The use of liquid swine manure for the control of potato cyst nematode through soil disinfestation in laboratory conditions

Crop Protection 49 (2013) 1e7

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The use of liquid swine manure for the control of potato cyst nematode through soil disinfestation in laboratory conditions J. López-Robles a, *, C. Olalla a, C. Rad a, M.A. Díez-Rojo b, J.A. López-Pérez b, A. Bello c, R. Rodríguez-Kábana d a

Área de Edafología y Química Agrícola, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Bañuelos s/n, 09001 Burgos, Spain Centro Agrario Marchamalo, JCCM Camino San Martín s/n, 19180 Marchamalo, Guadalajara, Spain Sociedad Española de Agricultura Ecológica (SEAE), Spain d Department of Entomology and Plant Pathology, Auburn University, AL 36849, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 March 2012 Received in revised form 21 December 2012 Accepted 12 March 2013

In this laboratory study the effect of liquid swine manure (LSM) on Globodera rostochiensis was evaluated. The LSM was applied to 500 g nematode-infested soil at 0.4, 2.0, 4.0, and 6.0% (v/w), equivalent to 31, 155, 310 and 465 kg of N ha
1, in closed and open containers (microcosms sealed (S) and open (O)). The results showed that LSM in closed containers enriched the volatile fatty acids (VFAs) through partial anaerobic incubation process. Bacterial- and fungal-feeding nematodes predominated, while the least opportunistic groups had a very low occurrence. The LSM in (S) significantly decreased cyst nematode populations and percent egg hatching in contrast with opened ones (O), increasing some chemical parameters related to soil fertility. Hatching tests showed that individual VFA vary in their lethality to G. rostochiensis. Acetic and propionic acids were the most toxic, reducing hatching most effectively and irreversibly. Results suggest that soil disinfestation on laboratory tests with LSM, is an efficient way for the reduction of G. rostochiensis populations in acidic soils, due to the VFAs nematicidal action. To address management alternatives, field studies would be needed that compared these materials to the current management practices, and demonstrated that they are competitive in the field. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Biodisinfestation Globodera rostochiensis Integrated crop management Organic amendments Volatile fatty acids

1. Introduction The potato cyst nematode Globodera rostochiensis (Wollenweber) Behrens is one of the most important pests of the potato (Solanum tuberosum L.) crop in cool-temperate areas. This species is a quarantine pest (A2 list: No. 125) for the European Plant Protection Organization (EPPO) (OEPP/EPPO, 1981) with a particular Council Directive 2007/33/EC. The incorporation of organic matter (OM) into soils has been used since the beginning of agriculture, with beneficial effects on soil physical and chemical characteristics, increasing fertility and contributing to pathogen management, and consequently improvement in crop growth (García Álvarez et al., 2004). The various types of organic amendments applied could be grouped as ‘on site’ produced amendments (green manures, cover crops) or ‘exogenous’ amendments (slurries, biosolids, animal manure, compost) with typical application rates of 10e40 t ha
1 (Thoden et al., 2011). Organic amendments offer an alternative or

* Corresponding author. Tel.: þ34 947 258 811; fax: þ34 947 258 831. E-mail address: [email protected] (J. López-Robles). 0261-2194/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cropro.2013.03.004

additional control tactic to chemical or cultural control of nematode pathogens on agricultural crops. Some studies indicate that composts from different organic and inorganic material reduced the population of plant parasitic nematodes and supported the beneficial free-living nematodes (Bello et al., 2008). Using LSM as a soil amendment in agriculture is a most appropriate means for decomposing it and reduces the need for chemical fertilizers while lowering costs and avoiding pollution in some areas (Jacobs, 1989). Suspension of the registration of some of the more hazardous nematicides has emphasized the need for new methods to control nematodes (Akhtar and Malik, 2000). Biofumigation or biodisinfestation has been proposed as a valid alternative to the use of agrochemicals for the control of plant pests (Methyl Bromide Technical Options Committee, 2007). Biodisinfestation is a broader concept which aims to harmonize the available non-chemical alternatives with the production system and other agroecological criteria. This concept promotes the use of local resources to minimize adverse impacts on the environment and to reduce transport cost. The main difference between this technique and the traditional OM application is the state of the OM used. The OM must be easily decomposable, which is not the case

