Isolation, cultivation, and maintenance of Actinobacteria isolates
Wheat (Triticum aestivum L.), maize (Zea mays L.), tomato (Solanum lycopersicum L.) and cucumber (Cucumis sativus L.) roots with adhering soil were harvested from healthy plants grown in individual farms located in Alborz province, Iran in 2015. Three farms were considered for each crop and three root samples were randomly collected from each farm. Also, three soil samples, without plant residues (assigned as bulk soil) were randomly collected from margins (5 meters away from the last row of plants) of each farm. Excess soils were removed following gentle shaking of the roots and the soils that remained attached to the roots were considered as rhizosphere soils. In total, 36 rhizosphere and 36 bulk soil samples were collected. For isolation of Actinobacteria, 2 g soil was diluted in 100 ml of sterile saline solution (0.9% NaCl) and shaken for 30 min. Three serial dilutions (1:100, 1:1000 and 1:10000) were prepared using sterile saline solutions in a total volume of 1 ml. An aliquot of 0.1 ml of each dilution was plated on water agar (18 g/l pH 7.2). The plates were incubated at 29 °C, for 7 days. Representative colonies were selected and streaked on new plates of MYA medium (containing 10 g/l malt extract, 4 g/l yeast extract, 4 g/l glucose and 18 g/l agar, adjusted to pH 7.2) at 29 °C.
Antifungal activity
The procedure of Yuan and Crawford (1995) was used to examine the antagonistic activity of the Actinobacteria isolates against five plant pathogens including Phytophthora drechsleri, P. capsici, Pythium ultimum, Rhizoctonia solani and Fusarium oxysporum. A sterile needle was used to culture one individual bacterial cfu in the center of PDA (potato dextrose agar) plate. Plugs from the growing edge of each pathogen fresh culture were placed on the two sides of the PDA plate. Plates were incubated at 23 °C for 5 days. The pathogen P. capsici strain sa (GenBank accession number MG670447) was kindly provided by Dr. Azimi, Iranian Research Institute of Plant Protection (IRIPP), Tehran, Iran. The growth inhibition percentage was calculated using the formula n = (a − b)/a × 100, where ‘n’ is the growth inhibition percentage, ‘a’ is the fungal growth radius of a control culture (in cm) and ‘b’ is the distance of the pathogen growth in the direction of bacteria (in cm).
Cellulase activity
Carboxymethyl cellulase (CMCase) activity was determined by Mandels-Reese medium with carboxymethyl cellulose (CMC) as the sole carbon source (Majidi et al. 2011). The bacteria were grown on CMC agar containing 0.4 g/l KH2PO4, 0.02 g/l CaCl2, 0.02 g/l NaCl, 0.02 g/l FeSO4. 7H2O, 2.5 g/l CMC, and 15.0 g/l agar. The pH was adjusted to 7.2 with 1 M NaOH. The CMC agar plates were incubated at 29 °C for 7 days to allow the secretion of cellulase. At the end of the incubation, to visualize the hydrolysis zone, the agar medium was flooded with an aqueous solution of Congo red (1 mg/ml) for 20 min. The Congo red solution was then poured off, and the plates were further treated by flooding with 1 M NaCl for 15 min. To indicate the cellulase activity, the diameter of a clear zone around each colony was measured. The ratio of the clear zone diameter to colony diameter was calculated and recorded as cellulase activity.
Chitinase activity
Chitinase production was determined according to the method of Hsu and Lockwood (1975). Bacterial isolates were grown on chitin agar containing 0.4% colloidal chitin and 1.5% agar adjusted to pH 7.2. The colloidal chitin was prepared according to Berger and Reynolds (1958). Plates incubated for 5 days at 29 °C. The ability of chitinase production was shown by a clear halo around colonies. The ratio of the clear zone diameter to colony diameter was calculated and recorded.
Siderophore production
Siderophore production was evaluated according to Alexander and Zuberer (1991) on Chrome Azurol agar (CAS) medium. The CAS agar medium was prepared and distributed in Petri dishes then bacterial isolates were spot-seeded onto the center of the plate and incubated at 29 °C for 7 days. The colonies producing orange halo were considered as siderophore-producing isolates. After three days, the ratio of the halo zone diameter to colony diameter was calculated and recorded.
