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 (Zehnderet al., 2001).

Primarily Pseudomonas fluorescens is identified as an important organism with ability for plant growth promotion and effective disease management properties. Their applicability as biocontrol agents has drawn wide attention because of the production of secondary metabolites such as siderophores (Neilands and Leong, 1986), indole-3-acetic acid (IAA) (Patten andGlick, 2002) and multiple antibiotics such as phenazine-1-carboxylic acid (PCA)(Thomashow et al., 1990), phenazine-1-carboxamide (PCN) (Chin-A-Woenget al.,1998; Sunish Kumar et al., 2005), pyocyanin (Calhoun et al., 1972), 2,4-diacetylphloroglucinol (DAPG) (Mavrodiet al., 2001), pyoluteorin (PLT) (Howell and Stipanovic, 1980), pyrrolnitrin (PRN) (Hammer et al.,1997) oomycin A (Gutterson, 1990) , viscosinamide (Nielson et al., 1999) and tensin (Nielsen et al., 2000).

These antibiotics, which have been implicated in plant disease control, enabling the producing strain to serve as a biocontrol agent (Mavrodi et al., 1998; Maddula et al., 2008; Costa et al., 2009; Selinet al., 2010). Phenazines are heterocyclic nitrogen-containing secondary metabolites synthesized by Pseudomonas ﬂuorescens and a few other bacterial genera (Price-Whelan et al., 2006 ). Biocontrol by phenazines is connected with their ability to undergo oxidation-reduction transformations thus causing the accumulation of toxic superoxide radicals in the target cells (Kerr, 2000; LaursenandNielsen,2004; Price-Whelan et al., 2006 ). A number of naturally occurring, broad spectrum, colored phenazines have been reported in different studies. P. ﬂuorescens 2-79 is among the ﬁrst few strains from which puriﬁed phenazine compounds were shown to have antifungal activity (Gurusiddaiah et al., 1986 ). Different phenazine derivatives originate either from phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC) (Leisinger and Margraff,1979; Kerr, 2000 ). Genes encoding the phenazine biosynthetic enzymes are arranged in one core operon, phzABCDEFG, in most producing pseudomonads, including P. chlororaphis (aureofaciens)30-84 (Pierson and Thomashow 1992; Delaney et al.,2001).

Pythium spp. are the causal agents of pre- and post-emergence damping-off and root rot of many crops. Seeds rot before or shortly after germination (pre-emergence damping-off)and newly emerged seedlings collapse (post-emergence damping-off) (Smith et al., 1988).

The principal objectives of this paper were to study the production of phenazine-1-carboxylic acid (PCA), hydrogen cyanide (HCN), and siderophores from some indigenous and lab P. fluorescens strains from one side and proposinga relation between production of these secondary metabolites specially PCA from the other side. Also, we want to investigate suppression of Pythium ultimum causing cucumber damping-off.

MATERIALS AND METHODS:

Cultures and media.

Fluorescent pseudomonad strains, Pseudomonas fluorescens PF5, P. fluorescens CHA0, P. fluorescens 2–79 and P. fluorescens CHA89 (gac mutant), and Pythium ultimum strain were obtained from the collection of microbial culture (Department of Plant Protection, University of Mashhad, Mashhad, Iran). Strain CHA89 was screened as control. Stock cultures of bacteria were prepared for storage at -80°C in 1.5 mL vials by mixing equal volumes of 50% glycerol in a 24-h culture broth [from single colony inoculum, 25mL LB medium, 100mL flask, 130 rpm]. Fungal strains after growth on slants of potato dextrose agar (PDA) were maintained under liquid.

Isolation of P. fluorescens was made from rhizosphere of cucumber fields and greenhouses in Khorasan province of Iran. The 10 cm rhizosphere soil particles loosely adhering to the roots were removed gently and the roots were cut into small pieces and mixed well. The soil thus obtained was crushed in a sterile mortar and pestle and shaken with 100 mL of sterile distilled water for 10-20 min. to obtain standard soil suspension. Isolation of P. fluorescens was made by following the serial dilutions and pour plate method using the specific King’s B medium.

