INTRODUCTION:

The exploitation of biocontrol agent for the management of plant disease have achieved greater significance in recent times due to its readily available nature, antimicrobial activity, easy biodegradation, non-phytotoxicity, besides inducing resistance in host ¹. Among various bacterial and fungal biocontrol agents, Bacillus, Pseudomonas and Trichoderma were most frequently used against various plant diseases. Fluorescent Pseudomonads and Bacillus were isolated and screened in vitro for their plant growth promoting traits like production of indole acetic acid (IAA), salicylic acid, hydrogen cyanide (HCN) and siderophores². Production of hydrogen cyanide (HCN) is a major factor in the control of soil-borne diseases by Pseudomonas fluorescens CHA0^.3

Investigations were conducted to determine the role of salicylic acid (SA) in induced systemic (ISR) resistance against blue mold disease of tobacco elicited by plant growth-promoting rhizobacteria (PGPR). The observations indicate that SA accumulation in tobacco plants may play a role in ISR against tobacco blue mold by PGPR. ⁴ Siderophores sequesters ferric ions in the environment and the ferric siderophores are taken up in the microbial cells after specific recognition by membrane proteins. The production of siderophores is an important trait of PGPR in their ability to suppress soil-borne pathogens.

Competition for ferric iron between the PGPR and the plant deleterious microorganisms is considered the mode of action of these siderophores ⁵. Fluorescent Pseudomonads isolated from rhizophere showed biocontrol potential and the isolates displayed the production of siderophore, HCN and IAA.⁶ The present study is aimed to investigate the production of antimicrobial compounds from the bacterial isolates.

MATERIALS AND METHODS:

Collection and maintenance of bacterial antagonists :

Ten isolates of P. fluorescens and ten isolates of B. subtilis were collected from the Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore - 43 and pure cultures were maintained on King’s B agar and nutrient agar slants respectively.

Antimcirobial compounds of P. fluorescens and B. subtilis isolates:

Hydrogen cyanide (HCN) production :

Qualitative assay :

P. fluorescens and B. subtilis strains were grown on Trypticase soy agar (TSA) Filter paper soaked in picric acid solution (2.5 g of picric acid; 12.5 g of Na₂CO₃, 1000 ml of distilled water) was placed in the lid of each petridish. Dishes were sealed with parafilm and incubated at 28^oC for 48 hr. A change in the colour of the filter paper discs from yellow to light brown, brown or reddish brown was recorded as an indication of weak, moderate or strong in producing HCN by each strain, respectively.⁷

Quantitative assay:

Pseudomonas fluorescens and B. subtilis strains were grown on Trypticase soy broth (TSB) Filter paper was cut into uniform strips of 10 cm long and 0.5 cm wide saturated with alkaline picrate solution and placed inside the conical flasks in a hanging position. After incubation at 28 ± 2^oC for 48 h, the sodium picrate in the filter paper was reduced to a reddish compound in proportion to the amount of hydrocyanic acid evolved. The colour was eluted by placing the filter paper in a clean test tube containing 10 ml of distilled water and absorbance was measured at 625 nm. ⁸

Salicylic acid (SA) production:

The strains were grown in the standard succinate medium at 28 ± 2^oC for 48 h. Cells were collected by centrifugation at 6000 g for 5 min and were resuspended in 1 ml of 0.1 M phosphate buffer. Four ml of cell free culture filtrate was acidified with 1 N HCl to pH 2.0 and SA was extracted in CHCl₃ (2×2ml). Four ml of water and 5 µl of 2M FeCl₃ were added to the pooled CHCl₃phases. The absorbance of the purple iron-SA complex, which was developed in the aqueous phase, was read at 527 nm. A standard curve was prepared with SA dissolved in succinate medium and quantity of SA produced was expressed as mg ml.^-1 ⁹.

Siderophore production:

Detection of nature of siderophore:

Each isolates of P. fluorescens and B. subtilis was inoculated on King’s B broth and nutrient broth, respectively. It was incubated in a rotatory shaker at 120 rpm for 48 hours. The bacteria multiplied in the broth were used as the sample for the detection of the nature of the siderophore.

