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Winter cover crop enhances 2,4-D mineralization potential of surface and subsurface soil
PERGAMON
Soil Biology and Biochemistry 31 (1999) 849±857
Winter cover crop enhances 2,4-D mineralization potential of surface and subsurface soil P.J. Bottomley a, b, *, T.E. Sawyer a, L. Boersma a, R.P. Dick a, D.D. Hemphill c a
Department of Crop and Soil Sciences, Oregon State University, Corvallis, OR 97331-3804, USA b Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA c North Willamette Research and Extension Center, Oregon State University, Aurora, OR 97002-9543, USA Accepted 27 October 1998
Abstract A study was conducted to determine if the use of a winter cover crop in a summer vegetable crop rotational system might in¯uence the potential of surface and subsurface layers of a Willamette silt loam soil to mineralize the herbicide, 2,4dichlorophenoxyacetic acid (2,4-D). On three occasions between April and September 1994, and three occasions between February and June 1995, soil samples were recovered from the Ap horizon (0±20 cm), and from within the argillic B (80±100 cm) horizon of ®eld plots managed in either a summer vegetable crop±winter fallow rotation, or a summer vegetable crop± winter cover crop (cereal rye) rotation. Composite samples of soil were prepared from the four replicates of each of the two ®eld treatments, and the mineralization of 6 mg 2,4-D kg ÿ 1 examined under laboratory conditions. 2,4-D was mineralized more quickly in the 0±20-cm soil from the cover crop treatment than the winter fallow treatment on ®ve of the six sampling occasions. 2,4-D mineralization characteristics of the 80±100-cm soil diered between the cover crop and winter fallow treatments and also diered between sampling dates. In February, 2,4-D mineralization rates developed slowly and persisted at suboptimal rates for at least 10 d in both treatments. In April and June, daily 2,4-D mineralization rates increased more rapidly in soil from the cover crop treatment than in samples taken from the winter fallow treatment. Although no dierences were detected in mineralization characteristics of subsurface soil sampled from the two treatments while the sweet corn summer crop was growing (July), treatment dierences were again discernible immediately after the summer crop was harvested (September). Our ®ndings identi®ed a potential bonus of using a winter cover crop, i.e. enhancing the potential of subsurface soil micro¯ora to mineralize herbicides that might leach from the surface. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Because of the mild climate and rainfall pattern, an extremely diverse agriculture is practiced in western Oregon throughout the calendar year. As a consequence, fertilizers and herbicides are applied under a wide variety of environmental conditions. Because of recent concerns about run-o and leaching of nitrate into water supplies, fall-seeded cover crops have surfaced as a simple strategy to immobilize nitrogen in well-drained soils during the winter and early spring * Corresponding author. Fax: +1-541-737-0496; e-mail: [email protected]
months (Meisinger et al., 1991; Brandi-Dorn et al., 1997). The eects of cover cropping on the ecacy and fate of the herbicides used in these cropping systems are unknown. For example, while plants might enhance the populations of pesticide-degrading microorganisms adjacent to their roots, and thereby stimulate breakdown of soil-borne herbicides (Boyle and Shann, 1995; Haby and Crowley, 1996), plant roots can also provide a conduit to transport surface-applied pesticides through the soil to ground water (Anderson et al., 1993; Shimp et al., 1993). Although soil microbial biomass and enzyme activities can be greater in cover cropped than fallowed soil (Miller and Dick, 1995), it is unknown how far below the soil surface the
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 8 ) 0 0 1 8 4 - 9
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eect of a cover crop might extend, nor if it in¯uences herbicide-degrading microorganisms speci®cally. Detailed studies on microbiological transformation of pesticides in the subsurface are somewhat limited (Moorman and Harper, 1989; Moorman, 1990; Pothuluri et al., 1990; Locke and Harper, 1991; Mueller et al., 1992; Mallawatantri et al., 1996; Veeh et al., 1996). Although many studies have been published on the eects of soil physical and chemical properties on pesticide movement (Beck et al., 1993), and mathematical models have been developed to predict pesticide transport (Wagenet and Hutson, 1990; Boesten and van der Linden, 1991), fewer studies have attempted to link microbiological degradation with water ¯ow and chemical movement in the subsurface soil (Estrella et al., 1993; Pivetz and Steenhuis, 1995). With this longer-term goal in our minds, the immediate objectives were to determine the mineralization potential of 2,4-D at dierent depths in a soil pro®le and to determine if a winter cover crop in¯uenced the 2,4D mineralization properties associated with soil at those dierent depths. We chose to use 2,4-D as the herbicide of choice because it is water soluble and continues to be used extensively in a variety of agricultural and urban settings at relatively high rates (Johnson et al., 1995; Michel et al., 1995). Furthermore, an extensive literature has accumulated over the past 40 yr describing the properties of 2,4-D breakdown in soil (Sandemann et al., 1988) and the characteristics of 2,4D degrading soil microorganisms (Ka et al., 1994; Tonso et al., 1995; Xia et al., 1995).
The experimental design was a randomized complete block with four replications of each treatment. Soil samples were taken from the four replicates of two of the winter cover crop treatments namely, winter fallow and cereal rye. About 30 cores of soil were taken on 1m spacing from a 64 m grid laid out in each replicate plot. Soil cores were recovered from the 0±20, 20±40, 40±60, 60±80 and 80±100-cm depth intervals using an extendible tube auger of 2.5 cm diameter. Soil cores were broken up by hand, thoroughly mixed, placed in large ziploc bags and transported to the laboratory. Some simple precautions were taken to reduce the chances of contaminating the lower layers of soil with overlying material. (1) All samples were removed from a speci®c depth before moving to the next depth. (2) The inside surfaces of the bore holes were carefully reamed with the probe before sampling the next depth increment. (3) Loose soil on the top of the lower cores and extraneous soil on the auger were removed before bagging the samples. (4) The auger was surface sterilized with ethanol between sampling the dierent depths. Prior to experimentation, soil water content was determined (1058C, 48 h). Textural analysis was conducted on samples of soil using the hydrometer procedure of the soil physical analysis laboratory, Department of Crop and Soil Sciences, Oregon State University. Total organic-C was determined by dry combustion with a Dohrman DC-80 carbon analyzer. Saturated hydraulic conductivity measurements were made with a double-ring in®ltrometer by J. Selker, Department of Bioresources Engineering, Oregon State University.
2. Materials and methods
2.2. 2,4-D mineralization studies
2.1. Experimental site
Within 2±3 d of sampling from the ®eld, mineralization experiments were conducted on composite samples of soil prepared by mixing subsamples of soil (sieved <2 mm) from each of the four replicates of a ®eld treatment. Mineralization experiments were conducted in a continuous air ¯ow and trap system that allowed humidi®ed CO2-free air to ¯ow through 125 ml Erlenmeyer ¯asks at a rate of about 10 ml min ÿ 1. Twenty-g portions of soil were added to deionized water containing sucient 2,4-D to provide a ®nal water content of 300 g kg ÿ 1 oven dry soil and 6 mg 2,4-D kg ÿ 1 of soil. The 2,4-D solution was supplemented with approximately 0.1 mci (3.7 kBq) of [UL-14 C] ring labeled 2,4-D, (speci®c activity, 696 MBq mmol ÿ 1, >98% chemical purity, Sigma Chemical Co.). In tests, samples of gamma-irradiated soil (4 Mrad dose) were supplemented with the labeled 2,4-D solution and incubated as described above to evaluate the possibility of abiological decomposition of the herbicide. The radioactivity recovered in the alkali traps never exceeded the background counts that were
Soil samples were collected from a vegetable crop rotation experiment initiated in 1989 at the North Willamette Research and Extension Center (NWREC), Aurora, OR. The soil is a Willamette silt loam (Pachic Ultic Argixeroll), and the site characteristics were described by Brandi-Dorn et al. (1997) and Burket et al. (1997). Field treatments included three dierent winter cover crop treatments in a summer vegetable crop rotation that alternates two summer crops, sweet corn (Zea mays L. cv. Jubilee) and broccoli (Brassica oleracea L. Botrytis group cv. Gem). The summer crops were planted in late May±early June (corn 1994 and broccoli 1995), and were harvested in September. The winter cover crop treatments included winter fallow, fall planted cereal rye (Secale triticale) seeded at 40 kg ha ÿ 1, and a mixture of cereal rye and Austrian winter pea (Pisum sativum L.) seeded at 40 and 112 kg ha ÿ 1, respectively. The cover crops were seeded in late September and incorporated into the soil in mid April.
