Isolation and Characterization of Halophilic Plant Growth Promoting Rhizobacteria from Marine Sediment, Water and Coastal Sanddune Plant and It’s Screening for Plant Growth Regulators

Soils, marine water and marine sediments harbor high levels of biodiversity and support biogeochemical processes that are relating to life on earth. The soil and sediment function within food webs. The structure of these soil and sedimentary food webs, as well as their role in transforming carbon and nutrients, are often studied separately because of the habitat-centered organization of the research (Ward, et al., 2017). Ipomoea is the largest genus in the flowering plant family convol vulaceae, converting over 600 species. It is a large and diverse group, with common names morning glory, water convolvulus, sweet potato, bindweed, moonflower, etc. The soil is in contact with the plant’s roots, which may include microorganisms and their activities change its composition. Gram-negative Rhizobium fixes nitrogen in soil. In these bacteria, plant cells infiltrate root nodules; they transform ambient nitrogen into ammonia using nitrogenase and supply glutamine to the plant. Halotolerant and halophilic bacteria live in salty settings, while moderately halotolerant germs are more common (Srivastava and Rauniyar, 2020; Shidhi et al., 2021). Competence of the bacteria to respond and sustain in fluctuations of external osmolarity is note worthy function for endurance and propagation in diverse ecological niches acquired during evolution through expression of salt stress and large scale tolerant genes from the bacteria (Das et al., 2019). Isolation and classification of salt stress tolerant bacteria from the ocean environment has biotechnological significance through high production of functional bio molecules such as exopolysaccharides, hydrolytic enzymes, osmolytes, etc. Diverse halophilic and halotolerant bacteria have been found in aquatic environments and salt stress tolerant genes are being extracted. Isolation, functional screening, characterisation and molecular classification of marine salt-tolerant microorganisms are employed for agriculture (Niu et al., 2018). The food containing unabsorbed antimicrobials and secreted antimicrobial metabolites in the water and environmental sediment of salmon farming sites often retain their antimicrobial activity and can remain in the aquatic environment for variable period of time depending on their initial concentrations, biodegradability, physical and chemical characteristics (Watts et al., 2017). Studies of halo tolerant microorganisms can tolerate salinity stress and even survive in its absence. The study aimed to collect and select the most salt-tolerant microorganisms from sediment and water samples of marine environments for probing osmotic stress in the halo tolerance of crop plants. Coastal sand dunes plants are narrow and mounds of sand with clear bounds determined by the sea and landward sand movement restrictions. Globally, sand dunes encompass 6 x 10⁶ km² of land. This ecology is different from inland dunes and sea. These are natural barriers against sea waves; the seashore protects inland natural habitats or developed places. Sand dunes plants are crucial to the richness of our seashore ecosystems. These researches examine halo-tolerant rhizosphere bacteria from marine water, marine sediment and coastal sand dune ipomoea plants: PGPR, IAA, antibacterial activity and EPS (Chu et al., 2019) were isolated and characterized. Then the effects of all the strain on the above plants with respect to their protein and chlorophyll content were performed (Alejandro et al., 2020).

This experiment was conducted 2021 at the Department of Biotechnology, Dr. M.G.R. Education and Research Institute (Deemed to be University), Chennai, Tamil Nadu, India.
 
Procurement of samples
                                               
Twenty two marine sediment, marine water, coastal sandune plant samples were collected from coastal area of Marakkanam slattern, (12.1899°N, 79.9249°E) Tamil Nadu, INDIA at the depth of 10 cm. The samples were collected in sterile air tight bottles and labeled with date of collection and transported to the laboratory for further investigation.
 
Media ingredients
 
King’s B Medium: It is used for the production of pigments especially non-fluorescent pigment. Serial dilution 10 gm of soil sample diluted in 90ml of distilled water and kept in shaker for 60 mins. Further serial dilution methodology was carried out while plating for 10-3 diluted sample and 10-5 diluted sample was carried using kings B medium (Simon, et al., 2011).
 
Plant sample
 
Plant samples were taken from Marakkanam (12.2158°N, 79.9831°E). The plant sample was crushed and 95 ml of distilled water was added to 5 gm of the sample. Further serial dilutions were done and King’s B medium was used to spread and pour plates for the 10-3 diluted sample and the 10-5 diluted sample (Giuliano, et al., 2019; Gupta and Pandey, 2019).
 
Water sample
 
Marakkanam slattern, (12.1899°N, 79.9249°E) a different place was taken and serial dilution of 1gm of soil sample in 9 ml of distilled water and kept in the shaker for 60 mins. Further serial dilution methodology was carried out and spread method plating and pour method plating for 10^-3 and 10^-5 was carried out on Kings B medium (Giuliano et al., 2019; Gupta and Pandey, 2019).
 
