Cloning of phenazine carboxylic acid genes of Fusarium fujikuroi antagonists bacteria

Bakanae disease caused by Fusarium fujikuroi is an important diseases on rice. Pseudomonas fluorescens produces the broad-spectrum antibiotic phenazine-carboxylic acid (PCA), which is active against a variety of fungal root pathogens. In this study two genes from seven gene locus of phenazine were cloned in Escherichia coli DH5 (cid:1) . The contaminated rice samples were collected from infected farms of Guilan. 238 bacteria were isolated from the rhizosphere. The antagonistic ability of 12 of which, was demonstrated with dual culture method. From the biochemical and culture results, 8 isolated were identified as P . fluorescens . The two genes from seven gene locus of phenazine were cloned into E . coli DH5 (cid:1) . We speculate that P . fluorescens that produce 2,4-diacetylphloroglucinol (Phl) play an important role in the natural suppressiveness of this soil to causal agent of collar and root rot of rice. Curculigo pilosa is commonly used for herbal preparations as a purgative and also in the management and treatment of hernia, infertility and gonorrhea in Southwestern Nigeria. Owing to reported resistance of Candida albicans to toxic expensive anticandidal agents such as azoles and its implication for promoting opportunistic fungal infections of immunosuppressed patients, the anticandidal activity of C. pilosa was studied. The phytochemical screening of its powdered rhizomes was done using standard procedure. The extracts and essential oil were prepared using Soxhlet and Clavenger-type apparatus respectively. Ten C. albicans isolates from vagina cotton swabs were obtained from three hospitals in Ibadan, Nigeria. The isolates were tested against extracts and essential oil for any anticandidal activity using agar-well diffusion method. The minimum inhibitory concentration (MIC) was determined using broth dilution method. The phytochemicals found in C. pilosa were alkaloids, saponins, tannins, cardenolides and traces of anthraquinones. The ethanol extracts (500 mg/ml) and undiluted essential oil exhibited anticandidal activity while the water extract (1000 mg/ml) was inactive against isolates. The MIC exhibited by the ethanol extract against the tested isolates range between 0.020 and 1.500 mg/ml. The isolation and identification of the active compounds of C. pilosa could lead to the discovery of anticandidal phytomedicine. Candida albicans, phytochemical screening, extracts, essential oil, anticandidal activity.


INTRODUCTION
There is increasing interest in applying microorganisms to control soil-borne plant pathogens. Inconsistent performance of the microorganisms, however, has hampered commercial application. Combining several modes of action against plant pathogens in one single organism by genetic modification can improve the efficacy of biological control agents (Van Loon, 1998). Bacterial secondary metabolites play critical roles in many aspects of bacterium-host interactions. Secondary metabolites that function as virulence factors play a central role in disease by altering host tissues (Kimura et al., 2001;Rahme et al., 1995). Other secondary metabolites produced by beneficial bacteria can function to prevent infection by pathogens by altering the environment and improving the bacterium's ability to compete with pathogens, by inhibiting the activity of pathogens, or by triggering host defenses (Bloemberg and Lugtenberg, 2001;Raaijmakers et al., 2002). The antibiotics phenazine-1carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) are major determinants of biological control of soil-borne plant pathogens by various strains of fluorescent Pseudomonas spp. (Raaijmakers et al., 1997). The ability to produce phenazines is limited almost exclusively to *Corresponding author. E-mail: anvari@iaurasht.ac.ir. bacteria and has been reported in members of the genera Pseudomonas, Streptomyces, Nocardia, Sorangium, Brevibacterium, and Burkholderia (Turner and Messenger, 1986). Particularly among fluorescent Pseudomonas, the production of 2,4 diacetylphloroglucinol (DAPG), Plt (pyoluteorin), Prn (pyrrolnitrin) and different derivatives of phenazine has been described (Thomashow and Weller, 1996). Pseudomonas putida WCS358r was modified to produce the antifungal compound phenazine-1-carboxylic acid (PCA) (Thomashow et al., 1990). One possible approach to improve biological control may be the application of combinations of biocontrol agents (Duffy and Weller, 1995). By combining microorganisms, multiple antifungal traits can be combined and one may assume that at least one biocontrol mechanism will be functional under the conditions faced by the released biocontrol agents. Moreover, combinations of biocontrol strains are expected to result in a higher level of protection (Dunne et al., 1998), have reduced variability of biological control (Guetsky et al., 2001;Guetsky et al., 2002), and have potential to suppress multiple plant diseases (Jetiyanon and Kloepper, 2002). It has been demonstrated that natural suppressiveness of the Châteaurenard soil in France against Fusarium wilt is based on various mechanisms involving several microbial populations acting alone or together to limit the activity of the pathogen (Alabouvette,

