Fungal strain and cotton variety
V. dahliae Vd080 (Zhang et al. 2016) which was used in this study was kept on potato dextrose agar (PDA) medium. The mycelia of V. dahliae were obtained from growing the strain in potato dextrose broth (PDB) medium at 28 °C, 160 rpm for 12 days. The susceptible cotton variety (Jimian 11) was used.
Isolation of endophytic bacteria
Cotton plants were collected from Liu he agricultural research farm, Nanjing, China. Endophytic bacteria were isolated, following (Wen et al. 2011) method with some minor modifications. The plants were severed aseptically just above the soil line, washed under tap water and were sterilized with 70% ethanol for 1 min followed by 5% sodium hypochlorite (NaClO) for 5 min then, washed with dH2O for three times. The outer tissues were removed by a knife, and the remaining parts were cut into small fragments. The fragments were sterilized by 70% ethanol and again washed in dH2O for 3 times. The fragments were semi dipped in Luria Bertani (LB) agar medium in the Petri dish and incubated at 28 °C until the colonies appeared around the segments. Distinct colonies were identified based on the following traits: color, form, elevation, margin, diameter, surface, and texture. Every distinct colony was purified by streak culture in Petri dish on LB agar medium in three replicates. A similar procedure without surface sterilization was launched as a negative control. As a positive control, the LB agar medium was inoculated with 0.1 ml of the last rinsed water. The purified colonies were transferred to 2 ml centrifuge tubes had 1 ml LB liquid medium and incubated in a shaker at 37 °C, 160 rpm for 24 h. The isolates were stored at – 20 °C for a short time and in 40% glycerol at – 80°C for long-term storage.
Antagonistic activity and pathogenicity reduction test in plate assay
Antagonistic activity of the isolates was tested on PDA plates in modified agar diffusion assay (Han et al. 2015) with some major modifications. In details, 2 ml of V. dahliae mycelial suspension was mixed with 200 ml of semi-solid (45 °C) 1% PDA medium and was poured into the Petri dish. Two wells were punched into the PDA medium with a sterile Pipette tip. Ten microliters of the overnight culture of isolates were placed into the well, and only LB liquid medium was added as a control. The plates were incubated at 25 °C. Five days later, the zone of inhibition was measured, and the mycelia of fungi from the zone of interaction were removed from the plate carefully, mixed with ddH2O, and the changes in morphology was observed under BX53 Olympus microscope. In the meantime, the mycelia from the interaction zone (treated-mycelia) and the mycelia from isolate-free zone (untreated-mycelia) in Petri dish were transferred to separate tubes, mixed with ddH2O, and their virulence was examined as follows: 2-week-old cotton seedling’s leaves were placed on 0.30% water agar. Ten microliters droplets of bacterial treated or untreated mycelial suspension (106 ml−1) was spotted on the surface of the leaves. Seven days later, the viability of the spotted mycelia was examined under a light microscope, and their germination on the leaves and the necrosis of the spotted area was photographed. Each assay was performed in three replicates.
16S rRNA gene analysis
The isolate was identified by partial analysis of 16S rRNA gene as follows: the total genomic DNA was extracted using TIANamp bacteria DNA Kit (TIANGEN Biotech Co., Ltd., Beijing, China). Universal primers (F27; 5′-AGA GTT TGA TCC TGG CTC AG-3′ and R1492; 5′-GGT TAC CTT GTT ACG ACT T-3′) (Edwards et al. 1989) were used to amplify 16S rRNA gene using PCR (GeneMate series). A total volume of 25 μl reaction mixture was prepared into the PCR tubes as follows: 1 μl DNA template, 0.5 μl forward primer, 0.5 μl reverse primer, 12.5 μl rTaq (Sigma chemical company), and 10.5 μl dH2O. The amplification process was carried out in a thermal cycle with the following reaction conditions: the PCR process began initial denaturation at 95 °C for 1 min, primers annealing 53 °C for 30 s, and primer extension at 72 °C for 1 min. The PCR products were analyzed with 2% gel agarose. The amplified gene was sequenced by (Beijing Liuhe Huada Gene Technology Co. Ltd., China). The sequence was compared with other related sequences in the national center for biotechnology information (NCBI) and was aligned using Clustal X. The phylogenetic tree was constructed using mega X software by the maximum likelihood method with the maximum parsimony options and 1000 bootstrap replicates for the support estimation of each branch. Morphological properties were examined by a general protocol as described by Kamlage (1996). The physiological and biochemical characteristics were examined using the API 20NE system (bioMerieux, Inc., Marcy l’Etoile, France).
