Zbl. Bakt. Abt. II, Bd. 130, S. 725-731 (1975)
[Department of Weed Research, Plant Pathology, and Microbiology, Agriculture Research Centre, Orman, Giza, A.R.E.]
Efficiency of Rhizobium leguminosarum as Affected by Certain Herbicides and Nematocides M. S. Tewfik, M. S. Embabi, and Y. A. Hamdi Summary Broad beans, planted in pots, were treated with the herbicides Dinitramine and Methazol at the rates of y:!, 1, and 2 litersjfeddan, and the nematocides DBCP and Aldicarb at the rates of 10, 20, and 40 liters or kilogramejfeddan. The chemicals were added at the time of planting or 28 days after. With the exception of DBCP, which had no effect, application of the compounds at the time of planting caused a significant reduction in yield. Application of the chemicals 28 days after planting, however, had no significant harmful effect on the yield. Broad bean seedlings were also grown in agar tubes treated with 1, 10, and 100 ppm of Dinitramine or Methazol, or 20, 200, and 2,000 ppm of DBCP and Aldicarb. Nodulation was completely absent in all treatments with the exception of plants grown with 20 ppm Aldicarb, where four nodules were formed, compared with 22 on the control plants. Growth of shoots was stunted, particularly with Aldicarb and DBCP at 200 and 2,000 ppm and Dinitramine at all the concentrations tested. Growth of Rhizobium leguminosarum strains M 360, M 361, and M 362 in medium 79, containing 1, 10, and 100 ppm of Dinitramine and Methazol and 20, 200, and 2,000 ppm of Aldicarb and DBCP was estimated. In general, all strains were more tolerant to Dinitramine and Aldicarb, especially at low concentrations. DBCP and Methazol caused an inhibitory effect on the growth of the strains tested, except strain M 362 which was not affected by 200 ppm and stimulated by 20 ppm of DEep.
Zusammenfassung In GefaJ3e ausgepflanzte Pferdebohnen wurden mit y:!, 1 und 2ljFeddan der Herbizide "Dinitramin" und "Methazol" behandelt. Von den Nematoziden "DBCP" und "Aldicarb" wurden 10, 20 und 40 I bzw. kgjFeddan zugegeben. Die genannten Chemikalien wurden bei der Aussaat bzw. nach 28 Tagen verabreicht. AuJ3er DBCP fiihrte die Zugabe dieser Chemikalien bei der Ausssaat zu einer signifikanten Abnahme der Ertrage. Ihre Zugabe 28 Tage nach der Aussaat wirkte dagegen nicht signifikant ertragsmindernd. Des weiteren wurden Keimlinge von Pferdebohnen auch in Agarriihrchen angezogen und mit 1, 10 und 100 ppm Dinitramin bzw. Methazol oder 20, 200 und 2000 ppm DBCP bzw. Aldicarb behandelt. Bei allen Behandlungen war die Kniillchenbildung viillig gehemmt, auJ3er bei den mit 20 ppm Aldicarb aufwachsenden Pflanzen, auf deren \Vurzeln sich 4 Kniillchen bildeten, wahrend die Kontrollpflanzen 20 Kniillchen besaJ3en. Das Schotenwachstum wurde durch 200 und 2000 ppm Aldicarb und DBCP sowie durch aile Dinitramin·Konzentrationen gehemmt. Weiterhin wurde das Wachstum der Rhizobium leguminosarum·Stamme M 360, M 361 und M 362 in Nahrliisung 79, die 1, 10 und 100 ppm Dinitramin und Methazol bzw. 20, 200 und 2000 ppm Aldicarb und DBCP enthielt, bestimmt. 1m allgemeinen waren aile Stamme toleranter gegeniiber Dinitramin und Aldicarb, besonders bei niedrigen Konzentrationen. DBCP und Methazol wirkten hemmend auf das Wachstum der untersllchten Stamme - auJ3er Stamm 362, der durch 200 ppm DBep nicht negativ beeinfluJ3t und durch 20 ppm gefiirdert wurde.
726
M. S.
TEWFIK,
J\L S.
EMBABI,
and Y. A.
HAMDI
With the increasing use of pesticides in agriculture, added interest in the effect of these compounds on rhizobia and rhizobia-legume systems has been noted. GROSSBARD (1970) classified the reported studies of the effect of herbicides on various species of Rhizobium as follows: 1) no effect, e.g. dalapon , simazine, prometryne; 2) inhibitory, bacteriostatic, or bactericidal at levels from several thousand to 100 ppm, e.g. phenoxyacetic and phenoxybutyric acids, DNOC, and pyrazon; 3) inhibitory below 100 ppm, e.g. dinoseb acetate and linuron; and 4) stimulatory, as with some chlorosubstituted aliphatic acids. F ew reports have evaluated the effect of nematocides on rhizobia or rhizobialegume symbiosis. Epps and CHAMBERS (1964) reported that DBCP, applied as a seed treatment at rates ranging from l/S-l/Z gal/acre, caused a reduction in both stand and yield of soybean in some trials. Tu (1972) evaluated the effect of the nematocides Dasanit, Carbofuran, D-D, and Vorlex on ammonification, nitrification, and sulfur-oxidation. ZAYED et al. (1968) reported that D-D did not affect the activities of ammonifiers, while the activities of the nitrifiers were greatly affected for a period of 30-45 days, after which the toxic effect of the nematocides gradually disappeared. The present work aimed at studying the effect of the herbicides Dinitramine and Methazol and the nematocides Aldicarb and DBCP on: a) growth and yield of broad beans, b) symbiotic association between R. leguminosarum and broad bean plants, and c) growth of R . leguminosarum strains in vitro.
