Bacterial isolates
The two bacterial isolates used in this study (SJ4 and SJ19) were isolated in 2018 from the rhizospheric soil of healthy tomato plants cultivated in unheated greenhouses near Jijel, Algeria. Isolation, purification and storage were performed according to Akter et al. (2015). Their identification was carried out on the basis of their molecular characterization. For this, their DNA was extracted and the amplification of the 16S rDNA was carried out using the universal primers fd1 and S17 as described by Bouaoud et al. (2018). The PCR results were sent for sequencing. The NCBI database (https://www.ncbi.nlm.nih.gov) and the EzBioCloud platform (https://www.ezbiocloud.net) were used to compare the sequences and determine the species. Based on this analysis, the bacterial isolates SJ4 and SJ19 were assigned, respectively, to the species Bacillus safensis (GenBank accession number: OK562384) with 100% of similitude and to Acinetobacter calcoaceticus (GenBank accession number: OK562383) with 99% of similitude.
Botrytis cinerea strains
Five strains of B. cinerea were used in this study. Four strains (BCJ1, BCJ2, BCJ3 and BCJ4) were isolated in the Jijel District from tomato plants cultivated in unheated greenhouses and exhibiting grey mould symptoms. Strain BC21 was provided by the Mycology Laboratory of INRAE Avignon, France. All the strains were stored at − 20 °C. For revivification, culture and inoculum production, PDA medium (Difco Laboratory Detroit, USA) was used according to Bouaoud et al. (2018).
Tomato plants
Tomato plants (variety Clodano; Syngenta Seeds, Switzerland) were grown in individual pots in a heated greenhouse and used 6 weeks after sowing, for in planta biocontrol assays.
In vitro antagonistic effect of the bacteria against Botrytis cinerea
Direct confrontation
To evaluate the direct in vitro antagonistic effect against five strains of B. cinerea, the bacterial isolates were placed in contact with the fungal pathogen on the surface of PDA medium (dual culture assay) as described by Xu and Kim (2014). Petri dishes containing only the fungus without bacteria were prepared in parallel with the other plates. After 72 h of incubation at 22 °C, the percentage of mycelial growth inhibition was calculated according to Whipps (1987). Three plates were used per bacterial isolate, and the experiment was repeated three times independently.
Indirect confrontation
The two bacteria were assessed for their ability to produce volatile antifungal compounds according to the protocol described by Barakat et al. (2014). Strain BC21 of B. cinerea was used in these tests. To determine the percentage of mycelial growth inhibition, control plates without the bacteria were prepared and incubated together with the other plates at 22 °C for 72 h. Three plates were used per bacterium, and three independent repetitions of the whole experiment were carried out.
In planta biocontrol assays
To evaluate the in planta antagonistic activity of the two bacterial isolates against B. cinerea, 6-week old tomato plants were used. Two leaves were removed from each plant, and the wounds were inoculated using 10-µl aliquots of a spore suspension of B. cinerea (strain BC21) at 106 spores/ml. The wounds were then immediately treated either with a bacterial suspension (10 µl per wound; 109 CFU /ml) or left untreated as control plants. The two bacterial isolates were applied either singly, or in combination using a 1:1 (V/V) mixture of the cell suspensions. To evaluate a possible effect of the time of application of the bacteria on their protective efficacy, additional modalities were used consisting of plants with an application of the bacteria on the wounds either 1 h before or after the inoculation with B. cinerea. Thus, in total, the biocontrol assay entailed 10 modalities of treatments, and 5 replicated tomato plants were used per modality. The whole assay was carried out three times independently.
Following inoculation, all the plants were incubated in a growth chamber (14-h photoperiod; 22 °C; 90% RH) for 7 days (Bouaoud et al. 2018). The development of disease from each inoculated wound was monitored by measuring the length of stem lesions between the 3rd and the 7th day after inoculation. Then, an area under the disease progress curve (AUDPC) was computed for each wound. The percentage of protection conferred by each bacterial isolate was assessed as follows: Protection (%) = 100 × (average AUDPCcontrol – average AUDPCbacterial treatment)/average AUDPCcontrol (Decognet et al. 2009).
Pathogenicity and induction of hypersensitive response (HR)
In conjunction with the biocontrol assays described above, batches of tomato plants were used to evaluate a possible deleterious effect of the bacterial isolates. For this, leaves were removed from the plants as described above, but the wounds were not inoculated with B. cinerea. Instead, 10-µl aliquots of the bacterial suspensions were applied to the wounds and the plants were incubated in a growth chamber as described above. After 7 days of incubation, the wounds were examined for possible symptoms. Five plants were used for each bacterial isolate, and the whole experiment was carried out three times independently.
Another test was used to evaluate the ability of the two bacteria to induce a hypersensitivity response. Tobacco (Nicotiana tabacum) leaves were infiltrated with suspensions of the two bacterial isolates (109 CFU/ml) according to Morris et al. (2010). Control plants were infiltrated with a suspension of Pseudomonas syringae strain CC94 as a positive control, while plants infiltrated with sterile distilled water were used as a negative control. The plants were then incubated for 48 h in a growth chamber at 22 °C with a 14-h photoperiod, and the plants were examined for possible necroses around the inoculation point, as a sign of hypersensitive response.
Tomato growth promotion
To assess the potential of the two bacteria to enhance tomato growth, tomato seeds (variety Clodano; Syngenta Seeds, Switzerland) were surface-sterilized by tipping its 3 min in 70% ethanol and 4 min in 0.9% (v/v) sodium hypochlorite solution. The seeds were then washed three times using sterile distilled water (Boukaya et al. 2018).
Batches of 25 seeds were then soaked for 2 h at room temperature in a suspension (109 cells/ml) of either SJ4 or SJ19. Control batches were soaked in sterile water. The seeds were then sown in sterile potting soil and placed in a heated glasshouse. Seven days after sowing, seed germination was assessed for each seed batch, and the seedlings were carefully uprooted and washed. Seedling growth was assessed, and a seed vigor index was computed according to Syed-Ab-Rahman et al. (2018) as the mean root length × percentage of seed germination. This assessment was carried out on 20 seedlings for each modality of seed treatment. The seven-day old seedlings were then transplanted to individual (10 × 15 cm) pots containing a sterile potting soil and grown in the glasshouse. Five seedlings were transplanted for each modality of seed treatment. Two days after transplanting, each seedling received a drench treatment with 5 ml of a similar bacterial suspension as the seed it had germinated from (drench with sterile distilled water for the control seedlings) (Mohamed et al. 2020). Four weeks after this treatment, the following morphometric parameters were measured on the plants: root and shoot lengths (cm), stem diameter above the second leaf (mm), number of leaves, and fresh weight of roots and shoots (g). The chlorophyll content of the leaves was measured with the help of a portable chlorophyll meter (Konica Minolta SPAD 502). The whole experiment was carried out three times independently.
Statistical analyses
Analysis of variance (ANOVA) with post hoc test (Newman–Keuls multiple range test) was used to test the growth-promoting in vitro and in planta effects of the two bacterial isolates. However, a Mann–Whitney test was used to check the effect of the two bacterial strains on seed germination. Statistica (StatSoft Inc., Tulsa, USA) was used to conduct all the statistical analyses.