Isolation, Characterization and Antagonistic Activity of Fluorescent Pseudomonads

Rhizobacteria that exert beneficial effects on plant growth and development are referred to as plant growth promoting rhizobacteria (PGPR) (Ashrafuzzaman et al., 2009). PGPR promote plant growth by various factors like ability to produce plant growth regulators, asymbiotic N2 fixation, antagonism against phytopathogenic microorganisms by production of siderophores, antibiotics and cyanide, solubilization of mineral phosphates and other nutrients (Sarvanakumar et al., 2007). PGPR may use more than one of above mechanisms to enhance plant growth as experimental evidence suggests that the plant growth stimulation is the net result of multiple mechanisms that may be activated simultaneously (Martinez et al., 2010). Pseudomonas is diverse genus that occupies many different niches and exhibits versatile metabolic capacity (Clarke, 1982).


Introduction
Rhizobacteria that exert beneficial effects on plant growth and development are referred to as plant growth promoting rhizobacteria (PGPR) (Ashrafuzzaman et al., 2009). PGPR promote plant growth by various factors like ability to produce plant growth regulators, asymbiotic N 2 fixation, antagonism against phytopathogenic microorganisms by production of siderophores, antibiotics and cyanide, solubilization of mineral phosphates and other nutrients (Sarvanakumar et al., 2007). PGPR may use more than one of above mechanisms to enhance plant growth as experimental evidence suggests that the plant growth stimulation is the net result of multiple mechanisms that may be activated simultaneously (Martinez et al., 2010). Pseudomonas is diverse genus that occupies many different niches and exhibits versatile metabolic capacity (Clarke, 1982).
They produce secondary metabolites with antibiotic properties such as phytohormones, volatile compounds, hydrogen cyanide (HCN) and siderophores. Plant growth-promoting ability of these bacteria is mainly due to the production of indole-3-acetic acid (IAA), siderophores and antibiotics (Lautenberg et al., 2001). Such strains can protect plants from various soil borne pathogens and/or stimulate plant growth (Haas and Defago, 2005). The abundance of literature on genus Pseudomonas is due to their elevated metabolic versatility capable of utilizing a wide range of simple and complex organic compounds and holding an important position in biosphere ecology (Scarpellini et al., 2004).
Antagonistic activity of Pseudomonas fluorescens and Pseudomonas putida in the rhizosphere has been recognized as major factor in the suppression of many phytopathogens. Bacteria of the genus Pseudomonas comprise a large group of the active biocontrol strains as a result of their general ability to produce a diverse array of potent antifungal metabolites.

Isolation of fluorescent pseudomonads
A total of 37 soybean rhizosphere soils were collected from major soybean growing areas of Dharwad (15) and Belagavi (22) districts of Karnataka at crop maturity stage and used for the isolation of fluorescent pseudomonads on King's B medium using serial dilution and spread plate technique.

Characterization of FP isolates
The colonies showing greenish to yellowish pigmentation on King's B medium were picked up and observed for fluorescence under UV light. The isolates were also studied for their colony morphology, cell shape and gram reaction as per the standard procedure given by Barthalomew and Mittewar (1950) and Anonymous (1957). The isolates were subjected to biochemical characterization by employing the standard procedures given by Cappuccino and Sherman (1992). Different biochemical tests performed were denitrification, starch hydrolysis, arginine hydrolysis, gelatin liquification and Oxidase test.

Functional Characterization of FP isolates
The isolates were tested for their ability to solubilise P, HCN production, Siderophore production and ability to produce plant growth promoting substances like IAA and GA.
In vitro screening of fluorescent pseudomonad isolates against fungal pathogens of soybean All the 62 FP isolates were studied for their antagonistic activity against two fungal pathogens viz., Sclerotium rolfsii Sacc., causing collor or root rot (soil borne) and Colletotricum truncatum causing anthracnose (foliar pathogen) in soybean plant under in vitro condition, these pathogens also causes variety of diseases in many crops.
The pure cultures of S.rolfsii and C. truncatum were obtained from the Department of Plant Pathology, University of Agricultural Sciences, Dharwad. The dual inoculation technique of Sakthivel and Gnanamanickam (1987) were followed to study the antagonistic activity of the fluorescent pseudomonads against soil borne and foliar plant pathogens.

