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Johnry S. Maloles
University of the Philippines, Los Baños, Laguna, 4031
31 March 2013
Plant growth promoting bacteria (PGPB) could be used for growth promotion and increase in nutrient uptake of agricultural crops. In this study, PGPBs were isolated from soil using selective and differential medium to screen for their capability to fix nitrogen and solubilize phosphate. Four unknown bacterial strains were obtained and showed positive results for nitrogen-fixation and only one for phosphate solubilization, namely R-C1, R-C2, F-C1, F-C2, and PSB, respectively. F-C2 was chosen for further studies since it can rapidly grow on the Dobereiner’s medium alongside with the PSB. The carrier based PGPB consortium with unknown isolates of F-C2 and PSB was prepared and the shelf life for each inoculant was studied up to four weeks of storage at 28 °C. F-C2 initial count was 2.24 x 106 CFU g-1 and finished with 9.33 x 106 CFU g-1 after 4 weeks of storage, indicating that cell viability was retained with significant percent increase in cell count. On the other hand, PSB started with a higher cell count of 2.87 x 108 CFU g-1. With increase in time of storage, viable cell count dramatically decreased reaching a percent cell viability of less than 1% (2.92 x 106 CFU g-1) after 4 weeks, indicating a 100-fold decrease in cell concentration. It can be concluded that the carrier was best for the unknown F-C2 bacterial strain and not for the PSB strain. The present investigation confirms that these chosen bacterial strains are still not ready for commercial production and needs further study. ______________________________________________________________
Production of inoculant for leguminous plants even started in the early 1890’s in the UK and the USA (22). The USA is probably still the largest producer of legume inoculants in the world (13). With the growth of the industry for commercial production of inoculants, considerable requirements have been brought up and extensive research was initiated for the vast improvement of the development process of the booming industry (25). And now, inoculant production is widespread worldwide, where commercial production is carried out in all continents including the biggest producing countries Australia, India, Brazil, Uruguay, Argentina, and other European countries (13). The key points on where development of inoculants can be successful depends on two key factors, namely the isolate to be cultured for mass production and the carrying material to be used. The actual steps for production of inoculants involve isolation of microorganism, mass culture production, and preparation of inoculants along with inoculant quality control. Individual organism can be mass multiplied using specific media either as small scale or as large-scale commercial production procedure using fermenters. The desired growth of organisms is then mixed with carrier materials and sealed in culture packets. Quality check of inoculant
is regularly checked prior to distribution of individual biofertilizer culture (11). In considering the microorganism to be used, a significant number of bacterial species are able to exert a beneficial effect upon plant growth. Mostly they are associated with the plant rhizosphere, so they are called as rhizobacteria. This group of bacteria has been termed plant growth promoting rhizobacteria, and among them are strains from genera such as Alcaligenes, Acinetobacter, Arthrobacter, Azospirillum, Bacillus, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Paenibacillus, Pseudomonas, Rhizobium, and Serratia (20, 12). Bacteria associated with plants can be either harmful or beneficial. PGPR may promote growth directly by fixing atmospheric nitrogen, solubilizing minerals such as phosphorus, producing of siderophores that solublize and sequester iron, or by producing plant growth regulators such as phytohormones (15, 23).Indirect growth promotion occurs when PGPB promote plant growth by improving growth restricting conditions (10). This can happen directly by producing antagonistic substances, or indirectly by inducing resistance to pathogens (9). A specific bacterium can affect plant growth by one or more of these mechanisms, and also use different abilities for
