Bacterial strain
For the bacterial treatments the strain BBC047 from the collection of the Laboratory of Applied Microbiology at CEAZA (Chile) was applied. This strain belongs to the species Bacillus velezensis (formerly Bacillus amyloliquefaciens) and was isolated in the Coquimbo Region from lettuce rhizosphere. Its main characteristics include its biocontrol capacity against several fungal phytopathogens, such as Botrytis cinerea, and the production of IAA12. Strain BBC047 was cultured in LB medium for 36 h at 30° C and 150 rpm, until reaching a concentration of ~ 109 CFU/ml. Bacterial cells were collected and resuspended in sterile water, adjusting concentration to ~ 106 CFU/ml for seedling inoculation or ~ 107 CFU/ml for applications during transplantation and post-transplant.
Nursery experiment with horticultural crops
For the nursery experiment six common horticultural crops were used: lettuce (Lactuca sativa L, cv Corona), broccoli (Brassica oleracea var. italica Plenck, cv Imperial), cabbage (Brassica oleracea L, cv Rinda), basil (Ocimum basilicum L, cv Italiana), tomato (Solanum lycopersicum cv. Cal Ace), and bell pepper (Capsicum annuum L. cv. “Correntin”). For each crop, 2 plug trays (420 cavities for lettuce, 240 cavities for the other crops) of a commercial sowing on peat as substrate were selected randomly and strain BBC047 was applied as follows: the first application when the first pair of true leaf were extended, a second and third application was carried out each separated by 7 days. In each application, approximately 500 ml per plug of the bacterial solution of ~ 106 CFU/ml was used. The other plug trays of the commercial sowing per crop were considered “control”. Seedlings were maintained in the horticulture nursery Servicios y All-macigos S.A., located in Coquimbito (Valle del Elqui) a 11 km de La Serena (S 29°54′27.92″, W 71° 9′41.17″). The trial was carried out under the management established in the nursery company (for details regarding irrigation, fertilization and pest management program see Tables S1).
Evaluation of growth parameters was carried out when seedlings reached transplant size (lettuce 30 days, broccoli 45 days, cabbage 45 days, basil 30 days, tomato 35 days, bell pepper 35 days). For each crop, the height, root length, aerial and root fresh weight of 20 seedlings (randomly picked) per treatment were quantified.
Bell pepper farming experiment
In the greenhouse of the farming and commercial company All-Fresh Ltda, located in Coquimbito (Valle del Elqui) a 11 km de La Serena (S 29°54′27.92″, W 71° 9′41.17″), the bell pepper farming experiment with 4 treatments was installed. With a completely randomized design, where each treatment was composed by 4 replicates with 10 plants each. Treatments were established as follows: T1 = control, plants without application of the bacteria; T2 = plants with application only in the nursery and not after transplanting; T3 = plants with application of the bacteria from transplant to the first harvest; T4 = plants with application of the bacteria from nursery to the first harvest.
During transplantation, seedlings for treatment T3 and T4 were immersed for 1 h in a solution of strain BBC047 (~ 107 CFU/mL), the others in seedlings in water (T1 and T2). The seedlings of the previous trial were transplanted to the greenhouse soil, whose physicochemical characteristics are presented in Table 1. The plantation density was 33,000 plants/hectare.
The treatments T3 and T4 received 2 more bacterial applications via irrigation, 30- and 60-days post-transplant. Per plant, 50 mL of a bacterial solution with 107 CFU/ml were administered 30 days post-transplant and 100 mL 60 days post-transplant. The trial was conducted under the management established in the agricultural company (for details regarding irrigation, fertilization and pest management program see Tables S1).
Growth parameters
In the nursery, the height of the seedlings was evaluated 21 days after sowing (second application). At the time of the transplant, height, leaf area, fresh weight of root and aerial part of 20 seedlings per treatment were quantified.
