Toksyczne substancje biologiczne aktywne pochodzenia grzybowego i ich wpływ na środowisko glebowe

R O C Z N IK I G L E B O Z N A W C Z E T. X X V , D O D A T E K , W A R S Z A W A 1974

B O L E S Ł A W S M Y K

B IO L O G IC A L L Y A C T IV E T O X IC S U B S T A N C E S OF F U N G A L O R IG IN A N D TH E IR IN F L U E N C E U P O N TH E SO IL H A B IT A T

Institute of Soil Science, A gricu ltu ral Chem istry and M icrobiology, A gricu ltu re U n iversity of Cracow

In world literature w e often meet works in the field of biochemistry of soils and ecological microbiology, informing of the appearance of the pathological phenomenon in the soils agriculturally exploited, decreasing the fertility o f soils 'known in the agricultural practice under the name of soil-sickness [3-9, 11, 12].

The results of F A O data show that soil-sickness causes heavy losses in the vegetable production of the globe (Table

1

The above mentioned fact of soil-sickness which is a result of breaking the biological balance in nature by man is potentielly the largest danger threatening contemporary world agriculture [11, 12, 14].

A t the present time of chemicalisation of agriculture (the wholesale applying of pesticides and so on, of the facilities of plant protection as w ell as controllers of plant growth and many other phytochemithera- peutical preparations) and at the intensification of agriculture (among other things, using large amounts of nitrogen fertilizers, especially at non-observing biological foundations of the succession of plants in ro­ tational structures; the increase of the level of mechanization in agri­ culture and so on) the necessity arises for recognizing the influence of the above mentioned factors upon the ibiology of soils and the biological productivity of agrocenose and other ecosystems, found in the immediate range of the economic activity of man.

T a b l e

1

Losses in vegetable production in agriculture caussd by the soil-sickness of arable lands*

/expressed proportionally in relation to general biological damage/

E U R O P E % Belgium 20 Czechoslovakia 13 Denmark 24 France 22 Holland 26

German Democratic Republic 24 German Federal Republic 52

Poland "*5 mainly in the growing of tobacco,the family Papilionaceae '.vegetable and in cultivation of meadows

Switzerland 20

Sweden 12

A F R I С A

Kenya 50 V _{nainly in the cultivation of cotton, coff-э,} Tanzania 25 К wegctables and other subtropical and

Uganda 24

J

Eevp- 55

N O R T H A M E R I C A ₁

The United States of America 28 i

Canada 21 I

S O U T H A M E R I C A

Argentina 18

Brazil 50

* According FAO data for 1970

Numerous examples of the annihilation of enormous cultivations accomplished by man in such agricultural countries as Africa, Europe, South America (Brazil), North America (The United States of America and Canada) and also Poland-indicate the necessity of closer cognition of these facts threatening the natural soil habitat.

The aim of this work was the recognition of the prevalence of Fungi with toxin-creative capabilities in the soils 'of chosen sinusion and plant communities coming into the groups (association) of agroecosystems and also grassy and forest ecosystems as w ell as in the soil habitat found in the reach of intensive economic activity of man (with special regard to soil-sickness, as, for instance, clover and alfa-alfa soil-sickness, soil- sickness in berseem and Persian clover, tobacco-soil^sickness and others) together determining the influence of the products o f their metabolism (mycotoxins) upon the chosen soil microorganisms and soil habitat.

B iologically active toxic substances... ₉₃

M E T H O D O L O G Y OF W O R K

The research work connected with the subject of the present study was carried out in the Department of Agricultural Microbiology at the University of Agriculture in Cracow (Poland) n ow known as the Institute of Soil Science, Agricultural Chemistry and Microbiology of the U ni­ versity of Agriculture in Cracow, in the years 1960-1972. Some taxonomic designations of fungi, as w ell as analytic examination in the range of toxic substances (mycotoxins) w ere performed at the Microbiological Institute of the Federal Technical University in Zürich and at the Insti­ tute of Applied Microbiology in the Agricultural University in Vienna. The isolation of toxin-producing fungi w ere selected from different soils of chosen plant communities, entering into the composition of nat­ ure and changed by the economic activity of man (agrocenoses), grassy and forest ecosystems situated in a specified area of Southern Poland. This isolation was done by the Koch’s plate cultural method using mo­ dified medium of Czapek [

2

The estimation of the toxicity of isolated fungi in relation to the chosen soil microorganisms was examined with the use to modern mi­ crobiological and biological methods [1, 2, 5,

6

8

nicillia by Raper, Thom and Fennel, 1949; The Genus Aspergillus by

Raper, 1965; The Fungi by W o lf and W olf, 1969; Pilze aus Agrarböden by Domsch and Gams, 1970).

