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Steinernema feltiae activity and infectivity in response to herbicide exposure in aqueous and soil environments
JOURNAL
OF INVERTEBRATE
PATHOLOGY
55,
375-379 (I!?%)
Steinernema feltiae Activity and Infectivity in Response to Herbicide Exposure in Aqueous and Soil Environments BRIANT.FORSCHLER,JOHN Department
of Entomology,
N. ALL,AND~AYNE
University of Georgia Georgia Station, Griffin,
College Georgia
A. GARDNER
of Agriculture 30223-I 797
Experiment
Stations,
Received May 15, 1989; accepted September 14, 1989 Laboratory
bioassays determined the effect of herbicide exposure on the nematode Steinernema carpocapsae). AIachIor, sethoxydim, 2,4-D, and gfyphosate were tested at three concentrations in Petri dishes including the recommended application rate and one log dose higher and one log dose lower than the recommended rate for each of three exposure periods of 24, 48, and 120 hr. Alachlor was most deleterious reducing dauer activity by 44 to 90% in aqueous solution. Only 19 to 30% of the nematodes exposed to the other three herbicides were inactive. Bioassays of nematodes extracted from these suspensions and subsequently washed 3 x in water against Galleria mellonella last-instar larvae yielded median lethal doses (LD,s) of 45.2, 1.1, 0.9, and 1.1 nematodes/larva for the alachlor, 2,4-D, glyphosate and sethoxydim treatments, respectively. Control treatments with water yielded an LD,, of 2.8 nematodes/larva. Nematode infectivity in soil was reduced by exposure to 2,4-D and alachlor. At the recommended application rate, the LD,,s for the alachlor, 2,4-D, and control treatments were 26.7, 8.8, and 2.7 nematodes/larva, respectively. 6 1590 Academic press, Inc. KEY WORDS: Steinernema .feltiae; bioassay: herbicide; nematode; alachlor; 2,4-D; glyphosate; sethoxydim.
feltiae
(=Neoapfectana
INTRODUCTION
Entomogenous nematodes could be effective in many integrated pest management (IPM) programs as partial pest suppression agents used in conjunction with other control methods (Fuxa, 1987). Precluding the successful use of insect parasitic nematodes in such systems is knowledge of the possible interactions of the nematodes with other production practices, including use of chemical pesticides. Effects of selected pesticides on entomogenous nematodes have been reported in aqueous solution (Prakasa et al., 1975; Kovats, 1982; Hara and Kaya, 1983a; Das and Divakar, 1987; Heungens and Buysse, 1987), in an artificial medium and in insect hosts following pesticide exposure (Hara and Kaya, 1982, 1983b), and in an agar medium incorporated with pesticides (Kamionek, 1979; Fedorko et al., 1977a, b). Investigations by Welch (1971), Dutky (1974), and Poinar (1986) demonstrated no effect by selected herbicides on nematode
survival. Welch (1971) and Dutky (1974) evaluated herbicides that are no longer in use, while Poinar (1986) tested atrazine and alachlor. Of 17 herbicides tested in water solution, only trifluralin and pendimethalin adversely affected nematode activity in a study by Kovacs (1982). Fedorko et al. (1977a) reported significant nematode mortality (70%) after a 48-hr exposure of nematodes to water agar impregnated with technical grade monolinuron. Applications of entomegenous nematodes for the suppression of soil-inhabiting insect pests could expose them to herbicides that are commonly used in production systems. In this study, the quantitative effect of several commonly recommended herbicides on the activity and infectivity of the nematode Steinernema feltiae (= Neoaplectana carpocapsae) was tested in aqueous and soil environments. MATERIALS
AND METHODS
S. feftiae. (All strain) nematodes reared in 375 0022-2011190 $1.50 Copyright 6 1990 by Academic Press, Inc. AD rights of reproduction in any form resetvut.