Phosphate-solubilizing activity
Pikovskaya’s medium (PVK) was used to measure calcium phosphate [Ca3(PO4)2]-solubilizing activity. Sterilized PVK medium with pH 7.2 was poured into the sterilized Petri plates. After solidification of the media, bacterial isolates were spot-seeded onto the center of the plate and incubated at 29 °C for 7 days. Solubilization index was evaluated according to the ratio of the clear zone diameter to colony diameter (Soltani et al., 2010).
Presumptive nitrogen fixation (free-living putative diazotrophs)
The ability of the isolates to growth on solid nitrogen-free medium (NFM) was evaluated according to Dahal et al. 2017. The bacteria were grown on NFM containing K2HPO4 (0.2 g/l), KH2PO4 (0.5 g/l), MgSO4.7H2O (0.2 g/l), FeSO4.7H2O (0.1 g/l), Na2MoO4.2H2O (0.005 g/l), NaCl (0.2 g/l), glucose (10g/l) and 15.0 g/l agar. After 7 days incubation at 29 °C, the growth of each isolate was evaluated and compared with growth on the MYA medium as a positive control.
Indole-3-acetic acid (IAA) productionThe production of IAA was determined based on the method described by Patten and Glick (2002). Actinobacteria isolates were inoculated in 100 ml flasks containing 25 ml TSB (Tryptic soy broth) medium supplemented with 2 mg/ml L-tryptophan at 29 °C for 5 days on a rotary shaker (150 rpm). Bacterial cells were then collected by centrifugation at 10,000 g for 15 min. Two ml of Salkowsky reagent was added to one ml of the supernatant and absorbance of the solution was read at 535 nm in a UV–Vis Spectrophotometer (Cary 300). The IAA concentration was determined using a standard IAA (Sigma-Aldrich) calibration curve.
Genotypic characterization
Isolation of DNA was performed according to the method of Tripathi and Rawal (1998). PCR amplification of the 16S rRNA gene was carried out as described by Chun and Goodfellow (1995). The almost-complete 16S rRNA gene sequences (1400 nt) were deposited in the GenBank database under the accession numbers MG995004 (strain 407), MG995003 (strain 405), MN888928 (strain 432), MN888932 (strain 515), MN888935 (strain 3637), MG984768 (strain 3415), MH266469 (strain 3513), MG984616 (strain 1119), MG995001 (strain 1118), MG995005 (strain 1331), MN493628 (strain 1317), MN888939 (strain 614) and MN888940 (strain 615). The sequences aligned manually with corresponding sequences of available Actinobacteria species drawn from the GenBank, EMBL and DDBJ databases by using BLAST (Altschul et al. 1997). Phylogenetic tree was constructed using the MEGA 5.0 software package (Tamura et al. 2011) based on the method of neighbor-joining. Bootstrap analyses were used to evaluate the stability of relationships based on 1000 resampling.
Greenhouse experiments
Experiment 1: evaluation of plant growth promotion
Surface sterilized cucumber seeds (C. sativus L. Soltan cultivar) were pre-germinated at room temperature for 72 h. Thirty-five seedlings were placed into a 35-cell plug tray (25 × 25 × 2.5 cm deep), with one seedling occupying each cell. The trays were filled with a sterile mixture of field soil and peat moss (1:2). For bacterial treatments, bacterial spore suspension in sterile saline solution was added to autoclaved sand and final cfu/g adjusted to 106. A mixture of sand and sterile saline solution was used as untreated control. Bacterial formulation (one-gram sand/seedling) was added to the mixture of soil and peat moss just before filling the trays. The trays were kept in a greenhouse at 27 °C and 16h/8h brightness/darkness. Soil irrigated with tap water and drain water from each tray was added to the soil. There were four replicates (4 trays) of each bacterial treatment and untreated control and the experiment were arranged in a randomized complete design (CRD). The seeds were watered every 2 days. To evaluate the effects of the bacterial treatment on plant growth, dry and fresh weight of root and shoot and plant height were measured 30 days after planting.