The estimation of the contents of mycotoxins in the cultures of isolated fungi w ere determined with the following methods: spectrophotometric (Nabney and Nesbit, 1965) and the chromatographic method [1, 5, 7,

8

I received from dr A. Campbell of the Food and D rug Institute in Washington (U S A ) standards of aflatoxims and some mycotoxins as ochra- toxins A and B,F-2 (zearalenone), penicillic acid, mycophenolic acid, citrinin and citreoviridin— for which, at this time— I w ant to express my warmest words of acknowledgement.

Other mycotoxins were received from the following chemical firms: Calbioch., Los Angeles, Calif.; Sigma Chemical Co., St. Louis, Mo.; K. & K. Laboratories Plainview, N .Y .— U S A .

A part of the mycotoxins was determined in foreign laboratories (Inst, of Applied Microbiology at the Agricultural University in Vienna).

T H E G E N E R A L R E S U LT S OF TH E STU D IE S

The results from studies carried out in the years 1960-1972, disclose that mycological examinations (ecological and synecological studies) of

chosen, differing soil haibitats from which toxin-producing fungi from the class Fungi imperfecti were separated out, posses a large capacity in the range of mycotoxin synthesis, substances which are biologically active (Tables

2

The metabolites of examined soil fungi are characterized by a direct toxic (influence upon soil microorganisms (active in nitrogen and carbo­ hydrate metabolism in soil) and some cultivated plants. Moreover it appears from the obtained results of examinations and chemical de­ signations that the toxic products of metabolism, examined in vitro Fungi, are represented by the following groups of chemical compounds :

— aliphatic metabolites : epoxides and epoxialcanes (epoxysuccinic acid

T a b l e

2

Ieolation of mycotoxin-producing fungi from agricultural soils in Poland

.Fungus Kycotoxj.n

Occurence in ecosystems: grassy forest agroceno-_ae

Aspergillus chevalieri, Thom and Church

Gliotoxin and other

Epipolythiadioxopipera-zines* + + +

Aspergillus flavus, Link Aflatoxin +

-Aspergillus ochxaceus, Wilh. Ochratoxin - -

-Aspergillus terreus, Thon. Citrinin + + +

Aspergillus versicolor, /Vuill./

Tirab. Sterigcatocys tin -

-Fusarium nivale, /Fr./Сеs. Nivalenol +

-Fusarium graminearum, Schwabe F-2 /zearalenone/** + -

-Fusarium scirpi, Lamb, and Fautr. ßcirpenol + + + Fusarium 3porotrichicddes, Sherb. Sporofusarin - - +

Pénicillium brunneun, Biource Rugulosin + - -f

Pénicillium citreoviride,

Biourge j Citreoviridin

* _

4-Ferdcillium citrinum, Th. j Citrinin + ii +

Pénicillium cyclcpium, Westlin£. | Cyclopiazonic acid and

penicillic acid ... +

Pénicillium islandicum, Sopp« Luteoskyrin + + Pénicillium puborulum, Bein. Penicillic acid and

other carcinogenic

lactones*** ♦

Pénicillium rubrum, Stol. Rubratoxin I + - ■f

Pénicillium rugulosum, T. Rugałosin 1 - *

Pénicillium tardum, Thom Rugulosin - + +

Pénicillium variabile, Sopp. Aflatoxin -

-Alternaria longipes, T.aW. Alternariol + + +

Thielaviopsis basicola, Berk.et

Br. /Ferr./ Thielaviotin - - ♦

After:

* A.Taylor - 1971

** C.J. Mirocha et al. - 1971 *** A.Ciegler et al. - 1971

B iologically active toxic substances... 95

T a b l e

3

The influence of extracts of Thielaviopsis basicola Berk. Br. upon the chosen soil microorganisms

Chlorophormic extract Soil bacteria

of 14- days of 20 days

old old

mycelium culture

Microbiological method Inhibitionzone - mm

1 Arthrobacter globiformis, Conn a.Dimmick 10 8 2 Arthrobacter terregens, Lochhead a. Burton 12 10 Arthrobacter sp.? strain "SB" 10 10

