Pseudomonas putida – Induced response in phenolic profile of tomato seedlings (Solanum lycopersicum L.) infected by Clavibacter michiganensis subsp. michiganensis

Accepted Manuscript Pseudomonas putida - Induced Response in Phenolic Profile of Tomato Seedlings (Solanum lycopersicum L.) Infected by Clavibacter michiganensis subsp. michiganensis Hasan Murat Aksoy, Yilmaz Kaya, Murat Ozturk, Zafer Secgin, Hasan Onder, Ahmet Okumus PII: DOI: Reference:

S1049-9644(16)30218-3 http://dx.doi.org/10.1016/j.biocontrol.2016.11.001 YBCON 3505

To appear in:

Biological Control

Received Date: Revised Date: Accepted Date:

24 February 2016 4 November 2016 5 November 2016

Please cite this article as: Murat Aksoy, H., Kaya, Y., Ozturk, M., Secgin, Z., Onder, H., Okumus, A., Pseudomonas putida - Induced Response in Phenolic Profile of Tomato Seedlings (Solanum lycopersicum L.) Infected by Clavibacter michiganensis subsp. michiganensis, Biological Control (2016), doi: http://dx.doi.org/10.1016/ j.biocontrol.2016.11.001

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Pseudomonas putida - Induced Response in Phenolic Profile of Tomato Seedlings (Solanum lycopersicum L.) Infected by Clavibacter michiganensis subsp. michiganensis Hasan Murat Aksoya*, Yilmaz Kaya b, Murat Ozturka, Zafer Secginb, Hasan Onderc, Ahmet Okumusb a

Ondokuz Mayis University, Agricultural Faculty, Plant Protection Department, Samsun,

55139, Turkey; b

Ondokuz Mayis University, Agricultural Faculty, Plant Biotechnology Department, Samsun,

55139, Turkey; c

Ondokuz Mayis University, Agricultural Faculty, Animal Science Department, Samsun,

55139, Turkey

ABSTRACT Efficacy of Pseudomonas putida (CKPp9) was tested under in vitro and in vivo conditions for their ability to plant growth promoting characteristics in tomato seedlings against Clavibacter michiganensis subsp. michiganensis (CmmGo2) infection. A significant difference was observed between bacterized tomato seedlings with P. putida (CKPp9) and the other treatments (P<0.01). Also, CmmGo2 was detected significant difference the least plant growth parameters for tomato seedlings (P <0.01). To correlate the induction of phenolic compounds by the CKPp9 with disease resistance, qualitative and quantitative modifications of phenolic compounds in tomato seedlings were observed in different treatments. High performance liquid chromatographic (HPLC) analysis of the leaves of the CKPp9-treated, CmmGo2-treated,

and

non-treated

(control)

plants

showed

the

presence

of

chlorogenic acid, caffeic acid, catechin and rutin with varied amounts in the CKPp9 treated as well as CmmGo2-treated and non-treated (control) plants. Maximum accumulation of catechin was observed in plants treated with the CKPp9 strain+CmmGo2 strain which was almost 10 times higher than the CKPp9-treated plants and also significantly high when compared to other treatments. A direct relationship between the level of catechin and seedling survivability was observed. P. putida-mediated induction of phenolic compounds as a biochemical barrier in tomato seedlings against C. michiganensis subsp. michiganensis infection is envisaged. Keywords: Tomato, Induced Response, Phenolic Compounds, Pseudomonas putida, Clavibacter michiganensis subsp. michiganensis 1

1. Introduction Tomato (Solanum lycopersicum L.) which belongs to family Solanaceae is one of the most important crops in the world. Tomatoes have endogenous defense mechanisms which include oxidative enzymes Peroxidase (PO) and Polyphenol Oxidase (PPO) that are generally produced in response to pathogens (Bhonwong et al., 2009). These enzymes catalyze the formation of lignin and other oxidative phenols that contribute to the formation of defense barriers for reinforcing the cell structure (Avdiushko et al., 1993). One of the non-pathogenic bacteria is fluorescent Pseudomonas isolates and several isolates of it is known to suppress the soil-borne pathogens through rhizosphere colonization (Elad and Chet, 1987). Bacterial canker of tomato is caused by the Clavibacter michiganensis subsp. michiganensis (Smith 1910) Davis et al. (1984) is an emerging tomato disease in the world. Also the bacteria as cause of bacterial wilt of tomato, is considered to be one of the most important source of pathogens worldwide (Blank et al., 2015). Due to the presence of phenolic compounds, regular consumption of tomatoes and their products can reduce the risk of the bacterial wilt and canker disease. Tomato bacterial wilt and canker agent, C. michiganensis subsp. michiganensis a very infectious and destructive disease in tomato crop both under glasshouse and field cultures (Utkhede and Koch, 2004). The bacterial wilt and canker of tomato can cause significant damage which may go up to the destruction of 100% crop (Gitaitis et al., 1991; Chang et al., 1992). Until now, many investigations have been performed to find suitable methods for the control of C. michiganensis subsp. michiganensis, but none has been found to be completely effective (de León et al., 2011). The biocontrol is an encouraging way and many researches have been made worldwide against C. michiganensis subsp. michiganensis (Boubyach et al., 2001; Daferera et al., 2003; Boudyach et al., 2004; Amkraz et al., 2010). In the current situation, unfortunately, resistant or highly tolerant cultivars of tomato are still not commercially available, and research/studies focusing on the chemical control of C. michiganensis subsp. michiganensis are scarce and have shown variable results (Gleason et al., 1993; Hausbeck et al., 2000; Theodoro and Maringoni, 2000; de León et al., 2008) In some of these researches, flourescent Pseudomonas isolates were used against C. michiganensis subsp. michiganensis for their biocontrol (Boudyach et al., 2004; Amkraz et al., 2010). Fluorescent Pseudomonas isolates are one of the most studied bacteria within 2

