain the coniferous forests of Kashmir Himalaya, India.Morphological and molecular characters were used forcorrect identification of these species. The speciesidentification at the molecular level involved sequencingof internal transcribed spacer (ITS) region of the nuclear

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ribosomal genes (rDNA). For this, genomic DNA wasisolated from sporocarps of collected species by Cetyltrimethylammonium bromide (CTAB) method (Doyle &Doyle 1990). The ITS region of rDNA was amplified bypolymerase chain reaction (PCR) with ITS1 and ITS4primers (White et al. 1990). The 50-μl reaction mixturefor PCR amplification contained 300 ng template DNA,1 × PCR buffer, 1 mM DNTps, 3 pmol of each primer,and 1 unit/50 ul of Taq polymerase. Amplifications wereperformed in a thermal cycler with an initial denaturationstep of 94°C for 5 min followed by 30 cycles of 94°C for1 min, 54°C for 1 min, and 72°C for 1 min, and a finalextension of 72°C for 8 min. The purified PCR productswere then sent for sequencing to Scigenome from Kochi,India. A Basic Local Alignment Search Tool (BLAST)search was performed to find the possible sister groups ofthe sequenced taxa.

Pure cultures used in the present study (Table 1)were isolated from the sporocarps of all these speciesand purity of isolated cultures was confirmed by com-paring Restriction fragment length polymorphism(RFLP) patterns of amplified ITS regions of culturewith that of fruit body. Two restriction enzymes, namelyAlu1 and BamH1 were used for RFLP comparison.Modified Melin Norkrans (MMN) agar medium wasused as the basic medium for isolation of pure cultures.Stock cultures of all species were kept on MMN slants at4°C and are maintained in University of Kashmir.

Utilization of the nitrogen and carbon sources

The utilization of nitrogen and carbon sources by ECMfungi was tested in broth MMN medium. For this, 2 mlmycelial suspension was inoculated into 75 ml liquidmedium in a 100 ml Erlenmeyer flask. The broth MMNmedium was supplemented with different carbon andnitrogen sources. The carbon and nitrogen sources wereadded individually to the medium in such a manner thatC/N ratio remained constant.

To study carbon source utilization, glucose in the basicculture medium was replaced separately with sucrose,trehalose, or maleic acid. The concentration of eachcarbon source was adjusted so that the amount of carbonin each medium was equal to the amount of carbon in the

basic medium. The pH of the medium was adjusted to 5.5with 1N HCl.

To study nitrogen source utilization, di-ammoniumhydrogen orthophosphate, the basic culture mediumwas replaced with potassium nitrate, L-asparagine, andL-alanine. The concentration of each nitrogen source wasadjusted so that the amount of nitrogen in each mediumwas equal to the amount of nitrogen in the basic medium.The pH of the media was maintained at 5.5 with1N HCl.

Mycelial growth was evaluated by dry weight deter-mination. The cultures were grown for a maximum of40 days. There were 12 replicate flasks of each combina-tion of species and nitrogen or carbon source. The flaskswere incubated at constant temperature of 25°C. Con-tents of three replicate flasks of each treatment combina-tion were harvested after 10, 20, 30, and 40 days ofincubation. The contents were filtered through What-man’s filter paper and the fungal mat was carefullytransferred to pre-weighed pieces of aluminum foil.These were oven dried at 60°C and the fungal dryweights were recorded. The average mycelial growthwas calculated from the value of three replicate flasks foreach medium and analyzed statistically using analysis ofvariance with P = 0.05.

Effect of glucose and di-ammonium hydrogenorthophosphate concentrations on the mycelialgrowth of fungi

Each species was cultured in a media of varying glucoseconcentrations [2 g/l, 5 g/l, 10 g/l (basic medium), 20 g/l,and 40 g/l] and varying di-ammonium hydrogen ortho-phosphate concentrations [2 g/l, 5 g/l (basic medium),10 g/l, and 20 g/l]. The pH of the media was maintainedat 5.5 with 1N HCl. The cultures were grown for amaximum of 20 days. There were four replicate flasks ofeach glucose or di-ammonium hydrogen orthophosphateconcentration. The flasks were incubated at constanttemperature of 25°C. The replicate flasks were harvestedafter 20 days of incubation. The contents were filteredthrough Whatman’s filter paper and the fungal mat wascarefully transferred to pre-weighed pieces of aluminumfoil. These were oven dried at 60°C and the fungal dryweights were recorded.

Table 1. Details of ECM fungal species cultures used in this study.

