- Email: [email protected]
Response of a soil nematode community to liquid hog manure and its acidification
Applied Soil Ecology 43 (2009) 75–82
Contents lists available at ScienceDirect
Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Response of a soil nematode community to liquid hog manure and its acidification A. Mahran a,b, M. Tenuta b,c,*, R.A. Lumactud b, F. Daayf a a
Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 Department of Soil Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 c Canada Research Chair in Applied Soil Ecology, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 September 2008 Received in revised form 31 May 2009 Accepted 12 June 2009
A study was conducted to determine why the number of opportunistic bacterial-feeding nematodes increase following addition of liquid hog manure and acidified mixtures of the manure to soil. A sandy loam soil harboring a wide range of nematode taxa, representing various trophic and colonizer–persister groups (c–p 1 through 5) and augmented with the plant-parasitic nematode, Pratylenchus spp., was used in microcosm experiments. Treatments were additions of liquid hog manure (0.15 v v 1 soil water), mixtures of manure (0.05, 0.10, and 0.15 v v 1 soil water) and sulfuric acid, as well as a sulfuric acid alone and a non-treated control. Three days post-treatment and during the presence of non-ionized volatile fatty acids from manure in soil, numbers of plant-parasitic nematodes, including Pratylenchus spp., decreased by at least 50% for all manure treatments compared to non-manure treatments. Thereafter, c– p 1 and c–p 2 nematode numbers increased in all manure but not in non-manure treatments. At week 4, c–p 1 and c–p 2 nematode numbers were greater by at least 6000 and 5000 individuals kg 1 soil, respectively, compared to non-manure treatments. In contrast, numbers of c–p 3–5 nematodes were not affected by the treatments. At week 4, the enrichment index, an assessment of the abundance of opportunistic c–p 1 and some c–p 2 nematodes compared to all c–p 2 nematodes, was around 70% for all manure treatments and lower, 32 and 29, respectively, for acid alone and non-treated control treatments. The increase in opportunistic nematodes following manure treatment was likely due to the increase in their food resources associated with the enrichment of the soil environment with readily degradable compounds. Volatile fatty acids present in the manure persisted in the soil for only four days before biological degradation. We conclude liquid hog manure is effective in killing plant-parasitic nematodes while increasing bottom-up food web interactions but not, as with soil fumigants, decimating top-down trophic interactions. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Acidity Food web Liquid hog manure Nematode communities Nematode faunal analysis Pratylenchus Root-lesion nematode Volatile fatty acids
1. Introduction Animal manures and wastes are the sources of plant nutrients and organic matter in soils of many agricultural systems. Anaerobically stored animal manures and wastes are capable of killing plant pathogens (Conn and Lazarovits, 1999) and pests such as plant-parasitic nematodes (Valocka et al., 2000; Jothi et al., 2003; Timper et al., 2004; Min et al., 2007). Acidic conditions generating non-ionized forms of short-chain volatile fatty acids (acetic, propionic, n-butyric, isobutyric, n-valeric, isovaleric and n-caproic acids) in liquid hog manure suppress the wilt fungus, Verticillium dahliae Kleb. (Tenuta et al., 2002; Conn et al., 2005). More recently, using solution exposure studies, Mahran et al. (2008a) concluded
* Corresponding author at: Canada Research Chair in Applied Soil Ecology, Department of Soil Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2. Tel.: +1 204 474 7827; fax: +1 204 474 7642. E-mail address: [email protected] (M. Tenuta). 0929-1393/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2009.06.003
that volatile fatty acids account for most of the lethal effect of liquid hog manure to Pratylenchus penetrans (Cobb) Chitwood and Oteifa under acidic conditions. Also, volatile fatty acids added to soil in pot studies (McElderry et al., 2005; Xiao et al., 2007, 2008) and present in liquid hog manure (Mahran et al., 2008b), reduced numbers of plantparasitic nematodes in field studies. After the application of acidified liquid hog manure, we observed large numbers of opportunistic bacterial-feeding nematodes of the Rhabditidae (unpublished data). These nematodes flourish under conditions of abundant bacterial growth that can occur following application of broad-spectrum pesticides, such as soil fumigants (Yeates et al., 1991; Wang et al., 2006) or following addition of readily decomposable materials to soil (Ferris and Matute, 2003; Nahar et al., 2006). Anaerobically stored animal manures contain large amounts of easily decomposable organic C, including volatile fatty acids (Zhu, 2000), amino acids (Dewes and Hunsche, 1998), and dissolved organic carbon (Royer et al., 2007). These materials are rapidly utilized by soil microorganisms as a source of energy (Sorensen,
76
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
1998). Kirchmann and Lundvall (1993) showed that volatile fatty acids in hog and cattle slurries decomposed in soil at 25 8C within two days of application. The amount of N immobilized in microbial biomass was correlated with the amount of volatile fatty acids applied, suggesting that these compounds are a source of energy for soil microorganisms. The efficacy of any compound in killing soil organisms, either beneficial or detrimental to plant growth, depends on dosage, duration of exposure and sensitivity of the organism (Rozman and Doull, 2000). Persistence of added toxic compounds is desired, in the short-term, to provide the concentration time product necessary for killing the target organism. However, long-term persistence is undesirable as it increases the probability of harm to non-target organisms (Haydock et al., 2006). The persistence, or the sorption and volatilization, of volatile fatty acids in acidified liquid hog manure added to soil is unknown but clearly long-term persistence increases the probability of negative effects on soil food webs. Nematode faunal analysis is an excellent tool for assessing the structure and function of soil food webs and their response to soil perturbations (Bongers and Ferris, 1999) such as addition of liquid hog manure. Nematodes (a) have high diversity and abundance in soil environments with complex food webs; (b) have a permeable cuticle in contact with dissolved constituents of soil water or volatiles in soil atmosphere; (c) respond to a proliferation of microbes following an increase in available carbon substrates; (d) differ in sensitivity and response to disturbance; (e) are relatively easy to isolate and identify to genus; (f) occupy key trophic guilds in soil food webs; and (g) there is a clear relationship between their feeding structures and food resource. The current study was conducted to determine: (i) if liquid hog manure and its acidification increases opportunistic nematodes through addition of readily decomposable materials, microbial growth after chemical sterilization of soil or reduction of ‘‘topdown’’ population control, (ii) if persistence of volatile fatty acids in soil receiving acidified liquid hog manure is of short or long duration, and (iii) if loss of volatile fatty acids in soil is due to biological degradation. 2. Materials and methods 2.1. Soil A cattle-grazed, native, mixed-grass prairie soil harboring a diverse range of nematode genera and trophic guilds was collected in the summer of 2007 from the Yellow Quill Prairie Preserve near the town of Shilo, in south central Manitoba, Canada. The soil was collected as three separate blocks (50 cm 50 cm 15 cm h) separated by about 10 m. Each soil block was placed in a polyethylene bag and transported to the laboratory in an ice chest, where it was stored (<1 week) at 5 8C until used in microcosm experiments. The soil was a sandy loam with 6.7% organic matter, 16.5% gravimetric moisture and pH (1:2 soil:H2O ratio) of 7.5. 2.2. Liquid hog manure Liquid hog manure was collected from an earthen storage lagoon at a commercial finisher hog farm in south eastern Manitoba and had a pH of 7.2, 2.7% dry matter, 0.6% total N, 0.01% P and 0.25% K. The manure was centrifuged (10 min at 3400 g) to remove particulates. The resulting solution had a total volatile fatty acid concentration (ionized plus non-ionized forms) of 347 mM (acetic = 190 mM, propionic = 51 mM, isobutyric = 23 mM, n-butyric = 54, isovaleric = 13 mM, n-caproic = 9 mM and n-valeric = 9 mM). It was frozen at 20 8C until used in microcosm experiments.
2.3. Pratylenchus spp. A soil naturally infested with high numbers of two root-lesion nematode species, P. penetrans and Pratylenchus crenatus Loof, was collected from a commercial potato field in the province of Prince Edward Island and reared on common mint (Mentha spicata L.) in a greenhouse. Pratylenchus spp. present in the soil were identified using species-specific polymerase chain reaction (PCR) primers (Al-Banna et al., 2004), confirmed by molecular sequencing of PCR products and sequence comparison with the GenBank BLAST database (Mahran et al., 2008b). Nematodes were extracted using Cobb’s sieving and decanting followed by sugar flotation (Ingham, 1994) 24 h prior to commencing the experiment. The density of the resulting mixed population of juveniles and adults of Pratylenchus spp. was adjusted in dH2O and added to be 1000 nematodes kg 1 dry soil equivalent. P. penetrans and P. crenatus, are common pests of potato in central and eastern Canada though not present in Manitoba. They were added to the soil to provide levels of a common nematode pest of great economic importance. 2.4. Microcosm Each of the three soil collections served as independent replicates in soil microcosm experiments. The soil was passed through a 2 mm mesh screen (USA Standard Test Sieve) to remove stones, roots and debris. Soil microcosms were prepared by adding 90 g fresh soil to 118 ml polyethylene specimen storage containers (6.4 cm d 6.4 cm h) (Fisher Scientific Canada Ltd., Edmonton, Canada). To prevent anaerobic conditions during incubation, 20 0.1 cm diameter holes were punched into container lids. Different combinations of 98% sulfuric acid (equivalent to 4.9 mg H2SO4 ml 1) 1) (Fisher Scientific Canada Ltd.) and liquid-fraction of manure were applied to microcosms to provide the following treatments: nontreated control, acid (initial soil pH reduced to 5.5 using sulfuric acid 98%), liquid hog manure (15% v v 1 soil water volume at 30% field capacity equivalent to 40,500 l ha 1) and acidified liquid hog manure to 5%, 10%, and 15% v v 1 soil water volume. At an assumed soil bulk density of 1.3 Mg m 3, a typical depth of field application (15 cm), and the moisture content of the soil, acidified liquid hog manure added at 5%, 10%, and 15% rates was equivalent to 13,500, 27,000, and 40,500 l ha 1, respectively. The amounts of sulfuric acid needed to bring initial soil pH and the liquid hog manure down to pH 5.5 was determined by titrating soil and liquid hog manure with various amounts of acid. Following application, the soil gravimetric moisture content was adjusted to 30% of field capacity (Cassel and Nielsen, 1986) using dH2O. Microcosms were arranged in a completely randomized design having three independent replicates per treatment and were placed at 22 8C in the dark (Iso-temp Incubator 304; Fisher Scientific Canada Ltd.). Soil samples were extracted 24 h, three days, and one, two, three and four weeks following experimental setup to examine changes in nematode communities as detailed by Forge and Tenuta (2008), and to determine volatile fatty acids concentration in soil. Fifty grams of soil from each microcosm was used for the extraction of nematodes by Cobb’s sieving and decanting followed by sugar flotation (Ingham, 1994). Live nematodes were collected in water in a 15 ml centrifuge tube, excess water was removed, and an equal volume of hot (80 8C) buffered formalin solution (pH 7.0) (Humason, 1972) was added to the nematode suspension to preserve the specimens for identification. Total number of nematodes was determined using a stereo microscope (40 magnification). The first 100 nematodes encountered in each sample were then identified to genus, according to diagnostic keys of Bongers (1994), using an inverted compound microscope (400 magnification), the abundance of each genus in a microcosm was
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
calculated by multiplying the fraction of nematodes of that genus in the identified 100 nematodes by the total number of nematodes. Nematode genera were assigned colonizer–persister values (c–p value) according to Bongers (1990) on a scale ranging from 1 to 5. Opportunistic bacterial-feeding nematodes having similar characteristics to r-strategists were assigned to group, c–p 1. Higher c– p groups were ordered based on increasing k-strategy life-history attributes. 2.5. Food web indices Food web indices computed included the enrichment and structure indices (Ferris et al., 2001). The structure trajectory is a weighted abundance of larger-, slower-reproducing bacterivore, fungivore, omnivore and carnivore c–p 3–5 value nematodes. The structure index was calculated as 100 (s/(s + b)), with b calculated as Skbnb, where kb are the weightings assigned to guilds that indicate basal characteristics of the food web and nb are the abundances of nematodes in those guilds, the s component was calculated similarly, using those guilds having weightings assigned to the structure index. The enrichment trajectory is a weighted abundance of opportunistic bacteriovorous c–p 1 and fungivorous c–p 2 nematodes species that respond rapidly to prey resources. The enrichment index was calculated as 100 (e/(e + b)). The e component was calculated as previously mentioned using those guilds indicating enrichment.