Pseudomonas, and they are ubiquitous bacteria that are common inhabitants of rhizosphere (Weller, 1988; Aksoy, 2006). Certain isolates of this fluorescent Pseudomonas, mainly P. fluorescens and P. putida strains, can stimulate the growth of several crops and by this way, increase their yield. They are known to protect plants from pathogens through various mechanisms such as viz., induced systemic resistance in the host (Maurhofer et al., 1994), antibiotic production (Maurhofer et al., 1995), growth promotion (Schippers et al., 1987) and competition for nutrients (Leeman et al., 1996). Phenolic compounds are natural constituents of all plants and antibiotic phenols have been implicated in plant defense mechanisms (Nicholson and Hammerschmidt, 1992; Kuc, 1995; Baker et al., 2005). Similarly, a pathogeninduced time-dependent increased in soluble phenols such as chlorogenic and cell-bound cinnamic acids in tomato leaves against Clavibacter spp.were shown by Beimen et al. (1992). In recent years, biological control of the bacteria including use of plant extracts (Ameziane et al., 2007; Talibi et al., 2011) and use of plant growth promoting microorganisms such as fluorescent Pseudomonas isolates, Bacillus (Weller, 1988; Verma et al., 2007; Ahmad et al., 2008; Trivedi et al., 2008; Amkraz et al., 2010) become a potential method for the control of tomato diseases. The purpose of this study is to determine the effect of the potential plant growth promoting fluorescent Pseudomonas isolates and C. michiganensis subsp. michiganensis on the level of phenolic compounds of tomato.

2. Material and methods 2.1. Bacterial isolation Two different bacterial groups were used in this study. The first bacterial group, C. michiganensis subsp. michiganensis, named CmmGo2 was provided by the Ondokuz Mayis University, Agricultural Faculty, Plant Protection Department, Bacteriology Lab. (Samsun, Turkey). The second bacterial group was isolated from tomato fields. For this purpose, a total of 26 soil samples were collected from tomato fields in the city of Samsun in Turkey between 20013-2014. About one kg of soil sample was taken to a depth of 0–20 cm per location. The soil samples were stored in sterile polyethylene bags at + 4°C until they are processed in the lab. These soil samples were used for the isolation of fluorescent Pseudomonas isolates. Each soil sample was then sieved through a 1-mm mesh sieve, mixed at a ratio of 1:10 with sterile distilled water and shaken thoroughly for 60 min and then serial dilutions (10 3–104) were prepared. Sample dilutions were placed on King's B medium and were incubated at 24±2°C 3

for 24–48 h. Bacterial colonies on King's B medium were examined under under UV light (366 nm) to identify fluorescent isolates.

2.2. Bacterial identification The morphological, physiological and biochemical characteristics of the fluorescent isolates were determined according to the standardized methods recommended in Bergey’s Manual of Systematic Bacteriology (Lelliott and Stead, 1987; Braun-Kiewnick and Sands, 2001). The isolates were subjected to PCR assay using specie specific primers to confirm phenotypic identification of the isolates. For species-specific PCR, a single colony of each of the fluorescent isolate was used as a source of template DNA. All primers were designed using Geneious 7 software (Biomatters, Auckland, New Zealand) based on identification of Pseudomonas species genoms sequences alignment. BLAST analysis was also performed to confirm the specificities of the primer sets in NCBI database. Details of primer sequences, and predicted size of the amplified product are given in Table 1. The PCR mixture contained 1 µl of DNA in a total volume of 10 µl containing 200 nM concentrations of each primer constituted in 1x BioMix Red (Bioline, Boston, Massachusetts, United States) PCR reaction buffer. The PCR mixture contained 1 µl of DNA in a total volume of 20 µl containing 200 nM of each primer in 1× reaction buffer. PCR amplifications were performed in a T100 thermal cycler (Bio-Rad) with the following touchdown cycling conditions: 4 min at 94°C, 24 cycles of 0.30 min at 94°C, 0.30 min at 65°C with a 1°C decrease per cycle, and 1.30 min at 72°C, followed by 10 cycles of 0.30 min at 94°C, 0.30 min at 56°C, and 1.30 min at 72°C, ending with a 16°C hold. PCR products were monitored on a 1.0% agarose gel in 1x TAE buffer at 100V for about 60 min and checked for their quality, size and yield.

2.3 Disc diffusion test

In vitro to identify antibacterial activity of fluorescent Pseudomonas isolates against CmmGo2 were determined using Kirby-Bauer disc diffusion method as described by Tortora et al. (2001). All the bacterial suspensions were prepared from the overnight-grown culture, 4

and the turbidity of the suspension was adjusted to an optical density at 600 nm (OD600) of 0.7 (∼1 × 109 CFU/ml). 100 µl of the inoculum was aseptically introduced on to the surface of King's B medium plates and sterilized cotton swabs were used for even distribution of the inoculum. Then filter paper discs (Oxoid) were impregnated for each of the fluorescent Pseudomonas isolates. The impregnated discs were placed on King's B medium in petri dishes, and then the plates were incubated at 26±2°C for 24 h. The diameter of the zone (mm) of inhibition produced by each the bacterial isolates were measured with a ruler. The experiment was performed in triplicates.

