Effect of bio-inoculants (Azotobacter and PSB) on rhizosphere microbial and enzymatic status in Chilli (Capsicum annuum L.) crop

Introduction

Biofertilizers are microorganisms that enrich the soil by enhancing the availability of nutrients for crops. In short, Biofertilizers are the replacement of chemical fertilizers (Bhattacharya and Mishra) [4] . They are economically and ecologically better than chemical fertilizers at improving the quality and productivity of crops by producing plant growthpromoting hormones and phytoalexins. They also reduce the application for chemical fertilizers, ensuring environmental safety. The most commonly used Biofertilizers in horticulture crops are Azotobacter (Afzal and Bano) [1] , Bacillus (Orhan et al.,) [15] , Azospirillum, and Pseudomonas (Naiman et al.,) [12] . Azotobacter is a free-living, aerobic, nitrogen-fixing, non-symbiotic heterotrophic bacteria found in soil. They can fix an average of 20 kg N -1 ha -1 year. Azotobacter also produces biologically active compounds such as phytohormones like auxins, IAA, and GA (Ahmad et al.) [2] , they also produce Siderophores and antifungal substances, thereby stimulating plant growth (Oblisami et al.) [14] . Biofertilizers Azotobacter is known to fix a substantial amount of atmospheric nitrogen (Ghoname and Shafeek) [8] . Phosphate solubilizing bacteria (PSB) are important biofertilizer that enhance the availability of phosphorus (P) to plants by converting insoluble forms of phosphorus into soluble forms that plant can absorb. Currently, PSB has attracted the attention of agriculturists as soil inoculums to improve plant growth and yield. Several studies have shown that Pseudomonas fluorescens as a soil bioinoculants improves the growth of plants by various mechanisms, like the production of antifungal compounds, siderophore production, plant hormone production and phosphate solubilization. Strains from the genera Pseudomonas, Bacillus and Rhizobium are among the most powerful phosphate solubilizers (Rodriguez) [16] . Phosphate solubilizing bacteria (PSB) and Vasicular arbuscular mycorrhizae (VAM) make more available phosphate in soil, which is supplied to the crop, enhance fertilizer use efficiency, soil fertility status, and ensure partial savings of fertilizers, which in turn reduce the cost of cultivation. The present investigation, we tried to study developing an efficient nutrient management system involving biofertilizers i.e., using beneficial Azotobacter and PSB strains to maintain soil fertility and for better crop production.