Isolation of F. fujikuroi
Rice bakanae disease was collected from infected farming in different areas as Rasht, Lahijan, Foman, Anzaly, Talesh and Astara in the Guilan province, Iran. For isolation of F. fujikuroi, small pieces of infected root with bakanae disease, was washed and surface sterilized with 5% sodium hypochlorite for 10 min. The infected tissues were cultured on acidified potato dextrose agar (PDA). The plates were incubated at room temperature 26°C for a week. The growing colonies of fungi were transferred to new plates for purification and identification.

Isolation of antagonistic bacteria isolates and identification
Antagonistic bacteria which colonized rice rhizosphere, one gram of exercised roots were shaked at 100 rpm in 100 mL of sterile distilled water for 25 min. Fluorescent pseudomonads under UV light (l = 356 nm) were isolated on King's medium B (KB). According to the methodology of Schaad et al. (2001), antagonistics isolates of bacteria were identified by biochemical, physiological, biological tests and PCR.

Screening for antifungal activity
Screening for antifungal activity was performed on PDA medium. Under this condition, fungal growth inhibition could be due to production of antifungal metabolites. Pathogen used was F. fujikuroi. An agar plug (5 mm diameter) taken from an actively growing fungal culture was placed on the surface of the PDA plate. Simultaneously, P. fluorescens strains were streaked 3 cm away from the agar plug at sides towards the edge of Petri plates. Plate inoculated with fungal agar plugs alone was used as control. The plates were incubated at 27°C until fungal mycelia completely covered the agar surface in control plate. Strains that inhibited mycelial growth of fungus were tested. Ability of antagonistic bacteria to production of volatile antibiotic, secretion of extracellular and production of diffusible antibiotic were tested according to Montealegro et al. (2003).
Results are expressed as means of inhibition (%) of the growth of F. fujikuroi in the presence and absence of any bacterial isolate. Percent inhibition was calculated using the fallowing formula (Montealegro et al., 2003).

Bacterial strains, plasmids and primers
The bacterial strains plasmids and primers used in this study are described in Table 1. Pseudomonas strains were grown at 28°C in king's B, 23 YT broth (Sambrook et al., 1989), while E. coli strains were grown in Luria-Bertani or 23 YT broth at 28 or 37°C.

DNA manipulations
Standard methods were used for DNA purification, restriction enzyme digestion, agarose gel electrophoresis, and ligation . Genomic DNA was isolated and purified by a cetyltrimethylammonium bromide (CTAB) miniprep procedure. A 6.4-kb DNA probe containing the entire phz locus from P. fluorescens F15 was generated by PCR performed with oligonucleotide primers phz-up and phz-low (Table 1). The amplification was carried out by using a 50 l reaction mixture containing 1x eLONGase buffer (Life Technologies, Inc., Rockville, Md.), 2 mM MgSO4, 3.0% dimethyl sulfoxide, 200 M (each) dGTP, dATP, dTTP, and dCTP, 10 pmol of each primer, 0.7 l of eLONGase enzyme mixture (cinagene, Inc.), and 20 ng of purified genomic DNA from isolated strains. All amplifications were performed with a PTC-200 thermal cycler. Amplification was performed in a thermal cycler programmed The reaction conditions are: a initial denaturation of 94°C for 2 min followed by 37 cycles of 94°C for 1 min, 64°C for 1 min, and 72°C for 1 min. A final extension step of 72°C for 10 min finishes the reaction. Amplified DNA fragments were examined by horizantal electrophoresis in 1.5% agarose gel in TBE buffer 90 mM Trisborate, 2 mM EDTA [pH 8.3]), with 8 L aliquots of PCR products. Gels were stained with ethidium bromide and were photographed under UV light (312 nm).