77
for 30 min at 2000 rpm at 5 8C using a refrigerated incubator shaker (New Brunswick Scientific Co., Edison, USA). The tubes were then centrifuged (5 min at 5000 g) and the supernatant analyzed for volatile fatty acids concentration. Individual volatile fatty acid compounds from C2 (acetic) to C6 (n-caproic) were determined using chemical-suppression ion-exclusion chromatography and conductivity detection (Tenuta et al., 2002) with a Dionex ion chromatography system, ICS–1000 (Dionex Corp., Sunnyvale, USA). The chromatograph was equipped with an IonPac ICE-AS1 (9 mm 250 mm) analytical column and anion micro-membrane suppressor (AMMS-ICE II). Soil extracts contained in 1 ml vials with filter caps (Dionex Corp.) were injected (25 ml) with an automated sampler (AS40, Dionex Corp.). For analysis, 1 mM heptaflurobutyric acid (Acros Organics USA, Morris Plains, USA) with 5% acetonitrile (v v 1) was used as the eluent. Commercially available individual volatile fatty acids (99% purity; acetic acid, Fisher Scientific Canada Ltd.; propionic acid, isobutyric acid, nbutyric acid, isovaleric acid, and n-valeric acid, Sigma–Aldrich; and n-caproic acid; Acros Organics) were used to calibrate the response of the conductivity cell to individual volatile fatty acid concentration using eight point response curves. For each soil sample, the concentration of volatile fatty acid in solution was determined by multiplying its concentration in the supernatant by a dilution factor estimated based on the soil moisture content of soil and the volume of water added to it (15 ml). 2.8. Statistical analysis
2.6. Volatile fatty acids persistence and fate in soil The persistence of volatile fatty acids in soil, and the speed of their biological degradation and sorption, was examined in a separate microcosm experiment. The same soil used in the previous experiment was split into two parts, one part (2 kg) was used without sterilization and the other part (2 kg) was autoclaved twice, three days apart, (at 121 8C and 15 psi) for 45 min. The same liquid hog manure used in the previous microcosm experiment was used here and added to non-sterile treatments. For non-sterile soil treatments, liquid hog manure was added without sterilization to non-sterile soil. Liquid hog manure was sterilized by centrifugation (30 min at 11,500 g) to remove particulates, the supernatant filtered through Whatman binder-free glass microfiber filters (9.0 cm d; Fisher Scientific Canada Ltd.) to remove fine particulates, then the filtrate was sterilized using Nalgene disposable sterile analytical filter units (pore size 0.2 mm; Fisher Scientific Canada Ltd.). The sterile liquid hog manure was then stored at 5 8C in sterile polyethylene 50 ml conical tubes until application to the soil. Microcosms were set up as described previously except that 30 g fresh soil was used and all polyethylene containers were sterilized using 95% ETOH (Fisher Scientific Canada Ltd.) prior to use. Treatments were non-treated control, acid (initial soil pH reduced to 5.5 using sulfuric acid 98%), and sterile acidified liquid hog manure (initial soil pH reduced to 5.5 and 15% v v 1 soil water at 30% field capacity) treatments. Sterile water was added to all treatments to bring soil moisture to 30% of field capacity. Microcosms were destructively sampled after setup (0 h) and every 24 h for the subsequent seven days to determine volatile fatty acid concentration in soil. Persistence of volatile fatty acids with the sterile acidified liquid hog manure treatment indicated lack of biological degradation, sorption and volatilization. In contrast, lack of persistence of volatile fatty acids in the non-sterile treatments only indicated biological degradation of the compounds. 2.7. Volatile fatty acids determination and concentration in soil Fifteen milliliters of dH2O was added to 15 g moist soil in 50 ml polyethylene conical tubes. The tubes were shaken, on their sides,
Results for both microcosm experiments were tested for normality using the Shapiro–Wilk test prior to the analysis of variance using the Proc GLM procedure of the Statistical Analysis Software (SAS Institute Inc., Cary, USA). Treatment means were compared using Tukey’s multiple comparison test (P < 0.05). Data for nematode indices were square-root transformed prior to the analysis of variance and comparison of means. 3. Results 3.1. Nematode abundance The total number of nematodes declined over the course of the first microcosm experiment in the non-treated control and acid treatments. Decline in the acid treatment was least of all treatments (Fig. 1A). For the liquid hog manure treatment, total number of nematodes declined over the first seven days following application then increased steadily; by week 4, it had the most nematodes of all treatments (Fig. 1A). Acidified liquid hog manure resulted in a decline in total number of nematodes by day 3, with lowest numbers at the highest liquid hog manure rate. Thereafter, total number of nematodes increased in acidified manure treatments but was lower than for the liquid hog manure alone treatment at week 4 (Fig. 1A). 3.1.1. c–p 1 nematodes The number of opportunistic (c–p 1) nematodes decreased from about 1500 to 250 individuals kg 1 soil by the end of the experiment in the acid only treatment (Fig. 1B). Numbers in the non-treated control increased by day 3 and thereafter declined to a level similar to that in the acid alone treatment (Fig. 1B). Numbers increased in the liquid hog manure treatment to 11,000 individuals kg 1 soil at week 4, a 25-fold increase than in the non-treated control. Individuals in the genera Diploscapter and Mesorhabditis mainly contributed to the increase in number of c–p 1 nematodes observed in the liquid hog manure treatment. In the acidified liquid hog manure treatment, addition to 5% resulted in a higher number of nematodes after one day followed by a decline to the level of all other
78
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
Fig. 1. Number of (A) total, (B) c–p 1, (C) c–p 2, (D) c–p 3–5, (E) plant-parasitic, and (F) Pratylenchus spp. nematodes. Treatments are control (non-treated), acid alone, liquid hog manure (LHM) 15% (v v 1 soil water) and acidified liquid hog manure 5%, 10%, and 15%. The mean of three independent replicates 1 standard error are shown. Means at day 28 followed by different letters are significantly different from one another (P < 0.05) as determined by Tukey’s multiple comparison test.
treatments by one week (Fig. 1B). Thereafter, numbers of individuals of the genera Diploscapter and Mesorhabditis increased for all acidified liquid hog manure treatments to a level similar (P > 0.05) as the liquid hog manure alone treatment. 3.1.2. c–p 2 nematodes Numbers of c–p 2 nematodes tended to be numerically higher in the non-treated control compared to acid alone treatment, though statistically similar (P > 0.05) by the end of the experiment (Fig. 1C). In the liquid hog manure treatment, c–p 2 nematodes declined over the first seven days of the experiment and then
increased to 20,000 individuals kg 1 by the end of the experiment (Fig. 1C). Bacterial-feeding nematodes of the genera Chiloplacus, Eucephalobus, Acrobeles, and the family Plectidae, and fungalfeeding nematodes of the genera Aphelenchus and Aphelenchoides, were the main component of the response to liquid hog manure treatment. There was a sharp increase in the numbers of c–p 2 nematodes at week 3 due to an increase in number of Chiloplacus and to a lesser extent, Acrobeles. A similar trend in numbers occurred with acidified liquid hog manure treatments except being intermediate of manure alone, non-treated control, and acid alone treatments.
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
79
3.1.3. c–p 3–5 nematodes Compared to c–p 1 and c–p 2 nematodes, numbers of c–p 3–5 nematodes collectively were not as responsive to treatments (Fig. 1D). There were no differences (P > 0.05) in their numbers among treatments by week 4. Two trends however, were evident; acid alone resulted in fewer nematodes during the two weeks following treatment with numbers remaining numerically lower than other treatments by the end of the experiment. Acidified liquid hog manure 15%, resulted in fewer c–p 3–5 nematodes by week 1 but a higher number than all treatments by the end of the experiment. The genera of nematodes comprising the colonizer–persister groups c–p 3–5 were the bacterial-feeding genera, Rhabdolaimus, Teratocephalus and Ethmolainus, the fungal-feeding genus, Tylencholaimus, and omnivorous nematodes of the family Qudsianematidae. 3.1.4. Plant-parasitic nematodes Numbers of plant-parasitic nematodes steadily declined over the first two weeks of the experiment in the non-treated control and acid alone treatments (Fig. 1E). There were fewer numbers of plant-parasitic nematodes in the acid alone than in the non-treated control over the course of the experiment. All liquid hog manure treatments had fewer plant-parasitic nematodes by day 3. Numbers increased after one week, then decline to the end of the experiment. The increase and subsequent decline in number was due to tylenchid nematodes, of the genera Filenchus and Tylenchus. By the end of the experiment, numbers of plant-parasitic nematodes were similar for all treatments (P > 0.05). 3.1.5. Pratylenchus spp. Pratylenchus spp. steadily declined over the course of the experiment in the non-treated control (Fig. 1F). In the acid treatment, Pratylenchus spp. declined over the first three days before increasing at week 2 then declining to the end of the experiment. Addition of liquid hog manure alone resulted in lower numbers of Pratylenchus spp. compared to the non-treated control throughout the experiment. 3.2. Nematode faunal analysis At 24 h following treatment, food webs in all treatments were moderately enriched and highly structured as indicated by nematode faunal analysis. At week 4, the enrichment index was lower in the non-treated control while the structure index
Fig. 2. Results of nematode faunal analysis showing nematode community structure and enrichment conditions of the soil food web at 24 h and 28 days following application of treatments. Treatments are control (non-treated), acid alone, liquid hog manure (LHM) 15% (v v 1 soil water) and acidified liquid hog manure 5%, 10%, and 15%. The mean of three independent replicates 1 standard error are shown.
