Isolation and characterization of drought stress tolerant plant growth promoting rhizobacter from chilli crop

Received: 15-10-2020 Accepted: 07-11-2020 Abstract: Plant growth promoting rhizobacter (PGPR) are adapted to adverse conditions and protect plants from the deleterious effects of some environmental stresses. The aim of the present study, the drought tolerant plants growth promoting bacteria (PGRB) were isolate and characterized from chilli crop (CapsicumannumL). From the rhizosphere soil twelve bacteria were isolated and characterized based on their biochemical and sugar fermentation. The drought stress tolarent stains were screened by production of exo polysaccharides and the plant growth promoting activities like production of IAA and PO4 solubilization were screened. Therefore the present study report that PGRB showed good stress tolerance under drought condition in chilli crop.


Introduction
Nowadays, Plants are constantly exposed to a wide range of biotic and abiotic stresses which limit plant productivity. In world-wide Drought considered one of the most important multi dimensional stress inducing environmental factors that affect plant growth and yield, particularly in arid and semiarid regions. So, most of the work has been focused in understanding the mechanisms behind plant responses to various environmental stresses [1,2].
Approximately, one-third of the world land area is prone to drought stress which poses severe threat to plant growth and food security [3]. In India where large populations depend on agriculture for their livelihoods, growth of agriculture assumes greater importance. The Indian agriculture mainly depend on two monsoon rains namely South West Monsoon and North East Monsoon. The recent failure in monsoon rain leaves 45% of the cropping area under drought (Indian Meteorological Department 2009). Thus drought negatively affects quantity and quality of growth in plants [4]. At the initial phase of plant growth and establishment, it negatively affects seed germination, stem elongation and expansion [5]. Reduced leaf growth and in turn the leaf areas caused a marked reduction in photosynthetic rate which subsequently decreased plant growth and yield [6]. Jaleel et al., 2009 reported that severe water stress produced deleterious effects on plant water relations, photosynthesis, ion uptake and nutrient metabolism as well as assimilates partitioning [7]. Therefore, world-wide extensive research is being carried out to develop strategies to cope with drought stress through development of drought tolerant varieties, shifting the crop calendars, resource management practices and most of these technologies are cost intensive. Recent studies indicate that microorganisms can also help plants to cope with drought stress. Plant growth promoting rhizobacter (PGPR) are adapted to adverse conditions and protect plants from the deleterious effects of some environmental stresses [8][9][10]. The rhizobacteria assemblages of many agricultural crops have been studied and the use of PGPR holds promise for plant growth promotion and alleviation of plant drought stress [11][12][13]. However, the drought-tolerant bacteria associated with crop species which are naturally adapted to drought, such as Chili have not been explored. Thus, the identification of beneficial microorganisms which mediate plant growth promoting activities and drought resistance in crop plants assumes greater importance. In the current study, the drought tolerant plant growth-promoting bacteria (PGPB) were isolate and characterized from chili crop (Capsicum annum L) with following objectives.

General Methods
The following procedures were adopted for conducting various experiments during the research periods.

Cleaning of glass wares
The glass wares used in the present studies were soaked in cleaning solution (100 g of Potassium dichromate was added to 1000 ml of distilled water followed by addition of 500 ml of concentrated Sulphuric acid) for about 12 hours and washed in tap water. Finally, they were cleaned in distilled water, dried and used for the study.

Sterilization
The cleaned glass wares were sterilized at 180 ͦ C for 3 hours in a hot air oven. The media were sterilized in autoclave at 15lbs pressure (121 ͦ C) for 20 minutes.

Chemical and water
All the chemicals used in the present studies were of Analytical Reagent (AR) grade and double distilled water was used throughout the studies.

Collection of soil sample
The samples of Chilli rhizosphere soil were collected from four different locations like Cheyyar, Madipakkam, Perungalathur, Kiliyathur.

Estimation of microorganism from chilli rhizosphere soil by serial dilution technique
After, 10 grams of chilli rhizosphere soil sample was mixed with 100ml of sterilized distilled water to get 10 -1 dilution, transferred 1ml of 10 -1 to 9 ml sterilized water blank to get 10 -2 dilution were continued till 10 -6 to 10 -7 dilution. Transferred 1 ml aliquot from required dilutions to pertriplates, 10 -6 and 10 -7 dilutions were used of bacteria. The melted and cooled media was transferred to respective pre labeled plates like nutrient agar media for bacterial plates. After plating, plates were kept for incubation about 24 to 48 hours for bacteria maintained at temperature of 35ºC. From the total bacterial count plates, isolates which showed distinct colony characters were Bull. Sci. Res. 1-12 | 3 isolated in nutrient agar plates. The pure culture was obtained by streak plate method.