2.4. Pathogenicity test To test whether the isolates were pathogenic to plants, we assessed plant responses after infiltration of bacterial suspensions at 106-10 7 CFU/ml into the leaves of tomato (Solanum lycopersicum) and tobacco (Nicotiana benthamiana) plants, as described by Oh et al. (2006). To assay non-host hypersensitivity responses, CmmGo2 and sterile distilled water were used as a positive and negative control, respectively.

2.5. Effect of the bacteria on growth of tomato plants To determine the phenolic component and antagonistic activity of Pseudomonas isolates against CmmGo2 in tomato plants, 10 ml bacterial suspensions adjusted to an optical density at 600 nm (OD600) of 0.7 (∼1 × 109 CFU/ml) were used to drench four-week-old tomato seedlings. There were four treatments including (i) tomato seedlings bacterization with fluorescent Pseudomonas isolate (CKPp9)+CmmGo2 (Cmm) (ii) tomato seedlings bacterization with fluorescent Pseudomonas isolate (CKPp9) (iii) tomato seedlings bacterization with CmmGo2 (iv) tomato seedlings with deionized sterile water (control). To fluorescent Pseudomonas isolate applications, the lateral roots of the genotypes (∼5 weeksold) were pruned with a scalpel to facilitate entry of the isolates in order to induce systemic resistance. The pruned roots of the tomato seedlings were soaked in 50 ml of fluorescent Pseudomonas isolate suspension containing 109 CFU/ml for 2 h at room temperature for the treatments (i) and (ii). For the treatment (i), 7 days after inoculation tomato 5

seedling with fluorescent Pseudomonas isolate, CmmGo2 was inoculated onto the same tomato seedlings by the cotyledon clipping method (Xu et al., 2010). For the treatment (iii), CmmGo2 isolate was inoculated onto ∼5-week-old tomato seedlings by the cotyledon clipping method. For the positive control treatment (IV) The cotyledon of the tomato seedlings were clipped with method deionized sterile water. Then, tomato seedlings were planted into 250 ml pots containing a mixture of soil (1:1, soil: sand, by weight). Afterwards, the pots were placed in sterilized plastic saucers that placed on greenhouse benches to avoid contamination by water splashing between pots. The pots were randomly placed in growing chambers set at 24 – 26°C with 8 h dark and 16 h light photoperiod and 70% relative humidity and watered as needed.

2.6. Extraction of phenolic compounds One week after the bacterial applications, a couple of tomato seedlings leaves from each treatment were harvested to determine which phenolic compound released. Then, the samples were transferred quickly in liquid nitrogen tank and stored at -86°C before HPLC analysis of phenolic component. Extraction of phenolic compositions was performed following the standard method with slight modifications (Yang et al., 2015). Each sample (1 g) was mixed thoroughly with 20 ml of 80% methanol-water mixture used as an extraction liquid and vortexed for 10 min. After that, the extract solution was incubated for 1 h at 50˚C with vortexing every hour. Subsequently, it was centrifuged at 12,000 rpm at 4˚C for 10 min. The supernatant was filtered through PTFE hydrophilic syringe filter (0.45 µm pore size, 13 mm diameter) prior to the quantification in HPLC. Dried samples were re-suspended in 1.0 ml HPLC grade methanol by vortexing and stored at 4°C for further analysis.

2.7. HPLC analysis High performance liquid chromatography (HPLC) of the samples was performed according to Singh et al. (2001). The HPLC system (Perkin Elmer Flexar, USA) for accurate LC-MS/MS analysis was analyzed by 6420 triple quadrupole LC-MS/MS system (Agilent Technologies, Santa Clara, CA). Reverse phase chromatographic analysis was carried out using a C-18 reverse phase HPLC column (250 x 4.6 mm id, particle size 5 µm Luna 5 µm C18(2), (Phenomenex, Torrance, USA) at 25°C under isocratic conditions where the 6

concentration of mobile phase was constant throughout the run. Running conditions included an injection volume of 5 µm, mobile phase methanol-0.4% acetic acid (80: 20 v/v), flow rate 1 ml min–1, attenuation 0.03 and detection at 280 nm and 370 nm wavelength. Samples were filtered through PTFE hydrophilic syringe filter (0.45 µm pore size, 13 mm diameter) prior to injection in the sample loop. Caffeic, catechin, chlorogenic, cinnamic, ferulic, gallic, oxalic, rosmarinic, rutin, salicylic, tannic and vanillic acids were used as internal and external standards. Phenolic compounds present in the samples were identified by comparing retention time (Rt) of standards and by coinjection. Concentrations were calculated by comparing peak areas of reference compounds with those in the samples run under the same elution conditions. External standards were used for identification and quantification, and results were presented as microgram per gram of dry weight. All the samples were running three times.

2.8. Statistical analyzing

Kolmogorov-Smirnov One Sample Test results showed that all traits could be assumed normally distributed (P>0.05). Levene variance homogeneity test results indicated that all traits had homoscedasticity (P>0.05). Then, One-Way ANOVA test was applied to the all data, Duncan multiple comparison tests were used to compare the means. Relations between traits were examined with Pearson Correlation Analyses. All analyses were evaluated using SPSS v20.0 with the license of Ondokuz Mayis University.

