Effect of Carbon Sources on the Growth of Ectomycorrhizal Fungi

Hemalatha S.¹, Mohan V. ²and Sujatha K.³

¹Department of Biotechnology, Vels University, Chennai-117

²Forest Protection Division, Institute of Forest Genetics and Tree Breeding, Coimbatore

³Department of Zoology, Government Arts College, Coimbatore

ABSTRACT:

The ectomycorrhizal fungi show higher tolerance to high soil temperatures, various soil and root borne pathogens and heavy metal toxicity. Hence an attempt has been made to investigate the growth characteristics of different ectomycorrhizal fungi on different carbon sources. The effect of different carbon sources viz., lactose, maltose, mannitol, and sucrose on the growth of Scleroderma citrinum, Suillus brevipes on day 30 and 45 were carried out. Scleroderma citrinum showed maximum growth on sucrose and lactose whereas Suillus brevipes showed maximum growth in lactose and mannitol in all the day intervals (30^th and 45^th days). On day 30 and 45 change in pH in the culture filtrate of Scleroderma citrinum and Suillus brevipes were also determined. The study revealed about the suitable carbon source for the mass cultivation of the selected ectomycorrhizal fungi.

KEYWORDS: Scleroderma citrinum, Suillus brevipes, biomass, carbon source.

INTRODUCTION:

Carbon is one of the essential elements required by living organisms and it takes part in major functions in the metabolism of fungi. Simple sugars, sugar acids, sugar alcohols, short and long chain polymers obtained from these subunits serve as carbon sources for the growth of fungi¹. Many fungi cannot utilize disaccharides, oligosaccharides or polysaccharides due to their inability to hydrolyze the larger molecules. So they utilize monosaccharides. The colonization of ectomycorrhizal fungi takes place effectively where there exists either a significant free concentration of sugars or where sugars or simple organic compounds are being released². The maximum growth of many ectomycorrhizal fungi were observed on glucose and complex carbohydrates were less useful for them³. Ectomycorrhizal fungi utilized complex carbohydrates⁴. The effect of various carbon compounds viz., glucose, mannitol, maltose, starch and dextrin on the growth of A. muscaria, B. luteus, B. variegatus, Lactarius deliciosis and Russula rubra in pure culture were checked. All fungi utilized maximum amount of glucose and very little amount of maltose ^{5a and b .}

The effects of various nutrient sources on the fungus Mycelium radicis-fagi were studied⁶. Growth was maximum in media containing saccharose, mannose, dextrose, gum and arabinose; intermediate in aesculin, cocoa butter whereas lactose, starch, maltose, asparagine, nucleic acid, malic acid, formic acid and tartaric acid were less favorable. Benzoic acid, urea and tannin completely inhibited the growth.

The growth of ectomycorrhizal fungi B. elegans and B. variegatus on various carbon sources namely glycerol, mannitol, dulcitol and sorbitol (alcohols); alkaline salts like acetic acid and formic acids (fatty acids); alkaline salts of lactic acid, malic, tartaric and citric acids (hydroxy acids); xylose and arabinose(pentoses); glucose(hexose); lactose and maltose (disaccharides); starch, dextrin (polysaccharides); arbutine and salicin (glucosides); tannin and humates of sodium were observed⁷. Among these carbon sources fungi utilized glucose, maltose and mannitol. B. elegans utilized starch and xylose; B. variegatus utilized lactose and dextrin. The responses of B. elegans to various carbon sources were observed⁸and found that it did not utilize cellulose and lignin but increased growth on alcohol washed pectin, however pectinases were not detected. Sucrose, maltose and glucose were equally utilized by Cenococcum graniforme and lactose to some extent⁹. Dextrin, starch and mannitol promoted the growth only in the presence of ‘start sugar’ glucose¹⁰. Glucose, mannose, maltose, trehalose, cellobiose and X-dextrin were effective carbon sources for Cenocccum graniforme. But inulin and cellulose didnot favour the growth¹¹.The utilization of sugars in Mikola’s medium by two isolates of C. graniforme was studied¹². Both isolates utilized mono and di than polysaccharides and they showed higher growth in maltose and starch was less effective. The growth of ectomycorrhizal fungi Astraeus hygrometricus, C. graniforme and Scleroderma bovista in various carbon sources were analysed and concluded that glucose, sucrose and mannitol were better sources but glycerin was an unsatisfactory source of carbon. The effect of 52 carbon sources on the growth of six ectomycorrhizal fungi viz., A. rubenscens , Russula emeticca, C. graniforme, R. roseolus, S.cothurnatus and S. punctipes were examined¹³. Among the carbon substrates, D-glucose, mannose and pectin increased the dry weight except in S. punctipes. Other isolates grew well on fructose, mannitol, sorbitol, cellobiose, maltose, trehalose, dextrin, glycogen, inulin and starch.