Morphological tests
The following morphological tests viz., cell shape and Gram reaction was carried out to characterize the isolates.

Cell shape
The purified cultures, at a log phase were observed microscopically for the cell morphological characters [14].

Gram staining
A loop full of the above culture was taken and smeared on the glass slide and heat fixed. After staining the smear was observed under oil immersion objective.

Biochemical characterization of test isolates
Physiological and metabolic characteristics of the microorganisms were assessed through biochemical tests.

Indole test
A loop full of the isolated culture was inoculated into the tryptone broth. The Kovac's reagent was added after the incubation periods of 24-48 hours to broth and shacked gently. Development of red color ring in the top layer of the tube was considered as positive result.

Methyl red reduction test
MR broth was inoculated with a test organism and incubated at 37 ͦ C for 24-48 hours. The red color was formed when the Methyl Red indicator was added in an evidence of positive result. Whereas there is no color change indicates a negative result [15].

Voges Proskauer test (VP)
VP broth was inoculated with the test organism and incubated at 37 ͦ C for 24-48 hours. The tube was shacked gently then allowed to remain undistributed for 10 to 15 minutes before the reaction is read. Formation of brownish red color indicated positive result. Whereas there is no color change indicates a negative result.

Citrate utilization test
Simmons citrate agar slant was streaked with isolated culture and incubated at 37 ͦ C for 24 to 48 hours. Color change from green to deep blue indicates positive result. The result was noted [15].

Carbohydrate fermentation test
The term sugar in microbiology denotes any fermentable substance. They may be monosaccharides, disaccharides, trisccharide, alcohols, glycosides and non-carbohydrate substances as inositol. The sugars were fermented by various types of bacteria and in that process acid and gas was produced which were indicated by the color indicated by the indicators for acid and gas collected into the inverted Durham's tube. The nutrient broth was prepared in 100 ml quantity with different carbon sources such as Dextrose, Fructose, Lactose, Maltose, Mannitol, Sucrose and Xylose transferred to test tubes with durham's tube and sterilized. The loopful of the test culture was inoculated to the broth and incubated color change from red to yellow indicates the acid production. Gas production is seen with the help of Durham's tube. If the medium reminds unchanges then the culture is a nonfermentative. Acid with gas or without gas reveals that the culture is fermentative.

Triple sugar iron test
The triple sugar iron agar medium was prepared, sterilized and dispensed into sterile test tubes. Slants were made and incubated with the test organism. The test tubes were incubated at 37 ͦ C for 24 hours. The result was observed and used for identification of the isolates.

Nitrate reduction test
Nitrate broth was prepared, sterilized and dispensed into sterile test tubes. The test organisms are inoculated into the nitrate broth. The test tubes were incubates at 37 ͦ C for 24 hours. The result was observed and used for identification of the isolates.

Catalase test
Catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water. 24 hours old test organism was taken in a sterile wooden stick or a glass rod and was immersed in a hydrogen peroxide solution. Development of immediate bubbling indicates that the organism posses catalase enzyme [16]. The result was observed and used for identification of the isolates [17].

Oxidase test
Some bacteria possess the enzymatic oxidase that forms the part of the electron transport system. The enzymes oxidase the reagent N-N tetra methyl paraphenyl diamine dihrdrochloride to coloured product indo phenols. When the growth of the organism was rubbed over the filter paper containing this reagent, purple color development. Oxidase disc was placed on the clean slide and 24 hours growth of culture was placed over the disc. The result were observed within 10 seconds .purple color indicate the positive result, no purple color change indicate negative result. The result was recorded and used for identification of the isolates.

Screening for Exo-polysaccharide
Rhizobacter isolates were grown on YMG agar medium were inoculated in YMG broth and pre incubated at 25 0 C for 24 hours. 200 μl of culture broth was inoculated into 50 ml of YMG broth and incubated at 25 0 C for 5 days at 120 rpm. Elimination of cells was followed by centrifugation (10,000 rpm for 20 min). The culture broth was mixed with 3 volumes of ethanol and after standing at 4 0 C for 24 hours, it was centrifuged at 10,000 rpm in 4 0 C for 20 min. The weight of the precipitated EPS, were measured after drying at 80 0 C for 3 days.

Screening for plant growth promoting activities of PGPR 2.7.1. Screening for IAA production
The Indole-3-acetic acid (IAA) producing rhizobacter isolates were grown in LB broth supplemented with 5 mM -1 tryptophan for 48 h and their IAA producing ability was detected according to Bric et al., 1991 [18]. The culture were centrifuged at 10,000 rpm for 15 min and 2 ml supernatant was transferred into a tube to which 100 μl of 10 mM O-phosphoric acid and 4 ml of Salkowaski reagent (1 ml of 0.5M FeCl3 in 50 ml 35% (v/v) HClO4) were added. The mixture was incubated at room temperature for 25 min. The tubes were observed for the development of pink color and their absorbance was measured at 530 nm. The IAA concentration was determined against standard curve constructed from different concentrations of indole-3-acetic acid.