3. Results and discussion 3.1. Bacterial isolates Five Pseudomonas putida isolates, named CDPp3, CDPp4, CDPp5, CKPp7, and CKPp9 were obtained from 26 soil samples after elimination of suspected colonies. Colonies of the P. putida isolates were whitish-grey, raised, and with diffusible, yellowish-green pigment, and they fluoresced under ultraviolet light (366 nm) on King’s B Medium. All of the isolates produced the expected bands of 222 bp with the primer pairs Pp2F/Pp2R.

7

3.2. Disc diffusion test In vitro to identify antibacterial activity of P. putida isolates against CmmGo2 was determined using Kirby-Bauer disc diffusion method. Only one strain, CKPp9 is determined as potential antibacterial agent based on its capacity to the formation of inhibition halo zone on King’s B medium (Fig. 1). Of the 5 P. putida isolates from soil screened for their ability to inhibit the pathogen. The inhibitory values (mm) of P. putida isolates against CmmGo2 are listed in Table 1. Of which 4 isolates, named CDPp3, CDPp4, CDPp5 and CKPp7 zones of inhibition measuring less than 10 mm in diameter around 25-µl discs. Only one of the five isolates isolates, named CKPp9 showed the highest inhibitory activity against CmmGo2 (Table 2).

3.3. Pathogenicity Test No disease symptoms and hypersensitivity responses were found on the control of tomato and tobacco leaves inoculated with P. putida isolates, and respectively while necrotic symptoms of bacterial canker were observed on CmmGo2-inoculated leaves (Fig. 2).

3.4. Effects of the Bacteria on Growth of Tomato Plants

Tomato seedlings were treated with P. putida CKPp9 and showed stimulatory effects on all plant vegetative parameters (plant height, fresh plant weights, root length, and root weights). A significant difference was observed between bacterized tomato seedlings with P. putida CKPp9 and the other treatments (P<0.01). In contrast, CmmGo2 isolate was detected significant difference the least plant growth parameters for tomato seedlings (P <0.01). Plant growth parameters were higher in bacterized tomato seedlings with P. putida CKPp9 than the other treatments. Analysis of data obtained from pot experiments reveals that the isolate, CKPp9 had significant effects on plant height, fresh plant weights, root length, and root weights as compared to that of the other treatments. Maximum plant height, fresh plant weights, root length, and root weights of 40.69 cm, 34.75 cm, 22.34 cm, 8.46 cm were recorded respectively in the CKPp9-treated tomato seedlings (P <0.01) (Fig. 3; Table 3).

8

3.5. Phenolic contents Quantitative variations were observed in the profile of phenolic compounds among the treatments CKPp9, CKPp9+CmmGo2, CmmGo2, and non-treated control. Quantitative estimation of the identified peaks showed significant variation among different treatments. Among all the four peaks, catechin was only recorded in plants treated with CKPp9 and CKPp9+CmmGo2 whereas it was completely absent in CmmGo2 and non-treated control. However, chlorogenic acid was consistently present in the leaves of all the CKPp9, CKPp9+CmmGo2 and CmmGo2-treated plants as well as non-treated control with varied amounts. The accumulation of catechin (25.65 µg/100 g dry weight, DW) was recorded highest in leaves treated with CKPp9+CmmGo2 which was 10 times higher than non-treated control plants. The accumulation of catechin in leaves induced by CKPp9 strain lower was very low level. In addition, consistent presence of chlorogenic acid, in leaves treated with CKPp9+CmmGo2, was significantly higher than CKPp9, CmmGo2 and non-treated control. Maximum accumulation of chlorogenic acid as observed in CKPp9+CmmGo2-treated leaves (20.98 µg/100 g dry weight, DW) which was 2 times higher than non-treated control plants. Its amount also varied significantly in all the other treatments (Fig. 4; Table 4). We have evaluated the effects of fluorescent Pseudomonas isolates and C. michiganensis subsp. michiganensis on growth of tomato plants and phenolics compounds of the plants. C. michiganensis subsp. michiganensis is a very important bacterial disease agent of tomato (Meletzus et al., 1993). Fluorescent Pseudomonas isolates have been studied for decades for their plant growth-promoting effects through effective suppression of plant pathogens (Marimuthu et al., 2013; Yin et al., 2013; Cabanás et al., 2014; Maksimov et al., 2015). The modes of action that play a role in disease suppression by these bacteria include siderophore-mediated competition foriron, antibiosis, production of lytic enzymes, and induced systemic resistance (ISR) (van Loon et al., 1998; Bakker et al., 2007). In addition, fluorescent Pseudomonas isolates cause greater accumulation of phenolic compounds in hosts tissues against pathogen entrance (van Peer et al., 1991; Wei et al., 1991; Maurhofer et al., 1994). Phenolic compounds are natural constituents of all plants and antibiotic phenols have been implicated in plant defense mechanisms (Kuc, 1995; Baker et al., 2005). Matta et al.(1988) also reported that there is a relationship between phenolic compounds and percentage of plant mortality Post-infection activation of phenol metabolism in the tomato. 9