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with stabilized OM (composts or ‘mature’ manures) commonly applied as organic amendments. It is also important to retain the gases produced during the biodecomposition process for at least two weeks; the effect of the gases is often biostatic and it is necessary to extend their action on pathogens (Piedra-Buena et al., 2006). Volatile fatty acids (VFAs) and ammonium (NHþ 4 ) are the two major constituents in swine manure, reported to be biocidal to pathogens (Conn and Lazarovits, 2000). The aim of the present work was to evaluate the effect of LSM on hatching of G. rostochiensis on nematode communities, and the role of VFAs. This information will contribute to the development of alternatives for the management of G. rostochiensis populations, taking advantage of the OM generated by swine production, and to resolve problems caused by the accumulation and potential contamination effect of these organic wastes, and significantly reduce the need for pesticides, fertilizers and other inputs. 2. Materials and methods 2.1. Soil collection and analysis The trial was carried out with a Calcic Luvisol (FAO, 1998) collected in a potato (S. tuberosum L.) field near the town of Salamanca, Spain, with a sandy loam texture (67% sand, 25% silt, 8% clay, 0.8% organic matter, pH 5.04, total nitrogen 0.137%, C/N ratio 10.9 and electric conductivity 0.460 dS m
1), naturally infested with the nematode G. rostochiensis. The soil was collected and taken to the laboratory for nematode identification. Multiplex polymerase chain reaction was used to identify the potato cyst nematodes in soil samples using primers (ITS5, PITSr3 and PITSp4), as described by Bulman and Marshall (1997). ITS products cloned into the ABI PRISMÒ 3100 Genetic Analyzer (Applied Biosystems) were subjected to a database search using BLAST (NCBI, National Centre for Biotechnology Information, to confirm the diagnosis (http://www. ncbi.nlm.nih.gov/)). A susceptible potato cultivar Desirée was grown in greenhouse on the field soil to increase the population of the potato cyst nematode. After three months, the plants were discarded and the soil sieved (2 mm mesh) and stored at 4  C. Prior to use, soils were air-dried for 1e2 days to reduce their moisture content to allow addition of specific amounts of water, or LSM. Gravimetric water content of soil samples was determined after soil drying at 105  C for 24 h. Air-dried soils were stored at 4  C. One day prior to set up of an experiment and the quantity of soil needed was removed from cold storage and placed at room temperature. 2.2. Manure collection and analysis The LSM was collected from an earthen storage lagoon at a commercial finisher hog farm in south eastern Burgos (Spain). The manure was centrifuged (3400g, 10 min) to remove particulates and analyzed according to the methods proposed by the FAO (Vermes, 1980). An aliquot of the supernatant was analyzed for its main VFAs constituents (C2eC6 including isomers) using chemical suppression ion exclusion chromatography and conductivity detection (Model 100; Dionex Corp., Sunnyvale, CA). The chromatograph was equipped with an IonPac ICE-AS6 analytical column and AMMS ICE II chemical suppressor (Dionex Corp.) according to Tenuta et al. (2002), while the remaining supernatant was frozen at
20  C for further use. Selected characteristics of the manure are reported in Table 1.

Table 1 Liquid swine manure composition (mean values of four replicates). Parameter

Mean (n ¼ 4)

Conductivity (dS m
1) Density (kg m
3) pH (1:2.5) Ash (g kg
1) Dry matter (g kg
1) Organic matter (g kg
1)
1 ) NHþ 4 -N (g kg Inorg-N (g kg
1)
1 Org-N (g kg ) Total-N (g kg
1) Inorg-P (g kg
1) Org-P (g kg
1) Total-P (g kg
1) C/N Tot (g kg
1) C/N Org (g kg
1) K (g kg
1) Mg (g kg
1) Ca (g kg
1) Na (g kg
1) Zn (mg kg
1) Cu (mg kg
1) Pb (mg kg
1) Mo (mg kg
1) Al (mg kg
1) Mn (mg kg
1) Fe (mg kg
1) VFAs Acetic (mM) Propionic (mM) n-Butyric (mM) Isobutyric (mM) Isovaleric (mM) n-Caproic (mM) n-Valeric (mM) Total (mM)

10.77 1250 7.33 8.98 36.13 27.03 1.42 2.47 0.70 3.07 0.67 0.11 0.78 4.56 28.55 0.74 0.23 1.21 0.23 24.4 13.5 0.31 0.36 36.80 12.50 81.50 190 51 54 23 13 9 9 349

