R. solanacearum isolate PHYRS3, the causal agent of potato wilt disease, was isolated in a previous work by Bereika (2008). In brief, samples of diseased potato plants showing wilt symptoms were washed 2–3 times by tap water, followed by sterile water and sterilized with 2% sodium hypochlorite solution; the disinfected plant samples were homogenized with 5 ml of sterile 0.05-M potassium phosphate buffer in a sterilized mortar and pestle. A loop of the resulted suspension was streaked onto 2,3,5-triphenyl tetrazolium chloride (TZC) agar medium in 9.0-cm Petri plates and then incubated at 27 °C for 48 h and examined daily for bacterial growth. The single colony technique was used to obtain pure cultures of the isolated bacteria by growing on the same media used and kept at 4 °C for further studies.
Trichoderma asperellum strain T34
T. asperellum strain T34 was obtained from Biocontrol Technologies S.L. Company Spain and reactivated and maintained on potato dextrose agar (PDA).
The endophytic bacteria were isolated from healthy potato plants, collected from Assiut Governorate, Egypt, during winter 2016. Segments of potato stems (2 cm) were surface-sterilized, using 2% sodium hypochlorite for 3 min, then with 70% ethanol for 30 s. The segments were rinsed by sterile distilled water 3 times and homogenized in 10 ml acetate buffer (pH 5.2). The homogenized plant tissue was taken by a loop and streaked on the surface of Petri dishes (9 cm) containing nutrient agar medium (NA). The plates were incubated at 28 °C for 48 h. Seven bacterial strains were recovered and their pure cultures were kept on NA slants and preserved at 4 °C for further use.
Evaluation of antagonistic potentially of the endophytic bacteria against R. solanacearum
The antagonistic and pathogenic bacteria were grown individually in 250-ml Erlenmeyer conical flasks containing 100 ml of nutrient sucrose broth and incubated at 28 °C for 48 h, and 150 rpm. After incubation, the bacterial growth was centrifuged at 10000×g in sterile microfuge tubes. The supernatants were excluded, and the bacterial cells were collected. Using spectrophotometer (at 600-nm), the bacterial cell density was adjusted to 108 cell/ml. The antagonistic activities of seven isolates of the endophytic bacteria were evaluated using the dual culture against R. solanacearum PHYRS3 based on the method described by Abo-Elyousr et al. (2012) with modification. Briefly, a suspension of R. solanacearum PHYRS3 (100 μl at 108 cell/ml) was spread over agar surface. After drying, 100 μl of each antagonistic isolate (108 cell/ml) were individually pipetted into 5-mm punches in the same agar inoculated with the pathogen. The antibiotic streptomycin (1.0 mg/ml) was used as positive control. After 2 days from incubation at 28 °C, the antibacterial effect of the strains was monitored by measuring the diameter of the inhibition zone (mm). The experiment was repeated twice with four replicates for each treatment.
Identification of the potent antagonistic bacterial strain
Based on the above test, the potent antagonistic bacterial strain was selected for identification via 16 s rRNA sequencing according the following protocol.
The genomic DNA was extracted from the endophytic bacterial strain using the genomic DNA Prep kit (SolGent, Daejeon, Korea) following to the manufacturer’s instructions. The extracted DNA was used as a template for PCR to amplify the 16S rRNA gene. Universal bacterial primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) were used to amplify the nearly complete 16S rRNA gene (Abd-Alla et al. 2012).
The PCR amplification was performed in a 25-μl reaction volume containing 0.4 μM of each primer, 0.75 U of EF-Taq DNA polymerase (SolGent, Daejeon, Korea), 0.2 mM of each d NTP, 10–50 ng of the template DNA, and 1 × EF-Taq reaction buffer. The thermo cycling conditions included an initial denaturation step at 95 °C for 15 min, followed by 30 cycles at 95 °C for 20 s, 50 °C for 40 s, and 72 °C for 1.5 min with a final extension step at 72 °C for 5 min. The PCR product was separated by 1.5% agarose gel electrophoresis containing ethidium bromide with buffer of 0.5× Tris-acetate-EDTA (TAE) and visualized using a UV illuminator (Abd-Alla et al. 2012).
The PCR product was purified according to the manufacturer’s instructions using a SolGent PCR purification kit (SolGent, Daejeon, Korea) (Sanger et al. 1977). The partial 16S rRNA gene sequence was compared to full sequences available in the GenBank database, using a BLAST search (NCBI) for identification of the bacterial strain. Sequences obtained with those retrieved from GenBank database and the sequenced data were deposited in the GenBank under a specific accession number.