Transformant screening and protein expression
Recombinant cells were identified by plating on to agar medium containing ampicillin, X-Gal and IPTG.
For protein expression E. coli DH5 harboring pUC18 was grown in LB broth to an optical density at 600 nm and induced with 0.5 mM Isopropylb-D-thiogalactopyranoside (IPTG). Cells were harvested 3 h later and total cellular protein was analyzed by electrophoresis in an SDS-10% polyacrylamide gel. Means followed by a common letter in a row are not significantly different according to LSD (T) test at P < 0.01.

Isolation of antagonistic bacteria
Two hundred thirty eight bacterial isolates were initially collected from the rhizoplane and rhizosphere of rice sheath blight disease in different farming of area of the Guilan province-Iran. Among them, thirteen isolates were found to inhibit growth of F. fujikuroi in vitro. Eight isolates; F1, F6, F12, F15, F16, F18, F21 and F25 were identified as Pseudomonas fluorescens biovar 3 according to the methodology of Schaad et al. (2001).

Identification of P. fluorescens isolates by direct PCR
All isolates of Pseudomonas fluorescens were identified by specific primers PCA1 and PCA2. On agarose gel electrophoresis 2%, isolates were produced a band 1110 bp (expected size). The bands of isolates were similarly with isolate standard of 2-79 RN ( Figure 1).

Mycelium inhibition assays
Two hundred thirty eight bacterial isolates were initially collected from the rhizoplane and rhizosphere of rice bakanae disease in different farming of area of the Guilan province-Iran. Among 268 isolates, two bacterial isolates (F15 and F16) were selected to the inhibit mycelium growth of F. fujikuroi greater than others ( Table 2).

Specificity of PCA primers
Primers phz-up and phz-low amplified the entire locus of P. fluorescens strain 2-79 RN (Figure 1). The specificity of PCA primers was reported in earlier study (Dimetri et al., 2001).

Cloning detection
The fragment (1110 bp) was cloned into pUC18, and positive clones, were identified by standard methods ( Ausubel et al., 1995). The antibiotic PCA is major determinant of biological control of soil-borne plant pathogens by strains of fluorescent Pseudomonas spp. (Thomashow and Weller, 1996).

DISCUSION
In several bioassays, strain F15 (pUC -PCA) and F16 (pUC -PCA) were able to suppress bakanae disease of rice by effectively of antifungal activity. In this study, we described variety of strains that inhibit mycellial growth of F. fujikuroi and screened a collection of phenazineproducing strains of P. fluorescens. The production of PCA increased antifungal activity of WCS358r and DAPG production resulted in an enhanced ability to inhibit growth of both fungi and bacteria. PCR and southern hybridization analysis were performed to determine the presence of genes involved in the biosynthesis of phenazine-derivatives described in P. fluorescens 2-79 and Pseudomonas aureofaciens 30 -84. Loci phzC, phzD (Mavrodi et al., 1998). For the presence of PCAgenes by direct PCR. Also we successfully cloned the entire locus of phenazine in E. coli DH5 with specific primers. Results indicated that phenazine biosynthesis is highly conserved among phenazine-producing strains of P. fluorescens. Cloning of different fragment of the locus can described the structure and function of the biosynthetic gene clusters from the isolated strains. Characterization of phenazine regulation by strains of P. fluorescens F15 and F16 has revealed many complexities in the activation of phenazine production, but prior to this study, genetic screens had not identified any negative regulators. We speculate that fluorescent Pseudomonas spp. that produce Phl play an important role in the natural suppressiveness of bakanae disease of rice. Because phenazine production by strain 2-79 RN contributes to its capacity in biological control, we tested the ability of F15 and F16 to inhibit F. fujikuroi. In in vitro plate assays, strain F15 and F16 were better at inhibiting mycelial growth of the fungus than wild type strain 2-79 RN ( Table  2). The environmental fitness of genetically modified microorganisms might be affected by the modification (De Leij et al., 1998). Future studies will determine the mechanism of PCA regulation of phenazine production and evaluate the long-term effect of the PCA mutation on bacterial colonization, persistence, and bakanae disease suppression on rice.