376
FORSCHLER,
ALL,
last-instar Galleria mellonella larvae were used in all tests. Only newly emerged dauer larvae (24 hr old) were used in the assays. Commercially formulated sethoxydim (Poast EC), 2,4-D (Weedar EC), glyphosate (Roundup EC), and alachlor (Lasso EC) were selected for the evaluations because of their widespread use as soil-applied agents and variety of recommended uses (French, 1988). Doses employed in the study were determined from recommended application rates. Commercially formulated herbicides were utilized in an effort to elucidate the field-use compatability of these pesticides with the nematode S. feltiae. Assays in aqueous suspensions. Nematode response to the herbicides was first determined in aqueous suspension, Treatments were arranged in a split-plot experimental design with the three exposure times as subplots of herbicide concentration for each of the four herbicides. Three dose levels for each herbicide were evaluated. These concentrations included the recommended field rate plus one log concentration higher and one log concentration lower than the recommended rate. Controls were exposed to deionized water only. Recommended rates were 4 ppm for 2,4-D, glyphosate, and alachlor and 1 ppm for sethoxydim. The respective herbicide solutions were added individually to Petri dishes (100 x 15 mm) in 20-ml aliquots. Two thousand dauer larvae were then added to each Petri dish. Nematodes were exposed to each herbicide concentration for one of three exposure times of 24, 72, or 120 hr. Each of these treatment combinations were replicated four times. Nematode activity was judged by response of the nematodes to mechanical stimulation with a probe at the appropriate exposure time. Twenty-five nematodes were selected at random from each Petri dish for evaluation of activity. Nematodes that responded to prodding by moving were considered active; those that did not move were recorded as inactive. The data were arcsin transformed and analyzed utilizing analysis of variance and least significant
AND
GARDNER
difference (LSD) procedures (SAS Institute, 1982). Infectivity of nematodes from these treatments also was evaluated in bioassays. After the prescribed exposure period, nematode suspensions from each Petri dish were centrifuged at 600 t-pm for 3 min. The resulting supernatant was decanted and replaced by deionized water. This procedure was repeated three times to remove the nematodes from contact with the herbicides. Infectivity of the washed nematodes was then evaluated in Petri dish bioassays using five nematode dose levels of 0,0.5, 1, 5, and 10 nematodes per host larva. Nematode doses were determined by actual counts using a binocular dissecting microscope. Ten last-instar G. mellonella larvae were exposed to the above doses in plastic Petri dishes (100 x 15 mm) lined with a moistened 9-cm diameter No. 1 Whatman filter paper. Each dose was replicated four times. Morality was recorded after 72 hr. Data were analyzed by probit analysis for treatment differences (SAS Institute, 1982). Soil assays. Nematode infectivity in response to herbicides also was evaluated in a Cecil coarse sandy loam soil (85% sand, 5% silt, and 10% clay). The soil (PH = 5.5) was sterilized with methyl bromide and ovendried before each assay. Bioassay arenas were constructed of polyvinyl chloride (PVC) pipe (lo-cm diam) cut into IO-cm lengths and sealed at the base with aluminum foil. Final bulk density and percentage moisture (w/w) of the test soil were 1.3 g/ cm3 and 22%, respectively. Twenty last-instar G. mellonella larvae were placed at the bottom of each test arena. Soil was then added to a depth of 5 cm to cover the larvae. Herbicide and nematode treatments were applied simultaneously to the soil surface of the appropriate arena. The herbicides alachlor and 2,4D were evaluated at 4 and 12 ppm and were applied evenly to the soil in 5 ml of solution/ arena. Controls were treated with an equivalent amount of deionized water. Nematodes were added to the appropriate arenas at levels of 0, 0.5, 1, 5, 10, 50, or 200 nem-
5. felfiae
AND HERBICIDE
atodesflarva. Nematode doses were determined by microscopic counts and were washed from a Petri dish lid onto the soil surface using a Pasteur pipet and deionized water. Bioassay arenas then were loosely covered with aluminum foil. Mortality was recorded 120 hr after exposure by excavating all G. mellonella from each arena. Four replicates of each nematode dose were performed at each herbicide concentration. Data were analyzed by probit analysis (SAS Institute, 1982). RESULTS AND DISCUSSION Assays in aqueous suspensions. All four herbicides evaluated decreased the activity of S. feltiae dauer larvae in aqueous solutions of the herbicides (Table 1). Alachlor was the most deleterious of the four herbicides yielding the highest mean percentage of inactive nematodes for both exposure time and herbicide concentration. Over 85% of the nematodes were inactive after exposure to the recommended field rate of alachlor. Only 22-23% of the nematodes were inactive after exposure to the recommended rate of the other three herbicides. For each herbicide tested, significantly (P < 0.05) more nematodes were inactive after exposure to 10x the recommended field rate than at lower concentrations. Length of exposure to the herbicides had little discernable effect on nematode activity (Table 1). Only alachlor significantly (P TABLE MEAN
