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Effects of insecticides on acetylene reduction by alfalfa, red clover and sweetclover
EFFECTS OF INSECTICIDES ON ACETYLENE REDUCTION BY ALFALFA, RED CLOVER AND SWEETCLOVER C. R. Departments
SMITH.
of Bacteriology
B. R. FLNU
and
J. T. SC.HL:L%
and Entomolog). North Fargo. ND 5X102 U.S.A.
Dakota
State L’niversit).
Summary Seven organophosphate and carbanate insecticides were tested for their effects on CzH,reduction by alfalfa (Medicuqo auriur L.), awectclo\cr (.24~/i/otus ~/ha L.) and red clover (T~/~/IMI pralvr~sr L.). Plants were grown it1 ri!ro for 21 daks on nonsterile vermiculite or soil substrates. Oftanoll”‘ was inhibitory to most plants on both soil and vermiculite. Carbofuran’s Inhibitory effect was almost entirely limited to soil-grown plants. Carbarql. alchcarb. chlorp!rlfos. and terhufos were inhibitor) IO certain plants under some conditions. Phosmet uas the least toxic. Significant difrerences wcrc observed in toxicit) which were dependent on the growth substrate used. The effects of carbamates tended to be restricted to plants grown on soil substrates rather than on vermiculite. Loss of C2HZreducing ability was usually correlated with mhibihon of nodule formation. The insecticides did not inhibit growth of Rkizohim mrlihi or R. frifidii in broth culture.
530”. Seeds were gerL.), “Vernal” and “Pioneer minated on moist filter paper in Petri dishes. The There has been some discussion of the potential seed and radiclc were inoculated by dipping into a adverse effects of pesticides on the Rhi_ohilrrll-leglIme slurry of peat-carrier type commercial inoculum symbiosis. Herbicides and fungicides have been the (Nitragin@ AB formulation for alfalfa and clovcrs) as main pesticides tested (Audus. 1970: Greaves ct trl.. instructed on the packet. All experiments contained 1976; Parr. 1974) but there have also been reports inoculated and uninoculated controls and there wcrc on effects of insecticides (Pareek and Gaur. 1970: 30 replicates per treatment. Plants were grown in Misra and Gaur. 1974; Braithwaitc c’f ul.. 1958; LichI6 x ISOmm test tubes under black plastic sheeting tenstein et ul.. 1962; Eno and Everett. 1958). There to shield them from diurnal light variation. Lighting are few reports on the use of the C,H,-reduction was by 40-W Sylvania GroLux’@l tluoresccnt lamps technique as a measure of the pesticide’s effect on approximatelq 20 cm above the test tube tops. Test the legume (Rode11 cf trl., 1977; Tu, 1977). tubes wcrc lillcd to a depth of ? cm with sand folOur purpose was to determine the effects ot lowed by an additional 7 cm of vermiculite which had selected insecticides on certain forage crops as deterbeen pulverized in ;I blender. or the I 3 mm fraction mined by C,Hz-reduction by laboratory-grown intact of air-dried soil. The soil was Bcardcn loam suppleDlants. These experiments were designed to avoid mented with lo”,, (H’ w) chopped Hax strau to inproblems previdusly encountered Tn greenhouse mobilize nitrate. We found that when using soil to studies in which the C,H,-reduction activity of grow plants in small undrnincd containers, nitrates excised nodules was tested (Rode11 er ~1.. 1977). can accumulate and inhibit nodulntion (Vincent. 1970). Also. soil should not be stored moist. but airdried within a felt hours after collection. because: MATE:Rli\LS AND METHODS nitrates can accumulate rapidly (Harding and Ross. 1964). The insecticides. in high-purity anal! tical forThe seven insecticides used were : terbufos. S-((( l- lmulations. were dissolved in acetone to deliver condimethyl-ethyl) thio)methyl) O.O-dirthyl phosphorocentrations of 5 parts. IO” (approximate field rate) and dithioate. (X5”,, a.i.): phosmet. N-(mcrcaptomethyl)50 parts IO” a.i. in 2 ml aliquots of the acctonc-insecphthalimide S-(O,O-dimethylphosphorodithioate. 95.7”,, ticide solution. C‘alculations to dctcrminc ports, IO” ai.): Oftanol@. (Chemagro Corp., Kansas City. MO). were based on the weight of moistened soil or \crmlI -methyl -ethyl - 2 - (((ethoxy( I - methylethyl)amino) culitc. Controls recci\cd 7 ml acetone. Test tuba conphosphinothioyl)oxy)-benzoate, (90”,, ai.); chlorpyrtaining vermiculite received 6 ml of Schwinghamcr’s ifos. O.O-diethyl 0-(X5.6-trichloro-2-pyridyl)-phosphor(1960) plant-nutrient solution. while test tubes conothioatc. (97.7”,, ai.); carbaryl. I-naphthyl N-mcthyltaining soil roccived 6 ml distilled wntcr. 4 \)ringc carbamate. (99.7”,, a.i.); carbofuran, 23dihvdro-2.2u Y-mm long needle was used to with dimethyl-7-ben7ofuranyl methyl carbamate: (YO.?‘,, cquippcd moisten soil. I ml wsater on top of the soil and the a.i.): and aldicarb. 2-methyl-2-(methjlthio) propionalremainder in,iected at the sand-sot1 interface. After 2 I dchydc 0-(methylcarbamoyl)oxime. (Y9”,, a.i.). The daqs. an air-tight serum bottle stopper was placed first four insecticides arc organophosphates and the on each test tube and I ml C2Hz injected. Replacelast three arc carbamates. with inert gases is The legumes tested were “Kenstar” red clover (‘TV;- ment of the normal atmosphere not necessary (Maggie and Burris. 1972). Test tuba fi,/iU/,? /Jrrrtc’rl.\c’ L.). “Arctic” sweetclover (i21&/ot~r.s The ulhtr L.). and two varieties of alfalfa (Metlrctr~go .s~~;I.LI wcrc incubated for I h at room tcmpcraturc. INTRODL’CTION