Fig. 3. Total (ionized plus non-ionized forms) volatile fatty acid (VFA) concentration (mM in soil water) in soil receiving the treatments, control (non-treated), acid alone, liquid hog manure (LHM) 15% (v v 1 soil water) and acidified liquid hog manure 5%, 10%, and 15%. The mean of three independent replicates 1 standard error are shown.
remained similar to that at 24 h. In the acid treatment, both the enrichment and the structure indices declined by week 4. In the liquid hog manure and acidified liquid hog manure treatments, the enrichment index increased (P < 0.05) and the structure index decreased (P < 0.05) at week 4 than after 24 h (Fig. 2). 3.3. Volatile fatty acids in soil No volatile fatty acids were detected in soil in the non-treated control and acid alone treatments (Fig. 3). The concentration (mM) of volatile fatty acids in soil at 0 h increased with increasing rate of liquid hog manure applied. Volatile fatty acids in soil declined steadily over time and were not detected at day 3 in the acidified liquid hog manure (5%, 10%, and 15%) treatments. Volatile fatty acids were almost undetectable at week 1 in the liquid hog manure 15% alone treatment.
Fig. 4. Total (ionized plus non-ionized forms) volatile fatty acid (VFA) concentration (mM in soil water) in soil receiving the treatments, control (non-treated), acid alone, and acidified liquid hog manure 15% (v v 1 soil water) under sterile and non-sterile conditions. The mean of three independent replicates 1 standard error are shown.
80
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
3.4. Volatile fatty acids persistence in soil Rates of volatile fatty acids applied with acidified and nonacidified liquid hog manure 15% were calculated to provide a concentration of 61.3 mM in soil solution. Immediately after setup of the second microcosm experiment, the recovery of volatile fatty acids in the acidified liquid hog manure 15% treatment was 59.0 mM under sterile conditions and 57.2 mM under non-sterile conditions (Fig. 4), being 96% and 93% of added volatile fatty acids, respectively. However, at day 4 volatile fatty acids were not recovered from soil treated with the acidified liquid hog manure (Fig. 4). Under sterile conditions, volatile fatty acids persisted throughout the experiment. Autoclaving of soil seems to have resulted in the generation of 3.0 and 2.3 mM volatile fatty acids in the non-treated control and acid alone treatments, respectively. These concentrations are similar to the increase in concentration of volatile fatty acids of sterile compared to non-sterile acidified liquid hog manure treatments (Fig. 4). 4. Discussion In this study we investigated whether an increase in opportunistic nematodes, following addition of liquid hog manure to control Pratylenchus spp., was a direct result of addition of readily available decomposable materials or indirect effects. Indirect effects being first the killing of soil organisms followed by proliferation of opportunistic organisms or the lack of ‘‘topdown’’ predation to control numbers of opportunistic nematodes. The decline in total numbers of nematodes following application of liquid hog manure and acidified liquid hog manure was most particular to plant-parasitic and Pratylenchus spp. nematodes. The number of plant-parasitic nematodes declined over the first three to seven days following application of liquid hog manure and acidified liquid hog manure. By the end of the experiment (28 days), numbers of plant-parasitic nematodes were not different between manure and the non-treated control and acid alone treatments. That could be attributed to the dependence of plantparasitic nematodes on plant hosts; in the absence of hosts, the nematodes starved (Viaene et al., 2006). Numbers of Pratylenchus spp. did rebound from initially depressed levels following acidified and non-acidified liquid hog manure by week 1 and 2 before declining again to the end of the experiment. This increase observed in number of Pratylenchus spp. in the non-acidified and acidified liquid hog manure treatments is possibly related to the hatching of eggs from gravid females added during setup of the experiment. The optimum pH for P. penetrans reproduction, and in turn egg hatching, is 5.2–6.4 (Morgan and MacLean, 1968; Willis, 1972). The pH of soil receiving these treatments lay within this optimum range for P. penetrans reproduction. The decline in total number of nematodes was greater with increasing liquid hog manure concentration and greatest for the liquid hog manure 15% (v v 1 soil water) rate. The decline in nematodes can be attributed to the presence of liquid hog manure as nematode numbers did not decline to the same extent in the non-treated control or acid alone treatments. The findings are consistent with volatile fatty acids being lethal to some soil-borne pathogens and pests (Tenuta et al., 2002; McElderry et al., 2005; Xiao et al., 2007; Mahran et al., 2008a). Volatile fatty acids in liquid hog manure, with the rates and at the pH used in this experiment, did not seem to act like a broadspectrum pesticide in eliminating all nematodes and causing a ‘‘biological vacuum’’ (Yeates et al., 1991; Wang et al., 2006). Total nematode numbers increased rapidly at week 1 after liquid hog manure and acidified liquid hog manure application, corresponding to loss of volatile fatty acids from soil. Opportunistic c–p 1 and
c–p 2 nematodes were most responsible for the increase in total nematode numbers in soil. Their increase in relation to acidified and non-acidified liquid hog manure treatment was likely in response to enrichment of the microbial communities in the soil environment by readily degradable compounds in liquid hog manure, including volatile fatty acids, and perhaps the mortality of some soil organisms. These enrichment-opportunist nematodes have increased in soil rapidly following the addition of decomposable materials or availability of organic resources from mortality of organisms (Ferris and Bongers, 2006). An increase in nematodes of c–p 1 indicates stimulation by ‘‘bottom-up’’ processes resulting from an increase in microbial biomass for feeding by enrichment-opportunistic nematodes. In contrast, number of higher trophic nematodes (c–p 3–5) did not change. Nematode taxa at different trophic and life-history groups responded differently to the application of acidified and nonacidified liquid hog manure. By week 4, opportunistic c–p 1 nematodes of the genera Diploscapter and Mesorhabditis were dominant in soil and the levels of the genus Monhystera declined. Bacterial-feeding c–p 2 nematodes of the genera Acrobeles and Chiloplacus increased in soil while individuals of the genus Plectus were not detected in soil in liquid hog manure and acidified liquid hog manure 10% and 15% treatments. Wilsonema generally was not affected by the treatments. Taxa of the persister nematodes, c–p 3–5, also varied in their response to the treatments. The number of the bacterial-feeding nematodes of the genus Ethmolaimus increased in soil in response to liquid hog manure treatments but the number of nematodes of the genus Tylencholaimus declined. The survival of higher trophic nematodes classified in colonizer–persister groups, c–p 3–5, was not significantly different between acidified and non-acidified liquid hog manure treatments by the end of the experiment. This observation is contrary to expectation as these nematodes have been shown to be more sensitive to different stressors such as: metals (Korthals et al., 1996, 2000), acidification (Ruess et al., 1996); nematicides (Smolik, 1983), and N compounds (Tenuta and Ferris, 2004). Perhaps the explanation lies in the short (three days) persistence of volatile fatty acids in soil. Tolerance to stress of some persister nematodes (carnivores and omnivores) has been observed elsewhere. In a field study, omnivore numbers increased in soil at week 4 following incorporation of the plant-parasitic-suppressive green manure, sunn hemp (Crotalaria juncea L.), into soil (Wang et al., 2004). Alternatively, it is possible that soil handling and preparation for microcosms could have killed higher c–p nematodes, sensitive to physical disturbance (Fiscus and Neher, 2002; Rahman et al., 2007), leaving more tolerant taxa. The majority of volatile fatty acids added to soil in liquid hog manure were recovered and thus sorption of volatile fatty acids to soil seems unimportant. Water was used as an extractant and thus only soluble volatile fatty acids are recovered and measured. Acetic acid, the major volatile fatty acid in liquid hog manure, has been shown to be sorbed to marine sediments (Shiba et al., 2001). However, in the soil used here, sorption does not seem to have been appreciable. Microbial degradation seems to have caused loss of volatile fatty acids within days of manure application. Various microorganisms are able to degrade volatile fatty acids in liquid hog manure and use them as a source of carbon and energy (Zhu, 2000). While some bacteria such as Acinetobacter calcoaceticus (Beijerinck) Bouvet and Grimont, Alcaligenes faecalis Castellani and Chalmers, and Arthrobacter flavescens Lochhead, are capable of degrading all types of volatile fatty acids in liquid hog manure (Bourque et al., 1987; Jolicoeur and Morin, 1987), others such as Corynebacterium glutamicum (Kinoshita) Abe and Micrococcus spp. can only degrade acetic and propionic acids which are
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
major volatile fatty acids in liquid hog manure (Bourque et al., 1987). Microbial biomass, enzyme activities and microbial respiration, increase upon application of anaerobically stored liquid animal manures to soil (Paul and Beauchamp, 1996; Zaman et al., 1999). Liquid animal manures to field plots increased microbial respiration for the week following application presumably due to the rapid oxidation of volatile fatty acids present in the slurry (Tenuta et al., 2000; Chantigny et al., 2001). Decomposition of liquid hog manure in field plots has been rapid; one-half of the total annual CO2 emissions from soil occurring within one week of application (Rochette et al., 2000). In conclusion, the increase in opportunistic nematodes in soil following application of liquid hog manure and acidified liquid hog manure was in response to enrichment of the soil environment likely resulting from readily decomposable materials from the manure and less so from mortality of some organisms. The large increase in abundance of opportunistic nematodes four weeks following application of the manure treatments, with no corresponding increase in high c–p nematodes, resulted in an apparently poorly structured food web indicating the lack of higher trophic links. The decrease in structure index was due to increase in numbers of c–p 2 numbers and not from decrease in number of c–p 3–5 nematodes. Why acidified liquid hog manure is as an effective method to control plant-parasitic nematodes including Pratylenchus spp. without having the same effect on other trophic guilds of nematode is worthy of further investigation. However, controlling plant-parasitic nematodes without killing higher trophic organisms is desirable in the promotion of soil health. Acknowledgements We thank Mervin Bilous and Oscar Molina for technical assistance and two anonymous reviewers and Howard Ferris for comments to previous versions of this manuscript. This research was funded by the Manitoba Agricultural Research and Development Initiative, Canada Research Chair Program in Applied Soil Ecology, and the Natural Sciences and Engineering Research Council of Canada Discovery Grant Program. References Al-Banna, L., Ploeg, A.T., Williamson, V.M., Kaloshian, I., 2004. Discrimination of six Pratylenchus species using PCR and species-specific primers. J. Nematol. 36, 142–146. Bongers, T., 1994. De nematoden van Nederland. Pirota Schoorl, Bibliotheek uitgave KNNV, Uitgeverij, Utrecht, Netherlands, 408 pp. Bongers, T., 1990. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia 83, 14–19. Bongers, T., Ferris, H., 1999. Nematode community structure as a bioindicator in environmental monitoring. Trends Ecol. Evol. 14, 224–228. Bourque, D., Bisaillon, J., Beaudet, R., Sylvestre, M., Ishaque, M., Morin, A., 1987. Microbiological degradation of malodorous substances of swine waste under aerobic conditions. Appl. Environ. Microbiol. 53, 137–141. Cassel, D.K., Nielsen, D.R., 1986. Field capacity and available water capacity. In: Klute, A. (Ed.), Methods of Soil Analysis. Part 1. Physical and Mineralogical Properties Including Statistics of Measurement and Sampling. American Society of Agronomy, Madison, WI, pp. 901–926. Chantigny, M.H., Rochette, P., Angers, D.A., 2001. Short-term C and N dynamics in a soil amended with pig slurry and barley straw: a field experiment. Can. J. Soil Sci. 81, 131–137. Conn, K.L., Lazarovits, G., 1999. Impact of animal manures on Verticillium wilt, potato scab, and soil microbial populations. Can. J. Plant Pathol. 21, 81–92. Conn, K.L., Tenuta, M., Lazarovits, G., 2005. Liquid swine manure can kill Verticillium dahliae microsclerotia in soil by volatile fatty acid, nitrous acid, and ammonia toxicity. Phytopathology 95, 28–35. Dewes, T., Hunsche, E., 1998. Composition and microbial degradability in the soil of farmyard manure from ecologically-managed farms. Biol. Agric. Hortic. 16, 251–268. Ferris, H., Bongers, T., 2006. Nematode indicators of organic enrichment. J. Nematol. 38, 3–12.
