Tomato is an economically and nutritionally important crop and is vulnerable to drought. Under drought, soil microbes provide beneficial effects to plants and alleviate stress. We suggest a reconstruction of the soil microbiome using biofumigation, an organic farming method, to protect tomatoes. In this study, we treated soil in four ways with varied concentrations: biofumigation (BF0.5, BF1.0, and BF1.5), green manure treatment (GM0.5, GM1.0, and GM1.5), autoclaving (AT), and non-treatment (NT). Tomatoes were grown in each treated soil, subjected to water shortages, and were rewatered. We investigated plant phenotypes and soil properties, focused on microbial communities using the Illumina MiSeq® System. Relative Water Content and malondialdehyde were measured as plant stress. The results showed that the 1% biofumigation treatment had 105% and 108.8% RWC during drought and after rewatering, compared to the non-treated soil. The highest concentration, the 1.5% treatment, lowered RWC due to an excess of NO3−, K+, Ca2+, and decreased alpha diversity. Through PLS-PM, bacterial alpha diversity was found to be the largest factor in the increase in RWC (coefficient = 0.3397), and both biofumigant and green manure significantly increased the Shannon index and observed species. In addition, biofumigation increased beneficial functional genes (purine metabolism, pyrimidine metabolism, carbon fixation pathways, and zeatin bio-synthesis) of soil microorganisms (p value < 0.05, <0.01, >0.05, and <0.05, respectively). The 1% biofumigation treatment enriched the core five genera of the fungal network (Enterocarpus, Aspergillus, Leucothecium, Peniophora, and Wallemia) of the fungal network which might suppress the most dominant pathogen, Plectosphaerella. In conclusion, biofumigation-derived soil microbiome alterations have the potential to lower plant stress under drought.
