R O C ZN 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
Z. KRĘŻEL, D. LESZCZYŃ SK A, Z. ŻU K O W SK A
HERBICIDAL EFFECT OF SOME MICROORGANISMS ON P L A N T S 1 Agricultural University of Wroclaw, Poland
It is known that in some case herbicides could disturb the biocenosis of soil [1, 2, 3, 4, 5, 6, 8]. Therefore, the attention is paid to the bio logical methods of weeds control. One of them might be the use of microorganisms as producers of phytotoxic substances.
The present work was designed to isolate microorganisms producing toxic metabolites, so-called ’’bioherbicides” , the effect of which on plants was similar to the synthetic herbicides.
METHODS
From roots and leaves of sugar beet 38 bacterial and 2 yeast strains were isolated. The isolation was performed on media with sugar beet and potato extracts, 3 synthetic media were adopted for the culture: (1) according to W i c . k e r h a m [11]; (2) after S p e n c e r [10]; (3) me dium composed of: glucose — 10 g, N aN 03 — 2 g, K 2H P 0 4 — 0,5 g, KC1 — 0,29 g, F eS04 — 0,01 g, yeast extract — 1 g, distilled water — 11.
Phytotoxic properties o f microorganisms were tested on sugar beet and weeds commonly occuring in the culture of this plant, namely
Galinsoga parvijlora, Raphanus raphanistrum, Chenopodium album, Chry santhemum segetum, Stellaria media, Echinochloa crus-galli, Senecio vulgaris. Moreover, Sinapis alba was adopted as an index plant for
biopreparations.
The phytotoxic effect of microorganisms was determined? as follows: — germination of seeds, previously soaked for 18 hours in' the culture of microorganisms and further growth during 2-3 weeks,
— spraying o f 5-8-day old plants with bacterial culture,
1 The work was supported by Grant No. FG-Po-249 from the US Department of Agriculture.
1 0 0 Z. Krężel, D. Leszczyńska, Z. Żukowska
— watering the plants with bacterial culture at a definite concen tration,
— vegetation experiments in micropots at planting germinated seeds into sand or soil, previously saturated with bacterial culture,
— inoculation of sugar beet leaves with bacterial suspension,
— inoculation of sugar beet and potato discs with bacterial suspension. The toxic substance from the bacterial culture (strain No. 101) was extracted with acetone. For this purpose, 1/2 1 of an 8-day bacterial culture were centrifuged for 15 minutes at 20 000 r.p.m. The super natant was treated with 4 volumes of acetone and left overnight at 4°C; the precipitate was dried in vacuum till a grey powder was recovered.
Thus obtained crude preparation was subjected to dialysis for 24-48 hours and the separated fractions were determined chemically using biuret, ninhydrin, Molisch, Fehling and antron tests. A part of the crude preparation (50 mg) was hydrolysed with 5N HC1 for 15 hours. The hydro- and dialysis products were determined by paper chromatography.
RESULTS
Among the 40 isolated bacterial strains, 8 were found to inhibit germination or growth of the tested plants. The reaction of the plants to some strains is shown in Figs 1, 2. A selective effect was exhibited by the strains: Corynebacterium sp. 101, Pseudomonas sp. 13 and Rho-
dotorula sp. 104.
Among the tested plants, Senecio vulgaris proved to be resistant to all strains under investigation. The reaction of other plants varied;
Fig. 1. The influence of microorganisms on the growth of Echinochloa crus-galli. Strain Nos: 101 — Corynebacterium sp., 103 — Bacillus spec., 104 — Rhodotorula sp.
Herbicidal effect of some microorganisms... 101
the strain No. 101 exerted an inhibitory effect not only on Echinochloa
crus-galli, Chrysanthemum segetum and Stellaria, but also on Beta vul garis and Sinapis alba. The germination of seeds as influenced by the
strain 101, was reduced by 10-30% and the growth by 30-60%, depending on plant. The strain was selected for further studies.
Although the strain 101 acted on most tested plants, their reaction varied; e.g. Echinochloa and Stellaria, in the presence of bacterial culture in the soil showed a weaker growth, but did not wither, while Sinapis,
Beta and Chenopodium withered and died as a rule, after 12-20 days.
