Plant Growth Promoting Characteristics in Some Flavobacterium spp. Isolated from Soils of Iran

Plant growth promoting rhizobacteria (PGPR) is referred to a heterogeneous group of beneficial rhizosphere bacteria that could enhance plant yield through one or more mechanisms. Flavobacterium has been noted as PGPR in almost all review articles. However, there are a few studies regarding plant growth promotion imposed by them. Some of Plant growth promoting characteristics such as Phosphate solubilizing capacity, ability to use of 1-Amino Cyclopropan-1-Carboxylate (ACC) as sole nitrogen source and production of auxin, siderophore, salicylic acid, chitinase and hydrogen cyanide were evaluated in forty-four flavobacteria isolated from rhizosphere of wheat. Results showed that none of the isolates were able to produce siderophore, salicylic acid and chitinase and they were not able to use ACC as well. Determining the siderophore showed that none isolates did not grow on Chrome Azurol S (CAS) Agar medium. The results of this part were further analyzed using CAS Agar Diffusion (CASAD) method, but the results were also negative. HCN production was observed in all isolates, but in lowest limit. Thirty-four isolates were capable to solubilize insoluble inorganic Phosphate (P) sources. The average rate of P-solubilization was 3.54 μg Pml, ranging from zero to 37.48 μgPml. There was a significant negative correlation (r = -0.81**) between solubilized P and the final pH of the growth medium. In this study, all the isolates were able to produce auxin, ranging from 0.27 to 12.03 μgml averaged by 2.03. Considering the ability of the isolates to produce auxin and for P-solubilization, it is necessary to evaluate their effect on growth and yield of different crop plants.


Introduction
The rhizosphere as defined by Boven and Rovira (1999) is a tender zone (about 1-3mm) of soil surrounding a plant root where living organisms are influenced by root vital activities (like compounds exuded by the root and respiration) qualitatively and quantitatively.Wide range of microorganisms lives in this area that their variation and accumulation is influenced by plant type.
Rhizobacteria have been classified into beneficial, deleterious and neutral according to their effect on host (Benizri et al., 2001).Classification of beneficial rhizobacteria to symbiotic and asymbiotic have been done on the base of their physical relationship with plant.The term PGPR was first used by Kloepper and Schroth (1978) and investigation on PGPR have been escalating at an ever increasing rate since then.PGPR can stimulate plant growth directly as they can improve the supply of nutrients, such as nitrogen (Dobbelaere et al., 2003) and phosphorous (Rashid et al., 2004) or by production of phytohormones (Choong et al., 2003;Stepanova et al., 2008) and ACC-deaminase synthesis (Arshad et al., 2007).Indirectly PGPR can also promote plant development by the suppression of pathogens mediated by different mechanisms such as antibiosis (Milner et al., 1996), iron sequestration by siderophores ( Bar-ness et al., 1992), HCN ( Keremer and Souissi, 2001), vitamin excretion (Streit et al., 1996), and cell wall degrading enzymes like chitinase (Ajit et al., 2006).So plant growth is promoted through reducing or neutralizing pathogen activity.
Azotobacter, Entrobacter, Bacillus, Burkholderia, Azospirillum, Pesudomonas, Acinetobacter, Arthrobacter, Alcaligenes, Serratia, Erwinia and Flavobacterium are some of the common PGPRs.(Sturz and Nowak, 2000;Bloemberg and Lugtenberg, 2001;Mayak et al., 2004).Flavobacterium is related to chlorobia group super phylum, Bacteriodete phylum, Flavobacteria class, Flavobacterials order and Flavobacteriaceae family.These bacteria are isomorphic, baciliform, aerobic and gram-negative.Lack of poly-β-hydroxybutyrate granules, endospore and fellagelin have been seen in these bacteria (Krieg and Holt, 1984).Plant growth promoting qualities of this bacterium is investigated despite it is plant growth stimulating bacteria.The most important growth promoting qualities like auxin production, the ability of using of ACC as nitrogen source, P-solubilization, siderophore, salicylic acid, chitinase and HCN production of Flavobacterium isolates were evaluated in microbial collection of soil biology department of soil and water institute.

Isolation of Flavobacterium
44 isolates of Flavobacterium were isolated from wheat rhizosphere.Fluorescent Pseudomonad, strain PA14 was investigated for auxin, salycilic acid, siderophore, chitinase and HCN production and P-solubilisation and PA25 was evaluated for ACC-deaminase production as control isolate.

Quantification of IAA
Production of IAA was assayed based on the method described by Patten and Glick (2002).Strains were grown at 28 º C for 48h on a rotary shaker in 100ml flasks containing 50ml TSB medium.Cells were then collected by centrifugation at 10000g for 15min.Finally 2ml of salkowsky was added to the supernatant.The absorbance of the pink-auxin complex, was read at 535nm in a Spectrophotometer.A standard curve was prepared with auxin dissolved in TSB medium.The quantity of auxin in the culture was expressed as gml -1 .