PERCENTAGE
INACTIVE
S. feltiae
EXPOSURE
377
< 0.05) affected nematode activity with a greater percentage of nematodes being inactive after 48 and 120 hr of exposure than after 24 hr of exposure. The effects of alachlor and glyphosate on S. feltiae in aqueous suspensions have been reported by Poinar (1986) and Kovacs (1982). Poinar (1986) reported no adverse effects following nematode exposure to a 1:200 dilution of Lasso 4E (alachlor) for 168 hr. Kovacs (1982) reported no effect on nematode activity after a %-hr exposure to glyphosate at concentrations of 10,000 ppm. Our data appear counter to these studies; however, comparisons are difficult since procedures were not outlined by Poinar (1986), and Kovacs (1982) used different techniques in evaluating herbicide effects. Bioassays of nematodes collected from the aqueous suspensions in our study yielded median lethal doses (LD,,s) of 45.24, 1.14, 0.97, 1.12, and 2.80 nematodes per larva for alachlor, 2,4-D, glyphosate, sethoxydim, and the untreated control, respectively (Table 2). These data indicate that reductions in nematode activity after exposure to glyphosate, sethoxydim, and 2,4-D are not accompanied with concomitant reductions in infectivity. Perhaps, nematodes exposed to these three herbicides become quiescent but, after being removed from contact with the herbicides, became active again and are capable of in1
NEMATODES RESULTING IN AQUEOUS SOLUTION
FROM EXPOSURE
TO SELECTED
HERBICIDES
Exposure times (hr)
Herbicide concentration= Herbicide
l/10
FR
10x
24
48
120
Alachlor Glyphosate Sethoxydim 24-D Control
44.7a 20.4a 20.8a 19.6a
85.lb 23.5ab 23.la 22.7a 4.4
9O.Ob 25.0b 28.8b 30.0b
63.9ab 23.6ab 23.3a 23.la O.Oa
75.6b 20.la 25.8a 22.9a 3.8a
80.2b 25.4b 23.7a 26.4a 9.3a
L1Concentrations included recommended field rate (FR); 1 log dose lower than FR (l/10); 1 log dose higher than FR (10X). Field rates were 4 ppm for 2,4-D, glyphosate, and alachlor and 1 ppm for sethoxydim. b Numbers followed by the same letter within a row for exposure times or herbicide concentration are not significantly different (LSD: P < 0.05).
378
FORSCHLER,
DOSE/MORTALITY
RESPONSE
Herbicide
LDm
Ala&or 2,4-D Glyphosate Sethoxydim Control
45.24 1.14 0.97 1.12 2.80
ALL,
RESPONSE
WITH
95%
Slope
CI
28.7-86.9 1.05-1.25 0.69-l .32 1.03-1.22 0.80-5.30
TABLE 3 OF G. mellonella LARVAE IN SOIL HERBICIDE AND S. feltiae 95% CI
26.70 8.81 2.71
Recommended field 13.20-102.1 4.77-16.77 2.14-3.41
Alachlor 2,4-D Control
391.4 28.62 2.10
3 X recommended field 129.3-500.2 8.1M6.03 2.63-5.37 alachlor
and 2,4-D
95% CI 0.88-1.08 2.17-2.21 2.28-2.34 2.26-2.30 2.08-2.16
These tests provide important information on the compatibility of herbicides and S. feltiae. Alachlor was extremely deleterious to the dauernematodesin aqueoussuspensionsand in soil arenas.However, the activity assays indicated that storage of nematodesin solutions of the other three herbicidesprior to application would have little effect on nematodeactivity after 24hr. Although 2,4-D, glyphosate, and sethoxydim reducednematodeactivity in aqueous suspensions,these herbicides did not reduce the nematodes’ ability to infect G. mellonella larvae oncethe nematodeswere washed and removed from those herbi-
Alachlor 2,4-D Control
rate for
0.97 2.19 2.31 2.28 2.12
NEMATODES
DISCUSSION
Wm
field
S. feltiae
of 2.1 nematodes/larvafor the untreated check was significantly (P < 0.05)less than the LDSo of either herbicide treatment. While alachlor was the more deleteriousof the two herbicides,2,4-D did reduce infectivity of S. feltiae in soil, perhapsby reducing nematodeactivity.
Herbicide
a Recommended
GARDNER
TABLE 2 OF G. mellonella IN PETRI DISH BIOASSAYS EXPOSED TO HERBICIDES
fecting susceptibleinsect hosts. This mechanism may be similar to the cryptobiotic state observedin Aphelenchus avenue exposedto low oxygen tensions(Cooperand Van Grundy, 1970).Nematodesexposedto alachlor, however,do not fully recover and are significantly less infective than nematodes exposedto the other herbicides. Soil assays. Results of the soil bioassays with alachlor and2,4-D further substantiate this hypothesis. LD+ of 26.7 and 8.81 nematodes/larvawere obtained with treatmentsat the recommendedratesof alachlor and 2,4-D, respectively (Table 3). These levels were significantly (P < 0.05)higher than those of the control groups, which yielded an LD,, of 2.71 nematodes/larva. Soil bioassayswith 3~ the recommended herbicide rate yielded LD,,s of 391.4 and 28.6 nematodes/larvafor alachlor and 2.4D, respectively. These were more than threefoldhigherthanthe LD,,,s obtainedafter exposureof the nematodesto the recommendedrate of the herbicides.The LD5,, DOSE/MORTALITY
AND
was 4 ppm.