Hydroxamate nature: It was examined by tetrazolium test. Instant appearance of deep red color by the addition of siderophore sample to tetrazolium salt under alkaline conditions indicated the presence of hydroxamate nature.¹⁰

Carboxylate nature: It was detected by vogel chemical tests where the disappearance of pink color on addition of phenolphthalein to sample under alkaline condition indicated the presence of carboxylate nature.¹¹

Quantitative detection of siderophore:

The P. fluorescens and B. subtilis strains were grown in KB broth and nutrient broth respectively for 7 days and centrifuged at 10000 rpm for 20 min. The supernatant was used for the estimation of catecholate type and salicylate type of siderophore. The pH of the supernatant was adjusted to 2.0 with 1 N HCl and equal quantity of ethyl acetate was added in a separatory funnel, mixed well and ethyl acetate fraction was collected. This process was repeated three times to bring the entire quantity of siderophore from the supernatant. The ethyl acetate fractions were pooled, air dried and dissolved in 5 ml of ethanol (50%). Five ml of ethyl acetate fraction was mixed with 5 ml of Hathway’s reagent (1.0 ml of 0.1 M FeCl₃ in 0.1 N HCl to 100 ml of distilled water + 1.0 ml of 0.1 M potassium ferricyanide). The absorbance was read at 560 nm ¹³ A standard curve was prepared using sodium salicylate for the estimation of salicylate type of siderophore .The quantity of siderophore synthesized was expressed as mg ml^-1of culture filtrate.

To measure the catechol type of siderophore, 5 ml of ethylacetate fraction was treated with 5 ml of Hathway reagent and absorbance was measured at 700 nm with 2, 3- dihydroxy benzoic acid as a standard. The quantity of siderophore synthesized was expressed as mg ml^-1of culture filtrate.

Indole acetic acid (IAA) :

Tripticase soy broth (TSB) with tryptophan as a precursor (100 g/ml) was inoculated with P. fluorescens and B. subtilis strains and incubated on a rotary shaker for 30 h. Supernatants from the cultures were collected after centrifugation at 2000 rpm for 10 min. To one ml of cell free culture filtrate, 2 ml Salkowsky reagent (1 ml of 0.5 M FeCl₃in 50 ml of 35% HClO₄) was added and incubated at 28 ± 2°C for 30 min. The absorbance was measured at 530 nm. A standard curve was prepared with IAA and quantity of IAA produced was expressed as mg ml^{-1 13}

Statistical Analysis:

All the experiments were repeated once with similar results. The data were statistically analyzed ¹⁴ and the treatments were compared by Duncan’s Multiple Range Test (DMRT). The package used for analysis was IRRISTAT version 92 developed by the International Rice Research Institute, Biometrics Unit, The Philippines.

RESULTS:

Antimicrobial compounds of P. fluorescens and B. subtilis isolates:

The effective isolates of P. fluorescens AUPF3 and AUPF4 and B. subtilis isolates AUB2 and AUB8 were selected for analyzing the antifungal compounds such as hydrogen cyanide, salicylic acid, siderophore and indole acetic acid.

Hydrogen cyanide (HCN) production:

HCN produced by the effective isolates of P. fluorescens and B. subtilis was assayed qualitatively and quantitatively. Among the four isolates, AUPF3 and AUPF4 recorded moderate HCN production whereas B. subtilis isolates AUB2 and AUB8 recorded weak production of HCN (Table 1).

Table 1: HCN production by antagonistic bacterial isolates

S. No.	Antagonistic bacterial isolates	HCN production*
S. No.	Antagonistic bacterial isolates	Qualitative Analysis	Quantitative Analysis (OD at 625 nm)
1.	AUPF3	Moderate	0.067^a
2.	AUPF4	Moderate	0.060^b
3.	AUBS2	Weak	0.041^c
4.	AUBS8	Weak	0.040^c

* Mean of three replications

In a column, means followed by a common letter(s) are not significantly different (P=0.05) by DMRT.

Quantitative analysis also revealed that among four isolates, AUPF3 produced higher amount of HCN (0.067 OD at 625 nm). This was followed by AUPF4 (0.060 OD at 625 nm), AUBS2 (0.041 OD at 625 nm) and AUBS8 (0.040 OD at 625 nm).

Salicylic acid production:

The results of present study revealed that among the four isolates, AUPF3 recorded maximum production of salicylic acid (26.97 mg/ml). This was followed by AUBS2, AUPF4 and AUBS8, which recorded 25.67, 24.5 and 20.44 mg/ml respectively (Table 2).

Table 2: Salicylic acid production by antagonistic bacterial isolates

S. No.	Antagonistic bacterial isolates	*Salicylic acid production (**mg/ml)
1.	AUPF3	26.97^a
2.	AUPF4	24.5^c
3.	AUBS2	25.67^b
4.	AUBS8	20.44^d

* Mean of three replications

In a column, means followed by a common letter(s) are not significantly different (P=0.05) by DMRT.

Siderophore production:

Analysis of nature of siderophore produced by antagonistic bacterial isolates revealed that all the isolates were known to produce hydroxymate type of siderophore. Quantitative analysis reported that AUPF3 and AUPF4 produced maximum amount of siderophore, which recorded 43.66 and 42.74 mg/ml respectively. This was followed by AUBS2 (19.47mg/ml) and AUBS8 (16.33 mg/ml) (Table 3).