81
Ferris, H., Bongers, T., De Goede, R.G.M., 2001. A framework for soil food web diagnostics: extension of the nematode faunal analysis concept. Appl. Soil Ecol. 18, 13–29. Ferris, H., Matute, M.M., 2003. Structural and functional succession in the nematode fauna of a soil food web. Appl. Soil Ecol. 23, 93–110. Fiscus, D.A., Neher, D.A., 2002. Distinguishing nematode genera based on relative sensitivity to physical and chemical disturbances. Ecol. Appl. 12, 565–575. Forge, T., Tenuta, M., 2008. Indicators of soil food web properties. In: Carter, M.R., Gregorich, E.D. (Eds.), Soil Sampling and Methods of Analysis. 2nd ed. Canadian Society of Soil Science, CRC Press, Boca Raton, FL, USA, pp. 577– 588. Haydock, P.P.J., Woods, S.R., Grove, I.G., Hare, M.C., 2006. Chemical control of nematodes. In: Perry, R.N., Moens, M. (Eds.), Plant Nematology. CAB International, Wallingford, UK, pp. 392–410. Humason, G.L., 1972. Animal Tissue Techniques, 3rd ed. W.H. Freeman and Company, San Francisco, pp. 641. Ingham, R.E., 1994. Nematodes. In: Weaver, R.W., Angle, S., Bottomley, P., Bezdicek, B., Smith, S., Tabatabai, A., Wollum, A. (Eds.), Methods of Soil Analysis. Part 2. Microbiological and Biochemical Properties no. 5. Soil Society of America, Madison, WI, USA, pp. 459–490. Jolicoeur, P., Morin, A., 1987. Isolation of Acinetobacter calcoaceticus strains degrading the volatile fatty acids of swine wastes. Biol. Wastes 19, 133–140. Jothi, G., Pugalendhi, S., Poornima, K., Rajendran, G., 2003. Management of root-knot nematode in tomato Lycopersicon esculentum, Mill., with biogas slurry. Bioresour. Technol. 89, 169–170. Kirchmann, H., Lundvall, A., 1993. Relationship between N immobilization and volatile fatty acids in soil after application of pig and cattle slurry. Biol. Fertil. Soils 15, 161–164. Korthals, G.W., Van de Ende, A., Van Megen, H., Lexmond, T.M., Kammenga, J.E., Bongers, T., 1996. Short-term effects of cadmium, copper, nickel, and zinc on soil nematodes from different feeding and life-history strategy groups. Appl. Soil Ecol. 4, 107–117. Korthals, G.W., Bongers, M., Fokkema, A., Dueck, T.A., Lexmond, T.M., 2000. Joint toxicity of copper and zinc to a terrestrial nematode community in an acid sandy soil. Ecotoxicology 9, 219–228. Mahran, A., Tenuta, A., Hanson, M.L., Daayf, F., 2008a. Mortality of Pratylenchus penetrans by volatile fatty acids from liquid hog manure. J. Nematol. 40, 119– 126. Mahran, A., Conn, K.L., Tenuta, M., Lazarovits, G., Daayf, F., 2008b. Effectiveness of liquid hog manure and acidification to kill Pratylenchus spp. in soil. J. Nematol. 40, 266–275. McElderry, C.F., Browning, M., Amador, J.A., 2005. Effect of short-chain fatty acids and soil atmosphere on Tylenchorhynchus spp. J. Nematol. 37, 71–77. Min, Y.Y., Sato, E., Shirakashi, T., Wada, S., Toyota, K., Watanabe, A., 2007. Suppressive effect of anaerobically digested slurry on the root-lesion nematode Pratylenchus penetrans and its potential mechanisms. Jpn. J. Nematol. 37, 93–100. Morgan, G.T., MacLean, A.A., 1968. Influence of soil pH on an introduced population of Pratylenchus penetrans. Nematologica 14, 311–312. Nahar, M.S., Grewal, P.S., Miller, S.A., 2006. Differential effects of raw and composted manure on nematode community, and its indicative value for soil microbial, physical and chemical properties. Appl. Soil Ecol. 34, 140–151. Paul, J.W., Beauchamp, E.G., 1996. Soil microbial biomass C, N mineralization and N uptake by corn in dairy cattle slurry and urea-amended soils. Can. J. Soil Sci. 76, 469–472. Rahman, L., Chan, K.Y., Heenan, D.P., 2007. Impact of tillage, stubble management and crop rotation on nematode populations in a long-term field experiment. Soil Till. Res. 95, 110–119. Rochette, P., Angers, D.A., Cote, D., 2000. Soil carbon and nitrogen dynamics following application of pig slurry for the 19th consecutive year. I. Carbon dioxide fluxes and microbial biomass carbon. Soil Sci. Soc. Am. J. 64, 1389–1395. Royer, I., Angers, D.A., Chantigny, M.H., 2007. Dissolved organic carbon in runoff and tile-drain water under corn and forage fertilized with hog manure. J. Environ. Qual. 36, 855–863. Rozman, K.K., Doull, J., 2000. Dose and time as variables of toxicity. Toxicology 144, 169–178. Ruess, L., Sandbach, P., Cudlin, P., Dighton, J., Crossley, A., 1996. Acid deposition in a spruce forest soil: effects on nematodes, mycorrhizas, and fungal biomass. Pedobiologia 40, 51–66. Shiba, T., Harada, H., Furushita, M., Maeda, T., 2001. Adsorption of acetate to coastal sediment particles. J. Natl. Fish. Univ. (Jpn) 49, 117–121. Smolik, J.D., 1983. Effect of nematicide treatments on non-target nematode populations associated with corn. Plant Dis. 67, 28–31. Sorensen, P., 1998. Carbon mineralization, nitrogen immobilization and pH change in soil after adding volatile fatty acids. Eur. J. Soil Sci. 49, 457–462. Tenuta, M., Bergstrom, D.W., Beauchamp, E.G., 2000. Denitrifying enzyme activity and carbon availability for denitrification following manure application. Comm. Soil Sci. Plant Anal. 31, 861–876. Tenuta, M., Conn, K.L., Lazarovits, G., 2002. Volatile fatty acids in liquid swine manure can kill microsclerotia of Verticillium dahliae. Phytopathology 92, 548– 552. Tenuta, M., Ferris, H., 2004. Sensitivity of nematode life-history groups to ions and osmotic tensions of nitrogenous solutions. J. Nematol. 36, 85–94. Timper, P., Newton, G.L., Johnson, A.W., Gascho, G.J., 2004. Nematode densities in year-round forage production systems utilizing manure fertilization. Nematropica 34, 219–227.
82
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
Valocka, B., Dubinsky, P., Papajova, I., Sabova, M., 2000. Effect of anaerobically digested pig slurry from lagoon on soil and plant nematode communities in experimental conditions. Helminthologia 37, 53–57. Viaene, N., Coyne, D.L., Kerry, B.R., 2006. Biological and cultural management. In: Perry, R.N., Moens, M. (Eds.), Plant Nematology. CAB International, Wallingford, UK, pp. 346–369. Wang, K.H., McSorley, R., Marshall, A.J., Gallaher, R.N., 2004. Nematode community changes associated with decomposition of Crotalaria juncea amendment in litterbags. Appl. Soil Ecol. 27, 31–45. Wang, K.H., McSorley, R., Kokalis-Burelle, N., 2006. Effects of cover cropping, solarization, and soil fumigation on nematode communities. Plant Soil 286, 229–243. Willis, C.B., 1972. Effects of soil pH on reproduction of Pratylenchus penetrans and forage yield of alfalfa. J. Nematol. 4, 291–295.
Xiao, J., Zhu, J., Chen, S., Ruan, W., Miller, C., 2007. A novel use of anaerobically digested liquid swine manure to potentially control soybean cyst nematode. J. Environ. Sci. Health 42, 749–757. Xiao, J., Chen, S., Zhu, J., Ruan, W., 2008. Effect of liquid swine manure on hatch and viability of Heterodera glycines. J. Nematol. 40, 152–160. Yeates, G.W., Bamforth, S.S., Ross, D.J., Tate, K.R., Sparling, G.P., 1991. Recolonization of methyl bromide sterilized soils under four different field conditions. Biol. Fertil. Soils 11, 181–189. Zaman, M., Di, H.J., Cameron, K.C., Frampton, C.M., 1999. Gross nitrogen mineralization and nitrification rates and their relationships to enzyme activities and the soil microbial biomass in soils treated with dairy shed effluent and ammonium fertilizer at different water potentials. Biol. Fertil. Soils 29, 178–186. Zhu, J., 2000. A review of microbiology in swine manure odor control. Agric. Ecosys. Environ. 78, 93–106.