INTRODUCTION
The prevalence of Candida albicans in candidiasis has been reported by many authors. Osho (2000) studied the antimicrobial effects of some medicinal plants on Candida species isolated from human oral mucosa and reported that C. albicans constituted 64.8% of the 128 isolates in the six species of Candida obtained by him. Other species encountered in the study were C. tropicalis, C. glabrata, C. krusei, C. stellatoidea and C. parapsilosis. The species most frequently causing human candidiasis are C. albicans, C. tropicalis and C. glabrata whilst the others may also be of medical importance (Jones, 1985). The global human immunodeficiency virus (HIV) epidemic has resulted in an increase in severely ill *Corresponding author. E-mail: gita4me2004@yahoo.com. Tel: 08035505173 / 070561114030.
immunocompromised hospitalized patients, accompanied by more reports of fungal infections. The most common fungal pathogens associated with invasive disease in humans are opportunistic yeasts (e.g. Candida albicans) (Toscano and William, 1999). Unfortunately the limited number of antifungal agents available in the market is toxic, expensive and C. albicans has developed resistance to commonly used antifungals (Perea et al., 2001). Due to this reason, there has been a search for newer generation of drugs to combat such complex mycotic pathogens. This has attracted the researchers to search for new antifungal agents of herbal origin which are relatively economically affordable, safer and easily available to common men (Rai et al., 2003). Curculigo pilosa belongs to Hypoxidaceae and is an herbaceous plant with stout, erect rhizomes bearing a cluster of grass-like leaves to 60 cm long and flower shoots to 20 cm at the end of the dry season. It is found in seasonally marshy savanna. It is widely dispersed from Senegal to West Camerouns and over much of tropical Africa and Madagascar (Burkill, 1985). In Nigeria, it is found in Mubi, Abuja, Igboho and Erin-odo (UIH). In the Yoruba traditional medicine of Southwestern Nigeria C. pilosa is used as a purgative as well as for the management and treatment of hernia, infertility, genital infections and sexually transmitted infections especially gonorrhea.
A survey of literature indicates that many investigators have studied herbal anticandidal agents in recent past. Giordani et al. (2001) reported the in-vitro susceptibility of C. albicans to Euphorbia characias latex using the macrobroth dilution method. Runyoro et al. (2006) reported that twenty-eight (28) out of the sixty-three (63) aqueous methanolic extracts, belonging to 27 plant species and constituting 48% of the Tanzanian medicinal plants collected exhibited activity against C. albicans. Ajaiyeoba and Sama (2006) reported that the leaf and stem redistilled hexane and ethanol extract of Capparis thonningii showed inhibitory activity against C. albicans and Aspergillus flavus. The concentrations of extract used were 250, 500 and 1000 mg/ml. This work examined the anticandidal activity of C. pilosa against 10 clinical isolates of C. albicans, to produce scientific insight for the use of the plant in ethnobotany and widen the spectrum of activity against Candida.

Plant material
Fresh rhizomes of C. pilosa were purchased from a local market in Ibadan, Nigeria in the month of July and were identified in the University of Ibadan Herbarium (UIH). The rhizomes were thoroughly washed with tap water, air-dried, ground into powder, weighed and stored in an air-tight glass container for further use.

Phytochemical screening
The powdered plant material was screened for the presence of natural products using standard procedures in the laboratory of the Department of Pharmacognosy, University of Ibadan, Ibadan, Nigeria.

Preparation of extracts and essential oil
Water extract: 200.0 g of the dried powdered rhizome was soaked in 1000 ml of sterile distilled water for 48 h. The mixture was filtered and the filtrate was freeze dried. 5 g of the extract was reconstituted in 5 ml sterile distilled water to obtain a solution of 1000 mg/ml, which was used for the anticandidal screening.