Experiment 2: evaluation of biocontrol activity
The PGPR isolates were selected based on the results of the first experiment and the second experiment was repeated as experiment 1 with 8 replications (8 trays) for each bacterial treatment and control.
After 14 days of bacterial treatment, trays were divided into two groups inoculated and uninoculated. Cucumber seedlings in the inoculated group were inoculated artificially with the pathogen. A plug of the P. capsici fresh culture on PDA media was positioned on the surface of each cell (at a distance of 0.5 cm from the crown) and disease signs were monitored daily. The inoculated seedlings were checked daily and the number of diseased seedlings (with a thin and brown stem or completely death) was recorded. Final disease incidence was determined 16 days after inoculation based on total diseased seedlings percentage. At the end of the experiment (30 days after planting) the plants of the uninoculated group were harvested and the fresh and dry weight of roots and shoots were measured.
Experiment 3: Performance evaluation of strain 1119 at the commercial greenhouse conditions
Greenhouse experiments were conducted in spring 2019 in a 1250 square meter soil-based typical solar commercial greenhouse located in Mohammadabad-e Arab a village in Varamin County, Tehran Province, Iran. The experimental design was a randomized complete block (RCB) with 2 treatments and four replications. Each replicate was a 20-m-long row containing 30 plants. Treatments consisted strain 1119 and untreated as control. Greenhouse soil preparation, use of animal manure, seed (C. sativus L. Soltan cultivar) germination and transplanting in the soil, irrigation, light and temperature management, weed removal, use of fungicide and pest management were carried out according to the typical methods in the area. The deficiency of minor elements was identified by the specialist during the plant growth and development and was treated with foliar spray. Three macronutrients of potassium (K), phosphate (P) and nitrogen (N) were added periodically to the irrigation water according to the expert opinion. Ten days after plants transfer to soil, treatment with 1119 was done. Fifteen days after treatment, the leaves of four plants from each replicate were harvested and pooled, frozen in liquid nitrogen and kept at -80 °C for more analysis. One hundred days after treatment, 15 times (with a two-day interval), fruits were harvested and their fresh weight was recorded.
Fruit quality, dry weight percent and content of sugar and nitrate
To determine the percentage of dry weight, 100 g of the fresh cucumber (4 fruits were selected from each replicate and then 25 g of each fruit was pooled) was cut into 5 mm pieces and dried in an oven at 55°C. For sensorial evaluation, the samples were presented to 40 untrained panelists, (25 males and 15 females in range of 24-50 years old). Each participant was asked to evaluate fruits by scoring characteristics (bitterness, fragility, aroma, juiciness, appearance, flavor and overall acceptance) with grades ranged from 1 to 5, where 1 = weakly accepted and 5 = excellent quality
To estimate total soluble sugar, 200 mg of frozen fruit was added to a centrifuge tube and homogenized with 1.5 mL of 80% ethanol solution in a vortex for 50 s. The sample was centrifuged at 5000 × g for 10 min and the supernatant was put in an oven at 50°C to evaporate ethanol. Respectively, 10 mL of deionized water, 0.47 mL of 0.3 N BaOH and 0.5 mL of 5% Zn (SO4)2 solution was added to the sample. The tube containing sample was centrifuged at 5000 × g for 10 min. Phenol (5%, 0.5 mL) and sulphuric acid (98%, 2.5 mL) were added to 1mL of the supernatant. After 45 min, the absorbance of solutions was read at 485 nm using a spectrophotometer (Cary 300, Agilent, USA). Soluble sugar content was calculated using glucose as a standard curve (Dubois et al., 1956).
To evaluate the nitrate content, 100 mg of frozen fruit was added to a centrifuge tube and homogenized with 1 mL of deionized water and placed in Bain-Marie at 45°C for 60 min. The homogenate was centrifuged at 8000 × g for 10 min and 100 μL of supernatant was mixed to 400 μL deionized water, then 100 μL of the mixture was added to 400 μL salicylic acid (5% salicylic acid in sulfuric acid). After 20 min, 9.5 mL NaOH 2M was added and the absorbance of solution was read at 410 nm using the spectrophotometer. Potassium nitrate (KN03) was used to prepare a standard curve (Cataldo et al., 1975).