TREATED
SIMULTANEOUSLY
Slope
WITH
95%
CI
rate’ 1.00 1.10 1.48
0.94-l .06 1.05-1.1s 1.44-1.52
0.75 0.53 1.42
0.57-O-93 0.45-0.61 1.21-1.63
rate
S. feltiae AND HERBICIDE
tides. Alachlor, on the other hand, signiticantly reduced nematode activity and infectivity in those assays. Within the soil arenas, 2,4-D and alachlor significantly reduced nematode infectivity in comparison to the untreated checks. In the soil assays, nematode searching ability was an important prelude to host infection. The hostfinding sequence may have been disrupted by the herbicide, or perhaps the constant exposure of the nematodes to the herbicides in the soil reduced activity and, therefore, nematode infectivity. These data indicate that simultaneous application of selected herbicides and S. feltiue may reduce the efficacy of the nematode treatment. These studies further illustrate the importance of using a series of bioassays when examining pesticide effects on entomogenous nematodes. Petri dish bioassays of activity and infectivity provide pertinent information concerning the actual physical interactions between pesticides and nematodes. However, soil bioassays can indicate effects not illuminated by the Petri dish tests. Pesticide testing with entomogenous nematodes should include bioassays within the soil environment to mimic the intended field conditions to fully elucidate physical as well as behavioral interactions. REFERENCES JR., AND VAN GRUNDY, S. D. 1970. Metabolism of glycogen and neutral lipids by Aphelenchus avenue and Caenorhabditis sp. in aerobic, microaerobic, and anaerobic environments. J. Nematol., 2, 305-315. DAS, J. N., AND DIVAKAR, B. J. 1987. Compatibility of certain pesticides with DD-136 nematode. Plant Prot. Bull., 39, 2&21. DUTKY, S. R. 1974. Nematode parasites. In “Proceedings of the Summer Institute on Biological Control of Plant Insects and Diseases” (F. G. Maxwell and F. A. Harris, Eds.), pp. 576-490. Univ. Press of Mississippi, Jackson. FEDORKO, A., KAMIONEK, M., KOZLOWSKA, J., AND MIANOWSKA, E. 1977a. The effects of some carbamide herbicides on nematodes from different ecological groups. Pal. Ecol. Stud., 3, 23-28. COOPER,
A. F.,
EXPOSURE
FEDORKO, A., KAMIONEK, MIANOWSKA, E. 1977b.
379
M., KOZLOWSKA, J., AND The effects of some insecticides on nematodes of different ecological groups. Pol. Ecol. Stud., 3, 79-88. FRENCH, C. M. (Ed.). 1988. “Georgia Pest Control Handbook.” Cooperative Extension Service, University of Georgia, Athens. FUXA, J. R. 1987. Ecological considerations for the use of entomopathogens in IPM. Annu. Rev. Entomol., 32, 225-251. HARA, A. H., AND KAYA, H. K. 1982. Effects of selected insecticides and nematicides on the in vitro development of the entomogenous nematode, Neoaplectana carpocapsae. .I. Nematol., 14, 48w91. HARA, A. H., AND KAYA, H. K. 1983a. Toxicity of selected organophosphate and carbamate pesticides to infective juveniles of the entomogenous nematode Neoaplectana carpocapsae (Rhabditida: Steinemematidae). Environ. Entomol., 12, 4!%-501. HARA, A. H., AND KAYA, H. K. 1983b. Development of the entomogenous nematode, Neoaplectana carpocapsae (Rhabiditida: Steinemematidae), in insecticide-killed beet armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol., 76, 423-426. HEUNGENS, A., AND BUYSSE, G. 1987. Toxicity of several pesticides in water solution on Heterorhabditis nematodes. Med. Fat. Landbouww. Rijksuniv. Gent., 52, 631-638. KAMIONEK, M. 1979. The influence of pesticides on the morality and effectiveness of Neoaplectana carpocapsae Weiser. In “Proceedings of the Intemational Colloquium on Invertebrate Pathology and 1 lth Annual Meeting, Society for Invertebrate Pathology. Praque, Czech” (J. Weiser, Ed.). pp. 8788. KOVACS, A. 1982. Sensibilita de1 nematode entomopathogens Neoaplectana carpocapsae Weiser ad antiparassitari. “Atti Giomate Fitopathologiche,” pp. 11-19. POINAR, G. O., JR., 1986. “ ‘Entomophagous Nematodes’ in Biological Plant and Animal Protection.” G. Fisher Verlag, Stuttgart. PRAKASA RAO, P. S., DAS, P. K., AND PADHI, G. 1975. Note on compatibility of DD-136 (Neoaplectuna dutkyi), an insect parasitic nematode with some insecticides and fertilisers. Indian J. Agric. Sci., 45, 275-277. SAS Institute. 1982. “SAS User’s Guide: Statistics.” SAS Institute, Cary, NC. WELSH, H. E. 1971. Various target species: Attempt with DD-136. In “Biological Control Programmes against Insects and Weeds in Canada, 1959-1968.” Tech. Commun., CIBC 4, pp. 6266.