Isolation of rhizobacteria
 
The plates were incubated at 37°C for 24 h. Obtained colonies were further sub cultured to get pure colonies. Secondary screening of fluorescent pseudomonades producing bacteria Kings B production medium was again prepared and poured into Petri plates and colonies were streaked and incubated for two days (Giuliano et al., 2019; Gupta and Pandey, 2019).
 
Screening of bacterial strains for the production of iaa
 
The selected bacterial cultures were inoculated in to nutrient broth medium with 1% of tryptone and incubated at room temperature for 2 days. The role of tryptone acts as precursor for Indole acetic acid biosynthesis. After 2 days of incubation, the grown culture was centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected to which salkowski reagent was added. IAA positive culture supernatants should give dark pink colour (Niu et al., 2018). Salkowski reagent is a mixture of 0.5 M ferric chloride (FeCl₃) and 35% perchloric acid (HClO₄) which upon reaction with IAA yields pink color, due to IAA complex formation with and reduction of Fe³⁺. After addition of reagent, the optical densities of 409 samples were recorded at 530nm using UV visible Spectrophotometer. Blank was also maintained as the negative control (Kumar et al., 2021).
 
Extracellular polysaccharides
 
Out of 480 strains, 79 strains were selected on the basis of their mucoid or sticky appearance (Surya et al., 2019). These 79 strains were then inoculated into the labelled test tubes respectively that consist of Yeast and Malt Extract with Glucose (YMG) broth. After inoculation, test tubes were kept for 4 days incubation at room temperature (Ragavan Das, 2019).
 
Antimicrobial activity
 
480 samples were sub cultured in King’s B media and was kept for overnight incubation. Vibrio culture was swabbed on 120 Nutrient agar plates in sterile environment using cotton swab (Bailey, et al., 2013). Then the sub cultured strains were inoculated on nutrient agar plates. (4 strains were inoculated on one nutrient agar plate) 4 x 120= 480. After inoculating strains, Ampicillin disc was placed at the centre on each agar plates. Allow it for incubation (37oC for 24 hrs). On overnight incubation, measure the zone of inhibition of all strains against Vibrio pathogen (Giuliano, et al., 2019).
 
Sample collection
                                                    
Samples were collected from Marakkanam (12.1899°N, 79.9831°E) Tamil Nadu, India at depth of 10 cm (Fig 1).


 
Isolation of Rhizobium bacteria
 
Colony characteristics
 
Bacteria show characteristic growth on solid media under appropriate cultural condition. The colonies may be varying in diameter, in outline (circular, wavy, rhizoid, etc.) as shown in Fig 2; elevation (flat, raised, convex, etc.) and translucency (transparent, translucent and opaque). The colony color may be varying (yellow, brown, white, etc.). In some bacteria the background (medium) may get a characteristics color (Srivastava and Rauniyar, 2020).


 
Secondary screening of rhizobium producing bacteria
 
Among the 530 isolated screened 480 isolated showed prominent pseudomonas productions in primary screening. They were subjected to secondary screening on same enrichment agar media for the consistence of the isolate rhizobium producing ability. Based on the secondary screening of the rhizobium production isolated of 480 showed prominent activities in both of the screening (Srivastava and Rauniyar, 2020). Hence the isolate 480 was taken for further analysis (Fig 3).


 
Screening of bacterial strains for the production of iaa
 
The selected bacterial cultures were injected into 1% tryptone nutrition broth for 2 days (Chu et al., 2019). Tryptone helps biosynthesize Indole acetic acid. The 2-day-old culture was centrifuged at 10,000 rpm for 10 minutes. Salkowski reagent was added to supernatant (Surya et al., 2019). 530 nm UV visible Spectrophotometer optical density of 409 samples (Chu et al., 2019). Blank was the negative control (Fig 4).


 
Extracellular polysaccharides
 
79 strains out of 480 were chosen for their mucoid or sticky look. 79 strains were put into YMG broth test tubes (Chu, et al., 2019). Test tubes were incubated at room temperature for 4 days after inoculation. Capsular EPS protects bacterial cells from unfavourable circumstances. EPS-producing bacteria hold and circulate free phosphorus to the plant. EPS-producing bacteria buffer from desiccation, adhere to surfaces and trigger plant defence in plant-microbe interactions (Simon et al., 2011).

In 480 (Fig 5) strains we get 14 potential strains which have IAA (Chu et al., 2019). EPS (Myo et al., 2019) and Antimicrobial Activity (Kose et al., 2019).


 
The potential strains have IAA, EPS and antimicrobial activity
 
The following conclusions have been drawn on the basis of present piece of work. Most of studies of pseudomonas are form bacterial but plant also found to be good source for the isolation of rhizobium. The bacteria will have better growth agent than bacterial of the pseudomonas since it has many side effects (Chu et al., 2019; Myo et al., 2019; Kose et al., 2019).

