PHENAZINES PRODUCING PSEUDOMONAS ISOLATES DECREASE ALTERNARIA TENUISSIMA GROWTH, PATHOGENICITY AND DISEASE INCIDENCE ON CARDOON

Phenazines, secondary metabolites of fluorescent Pseudomonas, represent a group of heterocyclic nitrogen-containing compounds showing a broad spectrum of antibiotic properties. Phenazines producing fluorescent Pseudomonas species are studied extensively for their application in plant disease management. In this study, we examined the antifungal activity of different indigenous Pseudomonas isolates (Q16, B25 and PS2) against the phytopathogenic fungus Alternaria tenuissima, which had infected cardoon (Cynara cardunculus L., Asteraceae). An in vitro experiment demonstrated the antifungal activity of selected indigenous isolates. In addition, an in vivo experiment under gnotobiotic conditions showed suppression of C. cardunculus disease caused by A. tenuissima. The quantification of phenazines revealed significant amounts of phenazine-1-carboxylic acid (PCA) and 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA). PCR analysis confirmed the presence of PCA genes in all examined indigenous Pseudomonas isolates. Based on our results, we assume that these Pseudomonas isolates have potential in controlling plant diseases caused by A. tenuissima.


INTRODUCTION
Plant diseases represent one of the biggest problems of modern agriculture.Phytopathogenic fungi, the most common plant pathogens, are capable of infecting different types of plant tissues.Among the main aims in agriculture is finding adequate strategies for their suppression.One of these strategies is biological control (biocontrol) of plant diseases that relies on the use of natural antagonists of phytopathogenic fungi (Heydari and Pessarakli, 2010).
A special place among the natural antagonists of phytopathogenic fungi belongs to rhizobacteria that show beneficial effects on plant growth.They are referred to as plant growth promoting rhizobacteria -PGPR (Zehnder et al., 2001).These bacteria use various mechanisms for their action: production of plant hormones, asymbiotic fixation of N 2 , antagonism towards phytopathogenic microorganisms and the ability to solubilize mineral phosphates and other nutrients (Cattelan et al., 1999).Different isolates of fluorescent Pseudomonas species take prominent place in this respect.Consequently, these isolates have been intensively studied.Fluorescent Pseudomonas species are present in temperate and tropical soils, often dominant among rhizobacteria (Ayyadurai et al., 2007).They belong to PGPR because of the ability to colonize the roots of plants and stimulate growth by decreasing the frequency of diseases.Suppression of diseases includes the inhibition of pathogens by competition and/or by antagonism (Couillerot et al., 2009).The prominent feature of fluorescent Pseudomonas species is the production of antibiotics as inhibitory compounds that play a role in the suppression of diseases caused by phytopathogenic fungi (Haas and Défago, 2005).
Fluorescent Pseudomonas species are capable of inhibiting the phytopathogenic fungus Alternaria tenuissima.This common plant pathogen in several regions of the world (USA, South Africa, Europe) infects various crops such as cereals, vegetables (carrot, cauliflower, potato, etc.) and fruits (lemon, apple, etc.) (Gannibal et al., 2007).A significant characteristic of this phytopathogenic fungus is causing secondary infections and producing spores in conditions of favorable moisture (rains, large amounts of dew) and temperature (19-23°C).Symptoms of infection are represented by dark necrotic spots on leaves, which often wilt and fall off (Blodgett and Swart, 2002).
One of the hosts of A. tenuissima is cardoon (Cynara cardunculus L., Asteraceae).On the leaves of plants infected with A. tenuissima, disease is manifested by the appearance of necrotic spots (Yang et al., 1988).C. cardunculus is a perennial herbaceous plant that grows in many regions of the world (Eu-rope, North Africa, South America), often in harsh conditions (high temperatures and water stress in summer) on unproductive and stony soils (Quilhó et al., 2004).Apart from its use in nutrition, C. cardunculus is also used in medicine because of its antioxidant properties (Ceccarelli et al., 2010).
A. tenuissima is a very aggressive plant pathogen of large numbers of medicinal plants.A. tenuissima isolated from C. cardunculus was the most aggressive compared with those isolated from other medicinal plants in our earlier investigations.The aim of this study was to examine the antifungal activity of different indigenous phenazines producing Pseudomonas isolates (Q16, B25 and PS2) against the phytopathogenic fungus Alternaria tenuissima which had infected cardoon (Cynara cardunculus L., Asteraceae).