Pour plate method.

King’s B medium, a selective one (Kings etal., 1954) was used for the isolation of P. fluorescens. One mL of soil suspension from aliquot dilutions (105 to 108) was aseptically added to sterile Petri plates containing 20 mL of sterile medium and incubated at 28±20°C for 48 h. After incubation, well separated individual colonies with yellow green and blue white pigments were marked and detected by viewing under UV light. The individual colonies were picked up with sterile loop and transferred to fresh King’s B slants and the pure cultures so obtained were stored in refrigerator at 40°C for further use. The test tubes containing sterilized Kings B medium were inoculated with the isolate of

Pseudomonas spp. incubated for five days and observed. Yellowish green fluorescent pigment observed under UV light (365 nm) indicated positive results.

Antagonistic activity of P. fluorescens strains against P. ultimum.

150 strains of bacteria were tested for antagonistic activity in vitro against Pythium ultimum according to the method of Keel et al. (1997). Bacterial suspension of each purified isolates were spotted with 5 mL space from the three edges of Petriplates containing potato dextrose agar medium (PDA) and kingB medium (KB) for Pseudomonas fluorescens and were incubated at 25ºC. After 48 hours, a 6 mm block of a three-day-old culture of P. ultimum was placed in the center of each plate and incubated at 25ºC. Inhibition zone of fungal growth was determined daily for 3-5 days. Strains with maximum inhibition zone were identified based on standard bacteriological tests and were selected for further studies (schaad et al., 2001 and Bosis et al., 2000). The percentage growth inhibition was calculated using the following formula.

% Inhibition = [1-(fungal growth/control growth)]×100

Antibiotic production.

To study antibiotic production, 1 mL of bacterial suspension (10⁸ CFU mL^-1) was flooded on PDA plate and incubated at 25ºC. After 72 hours the colonies were removed by sterile cotton swab and exposed to chloroform vapor for 30 min (Lindberg, 1981). Blocks (5 mm) of 3-day-old culture of Pythium ultimum was placed in the center of plates and incubated at 25ºC. The growth of P. ultimum was monitored and the percentage of inhibition of mycelium growth was determined for 5 days (Kraus and Loper, 1990).

Siderophore production.

For siderophore production, iron free SM medium with pH 7.0 consisting of K₂HPO₄(6.0gL^-1), KH₂PO₄(3.0gL^-1), MgSO₄7H₂O (0.2gL^-1), (NH₄)₂SO₄(1.0gL^-1), and succinic acid (4.0gL^-1) was used to inoculate 24 h old cultures of P.fluorescens at the rate of 1% (v/v) inoculum. It was incubated for 24-30 h at 29°C with constant shaking at 120 rpm. Following the incubation, fermented broth was centrifuged (10000 rpm for 15 min) and cell free supernatant was subjected to detection and estimation of siderophores based on their absorbance at 400 nm (Castaneda, et al., 2005).

HCN production.

Production of HCN was assessed on King’ S B medium (KB) containing 4.4g/L of glycine with indicator paper (whatman soaked in 0.5% (w/v) picric acid and 2% (w/v) sodium carbonate) and plates incubated at 27؛°C for 48h to 72h. Any positive response caused the indicator paper to turn from yellow to cream, light brown, dark brown, and brick scaled 1-4 (Alstrom and Burns, 1989)

Qualification of PCA production.

All pseudomonads in the collection were assessed for the production of phenazine-1-carboxylic acid as described by Thomashow and Weller (1988). Isolates were grown over-night on TSBA before being streaked onto PDA and incubated at 28^؛C for 4 days. Dark green pigmentation or crystalline deposits in the centre of colonies was indicative of phenazine production.

PCR detection of the gene for phenazine-1-carboxylic acid.

DNA samples were isolated from the overnight cultures of the examined indigenous Pseudomonas isolates. 200 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. Afterward, the samples were incubated for 5 min at -20°C and centrifuged (13000rpm, 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 (60s) 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).