Material and Methods

An experiment was conducted that included rhizosphere microbial soil analysis of the chilli crop as well as chemical and morphological characterization of the microorganisms Azotobacter and PSB (pseudomonas). The experiment was carried out at Department of Agricultural Microbiology, college of Agriculture Raipur with seven treatments and four replications. The treatment details are T1 (Control), T2 (Azoto-122), T3 (Azoto-146), T4 (Azoto-137), T5 (Azoto-122+PSB-126), T6 (Azoto-146 + PSB-126), T7 (Azoto-137 + PSB-126). The Azotobacter and PSB isolates were obtained from departmental culture collection bank of Department of Agricultural Microbiology, for this investigation. The isolates of Azotobacter and PSB (pseudomonas) were characterized in a laboratory setting using Gram staining, colony morphology features, starch hydrolysis, catalase, urease, oxidase, TSI (triple sugar iron) test and antibiotics. Gram staining, colony morphology and colony characters were performed on cultures that were 48 hours to 7 days old. On agar medium, the colony characteristics viz. margin, elevation, size and colour were noted. Further appropriate dilution i e. 1.0 ml of Azotobacter and PSB was transferred into the Petri plates containing Jensen's media and Pikovskaya's media respectively. For 2-3 days were spent incubating the plates at 28 ± 2°C. In the gram's staining a thin and smooth bacterial smear was made on glass slides. To which we, apply crystal violet followed by washing of slide with distilled water. Gram's iodine solution was applied to each slide then to remove the iodine solution; these slides were washed in 95 percent ethyl alcohol. The slides were cleaned and drained with distilled water once more. The slides were then poured with Safranin red for 30 seconds. They were then rinsed in distilled water. Gram positive cells were looked purple, while Gram negative cells were looked pink or red (Aneja) [3] . The isolates underwent a variety of biochemical tests, including the Starch hydrolysis test, Catalase test, Urease test, Oxidase test, TSI Test (triple sugar iron test) and antibiotic teat. The enzyme amylase, which breaks down starch is detected by the starch hydrolysis method. Amylase converts starch, a polysaccharide, to maltose, a disaccharide and some monosaccharide such as glucose. Isolates were streaked singly on starch agar media and incubated at 28± 2 o C for 48 hours. After that, the plates were flooded with iodine solution to test the isolates starch digestion capacity. A yellow zone around the colony indicated starch hydrolysis, while a blue/black area indicated starch presence. For catalase test a bacterial colony was transferred on the surface of a clean, dry glass slide using a loop or sterilized wooden stick. A drop of 3% H2O2 was dropped on the slide, and the combination of quick oxygen evolution (within 5-10 seconds) as proven by bubbling indicated a positive result, whereas no bubbles or simply a few scattered bubbles indicated a negative result. In Urease test Azotobacter and PSB cultures were added to the Urea agar slants. At 37°C for 24 hours, the slants were incubated. If the bacterial growth zone on the orange-colored urease slant turns pink, urease hydrolysis activity is confirmed. The oxidase test is used to see if the isolates can make cytochrome oxidase. Azotobacter and PSB isolates were inoculated to nutrient agar medium plates which were inoculated for 24-48 hours at 37°C. After that a commercially available oxidase disc containing the reagent is placed on the plates and waited for 10 seconds to see the colour to change. Deep purple colour change was deemed positive, while a change later than 10 seconds or no colour change was considered negative. Triple Sugar iron test is used for the separation of microorganisms by the virtue of their ability to determine the use of carbohydrate fermentation and hydrogen sulphide production. The microorganisms that ferment glucose change the medium's colour from red to yellow and produce a range of acids. The Triple Sugar Iron agar medium was set in the sloped form with a butt about 1inch long slants were stabbed, incubated at 28±2ºC for 48 hours and results were observed there after (Krumwiede and Kohn) [10] . The presence of alkaline (red) slant and an acid (yellow) butt after incubation indicates that microorganisms is a glucose fermentation but are unable to ferment lactose or sucrose (or both), in addition to glucose, in large amounts acids do produce. Gas production (CO2) is seen through the presence of cracks or bubbles in the medium, when the accumulated gas is saved. The antibiotic resistance of the strain was assayed with the help of agar diffusion method using filter paper disc impregnate with the test antibiotic in different concentration (Date and hurse) [5] . The actively growing culture of the test Azotobacter and PSB isolates were prepared in Jensen's and Pikovskaya's broth media.

Sterilized petri-plates containing solidified medium were surface inoculated with 0.5ml of actively growing culture of the test strain and spread uniformly by means of a sterilized spreader Application of Biofertilizers, Seed treatment and Seed sowing Liquid biofertilizers were given to chilli crops in pots according to treatment description through soil as well as seed treatment methods. Liquid biofertilizers of Azotobacter and PSB isolates separately applied to soil in the pot before sowing @ 5 ml pot -1 ; About 24 hours before sowing, followed by light irrigation. Seeds of chilli were treated with all Azotobacter and PSB isolates, for inoculation, matured broth of Azotobacter and PSB isolates was diluted with sterilized aqueous so that 1:4 dilution was attained. In this diluted broth sugar was added @ 0.5g 100ml -1 broth as a sticking agent. Four holes (2 to 3 cm deep) per pot were made with the help of sterilized glass rod maintaining equal distance from hole to hole. Sowing of 4 seeds was done by placing one seed in each hole with the help of sterilized forceps.

