Streptomyces lincolnensis strain SZ03 belonged to the bacterial collection of our research laboratory (Laboratoire de Biologie des Systèmes Microbiens – LBSM – ENS de Kouba – Algiers – Algeria). It was isolated and identified by Goudjal et al. (2013) from root of the Saharan native plant Zizyphus lotus, which was well adapted in the arid climatic conditions and poor sandy soil of the Algerian Sahara, where symbiotic bacteria may play a major effect in such adaptation. The strain SZ03 was selected through a preliminary study showing strong antagonistic activities against several soil-borne phytopathogenic fungi.
The pathogen R. solani was obtained from the fungal collection of our research Laboratory (LBSM, ENS-Kouba, Algeria). This strain was isolated and identified by Zamoum et al. (2017) from typical symptoms of the disease observed on the crowns and roots of tomato plants cv. Aïcha harvested in an infected tomato field in Algiers (Algeria).
Production of siderophores
Production of siderophores by strain SZ03 was carried out on chrome azurol (CAS) plates using a method of Sadeghi et al. (2012). Investigated actinobacterial strain was spot inoculated on CAS plates and incubated at 30°C for 7 days. The appearance of clear halo surrounding colonies was considered as positive results for siderophores production. Non-inoculated CAS plates were considered as control (Tamreihao et al. 2016).
Production of hydrogen cyanide (HCN)
Thestrain SZ03 was grown on the King’s B medium supplemented with glycine (4.4g l-1). A sterile filter paper (Whatman n°1) flooded with a 0.5% picric acid solution (prepared in 2% sodium carbonate for one minute) was stuck beneath the plate lid. The Petri dishes were then sealed with parafilm and incubated at 30°C until sporulation. Apparition of orange color on the filter paper indicated positive results for HCN production (Mahesh et al. 2015).
Extracellular enzymatic activities
The chitinolytic activity was investigated using colloidal chitin medium (Ramos-Solano et al. 2010). The strain SZ03 was spot inoculated onto the medium and incubated at 30°C for 5 days. Chitinolytic activity was evaluated by measuring the size of the hydrolytic halo surrounding the bacterial colony.
The cellulases production by strainSZ03 was determined using a modified methods of Sharma and Bajaj (2005) and Nirmala and Sindhu (2011). Actinobacterial strain was inoculated on ISP9 plates containing carboxymethyl cellulose (CMC) as a sole carbon source and then incubated at 30°C for 7 days. The activity of extracellular cellulase was revealed by flooding the colonies with 0.1 ml of red Congo for 15 min before washing three times with NaCl solution (0.1M). The appearance of clear halos surrounding colonies as well as their diameters was noted.
S. lincolnensis strain SZ03 was streaked on a skimmed milk agar plate and incubated at 37°C for 72h. The clear halo around the bacterial colonies was considered as positive result for protease production (Geetha et al. 2002).
The production of β-1,3-glucanase strain SZ03 was investigated according to the method of Singh andal. (1999). A 250 ml-conical flasks containing 50 ml of Tryptic Soya Broth (TSB) medium supplemented with 1% colloidal chitin are inoculated with 1 ml of the spore suspension (≈106ufc ml-1) and incubated at 30°C under permanent agitation (200rpm) for 5 days. The cultures were then centrifuged for 10 min at ×10,000g and 1 ml of the supernatant was mixed with 0.1 ml of a laminarine solution and deposited at 40°C for 1 hour. Three ml of 3,5-dinitrosalicylic acid was added to stop the reaction before boiling for 10 min. The color of was measured at 530 nm. The level of reducing sugars was then calculated from a calibration curve drawn by a glucose concentration gradient.