Quality control of the developed inoculant was monitored to determine percent cell viability of the plant growth-promoting bacteria. For nodulating legumes. 2 g. The unknown bacterial isolates were inoculated in Nutrient Agar (28 g Nutrient Agar in 1 L distilled water) and were streaked for isolation.1 mL of dilutions 10-4 to 10-6 were spread plated in triplicates on selective and differential medium of Dobereiner’s Medium ( composed of 5 g. Single colonies were observed and their cultural characteristics were noted including the size. Biological nitrogen fixation by capable bacteria is a significant source of available nitrogen for the utilization of agricultural crops (8). . margin. 2 mL. 1 mL. Bromthymol Blue. . pigmentation. CaPO4. Selected isolates from Dobereiner’s Medium and Pikovskaya medium were multiplied in large quantities in appropriate culture broths. 5% FeSO4·7H2O. 10% K2HPO4. It was washed again with water and let to air dry. MATERIALS AND METHODS Isolation. Morphological characteristics of the two unknown bacterial isolates were determined by the gram staining technique. Screening. 0. It was washed again with tap water and safranin was added as a counterstain for 2 min. 1 mL. and will continue to be important in future sustainable crop production systems (16). Cultural and morphological characteristics of the best nitrogen-fixer and phosphate-solubilizer were observed. forest soil.01 g. A smear of the F-C2 and PSB were prepared on a glass slide. 1M NH4Cl. decolorization was done to remove the excess stain. 1 mL. It was then stained with crystal violet solution for 1 min and washed with tap water. . Ten grams of each soil sample was weighed and diluted in dilution bottle containing 90 mL of saline water. Insoluble phosphate compounds can be solubilized by organic acids and phosphatase enzymes produced by plants and microorganisms For example. 1% MnSO4·H2O. consistency. both containing 100ppm of cycloheximide. and to further evaluate their capability to retain viability in a certain period of time in carrier mix.1% NaMoO4. Preparation of Starter Culture and Carrier. 10% MgSO4·7H2O. Their gram reaction and appearance under the microscope were noted. Peat also usually contains certain essential nutrients which not only help survival of the cultures but also allow further growth and multiplication during storage (18) The objective of this study was to screen for nitrogen-fixing and phosphate solubilizing bacteria from different soil samples. airdried and heat fixed. 4 g. Nitrogen is required for cellular metabolism and is therefore important in plant growth and production of food and feed. 0. 3 mL. This process of biological nitrogen fixation accounts for 65% of the nitrogen currently utilized in agriculture. form. A large fraction of soil microbes can dissolve insoluble inorganic phosphates present in the soil and make them available to the plants (28). Glucose. method. Laguna. Malic Acid. Bacterial strains were processed using standard gram staining procedure. 5 mL. Yeast Extract.251 g. The transformation of insoluble phosphate into soluble form is carried out by a number of microbes present in the soil. PSB have been shown to enhance the solubilization of insoluble P compounds through the release of organic acids and phosphatase enzymes (19). 10% NaCl.8)) and Pikovskaya Medium (composed of 1 g. Bacterial strains were isolated from soil from three different sampling sites at the University of the Philippines. and elevation. Agar plating method was carried out to select for putative plant growth-promoting bacteria (PGPB) through serial dilution. and Maintenance of Bacterial Isolates. and were incubated for 5 weeks at 28 °C. Characterization of Nitrogen-Fixers and Phosphate Solubilizer. Agar 1000 mL H2O (pH of 6. Inoculants normally consist of a mixture of an appropriate bacterial strain or strains and a carrier material. 0. .2 mL. Los Baños. plants can only absorb phosphate only in soluble form. A loopful of bacterial growth of nitrogen-fixer and phosphatesolubilizer were transferred to 15 mL of DM and PM. CaCl2. 2 g. Using 95% ethanol. Agar (g/L)).025 g. nitrogen can be provided through symbiotic fixation of atmospheric N2 by nitrogenase in rhizobial bacteroids.growth promotion at various times during the life cycle of the plant (10).05 g. incubated at 28 °C for 24 h. Soil samples from grassland area. 4 mL. Isolates that showed positive results for nitrogen fixation and phosphate solubilization were picked and streaked for isolation twice to ensure purity. MgSO4·7H2O. Soil suspension was serially diluted up to 10-6. KOH. 1 mL. 5 g. Yeast Extract. and rice paddy soil were aseptically collected prior to the isolation . Concerning the available phosphorus in soil. Iodine solution was dropped on the glass slides and let to stain for 1 min. A wide range of carriers has been used as a base for Rhizobium inoculants (4) but peat has remained the preferred material because of the protection it offers to cultures against high storage temperatures and its ability to maintain hydration of the cultures.5-6. 10% CaCl2. Isolates were maintained at Dobereiner’s and Pikovskaya slants stored at 4 °C.
