R O C Z N IK I G L E B O Z N A W C Z E T . X X X IV , N R 1—2, W A R S Z A W A 1983
I K R U N A M ISKOVIC. I I B R A N ISL A V A RASOYIĆ, | JERZY SO B IE SZ C Z A Ń SK I, LJUjSICA BRA NK O V
EFFECT OF DIFFERENT DOSES OF VENZAR ON Azotobacter chroococcum
PART I: MORPHOLOGICAL CHANGES
From th e A g ricu ltu ra l A cad em y, W rocław , P olan d and th e A gricu ltu re F acu lty, U n iv e r sity of N o v i Sad, Y u goslavia
Different types of herbicides are increasingly used in the world [7]. • .Venzar (3-cyclohexyl-5,6-trim ethylenouracil, C43H18N2O2) is used in va rious field and vegetable crops. In soil, this herbicide decomposes slowly. It is of interest to know its effect on quantitive and qualitative com position of microorganisms, especially their morphological and physiolo gical character.
Azotobacter is frequent in Yugoslav chernozem [12]. These bacteria according [8, 14] are extrem ely important in the soil because they fixate molecular nitrogen and produce biologically active substances. That is w h y we decided to study the effect of venzar on morphological and phy siological characters of this bacterium. This paper reports the morpho logical changes of Azotobacter as affected by venzar.
M A TER IALS A N D M ETH ODS
The strains were identified after Bred [1]. Pure cultures were grown on Fedorov medium [3]. A ll isolated strains belonged to the specie-; Azotobacter chroococcum.
The growth and morphology of Azotobacter strains of Fedorow m e dium were examined by the method of “fertile drops” [4], adding diffe rent doses of venzar. Two experim ents were conducted:
I. 0.5, 1.0, 2.0, 3.0 and 5.0 g of venzar added to a litre of medium, II. 5.0 and 10.0 g of venzar added to a litre of medium.
202 K. Miskovic and al. T a b l e 1 O r ig in s o f A z o to b a c te r c h ro o c o cc u n s t r a i n s S t r a i n d e s ig n O r i g i n S o i l R h iz o sp h e re P la n t 8 + w heat 21 + s u n flo w e r 23 ♦ + s u n flo w e r 58 a + s u g a r b e e t 59 +
-Samples in three replications were incubated at 28°C for 24 and 48 hrs. Fixated prepartions were stained after Gram. Ceil forme and struc tures w ere observed by a microscope fitted w ith an “Exacta” camera at the general laboratory of the Faculty of Agriculture, Nowi Sad. Micro- scopis prepartions, mounted on a “Reichert” microscope, were photo graphed at the photolaboratory of the Institute of Field and Vegetable Crops, Nowi Sad.
R ESU L TS
In is w ell known that herbicides affect microorganisms both stimula • tively and inhibitively [16, 17].
• T a b 1 © 2 P e rc e n ta g e o f " f e r t i l e d ro p s " a t d i f f e r e n t c o n c e n t r a t i o n « o f те п * а г V enzar S t r a i n e c o n c e n tr a t i o n g /1 8 21 23 58a 59 o f medium 24h 48h 24h 48* 24h 48h 24h 48» 24h 48h 0 .5 10 92 75 100 97 100 100 100 100 100 1 .0 90 100 100 100 100 100 100 100 100 100 2 .0 100 100 100 100 100 100 100 100 100 100 3 .0 100 100 100 100 93 100 100 100 100 100 5 . С 100 100 100 100 97 100 100 100 100 100
The results in Table 2 show that in experim ent I, which included five different concentrations of venzar, there were no negative effects on the growth of the examined strains, regardless of the concentration of the herbicide. It is probably related to the venzar was increased to
Effect of different doses of venzar 203
10 g/litre of medium, the effect of venzar in the growth of the examined strains becomes different, as shown in Table 3.
The results in Table 3 show that strain 8 was most sensitive; it grew only for 8.0% in 48 hrs. Strain 23 had a similar reaction w hile strains 21, 58a and 59 grew slow ly and resumed their growth after 48 hrs.
