Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue5.2000:

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Letter to the editor

Rhizobium in long-term metal contaminated soil

My paper ``Rhizobium in soils contaminated with copper and zinc following long-term application of sewage sludge and other organic wastes'' (Smith, 1997) has evoked a strongly critical response by colleagues at IACR-Rothamsted (McGrath and Chaudri, 1999). The authors essentially raise three criticisms of the work:

1. That the application of the methods of R.detection in soil were inappropriate and the statistical and microbiological interpretation of the results are therefore incorrect.

2. They disagree with the proposed explanation for the occurrence of a strain of Rhizobium leguminosarum biovar trifolii, ineective in N2-®xation, in soil at

the Woburn Market Garden Experiment.

3. That the paper ignores the main issue of metal tox-icity to rhizobia in soil.

A single 100-fold dilution was used to indicate pre-sence or abpre-sence of rhizobia in my survey of metal-contaminated soils. However, as McGrath and Chau-dri (1999) point out, it is possible for other microbes present in the soil extract to interfere with the nodula-tion process so results recorded as a negative may actually be positive. Vincent (1970) states that this is a condition that can be encountered in some soils, but it is not a characteristic of all soils, as implied by McGrath and Chaudri (1999). The example Vincent (1970, p. 72) quotes shows that 102is equally as sensi-tive as higher dilutions at detecting rhizobia by plant infection. From my experience with the plant infection test, if nodulation is not apparent at the 102 dilution, then neither does it occur at greater dilutions, resulting in a negative score. I am not aware of any laboratory reporting pathogen populations when detecting rhizo-bia in soil by the plant infection assay.

Rather than invalidating the results, to the contrary, this argument actually enforces the innate conserva-tism of the statistical models developed to assess the signi®cance of dierences in soil properties between samples with or without rhizobia. The models may be `wrong' in as much as some soils that were recorded negative may have contained rhizobia. However, this

could be regarded as an advantage because the in-terpretation of results concerning the signi®cance of the eects of potentially toxic metals on rhizobia in sludge-treated soil was inherently precautionary.

The purpose of my study was to conduct an initial screening exercise of a large number of soils for pre-sence of rhizobia to assess the overall signi®cance of the eects of metals on the occurrence of free-living bacteria in sludge-treated soil. In principal, a simple qualitative test of presence or absence of rhizobia in soil isuseful for assessing metal toxicity because rhizo-bial numbers appear to crash abruptly at the critical toxic metal concentration on a linear soil concen-tration scale. Chaudri et al. (1993) observed this beha-viour on the sludge-treated plots at Braunschweig where the rhizobial population was completely wiped-out above a certain critical soil concentration. If this is what happens in other sensitive sludge-treated soils then the use of a presence±absence assay, to indicate potential toxicity of metals to rhizobia in sludge-amended systems, is justi®ed because the step from presence to absence apparently occurs over a very narrow metal concentration range. Statistical tests can then be applied to the data to quantitatively compare, and to quantify the signi®cance of, important soil vari-ables according to the presence or absence of rhizobia in soil samples.

The statistical analysis of presence/absence data showed that the occasional absence of rhizobia from soil in the practical ®eld situation was generally a chance event unrelated to the concentrations of metals in soil. At one site, there was evidence of a statistically signi®cant increase in Zn concentration associated with the absence of rhizobia. However, the identi®ed Zn eect was actually not very convincing, though statisti-cally signi®cant, because Zn bioactivity was low due to the high pH value of this particular soil. There could well be some other plausible explanation for the indi-cated absence of rhizobia from the soil that was not considered in the statistical analysis. Indeed at another site, particularly enriched with Zn, a statistically sig-ni®cant eect of Zn was also identi®ed, but in this case presence was associated with the highest Zn concen-trations in soil and absence occurred at lower soil con-centration values.