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obtained when scintillation ¯uid alone was counted. Nonetheless, samples of sterile soil amended with radiolabeled 2,4-D, and radiolabel without soil were included routinely in each study to serve as controls. Triplicate samples of composite soil prepared from each of the two ®eld treatments were used for each experimental treatment. In the early stages of the study (1993 and February and April, 1994), a few experiments were carried out with two additional concentrations of 2,4-D (0.06 and 0.6 mg kg ÿ 1). Because the treatment eects on mineralization properties were found to be similar at both 0.6 and 6.0 mg kg ÿ 1, the majority of the experiments we describe here were conducted with 6 mg 2,4-D kg ÿ 1. 14 C labeled CO2 was collected by passing the euent air through 20 ml capacity vials containing 6 ml portions of 0.1 M NaOH. The vials were changed daily or at other intervals when appropriate. Ten-ml portions of Scintisafe Plus counting ¯uid (Fisher Scienti®c) were added to each vial and counts were determined in a Beckman LS3801 scintillation counter. Although NaOH was used to trap the liberated CO2, the eciency of counting was high (r95%). Presumably, a high percentage of the alkali in the traps was neutralized by the air-derived CO2 continuously passing through the vials, and by the CO2 derived from both 2,4-D and the background soil respiration. The percentage of 2,4-D mineralized was calculated by dividing the average dis min ÿ 1 recovered in the alkali traps by the dis min ÿ 1 added to each soil incubation. 2.3. Microbial biomass determinations Microbial biomass-C measurements were determined with the chloroform fumigation±extraction method (CFEM) described by Horwath and Paul (1994). Brie¯y, four 10-g portions of soil from each of the soil depths (0±20, 20±40, 40±60, 60±80 and 80±100-cm) were wetted to 30% (wt/vol) water content, incubated at 258C for 4 d, fumigated with chloroform vapor for 5 d, and extracted in 0.5 M K2SO4 (1:5(wt/vol), soil:solution). The supernatants were ®ltered through acidwashed Whatman ®lter paper and 100-ml portions were
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analyzed in a Dohrman carbon analyzer. A correction factor of 0.35 was used in the calculation to obtain biomass values. 2.4. Determination of the population of 2,4-D degrading microorganisms by a most probable number (MPN) analysis A procedure was developed which was based upon that of Lehmicke et al. (1979). Portions (10 g) of soil were serially diluted in a phosphate-buered mineral salts solution. Replicate (n = 4) 10-ml samples of appropriate dilutions were added to 50-ml serum vials containing 0.1 mmol of 2,4-D supplemented with 50 nci (1.85 kBq) of 14 C-ring labeled 2,4-D. The 2,4-D was added to the vials as a methanolic solution that was allowed to evaporate before the samples of soil dilutions were added. Wicks constructed of ¯uted ®lter paper were placed in small plastic bucket assemblies (Kontes of California), which were ®xed into the rubber septa of the vials and suspended above the soil dilutions. Portions (100 ml) of 0.1 M NaOH were injected through the rubber septa onto the wicks. Incubations were conducted at room temperature without shaking. After approximately 30 d, the radioactivity trapped in the NaOH was determined by scintillation counting. Any vial in which 10% or more of the original activity was recovered in the NaOH was considered positive. The sizes of the 2,4-D degrading populations in the soil samples were derived using a most-probable-number computer program described by Woomer et al. (1990). Tests showed that 28 d was an adequate incubation period to obtain the maximum number of positive vials from a dilution series. 3. Results Several physical, chemical and biological properties of the Willamette silt loam changed with depth at the experimental site (Table 1). Decreases in both organic matter content and saturated hydraulic conductivity, accompanied by a substantial increase in the clay con-
Table 1 Physical properties of Willamette silt loam soila Depth increment (cm)
Textural analysis (g kg ÿ 1) sand (>50 mm)
silt (50-2 mm)
clay (<2 mm)
0±20 20±40 40±60 60±80 80±100
313 328 298 286 374
540 499 476 490 450
147 173 226 224 175
a
See Section 2 for details of procedures.
Satd. hydr. cond., Ksat (10 ÿ 6 m s ÿ 1)
Total soil carbon (g kg ÿ 1)
pH (1:2 w/v soil:H2O)
60 N.D. 7 N.D. 0.7
18 18 9.4 6.5 4.7
6.3 N.D. 6.3 N.D. 6.2
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Table 2 Changes in microbial parameters with soil depth in the two cover crop treatmentsa,b Soil depth (cm)
0±20 20±40 40±60 60±80 80±100
Microbial biomass (mg C kg ÿ 1)
Respiratory rate (mmol CO2 kg ÿ 1 d ÿ 1)
No. (106) of 2,4-D degraders kg ÿ 1
WF
CC
WF
CC
WF
CC
140 (58)c 148 (62) 86 (35) 28 (12) undetectable
204 (100) 135 (88) 45 (30) 29 (19) undetectable
970 (400)c 276 (116) 352 (144) 60 (26) 80 (33)
610 (300) 260 (170) 90 (60) 150 (99) 70 (45)
(1.6) 4.6 (13.3)d (0.3) 0.9 (2.6) not determined (0.05) 0.13 (0.4) (0.05) 0.15 (0.4)
(7.4) 21.3 (61.5) (8.9) 25.8 (74.3) not determined (0.04) 0.10 (0.3) (0.10) 0.31 (0.9)
a See Section 2 for details of the procedures. bAbbreviations stand for winter fallow (WF) and cover crop (CC) treatments, respectively. Values in parentheses represent standard deviations of triplicate determinations. dValues in parentheses represent the upper and lower 95% con®dence limits of the population estimates.
c
tent of the soil at 40±60 cm delineated the upper limit of the argillic B horizon. Under both the winter fallow, and the cover crop treatments, microbial biomass-C declined abruptly at the 40±60-cm depth from about 140±200 mg C kg ÿ 1 soil in the surface 0±40-cm layer, to 45±86 mg C kg ÿ 1 soil at 40 cm depth and below (Table 2). Although microbial biomass could not be measured in soil from the 80±100-cm depth increment with either the chloroform fumigation±incubation or fumigation±extraction methods, the most-probablenumber procedure detected a population of 2,4-D degraders at densities of approximately 105 kg ÿ 1 below 40 cm in the pro®le (Table 2). Despite the experiment having been in place since 1989, no signi®cant dierences were detected among the treatments in amounts of microbial biomass throughout the pro®le, and in populations of 2,4-D degraders at the 60±100 cm depth in the pro®le. MPN estimates showed that the mean population sizes of 2,4-D degraders are between 5 and 20-fold greater in the 0±20 and 20±40 cm layers, respectively, of the cover crop treatment than in the winter fallow treatment. However, these population means are accompanied by rather large con®dence limits. In preliminary experiments conducted on soil recovered at 20 cm depth increments between 0 and 100 cm, the characteristics of 2,4-D mineralization (0.6 and 6.0 mg 2,4-D kg ÿ 1 of soil) were observed to change with soil depth (Fig. 1a and b). A distinctly longer period was required before mineralization of 0.6 mg 2,4-D kg ÿ 1 could be detected at depths of 40 cm and below (04 d) than at more shallow depths (1±2 d). With the higher concentration of 2,4-D (6 mg kg ÿ 1) the dierence between depths was more exaggerated because of the lower speci®c activity of the label and because the rate of mineralization did not increase in response to the increase in 2,4-D concentration (Fig. 1b). In subsequent experiments, comparisons of 2,4-D mineralization were made only between soil taken from the surface layer (0±20 cm), and from an increment of the
subsurface (80±100 cm) that lay within the argillic horizon, but could be reached from the surface with simple tools and minimal disturbance of the plot area. Over the course of the 2-yr study, we observed that the 2,4-D mineralization properties of surface and subsurface soil were in¯uenced by sampling time and by the winter cover crop treatment (Figs. 2±4). On
Fig. 1. Mineralization of 14 C ring-labeled 2,4-D applied at (a) 0.6 mg kg ÿ 1 and (b) 6.0 mg kg ÿ 1 to soil recovered from: w, 0±20 cm, Q, 20±40 cm, q, 40±60 cm, r, 60±80 cm and R, 80±100 cm depth increments from the winter fallow treatment in February, 1994. Absence of error bars on the data points indicates that the SE of the mean was less than the size of the symbol.
