باکتری‌های اندوفیت گوجه فرنگی بیماری پژمردگی باکتریایی ناشی از Ralstonia solanacearum را سرکوب کرده و متابولیت‌های دفاعی را فعال می‌کنند

Introduction

Ralstonia solanacearum Smith (1) is an important soil borne bacterial plant pathogen with a worldwide distribution and a wide host range. Some of its economically important plant hosts include tomato, potato, eggplant, pepper, tobacco, banana, chili and peanut (2). However, different strategies for the management of bacterial wilt have been used in the world, to date no effective control methods exist for bacterial wilt disease. Plant breeding, field sanitation, crop rotation and use of bactericides have had only limited success (3). Hence, biological control method has been studied for more than 60 years. Various recent studies have indicated that biological control of bacterial wilt disease could be achieved by using antagonistic bacteria (4, 5). One important group of antagonistic bacteria is bacterial endophytes that offer great potential for protection and have beneficial effects on plants. Bacterial endophytes, as the name indicates, are defined as microorganisms that colonize living internal tissues of plants without causing signs of plant diseases. (6, 7). Nawangsih et al. (8) isolated 49 endophytic bacteria, from healthy tomato stems and Staphylococcus epidermidis and Bacillus amyloliquefaciens were able to control the R. solanacearum. Results of other researchers indicated that potential bio-control agents against R. solanacearum are mostly species of Pseudomonas (9, 10, 11). Maji and Chakrabartty (11) showed that combination of Pseudomonas sp. with the pathogen, control the disease and also improved the fresh weight, dry weight and vigor index of the tomato seedlings.Growth promotion of plants by endophytic bacteria may be achieved by production of phytohormones, nitrogen fixation and phosphate solubilization which also protect host plants by the synthesis of antimicrobial substances, production of siderophores and by induction of plant defense mechanisms (12). Induction of plant’s defense genes by prior application of inducing agents is called induced resistance. The defense gene products include peroxidase (PO), polyphenol oxidase (PPO) that catalyze the formation of lignin and phenylalanine ammonia-lyase (PAL) that is involved in phytoalexins and phenolics synthesis (13). Abo-Elyousret al. (14) showed that application of acibenzolar-S-methyl (ASM) and Pseudomonas fluorescens (Pf2) alone or in combination could control the tomato bacterial wilt disease and significant changes in the activities of polyphenol oxidase, glucosidase and peroxidase in tomato after the application of ASM and Pf2 and inoculation with R. solanacearum. Bacterial wilt caused by R. solanacearum is one of the important diseases in field-grown potato and tomato production in Hamedan province. Considering the limitations of the different strategies for the management of bacterial wilt and due to recent awareness about human health and environment, biological method is preferred for management of R. solanacearum. Hence, we studied the effect of native endophytic bacteria against R. solanacearum and followed the changes in PO, PPO and total phenolic compounds as biochemical markers after treatment with certain bio-control agents, in order to correlate the levels of these enzymes to the level of tomato bacterial wilt.

Materials and methods

Isolation of endophytic bacteria: Samples of plant were randomly collected from tomato fields that had been cultivated with tomato for at least two consecutive years, at different villages of Hamedan province, Iran. The selected plants were healthy and their age was fewer than sixty days. Endophytic bacteria were isolated from the internal tissues of roots, leaf and stems. Samples were briefly washed under running tap water and cut in to 0.5-1 cm long pieces. These pieces were disinfected by soaking each in 3% sodium hypochlorite and then adding 1 ml of Tween 20 (polyoxy ethylene esorbitanmonolaurate) for 3–6 min, depending on the tissue then transferred to 70% ethanol for 1–2 min and finally rinsed three times in sterile distilled water. To check sterility, we plated aliquots of the sterile water which was used in the final rinse in 10% TSA (1.5 g/L of triptone, 0.5 g/L of soy peptone, 1.5 g/L of NaCl, 15 g/L of agar, pH 7.3) at 28 ºC for 15 days and checked the plates for microbial growth colony. The tissues were macerated with 6 mL of aqueous solution (0.9 % NaCl) by a sterile mortar and pestle. To allow the complete release of endophytic microorganisms from the host tissue, macerate was incubated at 28 ºC for 3 hours. A dilution serial was made and was placed on five 10% TSA plates for each dilution (10^–1 and 10^–2). The plates were incubated at 28ºC. Colonies were selected according to their growing time and morphology (color, size, shape) on 2, 5, 10, and 15 days post incubation. All bacterial colonies purified and pure isolates were maintained in sterile water at 4°C for further investigation (15).

