Abstract: The objectives of this study were to determine the suitability of transport medium (ice jells) and estimate the duration of viability of Pseudomonas in the transport medium. Bacteria of the genus Pseudomonas comprise a large group of the active biocontrol strains as a result of their general ability to produce a diverse array of potent antifungal metabolites. These include simple metabolites such as 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and pyrrolnitrin [3-chloro-4-(2-nitro-3-chlorophenyl)-pyrrole], as well as the complex macrocyclic lactone, 2, 3-de-epoxy-2, 3-didehydro-rhizoxin. Pyrrolnitrin is active against Rhizoctonia sp., Fusarium sp. and other pathogenic fungi and it has been used as a lead structure in the development of a new phenylpyrrole fungicide. The survival rates of four different pseudomonad strains after continuous incubation for 4 h in the cold temperature (4°C) were: 94.8% for P. putida strain CBD, 94.5% for P. aeruginosa No. BRCH and 62.1% for Pseudomomas species (fluorescent) with lowest survival rate of 33.5% for P. aeruginosa strain H. Since, there were no drastic reductions in the survival rates, the study findings suggest that the transport medium would be generally suitable for these cold-sensitive bacteria.
INTRODUCTION
Pseudomonas is a chemoorganotrophic Gram negative bacterium, that is known to cause bovine mastitis (Buxton and Fraser, 1977). The fluorescent pseudomonads are of particular interest because a number of these species are plant pathogens (Ligon et al., 1999) and at least one (P. aeruginosa) is a human pathogen (Howell and Stipanovic, 1980). It is now well established that some bacteria have the ability to produce antifungal compounds involving both the metabolites and enzymes. The biological mechanism underlying this phenomenon is known as bio-control and the bacteria of the genus Pseudomonas comprise a large group of active bio-control strains as a result of their general ability to produce a diverse array of potent antifungal metabolites. These include simple metabolites such as 2, 4-diacetylphloroglucinol, phenazine-1-carboxylic acid and pyrrolnitrin [3-chloro-4-(2-nitro-3-chlorophenyl)-pyrrole], as well as the complex macrocyclic lactone, 2, 3-de-epoxy-2, 3-didehydro-rhizoxin. The study of biochemistry and the mechanism of formation of these metabolites has proved useful in several ways. Pyrrolnitrin is active against Rhizoctonia sp., Fusarium sp., Pythium ultimum, Gaeumannomyces graminis var. trici and Thielaviopsis basicola and other pathogenic fungi and has been used as a lead structure in the development of a new phenylpyrrole fungicide (Weller and Cook, 1983; Ayers, 1960; Nickerson and Sinskey, 1973; Walker, 1975; Mirleau et al., 2005). In addition, pyrrolnitrin has been used for years as a model for the study of mechanisms involved in the chlorination of organic molecules. The Pseudomonas sp. have been investigated as potential biological control agents due to their ability to produce a battery antifungal compounds against a range of agronomically important fungal diseases (Misaghi and Grogan, 1969; Curtin et al., 1961; Weller and Cook, 1983; Walker, 1975). The fluorescent pseudomonads have also been considered as biological control agents against various root diseases (and in the degradation of oil spills (McGill et al., 1981; Ayers, 1960; Mirleau et al., 2005). The fluorescent pseudomonads have been frequently reported as contaminants in certain meat and dairy products, in the biodegradation of pesticides and chemical wastes and in the industrial fermentation of organic acids and have been suggested as indicators of water quality (Krieg and Holt, 1984; Howell and Stipanovic, 1979; Weller and Cook, 1983; McGill et al., 1981). Many Pseudomonas strains are not easily maintained at refrigerated temperature and relatively die within a short time at 4°C. Because many Pseudomonas strains are inactivated at low temperatures and are cold sensitive (Krieg and Holt, 1984; Wolf, 1972).
Haas et al. (2000) reported that root diseases caused by fungal pathogens can be suppressed by certain rhizobacteria that effectively colonize the roots and produce extracellular antifungal compounds. To be effective, biocontrol bacteria need to be present at sufficiently high cell densities. These conditions favor the operation of positive feedback mechanisms that control the production of antifungal compounds in biocontrol strains of fluorescent pseudomonads, via both transcriptional and post-transcriptional mechanisms. Landa et al. (2002) stated that the indigenous populations of 2, 4-diacetylphloroglucinol (2, 4-DAPG)-producing fluorescent Pseudomonas sp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)-1 over the entire 15 week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2, 4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2, 4-DAPG-producing strains.
Johansson and Wright (2003) reported that the influence of environmental factors during isolation on the composition of potential biocontrol isolates is largely unknown. Bacterial isolates that efficiently suppressed wheat seedling blight caused by Fusarium culmorum were found by isolating psychrotrophic, root-associated bacteria and by screening them in a bioassay that mimicked field conditions. Isolates in this group were identified as Pseudomonas sp., they were fluorescent on King's medium B and had characteristic crystalline structures in their colonies. Members of this morphological group grow at 1.5°C and produce an antifungal polyketide (2, 3-deepoxy-2, 3-didehydrorhizoxin [DDR]). In summary, this study described some isolation factors that are important for obtaining disease-suppressive bacteria in our system and is described a novel group of biocontrol pseudomonads.