Fig. 2. The influence of microorganisms on the growth of Chrysanthemum sege tum. Strain Nos: 100 — Bacillus oligonitrofilus, 103 — Bacillus sp., 104 —
Rhodo-torula, 13 — Pseudomonas sp.
Fig. 3. Sinapis alba seedlings 12 hours after the treatment with culture Coryne-bacterium sp. (101)
10 2 Z. K rężel, D. Leszczyńska, Z. Ż u k ow sk a
Withering and dying of plants were much quicker, if they were sprayed or watered; in this case withering occurred after 10-40 hours (Figs 3, 4).
Fig. 4. N ecrotic spots on leaves o f G a lin so ga p a rv iflo r a due to spraying w ith C o r y n e b a c te r iu m sp. (101)
Fig. 5. N ecrotic spots on sugar beet leaves due to spraying w ith C o r y n e b a c te r iu m sp. (101)
The active substance separated from the bacterial culture exhibited similar phytotoxic properties under retention of its selective effect. The isolated preparation, like the whole culture, brought about necrosis of leaves (Fig. 5) and roots of sugar beets (Fig. 6), without producing similar damage to the potato discs.
H erbicida l e ffe ct o f som e m icroorganism s... 10 3
Fig. 6. The necrosis of sugar beet root due to injection with Corynebacterium sp. (101)
The crude preparation proved to be a complex proteinopolysaccharide compound, the phytotoxic properties being linked with the polysaccha ride fraction. The fraction was composed of glucose, galactose, mannose, apparently of fueose and other nonidentified compounds with R f = 0.93 and 0.62; the protein fraction consisted of histidine, asparagine, alanine, glutamic acid.
The culture of Corynebacterium No. 101 showed, in addition to its phytotoxic properties, an antibiotic effect on some microorganisms, e.g.
Azotobacter chroococcum (Fig. 7) and Ervinia carotovora, the latter
being affected by the polypeptide fraction.
104 Z. K rężel, D. L eszczyń sk a, Z. Ż u k ow sk a
The use of the strain for experiments with Venzar was found to enhance the toxic effect of the herbicide by about 30% in a number of withering plants.
CONCLUSIONS
The experiments aiming at isolation of ’’bioherbicides” are very promising; the range of their effect on plants and microorganisms seems to be much wider than that of synthetic herbicides. Moreover, they might interfere with the latter. With the present tendency of combining herbicides in soil cultivation, a current use of synthetic preparations with the metabolites of bacteria should be taken into consideration, the more so as such combinations may occur in soil without our intervention.
R E F E R E N C E S [1] B a l i c k a N. , K r ę ż e l Z .: A b s t. V I C o n f. I t e r . P r o t P la n t, W ie n 1967. [2] B a l i c k a N. , K r ę ż e l Z .: W e e d Res. 9, 1969, 38. [3] B a l i c k a N. , S o b i e s z c z a ń s k i J .: A c ta m ic ro b . p o l. 18B, 1969, 3 a n d 7. [4] C u r l E. A. , F u n d e n b r u k H . H .: P ro g . R e p o rt P H S Res. G r a n t 5 R O l E F -00639-02, 1966, 1. [5] E n n i s W . B .: W e e d Res. 4, 1964, 93. [6] F l e t c h e r W . W .: P ro c . 8 th B r. W e e d C o n t. C o n f. 1966, 896. [7] K r a s i l n i k o w N . A .: M ic ro o rg a n , p o c z w y i w y s . ra s t. M o s k w a , Iz d . A N S S R , 1958. [8] K r ę ż e l Z., L e s z c z y ń s k a D .: M e d e l F a c u lt. L a n d b o u w . W e tte n s c h a p p e n , G e n t 35, 2, 1970, 655. [9] M i r c z i n k T . G .: M ik r o b io ło g ija 26, 1957, 1. [10] S p e n c e r J. F. T., G o r i n P. A . J .: C a n a d . J o u rn . o f M ic r o b io l. 7, 1961, 185.