ACC-deaminase assay
The ability of strains to utilize ACC as sole nitrogen source was assayed as described by Amico et al. (2005) with some modifications.The bacteria were cultured first in rich medium (TSB).Stock solution of ammonium sulphate (13.21 g of (NH 4 ) 2 SO 4 in 1000 ml of distilled H 2 O) and DF salts minimal medium (without nitrogen source) were prepared and sterilized by autoclaving and 3.0 mM solution of ACC (30.33 mg of ACC in 10 ml of distilled H 2 O) as the source of nitrogen was also filter-sterilized (0.2 µm).300µL of each bacterial culture were added to 50-ml flasks containing 15 ml of sterile DF salts medium (with out nitrogen source).Two hundreds micro liters of ACC and (NH 4 ) 2 SO 4 were added to the flasks.Cultures were incubated at 30C on a rotary shaker at 200 rpm for 48 h.After incubation for 48 h the density of the culture was measured at 405 nm and the ratio of density in the culture of DF +ACC medium and DF+ (NH 4 ) 2 SO 4 medium was evaluated.

Phosphorous assay
Sterilized Pikovskaya media (PKV) was poured in to sterilized Petri plates after solidification of media, a pinpoint inoculation of bacterial strains was made on the plates under aseptic conditions.The plates were incubated at 28C for 8 days and observed for colony diameter and diameter of solubilization zone regularly during 8 days.Solubilization index was evaluated according to the ratio of the total diameter (colony + halo Zone) and the colony diameter (Rashid et al., 2004).

CAS agar assay
Production of siderophores was determined by the method of Alexander and Zuberer, (1991).Isolates were grown on CAS agar plates.The presence of orange halos was recorded up to 7 days after incubation.

CASDA assay
Siderophore production in CASAD method was determined by the method of Shin et al., (2001).Strains were grown at 28oc for 48h on a rotary shaker in 100mL flasks containing 50mL succinate medium (succinic acid, 4.0g; K 2 HPO 4 , 6.0g; KH 2 PO, 3.0g; (NH 4 )SO 4 , 1.0g; MgSO 4 .7H 2 O, 0.2g; distilled water, 1000mL; pH: 7.0) Cells were then collected by centrifugation at 10000g for 10min.Punch the CAS medium with cork borer and pour 35 mL from supernatant to these sinks after absorbing.The equal amount of supernatant should be poured in sinks, read the halo diameter.

Production of Salicylic acid (SA)
Strains were grown at 28 o C for 48h on a rotary shaker in 100ml flasks containing 50ml succinate medium.Cells were then collected by centrifugation at 6000g for 5min and 4ml of cell free culture was acidified with 1N HCl to adjust the pH to 2.0 and SA was extracted in CHCl 3 (2×2ml).To the pooled CHCl 3 phases, 4ml of distilled water and 5µl of 2M FeCl 3 were added.The absorbance of the purple iron-SA complex, which was developed in the aqueous phase, was read at 527nm in a Spectrophotometer.A standard curve was prepared with SA dissolved in succinate medium.The quantity of SA in the culture was expressed as µgml -1 (Meyer et al., 1992).

Agar plate assay for chitinase
Chitinase production was determined in a defined medium composed of (gL -1 ) colloidal chitin (Berger and Reynolds, 1958).It was added to nutrient agar medium.12 mL of bacterial suspension was cultured in this medium and incubated for 120 hours in 28 o C. The ability of chitinase production was shown by clear halo around colonies (Robert and Cabib, 1988).

Cyanide production
Hydrogen cyanide (HCN) production from glycine was tested growing the bacteria in 10% trypic soy agar (TSA) supplemented with glycine (4.4gL -1 ) and cyanogenesis was revealed using picric acid and Na 2 CO 3 (0.5 and 2% respectively).Impregnated filter paper fixed to the underside of the Petridis lids.Results were read after five days of culture at 28 o C. A change in filter paper colors from yellow to orange-brown indicated production of HCN.(Yellow (1) limit cyanide production, orange (2) moderate cyanide production, light brown (3) relatively high cyanide production and brown (4) high cyanide production) (Donate-correa et al., 2004).

Results
44 isolates of Flavobacterium were isolated from wheat rhizosphere were tested for their plant growth promoting qualities.All of the strains could produce auxin.The average amount of auxin was 2.03 µgml -1 and its extent was changeable between 0.27 to 12.03.The high auxin activity was recorded for F9 and F32 respectively (Table 1).
None of the isolates produced salicylic acid (Table 1) when, the control isolate, PA14, produced 9.05 µgml -1 salicylic acid.Any isolate couldn't generate chitinase while the culture media was suitable for responding against chitinase and some of bacteria which were isolated from soil, made a bright halo around themselves (Table 1).None of the isolates could grow on CAS-agar media.CASAD method indicated, none of the isolates could produce siderophore (Table 1).