T a b l e j P e rc e n ta g e o f " f e r t i l e d ro p s '1 i n c o n c e n t r a t i e n e o f 5 .0 and 1 0 .0 g o f v e n z a r p e r l i t r e o f n e d iu a V enzar c o n c e n tr a t i o n g /1 o f medium S t r a i n 8 8 21 23 58a 59 24h 48h 24h 48h 24h 43* 24h 48h 24h 48h 5 .0 c o a l 1 e r 100 100 100 100 90 100 100 100 100 100 1 0 .0 b ig g e r 0 .0 8 . 0 20 96 0 .0 5 .3 48 100 40 9 0 .8
The observations of pigmentation, cell from, development of capsules and the staining by Gram method after 48 hrs, showed:
S t r a i n 8. — The dosage of 5.0 g of venzar per litre of medium did not inhibit the growth of Azotobacter because it grew equally (100%) in all drops (Fig. 1-C). It m ay be seen on the picture that the
drops merged because Azotobacter developed uninhibitedly. However,
this dosage caused the development of cysts as may be seen in 1-D. The dosage of 10.0 g of venzar per litre of medium accelerated the decompo sition of the cysts, as -shown in Fig. 1-E.
S t r a i n 21. — The dosage of 5.0 g of venzar per litre of medium did not inhibit the growth of Azotobacter. It grew in all fertile drops, retaining a partial ability of pigmentation. (Fig. 2-C). With this dosage, the from of Azotobacter cells was very little changed in relation to the control (Fig. 2-D). Higher dosage of venzar inhibited the growth of Azo tobacter although it grew in 96.0% fertile drops after 48 hours, as shown in Table 3, there were small, thinned out colonies which lost the ability of pigmentation (Fig. 2-C). The cells were smaller and paler then normal ones (Fig. 2-E).
S t r a i n 23. — W ith the dosage of 5.0 g of venzar per litre of m e dium, Azotobacter grew in all fertile drops (100%) after 48 hrs, as show in Table . The cells made groups of irregular form. The capsule was not emphasized, same as in the control (Fig. 3-B, 3-D). The dosage od 10.0 g of venzar per liter of medium inhibited the developm ent of Azotobacter. The percentage of colonies in fertile drops was low and th * colonies the m selves were thinned out. This dosage affected the form of cells — they were enlarged, forming a larger capsule, which was not the case with
Fig. 1. E ffect of venzar on the m orphology of colonies and cells of A z o t ob a c te r i u m
chroococcum , strain 8
A — control, В —I cell form control, С — effects of low er and hig h er dosage, D — cell form at low er dosage; cysts in A z o t o b a c t e r iu m cell are visible, E — cell form a t h igher
Fig. 2. E ffect of venzar on th e m orphology of colon ies and cells of A z o t o b a c t e r i u m c h r o o c o c c u m , strain 21
A — control, В — cell form in control, С — effects of lo w e r and* h ig h er dosage; w ith hig h er dosage, th e colonies in fe rtile drops are th in n ed out and w ith o u t p ig m en tatio n , D —
Fig. 3. E ffect of ven zar on the m orphology of colon ies and cells of A z o t o b a c t e r i u m c h r o o c o c c u m , strain 23
A — control, В — cell form in control, С — effects of low er and h ig h er dosage; w ith h igher dosage, th e colonies in fe rtilite drops a re th in n e d out in ten siv ely , D — cell form a t low er dosage, E — cell fo rm a t h ig h er dosage; cell are e n larg ed an d capsules m ore
Fig. 4. Effect of venzar on the morphology of colonies and cells of A zotobacterium chroococcum , strain 58a
A — control, В —i cell form in control, С — effects of low er and h ig h er dosage; w ith higher dosage, th e colonies in fe rtile drops are th in n ed out; D — cell form at low er dosage,
Fig. 5. E ffect of v en za r on th e m orp h ology of colon ies anjd cells of A z o t o b a c t e r i u m c h r o o c o c c u m, stra in 59
A —. control, B( — < cell fo rm in control, С — effects of low er and h ig h er dosage; w ith h ig h er dosage, th e colonies in fe rtile drops a re th in n e d out, D —< cell form at low er
204 K. M iskovic and al.
this strain in the control and the variant w ith lower dosage of venzar (Fig. 3-D, 3-D, and 3-E).
S t r a i n 58a. — With the dosage of 5.0 of venzar per litre of m e dium, Azotobaeter grew in all fertile drops (100%) as shown in Table 3. The pigmentation was lost. The cells were rodshąped in form, as shown in (Fig. 4-D) w ith rounded ends, somewhat smaller in relation to the control, had no capsula as in control (Fig. 4-B, 4-D). The dosage of 10.0 g of venzar per litre of medium inhibited the growth of Azotobaeter thin ned out colonies which lost the ability of pigmentation and the develop- m enf of capsules as w ell as cysts occurred (Fig.4-E). With this dosage, the rod-shaped w ith rounded ends were thinner (Fig, 4-E).