463
464
C. R. Shrlw.
B R. tl,xsl
reaction wab stopped b) addition of 3 ml 5y H2S0,. Samples of 0.5 ml of gas were removed through the rubber septum with ;I 1ml disposable s!ringc fitted with ;I 2.5 gauge x 4 mm needle. Gas samples were analyzed for C2H, on a gas chromatograph with flame ioniration detector using a 1.82 m x 7 mm i.d. glass column packed with Porapak R. 100 I20 mesh (Applied Science Laboratories). Operating conditions were: inlet 70 C. column 65 C. dctcctor line 90 c‘. detector 120 C. N, carrier gas flow SOml min ‘. HZ gas flow 45 ml min ‘, air flow 250 ml min I. carrier makeup N2 gas How 70 ml min ‘. For experiments with pure cultures. Rhixhrm wdihri I02F65 and R. !/+folii lh2P17 were obtained from J. C. Burton. Nitragin Company. Milwaukee, WI. The growjth medium was mannitol-yeast extract (MYE) broth (Vincent. 1970). supplemented with l.5”(, agar for solid media. Cells were suspended in 5 ml MYE broth. then 0.5 ml of the homogeneous suspension was inoculated into a 300-ml Erlenmeyer flask containing 50 ml MYE broth. The flask was shaken for 24 h at 30 C‘. then 0.5 ml of the culture was pipctted into a 250.ml nephclometer flask (5 Id. Bcllco Co.. Vineland. NJ) containing 50 ml MYE broth. The initial bacterial concentrations in each flask were adjusted by turbidity and confirmcd by duplicated plate counts. Insecticide (100 mg) was dissolved in 4 ml acetone and diluted to deliver concentrations of 5 and 50 parts!lO’ in 0. I ml aliquots. The acetoncinsecticide solution was aseptically added. at the time of inoculation. to each flask through a sterile 0.2 /tm Nucleporc membrane in a syringe filtration attachment. Control flasks received 0.1 ml amounts of acetone, Flasks were incubated for 33 h at 30 C on a rotary shaker. at which time the logarithmic phase of growth had ended. All trials wcrc performed in Table I.
Pioneer
variety
530 alfalfa.
and .I. T.
%HI r./
triplicate and population incrcasc was followed on a K lett Summerson colorimetcr at 420 nm. Final hatterial concentrations wcrc determined bl duplicated plate counts (MYE agar) for each flask.
KE.SI LTS
Considering tirst the results of 5 parts, 10” conccntrations at the (1.01 and 0.001 levels of significance. Oftanol’t, was ;I comparativclq toxic pcsticidc. It adversely affected C,H,-reduction by all four of the test plants on soil. and three of the four on vermiculite (Tables I. 7. 3 and 4). Carbofuran MRS inhibitory to C‘,H,-reduction on the soil substrate; wl~on plants were grown on vcrmiculitc only sweetclo\cr \I;IS atfcctcd. Carbaryl also afTectcd all four plants grown on soil; no vermiculite-grown plant was inhibited. Aldicarh affected onI1 soil-grown plants. two of the four tcstcd. In gcncral. the 5 parts IO” applications of the carbamatcs (carbofuran. carharyl and aldicarb) tended to depress CzHz reduction bq plants grown on soil (to 63”,, of normal activit!) and not on vcrnilculite (90”,, of normal activit!). Terhufos atl’cctcd onI1 red clo\cr. depressing CLH2-reduction significantl) on both substrates. Phosmet trcatmont did not ad\ersclh affect any plant. At SO parts;lO” the insecticides were more inhibitor). The carbamatcs and Oftanol’” almost uniformly deprcsscd C,H,-reduction. an exception being aldicarb on Vernal alblfri. Chlorpyrifos was inhibitor) to all plants on vcrmiculitc but not at all on soil. Terbufos affected onl) red clover. Phosmct inhibited C2H2-reduction 1~) red clover and sweetclover on \crmiculitc aubstratc. The swcctclovcr M;IS atlicctcd on soil
also.