Discussion
Auxin is the most investigated hormone between plant growth regulators.The most common, best characterized and physiologically most active auxin in plant is indole-3-acetic acid (IAA).IAA is known to stimulate both a rapid response (e.g.increased cell elongation) and a long-term response (e.g.cell division and differentiation) in plants (Ahmad et al., 2005).In this investigation, all Flavobacterium isolates have been able to produce IAA.Asghar et al. (2004) showed, S 58 and S 89 which were related to Flavobacterium genus could beget IAA.The amount of IAA was 24.03 µgml -1 & 2.27 µgml -1 for S58 and 24.6 & 4.6 µgml -1 for S89 in presence and absence of tryptophan, respectively.In the other research, Cattelan et al, (1999) reported, GW 2103 and LC 1118 isolates of Flavobacterium indologenes had the ability of IAA production.IAA production by PGPRs is different.Ahmad et al. (2005) indicated that the amplitude of IAA in different densities of tryptophan was changeable between 5.32-5.34µgml -1 .The extent of IAA in Flavobacterium was changeable and it was enranged from 0.27 to 12.03 µgml -1 in this research.P-solubilizing microorganisms (PSM) involve different character of microorganisms which turn insoluble organic compounds of phosphorous to soluble form (Raju and Reddy, 1999;Sundara et al., 2002).Bacillus and pseudomonas have been the most important P-solvent bacteria (Rashid et al., 2004;Bar-Yosef et al., 1999).Rashid et al, (2004) have shown that 10 isolates of different bacteria which were selected from rice rhizosphere, could solve Ca 3 (Po 4 ) 2 in pikovskaya medium.The results indicated 10 isolates couldn't solve Ca 3 (Po 4 ) 2 between 49 isolates.Cattelan et al, (1999) have shown that GW 2103 and LC 1118 isolates of F. indologenes couldn't solve it too.Remaining isolates had the low ability to solve it in this research.The highest solvability was 37.48 µgml -1 for F11.
In addition to the well-characterized mechanisms, such as auxin production and siderophore synthesis, that are employed by PGPR, it was recently proposed that many PGPR may stimulate plant growth through the activity of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase.Some bacteria degrade ACC to ammonia and α-ketobutyrate through ACC deaminase and finally it reduces ethylene (Penrose and Glick, 2003).Recent researches showed that some of pseudomonas could produce ACC deaminase (Safronova et al., 2006).Belimov et al. 2005 presented that different isolates of Variovorax paradoxus had the ability to make ACC deaminase but AY 197006 and AY 197009 isolates of Flavobacterium were unable to produce it.Cattelan et al. (1999) exhibited, GW 2103 isolate of F. indologenes were unable to produce ACC deaminase and LC 1118 isolate wasn't investigated due to its lack of growth.The result of our research demonstrated that no Flavobacterium isolate could make ACC deaminase too.
Siderophores are low-molecular-weight molecules that are secreted by many microorganisms in iron shortage condition.O'sullivan and O'Gara. (1992) revealed that many isolates of P.fluorescens have the ability of making siderophore.Belimov et al. (2005) reported, AY 197010 isolate of Pseudomonas and AY 197006 and AY 197009 isolates of Flavobacterium could manufacture siderophore.In this research none of the isolates could grow on CAS-Agar medium.Probabely HDTMA (Hexadecyl trimethyl ammonium bromide) toxicity cause growth insufficiency (Sung et al., 2001).The result of CASAD method have shown, any isolate couldn't make siderophore.Cattelan et al. (1999) reported, there was negligible production of siderophore by LC 1118 and GW 2103 of F. indologenes.
PGPR also activate plant defense resulting in systemic protection against plant pathogens, a phenomenon termed induced systemic resistance (ISR).Salicylic acid produced by PGPR in rhizosphere may be involved in ISR (Maurhofer et al., 1998).SA production by WC 374 , WCS 417r (Leeman et al.,1996) and CHAO (Maurhofer et al., 1994), P.fluorescens and also 7NSK 2 isolate of P.aeruginosa (Demeyer and Hofte, 1997) was reported.Any Flavobacterium isolate could not produce SA in our study.
Many different bacteria could produce HCN which is toxic for fungi (Blumer and Hass, 2000).HCN production by P.fluoresens, P.aeruginosa and Chromobacterium uiolaceum was reported by many researchers (Seddiqui et al., 2003).Some of Rhizobium isolates were introduced as HCN producers by Antoun et al. (1998).Any isolate couldn't generate HCN in this study.Cattelan et al. 1999indicated that LC 1118 and GW 2103 isolates of F.  Phosphorous solubilization (mgL -1 ) pH

Figure 1 .
Figure 1.Correlation between Phosphorous solubilization and pH

Table 3 .
Analysis of variance of different isolate of Flavobacterium in Auxin production and phosphorous solubilization