Preliminary Investigations of the Content And

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PRELIMINARY INVESTIGATIONS OF THE CONTENT AND LOCALIZATION OF CHITIN IN SELECTED SPECIES OF PILEUS FUNGI Janina Fiema, Janusz Kalbarczyk1, Bernarda Piskorz–Bińczycka2 The F. Górski Institute of Plant Physiology, Polish Academy of Sciences, ul. Niezapominajek 21, 30-239 Kraków, Poland 1 Fruits and Vegetables Processing Department, Agriculture Academy, ul. Skromna 8, 20-704 Lublin, Poland 2 Faculty of Biology and Agriculture, University of Rzeszów, ul. Ćwiklińskiej, Rzeszów, Poland

1. Introduction From literature data concerning the level of the content of radioactive cesium (137Cs) and potassium (40K) in the fungi it follows that Xerocomus badius was one of the more polluted species of fungi in Poland (data from the year 1998) [1]. It will be interesting to explain the existing differentiation as well as the protective mechanism among the various fungi against the environmental pollution.

From the authors’ own investigations [4, 5] as well as from other literature data it follows that looking for an answer should be concentrated on the investigation of the structure of the cell walls, which form a barrier between the inside of the cell and the outer environment especially on such parts of the component walls – certain polysaccharides such as chitin and chitosan,

From the recent reports it is already known that: 1) chitosan may be the sorbent of noble metals [2]. 2) absorption of toxic substances by the mycorrhize fungi is related with the composition of their cell walls [3]. 3) increased content of chitin in the cell walls of Aspergillus giganteus mut. alba in cultures grown in light [4, 5] protects the fungi against the detrimental influence of metals [6]. 4) Chitosan addition to the nutrient reduces the negative effect of nickel on the growth of Aspergillus giganteus mut. alba and simultaneously increases its dry weight by 16% (7). Thus the question arises whether it is possible via analogy to refer the above data also to the radioactive elements? These considerations inspired the author to study the problem concerning the content of chitin and its localization in the fruit bodies of some species of the pileus fungi.

Polish Chitin Society, Monograph XII, 2007 235 J. Fiema, J. Kalbarczyk, B. Piskorz-Binczycka Successive aspects of the dependence of chitin synthesis on light ...

2. Material and methods 2.1. Origin of fungi species 1) Fungi collected from natural stands in Malopolska Region; n king - bolete (Boletus edulis) n bay - bolete (Xerocomus badius) n birch - bolete (Leccinum scabrum) n saffrom – milk - cap (Lactarius deliciosus) n sticky bun (Suillus luteus) 2) Boletus edulis, Boletus edulis ssp. reticulatus, Xerocomus badius, species collected in Wielkopolska Region. 3) Ganoderma lucidum from Asia, at present is maintain in laboratory.

The method of growing Ganoderma lucidum was elaborated at the Department of Fruits and Vegetables Processing of the Agriculture Academy in Lublin, from where the fruits bodies were obtained. It is shown on Plate 1. No 2

The pileus is flat, spherical, up to 30 cm wide, shining, of red – brown color, with concentrated ridges, inedible. In laboratories, under controlled conditions this species is grown in containers on an artificially prepared substance [8].

2.2. Chitin determination Chitin was determined on the basis of the amount of glucosamine after a hydrolysis (carried out earlier) of the fungus material intended for the analysis [4].

Glucosamine was determined spectrophotometrically at the wave length 530 nm, according to Elson – Morgan method [9].

Plate 1. 1 – Boletus edulis, Plate 2. 1a, 1b – Xerocomus badius, 2a, 2b – Lactarius deliciosus, 2 – Ganoderma lucidum. 3a, 3b, 3c – Suillus luteus.

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Chitin was determined in the whole body of fungus, in the large and in the small body or in the particular parts of it: the skin, the upper and the lower parts of pileus and in the stem.

The obtained data are presented as the mean values derived from several fruit bodies.

3. Results and discussion For each examined species there are presented data concerning the general content of chitin in the fruit body and in the particular parts of it: the skin, the upper and the lower parts of pileus and the stem (Table 1).

Table 1. Chitin contents in the particular parts and in the whole fruit body for the examined species are presented as percent values (in dry weight) in large and small fruit bodies; • - small fruit body, small bracket - chitin content in the whole small pileus. See also Plate 1 and 2.

Examined species Chitin content in percent and Total content of chitin (particular parts and mean values in in large and small total fungus) large bodies small bodies bodies, % Lactarius deliciosus 1. skin 1.32 ● - 2. upper part of pileus 2.91 ● 2.10 2.23 3. bottom part of pileus 2.32 2.13 ● 2.16 4. stem 2.08 ● 2.30 5. total of fungus 2.42 ● 2.10

Boletus edulis 1. skin 3.71 ● – 2. upper part of pileus 4.67 ● 4.40 4.31 3. bottom part of pileus 3.20 4.03 ● 4.44 4. stem 4.56 ● 4.83 5. total of fungus 4.47 ● 4.10

Xerocomus badius 1. skin 2.02 ● – 2. upper part of pileus 3.45 ● 3.10 3.10 3. bottom part of pileus 2.80 3.09 ● 2.16 ● 3.10 4. stem 4.10 ● 4.04 5. total of fungus 3.12 ● -