All 1-14 strains incubated IAA production, EPS production and antibacterial activity (Table 1).

The selected bacterial cultures were inoculated to nutrient broth medium with 1% of tryptone and incubated at room temperature for 2 days. The role of tryptone acts as precursor for Indole acetic acid biosynthesis. After 2 days of incubation, the grown culture was centrifuged at 10,000 rpm for 10 minutes. Supernatant was added to Salkowski reagent. Positive IAA culture supernatants should be pink. Salkowski Reagent: Dissolve 2.03 g ferric chloride in 500 mL distilled water and add 300 mL H2S04. In 480 strains, 95 are pink. After adding reagent, 480 samples’ optical density was measured at 530 nm with a UV-Spectrophotometer. Blank was a negative control. 80% of Rhizosphere bacteria make IAA, a plant growth regulator. Tryptophan is IAA’s precursor. Based on extracellular Polymeric substance (EPS), 79 strains were selected based on their mucoid or sticky appearance. These 79 strains were then inoculated into the labeled test tubes respectively that consist of YMG broth. After inoculation, test tubes were kept for 4 days incubation at room temperature.

A total of 480 samples were sub cultured in King’s B media and was kept for overnight incubation. Vibrio culture was swabbed on 120 nutrient agar plates in sterile environment using cotton swab. Then the sub cultured strains were streaked on nutrient agar plates 4 x 120= 480 each plate we cultured 4 strains (Tabe 2).



After putting the strains on the agar plates, an ampicillin disc was put in the middle of each one. Allow it to sit for 24 hours at 37°C. Measure the zone of inhibition of all the Strains against the Vibrio pathogen after letting them sit for a day.

The present study is based on the rice rhizosphere population in the marine sediment, water, coastal sand dune plant (Ipomoea spp) in Kings B medium. We tested 480 pseudomonades spp strains from rice rhizosphere soils for IAA. IAA is a phytohormone, a form of auxins, vital for plant growth and development (Ward et al., 2019).

Bacterial cells produce capsular EPS to protect themselves against unfavorable environmental conditions. The EPS producing bacteria helps to hold the free phosphorous and circulating essential nutrient to the plant. EPS-producing bacteria protect plants from desiccation, plant invasion and plant defence in plant-microbe interactions. Phosphorus is a plant macronutrient. PSB solubilize inorganic phosphorus from insoluble materials. PSB are phosphate biofertilizers (Chu et al., 2019).

For determining the phosphate solubilizing ability, the isolated bacterial strains were spot inoculated in Pikovskaya’s medium. These cultures were incubated for 5 days at room temperature. Antibacterial Isolating and screening enzyme-producing bacteria from aquatic and terrestrial environments may yield plant growth promotion samples. Antimicrobial action kills disease-causing microorganisms. Antimicrobials are utilised. Antibacterial, antifungal, or antiviral are antimicrobials. They all suppress infection in distinct ways. A Zone of Inhibition Test, also termed a Kirby-Bauer Test, measures antibiotic resistance and the ability of solids and fabrics to prevent microbial growth. This test measures and compares antimicrobial activity in fabrics, surfaces and liquids. Using a sterile swab, one million single-strain cells are distributed on an agar plate and incubated with the antimicrobial item (ex: Ampicillin disk). If the bacterial or fungal strain is susceptible to the antimicrobial agent, then a zone of inhibition appears on the agar plate. If it is resistant to the antimicrobial agent, then no zone is evident (Kose et al., 2019).

Finally we concluded that the potential isolation is from the rice rhizosphere soil. This study indicate that the potential of the bacteria to produce antimicrobial compounds which can be useful for many therapeutically applications.
 

Thus the study concludes in the present study, out of 480 strains isolated from marine sediments, marine water and coastal sand dune plants only 14 strains showed resistance in IAA, EPS and antimicrobial activity. The selected (KBMT (Soil)-1, KBMT (Water)-2, KBMT (Soil)-3, KBMT (Soil)-5, KBMDK (soil)-1, KBMT (Root)-1, KBMT (Root)-2, KBMT (Root)-3, KBMDK (Water)-3, KBMDK (Water)-4, KBMDK (Water)-6, KBMT (Root)-2, KBK (Plant)-1, KBMTR (Root)-3. All these strains were isolated from the marine sediments, marine water and coastal sand dune plants, Marakkanam Chennai. These 14 selected strains from marine environment can be potentially used in therapeutic and plant growth applications.

We gratefully acknowledge Er. A.C.S. Arun Kumar, President, Dr. M.G.R. Educational and Research Institute (Deemed to be University), Chennai for providing the necessary facilities.

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