Tomato Seed Inoculation with Bacillus subtilis Biofilm Mitigates Toxic Effects of Excessive Copper in the Substrate

Accumulation of copper (Cu) in soils devoted to intensive agriculture due to anthropogenic additions is becoming a significant threat to plant productivity. Biological inoculants may play an important role in alleviating toxic effects of heavy metals on plants. The plant-growth-promoting rhizobacteria (PGPR) Bacillus subtilis subsp. spizizenii has demonstrated the ability to reduce harmful impacts of heavy metals on crops. This study aimed to evaluate the suitability of seed inoculation with biofilm produced by this bacterium to mitigate the severity of Cu toxicity on tomato. In the laboratory, first, B. subtilis was cultivated under increased Cu concentrations. Then, germination of inoculated and non-inoculated tomato seeds was tested for Cu concentrations of 0, 50, 100, 150, and 200 ppm. Next, a greenhouse experiment was conducted for four months to assess the effects of both inoculation and excess 150 ppm Cu in the substrate. The studied treatments included control, no inoculation and Cu surplus, inoculation and no Cu surplus, and inoculation plus Cu surplus. In the laboratory, first, the bacterium&rsquo:s ability to grow in a liquid medium containing Cu was confirmed. Thereafter, we verified that the germination of non-inoculated seeds was negatively affected by Cu, with higher concentrations leading to a more detrimental effect. However, seed inoculation with biofilm mitigated the adverse impact of Cu on germination. Under greenhouse conditions, excess Cu significantly reduced root dry weight, tomato number, and tomato yield compared with the control, whereas shoot dry weight, plant height, leaf area, and soluble solid concentration (Brix index) did not experience significant changes (p &lt: 0.05). However, seed inoculation mitigated the toxic effects of excess Cu, significantly enhancing all the aforementioned plant parameters, except plant height. Seed inoculation also significantly reduced the Cu contents in the fruits of tomato plants growing in the metal contaminated substrate. The biofilm of the B. subtilis strain used demonstrated its effectiveness as a bioinoculant, attenuating the detrimental effects induced by a substrate with excess Cu.