P.J. Bottomley et al. / Soil Biology and Biochemistry 31 (1999) 849±857
Fig. 2. Mineralization of 14 C ring-labeled 2,4-D applied at (a) 0.6 mg kg ÿ 1 and (b) 6.0 mg kg ÿ 1 to soil recovered in April, 1994 from either the winter fallow (circles) or the cereal rye cover crop (triangles) treatments. Open symbols represent surface (0±20 cm) soil and closed symbols represent subsurface (80±100 cm) soil.
sampling occasions in April and July, 1994 (Figs. 2 and 3) and February, April and June, 1995 (Fig. 5), 2,4-D mineralization could be detected in soil from the surface layer (0±20 cm) of the cover crop treatment about 1 d earlier than in soil taken from the winter fallow treatment. Only in September 1994 (Fig. 3b) did the mineralization characteristics of 0±20-cm samples of the cover crop and winter fallow treatments overlap. An eect of the winter cover crop treatment on 2,4-D mineralization was detected in the 80±100-cm layer. For example, in April 1994, although 2,4-D mineralization in the subsurface soil of the cover crop treatment was initiated at a similar time as the winter fallow treatment (3±4 d) (Fig. 2), mineralization of both 0.6 mg kg ÿ 1 (Fig. 2a) and 6.0 mg 2,4-D kg ÿ 1 (Fig. 2b) increased more rapidly in soil from the cover crop treatment. In July and September, 1994, however, 2,4D mineralization was initiated at the same time and progressed similarly in the 80±100-cm samples of both treatments (Fig. 3a and b). In 1995, dierences between the 2,4-D mineralization properties of the cover crop and winter fallow treatments were apparent in both April and June samplings of the 80±100-cm layer (Fig. 4b and c), but not in February (Fig. 4a) where mineralization proceeded slowly regardless of
853
Fig. 3. Mineralization of 14 C ring-labeled 2,4-D applied at 6 mg kg ÿ 1 to soil recovered from the winter fallow and cereal rye cover crop treatments in (a) July and (b) September 1994. Symbol de®nitions are the same as represented in Fig. 2.
treatment. The time required to detect 2,4-D mineralization in the 80±100-cm layer of the cover crop treatment decreased progressively from 4±6 d, to 3±4 d and to 2±3 d in the February, April and June samples, respectively. In contrast, the duration of the lag period never dropped below 4±5 d in samples from the 80± 100-cm layer of the winter fallow treatment. Mineralization data were also examined in the context of how the daily rates of 2,4-D mineralization changed throughout the incubation (Figs. 5 and 6). Regardless of time of sampling or treatment, the daily rates of 2,4-D mineralization in 0±20 cm soil reached a distinct maximum value within 2±5 d of incubation. The daily rates of mineralization in the 0±20-cm layer of cover crop treatment (up to its daily maximum) were almost always greater (except September 1994) than measured in the winter fallow treatment on any speci®c day of incubation. Nonetheless, the winter fallow treatment usually reached a similar daily rate of mineralization about 1 d later. In the 80±100-cm subsurface soil, the eects of the cover crop treatment on the daily mineralization rates varied with sampling time. For example, in April 1994, daily rates of mineralization in the cover crop treatment developed to a maximum value within 2 d of being detectable (3±5 d) (Fig. 5a). In the case of the winter fallow, daily rates
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Fig. 4. Mineralization of 14 C ring-labeled 2,4-D applied at 6 mg kg ÿ 1 to soil recovered from the winter fallow and cereal rye cover crop treatments in (a) February, (b) April and (c) June 1995. Symbol de®nitions are the same as represented in Fig. 2.
increased slowly to a daily rate that did not change signi®cantly from the day 5 through day 9 of incubation. By contrast, in July 1994, the daily mineralization rates of the 80±100-cm layer of both the winter fallow and cover crop treatments reached the same maximum daily rate on the same day (Fig. 5b). In September 1994, the increase in the daily rate of mineralization in the 80±100-cm layer of the cover crop treatment was again somewhat faster than in the same layer of the winter fallow treatment (Fig. 5c). In February 1995, daily rates of mineralization increased very slowly in subsurface soil from both treatments and never exceeded 500 ng g ÿ 1 d ÿ 1. In the case of the winter fallow treatment, the daily rate of mineralization was relatively stable between day 8 and day 13 of incubation, whereas the rate in the cover crop treatment continued to slowly increase (Fig. 6a).
Fig. 5. A comparison of the daily rates of mineralization of 2,4-D applied at 6 mg kg ÿ 1 to soil recovered from the 0±20 cm and 80± 100 cm layers of the winter fallow and cereal rye cover crop treatments in (a) April, (b) July and (c) September 1994. Symbol de®nitions are the same as represented in Fig. 2.
Indeed, tests conducted with subsurface soil recovered from the winter fallow treatment in February 1994 had given similar results (see Fig. 1b). Unfortunately, at that time comparisons were not made with the cover crop treatment. In April 1995, the increase in daily rate of 2,4-D mineralization in the 80±100-cm samples from the cover crop treatment was greater than observed in the winter fallow (Fig. 6b). In neither treatment, however, did the daily rate of mineralization increase rapidly enough to create the distinct peak in the daily rate of mineralization (r1200 ng g ÿ 1 d ÿ 1) invariably seen in the 0±20-cm soil samples, and in the 80±100-cm samples taken from the cover crop treatment in April, July and September, 1994 and in June 1995. Instead, daily mineralization rates of R500 ng g ÿ 1 d ÿ 1 were sustained for about 4±6 d. Although
P.J. Bottomley et al. / Soil Biology and Biochemistry 31 (1999) 849±857
Fig. 6. A comparison of the daily rates of mineralization of 2,4-D applied at 6 mg kg ÿ 1 to soil recovered from the 0±20 and 80±100 cm layers of the winter fallow and cereal rye cover crop treatments in (a) February, (b) April and (c) June, 1995. Symbol de®nitions are the same as represented in Fig. 2.