Contents lists available at ScienceDirect
Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Response of a soil nematode community to liquid hog manure and its acidification A. Mahran a,b, M. Tenuta b,c,*, R.A. Lumactud b, F. Daayf a a
Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 Department of Soil Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 c Canada Research Chair in Applied Soil Ecology, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 September 2008 Received in revised form 31 May 2009 Accepted 12 June 2009
A study was conducted to determine why the number of opportunistic bacterial-feeding nematodes increase following addition of liquid hog manure and acidified mixtures of the manure to soil. A sandy loam soil harboring a wide range of nematode taxa, representing various trophic and colonizer–persister groups (c–p 1 through 5) and augmented with the plant-parasitic nematode, Pratylenchus spp., was used in microcosm experiments. Treatments were additions of liquid hog manure (0.15 v v 1 soil water), mixtures of manure (0.05, 0.10, and 0.15 v v 1 soil water) and sulfuric acid, as well as a sulfuric acid alone and a non-treated control. Three days post-treatment and during the presence of non-ionized volatile fatty acids from manure in soil, numbers of plant-parasitic nematodes, including Pratylenchus spp., decreased by at least 50% for all manure treatments compared to non-manure treatments. Thereafter, c– p 1 and c–p 2 nematode numbers increased in all manure but not in non-manure treatments. At week 4, c–p 1 and c–p 2 nematode numbers were greater by at least 6000 and 5000 individuals kg 1 soil, respectively, compared to non-manure treatments. In contrast, numbers of c–p 3–5 nematodes were not affected by the treatments. At week 4, the enrichment index, an assessment of the abundance of opportunistic c–p 1 and some c–p 2 nematodes compared to all c–p 2 nematodes, was around 70% for all manure treatments and lower, 32 and 29, respectively, for acid alone and non-treated control treatments. The increase in opportunistic nematodes following manure treatment was likely due to the increase in their food resources associated with the enrichment of the soil environment with readily degradable compounds. Volatile fatty acids present in the manure persisted in the soil for only four days before biological degradation. We conclude liquid hog manure is effective in killing plant-parasitic nematodes while increasing bottom-up food web interactions but not, as with soil fumigants, decimating top-down trophic interactions. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Acidity Food web Liquid hog manure Nematode communities Nematode faunal analysis Pratylenchus Root-lesion nematode Volatile fatty acids
1. Introduction Animal manures and wastes are the sources of plant nutrients and organic matter in soils of many agricultural systems. Anaerobically stored animal manures and wastes are capable of killing plant pathogens (Conn and Lazarovits, 1999) and pests such as plant-parasitic nematodes (Valocka et al., 2000; Jothi et al., 2003; Timper et al., 2004; Min et al., 2007). Acidic conditions generating non-ionized forms of short-chain volatile fatty acids (acetic, propionic, n-butyric, isobutyric, n-valeric, isovaleric and n-caproic acids) in liquid hog manure suppress the wilt fungus, Verticillium dahliae Kleb. (Tenuta et al., 2002; Conn et al., 2005). More recently, using solution exposure studies, Mahran et al. (2008a) concluded
* Corresponding author at: Canada Research Chair in Applied Soil Ecology, Department of Soil Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2. Tel.: +1 204 474 7827; fax: +1 204 474 7642. E-mail address: [email protected] (M. Tenuta). 0929-1393/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2009.06.003
that volatile fatty acids account for most of the lethal effect of liquid hog manure to Pratylenchus penetrans (Cobb) Chitwood and Oteifa under acidic conditions. Also, volatile fatty acids added to soil in pot studies (McElderry et al., 2005; Xiao et al., 2007, 2008) and present in liquid hog manure (Mahran et al., 2008b), reduced numbers of plantparasitic nematodes in field studies. After the application of acidified liquid hog manure, we observed large numbers of opportunistic bacterial-feeding nematodes of the Rhabditidae (unpublished data). These nematodes flourish under conditions of abundant bacterial growth that can occur following application of broad-spectrum pesticides, such as soil fumigants (Yeates et al., 1991; Wang et al., 2006) or following addition of readily decomposable materials to soil (Ferris and Matute, 2003; Nahar et al., 2006). Anaerobically stored animal manures contain large amounts of easily decomposable organic C, including volatile fatty acids (Zhu, 2000), amino acids (Dewes and Hunsche, 1998), and dissolved organic carbon (Royer et al., 2007). These materials are rapidly utilized by soil microorganisms as a source of energy (Sorensen,
76
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
1998). Kirchmann and Lundvall (1993) showed that volatile fatty acids in hog and cattle slurries decomposed in soil at 25 8C within two days of application. The amount of N immobilized in microbial biomass was correlated with the amount of volatile fatty acids applied, suggesting that these compounds are a source of energy for soil microorganisms. The efficacy of any compound in killing soil organisms, either beneficial or detrimental to plant growth, depends on dosage, duration of exposure and sensitivity of the organism (Rozman and Doull, 2000). Persistence of added toxic compounds is desired, in the short-term, to provide the concentration time product necessary for killing the target organism. However, long-term persistence is undesirable as it increases the probability of harm to non-target organisms (Haydock et al., 2006). The persistence, or the sorption and volatilization, of volatile fatty acids in acidified liquid hog manure added to soil is unknown but clearly long-term persistence increases the probability of negative effects on soil food webs. Nematode faunal analysis is an excellent tool for assessing the structure and function of soil food webs and their response to soil perturbations (Bongers and Ferris, 1999) such as addition of liquid hog manure. Nematodes (a) have high diversity and abundance in soil environments with complex food webs; (b) have a permeable cuticle in contact with dissolved constituents of soil water or volatiles in soil atmosphere; (c) respond to a proliferation of microbes following an increase in available carbon substrates; (d) differ in sensitivity and response to disturbance; (e) are relatively easy to isolate and identify to genus; (f) occupy key trophic guilds in soil food webs; and (g) there is a clear relationship between their feeding structures and food resource. The current study was conducted to determine: (i) if liquid hog manure and its acidification increases opportunistic nematodes through addition of readily decomposable materials, microbial growth after chemical sterilization of soil or reduction of ‘‘topdown’’ population control, (ii) if persistence of volatile fatty acids in soil receiving acidified liquid hog manure is of short or long duration, and (iii) if loss of volatile fatty acids in soil is due to biological degradation. 2. Materials and methods 2.1. Soil A cattle-grazed, native, mixed-grass prairie soil harboring a diverse range of nematode genera and trophic guilds was collected in the summer of 2007 from the Yellow Quill Prairie Preserve near the town of Shilo, in south central Manitoba, Canada. The soil was collected as three separate blocks (50 cm 50 cm 15 cm h) separated by about 10 m. Each soil block was placed in a polyethylene bag and transported to the laboratory in an ice chest, where it was stored (<1 week) at 5 8C until used in microcosm experiments. The soil was a sandy loam with 6.7% organic matter, 16.5% gravimetric moisture and pH (1:2 soil:H2O ratio) of 7.5. 2.2. Liquid hog manure Liquid hog manure was collected from an earthen storage lagoon at a commercial finisher hog farm in south eastern Manitoba and had a pH of 7.2, 2.7% dry matter, 0.6% total N, 0.01% P and 0.25% K. The manure was centrifuged (10 min at 3400 g) to remove particulates. The resulting solution had a total volatile fatty acid concentration (ionized plus non-ionized forms) of 347 mM (acetic = 190 mM, propionic = 51 mM, isobutyric = 23 mM, n-butyric = 54, isovaleric = 13 mM, n-caproic = 9 mM and n-valeric = 9 mM). It was frozen at 20 8C until used in microcosm experiments.
2.3. Pratylenchus spp. A soil naturally infested with high numbers of two root-lesion nematode species, P. penetrans and Pratylenchus crenatus Loof, was collected from a commercial potato field in the province of Prince Edward Island and reared on common mint (Mentha spicata L.) in a greenhouse. Pratylenchus spp. present in the soil were identified using species-specific polymerase chain reaction (PCR) primers (Al-Banna et al., 2004), confirmed by molecular sequencing of PCR products and sequence comparison with the GenBank BLAST database (Mahran et al., 2008b). Nematodes were extracted using Cobb’s sieving and decanting followed by sugar flotation (Ingham, 1994) 24 h prior to commencing the experiment. The density of the resulting mixed population of juveniles and adults of Pratylenchus spp. was adjusted in dH2O and added to be 1000 nematodes kg 1 dry soil equivalent. P. penetrans and P. crenatus, are common pests of potato in central and eastern Canada though not present in Manitoba. They were added to the soil to provide levels of a common nematode pest of great economic importance. 2.4. Microcosm Each of the three soil collections served as independent replicates in soil microcosm experiments. The soil was passed through a 2 mm mesh screen (USA Standard Test Sieve) to remove stones, roots and debris. Soil microcosms were prepared by adding 90 g fresh soil to 118 ml polyethylene specimen storage containers (6.4 cm d 6.4 cm h) (Fisher Scientific Canada Ltd., Edmonton, Canada). To prevent anaerobic conditions during incubation, 20 0.1 cm diameter holes were punched into container lids. Different combinations of 98% sulfuric acid (equivalent to 4.9 mg H2SO4 ml 1) 1) (Fisher Scientific Canada Ltd.) and liquid-fraction of manure were applied to microcosms to provide the following treatments: nontreated control, acid (initial soil pH reduced to 5.5 using sulfuric acid 98%), liquid hog manure (15% v v 1 soil water volume at 30% field capacity equivalent to 40,500 l ha 1) and acidified liquid hog manure to 5%, 10%, and 15% v v 1 soil water volume. At an assumed soil bulk density of 1.3 Mg m 3, a typical depth of field application (15 cm), and the moisture content of the soil, acidified liquid hog manure added at 5%, 10%, and 15% rates was equivalent to 13,500, 27,000, and 40,500 l ha 1, respectively. The amounts of sulfuric acid needed to bring initial soil pH and the liquid hog manure down to pH 5.5 was determined by titrating soil and liquid hog manure with various amounts of acid. Following application, the soil gravimetric moisture content was adjusted to 30% of field capacity (Cassel and Nielsen, 1986) using dH2O. Microcosms were arranged in a completely randomized design having three independent replicates per treatment and were placed at 22 8C in the dark (Iso-temp Incubator 304; Fisher Scientific Canada Ltd.). Soil samples were extracted 24 h, three days, and one, two, three and four weeks following experimental setup to examine changes in nematode communities as detailed by Forge and Tenuta (2008), and to determine volatile fatty acids concentration in soil. Fifty grams of soil from each microcosm was used for the extraction of nematodes by Cobb’s sieving and decanting followed by sugar flotation (Ingham, 1994). Live nematodes were collected in water in a 15 ml centrifuge tube, excess water was removed, and an equal volume of hot (80 8C) buffered formalin solution (pH 7.0) (Humason, 1972) was added to the nematode suspension to preserve the specimens for identification. Total number of nematodes was determined using a stereo microscope (40 magnification). The first 100 nematodes encountered in each sample were then identified to genus, according to diagnostic keys of Bongers (1994), using an inverted compound microscope (400 magnification), the abundance of each genus in a microcosm was
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
calculated by multiplying the fraction of nematodes of that genus in the identified 100 nematodes by the total number of nematodes. Nematode genera were assigned colonizer–persister values (c–p value) according to Bongers (1990) on a scale ranging from 1 to 5. Opportunistic bacterial-feeding nematodes having similar characteristics to r-strategists were assigned to group, c–p 1. Higher c– p groups were ordered based on increasing k-strategy life-history attributes. 2.5. Food web indices Food web indices computed included the enrichment and structure indices (Ferris et al., 2001). The structure trajectory is a weighted abundance of larger-, slower-reproducing bacterivore, fungivore, omnivore and carnivore c–p 3–5 value nematodes. The structure index was calculated as 100 (s/(s + b)), with b calculated as Skbnb, where kb are the weightings assigned to guilds that indicate basal characteristics of the food web and nb are the abundances of nematodes in those guilds, the s component was calculated similarly, using those guilds having weightings assigned to the structure index. The enrichment trajectory is a weighted abundance of opportunistic bacteriovorous c–p 1 and fungivorous c–p 2 nematodes species that respond rapidly to prey resources. The enrichment index was calculated as 100 (e/(e + b)). The e component was calculated as previously mentioned using those guilds indicating enrichment.