Results and Discussion
Isolation of fluorescent pseudomonads from rhizosphere soil was carried out by employing serial dilution and plating on King's B selective media. It may be attributed to specific choice of media employed for isolation viz., Nutrient agar and King's B. Several others hence supported to use King's B medium for isolation of fluorescent Pseudomonas sp. (King et al., 1954). For the isolation of pseudomonads and fluorescent Pseudomonas sp., TSA and King's B agar medium were also used (Raaijmakers and Weller, 1998). Total of 62 FP isolates were obtained from the 37 rhizosphere soil samples collected from the major soybean growing areas of Dharwad and Belagavi district of Karnataka (Table 1).
All 62 isolates were used to study morphological traits. The colony morphology of isolates was found to be round to irregular shape and the irregular shaped colonies were found to be spreading type. All the 62 isolates exhibited fluorescence under UV light. However variations with respect to intensity of fluorescence and colony morphology were observed. Based on intensity of fluorescence under UV light, the isolates were classified as very good fluorescence (+++), medium fluorescence (++) and low fluorescence (+). Out of 62 isolates, 27 showed very good fluorescence (+++) and 32 isolated showed medium fluorescence (++) under UV light.
Fluorescence is an important trait for identification, characterization and grouping of fluorescent pseudomonads (Brown and Lowbury, 1968). Among the 62 isolates majority of the isolates produced light green to green pigmented colonies, while some of the isolates appeared to have yellowish orange colonies.
Colonies showing fluorescence under transilluminator were marked and a loopful of growth was picked in the laminar air flow for further purification. This could be a useful practice to obtain fluorescent pseudomonads from plate containing several isolated colonies from soil or root samples. It is reported from previous studies that some pigments such as carotenoids produced by pseudomonads species do not diffuse in to the medium and such colonies were found to have yellowish green colour which could resemble other fluorescing pigments (Indi, 2010). The cell morphological studies and the Gram reaction test revealed that all the 62 isolates were rod shaped and displayed a negative reaction for the Gram staining.
The biochemical characterization indicated that out of 62 isolates, 36 were positive for starch hydrolysis and 24 for arginine hydrolysis, 52 for oxidase test, 39 were positive for gelatin liquefaction and 36 exhibited the denitrification ability (Table 2). These results are in conformity with the work of Singh et al., (2007), who reported that out of seventeen isolates, seven isolates were identified to be Pseudomonas sp. on the basis of their cultural, morphological and biochemical characters. Similarly, Thirty five isolates of Pseudomonas fluorescens were isolated from the rhizosphere of rice fields by Meera and Balabaskar (2012). Among these, seven isolates which showed bright fluorescence under UV light were confirmed to be P. fluorescens after cultural and biochemical studies.
All the 62 isolates were also exercised to study the ability of the isolates to grow at 4 0 C and 41 0 C temperature. It was observed that there is an existence of variation among the isolates. Fifty two of the isolates were able to grow at 4 0 C temperature and 10 isolates showed growth at 41 0 C ( Table 3). The findings are in conformity with the work of Suman et al., (2016) who revealed that, among nineteen isolates, eight isolates showed growth at 4 0 C and eleven isolates at 42 0 C.

By
considering the morphological, biochemical characteristics and ability of the isolates to grow at 4 o C and 41 o C and utilize different carbon sources for their growth, 50 isolates were tentatively identified as P. fluorescens, 10 as P. aeruginosa and 2 isolates as P. aureofaciens strains based on Bergey's manual of Bacteriology. The results pertaining to characterization in this study are in line with the observation made by Paramageetham and Prasada Babu, 2012;Suman et al., 2016. All the isolates were also examined for their functional properties like, P-solubilization, production of plant growth promoting substances and their biocontrol potential ( Table 4). Out of 62 isolates, 52 were able to produce clear zone of P-solubilization (TCP) on Pikovskaya's agar medium. These isolates displayed wide variations in the diameter of the zone of solubilization, which varied from 5.31-21.71mm. The extent of zone of solubilization may or may not correlate with the amount of P solubilized (Rashid et al., 2004). Isolates of Pseudomonas fluorescens species differ in the ability to produce phosphatase enzyme and production of organic acids and hence showed different solubilization efficiency.