In vitro antagonistic activity: Antagonism test was done on nutrient agar medium by using agar-diffusion method (16). One-hundred microliters of R. solanacearum suspension containing 10⁸ CFU/ml was spread on NA plates and four holes of 7 mm diameter punched into the agar. In these holes 40 µl supernatant, obtained after centrifugation and filtration by 0.22 lm filter (Millipore) of a 48-h-grown culture of each test antagonist (10⁹ CFU/ml) was added and the plates incubated at 28 C° for 48 h. The effectiveness of strains was evaluated by measuring the radius of inhibition zone around antagonist bacteria. The experiment was performed with a completely randomized design with three replications and repeated twice.

Identification of potentialant agonistic bacteria:The selected antagonists were differentiated based on their reactions to standard biochemical tests from Bergey’s Manual of Systematic Bacteriology (17) and Schaad et al., (18). The antagonists were finally characterized by their 16S rDNA sequencing by Macrogen Inc. Korea. Universal primers 27(8F) and 1492R (19) were used for amplification of 16S rDNA region. The final reaction mixture for polymerase chain reaction (PCR) was 25 µl, contained of 2 µl of magnesium chloride [MgCl₂] (50 mM), 2.5 µl  PCR buffer (10 X), 0.5 µl of dNTPs (10 mM), 1 µl of each forward and reverse primers (10 pmol /μl),15.5 µl sterile deionized water, 1 µl template DNA and 0. 4µl of Taq DNA Polymerase 5unit/μl (CinnaGen, Iran). Amplifications were carried out in an automated thermal cycler (T Gradient-Biometra, Germany) with following conditions: an initial denaturation in 94̊C for 5 min, 35 cycles of denaturation (94̊C for 45s), annealing (60̊C for 50s), extension 72̊C for 2 min and final extension in 72̊C for 10 min. PCR product size was confirmed on 1% agarose gel electrophoresis and photographed under UV light. DNA sequences of antagonist strains were compared with sequences deposited at the National Center for Biotechnology Information (NCBI) using BLAST.The phylogenetic and molecular analyses were performed with all the closely related taxa according to procedure as described previously, using MEGA version 5.1 (20). The stability of the relationship was assessed by bootstrap analysis by performing 1000 re-samplings for the tree topology of the neighbor-joining method.

.Greenhouse evaluation of antagonist isolates for vascular wilt management:Greenhouse evaluation was carried out by using the root soaking method. Three strains of bacteria with the greatest inhibition in in vitro test were tested in the greenhouse to evaluate their ability to control bacterial wilt in planta. A potting medium that contains quartz sand (1:3) was sterilized at 121 °C for 20 min. Tomato seeds (Cv "CALL j N 3") were grown in this mixture soil in a greenhouse at 25-30 °C, watered daily with 1/2 Hoagland’s solution. One-month-old tomato seedlings, which had 5-6 leaves, were selected for experiments. Prior to planting, seedlings roots were dipped in a suspension (10⁹ CFU / ml) of endophytic bacteria for 60 min and transplanted immediately. The non-treated control plants were dipped in distilled water. One week after transplanting, treatments were drenched with pathogen at 10⁷ CFU/g soil. Infected control treatments were only inoculated with R. solanacearum. Distilled water inoculated plants served as control 2. Three replicates of each treatment were performed in a completely randomized design. The experiments were conducted under greenhouse conditions at 24–28 °C and 75–90% relative humidity in 12 h light and 12 h dark conditions. Disease index percentage was recorded using the following formula (21):

Where, ni= number of plants with respective disease rating; vi= disease rating; V= the highest disease rating (5); and N= the numberof plants observed.

Disease rating was calculated as following scale:

1=no symptoms. 2=one leaf wilted. 3=two to three leaves wilted. 4=four or more leaves wilted. 5=whole plant wilted.

Induction of defense mechanisms and experimental design:According to the greenhouse results, best antagonistic bacteria were selected for evaluation of induction of defense enzymes activity. Green house experimental design was performed as above-mentioned. In this test after pathogen inoculation, top leaves of tomatoes were harvested at different time intervals: 0, 18, 24, 48, 72 and 96 h after pathogen inoculation for determination of total phenolic compounds, guaiacol peroxidase(PO) and polyphenol oxidase (PPO) activities. Treatments were replicated three times and the pots were arranged in a completely randomized design.