Nevertheless our findings are also confirm Beiman et al. (1992) who absorved that a pathogen-induced time-dependent increase in soluble phenols such as chlorogenic and cellbound cinnamic acids in tomato leaves against Clavibacter spp. Amkraz et al. (2010) also suggested in their finding that some of flourescent Pseudomonas isolates were used against C. michiganensis subsp. michiganensis for its biocontrol (Amkraz et al., 2010). Until now, there are very few reports are known about the potential biocontrol agents of fluorescent Pseudomonas isolates for controlling bacterial canker of tomato (Boudyach et al., 2001; Umesha, 2006; Lanteigne et al., 2012). To date however, there have been no reports regarding to specifically demonstrate the contribution of antimicrobial metabolite production by fluorescent Pseudomonas isolates for controlling C. michiganensis subsp. michiganensis and preventing the development of the disease it causes for phenolic compounds. The main aim of this study is to determine the effect of the potential plant growth promoting fluorescent Pseudomonas isolates and C. michiganensis subsp. michiganensis on the level of phenolic compounds of tomato, and also the parameters were also investigated to understand the impact of fluorescent Pseudomonas isolates and C. michiganensis subsp. michiganensis on the plant growth. The disease suppressive activity of plant growthpromoting organisms including P. putida is exerted by eliciting a plant-mediated systemic resistance response. Our present study confirmed earlier data published by van Wees et al., (2008) and our finding that the systemic resistance triggered by beneficial microorganisms is generally related to overproduction of plant phenolic compounds from different classes and endows a broad-spectrum resistance to host plants that is effective against different types of attackers. To do this, P. putida CKPp9 was chosen the highest activity against C. michiganensis subsp. michiganensis based on the inhibition zone diameters obtained against the bacterial canker agent. It was treated in vitro anti-bacterial activity of fluorescent Pseudomonas isolates. This approach could be a promising antibacterial alternative to chemicals whose multiple disadvantages constitute a major constraint for both the consumer and the environment. According to our results, Tomato seedlings treatment with P. putida CKPp9 resulted in limited suppression of bacterial canker and wilt disease caused by C. michiganensis subsp. michiganensis. A close relationship between total phenolic concentration such as chlorogenic and cell-bound cinnamic acids in tomato leaves and percentage of plant mortality as observed in the present investigation is in conformity by Beimen et al. (1992). Similarly, this is in

10

agreement with our results. The specific phenolic components of tomato seedlings such as catechins and chlorogenic acid levels had increased to be effective antioxidants. As regards the catechins effect toward C. michiganensis subsp. michiganensis exercised by P. putida CKPp9, the results correlate well with those of our results. Similarly, Sharma et al. (2012) had reported that catechin is responsible for the antimicrobial activity against Bacillus subtilis, Micrococcus luteus, Pseudomonas fluorescens, Staphylococcus epidermidis, and Brevibacterium linens. By comparison, catechin only enhanced when used P. putida CKPp9, and did not enhanced tomato plant in any condition. In our results, the catechins effect on growth inhibitory power was significant, and showed that the catechin has a link with P. putida CKPp9. Also C. michiganensis subsp. michiganensis and P. putida CKPp9 in the tomato plant are known to promote catechin as a phenolic compounds. Also there are two bacteria that may interact through synergistic, antagonistic and additive mechanism relying on the applied plants that promote to increasing catechin dramatically. A positive correlation was found between the antibacterial activity and the content of flavonoids and phenols. These results are promising and may contribute to the future development of natural bio pesticides for the control of bacterial canker of tomato. Further studies are needed to complete and confirm these results in green house level in order to estimate the power of the selected plants to prevent and control the bacterial canker in tomato. Indeed, the possibility of combining biological methods remains an important factor in promoting the use of biological control. This approach can provide powerful and reliable tools for biological control against the major pest problems in crops.

Acknowledgements This study was supported by Ondokuz Mayis University, Project Management Office with project number of PYO.ZRT.1901.14.008. References Ahmad, F., Ahmad, I., Khan, M.S., 2008. Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol. Res. 163, 173–181. Aksoy, H.M., 2006. Toprak Kökenli Fungal Patojenlerin Fluoresan Pseudomonadlarla Biyolojik Mücadelesi. Ondokuz Mayıs Üniversitesi Ziraat Fakültesi Dergisi. 21 (3), 364– 369. 11

Amkraz, N., Boudyach, E.H., Boubaker, H., Bouizgarne, B., Ait Ben Aoumar, A., 2010. Screening for fluorescent pseudomonades, isolated from the rhizosphere of tomato, for antagonistic activity toward Clavibacter michiganensis subsp. michiganensis. World J. Microbiol. Biotechnol. 26 (6), 1059–1065. Ameziane, N., Boubaker, H., Boudyach, E.H., Msanda, F., Jilal, A., Ait Benaoumar, A., 2007. Antifungal activity of Moroccan plants against citrus fruit pathogens. Agron. Sustain. Dev. 27 (3), 73–278. Avdiushko, S.A., Ye, X.S., Kuc, J., 1993. Detection of several enzymatic activities in leaf prints of cucumber plant. Physiological and Molecular Plant Pathology. 42, 441–454. Baker, C.J., Whitaker, B.D., Mock, N.M., Rice, C., Deahl, K.L., Roberts, D.P., Ueng, P.P. and Averyanov, A.A., 2005. Differential induction of extracellular bioactive phenolics that are redox sensitive. Physiol. Mol. Plant Pathol. 66, 90–98. Bakker, P.A., Pieterse, C.M., van Loon, L.C., 2007. Induced systemic resistance by fluorescent Pseudomonas spp. Phytopathology. 97 (2), 239-43. Beimen, A., Bermpohl, A., Meletzus, D., Eichenlaub, R., Barz, W., 1992. Accumulation of phenolic compounds in leaves of tomato after infection with Clavibacter michiganes sp. michiganes strains differing in virulence. Zeityschrift fur Naturforschung, Sektion I. Biosciences. 47, 898–909. Bhonwong, A., Stout, M.J., Attajarusit J., Tantasawat, P., 2009. Defensive role of tomato polyphenol oxidases against cotton bollworm (Helicoverpa armigera) and beet armyworm (Spodoptera exigua). J. Chem. Ecol. 35, 28–38. Blank, L., Cohen, Y., Bomshein, M., Shulhani, R., Sofer, M., Shtienberg, D., 2015. Variables associated with severity of bacterial canker and Wilt caused by Clavibacter michiganensis