Number of the bacteria was estimated by performing simple windows test technique to check for the required turbidity of the mother culture. namely R-C1. Fifteen millilitres of the mother culture of the nitrogen-fixer and phosphate-solubilizer were aseptically transferred to the prepared sterile carriers by pipetting. After the cells were acclimatized on seed broths. incubated for 48 h at 28 °C. Percent cell viability was also computed for comparison. Carrier weighs 80 g each composed of 50% charcoal and 50% clay placed in a low density polythene bag. RESULTS Isolation and Screening. Phosphate-solubilizing bacteria in Pikovskaya Agar Plate after five weeks of incubation at 28 °C. 10 mL of the cultures were transferred to 200 mL of sterile Nutrient Broth (NB) and incubated for 6 d at 28 °C with shaking. Four isolates were found to exhibit nitrogen fixation capability based on the color change of the Dobereiner’s medium from light green to blue-green (Figure 1). The quality of the mother culture and inoculant were determined by performing spread plating in Nutrient Agar plates by serial dilution technique. . respectively. a total of five isolates were selected based on their cultural growths and reactions in the selective and differential medium. F-C1. The bags were thoroughly kneaded to ensure the absorption of the liquid culture into the carrier. and were incubated for 48 h at 28 °C without shaking. Bacterial isolates from rice paddy soil and forest soil labelled as R-C1. respectively. Two of the isolates were from the rice paddy soil and the other two were from the forest soil. Inoculants were let to cure and incubate at 28 °C. Plates were incubated for 24 h at 28 °C and total viable cell count was computed. The carriers were sterilized at 121 °C for 1 h for three consecutive days prior to mixing of the inoculant. Los Baños (UP-BIOTECH).Figure 1. and F-C2 streaked for isolation in Dobereiner’s medium. After 5 w of incubation of the spread plated agars in the Dobereiner’s and Pikovskaya Medium. R-C2. and F-C2. Initial count of the viable cell concentration was determined by performing spread plating technique. F-C1. Preparation of Final Inoculant and Quality Control. and was incubated for 4 d at ambient temperature in the rotary shaker. Five milliliters of the broth was transferred to 100 mL seed culture of DM and PM. Carrier used for this study was provided by the Agricultural Laboratory of the Institute of Molecular Biology and Biotechnology. Figure 2. Change in the medium were also consistently monitored in every step to ensure that the bacterium being investigated still possesses the capability to fix nitrogen or solubilize phosphate. R-C2. Only one isolate showed a zone of clearing in the Pikovskaya medium which indicated a positive result for phosphate solubilization. The phosphate solubilizer was isolated from the forest soil and was labelled as the PSB (Figure 2). The populations of individual plant growth promoting bacteria in the inoculant carriers were assessed at weekly intervals up to 1 month with three bags as representative for each.
Morphological characteristics of the two unknown strains were also investigated to have an idea of the possible identification of the isolates. making a more than ten-fold higher than the other. A starting 2. an entire and umbonate colony. and creamy consistency. Initial cell count of the mother cultures and total number of contaminants in the carrier after sterilization. Carrier had a total count of 1.7 x 109 CFU mL-1) mother culture than the nitrogen-fixer (1. with yellowish pigmentation. cocci bacterium. Three representative bags containing the mixed mother culture and carrier were monitored for a weekly interval up to four weeks with three replicates each.7 x 109 1. Fresh cultures were obtained and were subjected to standard gram staining procedure. the experiment was still carried out.24 x 106 CFU g-1 was obtained in the initial cell count after the mother culture and carrier were mixed. Gram staining reaction of unknown bacterial isolates F-C2 and PSB. Both of the mother cultures of the nitrogen-fixer and phosphate solubilizer strains reach the required minimum number of cells