Production, extraction and antifungal activity
The growth of strain SZ03 and production of antifungal compounds was carried out in a 500 ml-conical flasks containing 100 ml of ISP2 broth and inoculated with 2 ml spore suspension (≈10-6cfu ml-1) before incubation at 30°C with constant shaking (200rpm). The kinetic of growth was carried out by daily sampling of 2 ml of the culture in a 2 ml-Eppendorf tubes. After centrifugation for 10 min at ×4000g and washing with distilled water, the tubes were dried at 105°C for 4 h and the dry cell weight was measured. The antifungal activity was studied by the well method (50μl filtrate per well 6 mm in diameter) against R. solani. The zones of inhibition were measured daily (incubation at 25°C for 5 days) and the incubation period corresponding to the maximum antifungal activity was noted.
In order to determine the best extraction solvent, the antifungal compounds were extracted separately with n-hexane, dichloromethane, n-butanol and ethyl-acetate before antibiography test against R. solani. Thus, 60 ml of filtrate were extracted with 60 ml of the solvent having allowed the best extraction rate. The organic phase was recovered and dehydrated by passage through a filter containing anhydrous sodium sulphate to remove hydrophilic contaminants and dried under vacuum conditions.
Antibiography, bioautography and HPLC purification
The inhibitory activity of the organic phase was tested by antibiography. Eighty µl of the organic extract were deposited on 6 mm in diameter paper discs, dried at 37°C for 30 min then UV sterilized for 30 min. The discs were aseptically placed on the surface of the ISP2 plates (10g of agar l-1) previously inoculated with 6 mm diameter explants from an active culture of R. solani LRS1. Plates were then placed at 4°C for 2h and zones of inhibition were noted in millimeters after incubation at 25°C for 72h.
Thin layer chromatography (TLC) on silica gel (GF254, thickness 0.25 mm, Merck, Germany) was carried out by spot deposition of 60 μl of the organic extract, using the mobile phase B.A.E. (acetic acid/n-butanol/water at 1/3/1, v/v/v) until saturation of the tank. The plates were dried then observed under UV light at 254nm (absorption) to locate and delimit the visible spots. The method of Betina (1973) was used to localize the active spots from the organic extracts on the TLC plates and to determine their number and their frontal ratio (FR). The TLC plates were deposited for 12h at 37°C to completely evaporate the solvent and then placed on a glass support in a polyethylene box (22×24 cm). All the device was then UV sterilized for 30 min.
Fifty ml of supercooled ISP2 medium (10g of agar/l) was distributed as a uniform thin film on the plate. After solidification, 6 mm diameter explants of R. solani culture were placed on the plate margins. The cultures were placed at 4°C for 2h and then incubated at 30°C. After 72h, the zones of inhibition are noted and the FR's were calculated for the active spots according to the formula:
The HPLC purification was carried out in reverse phase: JASCO apparatus, 600 controller, 600 pump, Dual λ Absorption 2487 detector, column C18 (250×7.8 mm UP ODS), mobile phase (methanol-H2O gradient with linear growth from 50 to 100% in 39 min); flow rate of 0.8 ml min-1, UV detection at 220nm. The final purification was obtained after a second re-injection and the activity of the eluates corresponding to each peak was checked by antibiography against R. solani.
Formulation of biofungicides and in vivo biocontrol essay
Spore suspension of strain SZ03 was prepared as used by Goudjal et al. (2014). The strain was inoculated on ISP2 plates and incubated at 30°C for 10 days. Actinobacterial pores were recovered in a Tween20 solution (0.05%) and the spores concentration of was adjusted to ≈106 spores ml−1.
The method of Dhanasekaran et al. (2005) was used for the preparation of R. solani suspension. Potato Dextrose Agar (PDA) plates were inoculates with the pathogen and incubated at 25°C for 6 days. Mycelial fragments were recovered by scraping the surface of the PDA medium and recovered in sterile distilled water. The fungal suspension was adjusted to ≈105cfu ml-1 using the same method cited above.
Formulation of biofongicides
Spores of strain SZ03 were formulated as WTP, SAP and CAP. All of the formulation processes were done under sterile laboratory conditions.
Formulation of wettable-talcum powder
A modified method of Sabaratnam and Traquair (2002) was used for the preparation of WTP biofungicide. Twenty-five ml spore suspension (≈106 spores ml-1) was mixed with 100g of autoclaved talc powder (Fisher Scientific), 10g of CMC and 1.5g of calcium carbonate. The mixture was dried under a laminar flow hood for 12h and finely ground. The WTP biofungicide was weighted and stored in the dark at room temperature.