Sequence	Primer
5 – TTGCCAAGCCTCGCTCCAA- 3	PCA2a
5- CCGCGTTGCCTCGTTCAT – 3	PCA3b

Greenhouse experiments.

Preparation fungal and bacterial inoculums.

The Pythium ultimum inoculum was prepared by wetting 200 g millet seeds with 100 mL water twice autoclaving at 15 psi for 30 min, adding ten, 5.0 mm mycelial discs from a 3-day-old culture of P. ultimum on PDA, and incubating at 25؛C for one month. Raw soil was infested with seeds to obtain about 100 propagules of Pythium ultimum per gram of soil. Cells of strains were grown on KB agar and incubated at 25؛C for 48 h. Bacteria were harvested by scraping cells from medium and suspending them in 9 mL sterile water. Solutions were serially diluted and bacterial concentrations were determined. The bacterial suspensions were diluted in KB medium to about 10⁹ CFU mL^-1. These suspensions were used for seed coating and soil drenching.

Seed coating. After surface sterilization of cucumber seedswith 70% ethylene for 2 min, and sodium hypochlorite for 2 min , and washing with sterile water (Kita et al.,2005). They were soaked in the bacterial suspension containing 1.5% carboxyl methyl cellulose (CMC) and shaken for 1 h. For control treatment, the bacterial suspension were replaced by 1.5% CMC. The treatment seeds were allowed to dry on filter paper in a laminar flow cabinet. 3 seed were sown in each pot filled with soil infested with fungal inoculum. The untreated control seeds were planted in infested soil. A negative control using pathogen-free soil was included in each test. Population densities of the applied bacteria on seeds were about 10⁹ CFU mL^-1. Three replicates were applied for each of strains and controls in a complete randomized design. After 10 days of growth, reduction percentages of cucumber damping-off were assessed by counting the number of healthy plants. Plant growth was measured by determining fresh weights of roots and aerial parts after 2 weeks of growth.

Statistical analysis.

All data were analyzed by ANOVA using SAS (V6.12). Duncan´s Multiple Range test was used to determine differences between treatments at 5% significance level.

RESULTS

Antifungal activity against P. ultimum in vitro.

150 indigenous isolates isolated from cucumber rhizosphere plus 3 lab strains 2-79, Pf-5,and CHA0 showed inhibition percentage greater than 25% against mycelial growth of Pythium ultimum. Strain CHA89used as control don’t show any inhibition. Among them, strains CHA0 and PF-5 showed the most inhibition percentage with 67.33%and 67% respectively. Between all the indigenous isolates, three isolates showed inhibition greater than 50%,i.e. M-80-3, M-8-10, and F140. Antibiotic assay strains which are CHA0, PF-5, 2-79, and 8 indigenous isolates showed 100% inhibition against mycelial growth of P. ultimum. In both of these assays control plates which didn’t treated by bacteria didn’t show any inhibition and were covered by Pythium ultimum mycelia completely (Table 1).

Table 1.Mycelial growth inhibition of Pythium ultimum by strains and indigenous isolates of P.fluorescensin antagonistic and antibiotic assay.

Bacterial isolates and strains

% Mycelial growth

Antagonistic assay

Antibiotic assay

2-79

F140

SH-1

PF-5

F-10

CHN4

SH-2-11

M-80-3

T30-1

F66

M8-10

T37

F130

F141

CHA0

CH-E

CHA89

CH-U

CH-L-9

CH-K-2

T-1

F20

Y-21

CONTROL

41.66abcd

50.83abc

42abcd

67ab

46.66abc

57.16abc

40.33abcd

61.1abc

43.3abcd

43.6abcd

56.1abc

43.33abcd

52.16abc

56.1abc

67.33ab

30.53bcd

0.0d

37.8abcd

25.53bcd

26.35bcd

28.33bcd

35.53abcd

45abc

0.0d

100a

74bc

0.0g

100a

66bcd

32e

60d

100a

10fg

0.0g

78b

9fg

100a

18f

19f

100a

0.0g

*Each number is the mean of three replicates. Mean values followed by the same letter are not significantly different according to Duncan´s test at 5% significance level.

