Acta Hortic. 1270. ISHS 2020. DOI 10.17660/ActaHortic.2020.1270.10 Proc. IX International Symposium on Soil and Substrate Disinfestation Eds.: A. Gamliel et al. 97 Defining anaerobic soil disinfestation through changes in the microbiome J.C. Hong 1 , F. Di Gioia 2,3 , J.B. Jones 2 , W. Turechek 1 , N. Kokalis-Burelle 1 , C.W. Johns 4 , N.L. Finley 4 , M. Ozores-Hampton 5 , G. McCollum 1 and E.N. Rosskopf 1 1 USDA-ARS, United States Horticultural Research Laboratory, Fort Pierce, FL, USA; 2 University of Florida, Gainesville, FL, USA; 3 Pennsylvania State University, State College, PA, USA; 4 Miami University, Oxford, OH, USA; 5 University of Florida, Institute of Food and Agricultural Sciences, South West Florida Research and Education Center, Immokalee, FL, USA. Abstract The main driver of anaerobic soil disinfestation (ASD) is manipulation of the microbiome. This is achieved by introducing soil amendments and depleting the soil of oxygen by saturating with water under a plastic tarp. The current standard ASD method in Florida, USA, consists of applying composted poultry litter (13 Mg ha -1 ) and feed- grade sugarcane molasses (13,900 L ha -1 ), tarping with totally impermeable film (TIF), and saturating the soil with 5 cm water. ASD is applied 3 weeks prior to transplanting. Herein, we report on impacts of modifications of the ASD treatment regarding the soil microbiome. The study was conducted in the greenhouse and field, in which standard ASD, ASD with double application of molasses (ASD2×), and soil covered with TIF (control) were compared. Spatial and temporal soil samples were taken at 0-15 cm and stored at -80°C. Total DNA was extracted and microbial populations were identified using length heterogeneity PCR (LH-PCR) and next-generation sequencing of the 16S ribosomal DNA. Based on the redox potential data, ASD2× was more anaerobic than ASD in the greenhouse, and both were statistically more anaerobic than the control at both locations. Using LH-PCR, shifts in the microbiome were observed, and both ASD- treated soils differed from the control in both the greenhouse and field experiments. Based on 16S rDNA, members of the phyla Firmicutes and Proteobacteria increased compared with the control. From a panel of seven different organic acids, only isobutyric acid was detected in the control samples, while six of the seven acids were detected in the ASD-treated soils. ASD2× had a greater quantity of each organic acid than ASD. Keywords: microbial ecology, metabolites, next-generation sequencing, DNA fingerprinting, alpha and beta diversity INTRODUCTION Anaerobic soil disinfestation (ASD) has proven to be a sustainable alternative to chemical soil fumigation regarding control of soil pathogens and fruit yield in Florida, USA (Butler et al., 2012; Di Gioia et al., 2016). Application of ASD in Florida consists of working feed-grade molasses and composted poultry litter (CPL) into the soil. The soil is then tarped with a plastic mulch and saturated with water to field capacity. Three weeks after application of the amendments, holes are punched through the plastic and the crop is transplanted. In Florida, ASD has been used as a soil pre-plant treatment for cut flowers, citrus trees, and specialty crops including strawberry, cucumber, tomato, peppers, squash, eggplant, and pak choi. Market yield from plants planted in ASD-treated soil has been similar to or has exceeded the yield from crops planted in chemically fumigated soil (Rosskopf et al., 2015; Di Gioia et al., 2016; Guo et al., 2017). The ASD treatment selects for facultative anaerobes and anaerobic microbes (Momma et al., 2013; Mowlick et al., 2013a). Through DNA extraction of post-treatment soil samples and partial sequencing of the 16S rDNA, it has been observed that the soil microbiome was drastically changed in ASD-treated soil after the treatment. Most report an increased abundance of the phylum Firmicutes in the ASD-treated soils (Mowlick et al., 2013b;