Materials and Methods Str ains Locally isolated Rhizobium leguminosarum strains M 360, M 361, and M 362 were used. The rhizobia strains were grown in yeast extract mannitol medium, comm only known as medium 79 (ALLEN 1959). Herbicides and nematocid es The herbicide Methazol (2-(3' ,4' -dichlorophenyl)-4-methyl-l ,2,4-oxodiazolidine-3,5-dione), kn own commercially as probe or VCS-438; and t he lately developed h erbicide Dinitramine (N,N' -diethyl-2'di nitro.6.trifluoromethyl-m -phenylene diamine), known commercially as Cobex or F 21; and t he n ematocides DECP (1 ,2-dibromo-3-chloropropone), known commercially as Fumazone 121, and Aldicarb (2-methyl-2-(methylthio) propionaldehyde-O-(methyl-carbamyl oxime), known commercially as Temik 10 g were used. Effect o f the chemicals on the yie ld of broad beans Earthen pots, 30 em in diameter and containing approximately 5 kg of Nile silt, were planted with 10 pre-soaked seeds of broad beans (Vicia laba). Twenty ml of the mixed rhizobia cultures strains M 360, M 361, and M 362, grown separately and mixed just before use, were transferred into each pot. The plants were thinned down to 5 per pot two weeks after emergence. The herbicides and the nematocides were added to t he pots either at the time of planting or 28 days after planting. Methazol and Dinitramine wer e added at the rates of Yt, 1, and 2 kg or I/feddan , re~ pectively, while Aldicarb a nd DECP were added at the rates of 10, 20, and 40 kg or l/feddan, respectively. Each treatment was performed in 5 replicates. A set of plants was left wit hout chemical treatment as a control. The pots were placed at random on a greenhouse floor and watered with tap water as needed . The experiment was terminated after five months; the pods were collected, t h e seeds were extracted, air-dried, and weighed. Effect o f the chemicals on m ed ium R. leguminosarum strains M containing 1,10, and 100 ppm of of the nematocides DECP and
growt h of Rhizobium legumino sa rum strains in culture 360, M 361, and M 362 were inoculated into 20 ml medium 79, the herbicides Methazol and Dinitramine and 20,200, and 2,000 ppm Aldicarb. The respective dilutions of the ch emicals were made of
Efficiency of Rhizobium leguminosarum as Affected by Certain Herbicides etc.
727
concentrated stock solutions, using sterile distilled water. With Aldicarb, 2 grams of the granular material were extracted in 10 ml ethanol (96 %) and respective dilutions in water were made then. After 8 days of incubation at 28°C, 5 ml of each culture were suspended in 10 ml acetone, which dissolved the chemicals and left the cells suspended. Then optical density readings were immediately taken at 600 nm, using an Unicum spectrophotometer. Effect of the chemicals on the rhizobia legume system in tube cultures One hundred ml nitrogen-free seedlings agar (BROCKWELL 1963) was prepared in glass tubes, 35 mm in diameter and 30 cm long. Seeds of Vicia faba were surface-sterilized (ALLEN 1959), pregerminated, and transplanted into the tubes. One ml of 96-hour old cultures of R. leguminosarum strains M 360, M 361, and M 362 were inoculated into each tube. Then each tube received one ml of the appropriate dilution of the chemicals to give final concentrations of 1, 10, and 100 of the herbicides Methazol and Dinitramine, and 20, 200, and 2,000 ppm of the nematocides DBCP and Aldicarb. The tubes were incubated at 22°C in a controlled environment greenhouse for 8 weeks. The seedlings were uprooted then and examined for nodulation and for symptoms of phytotoxicity. The shoots were separated from the roots, dried, and weighed.
Results Dry weight of broad bean yield (seeds), as affected by the different levels of the herbicides and nematocides, is presented in Table 1. When the chemicals were applied at planting, a highly significant reduction of the yield occurred at all levels of the herbicides Dinitramine and Methazol. The reduction was proportional to the rate of the chemical applied. With nematocides, Aldicarb showed a significant depressing effect on the yield at all levels used, whereas DBep had no effect. Inspection of the two-week old seedlings (plants removed at thinning down) showed that in the presence of dinitramine at one and 2liters/feddan the growth Table 1. Yield (g of seeds/plant) of broad bean, treated with various levels of herbicides and nemstocides (Data are averages of 5 replicates) Treatment and Rate/feddan
Control Dinitramine 1 0.5 1.0 2.0 Methazol 0.5 1.0 2.0 Aldicarb kg 10 20 40 DBCP 10 20 40
Time of application at planting
4 weeks after planting
6.0
6.0
3.32 ) 2.9 2 ) 3.0 2 )
5.9 4.6 3.6
2.6 2 ) 2.6 2 ) 1. 72 )
4.5 3.6 2.3
4.41 ) 4.41 ) 4.01 )
5.8 5.4 5.0
5.9 5.9 5.2
6.6 6.4 5.6
1) Significant at 5 % level. 2) Significant at 1 % level.
