Crop specific plant growth promoting effects of ACCd enzyme and siderophore producing fluorescent Pseudomonas on okra and pumpkin

Introduction

The plant rhizosphere is an important soil ecological environment for plant microbe interactions. It involves colonization by a variety of micro-organisms in and around the roots which may result in symbiotic, associative, neutralistic or parasitic relations within the plant, depending on the type of microorganism, soil nutrient status, plant defense system and soil environment. Aerobic gram-negative Fluorescent Pseudomonas spp. has emerged as the largest and potentially most promising group of plant growth promoting rhizobacteria involved in the bio-control of plant diseases (Weller et al. 2002; Fravel 2005) [17,4] . A large number of secondary metabolites, growth hormones, antibiotics and chelating compounds such as siderophores (Choudhary et al. 2009; Beneduzi et al. 2012) [3,2] are known to be released by these fluorescent pseudomonads. This group of bacteria has been termed "plant growth promoting rhizobacteria" or PGPR. Plant growth promoting rhizobacteria (PGPR) can stimulate growth by one or more different mechanisms. The direct mechanisms include production of growth hormones like IAA, phosphate-solubilization and uptake of iron, whereas indirect mechanisms include check on phytopathogens by the release of HCN, antibiotics and siderophores (O'Sullivan and O'Gara, 1992) [6] . Antagonistic rhizobacteria, more specifically, fluorescent pseudomonads, are well known to suppress fungal root diseases of agronomic crops, such as seedling disease of cow pea and wheat caused by S. rolfsii. Considerable progress has been made over the past decades to elucidate the mechanisms by which fluorescent pseudomonads suppress disease. It is apparent that a large variety of antifungal metabolites are produced by fluorescent pseudomonads. Hydrogen cyanide, phloroglucinol, pyoluteorin and pyrrolnitrin are produced by many of these bacteria, and attempts have been made to quantify the importance of each in disease suppression. Characteristics, especially biochemical characteristics, of fluorescent Pseudomonas has an important role to determine its potential level in inhibition of plant pathogens but the characterization never been done. The characteristics should be tested in order to know the field application of antagonist. [11] . Selected 21 candidate potential isolates were then used to screen the isolates for phosphate solubilization, siderophore producing and plant growth promoting ability. These isolates were isolated from soil samples of different geographical locations of Chhattisgarh. During the course of study, King'B medium was used for maintaining the cultures of Pseudomonas.

Biochemical characterization of bacterial isolates

The biochemical characterization of fluorescent Pseudomonas isolates will be carried out to characterize all the isolates of Fluorescent Pseudomonas. A rapid antibiotic sensitivity test was used to distinguish different species of fluorescent Pseudomonas. Isolates will be then characterized based on biochemical tests as per the procedures outlined in Bergey's Manual of Systematic Bacteriology (Sneath et al., 1984) [12] . The siderophore production was determined qualitatively by CAS agar plate method and specific tests were carried out for identification of hydroxamate and Catecholate types of siderophores following the standard methods (Arnow, 1937) [1] . Qualitative screening of phosphate solubilizing fluorescent Pseudomonas was performed on Pikovskayas agar medium (Hi media) containing tricalcium phosphate as a phosphate source and Bromo Cresol Purple (0.1 g/l) as a pH indicator for acidification (Vazquez et al. 2000) [15] . HCN production was determined on KM plates supplemented with 4.4 g L-1 glycine as described by Wei et al. (1991) [16] . ACC deaminase activity was determined by measuring the production of a-ketobutyrate and ammonia generated by the cleavage of ACC by ACC deaminase (Honma and Shimomura 1978) [5] . Based on the production of siderophores, HCN and phosphate solubilization candidate isolates were selected for experiments and therefore was also characterized for other plant growth promoting potentials.

In vitro Screening for antagonistic activity against soil borne phytopathogens

The twenty isolates were tested for their efficacy as biocontrol agent against the phytopathogens Sclerotium rolfsii. Equal volume of sterilized potato dextrose agar (PDA) and King's B medium was mixed and poured in sterilized petridishes. A heavy inoculum from an actively growing fluorescent Pseudomonas was streaked at 1 cm away from the edges of the plate and the mycelia disc of the pathogens were placed at the centre of petriplates. Control plates were inoculated only with phytopathogens but not with Pseudomonas isolates. Percent inhibition of pathogens by Pseudomonas isolates over control was calculated by using the formula:

[(Growth of pathogen in control -Growth of pathogen with Pseudomonas isolate)/ Growth of pathogen in control] x 100

Host seeds

Seeds of Okra (Var. ADITYA) and Pumpkin (var. HARITA) were obtained from Research cum Instructional farm, IGKV, Raipur, India.

