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Pig slurry reduces the survival of Ralstonia solanacearum biovar 2 in soil A. Gorissen, L.S. van Overbeek, and J.D. van Elsas

Abstract: The effect of added pig slurry and solarization on the survival of Ralstonia solanacearum biovar 2 strain 1609 in soil was analysed in soil microcosms and field plots. In addition, the invasion of potato plants by R. solanacearum and the development of disease symptoms were determined, as measures of induced disease suppressiveness. In untreated soil, R. solanacearum showed slow population declines in both microcosms and the field from, initially, 106–107 to 103–104 CFU·(g dry soil)–1 in about 9 weeks. The suppressiveness assays of these untreated soils after this period revealed that most of the plants that were used developed wilting symptoms and (or) contained the pathogen in their lower stem parts, as shown by immunofluorescence colony staining and PCR. The addition of pig slurry resulted in a significantly lower population size of R. solanacearum as well as reduced numbers of infected and (or) diseased plants in the soil suppressiveness tests. On the other hand, solarization of soil also decreased R. solanacearum survival but did not enhance soil suppressiveness as measured by development of disease symptoms and (or) plant invasion after 9 weeks. Combined soil solarization and pig slurry addition showed an additive effect of both treatments. Healthy-looking plants, primarily from soils treated with pig slurry and solarization, incidentally revealed the latent presence of R. solanacearum in the lower stem parts. The mechanism behind the enhanced population declines and disease suppressiveness induced by pig slurry is unclear but shifts in community profiles were clearly discernible by PCR – denaturing gradient gel electrophoresis 9 weeks after pig slurry addition in the field experiment, indicating induced changes in the bacterial community structure. Key words: soil suppressiveness, organic amendment, solarization, DGGE analysis, immunofluorescence colony staining. Résumé : Nous avons analysé l’impact de l’ajout de lisier de porc et de la solarisation sur la survie dans le sol de la souche 1609 de Ralstonia solanacearum biovar 2, dans des microcosmes du sol et des placettes de champs. De plus, nous avons déterminé la capacité à supprimer les maladies induites en mesurant l’invasion des plants de pommes de terre par R. solanacearum et le développement des symptômes de maladies. Dans le sol non traité, la population de R. solanacearum a lentement décliné dans les microcosmes et dans le champ, passant au départ de 106–107 à 103–104 UFC par gramme de sol sec dans une intervalle d’environ 9 semaines. Les analyses des capacités suppressives de ces sols non traités à la suite de cette période ont révélé que la plupart des plants utilisés ont développé des symptômes de flétrissement et/ou contenaient des pathogènes dans les parties inférieures de leur tige, tel que vu par coloration immunofluorescente des colonies (CIF) et par PCR. L’ajout de lisier de porc a permis d’obtenir une taille de population de R. solanacearum significativement inférieure de même qu’un nombre inférieur de plantes infectées et/ou malades dans les tests de capacités suppressives des sols. En outre, la solarisation des sols a également diminué la survie de R. solanacearum mais n’a pas augmenté la capacité suppressive des sols tel que mesurée par le développement de symptômes de maladies et/ou par l’invasion des plantes après neuf semaines. La combinaison de la solarisation des sols et de l’ajout de lisier de porc a entraîné un effet additif sur les deux traitements. Des plants d’apparence saine, provenant principalement de sols traités avec du lisier de porc et avec la solarisation, ont incidemment révélé la présence latente de R. solanacearum dans les parties inférieures des tiges. Les mécanismes responsables du déclin accéléré des populations et de la suppression des maladies induits par le lisier de porc sont nébuleux, mais il fut possible de discerner dans l’expérience dans le champ des déplacements dans les profils des communautés par PCR-DGGE, neuf semaines après l’ajout de lisier de porc, ce qui signale des changements induits dans la structure des communautés bactériennes. Mots clés : capacité suppressive des sols, enrichissement organique, solarisation, analyse par DGGE, coloration immunofluorescente des colonies. [Traduit par la Rédaction]

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Received 23 June 2003. Revision received 30 March 2004. Accepted 26 April 2004. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 29 September 2004. A. Gorissen and L.S. van Overbeek.1 Crop and Production Ecology, Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands. J.D. van Elsas. University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. 1

Corresponding author (e-mail: [email protected]).