Ethanol extract
500 g of powdered sample was extracted in 1.5 litre of ethanol (95 % w/v) for 24 h using Soxhlet apparatus. The extract was transferred into sample holder of the rotary vacuum evaporator, where the extract was concentrated to dryness at 50°C and then Gbadamosi and Egunyomi 1237 air-dried to constant weight. The extract was refrigerated at 4°C prior to use. 5 g of the extract was reconstituted in 10 ml sterile distilled water to obtain a solution of 500 mg/ml, which was used for the anticandidal screening.

Essential oil
Essential oil was extracted from 300 g of the plant sample (4 h) by hydrodistillation using a Clavenger -type apparatus designed to the British pharmacopoeia specification (1980). The essential oil was stored in the refrigerator at 4°C prior to use. The undiluted oil was used for the anticandidal screening.

Identification of C. albicans isolates
The C. albicans isolates were identified according to the methods used by Gbadamosi and Egunyomi (2008).

Screening of plant extract for anticandidal activity
The extracts were tested for their anticandidal activity using agar well diffusion method. Each was suspended in sterile malt extract broth (Difco Laboratories, USA), incubated at 35 ± 2°C for 18 h. Different concentrations of each isolate were prepared from the broth in sterile distilled water to give a range of concentrations at 10 -1 to 10 -6 colony forming unit (cfu) per ml. One millilitre of each concentration was added and thoroughly mixed with 19 ml of sterile liquid Mueller Hilton agar (LAB M, UK.) and poured into sterilized Petri dishes (100 mm in diameter). The agar was left to solidify, from each of these plates 9 mm diameter wells were cut out from the agar using sterile cork-borer. Each of these wells was filled with 50 µl of plant extract using a micro pipette. The plates were left at room temperature, long enough for diffusion of the extract into agar. Subsequently, the plates were incubated at 35 ± 2°C for 18 -36 h. Zones of inhibition were measured in millimetres. A control plate containing the test organism without any plant extract was also incubated. Each examination was carried out in triplicates for all isolates.

Minimum inhibitory concentration (MIC) of ethanol extract
The MIC was also determined using broth dilution method. The dilutions of the ethanol extract to be tested were prepared in 5.0 ml volumes of sterile nutrient broth to give a range of concentration from 5,000 to 0.020 mg/ml. After preparation of suspensions of test organisms Ca. 10 -6 organisms per ml, 0.1 ml was added to the extract/broth dilutions (Atalay et al., 1998). For control experiment, 200 mg tablet of metronidazole (May and Baker, Nigeria) was dissolved in 200 ml of sterile distilled water to give a concentration of 1 mg/ml, The dilutions of metronidazole to be tested were prepared in 5.0 ml volumes of sterile nutrient broth to give a range of concentration from 1 to 0.020 mg/ml, that was used for the MIC test. After 18 h incubation at 35 ± 2°C, the tubes were then examined for growth.

Assay of essential oil by agar-well diffusion method
All overnight cultures of isolates were grown in malt extract broth at 35 ± 2°C for 18 h. The inoculum load was adjusted to 1 x 10 -6 organisms per ml using serial dilution method prior to use. 1 ml of this concentration of inoculum was added and thoroughly mixed with 19 ml of sterile liquid. Mueller Hilton agar and poured (aseptically) into sterilized Petri-dishes. The agar was allowed to solidify. From each plate 9 mm diameter wells (two wells per Petri   dish) were cut from the agar using sterile cork-borer, each of these wells was aseptically filled with 50 µl of undiluted essential oil of plant samples or sterile nutrient broth (control). The plates were incubated at 35 ± 2°C for 18 -38 h and zones of inhibition were recorded in millimetres (mm) (Jennie et al., 2003).

