Wpływ CIPC na komórki drożdży Rhodotorula glutinis

R O C Z N IK I G L E B O Z N A W C Z E T . X X V I , Z . 2, W A R S Z A W A 1975

3 . KOSINKIEW ICZ, J. LU BCZYŃ SKA, M. STA N KIE W ICZ

THE EFFECT OF THE HERBICIDE CIPC ON RHODOTORULA GLUTINIS CELLS 1

Institute of Agricultural Chemistry Soils Science and Microbiology Agricultural

University of Wrocław, Poland

It had been known before [2, 8, 9, 10] that the herbicides of the carbamates group strongly affect physiology of microorganisms. The aim of the present work was to gain an insight into the action of the herbicide CIPC (isopropyl-N/3-chlorphenyl carbamate) on the cells of soil yeast Rhodotorula glutinis, as a model of soil microflora. Attention has been paid to the following questions: the effect of the CIPC on the growth of the cells, the pigment production by yeast, the content of nucleic acids and lipids, the sorption of some dyes by the cells and the penetration of the CIPC into the cells. The action of the herbicide CIPC on the yeast cells with the action of the detergent of sodium taurocho- late has been compared.

METHODS

The yeast strain Rhodotorula glutinis, isolated from soil [10] in the Rider medium, during 1-6 weeks, was cultivated at 30°C. The herbicide CIPC, technically pure (50% of active ingredient), in the dose of 100 ppm, or CIPC analytically pure, in the dose of 50 ppm, or sodium taurocholate o f 10 ppm was added to the culture medium. The growth of yeast cells was determined nephelometrically.

The pink pigment produced by yeast has been obtained from the cells by means of extraction with 96% ethanol and determined spectro- photometrically.

1 The work supported partially by the Committee of Microbiology, Ilnd Department of Polish Academy of Sciences.

108 В. K osin k iew icz, J. L u bczyń ska, M. Stan k iew icz

The determinations of nucleic acids content in the yeast cells with different methods were carried out. The yeast cells after 2-4 weeks of incubation were harvested by centrifugation, washed twice with phosphate buffer pH 7, once with distilled water and dried in vacuo at 0°C. Dried cells were disintegrated mechanically by grinding with acid-washed sand for 20 min at 5°C, then DNA was isolated according to the K i r b y method [7] and determined gravimetrically. The nucleic acids (RNA + DNA) were also isolated by the method of M a r m u r [12] or the method of S c h n e i d e r [15]. Then the RNA amount was de­ termined colorimetrically by the method of M e j b a u m [13] and DNA by the method of D i s e h e [6] modified by B u r t o n {4]. The content of nucleic acids (RNA + DNA) was determined spectrophotometrically. The microscopic observations of nucleoids of the yeast cells have been carried out according to the method o f Tulasne Vendrely [3].

Intracellular lipids of the yeast cells were obtained using the method described by D e i n e m a [5] and determined gravimetrically.

The effect of CIPC and sodium taurocholate on the sorption of some dyes by the yeast cells was determined by the following method :

The cells were harvested by centrifugation, washed twice with phos­ phate buffer of pH 7 and suspended in the water solution (0,001%) of a dye. Each sample contained the same number of yeast cells. After half an hour the cells were centrifuged and the quantity of a dye in the supernatant was determined spectrophotometrically. The following dyes have been used: Janus green, methylene violet, acridine orange, neutral red, trypaflavine, methylene blue, erythrosine and eosine. In the next ex­ periment the yeast cells in the medium without the herbicide, were incubated for 16 days. Then the yeast cells were centrifuged and suspend­ ed in the suspension of the CIPC technically pure in the doses of 1000, 10 and 0,1 ppm, or in the solution of sodium taurocholate in the doses of 1000 and 100 ppm. After an hour the yeast cells were centrifuged and their sorption ability of methylene blue was determined. Simul­ taneously the microscopic observations of the yeast cells coloured with methylene blue were carried out.

To test the penetration of CIPC into the yeast cells, the yeast have been incubated in the media containing CIPC technically or analytically pure in doses 100 and 50 ppm, respectively. After 1-2 weeiks of in­ cubation the cells were centrifuged and washed twice with ethanol (96%), to remove the CIPC from their surface. Then, the air-dry cells were weighed and the quantity of CIPC were determined according to the Lowen’s method [10]. The presence of CIPC in the yeast cells was

The effect of the herbicide CIPC.., ₁₀₉

determined biologically with buckwheat test by the method described by S o b i e s z c z a ń s k i [14] and by thin layer chromatography accord­ ing to the method of A b b o t [1].