(8.0 cm day  10.0 cm h). Different concentrations of liquid-fraction of manure were applied to microcosms to provide the following treatments: liquid swine manure (LSM) 0.4, 2.0, 4.0 and 6.0% (volume/mass soil). Following application, the soil gravimetric moisture content was adjusted to 100% of field capacity using dH2O. The treatments were equivalent to 10,000; 50,000; 100,000 and 150,000 L ha
1 (31, 155, 310 and 465 kg of N ha
1), assuming a soil bulk density of 1250 kg m
3 and a typical field application depth of (0.2 m). Control microcosms received only dH2O to 100% of field capacity. The retention of gasses during incubation was assessed by doubling the number of plastic pots; with one half of the pots closed by heat sealing (S) to retain the gasses, and the other half open (O). Microcosms were arranged in a completely randomized design with eight replicates per treatment and type of cover. All pots were incubated (Iso-temp Incubator 304; Fisher Scientific Canada Ltd.) in the dark at 30  C for 30 days and the experiment was terminated. 2.4. VFAs in soil Immediately after LSM application and at the end of the incubation period, VFAs were quantified in soil samples by shaking 5 g of moist soil with 15 mL of cold water (4  C) for 30 min in 50 ml centrifuge tubes. The extracts were centrifuged (16,000g for 10 min), filtered at 0.45 mm and stored frozen until analyzed by gas chromatography as described by Chantigny et al. (2002).

2.3. Microcosm

2.5. Effect of individual VFAs on G. rostochiensis egg hatching

Soil microcosms were prepared by adding 500 g dry weight equivalent soil to 500 cm3 polyethylene specimen storage pots

Bioassays to determine the sensitivity of G. rostochiensis to individual VFAs present in the LSM were conducted. Stock solutions

J. López-Robles et al. / Crop Protection 49 (2013) 1e7

of VFAs (acetic, propionic, n-butyric, isobutyric, isovaleric, n-caproic and n-valeric) were prepared in a citric acideNaOH buffer solution at pH 4.5 with a concentration as in the LSM collected. A pH of 4.5 was used because at this pH the majority of the VFA will be in their toxic, non-ionized VFA forms (e.g., acetic acid instead of acetate). All stocks were stored at 5  C and standard solutions were prepared by serial dilutions to give a final concentration equal to those of the LSM treatments. To bioassay the toxicity of VFAs on nematode, eggs of G. rostochiensis were collected from cysts and crushed to release the eggs. The collected eggs were stored in tap water until use. Batches of 1000 egg were placed on fine nylon mesh in solid watch glasses containing 5 ml freshly prepared VFA solution covered and incubated at 28  C during 20 days. Distilled water was taken as control. After the time of incubation the egg masses were washed free of VFA solution and further hatching was allowed in distilled water for another 7 days to observe resumption of hatching. Each treatment was replicated twenty times. 2.6. Hatch percentage and mortality of G. rostochiensis A 100 g soil subsample was taken from each pot after incubation period and cysts nematodes were extracted by the flotation method (Fenwick, 1940). For each replicate, cyst were placed in a well of 24well tissue culture plates (ten cysts per well), 2 ml of a solution 4 mM ZnCl2 (a hatching stimulant) was added to the tissue culture plates and incubated at 22  C. The number of hatched second-stage juveniles (J2) was counted every 24 h, during 21 days. The hatched J2 were removed from the well tissue culture plates, along the successive counts. After the last count, the total of hatched J2 was determined and, to calculate the cumulative hatching percentage, the cysts were crushed and the total egg content was determined. To determine the percentage egg mortality another 100 g soil subsample was taken from each pot, and cysts nematodes were extracted by the same method. The cysts were then crushed with an Eppendorf homogenizer to liberate the contents, the egg suspension was centrifuged and the supernatant removed. The eggs were resuspended in 400 ml 0.05% (w/v) aqueous Meldola’s blue solution (Shepherd, 1962) and incubated for 5 days at 20  C. The solution was then removed by centrifugation and the eggs were resuspended in 400 ml water for 24 h. The percentage of mortality (non-viable) eggs was estimated by removing an aliquot (20 ml volume) from each sample and counting the numbers of stained (non-viable) and non-stained (viable) eggs. Only completely stained dark blue eggs were considered non-viable (Devine and Jones, 2001). 2.7. Free-living nematodes To study the changes in nematode communities, nematodes were extracted from 100 g soil with sugar centrifugation, at the end of the incubation process, (Nombela and Bello, 1983). The specimens were heat-killed (65e70  C), preserved with 4% formalin and mounted in glycerine in permanent microscope slides, followed by observation of individuals using a stereomicroscope (Leica). The different nematodes were counted and assigned to five trophic groups: bacterial-feeders, fungal-feeders, phytophages, predators and omnivores (Yeates et al., 1993).

