Inoculum handling alters the strength and direction of plant–microbe interactions

The pooling of soil samples in plant–microbe interaction studies is commonly employed, but the impact of sample handling has rarely been explored experimentally. Concerns have been raised that sample pooling may reduce biological variation leading to inflated type I errors or may alter the magnitude of microbial effects observed, invalidating the results achieved. To assess the impact of inocula pooling on plant–microbe interactions, we examined the reciprocal influence of unpooled and pooled soil microbial inocula on growth of Solidago altissima and Schizachyrium scoparium, with and without inoculum sterilization. Soil pooling had no effect on the variance among replicates in either plant species. However, pooling dramatically altered the magnitude and direction of microbial impacts on plant performance. Pooling of Solidago altissima soil increased the antagonistic effects on growth of both target species. In contrast, pooling of Schizachyrium scoparium soil shifted impacts on Solidago altissima from effectively neutral to slightly positive. Pooling in this system altered both the strength and direction of plant–microbe interactions relative to unpooled soils. Therefore soil mixing should be avoided when the research goal is to determine naturally occurring interaction strengths, even within a single habitat. One of the major ecological developments over the last two decades is a comprehension of the critical role that soil microbial communities play in determining plant success and community structure (Bever 1994, Van Der Heijden et al. 2008, Brinkman et al. 2010, van der Putten et al. 2013, Smith‐Ramesh and Reynolds 2017). As soils are highly heterogeneous, studies typically include some degree of pooling of individual samples (Robertson et al. 1999) to provide a better view of soil parameters such as organic matter or nutrient concentrations with limited analytical effort. Plant–microbe interaction work has often followed this practice of sample pooling, by combining samples of field experimental units (e.g., plots, sites) per treatment and then performing assays on the mixture in place of the original independent soil samples (e.g., Reinhart and Rinella 2016). Some feel that performing assays on mixtures of samples, in place of independent samples, is a simpler means of conducting plant–soil interactions research (e.g., Cahill et al. 2017, Gundale et al. 2017, 2019, Teste et al. 2019). However, others have warned that performing assays on a mixture of samples in place of the original samples is likely to produce biased inferences (Reinhart and