subsp.

michiganensis

in

tomato

greenhouse.

Phytopathology.

http://dx.doi.org/10.1094/PHYTO-07-15-0159-R. Boubyach, E.H., Fatmi, M., Akhayat, O., Benizri, E., Ait Ben Aoumar, A., 2001. Selection of antagonistic bacteria of Clavibacter michiganensis subsp. michiganensis and evaluation of their efficiency against bacterial canker of tomato. Biocontrol. Sci. Tech. 11, 141–149. Boudyach, E.H., Fatmi, M., Boubaker, H., Ait Ben Aoumar, A., Akhayat, O., 2004. Effectiveness of fluorescent Pseudomonads strains HF22 and HF142 to control bacterial canker of tomato. J. Food. Agric. Environ. 2 (34), 115–120. Braun-Kiewnick, A., Sands, D.C., 2001: II. Gram-negative bacteria: Pseudomonas. In: N.W.

Schaad, J.B. Jones, W. Chun (eds.): Laboratory Guide for Identification of Plant 12

Pathogenic Bacteria, pp. 84-119. APS Press, St. Paul, MN, USA. 84–120. Cabanás, C.G.L., Schilirò, E., Corredor, A.V., Blanco, J.M., 2014. The biocontrol endophytic bacterium Pseudomonas fluorescens PICF7 induces systemic defense responses in aerial tissues upon colonization of olive roots. Frontiers in Microbiology. 5, 1-14. Chang, R.J., Ries, S.M., Pataky, J.K., 1992. Reduction in yield of processing tomatoes and incidence of bacterial canker. Plant Disease. 76, 805–809. Daferera, D.J., Ziogas, B.N., Polissiou, M.G., 2003. The effectiveness of plant essential oils on the growth of Botrytis cinerea, Fusarium sp. And Clavibacter michiganensis subsp. michiganensis. Crop Prot. 22 (1), 39–44. Davis, M.J., Gillaspie, A.G.Jr, Vidaver, A.K., Harris, R.W., 1984. Clavibacter: a new genus containing some phytopathogenic Coryneform bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. & Clavibacter xyli subsp. cynodontis subsp. nov., pathogens that

cause Ratoon stunting disease of sugarcane and Bermudagrass stunting disease. International Journal of Systemic Bacteriology. 34, 107–17.e León, L., Siverio, F., López, M.M., Rodríguez, A., 2008. Comparative efficiency of chemical compounds for in vitro and in vivo activity against Clavibacter michiganensis

subsp. michiganensis, the

causal agent of tomato bacterial canker. Crop Prot. 27, 1277–1283. de León, L., Siverio, F., Lopez, M.M., Rodriguez, A., 2011. Clavibacter michiganensis subsp. michiganensis, a seedborne tomato pathogen: Healthy seeds are still the goal. Plant Disease. 95, 1328–1338. Elad, Y., Chet, I., 1987. Possible role of competition for nutrient in biocontrol of Pythium damping-off by bacteria. Phytopathology. 77, 190–195 Gleason, M.L., Gitaitis, R.D., Ricker, M.D., 1993. Recent progress in understanding and controlling bacterial canker of tomato in eastern North America. Plant Dis. 77, 1069– 1076. Gitaitis, R.D., Beaver, R., Voloudakis, A., 1991. Detection of Clavibacter michiganensis subsp. michiganensis in symptomless tomato transplants. Plant Disease. 75, 834–838. Hausbeck, M.K., Bell, J., Medina-Mora, C., Podolsky, R., Fulbright, D.W., 2000. Effect of bactericides on population sizes and spread of Clavibacter michiganensis subsp. michiganensis on tomatoes in the greenhouse and on disease development and crop yield in the field. Phytopathology. 90, 38–44. Kuc, J., 1995. Induced systemic resistance-an overview. In: Hammerschmidt R, Kuc J, editors. Induced resistance to disease in plants. Amsterdam, The Netherlands: Kluwer 13

Publishers. 169–175. Lanteigne, C., Gadkar, T., Wallon, V.J., Novinscak, T.A., Filion, M., 2012. Production of DAPG and HCN by Pseudomonas sp. LBUM300 Contributes to the Biological Control of Bacterial Canker of Tomato. Biological Control. 102, 967–973. Leeman, M., Den Ouden, F.M., Van Pelt, J.A., Dirkx, F.P.M., Stejil, H., Bakker, P.A.H.M., Schippers, B., 1996. Iron availability affects induction of systemic resistance to Fusarium wilt of radish by Pseudomonas fluorescens. Phytopathology. 86, 149–155. Lelliott, R.A., Stead, D.E., 1987. Methods for Diagnosis of Bacterial Diseases of Plants. Blackwell,

Oxford,

United

Kingdom.