3

mechanically shaking the fresh samples for an hour at a solid:water ratio of 1:10 (w/v, dry weight basis). About 5.0 ml of each extract was pipetted into a sterilized plastic petri dish lined with a Whatman #2 filter paper. Ten cress seeds were evenly placed on the filter paper and incubated at 25  C in the dark for 48 h. Eight replicates were analyzed for each treatment. Treatments were evaluated by counting the number of germinated seeds, and measuring the length of roots. The responses were calculated by a germination index (GI) that was determined according to the following formula (Zucconi et al., 1981): Germination index (%) ¼ 100  Seed germination (%)  root length of treatment/Seed germination (%)  root length of control 2.9. Statistical analysis ANalysis Of VAriance (ANOVA) and Least Significant Difference (LSD) were applied to all data to evaluate differences between means. Differences at levels of P < 0.05 were considered significant. All statistical analysis was performed using SPSS 17.0. for Windows (Statistical Package for the Social Sciences 2007). 3. Results and discussion 3.1. VFAs in soil Acetic acid was the predominant VFA in LSM constituting 54.4% of the total VFAs in the LSM; n-butyric, propionic and isobutyric acids were present in lesser amounts at 15.5, 14.6 and 6.6% of the total VFAs present, respectively; isovaleric, n-caproic and n-valeric acids also were present in very low levels with each <4% of the total VFAs. Formic acid was not detected (Table 1). The concentrations of individual VFA can vary, according to the pigs’ diet, storage conditions (aerobic or anaerobic), and the number and the age of animals in the barn. However, the relative concentration of individual VFA is generally consistent, with acetic and n-butyric acids dominating, followed by propionic and isobutyric acids at intermediate concentrations and isovaleric, n-valeric and n-caproic acids at lower concentrations. These results agreed with those of Tenuta et al., (2002). VFAs in soils were measured immediately after LSM application and at the end of the incubation period. Total VFAs concentrations increased in sealed microcosms to final values of 2.55, 13.44, 21.01, and 33.74 (mM) for the LSM treatments of 0.4, 2.0, 4.0 and 6.0% respectively. In all cases, individual VFAs values also increased; on the other hand no VFAs were detected after 30 days in open microcosms (Table 2). These results were attributed to the production of VFAs by microbial fermenters under partially anaerobic conditions in soil microcosms and inhibition of its volatilization. A similar VFAs concentration in soil was measured in the manure digestion under anaerobic conditions (Xiao et al., 2007). A remarkable 77% increase in VFAs level in the anaerobically treated LSM was observed on around day 20 (Ndegwa et al., 2002). All this information, together with the results from this study, clearly demonstrates that LSM can be enriched with VFA through anaerobic digestion process.

2.8. Effects on soil fertility and phytotoxicity

3.2. Characterization of individual VFAs effect on G. rostochiensis hatch

The remaining soil of each replicate was dried at room temperature (20  2  C) for one week, and ground in a mortar for chemical analysis (MAPA, 1994). To determine the phytotoxicity index, a cress (Lepidium sativum L.) seed germination test was done. Seed germination and root length tests were carried out on water extracts by

In general, exposure of G. rostochiensis eggs to different VFA solutions suppressed nematode hatch (Fig. 1). At the lowest concentration tested, acetic and propionic acids were most effective. For acetic, propionic, n-butyric and isobutyric acids there was a significant and progressive decrease in eggs hatching in response to

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Table 2 Concentrations of VFAs in (mM) immediately after application (I) and after 30 days (O) opened microcosm, (S) sealed microcosm (mean values of eight replicates). VFAs

Control

Acetic Propionic n-Butyric Isobutyric Isovaleric n-Caproic n-Valeric Total

LSM 0.4%

LSM 2.0%

LSM 4.0%

LSM 6.0%

(I)

(O)

(S)

(I)

(O)

(S)

(I)

(O)

(S)

(I)

(O)

(S)

(I)

(O)

(S)

e e e e e e e e

e e e e e e e e

e e e e e e e e

0.76 0.20 0.21 0.09 0.05 0.03 0.03 1.37

e e e e e e e e

0.95 0.45 0.48 0.21 0.17 0.15 0.14 2.55

3.80 1.02 1.08 0.46 0.26 0.18 0.18 6.98

e e e e e e e e

4.05 2.11 2.17 1.54 1.32 1.15 1.10 13.44

7.60 2.04 2.16 0.92 0.52 0.36 0.36 13.96

e e e e e e e e

8.02 3.16 3.29 2.06 1.65 1.43 1.40 21.01

11.40 3.06 3.24 1.38 0.78 0.54 0.54 20.94

e e e e e e e e

13.70 5.21 5.52 3.41 2.06 1.98 1.86 33.74

e: not detected.

