Mirta Esther Galelli

The negative effects of soil heavy metal contamination on food production could be mitigated using nature-based solutions, i.e., plant-growth-promoting rhizobacteria (PGPR). Yield of Lactuca sativa L. has been shown to increase by seed inoculation with biofilm of Bacillus subtilis subsp. spizizenii. The aim of this work was to assess whether this promoting effect occurs even in the presence of toxic concentrations of copper (Cu) and zinc (Zn). First, germination rates of lettuce seeds with increasing Cu and Zn concentrations were assessed. Then, lettuce plants growing from inoculated and non-inoculated seeds were cropped on substrates with excess Cu and Zn. Above- and below-ground lettuce variables were measured, and leaf macro- and microelements were determined. Germination was more severely affected by Cu overload than by Zn overload, while this trend was reversal for plant growth. Seed inoculation enhanced germination and increased plant growth assessed by root and shoot biomass, plant height and leaf area. For example, seed inoculation increased lettuce root and aerial biomass of lettuce growing on a metal- free substrate by 68% and 62%, respectively. This practice also promoted lettuce growth in metal-overloaded substrates, increasing aerial and root biomass by 32% and 29%, respectively, in connection with Cu, and by 260% and 183% when it came to Zn. Both Cu or Zn accumulated in the edible parts of lettuce growing on contaminated substrates, but seed inoculation did not mitigate metal uptake in any case. Except for Cu and Zn, macronutrients, micronutrients and heavy metal levels in lettuce leaves were affected neither by excess metal nor by seed inoculation. Altogether, B. subtilis biofilm has been proven to be an effective seed inoculant promoting seed germination and plant growth even in the presence of heavy metals. Adverse health effects due to metal accumulation in the lettuce edible parts are not expected to increase following seed inoculation.

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’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 < 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.

The plant growth-promoting bacterium, B. subtilis subsp. Spizizizenii, has been proven to develop a biofilm under certain culture conditions, which can be applied as an efficient bioinoculant. Biofilm can be produced cost-effectively using biodiesel byproduct glycerol as a carbon source. Soils from urban peripheries may contain very high lead (Pb) levels. The main aim of this study was to assess the impact of biofilm seed inoculation on plant development and fruit quality of tomatoes growing on a Pb-contaminated substrate. Also, effects of excess Pb on biofilm production, stability, and seed germination were analyzed. B. subtilis biofilm was produced with Pb concentrations ranging from 0 to 300 ppm. Biofilm stability was tested at 4 °C and 25 °C. The impacts of Pb and inoculation on seed germination were evaluated in laboratory conditions, while the impacts on plant agronomic parameters were assessed via a greenhouse assay. Adding Pb to the culture medium increased biofilm production by about 20%. Regardless of Pb level, biofilms were more stable at 4 °C than at 25 °C. Beneficial effects of biofilm on germination were greater on seeds exposed to 200 and 300 ppm Pb. Excess Pb significantly reduced plant biomass and tomato yield. However, biofilm inoculation significantly increased plant aboveground and root biomass, plant height, leaf area, fruit number, and fruit size, regardless of substrate Pb excess. Tomato fruits of plants grown in the metal-contaminated substrate showed no significant increases in Pb concentration with respect to the control. In summary, the biofilm produced by B. subtilis subsp. spizizenii proved to be an effective bioinoculant to counteract the negative effects of substrate excess Pb on tomato germination, growth, and production.

Biofilm obtained from Bacillus subtilis subsp. spizizenii inoculated on vegetable seeds has been shown to have plant growth-promoting capacity. Seed inoculation with biofilm produced by this strain could also reduce the adverse effects on plant growth caused by soil or substrate heavy metal overabundance. Therefore, the objective of this work was to evaluate the impact of biofilm inoculated on tomato (Solanum lycopersicum L.) seeds, which were planted on a substrate with artificially added zinc. First, seeds of the Río Grande tomato variety were exposed to increasing zinc concentrations, namely: 50, 100, 200, and 400 ppm, with and without bacterial biofilm inoculation. Zinc addition and seed inoculation affected germination parameters. For example, an extra 200 and 400 ppm of zinc led to high toxicity. Biofilm inoculation, however, reduced the noxious effects of excess zinc, bringing acute toxicity down to moderate. Then, tomato plants growing from inoculated and non-inoculated seeds were cropped for 4 months in both substrates with 400 ppm zinc and without added zinc. Extra zinc addition significantly (p < 0.05) reduced tomato root and shoot biomass, plant height, and fruit number at harvest time. However, seed biofilm inoculation avoided the harmful effect of zinc on plant growth parameters, fruit yield, and fruit quality. The roots and shoots of plants growing on contaminated substrates showed very noticeable increases in zinc levels compared to the control, while fruits only showed a much weaker zinc gain, even if this was significant (p < 0.05). Moreover, root shoot and fruit concentrations of elements other than zinc, (nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese, copper, lead, and cadmium) were not or only weakly affected by the addition of this metal to the substrate. In summary, the biofilm of B. subtilis proved to be effective as a bioinoculant to alleviate negative effects on tomatoes cropped in a substrate with excess zinc.