Soil microbial analysis

Soil microbial analysis viz, estimation of microbial population, dehydrogenase test in soil, phosphatase activity test and basal soil respiration study were conducted in microbiology laboratory. Rhizosphere soil samples from different treatments were collected for enumeration of Azotobacter and PSB. The plating and serial dilution methods were used for enumeration (Subba Rao) [18] . Following colony counting, the populations were converted to cfu g -1 of dry soil using the formula (Schmidt and Caldwell) [17] . Dehydrogenase activity was determined with adding 0.2 ml of 3% Triphenyl Tetrazolium Chloride solution was added in each of the tubes to saturate the soil. The tubes were incubated for 24 hrs at 37ºC and then 10 ml of methanol was added to them. The tubes were shaken and left to stand for 6 hrs. Clear pink coloured supernatant was withdrawn and readings were taken with a colorimeter. (Klein et al.) [9] . The acid phosphatase activity was calculated with the experimental soil of the chilli pots' pH of 6.9 in mind. Out of two sets one was used as control. 4 ml of modified universal buffer and 0.2 ml of toluene were added. To one set of samples, 1 ml of p-nitrophenyl phosphate solution was added. After that, the flasks were incubated for an hour at 37ºC. Four ml of 0.5 M NaOH and one ml of 0.5 M CaCl2 were added following incubation. The remaining set (control) of samples received 1 ml of p-nitrophenyl phosphate. The yellow colour intensity of the filtrate was measured with a blue filter. The result obtained using the MUB (Modified Universal Buffer) of pH 6.5 was the measure of acid phosphatase (Evazi and Tabatabai) [9] . The basal respiration of soil was determined by measuring the rates of CO2 evolution. One litter conical flask was filled with 100 g of oven-dried soil. 20 ml of 0.5N NaOH were placed in test tubes. Rubber stoppers were used to keep the flasks airtight while the tubes were hung inside the conical flasks using thread. The flasks were incubated at 28°C for about 20 hrs. The NaOH solution was promptly moved from the test tube to a 150 ml conical flask following incubation. A few drops of phenolphthalein indicator were added to 5 ml of 3N BaCl2 solution. Titrate the material gradually with regular 0.5N H2SO4 until the pink hue simply goes away.

Results and Discussion

Retrieval of different strains of Azotobacter and PSB isolates

Azotobacter and PSB isolates cultures were revived by inoculating Azotobacter in Jensen's medium and PSB in Pikovskaya's medium of pH (7.0) and temperature (28±2⁰C) each. After two days of incubation, bacterial colonies were obtained, and pure cultures were preserved as slant in the refrigerator. Active culture broths of isolates were prepared and subjected to morphological and biochemical characteristics.

Colony morphology and Gram staining

The Azotobacter isolates produced translucent, nearly round, and gummy colonies which varied in size between 1.5 to 2.0 mm and were whitish in colour (Table -1). Azotobacter colony morphological characteristics were examined which revealed. After the gram-staining, the bacteria assumed a red colour, which indicated that it was a Gram (-ve) stain. Phosphorous solubulizing bacterial isolates were found capable of forming a clear zone on solid Pikovskaya's media. These were further studied for colony morphology, Gram staining. The colonies of all the isolates were found to be round and yellowish in colour. All the isolates were gram-negative. Based on the biochemical and morphological tests, the isolated bacteria were identified in consultation with Bergey's Manual of Systematic Bacteriology. PSB isolate was found to belong to Pseudomonas sp. The identification studies are also in coordination with the works done by Gadagi and Sa [7] and El-Dsouky et al. [6] .

with T7 having the highest activity (12.49 μg TPF g -1 soil h - 1 ). Above observations were in close agreement with Nowark [13] and Wyszkowska and Kucharski [19] . Who claimed that dehydrogenase activity is a reflection of the biological status of soil.

Phosphatase test

The result on Acid phosphatase activity (μg PNP g -1 soil hr - 1 ) in rhizosphere soil of pot grown chilli crop after harvesting were presented in Table-4. were significant. After harvest treatment T7 (Azoto-137+PSB-126) was recorded significantly higher acid phosphatase activity (15.11 μg PNP g -1 soil hr -1 ) followed by T6 (13.44 μg PNP g - 1 soil hr -1 ). The lower acid phosphatase activity was recorded in the treatment T1 (Control) (2.23 μg PNP g -1 soil hr -1 ). Similar findings were reported by Linu et al. [11] .

Basal soil respiration test

Basal soil respiration rate (BSR) after harvest the treatment T7 (Azoto-137+PSB-126) was recorded highest Basal soil respiration (1.07) followed by treatment T3 (0.97. The minimum Basal soil respiration was recorded in treatment T1 (Control) (0.55) presented in Table 4.

