2.1. Plant growth-promoting rhizobacteria (PGPR) strain cultures
A mixture of three PGPR strains, i.e. Bacillus subtilis str. B10 (KT921327), B. thuringiensis str. B2 (KU158884) and Enterobacter cloacae str. B16 (KT921429), was used in the present study. They were originally recovered from the rhizosphere of visibly healthy tomato plants. They were previously subjected to biochemical characterization and molecular identification (Ouhaibi-Ben Abdeljalil et al. 2016a). They were selected based on their antifungal and/or plant growth-promoting abilities (Ouhaibi-Ben Abdeljalil et al. 2016b). Their specific traits are summarized in Table 1. They were routinely cultured on Nutrient Agar medium and stock cultures were maintained at -20°C in Luria Bertani (LB) broth amended with 15% glycerol.
2.2. Pythium oligandrum strain inocula
Inocula of the P. oligandrum Po37 strain (oospores-mycelium homogenates) used in planta biocontrol assays were prepared and kindly provided by Biovitis (Saint Etienne de Chomeil, France). Their concentration was adjusted to 104 oospores/mL before being applied in planta.
2.3. Sclerotinia sclerotiorum strain and culture conditions
The target pathogen was originally recovered from tomato plants exhibiting typical symptoms of white mold and/or stem rot disease at the Plant Pathology Laboratory of the Regional Research Centre on Horticulture and Organic Agriculture of Chott-Mariem in Tunisia, as previously described in Ouhaibi et al. (2016a).
For planta assays, the S. sclerotiorum isolate was cultured on Potato Dextrose Agar (PDA) medium amended with Streptomycin sulsulfate00 mg/L) (w/v) and incubated at 28°C duriforays. Mycelia were scraped at the surface of 10 Petri dishes and then mixed in 1 L of sterile distilled water (SDW) (Ouhaibi el al. 2016b). Mycelial fragment density was assessed using a hemacytometer and adjusted to 108 mycelial fragments/mL.
2.4. Assessment of the in vitro antifungal activity of tested microbial agents
2.4.1. Potential of PGPR strains
The ability of the three rhizobacterial strains to suppress S. sclerotiorum mycelial growth was evaluated using the dual culture technique. A loopful of each bioagent (48 hr old culture) was addedina 100 mL Nutrient Broth (NB) medium, then placed on a rotary shaker (150 rpm) and incubated at 28 ± 2°C for 2 days. A 5-day-old fungal plug (6 mm in diameter) was placed on the side of a 90 mm-diameter Petri plate containing a PDA medium. Then, 10 μL taken from 48-h-old bacterial suspension (~108 cells/mL) was deposited into a well (6 mm in diameter, 3 mm in depth) on the opposite side. Plates inoculated with fungal agar plugs and treated with the same volume of SDW served as control. The assays were performed in triplicate.
After incubation at 28°C for 7 days, the diameter of the pathogen colony and the inhibition zone were measured and compared with the untreated control. The percentage of fungal inhibition (FI) was calculated according to Rostami et al. (2013) as follows: GI (%) = (C- t)/ C ×100; where C is the diameter of the pathogen colony in control plates and t is the colony diameter in treated plates.
2.4.2. Potential of Pythium oligandrum
The dual culture technique was also used in this antagonism study. A mycelial plug (6 mm in diameter) taken from 7-day-old P. oligandrum Po37 culture was placed at one side of the Petri plate (90 mm in diameter) and another of S. sclerotiorum (6 mm in diameter), removed from a 5-day-old culture, was placed at the opposite side. For control plates, only pathogen plugs were placed on the PDA medium. Then, the plates were incubated at 25 °C for 5 days.
The diameter of S. sclerotiorum colony was measured and the mycelial growth inhibition percentage was calculated as described above. For the elucidation of hyphal interactions between the antagonist and the target pathogen, samples of mycelium were taken from the zone of interaction of the two agents and examined under an optical microscope followed by the taking of micrographs (Horner et al. 2012).
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2.5. Combined biocontrol treatment in pot experiments
2.5.1. Plant material and growth condition
Tomato cv. Rio Grande seedlings, a cultivar known for its susceptibility to S. sclerotiorum infection, were used for all the in vivo trials. Seeds were surface sterilized with 3% sodium hypochlorite for 3 min and immediately rinsed with SDW three times. Next, they were sowed in alveolus plates (7 × 7 cm) filled with sterilized peat. Plates were placed under controlled conditions with day and night photoperiod and temperatures ranging between 21–18 ± 2°C, respectively. They were watered regularly, to avoid water stress, until reaching the two-true-leaf growth stage.