728
M. S.
TEWFIK,
M. S.
EMBABI,
and Y. A.
HAlIfDI
was stunted and the roots were abnormally thickened. No abnormal symptoms were observed, however, with the lowest rate of this chemical or with the other three chemicals at any of the levels used. Application of the chemicals 4 weeks after planting had no significant effect on the yield. With the herbicides, however, there was some reduction in yield, but the reduction was not statistically significant. The effect of the different herbicides and nematocides on the growth and nodulation of broad beans in tube cultures is presented in Table 2. Nodulation failed in all treated plants with the exception of those plants grown with Aldicarb at 20 ppm where 4 nodules were formed, compared with 22 nodules on the control plants. Table 2. Effect of certain herbicides and nematocides on growth and nodulation of broadbean plants, grown in tube cultures (Data are averages of 4 replicates) Treatment ppm
Length of shoots (cm)
Dry weight of shoots (mg)
Number of nodules
Non·inoculated control Inoculated control Dinitramine 100 10
26.3
293 360 20 20 220 140 170 172 50 280 338 10 21 216
None 22 None None None None None None None None 4 None None None
Methazol
100 10
Aldicarb
2,000 200 20 2,000 200 20
DBCP
:~5.0
1.5 1.0 1.0 14.5 15.0 21.0 1.0 2.5 25.0 0.5 2.5 20.0
Table 3. Growth (O.D. X 100) of R. leguminosarum strains, grown in medium 79, containing various levels of herbicides and nematocides Treatment ppm Control Dinitramine
R. leguminosarum strains M·360 100 10
Methazol
100 10
Aldicarb
2,000 200 20 2,000 200 20
DEep
M·361
M·362
10.3 2.3 :1.0 15.3 4.5 6.5 6.0 12.5 18.0 17.0 3.0 6.5 5.5
14.0 1.0 11.5 14.5 12.0 10.0 10.5 3.0 9.0 12.0 2.5 2.0 2.0
29.0 14.5 26.0 30.5 16.5 16.0 19.0 U.5 16.0 26.0 7.5 25.0 48.0
Efficiency of Rhizobium leguminosarum as Affected by Certain Herbicides etc.
729
Aldicarb at 20 ppm had no effect on the growth of broad bean shoots; otherwise, all chemicals at all levels had a clear stunting effect, as compared with the inoculated control. Symptoms of phytotoxicity were apparent with 2,000 ppm Aldicarb, 200 and 2,000 ppm DBCP, and with Methazol and Dinitramine at 1, 10, and 100 ppm. The cotyledons were necrotic and discoloured, the roots thickened and abnormally branched. Growth of R. leguminosarum strains M 360, M 361, and M 362 in medium 79, treated with the various levels of herbicides and nematocides, is presented in Table 3. With Aldicarb, strain M 360 was significantly stimulated at 20 and 200 ppm, but with 2,000 ppm there was only a slight stimulation. Twenty ppm of this nematocide did not appreciably affect the growth of strains M 361 and M 362, but concentrations of 200 and 2,000 ppm were inhibitory. DBCP was highly toxic to strains M 360 and M 361 at all levels. Strain M 362, however, was stimulated by 20 ppm, tolerant to 200 ppm, and inhibited with 2,000 ppm. Growth of strains M 361 and M 362 was not affected by the herbicide Dinitramine at 1 and 10 ppm levels, but was reduced by the 100 ppm level. Strain M 360 was only tolerant to 1 ppm Dinitramine, but was inhibited by 10 and 100 ppm. Methazol was inhibitory to strains M 360 and M 362 at all levels used. Growth of strain M 361, however, was only slightly reduced by the various levels of this herbicide.
Discussion Earlier studies on the effect of Trifluralin on nodulation of broad beans, inoculated with R. leguminosarum, have shown that the application of 0.5, 1.0,2.0, and 51jfeddan at the time of planting inhibited nodulation and caused root deformation (GAFAR et al. 1969). Application of this herbicide 21 days after planting caused a stimulation of growth by 0.5-2.0 ljfeddan, whereas 5ljfeddan inhibited the growth appreciably. Similarly, nodulation and growth of cowpea plants were inhibited upon the application of 1, 3, and 61/feddan of Trifluralin at the time of planting (HAMDI and TEWFIK 1969). Application of 11/feddan of this herbicide 27 days before planting, however, had no inhibitory effect on the growth of cowpea. In agreement with these findings, the yield of broad bean in the present studies was not affected by the application of Dinitramine, Methazol, Aldicarb, and DBCP when applied 4 weeks after planting. With the application of these compounds at the time of planting, however, the yield was significantly reduced with the herbicides Methazol and Dinitramine and the nematocide Aldicarb. DBCP also caused some reduction in yield, but the reduction was not significant. In tube cultures, after 3 weeks, phytotoxic symptoms were apparent with Methazol and Dinitramine at all levels, but only with higher levels of the nematocides. Phytotoxic symptoms were also apparent with the higher levels of Dinitramine in two-week old plants grown in pots. With progress of time, however, the plants recovered, particularly those grown in the presence of the lower concentrations with the exception of those treated with Dinitramine. The recovery of the plants may be due to chemical and biological degradation of the added chemicals in soil as has been noted with other chemicals, e.g. 3,5 dinitro-o-cresol (DNOC) (TEWFIK and EVANS 1966), DNEP and nitrophenols (TEWFIK 1966), Trifluralin (HAMDI and TEWFIK 1969 a and b), or to the ability of the plants to degrade the chemicals to non- toxic compounds. TEWFIK and ZAKI (1969) reported that broad bean plants metabolized DNOC. Also
730
lVI.