Siderophore and HCN assays.

HCN production screened by changing indicator paper from yellow to brick that scaled 1 to 4.( Table2).

All isolates and strains gave positive response to HCN production except strain CHA89 and F140. CHA0 and CHN4Changed indicator paper color from yellow to brick which indicates the highest level of HCN production.

Siderophore production determined by color change of succinate medium.

All of 19 indigenous isolates and four lab strains produce siderophore in succinate medium. Among them, F141, F140, CHA0, CHA89, andF66 showed high level of siderophore production (Table2).

Table2. Quantity of siderophore production (µmol/L) and qualify of HCN production in P. fluorescens strains and indigenous isolates HCN production scaled 1-4,cream=1, light brown=2, dark brown=3 and brick=4

Bacterial isolates and strains

Siderophore (µmolL^-1)

HCN

2-79

F140

SH-1

PF-5

F-10

CHN4

SH-2-11

M-80-3

T30-1

F66

M8-10

T37

F130

F141

CHA0

CH-E

CHA89

CH-U

CH-L-9

CH-K-2

T-1

F20

Y-21

CONTROL

5fg

89a

24.5c

24.5C

19cde

9.5efg

20cde

17.5cdef

19.5cde

67.5c

22cd

14.5cdef

20.5cde

93a

81b

12cdef

81b

15cdef

12cdefg

17.5cdef

13cdefg

0.0g

*Each number is the mean of three replicates. In siderophore data mean values followed by the same letter are not significantly different according to Duncan´s test at 5% significance level.

Detection of PCA gene by gene-specific primer. The results of the PCR analysis using primers pca2a and pca2b showed that a DNA fragment approximately 1150 bp in size, as predicted from the known PCA gene sequence, was amplified in2-79, F141, CHN4, Y-21, F10, M8-10,M-80-3, T37, T30-1. Strain 2-79 which was used as a positive standard, known from previous work (Raajmakers et al., 1997) to have the PCA gene. 1150 bp

Screening of PCA production.

Among isolates and strains of P. fluorescens that had PCA gene only F10, F141, 2-79, and Y-21 were able to produce crystalline deposited on PDA medium.

Antifungal activity on Pythium ultimum in greenhouse. After ten days of plant growth, strains, PF-5, CHA0, CHA89, and 2-79 and 15 indigenous isolated reduced damping-off in soil inoculated with P. ultimum as compared to the inoculated, but not bacterially treated control. Disease percentage reduction measured in successful strains and isolates. Strains CHA0 and PF-5 reduced disease by 90% and 85% respectively (Fig.2). Two weeks after the treatment, the average plant fresh weights after seed coating were higher in the case of CHA0 treatment comparing to the other treatments.

(Fig. 3)

Fig.1. PCR amplification of PCA gene. Lane M, 1-kb ladder; lane 1, Y-21; lane 2, T30-1; lane 3, CHN4; lane 4, 2-79; lane 5, F10; lane 6, M-8-10; lane 7, T-37; lane 8, F141; lane 9, M-80-3; C, negative control (lysis buffer).

DISCUSSION

Pythium spp. are the causal agents of pre-and post-emergence damping-off of a number of crops. Seeds rot before or shortly after germination (pre-emergence damping-off) and newly emerged seedlings collapse (post-emergence damping-off) (Smith, et al., 1988). Antagonistic root-associated bacteria are important for the control of soil-borne pathogens. (Weller, 1988; Sorensen, 1997). In recent years, fluorescent pseudomonads have drawn worldwide attention because of their ability in production of secondary metabolites such as siderophores, antibiotics like phenazine-1- carboxylic acid, HCN, enzymes, and phytohormones. P. fluorescens showed the ability to suppress cucumber damping-off caused by Pythium spp.(Brisbane and Janic, 1987; Gurusiddaiah, et al., 1986). Phenazine-1-carboxylic acid (PCA) is the major determinant of biological control of soil-borne plant pathogens by strains of fluorescent pseudomonads (Tomashow and Weller, 1996).