Post-transplant to the greenhouse, evaluations were made according to the phenological state of the plant, measuring the height of plants from the stem to the bud on the highest branch. At the beginning of flowering, the fully open fertile flowers were counted, excluding floral buds and already fertilized flowers. During the fruit set those fruits formed with a diameter > 2 cm were counted, excluding freshly fertilized flowers. In addition, the survival rate per treatment was recorded 22 days after transplantation. Evaluations were carried out in 40 plants per treatment (corresponding to 4 replicates with 10 plants each) until the end of the trial.
For time reference we use DPI (days post- inoculation, referring to the first inoculation realized to seedlings in the nursery).
Growth kinetics
Using the height data set, three mathematical models were applied to characterize the growth dynamics in each treatment (Gombertz, Logistic, and Weibull Model, details in Table S2), selecting that model with the best fit. Analyses were performed with the statistical package SigmaPlot version 11.0. (Systat Software, Inc., San Jose California USA, www.systatsoftware.com). The goodness of fit of each model was evaluated by statistical tests that include the sum of squared error (SSE), chi-square (χ2) and the root of the mean square error (RMSE) where the lowest values of SSE, χ2 and RMSE were considered (Table S2). The studies of the parameters were performed in triplicate.
Yield
During the trial, 4 times fruits with commercial size and quality (parameters: length, diameter and color) were harvested. In each harvested fruit, length, diameter (both measured with digital Caliper brand Mitutoyo, Japon) and weight were registered. The first harvest considered fruits “in green” and the three remaining “in red” with 80% to 100% of surface turned red. From the first harvest (= harvest 2) “in red” 10 fruits per block were randomly selected for laboratory processing and determination of vitamin C.
Vitamin C content
The content of vitamin C was determined as ascorbic acid, in fruits of the first “red” fruit harvest, according to Islam et al.13 with minor modifications. From each treatment, 10 fruits were lyophilized. Subsequently, in a precipitated beaker, 1.0 g of lyophilized fruit, 0.2 g of oxalic acid (Fluka, Germany) and 30 ml of deionized water were added. The volume obtained was homogenized in Ultraturrax (IKA, model T18, Staufen, Germany) at 9000 RPM for 2 min, then 20 mL of diethyl ether was added (Merck KGaA, Darmstadt, Germany). Finally, the mixture was homogenized for 30 s and centrifuged at 4472 g for 15 min at 4° C.
Then, a 5 mL aliquot of the aqueous phase was taken and mixed with 5 mL of a 4% w / v solution of potassium iodide (KI, Sigma Aldrich, Saint Louis, USA), 2 mL of a solution of acetic acid 10% v / v (CH3COOH Merck KGaA, Darmstadt, Germany) and three drops of a 1% starch solution prepared at the time. Afterwards, a titration was carried out with NBS (N-Bromosuccinimide; C4H4BrNO2, Merck KGaA, Darmstadt, Germany), which was prepared by dissolving 0.2 g of NBS in 1.0 L of deionized water. At the same time, a standard vitamin C solution (Fluka, Germany) was prepared by weighing 51.12 mg of the standard and 2.0 g of oxalic acid, which were dissolved in 250 mL of deionized water. The solutions remained in darkness during their use. The results were expressed in mg Vitamin C / 100 g DW (mg of Vit C / 100 g of dry matter).
Statistical analysis
The comparisons between treatments during the nursery stage were carried out using the Student t test. The statistical analysis of the greenhouse data was performed by one-way ANOVA for the normal data, applying the Kolmogorov–Smirnov test. Subsequently a Fisher LSD test was used for the comparison of means. When the distribution of the data was not normal, the Kruskal–Wallis test was used and later a multiple comparison test of the Agricolae package in R. All analyses were performed using the Agricolae package in R14.
Ethical statements
The study was carried out complying with local and national regulations. In this study was carried out in collaboration with the mentioned farmers, who permitted us to work in their property, installations (nursery, greenhouse) and collect samples from their production (seedlings/ fruits). All plant material was provided by these farmers. No additional permissions or licenses were required.