.Preparation of crude enzyme extracts:Enzyme extract: Leaf tissue from treated and pathogen- inoculated tomato plants was immediately homogenized with liquid nitrogen and mechanically homogenized using a mortar in 4 ml of 50 mM potassium phosphate buffer (pH 7.0) with 0.1 mM EDTA and (4 % (w/v) PVP (poly-vinyl-pyrrolidone). The homogenate was centrifuged at 15,000 g for 30 min at 4 °C. The supernatants were stored at −80 °C or immediately used for determination enzymes activities and total protein. For all enzymes under investigation, each treatment consisted in four replications (plants) and two spectrophotometric readings were taken per replication using a spectrophotometer (WARIAN® CARY 100Conc).

Protein contents of the extracts were determined according to standard procedure of Bradford, (22).To each 50 μl of supernatant, 2.5 ml of Bradford reagent was added and mixed by vortex. After 5 min, readings were performed at 595 nm using a spectrophotometer. Protein concentration, expressed in mg per ml of sample (mg protein ml⁻¹), was determined using Bovine serum albumin (BSA) as the standard curve.

Enzyme activity assaysPeroxidase assay:Guaiacol peroxidase (GPOX) activity was determined at 30 °C by measuring the conversion of guaiacol to tetraguaiacol at 470 nm. The reaction mixture consisted of 2.5 ml of a mixture containing 0.25 percent (v/v) guaiacol in 10mM, 0.1 M phosphate buffer (pH 7.0), and 0.1M hydrogen peroxide H2O2 (2 mM). Enzyme extract (50 µl) was added to initiate the reaction, which was followed colorimetrically at 470 nm at 30s intervals up to 3 min in a spectrophotometer. Activity was expressed as the increase in absorbance at 470 nm (min^-1 mg^-1 of protein). The boiled enzyme extract was used as blank (23).

.Polyphenol oxidase assay (PPO):Polyphenol oxidase activity was determined as per the procedure given by Mayer et al. (24). The reaction mixture consisted of 200 μl of the enzyme extract and 1.5 ml of 0.1 M sodium phosphate buffer (pH 6.5). To start the reaction 200 μl of 0.01 M catechol was added and the activity was expressed as changes in absorbance at 495 nm at 30s intervals for 3 min (min^-1 mg^-1 of protein).

.Extraction and Determination of total phenolic compounds:The amount of total phenolics in extracts was determined with the Folin- Ciocalteu reagent.

0.5 g sample was grinded in 80% ethanol containing HCL 10%. The homogenate was centrifuged at 10000 rpm for 20 minutes and the supernatant was saved. The residue was re-extracted in 80% ethanol, centrifuged and the supernatant was evaporated to dryness using water bath set at 60°C. To determine total phenolic compounds, one ml of the ethanolsupernatant, prepared as described above, was added to 5 ml distilled water and 250 μl Folin-Ciocalteu reagent (Merck) and the solution was incubated at room temperature. After 3 min, 1 ml of a saturated solution of Na₂CO₃ and 1 ml distilled water was added and the reaction mixture was incubated for 1 h. The absorbance of the developed blue color was measured at 725 nm using a blank of water and reagent only. Gallic acid was used as a standard and the total phenolic compounds were expressed as milligram gallic acid per g of leaf fresh weight (25).

Statistical analysis:The data obtained in this study was subjected to the analysis of variance (ANOVA), using the SAS 9.1.3 statistical software, for a completely randomized design and completely randomized block design. The means were separated by Duncan’s multiple range tests with P< 0.05 being accepted as significant.

Results.

In vitro antagonistic activity assays: All bacteria were screened against R. solanacearum by agar-diffusion method. The results of test indicated FS67, FS167 and FS184 strains had maximum inhibition zone and they were selected and used for further studies (Table 1).

Table1- In vitro inhibition of growth of Ralstonia solanacearum by antagonist isolates (FS67, FS167 and FS184) in agar diffusion method.

*The values given are mean (n= 9) with standard deviation. Values with different letter indicate significant differences (P< 0.05) according to Duncan’s multiple range tests.