Effect of insecticide treatment on proportional C,Hz-rcdnction (latter in parentheses). (Control plants = 1.00) Vcrmicnlitc
lnsectlcide
5 parts.
Carbofuran Aldicarb Carbaryl Oftanol Terbufos Phosmct Chlorpyrifos
IO”
I .OZ(O.S5)
Tahlc
2. Vernal
alfalfa.
I-test.
(C,H?-rcdnction
Effect
parts.10“
5
1.14(0.69) 0.X6(0.84) 0.12(0.30)*** 0.53(0.s0)*** 0.93(0X9) 1.02(l.OY) 0.27(0.57)***
0.63(0.97)**
of significance.
fgurcs):
*0.05:
of insecticide treatment in parentheses).
parts IO”
S parts,‘1 0”
Carbofuran Aldicarb Carbaryl Oftanol Terbufos Phosmet Chlorpyrlfos Levels
of slgmficance.
r-t&.
(C,H,-reduction
on proportional (Control plants
C‘>H,-reduction = 1.00)
IO”
0.53(0.53)*** 0.7x(0.75)** 0.23(0.33)*** 0.35(0 40)*** 0.76(0.X5)’ 0.97( I .04) O.Y2( I .W)
and
nodule
ntlmbcl-s
(latter
Soil S parts/l
0.98(0.61 ) l.l2(1.26) 0.31(0.36)*** 0.76(0.61) 0.X9(0.87) I .09(0.X4) 0.36(0. IX)*** f&w\):
numhw
**O.Ol : ***O.OOl.
50 parts.lOh
0.X9(0.77) 0.99( 1.27) 0.8 l(0.92) 0.81(0.80) I .05(O.Y I ) I .04(0.X I ) 0.40(0.64)***
XI parts
0.58(0.67)*** 0.77(0.97)** 0.43(0.45)*** 0.52(0.63)*** 0.X4(0.96) 0.99( 1.24) O.YO(O.95)
Vermiculite Insecticide
nodule
Soil
so
O.Y8(0.68) 0.8x(0.82) I .03(0.64) 0.65(0.69,*** l.OO(O.85)
Levels
and
*&OS: **O.OI
0”
0.57(0.54)** O.Y9(0.88) 0.26(0.31)*** 0.47(0.43)*** O.YX(O.95) O.Y5(O.SX) 0.99(0.9 I )
: ***O.OOl
50 parts IO” 0.45(0.37)*** 0.93(0.‘)0) 0.20(0.20)*** 0.19(0.20)*** 0.96(0.96) O.YO(O.93) 0.9h(O.Y I )
Ell’ects of inscctictdes on acetylene reduction Table 3. Kenstar
red clover, Effect of insecticide treatment in parentheses).
on proportional
(Control
465
C,H?-reduction
Vermiculite
Carbofuran Aldicarb Carbaryl Oftanol Tcrbufos Phosmet Chlorpyrifos Levels of significance,
0.90(0.87) 0.37(0.76,*** 0.56(0.77)* 0.33(0.5X)*** 0.72(0.62)*** 0.19(0.17)*** 0. I8(0.34)***
I.OX(1.14) 0.93(0.74) 0.67(0.85) 0.52(0.93)*** 0.66(0.74)*** 0.86(0.65) 0.71(1.29) t-test. (CzHz-reduction
Table 4. Arctic sweetclover.
figures): *0.5:
**O.Ol:
Carbofuran Aldicarb Carbaryl Oftanol Terbufos Phosmet Chlorpyrifos Levels of significance,
0.70(0.69)*** 0.79(0.81)* 0.98(0.62) 0.51(0.68)*** 0.72(0.70)* 0.65(0.95)* 0.8 l(0.97) t-test. (CZHZ-reduction
0.6?(0.54)*** 0.71(0.76)*** 0.48(0.63)*** 0.60(0.52)*** 0.25(0.31)*** 0.96(0.89) 0.X5(0.64)