Fungal species Culture name Date of collection Location

S. citrinum KMR-H 005 15th September 2010 Kashmir, IndiaS. luteus KMR-H 009 5th August 2010 Kashmir, IndiaL. laccata KMR-H 012 25th September 2010 Kashmir, IndiaC. fulvoconicus KMR-H 014 3rd October 2010 Kashmir, IndiaC. flexipes KMR-H 018 15th September 2010 Kashmir, IndiaT. aurantium KMR-H 019 18th September 2010 Kashmir, India

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Results

Species identification

Morphological and molecular analysis of the collectedsporocarps revealed that these belonged to six differentECM species. These are S. citrinum, S. luteus, L. laccata,C. fulvoconicus, C. flexipes, and T. aurantium. The PCRproduct amplified with ITS1 and ITS4 primers was 650–700 bp in length in all six species. The BLAST at GenBank was used for determining sequence similarities ofamplified rDNA sequences; those with high similaritiesabove (97%) were considered the same taxon. Thesearch for similar sequences in the Gen Bank using theBLAST program showed that the amplified rDNAsequences showed sequence similarity values with sixdifferent ECM fungal species. The Gen Bank accessionnumber (of species with which our species showedmaximum identity) query coverage and similarity valuesare given in Table 2. RFLP of sporocarps and isolatedpure cultures was used to confirm purity of isolatedcultures and it was found that RFLP pattern of culturesmatched significantly with corresponding sporocarps.Pure cultures isolated were isolated from the sporocarpsof all these species on MMN agar medium.

Utilization of nitrogen sources

The utilization of various nitrogen sources was tested forsix ECM fungal species and the mycelial dry weightyields after 10, 20, 30, and 40 days of incubation areshown in Figure 1. It was observed that all the six ECMspecies grew well in MMN medium containing nitrogenin ammonical form than in nitrate form suggesting thatammonium is the preferable nitrogen source for theseECM fungi as compared with nitrate. For S. luteus andL. laccata, the mycelial growth in medium with ammo-nium was significantly better than that in media withother three nitrogen sources. For T. aurantium, S. citrinum,and C. flexipes, growth in the medium with ammoniumappeared to be best but there was no significant differenceamong different nitrogen sources tested. For almost all

six ECM fungal species, growth in media containinginorganic nitrogen sources was higher than in mediacontaining organic nitrogen sources, suggesting thatECM species mostly utilize inorganic than organiccombinations of nitrogen in culture medium. C. fulvoco-nicus and S. citrinum showed significantly higher mycelialgrowth in MMN medium containing L-asparagine thanL-alanine, whereas S. luteus, L. laccata, T. aurantium, andC. flexipes showed better mycelial growth in L-alaninecontaining MMN medium than L-asparagine.

Utilization of carbon sources

Six species of ECM fungi were tested to investigate theutilization of various carbon sources and the mycelialdry weight yields after 10, 20, 30, and 40 days ofincubation are shown in Figure 2. All the six ECMisolates investigated during the present study showedreduced growth in the MMN medium containing maleicacid as carbon source, suggesting that maleic acid is theleast preferable carbon source for these ECM fungi ascompared with other carbon sources in culture media.The investigated species did not show a uniform responsevis-à-vis other three carbon source and different spe-cies preferred different carbon sources. For instance,S. citrinum and C. fulvoconicus grew equally well onmedium containing glucose or sucrose or trehalose ascarbon source. These two ECM species showed almostsimilar mycelial growth whether the MMN medium wassupplemented with glucose, sucrose, or trehalose, sug-gesting that in these two ECM species either of thecarbon sources can be used for mass cultivation. S. luteusand C. flexipes grew equally well in media containingsucrose or glucose as carbon source and mycelial growthon these two carbon sources was higher than on othertwo carbon sources tested. T. aurantium showed bestmycelial growth in media containing glucose than otherthree carbon sources tested, so glucose can be used ascarbon source for T. aurantium when mass cultivation ofthis species is required for forest inoculation or for otheruses. L. laccata showed better mycelial growth inmedium containing trehalose than other three carbonsources tested, suggesting that for mass cultivation ofthis species trehalose is the best carbon source.

Effect of glucose concentrations on themycelial growth

In media containing glucose concentrations of 2 g/l,mycelial growth for all six species was minimal.Generally, a large difference in mycelial growth in mediawith different concentrations of glucose was observedwith di-ammonium hydrogen orthophosphate concentra-tions of 5 g/l (Figure 3). S. luteus, S. citrinum, L. laccata,and T. aurantium showed maximal mycelial growth at20 g/l of glucose concentration and for all these species

Table 2. National Center for Biotechnology Information(NCBI) accession number (of species with which our speciesshowed maximum identity), maximum identity, query cover-age, and taxonomic information about the species.

NCBIaccession no. Species name

Querycoverage

(%)

Maxidentity(%)

JX679368.1 S. citrinum 96 98KC339728.1 S. luteus 97 100JX504147.1 L. laccata 96 99AY669677.1 C. fulvoconicus 97 97AY669683.1 C. flexipes 96 98JN019587.1 T. aurantium 96 99

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mycelial growth decreased with increasing glucoseconcentration further (Figure 3).