This work demonstrates the effect of PCA in biocontrol of cucumber pre and post-emergence damping-off. Greenhouse experiments proved all nine strain contained PCA gene showed suppression against P. ultimum. In addition, four strains, including 2-79, F10, F141, and Y-2 which were able to induce PCA gene expression by producing crystalline deposit in PDA medium, showed better inhibition in disease index which denotes better effect on pre and post-emergence. Five other isolates, which had PCA but didn’t express that showed acceptable control in greenhouse condition and inhibited damping-off. This point reveals that maybe gene expression was not good enough to produce crystalline deposits. Also, other phenazine derivatives might be produced (Chin-A-Woeng, et al., 2003). Role of other phenazine derivatives has proved in plant pathogens biocontrol (Hernandez and Kappler, 2004).

Pseudomonads strains capable of producing hydrogen cyanide (HCN), compete with a pathogen for niches and nutrients or induce systemic acquired resistance in the plant after successful colonization of the roots (O’Sullivan and O’Gara, 1992).

Strains including CHAO, CHN4, T37 and F140 showed high level of HCN production. They were all effective in reducing disease index. So, it can be concluded that there is a relation between HCN production and reducing mycelial growth of P. ultimum.

Siderophores are low molecular weight compounds with high affinity for Fe⁺³ (Nielands, 1981), which are produced under limiting concentration of iron.

These compounds are able to transport this element inside the cell for metabolic functions (Press et al., 2001). Microorganisms that produce siderophore show competitive advantage over those who do not. Siderophore improve biocontrol of Pythium disease by P. fluorescens in rhizosphere of wheat (Becker and cook, 1988).

P. fluorescens 3551, siderophore mutant showed low biocontrol ability against cotton damping-off caused by Pythium spp. (Loper, 1988). Our screening demonstrated that siderophore has a great effect on post-emergence damping-off and fresh weight of cucumber plants. Strains F140, F141, CHA0, CHA89 and F66 showed better production of this metabolite in comparing to other isolates and strains. Among the mentioned strains, F141 produced siderophore more than others. Although, it didn’t reduce disease index like strain PF-5, but it had better effect on plant fresh weight comparing to PF-5. In addition, strain CHA89, which can’t produce any antifungal metabolites except siderophore because of the lack of Gac sensor, was effective on reducing disease index and plant fresh weight.

It is proved that siderophore can effectively inhibit mycelial growth of P. ultimum and help plant to grow better and produce more roots. In this way, the evolution of pathogen can be delayed.

The results of this study suggest that strains with ability to produce PCA can inhibit pre and post cucumber damping-off caused by Pythium ultimum. Also, it’s found out that some metabolites like siderophore and HCN can interrupt mycelial growth and make a better condition for plant growth and help biocontrol agents to successfully defeating pathogens. But all of these favorable events happen in proper combination of factors like pH, existing of some mineral and nutrient material, temperature and some other important factors. To demonstrate the role of each metabolites, each of them should be extracted and apply against pathogens directly. This study also provides evidence that in vitro assay for the activity of a microbial metabolite against a target pathogen maybe useful in a program to predict biocontrol potential of the antifungal metabolites in vivo.

In conclusion, further investigations should be done in order to identify special effects of these metabolites on pathogens. On the other hand, some investigation should be conducted to establish soil conditions which let fluorescent pseudomonads to have the highest level of suppression against plant pathogens.

ACKNOWLEDGEMENTS

The authors are grateful to Drs. Ahmadzadeh M. and Saberi R. for their advisements. We thanks to Iran, Mashhad, Ferdowsi University, Plant Pathology Department for providing pure cultures and stocks of strains.

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Received on 22.11.2013 Modified on 02.01.2014

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