Identification of potentialantagonistbacteria to R. solanacearum:Based on standard biochemical methods (Table 2) and molecular identification (BLAST search), antagonist bacteriaFS67, FS167 and FS184 were identified as Pseudomonas mosselii, P. fluorescens and P. brassicacearum respectively. The sequences of the amplified products (universal primers 8F and 1492R for 16srRNA) then, were deposited in the GenBnk database and assigned accession numbers: FS67:KY231157, FS167: KY231156 and FS184: KY231158. The Phylogenetic tree showing inter-relationship of these strains with closely related species of the genus Pseudomonas inferred from aligned unambiguous sequences of 16S rRNA gene. FS67, FS167 and FS184 clustered with and belonged to the genus Pseudomonas (Figure 1). Because  the present work is the first report of isolation and antagonistic activity of P. mosselii from tomato plants from Iran, we indicated some important biochemical characteristics of it in Table 2.

Fig. 1- Phylogenetic tree analysis of antagonist strains, FS67, FS167 and FS184 based on the nucleotide sequence of 16S rRNA gene. The branching pattern was generated by the neighbor joining (NJ) method. Bootstrap probability values are indicated at branch-points (tree re-sampled for 1,000 times). Accession numbers are indicated in parentheses.

Table 2-Some important biochemical characteristics of Pseudomonas mosselii strains.

*(+) 90% or more of the strains are positive; (-) negativereaction; (d) 11±89% of the strains are positive.

Greenhouse evaluation of antagonist isolates for bacterial-wilt management:In greenhouse evaluation, FS67and FS167 isolates reduced disease severity of bacterial-wiltwith compared pathogen inoculated treatment significantly (P < 0.05). Disease index (%) was 40, 57.5 and 75.6%in antagonist treatments, FS167, FS67 and FS184 respectively. Disease severity in FS184 treatment was not significantly different from infected control (81.5%, Figure 4).

Fig. 2- Effect of antagonist strains FS67, FS167 and FS184 on disease incidence of Ralstonia solanacearum in greenhouse. R: of Ralstonia solanacearum. Bars represent the mean ± standard error values.

Assay of defense related enzymes

PPO, PO and total phenolic compounds activities analysis in tomato leaves: All antagonistic bacteria influenced the changes in defensive related enzymes and phenolic compounds activities, and the maximum activity of these enzymes occurred in different stages. Data in Figure 3 showed that all treatments indicated significant increases in the activity of PPO when compared to control plants. The activity of PPO were higher than non-inoculated control and reached maximum at 48 h after pathogen inoculation (Figure 3), but it started to decrease only in pathogen-inoculated treatment at 48 h. All antagonistic treatment started decreasing after 72 h. The PO activities of tomato treated with antagonistic bacteria were increased rapidly at 18 h after pathogen inoculation. It declined in all treatments 24 h after pathogen inoculation, except for P.fluorescens treatment; however, it was high level in all antagonistic treatment compared to only pathogen-inoculated control. Maximum PO activity was noticed in the P.fluorescens and R. solancearum inoculated plants compared to all treatments and kept higher level from 18 to 72 h and got to maximum (1.6U/mg protein ml⁻¹⁾) at 72 h(Figure 4).

There was a progressive and significant increase in total phenols activity in plants treated with antagonistic bacteria and pathogen. Its activity dropped at 18 until 72 h after pathogen challenge and then declined at 96 h. Maximum level of total phenols in only pathogen-inoculated plants was at 24 h but its decline was at 48 h after the pathogen inoculation (Figure 5). Total phenols activity was significantly higher in plants treated with P. fluorescens, resulted in the maximum accumulation of phenol (7.16 (mg/gFW)) when compared to the pathogen inoculated control (1.46 (mg/gFW)). 

Fig. 3- Induction of activities of polyphenol oxidase in tomato plants treated with bio-control agents (FS167 and FS67) 0-96 h after R. solancearum inoculation. C: non-inoculated and R: only pathogen inoculated plants.

Fig. 4- Induction of activities of peroxidase in tomato plants treated with bio-control agents (FS167 and FS67) 0-96 h after R. solancearum inoculation. C: non-inoculated and R: only pathogen inoculated plants.

Fig. 5- Induction of activities of total phenols in tomato plants treated with bio-control agents (FS167 and FS67) 0-96 h after R. solancearum inoculation. C: non-inoculated and R: only pathogen inoculated plants.