Effect of E. cloacae PS14 and T. asperellum T34 on diseases severity under greenhouse conditions
The greenhouse trials were conducted at the Experimental Greenhouse of Department Plant Pathology, Assiut, Egypt, during the 2017 growing season. Healthy potato tubers (Solanum tuberosum L.) cv. Berema were surface-sterilized by soaking in 2% sodium hypochlorite for 3 min, washed thoroughly with sterilized distilled water and planted directly in sterilized 25-cm diameter pots. Pots and soil were sterilized by 5% formalin and left for 15 days before planting. Pots filled with 5-kg sterilized sandy-clay soil (3:1 w/w) were kept in the greenhouse under natural temperatures and photoperiods during the growing season. Plants were fertilized every 15 days with (20 g/pots) of 46% urea and irrigated with water when necessary. Forty-five days after planting, potato cultivar was inoculated with R. solanacearum PHYRS3 (the pathogen) suspended in water at 108 cell/ml, by using an alcohol knife which was inserted 4–5 cm into the soil to cut the roots along two sides and 20 ml of R. solanacearum PHYRS3 suspension was added to each plant around the basis of each plant (Bereika 2008). Infected control plants were inoculated with the pathogen only and treated with 20 ml sterile distilled water, while healthy control did not receive any treatment (without infection and without treatment). After inoculation, the potato cultivar was kept in a moist chamber at 25 °C for 2 days before being transferred to the greenhouse at 25–30 °C. Two days after inoculation, 20 ml from each agent suspension (E. cloacae PS14 or T. asperellum T34) was added surrounding the bases of plants. Four replicates were used for each treatment. After 6 weeks, the development of bacterial wilt symptoms was observed. The disease severity was monitored and the percentage of disease severity (DS%) was estimated using the formula suggested by Kempe and Sequeira (1983).
Disease severity was recorded, using the scale of Kempe and Sequeira (1983) as follows:
0 = no symptoms, 1 = 1–25% of leaves wilted, 2 = 26–50% of leaves wilted, 3 = 51–75% of leaves wilted, 4 = more than 75% and less than 100% of leaves wilted, and 5 = all leaves wilted and died. Disease severity percentage was calculated by the following equation:
DS% = [Σd/d (max × n)] × 100
where “d” is the disease rating on each plant, “d max” is the maximum disease rating possible, and “n” is the total number of plants examined in each replicate.
Application of E. cloacae PS14 and T. asperellum T34 under field conditions
The experiments were carried out at the farm of Faculty of Agriculture, Assiut University, Egypt. Four replicates were used for each treatment and the treatments were distributed in a completely randomized block design. The experimental plot area was 25-m2 containing 5 rows, each row was 4.5-m in length and the distance between each row was 0.5 m. Potato seed tubers (cv. Berema) were sown on the middle of the ridge at 0.4-m apart. After one month from planting, each treatment was added singly to the plant’s soil by drenching around the plants at 20-ml from each microorganism before 48 h of inoculation with R. solanacearum PHYRS3 (108 cell/ml) as described in greenhouse experiment. The control plants were treated by 20-ml distilled water after cutting (Winstead and Kelman 1952) and the disease severity was recorded after 6 weeks after inoculation according to Kurabachew and Wydra (2013). At harvest time (110 days after planting), potato plants of 6 plants from each replicate were pulled for measuring a total tuber yield (kg) per replicate and the yield was expressed as ton/hectare.
The effects of E. cloacae PS14 and T. asperellum T34 on biochemical changes of the inoculated potato plants by R. solanacearum PHYRS3 were investigated. Leaves samples were taken at zero time and at 2, 4, 6, and 8 days after inoculation for determination of total phenoland salicylic acid contents and enzyme activities.
Determination of total phenol and salicylic acid contents
One gram of potato plant’s leaves was crushed in liquid nitrogen and homogenized in 10-ml of 80% methanol. The homogenate was centrifuged at 10,000 g for 30 min at 4 °C. The pellets were wasted after adding ascorbic acid (0.1 g/5 ml). The homogeneous product was evaporated at 65 °C in a rotary evaporator and the process was repeated 3 times each for 5 min. The residues were dissolved in 5 ml of 80% methanol. For each treatment, 4 replicates were used (Rapp and Ziegler 1973). The method of Abo-Elyousr et al. (2008) was used to determine phenol content as milligrams gallic acid/gram plant material. Salicylic acid content was estimated, using a modified method by Dat et al. (1998) as micrograms salicylic acid/gram plant material.
For determination of activities of peroxidase (PO), polyphenol oxidase (PPO), and lipoxygenase (LO), 1 g of fresh potato plants’ leaves was treated with liquid nitrogen and homogenized with 10-ml of 0.1 M Na-acetate buffer (pH 5.2). The mixture was centrifuged at 10,000 g for 30 min at 4 °C and the enzyme activity was determined in the supernatants. Four replicates were used for each treatment. Total protein was estimated based on the method described by Bradford (1976) using Bradford reagent spectrophotometrically at 595-nm using Bovine serum albumin as standard.
Peroxidase (PO) activity
The enzyme activity was determined spectrophotometrically by the method of Putter (1974), using guaiacol as a substrate. The reaction mixture was composed of 0.2 ml supernatant, 1 ml of 0.1 M Na-acetate-buffer (pH 5.2), 0.2 ml of 1% H2O2 and 0.2 ml of 1% guaiacol. The mixture was incubated for 5 min at 25 °C and then measured at 436 nm. The blank was used with extraction buffer. The PO activity was calculated according to the change in absorbance and expressed as enzyme per 1 mg protein.