per mL of 108 (Table 1). Nitrogen fixer F-C2 had a diameter of about 1 mm after 24 h of incubation.5 x 108 CFU mL-1). After one week of storage at 28 °C.5 x 108 CFU/g 1.5 x 103 2 0 1 Quality Check of Mixed Inoculant and Carrier.24 x 106 to 7.Figure 3. Higher cell density was recorded for the phosphate solubilizer (8. The nitrogen fixer F-C2 was a gram-negative. The two cultures were streaked for isolation and their cultural characteristics were observed for characterization. .5 mm in diameter after 24 h of incubation. Cultural and Morphological Characterization The unknown F-C2 isolate was considered for further study since it can rapidly fix nitrogen and it can propagate very rapidly alongside with the phosphate solubilizer unknown bacterial strain. which is a relatively low count and makes the carrier a good material for the inoculant production to proceed.5 x 103 CFU g-1 of bacteria. The quality of the prepared mother culture in nutrient broth and carrier were monitored by simple serial dilution and spread plating technique. however. This initial count of bacterial cells did not reached the required minimum CFU g -1 of 108. The phosphate solubilizer had a growth of about 1. short rods bacterium that usually come in pairs (Figure 3). and creamy in color and consistency. while the phosphate solubilizer was a gram-positive.91 x 106 CFu g-1. Starter Culture and Carrier Quality Check. The average of the three bags was taken to compute for the percent cell viability at each sampling point as can be seen on Figure 4. an entire and umbonate colony. Table 1. cell count increased by three-fold from 2. Nitrogen-Fixer. Seed Inoculant and Carrier Quality 10-6 PSB 879 863 810 F-C2 27 14 5 10 carrier -2 10-7 96 80 86 1 4 1 10-3 1 0 0 CFU/mL 8.
Most of the bacterial cells probably did not survive or acclimatized with the carrier during the curing process and resulted to less than 1% of viable cells at the end of the four weeks monitoring period. as supported by the exponential average.78 x 107 CFU g-1. (Average) .28E+08 4. respectively. stored at 28 °C. viable cell count reached up to 3.62E+08 3. Growth profile of the phosphate solubilizing PSB unknown bacterial strain inoculated in the carrier showing exponential average with weekly sampling interval up to 1 mo. reaching up to only . The nitrogen-fixing bacteria might be compatible to the carrier which best suited for the acclimitazation of the inoculant. On the second and third week of sampling. A decreasing trend of cell count was observed for all the sampling bags containing the carrier and inoculant. stored at 28 °C. Sample 1 Viable Cell Count (CFU/g) 2. (Average) 6. Phosphate Solubilizer.40E+04 8.98% cell viability with 2.28E+07 4.10E+07 5.97 x 108 CFU g-1 was determined for the F-C2 inoculant mixed in the carrier (Figure 5). This erratic observation on the trend of the cell concentration per gram of the carrier might be caused by the uneven distribution of the inoculant to the carrier during the mixing process.97 x 108 to 2.12E+06 6.62E+09 3.29 x 107(1022%) CFU g-1. Week two almost have a ten-fold decrease in viable cell count with only 3.01 x 107 (1342%) and 2. corresponding to a 416% cell viability. This trend of decreasing cell count continued up to week 4. computed cell concentration of 9. 0 1 Sample 2 Sample 3 Expon. A starting 2.33 x 106 CFU g-1 was obtained.92 x 106 CFU g-1. Three bags with three replicates each for sampling was monitored weekly for one month and all were averaged for easy monitoring. Sampling procedure might also have affected the total number of cell count in each sampling time.40E+05 8. From week zero to week one. corresponding to a 352.10 x 108 CFU g-1) remaining. forth week.12E+05 Sample B Sample C Expon. Growth profile of the nitrogen fixing F-C2 unknown bacterial strain inoculated in the carrier showing exponential average with weekly sampling interval up to 1 mo. a notable decrease was already recorded with only 70% of computed cell viability (2. corresponding to a 12.98% cell viability.72% cell viability.00E+03 0 1 2 3 4 Time (week) Figure 4.10E+06 5.00E+04 2 3 4 Time (week) Figure 5.Sample A Viable Cell Count (CFU/g) 2. And on the last sampling.