[11] W i с к e r h a m L . J. — a fte r K r a s iln ik o w N . A .: M ie to d y izu cz. pocz. m ik ro o r g . i ic h m e ta b o l. M o s k w a , Iz d . M o s k . U n iw ., 1966. Z . K R Ę ŻE L, D. L E S Z C Z Y Ń S K A , Z. Ż U K O W S K A H E R B IC Y D O W E D Z I A Ł A N I E N IE K T Ó R Y C H M IK R O O R G A N IZ M O W N A R O Ś L IN Y A k a d e m ia R o ln ic z a w e W r o c ła w iu S t r e s z c z e n i e W p o s z u k iw a n iu m ik ro o r g a n iz m ó w , p ro d u k u ją c y c h s u b s ta n c je to k s y c z n e d la c h w a s tó w , w y o d rę b n io n o z k o r z e n i i liś c i b u ra k a c u k ro w e g o 38 szczep ów b a k t e r ii i 2 szczepy d ro ż d ż y . W ła ś c iw o ś c i fito to k s y c z n e ty c h m ik ro o r g a n iz m ó w b a d a n o n a b u r a k u c u k r o w y m o ra z na c h w a s ta c h w y s tę p u ją c y c h n a jc z ę ś c ie j w u p r a w ie t e j
H erbicidal e ffe ct o f som e m icroorganism s.., 105
ro ś lin y . N a te j p o d s ta w ie w y s e le k c jo n o w a n o 8 szczep ów m ik ro o r g a n iz m ó w o d z ia ła n iu w y b ió r c z y m w s to s u n k u do c h w a s tó w . N a jb a r d z ie j in te re s u ją c e b y ły Coryne bacterium sp. 101, Pseudomonas sp. 13 i Rhodotorula sp. 104.
Z k u lt u r y Corynebacterium sp. 101 w y o d rę b n io n o s u b s ta n c ję a k ty w n ą ” b io - h e r b ic y d ” . O k re ś lo n o je j p ro te in o w o -p o lis a c h a r y d o w y c h a ra k te r. U z y s k a n y s u ro w y p re p a ra t w y k a z y w a ł s e le k ty w n e d z ia ła n ie w s to s u n k u do b a d a n y c h c h w a s tó w , p o d o b n ie ja k c a ła k u lt u r a . S tw ie rd z o n o ró w n ie ż , że szczep Corynebacterium sp. 101 p ro d u k u je s u b s ta n c ję a n ty b io ty c z n ą w s to s u n k u do Azotobacter chroococcum i Ervinia carotovora. 3 . К Р Е Н Ж Е Л Ь , Д. Л Е Щ И Н Ь С К А , 3 . Ж У К О В С К А Г Е Р Б И Ц И Д Н О Е Д Е Й С Т В И Е М И К Р О О Р Г А Н И З М О В Н А Р А С Т Е Н И Я 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 л и с т ь е в и к о р н е й с а х а р н о й с в е к л ы и з о л и р о в а л и 38 ш т а м м о в б а к т е р и й и 2 ш т а м м а д р о ж ж е й . Ф и т о т о к с и ч е с к и е с в о й с тв а м и к р о о р га н и з м о в о п р е д е л я л и на с а х а р н о й с в е кл е и т е х с о р н я к а х , к о т о р ы е ч а щ е в с е го п о я в л я ю т с я в э то й к у л ь т у р е . Н а этом о с н о в а н и и в ы б р а н о 8 ш т а м м о в м и к р о о р га н и з м о в д е й с т в у ю щ и х с е л е к т и в н о на с о р н я к и . С а м ы м и п е р с п е к т и в н ы м и б ы л и : Corynebacterium sp. 101 r Pseudomonas sp. 13, Rhodotorula sp. 104. И з к у л ь т у р ы Corynebacterium sp. 101 б ы л о в ы д е л е н о ф и т о т о к с и ч е с к о е в е щ е с тв о , д е й с т в у ю щ е е к а к „б и о ге р б и ц и д ” . П р е п а р а т и м ее т п р о т е и н о -п о л и с а х а р и д н ы й состав. Ф и т о т о к с и ч е с к и е с в о й с тв а с в я з а н ы с п о л и с а х а р и д н о й ф р а к ц и е й .
К р о м е т о го э то т ш т а м м п р о и з в о д и л а н т и б и о т и к д е й с т в у ю щ и й н а Ervinio carotovora и Azotobacter chroococcum.