OF INVERTEBRATE
PATHOLOGY
55,
375-379 (I!?%)
Steinernema feltiae Activity and Infectivity in Response to Herbicide Exposure in Aqueous and Soil Environments BRIANT.FORSCHLER,JOHN Department
of Entomology,
N. ALL,AND~AYNE
University of Georgia Georgia Station, Griffin,
College Georgia
A. GARDNER
of Agriculture 30223-I 797
Experiment
Stations,
Received May 15, 1989; accepted September 14, 1989 Laboratory
bioassays determined the effect of herbicide exposure on the nematode Steinernema carpocapsae). AIachIor, sethoxydim, 2,4-D, and gfyphosate were tested at three concentrations in Petri dishes including the recommended application rate and one log dose higher and one log dose lower than the recommended rate for each of three exposure periods of 24, 48, and 120 hr. Alachlor was most deleterious reducing dauer activity by 44 to 90% in aqueous solution. Only 19 to 30% of the nematodes exposed to the other three herbicides were inactive. Bioassays of nematodes extracted from these suspensions and subsequently washed 3 x in water against Galleria mellonella last-instar larvae yielded median lethal doses (LD,s) of 45.2, 1.1, 0.9, and 1.1 nematodes/larva for the alachlor, 2,4-D, glyphosate and sethoxydim treatments, respectively. Control treatments with water yielded an LD,, of 2.8 nematodes/larva. Nematode infectivity in soil was reduced by exposure to 2,4-D and alachlor. At the recommended application rate, the LD,,s for the alachlor, 2,4-D, and control treatments were 26.7, 8.8, and 2.7 nematodes/larva, respectively. 6 1590 Academic press, Inc. KEY WORDS: Steinernema .feltiae; bioassay: herbicide; nematode; alachlor; 2,4-D; glyphosate; sethoxydim.
feltiae
(=Neoapfectana
INTRODUCTION
Entomogenous nematodes could be effective in many integrated pest management (IPM) programs as partial pest suppression agents used in conjunction with other control methods (Fuxa, 1987). Precluding the successful use of insect parasitic nematodes in such systems is knowledge of the possible interactions of the nematodes with other production practices, including use of chemical pesticides. Effects of selected pesticides on entomogenous nematodes have been reported in aqueous solution (Prakasa et al., 1975; Kovats, 1982; Hara and Kaya, 1983a; Das and Divakar, 1987; Heungens and Buysse, 1987), in an artificial medium and in insect hosts following pesticide exposure (Hara and Kaya, 1982, 1983b), and in an agar medium incorporated with pesticides (Kamionek, 1979; Fedorko et al., 1977a, b). Investigations by Welch (1971), Dutky (1974), and Poinar (1986) demonstrated no effect by selected herbicides on nematode
survival. Welch (1971) and Dutky (1974) evaluated herbicides that are no longer in use, while Poinar (1986) tested atrazine and alachlor. Of 17 herbicides tested in water solution, only trifluralin and pendimethalin adversely affected nematode activity in a study by Kovacs (1982). Fedorko et al. (1977a) reported significant nematode mortality (70%) after a 48-hr exposure of nematodes to water agar impregnated with technical grade monolinuron. Applications of entomegenous nematodes for the suppression of soil-inhabiting insect pests could expose them to herbicides that are commonly used in production systems. In this study, the quantitative effect of several commonly recommended herbicides on the activity and infectivity of the nematode Steinernema feltiae (= Neoaplectana carpocapsae) was tested in aqueous and soil environments. MATERIALS
AND METHODS
S. feftiae. (All strain) nematodes reared in 375 0022-2011190 $1.50 Copyright 6 1990 by Academic Press, Inc. AD rights of reproduction in any form resetvut.