Effect of di-ammonium hydrogen orthophosphateconcentrations on the mycelial growth

The concentration of di-ammonium hydrogen orthopho-sphate also affected mycelial growth of six ECM

investigated during the present study. The mycelialgrowth in all the species was good at concentrations ofdi-ammonium hydrogen orthophosphate greater than2 g/l and at 2 g/l of di-ammonium hydrogen orthopho-sphate concentration all the species showed low levels ofmycelial growth. Generally, a large difference in mycelialgrowth in media with different concentrations of di-ammonium hydrogen orthophosphate was observed with

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Days of incubation

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Laccaria laccata Tricholoma aurantium

Cortinarius flexipes Cortinarius fulvoconicus

Figure 1. Yield (mg) of various ECM fungi in liquid media containing different nitrogen sources (mean ± SEM is based on threereplicates).

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Figure 2. Yield (mg) of various ECM fungi in liquid media containing different carbon sources (mean ± SEM is based on threereplicates).

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glucose concentrations of 10 g/l (Figure 4). S. luteus,L. laccata, and C. fulvoconicus showed maximalmycelial growth at 10 g/l of di-ammonium hydrogenorthophosphate concentration which is twice than basic(5 g/l) of di-ammonium hydrogen orthophosphate con-centration. T. aurantium and C. flexipes showed maximalmycelial growth at basic (5 g/l) of di-ammonium hydro-gen orthophosphate concentration. S. citrinum showedmaximal mycelial growth increased with increasing di-ammonium hydrogen orthophosphate concentration andwas maximum at the highest (20 g/l) concentration of di-ammonium hydrogen orthophosphate.

Discussion

Effect of different nitrogen and carbon sources andconcentrations on the mycelial growth of six differentECM fungi was studied in the present study. The effectof nitrogen and carbon sources on the mycelia growthdepends on species, culture media, and growth condi-tions supported by work of Lin and Yang (2006) who

also reported similar findings. All the six species studiedshowed better mycelial growth (measured as dry mass)when nitrogen was supplied in the ammonical forminstead as nitrate. Ammonium is generally recognized asthe most readily utilizable N source for the most of ECMfungi (Rangel-Castro et al. 2002; Sangtiean & Schmidt2002), and our results from the present study of all sixECM fungi studied also support this assertion. The obser-vations of our results also draw support from similarfindings reported by Abuzinadah and Read (1986),Finlay and Read (1986), Finlay et al. (1992), andRangel-Castro et al. (2002). Our results are in contraryto those of Kibar and Pekşen (2011) who reported lowestmycelial growth for Lactarius pyrogalus and L. con-troversus in (NH4)2 HPO4 media. Growth on nitratecontaining medium was low in all species as most ECMfungi are unable to utilize nitrate-nitrogen though nitratereductase enzyme (nar gene) is present in vast majorityof ECM fungi (Nygren et al. 2008). The presence ofnitrate ion has a negative effect on the development ofsome ECM fungi (Griftin 1994). The growth of the all

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six isolates varied in response to organic nitrogensources; growth, in general, was higher when nitrogenwas supplied in an inorganic combination than in anorganic combination. Similar results have been obtainedby Sarjala (1999) while studying the effect of organicand inorganic nitrogen sources on endogenous poly-amines and growth of ECM fungi in pure culture. Theuptake of organic nitrogen by the trees Pinus sylvestrisand Picea abies having ECM type of mycorrhizationwas studied using glycine as organic nitrogen source.These plants absorb intact glycine from the organic layerof a boreal coniferous forest, thereby bypassing thecommon mineralization pathway (Näsholm et al. 1998).

The ECM isolates investigated during the presentstudy showed reduced growth in the medium containingmaleic acid as carbon source. Maximum growth did notshow a uniform response vis-à-vis carbon source anddifferent species preferred different carbon sources. Forinstance, S. citrinum and T. aurantium grew equally wellon medium containing glucose or sucrose or trehalose as

carbon source. S. luteus grew equally well on mediacontaining sucrose or glucose as carbon source. Hata-keyama and Ohmasa (2004) also obtained similar resultswhile studying mycelial growth of Suillus and Boletinusspecies in media with a range of carbon and nitrogensources. Besides, present observations are also supportedby the work of Daza et al. (2006) who reported thatglucose yielded the highest mycelium dry weights of allthe strains of Amanita caesarea studied and also by workof Guler and Ozkaya (2008) who reported that bettergrowth in ECM fungi occurred on defined mediacontaining glucose, sucrose, maltose, and starch. Akataet al. (2012) also obtained similar results while studyinggrowth of the mycelium of three ECM macrofungi,Infundibulicybe geotropa, Tricholoma anatolicum, andLactarius deliciosus in culture media containing variouscarbon sources.