***O.OOl.
EtTect of insecticide treatment on proportional C,HL-reduction in parentheses). (Control plants = 1.00)
5 parts/l 0”
(latter
50 parts! IOh
0.67(0.52)*** 0.9 I(0.96) 0.77(0.x2)** 0.55(0.47)*** 0.35(0.40)*** 0.9X( 1:08) 0.9 l(O.98)
Vermiculite Insecticide
numbers
Soil 5 partsilO”
50 parts/lOh
5 parts/IO”
Insecticide
and nodule
plants = 1.00)
and nodule numbers
(latter
Soil 50 partsjlOh
5 parts. IOh
0.73(0.66)*** 0.77(0.58)* O.l2(0.30)*** 0.10(0.12)*** 0.7 1(0.68)* 0.31(0.63)*** 0.42(0.64)*** figures): *0.5
At 50 parts/lOh. phytotoxic effects such as stunted growth and browned edges or yellowing of lcaves were observed. Chlorpyrifos. carbaryl and Oftanol@ caused such plant damage. which was correlated with poor C,H,-reduction. Red clover was similarly affected by carbofuran. Tests to determine effects of insecticides on growth of R. rwliloti and R. tr$dii in broth cultures showed no significant inhibition. DISCUSSION
The results were more satisfactory, particularly in reproducibility. than in our previous attempt to assess pesticide effects on legumes by C,H,-reduction (Rode11 it al.. 1977). The laboratory conditions under continuous lighting minimized diurnal and temperature effects (Mague and Burris, 1972). The larger numbers of replicates made possible by smaller containers minimized the variations inherent in a plant cultivar (Lindsay and Jordan, 1976). The use of intact plants avoided the problems reported with excised nodules (van Straten and Schmidt, 1975). If C2H,reduction is used as a criterion of phytotoxicity by pesticides, these factors should be taken into consideration. Comparison with previous work, in which certain of the same pesticides were used, shows that carbaryl (Sevin) at application rates comparable to ours inhibited growth of peas grown on sand (Lichtenstein et ul., 1962). These workers suggested that growth substrates such as sand might not be directly comparable to field soil. Our data indicate that significant differences do exist between soil and nonsoil substrates in tests for plant-pesticide interactions. Reduced nitro-
**0.01:
50 parts; lOh
0.73(0.77)*** 0.39(0.46)*** 0.44(0.40)*** 0.51(0.57)*** 0.83(0.82) 0.93(0.92) 0.91(1.00)
0.69(0.59)*** 0.36(0.44)*** 0.26(0.23)*** 0. I7(0. I2)*** 0.X6(0.83) 0.74(0.x7)**. 0.87(0.86)
***0.001.
genase activity of soybeans grown on sand after application of a fungicide-chlorpyrifos mixture was reported by Tu (1977). On the other hand. there have been reports of applications of carbaryl or carbofuran which resulted in no adverse effects on various legumes (Kapusta and Rouwenhorst, 1973: Kulkarni cf al.. 1974; Shehane and Bass, 1974; Eckenrode et al., 1973). Even when plants, pesticides and growth conditions are identical, comparisons can be complicated by variations in criteria for adverse affects of pesticides on plants, such as. weight, numbers and leghemoglobin content of nodules (Kulkarni et (II.. 1974; Eno and Everett. 1958): plant size or weight (Nash and Harris. 1973; Shehane and Bass. 1974; Lichtenstein c)t ul., 1962). or stem length and stem elongation (Fick. 1977). Ack,lou/rdyr,ttt~lf Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article 870.
REFERENCES
ALIDUS L. J. (1970) The action of herbicides and pesticides on the microflora. M&d. Rijksfac. Landhouwwrt. Grnf. 35. 465-492. BRAITHWAITF B. M.. JANE A. and SWAIN F. G. (1958) Effects of insecticides on sod sown sub-clovers. J. Auf. Iusr. uyric. Sci. 24. 155-157. ECK~NROUE C. J.. GAUIIUFR N. L.. DANIELSON D. and W~RB D. R. (1973) Seedcorn maggot: seed treatments and granule furrow applications for protecting beans
and sweet corn. J. ccm.
Ent. 66. I 191
I 194.
466
C. R. SMITH. B. R. FLINKI: and .I. T. SCH~ L/
ENO C. F. and Evt~tr-r P. H. (1958) Effect of soil applications of 10 chlorinated hydrocarbon insecticides on soil microorganisms and the growth of stringless Black Valentine beans. Soil SC;. Sot. AN. Proc. 22. 235-238. FISK G. W. (1977) Methods for evaluating insecticide effects on alfalfa growth. J. rnrir. Quul. 6. 443-445. GRI.AVES M. P.. DAVIES H. A.. MARSH .I. A. P. and WINGFI~ILI)G. I. (1976) Herbicides and soil microorganisms. Crir. Rer. Micro&o/. 5. l-38. HARINNG D. E. and Ross D. J. (1964) Some factors in low-temperature storage influencing the mineralisablenitrogen in soils. J. Sci. Fd. Agric. IS. X29-834. KAP~JSTA G. and R~UWENHORST D. L. (1973) Interaction of selected pesticides and Rhizohiuw jupo~~icum in pure culture and under field conditions. Ayron. J. 65, I I2 I 15. K~.LI(ARNI J. J.. SARIXSHPANIX J. S. and BAGYARAJ D. J. (1974) Effect of four soil-applied insecticides on symbiosis of Rhix~hiurn sp. with Arachic hypcyara Linn. PI. Soil 40. I69- 172. LI~HTENSTFIN E. P.. MILLINGTON W. F. and C~WLEY G. T. (1962) Effect of various insecticides on growth and respiration of plants. J. ayric. Fd. chern. 10. 25 I-256. LINIXAY C. R. and JORI)AN D. C. (1976) Use of stem cuttings to reduce plant variation in Rki:ohiutn-legume investigations. Can. J. Soil Sci. 56. 495-497. MAGU~ T. H. and BURRIS R. H. (1972) Reduction of acetylene and nitrogen by field-grown soybeans. Nrw PhJ,rol. 71. 275-286.