376
FORSCHLER,
ALL,
last-instar Galleria mellonella larvae were used in all tests. Only newly emerged dauer larvae (24 hr old) were used in the assays. Commercially formulated sethoxydim (Poast EC), 2,4-D (Weedar EC), glyphosate (Roundup EC), and alachlor (Lasso EC) were selected for the evaluations because of their widespread use as soil-applied agents and variety of recommended uses (French, 1988). Doses employed in the study were determined from recommended application rates. Commercially formulated herbicides were utilized in an effort to elucidate the field-use compatability of these pesticides with the nematode S. feltiae. Assays in aqueous suspensions. Nematode response to the herbicides was first determined in aqueous suspension, Treatments were arranged in a split-plot experimental design with the three exposure times as subplots of herbicide concentration for each of the four herbicides. Three dose levels for each herbicide were evaluated. These concentrations included the recommended field rate plus one log concentration higher and one log concentration lower than the recommended rate. Controls were exposed to deionized water only. Recommended rates were 4 ppm for 2,4-D, glyphosate, and alachlor and 1 ppm for sethoxydim. The respective herbicide solutions were added individually to Petri dishes (100 x 15 mm) in 20-ml aliquots. Two thousand dauer larvae were then added to each Petri dish. Nematodes were exposed to each herbicide concentration for one of three exposure times of 24, 72, or 120 hr. Each of these treatment combinations were replicated four times. Nematode activity was judged by response of the nematodes to mechanical stimulation with a probe at the appropriate exposure time. Twenty-five nematodes were selected at random from each Petri dish for evaluation of activity. Nematodes that responded to prodding by moving were considered active; those that did not move were recorded as inactive. The data were arcsin transformed and analyzed utilizing analysis of variance and least significant
AND
GARDNER
difference (LSD) procedures (SAS Institute, 1982). Infectivity of nematodes from these treatments also was evaluated in bioassays. After the prescribed exposure period, nematode suspensions from each Petri dish were centrifuged at 600 t-pm for 3 min. The resulting supernatant was decanted and replaced by deionized water. This procedure was repeated three times to remove the nematodes from contact with the herbicides. Infectivity of the washed nematodes was then evaluated in Petri dish bioassays using five nematode dose levels of 0,0.5, 1, 5, and 10 nematodes per host larva. Nematode doses were determined by actual counts using a binocular dissecting microscope. Ten last-instar G. mellonella larvae were exposed to the above doses in plastic Petri dishes (100 x 15 mm) lined with a moistened 9-cm diameter No. 1 Whatman filter paper. Each dose was replicated four times. Morality was recorded after 72 hr. Data were analyzed by probit analysis for treatment differences (SAS Institute, 1982). Soil assays. Nematode infectivity in response to herbicides also was evaluated in a Cecil coarse sandy loam soil (85% sand, 5% silt, and 10% clay). The soil (PH = 5.5) was sterilized with methyl bromide and ovendried before each assay. Bioassay arenas were constructed of polyvinyl chloride (PVC) pipe (lo-cm diam) cut into IO-cm lengths and sealed at the base with aluminum foil. Final bulk density and percentage moisture (w/w) of the test soil were 1.3 g/ cm3 and 22%, respectively. Twenty last-instar G. mellonella larvae were placed at the bottom of each test arena. Soil was then added to a depth of 5 cm to cover the larvae. Herbicide and nematode treatments were applied simultaneously to the soil surface of the appropriate arena. The herbicides alachlor and 2,4D were evaluated at 4 and 12 ppm and were applied evenly to the soil in 5 ml of solution/ arena. Controls were treated with an equivalent amount of deionized water. Nematodes were added to the appropriate arenas at levels of 0, 0.5, 1, 5, 10, 50, or 200 nem-
5. felfiae
AND HERBICIDE
atodesflarva. Nematode doses were determined by microscopic counts and were washed from a Petri dish lid onto the soil surface using a Pasteur pipet and deionized water. Bioassay arenas then were loosely covered with aluminum foil. Mortality was recorded 120 hr after exposure by excavating all G. mellonella from each arena. Four replicates of each nematode dose were performed at each herbicide concentration. Data were analyzed by probit analysis (SAS Institute, 1982). RESULTS AND DISCUSSION Assays in aqueous suspensions. All four herbicides evaluated decreased the activity of S. feltiae dauer larvae in aqueous solutions of the herbicides (Table 1). Alachlor was the most deleterious of the four herbicides yielding the highest mean percentage of inactive nematodes for both exposure time and herbicide concentration. Over 85% of the nematodes were inactive after exposure to the recommended field rate of alachlor. Only 22-23% of the nematodes were inactive after exposure to the recommended rate of the other three herbicides. For each herbicide tested, significantly (P < 0.05) more nematodes were inactive after exposure to 10x the recommended field rate than at lower concentrations. Length of exposure to the herbicides had little discernable effect on nematode activity (Table 1). Only alachlor significantly (P TABLE MEAN