The seeds were washed, sterilized with 5.5% sodium hypochlorite (NaClO) for 1min, then washed with ddH2O for three times and were kept in ddH2O for 24 h for germination initiation. To make the bacterial solution, a fresh culture of bacteria was centrifuged, and the pellets were washed by ddH2O for three times. Then, the pellets were dissolved in 0.5% carboxy methylcellulose (CMC) solution, optical density (OD) = 0.3 for seed treatment. The pellets were dissolved in ddH2O (7 × 109 cell ml−1) for soil drench method. For seed treatment, the seeds were dipped in bacterial solution for 4 h. As a control, the seeds were dipped in the same volume of CMC solution without bacteria and were then grown in 300 ml plastic cups filled with sterile vermiculite medium and watered accordingly. Five days after the treatment, the development of the lateral roots were randomly recorded for 10 plants from each treatment. As a soil drench method, 10 ml of the bacterial solution was added to every cup of 1-week-old seedlings grown in 300 ml plastic cups or the same volume of only dH2O was added (control) following the procedure of Selim et al. (2017).
The seedlings were challenged inoculated at the 6th true leaves stage with V. dahliae spore solution as described by Wang et al. (2015) with some modification. 0.5 ml of fungal spore was inoculated in 200 ml PDB medium in 500 ml flask and incubated in 150 rpm, 37 °C for 7 days. Ten milliliters of spore solution (6.107 ml−1) was applied to the soil around the plant in every cup. Fifteen days after the inoculation, the disease severity was assessed for each plant. A scale from 0 to 4 was used to classify plants according to the percentage of plant leaves effected by chlorosis, necrosis or defoliation (0 = no symptoms, 1 = slight marginal chlorosis, 2 = moderate marginal chlorosis, 3 = moderate wilt and visible necrosis, 4 = severe wilt and defoliation). The leaf wilt index (LWI) was calculated as follows: leaf wilt index = Σ (number of plants with a disease scale × value of the corresponding scale)/(total plants rated × the highest scale value) (Li et al. 2012). Biocontrol efficacy was calculated as follows: Biocontrol efficacy = [(LWI of control plants–LWI of treated plants)/disease incidence of control] × 100 (Huang et al. 2006). The experiment was designed in three replicates with 19 to 25 plants (each cup had 1 or 2 seedlings).
Hydrogen peroxide (H2O2) staining in the leaves
Overnight culture of the isolate was centrifuged at 12000 rpm for 10 min, the supernatants were discarded, the pellets were mixed with ddH2O and centrifuged again in 12000 rpm for 10 min (repeated this step for 3 times). Finally, the pellets were mixed with ddH2O (OD = 0.2). In the 6th true leave stage, the bacterial solution was sprayed on the whole plant (only ddH2O was sprayed on the CK group). Five days later, ROS (H2O2) was assessed as described by Kumar et al. (2009). The experiment was designed in three replicates.
Hydrogen peroxide (H2O2) staining in the roots
For hydrogen peroxide staining and to visualize bacterial internalization and their infections in the roots, the cotton seeds were sterilized with NaClO and were treated with the bacterial solution as described above. The seeds were then grown on a 0.5% water agar medium. One-week-old seedlings roots were stained with a solution of 100 mM potassium phosphate buffer, pH 7.0, 2.5 mM 3, 3′-diaminobenzidine tetrachloride (DAB), and 5 units/mL purpurogallin of horseradish peroxidase (Type VI, Sigma Chemical Company) (Pick and Keisari 1980). The roots were excised, placed on a slide contained aniline blue/lactophenol (0.012 g aniline blue dye, 5 g phenol crystals, 10 ml glycerol, 5 ml lactic acid, 5 ml H2O), and examined under bright field microscopy.
Transmission electron microscope
To explore systemic colonization and the precise location of the endophytic isolate, the seeds were dipped in bacterial solution for 4h. As a control, the seeds were dipped in the same volume of CMC solution without bacteria as described previously. Then, the seeds were sown in 500 ml glass flask contained sterilized vermiculite medium (all the procedure was performed under laminar flow hood). After 1 week, the seedlings were harvested, the roots were cut into small pieces and kept in 2.5% glutaraldehyde solution in potassium phosphate buffer (PB), pH 7 until transmission electron microscope analysis (Quadt-Hallmann et al. 1997).