Fluorescent
pseudomonads offer an interesting biological system with their ability to promote plant growth directly through production of plant growth promoting substances (IAA and GA) and indirectly through control of plant pathogens and deleterious organisms or both (Bakthavatchalu et al., 2012).
Seed bacterization with such organisms has emerged as a powerful technology to enhance plant growth and yield, besides providing protection against diseases. Earlier, Suneesh (2004) and Megha et al., (2007a) made an attempt to characterize PGPR isolates of Western Ghats and studied their functional diversity. Their efforts helped in identifying several PGPR with novel traits useful in agriculture.
The present study is complimented with the previous work done. All 62 isolates were screened for their ability to produce IAA and GA. All the fluorescent pseudomonads in the present study produced significantly varying quantities of IAA (3.90 g to 28.89 g IAA/25 ml of broth) (Table 4). Among the isolates, DFP48 recorded highest IAA production of 28.89 g/25ml.
Isolates from the rhizosphere are more efficient auxin producers than isolates from the bulk soil (Sarwar and Kremer, 1992) and IAA production by PGPR can vary among different species and strains, and it is also influenced by culture condition, growth stage and substrate availability (Mirza et al., 2001). The results obtained in this study are in line with the observation made by Khakipour et al., (2008), who reported that the IAA produced by P. fluorescens and P. putida strains varied from 0 to 31.6 mg/l and 0 to 24.08 mg/l, respectively.     Note: (+) slight production, (++) moderate production, (+++) maximum production (-) no production  The variations in IAA production could be an inherent metabolic variability among the isolates (Leinhos and Vacek, 1994). The level of expression of IAA depended on the biosynthetic pathway, the location of genes involved and the presence of enzymes that could convert active free IAA into an inactive conjugated form (Patten and Glick, 1996).
Gibberellic acid is a class of phytohormone most commonly associated with modifying plant morphology by the extension of plant tissue, particularly the stem tissue (Salisbury, 1994). The amount of GA production by different FP isolates ranged from 0.56 to 18.52 g/25ml of broth. The isolate DFP54 was found to produce maximum amount of GA (18.52 µg per 25 ml broth) (Table 4). These results could be compared with those reported earlier by Lenin and Jayanti (2012), who observed the production of GA 3 by isolates of Pseudomonas ranged from 6.21 to 6.80 µg per 25 ml broth. Similarly, Suneesh (2004) reported that all the 48 fluorescent Pseudomonads isolated from the moist deciduous forests produced GA in the range of 0.72 to 5.27 µg per 25 ml of broth.
The fluorescent pseudomonads have been the most widely studied group of PGPR with respect to biocontrol of soil borne plant pathogens. The increased interest in the fluorescent Pseudomonas sp. in worldwide as biocontrol agents gained momentum after the initial studies conducted at the University of California, Berkeley, during the late 1970s (Weller et al., 1988). In addition to all the beneficial traits as discussed earlier, 62 isolates were used to study their antagonistic potential against two fungal pathogens (S. rolfsii and C. truncatum) of soybean under in vitro condition using dual plate technique (Vincent, 1947).
The siderophore production by antagonistic microorganisms is believed to be a mechanism of pathogen suppression. Siderophore production test using CAS agar plate has been used for rapid screening of potential beneficial bacterial isolates (Schwyn and Neiland, 1987). Bacteria with the ability to produce siderophore can enhance plant growth by increasing the availability of iron near the roots for plant uptake (Alexander and Zuberer, 1991). Although, all 52 antagonistic isolates of this study produced siderophores, the zone of clearance on CAS agar ranged from 6 to 36.37 mm (Table 4). Twenty one isolates produced the zone of clearance between 5 to 10 mm, which were significantly superior to the remaining isolates. Similarly, among the isolates tested, 15 isolates recorded 10-15 mm zone of clearance, 3 isolates exhibited the zone of clearance between 25 to 30 mm. The isolates DFP54 recorded highest zone of clearance of 36.36 mm and three isolates did not show any clearance zone.
The siderophores are usually produced by various beneficial soil microbes. Among them fluorescent pseudomonads are also involved in inhibition of S. rolfsii which is positively correlated (r = +0.336**) with production of siderophores by fluorescent pseudomonads (Indi, 2010). The concept of disease suppression by siderophores and the role of siderophores produced by fluorescent pseudomonads in plant growth promotion were explained for the first time by Kloepper et al., (1980). This study resulted in obtaining 62 fluorescent pseudomonads from the soybean rhizosphere samples collected from Belagavi and Dharwad districts of Karnataka. Some of these isolates exhibited MPS activity, PGPR production, HCN production and siderophore production. They also showed antagonistic activity against S. rolfsii and C. truncatum under in vitro condition. Application of these microbes for diseases management and their practical use requires further investigation under field conditions.