S t r a i n 59. — W ith the dosage of 5.0 g of venzar per litre of m e dium, Azotobaeter grew on all fertile drops, but the pigmentation was lower. The cells w ere much enlarged in relation to the control (Fig. 5-Br and 5-D). The m ajority of cells was spherical and glued together. The capsules were larger than in the control (Fig. 5-B). W ith the dosage o f
10.0 g of venzar per litre of medium, the colonies were thinned out, smal ler, and had no pigment w hile the cells were much smaller, in relation to the control and the variant w ith 5.0 g of venzar (Fig. 5-E). The cells could not be stained so w ell and had small capsules. A number of very small cells from decomposed cysts was observed (Fig. 5-E).
It may be concluded that venzar in the doses of 10.0 g per litre of m e dium inhibited the growth of Azotobaeter. The inhibition was particular ly intensive with strains 8 and 23 which almost did not grew. The other three strains (21, 58a and 59) had very thin colonies. The high dosage caused a lower pigmentation with the latter three strains while the for mer two strains failed to pigment at both concentrations of venzar. The consistency of the colonies of the examined strains was not affected by venzar. A ll strains remained grammnegative at both concentration of venzar, only strain 21 could not be stained so w ell.
DISCUSSION
Studing effects of different dosages of tilam and atrazine S o b i e s z - c z a ń s k i [15], found that the genera Arthrobacter and Corynebacter underwent morphological changes under the effect of these herbicides, [2] stated that Azotobaeter chroococcum sufferend morphological chan ges as affected by chloramphenicol.
This study showed that high dosages of venzar caused changes in cell from stimulafed or inhibited the occurrence of pigments and capsules of the exam ined Azotobaeter strains. The effect of venzar differend from strain to strain. The herbicide had a smaller effect on strains 8 and 21
Effect of different doses of venzar ... 205
than on the other three strains which were much more sensitive to ven zar. It means that the effect of venzar on morphological changes of Azo- tobacter depends on the bacterial strain.
It was observed in this study that in the presence of venzar, some strains formed gigantic cells (strains 59 and 23) small cells (strains 8 and 59), as w ell as cysts (strains 8, 58 a and 59). R u b e n ć i k [13] described such occurrences, when several drops of 15-day-old culture of B. mycoi- des were added to the Azotobacter culture. K r a s i l n i k o v [5] descri bed the occurrence of gigantic spindle-shaped cells of Azotobacter chroo-
coccum on malt agar w ith 0.25*/o glucose, P о c h o n et al. [10] obtained
such forms of Azotobacter at elevated temperatures (36— 39°C) or by witholding m agnesium and phosphorus from the medium. K r a s i l n i k o v [6] and P o c h o n [11] decided that this occurrence was related to unfavorable living conditions of the bacteria. R u b e n ć i k [13] stated that gigantic Azotobacter cells should not be considered as stage of ontogenic development but as reactive forms, occurring due to the effect of unfavorable factors.
Dwarf coccoid cells, although encountered frequently, should be con sidered as an abnormal form of Azotobacter [13]. The other authors [6, 18], pointed at he formation of cysts. Their papers showed that different factors of nutritive medium change morphological characters of Azoto bacter.
However, other author [2] stated that physiological characters of Azotobacter may be changed under the effect of some factors, e.g. chlo ramphenicol. P i e t r [9] found that venzar changes the nitrogenase activity of Azotobacter. It means that this herbicide affects also the physiological characters of Azotobacter.
C O N CLUSIO N S
The following conclusions may be drawn on the basis of obtained results.
— Low dosages of venzar m ostly did not affect negatively the growth of the examined strains of Azotobacter but still changed the morpholo gical form of cells, intensity of pigmentation, and the development of capsules.
— High dosages of venzar changed the morphological form of the examined strains, leading us to suspect the effect of venzar on the phy siological characters of Azotobactea:.
— Effects of both-high and low dosages of venzar depended on the strain of Azotobacter.
