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Microbial community response to copper oxychloride contamination in acidic, sandy loam soils
Keith du Plessis1, Kobus Conradie1, Alf Botha2
Keywords: Copper oxychloride, metabolic potential, microbial community, protozoa, soils.
INTRODUCTION
Copper oxychloride is applied annually to vineyards as a fungicide to control a significant number of plant diseases (Nel et al., 1999). Some of this copper (Cu) ends up in vineyard soil, and if present, in the adjacent pristine natural vegetation. In many cases these soils are slightly acidic, making Cu more mobile and bio-available than in alkaline soils (McBride, 1994). The accumulation of Cu results in changes in soil microbial community composition (Chaphekar, 1978) and diversity (Atlas, 1984). These changes in soil microbial diversity may impact on the capacity of agricultural land to remain productive (Naeem et al., 1995). In a study by Conradie (2004) that was performed in a nursery, soil Cu levels adjusted to as high as 100 mg/kg, had no negative affect on percentage take, cane mass and root development of grapevines. The author also found that increased soil Cu levels caused an increase in the levels of Cu in the grapevine roots, and suppressed the uptake of iron when it exceeded 50 mg/kg.
Heavy metals such as Cu affect the structure and function of microbial communities, thereby impacting negatively on processes important for soil fertility (Giller et al., 1998). Heavy metal contamination inhibits the mineralization of organic material and nitrogen fixation, and may lead to a reduction in total microbial biomass, to a decrease in numbers of specific populations, or to shifts in microbial community structure (Sandaa et al., 1999). Bacteria are more sensitive to heavy metals than fungi (Frostegård et al., 1996). Heavy metals cause growth abnormalities in some protozoa and may even reduce the growth rate of these organisms (Foissner, 1994). This sensitivity of protozoa to heavy metals has been utilized in the development of bioassays to determine the bioavailability of heavy metals in soils (Forge et al., 1993).
The aim of this study was therefore to determine the microbial response to copper oxychloride in soil microcosms derived from acidic sandy loam soil from cultivated viticultural land and uncultivated land.
MATERIALS AND METHODS
The soil that was used to prepare the microcosms was collected from two wine farms near Stellenbosch and one near Somerset West in the Western Cape, South Africa (Table 1). The sampling sites were covered with vines, grasses or indigenous fynbos vegetation. The soils were classified according to the Soil Classification Working Group (1991), while soil texture was determined using the hydrometer method (Van der Watt, 1966). The chemical characteristics and exchangeable cations were determined as previously described (Du Plessis et al., 2005).
The first series of soil microcosms was prepared from sample I by adding nine different amounts of copper oxychloride to each of nine sub-samples of soil at the concentrations shown in Table 2. Each of these nine soil + Cu treatments was subdivided into triplicate microcosms in polythene bags, each containing ca. 2 kg of soil, equivalent to a soil column, 10 cm in diameter and 20 cm in height. Microcosms prepared from samples II-V were similar to the first series, but received fewer treatments (Table 2). Sterile distilled water was added to each of the microcosms to establish a soil moisture content of 15% (v/w). Thereafter 200 ml water was added to each microcosm every 2 weeks.
Impact of Cu on microbial numbers in soil sample I
Routine monitoring of the numbers of actinomycetes was done using actinomycete isolation agar (Oxoid), while fluorescent bacteria, heterotrophic micro organisms and Pseudomonas strains were enumerated on King’s medium B (Biolab), tryptone soy agar (Biolab), and Pseudomonas CFC medium (Oxoid), respectively. These counts were performed on days 1, 3, 7, 14, 70 and 245 days in sample I.
Impact of Cu on whole community metabolic profiles
On days 70 (samples II-V) and 245 (sample I), measurement of the metabolic potential of the microbial community were performed using BiologTM Eco microplates (Garland, 2000). These measurements were done on the microcosms that received 0, 30, 100, and 1000 mg/kg Cu.
Impact of Cu on protozoan numbers
Protozoan counts were performed after 70 days on samples I-V, and also on day 245 for sample I, according to the method of Griffiths and Ritz (1988).