Bergsma-Vlami et al. (2005) found that the genotypic diversity of antibiotic-producing Pseudomonas sp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. They developed a simple and rapid method to determine the presence and genotypic diversity of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strains in rhizosphere samples. Denaturing Gradient Gel Electrophoresis (DGGE) of 350 bp fragments of phlD, a key gene involved in DAPG biosynthesis, allowed discrimination between genotypically different phlD+ reference strains and indigenous isolates. Collectively, these results demonstrated that DGGE analysis of the phlD gene allows identification of new genotypic groups of specific antibiotic-producing Pseudomonas with different abilities to colonize the rhizosphere of sugar beet seedlings.
The objectives of this study were to determine the suitability of transport medium (ice jells) and estimate the duration of viability of Pseudomonas in the transport medium.
MATERIALS AND METHODS
Four Pseudomonas strains such as Pseudomonas aeruginosa strain BRCH, Pseudomonas aeruginosa strain H, Pseudomonas putida strain BCD and Pseudomonas sp. (fluorescent) were obtained from American Type Culture Collection (ATCC). Each strain was grown in nutrient broth (Difco) in 125 mL Erlenmeyer flask and incubated in a gyratory water bath shaker at room temperature (23°C). After 48 h, 1 mL of the incubated culture was transferred to another set of tubes containing 5 mL of skim milk broth (Difco). At time zero, a series of dilutions ranging from 10-1 to 10-7 were done by pipetting 1 mL of the skim milk culture mixture into 9 min nutrient broth (dilutent). Spread plating was performed in duplicate by pipetting 0.1 mL of each of the final dilutent on Proteose Glycerol Agar (PPG) plates (Difco). However, delayed pipetting was avoided to eliminate the cell's attachment to the pipettes. The above procedures were repeated after each one hour (up to 4 h) incubation in a small ice chest containing 3 jell ice bags and PPG plates were incubated up to 24 h at room temperature (23°C). The standard plate counts (Krieg and Holt, 1984) were used to monitor viable cell counts of four different Pseudomonas strains. Only those plates with 30-300 colonies were selected. The valid counts were multiplied by the dilution factor of each plate to determine the total plate counts which represented the original number of bacteria per milliliter of milk. The survival percent of a strain was computed by dividing the number of viable cells after incubation for 4 h by the number of viable cells at zero time incubation. This experiment was designed to assure that if Pseudomonas is isolated, then the transportation medium, temperature and time to laboratory would not affect isolation negatively.
RESULTS AND DISCUSSION
The results showed that the viability of four different Pseudomonas strains was considerably affected by the incubation time (Table 1). The Pseudomonas aeruginosa strain BRCH count declined gradually from 9.1x108 at zero hour to 8.9x108 at 3 h and 8.6x108 at 4 h of incubation. The P. aeruginosa strain H showed marked decline in the colony count from 2x109 to 6.7x108 at the end of continuous incubation for 4 h. The P. putida strain CBD showed gradual decrease in the colony count from 5.8x108 to 5.6x108 at 2 h and further decreased to 5.5x108 after continuous incubation for 4 h. The Pseudomonas sp. (fluorescent) showed moderate decline in the colony count from 6.6x108 at 0 h, to 6.5x10°, 5.8x10°, 4.7x108 and 4.1x108 at 1, 2, 3 and 4 h incubation period, respectively. However, survival rates of four different pseudomonad strains after continuous incubation for 4 h in the cold temperature (4°C) were 94.8% for P. putida strain CBD (the highest survival rate), 94.5% for P. aeruginosa No. BRCH and 62.1% for Pseudomonas species (fluorescent). On the other hand, the lowest survival rate of 33.5% was for P. aeruginosa strain H.
| Table 1: | Viable cell count of four different strains of Pseudomonas sp. at dilution 10-6 of skim milk1 |
| 1Plating factor =10 | |
Since there were no drastic reductions in the survival rate of different strains which suggests that the transport method would be suitable even for these cold-sensitive bacteria. The experimental findings were comparable to those reported by Johansson et al. (2003), who reported that the influence of environmental factors during isolation on the composition of potential biocontrol isolates is largely unknown. They identified Isolates in this group as Pseudomonas sp., they were fluorescent on King's medium B and had characteristic crystalline structures in their colonies. Members of this morphological group grow at 1.5°C and produce an antifungal polyketide (2, 3-deepoxy-2, 3-didehydrorhizoxin [DDR]).
However, the study results do not agree with those of Krieg and Holt (1984) and Wolf (1972) who stated that many Pseudomonas strains are not easily maintained at refrigerated temperature and relatively die within a short time at 4°C. Because many Pseudomonas strains are inactivated at low temperatures and are cold sensitive.
CONCLUSIONS
The viability of four different Pseudomonas strains was considerably affected by the incubation time. The survival rates of four different pseudomonad strains after continuous incubation for 4 h in the cold temperature (4°C) were 94.8% for P. putida strain CBD (the highest survival rate), 94.5% for P. aeruginosa No. BRCH and 62.1% for Pseudomonas species (fluorescent). However, the lowest survival rate of 33.5% was obtained for P. aeruginosa strain H. Since, there were no drastic reductions in the survival rate of different strains which suggests that the transport medium would be suitable even for these cold-sensitive bacteria.
All the strains gave more than 300 and less than 30 colony forming units at dilution 10-4, 10-5 and at dilution 10-7, respectively, when incubated up to 4 h in ice chest.
ACKNOWLEDGMENT
The authors gratefully appreciate Mr. Abdul Latif Alsagar for assistance and help in research.