Can. J. Microbiol. 50: 587–593 (2004)

doi: 10.1139/W04-042

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Introduction Wilting disease in potato caused by Ralstonia solanacearum biovar 2 is an enormous risk for potato cropping in tropical and temperate climate regions (Hayward 1991; Janse et al. 1998; Prior et al. 1998). The increasing occurrence of this organism in cold regions has increased the fear that adaptation to lower temperatures (Hayward and Hartmann 1994; Elphinstone 1996; Poussier et al. 1999) threatens the supply of potato production in these regions, some of which represent major potato-growing areas. Thus, efforts have been made to develop and apply soil management methods that reduce the threat posed by R. solanacearum in soil (Schönfeld et al. 2003). Simple and cost-effective soil management strategies, such as addition of compost or manure (Hoitink and Fahy 1986; Hoitink and Boehm 1999; Schönfeld et al. 2003) or solarization, i.e., coverage of (sun-exposed) soil with plastic foil (Katan 1981; Stapleton 2000), have been shown to induce pathogen-suppressive conditions in the soil (Craft and Nelson 1996; Hoitink and Fahy 1986; Schönfeld et al. 2003). Other soil amendments that have been used against R. solanacearum are urea and calcium oxide; these showed variable results (Michel et al. 1997; Michel and Mew 1998). The suppressiveness of soils, defined as the capacity to reduce survival and activity of plant pathogens (van Bruggen and Semenov 2000), has been suggested to be related to a stimulated antagonistic microbial population induced by soil amendments (Dixon and Walsh 1998). On the other hand, the stimulation of a pathogen population by organic amendments to soil has also been shown to occur (Tuitert et al. 1998; Nasir et al. 2003). Abiotic factors such as soil texture, organic matter content, pH, ammonium, and nitrite may strongly affect the interactions between pathogens and antagonistic populations in soil (Hoitink and Fahy 1986; Michel and Mew 1998). Thus, as a result of the inherent complexity of the system, the mechanisms of enhanced disease suppression often remain unresolved. Whereas compost-like organic additions are currently reasonably well accepted as means to induce soil suppressiveness, another potentially promising and simple strategy, the addition of pig slurry, has been less explored. Pig slurry is known to bring about major changes in soil (Gótz and Smalla 1997). Moreover, there is a continuous overproduction of pig slurry in The Netherlands and other parts of northwest Europe, and it has been a challenge to look for useful applications of this by-product of pig husbandry. Solarization of the soil is another soil management strategy, whether or not in combination with organic matter additions (Katan 1987). This treatment may change the soil and rhizosphere microflora by stimulating microbial groups such as actinomycetes and fluorescent pseudomonads (Gamliel and Katan 1991), thus possibly evoking suppressiveness to soil pathogens. However, this method may also enhance the incidence of plant disease, as was reported for Fusarium wilt in banana by Nasir et al. (2003). In this study, we tested the effect of the addition of pig slurry, whether or not in combination with soil solarization, in microcosms as well as in field plots on the survival of R. solanacearum in soil. The fate of R. solanacearum was monitored using a combination of cultivation-based (immunofluorescence colony staining (IFC); van Vuurde and

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van der Wolf 1995; van der Wolf et al. 1998) and cultivationindependent techniques (specific PCR amplification from microbial community DNA; Schönfeld et al. 2003). In addition, soil suppressiveness to R. solanacearum activity was assessed in bioassays and changes in the bacterial microflora were recorded.