206 K. M iśković and al.
REFERENCES
[1] B r e d R. S., M u r r a y E. G. D. and S m i t h N. R.: B e r g e y ’s M anual of D eterm in a tiv e B acteriology. S ev e n th Ed. B altim ore, U S A 1957.
[2] B r o t o n e g o r o S.: N itrig en fix a tio n and n itro g en a se a c tiv ity of A zo to bacter chroococcum . M ad ed elin gen L an d b ou w h ogesch ool, 1974, 74, 10 W age- n ingen.
[3] F e d o r o v M. V.: R u k ovod stvo к p ra k tićesk im zan atijam po m ikrob iologii. M oskva 1957.
[4] J o h n s o n C. E.: In flu en ce of S tu b le M ulching of th e N um ber of A zotob acter in th e Soil. M aster th esis, U n iv. of N ebrasca, 1953, Soil Sei, 1958, 56, 2. [5] K r a s i l n i k o v N. A.: Istorija r a zv itija A zotob acter v sv ja zi s problem oj
polim orfizm a. M ikrobiol. 2 u rn . 1 2, 1931.
[6] K r a s i l n i k o v N. A.: P o cv en ie m ik roorgan izm i i v is ie rasten ija. Izd -tv o A N SSSR , M oskva 1958.
[7] K u n e F.: C ontrol of P esticid e P ersisten ce in S o il w ith S p ecial R esp ect to M icrobial activ ity . Zbl. Bact. A bt. II 1975, 130, 82— 103.
[8] M d ś u e t i n E. N., S i 1 j n i к o v a V. K.: B io lo g ićesk a ja fik sa cija a tm o sfern o - go azota. Str. 168. Izd -tv o A N SSS R , M oskva 1968.
[9] P i e t r S.: The in flu e n c e of som e herbicides on n itro g en a se a c tiv ity of
A z o t o b a c t e r c h r o o c o c c u m . Int. Sym . “ T he In tera ctio n of S o il M icroflora and E n viron m en tal P o llu tio n s”. 1, 1977, 205— 210, P u la w y .
[10] P o c h o n J., T c h a n Y. T. et W a n g T. L.: R ech erch es sur le cycle m o rp h o logiq u e et l ’appareil n u clesire des A zotobacter. A nn. Inst. P a steu r 1944, 74, 182.
[11] P o c h o n J., C h a l a u s t R. et T c h a n Y. T.: In ocu lation d ’A zotob acter dans les sol. A cad. de F rance 1948, 34, 791.
[12] R a ś о v i ć B.: U ticaj rdzosferne i zem ljiśn e b ak terio flo re p śen ice, kukuruza, śeććrn e repe i soje na p op u laciju azotobactera u zem ljistu . D okt. dis. U n iv. N ovi Sad, 1971, 1— 141.
[13] R u b e n c i k L. I.: A zotob acter i ego p rim en en ie v seljsk o h o zja istv o . Izd -tv o A N SSSR , K ie v 1960.
[14] S o b i e s z c z a ń s k i J., N i e w i a d o m a T.: The In flu en ce M etab olites of A zotob acter upon th e grow th and D ev a lo p m en t of P la n ts. IX Int. C ongress of M icrobiology, 285, M oskva 1966.
[15] S o b i e s z c z a ń s k i J.: T he E ffect of H erb icid es on G row th and M orphology of S om e S p ecies of B acteria. A cta M icrobiol. P olon ica, X V III, Ser. В/1, 1969, 18 N, 3—4, 99— 104.
[16] S o b i e s z c z a ń s k i J., M i s k o v i é K., and R a ś o v i ć B.: B iod égrad ation of V enzar in Soil. P art I: D eg ra d a tio n T im e of V enzar in d ifferen t typ es of S oil by p erco la tio n m ethod. The Int. S y m . „The in tera ctio n of S o il m icroflora and en v iro n m en ta l p o llu tio n s”. 1977, 234— 242, P u ła w y .
[17] T a t e R. L., and A l e x a n d e r M.: M icrobial fo rm a tio n and degrad ation of dim eth yam in e. App. and E n v iro n m en ta l M crobiology 314 1976, 399— 403. [18] W i n o g r a d s k y S.: E tude sur la m icrob iologie du sol. N eu v ièm e m em oire,
Sur la m orphologie et eco lo g ie des A z o t o b a c t e r. Ann. Inst. P asteu r, 60, 1938, 3.
