Mg2+ is one of the essential elements for bacterial cell growth. The presence of the magnesium cation (Mg2+) in various concentrations often affects cell growth restoration in plant-associating bacteria. This study attempted to determine whether Mg2+ levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is. S. yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings. S. yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves. Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation. A concentration of ca. 0.10 mM Mg2+ or more allowed S. yanoikuyae EC-S001 to grow in potato-dextrose broth medium. Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg2+ requirements. For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg2+-free Hoagland's no. 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg2+ for its growth. In contrast, S. yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg2+ to Mg2+-free HSG medium. Our studies concluded that Mg2+ is more than just the essential trace element needed for cell growth restoration in S. yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg2+ or another specific essential element for their growth.