Abstract: Background: Tobacco bacterial wilt caused by Ralstonia solanacearum is a devastating disease in Solanaceae crops and the pathogen is a notorious pathogen worldwide. Methodology: The phenotypic characterization of R. solanacearum under various osmolytes and pH environments were analyzed by using biolog Phenotype Microarray system (PMs). Using PM plates 9 and 10, 192 different assays were tested, including 96 different osmolytes and 96 pH environments. Results: Result presented that all four isolates of R. solanacearum exhibited similar phenotypic fingerprints. They had wide range adaptabilities in osmolytes with up to 4% sodium chloride, up to 20% ethylene glycol, up to 4% urea, up to 2% sodium lactate, up to 200 mM sodium phosphate (pH 7), up to 50 mM ammonium sulfate (pH 8), up to 100 mM sodium nitrate and up to 80 mM sodium nitrite. The bacterium did not grow at other osmolytes, including sodium chloride ranging from 5.5-10%, sodium formate ranging from 3-6%, urea ranging from 5-7%, sodium lactate ranging 7-12% and sodium benzoate (pH 5.2) ranging from 100-200 mM. They also exhibited active metabolism under pH values between 5 and 10, with an optimal pH value of around 6. The R. solanacearum showed active deaminase activity, while no decarboxylase activity in the presence of various amino acids. Conclusion: This study increased understanding on the bacterium, especially in survivability of the bacterium in the environment and would provide valuable profiles in developing practical ideas and methods for the disease control.
INTRODUCTION
Tobacco bacterial wilt caused by Ralstonia solanacearum (Smith) causing is a notorious soil-borne bacterial disease. Its pathogen has a large host range of more than 200 species in 50 families and a worldwide distribution1. It is a major threaten in the production of several important economy crops, including tobacco, tomato and potato in the world2. Recently, R. solanacearum is one of the wide spread and most important bacterial phytopathogens in flue-cured tobacco3. Symptoms of bacterial wilt include leaf wilting, yellowing and necrosis4. The bacterium spreads through contaminated irrigation water and infected planting material and survives in infested soil, infected plants, susceptible weed hosts and volunteer crops5.
Ralstonia solanacearum presents great diversities in geographical distribution, host range, physiological properties and pathogenicity6,7. Most bacterial wilt in host of tobacco in China caused by R. solanacearum belongs to Race 1 biovar 3 (R1Bv3)8-10. With environment variation of soil in the growing season, soil osmolytes and pH environments may change greatly. This fluctuation may influence the survival of the soil pathogen R. solanacearum. However, to the best of our knowledge, the survival ability of R. solanacearum under various osmolytes and pH environments is still unclear and will also affect the control of bacterial wilt.
Recently, a high throughput Phenotypic Microarray (PM)/Omnilog system, was developed by the company Biolog (Hayward, CA) to assay osmotic and pH metabolic phenotypes of many microorganism11. In the system, microorganism cells in test medium including a tetrazolium dye are inoculated into 96-well plates, where the plates include various substances to analyze for varying effects of osmotic, ionic and pH environments. A software sketches kinetic data of color formation against time for each well and ascribes artificial color to each plot.
In this study, phenotypic characterizations of R. solanacearum under various osmolytes and pH environments were characterized by phenotypic microarray system. The characterizations were also compared between different strains to explore their phenotypic diversities. Though nearly no effective management methods are available to control bacterial wilt in tobacco, a detailed understanding of the phenotypes of R. solanacearum will be valuable to develop management practices to decrease the impacts of the disease.
MATERIALS AND METHODS
Origin and collection of Ralstonia solanacearum isolates: Four isolates (R1, R2, R3 and R4) of R. solanacearum used in this study were collected from infected tobacco stem with typical bacterial wilt symptoms in 2011 during the disease epidemic season in Guizhou province of China. They were incubated on a semi-selective medium (SMSA)12 at 30°C in the dark. All strains were stored as suspensions in sterile water at 25°C for long-term storage and retrieved at 28°C using the YPGA medium (peptone 5 g L–1, yeast extract 5 g L–1, glucose 10 g L–1 and agar 16 g L–1, pH 7.2).