s. TEWFIK, lVI. S. EMBABI,
and Y. A. HAMDI
nodular and plant tissues of bird's foot trefoil were reported to metabolize 2,4 DB (GARCIA and JORDAN 1969). Growth of R.leguminosarum strains M360, M 361, and M 362 was affected differently with the various compounds. KAZUBIAK (1968a and b) studied the effect of the herbicides afalon, aretit, and lirobetarex on R. meliloti and R. lupini. He showed that resistance to these herbicides may be connected with permeability, dehydrogenase activity, or the development of alkalinity in the culture media. Resistance of rhizobia to certain herbicides may also be due to the ability of these organisms to degrade such chemicals. Such mechanism was observed in the case of DNOC which was degraded by certain strains of rhizobia and Azotobacter (HAMDI and TEWFIK 1970). In the present studies the nature of resistance of some strains of rhizobia to certain levels of herbicides and nematocides was not examined and awaits further investigation. In general, these studies indicate that toxic effects of the herbicides and the nematocides are exerted both on the organisms and the symbiont. The application of nematocides is usually recommended to be completed two weeks before planting (MOUNTAIN 1965). In the present work, however, the use of DBCP even at the time of planting was harmless. With regard to the herbicides, it is recommended to avoid the use of these compounds with broad beans. Even rates or residues of Ij2ljfeddan at the time of planting would be harmful. Application of these herbicides at this rate 4 weeks after planting, however, was fairly harmless. References ALLEN, O. N.: Experiments in Soil Bacteriology. Burgess Publ. Co. Minneapolis, Minnesota, U.S.A. 1959. BROCKWELL, J. : Accuracy of a plant infection technique for counting populations of Rhizobium trifolii. Appl. Microbiol. II (1963), 377 - 383. Epps, 1. M., and CHAMBERS, A. Y.: Nematocidal seed treatment for control of Heterodera glycine8 in soybeans. Phytopathology 54 (1964), 622 (Abstract). GAFAR, ZEINAB, A., HAiVIDI, Y. A., and TEWFIK, M. S.: Effect of trifluralin on the nitrogen fixed in broad beans. First Arab Conf. of Physiological Sciences, Cairo 1969. GARCIA, M. M., and JORDAN, D. C.: Action of 2,4.DB and dalapon in the symbiotic properties of L. corniculatu8. Plant and Soil 30 (1969), 317 -336. GROSBARD, E.: Effect of herbicides on the symbiotic relationship between R. trifolii and white clover. Occasional Symposium No.6., British Grassland Soc., 1970. HAMDI, Y. A., and TEwFIK, M. S.: Effect of the herbicide trifluralin on nitrogen fixation in Rhizobium and Azotobacter and on nitrification. Acta Microbiol. Polon. 1 (18) (1969a), 53-58. - Decomposition of the herbicide trifluralin by a pseudomonad. Acta Microbio!' Polon. 1 (1969b), 83-84. - The degradation of the herbicide DNOC by rhizobia and Azotobacter. Soil. BioI. Biochem. 2 (1970),163-166. KAZUBIAK, H.: Effect of herbicides on Rhizobium. II. Adaptation of Rhizobium to afalon, aretit, and lirobetarex. Acta Microbiol. Polon. 17 (1968 a), 41- 50. MOUNTAIN, W. B.: Nematocides. Agric. lnst. Rev., March, April 1965. Canada Dept. of Agric., Vineland Station, Ontario, Canada. TEWFIK,]\I. S.: The metabolism of certain aromatic nitroherbicides by soil micro-organisms. Ph. D. Thesis University of Wales, Bangor 1966. and EVANS, 'V. C.: The metabolism of 3,5-dinitro-o-cresol (DNOC) by soil micro-organisms. Biochem. J. 99 (1966), 31 pp. and ZAKI, M. A.: The reduction of DNOC by plant tissues in vitro. 6th Scientific Arab Congress, Damascus, 1969, Part 2, 46:~-465.
Efficiency of Rhizobium legumino8arum as Affected by Certain Herbicides etc.
731
Tu, C. M.: Effect of four nematocides on activities of microorganisms in soil. Appl. Microbiol. 23 (1972),498-501. ZAYED, M. N., ABD-EL-NAssER, M., ABD-EL-MALEK, Y., and MONIB, M.: Effect of DD on nitrogen transformations in soil. Zbl. Bakt. II 122 (1968), 528-532. Authors' address: Dr. M. S. TEWFIK, M. S. EMBABI, and Dr. Y. A. HAMDI, Agriculture Research Centre, Dept. of Weed Research, Orman, Giza, A.R.E.
