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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
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Methods: Twenty two marine sediments, 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.
Result: These 14 selected strains from marine environment can be potentially used in therapeutic and plant growth applications.
MATERIALS AND METHODS
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.
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 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).
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).
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).
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).
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
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).
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).
RESULTS AND DISCUSSION
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.
Conflict of interest
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