This article was downloaded by: [University of Toronto Libraries]On: 25 February 2013, At: 04:35Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Toxicological & EnvironmentalChemistryPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/gtec20

Compatibility of a soybean peatinoculant with some seed appliedfungicides and microgranularinsecticidesGérard Catroux a & Fernand Arnaud ba INRA, Laboratoire de Microbiologie des Sols, BP 1540 , F.21034 , Dijon Cédexb CETIOM, 174 avenue Victor Hugo, 75116, ParisVersion of record first published: 19 Sep 2008.

To cite this article: Gérard Catroux & Fernand Arnaud (1991): Compatibility of a soybeanpeat inoculant with some seed applied fungicides and microgranular insecticides,Toxicological & Environmental Chemistry, 30:3-4, 229-239

To link to this article: http://dx.doi.org/10.1080/02772249109357661

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes.Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expresslyforbidden.

The publisher does not give any warranty express or implied or make anyrepresentation that the contents will be complete or accurate or up to date. Theaccuracy of any instructions, formulae, and drug doses should be independentlyverified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand, or costs or damages whatsoever orhowsoever caused arising directly or indirectly in connection with or arising out ofthe use of this material.

Toxicological and Environmental Chemistry, Vol. 30, pp. 229-239 © 1991 Gordon and Breach Science Publishers S.A.Reprints available directly from the publisher Printed in the United KingdomPhotocopying permitted by license only

COMPATIBILITY OF A SOYBEAN PEATINOCULANT WITH SOME SEED

APPLIED FUNGICIDES ANDMICROGRANULAR INSECTICIDES

GÉRARD CATROUX

INRA, Laboratoire de Microbiologie des Sols, BP 1540 F. 21034 Dijon Cédex.

and

FERNAND ARNAUD

CETIOM, 174 avenue Victor Hugo, 75116 Paris.

Soybean cropping is being developed in Italy and France, and the practices of disinfection of seeds withfungicides and the treatment of soil with microgranular insecticides are increasing. Since farmers intendto combine seed and soil treatments with inoculation using Bradyrhizobium japonicum, several questionsconcerning the compatibility of soybean inoculants with these practices have arisen.

We describe the procedure we are using to assess the possible effects of both fungicides and micro-granular insecticides on survival of Bradyrhizobium japonicum during both inoculation and early nodula-tion of the soybean in the glasshouse. Reliability and possible conclusions are discussed.

KEY WORDS: Bradyrhizobium japonicum, soybean, inoculants, fungicides, insecticides.

1 INTRODUCTION

Soybean cropping is now developing in Italy and France. When soybean is grownfor the first time in European soil, the plant generally must be inoculated, since thebacteria, Bradyrhizobium japonicum, is not indigenous.

Inoculation is practised by farmers using peat inoculants containing high numbersof viable B. japonicum—more than 109 per g—and no contaminants. For France,these inoculants are applied in two ways:

—directly on the seed by the classical "slurry" method, with or without additives("stickers", micronutrients... ), using a concrete-mixer.

—indirectly, by seed-bed inoculation, mixing the peat inoculant with mineralmicrogranules of 0.2-0.8 mm size and dispensing throughout the seed-bed, as is donefor microgranular pesticides.

This last method is easier to use since farmers mix the inoculant with 10 kgmicrogranules per ha instead of mixing with 100-150 kg seeds per ha for the "slurry"method.

Inoculation must furnish a maximal amount of viable B. japonicum per seed toallow good nodulation. It was clearly demonstrated by field experiments on soil

229

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

230 G. CATROUX AND F. ARNAUD

previously free of B. japonicum, that the rate of inoculation influences nodulation i.e.,number and weight of nodules and often seed yield1"4. In France, the minimumnumber of B. japonicum supplied by inoculants must be 106 per seed, which generallyallows a good nodulation.

Care must be taken with respect to possible losses of bacteria during the inocula-tion. Losses of 50 to 90% of viable B. japonicum on the seed were found duringinoculation and sowing (unpublished results). These losses are probably due to:

—death of bacteria during desiccation of the seed

—inoculant not firmly fixed on the seed.

Another possible loss of viable B. japonicum may arise from adverse effects ofpesticides. For example, the fungicides captan and carboxine were found to inhibitthe survival of B. japonicum on inoculated seed and also to inhibit soybeannodulation5'6. Captan was also found to depress seed yield in field experiments7.