[Department of Weed Research, Plant Pathology, and Microbiology, Agriculture Research Centre, Orman, Giza, A.R.E.]
Efficiency of Rhizobium leguminosarum as Affected by Certain Herbicides and Nematocides M. S. Tewfik, M. S. Embabi, and Y. A. Hamdi Summary Broad beans, planted in pots, were treated with the herbicides Dinitramine and Methazol at the rates of y:!, 1, and 2 litersjfeddan, and the nematocides DBCP and Aldicarb at the rates of 10, 20, and 40 liters or kilogramejfeddan. The chemicals were added at the time of planting or 28 days after. With the exception of DBCP, which had no effect, application of the compounds at the time of planting caused a significant reduction in yield. Application of the chemicals 28 days after planting, however, had no significant harmful effect on the yield. Broad bean seedlings were also grown in agar tubes treated with 1, 10, and 100 ppm of Dinitramine or Methazol, or 20, 200, and 2,000 ppm of DBCP and Aldicarb. Nodulation was completely absent in all treatments with the exception of plants grown with 20 ppm Aldicarb, where four nodules were formed, compared with 22 on the control plants. Growth of shoots was stunted, particularly with Aldicarb and DBCP at 200 and 2,000 ppm and Dinitramine at all the concentrations tested. Growth of Rhizobium leguminosarum strains M 360, M 361, and M 362 in medium 79, containing 1, 10, and 100 ppm of Dinitramine and Methazol and 20, 200, and 2,000 ppm of Aldicarb and DBCP was estimated. In general, all strains were more tolerant to Dinitramine and Aldicarb, especially at low concentrations. DBCP and Methazol caused an inhibitory effect on the growth of the strains tested, except strain M 362 which was not affected by 200 ppm and stimulated by 20 ppm of DEep.
Zusammenfassung In GefaJ3e ausgepflanzte Pferdebohnen wurden mit y:!, 1 und 2ljFeddan der Herbizide "Dinitramin" und "Methazol" behandelt. Von den Nematoziden "DBCP" und "Aldicarb" wurden 10, 20 und 40 I bzw. kgjFeddan zugegeben. Die genannten Chemikalien wurden bei der Aussaat bzw. nach 28 Tagen verabreicht. AuJ3er DBCP fiihrte die Zugabe dieser Chemikalien bei der Ausssaat zu einer signifikanten Abnahme der Ertrage. Ihre Zugabe 28 Tage nach der Aussaat wirkte dagegen nicht signifikant ertragsmindernd. Des weiteren wurden Keimlinge von Pferdebohnen auch in Agarriihrchen angezogen und mit 1, 10 und 100 ppm Dinitramin bzw. Methazol oder 20, 200 und 2000 ppm DBCP bzw. Aldicarb behandelt. Bei allen Behandlungen war die Kniillchenbildung viillig gehemmt, auJ3er bei den mit 20 ppm Aldicarb aufwachsenden Pflanzen, auf deren \Vurzeln sich 4 Kniillchen bildeten, wahrend die Kontrollpflanzen 20 Kniillchen besaJ3en. Das Schotenwachstum wurde durch 200 und 2000 ppm Aldicarb und DBCP sowie durch aile Dinitramin·Konzentrationen gehemmt. Weiterhin wurde das Wachstum der Rhizobium leguminosarum·Stamme M 360, M 361 und M 362 in Nahrliisung 79, die 1, 10 und 100 ppm Dinitramin und Methazol bzw. 20, 200 und 2000 ppm Aldicarb und DBCP enthielt, bestimmt. 1m allgemeinen waren aile Stamme toleranter gegeniiber Dinitramin und Aldicarb, besonders bei niedrigen Konzentrationen. DBCP und Methazol wirkten hemmend auf das Wachstum der untersllchten Stamme - auJ3er Stamm 362, der durch 200 ppm DBep nicht negativ beeinfluJ3t und durch 20 ppm gefiirdert wurde.
726
M. S.
TEWFIK,
J\L S.
EMBABI,
and Y. A.
HAMDI
With the increasing use of pesticides in agriculture, added interest in the effect of these compounds on rhizobia and rhizobia-legume systems has been noted. GROSSBARD (1970) classified the reported studies of the effect of herbicides on various species of Rhizobium as follows: 1) no effect, e.g. dalapon , simazine, prometryne; 2) inhibitory, bacteriostatic, or bactericidal at levels from several thousand to 100 ppm, e.g. phenoxyacetic and phenoxybutyric acids, DNOC, and pyrazon; 3) inhibitory below 100 ppm, e.g. dinoseb acetate and linuron; and 4) stimulatory, as with some chlorosubstituted aliphatic acids. F ew reports have evaluated the effect of nematocides on rhizobia or rhizobialegume symbiosis. Epps and CHAMBERS (1964) reported that DBCP, applied as a seed treatment at rates ranging from l/S-l/Z gal/acre, caused a reduction in both stand and yield of soybean in some trials. Tu (1972) evaluated the effect of the nematocides Dasanit, Carbofuran, D-D, and Vorlex on ammonification, nitrification, and sulfur-oxidation. ZAYED et al. (1968) reported that D-D did not affect the activities of ammonifiers, while the activities of the nitrifiers were greatly affected for a period of 30-45 days, after which the toxic effect of the nematocides gradually disappeared. The present work aimed at studying the effect of the herbicides Dinitramine and Methazol and the nematocides Aldicarb and DBCP on: a) growth and yield of broad beans, b) symbiotic association between R. leguminosarum and broad bean plants, and c) growth of R . leguminosarum strains in vitro.