MISRA K. C. and GAUR A. C. (1974) Influence
of simazine.
lindane and ceresan on different parameters of nitrogen fixation by groundnut. I~ltl. J. 4gric. SC,;. 44, X38 X40. NASH R. G. and HAHKIS W. G. (1973) Screening for phqtotoxic pesticide interactions. J. euvir. Quad 2. 493 497. PAR~EI< R. P. and GAIX A. C. (1970) Elect of dichlorodiphenyl-trichloro-ethane (DDT) on symbiosis of Rhinohiurn sp. with Pha.sc~ol~rs mrm~ (Green Gram). PI. Soil 33. 297 304. PARK J. F. (1974) Effects of pesticides on symbiotic nitrogen-fixing bacteria. In Pc\ticidu itI Soil trml Auto, (W. D. Guenzi, Ed.). pp. 315 3X6. Soil Sci. Sot. Am., Madison. WI. ROI)I:LL S.. FLIN~(~.B. R. and ScffuL% J. T. (1977) Effects of insecticides on acetylene reduction by d:orohuctc,~ vinulaudii and soybean nodules. PI. Soil 47, 375-382. SCHWIN~;HAMI.R E. A. (1960) Studies on induced variation in the rhizobia. 1. Defined media and nodulation test techniques. Appl. Miuohrol. 8. 349-352. SHEHANI, R. H. and BASS M. H. (1974) Growth and yield of soybeans following treatment with Phorate. Carbofuran. Methomyl and Disulfoton. Enur. EH~. 3, 574-575. VAN STRATLU J. and SCH.MII)X-E. L. (1975) Action of water in depressing acetylene reduction in detached nodules. Appl.
Microbial.
29. 432 434.
Tu C. M. (1977) ElTects of pesticides on soil nitrogen-fixing bacteria. Abstract.\ ofrbr Annuul Mrrriq offhe Amrrkcrn Sociefy ,for Microbiology p. 241. VINCENT J. M. (1970) 4 Mum~~rl for rhr Prutic(l/ Strrrl~ of Root-h’odtrk Buctcviu. Blackwells. Oxford.
SMITH.
of Bacteriology
B. R. FLNU
and
J. T. SC.HL:L%
and Entomolog). North Fargo. ND 5X102 U.S.A.
Dakota
State L’niversit).
Summary Seven organophosphate and carbanate insecticides were tested for their effects on CzH,reduction by alfalfa (Medicuqo auriur L.), awectclo\cr (.24~/i/otus ~/ha L.) and red clover (T~/~/IMI pralvr~sr L.). Plants were grown it1 ri!ro for 21 daks on nonsterile vermiculite or soil substrates. Oftanoll”‘ was inhibitory to most plants on both soil and vermiculite. Carbofuran’s Inhibitory effect was almost entirely limited to soil-grown plants. Carbarql. alchcarb. chlorp!rlfos. and terhufos were inhibitor) IO certain plants under some conditions. Phosmet uas the least toxic. Significant difrerences wcrc observed in toxicit) which were dependent on the growth substrate used. The effects of carbamates tended to be restricted to plants grown on soil substrates rather than on vermiculite. Loss of C2HZreducing ability was usually correlated with mhibihon of nodule formation. The insecticides did not inhibit growth of Rkizohim mrlihi or R. frifidii in broth culture.