increased VFA concentration, with the lowest hatching resulting from the highest concentration of acetic acid. Isovaleric, n-caproic and n-valeric acids had no significant effect on hatching activity. Similar results were obtained for the root-knot nematode Meloidogyne incognita (Bansal and Bajaj 2003). The VFA have also been found to be responsible for the control of the microsclerotia of Verticillium dahliae (Tenuta et al., 2002) and the root-lesion nematode Pratylenchus penetrans (Min et al., 2007) that interact synergistically with V. dahliae, causing early dying of potato. However, individual VFA varied in their toxicity to P. penetrans and V. dahliae. While for Min et al., (2007) butyric acid and propionic acid were found to be the most lethal to P. penetrans, n-caproic acid was the most lethal to V. dahliae. In addition, individual VFA vary in their lethality to the two organisms. Accordingly, the effectiveness of the LSM in controlling plant pathogens will vary according to the target organism and the VFA profile in the LSM (Tenuta et al., 2002). Our results demonstrate that non-ionized forms of VFA are the dominant lethal agent in the LSM to the cyst nematode G. rostochiensis, under acidic conditions and that individual VFA present in the LSM do not appear to have synergistic interactions between them. 3.3. Hatch percentage and mortality of G. rostochiensis eggs In the hatch assay in open microcosm, there were no significant differences between the LSM treatments. In closed microcosms however, there was a progressive decrease in hatching values in response to increased LSM concentration, with a value near 0 for LSM 6.0% (Fig. 2). Results are consistent with those obtained for the soybean cyst nematode Heterodera glycines (Xiao et al., 2007, 2008). 100

80 70

Acetic

60

Propionic n-Butiryc

50

Isobutiric

40

Isovaleric

30

n-Caproic

20

n-Valeric

10

70

d

b

60

Percentage hatch

Percentage J2 hatched

90

In all cases, egg mortality rates in opened microcosm were less than 50%; a progressive mortality increase was recorded from 12.5% for the control, to a maximum of 43.5% in LSM 6.0%. In closed microcosms (S), nematode mortality ranged from 24.0% for the control to 56.2% in LSM 0.4% and 99.4% in LSM 6.0% (Fig. 3). There is a significant difference in the two trials between open and closed microcosm, which suggests the need to retain the gases generated during the disinfection process to obtain efficient nematode control. Fresh LSM normally contains a large amount of proteins that can be converted via anaerobic digestion to VFAs and ammonium (NHþ 4 ). The VFAs and ammonia (NH3) have nematicidal activities (Rodríguez-Kábana, 1986). At pH ranges of neutral or higher VFAs are ionized and exist as non-toxic salts (e.g., sodium acetate) and NHþ 4 starts to be converted into NH3. Tenuta et al. (2002) confirmed that the VFAs in the LSM can kill microsclerotia of V. dahliae in acid soils, because acidity promotes the protonation and generation of non-ionized forms of short-chain VFAs in LSM and thus, VFAs would only be expected to reduce pathogen survival in acidic soils. In spite of a number of studies showing that swine manure may have potential for control of microbial pathogens of plants (Conn and Lazarovits, 2000; Tenuta et al., 2002; Conn et al., 2005), there is no research on LSM for management of G. rostochiensis. Our studies provide more insight on the value of LSM for disease and pest management. The present study showed the toxicity of the LSM to G. rostochiensis eggs, and its inhibitory effect on hatching in closed microcosms. This effect was not related to direct oxygen deficit, but to production of products of anaerobic OM decomposition (Ponnamperuma, 1972). Due to the low soil pH in this study VFAs are probably the major compounds in the LSM responsible for the nematicidal activity, inhibiting G. rostochiensis egg hatch and killing eggs. Information regarding the sensitivity of the target plant pathogen to non-ionized forms of VFA and analysis of the VFA composition of the LSM is essential to predict the effectiveness of

50

a

CONTROL LSM 0.4% LSM 2.0% LSM4.0% LSM6.0%

Treatments Fig. 1. Percentage of juveniles of G. rostochiensis hatched per egg mass after incubation with individual VFA (mean values of twenty replicates). Error bars indicate standard errors.

a

a

CONTROL LSM 0.4%

40

LSM 2.0% 30 20

c

LSM 4.0% b

LSM 6.0% b

10

0

a

a

0 (S) Sealed

(O) Opened

Fig. 2. Hatch percentage of G. rostochiensis after LSM treatments (mean values of eight replicates). Different letters above bars for each treatment (O) and (S) indicate significant differences (P  0.05). Error bars indicate standard errors.