Phenotypic characterization of Ralstonia solanacearum under various osmolytes and pH environments: Four isolates (R1, R2, R3 and R4) of R. solanacearum from tobacco were detected for this study. All materials, reagents and media for the phenotypic study were bought from Biolog. Isolates were streaked on Biolog Universal Growth medium plus blood agar (BUG+B) plates and incubated at 30°C in darkness for 48 h. Cells were scraped from the surface of the plates and re-suspended in appropriate medium containing Dye Mix A, 120 μL of a 1:200 dilution of an 85% transmittance cells suspension were added to each well of the PM plates. Plates 9-10, which test for phenotypes of varying effects of osmotic, ionic and pH environments, were used in this study. The IF-10 GN Base plus Dye Mix A was utilized for plates 9-10. After inoculation under clean beanth, plates were incubated in the OmniLog incubator for 72 h. Data were collected every 15 min by the Biolog incubator and analyzed using kinetic and parametric software (Biolog). Phenotype diversities were performed based on the area difference under the kinetic curve of dye formation.
RESULTS
Phenotypic characterization of R. solanacearum under various osmolytes and pH environments was conducted by using the PM plates 9 and 10. A total of 192 different growth conditions were analyzed, including 96 different osmolytes and 96 pH environments. All tested isolates of R. solanacearum from tobacco had nearly the same metabolic fingerprints.
| Table 1: | Metabolic profiling of Ralstonia solanacearum on Biolog PM 9 plate* |
| *: ‘+’ and ‘-’ means the substrate was metabolized and not metabolized by Ralstonia solanacearum examined using the Biolog PM 9 plate, respectively, aR is short for pathogen R. solanacearum | |
The R. solanacearum were able to utilize 53.13% of tested osmolytes, including sodium chloride ranged from 1-4%, ethylene glycol ranged from 9-20%, urea ranged from 2-4%, sodium lactate ranged 1-2%, sodium phosphate (pH 7) ranged from 20-200 mM, ammonium sulfate (pH 8) ranged from 10-50 mM, sodium nitrate ranged from 10-100 mM and sodium nitrite ranged from 10-80 mM. However, it could not grow at some other osmolytes, including sodium chloride ranged from 5.5-10%, sodium formate ranged from 3-6%, urea ranged from 5-7%, sodium lactate ranged 7-12% and sodium benzoate (pH 5.2) ranged from 100-200 mM (Table 1).
The pH range where R. solanacearum exhibited active growth was between 5 and 10, with an optimal pH value of around 6.0. When combined with various amino acids at the pH value of 4.5, R. solanacearum did not grow in all tests (plate PM10, Well B01-B12, C01-C12 and D01-D12). In comparison, when combined with various amino acids at the pH of 9.5, R. solanacearum presented actively growth in all tests (plate PM10, Well E01-E12, F01-F12 and G01-G12). The PM 10, wells B1-D12 and E1-G12, tested the decarboxylase and deaminase activities of the bacterium in the presence of amino acids at pH 4.5 and pH 9.5, respectively. In the presence of most amino acids, R. solanacearum showed active deaminase activity, while no decarboxylase activity (plate PM 10). Meanwhile, in the well of H01-H12 in plate PM 10, R. solanacearum also exhibited active growth under pressures of other compounds, including X-caprylate, X-α-D-glucoside, X-ί-D-glucoside, X-α-D-galactoside, X-ί-D-galactoside, X-α-D-glucuronide, X-ί-D-glucuronide, X-ß-D-glucosaminide, X-ß-D-galactosaminide and X-α-D-mannoside and X-PO4 and X-SO4 (Table 2).