Plant systemic resistant enzymes assessment in the roots
The seeds were surface sterilized by NaClO and were treated with a fresh culture of the bacterial solution and were sown in 300 ml plastic cups as described above. One week after cultivation, the seedlings were inoculated with fungal spores and the roots samples were collected at 0, 24, 72, 96, and 168 h time interval, ground in liquid nitrogen, and kept in − 70°C until use.
Determination of phenylalanine ammonia-lyase (PAL) activity
0.5 g nitrogen ground roots were homogenized with 1 ml of 0.1 M phosphate buffer (pH 7.0), centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatants were used as an enzyme source. One hundred microliters of enzyme extract was mixed with 1.2 ml of 0.1 M borate buffer (pH 9) and 1.5 ml 12 M L-phenyl alanine and was incubated at 30 °C for 30 s. 0.1 ml of HCl at a concentration of 5 M was added to terminate the reaction. The absorbance at OD290 was measured by a spectrophotometer. Enzyme activity was expressed as mmol trans-cinnamic acid min−1 gr−1 of tissues (Dickerson et al. 1984). The experiment was set up for three replicates, and each replicate was measured in parallel for three times.
Determination of peroxidase (POD) activity
0.5 g nitrogen ground roots were homogenized with 1 ml of 0.1 M phosphate buffer (pH 7.0). The mixture was shaken and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatants were used as an enzyme source and were transferred to another centrifuge tube. 1.5 ml of 0.05 M pyrogallol at a concentration of 0.05 M and 0.5 ml of 1% H2O2 were added to 0.5 ml of enzyme extract. The changes in absorbance (OD) at 420 nm were recorded every 60 s for 5 min. The enzyme activity was expressed as changes in OD of the reaction mixture min−1 gr−1 as enzyme activity (Mayer et al. 1966). The experiment was set up three times and every time the optical density was also measured three times.
Determination of polyphenol oxidase (PPO) activity
0.5 g of the stored ground roots were mixed with 1 ml of 0.1 M sodium phosphate buffer (pH 6.5), Shaken and mixed. Afterward, centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatants were transferred to another centrifuge tube and were used as an enzyme source. The reaction mixture contained 200 μl enzyme extract and 1.5 ml of 0.1M sodium phosphate buffer (pH 6.5). To start the reaction, 200 μl of 0.01 M catechol was added, after mixing, the absorbance was measured immediately at 495 nm every 1 min for 5 min. The changes in absorbance at 495 nm min−1 g−1 was used as enzyme activity unit (U) (Hammerschmidt et al. 1982). The experiment was set up for three repetitions and optical density (OD) measurements were also repeated three times.
Total phenolic contents
The total phenolic contents in the roots were assessed as described by Zieslin and Ben-Zaken (1993). In detail, 0.5 g of liquid nitrogen ground roots were homogenized with 5 ml of 80% methanol. One milliliter of methanolic extract was mixed with 5 ml of distilled water and 250 μl of Folin–Cioalteau reagent (1 N), and the solution was kept at 25 °C for 3 min. Then, 1 ml of sodium carbonate saturated solution and 1 ml of sterile distilled water were added and were incubated at 25 °C for 1 h. The OD was measured at 725 nm. The soluble total phenol contents were calculated based on a standard carve from Folin–Cioalteau reagents with the phenol solution and were expressed as catechol equivalent per gram of tissue weight.
Hydrolytic enzymes activity test
To examine cellulase activity of the strain, water agar plates containing 1% CMC were prepared. The strain was spotted in the middle of the plate and incubated at 30 °C for 2 days. The plates were flooded with 0.1% Congo red, and 20 min later, the Congo red was poured off and washed with 1 M NaCl for 10 min. To examine chitinase activity, colloidal chitin was prepared as described by Joe and Sarojini (2017). Agar plates containing 1% colloidal chitin was prepared, and the strain was spotted in the middle of the plate and incubated at 30 °C for 3 days.
Isolation and antifungal activity of the secondary extract
The antifungal compounds produced by the isolate were extracted from the supernatant’s fluid as follows: 500 μl of the bacteria was inoculated in 1 L LB medium and incubated at 37 °C, 160 rpm. The culture was harvested after 48, 72, 96, and 120 h of incubation by centrifugation at 12,000rpm for 15 min. The antifungal ability of the supernatant was tested in Petri dish. Subsequently, the extracts were precipitated by ammonium sulfate 80% saturation and were kept at 4 °C overnight. After centrifugation at 12,000 rpm for 20 min, the precipitates were collected and dissolved in methanol. The methanol was evaporated, and the extracts were dissolved in ddH2O and were freeze-dried. The antifungal activity of the enzyme extracts was examined by agar well diffusion method as described by Balouiri et al. (2016) with some modifications. In details, Petri dishes were filled with PDA medium. Approximately (107 ml−1) V. dahliae spores were spread on the surface of the media with a glass spreader, wells were punched with a sterilized 0.5 cm in diameter Pipette tip. The extracts were filtered (0.2 μm) and were applied to the wells. Only liquid LB medium was applied to the wells as a control. The plates were incubated at 28 °C; 3 days later, the diameter of the inhibited clear zone around the wells was measured and was directed as antifungal activity of the secondary extract. To determine the specific active fractions of the secondary extract, cellulase, and chitinase specific activities were assessed.