Protein, MDA (malondialdehyde) and H2O2 content and antioxidant enzymes activity
Frozen leaves (500 mg fresh weight) were homogenized in Na-Pi buffer containing 10 mg polyvinylpyrrolidone. The homogenate was centrifuged at 8000 × g for 30 min at 4°C and the supernatant was used as a crude enzyme extract (CEE) to determine total protein content (Bradford, 1976) and activity of ascorbate peroxidase (APX: EC 1.11.1.11), catalase (CAT: EC 1.11.3.6) and peroxidase (POX: EC 1.11.1.7). To measure CAT activity, a reaction mixture consisting of 100 mM potassium phosphate buffer (pH 7), deionized water and 70 mM H2O2 diluted in 100 mM potassium phosphate buffer (pH 7) was prepared. The reaction was initiated after adding 20 µL of the CEE, and the enzyme activity was measured by the rate of H2O2 decomposition at 240 nm using spectrophotometer for 3 min at 25°C. (Cakmak and Horst, 1991). The APX activity was determined following the method of Cakmak and Marschner (1992). The reaction mixture (in a volume of 980 µL) composed of 100 mM potassium phosphate buffer (pH 6.8), 2 mM H2O2, and 0.5 mM ascorbate was added to 20 µL of CEE to start the reaction. Ascorbate oxidation at 290 nm was measured to determine the APX activity. The POX activity was assayed following the colorimetric determination of pyrogallol oxidation according to Hasan et al. (2011). The reaction mixture (in a volume of 980 µL) contained 100 mM potassium phosphate (pH 6.8), 20 mM pyrogallol, and 70 mM H2O2 was added to 20 µL of CEE to start the reaction. Enzyme activity was measured following a record of absorbance of colored purpurogallin at 420 nm for 3 min at room temperature. The specific activity of the enzymes was expressed as U/mg protein.
To measure MDA content, lyophilized tissue (100 mg) was extracted with 0.1% trichloroacetic acid (TCA). After centrifuging at 8000 × g for 30 min, 250 µL of supernatant was added to 1 mL of 0.5 % thiobarbituric acid (TBA) prepared in 20 % TCA solution. The mixture was incubated at 95 °C for 30 min, cooled in an ice bath, and then centrifuged at 10,000×g for 15 min. The absorbance of the supernatant was measured at 532 nm and non-specific absorbance was measured at 600 nm. The MDA concentration was defined by its extinction coefficient of 155 mM-1cm-1 (Stewart and Bewley, 1980).
The H2O2 concentration was measured on lyophilized tissue crushed in 5 ml of cold 0.1 % (w/v) TCA and then centrifuged for 15 min at 12000×g in a refrigerated centrifuge. The supernatant (0.5 mL) was added to 100 mM phosphate buffer (pH 7.0) and 1 M of iodate potassium (KI) solution and the absorbance was measured at 390 nm (Loreto and Velikova, 2001). The amount of H2O2 was calculated using a standard curve prepared with known concentrations of H2O2.
qRT-PCR analysis of the defense-related genes
Total RNA was isolated from fresh shoots using TRIzol® (Invitrogen). One microgram RNA was used for synthesizing cDNA after treating with RNase-free DNase I (BioLabs) using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). Gene expression was assayed using Roche LightCycler® 96 real-time PCR system and iQ SYBR Green Supermix kit (BioRad), according to the manual description.
Transcription of each gene was studied by RT-PCR with 0.5 μL of 10 pM of each forward and reverse specific primer designed in this study (Table 1) and 1 μL of template cDNA (100 ng). The following PCR profile was used: 5 min at 95°C; 40 cycles (30 s at 95°C, 30 s at 64°C, 30 s at 72°C); 10 min at 72 °C and recording melting curve. The transcription of the EF (elongation factor) gene was used as an internal control. Gene expression ratio was calculated using the REST 2009 software (Pfaffl et al., 2002).
Statistical analysis
Statistical analysis was performed using analysis of variance (ANOVA) by SPSS windows version 16.0 (SPSS Inc., Chicago, IL, USA). The significance of difference between treatments were analyzed using Duncan test at level of P ≤ 0.05.