Materials and Methods Str ains Locally isolated Rhizobium leguminosarum strains M 360, M 361, and M 362 were used. The rhizobia strains were grown in yeast extract mannitol medium, comm only known as medium 79 (ALLEN 1959). Herbicides and nematocid es The herbicide Methazol (2-(3' ,4' -dichlorophenyl)-4-methyl-l ,2,4-oxodiazolidine-3,5-dione), kn own commercially as probe or VCS-438; and t he lately developed h erbicide Dinitramine (N,N' -diethyl-2'di nitro.6.trifluoromethyl-m -phenylene diamine), known commercially as Cobex or F 21; and t he n ematocides DECP (1 ,2-dibromo-3-chloropropone), known commercially as Fumazone 121, and Aldicarb (2-methyl-2-(methylthio) propionaldehyde-O-(methyl-carbamyl oxime), known commercially as Temik 10 g were used. Effect o f the chemicals on the yie ld of broad beans Earthen pots, 30 em in diameter and containing approximately 5 kg of Nile silt, were planted with 10 pre-soaked seeds of broad beans (Vicia laba). Twenty ml of the mixed rhizobia cultures strains M 360, M 361, and M 362, grown separately and mixed just before use, were transferred into each pot. The plants were thinned down to 5 per pot two weeks after emergence. The herbicides and the nematocides were added to t he pots either at the time of planting or 28 days after planting. Methazol and Dinitramine wer e added at the rates of Yt, 1, and 2 kg or I/feddan , re~ pectively, while Aldicarb a nd DECP were added at the rates of 10, 20, and 40 kg or l/feddan, respectively. Each treatment was performed in 5 replicates. A set of plants was left wit hout chemical treatment as a control. The pots were placed at random on a greenhouse floor and watered with tap water as needed . The experiment was terminated after five months; the pods were collected, t h e seeds were extracted, air-dried, and weighed. Effect o f the chemicals on m ed ium R. leguminosarum strains M containing 1,10, and 100 ppm of of the nematocides DECP and
growt h of Rhizobium legumino sa rum strains in culture 360, M 361, and M 362 were inoculated into 20 ml medium 79, the herbicides Methazol and Dinitramine and 20,200, and 2,000 ppm Aldicarb. The respective dilutions of the ch emicals were made of
Efficiency of Rhizobium leguminosarum as Affected by Certain Herbicides etc.
727
concentrated stock solutions, using sterile distilled water. With Aldicarb, 2 grams of the granular material were extracted in 10 ml ethanol (96 %) and respective dilutions in water were made then. After 8 days of incubation at 28°C, 5 ml of each culture were suspended in 10 ml acetone, which dissolved the chemicals and left the cells suspended. Then optical density readings were immediately taken at 600 nm, using an Unicum spectrophotometer. Effect of the chemicals on the rhizobia legume system in tube cultures One hundred ml nitrogen-free seedlings agar (BROCKWELL 1963) was prepared in glass tubes, 35 mm in diameter and 30 cm long. Seeds of Vicia faba were surface-sterilized (ALLEN 1959), pregerminated, and transplanted into the tubes. One ml of 96-hour old cultures of R. leguminosarum strains M 360, M 361, and M 362 were inoculated into each tube. Then each tube received one ml of the appropriate dilution of the chemicals to give final concentrations of 1, 10, and 100 of the herbicides Methazol and Dinitramine, and 20, 200, and 2,000 ppm of the nematocides DBCP and Aldicarb. The tubes were incubated at 22°C in a controlled environment greenhouse for 8 weeks. The seedlings were uprooted then and examined for nodulation and for symptoms of phytotoxicity. The shoots were separated from the roots, dried, and weighed.
Results Dry weight of broad bean yield (seeds), as affected by the different levels of the herbicides and nematocides, is presented in Table 1. When the chemicals were applied at planting, a highly significant reduction of the yield occurred at all levels of the herbicides Dinitramine and Methazol. The reduction was proportional to the rate of the chemical applied. With nematocides, Aldicarb showed a significant depressing effect on the yield at all levels used, whereas DBep had no effect. Inspection of the two-week old seedlings (plants removed at thinning down) showed that in the presence of dinitramine at one and 2liters/feddan the growth Table 1. Yield (g of seeds/plant) of broad bean, treated with various levels of herbicides and nemstocides (Data are averages of 5 replicates) Treatment and Rate/feddan
Control Dinitramine 1 0.5 1.0 2.0 Methazol 0.5 1.0 2.0 Aldicarb kg 10 20 40 DBCP 10 20 40
Time of application at planting
4 weeks after planting
6.0
6.0
3.32 ) 2.9 2 ) 3.0 2 )
5.9 4.6 3.6
2.6 2 ) 2.6 2 ) 1. 72 )
4.5 3.6 2.3
4.41 ) 4.41 ) 4.01 )
5.8 5.4 5.0
5.9 5.9 5.2
6.6 6.4 5.6
1) Significant at 5 % level. 2) Significant at 1 % level.