77
for 30 min at 2000 rpm at 5 8C using a refrigerated incubator shaker (New Brunswick Scientific Co., Edison, USA). The tubes were then centrifuged (5 min at 5000 g) and the supernatant analyzed for volatile fatty acids concentration. Individual volatile fatty acid compounds from C2 (acetic) to C6 (n-caproic) were determined using chemical-suppression ion-exclusion chromatography and conductivity detection (Tenuta et al., 2002) with a Dionex ion chromatography system, ICS–1000 (Dionex Corp., Sunnyvale, USA). The chromatograph was equipped with an IonPac ICE-AS1 (9 mm 250 mm) analytical column and anion micro-membrane suppressor (AMMS-ICE II). Soil extracts contained in 1 ml vials with filter caps (Dionex Corp.) were injected (25 ml) with an automated sampler (AS40, Dionex Corp.). For analysis, 1 mM heptaflurobutyric acid (Acros Organics USA, Morris Plains, USA) with 5% acetonitrile (v v 1) was used as the eluent. Commercially available individual volatile fatty acids (99% purity; acetic acid, Fisher Scientific Canada Ltd.; propionic acid, isobutyric acid, nbutyric acid, isovaleric acid, and n-valeric acid, Sigma–Aldrich; and n-caproic acid; Acros Organics) were used to calibrate the response of the conductivity cell to individual volatile fatty acid concentration using eight point response curves. For each soil sample, the concentration of volatile fatty acid in solution was determined by multiplying its concentration in the supernatant by a dilution factor estimated based on the soil moisture content of soil and the volume of water added to it (15 ml). 2.8. Statistical analysis
2.6. Volatile fatty acids persistence and fate in soil The persistence of volatile fatty acids in soil, and the speed of their biological degradation and sorption, was examined in a separate microcosm experiment. The same soil used in the previous experiment was split into two parts, one part (2 kg) was used without sterilization and the other part (2 kg) was autoclaved twice, three days apart, (at 121 8C and 15 psi) for 45 min. The same liquid hog manure used in the previous microcosm experiment was used here and added to non-sterile treatments. For non-sterile soil treatments, liquid hog manure was added without sterilization to non-sterile soil. Liquid hog manure was sterilized by centrifugation (30 min at 11,500 g) to remove particulates, the supernatant filtered through Whatman binder-free glass microfiber filters (9.0 cm d; Fisher Scientific Canada Ltd.) to remove fine particulates, then the filtrate was sterilized using Nalgene disposable sterile analytical filter units (pore size 0.2 mm; Fisher Scientific Canada Ltd.). The sterile liquid hog manure was then stored at 5 8C in sterile polyethylene 50 ml conical tubes until application to the soil. Microcosms were set up as described previously except that 30 g fresh soil was used and all polyethylene containers were sterilized using 95% ETOH (Fisher Scientific Canada Ltd.) prior to use. Treatments were non-treated control, acid (initial soil pH reduced to 5.5 using sulfuric acid 98%), and sterile acidified liquid hog manure (initial soil pH reduced to 5.5 and 15% v v 1 soil water at 30% field capacity) treatments. Sterile water was added to all treatments to bring soil moisture to 30% of field capacity. Microcosms were destructively sampled after setup (0 h) and every 24 h for the subsequent seven days to determine volatile fatty acid concentration in soil. Persistence of volatile fatty acids with the sterile acidified liquid hog manure treatment indicated lack of biological degradation, sorption and volatilization. In contrast, lack of persistence of volatile fatty acids in the non-sterile treatments only indicated biological degradation of the compounds. 2.7. Volatile fatty acids determination and concentration in soil Fifteen milliliters of dH2O was added to 15 g moist soil in 50 ml polyethylene conical tubes. The tubes were shaken, on their sides,
Results for both microcosm experiments were tested for normality using the Shapiro–Wilk test prior to the analysis of variance using the Proc GLM procedure of the Statistical Analysis Software (SAS Institute Inc., Cary, USA). Treatment means were compared using Tukey’s multiple comparison test (P < 0.05). Data for nematode indices were square-root transformed prior to the analysis of variance and comparison of means. 3. Results 3.1. Nematode abundance The total number of nematodes declined over the course of the first microcosm experiment in the non-treated control and acid treatments. Decline in the acid treatment was least of all treatments (Fig. 1A). For the liquid hog manure treatment, total number of nematodes declined over the first seven days following application then increased steadily; by week 4, it had the most nematodes of all treatments (Fig. 1A). Acidified liquid hog manure resulted in a decline in total number of nematodes by day 3, with lowest numbers at the highest liquid hog manure rate. Thereafter, total number of nematodes increased in acidified manure treatments but was lower than for the liquid hog manure alone treatment at week 4 (Fig. 1A). 3.1.1. c–p 1 nematodes The number of opportunistic (c–p 1) nematodes decreased from about 1500 to 250 individuals kg 1 soil by the end of the experiment in the acid only treatment (Fig. 1B). Numbers in the non-treated control increased by day 3 and thereafter declined to a level similar to that in the acid alone treatment (Fig. 1B). Numbers increased in the liquid hog manure treatment to 11,000 individuals kg 1 soil at week 4, a 25-fold increase than in the non-treated control. Individuals in the genera Diploscapter and Mesorhabditis mainly contributed to the increase in number of c–p 1 nematodes observed in the liquid hog manure treatment. In the acidified liquid hog manure treatment, addition to 5% resulted in a higher number of nematodes after one day followed by a decline to the level of all other
78
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
Fig. 1. Number of (A) total, (B) c–p 1, (C) c–p 2, (D) c–p 3–5, (E) plant-parasitic, and (F) Pratylenchus spp. nematodes. Treatments are control (non-treated), acid alone, liquid hog manure (LHM) 15% (v v 1 soil water) and acidified liquid hog manure 5%, 10%, and 15%. The mean of three independent replicates 1 standard error are shown. Means at day 28 followed by different letters are significantly different from one another (P < 0.05) as determined by Tukey’s multiple comparison test.
treatments by one week (Fig. 1B). Thereafter, numbers of individuals of the genera Diploscapter and Mesorhabditis increased for all acidified liquid hog manure treatments to a level similar (P > 0.05) as the liquid hog manure alone treatment. 3.1.2. c–p 2 nematodes Numbers of c–p 2 nematodes tended to be numerically higher in the non-treated control compared to acid alone treatment, though statistically similar (P > 0.05) by the end of the experiment (Fig. 1C). In the liquid hog manure treatment, c–p 2 nematodes declined over the first seven days of the experiment and then
increased to 20,000 individuals kg 1 by the end of the experiment (Fig. 1C). Bacterial-feeding nematodes of the genera Chiloplacus, Eucephalobus, Acrobeles, and the family Plectidae, and fungalfeeding nematodes of the genera Aphelenchus and Aphelenchoides, were the main component of the response to liquid hog manure treatment. There was a sharp increase in the numbers of c–p 2 nematodes at week 3 due to an increase in number of Chiloplacus and to a lesser extent, Acrobeles. A similar trend in numbers occurred with acidified liquid hog manure treatments except being intermediate of manure alone, non-treated control, and acid alone treatments.
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
79
3.1.3. c–p 3–5 nematodes Compared to c–p 1 and c–p 2 nematodes, numbers of c–p 3–5 nematodes collectively were not as responsive to treatments (Fig. 1D). There were no differences (P > 0.05) in their numbers among treatments by week 4. Two trends however, were evident; acid alone resulted in fewer nematodes during the two weeks following treatment with numbers remaining numerically lower than other treatments by the end of the experiment. Acidified liquid hog manure 15%, resulted in fewer c–p 3–5 nematodes by week 1 but a higher number than all treatments by the end of the experiment. The genera of nematodes comprising the colonizer–persister groups c–p 3–5 were the bacterial-feeding genera, Rhabdolaimus, Teratocephalus and Ethmolainus, the fungal-feeding genus, Tylencholaimus, and omnivorous nematodes of the family Qudsianematidae. 3.1.4. Plant-parasitic nematodes Numbers of plant-parasitic nematodes steadily declined over the first two weeks of the experiment in the non-treated control and acid alone treatments (Fig. 1E). There were fewer numbers of plant-parasitic nematodes in the acid alone than in the non-treated control over the course of the experiment. All liquid hog manure treatments had fewer plant-parasitic nematodes by day 3. Numbers increased after one week, then decline to the end of the experiment. The increase and subsequent decline in number was due to tylenchid nematodes, of the genera Filenchus and Tylenchus. By the end of the experiment, numbers of plant-parasitic nematodes were similar for all treatments (P > 0.05). 3.1.5. Pratylenchus spp. Pratylenchus spp. steadily declined over the course of the experiment in the non-treated control (Fig. 1F). In the acid treatment, Pratylenchus spp. declined over the first three days before increasing at week 2 then declining to the end of the experiment. Addition of liquid hog manure alone resulted in lower numbers of Pratylenchus spp. compared to the non-treated control throughout the experiment. 3.2. Nematode faunal analysis At 24 h following treatment, food webs in all treatments were moderately enriched and highly structured as indicated by nematode faunal analysis. At week 4, the enrichment index was lower in the non-treated control while the structure index
Fig. 2. Results of nematode faunal analysis showing nematode community structure and enrichment conditions of the soil food web at 24 h and 28 days following application of treatments. Treatments are control (non-treated), acid alone, liquid hog manure (LHM) 15% (v v 1 soil water) and acidified liquid hog manure 5%, 10%, and 15%. The mean of three independent replicates 1 standard error are shown.