it took longer to initiate mineralization in the 80±100cm samples from the winter fallow treatment than the cover crop treatment in June 1995 (Fig. 6c), large increases in the daily rates of mineralization were observed in both treatments. 4. Discussion Although numerous papers have been written on the subject of 2,4-D degrading bacteria and the fate of 2,4D in soil (Loos, 1969; Wilson and Cheng, 1976, 1978; Sandemann et al., 1988; Smith and Lafond, 1990), our ®ndings add new insights into the role that cover crops might play in the fate of pesticides that penetrate into the subsurface soil environment. The data presented in
855
this manuscript clearly indicate that a cover crop of cereal rye can have a measurable eect on the 2,4-D mineralization potential of surface and subsurface soil. While it is well known that microorganisms in subsurface soil have the potential to mineralize herbicides (Harris et al., 1966; Lavy et al., 1973; Bouchard et al., 1982; Moorman, 1990) mineralization responses with soil depth have been quite variable. For example, while a signi®cant decline in the rate of metribuzin transformation occurred in soil samples recovered from >10 cm below the surface of a Dundee silt loam (Moorman and Harper, 1989; Locke and Harper, 1991), Kempson-Jones and Hance (1979) measured similar rates of metribuzin transformation in samples of soil recovered between the surface and 50±60 cm depth. Veeh et al. (1996) measured dierences in 2,4-D mineralization between 0±30, 30±60 and 60±120 cm in one soil, whereas no dierences were detected in the 30±60 and 60±120-cm layers of another soil. In the latter study, the authors showed that 2,4-D half life increased from 5 to 25 d as soil depth increased, and the increase in half life correlated with a decline in soil organic-C with soil depth from 1 to 0.4 wt%. By contrast, in our study, we found that the 2,4-D mineralization potential of soil was relatively constant on any particular sampling date between 40 and 100 cm depth in the pro®le, despite the fact that soil organic-C contents declined from 1 to 0.4 wt% over this depth increment. Furthermore, we could not detect a signi®cant winter cover crop eect on total soil organic-C contents below 40 cm in the pro®le (data not shown), despite showing repeatedly that the 2,4-D mineralization potential of the subsurface diered between the winter fallow and cover crop treatments. It is of interest to speculate about how a cover crop might be in¯uencing the herbicide-degrading potential of the soil. It is reasonable to believe that the cover crop eect detected in the surface soil might be attributed to the somewhat higher population density of 2,4D degraders found in surface soil of the cover crop than in the winter fallow treatment. This idea would be consistent with the cover crop treatment supporting a higher population density of bacteria because of the inputs of cover crop residue. However, we need to bear in mind that a traditional MPN estimate of a population size is accompanied by rather large con®dence limits, and population density dierences of less than 10-fold are suspect (Woomer et al., 1990). Because nonlinear increases in daily rates of 2,4-D mineralization are consistent with growth of the 2,4-D degrading community in response to the substrate, we conclude that cover cropped subsurface soil is superior to the winter fallow treatment in supplying the supplementary resources needed to support microbial growth on 2,4-D. The exceptions to the rule were samples recovered in February and July. Because we
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have observed that cover crop growth at this site increases signi®cantly during March and April, as daylength and temperatures increase, support for the degrading community might be coming directly from the roots of the cover crop (Haby and Crowley, 1996). However, the situation is complicated by the fact that the cover crop is incorporated into the soil in midApril, and its eect on 2,4-D mineralization was sustained into June. Further work is needed to determine to what extent the stimulatory eect of the cover crop on 2,4-D mineralization potential in the subsurface soil can be attributed to active cover crop growth, versus decomposition of cover crop residues after soil incorporation. In the context of speculating about how a cover crop or its residues might in¯uence the subsurface soil environment, Mallawatantri et al. (1996) showed that macropore surface coatings in a subsurface argillic layer of a Thatuna silt loam were enriched in organic carbon and clay relative to the surrounding soil matrix. Furthermore, these coatings expressed greater 2,4-D mineralization potential than soil from either the surrounding Bt layer, or the overlying E horizon. They speculated that macropore linings might play a signi®cant role in 2,4-D immobilization and mineralization in the subsurface, especially if preferential saturated ¯ow directs herbicide through these macropores. Clearly, in our situation there is a need to determine if the cover crop treatment has improved the subsurface hydraulic conductivity of the argillic horizon soil, and facilitated movement of soluble C from the surface into the subsurface. In addition, we should determine to what extent the enhanced 2,4-D mineralization of the subsurface layer is uniformly distributed versus being associated with heterogeneities such as macropores and their linings. Despite 2,4-D being easily biodegradable in most soils, water availability, soil temperature and 2,4-D concentration are known to modify the kinetics of its decomposition (Ou et al., 1978; Parker and Doxtader, 1982, 1983; Stott et al., 1983; Ou, 1984; Estrella et al., 1993; Veeh et al., 1996). In this context, it has been speculated that dierent members of the soil microbial community might be responsible for degrading 2,4-D under dierent conditions (Fournier, 1980; Fournier et al., 1981; Parker and Doxtader, 1982; Soulas, 1993). Throughout the past few years several reports have appeared describing the great diversity of soil bacteria that are capable of degrading 2,4-D (Miwa and Kuwatsuka, 1991; Ka et al., 1994; Tonso et al., 1995; Xia et al., 1995). Considering the changes that were observed in the 2,4-D mineralization characteristics of subsurface soil between the months of February and July, it would be interesting to examine the possibility that dierent members of the 2,4-D degrading community might be responding to the presence of
2,4-D under the dierent management and seasonal conditions.
Acknowledgements This is Technical Paper No. 10,882 of the Oregon Agricultural Experiment Station. Support for this research was partially defrayed by grants from USDACSRS and by the Oregon Agricultural Experiment Station. We gratefully appreciate the assistance of David Phillips, Khrys Duddleston and Ieda Mendes in ®eld sampling, and Dr. W. Horwath for use of the carbon analyzer for biomass determinations.
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P.J. Bottomley et al. / Soil Biology and Biochemistry 31 (1999) 849±857 teria isolated from 2,4-D-treated ®eld soil. Applied and Environmental Microbiology 60, 1106±1115. Kempson-Jones, G.F., Hance, R.J., 1979. Kinetics of linuron and metribuzin degradation in soil. Pesticide Science 10, 449±454. Lavy, T.L., Roeth, F.W., Fenster, C.R., 1973. Degradation of 2,4-D and atrazine at three soil depths in the ®eld. Journal of Environmental Quality 2, 132±137. Lehmicke, L.G., Williams, R.T., Crawford, R.L., 1979. 14 C-mostprobable number method for enumeration of active heterotrophic microorganisms in natural waters. Applied and Environmental Microbiology 38, 644±649. Locke, M.A., Harper, S.S., 1991. Metribuzin degradation in soil. I. Eects of soybean residue amendment, metribuzin level and soil depth. Pesticide Science 31, 221±237. Loos, M. A. 1969. Phenoxyalkanoic acids. In: Kearney, P.C., Kaufman, D.D. (Eds.), Degradation of Herbicides. Dekker, New York, pp. 1±49. Mallawatantri, A.P., McConkey, B.G., Mulla, D.J., 1996. Characteristics of pesticide sorption and degradation in macropore linings and soil horizons of Thatuna silt loam. Journal of Environmental Quality 25, 227±235. Meisinger, J.J., Hargrove, W.H., Mikkelsen, R.L., Williams, J.R., Benson, V.P., 1991. Eect of cover crops on groundwater quality. In: Hargrove, W.L. (Ed.), Cover Crops for Clean Water. Soil Conservation Society, Ankeny, pp. 57±68. Michel, F.C., Reddy, C.A., Forney, L.J., 1995. Microbial degradation and humi®cation of the lawn care pesticide 2,4-dichlorophenoxyacetic acid during the composting of yard trimmings. Applied and Environmental Microbiology 61, 2566±2571. Miller, M., Dick, R.P., 1995. Thermal stability and activities of soil enzymes as in¯uenced by crop rotations. Soil Biology & Biochemistry 27, 1161±1166. Miwa, N., Kuwatsuka, S., 1991. Characteristics of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading microorganisms isolated from dierent soils. Soil Science and Plant Nutrition 37, 575±581. Moorman, T.B., 1990. Adaptation of microorganisms in subsurface environments: signi®cance to pesticide degradation. In: Racke, K.D., Coats, J.R. (Eds.), Enhanced Biodegradation of Pesticides in the Environment. American Chemical Society, Washington, DC, pp. 167±180. Moorman, T.B., Harper, S.S., 1989. Transformation and mineralization of metribuzin in surface and subsurface horizons of a Mississippi delta soil. Journal of Environmental Quality 18, 302± 306. Mueller, T.C., Moorman, T.B., Snipes, C.E., 1992. Eects of concentration, sorption and microbial biomass on degradation of the herbicide ¯uometuron in surface and subsurface soils. Journal of Agriculture and Food Chemistry 40, 2517±2522. Ou, L-T., 1984. 2,4-D degradation and 2,4-D degrading microorganisms in soils. Soil Science 137, 100±107. Ou, L-T., Rothwell, D.F., Wheeler, W.B., Davidson, J.M., 1978. The eect of high 2,4-D concentrations on degradation and carbon