Possible effects of pesticides on B. japonicum and on soybean—B. japonicumsymbiosis are well documented. However, data are difficult to interpret since not allthe possible effects on symbiosis have been investigated and the laboratory resultshave not been checked in the field experiments.

Effects of pesticides on B. japonicum were assessed in different ways:

—inhibition of bacterial growth was detected by a test on agar medium using filterpaper disks with different pesticide concentrations8'9 or a gradient method10 or aporcelain bead method11. B. japonicum cultures in liquid media with increasingpesticide concentrations were also used10.

—survival of B. japonicum in the presence of pesticides may be studied in bacterialsuspensions9 or directly with inoculated (treated) seeds,5'11.

—effects on nodulation, plant growth and N2 fixation are more often studied inpots in glasshouses or in growth chambers5 '6 '8 '11"13. The same kind of study wasalso performed in field trials7 '10 '14.

Actually, in France, seed disinfection by means of fungicides and soil treatmentsby means of microgranular insecticides are increasing and should become compulsoryin the future.

Two main questions arise at the farmer's level:

—are the usual seed disinfectants (not registered for soybean) compatible with B.japonicum inoculation?

—are the insecticides microgranules used in soil treatment useful as inoculantcarrier for the seed bed inoculation method?

The purpose of this report was to assess the possible negative effects of severalfungicides and insecticides on B. japonicum and soybean—B. japonicum symbiosis. Asunderlined by Diatloff1 i, we tried to consider the different step of symbiosis establish-ment: (a) Survival of B. japonicum, (b) infection process, (c) nodule formation, (d) N 2

fixation estimated by seed yield.Results of laboratory, glasshouse and field experiments are discussed.

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

COMPATIBILITY OF A SOYBEAN PEAT INOCULANT 231

2 MATERIAL AND METHODS

2.1 Chemicals

10 commercial products were used depending on whether seed or soil were treated.For the seed treatment, we used 4 fungicides, metalaxyl (Apron'), iprodione (Rovral1),carbendazime (Bavistiner), carbendazime + Cu oxyquinoleate (Quinolate pror), and1 insecticide, diethion (Hylemoxr). For the soil treatment, we used the insecticidemicrogranules of carbofuran (Curaterr), chlorfenvinphos (Birlaner), terbuphos (Coun-ter'), furathiocarbe (Deltanetr), tefluthrine (Forcer). Commercial clay microgranulesdesigned for inoculation were used for the controls.

2.2 Effects of pesticides on the growth of B. japonicum

Plates of solid culture medium were inoculated with 106 viable B. japonicum cellseach. The strain used was MSDJ G49 (IARI SB 16), which is the registered strain forsoybean inoculation in France. Soybean {Glycine max (L.) Merr.) cv Weber, weretreated following the recommended level as indicated in Table 1.

2 treated seeds were placed in a plate and the plates were incubated at 28°C. After2 days, the roots which emerged were aseptically cut and the diameter of the inhibitionzone was measured after 6 days growth. Only 3 microgranular insecticides were tested(carbofuran chlorfenvinphos and terbuphos) placing 16 mg pesticides on the sameplates as described above.

2.3 Survival of inoculated B. japonicum on treated seeds

Soyben (Glycine max (L.) Merr.) seeds, cv Labrador, were treated at levels indicatedin Table 1. 200 g of treated seeds were inoculated by the slurry method using acommercial inoculant (1 g inoculant = 2,14 109 viable B. japonicum mixed with 2 mlsterile water). Inoculated seeds were stored in a closed 500 ml glass bottle at 20°Cfor the experiment.

Samples of 50 seeds were analyzed after 1, 4 and 24 hours storage (4 replicates).Viable B. japonicum were recovered after agitation of 50 seeds in 100 ml of sterile

Table 1 Seed treatment levels used.

Fungicides

metalaxyliprodionecarbendazimeCarbendazime + Cu oxyquinoleate

Insecticides

diethion

g. commercial product/WO kg seed

400300150300

1,200

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

232 G. CATROUX AND F. ARNAUD

water and counted after growth on usual solid media. Two distinct experiments wereconducted.

2.4 Survival of inoculated B. japonicum on microgranules

Microgranules of pesticides were inoculated in the same ratio as for the "seed bed"inoculation, adding 4 g peat inoculant without water, to 100 g of microgranules andmixing carefully.