530”. Seeds were gerL.), “Vernal” and “Pioneer minated on moist filter paper in Petri dishes. The There has been some discussion of the potential seed and radiclc were inoculated by dipping into a adverse effects of pesticides on the Rhi_ohilrrll-leglIme slurry of peat-carrier type commercial inoculum symbiosis. Herbicides and fungicides have been the (Nitragin@ AB formulation for alfalfa and clovcrs) as main pesticides tested (Audus. 1970: Greaves ct trl.. instructed on the packet. All experiments contained 1976; Parr. 1974) but there have also been reports inoculated and uninoculated controls and there wcrc on effects of insecticides (Pareek and Gaur. 1970: 30 replicates per treatment. Plants were grown in Misra and Gaur. 1974; Braithwaitc c’f ul.. 1958; LichI6 x ISOmm test tubes under black plastic sheeting tenstein et ul.. 1962; Eno and Everett. 1958). There to shield them from diurnal light variation. Lighting are few reports on the use of the C,H,-reduction was by 40-W Sylvania GroLux’@l tluoresccnt lamps technique as a measure of the pesticide’s effect on approximatelq 20 cm above the test tube tops. Test the legume (Rode11 cf trl., 1977; Tu, 1977). tubes wcrc lillcd to a depth of ? cm with sand folOur purpose was to determine the effects ot lowed by an additional 7 cm of vermiculite which had selected insecticides on certain forage crops as deterbeen pulverized in ;I blender. or the I 3 mm fraction mined by C,Hz-reduction by laboratory-grown intact of air-dried soil. The soil was Bcardcn loam suppleDlants. These experiments were designed to avoid mented with lo”,, (H’ w) chopped Hax strau to inproblems previdusly encountered Tn greenhouse mobilize nitrate. We found that when using soil to studies in which the C,H,-reduction activity of grow plants in small undrnincd containers, nitrates excised nodules was tested (Rode11 er ~1.. 1977). can accumulate and inhibit nodulntion (Vincent. 1970). Also. soil should not be stored moist. but airdried within a felt hours after collection. because: MATE:Rli\LS AND METHODS nitrates can accumulate rapidly (Harding and Ross. 1964). The insecticides. in high-purity anal! tical forThe seven insecticides used were : terbufos. S-((( l- lmulations. were dissolved in acetone to deliver condimethyl-ethyl) thio)methyl) O.O-dirthyl phosphorocentrations of 5 parts. IO” (approximate field rate) and dithioate. (X5”,, a.i.): phosmet. N-(mcrcaptomethyl)50 parts IO” a.i. in 2 ml aliquots of the acctonc-insecphthalimide S-(O,O-dimethylphosphorodithioate. 95.7”,, ticide solution. C‘alculations to dctcrminc ports, IO” ai.): Oftanol@. (Chemagro Corp., Kansas City. MO). were based on the weight of moistened soil or \crmlI -methyl -ethyl - 2 - (((ethoxy( I - methylethyl)amino) culitc. Controls recci\cd 7 ml acetone. Test tuba conphosphinothioyl)oxy)-benzoate, (90”,, ai.); chlorpyrtaining vermiculite received 6 ml of Schwinghamcr’s ifos. O.O-diethyl 0-(X5.6-trichloro-2-pyridyl)-phosphor(1960) plant-nutrient solution. while test tubes conothioatc. (97.7”,, ai.); carbaryl. I-naphthyl N-mcthyltaining soil roccived 6 ml distilled wntcr. 4 \)ringc carbamate. (99.7”,, a.i.); carbofuran, 23dihvdro-2.2u Y-mm long needle was used to with dimethyl-7-ben7ofuranyl methyl carbamate: (YO.?‘,, cquippcd moisten soil. I ml wsater on top of the soil and the a.i.): and aldicarb. 2-methyl-2-(methjlthio) propionalremainder in,iected at the sand-sot1 interface. After 2 I dchydc 0-(methylcarbamoyl)oxime. (Y9”,, a.i.). The daqs. an air-tight serum bottle stopper was placed first four insecticides arc organophosphates and the on each test tube and I ml C2Hz injected. Replacelast three arc carbamates. with inert gases is The legumes tested were “Kenstar” red clover (‘TV;- ment of the normal atmosphere not necessary (Maggie and Burris. 1972). Test tuba fi,/iU/,? /Jrrrtc’rl.\c’ L.). “Arctic” sweetclover (i21&/ot~r.s The ulhtr L.). and two varieties of alfalfa (Metlrctr~go .s~~;I.LI wcrc incubated for I h at room tcmpcraturc. INTRODL’CTION
463
464
C. R. Shrlw.
B R. tl,xsl
reaction wab stopped b) addition of 3 ml 5y H2S0,. Samples of 0.5 ml of gas were removed through the rubber septum with ;I 1ml disposable s!ringc fitted with ;I 2.5 gauge x 4 mm needle. Gas samples were analyzed for C2H, on a gas chromatograph with flame ioniration detector using a 1.82 m x 7 mm i.d. glass column packed with Porapak R. 100 I20 mesh (Applied Science Laboratories). Operating conditions were: inlet 70 C. column 65 C. dctcctor line 90 c‘. detector 120 C. N, carrier gas flow SOml min ‘. HZ gas flow 45 ml min ‘, air flow 250 ml min I. carrier makeup N2 gas How 70 ml min ‘. For experiments with pure cultures. Rhixhrm wdihri I02F65 and R. !/+folii lh2P17 were obtained from J. C. Burton. Nitragin Company. Milwaukee, WI. The growjth medium was mannitol-yeast extract (MYE) broth (Vincent. 1970). supplemented with l.5”(, agar for solid media. Cells were suspended in 5 ml MYE broth. then 0.5 ml of the homogeneous suspension was inoculated into a 300-ml Erlenmeyer flask containing 50 ml MYE broth. The flask was shaken for 24 h at 30 C‘. then 0.5 ml of the culture was pipctted into a 250.ml nephclometer flask (5 Id. Bcllco Co.. Vineland. NJ) containing 50 ml MYE broth. The initial bacterial concentrations in each flask were adjusted by turbidity and confirmcd by duplicated plate counts. Insecticide (100 mg) was dissolved in 4 ml acetone and diluted to deliver concentrations of 5 and 50 parts!lO’ in 0. I ml aliquots. The acetoncinsecticide solution was aseptically added. at the time of inoculation. to each flask through a sterile 0.2 /tm Nucleporc membrane in a syringe filtration attachment. Control flasks received 0.1 ml amounts of acetone, Flasks were incubated for 33 h at 30 C on a rotary shaker. at which time the logarithmic phase of growth had ended. All trials wcrc performed in Table I.