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dioxide evolution in soils. Journal of Environmental Quality 7, 241±246. Parker, L.W., Doxtader, K.G., 1982. Kinetics of microbial decomposition of 2,4-D in soil: eects of herbicide concentration. Journal of Environmental Quality 11, 679±684. Parker, L.W., Doxtader, K.G., 1983. Kinetics of the microbial degradation of 2,4-D in soil: eects of temperature and moisture. Journal of Environmental Quality 12, 553±558. Pivetz, B.E., Steenhuis, T.S., 1995. Soil matrix and macropore biodegradation of 2,4-D. Journal of Environmental Quality 24, 564± 570. Pothuluri, J.V., Moorman, T.B., Obenhuber, D.C., Wauchope, R.D., 1990. Aerobic and anaerobic degradation of alachlor in samples from a surface-to-groundwater pro®le. Journal of Environmental Quality 19, 525±530. Sandemann, E.R.I.C., Loos, M.A., van Dyck, L.P., 1988. The microbial degradation of 2,4-dichlorophenoxyacetic acid in soil. Reviews in Environmental Contamination and Toxicology 101, 1± 53. Shimp, J.F., Tracy, J.C., Davis, L.C., Lee, E., Huang, W., Erickson, L.E., 1993. Bene®cial eects of plants in the remediation of soil and groundwater contaminated with organic material. Critical Reviews in Environmental Science and Technology 23, 41±77. Smith, A.E., Lafond, G.P., 1990. Eects of long-term phenoxyalkanoic acid herbicide ®eld applications on the rate of microbial degradation. In: Racke, K.D., Coats, J.R. (Eds.), Enhanced Biodegradation of Pesticides in the Environment. American Chemical Society, Washington, DC, pp. 14±22. Soulas, G., 1993. Evidence for the existence of dierent physiological groups in the microbial community responsible for 2,4-D mineralization in soil. Soil Biology & Biochemistry 25, 443±449. Stott, D.E., Martin, J.P., Focht, D.D., Haider, K., 1983. Biodegradation, stabilization in humus and incorporation into soil biomass of 2,4-D and chlorocatechol carbons. Soil Science Society of America Journal 47, 66±70. Tonso, N.L., Matheson, V.G., Holben, W.E., 1995. Polyphasic characteristics of a suite of bacterial isolates capable of degrading 2,4-D. Microbial Ecology 30, 3±24. Veeh, R.H., Inskeep, W.P., Camper, A.K., 1996. Soil depth and temperature eects on microbial degradation of 2,4-D. Journal of Environmental Quality 25, 5±12. Wagenet, R.J., Hutson, J.L., 1990. Quantifying pesticide behavior in soil. Annual Review of Phytopathology 28, 295±319. Wilson, R.G., Cheng, H.H., 1976. Breakdown and movement of 2,4D in the soil under ®eld conditions. Weed Science 24, 461±466. Wilson, R.G., Cheng, H.H., 1978. Fate of 2,4-D in a Na silt loam soil. Journal of Environmental Quality 7, 281±286. Woomer, P., Bennett, J., Yost, R., 1990. Overcoming the in¯exibility of most probable number procedures. Agronomy Journal 82, 349± 353. Xia, X., Bollinger, J., Ogram, A.V., 1995. Molecular genetic analysis of the response of three soil microbial communities to the application of 2,4-D. Molecular Ecology 4, 17±28.
Soil Biology and Biochemistry 31 (1999) 849±857
Winter cover crop enhances 2,4-D mineralization potential of surface and subsurface soil P.J. Bottomley a, b, *, T.E. Sawyer a, L. Boersma a, R.P. Dick a, D.D. Hemphill c a
Department of Crop and Soil Sciences, Oregon State University, Corvallis, OR 97331-3804, USA b Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA c North Willamette Research and Extension Center, Oregon State University, Aurora, OR 97002-9543, USA Accepted 27 October 1998
Abstract A study was conducted to determine if the use of a winter cover crop in a summer vegetable crop rotational system might in¯uence the potential of surface and subsurface layers of a Willamette silt loam soil to mineralize the herbicide, 2,4dichlorophenoxyacetic acid (2,4-D). On three occasions between April and September 1994, and three occasions between February and June 1995, soil samples were recovered from the Ap horizon (0±20 cm), and from within the argillic B (80±100 cm) horizon of ®eld plots managed in either a summer vegetable crop±winter fallow rotation, or a summer vegetable crop± winter cover crop (cereal rye) rotation. Composite samples of soil were prepared from the four replicates of each of the two ®eld treatments, and the mineralization of 6 mg 2,4-D kg ÿ 1 examined under laboratory conditions. 2,4-D was mineralized more quickly in the 0±20-cm soil from the cover crop treatment than the winter fallow treatment on ®ve of the six sampling occasions. 2,4-D mineralization characteristics of the 80±100-cm soil diered between the cover crop and winter fallow treatments and also diered between sampling dates. In February, 2,4-D mineralization rates developed slowly and persisted at suboptimal rates for at least 10 d in both treatments. In April and June, daily 2,4-D mineralization rates increased more rapidly in soil from the cover crop treatment than in samples taken from the winter fallow treatment. Although no dierences were detected in mineralization characteristics of subsurface soil sampled from the two treatments while the sweet corn summer crop was growing (July), treatment dierences were again discernible immediately after the summer crop was harvested (September). Our ®ndings identi®ed a potential bonus of using a winter cover crop, i.e. enhancing the potential of subsurface soil micro¯ora to mineralize herbicides that might leach from the surface. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Because of the mild climate and rainfall pattern, an extremely diverse agriculture is practiced in western Oregon throughout the calendar year. As a consequence, fertilizers and herbicides are applied under a wide variety of environmental conditions. Because of recent concerns about run-o and leaching of nitrate into water supplies, fall-seeded cover crops have surfaced as a simple strategy to immobilize nitrogen in well-drained soils during the winter and early spring * Corresponding author. Fax: +1-541-737-0496; e-mail: [email protected]
months (Meisinger et al., 1991; Brandi-Dorn et al., 1997). The eects of cover cropping on the ecacy and fate of the herbicides used in these cropping systems are unknown. For example, while plants might enhance the populations of pesticide-degrading microorganisms adjacent to their roots, and thereby stimulate breakdown of soil-borne herbicides (Boyle and Shann, 1995; Haby and Crowley, 1996), plant roots can also provide a conduit to transport surface-applied pesticides through the soil to ground water (Anderson et al., 1993; Shimp et al., 1993). Although soil microbial biomass and enzyme activities can be greater in cover cropped than fallowed soil (Miller and Dick, 1995), it is unknown how far below the soil surface the
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 8 ) 0 0 1 8 4 - 9
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eect of a cover crop might extend, nor if it in¯uences herbicide-degrading microorganisms speci®cally. Detailed studies on microbiological transformation of pesticides in the subsurface are somewhat limited (Moorman and Harper, 1989; Moorman, 1990; Pothuluri et al., 1990; Locke and Harper, 1991; Mueller et al., 1992; Mallawatantri et al., 1996; Veeh et al., 1996). Although many studies have been published on the eects of soil physical and chemical properties on pesticide movement (Beck et al., 1993), and mathematical models have been developed to predict pesticide transport (Wagenet and Hutson, 1990; Boesten and van der Linden, 1991), fewer studies have attempted to link microbiological degradation with water ¯ow and chemical movement in the subsurface soil (Estrella et al., 1993; Pivetz and Steenhuis, 1995). With this longer-term goal in our minds, the immediate objectives were to determine the mineralization potential of 2,4-D at dierent depths in a soil pro®le and to determine if a winter cover crop in¯uenced the 2,4D mineralization properties associated with soil at those dierent depths. We chose to use 2,4-D as the herbicide of choice because it is water soluble and continues to be used extensively in a variety of agricultural and urban settings at relatively high rates (Johnson et al., 1995; Michel et al., 1995). Furthermore, an extensive literature has accumulated over the past 40 yr describing the properties of 2,4-D breakdown in soil (Sandemann et al., 1988) and the characteristics of 2,4D degrading soil microorganisms (Ka et al., 1994; Tonso et al., 1995; Xia et al., 1995).