J. López-Robles et al. / Crop Protection 49 (2013) 1e7

e Percentage nematode mortality

100

d

90 c

80 70

b

CONTROL

60 d

50 40 30

d

LSM 0.4%

c

LSM 2.0%

b

a

LSM 4.0%

a

20

LSM 6.0%

10 0 (S) Sealed

(O) Opened

Fig. 3. Percentage G. rostochiensis mortality after LSM treatments (mean values of eight replicates). Different letters above bars for each treatment (O) and (S) indicate significant differences (P  0.05). Error bars indicate standard errors.

the LSM in controlling the target pathogen prior to manure application under field conditions (Mahran et al., 2009). 3.4. Free-living nematodes The main nematode trophic groups found in soil that are most relevant to this study are bacterial-feeders (Rhabditidae), fungalfeeders (Aphelenchidae), plant-feeders (Tylenchidae), and omnivores (Aporcelaimidae). Of these, the bacterial-feeders always appeared as the major group present, comprising at least 50% of the total nematode population, and at the other extreme there were the predators (Mononchidae), which were completely absent, plantfeeders and omnivores showed very low values of relative abundance (Fig. 4). In opened microcosm (O) bacterial- and fungal-feeders increased significantly as LSM dosages increased, with a ten-fold multiplication with LSM 4.0%, declining slightly in microcosms

5

receiving a 6.0% LSM dosage. In sealed microcosm (S) there was a similar evolution on bacteria- and fungal-feeders, with the highest nematode number in LSM 2.0%, declining sharply with increasing dose; however, the number of nematodes in all the trophic groups was ten-fold lower than in opened trials. By the end of the experiment (30 days), phytonematodes did not increase at all in response to treatments in either O or S experiments. This may be attributed to the dependence of phytonematodes on plant hosts; in the absence of hosts, the nematodes starved (Viaene et al., 2006). Compared to cep 1 (Rhabditidae) and cep 2 (Aphelenchidae) nematodes, numbers of cep 5 nematodes (Aporcelaimidae) were very low, constant in all treatments and without significant differences between treatments. The number of cep 5 nematodes was three-fold lower in close containers; the explanation to this difference could be the short persistence of volatile fatty acids in open pots. These results are contrary to expectation as these nematodes have been shown to be more sensitive to different stressors such as N compounds; it is possible that soil handling and preparation for microcosms could have killed higher cep nematodes, sensitive to physical disturbance, leaving more tolerant taxa (Mahran et al., 2009). Within this group, Aporcelaimellus obtusicaudatus (Bastian) was the only species in the soil tested, very tolerant to the N fertilization (Cobacho, 2003), therefore, this could be the reason in the survival of these cep 5 nematodes. This form of organic input is ultimately accompanied by a marked increase in bacterial-feeding nematodes and also by a similar increase in fungal-feeding nematodes. Bacterial-feeders are enrichment opportunists with a low cep-value (Bongers, 1990), and are indicative of organic nutrient inputs, especially benefited from the provision of fresh organic matter. Consequently, an increase of Rhabditidae, Panagrolaimidae or Diplogastridae, all bacterial-feeding nematode families favored by nutrient-enriched conditions, has been reported by several authors (Nahar et al., 2006; Yeates et al., 2006; Hu and Qi, 2010). Sometimes, a ten-fold multiplication of enrichment opportunists could ultimately be

(O) Opened

Nematodes / 100 g of soil

1600 1400 1200

Plant-feeders

1000

Fungal-feeders

800

Bacterial-feeders

600

Predators

400

Omnivores

200 0 CONTROL

LSM 0.4%

LSM 2.0%

LSM 4.0%

LSM 6.0%

Liquid hog m anure concentration

(S) Sealed Nematodes / 100 g of soil

140 120 100

Plant-feeders Fungal-feeders

80

Bacterial-feeders

60

Predators 40

Omnivores

20 0 CONTROL

LSM 0.4%

LSM 2.0%

LSM 4.0%

LSM 6.0%

Liquid hog manure concentration Fig. 4. Nematological trophic groups analysis after LSM treatments (mean values of eight replicates). (O) Open microcosm, (S) closed microcosm. Error bars indicate standard errors.