728
M. S.
TEWFIK,
M. S.
EMBABI,
and Y. A.
HAlIfDI
was stunted and the roots were abnormally thickened. No abnormal symptoms were observed, however, with the lowest rate of this chemical or with the other three chemicals at any of the levels used. Application of the chemicals 4 weeks after planting had no significant effect on the yield. With the herbicides, however, there was some reduction in yield, but the reduction was not statistically significant. The effect of the different herbicides and nematocides on the growth and nodulation of broad beans in tube cultures is presented in Table 2. Nodulation failed in all treated plants with the exception of those plants grown with Aldicarb at 20 ppm where 4 nodules were formed, compared with 22 nodules on the control plants. Table 2. Effect of certain herbicides and nematocides on growth and nodulation of broadbean plants, grown in tube cultures (Data are averages of 4 replicates) Treatment ppm
Length of shoots (cm)
Dry weight of shoots (mg)
Number of nodules
Non·inoculated control Inoculated control Dinitramine 100 10
26.3
293 360 20 20 220 140 170 172 50 280 338 10 21 216
None 22 None None None None None None None None 4 None None None
Methazol
100 10
Aldicarb
2,000 200 20 2,000 200 20
DBCP
:~5.0
1.5 1.0 1.0 14.5 15.0 21.0 1.0 2.5 25.0 0.5 2.5 20.0
Table 3. Growth (O.D. X 100) of R. leguminosarum strains, grown in medium 79, containing various levels of herbicides and nematocides Treatment ppm Control Dinitramine
R. leguminosarum strains M·360 100 10
Methazol
100 10
Aldicarb
2,000 200 20 2,000 200 20
DEep
M·361
M·362
10.3 2.3 :1.0 15.3 4.5 6.5 6.0 12.5 18.0 17.0 3.0 6.5 5.5
14.0 1.0 11.5 14.5 12.0 10.0 10.5 3.0 9.0 12.0 2.5 2.0 2.0
29.0 14.5 26.0 30.5 16.5 16.0 19.0 U.5 16.0 26.0 7.5 25.0 48.0
Efficiency of Rhizobium leguminosarum as Affected by Certain Herbicides etc.
729
Aldicarb at 20 ppm had no effect on the growth of broad bean shoots; otherwise, all chemicals at all levels had a clear stunting effect, as compared with the inoculated control. Symptoms of phytotoxicity were apparent with 2,000 ppm Aldicarb, 200 and 2,000 ppm DBCP, and with Methazol and Dinitramine at 1, 10, and 100 ppm. The cotyledons were necrotic and discoloured, the roots thickened and abnormally branched. Growth of R. leguminosarum strains M 360, M 361, and M 362 in medium 79, treated with the various levels of herbicides and nematocides, is presented in Table 3. With Aldicarb, strain M 360 was significantly stimulated at 20 and 200 ppm, but with 2,000 ppm there was only a slight stimulation. Twenty ppm of this nematocide did not appreciably affect the growth of strains M 361 and M 362, but concentrations of 200 and 2,000 ppm were inhibitory. DBCP was highly toxic to strains M 360 and M 361 at all levels. Strain M 362, however, was stimulated by 20 ppm, tolerant to 200 ppm, and inhibited with 2,000 ppm. Growth of strains M 361 and M 362 was not affected by the herbicide Dinitramine at 1 and 10 ppm levels, but was reduced by the 100 ppm level. Strain M 360 was only tolerant to 1 ppm Dinitramine, but was inhibited by 10 and 100 ppm. Methazol was inhibitory to strains M 360 and M 362 at all levels used. Growth of strain M 361, however, was only slightly reduced by the various levels of this herbicide.
Discussion Earlier studies on the effect of Trifluralin on nodulation of broad beans, inoculated with R. leguminosarum, have shown that the application of 0.5, 1.0,2.0, and 51jfeddan at the time of planting inhibited nodulation and caused root deformation (GAFAR et al. 1969). Application of this herbicide 21 days after planting caused a stimulation of growth by 0.5-2.0 ljfeddan, whereas 5ljfeddan inhibited the growth appreciably. Similarly, nodulation and growth of cowpea plants were inhibited upon the application of 1, 3, and 61/feddan of Trifluralin at the time of planting (HAMDI and TEWFIK 1969). Application of 11/feddan of this herbicide 27 days before planting, however, had no inhibitory effect on the growth of cowpea. In agreement with these findings, the yield of broad bean in the present studies was not affected by the application of Dinitramine, Methazol, Aldicarb, and DBCP when applied 4 weeks after planting. With the application of these compounds at the time of planting, however, the yield was significantly reduced with the herbicides Methazol and Dinitramine and the nematocide Aldicarb. DBCP also caused some reduction in yield, but the reduction was not significant. In tube cultures, after 3 weeks, phytotoxic symptoms were apparent with Methazol and Dinitramine at all levels, but only with higher levels of the nematocides. Phytotoxic symptoms were also apparent with the higher levels of Dinitramine in two-week old plants grown in pots. With progress of time, however, the plants recovered, particularly those grown in the presence of the lower concentrations with the exception of those treated with Dinitramine. The recovery of the plants may be due to chemical and biological degradation of the added chemicals in soil as has been noted with other chemicals, e.g. 3,5 dinitro-o-cresol (DNOC) (TEWFIK and EVANS 1966), DNEP and nitrophenols (TEWFIK 1966), Trifluralin (HAMDI and TEWFIK 1969 a and b), or to the ability of the plants to degrade the chemicals to non- toxic compounds. TEWFIK and ZAKI (1969) reported that broad bean plants metabolized DNOC. Also
730
lVI.