2,

216.

Maksimov,

I.V., Veselova, S.V.,

Nuzhnaya, T.V., Sarvarova, E.R., Khairullin, R.M., 2015. Plant growth-promoting bacteria in regulation of plant resistance to stress factors. Russian Journal of Plant Physiology. 62, 715–726. Marimuthu, S., Ramamoorthy, V., Samiyappan, R., Subbian, P., 2013. Intercropping System with Combined Application of Azospirillum and Pseudomonas fluorescens Reduces Root Rot Incidence Caused by Rhizoctonia bataticola and Increases Seed Cotton Yield. Journal of Phytopathology. 161 (6), 405-411. Matta, A., Ferraris, L., Abbattista, G.I., 1988. Variations of phenoloxidase activities and the consequence of stress induced resistance to Fusarium wilt of tomato. Phytopathology. 122, 45-53. Maurhofer, M., Hase, C., Meuwly, P., Metraux, J.P., Defago, G., 1994. Induction of systemic resistance of tobacco to tobacco necrosis virus by the root-colonizing Pseudomonas fluorescens strain CHA0: Influence of the gacA gene and of pyoverdine production. J Phytopathol. 84, 139–146. Maurhofer, M., Keel, C., Haas, D., Defago, G., 1995. Influence of plant species on disease suppression by Pseudomonas fluorescens CHA0 with enhanced antibiotic production. Plant Pathol. 44, 44–50. Meletzus, D., Bermpohl, A., Dreier, J., Eichenlaub, R., 1993. Evidence for plasmid – encoded virulence

factors

in

the

phytopathogenic

bacterium Clavibacter

michiganensis subsp. michiganensis NCPPB382. J. Bacteriol. 175, 2131–2136.

Nicholson, R.L., Hammerschmidt, R., 1992. Phenolic compounds and their role in disease resistance. Ann. Rev. Phytopathol. 30, 369–389. Oh, S.K., Lee, S., Chung, E., Park, J.M., Yu, S.H., Ryu, C.M., Choi, D., 2006. Insight into Types I and II nonhost resistance using expression patterns of defense-related genes in 14

tobacco. Planta. 223, 1101–1107. Sharma, A., Gupta, S., Sarethy, I.P., Dang, S., Gabrani, R., 2012. Green tea extract: Possible mechanism and antibacterial activity on skin pathogens. Food Chemistry. 135, 672–675. Schippers, B., Bakker, A.W., Bakker, P.A.H.M., 1987. Interactions of deleterious and beneficial rhizosphere microorganisms and the effect of cropping practices. Ann. Rev. Phytopathol. 25, 339–358. Smith, E.F., 1910. A new tomato disease of economic importance. Science (N.S.) 31, 794796.Talibi, I., Amkraz, N., Askarne, L., Msanda, F., Saadi, B., Boudyach, E.H., Ait Ben Aoumar, A., 2011. Antibacterial activity of Moroccan plants extracts against Clavibacter michiganensis subsp. michiganensis, the causal agent of tomatoes’ bacterial canker. J. Med. Plants Res. 5, 4332–4338. Theodoro, G., Maringoni, A.C., 2000. In vitro and in vivo action of chemicals on Clavibacter michiganensis subsp. michiganensis, causal agent of the bacterial canker of tomato. Sci. Agric. 57, 439-443. Trivedi, P., Pandey, A., Palni, L.M.S., 2008. In vitro evaluation of antagonistic properties of Pseudomonas corrugates. Microbiol. Res. 163, 329–336. Tortora, G.J., Funke, B.R., Case, C.L., 2001. Microbiology: An Introduction. 7th Edn., Benjamin Cummings, UK. Umesha, S., 2006. Occurrence of bacterial canker in tomato fields of Karnataka and effect of biological seed treatment on disease incidence. Crop Prot. 25, 375–381. Utkhede, R., Koch, C., 2004. Biological treatments to control bacterial canker of greenhouse tomatoes. Bio. Control. 4 (9), 305–313. van Loon, L.C., Bakker, P.A.H.M., Pieterse, C.M.J., 1998. Systemic resistance induced by rhizosphere bacteria. Annu. Rev. Phytopathol. 36, 453–83.van Peer, R., Niemann, G.J., Schippers, B., 1991. Induced resistance and phytoalexin accumulation in biological control of Fusarium wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopathology. 81, 728–734. van Wees, S.C.M., van der Ent, S., Pieterse, C.M.J., 2008. Plant immune responses triggered by beneficial microbes. Current Opinion in Plant Biology. 11, 443–448. Wei, G., Kloepper, J.W., Tuzun, S. 1991. Induction of systemic resistance of cucumber to Colletotrichum orbiculare by select strains of plant growth-promoting rhizobacteria. Phytopathology. 81, 1508–1512. Weller, D.M., 1988. Biological control of soil borne pathogens in the rhizosphere with 15

bacteria. Ann. Rev. Phytopathol. 26, 379–407. Verma, R., Naosekpam, A.S., Kumar, S., Prasad, R., Shanmugam, V., 2007. Influence of soil

reaction on diversity and antifungal activity of fluorescent pseudomonads in crop rhizospheres. Bioresour Technol. 98, 1346–1352. Xu, X., Miller, S.A., Baysal-Gurel F., Gartemann, K.H., Eichenlaub, R., Rajashekara, G., 2010. Bioluminescence imaging of Clavibacter michiganensis subsp. michiganensis infection of tomato seeds and plants. Appl. Environ. Microbiol. 76, 3978–3988.