2.5.2. Combined treatment preparation and co-inoculation assessment
Stock cultures of rhizobacteria were cultured onto Nutrient Agar (NA) medium and incubated at 28°C for 48 h. After the incubation period, a bacterial colony of each strain was suspended in NB (300 mL) and incubated in a rotary shaker (175 rpm) for two days at 28°C. Then, the 48h-old cell culture was diluted in 1L of SDW and adjusted to 108cells/mL (Wu et al. 2014). For the preparation of mixed biocontrol formulations, equal volumes of each rhizobacterial cell suspension were mixed, and the consortium obtained (3B) was tested alone or in a mixture with the P. oligandrum inoculum (3B+Po37).
Tomato seedlings used for the trial were deprived of water two days before the bioassay. Seedling treatment was performed as substrate drench around the stem using 30 mL of the rhizobacterial consortium alone or in a mixture with P. oligandrum. After one week, S. sclerotiorum inoculum (30 mL) was sprayed at the same level on each seedling. The next day, seedlings were transplanted into pots (16 cm in diameter) containing pathogen-infected peat (Benchabane et al. 2000; Le Foch et al. 2003).
Negative controls (uninoculated control seedlings) were treated with SDW only, while positive control plants were inoculated with S. sclerotiorum and treated with SDW or with a commercial fungicide, i.e. Previcur® (Bayer, France, propamocarb hydrochloride 722 g/L) applied at 0.5 mL/L. Uninoculated seedlings challenged with the rhizobacterial consortium and/or P. oligandrum were also used for comparison and the elucidation of their plant growth-promoting potential. This method of inoculation was chosen to avoid any trauma to tomato seedlings following root or stem injury.
Pots were grown under controlled conditions (60-70% relative humidity, 13/11 h light/dark photoperiod at 21/18 ± 2°C light/dark temperature) and the whole experiment was repeated for two consecutive years (2012 and 2013).
2.5.3 Experimental layout
For the experiment, 135 tomato seedlings were used and distributed between nine treatments. The experimental design consisted of a randomized complete block design with 15 seedlings per individual treatment, under the two trials (2012 and 2013). The different treatments were: (i) C: untreated control, (ii) Sc: inoculated with S. sclerotiorum and untreated, (iii) Sc+f: S. sclerotiorum-inoculated and treated with a commercial fungicide, i.e. Previcur®, (iv) Po37: uninoculated and treated with P. oligandrum Po37, (v) Sc+Po37: inoculated with S. sclerotiorum and treated with P. oligandrum, (vi) 3B: uninoculated and treated with the rhizobacterial consortium, (vii) 3B+Po37: uninoculated and treated with P. oligandrum and the rhizobacterial consortium, (viii) Sc+3B: inoculated with S. sclerotiorum and treated with the rhizobacterial consortium, (ix) Sc+3B+Po37: inoculated with S. sclerotiorum and treated with P. oligandrum and the rhizobacterial consortium.
2.5.4. Assessment of the disease suppression ability and the plant growth-promoting potential of tested treatments
At the end of the experiment (two months after the pathogen challenge), plants were uprooted, washed under running tap water to remove peat traces, and air-dried on filter papers. Parameters measured were plant height (cm) and fresh weight of aerial parts and roots (g) (Hassen and Labuschagne 2010). Disease severity on roots was scored using a 0-5 scale where 0= no symptoms, 1= 0-25% root browning, 2 = 26-50% root browning, 3 = 51-75% root browning, 4= 76-100% root browning and 5= plant dieback (Takenaka et al. 2008). Disease incidence (DI) percentage was determined using the following equation:
DI % = (Number of symptomatic plants /The total number of scored plants) ×100.
2.5.5. Assessment of the shifts in the microbial communities following treatments
Characterization of the microbial (fungi and bacteria) communities’ structure and diversity was performed using profiles obtained by the CE-SSCP method, as previously described by Gerbore et al. (2014).
2.5.6. Sampling and DNA extraction
At the end of the experiment and the scoring of growth and severity parameters, 15 plants of each treatment were used for the characterization of the microbial community of roots following tested treatments.
Plants were uprooted gently from each pot to preserve the small feeder roots and were shaken to remove clumps of peat around the roots. Roots were cut into small fragments and crushed until further use for microbial and molecular analyses.