Determining the effect of different indigenous Pseudomonas isolates on the germination of conidia of Alternaria tenuissima isolated from cardoon (Cynara cardunculus)
The  (Matijević and Gavran, 1993).

Determination of the antagonistic action of different indigenous Pseudomonas isolates toward Alternaria tenuissima isolated from cardoon (Cynara cardunculus)
Determination of the antagonistic actions of the examined Pseudomonas isolates Q16, B25 and PS2 toward A. tenuissima were conducted on two nutrient media -King B and Waksman agar plates.Overnight cultures of Pseudomonas isolates Q16, B25 and PS2, optimized to 1•10 7 cfu/ml were used: a) to examine the influence of extracellular metabolites on cells (1 ml of cultures was centrifuged at 13000 rpm for 10 min and resuspended in the same volume of sterile saline solution); b) thermostable extracellular metabolites -heat stable antifungal factors (HSAF) influence (1 ml of cultures was centrifuged at 13000 rpm for 10 min, supernatant was filtered and filtrate was incubated at 70°C for 30 min).
The assay on every examined isolate was conducted in four variants: cell free without extracellular metabolites in (1) King B and (2) Waksman agar plates, suspension of thermostable extracellular metabolites (HSAF) added to (3) King B and (4) Waksman agar plates.Sowing 10 μl of these two agents was done near the edges of Petri dishes and mycelia of A. tenuissima were placed in the centre.Control variants contained only mycelia of A. tenuissima on King B and Waksman agar plates.Observation and the measuring of zones of growth inhibition of mycelia around bacterial colonies were performed after seven and fourteen days of incubation at 25°C (Nair and Anith, 2009).The percentage of growth inhibition of A. tenuissima mycelia was calculated by the formula: % Inhibition = [(Control -Treatment)/Control] x 100 (Ogbebor and Adekunle, 2005).

Experiment under gnotobiotic conditions
In vivo screening for suppression of C. cardunculus disease (caused by A. tenuissima) with indigenous Pseudomonas isolates Q16, B25 and PS2 was performed under gnotobiotic conditions.Uninfected seedlings of C. cardunculus and seedlings infected with A. tenuissima were used in the experiment.In-fected seedlings were planted fifteen days after infection.The controls included (1) plants developed from uninfected seedlings, (2) infected seedlings that had not been inoculated with the examined Pseudomonas isolates (Q16, B25 and PS2), and (3) infected seedlings treated with fungicide DACOFLO 0.2%.Every variant of the experiment was set in eight repetitions.Inoculation with 1 x•10 9 cfu of Pseudomonas isolates Q16, B25 and PS2 was performed at the base of the plant two times during the experiment (the 4th and the 14th day).Based on the developed symptoms forty days after planting, the percentage of C. cardunculus leaves infected by A. tenuissima was calculated using the formula: % of leaf incidence = (no. of leaves infected with A. tenuissima / no. of leaves observed) x 100 (Portisanos et al., 2006); the efficiency was calculated using the formula: Efficiency (%) = [(Control -Variant) / Control] x 100 (Gado, 2007).

PCR detection of the gene for phenazine-1-carboxylic acid
DNA samples were isolated from the overnight cultures of the examined indigenous Pseudomonas isolates Q16, B25 i PS2.Two hundred μl of the overnight cultures of all the examined isolates were resuspended in 500 μl of sterile distilled water and incubated for 10 min at 95°C.The samples were then incubated for 5 min at -20°C and centrifuged (13000 rpm, 5 min).The supernatant was stored at -20°C.Dream Taq Green PCR Master Mix and PCA2a/ PCA3b primers were used for PCR reaction according to Raaijmakers et al. (1997).The temperature profile of PCR amplification of initial denaturation of DNA at 94°C (2 min), 30 cycles (denaturation at 94°C (60 s), annealing at 67°C (45 s), extension at 72°C (60 s)) and final extension at 72°C (8 min), was applied.PCR products were separated on 1% agarose gel in 1xTBE buffer, stained with ethidium bromide, visualized under the UV light of a transilluminator and photographed (Raaijmakers et al., 1997).
Statistical analyses were performed by the Duncan multiple test.