PERCENTAGE
INACTIVE
S. feltiae
EXPOSURE
377
< 0.05) affected nematode activity with a greater percentage of nematodes being inactive after 48 and 120 hr of exposure than after 24 hr of exposure. The effects of alachlor and glyphosate on S. feltiae in aqueous suspensions have been reported by Poinar (1986) and Kovacs (1982). Poinar (1986) reported no adverse effects following nematode exposure to a 1:200 dilution of Lasso 4E (alachlor) for 168 hr. Kovacs (1982) reported no effect on nematode activity after a %-hr exposure to glyphosate at concentrations of 10,000 ppm. Our data appear counter to these studies; however, comparisons are difficult since procedures were not outlined by Poinar (1986), and Kovacs (1982) used different techniques in evaluating herbicide effects. Bioassays of nematodes collected from the aqueous suspensions in our study yielded median lethal doses (LD,,s) of 45.24, 1.14, 0.97, 1.12, and 2.80 nematodes per larva for alachlor, 2,4-D, glyphosate, sethoxydim, and the untreated control, respectively (Table 2). These data indicate that reductions in nematode activity after exposure to glyphosate, sethoxydim, and 2,4-D are not accompanied with concomitant reductions in infectivity. Perhaps, nematodes exposed to these three herbicides become quiescent but, after being removed from contact with the herbicides, became active again and are capable of in1
NEMATODES RESULTING IN AQUEOUS SOLUTION
FROM EXPOSURE
TO SELECTED
HERBICIDES
Exposure times (hr)
Herbicide concentration= Herbicide
l/10
FR
10x
24
48
120
Alachlor Glyphosate Sethoxydim 24-D Control
44.7a 20.4a 20.8a 19.6a
85.lb 23.5ab 23.la 22.7a 4.4
9O.Ob 25.0b 28.8b 30.0b
63.9ab 23.6ab 23.3a 23.la O.Oa
75.6b 20.la 25.8a 22.9a 3.8a
80.2b 25.4b 23.7a 26.4a 9.3a
L1Concentrations included recommended field rate (FR); 1 log dose lower than FR (l/10); 1 log dose higher than FR (10X). Field rates were 4 ppm for 2,4-D, glyphosate, and alachlor and 1 ppm for sethoxydim. b Numbers followed by the same letter within a row for exposure times or herbicide concentration are not significantly different (LSD: P < 0.05).
378
FORSCHLER,
DOSE/MORTALITY
RESPONSE
Herbicide
LDm
Ala&or 2,4-D Glyphosate Sethoxydim Control
45.24 1.14 0.97 1.12 2.80
ALL,
RESPONSE
WITH
95%
Slope
CI
28.7-86.9 1.05-1.25 0.69-l .32 1.03-1.22 0.80-5.30
TABLE 3 OF G. mellonella LARVAE IN SOIL HERBICIDE AND S. feltiae 95% CI
26.70 8.81 2.71
Recommended field 13.20-102.1 4.77-16.77 2.14-3.41
Alachlor 2,4-D Control
391.4 28.62 2.10
3 X recommended field 129.3-500.2 8.1M6.03 2.63-5.37 alachlor
and 2,4-D
95% CI 0.88-1.08 2.17-2.21 2.28-2.34 2.26-2.30 2.08-2.16
These tests provide important information on the compatibility of herbicides and S. feltiae. Alachlor was extremely deleterious to the dauernematodesin aqueoussuspensionsand in soil arenas.However, the activity assays indicated that storage of nematodesin solutions of the other three herbicidesprior to application would have little effect on nematodeactivity after 24hr. Although 2,4-D, glyphosate, and sethoxydim reducednematodeactivity in aqueous suspensions,these herbicides did not reduce the nematodes’ ability to infect G. mellonella larvae oncethe nematodeswere washed and removed from those herbi-
Alachlor 2,4-D Control
rate for
0.97 2.19 2.31 2.28 2.12
NEMATODES
DISCUSSION
Wm
field
S. feltiae
of 2.1 nematodes/larvafor the untreated check was significantly (P < 0.05)less than the LDSo of either herbicide treatment. While alachlor was the more deleteriousof the two herbicides,2,4-D did reduce infectivity of S. feltiae in soil, perhapsby reducing nematodeactivity.
Herbicide
a Recommended
GARDNER
TABLE 2 OF G. mellonella IN PETRI DISH BIOASSAYS EXPOSED TO HERBICIDES
fecting susceptibleinsect hosts. This mechanism may be similar to the cryptobiotic state observedin Aphelenchus avenue exposedto low oxygen tensions(Cooperand Van Grundy, 1970).Nematodesexposedto alachlor, however,do not fully recover and are significantly less infective than nematodes exposedto the other herbicides. Soil assays. Results of the soil bioassays with alachlor and2,4-D further substantiate this hypothesis. LD+ of 26.7 and 8.81 nematodes/larvawere obtained with treatmentsat the recommendedratesof alachlor and 2,4-D, respectively (Table 3). These levels were significantly (P < 0.05)higher than those of the control groups, which yielded an LD,, of 2.71 nematodes/larva. Soil bioassayswith 3~ the recommended herbicide rate yielded LD,,s of 391.4 and 28.6 nematodes/larvafor alachlor and 2.4D, respectively. These were more than threefoldhigherthanthe LD,,,s obtainedafter exposureof the nematodesto the recommendedrate of the herbicides.The LD5,, DOSE/MORTALITY
AND
was 4 ppm.