Inoculated microgranules were stored in a closed 250 ml glass bottle at 20°C forthe experiment.

Samples of 5 g were analyzed after 1, 4 and 24 hours storage (3 replicates) as forinoculated seeds.

2.5 Effect of pesticides on the short term nodulation

Seeds used for enumeration of surviving B. japonicum 1, 4 and 24 hours afterinoculation were sown in 2,5 1 plastic pots filled with perlite. 4 replicates with 4 seedseach were grown using N free-nutrient solution in randomized blocks in glasshouse.After 3 weeks, the plants were harvested and nodules counted. As in the survivalstudy, two distinct experiments were conducted.

Samples of 5 g inoculated microgranules, stored for 1, 4 and 24 hours, were usedto inoculate seeds (cv, Labrador), which were sown as were the inoculated seeds. Theamount of microgranules per seed was 17 mg per seed, which is in the same ratio asrecommended by manufacturers (10 kg per ha = 10 kg per 600 000 seeds).

2.6 Field trial

A field trial consisting of four replicates in completely randomized block design wasconducted over one year in a loamy soil with the soybean line Weber. Comparisonswere made between the two methods of inoculation: namely, "slurry" or "seed bed"inoculation. Pesticides used were metalaxyl, iprodione, carbendazime, carbendazi-me + Cu oxyquinoleate and diethion.

3 RESULTS

3.1 Seed treatments

Effect of pesticides on the growth of B. japonicum There were no inhibition zonesdetected on inoculated plates around treated seeds for the 7 pesticides (metalaxyl,iprodiome, carbendazime, carbendazime + Cu oxyquinoleate, carbofuran and terbu-phos).

Survival of B. japonicum on treated seeds Numbers of viable bacteria recovered 1, 4and 24 hours after inoculation are shown in Figures 1 and 2. Results indicate

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

COMPATIBILITY OF A SOYBEAN PEAT INOCULANT 233

Log number of recovered7 T B . japonicum

CONTROL

BMETALAXYL

D IPRODIONE

CV:2.6%24

CV:1.9%

hours afterinoculation

—treatment from same sampling time followed by the same letterare not statistically different (P-0.06)

Figure 1 Survival of B. japonicum on treated seeds.—Treatments from same sampling time followed bythe same letter are not statistically different at p:0.05.—Coefficient of variation at 1, 4 and 24 hours: 2.6,1.3, 1.9 p. cent.

significantly lower survival (P = 0.05) than for the control for metalaxyl, carbenda-zime and diethion after 1 and 4 hours and for metalaxyl, iprodione, carbendazimeand diethion after 24 hours. No detectable effects were found for Carbendazime + Cuoxyquinoleate.

The decrease of viable B. japonicum is less than 1 log number for the active productswith the exception of metalaxyl (2 log at 24 hours).

Effects of pesticides on short term nodulation Numbers of nodules after 3 weeksgrowth in glasshouse of treated seeds used in survival experiments are shown inFigures 3 and 4.

Results indicate significantly lower nodulation (p = 0.05) than for the control formetalaxyl treated seed sown 1,4 and 24 hours after inoculation. The same decreasingeffect was found for iprodione treated seeds sown 4 and 24 hours after inoculation.No detectable effects on nodulation were found for carbendazime products anddiethion.

TOXI.-E

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

234 G. CATROUX AND F. ARNAUD

7 Log number of recoveredB. Japonicum

CV:1.0%24

CV:1.0%

• CONTROL

Q CARBEKDAZTJÍE +OXYQUDÍOLEATEDE CU

0 CARBENDAZIME

DDIETHION

hours afterInoculation

—treatment from same «ampling time followed by the same letterere not statistically different (P=O.OS)

Figure 2 Survival of B. japonicum on treated seeds.—Treatments from same sampling time followed bythe same letter are not statistically different at p:0.05.—Coefficient of variation at 1, 4 and 24 hours: 1.0,1.0, 1.0 p. cent.

Field trial Average nodule numbers and grain yields are indicated in Table 2. Thereare no significant differences in nodule numbers and grain yields between treatmentswhen inoculant is used by the seeds bed method (treatments 2 to 7). On the contrary,when the inoculant is used by the slurry method on treated seeds, significant decreasesin nodule numbers are found for metalaxyl, the two carbendazime products andiprodione. No significant difference was found for diethion.