s. TEWFIK, lVI. S. EMBABI,
and Y. A. HAMDI
nodular and plant tissues of bird's foot trefoil were reported to metabolize 2,4 DB (GARCIA and JORDAN 1969). Growth of R.leguminosarum strains M360, M 361, and M 362 was affected differently with the various compounds. KAZUBIAK (1968a and b) studied the effect of the herbicides afalon, aretit, and lirobetarex on R. meliloti and R. lupini. He showed that resistance to these herbicides may be connected with permeability, dehydrogenase activity, or the development of alkalinity in the culture media. Resistance of rhizobia to certain herbicides may also be due to the ability of these organisms to degrade such chemicals. Such mechanism was observed in the case of DNOC which was degraded by certain strains of rhizobia and Azotobacter (HAMDI and TEWFIK 1970). In the present studies the nature of resistance of some strains of rhizobia to certain levels of herbicides and nematocides was not examined and awaits further investigation. In general, these studies indicate that toxic effects of the herbicides and the nematocides are exerted both on the organisms and the symbiont. The application of nematocides is usually recommended to be completed two weeks before planting (MOUNTAIN 1965). In the present work, however, the use of DBCP even at the time of planting was harmless. With regard to the herbicides, it is recommended to avoid the use of these compounds with broad beans. Even rates or residues of Ij2ljfeddan at the time of planting would be harmful. Application of these herbicides at this rate 4 weeks after planting, however, was fairly harmless. References ALLEN, O. N.: Experiments in Soil Bacteriology. Burgess Publ. Co. Minneapolis, Minnesota, U.S.A. 1959. BROCKWELL, J. : Accuracy of a plant infection technique for counting populations of Rhizobium trifolii. Appl. Microbiol. II (1963), 377 - 383. Epps, 1. M., and CHAMBERS, A. Y.: Nematocidal seed treatment for control of Heterodera glycine8 in soybeans. Phytopathology 54 (1964), 622 (Abstract). GAFAR, ZEINAB, A., HAiVIDI, Y. A., and TEWFIK, M. S.: Effect of trifluralin on the nitrogen fixed in broad beans. First Arab Conf. of Physiological Sciences, Cairo 1969. GARCIA, M. M., and JORDAN, D. C.: Action of 2,4.DB and dalapon in the symbiotic properties of L. corniculatu8. Plant and Soil 30 (1969), 317 -336. GROSBARD, E.: Effect of herbicides on the symbiotic relationship between R. trifolii and white clover. Occasional Symposium No.6., British Grassland Soc., 1970. HAMDI, Y. A., and TEwFIK, M. S.: Effect of the herbicide trifluralin on nitrogen fixation in Rhizobium and Azotobacter and on nitrification. Acta Microbiol. Polon. 1 (18) (1969a), 53-58. - Decomposition of the herbicide trifluralin by a pseudomonad. Acta Microbio!' Polon. 1 (1969b), 83-84. - The degradation of the herbicide DNOC by rhizobia and Azotobacter. Soil. BioI. Biochem. 2 (1970),163-166. KAZUBIAK, H.: Effect of herbicides on Rhizobium. II. Adaptation of Rhizobium to afalon, aretit, and lirobetarex. Acta Microbiol. Polon. 17 (1968 a), 41- 50. MOUNTAIN, W. B.: Nematocides. Agric. lnst. Rev., March, April 1965. Canada Dept. of Agric., Vineland Station, Ontario, Canada. TEWFIK,]\I. S.: The metabolism of certain aromatic nitroherbicides by soil micro-organisms. Ph. D. Thesis University of Wales, Bangor 1966. and EVANS, 'V. C.: The metabolism of 3,5-dinitro-o-cresol (DNOC) by soil micro-organisms. Biochem. J. 99 (1966), 31 pp. and ZAKI, M. A.: The reduction of DNOC by plant tissues in vitro. 6th Scientific Arab Congress, Damascus, 1969, Part 2, 46:~-465.
Efficiency of Rhizobium legumino8arum as Affected by Certain Herbicides etc.
731
Tu, C. M.: Effect of four nematocides on activities of microorganisms in soil. Appl. Microbiol. 23 (1972),498-501. ZAYED, M. N., ABD-EL-NAssER, M., ABD-EL-MALEK, Y., and MONIB, M.: Effect of DD on nitrogen transformations in soil. Zbl. Bakt. II 122 (1968), 528-532. Authors' address: Dr. M. S. TEWFIK, M. S. EMBABI, and Dr. Y. A. HAMDI, Agriculture Research Centre, Dept. of Weed Research, Orman, Giza, A.R.E.