Effect of different doses of venzar.. 207 К. М И Ш КО В И Ц Ъ, Б. РА Ш О ВИ Ц Ъ, E. С О БЕЩ А Н ЬС К И , Л . ЬРА Н КО В ВЛИЯНИЕ РАЗНЫХ ДОЗ ВЕНЗАРА НА AZOTOBACTEP CHROOCOCCUM ЧАСТЬ 1-Я. МОРФОЛОГИЧЕСКИЕ ИЗМЕНЕНИЯ Институт Растениеводства и овощеводства Сель-хоз. Факультет Новый Сад (Югославия) Сольскохозайственная акадения — Вроцлав Р е з ю м е Испытывали влияние вензора (3-cyklo-hexyl-5,6-trimetyJenuracil, C*3H i80 2) на рост, морфологию колоний и поведение клеток пяти штаммов A zo tobacter chroococcum. Штаммы были обособлены из югославянского чернозема, залегающего вблизи г. Нови Сад. Их суспензию высевали по методу ’’fertil drops,, на питательную смесь Федорова, обо гащенную 0,5 до 5 г ’’низкая доза,, и 10 г ’’высокая доза,, вензара. Время инкубирования: 24 и 48 часов при 28° по Ц. Анализы были проведены в трех повторностях. Развившиеся колонии азотобактера и его клетки были описаны и сфотогра фированы. Выявлено, что ’’низкие дозы,, вензора в общем не ограничивали роста исследо ванных штаммов, однако клетки бактерий меняли свои формы, теряли способность созда- вания слизистых оболочек и меланиновых пигментов; повышалось число образованных цист. ’’Высокая доза,, ве; зора сильно тормозила рост некоторых штаммов (8 и 23), остальные штаммы (21,58 и также 59) росли медленнее и формировали ничтожные бесцветные колонии. Цисты образованные некоторыми штаммами азотобактера подвергались распаду под вли янием высоких доз вензора. К . M IS K O VII С, в. R A S O V IC , J . SO B IE SZC ZA IN T SK I, L . B R A N K O V
W PŁYW RÓ ŻNYCH DAW EK V EN ZA R U N A A Z O T O B A C T E R C H R O O C O C C U M
CZĘŚĆ I. ZM IA N Y M ORFOLOGICZNE
A k ad em ia R olnicza w e W rocław iu U n iw e r sy te t w N o w y m S adzie, J u g o sła w ia
S t r e s z c z e n i e
B adano w p ły w ven zaru (3 -cy k lo -h ex y l-5 ,6 -trim ety len u ra cil, C43H 180 2) na w zrost i m orfologię k o lo n ii oraz zach ow an ie się k om órek p ięciu szczep ów A z o t o b a c t e r c h r o o c o c c u m .
S zczep y poch od ziły z czarn oziem u ju g o sło w ia ń sk ieg o w pobliżu N o w eg o Sadu Ich za w iesin ę za siew a n o m etodą „fertil d rop s” na p o ży w k ę F iod orow a, w zb ogacon ą od 0,5 do 5 g (niska daw ka) i 10 g (w ysok a daw ka) venzaru.
C zas in k u b a cji w y n o sił 24 i 48 god zin w 28°C. A n a lizy w y k o n y w a n o w trzech p ow tórzen iach . W yrosłe k o lo n ie azotob ak teria i kom órk i o p isyw an o i fo to g ra fo w a no. U jaw n ion o, że n isk ie d aw k i v en zaru n ie ogran iczały na ogół w zro stu b ada n ych szczepów , n a to m ia st kom órki b a k terii zm ien ia ły sw o je k szta łty , tra ciły zdol n ość do w y tw a rza n ia otoczek ślu zo w y ch i b a rw n ik ó w m ela n in o w y ch ; zw ięk sza ła się liczba w y tw a rza n y ch cyst.
208 K. M iskovic and al.
W ysoka daw ka v en zaru siln ie h am ow ała w zrost n iek tó ry ch szczep ów (3 i 23), p ozostałe szczepy: 21, 58 oraz 59, rosły w o ln iej i w y tw a r z a ły bardzo n ik łe b ez barw ne k olonie. C ysty w y tw o rzo n e przez n iek tóre szczep y azotobaktera rozpadały się pod w p ły w em w y so k ich d aw ek venzaru.
P r o f . dr J e r z y Sob ie s zc z ań s k i A k a d e m i a R o ln ic z a