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Table 3 Soil chemical analysis after biodisinfectant treatment (mean values of eight replicates). Treatment

Total N %

Organic C %

P2O5a

Caa

Mga

Ka

Naa

pH

GI %

LSM 0.4% LSM 2.0% LSM 4.0% LSM 6.0% Control

0.152ab 0.154ab 0.161b 0.163b 0.137a

2.23b 2.35bc 2.68c 2.85d 2.09a

106a 109ab 109ab 111b 106a

584a 586a 595b 596b 583a

63.0a 63.1a 63.2a 63.5a 63.0a

183b 203c 240d 251e 165a

69.8a 69.5a 70.1a 70.3a 69.3a

5.04a 5.04a 5.05a 5.06a 5.04a

99d 90c 82b 71a 100d

Comparisons of means according to Least Significant Difference method. Numbers in a column followed by the same letter are not significantly different (P  0.05). a mg 100 g
1.

observed after addition of slurries even if initially, shortly after application, their numbers decreased due to nematicidal effects (Min et al., 2007; Mahran et al., 2009). 3.5. Effects on soil fertility and phytotoxicity Results obtained in the chemical analysis of soil are shown in Table 3; LSM resulted in a slight but significant increase in the N level, and more clearly in the organic C level, from 2.09% in control pots to 2.85% in LSM 6.0%. The K, P and Ca levels also increased proportionally as LSM dosages increased, with significant differences between the control and the LSM 6% pots. The levels of Na and Mg were not affected by the treatments. The pH increased slightly but without significant differences between the control and the LSM pots. In general terms, LSM has a beneficial effect on soil fertility, contributing to maintaining soil nutrient levels, therefore reducing the need for chemical fertilizers, and without negative effects on the following crops (Jacobs, 1989). All samples taken after the incubation period had GI values greater than 70%, which, according to Zucconi et al. (1981), indicates a phytotoxic-free extract. 4. General conclusion In conclusion, this study showed that using LSM to disinfest soil on laboratory tests, is an efficient way for the reduction of G. rostochiensis populations in acidic soils. In sealed microcosms we found a significant reduction of the pathogen population density; this effect was due to production of VFAs and inhibition of its volatilization. The manure anaerobically incubated increases the VFAs. Individual VFA vary in their lethality to G. rostochiensis. Acetic and propionic acids were the most toxic, reducing hatching most effectively and irreversibly. The LSM application as biodisinfestation material has additional advantages: it avoids organic residue accumulation and decreases contamination problems, To address management alternatives, field studies under different soil conditions would be needed that compared these materials to the current management practices, and demonstrated that they are competitive in the field. Acknowledgments To the members of the Department of Agroecology, CCMA-CSIC (Madrid, Spain). This work is a part of the Project CTM2006-07309 funded by the Ministry of Science and Technology of Spain and INCRECYT Contract (Fundación Parque Científico y Tecnológico de Albacete). References Akhtar, M., Malik, A., 2000. Roles of organic soil amendments and soil organisms in the biological control of plant-parasitic nematodes: a review. Bioresour. Technol. 74, 35e47.