The experimental design was a randomized complete block with four replications of each treatment. Soil samples were taken from the four replicates of two of the winter cover crop treatments namely, winter fallow and cereal rye. About 30 cores of soil were taken on 1m spacing from a 64 m grid laid out in each replicate plot. Soil cores were recovered from the 0±20, 20±40, 40±60, 60±80 and 80±100-cm depth intervals using an extendible tube auger of 2.5 cm diameter. Soil cores were broken up by hand, thoroughly mixed, placed in large ziploc bags and transported to the laboratory. Some simple precautions were taken to reduce the chances of contaminating the lower layers of soil with overlying material. (1) All samples were removed from a speci®c depth before moving to the next depth. (2) The inside surfaces of the bore holes were carefully reamed with the probe before sampling the next depth increment. (3) Loose soil on the top of the lower cores and extraneous soil on the auger were removed before bagging the samples. (4) The auger was surface sterilized with ethanol between sampling the dierent depths. Prior to experimentation, soil water content was determined (1058C, 48 h). Textural analysis was conducted on samples of soil using the hydrometer procedure of the soil physical analysis laboratory, Department of Crop and Soil Sciences, Oregon State University. Total organic-C was determined by dry combustion with a Dohrman DC-80 carbon analyzer. Saturated hydraulic conductivity measurements were made with a double-ring in®ltrometer by J. Selker, Department of Bioresources Engineering, Oregon State University.
2. Materials and methods
2.2. 2,4-D mineralization studies
2.1. Experimental site
Within 2±3 d of sampling from the ®eld, mineralization experiments were conducted on composite samples of soil prepared by mixing subsamples of soil (sieved <2 mm) from each of the four replicates of a ®eld treatment. Mineralization experiments were conducted in a continuous air ¯ow and trap system that allowed humidi®ed CO2-free air to ¯ow through 125 ml Erlenmeyer ¯asks at a rate of about 10 ml min ÿ 1. Twenty-g portions of soil were added to deionized water containing sucient 2,4-D to provide a ®nal water content of 300 g kg ÿ 1 oven dry soil and 6 mg 2,4-D kg ÿ 1 of soil. The 2,4-D solution was supplemented with approximately 0.1 mci (3.7 kBq) of [UL-14 C] ring labeled 2,4-D, (speci®c activity, 696 MBq mmol ÿ 1, >98% chemical purity, Sigma Chemical Co.). In tests, samples of gamma-irradiated soil (4 Mrad dose) were supplemented with the labeled 2,4-D solution and incubated as described above to evaluate the possibility of abiological decomposition of the herbicide. The radioactivity recovered in the alkali traps never exceeded the background counts that were
Soil samples were collected from a vegetable crop rotation experiment initiated in 1989 at the North Willamette Research and Extension Center (NWREC), Aurora, OR. The soil is a Willamette silt loam (Pachic Ultic Argixeroll), and the site characteristics were described by Brandi-Dorn et al. (1997) and Burket et al. (1997). Field treatments included three dierent winter cover crop treatments in a summer vegetable crop rotation that alternates two summer crops, sweet corn (Zea mays L. cv. Jubilee) and broccoli (Brassica oleracea L. Botrytis group cv. Gem). The summer crops were planted in late May±early June (corn 1994 and broccoli 1995), and were harvested in September. The winter cover crop treatments included winter fallow, fall planted cereal rye (Secale triticale) seeded at 40 kg ha ÿ 1, and a mixture of cereal rye and Austrian winter pea (Pisum sativum L.) seeded at 40 and 112 kg ha ÿ 1, respectively. The cover crops were seeded in late September and incorporated into the soil in mid April.
P.J. Bottomley et al. / Soil Biology and Biochemistry 31 (1999) 849±857
obtained when scintillation ¯uid alone was counted. Nonetheless, samples of sterile soil amended with radiolabeled 2,4-D, and radiolabel without soil were included routinely in each study to serve as controls. Triplicate samples of composite soil prepared from each of the two ®eld treatments were used for each experimental treatment. In the early stages of the study (1993 and February and April, 1994), a few experiments were carried out with two additional concentrations of 2,4-D (0.06 and 0.6 mg kg ÿ 1). Because the treatment eects on mineralization properties were found to be similar at both 0.6 and 6.0 mg kg ÿ 1, the majority of the experiments we describe here were conducted with 6 mg 2,4-D kg ÿ 1. 14 C labeled CO2 was collected by passing the euent air through 20 ml capacity vials containing 6 ml portions of 0.1 M NaOH. The vials were changed daily or at other intervals when appropriate. Ten-ml portions of Scintisafe Plus counting ¯uid (Fisher Scienti®c) were added to each vial and counts were determined in a Beckman LS3801 scintillation counter. Although NaOH was used to trap the liberated CO2, the eciency of counting was high (r95%). Presumably, a high percentage of the alkali in the traps was neutralized by the air-derived CO2 continuously passing through the vials, and by the CO2 derived from both 2,4-D and the background soil respiration. The percentage of 2,4-D mineralized was calculated by dividing the average dis min ÿ 1 recovered in the alkali traps by the dis min ÿ 1 added to each soil incubation. 2.3. Microbial biomass determinations Microbial biomass-C measurements were determined with the chloroform fumigation±extraction method (CFEM) described by Horwath and Paul (1994). Brie¯y, four 10-g portions of soil from each of the soil depths (0±20, 20±40, 40±60, 60±80 and 80±100-cm) were wetted to 30% (wt/vol) water content, incubated at 258C for 4 d, fumigated with chloroform vapor for 5 d, and extracted in 0.5 M K2SO4 (1:5(wt/vol), soil:solution). The supernatants were ®ltered through acidwashed Whatman ®lter paper and 100-ml portions were
851
analyzed in a Dohrman carbon analyzer. A correction factor of 0.35 was used in the calculation to obtain biomass values. 2.4. Determination of the population of 2,4-D degrading microorganisms by a most probable number (MPN) analysis A procedure was developed which was based upon that of Lehmicke et al. (1979). Portions (10 g) of soil were serially diluted in a phosphate-buered mineral salts solution. Replicate (n = 4) 10-ml samples of appropriate dilutions were added to 50-ml serum vials containing 0.1 mmol of 2,4-D supplemented with 50 nci (1.85 kBq) of 14 C-ring labeled 2,4-D. The 2,4-D was added to the vials as a methanolic solution that was allowed to evaporate before the samples of soil dilutions were added. Wicks constructed of ¯uted ®lter paper were placed in small plastic bucket assemblies (Kontes of California), which were ®xed into the rubber septa of the vials and suspended above the soil dilutions. Portions (100 ml) of 0.1 M NaOH were injected through the rubber septa onto the wicks. Incubations were conducted at room temperature without shaking. After approximately 30 d, the radioactivity trapped in the NaOH was determined by scintillation counting. Any vial in which 10% or more of the original activity was recovered in the NaOH was considered positive. The sizes of the 2,4-D degrading populations in the soil samples were derived using a most-probable-number computer program described by Woomer et al. (1990). Tests showed that 28 d was an adequate incubation period to obtain the maximum number of positive vials from a dilution series. 3. Results Several physical, chemical and biological properties of the Willamette silt loam changed with depth at the experimental site (Table 1). Decreases in both organic matter content and saturated hydraulic conductivity, accompanied by a substantial increase in the clay con-
Table 1 Physical properties of Willamette silt loam soila Depth increment (cm)
Textural analysis (g kg ÿ 1) sand (>50 mm)
silt (50-2 mm)
clay (<2 mm)
0±20 20±40 40±60 60±80 80±100
313 328 298 286 374
540 499 476 490 450
147 173 226 224 175
a
See Section 2 for details of procedures.