Total DNA was extracted as reported by Godon et al. (1997) with slight modifications. Briefly, root fragments were transferred into 2 ml polypropylene microcentrifuge tubes and kept frozen in a -80°C freezer rack, then lyophilized for 12 h before DNA extraction.
DNA was extracted from 60 mg of ground lyophilized root fragments. A volume of 600 μL of lysis buffer CTAB (1x) was added to each 2 mL tube and incubated at 65°C/1 h. To remove proteins, 400 μL of chloroform-isoamyl alcohol (24 :1, v/v) were added and tubes were shaken at 200 rpm for10 min, then centrifuged at 13,000 rpm for 10 min at 4°C. Aqueous phases were transferred to new 2 ml tubes. Nucleic acids were precipitated by the addition of 330 mL of cold isopropanol and then kept at -20°C overnight. Nucleic acids were recovered by centrifugation at 13,000 rpm at 4°C for 10 min.
Supernatants were discarded and DNA finally were pellets washed with 800 μL of ethanol 70%. After centrifugation at 13,000 rpm at 4°C for 10 min, ethanol was discarded. Then DNA pellets were air-dried and re-suspended into 50 μL of SDW. DNA extracts were then quantified with a nanodrop (ND-1000, Thermo scientific, Labtech) and normalizedat 10 ng/μL.
2.5.7. PCR-SSCP Analyses
For fingerprinting analyses using Single-Strand Conformation Polymorphism (SSCP), pairs of primers recognizing the V5–V6 region of the 16S rRNA gene, i.e. 799f /1115r (Redford et al. 2010), and the mitochondrial large subunit rDNA gene, i.e.ML1/ML2 (White et al.1990) were used respectively for bacteria and fungi.
DNA was amplified by PCR in a reaction mixture (25 μL final volume) consisting of 1 μL of DNA template (10 ng/μL), 2.5 μL of Pfu buffer (10x), 2.5 μL of BSA (Bovine Serum Albumin) at 10 μg/μL (BioLabs), 1 μL of dNTP (10 mM), 0.5 μL of each primer, 0.5 μL of PfuTurboo (Stratagene) and 16.5 μl of sterile distilled water.
The cycling conditions for bacteria were: enzyme activation at 95°C for 2 min; 25 cycles of denaturation at 95°C for 45 s; hybridization at 54°C for 30 s; extension at 72°C for 1 min; and a final extension at 72°C for 10 min. For fungi, the cycling parameters were 95°C for 2 min, followed by 35 cycles at 95°C for 30 s, 58°C for 30 s, 72°C for 1 min and a final extension at 72°C for 10 min. The PCR products were visualized by 2% Tris-Borate-EDTA (TBE) agarose gel electrophoresis before SSCP analysis. The lengths of the fragments yielded by amplification were 250 bp and 350 pb for fungi and bacteria, respectively.
Single-Strand Conformation Polymorphism analyses were performed on an ABI PRISM 3130 genetic analyser (Applied Biosystems) equipped with four 36-cm-long capillaries. One microliter of a PCR product was mixed with 18.8 µl Hi-Di formamide (Applied Biosystems) and 0.2 µl of the Genescan 400 HD ROX standard internal DNA molecular size marker (Applied Biosystems). The sample mixture was denatured at 95°C for 5 min, instantly iced (10 min) and then placed onto the instrument. SSCP is based on the electrophoretic mobility of single-stranded DNA fragments. This mobility is different according to their three-dimensional conformation. Samples were allowed to co-migrate with the fluorescent size standard (GeneScan 400 ROX) to allow the comparison of migration profiles between samples.
2.6. Statistical analyses
Analysis of experimental data was achieved by using one-way analysis of variance (ANOVA) with Statistical Package for the Social Sciences (SPSS) software for Windows version 16.0.
Each of the in vitro or in vivo experiments was repeated twice in time. Data were analyzed according to a completely randomized design in which 9 treatments were tested (i.e. 15 seedlings per individual treatment).
The means were separated using the Student-Newman-Keuls test to identify significant pair-wise differences at P ≤ 0.05. Correlations between disease severity and plant growth parameters were analyzed using the bivariate Pearson’s test at P < 0.01.
SSCP patterns were aligned with Stat Fingerprints (version 2.0) (Michelland et al. 2009) and were gathered in a single numerical database before being statistically described by a global PCA using R software (version 2.15.2).