Materials and methods Bacterial strain Ralstonia solanacearum biovar 2 (race 3) strain 1609 was used in the soil microcosm and field experiments. This strain was isolated in 1995 from Solanum tuberosum L. ‘Bartina’ by the Dutch Plant Protection Service. Cultures were kept either in 20% glycerol at –80 °C for long-term storage or in pure water at room temperature. The strain was regularly tested for aggressiveness using tomato plants (Lycopersicon esculentum Mill. ‘Moneymaker’) and consistently caused bacterial wilt within about 2 weeks. Prior to use in the experiments, R. solanacearum was grown at 27 °C until the midexponential phase under rotational shaking at 280 rpm in 10% strength trypticase soy broth (TSB) (3 g·L–1) supplemented with 0.1% sucrose. The cells were harvested by centrifugation (7000g for 15 min) and suspended and diluted in sterile Millipore membrane filtered water. Soil and pig slurry Soil was collected at a noninfested area adjacent to an agricultural field near Hellendoorn (The Netherlands) in which an outbreak of bacterial wilt in potato had been recorded in 1997 (Schönfeld et al. 2003; van Elsas et al. 2000). Plots in the same field were also used for the field work. The soil was a loamy sand with 4.8% organic C and a pH-KCl of 5.2. For the microcosm experiments, soil was sieved (mesh size 5 mm) to remove large root and plant parts as well as stones before drying at room temperature to about 17% moisture (w/w) on a dry weight basis. Characteristics of the pig slurry used were as follows: dry matter 13.4%, total C 379 g·kg–1, total N 21.9 g·kg–1, NO3-N 104 CPU·(g stem tissue–1)). A theoretical disease index was then determined for each treatment, as described under Statistics.

1999; Muyzer et al. 1993). Briefly, gradients of 45–65% denaturants were used in an Ingeny (Goes, The Netherlands) DGGE apparatus with running time of 16 h at 100 V. After electrophoresis, gels were stained with SYBR gold and images were captured using a laboratory imaging system. The images were analysed using cluster analysis (UPGMA, Dice coefficient of similarity) of the Molecular Analyst (BioRad, Veenendaal, The Netherlands).

Soil DNA extraction Direct DNA extraction on the basis of harsh bead beating cell lysis was used on soil samples (1 g) obtained from all treatments (Smalla et al. 1993; van Elsas et al. 1997). The method included phenol and phenol–chloroform extractions, CsCl precipitation, and purification using the Wizard DNA spin column cleanup (Promega). The DNA extracts were checked for yield, molecular size, and purity (absence of brown humics and RNA from soil) of the DNA using electrophoretic separation in 0.8% agarose gels (Sambrook et al. 1989). The extracts were sufficiently pure to run PCR amplifications for specific detection of R. solanacearum as well as for profiling of the bacterial communities by PCR – denaturing gradient gel electrophoresis (DGGE). PCR for detection of R. solanacearum The R. solanacearum division 2 specific primers D2 and B described by Boudazin et al. (1999) were used for detection of R. solanacearum biovar 2 strain 1609. The PCR amplification was performed with AmpliTaq Stoffel fragments in the buffer supplied by the manufacturer (Applied Biosystems, Foster City, Calif.). The primers yielded PCR products of 650 bp with the specific target genomic DNA of R. solanacearum strain 1609. PCR was performed in a Peltier thermal cycler (MJ Research, Biozym, Landgraaf, The Netherlands) using a hot start (5 min at 95 °C) followed by 35 cycles of 20 s at 94 °C, 20 s at 66 °C, and 30 s at 72 °C and a final extension for 10 min at 72 °C. PCR–DGGE for fingerprinting of bacterial communities PCR–DGGE for fingerprinting of soil bacterial communities was performed as described previously (Gelsomino et al.

Statistics The experiment consisted of 2 approach levels (microcosm and field plot) each containing the following treatments: 2 R. solanacearum levels (±), 2 pig slurry levels (±), and 2 solarization levels (±). Each treatment combination had 3 replicates, resulting in 24 microcosms and 24 field plots. To assess differences between treatments, analysis of variance was carried out using Genstat-5, release 4.1 (Rothamsted Experimental Station, Harpenden, UK). A theoretical disease index was calculated as the average of the individual scores (1 = healthy, R. solanacearum not detected in lower stem part; 2 = healthy, latent presence of R. solanacearum in lower stem part; 3 = wilted) of 6 potato plants per treatment in the suppressiveness test for the microcosms and the field plots. Differences were considered significant when P values were lower than 0.05.