3.2 Soil treatment

Effect of pesticides on the growth of B. japonicum On the 3 microgranular insecticidesstudied, only chlorfenvinphos has shown inhibitory effect on the growth of B.japonicum on solid medium (inhibition zone diameter = 1 5 mm).

Survival ofB. japonicum on pesticides microgranules Numbers of viable B. japonicumrecovered 1, 4 and 24 hours after inoculation are represented in Figure 5.

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

25T-Number of nodules per plant

20--

1 5 - -

235

1 0 - -

• CONTROL

Q METALAXYL

D IPRODIONE

CV:13.1%21

CV:15.6%

hours afterinoculation

—treatment from same sampling time followed by the same letterare not «taUiticoBj- different (F-0.06)

Figure 3 Effect of seed treatments on short-term nodulation.—Treatments from same sampling timefollowed by the same letter are not statistically different at p:0.05.—Coefficient of variation at 1, 4 and24 hours: 13.1, 17.3, 15.6 p. cent.

Table 2 Average nodules numbers and grain yields in the field trial.

Treatment

1 Control—non inoc

"Slurry" inoculation

2 control—inoc3 metalaxyl—inoc4 iprodione—inoc

5 carbendazime—inoc6 carbendazime + Cu oxyquinolate—inoc7 diethion

"Seed bed" inoculation

8 control—inoc9 metalaxyl—inoc

10 iprodione—inoc11 carbendazime—inoc12 carbendazime + Cu oxyquinoleate—inoc13 diethion

Least significant difference (P = 0.05)

Nodule Numberper plant(flowering)

0.0

59.5204.5

10.521

1614.519.58

3924

42.1

Grain Yieldtons/ha

1.795

2.3021.7751.8202.0511.8442.146

2.4292.4302.4672.2262.2542.340

0.247

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

236 G. CATROUX AND F. ARNAUD

25-1-Number of nodules per planta

CONTROL

B CARBENDAZIME +OXYQUINOLEATEDE CU

0 CARBENDAZIME

DDIETfflON

CV:15.4%24

CV:15.8%

hours otterinoculation

—treatment from mazne sampling time followed by the •ame letterarts not statistically different (P-0.05)

Figure 4 Effect of seed treatments on short-term nodulation—Treatments from same sampling timefollowed by the same letter are not statistically different at p:0.05.—Coefficient of variation at 1, 4 and24 hours: 15.4, 14.4, 15.8 p. cent.

Results indicate significantly lower recovery (P = 0.05) related to control forchlorfenvinphos and terbuphos after 1, 4 and 24 hours.

After 4 hours, Terbuphos decreases the number of viable cells of 3 log and thereis no more detectable B. japonicum after 24 hours.

Effect of pesticides on short term nodulation Numbers of nodules after 3 weeksgrowth in glasshouse of seeds inoculated with microgranules are shown in Figure 6.

Results indicate significantly lower nodulation (P = 0.05) related to control onlyfor terbuphos after 1, 4 and 24 hours. Nodulation is nearly suppressed after 4 hoursstorage of inoculated microgranules of terbuphos.

4 DISCUSSION-CONCLUSION

Inhibitory or depressive effects were found with the different methods used for severalpesticides.

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

COMPATIBILITY OF A SOYBEAN PEAT INOCULANT 237

g Log number of recoveredB. japonicum

• CONTROL

3 CARBOFURAN

Q CHLORFENVINPHOS

Q TERBUPHOS

H FURATHIOCARBE

• TEFLUTHRINE

hours afterinoculation

—treatment from same sampling time followed by the same letterare not «UtlatlcaDy different (P-0.05)

Figure 5 Survival of B. japonicum on microgranular insecticides.—Treatments from same sampling timefollowed by the same letter are not statistically different at p:0.05.—Coefficient of variation at 1, 4 and24 hours: 1.5, 1.8, 2.0 p. cent.

The results are summarized in Table 3.There are some similarities between B. japonicum survival, short term nodulation

and field experiment. Of the ten pesticides tested, only six have negative effects onB. japonicum survival (metalaxyl, iprodione, carbendazime, diethion, chlorfenvinphos,terbuphos).

It is interesting to note, that only one of these active products has a negative effecton B. japonicum growth (Chlorfenvinphos). Therefore, it may be concluded thattesting of growth inhibition is not suitable to assess the possible effect of pesticideson soybean-2?. japonicum symbiosis.