Determining the effect of different indigenous Pseudomonas isolates on the germination of conidia of Alternaria tenuissima isolated from cardoon (Cynara cardunculus)
The toxicity and slope of the regression lines were calculated from the obtained results of the number of germinated conidia of A. tenuissima under the influence of different concentrations of the examined Pseudomonas isolates (Q16, B25 and PS2).These values are shown in Table 1.
Based on these results it is evident that the Pseudomonas isolate Q16 showed the highest toxicity on the conidial germination of A. tenuissima, i.e. 5.219•10 4 cfu/ml.The indicator of the activity of Pseudomonas isolates Q16, B25 and PS2 in the inhibition of conidial germination is the slope of the regression lines.The lowest value of 0.29 for Pseudomonas isolate B25 shows that this isolate has a delayed action on the conidial germination of A. tenuissima compared with isolate PS2, whose regression line slope was the highest (0.33).The impact of different concentrations of Pseudomonas isolates Q16, B25 and PS2 is shown in Fig. 1.Also, the treatment with fungicide DACOFLO 0.2% caused total inhibition of A. tenuissima conidial germination of (Fig. 2).
The results are consistent with the study of Mishra et al. (2011) that showed that P. fluorescens isolate MA-4 inhibits the spore germination of the phytopathogenic fungus Alternaria alternata (in the range 40.3-82.7%).Kumaresan et al. (2005) showed that P. chlororaphis isolate PA23 inhibits spore germination of A. solani as a consequence of phenazine action (phenazine caused a high percentage of inhibition of spore germination in vitro, while germination was not inhibited in untreated controls).Different concentrations of suspensions of P. fluorescens isolates A-5, C-03, CRM-3, L-5 and Pf4-1 exhibited antifungal activity against A. cajani (by inhibition of sporulation) in the study of Srivastava and Shalini (2008), where all five isolates showed different levels of inhibition of spore germination: the highest concentration of suspension of these isolates exhibited the highest percentage of inhibition.

Determination of the antagonistic action of different indigenous Pseudomonas isolates toward Alternaria tenuissima isolated from cardoon (Cynara cardunculus)
The impact of cells free of extracellular metabolites and heat stable antifungal factors (HSAF) of Pseudomonas isolates Q16, B25 and PS2 on growth inhibition of A. tenuissima isolates from C. cardunculus is shown in Table 2.In the case of cells devoid of extracellular metabolites, the highest percentage of growth inhibition of A. tenuissima on Waksman agar plates was caused by Pseudomonas isolate PS2 (51.5%), whereas the lowest percentage of inhibition was that of Pseudomonas isolate Q16 (43.7%).On King B agar plates, the highest inhibitory effect was caused by Pseudomonas isolate B25 (80.0%) and the lowest by Pseudomonas isolate Q16 (58.7%).All values of percentages of inhibition on the Waksman agar plates were statistically significant, but on the King B agar plates the only statistically significant value was obtained in the case of isolate B25.The HSAF of isolates Q16 and PS2 showed a stronger effect on the growth inhibition of A. tenuissima (53.7%) compared with isolate B25 (37.7%).Fungal growth inhibition of 80% caused by B25 cells and the low inhibition caused by HSAF suggest a high enzymatic activity of the B25 isolate growing in the KB medium.

Experiment under gnotobiotic conditions
The infection percentages by A. tenuissima of C. cardunculus plants are presented in Table 3. Inoculation with Pseudomonas isolates Q16 and B25 showed a higher percentage of reduction of symptoms caused by A. tenuissima compared with isolate PS2.The lowest efficiency, with the lowest statistical significance, was that of PS2 (18.99%).Ramjegathesh et al. (2011) reported that P. fluorescens reduces leaf blight disease of onion caused by Alternaria alternata by 34.90%.The results of Tabarraei et al. (2011) show that P. putida, P. aeruginosa and P. fluorescens have poten-  tial for decreasing diseases caused by Phytophthora drechsleri.The treatment of soil with bacterial suspensions of different isolates of these bacteria reduces frequency of disease (30.0-83.7%)compared with the control, and treatment of the seeds with bacterial suspensions reduces disease incidence in the range 36.0-85.0%.The obtained results are significant since there is little known data about the biocontrol of A. tenuissima by fluorescent Pseudomonas in gnotobiotic and field conditions.
The research of Portisanos et al. (2006) on the Pseudomonas chlororaphis isolate PA23 showed that this isolate provides significant protection against leaf infection by the fungus Sclerotinia sclerotiorum, highly reducing the intensity of the disease.In the untreated control, there were no infected leaves, in plants treated with isolate PA23 about 10% and in the positive control about 75% of diseased leaves.The study of Selin et al. (2010) of P. chlororaphis PA23 also showed biocontrol activity of this isolate towards S. sclerotium, because this bacterium produces phenazine, which inhibits the growth of S. sclerotiorum: isolate PA23-63, with deficiency in the production of this antibiotic, is equivalent to a wild-type isolate in its ability to control fungal infection and reduce disease intensity.Compared with the positive control, there was no difference in leaf incidence.This indicates that phenazines can, to a certain degree, inhibit phytopathogenic fungi, even though they have a minor role in disease suppression, which is consistent with the results obtained in this study.