4 Azotobacter chroococcum, Beijerinck 12 10

5 Bacillus subtilis, Cohn 10 9

6 Bacillus cereus, Frankl. 15 11

7 Rhizobium leguminosarum, Frank. 10 8

8 Rhizobium meliloti, Dangeard 10 10

9 Rhizobium trifolii, Dangeard. 12 10

10 Rhizobium lupini, Eckl. 10 8

* Cultures of Bacteria mentioned in the position 1, 2, 5, 6 come from the National Coll. of Industr. Bacteria Aberdeen; whereas cultures mentioned in position 4 r 7, 8, 9, 10 -come from Rothamsted Expt. Agricult. Station, HarDenden /Herts./.

= l-trans-oxidoethylene-a-/?-dicarboxylic acid: tetronic acids); compounds of the furan rings as: sterigmatocystin and 2-hydroxym ethyl-furan-5- -canboxylic acid; penicillic acid; out of the group of compounds of a- -pyrone ring: kojic acid, patulin, citrinin and citreoviridin;

— aromatic metabolites: aflatoxins, ochratoxins, mycophenolic acid; cyclopolic acid; tropolones (as puberulic and puiberulonic acid); dianthra- quinones (for instance: skyrin and iridoskyrin, rubroskyrin, luteoskyrin, rugulosin); antraquinones (for instance: emodic acid); hemiquinones (for instance: fuscin and purpurogenone) ;

— metabolites containing nitrogen (for instance: /?-nitropropianic acid); — metabolites containing sulphur (for instance : gliotoxin) ;

— metabolites containing chlorine (for instance: nidulin, griseofulvin) and also

— lactones (for instance: curvularin, zearalenone).

The above mentioned metabolites of fungi (mycotoxins) are charac­ terized by large biological activity. Some of them, as for instance alfa- toxin's, belong to the strongest carcinogens of the animal world. A lfa - toxins and the other above mentioned mycotoxins operate also as tera­ togens (teratogenetically). A ll of the above mentioned metabolites of the examined soil fungi are distinguished by strong phytotoxic activity. The

majority of examined mycotoxins (especially from the group of aromatic metabolites) belongs to the group of strong inhibitors of the synthesis of R N A and D N A [5, 6]. According to F i s h b e i n , F l a m m and F a l k [6], some mycotoxins (alfatoxins, ochratoxins, patulin, skyrins, rubro- skyrins, and luetoskyrins) produced by the soil fungi, are marked also by specific mutagens (mutagenal) activity both in regard to microorga­ nisms and also to higher organisms. The attendance of the induced m u­ tation with the help of chemical mutagens at procaryotic and eucaryotic microorganisms is much higher than when using physical mutagens [6].

To a group of these compounds belong, among other, alkanes, epo- xialcanes, lactones and also alkylating agents (for instance: epoxides, epichlorohydrin. aldehydes). Moreover to chemical mutagens are included also Nitrosamines, Aziridines and related derivates and the majority of pesticides (within it also some herbicides) and miscellaneous mutagens and thus chemical compounds or products of their biodégradation— put commonly into practice in agriculture and forestry.

C O N C LU S IO N S

On the basis of obtained results from the studies carried out in the years 1960-1972 of mycological examinations of soils of chosen field ecosystems (agrocenose), of forest and grassy ecosystems and chemical investigations at toxic substances biologically active and their influence at the microflora of soil habitat— we may draw the following very gen­ eral conclusions:

1. Out of different soil habitats of chosen vegetable communities entering into the composition of examined agro-ecosystems, forest and grassy ecosystems and also the soils situated within the direct reach of the intensive economic activity of man 21 species of fungi, mycotoxin- creating being dominate were separated out from the soils of examined vegetable sinusion. It was observed that in examined arable lands— es­ pecially the so called soil-sickness of known chemical activity (pestici­ des) and biological activity (mycotoxins and other biotic factors) of the casual factors (of definite vegetable sinusion) toxin producing fungi were found to be dominant, creating in vivo as w ell as in vitro biologically toxic substances (mycotoxins).

On the other hand, in the soil habitat of examined forest ecosystems

(Fagetum carpaticum and Tilio-Carpinetum) and grassy ecosystems (A r - rhenatheretum elatioris and Gladiolo-Agrostidetum)— the above mention­

ed myco toxin-producing fungi, occur sporadically.