Yang, S.C., Arasu, M.V., Chun, J.H., Jang, Y.S., Lee, Y.H., Kim, I.H., Lee, K.T., Hong, S.T., Kim, S.J., 2015. Identification and Determination of Phenolic Compounds in Rapeseed Meals (Brassica napus L.). Journal of Agricultural Chemistry and Environment. 4, 14–23.

Yin, D., Wang, N., Xia, F., Li, Q., Wang, W., 2013. Impact of biocontrol agents Pseudomonas fluorescens 2P24 and CPF10 on the bacterial community in the cucumber rhizosphere. European Journal of Soil Biology. 59, 36–42.

16

Mean diameter zone of inhibition (mm)

50 40 30 20 10 0

a

b

b

b

b

CDPp3 CDPp4 CDPp5 CKPp7 CKPp9 Pseudomonas putida isolates a

b

Fig. 1. a. Antibacterial activity of Pseudomonas putida isolates against CmmGo2 [ Mean values ± SE (Standart Error of the mean) with same letters are not significantly different; Duncan’s Multiple Comparison Test; P<0.01] and b. Antibacterial activity of Pseudomonas putida isolates

against CmmGo2 on King’s B medium under UV transilluminator.

Fig.2. a. Hypersensitive response in tobacco leaves and b. Necrotic symptoms in tomato leaves by Clavibacter michiganensis subsp. michiganensis, CmmGo2.

40

aPlant fresh weight/Root weight

35 Weight (g)

30

b

25 c

20

d

15 10

a

b

Plant fresh weight c

5

d

Root weigth

0

Treatment

a a

45 Height/Length (cm)

40

Plant height/Root length

b

35

c

30 25 20

b

d

a c

d

15 10

Plant heigth Root length

5 0

Treatment

b Fig. 3. The effect of Pseudomonas putida CKPp9 alone or in combination with CmmGo2 on: a. Plant fresh weight/Root weight; b. Plant height/Root height ((Different letters above the bars are significant at P<0.01by Duncan’s Multiple Range Test)

30 a

µg/100 g dr wt.

25

a

20 Control

15

b

b

CKPp9

b

10

b 5

d

c c

0

b

bc

c

b

Caffeic acid

CKPp9+CmmGo2

a

a

CmmGo2

c

Chlorogenic acid Catechin Phenolic Contents

Rutin

a

µg /100 g dry weight

30

A A

25 20 15 A

10 5 0

A

A

B

B B C

BC

C

B

Caffeic acid

B B

B

Chlorogenic acid Catechin

C

Rutin

Treatment

b

Fig. 4. Effects of different treatments on occurrence of phenolic compounds in tomato seedlings (The values are expressed in mean ± standard deviation. a,b: Different letters indicates the statistical difference at significance of 0.05; A,B: Different letters indicates the statistical difference at significance of 0.05).

Table 1 Characteristics of specific primers for Pseudomonas aeruginosa, P. fluorescens, P. putida.

Product size Primer Sequence* Pair (bp) Pa1 F 5’-GTCCCATCGCAGACCCAC -3’ 171 R 5’-ATCGATGCTGGGTTCTTCCG-3’ Pseudomonas aeruginosa Pa2 F 5’-AGAACTTCGTCGAGGGCAAG-3’ 597 R 5’-CTTCCAGTTCACGCACGTTG-3’ Pa3 F 5’-ATCTTCTCCGCGATGAGCTG-3’ 2259 R 5’-GTCCACCAGCTTCGAAGTGA-3’ Pf1 F 5’-TGCGCTCAAGCACACCAGCT-3’ 121 R 5’-ACCGTGCTTGGGGTTGTCGG-3’ Pseudomonas fluorescens Pf2 F 5’-CCTGTGCTGCGCTGTCGGAT-3’ 222 R 5’-CCACATGCAGGTTGCCCGGT-3’ Pf3 F 5’-TGCGTGATGAGCTGCCTGCC-3’ 141 R 5’-GCACGCGCCCCAGGTACTTT-3’ Pp1 F 5’-CGCGATGAACTGCCTGCCCA-3’ 181 R 5’-GGGCTGGTGCAATGCCGCTA-3’ Pseudomonas putida Pp2 F 5’-AGCCGAAGGCGACGAGTTGC-3’ 222 R 5’-TGAGTCTGCGCCAGCGAAGC-3’ Pp3 F 5’-TCGGGCGTACCACGATCGGT-3’ 181 R 5’-GTCAGCGCGCTCGAAGAGCA-3’ * Position at 5’ end of direct and reverse primers for each PCR primer pair. F and R, forward and reverse primers, respectively. Pseudomonas Species

Efficacy of Pseudomonas putida on the plant growth Biological control of C. michiganensis subsp. michiganensis P. putida caused plant phenolic compounds changes