References

1. Afzal A, Bano A. Rhizobium and phosphate solubilizing bacteria improve the yield and phosphorus uptake in wheat (Triticum aestivum). J Int Agric Bio. 2008;10:85-88.
2. Ahmad F, Ahmad I, Khan M. Indole acetic acid production by the indigenous isolates of Azotobacter and fluorescent Pseudomonas in the presence and absence of tryptophan. Turkish Journal of Biology. 2005;29:29-34.
3. Aneja KR. Gram staining of bacteria. In: Experiments in Microbiology, Plant Pathology and Biotechnology. New Delhi: New Age International (P) Ltd.; 2003. p.102-105.
4. Bhattacharya P, Mishra UC. A book on biofertilizer for extension workers. Ghaziabad: National Biofertilizer Development Centre, Department of Agriculture and Cooperation, Government of India; 1994.
5. Date RA, Hurse LS. Intrinsic antibiotic resistance and serological characterization of populations of indigenous Bradyrhizobium isolated from nodules of Desmodium intortum and Macroptilium atropurpureum in three soils of SE Queensland. Soil Biology and Biochemistry. 1991;23(6):551-561.
6. El-Dsouky MM, Farida BH, Sadiek HS, Abo Baker AA. Isolation, characterization and selection of rhizobacterial strains from plant rhizosphere for use in inoculation tests. Assiut Journal of Agricultural Sciences. 2003;34(6):89-107.
7. Gadagi RS, Sa T. New isolation method for microorganisms solubilizing iron and aluminum phosphates using dyes. Soil Science and Plant Nutrition. 2002;48(4):615-618.
8. Ghoname A, Shafeek MR. Growth and productivity of sweet pepper (Capsicum annuum L.) grown in plastic houses as affected by organic, mineral and bio-N fertilizer. J Agron. 2005;4:369-372.
9. Klein DA, Loh TC, Goulding RL. A rapid procedure to evaluate the dehydrogenase activity of soils low in organic matter. Soil Biology and Biochemistry. 1971;3:385-387.
10. Krumwiede Jr C, Kohn LA. A triple-sugar modification of the Russell double-sugar medium. The Journal of Medical Research. 1917;37(2):225.
11. Linu MS, Aju K Asok, Meenu Thampi, Sree Kumar J, Jisha MS. Plant growth promoting traits of indigenous phosphate solubilizing Pseudomonas aeruginosa isolates from chilli (Capsicum annuum L.) rhizosphere. Communications in Soil Science and Plant Analysis. 2019;50(4):444-457.
12. Naiman AD, Latronico A, Garcia de Salamone IE. Inoculation of wheat with Azospirillum brasilense and Pseudomonas fluorescens: impact on the production and culturable rhizosphere microflora. Eur J Soil Biol. 2009;45:44-51.
13. Nowark J. Interactions between biodegradation tetrachlorwin-fosu and chlorfenwin-fosu but in different conditions amount of alive biomass mikroorganizmow tempearatury and humidity of soil. Zesz Sciences Stettin AR. 1996;173(63):191.
14. Oblisami G, Santanakrishna P, Pappiah CM, Shabnugavelu KG. Effect of Azotobacter inoculants and growth regulators on the growth of cashew. Acta Horticulturae (ISHS). 1979;108:44-49.
15. Orhan E, Esitken A, Ercisli S, Turan M, Sahin F. Effects of plant growth promoting rhizobacteria (PGPR) on yield, growth and nutrient contents in organically growing raspberry. Scientia Horticulturae. 2006;111(1):38-43.
16. Rodriguez H, Reynaldo F. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances. 1999;17:319-339.
17. Schmidt EL, Caldwell AC. A practical manual of soil microbiology laboratory methods. Rome: Food and Agriculture Organization of the United Nations; 1967. p.72-75.
18. Subba Rao NS. Prospect of bacterial fertilization in India. Fert News. 1974;12:11-12.
19. Wyszkowska J, Kucharski J. Biochemical and physicochemical properties of soil contaminated with herbicide Triflurotox 250 EC. Polish Journal of Environmental Studies. 2004;13(2):223-231.