2. Mycotoxins— -as products of metabolism of examined soil fungi out of the class Fungi imperfecti (Table 2) are characterized by large

Biologically active toxic substances... 97

biological activity in relation to soil microorganisms (Table 3) and culti­ vated plants. Some of the determined mycotoxins, produced by the eli­ minated soil fungi are characterized also by distinct mutagens (mutagenal) activity in relation to soil michroorganisms as w ell as higher organisms [5, 6, 9, 1].

3. From the above mentioned data it results that mycotoxins— as metabolites of examined toxinnproducing fungi are characterized b y large biological activity (among others they are distinguished by mutagenal and carcinogenal activity, bacteri- and fungicidal and also phytoxic ac­ tivity) they can influence, in definite ecological conditions, a destructive effect upon the biological balance of a definite soil habitat— biocenose.

Consedering that the soil habitat is influenced by— besides the above mentioned mycotoxins— pesticides (herbicides, fungicides, insecticides and so on-chemical compounds for the protection of plants) fill the soil from several to more than ten months or several to more than ten years. The final effect may lead to essential troubles in the biological balance of the soil community of agroeeosystems. It refers to troubles in the metabolic processes as w ell as the biocenotic soil microorganisms and also physiopathological changes of succesion crops within definite — crop rotations systems.

REFERENCES

[il] A j 1 S. J., C i e g l e r A., K a d i s S., M o n t i e T. C., W e i n b a u m G.: M icrobial Toxins — A Com prehensive Treatise, N e w Y o rk — London 1970-1972, Academ ic Press, vol. 1-8.

[2] A i n s w o r t h G. C., S u s s m a n A. S.: The Fungi — A n Advanced Treatise, N e w Y o rk — London 1965, 1966, 1968, Academ ic Press, vol. 1-3.

[3] A l e k s a n d r o w i c z J., S m y k В.: M ycotoxins and their role in onco­ genesis w ith special references to blood diseases, Polish M edical Science Bull. 14, 1971, 25-30.

[4] D o m s c h K. H.: M icrobial Stim ulation and Inhibition of P lan t Growth, 9th Intern. Congress of Soil Sei. 2, 1968, 455-464.

[5] F i s h b e i n L.: Chrom atography of Environm ental Hazards. I. Carcinogens, Mutagens and Teratogens. Am sterdam — London — N e w Y o rk 1972, E lsevier P u ­ blishing Company, pp. 499.

[6] F i s h b e i n L., F 1 a m m G. W., F a l k H. L.: Chem ical Mutagenes — E n v i­ ronm ental Effects on B iological Systems. N ew Y o rk — London 1970, Academ ic Press, pp. 306.

[7] I s v a r a n V., H a r r i s J. R.: M icrobiological Aspects of P h ytotoxicity in Soil. The Madras A gri. Journal 55 (9), 1968, 401-405.

[8] M a g g o n K. K., V i s w a n t h a n L., V e n k i t a u s b r a m a n i a n T. A., M u k e r j i H. G., A fla to x in Production by Some Indian Strains of A spergillus flavus Lin k ex Fries. J. Gen. Microb. 59, 1969, pp. 119-124.

[9] M c C a l l a Т. M., H a s k i n s F. A.: Ph ytotoxic Substances from Soil M icro ­ organisms and Crop Residues. Bact. Rev. 28, 1964, 181-207.

[10] N o r r i s J. R., R i b b o n s D. W .: Methods in M icrobiology. N e w Y o rk and London 1969-1972, Academ ic Press, vol. 1-7B.

[11] S m y k В.: Zm ęczenie gleb uprawnych w św ietle badań m ikrobiologicznych i agrobiologicznych. Post. M ikrob. 8, 1969/70, 2, 205-224.

[12] S m y k B.: Tren dy w podnoszeniu żyzności gleb, efek ty gospodarcze i kon­ sekwencje eu trofizacji środowiska glebow ego w roln ictw ie i w leśnictwie. P A N Zesz. probl. Post. Nauk roi. 155, 1974.

[13] T u r n e r W. B.: Fungal Metabolites. London 1971, Academ ic Press, p. 456. [14] V i r t a n e n A. I.: Antim ikrobiologische Substanzen in unseren Kulturpflanzen

und ihre Bedeutung fü r die Pflan zen und fü r die Ernährung des Menschen und des Tiere. Path, et Microb. 21, 1963, 970-979.