Detection of cellulase
The specific activity of the total cellulase was determined by measuring the amount of reducing sugar produced during the enzymatic reaction by dinitrosalicylic acid (DNS) as described by Miller (1959). A reaction mixture contained 0.5 ml enzyme extract, 0.5 ml 0.05 M citrate-phosphate buffer (pH 7.0) and 1.0 ml of 1% (w/v) CMC (Sigma-Aldrich) in 0.05 M citrate-phosphate buffer (pH 7.0) was prepared and incubated in 37 °C for 15 min. 1.5 ml DNS reagent was added to terminate the reaction, boiled for 15 min in a water bath, and the absorbance was measured at 540 nm. The mixture without the reaction enzyme was used as a blank (Saowapar et al. 2014). Each unit of cellulase activity was determined as the amount of the enzyme required for releasing 1 μmole of glucose produce per min per ml of the enzyme solution.
Detection of chitinase
For chitinase activity measurement, colloidal chitin was used as a substrate and was examined by the release of glucose. One milliliter of enzyme solution was mixed with 1 ml of substrate solution (1% colloidal chitin in phosphate buffer, pH 7.0). The reaction mixture was incubated in 50 °C for 60 min, and then 2 ml of 1% 3, 5-dinitrosalicylic acid (DNS) (sigma) was added to stop the reaction. The mixture was boiled in a water bath for 15 min and was cooled to room temperature. The absorbance was measured at 530 nm.
Antagonistic potential of the secondary extract
In the absence of cotton
Fifty milliliters of liquid LB medium was prepared in 150 ml flasks containing 25 μ/ml and 50 μ/ml concentration of the extract. Only dH2O was added in control. The flasks were inoculated with 50 μl of V. dahliae mycelia and incubated at 28 °C, 80 rpm. Six days later, the number of living mycelia was counted using hemocytometer.
In the presence of cotton plant
The greenhouse experiment was conducted to investigate the antagonistic potential of the extracts against V. dahliae mycelia in the presence of cotton. Sterile soil was mixed with V. dahliae microsclerotia with a concentration of 2% (w/w) and placed in 250 ml plastic cups. Twenty-five micrograms per milliliters and 50 μg/ml concentration of the enzyme extract was dissolved in dH2O and were used as the secondary extract solution. Cotton seeds were surface sterilized as above and were grown in the soil in three replicates then were placed at 27 °C with 14 h photoperiod. After germination, 20 ml of the extract solution was poured into every cup (only dH2O was added for control). Diseases severity and leaf wilt index (LWI) were calculated at 6th true leaves stage as described above. The experiment was designed in 3 replicates with 20 plants (every cup had 1 or 2 plants). Subsequently, 4 cups from each treatment were selected randomly and investigated for the quantification of microsclerotia in the soil and their extension into the plants as follows: 100 mg of the soil was pestled in 1 ml of 0.85% NaCl solution and then was diluted with the same concentration of NaCl solution. The mixture was placed on PDA medium amended with 100 μg ml−1 hygromycin B and the number of microsclerotia was assessed following the procedure of Guo et al. (2011). To recover V. dahliae from the plants, the stem was sectioned in 2-cm-long fragments from the base to the first node and washed under tap water. The fragments were surface sterilized with 75% ethanol, then in 1% NaClO for 1 min, followed by 3 washes with dH2O. The fragments were then cut into 2–3 mm cross-sectional cuttings. Ten cuttings were placed on every PDA plate amended with 100 μg ml−1 hygromycin B and incubated at 28 °C for 5 days. Plant colonization ability of V. dahliae where the soil was amended with different concentrations of the extract was calculated by comparing the number of the stem cuttings from which the fungus germinated with those which the fungus was not germinated (Zhang et al. 2011).
Statistical analysis was performed using ANOVA, and the significance of the difference between the treatments was determined by fisher’s protected least significant difference PLSD (statView 5.0.1). P < 0.05 was considered significant.