The carriers were autoclaved for three consecutive days at 121 °C for 1 h to eradicate all the vegetative cells and possible spores that were present in the inoculant. Percent cell viability on the forth week was less than one percent. Soils from different environments were processed for the isolation of nitrogen-fixing and phosphate solubilizing bacteria because they contain different populations of bacteria. which are generally influenced by the soil properties like pH. slurries. These trends in the cell concentration of bacterial cells can be linked to two major factors when considering development of inoculants. Other carriers that can be used can be of plant waste material origin. It is very important to sterilize the carrier to make sure that the longevity of the inoculant be achieved. A method of autoclaving peat for 4 h at 121 °C was also found to be a satisfactory method (3). powders. the quality and shelf-life of the inoculant would be very much affected (2). chemical composition. The use of non-sterile carriers is much cheaper. the starting initial count already did not pass the required number of viable cell count of 1 x 108 CFU g-1. however. oxidase test. no known universal carrier or formulation is presently available for all types of microorganisms (24).DISCUSSION Nitrogen-fixing and phosphate solubilizing bacteria were isolated from the rice paddy and forest soils. granular. A good carrier should have one essential characteristic which is the capacity to deliver the right number of viable cells in good physiological condition at the right time (7). Molecular techniques for identification of the bacterial strains would be a much better option where DNA can be extracted and 16S DNA could be amplified using PCR and be sequenced and would give the most probable identities of the unknown isolates. which all depend on the market availability. On the other hand.24 x 106 CFU g-1. Mode of action of the bacteria to be positive on the Dobereiner’s medium was to fix nitrogen from the atmosphere since the media was lacking of a nitrogen source. Other methods that can be employed are flash-drying. In summary. This was not expected because nitrogen-fixers can be isolated from tropical grasses that have associative symbioses with the microorganisms (6). probable identification of the unknown isolates cannot be deduced. In cases when the seed broth or mother culture do not reach the required 1 x 108 CFU g-1. cell concentration was observed to decrease dramatically reaching up to only 2. catalase test. the prepared inoculum should be discarded and a new build-up culture should be made. It is better to have the identity of the nitrogen-fixer or . and coarseness of the soil particles (11). Choosing the right carrier is really important because it takes the major portion of the inoculant. and none of the isolates from the grassland area was positive for both determinative tests. Based on the observed cultural and morphological characteristics of the F-C2 and PSB isolates. Developed inoculant lacking the capability to retain its viability for a long period of time is generally unacceptable in the agricultural market (14). A positive phosphate solubilizer is indicated by a clear zone surrounding the isolated colony. salinity. There are other ways on how to sterilize carriers which depends mainly on the type of the container in which the carrier is packed but also on the facilities available. the selection of the bacterium and the proper carrier to be used (2). methyl red test. voges-prokauer test. where the tricalcium phosphate in the media is solubilized by the microorganism to get access of the phosphate ion (17). Pikovskaya medium was used for the detection of phosphate solubilizing PGPB. To date. One of the major problems encountered in this experiment was the loss of viability of the bacterial cells in the inoculant after sometime. and liquids. On the other hand. For the nitrogen-fixer F-C2. Inoculants have to be designed to provide a dependable source of beneficial bacteria that survive in their corresponding carriers. Accumulation of ammonia (NH3) on the medium due to the fixing capability of the bacteria results to the increase in pH making the media turn from light green to blue-green due to the indicator dye bromthymol blue. Phosphate dissolving soil microorganisms play a part in correcting the phosphorus balance of crop plants. cell concentration was maintained higher than the initial cell count of 2. More morphological tests should be done to identify the unknown isolates of bacteria like oxidative-fermentative test.92 x 106 CFU g-1. gamma irradiation. The most commonly used carrier since 1950’s up to date is finely ground peat although soils high in organic matter and mixtures of soil plus peat or compost or lucerne meal have been used successfully (26). and the needs of a particular crop under specific environmental conditions (21).97 x 108 CFU g-1. However. and chemical sterilants (5). cost. On the monitoring of the cell viability from week zero to week four. the initial cell concentration from the carrier with the PSB inoculant reached the minimum viable cell count required of 2. the selection of an isolate with superior characteristics compatible with a specific carrier should be properly chosen and be optimized. Cell concentration of F-C2 and PSB had a different exponential trend which may be due to some factors associated with the proper selection of bacterial strain and carrier. and motility test (27).
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