Results Survival of R. solanacearum strain 1609 in treated soils The population dynamics of R. solanacearum strain 1609 in soil was monitored by IFC, which specifically allows the detection of culturable cells (van Vuurde and van der Wolf 1995). The IFC assay of the control soils showed that both the soil and pig slurry used in the experiments were free of R. solanacerarum in (data not shown). The population dynamics of the introduced R. solanacerarum in the soil microcosms and field plots are shown in Figs. 1 and 2, respectively. In the soil microcosms and field plots, R. solanacearum declined progressively during the course of the experiment, with initial higher average decline rates in © 2004 NRC Canada

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Table 1. Suppressiveness of soils treated with pig slurry (ps), application of solarization (s), and pig slurry and solarization (ps-s) towards infection of potato plants by Ralstonia solanacearum biovar 2 strain 1609. % healthy plants Treatment

Disease index*

Microcosm (n = 6)

Field (n = 6)

Overall

Statistics†

Remarks

Untreated ps s ps-s

1.8 1.3 2.1 1.3

33 100 33 67

17 50 17 67

25 75 25 67

a b a b

Only a few plants with pathogen; latency Most plants infested by strain 1609 Latency

*Disease index based on the average of the individual scores (1 = healthy, R. solanacearum not detected in lower stem part; 2 = healthy, latent presence of R. solanacearum in lower stem part; 3 = wilted). † Different letters indicate significantly different overall values (LSD0.05 = 0.46).

the microcosms than in the field plots. The soil treatments had a similar declining effect in the microcosms and field plots. In the untreated controls of soil microcosms and field plots, the dynamics of R. solanacearum showed the lowest decline rates: in the microcosms, the counts fell from about log 6.8 to log 2.9 CFU·(g dry soil)–1 after 9 weeks and in the field plots from about log 6.5 to about log 3.0 CFU·(g dry soil)–1. Up to day 14, the declines in the treatments were similar to those in the controls. However, from 2 weeks onwards, a strong decline in the R. solanacearum IFC counts was observed in both microcosms and field plots on day 14 (P < 0.01), day 28 (P < 0.01), and day 62 (P < 0.02) in the soils treated with pig slurry as compared with the controls. Solarization also induced a significantly stronger decline on day 14 (P < 0.05), day 28 (P < 0.05), and day 62 (P < 0.01) than the declines observed in the controls. No interactions were observed between the pig slurry and solarization treatments. PCR with the R. solanacearum division 2 specific primers on the basis of total community DNA from soil showed that, with exception of the samples from pig slurry treated plots at day 62, 650-bp amplicons typical for R. solanacearum strain 1609 were obtained throughout the experiments from all treatments until the end of the experiments (data not shown). The uninoculated soil as well as the pig slurry used did not yield 650-bp amplicons. Suppressiveness of treated soils towards R. solanacearum strain 1609 At the end of the experiments, soils from the microcosms and field plots as well as control (uninoculated) soil were used to assess the suppressiveness towards R. solanacearum strain 1609. Plants grown in uninoculated soil did not show any disease symptoms, nor were R. solanacearum CFUs found in their stem parts. Overall, the disease indices (Table 1) obtained after inoculation showed that few of the potato plants that had grown in the untreated soils (25% for combined results of microcosms and field plots) as well as those grown in soils treated by solarization only (25%) remained healthy and had not been invaded by R. solanacearum, as evidenced via IFC and PCR. PCR was mainly used to check negative IFC results to confirm the presence–absence of nonculturable CFUs in the stems. In contrast, in both the microcosms and the field plots, the addition of pig slurry brought about a significantly enhanced disease-suppressive condition in the soil, as 75% and 67% of the test plants

grown in respectively pig slurry treated or pig slurry and solarization treated soils remained healthy and pathogen free (see Table 1). The remainder of these plants (25% and 33%, respectively) were also disease free but showed the presence of R. solanacearum in their lower stem parts.