Soil Biology & Biochemistry 32 (2000) 729±731

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We have consistently argued that ineectiveness in N2-®xation was a unique feature of the Woburn site

(Smith and Giller, 1992). I agree entirely with McGrath and Chaudri (1999) that the presence of an ineective strain is ``a rare chance event and not much emphasis should be placed on it''. The ®nal clari®ca-tion of this has been a useful outcome from the corre-spondence and, as we agree, there seems little point in discussing it further. Nevertheless, the speculative ex-planation proposed for the occurrence of the ineec-tive strain was based on the known association between acid soil conditions, ineectiveness and toler-ance to certain metals (Holding and King, 1963; FaÊh-raeus and Ljunggren, 1968; Nutman and Ross, 1970; Holding and Lowe, 1971; Hagedorn et al., 1983; Thornton and Davey, 1983).

Eectiveness in N2-®xation was a relatively minor

component of the study included because ineective rhizobia were isolated from a single soil sample. It is unreasonable to suggest that this was intended as a diversionary tactic to the main issue of toxicity. That healthy, diverse populations of rhizobia existed before sludge was applied at Woburn and Braunschweig and that metal contamination subsequently killed o non-tolerant forms is not in dispute. I think it is unreason-able of McGrath and Chaudri (1999) to suggest that this point was ignored, since it is central to the entire investigation. They also state incorrectly that the paper disregards some other important points relating to these ®eld trials, such as dierentiating between results obtained with salt-amended and unamended sludges. For example, I clearly explain (p. 1476) that ``rhizobial numbers were also reduced on plots receiving unspiked sludges'' at Braunschweig. The main objective spelt out in the paper was to assess whetherRhizobiumwere lost from sludge-treated soils in the practical ®eld situ-ation and whether this could be attributed to metal toxicity. However, in practice I found that this was not the case.

My account of metal toxicity to rhizobia in the plots at Woburn implicates Cd as one of the critical el-ements of interest in soil from that ®eld experiment. Surprisingly, McGrath and Chaudri (1999) barely mention this controversial point in contrast to their detailed comments on the relatively minor issue of ineectiveness and soil pH. I raise this here because McGrath and Chaudri (1999) draw attention to the fact that no eects of Cd were detected from the pre-sence/absence analysis. However, it should be empha-sised that this was expected because the investigated sites were selected speci®cally for low Cd content (usually <3 mg kgÿ1 _{in soil), to separate the}

poten-tially confounding toxic eects of this element from those of Zn. Cadmium is an important issue because in my view, and that of the Independent Scienti®c Committee (MAFF/DoE, 1993), high soil Cd may

reduce the value of the Woburn trial. The research as-sociated with the Woburn experiment (McGrath et al., 1988; Giller et al., 1989; Chaudri et al., 1992; Hirsch et al., 1993) is unarguably pioneering. However, trade euent control has signi®cantly improved the quality of sewage sludge for agricultural use and has dramati-cally reduced its Cd content (Smith, 1996) compared to the highly contaminated material applied at Woburn in the 1940s to 1960s (McGrath, 1984). Therefore, extrapolating evidence from Woburn to the current agricultural use of sewage sludge should be undertaken cautiously for the purposes of developing national soil limits for the simple reason that high Cd soils do not occur in practice. This does not dismiss the possibility that Zn may also be involved at Woburn as is indicated by evidence from the single ®eld trial site at Braunschweig. However, the apparent similarity with Braunschweig in the critical Zn concentrations aecting rhizobia (180±200 mg Zn kgÿ1_{) (McGrath et}

al., 1995) may be simply a coincidence, partially at-tributable to an artefact of the large Cd content (6.0 mg Cd kgÿ1_{), which is potentially toxic to rhizobia}

(Chaudri et al., 1992), that occurs simultaneously at this Zn concentration range in Woburn soil.