RESULTS AND DISCUSSION

CIPC technically pure in the dose of 100 ppm inhibited the growth of yeast, while the CIPC analytically pure in the dose of 50 ppm de­ layed it (Fig. 1). Sodium taurocholate in the dose of 10 ppm showed only a slight effect on the growth of yeast.

Fig. 1. The effect of CIPC and sodium taurocholate on the growth of yeast 1 — control cells. Cells influenced b y : 2 —

CIPC technically pure in the dose o f 100 ppm ,

3 — CIPC analytically pure in the dose of

50 ppm , 4 — sodium taurocholate in the dose of 10 ppm

It was observed that the yeast cells in the presence of CIPC, or detergent in the dose of 100 ppm, did not produce the pink pigment very characteristic for the control cells (Fig. 2). The yeast cells in the medium with sodium taurocholate in the dose of 10 ppm produced this pigment, but after 4 weeks of incubation they lost it, while the loss of pigment in the control culture was noted after about 6 weeks.

The amount of the nucleic acids, isolated from the yeast cells in­ fluenced by CIPC analytically pure, was about 4 times less than the control (Fig. 3). The quantitative determinations o f RNA and DNA show­ ed that in the cells incubated in the presence of CIPC there were less RNA and less DNA (Table 1). The microscopic observations confirmed it.

The determination of intracellular lipids of the yeast cells showed, that the CIPC caused the decrease of the lipids content (Table 2). It was also found that the cells influenced by CIPC were about 2,5 times lighter than the control cells (Fig. 4). This could be explained by the change

110 В. K osin k iew icz, J. L u bczyń sk a , M. Stan k iew icz E

ЦЗ-0,2

-

0,1-Fig. 2. The effect of CIPC and sodium taurocholate on pigment production by

yeast explanation as in Fig. 1 380 400 № Ш Wavelenght (nm) En 0,8- 0,7- oß-45- 0,4- 0,3-o?- 0,1 -240 260 280 300 Wavelenght (nm) 'x b. Ъ 1 в 1 The amount of R1ÎA cLod DNA in the yeast cells

Г

Cells influenced b y CIPC analytically pure 74,4 17,4

Cells influenced b y CIPC technically pure 96,0 21,5

Fig. 3. The effect of CIPC on the nucleic acids content in yeast cells

1 — control cells, 3 — cells influenced b y C I P C analytically p u r e in the d o s e of 50 p p m

T h e e ffe ct o f the h e rb icid e CIPC... _Ill

T a b l e The amount o f in tr a c e llu la r li p id s in the yeast c e lls

Objećt Intracellular lipids /£/ 1 g wet weight of cells % Control cells 0,0326 100

Cells influenced by CIPC analytically pure 0,0081 25

Cells influenced by CIPC technically pure С ,0143 L'A

of cellular structure caused by this herbicide. The cells influenced by CIPC absorbed more of some dyes than the control cells. The methylene blue was absorbed to the most extent (Fig. 5). The number of living cells incubated in the presence o f CIPC was not smaller than the number of control cells, so the increase of absorbed dye by the cells influenced by CIPC could be concidered as a result o f this herbicide action. It was worth noting that all the dyes absorbed by the cells to a greater degree

Fig. 4. The efect of CIPC on dry weight of yeast cells

l — control cells. Cells influenced b y : 2 —

CIPC technically pure in the dose of 100 ppm ,

3 — CIPC analytically pure in the dose of

50 ppm

belonged to the basic dyes. Two acid dyes: eosine and erythrosine were not absorbed by the cells incubated in the medium with CIPC. This could be explained by some changes of electrostatic surface charge of the cells influenced by the herbicide. The quantity of the methylene — blue absorbed by the cells depended on the age of the cells (Fig. 6). Maximal methylene blue sorption was after 2 weeks of incubation, but the cells influenced by CIPC absorbed 20% more of the dye, while the cells influenced by sodium taurocholate 15% more then control cells. The

11 2 В. Kosinkiewicz, J. Lubczyńska, M. Stankiewicz 120 ^3 о / /0 Qi 1 ■§ 110

I

-F i g . 5. Sorption of various dyes by yeast cells

1 — dyes absorbed by control cells. Dyes absorbed by cells influenced by CIPC techni­ cally pure in the dose o f 100 p p m : 2 — Janus green, 3 — m ethylene violet, 4 — acridine

orange, 5 — neutral red, 6 — trypaflavine, 7 — m ethylene blue

Fig. 6. Sorption of methylene — blue by yeast cells after different incubation time

explanation as in Fig. 1

yeast cells incubated in the control medium without the herbicide and treated with CIPC or sodium taurocholate for an hour, showed also the increase in the methylene blue sorption, but to a smaller degree (Fig. 7).

Generally, the effect of CIPC on the dye sorption by the yeast cells was similar to the effect of the sodium taurocholate, but the herbicide was affected stronger than the detergent.