в. смык Т О К С И Ч Е С К И Е Б И О Л О Г И Ч Е С К И А К Т И В Н Ы Е В Е Щ Е С Т В А ГР И Б Н О ГО П Р О И С Х О Ж Д Е Н И Я И И Х В Л И Я Н И Е Н А П О Ч В Е Н Н У Ю С Р Е Д У Институт почвоведения, агрохимии и микробиологии Сельскохозяйственной академии в Кракове Р е з юме Среди вы бранных к ул ьту р н ы х почв и почв входящ их в состав травяны х и лесн ы х экосистем A rrh en a teretu m elatioris— G la d iolo-A g rostid etu m и соответ­ ственно T ilio -C a rp in e tu m — Fagetum carpaticum бы л вы делен 21 вид токсино­ генны х грибов, м. пр. A spergillus, Fusarium , P é n icilliu m , Thielaviopsis, класса F u n gi im p erfecti. Экологические исследования показали, что в к ульту р н ы х почвах, а осо­ бенно в „утом лен н ы х” почвах, преобладают вы ш еуказанные токсиногенные гри­ бы. В почвенны х ж е средах исследуем ы х лесн ы х и травяны х экосистем уп о­ мянутые грибы встречаются лиш ь в отдельны х случаях. Выделенны е токсиногенные грибы образуют как in v itro так и in v iv o (т.е. в почвенной среде) биологически активные вещества — микотоксины, в частности: глиотоксины, афлатоксины, охратоксины, цитронаты, F-2 (zeara- lenon), стеригматоцистин, ругулозин, цитреовиридин, пеницилловую кислоту, рубратоксин, спорофузарин и др. М икотоксины как м етаболиты исследуем ы х почвенны х грибов отличаются высокой биологической активностью (м. пр. их характеризует фитотоксинное и »мутагенное, а такж е бактерио- и фунгицидное и ко-карциногенное воздействие) могут оказывать в определенны х эк ологи ­ чески х услови я х почвенной среды деструктивное влияние на биологическое равновесие почвенны х биоценозов. В. SM YK S U B S T A N C E S B IO L O G IQ U E S T O X IQ U E S A C T IV E S P R O V E N A N T DE C H A M P IG N O N S E T L E U R IN F L U E N C E SU R L E M IL IE U DE S O L

Institut de Pedologie, de Chim ie agricole et de M icrobiologie, U niversité Agronom ique de Cracovie

R é s u mé

On a isolé des sols cultivables et des sols qui composent des écosystèmes herbeux A rrh en a th eretu m elatioris et G la d ioloA g rostid etu m et forestiers T ïlio

-Biologically a ctive toxic substances... ₉₉

- C arpinetum et Fagetum carpaticum 21 sortes de champignons toxiques de genres: Aspergillus, Fusarium , P é n ic illiu m et Thielaviopsis de la classe F u n g i im p erfecti.

A u cours les examens écologiques on a observe que dans les sols cultivables et surtout dans les sols fatigués des champignons toxiques mentionnés ci-dessus dominent.

Cependant dans les m ilieu x de sols des écosystèmes exam inés forestiers et herbeux les champignons mentionnés sont sporadiques. Les champignons toxiques isolés produisent in v itro comme in v iv o (c’est à dire au m ilieu du sol) des sub­ stances biologiquem ent actives m ycotoxines à savoir: gliotoxines, aflatoxines, ochra- toxines, citriniens, F-2 (zearalenone), sterigmaticistine, rugulosine, citreoviridine, acide de pencilline, rubratoxine, sporofusarine etc. Les m ycotoxines comme des m etabolites des champignons de sols examinés en se distinguant (entre autres) par une grande activité biologique sont caractérisés par l’ action phytotoxique l'action mutagène, la réaction bacterio- et fungicide.

Dans des conditions écologiques déterminées du m ilieu du sol peuvent exercer une influence destructive sur l ’équilibre biologique des biocenoses du sol.

