Survival of a Genetically Modified Root-Colonizing Pseudomonad and Rhizobium Strain in an Acidic Soil

Maintaining threshold populations of inoculum microorganisms in the soil environment is important for such practical applications as biocontrol, plant growth-promotion, bioremediation, and nodulation. However, because of both technical and labor constraints in monitoring bacterial viability in nonsterile soils, few studies have reported on survival kinetics, particularly in relation to subtle alterations in soil acidity-related factors. A genetically modified strain Pseudomonas putida R20/lacZY or Rhizobium leguminosarum bv. trifolii 162S7a/ gusA was introduced into conditioned, nonsterile Gilpin (fine loamy, mixed mesic, Typic Hapludult) silt loam soil, limed at four low levels (pHw 4.71, 4.81, 4.92, and 4.99) or derivative soil solutions with highly correlated (R $ 0.81) chemical properties. Immediate declines in viability of both strains were found in all soils, reaching 0.1 to 1% initial colony-forming unit (CFU) g soil in 35 h for P. putida and in 68 h for R. leguminosarum bv. trifolii. Death rate constants (kd) for both strains were directly related to lime level (soil pH), although differences were not significant (P . 0.05) for the rhizobium. Use of soil solutions gave similar responses for both strains, but over much shorter incubation times. As with soils, kd values for both strains in soil solutions were directly related to lime level (solution pH). In both soil and soil solution experiments, survival (kd) was negatively correlated (R $ 20.914) with pH and basic cation (Ca and Mg), and positively correlated (R $ 0.933) with Al, concentrations. This relationship of viability to soil solution chemistry was broadly confirmed for both bacterial strains by use of fluorescent probes, suggesting increased cell membrane damage at lower pHs. These results demonstrate not only the alternative utility of using soil solutions, rather than nonsterile soils, for bacterial viability assessments, but also the positive effect of low-level liming (|0.28 pH unit increase) on survival of beneficial root-colonizing bacteria in acidic soils. INTRODUCING BENEFICIAL BACTERIA into the soil environment has been of interest to microbiologist for many years, most notably the use of rhizobia as inocula for legumes. More recently, other bacteria have become important for purposes of enhancement of crop growth, bioremediation, and biocontrol. For all these uses, threshold populations are typically required. As a consequence, there has been renewed interest in developing methods for predicting their survivability, that is, their saprophytic competency, based on the properties of both the bacterium and soil. It has often been observed that laboratory-grown, heterotrophic bacteria, introduced into soils, very seldom grow, and typically decline in number over time. In the absence of soil toxicity factors (and other stresses such as temperature, moisture, etc.), the major reason appears to be the lack of an easily oxidizable C source for growth, that is, the oligotrophic state of most natural soils (Williams, 1985; Roszak and Colwell, 1987; Morita, 1988). And, when toxic soils (here defined as being low in pH/Ca and possibly high in Al) are employed, populations decline more rapidly. This detrimental effect of acidic, nonsterile soils on survival has been amply demonstrated for such root-colonizing bacteria as the saprophytic pseudomonads (Zechman and Casida, 1982; Van Elsas et al., 1986, 1991; Acea et al., 1988; Compeau et al., 1988; Heijnen et al., 1993; Gu and Mazzola, 2001) and symbiotic clover rhizobia (Heynen et al., 1988; Postma et al., 1991; Heijnen et al., 1992, 1993; Hirsch, 1996; Watkin et al., 2000). Although there is considerable literature on the beneficial effects of liming acidic soils on legume nodulation, no information exists on liming (essentially, Ca and pH increases) effects on rhizobia survival, per se, in soils. But, there is a large body of literature on pH and Ca (and Al) effects on rhizobia growth. Nearly all of these studies, however, have been done with solution growth cultures (i.e., amended with some easily oxidizable C source), rather than in soils (Munns, 1968; Wood et al., 1984a, 1984b; Howieson et al., 1992; Flis et al., 1993; Reeve et al., 1993; Glenn et al., 1997; Watkin et al., 1997; Dilworth et al., 1999). For pseudomonads, no literature exists on the effect of liming on survival in soils, nor on pH and/or Ca effects in solution growth cultures. The generalization from these studies, applied to soils, is that the more acidic the soil, and the less Ca present, the more toxic it is to introduced rhizobia (and, presumably to pseudomonads), and that the presence of Al exacerbates the situation. To the best of our knowledge, no reports, other than a cursory examination by Staley and Voigt (2000), have appeared in the literature concerning the effect of subtle (low-level) liming of acidic soils on pseudomonad and rhizobium survival kinetics. Also, there appears to be no information available on their survival kinetics in soils, compared with soil solutions (as opposed to solution growth cultures). Determining this relationship is important because of the heterogeneous nature of soil, since soil may present a significantly different chemistry to introduced bacteria than that to which they are exposed in soil solutions derived from them (Van Elsas and Van Overbeek, 1993). To address these issues, we employed two test systems. The first system consisted of an acidic (pHw 4.71; moderate Al concentration) soil, typical of abandoned pasture soils of the Appalachian hill-land region, in T.E. Staley, USDA-ARS, Appalachian Farming Systems Research Center, 1224 Airport Rd., Beaver, WV 25813-9423; D.K. Brauer, USDA-ARS, Dale Bumpers Small Farms Research Center, 6883 S. State Hwy. 23, Booneville, AR 72927-8209. Received 23 Feb. 2005. *Corresponding author ([email protected]). Published in Soil Sci. Soc. Am. J. 70:1906–1913 (2006). Soil Biology & Biochemistry and Soil Fertility & Plant Nutrition doi:10.2136/sssaj2005.0056 a Soil Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: CFU, colony-forming unit; PSS, phosphate-salt solution. R e p ro d u c e d fr o m S o il S c ie n c e S o c ie ty o f A m e ri c a J o u rn a l. P u b lis h e d b y S o il S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . 1906 Published online September 20, 2006