DISCUSSION
Despite many molecular biology, genetic and some phenotypic characters studies have been conducted to the sequenced pathogen R. solanacearum, the phenotypic characterization of this bacterium under various osmolytes and pH environments is still unclear. Direct high-throughput testing of phenotypes using the Phenotype Microarray (PM) system (Biolog)11 has provided a much easy way for biology, genomic and population studies of microorganisms13-16 and was used here to detect the metabolic abilities of R. solanacearum under various osmolytes and pH environments. Significant metabolic diversity was found.
| Table 2: | Metabolic profiling of Ralstonia solanacearum on Biolog PM 10 plate* |
| *: ‘+’ and ‘-’ means the substrate was metabolized and not metabolized by Ralstonia solanacearum examined using the Biolog PM 10 plate, respectively, aR is short for pathogen R. solanacearum | |
Ralstonia solanacearum is a notorious bacterium that has a fair ability to colonize both plants and soils17. Bacterial wilt caused by R. solanacearum is a great threat to many important crops in China, especially for tobacco and tomato18,19. Huge economic losses happened each year for tobacco and tomato throughout the country18-20. Due to high prevalence and varies of the pathogen, many strategies for bacterial wilt management showed very limited success. Phenotypic diversities of R. solanacearum might be one of the reasons for the difficulty. The results in this study may help us to know the great survival ability of the pathogen in various soil conditions. The phenotypic character of wide range of osmolytes and pH environments found in this study has great adaptive value. These characters indicate a general view of the lifestyle of the pathogen. Meanwhile, since the characters tested including different environmental stressors, it becomes possible to determine whether it has a chance for bacterial wilt management in agriculture. Changing the pH values of the soil or irrigating tobacco plant with sodium chloride or sodium lactate to get more than 5.5 or 7% osmotic pressure may decrease the survival ability of R. solanacearum and thus may reduce the damage caused by bacterial wilt in the field. These imagines could be proved in the next study in the near future.
In this study, the soil pathogen R. solanacearum had wide range adaptabilities in osmolytes and pH conditions. These adaptabilities can be rationalized as adaption of the bacterium to the variation in soil pH and osmolytes due to varying conditions of the season. Decarboxylase of the bacterium generates alkaline amines by the catabolism of amino acids, which help to counteract an acidic pH. On the other hand, a high pH can be counteracted by deaminases that generate acids21. The R. solanacearum showed effective deaminase activity while no decarboxylase activity. Consequently, phenotypic characters for widerange adaptabilities of R. solanacearum increased understanding on the bacterium, especially in plant-microbe interaction and survivability of the bacterium in the soil of Solanaceae crops and would provide valuable profiles in develop practical ideas and methods for the disease control.
CONCLUSION
Four isolates of R. solanacearum exhibited similar phenotypic fingerprints in 192 different assays concerning osmolytes and pH environments. They had wide range adaptabilities in osmolytes with up to 4% sodium chloride, up to 20% ethylene glycol, up to 4% urea, up to 2% sodium lactate, up to 200 mM sodium phosphate (pH 7), up to 50 mM ammonium sulfate (pH 8), up to 100 mM sodium nitrate and up to 80 mM sodium nitrite. The bacterium did not grow at other osmolytes, including sodium chloride ranging from 5.5-10%, sodium formate ranging from 3-6%, urea ranging from 5-7%, sodium lactate ranging 7-12% and sodium benzoate (pH 5.2) ranging from 100-200 mM. They also exhibited active metabolism under pH values between 5 and 10, with an optimal pH value of around 6. The R. solanacearum showed active deaminase activity while no decarboxylase activity in the presence of various amino acids. Phenotypic characterizations of R. solanacearum increased understanding on the survivability of the bacterium in the environment.
ACKNOWLEDGMENT
This study was supported by National Natural Science Foundation of China (31360448, 31501679) and Guizhou Tobacco Company (201305, 201336, 201436). We also thank the anonymous reviewers for critical reviews of the manuscript.
SIGNIFICANCE STATEMENT
Tobacco bacterial wilt is difficult to control in the world. Various osmolytes and pH environments might influence the survival ability of the pathogen R. solanacearum. Various studies have shown the metabolic characterization of this bacterium. Unfortunately, such researches are not systemic and lots are still unclear. The present study provides dynamic data relating metabolic ability of R. solanacearum in 192 different osmolytes and pH environments. It helps to know the survival ability of the bacterium.