Bansal, R.K., Bajaj, A., 2003. Effect of volatile fatty acids on embryogenesis and hatching of Meloidogine incognita eggs. Nematol. Medit. 31, 135e140. Bello, A., Porter, I., Díez-Rojo, M.A., Rodríguez-Kábana, R., 2008. Soil biodisinfection for the management of soil-borne pathogens and weeds. In: Third International Biofumigation Symposium, 21e25 July 2008, Canberra, Australia, 35 pp. Bongers, T., 1990. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia 83, 14e19. Bulman, S.R., Marshall, J.W., 1997. Differentiation of Australasian potato cyst nematode (PCN) populations using the polymerase chain reaction (PCR). N. Z. J. Crop Hortic. Sci. 25, 123e129. Chantigny, M.H., Angers, D.A., Rochette, P., 2002. Fate of carbon and nitrogen from animal manure and crop residues in wet and cold soils. Soil Biol. Biochem. 34, 509e517. Cobacho, S., 2003. Estudio de los nematodos depredadores (Orden Mononchida y Dorylaimida) en suelos de uso agrícola de España. Ph.D. Thesis. Faculty of Sciences, University of Córdoba, Spain, 459 pp. Conn, K.L., Lazarovits, G., 2000. Soil factors influencing the efficacy of liquid swine manure added to soil to kill Verticillium dahliae. Can. J. Plant Pathol. 22, 400e 406. 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, 28e35. Devine, K.J., Jones, P.W., 2001. Effects of hatching factors on potato cyst nematode hatch and in-egg mortality in soil and in vitro. Nematology 3, 65e74. FAO, 1998. World Reference Base for Soil Resources. World Soil Resource Reports 84. Food and Agricultural Organization of the United Nations, Rome, Italy. Fenwick, D.W., 1940. Methods for the recovery and counting of cysts of Heterodera schachtii from soil. J. Helminth. 18, 155e172. García Álvarez, A., Díez-Rojo, M.A., López-Pérez, J.A., Bello, A., 2004. Materia orgánica, biofumigación y manejo de organismos del suelo patógenos de vegetales. In: Labrador, J. (Ed.), Conocimientos, Técnicas y Productos para la Agricultura y la Ganadería Ecológica. SEAE, Valencia, pp. 71e76. Hu, C., Qi, Y., 2010. Abundance and diversity of soil nematodes as influenced by different types of organic manure. Helminthologia 47, 58e66. Jacobs, L.W., 1989. Utilizing manure is better than disposing of waste. Pigs-Misset 89, 2. Mahran, A., Tenuta, B., Lumactud, R.A., Daayf, F., 2009. Response of a soil nematode community to liquid hog manure and its acidification. Appl. Soil Ecol. 43, 75e82. MAPA, 1994. Métodos oficiales de análisis vol. 3. ed.. MAPA, Madrid, 662 pp. Methyl Bromide Technical Options Committee, 2007. 2006 MBTOC Assessment Report. UNEP, Nairobi, Kenya, 437 pp. 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, 93e 100. Nahar, M.S., Grewal, P.S., Miller, S.A., Stinner, D., Stinner, B.R., Kleinhenz, M.D., Wszelaki, A., Doohan, D., 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, 140e151. Ndegwa, P.M., Zhu, J., Luo, A., 2002. Effects of solids separation and time on the production of odorous compounds in stored pig slurry. Biosys. Eng. 81, 127e133. Nombela, G., Bello, A., 1983. Modificaciones al método de extracción de nematodos fitoparásitos por centrifugación en azúcar. Bol. Serv. Plagas 9, 183e189. OEPP/EPPO, 1981. Data sheets on quarantine organisms No. 125, Globodera rostochiensis. Bull. OEPP/EPPO 11 (1). Piedra-Buena, A., García-Álvarez, A., Díez-Rojo, M.A., Bello, A., 2006. Use of crop residues for the control of Meloidogyne incognita under laboratory conditions. Pest Manag. Sci. 62, 919e926. Ponnamperuma, F.N., 1972. The chemistry of submerged soils. Adv. Agron. 24, 29e96. Rodríguez-Kábana, R., 1986. Organic and inorganic nitrogen amendments to soil as nematode suppressants. J. Nematol. 18, 129e135. Shepherd, A.M., 1962. New Blue R. A stain that differentiates between living and dead nematodes. Nematologica 8, 201e208. Tenuta, M., Conn, K.L., Lazarovits, G., 2002. Volatile fatty acids in liquid swine manure can kill microsclerotia of Verticillium dahliae. Phytopathology 92, 548e 552. Thoden, T., Korthals, G.W., Termorshuizen, A.J., 2011. Organic amendments and their influences on plant-parasitic and free-living nematodes: a promising method for nematode management? Nematology 13, 133e153.

J. López-Robles et al. / Crop Protection 49 (2013) 1e7 Vermes, L., 1980. Recommended Analytical Methods for the First Priority Components of Liquid Manure. Information Bulletin from the FAO European Network on Animal Waste Utilization. Subnetwork 4, Budapest, pp. 63e84. 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. 346e369. 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 B 42, 749e757.

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Xiao, J., Chen, S., Zhu, J., Ruan, W., 2008. Effect of liquid swine manure on hatch and viability of Heterodera glycines. J. Nematol. 40, 152e160. Yeates, G.W., Wardle, D.A., Watson, R.N., 1993. Relationships between nematodes, soil microbial biomass and weed-management strategies in maize and asparagus cropping systems. Soil Biol. Biochem. 25, 869e876. Yeates, G.W., Speir, T.W., Taylor, M.D., Clucas, L., Schaik, A.P., 2006. Nematode responses to biosolids incorporation in five soil types. Biol. Fertil. Soils 42, 550e555. Zucconi, F., Pera, A., Forte, M., de Bertoldi, M., 1981. Evaluating toxicity of immature compost. BioCycle 22, 54e57.