Submitted to: Soil Science Society of America Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2003
Publication Date: 11/15/2003
Citation: Delorme, T.A., Gagliardi, J.V., Angle, J.S., Van Berkum, P.B., Chaney, R.L. 2003. Phenotypic and genetic diversity of rhizobia isolated from nodules of clover grown in a zinc and cadmium contaminated soil. Soil Science Society of America Journal. 67(6):1746-1754. Interpretive Summary: Heavy metal contamination of soils may cause phytotoxicity (toxicity to plants), or adverse effects to soil microbes depending on metal concentration, soil pH, and some other factors. Others have reported that the rhizobium specific for white clover and similar species was harmed by soil Zn before the plants were harmed. But our previous studies have indicated that low soil pH had a more adverse effect on this microbe than did the Zn or Cd accumulation in field soils amended with biosolids. This species is considered an important microbe needed for nitrogen fixation, so understanding the nature of soil metal risk is important. With the availability of new techniques which can be used to characterize the genetic nature of rhizobium, we undertook a testing of the Zn and Cd tolerance of white clover rhizobium collected from soils contaminated by long term emissions from a Zn-smelter at Palmerton, PA. Strains of the rhizobium were collected from nodules on red clover plants growing in the different fields, or were extracted from the soil. Examination of the patterns of genetic diversity and metal tolerance indicated that there was a slight increase in genetic diversity of the strains which nodulated clover in the more contaminated soil. But all strains studied formed nodules with red clover and effectively fixed nitrogen, in strong contrast with results from one site in the UK where only ineffective rhizobium survived in a field treated with biosolids several decades ago. Our results also differed strongly from those of studies which added metal salts to soils and evaluated survival of rhizobium. We believe that the slow accumulation of Zn and Cd in smelter contaminated soils gives the microbes time to adapt. And if soil pH is near that recommended for growing clovers (near neutral pH), the rhizobium adaptation causes somewhat greater genetic diversity, in strong contrast with the UK site where only one ineffective genotype of the rhizobium survived. Because industrial pretreatment of industrial discharge of metals has allowed production of biosolids with low levels of Zn and Cd, and biosolids are applied only at fertilizer rates, these results indicate that Zn and Cd in modern biosolids are not a threat to nitrogen fixation by the most Zn-sensitive rhizobium strain needed for nodulation of white and red clover.
Technical Abstract: Metal contamination can affect the diversity of microbes living in soil. We compared phenotypic and genetic characteristics of Rhizobium leguminosarum bv. trifolii isolated from clovers (Trifolium pratense) found at a metal contaminated and a control site, or isolated from nodules of clovers used in most probable number (MPN) determinations of rhizobia in the metal contaminated soil. Analysis of variations in genetic patterns using BOX-PCR was used for genetic characterization. Zinc and Cd tolerance of each isolate was also determined. Rhizobia isolated from the control soil compared with the metal contaminated soil differed both genetically and phenotypically. Genetic diversity, measured by AMOVA, was high and not related to the presence or the absence of high metal concentrations. Isolates originating from the metal contaminated soil were more tolerant to both Cd (minimal inhibitory concentration [MIC50]= 2.5 'M) and Zn (MIC50 = 92.9 'M) compared with those observed for isolates of control soil (Cd MIC50 = 2.0 'M and Zn MIC50 = 41.9 'M). Rhizobia originating from the metal contaminated soil expressed a higher number of metal phenotypes (9) compared with isolates of control soils (6). Slow rates of metal accumulation over the years favored an adaptation of the rhizobia to the metal rather than elimination of metal sensitive organisms and the selection of a few preexisting metal tolerant organisms.