Discussion This study clearly showed a strong negative effect of pig slurry on R. solanacearum strain 1609 survival in soil. In addition, solarization also reduced the abundance of the pathogen in soil. The combination of pig slurry amendment with solarization showed an additive effect (no interaction was observed). This combined treatment may be most promising for testing under a range of different conditions with respect to soil type and other abiotic factors. The effects of pig slurry and solarization were detectable in both the soil microcosms and the field plots and so might be robust in the light of the clear differences between both types of systems. Most importantly, the pig slurry treatment also lowered the invasion by R. solanacearum strain 1609 of potato plants growing in the soils after about 9 weeks. Both effects indicated that the suppressiveness of the soil towards the pathogen, in terms of limiting its survival and activity, was enhanced. Hence, in line with the previously reported effects of compost addition to the same soil (Schönfeld et al. 2003), pig slurry can also reduce the survival of R. solanacearum in soils. Manipulation of natural communities of antagonistic microorganisms in soil through organic amendments such as manure or compost thus may provide a potentially effective form of biological control of soil-borne plant pathogens like R. solanacearum (Nielsen and Todd 1945; Hoitink and Boehm 1999). Soil characteristics also have an important influence on the efficacy of soil amendments (Michel and Mew 2003), and the soil-specific fate of pig slurry should be further investigated. To enable prediction of the effects, the causative agent(s) in the slurry as well as its quality and stability are crucial. Pig slurry may be variable in quality (in terms of, e.g., organic matter content, inorganic nitrogen content, C:N ratio, and pH), and, owing to our ignorance of which parameters in the slurry reduce the survival, we cannot yet extrapolate the current findings to other slurries. The effect of soil solarization on the survival of R. solanacearum and its activity was striking, as the clear survival-depressing effects were not reflected in the suppressiveness assays with freshly added inoculum. Most likely, solarization exerted a direct (debilitating) effect on the © 2004 NRC Canada

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pathogen by virtue of, for instance, the temperature increase, although this was not controlled in the field study. On the other hand, the soil characteristics that play a key role in the infection process and establishment of suppressiveness may have remained largely unaffected. This was, however, not true for the combined treatment in which clear effects on pathogen survival and activity were seen. Katan (1981) and Stapleton (1999) described the effects of solarization of soil on a range of plant pathogens. The main factors involved in the suppressive effect were temperature increase and the presence of enhanced concentrations of volatile toxic compounds. Gamliel et al. (2000) reported that solarization combined with varying organic amendments reduced the pressure of plant pathogens under field conditions, changing the soil microflora and even suppressing the reestablishment of plant pathogens. Our results show that a combination of pig slurry and solarization may have an effect akin to that found by Gamliel et al. (2000) on the survival and suppression of R. solanacearum in soil. Unfortunately, pig slurry evokes strong negative sentiments because, as a by-product of pig husbandry, it is produced in excessive amounts, especially in The Netherlands. It may seem odd to propose the use of pig slurry for plant protection, but on the other hand, when applied as an inducing agent for suppressiveness against R. solanacearum, pig slurry may actually provide a relevant new application area, in particular in combination with solarization. The bacterial community in the control and pig slurry treated field soils was analysed at the end of the experiment using bacterial PCR–DGGE of duplicate samples. A clear clustering of the bacterial community profiles along treatment (not shown) was found. Several bands were unique for the field plots treated with pig slurry. In other words, pig slurry treatment induced shifts in the bacterial community structures of the field soils that were visible about 9 weeks after treatment. It would be premature to pinpoint the changed bacterial community as the cause of the enhanced suppressiveness, but the pig slurry induced bands might relate to organisms favoured by pig slurry addition. Götz and Smalla (1997) showed that the copiotroph Pseudomonas putida, an organism that harbours a wealth of suppressive strains, was strongly stimulated by pig slurry. A related species, Pseudomonas fluorescens, recently indeed showed suppression of R. solanacearum in the rhizosphere of young tomato plants (van Overbeek et al. 2002), indicating that this is a challenging area that warrants future research.

Acknowledgements This study was supported by the EU-FAIR project FATE (FAIR 3632) as well as by the Dutch Ministry of Agriculture, Nature Management and Fisheries, division DWK (Agrobiodiversity programme 352). We thank Ineke de Vries, Rogier Doornbos, and Remi Hillekens for their help during the experiments, Joeke Postma for help with the analysis of plant disease data, and Hendrik Terburg for linguistic corrections.

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