Polyphenol oxidase (PPO) activity
The enzymatic activity was determined, using the method described by Batra and Kuhn (1975). The reaction mixture was 0.5 ml of the supernatant, 2 ml of 50 mM Sorensen phosphate buffer (g/l: KH2PO4, 6.8; Na2HPO4·2H2O, 8.99; EDTA, 0.372 and distilled water up to 1000 ml, pH was adjusted to 6.5) and 0.5 ml of the substrate Bren catechol (Sigma Aldrich). The mixture was incubated for 2 h in water bath at 37 °C and measured at 410 nm. Activity of PPO = OD at 410 nm/mg protein.
Lipoxygenase (LOX) activity
The enzymatic activity was evaluated according to the protocol of Axelred et al. (1981), the increasing in lipoxygenase activity was measured according to the increase in absorbance at 234-nm resulting from the conjugated double-bond system in the hydroperoxide produced from the substrate, linoleic acid (10-mM sodium linoleate; pH 9). The mixture contained 10 μl of mixture, 20 μl of the substrate, and 1 ml of 50 mM sodium phosphate buffer (pH 6). Absorbance readings were made spectrophotometrically for 3 min at room temperature. A mixture containing buffer and substrate was used as a blank. The activity was measured from the extinction coefficient of 25 mM−1 cm−1.
Possible mode of action exerted by E. cloacae PS14 and T. asperellum T34
Salicylic acid production
Salicylic acid (SA) produced by E. cloacae PS14 and T. asperellum T34 were determined, following the method described by Meyer and Abdallah (1978). The microorganisms were grown in the standard succinate medium at 28 °C for 48-h components from K2HPO4 (11.5 g/l), (NH4)2 SO4 (28.7 g/l), and distilled water up to 1000 ml. The pH was adjusted to 7.0 with 20% NaOH prior to autoclaving. Cells were collected by centrifugation at 8000×g for 5 min and re-suspended in 1 ml of 0.1 M phosphate buffer. A 4-ml of cell-free culture supernatant was acidified with 1-N HCl to pH 2.0 and SA was extracted in CHCl3. Four milliliters of water and 5 μl of 2-M FeCl2 were added to the pooled CHCl3 phases. The absorbance of the purple iron-SA complex, developed in the aqueous phase, was read at 527 nm. A standard curve was prepared with SA dissolved in succinate medium and quantity of SA produced was calculated.
Indole acetic acid production
E. cloacae PS14 and T. asperellum T34 were inoculated by TSB with tryptophan as a precursor (100 μg/ml) on a shaker for 30 min. The culture’s supernatants were collected after centrifugation for 10 min at 2000 rpm and 1 ml of cell-free culture filtrate was mixed with 2 ml of Salkowski reagent (1 ml of 0.5 M FeCl in 50 ml of 35% perchloric acid) and incubated at 28 °C for 30 min. Quantification was done calorimetrically at 530 nm comparing to indole acetic acid production (IAA) standard curve (Nandhini et al. 2012).
Quantitative detection of hydrogen cyanide and siderophore
The antagonistic microorganisms were grown in trypticase soy broth (TSB, Hi media, India). Filter paper was cut into uniform strips of 10-cm-long and 0.5-cm-wide saturated with alkaline picrate solution and placed inside the test tubes in hanging positions. After incubation for 48 h at 28 °C, the sodium picrate in the filter paper was reduced to a reddish compound in proportion to the quantity of hydrocyanic acid evolved. The color was eluted by placing the filter paper in a clean glass test tube containing 10 ml of distilled water and absorbance was measured at 625 nm (Sadasivam and Manickam 1992).
The quantitative estimation of siderophore produced by bioagents was done by CAS-shuttle assay (Schwyn and Neilands 1987). The strains were grown in a succinate medium and incubated at 28 °C for 24 to 30 h and shaking at 120 rpm. After incubation, the fermented broth was taken and centrifuged at 10,000×g for 10 min at 4 °C and the cell-free supernatant was mixed by 0.5 ml CAS solution. The color obtained was determined at 630 nm after 20 min from incubation with reference containing 0.5 ml CAS solution with 0.5 ml un-inoculated succinate medium. The percentage of siderophore units was determined as the proportion of CAS color shifted using the formula% Siderophore units = (Ar − As/Ar) × 100, where Ar = Absorbance of reference at 630 nm (CAS reagent) and AS = Absorbance of sample at 63 nm
Data were initially examined for their normal distribution of errors using Shapiro-Wilk’s W test and for homogeneity of variances using Levene’s test. All statistical analyses were performed with SPSS 22.0 software. The data were analyzed for significance of variation using one-way analysis of variance (ANOVA) for antagonistic capability and for the rest of experiments two-way analysis were used. The least significant difference (LSD) test was used at P < 0.05 to identify the significant differences among the means of the treatments according to Gomez and Gomez (1984).