S Shahab, N Ahmed
acinetobacter lwofii, auxin production, bacilllus thuringiensis, indole acetic acid, indole butyric acid, phosphate solubilization, plant growth, pseudomonas aeruginosa
S Shahab, N Ahmed. Growth Promotion Of Chick Pea By Native Phosphate Solubilizing And Auxin Producing Bacteria. The Internet Journal of Microbiology. 2009 Volume 9 Number 1.
Solubilization of insoluble organic phosphate has been the focus of many studies as it increases the availability of phosphorus to vegetation and improves plant growth. The aim of this study was to study those bacterial strains which were positive for phosphate solubilization in plate assay as well as in liquid media. A total of 70 metal solubilizing indigenous bacterial strain were isolated. Ten efficient phosphate solubilizing bacterial stains were investigated for phosphate solubilization in liquid media. Growth substances produced by these ten bacterial stains were determined via bioassay. Three bacterial strainsCMG851,CMG857 and CMG860 which found positive to auxin production were further investigated for indole acetic acid and indole butyric acid production .It was found that Indole acetic acid , indole butyric acid were produced by these bacterial strains in varying concentration with and with out the addition of tryptophan. These bacterial strains showed stimulatory effects on the growth of root and shoot elongation of chick pea. Three promising bacterial strains CMG854, CMG857 and CMG860 were investigated to establish the effect on plant growth.
Plant growth promoting rhizobacteria (PGPB) are considered to promote plant growth directly or indirectly. PGPB can exhibit a variety of characteristics responsible for influencing plant growth. The common traits include production of plant growth regulators (auxin, gibberellin, ethylene etc.), siderophores, HCN and antibiotics (Arshad, 1992 ). Indole acetic acid (IAA) is one of the most physiologically active auxins. IAA is a common product of L-tryptophan metabolism by several microorganisms including PGPR (Lynch,1985; Frankenberger,1983). Microorganisms inhabiting rhizospheres of various plants are likely to synthesize and release auxin as secondary metabolites because of the rich supplies of substrates exuded from the roots compared with non rhizospheric soils (Kampert etal 1975). Plant morphogenic effects may also be a result of different ratios of plant hormones produced by roots as well as by rhizosphere bacteria (Strzelczyk 1984). Diverse soil microorganisms including bacteria ( Muller et al 1989), fungi (stein et al 1990) and algae (Finnie and Staden 1985) are capable of producing physiologically active quantities of auxins, which may exert pronounced effects on plant growth and establishment. Azotobacter paspali secreted IAA into culture media and significantly increased the dry weight of leaves and roots of several plant species following root treatment ( Barea and Brown 1974). It was found that inoculation of wheat seedlings with Azospirillum brazilance increased the number and length of lateral roots (Barbieri etal,1986). Inoculation of canola seeds with
Indole-3-acetic acid (IAA) is the main auxin in plants, controlling many important physiological processes including cell enlargement and division, tissue differentiation, and responses to light and gravity. It is now generally agreed that indole-3-acetic acid (IAA) is the major and most abundant auxin in plants. IAA plays a key role in the regulation of plant growth and development (Moore, 1989; Luthen et al 1999; Davies 1995) and help in the elongation of the primary root, formation of lateral and adventitious roots, (Finnie and Staden 1985). Over the last few years significant progress has been made in understanding the IAA-induced signal transduction pathway (Napier RM, Venis 1995 ;Venis and Napier 1991.). Although other auxins, such as indole-3-acetic acid) indole 3 butyiic acid (IBA) and phenyl acetic acid (PAA) have also been identified in plants (Normanly1997), little is known about their physiological function.
It is presumed that PGPB producing plant growth regulators play a critical role in plant growth promotion. To assess this hypothesis, local isolates of PSBs were screened for their intrinsic ability to produce IAA in the presence of L-tryptophan and their effect on root elongation of germinating seeds of test plants. Inoculations with PSBs. have increased shoot length and root length of plants in both green house. In the experiments reported here we studied phytohormone production in PSBs in association to determine whether the bacteria might enhance plant growth by this mechanism.
Materials And Methods
Isolation, purification and preservation
Soil samples for isolation of phosphate solubilizing bacterial strains were taken from various localities of Karachi. PSBs were isolated by plating serial dilutions of this soil in the tris minimal medium as described by Shahab and Ahmed 2008. Bacterial strains producing clear and large halos were selected for future studies and were preserved in 20% glycerol.