в. SM YK

B IO L O G IS C H A K T IV E T O X IS C H E S TO FFE V O N P IL Z E N H E R K U N F T U N D D E R E N E IN F L U S S A U F D A S B O D E N M IL IE U

Institut fü r Bodenkunde, Agrikulturchem ie und M ikrobiologie der Landw irtschaftlichen U niversität in K ra k ów

Z u s a m m e n f a s s u n g

Von den erwählten Agrarböden und den Böden, die in die Zusammensetzung der Gras und W aldökosystem e Arrhervatheretum elatioris und G la d io lo -A g ro s tid e - tu m ; T ü io -C a rp in e tu m und Fagetum carpaticum hineingehen, wurden 21 toxin b il- dende Pilzarten u.a.: Aspergillus, Fusarium , P é n ic illiu m und Thielaviopsis, aus der Klasse von F u n gi im p erfecti, abgesondert.

Die ökologischen Untersuchungen haben gezeigt, das in den untersuchten A g r a r ­ böden, und besonders den ’’ermüdeten” Böden, die obenerwähnten toxinbildenden P ilz e vorherrschen. Dagegen in den Bodenmilieus der untersuchten W ald - und Grasökosysteme treten die genannten P ilze vereinzelnd auf.

Von den aggesonderten toxinbildenden P ilzen wurden sowohl in v itro als auch in v iv o (d.h. im Bodenm ilieu) biologisch aktive S toffe — M ykotoxine, und zw ar: G liotoxine, A flatoxin e, Ochratoxine, Citrinin, F-2 (Zearalenon), Sterigm a- tocystin, Rugulosin, C itreoviridin, Penicillinsäure, Rubratoxin, Sporofusarin u.dgl., abgesondert. D ie M ykotoxinen als M etabolite von untersuchten Bodenpilzen zeichnen sich m it hoher biologischen A k tiv itä t aus, indem sie u.a. phytotoxische sowie und stärke mutagene W irkung auf manche Bodenm ikroorganism en sowie auch auf höhere Organismen, fern er eine bakterien- und pilztötende W irkung aufweisen und können in bestimmten ökologischen Bedingungen des Bodenmilieus einen destrukti­ ven Einfluss auf das biologische G leichgew icht von Bodenbiozönosen ausüben.

в. SMYK

T O K S Y C Z N E SU B S TA N C JE B IO L O G IC Z N IE A K T Y W N E

P O C H O D Z E N IA G R Z Y B O W E G O I IC H W P Ł Y W N A Ś R O D O W IS K O G LE B O W E Instytut Gleboznawstwa, Chem ii Rolnej i M ikrobiologii

Akadem ii R olniczej w K ra k ow ie

S t r e s z c z e n i e

Z w ybranych gleb uprawnych i z gleb wchodzących w skład ekosystemów trawiastych A rrh en a th eretu m elatioris i G la d iolo-A grostid etu m oraz leśnych T ilio - C arpinetum i Fagetum carpaticum w yodrębniono 21 gatunków grzybów toksyno- twórczych z rodzajów : A spergillu s, Fusarium , P é n ic illiu m i Thielaviopsis — klasy F u n gi im p erfecti.

W badaniach ekologicznych zaobserwowano, że w glebach uprawnych, a z w ła ­ szcza w zmęczonych, dominują w ym ien ion e grzyb y toksynotwórcze. Natom iast w środowiskach glebow ych badanych ekosystem ów leśnych i trawiastych w spom ­ niane grzyby występują sporadycznie.

W yodrębnione grzyby toksynotwórcze w ytw a rza ją in v itro , jak i in v iv o (tzn. w środowisku glebow ym ) substancje biologicznie aktyw ne — mikotoksyny, a m ia ­ now icie: gliotoksyny, aflatoksyny, ochratoksyny, cytryniny, F-2 (zearalenon), ste- rygm atocystynę, regulozynę, citreow irydynę, kwas penicylinow y, rubratoksynę, spo- rofuzarynę i inne. M ikotoksyny, jako m etabolity badanych grzyb ów glebowych, odznaczając się dużą aktywnością biologiczną (m. in. cechuje je działanie fitoto- ksyczne i mutagenne, oddziaływ anie bakterio- i grzybobójcze oraz ko-karcino- genne) mogą w yw iera ć w określonych warunkach ekologicznych środowiska g le ­ bowego destruktywny w p ły w na rów now agę biologiczną biocenoz glebowych.

P r o f . d r h a b. B o le s ła w S m y k In s ty t u t G le b o z n a w s t w a , C h e m ii R o l n e j i M ik r o b io lo g ii A . R . K r a k ó w , ul. M ic k ie w ic z a 24128