In the practical ®eld situation, rhizobial populations are aected by many site, edaphic, ecological and en-vironmental factors and these exert a major in¯uence on the distribution of R. in soil (Nutman, 1975). Indeed, Giller et al. (1998) were unable to detect rhizo-bia in uncontaminated soil samples from a ®eld adja-cent to the Woburn experiment and presumed that the absence of the bacteria was due partly to chance and also previous land-use, particularly the lack of cultiva-tion of clover on the soil. However, soil populacultiva-tions rapidly recover and rhizobia are always found in soil with the host plant irrespective of previous land man-agement, environmental conditions or the extent of soil contamination with heavy metals (Smith and Gil-ler, 1992; Obbard and Jones, 1993; Smith, 1997). The presence/absence analysis and occurrence of free-living rhizobia (i.e. without the host plant) above current soil limit values suggests that these mechanisms are prob-ably much more important than soil metal concen-trations at in¯uencing the distribution of rhizobia in most sludge-treated ®elds in practice.

In summary, none of the criticisms raised by McGrath and Chaudri (1999) devalue the results or alter the conclusions of the study that showed the pre-sence of rhizobia in soils exceeding the current statu-tory limit values of heavy metals, including Zn. Moreover, McGrath and Chaudri (1999) have inadver-tently emphasised the apparent conservatism of the in-terpretation relating to the signi®cance of metal contamination on rhizobial survival in soil. Conse-quently, the presence/absence analysis shows that the UK advisory limit for Zn (200 mg Zn kgÿ1_{, pH 5±7;} Letter to the editor / Soil Biology & Biochemistry 32 (2000) 729±731

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DoE, 1996) is a highly precautionary value. The exper-imental techniques employed were appropriate, rigor-ous and the data produced were robust and help provide a balanced assessment and account of the eects of heavy metals on the fertility of sludge-treated soil. The results show that there is no need for panic measures to radically alter maximum permissible soil concentrations in the EU Directive (CEC, 1986) to protect sensitive soil microorganisms when sewage sludge is used in agriculture. However, I agree with McGrath and Chaudri (1999) that eects of metals on sensitive microbial indicators in real-world situations require rigorous and quantitative assessment. This is essential to provide the sound technical and pragmatic basis to soil limits that protect the environment and fa-cilitate the recycling, rather than the disposal, of the valuable and useful resources in sludge. A consistent approach to soil protection should also be extended to all potentially contaminated wastes applied on farm-land including, for example, the farm-land spreading of cer-tain types of industrial and livestock waste.

References

CEC (Council of the European Communities), 1986. Council Directive of 12 June 1986 on the protection of the environment, and in particular of the soil, when sewage sludge is used in agri-culture (86/278/EEC). Ocial Journal of the European Communities L 181, 6±12.

Chaudri, A.M., McGrath, S.P., Giller, K.E., 1992. Survival of the indigenous population ofRhizobium leguminosarumbiovartrifolii

in soil spiked with Cd, Zn, Cu and Ni salts. Soil Biology & Biochemistry 24, 625±632.

Chaudri, A.M., McGrath, S.P., Giller, K.E., Rietz, E., Sauerbeck, D.R., 1993. Enumeration of indigenousRhizobium leguminosarum

biovartrifolii in soils previously treated with metal-contaminated sewage sludge. Soil Biology & Biochemistry 25, 301±309. DoE (Department of the Environment), 1996. Code of Practice for

Agricultural Use of Sewage Sludge. DoE, London.

FaÊhraeus, G., Ljunggren, H. 1968. Preinfection phases of the legume symbiosis. In: Gray, T.R.G., Parkinson, D. (Eds.), The Ecology of Soil Bacteria: an International Symposium. Liverpool University Press, Liverpool, pp. 396±421.

Giller, K.E., McGrath, S.P., Hirsch, P.R., 1989. Absence of nitrogen ®xation in clover grown on soil subject to long-term contami-nation with heavy metals is due to survival of only ineective

Rhizobium. Soil Biology & Biochemistry 21, 841±848.