Quantification of phenazine-1-carboxylic acid and 2-hydroxy-phenazine-1-carboxylic acid
The established concentrations of PCA and 2-OH-PCA (Table 4) indicate that all examined isolates produced both types of phenazine derivatives, but isolate Q16 proved to be the best producer of PCA and 2-OH-PCA.These results are consistent with the study of Mavrodi et al. (1998) which found that PCA can be accumulated in media up to a concentration of 1 g/l.Furthermore, Timms-Wilson et al. (2000) identified PCA as an inhibitory agent for P. ultimum.Maddula's et al. (2008) quantification of PCA and 2-OH-PCA in their study of the P. chlororaphis isolate 30-84 revealed a production of 28.5 μg/ ml of PCA and 6.7 μg/ml of 2-OH-PCA.This study showed that isolates with no phenazine production do not inhibit the growth of fungus -the loss of 2-OH-PCA production results in a significant reduction of inhibition of the fungal pathogen Gaeumannomyces graminis var.tritici.

PCR detection of the gene for phenazine-1-carboxylic acid
Electrophoresis on agarose gel confirmed the amplification of the gene for phenazine antibiotic PCA of all three examined indigenous Pseudomonas isolate.Q16, B25 and PS2.This result is consistent with the study of Raaijmakers et al. (1997), where the primers PCA2a and PCA3b amplified the predicted DNA fragment of P. fluorescens isolate 2-79.In addition, their study confirmed the amplification of the same sized fragment from the DNA of all the other examined phenazine-producing isolates (P.aureofaciens 30-84, PGS12, AP9, 13985, TAMOak81, Pseudomonas spp.BS1391, BS1393).
The study of Djurić et al. (2011) shows that the indigenous Pseudomonas isolate PS2 is an efficient antifungal agent due to production of extracellular enzymes (chitinases and lytic enzymes) and siderophores.Because of this, isolate PS2 could be further investigated for its application as a biocontrol agent.In our study, indigenous Pseudomonas isolates Q16, B25 and PS2 showed similar properties, indicating that such investigations should be carried out on isolates Q16 and B25.Apart from the detected antibiotics (PCA and 2-OH-PCA) from the phenazine group, the ability of indigenous Pseudomonas isolates Q16, B25 and PS2 to produce other antibiotics (whose role in biocontrol has been proved to be very significant) could be further examined.

CONCLUSIONS
Biological control of A. tenuissima, the most aggressive isolate from medicinal plants in Serbia, isolated from C. cardunculus, is an ecological method of plant protection.Our investigation confirmed the antifungal activity of indigenous Pseudomonas isolates Q16, B25 and PS2.They had an inhibitory effect on the conidial germination and mycelial growth of A. tenuissima and decreased the symptoms of the disease caused by this fungus.PCR analysis confirmed the presence of the gene for PCA in all three isolates.Quantification of PCA and 2-OH-PCA showed that the isolates Q16, B25 and PS2 are good producers, with Q16 as the best of them.Phenazine productions classify our isolates as a promising group of PGPR.Further analyses of other antifungal metabolites are needed to support their role in the biocontrol of C. cardunculus disease caused by A. tenuissima.

Table 2 .
Inhibition of A. tenuissima depending on the type of inhibitory agent.

Table 3 .
The degree of ilness in C. cardunculus infected with A. tenuissima and disease suppression efficiency by treatment with Pseudomonas sp.

Table 4 .
Production of PCA and 2-OH-PCA by isolates of Pseudomonas sp.