Satd. hydr. cond., Ksat (10 ÿ 6 m s ÿ 1)
Total soil carbon (g kg ÿ 1)
pH (1:2 w/v soil:H2O)
60 N.D. 7 N.D. 0.7
18 18 9.4 6.5 4.7
6.3 N.D. 6.3 N.D. 6.2
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Table 2 Changes in microbial parameters with soil depth in the two cover crop treatmentsa,b Soil depth (cm)
0±20 20±40 40±60 60±80 80±100
Microbial biomass (mg C kg ÿ 1)
Respiratory rate (mmol CO2 kg ÿ 1 d ÿ 1)
No. (106) of 2,4-D degraders kg ÿ 1
WF
CC
WF
CC
WF
CC
140 (58)c 148 (62) 86 (35) 28 (12) undetectable
204 (100) 135 (88) 45 (30) 29 (19) undetectable
970 (400)c 276 (116) 352 (144) 60 (26) 80 (33)
610 (300) 260 (170) 90 (60) 150 (99) 70 (45)
(1.6) 4.6 (13.3)d (0.3) 0.9 (2.6) not determined (0.05) 0.13 (0.4) (0.05) 0.15 (0.4)
(7.4) 21.3 (61.5) (8.9) 25.8 (74.3) not determined (0.04) 0.10 (0.3) (0.10) 0.31 (0.9)
a See Section 2 for details of the procedures. bAbbreviations stand for winter fallow (WF) and cover crop (CC) treatments, respectively. Values in parentheses represent standard deviations of triplicate determinations. dValues in parentheses represent the upper and lower 95% con®dence limits of the population estimates.
c
tent of the soil at 40±60 cm delineated the upper limit of the argillic B horizon. Under both the winter fallow, and the cover crop treatments, microbial biomass-C declined abruptly at the 40±60-cm depth from about 140±200 mg C kg ÿ 1 soil in the surface 0±40-cm layer, to 45±86 mg C kg ÿ 1 soil at 40 cm depth and below (Table 2). Although microbial biomass could not be measured in soil from the 80±100-cm depth increment with either the chloroform fumigation±incubation or fumigation±extraction methods, the most-probablenumber procedure detected a population of 2,4-D degraders at densities of approximately 105 kg ÿ 1 below 40 cm in the pro®le (Table 2). Despite the experiment having been in place since 1989, no signi®cant dierences were detected among the treatments in amounts of microbial biomass throughout the pro®le, and in populations of 2,4-D degraders at the 60±100 cm depth in the pro®le. MPN estimates showed that the mean population sizes of 2,4-D degraders are between 5 and 20-fold greater in the 0±20 and 20±40 cm layers, respectively, of the cover crop treatment than in the winter fallow treatment. However, these population means are accompanied by rather large con®dence limits. In preliminary experiments conducted on soil recovered at 20 cm depth increments between 0 and 100 cm, the characteristics of 2,4-D mineralization (0.6 and 6.0 mg 2,4-D kg ÿ 1 of soil) were observed to change with soil depth (Fig. 1a and b). A distinctly longer period was required before mineralization of 0.6 mg 2,4-D kg ÿ 1 could be detected at depths of 40 cm and below (04 d) than at more shallow depths (1±2 d). With the higher concentration of 2,4-D (6 mg kg ÿ 1) the dierence between depths was more exaggerated because of the lower speci®c activity of the label and because the rate of mineralization did not increase in response to the increase in 2,4-D concentration (Fig. 1b). In subsequent experiments, comparisons of 2,4-D mineralization were made only between soil taken from the surface layer (0±20 cm), and from an increment of the
subsurface (80±100 cm) that lay within the argillic horizon, but could be reached from the surface with simple tools and minimal disturbance of the plot area. Over the course of the 2-yr study, we observed that the 2,4-D mineralization properties of surface and subsurface soil were in¯uenced by sampling time and by the winter cover crop treatment (Figs. 2±4). On
Fig. 1. Mineralization of 14 C ring-labeled 2,4-D applied at (a) 0.6 mg kg ÿ 1 and (b) 6.0 mg kg ÿ 1 to soil recovered from: w, 0±20 cm, Q, 20±40 cm, q, 40±60 cm, r, 60±80 cm and R, 80±100 cm depth increments from the winter fallow treatment in February, 1994. Absence of error bars on the data points indicates that the SE of the mean was less than the size of the symbol.
P.J. Bottomley et al. / Soil Biology and Biochemistry 31 (1999) 849±857
Fig. 2. Mineralization of 14 C ring-labeled 2,4-D applied at (a) 0.6 mg kg ÿ 1 and (b) 6.0 mg kg ÿ 1 to soil recovered in April, 1994 from either the winter fallow (circles) or the cereal rye cover crop (triangles) treatments. Open symbols represent surface (0±20 cm) soil and closed symbols represent subsurface (80±100 cm) soil.
sampling occasions in April and July, 1994 (Figs. 2 and 3) and February, April and June, 1995 (Fig. 5), 2,4-D mineralization could be detected in soil from the surface layer (0±20 cm) of the cover crop treatment about 1 d earlier than in soil taken from the winter fallow treatment. Only in September 1994 (Fig. 3b) did the mineralization characteristics of 0±20-cm samples of the cover crop and winter fallow treatments overlap. An eect of the winter cover crop treatment on 2,4-D mineralization was detected in the 80±100-cm layer. For example, in April 1994, although 2,4-D mineralization in the subsurface soil of the cover crop treatment was initiated at a similar time as the winter fallow treatment (3±4 d) (Fig. 2), mineralization of both 0.6 mg kg ÿ 1 (Fig. 2a) and 6.0 mg 2,4-D kg ÿ 1 (Fig. 2b) increased more rapidly in soil from the cover crop treatment. In July and September, 1994, however, 2,4D mineralization was initiated at the same time and progressed similarly in the 80±100-cm samples of both treatments (Fig. 3a and b). In 1995, dierences between the 2,4-D mineralization properties of the cover crop and winter fallow treatments were apparent in both April and June samplings of the 80±100-cm layer (Fig. 4b and c), but not in February (Fig. 4a) where mineralization proceeded slowly regardless of
853
Fig. 3. Mineralization of 14 C ring-labeled 2,4-D applied at 6 mg kg ÿ 1 to soil recovered from the winter fallow and cereal rye cover crop treatments in (a) July and (b) September 1994. Symbol de®nitions are the same as represented in Fig. 2.
treatment. The time required to detect 2,4-D mineralization in the 80±100-cm layer of the cover crop treatment decreased progressively from 4±6 d, to 3±4 d and to 2±3 d in the February, April and June samples, respectively. In contrast, the duration of the lag period never dropped below 4±5 d in samples from the 80± 100-cm layer of the winter fallow treatment. Mineralization data were also examined in the context of how the daily rates of 2,4-D mineralization changed throughout the incubation (Figs. 5 and 6). Regardless of time of sampling or treatment, the daily rates of 2,4-D mineralization in 0±20 cm soil reached a distinct maximum value within 2±5 d of incubation. The daily rates of mineralization in the 0±20-cm layer of cover crop treatment (up to its daily maximum) were almost always greater (except September 1994) than measured in the winter fallow treatment on any speci®c day of incubation. Nonetheless, the winter fallow treatment usually reached a similar daily rate of mineralization about 1 d later. In the 80±100-cm subsurface soil, the eects of the cover crop treatment on the daily mineralization rates varied with sampling time. For example, in April 1994, daily rates of mineralization in the cover crop treatment developed to a maximum value within 2 d of being detectable (3±5 d) (Fig. 5a). In the case of the winter fallow, daily rates
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Fig. 4. Mineralization of 14 C ring-labeled 2,4-D applied at 6 mg kg ÿ 1 to soil recovered from the winter fallow and cereal rye cover crop treatments in (a) February, (b) April and (c) June 1995. Symbol de®nitions are the same as represented in Fig. 2.
increased slowly to a daily rate that did not change signi®cantly from the day 5 through day 9 of incubation. By contrast, in July 1994, the daily mineralization rates of the 80±100-cm layer of both the winter fallow and cover crop treatments reached the same maximum daily rate on the same day (Fig. 5b). In September 1994, the increase in the daily rate of mineralization in the 80±100-cm layer of the cover crop treatment was again somewhat faster than in the same layer of the winter fallow treatment (Fig. 5c). In February 1995, daily rates of mineralization increased very slowly in subsurface soil from both treatments and never exceeded 500 ng g ÿ 1 d ÿ 1. In the case of the winter fallow treatment, the daily rate of mineralization was relatively stable between day 8 and day 13 of incubation, whereas the rate in the cover crop treatment continued to slowly increase (Fig. 6a).