Screening for phosphate solubilization
The phosphate solubilization of the rhizobacter isolates were characterized by using Pikovskaya's medium [19]. The

Bull. Sci. Res. 1-12 | 5
Pikovskaya agar plates were prepared and inoculate with 10μl of rhizobacter isolates grown in Pikovskaya broth to the exponential phase. After inoculation plates were incubating at 30°C for 3 days, formation of a clear zone around the spot was checked.

Results and discussions 3.1. Collection of rhizosphere soil
The sample of Chilli rhizosphere soils were collected from different location near by Cheyyar, Madipakkam, Perungalathur and Kiliyathur

Estimation of microorganism from chilli rhizosphere soil by serial dilution technique
The amount of bacteria present in rhizosphere soils were estimated by pour plate method and results were showed in Table 1. Among the four samples Perungalathur rhizosphere soil showed highest population bacteria (5X10 7 CFU/g), followed by Mamandur soil (4X10 7 CFU/g) and least amount of population was recorded by Cheyyar soil (1X10 7 CFU/g).

Identification of the bacterial isolates by biochemical and sugar fermentation
The isolated 12 bacterial isolates were characterized based on their biochemical and sugar fermentation.
The morphology of isolates were studied by Gram staining techniques, from the results five isolates were showed Gram negative Rods, four isolates Gram positive Cocci and three isolates gram positive rods viewed in    Table 2 Morphological and biochemical characterization of bacterial isolates Characters  ISO-1  ISO-2  ISO-3  ISO-4  ISO-5  ISO-6  ISO-7  ISO-8  ISO-9  ISO-10  ISO-11  ISO-12   1 Gram staining  The drought tolerant isolates were screened by production of exo-polysaccharide and the results were showed in Table 3. Among the 12 isolates, only 9 isolates exhibited exopolysaccharide production. The highest

Screening of plant growth promoting activities from Isolates
The plant growth promoting activities like production of IAA and PO4 solubilization were screened from the Isolates by different methods.

Estimation of IAA production from isolates
The plant growth hormone IAA (1,3-Indole Acetic Acid) producing isolates were screened and estimated their ability. The results were showed in Table.4. Among the 12 isolates, only 9 isolates can able to producing IAA. From the 9 isolates, Bacillus sp (1.36 µg\ml) exhibited highest amount of production followed by Azotobacter sp (1.29 µg\ml) and least amount of IAA production was recorded by Enterobacter sp (0.63 µg\ml)

Estimation of Phosphate solubilization from isolates
The plant growth promoting activities like PO4 solubilization was determined from the isolates and the results were tabulated in Table  5. From 12 isolates, 9 isolates only recorded zone of inhibition. Among the nine isolates Pseudomonas sp (18mm) showed highest zone of inhibition followed by Bacillus sp (17 mm) and least zone of inhibition was recorded by Enterobacter sp (01 mm) the results also shown in the     Bacillus sp 17 8 Pseudomonas sp 18 9 Pseudomonas sp 17 10 Asospirillum sp 08 11 Azotobacter sp 06 12 Enterobacter sp 01

Conclusion
The present investigation was undertaken to study the isolation and characterization of drought stress plant growth promoting rhizobacter form chili plant. There are 12 bacterial isolates were isolated and characterized based on their biochemical test and identified as Klebsiella sp, Staphylococcus sp, Bacillus sp, Serratia sp, Staphylococcus sp, Streptococcus sp, Pseudomonas sp, Streptococcus sp, Asospirillum sp, Azotobacter sp and Enterobacter sp. The drought tolerant character was screened by production of Exopolysaccharide. The highest amount was recorded by Klebsiella sp (0.41 mg/5ml) followed by Pseudomonas sp (0.33 mg/5ml) and lowest amount of Serratia sp (0.02 mg/5ml). The PGPR activities were screened by production of IAA and PO4 solubilization. The highest amount of plant growth hormone like IAA was exhibited by Bacillus sp (1.36 µg\ml) and least amount was recorded by Enterobacter sp (0.63 µg\ml). The PO4 solubilization was studied. The highest zone of inhibition was recorded by Pseudomonas sp (18mm) followed by Bacillus sp (17 mm) and least zone of inhibition was recorded by Enterobacter sp (01 mm). Therefore the present study reported that PGPR showed good stress tolerate under drought conditions in chilli crops.