Pioneer
variety
530 alfalfa.
and .I. T.
%HI r./
triplicate and population incrcasc was followed on a K lett Summerson colorimetcr at 420 nm. Final hatterial concentrations wcrc determined bl duplicated plate counts (MYE agar) for each flask.
KE.SI LTS
Considering tirst the results of 5 parts, 10” conccntrations at the (1.01 and 0.001 levels of significance. Oftanol’t, was ;I comparativclq toxic pcsticidc. It adversely affected C,H,-reduction by all four of the test plants on soil. and three of the four on vermiculite (Tables I. 7. 3 and 4). Carbofuran MRS inhibitory to C‘,H,-reduction on the soil substrate; wl~on plants were grown on vcrmiculitc only sweetclo\cr \I;IS atfcctcd. Carbaryl also afTectcd all four plants grown on soil; no vermiculite-grown plant was inhibited. Aldicarh affected onI1 soil-grown plants. two of the four tcstcd. In gcncral. the 5 parts IO” applications of the carbamatcs (carbofuran. carharyl and aldicarb) tended to depress CzHz reduction bq plants grown on soil (to 63”,, of normal activit!) and not on vcrnilculite (90”,, of normal activit!). Terhufos atl’cctcd onI1 red clo\cr. depressing CLH2-reduction significantl) on both substrates. Phosmet trcatmont did not ad\ersclh affect any plant. At SO parts;lO” the insecticides were more inhibitor). The carbamatcs and Oftanol’” almost uniformly deprcsscd C,H,-reduction. an exception being aldicarb on Vernal alblfri. Chlorpyrifos was inhibitor) to all plants on vcrmiculitc but not at all on soil. Terbufos affected onl) red clover. Phosmct inhibited C2H2-reduction 1~) red clover and sweetclover on \crmiculitc aubstratc. The swcctclovcr M;IS atlicctcd on soil
also.
Effect of insecticide treatment on proportional C,Hz-rcdnction (latter in parentheses). (Control plants = 1.00) Vcrmicnlitc
lnsectlcide
5 parts.
Carbofuran Aldicarb Carbaryl Oftanol Terbufos Phosmct Chlorpyrifos
IO”
I .OZ(O.S5)
Tahlc
2. Vernal
alfalfa.
I-test.
(C,H?-rcdnction
Effect
parts.10“
5
1.14(0.69) 0.X6(0.84) 0.12(0.30)*** 0.53(0.s0)*** 0.93(0X9) 1.02(l.OY) 0.27(0.57)***
0.63(0.97)**
of significance.
fgurcs):
*0.05:
of insecticide treatment in parentheses).
parts IO”
S parts,‘1 0”
Carbofuran Aldicarb Carbaryl Oftanol Terbufos Phosmet Chlorpyrlfos Levels
of slgmficance.
r-t&.
(C,H,-reduction
on proportional (Control plants
C‘>H,-reduction = 1.00)
IO”
0.53(0.53)*** 0.7x(0.75)** 0.23(0.33)*** 0.35(0 40)*** 0.76(0.X5)’ 0.97( I .04) O.Y2( I .W)
and
nodule
ntlmbcl-s
(latter
Soil S parts/l
0.98(0.61 ) l.l2(1.26) 0.31(0.36)*** 0.76(0.61) 0.X9(0.87) I .09(0.X4) 0.36(0. IX)*** f&w\):
numhw
**O.Ol : ***O.OOl.
50 parts.lOh
0.X9(0.77) 0.99( 1.27) 0.8 l(0.92) 0.81(0.80) I .05(O.Y I ) I .04(0.X I ) 0.40(0.64)***
XI parts
0.58(0.67)*** 0.77(0.97)** 0.43(0.45)*** 0.52(0.63)*** 0.X4(0.96) 0.99( 1.24) O.YO(O.95)
Vermiculite Insecticide
nodule
Soil
so
O.Y8(0.68) 0.8x(0.82) I .03(0.64) 0.65(0.69,*** l.OO(O.85)
Levels
and
*&OS: **O.OI
0”
0.57(0.54)** O.Y9(0.88) 0.26(0.31)*** 0.47(0.43)*** O.YX(O.95) O.Y5(O.SX) 0.99(0.9 I )
: ***O.OOl
50 parts IO” 0.45(0.37)*** 0.93(0.‘)0) 0.20(0.20)*** 0.19(0.20)*** 0.96(0.96) O.YO(O.93) 0.9h(O.Y I )
Ell’ects of inscctictdes on acetylene reduction Table 3. Kenstar
red clover, Effect of insecticide treatment in parentheses).