Nodulation is reduced by only three of the six products active on B. japonicumsurvival (metalaxyl, iprodione and terbuphos). The results obtained with metalaxylare not in accordance with the data obtained by Diatloff1 * who worked with anothersoybean line and another B. japonicum strain.

Carbendazime + Cu oxyquinoleate has no effect on survival and short termnodulation yet is found to be active in the field experiment. Carbendazime alone,

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

238 G. CATROUX AND F. ARNAUD

35-T-Number of nodules per plant

30--

ab

CV:24.3%4

CV-.30.5X24

CV.22.9%

M CONTROL

Q CARBOFURAN

E3 CHLORFENVINPHOS

H TERBUPHOS

0 FURATHIOCARBE

D TEFLUTHRINE

hours afterinoculation

—treatment from asme campling time followed by the tameletter ere not «tAtlsUcaUy different (P=0.05)

Figure 6 Effect of microgranular insecticides on short-term nodulation.—Treatments from same samplingtime followed by the same letter are not statistically different at p:0.05.—Coefficient of variation at 1, 4and 24 hours: 24.3, 30.5, 22.9 p. cent.

Table 3 Summary of the detected effects of pesticides on soybean—B. japonicum symbiosis, (n.d.) notdetermined; (—) no effect; (<) depressing or inhibitory effect.

B. japonicum B. japonicum Glass house Field experimentgrowth Survival Nodulation inoculated treated seed

nodulation yield

Seed treatment

metalaxyliprodionecarbendazimecarbendazime + Cu oxyq.diethion

Soil treatment

carbofuranchlorphenvinphosterbuphosfurathiocarbetefluthrine

ndnd

ndndndndnd

ndndndndnd

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013

COMPATIBILITY OF A SOYBEAN PEAT INOCULANT 239

active on survival but not on short term nodulation, is also active in the fieldexperiment.

From a methodological point of view, it may be concluded that if a product isactive on B.japonicum survival and short term nodulation, there is a risk of depressiveaction, in the field, on the nodulation and the yield. But products inactive in the twotests like carbendazime + Cu oxyquinoleate may depress, yield in the field.

From a practical point of view, more data are needed to conclude but metalaxyland iprodione are probably not compatible with the "slurry" inoculation of treatedseeds. On the other hand, all products are compatible with the seed bed inoculationmethod (Table 2).

Several microgranular insecticides are compatible with the inoculant and nodula-tion (carbofuran, Furathiocarbe, Tefluthrine) but care must be taken as thesemicrogranules are not designed to fix peat inoculants and data from field trials areneeded.

References

1. R. W. Weaver and L. R. Frederick, 1972. Agron. J., 64, 597-599.2. B. Lagacherie and M. Obaton, 1973. C. R. Acad. Agric., 1, 67-79.3. R. S.'Smith, M. A. Ellis, and R. E. Smith, 1981. Agron. J., 73, 505-508.4. J. Brockwell, R. R. Gault, D. L. Chase, G. L. Turner and F. J. Bergersen, 1985. Aust. J. Agric. Res.,

36, 397-409.5. R. L. Curley and J. C. Burton, 1975. Agron. J., 67, 807-808.6. K. Tesfai and M. A. B. Mallik, 1986. Bull. Environ. Contam. Toxicol., 36, 819-826.7. A. M. Chamber and F. J. Montes, 1982. Plant and Soil, 66, 353-360.8. C. M. Tu, 1977. Bull. Environ. Contam. Toxicol., 18, 190-199.9. M. A. B. Mallik and K. Tesfai, 1983. Bull. Environ. Contam. Toxicol. 31, 432-437.

10. G. Kapusta and D. L. Rouwenhorst, 1973. Agron. J., 65, 112-115.11. A. Diatloff, 1986. Soil Biol. Biochem., 18, 121-122.12. C. M. Tu, 1981. Protection Ecology, 3, 41-46.13. M. A. B. Mallik and K. Tesfai, 1985. Plant and Soil, 85, 33-41.14. R. J. Rennie and S. Dubetz, 1984. Agron. J., 76, 451-454.

Dow

nloa

ded

by [

Uni

vers

ity o

f T

oron

to L

ibra

ries

] at

04:

35 2

5 Fe

brua

ry 2

013