Formulation of propagules
SAP was prepared using a modified method of Zacky and Ting (2015). The sodium alginate (Sigma-Aldrich) solution (2%) was prepared with sterile distilled water and dissolved under agitation at 45°C. A 25 ml spore suspension (≈10-6cfu ml-1) was mixed with 200 ml sodium alginate solution and pumped using a peristaltic pump through two hypodermics syringes (45 mm length×0.6 mm diameter) and dropped into sterile CaCl2 solution (2.5%) to form and solidify the propagules (0.6-0.8 mm in diameter). Propagules were then dried for 12h under a flow hood, weighted and stored at room temperature.
CAP was prepared using the same process as described above. A 200 ml alginate solution was mixed with 50g autoclaved green clay (0.2 mm in particle diameter). The mixture was dropped through hypodermic syringes (45 mm length×1.2 mm diameter) into the same CaCl2 solution to form propagules (0.8-1.0 mm in diameter), which were dried as described above.
Viability of spores and purity of biofungicides
The viability of spores and purity of the formulated biofungicides were checked before the biocontrol essay and after one year storage at room temperature for the high effective biofungicide. Decimal suspension-dilutions were prepared from each biofungicide and inoculated on ISP2 plates. After incubation at 30°C for 7 days, the SZ03 colonies were authenticated and the possible growth of contaminant microbes was checked.
In vivo biocontrol trials
The tomato (Solanum lycopersicum L.) cv. Aïcha was selected to investigate the potential of formulated biofungicides in the biocontrol of R. solani damping-off. This variety is very wide cultured in fields and presents a great sensitivity to soil-borne phytopathogenic fungi.
In vivo trials were conducted thrice to ensure reproducibility. Five treatments were retained for the in vivo biocontrol essay both in autoclaved and non-autoclaved sandy soils. Negative control (NC): surface-disinfected tomato seeds were sown in sandy soils without any treatment to check the germination rate of the seeds. Positive control (PC): surface-disinfected seeds were sown in sandy soils infected with R. solani. This treatment confirms the fungus virulence. Biocontrol treatment (BT): the formulated biofungicides were used to control the damping-off of seedling in infected soils. Treatments with marketed chemical agent (AT): Efficacy of the formulated biofungicides was compared with commercially available fungicides: Acil 060FS® (with 60g l-1 of Tebuconazole belonging to the Triazoles family). Treatment with the marketed biocontrol agent (ST): Serenade® WP(based on spores of Bacillus subtilis strain QST713). These two marketed fungicides are commonly used against several soil-borne fungi such as Pythium, Fusarium, and Rhizoctonia.
The in vivo biocontrol assay was performed in a five repetitions of complete randomized block design. The experimental unit is represented by a pot containing 6 tomato seeds.
The sandy soil was treated with the formulated biofungicides. SAP was added at a rate of 1g Kg-1 of soil then homogenized using a soil propeller. The CAP was added at the rate of 5g Kg-1 of soil. The WTP was liquefied in sterile distilled water (50g l-1).
The R. solani symptoms were noted using a 5-class scale (Dhanasekaran et al. 2005): no symptom (0); 0–25% of root browning (1); 26–50% of root browning (2); 51 – 75% of root browning (3); 76–100% of root browning (4) and plant death (5). Annotations were converted to DSI using the following equation:
Where: R is the disease rating, N is the number of plants with this rating, H is the highest rating category and T is the total number of plants counted.
In the aim to evaluate the effect of the three formulated products on the plant growth, the root and shoot lengths, and the dry weight were measured for healthy seedlings. To answer the stability and efficacy, the high effective biofungicide was stored at room for one year and in vivo trials were then reproduced as described above.
Data were subjected to variance analysis (ANOVA). Significant differences between means were compared using Fisher’s protected LSD test at P=0.05. Differences were considered significant when P<0.05.