Suillus luteus 1. skin 2.80 ● 3.16 2. upper part of pileus 6.12 ● 2.50 3.92 3. bottom part of pileus 4.64 4.50 ● - ● 2.72 4. stem 4.44 ● - 5. total of fungus 4.55 ● 2.50

Leccinum scabrum 1. skin 4.24 2. upper part of pileus 4.80 4.95 3. bottom part of pileus 4.95 4. stem 5.80 5. total of fungus -

Ganoderma lucidum 1. skin - 2. upper part of pileus 3.56 3.30 3. bottom part of pileus 3.48 3.29 4. stem 2.84 5. total of fungus 3.30

The results obtained from the determination of chitin in the whole fruit body were in agree- ment with the chitin determination from the particular parts of the fruit body, which is a confirmation of the accuracy of determinations. In the global amount of chitin there are contained all the particular data both for the large and the small fruit bodies.

Chitin content in the large and the small fruit bodies does not substantially differ (except Suillus luteus). The total content of chitin in fungi collected in Wielkopolska Region essentially does not differ from the amount in the remaining fungi: for Boletus edulis = 3.24% for Boletus edulis ssp. reticulatus = 3.7%, Xerocomus badius = 3.1%.

When analyzing the results given in the particular sections (Table 1) one can notice the differences between the species not only in the overall content of chitin in the dry mass of fungi, buy also in the particular component parts. Especially distinct is the connection of the total content of chitin – with the chitin content in the skin which increases parallely. Thus it is possible to systematize the species according to the increasing content of chitin as follows (Table 2);

Table 2. Total content of chitin, and chitin in the skin in the examined fungi species.

Content of chitin, % Fungi species total in skin Lactarius deliciosus 2.20 1.32 Xerocomus badius 3.10 2.02 Ganoderma lucidum 3.30 - Suillus luteus 3.61 2.80 Boletus edulis 4.40 3.71 Leccinum scabrum 4.95 4.24

This dependence is reflected in the overall strength and brittleness – observed in practice – in the particular species. (see: chitin content in Lactarius deliciosus – the most brittle and of little strength species in comparison with the considerable strength of Boletus edulis and of the other species, which contain more chitin). Also the level of chitin content in the skin confirms its strength properties, e.g. the brittleness ofLactarius deliciosus demonstrates the poor strength properties of its skin, and the elasticity of the skin in Suillus luteus illustrates its strength.

Determination of chitin in the particular parts of the fruit bodies was intended to demonstrate whether there is a place inside them – especially privileged with respect to chitin content. It has been found that there exist such places: the stem and the upper layer of pileus. Only Lac- tarius deliciosus shows uniform distribution of chitin in the whole fruit body. Generally, the least amount of chitin is found in the skin and it differs in the particular species. Among the examined fungi (apart from Lactarius deliciosus), Xerocomus badius had the least amount of chitin; (maybe that is why it was one of the more polluted species?). This would be a confirmation of the assumption, suggested in the Introduction that chitin may play an important role in the protection mechanism of the fungi against environmental pollution.

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The differences in the level of the fungi pollution by radioactive metals [1] may be also con- nected with the structure of the cell walls and the different content of chitin there.

Some data [3] suggest that some fungi, in order to protect themselves against environmental pollution, produce transverse intervals inside the cell walls, cutting off hyphae containing the toxic layer of metals, in order to protect the remaining part of mycelium. Therefore, in the further procedure there will be taken into consideration the analysis of mycelium with respect to the chitin content – so as to explain the differences in the chitin content in the species of fungi investigated at present.

4. References 1. Skażenia promieniotwórcze środowiska i żywności w Polsce w 1998 r. Raport CLOR nr 139, Centralne Laboratorium Ochrony Radiologicznej, Warszawa, 1999, in: Piotr Jaracz: Promienio- wanie jonizujące w środowisku człowieka copyright by Wydawnictwa Uniwersytetu Warszaw- skiego 2001. 2. Kula K., Jaworska M., Guibal E.; Nowe aspekty w chemii i zastosowaniu chityny i jej pochod- nych, 2002, 11 3. Turnau K.; Ekofizjologiczne aspekty reakcji roślin na działanie abiotycznych czynników streso- wych, 1996 Kraków, 97-101. 4. Fiema J.; Acta Physiol. Plant., 5 (3), 1983, 123. 5. Fiema J., Gołębiewska T.; Acta Biol. Crac. Ser. Bot. 23, 1981, 1. 6. Fiema J., Piskorz – Bińczycka B.; The effect of heavy metals on the mycelium of Aspergillus giganteus mut. alba in various light conditions: presented on the 6th International Conference on: Eko – physiological aspects of plant responses to stress factors, Kraków, 2005. 7. Piskorz – Bińczycka B., Fiema J.; Obieg pierwiastków w przyrodzie, Monografia, Instytut Ochrony Środowiska, red. B. Gworek, t. III, 2005, 524 – 527. 8. Fiema J., Kalbarczyk J.; Żywienie a zdrowie – Interakcje, Materiały Konferencji Naukowej, Kraków, 2005, 45. 9. Rondle C. J., Morgan W. T. J.; Biochem. J., 61, 1955, 586.

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