Fig. 3. Total (ionized plus non-ionized forms) volatile fatty acid (VFA) concentration (mM in soil water) in soil receiving the treatments, control (non-treated), acid alone, liquid hog manure (LHM) 15% (v v 1 soil water) and acidified liquid hog manure 5%, 10%, and 15%. The mean of three independent replicates 1 standard error are shown.
remained similar to that at 24 h. In the acid treatment, both the enrichment and the structure indices declined by week 4. In the liquid hog manure and acidified liquid hog manure treatments, the enrichment index increased (P < 0.05) and the structure index decreased (P < 0.05) at week 4 than after 24 h (Fig. 2). 3.3. Volatile fatty acids in soil No volatile fatty acids were detected in soil in the non-treated control and acid alone treatments (Fig. 3). The concentration (mM) of volatile fatty acids in soil at 0 h increased with increasing rate of liquid hog manure applied. Volatile fatty acids in soil declined steadily over time and were not detected at day 3 in the acidified liquid hog manure (5%, 10%, and 15%) treatments. Volatile fatty acids were almost undetectable at week 1 in the liquid hog manure 15% alone treatment.
Fig. 4. Total (ionized plus non-ionized forms) volatile fatty acid (VFA) concentration (mM in soil water) in soil receiving the treatments, control (non-treated), acid alone, and acidified liquid hog manure 15% (v v 1 soil water) under sterile and non-sterile conditions. The mean of three independent replicates 1 standard error are shown.
80
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
3.4. Volatile fatty acids persistence in soil Rates of volatile fatty acids applied with acidified and nonacidified liquid hog manure 15% were calculated to provide a concentration of 61.3 mM in soil solution. Immediately after setup of the second microcosm experiment, the recovery of volatile fatty acids in the acidified liquid hog manure 15% treatment was 59.0 mM under sterile conditions and 57.2 mM under non-sterile conditions (Fig. 4), being 96% and 93% of added volatile fatty acids, respectively. However, at day 4 volatile fatty acids were not recovered from soil treated with the acidified liquid hog manure (Fig. 4). Under sterile conditions, volatile fatty acids persisted throughout the experiment. Autoclaving of soil seems to have resulted in the generation of 3.0 and 2.3 mM volatile fatty acids in the non-treated control and acid alone treatments, respectively. These concentrations are similar to the increase in concentration of volatile fatty acids of sterile compared to non-sterile acidified liquid hog manure treatments (Fig. 4). 4. Discussion In this study we investigated whether an increase in opportunistic nematodes, following addition of liquid hog manure to control Pratylenchus spp., was a direct result of addition of readily available decomposable materials or indirect effects. Indirect effects being first the killing of soil organisms followed by proliferation of opportunistic organisms or the lack of ‘‘topdown’’ predation to control numbers of opportunistic nematodes. The decline in total numbers of nematodes following application of liquid hog manure and acidified liquid hog manure was most particular to plant-parasitic and Pratylenchus spp. nematodes. The number of plant-parasitic nematodes declined over the first three to seven days following application of liquid hog manure and acidified liquid hog manure. By the end of the experiment (28 days), numbers of plant-parasitic nematodes were not different between manure and the non-treated control and acid alone treatments. That could be attributed to the dependence of plantparasitic nematodes on plant hosts; in the absence of hosts, the nematodes starved (Viaene et al., 2006). Numbers of Pratylenchus spp. did rebound from initially depressed levels following acidified and non-acidified liquid hog manure by week 1 and 2 before declining again to the end of the experiment. This increase observed in number of Pratylenchus spp. in the non-acidified and acidified liquid hog manure treatments is possibly related to the hatching of eggs from gravid females added during setup of the experiment. The optimum pH for P. penetrans reproduction, and in turn egg hatching, is 5.2–6.4 (Morgan and MacLean, 1968; Willis, 1972). The pH of soil receiving these treatments lay within this optimum range for P. penetrans reproduction. The decline in total number of nematodes was greater with increasing liquid hog manure concentration and greatest for the liquid hog manure 15% (v v 1 soil water) rate. The decline in nematodes can be attributed to the presence of liquid hog manure as nematode numbers did not decline to the same extent in the non-treated control or acid alone treatments. The findings are consistent with volatile fatty acids being lethal to some soil-borne pathogens and pests (Tenuta et al., 2002; McElderry et al., 2005; Xiao et al., 2007; Mahran et al., 2008a). Volatile fatty acids in liquid hog manure, with the rates and at the pH used in this experiment, did not seem to act like a broadspectrum pesticide in eliminating all nematodes and causing a ‘‘biological vacuum’’ (Yeates et al., 1991; Wang et al., 2006). Total nematode numbers increased rapidly at week 1 after liquid hog manure and acidified liquid hog manure application, corresponding to loss of volatile fatty acids from soil. Opportunistic c–p 1 and
c–p 2 nematodes were most responsible for the increase in total nematode numbers in soil. Their increase in relation to acidified and non-acidified liquid hog manure treatment was likely in response to enrichment of the microbial communities in the soil environment by readily degradable compounds in liquid hog manure, including volatile fatty acids, and perhaps the mortality of some soil organisms. These enrichment-opportunist nematodes have increased in soil rapidly following the addition of decomposable materials or availability of organic resources from mortality of organisms (Ferris and Bongers, 2006). An increase in nematodes of c–p 1 indicates stimulation by ‘‘bottom-up’’ processes resulting from an increase in microbial biomass for feeding by enrichment-opportunistic nematodes. In contrast, number of higher trophic nematodes (c–p 3–5) did not change. Nematode taxa at different trophic and life-history groups responded differently to the application of acidified and nonacidified liquid hog manure. By week 4, opportunistic c–p 1 nematodes of the genera Diploscapter and Mesorhabditis were dominant in soil and the levels of the genus Monhystera declined. Bacterial-feeding c–p 2 nematodes of the genera Acrobeles and Chiloplacus increased in soil while individuals of the genus Plectus were not detected in soil in liquid hog manure and acidified liquid hog manure 10% and 15% treatments. Wilsonema generally was not affected by the treatments. Taxa of the persister nematodes, c–p 3–5, also varied in their response to the treatments. The number of the bacterial-feeding nematodes of the genus Ethmolaimus increased in soil in response to liquid hog manure treatments but the number of nematodes of the genus Tylencholaimus declined. The survival of higher trophic nematodes classified in colonizer–persister groups, c–p 3–5, was not significantly different between acidified and non-acidified liquid hog manure treatments by the end of the experiment. This observation is contrary to expectation as these nematodes have been shown to be more sensitive to different stressors such as: metals (Korthals et al., 1996, 2000), acidification (Ruess et al., 1996); nematicides (Smolik, 1983), and N compounds (Tenuta and Ferris, 2004). Perhaps the explanation lies in the short (three days) persistence of volatile fatty acids in soil. Tolerance to stress of some persister nematodes (carnivores and omnivores) has been observed elsewhere. In a field study, omnivore numbers increased in soil at week 4 following incorporation of the plant-parasitic-suppressive green manure, sunn hemp (Crotalaria juncea L.), into soil (Wang et al., 2004). Alternatively, it is possible that soil handling and preparation for microcosms could have killed higher c–p nematodes, sensitive to physical disturbance (Fiscus and Neher, 2002; Rahman et al., 2007), leaving more tolerant taxa. The majority of volatile fatty acids added to soil in liquid hog manure were recovered and thus sorption of volatile fatty acids to soil seems unimportant. Water was used as an extractant and thus only soluble volatile fatty acids are recovered and measured. Acetic acid, the major volatile fatty acid in liquid hog manure, has been shown to be sorbed to marine sediments (Shiba et al., 2001). However, in the soil used here, sorption does not seem to have been appreciable. Microbial degradation seems to have caused loss of volatile fatty acids within days of manure application. Various microorganisms are able to degrade volatile fatty acids in liquid hog manure and use them as a source of carbon and energy (Zhu, 2000). While some bacteria such as Acinetobacter calcoaceticus (Beijerinck) Bouvet and Grimont, Alcaligenes faecalis Castellani and Chalmers, and Arthrobacter flavescens Lochhead, are capable of degrading all types of volatile fatty acids in liquid hog manure (Bourque et al., 1987; Jolicoeur and Morin, 1987), others such as Corynebacterium glutamicum (Kinoshita) Abe and Micrococcus spp. can only degrade acetic and propionic acids which are
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
major volatile fatty acids in liquid hog manure (Bourque et al., 1987). Microbial biomass, enzyme activities and microbial respiration, increase upon application of anaerobically stored liquid animal manures to soil (Paul and Beauchamp, 1996; Zaman et al., 1999). Liquid animal manures to field plots increased microbial respiration for the week following application presumably due to the rapid oxidation of volatile fatty acids present in the slurry (Tenuta et al., 2000; Chantigny et al., 2001). Decomposition of liquid hog manure in field plots has been rapid; one-half of the total annual CO2 emissions from soil occurring within one week of application (Rochette et al., 2000). In conclusion, the increase in opportunistic nematodes in soil following application of liquid hog manure and acidified liquid hog manure was in response to enrichment of the soil environment likely resulting from readily decomposable materials from the manure and less so from mortality of some organisms. The large increase in abundance of opportunistic nematodes four weeks following application of the manure treatments, with no corresponding increase in high c–p nematodes, resulted in an apparently poorly structured food web indicating the lack of higher trophic links. The decrease in structure index was due to increase in numbers of c–p 2 numbers and not from decrease in number of c–p 3–5 nematodes. Why acidified liquid hog manure is as an effective method to control plant-parasitic nematodes including Pratylenchus spp. without having the same effect on other trophic guilds of nematode is worthy of further investigation. However, controlling plant-parasitic nematodes without killing higher trophic organisms is desirable in the promotion of soil health. Acknowledgements We thank Mervin Bilous and Oscar Molina for technical assistance and two anonymous reviewers and Howard Ferris for comments to previous versions of this manuscript. This research was funded by the Manitoba Agricultural Research and Development Initiative, Canada Research Chair Program in Applied Soil Ecology, and the Natural Sciences and Engineering Research Council of Canada Discovery Grant Program. References Al-Banna, L., Ploeg, A.T., Williamson, V.M., Kaloshian, I., 2004. Discrimination of six Pratylenchus species using PCR and species-specific primers. J. Nematol. 36, 142–146. Bongers, T., 1994. De nematoden van Nederland. Pirota Schoorl, Bibliotheek uitgave KNNV, Uitgeverij, Utrecht, Netherlands, 408 pp. Bongers, T., 1990. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia 83, 14–19. Bongers, T., Ferris, H., 1999. Nematode community structure as a bioindicator in environmental monitoring. Trends Ecol. Evol. 14, 224–228. Bourque, D., Bisaillon, J., Beaudet, R., Sylvestre, M., Ishaque, M., Morin, A., 1987. Microbiological degradation of malodorous substances of swine waste under aerobic conditions. Appl. Environ. Microbiol. 53, 137–141. Cassel, D.K., Nielsen, D.R., 1986. Field capacity and available water capacity. In: Klute, A. (Ed.), Methods of Soil Analysis. Part 1. Physical and Mineralogical Properties Including Statistics of Measurement and Sampling. American Society of Agronomy, Madison, WI, pp. 901–926. Chantigny, M.H., Rochette, P., Angers, D.A., 2001. Short-term C and N dynamics in a soil amended with pig slurry and barley straw: a field experiment. Can. J. Soil Sci. 81, 131–137. Conn, K.L., Lazarovits, G., 1999. Impact of animal manures on Verticillium wilt, potato scab, and soil microbial populations. Can. J. Plant Pathol. 21, 81–92. Conn, K.L., Tenuta, M., Lazarovits, G., 2005. Liquid swine manure can kill Verticillium dahliae microsclerotia in soil by volatile fatty acid, nitrous acid, and ammonia toxicity. Phytopathology 95, 28–35. Dewes, T., Hunsche, E., 1998. Composition and microbial degradability in the soil of farmyard manure from ecologically-managed farms. Biol. Agric. Hortic. 16, 251–268. Ferris, H., Bongers, T., 2006. Nematode indicators of organic enrichment. J. Nematol. 38, 3–12.