The effect of glucose, sucrose, maltose and glycerin on the growth of Astraeus hygrometricus, C. graniforme and Scleroderma bovista in Hagem’s medium were studied¹⁴. Astraeus hygrometricus and C. graniforme utilized glucose followed by maltose and sucrose, whereas S. bovinus utilized sucrose than maltose and glucose. None of the fungi utilized glycerin and he concluded that most of the fungi prefer simple carbohydrates.

The ability of utilizing various carbon source by 21 ectomycorrhizal fungi viz., Amanita muscaria, A. rubensis, Lactaruis popperatus, Leccinum Scabrum, Lepista nuda, Pisolithus tinctorius, R. luteolus, R. roseolus, Suillus aeruginascens, S. bovinus, S. granulatus, S. grevillei, S. luteus, Thelephora luciniata, T.terrestris, Xerocomus subtomentosus and other five unidentified fungi, each with a single isolate were studied¹⁵. Almost all isolates utilized D-glucose, D-mannose, fructose, cellobiose, trehalose, sucrose, dextrin, glycogen, starch and pectin.

Association of certain fungi with ectomycorrhizal fungi has physiological differences like abilities for utilization of different carbon sources. The glucose which is actively taken up by the fungus is immediately converted to trehalose, mannitol and glycogen within the fungus and this maintains the density gradient were reported¹⁶. Therefore, the present work aims the effect of carbon sources on the growth of ectomycorrhizal fungi.

MATERIALS AND METHODS:

General laboratory techniques recommended by ^{17,18 and19} were followed for the preparation of media, inoculation and maintenance of cultures.

STERILIZATION:

Media were sterilized in an autoclave at 15 lbs / sq.inch pressure for 20 minutes. The glasswares were sterilized in hot air oven at 160^oC for three hours.

CHEMICALS:

All the chemicals used were of ‘Analar’ grade obtained from Hi-Media, India and Sigma chemicals, USA.

ISOLATION OF ECTOMYCORRHIZAL FUNGI:

Pure cultures of the ectomycorrhizal fungi were obtained from the spores and tissues obtained from the fresh basidiomata (Plate 1). The basidomata of the fungus collected from the field was surface sterilized with alcohol. The fruit body was cut into two by using sterile blade and the tissue was taken and placed on a sterile Potato Dextrose agar medium (Peeled Potatoes-200 g, Dextrose- 20 g, Agar- 20g, Distilled water- 1000 ml, pH 5.5- 5.7). The fungi were allowed to grow for a week (Plate 2 and Plate 3). The pure culture of the fungal isolate were identified as Scleroderma citrinum and Suillus brevipes and maintained on Potato Dextrose Agar slants for further use. All the ectomycorrhizal cultures were maintained on Potato Dextrose Agar / Modified Melin Norkrans Medium (MMN) slants at room temperature (27±1)⁰C in diffused light. The cultures were subcultured in PDA slants periodically and stored at 4^o C.

Plate 1. Basidiomata of an ectomycorrhizal fungus,

Suillus brevipes

Plate 2. Scleroderma citrinum, an ectomycorrhizal fungus grown in Potato Dextrose Agar medium

Plate 3. Suillus brevipes, an ectomycorrhizal fungus grown in Potato Dextrose Agar medium

Preparation of Inoculum:

The different isolates of the ectomycorrhizal fungi, Scleroderma citrinum and Suillus brevipes were maintained on PDA plates for 10 days. These served as the source of inoculum for the experiment.

Biomass weight method:

Modified Melin Norkans medium (Malt extract-3 g, D-Glucose-10 g, (NH₄)₂HPO₄-0.25 g, KH₂PO₄- 0.5 g, MgSO₄.7H₂O - 0.15 g, CaCl₂- 0.05 g, NaCl- 0.025 g, FeCl₃(1 % solution)- 1.2 ml, Thiamine HCl- 1 mg, Distilled water- 1000 ml, pH- 5.8) containing different carbon sources such as Glucose, Lactose, Maltose, Mannitol, Sucrose were prepared and autoclaved. To the sterilized medium 15–30 days old 5 mm diameter mycelial mat was inoculated and incubated at 27±1^oC. For each treatment six replicate flasks were maintained. The ectomycorrhizal fungi were checked periodically for the growth of the fungus for a period of about 30 days and 45 days.