Quantification of free phosphate via Calorimetric assay
The bacterial strains which showed efficient solubilization of zinc phosphate via plate assay method were selected for study of phosphate release (PO4) in liquid media. The bacterial strains were grown in liquid tris minimal medium amended with insoluble phosphate compound (5 mM zinc phosphate) and incubated at 30C at 100rpm. A 10 ml aliquot was aseptically removed from each flask at each intervals of time (0, 1,2,3,4,5,6,7,8,9,10,12 days) and centrifuged at 5,000g for 15 min. the supernatant was filtered through a sterilized 0.45µm Millipore filters and assessed for free phosphate contents in the filtrate using spectrophotometry method of Murphy and Riley (1962). Distilled water 0.95ml and 0.67ml sodium molybdate was added into test tube containing 0.05ml of samples .The phosphate in the solution was visualized by adding 4ml of SnCl2 with the estimation of blue complex at 720 nm(Pharmacia LKB novaspec.11 spectrophotometer) with reference to a standard curve similarly prepared for a period of 10 days.
Bio assay for IAA
IAA was determined in vitro by the method of Salkowski (13). All the test strains were screened for IAA production (15). Briefly, test bacterial culture was inoculated in the nutrient broth with tryptophan.(0.1g/l) or without tryptophan incubated at 30°C. Cultures were centrifuged at 3000 rpm for 30 min. Two milliliters of the supernatant was mixed with 2 drops of orthophosphoric acid and 4 ml of reagent Salkowski (50 ml, 35% perchloric acid; 1 ml 0.5 FeCl3).
Determination of indole acetic and indole butyric acid via HPLC
HPLC chromatograms were produced by injecting 10μl of the filtered extracts onto a – (C18, 5 μm 25x0.46 cm) in a chromatograph equipped with a differential ultraviolet detector absorbing at 280 nm. Mobile phase was methanol and water (80:20 [vol/vol]), flow rate was 1.5 ml/min, Retention times for peaks were compared to those of authentic standards added to the medium and extracted by the same procedures used with bacterial cultures. Quantification was done by comparison of peak heights.
Response to plant growth
Seeds of chick pea were surface sterilized with 95% ethanol and 2.5% sodium hypochlorite. Then washed with distilled water repeatedly up to 10 minutes. Similar sized seeds of chick pea were selected and Seeds dressing were done via O/N cultures then seeds were put onto Petri plate having underlined soaked filter paper and incubated for 4hrs at 37° C. After 4 hrs seeds, ten seeds per pot were sown at equal depth 1-cm2 sections in to plastic pots having 200g autoclaved soil. Soil in each treatment was moistened with an equal volume of autoclaved distilled water for daily watering. Lateral root and root hair formation was examined. After 22 days root and shoot length of the plants and controls were measured by using centimeter scale.
Results And Discussions
Selection of bacterial strains
A total of 70 metal solubilizing bacterial strains were isolated from soil and tentatively identified on the basis of API kit. We selected the strain CMG851, CMG857 and CMG860 because they released maximum amount of free phosphate in the liquid media and among all the other PSBs isolated, they produced the largest halos, of approximately 20-40 mm within 3-4 days of incubation According to de Freitas et al. (1997), good solubilizers produce halos around their colonies with diameters higher than 15 mm. Since it has been reported that some strains loose their solubilizing capability after several cycles of inoculation, we corroborated the persistence of this trait in all three bacterial strains by successive subcultures.
16S ribosomal RNA
CMG851, CMG857 and CMG860 were finally identified by 16S ribosomal RNA gene sequence homology. Genomic DNA was obtained from bacteria grown overnight at 37C in 10 ml Luria -Bertani medium .Genomic DNA was isolated by Qiagen-kit according to vendor recommendation. PCR amplification was performed with a Epicentre thermocycler (Epicentre ). Colony PCR was performed which consisted of repective bacterial colony, 2.5 µl (20 pico mole concentration) of each forward and backward primer, MasterAmp
Phosphate solubilization in plate assay
Determination of phosphate solubilization via plate assay method was performed for 20 days. It was noted that CMG851, CMG854, CMG857 and CMG860 showed best solubilization efficiency and they continue their solubilization trait up to 20 days. CMG851 and CMG854 showed 221% and 250% phosphate solubilization efficiency after 20 days of incubation in plate assay while CMG860 showed gradual increase in the efficiency of phosphate solubilization and it showed maximum (375%) phosphate solubilization efficiency after 20 days of incubation in plate assay (Table 3). Nguen (1992) and Nautiyal (1999) also reported the solubilization of phosphate in plate assay Seshadre, 2000 reported the solubilization of phosphate up to 150 % by pseudomonas species.