It was found that CIPC penetrated into the yeast cells. After 16 days of incubation the yeast cells contained 9.5 \ig of CIPC technically pure or 5.0 jxg of CIPC analytically pure in 10 mg of dry weight. The presence of CIPC in the yeast cells was also indicated by thin layer chromato­ graphy and biologically with buckwheat test (Fig. 8).

The effect of the herbicide CIPC.., ₁₁₃

HO

-Fig. 7. Sorption of methylene blue by yeast cells treated for an hour with CIPC technically pure or sodium tauro­

cholate. Cells influenced by 1 — CIPC in the dose o f 0,1 ppm , 2 — CIPC

in the dose of 10 ppm , 3 — CIPC in the dose of 1000 ppm , 4 — sodium taurocholate in the dose o f 1000 ppm , 5 — sodium taurocholate in

the dose o f 100 ppm

90-Fig. 8. Penetration of CIPC into yeast cells determined by buckwheat test. Cells

treated with

1 — ether extract from control cells, 2 — ether

solution o f CIPC technically pure in the dose o f 100 ppm , 3 — ether solution o f CIPC an aly­ tically pure in the dose o f 50 ppm , 4 — ether extract from the cells influenced b y CIPC tech­ nically pure in the dose o f 100 ppm , 5 — ether extract from the cells influenced b y CIPC ana­ lytically pure in the dose o f 50 ppm , ß — water

Dilutions

CONCLUSIONS

1. The CIPC technically pure, which is being applied in agriculture, affects stronger the growth of yeast than the CIPC analytically pure.

2. The yeast cells in the presence of the CIPC, both technically and analytically pure, do not produce the pigment.

3. The CIPC, both technically and analytically pure, causes the changes in the cellular structure.

4. The action of the herbicide CIPC technically pure on the sorption of methylene blue by yeast cells is similar to the action of the de­ tergent, however, the herbicide action is stronger.

5. The CIPC penetrates in the cells but CIPC technically pure does that to a greater degree.

114 В. K osin k iew icz, J. L u bczyń sk a, M. Stan kiew icz REFERENCES

[1] A b b o t D. C., B l a k e W. K., T a r r a n t K. R., T h o m s o n J.: J. of Chrom. 30, 1967, 136.

[2] B a l i c k a N., G o ł ę b i o w s k a J.: P ostępy Mikrobiologii X I, 21, 1972, 117. [3] B a s s a l i k - C h a b i e l s k a L.: Ćwiczenia z mikrobiologii. Praca zbiorowa,

PWN, Warszawa 1971.

[4] B u r t o n K.: Biochem. J. 62, 1956, 315.

[5] D e i n e m a M. T.: Mededel. van de Lanbonw. Wageningen, 61, 1961, 2. [6] D i s с h e Z.: Microchemie 8, 1930, 4.

[7] K i r b y K. S.: Biochim. Biohys. Acta 18, 1955, 575.

[8] K o s i n k i e w i c z B.: Acta microb. poi., Ser. В 5, 1973, 22, 145.

[9] K r ę ż e l Z., K o s i n k i e w i c z B.: Acta microb. poi. Ser. B, 4, 1972, 21, 3. [10] L e s z c z y ń s k a D.: Mededel. Fac. Landbouw, Gent, 2, 1970, 35.

[11] L ö w e n W. K., В l e i d n e r W. E., K i r k l a n d J. J., P e a s e H. S.: Annal, methods for pesticides plant growth regulators and food additives. Ed. by G. Zweig, N. York— London lß64.

[12] M a r m u r J.: Methods in Enzymology Collovick Kaplan N. VI, 1963, 726. [13] M e j b a u m W .: Z. Physiol. Chem. 117, 1939, 258.

[14] S o b i e s z c z a ń s k i J.: Mededel. Fac. Lanbonw. Gent, 36, 2, 1970, 681. [15] S c h n e i d e r W. C., H e g e b o o m G. H., R o s s H. E.: J. Nat. Can. Inst. 10,

1950, 977.

B. K O S IN K IE W IC Z , J. L U B C Z Y Ń S K A , M. S T A N K IE W IC Z

W P ŁY W CIPC N A KOM ÓRKI DROŻDŻY RHODOTORULA GLUTINIS

Instytut Chemii Rolnej, Gleboznawstwa i Mikrobiologii Akademii Rolniczej we Wrocławiu

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

Badano wpływ herbicydu CIPC — preparatu technicznie czystego (50% sub­ stancji aktywnej) i analitycznie czystego, na komórki drożdży glebowych Rhodo-

torula glutinis.

Działanie herbicydu porównywano z działaniem detergentu taurocholanu sodu. Stwierdzono, że CIPC technicznie czysty w ilości 100 ppm hamował wzrost drożdży, podczas gdy CIPC analitycznie czysty oraz detergent opóźniały podział komórek.