Over-fertilization and agrochemicals adversely affect soil quality and agricultural ecosystem sustainability. Tomato (Solanum lycopersicum) is ranked as an important crop due to its high profitability and nutritional value. In Argentina, tomato is mainly produced in horticultural belts at peri-urban areas, whose soils frequently are contaminated by heavy metals and/or agrochemicals. To explore safer alternatives, we investigated the effects of seed inoculation with a common plant-growth-promoting rhizobacteria (PGPR), i.e., Bacillus subtilis subsp. spizizenii, on development at various growth stages of two tomato varieties, “Platense” and “Río Grande”, and on production and fruit quality at harvest time of the “Río Grande” variety. The experimental design consisted of three treatments per variety: a control versus traditional planktonic or biofilm inoculation, with three replicates per treatment. Germination at 10 days and seedling agronomical parameters showed that the response to seed inoculation was superior in the “Río Grande” variety. At harvest time, and irrespective of the inoculant, several agronomical parameters of the “Río Grande” variety were significantly enhanced with respect to the control. The biofilm significantly increased tomato production, as quantified by fruit number and weight, compared to the planktonic inoculum. This case study demonstrates that the incorporation of bio-inoculants is relevant in sustainable agriculture to promote crop growth and quality.

In Argentina, the excessive use of fertilizers is common in intensively cultivated zones around highly populated areas. Bioinoculants based on plant growth promoting rhizobacteria (PGPR) could be effective for crop production improvement without negative effects on the environment. The objective of this work was to evaluate an alternative inoculation method, namely the application of the biofilm produced by Bacillus subtilis as a growth promoter on seeds of three varieties of Lactuca sativa, and to compare it with the common planktonic approach. Biofilm was obtained under static culture conditions, while planktonic inoculum was produced at 150 rpm. The major biofilm effects were observed with Bacillus subtilis subsp. spizizenii, that showed antifungal activity against phytopathogens, synthesized plant growth regulators (IAA, cytokinin and ABA) and solubilized phosphates. The Grand Rapid variety inoculated with biofilm showed the best results, with 30% and 37% higher aerial and root biomass, respectively, compared to the planktonic form. Moreover, the biofilm positive effects were observed through successive plant development stages until harvest, when the bacterium was recovered from the interior of the roots. The biofilm of B. subtilis subsp. spizizenii behave as a superior growth-promoting inoculant compared to the traditional planktonic inoculation technique.

En Argentina, gran parte de la horticultura se desarrolla alrededor de los principales núcleos urbanos, en los llamados Cinturones Hortícolas. Sin embargo, debido a la acción antrópica, estos suelos sufren distintos tipos de contaminación, tanto por compuestos orgánicos como por metales. Estos últimos se acumulan en los primeros centímetros del suelo quedando accesibles para ser absorbidos por las raíces de las plantas. Dada esta problemática, toman relevancia los estudios tendientes al uso de Plant Growth Promoting Rhizobacteria (PGPR) que resulten resistentes a la presencia de metales tóxicos. El género Bacillus ha sido reconocido por su potencial como PGPR, a través de su acción como biofertilizante, bioestimulante y agente de control biológico. Bacillus subtilis subsp. spizizenii crecido en condiciones estáticas en un medio mínimo de sales, glicerol 1% y ácido L-glutámico 55 mM genera un biofilm, cuya adherencia permite su aplicación sobre semillas. El objetivo de este trabajo fue evaluar si el biofilm producido por Bacillus subtilis subsp. spizizenii al ser aplicado sobre semillas de Lactuca sativa variedad Grand Rapid actúa como promotor del crecimiento en plantas crecidas en un sustrato con elevados niveles de cobre. Sobre semillas (con y sin biofilm) y expuestas a concentraciones de cobre entre 50 y 200 ppm Cu (II) se evaluó el porcentaje de germinación GR%, la variación porcentual de la longitud radicular (ER%) y el índice de germinación porcentual relativo (IG%). Los resultados mostraron que, a elevadas concentraciones del metal, el biofilm causó un mayor desarrollo radicular y protegió a la semilla dado que no se observaron signos de toxicidad aguda. En los bioensayos realizados hasta tiempo de cosecha, la inoculación de las semillas con el biofilm mostró un aumento del 72 % en la biomasa radicular y del 62 % en la biomasa aérea con respecto a las plantas no inoculadas. Este efecto positivo se mantuvo aún en presencia de 150 ppm de Cu (II); mostrando aumentos del 32% y 29% respectivamente. Además, todas las plantas inoculadas mostraron mayor altura y desarrollo foliar. El biofilm resultó ser una propuesta innovadora y eficaz como método de aplicación del microorganismo PGPR (Bacillus subtilis subsp spizizenii) para el crecimiento de Lactuca sativa var. Grand Rapid, tanto cuando la planta creció en un sustrato sin contaminar como en un sustrato con elevadas concentraciones de cobre.