Seed bacterization

Twenty isolates of fluorescent Pseudomonas were tested for their ability to promote plant growth in pot experiments. The bacterial cultures were inoculated in 25ml King's B broth and incubated at 28±2°C for 48 hrs. For seed soaking inoculums of all the isolates were diluted to final concentration of 10 8 -10 9 CFU mL -1 (600 nm). Seeds of chick pea variety were soaked in the diluted suspension for 1 hour. Seeds soaked in water served as control.

Pot experiment

The treated seeds were planted in pots containing soil mixed with sand and compost in the ratio of 3:1:1. Each treatment was replicated twice and the experiment was repeated two times. For taking measurement on growth parameters, three plants were randomly uprooted from each pot at 30 days after sowing (DAS). The following observations were taken -shoot and root length (cm), shoot and root fresh weight (gm) and dry weight (gm). Shoot and root dry mass was recorded after drying the samples in oven at 60 °C.

Results and Discussion

All the twenty isolates of fluorescent Pseudomonas showed positive response for siderophore production and phosphate solubilization (in Pikovskaya's agar medium with and without BCP indicator). Isolates showed variability for traits such as gelatin liquefaction, casein hydrolysis, lipolytic activity, nitrate reduction and antibiotic sensitivity test. In antibiotic sensitivity test, all twenty isolates were resistant to antibiotic carbenicillin amended medium. Siderophores are also thought to facilitate bio control by sequestering iron from pathogens, thus limiting their growth and for the solubilization of extracellular ferric iron by most bacteria and fungi. CAS assay is the universal assay for detection of siderophores. The principle of this assay is based on a colour change of CAS from blue to orange resulting from siderophoral removal of iron from the chrome azurol dye. All the isolates showed positive response for siderophore production on CAS agar plate i.e. orange colour halo was observed in all the isolates. All isolates exhibited positive response for its ability to produce HCN (Table 1). Overall, these isolates were found to be more efficient against S. rolfsii indicating that these isolates could therefore be exploited as potential candidates for the development of bio pesticides more specifically against these most devastating pathogens of crops.

Plant growth promotion activity of fluorescent

Pseudomonas on Okra (Var. ADITYA) and Pumpkin (var. HARITA) Efficacy of different isolates of Pseudomonas for mungbean okra plants varied to induce shoot and root length ranging from 9.05i±0.1 to 21.45a±0.9 cm and 14.025k±0.825 to 36.175a±0.975 cm respectively. Maximum shoot length (21.45a±0.9 cm) and root length (36.175a±0.975) were recorded when seeds were treated with P-11 and P-12, respectively as compared to control (Table 3, Fig. 1 and Fig. 2). Plants treated with P-30 and P-11 recorded highest shoot and root fresh weight (4.915a±0.017and 1.173a±0.018 gm, respectively) and maximum shoot and root dry weight (1.385a±0.015 and 0.137a±0.015 gm, respectively). Similarly in case of pumpkin efficacy of different isolates of Pseudomonas for pumpkin plants varied to induce shoot and root length ranging from 7.375h±0.125 to 17.425a±0.00 cm and 28.075i±5.575 to 76.9a±0.650 cm respectively. Maximum shoot length (17.425a±0.00 cm) and root length (76.9a±0.650 cm) were recorded when seeds were treated with P-15 and P-2, respectively as compared to control (Table 4, Fig. 1 and Fig. 2). Plants treated with P-1 and P-9 recorded highest shoot and root fresh weight (20.07a±1.073and 4.255a±0.215 gm) whereas P-1 and P-6 recorded maximum shoot and root dry weight (2.29a±0.107 and 1.52a±0.0403 gm, respectively.

Conclusion

This study illustrates the significance of rhizobacteria under in vitro conditions for multiple PGPR traits. This led to the selection of effective PGPR Isolates which as a result of their multiple PGPR traits, could prove effective in improving the seed germination rate, plant growth and vigor index of plants. Such type of study is necessary as it advocates that use of PGPR as inoculants or biofertilizers is an efficient approach to replace chemical fertilizers and these PGPR isolates may be used as biofertilizers to enhance the growth and productivity of for commercially grown plants under local agro-climatic conditions of Chhattisgarh.

Acknowledgement

AcknowledgmentsThe authors are thankful to the Dr. Anil S. Kotasthane, Department of Plant Molecular Biology and Biotechnology, Indira Gandhi Krishi Vishwa Vidyalaya, Raipur (C.G.) for his untiring help and assistance during the experiment and preparation of the manuscript.

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