81
Ferris, H., Bongers, T., De Goede, R.G.M., 2001. A framework for soil food web diagnostics: extension of the nematode faunal analysis concept. Appl. Soil Ecol. 18, 13–29. Ferris, H., Matute, M.M., 2003. Structural and functional succession in the nematode fauna of a soil food web. Appl. Soil Ecol. 23, 93–110. Fiscus, D.A., Neher, D.A., 2002. Distinguishing nematode genera based on relative sensitivity to physical and chemical disturbances. Ecol. Appl. 12, 565–575. Forge, T., Tenuta, M., 2008. Indicators of soil food web properties. In: Carter, M.R., Gregorich, E.D. (Eds.), Soil Sampling and Methods of Analysis. 2nd ed. Canadian Society of Soil Science, CRC Press, Boca Raton, FL, USA, pp. 577– 588. Haydock, P.P.J., Woods, S.R., Grove, I.G., Hare, M.C., 2006. Chemical control of nematodes. In: Perry, R.N., Moens, M. (Eds.), Plant Nematology. CAB International, Wallingford, UK, pp. 392–410. Humason, G.L., 1972. Animal Tissue Techniques, 3rd ed. W.H. Freeman and Company, San Francisco, pp. 641. Ingham, R.E., 1994. Nematodes. In: Weaver, R.W., Angle, S., Bottomley, P., Bezdicek, B., Smith, S., Tabatabai, A., Wollum, A. (Eds.), Methods of Soil Analysis. Part 2. Microbiological and Biochemical Properties no. 5. Soil Society of America, Madison, WI, USA, pp. 459–490. Jolicoeur, P., Morin, A., 1987. Isolation of Acinetobacter calcoaceticus strains degrading the volatile fatty acids of swine wastes. Biol. Wastes 19, 133–140. Jothi, G., Pugalendhi, S., Poornima, K., Rajendran, G., 2003. Management of root-knot nematode in tomato Lycopersicon esculentum, Mill., with biogas slurry. Bioresour. Technol. 89, 169–170. Kirchmann, H., Lundvall, A., 1993. Relationship between N immobilization and volatile fatty acids in soil after application of pig and cattle slurry. Biol. Fertil. Soils 15, 161–164. Korthals, G.W., Van de Ende, A., Van Megen, H., Lexmond, T.M., Kammenga, J.E., Bongers, T., 1996. Short-term effects of cadmium, copper, nickel, and zinc on soil nematodes from different feeding and life-history strategy groups. Appl. Soil Ecol. 4, 107–117. Korthals, G.W., Bongers, M., Fokkema, A., Dueck, T.A., Lexmond, T.M., 2000. Joint toxicity of copper and zinc to a terrestrial nematode community in an acid sandy soil. Ecotoxicology 9, 219–228. Mahran, A., Tenuta, A., Hanson, M.L., Daayf, F., 2008a. Mortality of Pratylenchus penetrans by volatile fatty acids from liquid hog manure. J. Nematol. 40, 119– 126. Mahran, A., Conn, K.L., Tenuta, M., Lazarovits, G., Daayf, F., 2008b. Effectiveness of liquid hog manure and acidification to kill Pratylenchus spp. in soil. J. Nematol. 40, 266–275. McElderry, C.F., Browning, M., Amador, J.A., 2005. Effect of short-chain fatty acids and soil atmosphere on Tylenchorhynchus spp. J. Nematol. 37, 71–77. Min, Y.Y., Sato, E., Shirakashi, T., Wada, S., Toyota, K., Watanabe, A., 2007. Suppressive effect of anaerobically digested slurry on the root-lesion nematode Pratylenchus penetrans and its potential mechanisms. Jpn. J. Nematol. 37, 93–100. Morgan, G.T., MacLean, A.A., 1968. Influence of soil pH on an introduced population of Pratylenchus penetrans. Nematologica 14, 311–312. Nahar, M.S., Grewal, P.S., Miller, S.A., 2006. Differential effects of raw and composted manure on nematode community, and its indicative value for soil microbial, physical and chemical properties. Appl. Soil Ecol. 34, 140–151. Paul, J.W., Beauchamp, E.G., 1996. Soil microbial biomass C, N mineralization and N uptake by corn in dairy cattle slurry and urea-amended soils. Can. J. Soil Sci. 76, 469–472. Rahman, L., Chan, K.Y., Heenan, D.P., 2007. Impact of tillage, stubble management and crop rotation on nematode populations in a long-term field experiment. Soil Till. Res. 95, 110–119. Rochette, P., Angers, D.A., Cote, D., 2000. Soil carbon and nitrogen dynamics following application of pig slurry for the 19th consecutive year. I. Carbon dioxide fluxes and microbial biomass carbon. Soil Sci. Soc. Am. J. 64, 1389–1395. Royer, I., Angers, D.A., Chantigny, M.H., 2007. Dissolved organic carbon in runoff and tile-drain water under corn and forage fertilized with hog manure. J. Environ. Qual. 36, 855–863. Rozman, K.K., Doull, J., 2000. Dose and time as variables of toxicity. Toxicology 144, 169–178. Ruess, L., Sandbach, P., Cudlin, P., Dighton, J., Crossley, A., 1996. Acid deposition in a spruce forest soil: effects on nematodes, mycorrhizas, and fungal biomass. Pedobiologia 40, 51–66. Shiba, T., Harada, H., Furushita, M., Maeda, T., 2001. Adsorption of acetate to coastal sediment particles. J. Natl. Fish. Univ. (Jpn) 49, 117–121. Smolik, J.D., 1983. Effect of nematicide treatments on non-target nematode populations associated with corn. Plant Dis. 67, 28–31. Sorensen, P., 1998. Carbon mineralization, nitrogen immobilization and pH change in soil after adding volatile fatty acids. Eur. J. Soil Sci. 49, 457–462. Tenuta, M., Bergstrom, D.W., Beauchamp, E.G., 2000. Denitrifying enzyme activity and carbon availability for denitrification following manure application. Comm. Soil Sci. Plant Anal. 31, 861–876. Tenuta, M., Conn, K.L., Lazarovits, G., 2002. Volatile fatty acids in liquid swine manure can kill microsclerotia of Verticillium dahliae. Phytopathology 92, 548– 552. Tenuta, M., Ferris, H., 2004. Sensitivity of nematode life-history groups to ions and osmotic tensions of nitrogenous solutions. J. Nematol. 36, 85–94. Timper, P., Newton, G.L., Johnson, A.W., Gascho, G.J., 2004. Nematode densities in year-round forage production systems utilizing manure fertilization. Nematropica 34, 219–227.
82
A. Mahran et al. / Applied Soil Ecology 43 (2009) 75–82
Valocka, B., Dubinsky, P., Papajova, I., Sabova, M., 2000. Effect of anaerobically digested pig slurry from lagoon on soil and plant nematode communities in experimental conditions. Helminthologia 37, 53–57. Viaene, N., Coyne, D.L., Kerry, B.R., 2006. Biological and cultural management. In: Perry, R.N., Moens, M. (Eds.), Plant Nematology. CAB International, Wallingford, UK, pp. 346–369. Wang, K.H., McSorley, R., Marshall, A.J., Gallaher, R.N., 2004. Nematode community changes associated with decomposition of Crotalaria juncea amendment in litterbags. Appl. Soil Ecol. 27, 31–45. Wang, K.H., McSorley, R., Kokalis-Burelle, N., 2006. Effects of cover cropping, solarization, and soil fumigation on nematode communities. Plant Soil 286, 229–243. Willis, C.B., 1972. Effects of soil pH on reproduction of Pratylenchus penetrans and forage yield of alfalfa. J. Nematol. 4, 291–295.
Xiao, J., Zhu, J., Chen, S., Ruan, W., Miller, C., 2007. A novel use of anaerobically digested liquid swine manure to potentially control soybean cyst nematode. J. Environ. Sci. Health 42, 749–757. Xiao, J., Chen, S., Zhu, J., Ruan, W., 2008. Effect of liquid swine manure on hatch and viability of Heterodera glycines. J. Nematol. 40, 152–160. Yeates, G.W., Bamforth, S.S., Ross, D.J., Tate, K.R., Sparling, G.P., 1991. Recolonization of methyl bromide sterilized soils under four different field conditions. Biol. Fertil. Soils 11, 181–189. Zaman, M., Di, H.J., Cameron, K.C., Frampton, C.M., 1999. Gross nitrogen mineralization and nitrification rates and their relationships to enzyme activities and the soil microbial biomass in soils treated with dairy shed effluent and ammonium fertilizer at different water potentials. Biol. Fertil. Soils 29, 178–186. Zhu, J., 2000. A review of microbiology in swine manure odor control. Agric. Ecosys. Environ. 78, 93–106.