All species used in the present study grew well ata glucose concentration of 20 g/l. C. fulvoconicus andC. flexipes showed best mycelial growth at 40 g/l of

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Figure 4. Change in mycelial growth of fungal species according to the di-ammonium hydrogen orthophosphate concentration in thebasic MMN medium. (A) S. citrinum, (B) Suillus luteus, (C) L. laccata, (D) C. flexipes, (E). T. aurantium, and (F) C. fulvoconicus.

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glucose (C) concentration. Rossi and Oliveira (2011)reported that Pisolithus microcarpus yielded maximumbiomass production when glucose concentration of MMNmedium was increased up to 40%. These observationssuggest that these fungal species can grow well atrelatively high glucose concentrations. This indicatesthat these fungal species frequently experience sugarconcentration conditions such as 20 g/l in the root systemof plants. As the concentration of nutrients in the apoplastis directly derived from the phloem sap, and sucrosepresent in the sap is efficiently transformed to glucoseand fructose by the plant invertase in the apoplast(Salzer & Hager 1991; Hampp et al. 1995; Nehls &Hampp 2000), the concentration of saccharides availableto mycorrhizal fungi may be within the same order ofmagnitude as the concentration of saccharides in thephloem sap solution. Fisher (1983) reported that theseasonal variation of sieve-tube sap osmolality in fieldspecimens of willow was 400–800 mosmole and thatsucrose accounted for virtually all of the sugars inexudates collected during the winter. The data reportedby Gall et al. (2002) for Norway spruce are alsoconsistent with our results.

All the species studied showed minimal mycelialgrowth at 2 g/l of di-ammonium hydrogen orthopho-sphate concentration with 10 g/l of glucose concentrationand with increasing di-ammonium hydrogen orthopho-sphate concentration keeping glucose concentration con-stant mycelial growth increased in all species up to acertain concentration of nitrogen source which indicatesthat nitrogen concentration was the limiting factor for themycelial growth at this carbon concentration (10 g/l).This shows that C/N ratio is very important for thegrowth of these ECM fungi. Wallander et al. (2003) andFransson et al. (2007) also considered that the C/N ratioof a medium is an important factor for the growth offungi and for regulation of fungal metabolites (Gharieb2000). For Suillus grevillei, Murata (1993) reported C/Nratios as 40 for one strain and below 80 for two otherstrains. Liangqing and Zhida (1998) reported a value of20 for a strain of S. luteus. Similarly, Rossi and Oliveira(2011) studied that P. microcarpus yielded maximumbiomass when C/N ratio of MMN medium was kept20/1. Figure 3(B)–(F) shows that for the S. luteus,L. laccata, C. flexipes, C. fulvoconicus, and T. aurantiumspecies, the mycelial growth increases with increasingdi-ammonium hydrogen orthophosphate concentrationup to a certain fixed concentration and decreases as theconcentration continues to increase. For S. citrinum,mycelial growth continues to increase with increasing di-ammonium hydrogen orthophosphate concentration andshows maximum mycelial growth at highest (20 g/l) ofdi-ammonium hydrogen orthophosphate concentrationindicating that these species have different requirementsof nitrogen for their growth.

The present study revealed that ECM fungi vary intheir ability to utilize various carbon and nitrogensources. Glucose and ammonium were found to be pre-ferred to carbon and nitrogen sources for all thesespecies studied. Growth of ECM species was also foundto be effected by concentration of carbon and nitrogensources. All the species showed maximal growth atdifferent concentrations of nitrogen keeping carbonconcentration constant and at different concentrationsof carbon keeping nitrogen concentration constant.

Conclusion

A detailed literature survey and the results obtained inthe present experimental study showed that the effect ofdifferent carbon and nitrogen sources as well as con-centrations on mycelium growth depends on the ECMfungal strain. Comparison of the growth of fungal strainsin media showed the presence of significant differencesand irregular growth patterns between them. Forexample, while the best carbon source was glucose forbiomass production in T. aurantium on the MMNmedium, trehalose was the best carbon source forbiomass production of L. laccata at the same conditions.The best nitrogen source for biomass production wasammonium for almost all species tested than nitrate butsignificant differences were reported. For C. fulvoconi-cus and S. citrinum, the best organic nitrogen source wasL-asparagine while for S. luteus, L. laccata, T. auran-tium, and C. flexipes, the best organic nitrogen sourcewas L-alanine at the same conditions. The future ofmetabolic engineering and physiological researches ofECM fungi is brilliant, but apparently researchers work-ing on ECM mushrooms will need more basic informa-tion on their physiological requirements.

FundingThe authors acknowledge the Department of Biotechnology,Government of India for financial support [grant number BT/PRO8072/AGR/21/215/2006].

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