Determination of fresh weight:

The mycelium was harvested on day 30 and 45 and washed in distilled water. The mycelium was pressed in between three- layered filter paper mat to remove the moisture and the fresh weight was measured.

Determination of dry weight:

On 30 and 45 days incubation, the mycelia duft was transferred to a prewashed, predried, preweighed filter paper and washed repeatedly with distilled water to make it free from any trace of adherent medium and dried in an oven at 80^oC to constant weight for 24 hours. The values obtained were used as an expression of growth index. The growths of the fungal mycelium were weighed and results were noted down. The ideal carbon source for the growth of the ectomycorrhizal fungi could be concluded from this study.

Effect of pH:

The changes in pH of the broth after fungal growth were measured at 30 and 45 days by using pH meter.

RESULTS AND DISCUSSION:

The growth of Scleroderma citrinum, Suillus brevipes in different carbon sources and the pH change in culture filterate on different day intervals viz., 30^th and 45^th day was determined. The mycelial dry weight of Scleroderma citrinum was decreased in control and mannitol in all the day intervals when compared with sucrose, maltose and lactose. On 30^th day, maximum mycelial dry weight was noticed in sucrose (125 mg) followed by maltose (115 mg) and lactose (101 mg) and minimum was found in control (79 mg) and mannitol (81 mg). Maximum dry weight of the mycelium was noticed in sucrose (137 mg) on 45^th day when compared with the other carbon sources (Fig.1).

Fig.1 Effect of different carbon sources on the mycelial dry weight of Scleroderma citrinum

The mycelial dry weight of Suillus brevipes was decreased in control, maltose and sucrose in all the day intervals when compared with lactose and mannitol (Fig.2). On 30^th day, maximum mycelial dry weight was observed in lactose(90 mg) and mannitol (83 mg).Maximum dry weight of the mycelium was noticed in lactose(105 mg) and mannitol(93 mg) on 45^th day and minimum was found in sucrose(46 mg).There was difference in growth rates of the selected ectomycorrhizal fungi on different carbon sources. The ectomycorrhizal fungi, Scleroderma citrinium preferred sucrose and Suillus brevipes preferred lactose for their maximum growth. Hence both utilized disaccharide rather than complex sugars. Carbohydrate movement preferably occurs from host to fungus in ectomycorrhizas²⁰. The specialized behavior of mycorrhizal fungi, their inability to use complex polysaccharides as sources of carbon and energy, their frequent requirements for various growth factors and inhibition by humus extract, identify them as typical rhizosphere fungi, despite the lack of a general study of a possible saprophytic behavior in the absence of the host and the ability of the same to decompose the litter²¹. Ectomycorrhizal fungi could not utilise polysaccharides in pure culture²². The utilization of simple sugars by various ectomycorrhizal fungi was reported ²³. Hence maximum dry weight of the mycelial mat was found due to the utilization of simple sugars.

Another experiment was conducted to investigate the pH changes in culture filterate on different day intervals viz, 30^th day and 45^th day which was maintained at optimum pH(5.8). Drastic change of pH in the culture filtrate of Scleroderma citrinum containing sucrose as a carbon source was noticed on 30^th and 45^th day comparing with the other carbon sources (Fig. 3). On 30^th and 45^th day change in pH was observed in culture filtrate of Suillus brevipes too(Fig. 4). Decreased pH resulted in decreased growth²⁴. In the present study, the pH of MMN broth culture filtrate decreased upto 2. Similar results were obtained²⁵ for Modified Melin Norkrans medium (MMN) and Palmer Hacskaylo Agar medium (PHA). During the growth, the fungi produce some organic acids which result in pH reduction of culture filtrate. This might be due to the oxidation of culture filtrate, which might be the result of oxidation of reducing groups or potential reducing groups and carbonyl groups to carboxyl groups²⁶.

CONCLUSION:

The suitable carbon sources for the maximum growth of ectomycorrhizal fungi were revealed through this study. Therefore the ectomycorrhizal fungi can be mass produced on the particular carbon containing medium and can be used as biofertilizers in forest nurseries for quality seedling production.

ACKNOWLEDGEMENTS:

Authors are grateful to The Director, Institute of forest Genetics and Tree breeding, Coimbatore for permitting us to carry out this work in the Institute.

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Received on 22.11.2010

Accepted on 25.12.2010

Research J. Science and Tech. 3(1): Jan.-Feb. 2011: 44-48