TREATED
SIMULTANEOUSLY
Slope
WITH
95%
CI
rate’ 1.00 1.10 1.48
0.94-l .06 1.05-1.1s 1.44-1.52
0.75 0.53 1.42
0.57-O-93 0.45-0.61 1.21-1.63
rate
S. feltiae AND HERBICIDE
tides. Alachlor, on the other hand, signiticantly reduced nematode activity and infectivity in those assays. Within the soil arenas, 2,4-D and alachlor significantly reduced nematode infectivity in comparison to the untreated checks. In the soil assays, nematode searching ability was an important prelude to host infection. The hostfinding sequence may have been disrupted by the herbicide, or perhaps the constant exposure of the nematodes to the herbicides in the soil reduced activity and, therefore, nematode infectivity. These data indicate that simultaneous application of selected herbicides and S. feltiue may reduce the efficacy of the nematode treatment. These studies further illustrate the importance of using a series of bioassays when examining pesticide effects on entomogenous nematodes. Petri dish bioassays of activity and infectivity provide pertinent information concerning the actual physical interactions between pesticides and nematodes. However, soil bioassays can indicate effects not illuminated by the Petri dish tests. Pesticide testing with entomogenous nematodes should include bioassays within the soil environment to mimic the intended field conditions to fully elucidate physical as well as behavioral interactions. REFERENCES JR., AND VAN GRUNDY, S. D. 1970. Metabolism of glycogen and neutral lipids by Aphelenchus avenue and Caenorhabditis sp. in aerobic, microaerobic, and anaerobic environments. J. Nematol., 2, 305-315. DAS, J. N., AND DIVAKAR, B. J. 1987. Compatibility of certain pesticides with DD-136 nematode. Plant Prot. Bull., 39, 2&21. DUTKY, S. R. 1974. Nematode parasites. In “Proceedings of the Summer Institute on Biological Control of Plant Insects and Diseases” (F. G. Maxwell and F. A. Harris, Eds.), pp. 576-490. Univ. Press of Mississippi, Jackson. FEDORKO, A., KAMIONEK, M., KOZLOWSKA, J., AND MIANOWSKA, E. 1977a. The effects of some carbamide herbicides on nematodes from different ecological groups. Pal. Ecol. Stud., 3, 23-28. COOPER,
A. F.,
EXPOSURE
FEDORKO, A., KAMIONEK, MIANOWSKA, E. 1977b.
379
M., KOZLOWSKA, J., AND The effects of some insecticides on nematodes of different ecological groups. Pol. Ecol. Stud., 3, 79-88. FRENCH, C. M. (Ed.). 1988. “Georgia Pest Control Handbook.” Cooperative Extension Service, University of Georgia, Athens. FUXA, J. R. 1987. Ecological considerations for the use of entomopathogens in IPM. Annu. Rev. Entomol., 32, 225-251. HARA, A. H., AND KAYA, H. K. 1982. Effects of selected insecticides and nematicides on the in vitro development of the entomogenous nematode, Neoaplectana carpocapsae. .I. Nematol., 14, 48w91. HARA, A. H., AND KAYA, H. K. 1983a. Toxicity of selected organophosphate and carbamate pesticides to infective juveniles of the entomogenous nematode Neoaplectana carpocapsae (Rhabditida: Steinemematidae). Environ. Entomol., 12, 4!%-501. HARA, A. H., AND KAYA, H. K. 1983b. Development of the entomogenous nematode, Neoaplectana carpocapsae (Rhabiditida: Steinemematidae), in insecticide-killed beet armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol., 76, 423-426. HEUNGENS, A., AND BUYSSE, G. 1987. Toxicity of several pesticides in water solution on Heterorhabditis nematodes. Med. Fat. Landbouww. Rijksuniv. Gent., 52, 631-638. KAMIONEK, M. 1979. The influence of pesticides on the morality and effectiveness of Neoaplectana carpocapsae Weiser. In “Proceedings of the Intemational Colloquium on Invertebrate Pathology and 1 lth Annual Meeting, Society for Invertebrate Pathology. Praque, Czech” (J. Weiser, Ed.). pp. 8788. KOVACS, A. 1982. Sensibilita de1 nematode entomopathogens Neoaplectana carpocapsae Weiser ad antiparassitari. “Atti Giomate Fitopathologiche,” pp. 11-19. POINAR, G. O., JR., 1986. “ ‘Entomophagous Nematodes’ in Biological Plant and Animal Protection.” G. Fisher Verlag, Stuttgart. PRAKASA RAO, P. S., DAS, P. K., AND PADHI, G. 1975. Note on compatibility of DD-136 (Neoaplectuna dutkyi), an insect parasitic nematode with some insecticides and fertilisers. Indian J. Agric. Sci., 45, 275-277. SAS Institute. 1982. “SAS User’s Guide: Statistics.” SAS Institute, Cary, NC. WELSH, H. E. 1971. Various target species: Attempt with DD-136. In “Biological Control Programmes against Insects and Weeds in Canada, 1959-1968.” Tech. Commun., CIBC 4, pp. 6266.