Giller, K.E., Witter, E., McGrath, S.P., 1998. Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: a review. Soil Biology & Biochemistry 30, 1389±1414. Hagedorn, C., Ardahl, A.H., Materon, L.A., 1983. Characteristics of

Rhizobium trifolii populations associated with subclover in Mississippi soils. Soil Science Society of America Journal 47, 1148±1152.

Hirsch, P.R., Jones, M.J., McGrath, S.P., Giller, K.E., 1993. Heavy metals from past applications of sewage sludge decrease the gen-etic diversity of Rhizobium leguminosarum biovar trifolii popu-lations. Soil Biology & Biochemistry 25, 1485±1490.

Holding, A.J., King, J., 1963. The eectiveness of indigenous popu-lations ofRhizobium trifoliiin relation to soil factors. Plant and Soil 18, 191±198.

Holding, A.J., Lowe, J.F., 1971. Some eects of acidity and heavy metals on the rhizobium±leguminous plant association. Plant and Soil Special Volume, 153±166.

MAFF/DoE (Ministry of Agriculture, Fisheries and Food/ Department of the Environment), 1993. Review of the Rules for Sewage Sludge Application to Agricultural Land: Soil Fertility Aspects of Potentially Toxic Elements. MAFF Publications, London (PB 1561).

McGrath, S.P., 1984. Metal concentrations in sludges and soil from a long-term ®eld trial. Journal of Agricultural Science, Cambridge 103, 25±35.

McGrath, S.P., Chaudri, A.M., 1999. Long-term eects of metal contamination on Rhizobium. Soil Biology & Biochemistry 31, 1205±1207.

McGrath, S.P., Brookes, P.C., Giller, K.E., 1988. Eects of poten-tially toxic metals in soil derived from past applications of sewage sludge on nitrogen ®xation byTrifolium repensL. Soil Biology & Biochemistry 20, 415±424.

McGrath, S.P., Chaudri, A.M., Giller, K.E., 1995. Long-term eects of metals in sewage sludge on soils, microorganisms and plants. Journal of Industrial Microbiology 14, 94±104.

Nutman, P.S. 1975.Rhizobiumin the soil. In: Walker, N. (Ed.), Soil Microbiology. Butterworth, London, pp. 111±131.

Nutman, P.S., Ross, G.J.S., 1970. Rhizobium in the soils of the Rothamsted and Woburn Farms. Rothamsted Experimental Station Report for 1969, Part 2. Rothamsted Experimental Station, Harpenden, pp. 148±167.

Obbard, J., Jones, K.C., 1993. The eect of heavy metals on dinitro-gen ®xation by Rhizobium±white clover in a range of long-term sewage sludge amended and metal-contaminated soils. Environmental Pollution 79, 105±112.

Smith, S.R., 1996. Agricultural Recycling of Sewage Sludge and the Environment. CAB International, Wallingford.

Smith, S.R., 1997.Rhizobiumin soils contaminated with copper and zinc following the long-term application of sewage sludge and other organic wastes. Soil Biology & Biochemistry 29, 1475±1489. Smith, S.R., Giller, K.E., 1992. Eective Rhizobium leguminosarum

biovartrifoliipresent in ®ve soils contaminated with heavy metals from long-term applications of sewage sludge or metal mine spoil. Soil Biology & Biochemistry 24, 781±788.

Thornton, F.C., Davey, C.B., 1983. Acid tolerance ofRhizobium tri-foliiin culture media. Soil Science Society of America Journal 47, 496±501.

Vincent, J.M., 1970. A Manual for the Practical Study of the Root-Nodule Bacteria. Blackwell, Oxford.

Stephen R. Smith* Department of Civil and Environmental Engineering, Imperial College, London SW7 2BU, UK E-mail address: s.r.smith@ic.ac.uk

* Tel.: +44-171-594-6051; fax: +44-171-823-9401.