Phosphate solubilization in liquid media
Various authors reported the solubilization of zinc phosphate by bacterial isolates (Fasim et al 2002., Uzair et al 2006, Gadd et al, 1999). Presence of soluble phosphate content in liquid media was determined at different time intervals. Results are shown in figure 1. CMG851 released maximum amount of phosphate after four days of incubation with concentration of 4709 ppm which drastically decline after 5 th day without any further revival while after 25 days incubation it reached minimum level of soluble phosphate i.e. 432ppm.This result is in agreement with Nautiyal 1999. CMG860 released maximum amount of phosphate after six days of incubation with concentration of 2580 ppm which gradually decline and after 25 days incubation it reached minimum level of soluble phosphate i.e. 482ppm.This result is in agreement with Nautiyal1999, Tripura 2007 and chen et al 2006. CMG857 released maximum amount of phosphate after eight days of incubation with concentration of 1646ppm which gradually decline and after 25 days incubation it reached minimum level of soluble phosphate i.e. 132 ppm. This result is in agreement with Nautiyal 1999 who had reported the similar pattern of phosphate solubilization in liquid media. Lin, 2006 also reported solubilization of phosphate by bacterial filtrate in the range of 160-200ug/ml while Katiyar and Goel 2003 reported the solubilization of calcium phosphate by
Indole acetic acid and indole butyric acid production
These bacterial isolates were screened for their ability to produce plant growth regulator, IAA. Varying levels of IAA production were recorded. The range of IAA production in PSBs isolates with tryptophan was 57 μg/ml-288μg/ml. while indole butyric acid was in range of 22-34μg/ml. Similar high level of IAA production is also recorded by other researchers (Glick 1995). However, reports demonstrating production of IAA by gram positive free living soil bacteria are still lacking. Synthesis of significant amounts of IAA by the gram positive phytopathogen
Production of IAA and phosphate solubilization by the PSBs were examined as possible contributing factors of chick pea growth promotion. Tests for production of the auxin IAA were positive for all test stimulant strains, suggesting a potential mechanism whereby these bacteria may regulate plant growth. This interpretation is in line with the well-known characteristic of certain phytohormones (e.g., auxin, ethylene) to elicit stimulatory effects on plant growth promotion. Induction of longer roots with increased number of root hairs and root laterals is a growth response attributed to IAA production by other rhizobacteria, which improves their nutrient uptake efficiency.
Response to plant growth
The symbiotic performance of three isolates (CMG851, CMG857 and CMG 860) with the chickpea shoot and root was evaluated in greenhouse experiments. At harvest, 22 days after inoculation, shoot length of plants inoculated with strain CMG860 ranked the highest and it was greater than that of the un inoculated plants. CMG860 showed significant root and shoot elongation. (Shahab and Ahmed unpublished data) Almost all lateral roots were densely covered by root hairs whereas very few or none developed on un inoculated control plants(Fig 2).
The findings of the present investigation highlighted that IAA producing bacteria from local soil could be easily isolated and may be exploited as a cheap mean of biofertilizer. However, further studies using IAA mutant strains of these bacterial strains are needed to explore the exact contribution of IAA production in the promotion of plant growth as well as the contribution of other PGP traits. There are numerous soil microflora involved in the synthesis of auxins in pure culture and soil (Barazani and Friedman 1999). Different plant seedlings respond differently to variable auxin concentrations (Sarwar and Frankenberger1994) and type of microorganisms. Substances produced by bacteria are released continuously, and, especially when they are produced on the surfaces or within the plant tissue since the bacteria grow there. It seems probable that plant growth substances produced by PSBs improve plant growth by their direct effects on metabolic processes. However, since they induce proliferation of lateral roots thus increase nutrient absorbing surfaces, this may lead to greater rates of nutrient absorption. This in turn would be expected to significantly increased the shoot and root length of the plants.
The root length increased in chik pea due to the inoculation with plant growth promoting bacteria was in the range of 50-175% as compared with un-inoculated control while increase in shoot length was in the range of 142%-180% as compared with un-inoculated control (Table 24; Figure 61, 62-63).
All three PGPRs, improves plant growth by various mechanisms which could not be mutually exclusive. It is likely that phosphate solubilizing strains might have helped in plant root proliferation and production of plant growth regulators by the bacteria at root interface which resulted in better water absorption and nutrients such as P by host plant (Barea