Fig. 5. A comparison of the daily rates of mineralization of 2,4-D applied at 6 mg kg ÿ 1 to soil recovered from the 0±20 cm and 80± 100 cm layers of the winter fallow and cereal rye cover crop treatments in (a) April, (b) July and (c) September 1994. Symbol de®nitions are the same as represented in Fig. 2.
Indeed, tests conducted with subsurface soil recovered from the winter fallow treatment in February 1994 had given similar results (see Fig. 1b). Unfortunately, at that time comparisons were not made with the cover crop treatment. In April 1995, the increase in daily rate of 2,4-D mineralization in the 80±100-cm samples from the cover crop treatment was greater than observed in the winter fallow (Fig. 6b). In neither treatment, however, did the daily rate of mineralization increase rapidly enough to create the distinct peak in the daily rate of mineralization (r1200 ng g ÿ 1 d ÿ 1) invariably seen in the 0±20-cm soil samples, and in the 80±100-cm samples taken from the cover crop treatment in April, July and September, 1994 and in June 1995. Instead, daily mineralization rates of R500 ng g ÿ 1 d ÿ 1 were sustained for about 4±6 d. Although
P.J. Bottomley et al. / Soil Biology and Biochemistry 31 (1999) 849±857
Fig. 6. A comparison of the daily rates of mineralization of 2,4-D applied at 6 mg kg ÿ 1 to soil recovered from the 0±20 and 80±100 cm layers of the winter fallow and cereal rye cover crop treatments in (a) February, (b) April and (c) June, 1995. Symbol de®nitions are the same as represented in Fig. 2.
it took longer to initiate mineralization in the 80±100cm samples from the winter fallow treatment than the cover crop treatment in June 1995 (Fig. 6c), large increases in the daily rates of mineralization were observed in both treatments. 4. Discussion Although numerous papers have been written on the subject of 2,4-D degrading bacteria and the fate of 2,4D in soil (Loos, 1969; Wilson and Cheng, 1976, 1978; Sandemann et al., 1988; Smith and Lafond, 1990), our ®ndings add new insights into the role that cover crops might play in the fate of pesticides that penetrate into the subsurface soil environment. The data presented in
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this manuscript clearly indicate that a cover crop of cereal rye can have a measurable eect on the 2,4-D mineralization potential of surface and subsurface soil. While it is well known that microorganisms in subsurface soil have the potential to mineralize herbicides (Harris et al., 1966; Lavy et al., 1973; Bouchard et al., 1982; Moorman, 1990) mineralization responses with soil depth have been quite variable. For example, while a signi®cant decline in the rate of metribuzin transformation occurred in soil samples recovered from >10 cm below the surface of a Dundee silt loam (Moorman and Harper, 1989; Locke and Harper, 1991), Kempson-Jones and Hance (1979) measured similar rates of metribuzin transformation in samples of soil recovered between the surface and 50±60 cm depth. Veeh et al. (1996) measured dierences in 2,4-D mineralization between 0±30, 30±60 and 60±120 cm in one soil, whereas no dierences were detected in the 30±60 and 60±120-cm layers of another soil. In the latter study, the authors showed that 2,4-D half life increased from 5 to 25 d as soil depth increased, and the increase in half life correlated with a decline in soil organic-C with soil depth from 1 to 0.4 wt%. By contrast, in our study, we found that the 2,4-D mineralization potential of soil was relatively constant on any particular sampling date between 40 and 100 cm depth in the pro®le, despite the fact that soil organic-C contents declined from 1 to 0.4 wt% over this depth increment. Furthermore, we could not detect a signi®cant winter cover crop eect on total soil organic-C contents below 40 cm in the pro®le (data not shown), despite showing repeatedly that the 2,4-D mineralization potential of the subsurface diered between the winter fallow and cover crop treatments. It is of interest to speculate about how a cover crop might be in¯uencing the herbicide-degrading potential of the soil. It is reasonable to believe that the cover crop eect detected in the surface soil might be attributed to the somewhat higher population density of 2,4D degraders found in surface soil of the cover crop than in the winter fallow treatment. This idea would be consistent with the cover crop treatment supporting a higher population density of bacteria because of the inputs of cover crop residue. However, we need to bear in mind that a traditional MPN estimate of a population size is accompanied by rather large con®dence limits, and population density dierences of less than 10-fold are suspect (Woomer et al., 1990). Because nonlinear increases in daily rates of 2,4-D mineralization are consistent with growth of the 2,4-D degrading community in response to the substrate, we conclude that cover cropped subsurface soil is superior to the winter fallow treatment in supplying the supplementary resources needed to support microbial growth on 2,4-D. The exceptions to the rule were samples recovered in February and July. Because we
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have observed that cover crop growth at this site increases signi®cantly during March and April, as daylength and temperatures increase, support for the degrading community might be coming directly from the roots of the cover crop (Haby and Crowley, 1996). However, the situation is complicated by the fact that the cover crop is incorporated into the soil in midApril, and its eect on 2,4-D mineralization was sustained into June. Further work is needed to determine to what extent the stimulatory eect of the cover crop on 2,4-D mineralization potential in the subsurface soil can be attributed to active cover crop growth, versus decomposition of cover crop residues after soil incorporation. In the context of speculating about how a cover crop or its residues might in¯uence the subsurface soil environment, Mallawatantri et al. (1996) showed that macropore surface coatings in a subsurface argillic layer of a Thatuna silt loam were enriched in organic carbon and clay relative to the surrounding soil matrix. Furthermore, these coatings expressed greater 2,4-D mineralization potential than soil from either the surrounding Bt layer, or the overlying E horizon. They speculated that macropore linings might play a signi®cant role in 2,4-D immobilization and mineralization in the subsurface, especially if preferential saturated ¯ow directs herbicide through these macropores. Clearly, in our situation there is a need to determine if the cover crop treatment has improved the subsurface hydraulic conductivity of the argillic horizon soil, and facilitated movement of soluble C from the surface into the subsurface. In addition, we should determine to what extent the enhanced 2,4-D mineralization of the subsurface layer is uniformly distributed versus being associated with heterogeneities such as macropores and their linings. Despite 2,4-D being easily biodegradable in most soils, water availability, soil temperature and 2,4-D concentration are known to modify the kinetics of its decomposition (Ou et al., 1978; Parker and Doxtader, 1982, 1983; Stott et al., 1983; Ou, 1984; Estrella et al., 1993; Veeh et al., 1996). In this context, it has been speculated that dierent members of the soil microbial community might be responsible for degrading 2,4-D under dierent conditions (Fournier, 1980; Fournier et al., 1981; Parker and Doxtader, 1982; Soulas, 1993). Throughout the past few years several reports have appeared describing the great diversity of soil bacteria that are capable of degrading 2,4-D (Miwa and Kuwatsuka, 1991; Ka et al., 1994; Tonso et al., 1995; Xia et al., 1995). Considering the changes that were observed in the 2,4-D mineralization characteristics of subsurface soil between the months of February and July, it would be interesting to examine the possibility that dierent members of the 2,4-D degrading community might be responding to the presence of
2,4-D under the dierent management and seasonal conditions.
Acknowledgements This is Technical Paper No. 10,882 of the Oregon Agricultural Experiment Station. Support for this research was partially defrayed by grants from USDACSRS and by the Oregon Agricultural Experiment Station. We gratefully appreciate the assistance of David Phillips, Khrys Duddleston and Ieda Mendes in ®eld sampling, and Dr. W. Horwath for use of the carbon analyzer for biomass determinations.
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