Statistical analysis
Analysis of variance and comparison of means were carried out on all data using Statistical Analysis System (SAS). Differences between means were assessed for significance at P 0.05 by Duncan , s multiple range test (DMRT).
The phytochemicals in C. pilosa extracts are shown in Table 1. All isolates were identified as C. albicans. The aqueous extract of C. pilosa showed no anticandidal activity. Table 2 shows the inhibitory activity of the ethanol extract on C. albicans isolates. The extract was active on 9 out of 10 tested isolates. The highest activity was on isolate C10 with an inhibition zone of 52.00 mm at 10 -6 cfu/ml inoculum load, the least activity was on isolate C6 with a diameter of inhibition of 12.50 mm at an inoculum concentration of 10 -2 cfu/ml. Thus the ethanol extract of C. pilosa was most active on isolate C10 and least active on isolate C6, while it was inactive on isolate C4 at all inoculum concentrations used. The result of the MIC tests is presented in Table 3. The essential oil of C. pilosa exhibited inhibitory activity against all screened isolates of C. albicans with inhibition zones of 31.00 -59.00 mm. The oil was most active on isolate C6 and least active on isolates C3 and C8 (Table 4).
The phytochemical analysis of the plant material revealed the presence of alkaloids, traces of anthraxquinones, cardenolides, saponins and tannins (Table 1). Many vegetable drugs owe their therapeutic action to phytochemical constituents (Oliver-Bever, 1986). Many well known puragative drugs such as aloes, senna and others contain di-tri or tetra-hydroxymethyl anthraquinones which occur in the plants either free or in the form of glycosides (Oliver, 1960). This finding justifies the use of C. pilosa as a purgative.
The extraction of the plant sample with water and 0.020 ± 0.00 0.100 ± 0.00 0.100± 0.00 0.100± 0.00 0.020 ± 0.00 0.100± 0.00 0.020 ± 0.00 0.020 ± 0.00 0.020 ± 0.00 1.500 ± 0.00 Metroni-dazole 0.040 ± 0.00 0.040 ± 0.00 0.040 ± 0.00 0.020 ± 0.00 0.040 ± 0.00 0.040 ± 0.00 0.040 ± 0.00 0.020 ± 0.00 0.020 ± 0.00 0.020 ± 0.00 Values represent Mean ± SD. (n = 3). ethanol gave different percentage yields of extracts, which did not have any relationship with the anticandidal activity of the plant. Although the yield of aqueous extract was higher, the extract was inactive on C. albicans. That the ethanol extract exhibited a relatively high degree of anticandidal activity while no activity was shown by the aqueous extract is significant. This finding can be correlated with the traditional preparation of herbs in which alcoholic drinks are used to extract the active plant components. Based on the results of antimicrobial screening, it is evident that the ethanol extract of C. pilosa was very active (90%) on C. albicans isolates ( Table 2). As shown in Table 3 the ethanol extract of C. pilosa gave the MIC values (0.02 -1.5 mg/ml) and metronidazole inhibited all the tested isolates with varied MIC values (0.02 -0.04 mg/ml). The MIC of ethanol extract of C. pilosa on C. albicans isolate C1 and C7 was 0.02 mg/ml, a value which was lower than the MIC of metronidazole (0.04 mg/ml). Also the essential oil showed varied degree of anticandidal activity. The oil of C. pilosa exhibited 100% anticandidal activity against all isolates (Table 4). A great many of essential oil have a slight antibiotic action and are used in the treatment of infections (Oliver, 1960).
Other pharmacological activities of C. pilosa have being reported. Palazzino et al. (2000) isolated two benzyl benzoate diglucosides, piloside A and piloside B and a glucosyl-fused norlignan, pilosidine, previously obtained as tetra-o-methyl derivative from the rhizome of C. pilosa. Pilosidine showed facilitating effect on adrenaline evoked contraction. Also Cometa et al. (2001) reported the reversible hypertensive effect of total extract of C. pilosa, its butanolic fraction (0.5 -100 microg) and the most active compounds structurally similar to adrenaline, pilosidine (10 µg -1 mg/kg) in anaesthetized rat.

Conclusion
The significant anticandidal activity exhibited by the ethanol extract and essential oil of C. pilosa is an indication that active compounds from this plant could be a source of anticandidal agent. Also tincture, ointment, cream and soap could be prepared from the plant for treatment of candidiasis and fungal infections of the skin. The results from this work form a basis for isolation and identification of phytochemical compounds responsible for the observed anticandidal activity.