Table 3: Siderophore production by antagonistic bacterial isolates

S. No.	Antagonistic bacterial isolates	*Siderophore production (**mg/ml)
1.	AUPF3	43.66^a
2.	AUPF4	42.74^a
3.	AUBS2	19.47^b
4.	AUBS8	16.33^c

* Mean of three replications

In a column, means followed by a common letter(s) are not significantly different (P=0.05) by DMRT.

Indole acetic acid (IAA) production:

In the present study, four isolates of antagonistic bacteria were evaluated for their efficacy in producing IAA. Among the four isolates, AUPF3 produced maximum amount of IAA (2.34 mg/ml). The isolates AUPF4, AUBS2 and AUBS8 recorded 1.97, 1.78 and 1.6 mg/ml respectively (Table 4).

Table 4: Indole acetic acid production by antagonistic bacterial isolates

S. No.	Antagonistic bacterial isolates	*Indole acetic acid production (**mg/ ml)
1.	AUPF3	2.34^a
2.	AUPF4	1.97^b
3.	AUBS2	1.78^bc
4.	AUBS8	1.6^c

* Mean of three replications

In a column, means followed by a common letter(s) are not significantly different (P=0.05) by DMRT.

DISCUSSION:

Pseudomonas and Bacillus species produces various secondary metabolites. Moreover, HCN and siderophores have been found to be inhibitory against different phytopathogens ¹⁵. It has been documented that HCN and cell wall degrading enzymes could be involved in antagonistic activity toward phytopathogens and contributes to the biocontrol of plant diseases ¹⁶. Study revealed that among the four isolates, AUPF3 and AUPF4 recorded moderate HCN production whereas isolates AUBS2 and AUBS8 recorded weak production of HCN.

Several authors have reported that hydrogen cyanide played an important role in disease suppression by Pseudomonas species.^17-20

Salicylic acid production:

A number of elicitors may be produced by the PGPR strains upon inoculation, including salicylic acid, siderophore, lipopolysaccharides and 2,3-butanediol and other volatile substances. ²¹The results of present study showed that among the four isolates, AUPF3 recorded maximum production of salicylic acid followed by AUBS2, AUPF4 and AUBS8.

Twelve isolates of P. fluorescens were tested for their ability to produce salicylic acid. Among the 12 isolates, PfUA 7 was resulted maximum amount of salicylic acid (159.7 mg/ml). This was followed by PfUA 6 (95.60 mg/ml) and PfUA 11 (73.80 mg/ml). The other isolates showed the production of salicylic acid ranging from 11.08 to 64.72 mg/ml²².

A study on four different B. subtilis isolates for salicylic acid production showed that the B. subtilis isolate BS13 produced maximum amount of salicylic acid accounting for 0.47 mg/ml. However other B. subtilis isolates (BS8, BS9 and BS11) produced less quantity compared to BS13.²³

Siderophore production:

Under conditions of low iron availability, most aerobic and facultative anaerobic microorganisms including fluorescent Pseudomonad spp. produce low molecular weight Fe³⁺ specific chelators, siderophores. Competition for ferric ion between these microorganism and plant pathogens is considered to be the mode of action of these siderophore ²⁴. Analysis of nature of siderophore produced by antagonistic bacterial isolates revealed that all the isolates were known to produce hydroxymate type of siderophore. Isolates AUPF3 and AUPF4 produced maximum amount of siderophore followed by AUBS2 and AUBS8.

Nine Pseudomonas fluorescens isolates were screened for their production of siderophore. The result indicated that all the fluorescent isolates produced siderophores and the concentration ranges between 13.6 to 196.3 μg/ml with the highest production by strain Psd 2 and lowest by strain Psd 9.²⁵

Indole acetic acid (IAA) production:

The ability to produce indole-3-acetic acid (IAA) is widespread among soil and plant-associated microorganisms ²⁶. The antagonistic bacteria were evaluated for their efficacy in producing IAA. Among the four isolates, AUPF3 produced maximum amount of IAA compared to the other isolates.

A report on the the ability of Pseudomonas isolates obtained from rice rhizosphere to produce IAA, was almost threefold higher than Pseudomonas isolates obtained from maize rhizosphere and they resulted in the higher ability to control fungal pathogen than isolates producing low amount of IAA.²⁷.

In the present study, among the various isolates of Pseudomonas fluorescens and Bacillus subtilis AUPF3, AUPF4, AUBS2 and AUBS8 showed maximum production of antimicrobial compounds such as HCN, IAA, salicylic acid and siderophores .The antagonistic bacterial isolates of Pseudomonas fluorescens and Bacillus subtilis can be used as a suitable candidate for the management various plant diseases caused by the fungal pathogens.

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Received on 14.12.2011 Modified on 28.12.2011

Asian J. Research Chem. 5(1): January 2012; Page 61-64

INTRODUCTION:

Statistical Analysis:

AUPF3

AUPF3

AUPF3

Antagonistic bacterial isolates

AUPF3