on proportional
(Control
465
C,H?-reduction
Vermiculite
Carbofuran Aldicarb Carbaryl Oftanol Tcrbufos Phosmet Chlorpyrifos Levels of significance,
0.90(0.87) 0.37(0.76,*** 0.56(0.77)* 0.33(0.5X)*** 0.72(0.62)*** 0.19(0.17)*** 0. I8(0.34)***
I.OX(1.14) 0.93(0.74) 0.67(0.85) 0.52(0.93)*** 0.66(0.74)*** 0.86(0.65) 0.71(1.29) t-test. (CzHz-reduction
Table 4. Arctic sweetclover.
figures): *0.5:
**O.Ol:
Carbofuran Aldicarb Carbaryl Oftanol Terbufos Phosmet Chlorpyrifos Levels of significance,
0.70(0.69)*** 0.79(0.81)* 0.98(0.62) 0.51(0.68)*** 0.72(0.70)* 0.65(0.95)* 0.8 l(0.97) t-test. (CZHZ-reduction
0.6?(0.54)*** 0.71(0.76)*** 0.48(0.63)*** 0.60(0.52)*** 0.25(0.31)*** 0.96(0.89) 0.X5(0.64)
***O.OOl.
EtTect of insecticide treatment on proportional C,HL-reduction in parentheses). (Control plants = 1.00)
5 parts/l 0”
(latter
50 parts! IOh
0.67(0.52)*** 0.9 I(0.96) 0.77(0.x2)** 0.55(0.47)*** 0.35(0.40)*** 0.9X( 1:08) 0.9 l(O.98)
Vermiculite Insecticide
numbers
Soil 5 partsilO”
50 parts/lOh
5 parts/IO”
Insecticide
and nodule
plants = 1.00)
and nodule numbers
(latter
Soil 50 partsjlOh
5 parts. IOh
0.73(0.66)*** 0.77(0.58)* O.l2(0.30)*** 0.10(0.12)*** 0.7 1(0.68)* 0.31(0.63)*** 0.42(0.64)*** figures): *0.5
At 50 parts/lOh. phytotoxic effects such as stunted growth and browned edges or yellowing of lcaves were observed. Chlorpyrifos. carbaryl and Oftanol@ caused such plant damage. which was correlated with poor C,H,-reduction. Red clover was similarly affected by carbofuran. Tests to determine effects of insecticides on growth of R. rwliloti and R. tr$dii in broth cultures showed no significant inhibition. DISCUSSION
The results were more satisfactory, particularly in reproducibility. than in our previous attempt to assess pesticide effects on legumes by C,H,-reduction (Rode11 it al.. 1977). The laboratory conditions under continuous lighting minimized diurnal and temperature effects (Mague and Burris, 1972). The larger numbers of replicates made possible by smaller containers minimized the variations inherent in a plant cultivar (Lindsay and Jordan, 1976). The use of intact plants avoided the problems reported with excised nodules (van Straten and Schmidt, 1975). If C2H,reduction is used as a criterion of phytotoxicity by pesticides, these factors should be taken into consideration. Comparison with previous work, in which certain of the same pesticides were used, shows that carbaryl (Sevin) at application rates comparable to ours inhibited growth of peas grown on sand (Lichtenstein et ul., 1962). These workers suggested that growth substrates such as sand might not be directly comparable to field soil. Our data indicate that significant differences do exist between soil and nonsoil substrates in tests for plant-pesticide interactions. Reduced nitro-
**0.01:
50 parts; lOh
0.73(0.77)*** 0.39(0.46)*** 0.44(0.40)*** 0.51(0.57)*** 0.83(0.82) 0.93(0.92) 0.91(1.00)
0.69(0.59)*** 0.36(0.44)*** 0.26(0.23)*** 0. I7(0. I2)*** 0.X6(0.83) 0.74(0.x7)**. 0.87(0.86)
***0.001.
genase activity of soybeans grown on sand after application of a fungicide-chlorpyrifos mixture was reported by Tu (1977). On the other hand. there have been reports of applications of carbaryl or carbofuran which resulted in no adverse effects on various legumes (Kapusta and Rouwenhorst, 1973: Kulkarni cf al.. 1974; Shehane and Bass, 1974; Eckenrode et al., 1973). Even when plants, pesticides and growth conditions are identical, comparisons can be complicated by variations in criteria for adverse affects of pesticides on plants, such as. weight, numbers and leghemoglobin content of nodules (Kulkarni et (II.. 1974; Eno and Everett. 1958): plant size or weight (Nash and Harris. 1973; Shehane and Bass. 1974; Lichtenstein c)t ul., 1962). or stem length and stem elongation (Fick. 1977). Ack,lou/rdyr,ttt~lf Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article 870.
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