Further details of the abovementioned experiments are described by Du Plessis et al. (2005).
RESULTS AND DISCUSSION
Different physiological groups reacted dissimilarly following the addition of Cu to the soil. During the initial 14 days of incubation of soil sample I, there were significant increases in the general microbial and fluorescent bacterial numbers in the soil microcosms (Figs 1 & 2). This phenomenon may be attributed to the re-wetting of the soil after it was dried for 2 weeks prior to initiation of the experiment (Franzleubbers et al., 2000). Increased Cu concentrations in the soil did not result in a decrease in the numbers of pseudomonads in the soil (data not shown). This indicates that other factors, in addition to the intrinsic abilities to resist high Cu concentrations, play a role in the regulation of pseudomonad populations under these conditions. For example, an increase in pseudomonad numbers may be a consequence of a negative impact of high Cu concentrations on the competitors and/or protozoan predators of pseudomonads. Actinomycete numbers remained similar to the control in all the microcosms that received copper oxychloride (results not shown).
Despite the considerable range of soils and soil chemical variables found in this study (Table 1), the microbial reactions to Cu in the various soils did not differ greatly from each other. The addition of copper oxychloride had a negative impact on both protozoan numbers (Fig. 3) and the metabolic potential of the soil microbial community. The number of carbon sources utilized by soil micro organisms on the BiologTM Eco microplates declined as the Cu concentration increased. Of the 32 carbon sources on the microplates, 29 were utilized by soil micro organisms when no Cu was added to the soil. Only 28, 25 and 19 carbon sources were, respectively, utilized when 30, 100 and 1000 mg/kg Cu were added to the soil. Of all the measured parameters, the protozoa were found to be the most sensitive to additional Cu. In some soils, as little as 15 mg/kg (Table 2) Cu had a significant negative impact on protozoan numbers (Figure 3).
CONCLUSION
The accumulation of Cu in soil may have detrimental effects on agriculture if it is not closely monitored. Of the micro organisms investigated protozoa were found to be most sensitive to elevated Cu levels. Protozoa in soil from uncultivated land were more abundant, and seemed more sensitive to additional Cu, than were the protozoa in soil originating from cultivated land. The pivotal role of protozoa in the mineralization process, and the negative impact of relatively low Cu concentrations on the populations of these microbes in soil, suggest that soil processes in these soils may be negatively affected at much lower levels of Cu than is commonly believed. Protozoan sensitivity to small increases in Cu concentrations demonstrates the vulnerability of soil ecosystems to Cu perturbations, especially as protozoa are important organisms in the flow of energy between trophic levels (feeding positions in a food chain such as primary producers, herbivores, primary carnivores, etc.).
ACKNOWLEDGEMENTS
The authors are grateful to Winetech and ARC Infruitec-Nietvoorbij for financial support. The staff members of the Microbiology Department at Stellenbosch University and the Soil Science division at ARC Infruitec-Nietvoorbij, respectively, are acknowledged for their technical support. Franscos Baron from ARC Infruitec-Nietvoorbij is singled out for his outstanding technical support.
For further information contact Keith du Plessis at (021) 809-3158 or e-mail dplessisk@arc.agric.za.
REFERENCES
Atlas, R.M., 1984. Use of microbial diversity measurements to assess environmental stress. In: Klug, M.J. and Reddy, C.A. (eds), Current perspectives in microbial ecology. American Society for Microbiology, Washington. pp. 540 - 545.
ABSTRACT
A study was carried out to determine the response of different microbial communities to copper oxychloride in acidic sandy loam soils. Culturable microbial populations were monitored over a period of 245 days in a series of soil microcosms spiked with copper oxychloride at different concentrations. Microbial populations responded differently to the applied copper (Cu). Number of protozoa and the metabolic potentials of the soil communities decreased. Metabolic potential was not significantly affected by =100 mg/kg additional Cu. In contrast, a negative impact on protozoa was observed in soil that contained only 15 mg/kg Cu. This negative impact of Cu on the number of protozoa was less severe in soils containing raised concentrations of phosphorous and zinc.
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