Komórki drożdży hodowane w obecności herbicydu lub detergentu w ilości 100 ppm nie produkowały różowego pigmentu, charakterystycznego dla kultury kontrolnej. Herbicyd powodował zmiany w budowie komórki, jak zmniejszenie się ilości kwasów nukleinowych, zwłaszcza DNA, oraz ilości lipidów wewnątrz­ komórkowych. Komórki drożdży hodowane w obecności herbicydu zarówno tech­ nicznie, jak i analitycznie czystego były około 2,5 raza lżejsze od komórek kon­ trolnych. Ponadto stwierdzono, że CIPC technicznie czysty powodował znaczne zwiększenie ilości sorbowanych przez komórki barwników zasadowych, a zwłaszcza błękitu metylenowego. Wpływ detergentu na zwiększenie się sorbcji błękitu m e­ tylenowego był około 10 razy słabszy.

T h e e ffe ct o f the h erb icid e CIPC.., ₁₁₅

S tw ie rd z o n o , że h e rb ic y d p rz e n ik a do k o m ó r k i d ro ż d ż y , p rz y c z y m p re p a ra t te c h n ic z n ie c z y s ty z n a jd o w a n o w ilo ś c i o k o ło 2 ra z y w ię k s z e j od ilo ś c i C IP C a n a lity c z n ie czysteg o. Б. К О С И Н К Е В И Ч , Я. Л Ю Б Ч И Н Ь С К А , М. С Т А Н К Е В И Ч В Л И Я Н И Е Г Е Р Б И Ц И Д А Н А К Л Е Т К И Д Р О Ж Ж Е Й R H O D O T O R U L A G L U T I N I S С е л ь с к о х о з я й с т в е н н а я а ка д е м и я , В р о ц л а в , П о л ь ш а Р е з ю м е И з у ч а л и в л и я н и е ге р б и ц и д а C IP С, т е х н и ч е с к и и а н а л и т и ч е с к и ч и с т о го на к л е т к и п о ч в е н н ы х д р о ж ж е й R h o d o r u l a g l u t i n i s . Д е й с т в и е ге р б и ц и д а с р а в н и в а л и с д е т е р ге н т о м т а у р о х о л а т н а т р и я . У с т а ­ н о в л е н о , ч то 100 p p m C IP C т е х н и ч е с к и ч и с т о го з а д е р ж и в а л и р о с т д р о ж ж е й , то гд а к а к а н а л и т и ч е с к и ч и с т ы й C IP C и д е т е р ге н т за м е д л я л и д е л е н ие к л е т о к . К л е т к и д р о ж ж е й и н к у б и р о в а н н ы е в п р и с у т с т в и и ге р б и ц и д а и л и д е т е р ге н т а с к о н ц е н т р а ц и е й 100 p p m не п р о и з в о д и л и р о з о в о го п и гм е н т а , о ч е н ь х а р а к т е р ­ н о го д л я э т и х м и к р о о р га н и з м о в . Г е р б и ц и д в ы з ы в а л и з м е н е н и я в с т р у к т у р е к л е т о к , в ч а с т н о с т и с н и ж а л к о л и ч е с т в о н у к л е и н о в ы х к и с л о т , в п е р в у ю о че ре д ь D N A , а т а к ж е л и п и д о в . В ес к л е т о к и н к у б и р о в а н н ы х в п р и с у т с т в и и ге р б и ц и д а , к а к т е х н и ч е с к и т а к и а н а л и т и ч е с к и ч и с т о го , б ы л на о ко л о 2,5 р аза м е н ь ш е , чем вес к о н т р о л ь н ы х к л е т о к . У с т а н о в л е н о т а к ж е , ч то т е х н и ч е с к и ч и с т ы й C IP C п о в ы ш а л к о л и ч е с т в о о с н о в ы х к р а с и т е л е й с о р б и р о в а н н ы х к л е т к а м и д р о ж ж е й , осо б ен н о м е т и л е н е в о й си н е й . Д е й с т в и е д е т е р ге н т а н а п о в ы ш е н и е с о р б ц и и м е т и л е н е в о й с и н е й о ка з а л о с ь н а о ко л о 10 р а з слабее. У с т а н о в л е н о , ч то ге р б и ц и д п р о н и к а е т в д р о ж ж е в у ю к л е т к у , о д н а к о к о л и ­ ч е с тв о т е х н и ч е с к и ч и с т о го п р е п а р а т а в к л е т к а х б ы л о на о ко л о 2 р а за б о л ь ш е , чем к о л и ч е с т в о а н а л и т и ч е с к и ч и с т о го C IP C .